UNIVERSIDADE ESTADUAL DO CEARÁ PRÓ-REITORIA DE PÓS-GRADUAÇÃO E PESQUISA
FACULDADE DE VETERINÁRIA PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS VETERINÁRIAS
CLÁUDIO AFONSO PINHO LOPES
UTILIZAÇÃO DA BIOTÉCNICA DE MANIPULAÇÃO DE OÓCITOS INCLUSOS EM FOLÍCULOS PRÉ-ANTRAIS PARA A AVALIAÇÃO
IN VITRO DO POTENCIAL DE ANTICORPOS ANTI-ZONA PELÚCIDA PARA A IMUNOESTERILIZAÇÃO DE CADELAS
FORTALEZA-CE 2008
CLÁUDIO AFONSO PINHO LOPES
UTILIZAÇÃO DA BIOTÉCNICA DE MANIPULAÇÃO DE OÓCITOS INCLUSOS EM FOLÍCULOS PRÉ-ANTRAIS PARA A AVALIAÇÃO
IN VITRO DO POTENCIAL DE ANTICORPOS ANTI-ZONA PELÚCIDA PARA A IMUNOESTERILIZAÇÃO DE CADELAS
Tese apresentada ao Programa de Pós-Graduação em Ciências Veterinárias da Faculdade de Veterinária da Universidade Estadual do Ceará, como requisito parcial para a obtenção do título de Doutor em Ciências Veterinárias. Área de Concentração: Reprodução e Sanidade Animal. Linha de Pesquisa: Reprodução e sanidade de carnívoros, onívoros, herbívoros e aves. Orientador: Prof. Dr. José Ricardo de Figueiredo
FORTALEZA-CE 2008
CLÁUDIO AFONSO PINHO LOPES
UTILIZAÇÃO DA BIOTÉCNICA DE MANIPULAÇÃO DE OÓCITOS INCLUSOS EM FOLÍCULOS PRÉ-ANTRAIS PARA A AVALIAÇÃO
IN VITRO DO POTENCIAL DE ANTICORPOS ANTI-ZONA PELÚCIDA PARA A IMUNOESTERILIZAÇÃO DE CADELAS
Tese apresentada ao Programa de Pós-Graduação em Ciências Veterinárias da Faculdade de Veterinária da Universidade Estadual do Ceará, como requisito parcial para a obtenção do título de Doutor em Ciências Veterinárias.
Aprovada em: _22_/_12_/_2008_ Conceito: Satisfatório “Com Louvor” Nota: 10 (Dez)
Banca Examinadora
_________________________________ Prof. Dr. José Ricardo de Figueiredo
Orientador – UECE
_______________________________ _______________________________ Profa. Dra. Maria Denise Lopes Profa. Dra. Lúcia Daniel Machado da Silva Examinadora-UNESP Examinadora-UECE
________________________________ ______________________________ Prof. Dr. Claudio Cabral Campello Prof. Dr. José Roberto Viana Silva Examinador-UECE Examinador-UFC
Aos animais
DEDICO
AGRADECIMENTOS
Agradeço à Universidade Estadual do Ceará (UECE) por toda a formação
profissional de qualidade proporcionada nos últimos doze anos, que culmina neste
momento com a conclusão do curso de doutorado em Ciências Veterinárias.
Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
pela bolsa de estudos para a realização do curso de doutorado, que foi muito importante
para o êxito deste trabalho.
À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) pela
bolsa para a realização de treinamento e estudos no exterior, que possibilitaram um
importante aprimoramento desta tese, além do incremento de minha qualificação e
experiência profissionais.
À Universidade de Brasília (UnB, Instituto de Biologia, Laboratório de
Microscopia Eletrônica) e ao Leibniz Institute for Zoo and Wildlife Research (IZW,
Berlim, Alemanha), pela oportunidade de treinamento e de realização de estudos.
Aos professores do Programa de Pós-Graduação em Ciências Veterinárias
(PPGCV) pelos ensinamentos e apoio prestados. Aos funcionários do PPGCV, e, em
especial, às secretárias Adriana Albuquerque e Cristina Sabóia do Nascimento, por toda
assistência sempre provida com atenção e cordialidade.
Agradeço a Deus pelo amor, força e orientação para prosseguir durante esta
jornada que se encerra e naquelas que estão por vir.
Aos meus amados pais Francisco Heine Maia Lopes e Suely Pinho Lopes por
todo o amor e dedicação sempre fundamentais.
À minha amada irmã Cláudia Valéria Pinho Lopes pelo amor, amizade,
orientação e alegria.
Ao meu querido orientador e amigo Prof. Dr. José Ricardo de Figueiredo por
todos os ensinamentos, pela confiança, pela atenção, pela compreensão, pelo apoio
incondicional, pela amizade, pelo exemplo de pesquisador competente e pessoa íntegra.
Aos Professores Dra. Sônia Nair Báo, Dra. Katarina Jewgenow e Dr. Claudio
Cabral Campello, pelos valiosos ensinamentos, pela confiança, pelo apoio e pela
amizade.
Aos Professores Dra. Maria Denise Lopes, Dra. Lúcia Daniel Machado da Silva
e Dr. José Roberto Viana Silva, por terem gentilmente aceito o convite para aprimorar
com sua competência este trabalho.
Aos companheiros do Laboratório de Manipulação de Oócitos e Folículos Pré-
antrais (LAMOFOPA) da UECE pela colaboração e pela amizade, e, em especial, a
Juliana Jalles de Holanda Celestino, Dra. Maria Helena Tavares de Matos, Márcia
Viviane Alves Saraiva, Ana Kelen Felipe Lima, Dra. Ana Paula Ribeiro Rodrigues,
Anelise Maria Costa Vasconcelos Alves, Mônica Aline Parente Melo, Fabrício de Sousa
Martins, Guilherme Ferreira de Sousa Meneses, Karla Daniely dos Santos Bastos, José
Erisvaldo Maia Júnior, Isabel Bezerra Lima-Verde, Dra. Liliam Mara Trevisan Tavares
e Jamily Bezerra Bruno.
Aos companheiros do Laboratório de Microscopia Eletrônica da UnB, Victor
Hugo da Silva Tibúrcio, Bruno Fiorillo, Bruno Arrivabene Cordeiro, Shélida Braz
Vasconcelos, Carolina Aquino Luque, Leonora Tavares Bastos, João Victor, Larissa
Cunha, Suzi, Elaine Porfírio, Débora, Khesller Name e Wellington.
Aos companheiros do IZW, Romy Waurich, Maxi Pöschmann, Birgit Bieber,
Karin Müller, Christiane Franz, Sigrid Holz, Jennifer Ringleb, Mirja Faβbender, Ulrike
Jakop, Sebastian Waldau, Antje Frank, Katrin Paschmionka, Marlies Rohleder, Beate
Braun, Yuyu Niu, Yvonne Meyer-Lucht, Aines Castro-Prieto, Rafael Prieto, Fabiano
Fernandes, Francisca Robles, Jan Axtner, Nina Schwensow, Jennifer Schön, Alexandra
Weyrich, Roland Frey, Simone Sommer, Joseph Saragusty, Heribert Hofer, Silke Ehle,
Beate Peters-Mergner, Wolfgang Richter, Steven Seet, Arne Ludwig, Wolfgang Tauche
e Steffen Berthold.
A todos que não foram aqui mencionados, mas que, direta ou indiretamente,
contribuíram para a realização deste trabalho.
RESUMO
O objetivo deste estudo foi avaliar o potencial de anticorpos anti-zona pelúcida para a
imunoesterilização de cães, através da análise da capacidade de antisoro produzido por
imunização de coelhos com zona pelúcida porcina (pZP) de promover a atresia de
folículos pré-antrais (FOPA). Para este propósito, utilizou-se a biotécnica de
manipulação de oócitos inclusos em folículos pré-antrais para se estabelecer um modelo
experimental in vitro, compreendendo métodos de preservação e cultivo in vitro de
FOPA caninos. Na Fase I, avaliaram-se os efeitos da temperatura (4, 20 ou 38°C), meio
(salina fisiológica ou Meio Essencial Mínimo – MEM) e tempo (2, 6, 12 ou 24 h) de
armazenamento sobre a morfologia e viabilidade folicular. As técnicas de histologia
clássica e microscopia eletrônica de transmissão, e a análise de viabilidade utilizando-se
Azul de Tripan e marcadores fluorescentes (calceína-AM e etídio homodímero-1) foram
utilizadas para essa finalidade. O uso de MEM a 4ºC apresentou-se como o método
mais eficiente, possibilitando a preservação de percentuais de folículos viáveis com
ultraestrutura íntegra similares ao controle fresco por até 12 h. Na Fase II, avaliou-se a
eficiência dos crioprotetores dimetil sulfóxido, etileno glicol, glicerol e 1,3-propanodiol
para a criopreservação de FOPA caninos através de congelação lenta. Observou-se que
o dimetil sulfóxido (DMSO) na concentração de 1,5 M possibilitou a preservação dos
maiores percentuais de FOPA viáveis e com ultra-estrutura intacta após a
descongelação. Na fase III, avaliou-se o efeito de soro anti-pZP sobre a viabilidade de
FOPA caninos cultivados in vitro durante 24 h, e também sobre a ligação de
espermatozóides caninos a óocitos homólogos. Utilizaram-se FOPA frescos ou
criopreservados com 1,5 M DMSO, que apresentaram resultados similares. Observou-se
que a adição de 10% de soro anti-pZP ao meio de cultivo não teve efeito sobre folículos
com diâmetro inferior a 100 µm em relação aos controles (ausência de soro ou adição de
10% de soro pré-imune). Entretanto, o soro anti-pZP causou a atresia de FOPA com
diâmetros superiores a 100 µm, conforme avaliado com calceína-AM e etídio
homodímero-1. Este soro a 10% mostrou-se capaz também de inibir a ligação de
espermatozóides à zona pelúcida canina. Desta forma, demonstrou-se in vitro o
potencial do soro anti-pZP para a imunoesterilização (através da eliminação de FOPA) e
para a imunocontracepção (através do bloqueio da ligação de espermatozóides aos
oócitos) de cães.
Palavras-chave: Imunocontracepção. Zona pelúcida. Folículos pré-antrais. Cães.
ABSTRACT
The aim of this study was do evaluate the potential of anti-zona pellucida antibodies for
immunosterilization in dogs, through analyzing the capability of anti-porcine zona
pellucida (pZP) antibodies to cause atresia of preantral follicles (PF). For this purpose,
the biotechnique of manipulation of oocytes enclosed in preantral follicles was
employed to establish an in vitro experimental model, comprising preservation and in
vitro culture methods for canine PF. In Phase I, the effects of storage temperature (4, 20
ou 38°C), medium (saline solution or Minimum Essential Medium – MEM) and time (2,
6, 12 ou 24 h) on follicular morphology and viability were evaluated. Classical
Histology and transmission electron microscopy, and viability analysis using Trypan
Blue and fluorescent labels (calcein-AM and ethidium homodimer-1) were used.
Storage in MEM at 4ºC was the most efficient method, which enabled preservation of
the percentages of viable follicles with intact ultrastructure similar to the fresh control
for up to 12 h. In Phase II, the efficiency of the cryoprotectants dimethyl sulfoxide,
ethylene glycol, glycerol and 1,2-propanediol for the cryopreservation of canine PF
through slow freezing was assessed. It was observed that dimethyl sulfoxide (DMSO) at
1.5 M provided the preservation of the highest percentages of viable PF with intact
ultrastructure after thawing. In phase III, the effects of anti-pZP serum on viability of
canine PF cultured in vitro for 24 h, and also on canine sperm binding to homologous
oocytes was evaluated. Fresh PF as well as PF cryopreserved in 1.5 M DMSO were
used and results between then were similar. Supplementation of culture medium with
10% anti-pZP serum had no effect on follicles smaller than 100 µm as compared to
controls (absence of serum or supplementation with pre-immune serum). However, anti-
pZP serum led PF with diameter larger than 100 µm to degenerate, as detected using
calcein-AM and ethidium homodimer-1. Anti-pZP serum also blocked sperm binding to
canine ZP. In conclusion, it was demonstrated in vitro the potential of anti-pZP serum
for immunosterilization (through induction of atresia to PF) and for
immunocontraception (through sperm binding blocking) in dogs.
Keywords: Immunocontraception. Zona pellucida. Preantral follicles. Dogs.
LISTA DE FIGURAS
Capítulo I
Figura 1. Esquema representativo do processo reprodutivo. 43
Capítulo II
Figura 1. Design of experiment I. 63
Figura 2. Histological features of stored canine ovarian fragments. 67
Figura 3. Effects of medium, temperature and time of storage on the
percentages of morphologically normal canine preantral
follicles. 68
Figura 4. Electron micrographs of normal follicles from control group and
stored in MEM at 4°C for 12 h. 70
Figura 5. Ultrastructure of follicles stored in MEM at 4°C for 24 h. 71
Figura 6. Electron micrographs of follicles stored in saline solution at 4°C
for 24 h. 72
Figura 7. Viability assessment of canine preantral follicles using trypan
blue dye exclusion test. 73
Figura 8. Viability assessment of canine preantral follicles using
fluorescent probes. 76
Figura 9. Percentages of viable canine preantral follicles in fresh ovaries
and after storage in MEM at 4ºC for 12 h or 24 h as assessed by
trypan blue dye exclusion test and labeling with calcein-AM and
ethidium homodimer. 77
Capítulo III
Figura 1. Percentages of morphologically normal preantral follicles in
ovarian tissue before (control) and after exposure and freezing-
thawing tests in medium containing 1.5 M EG or GLY. 90
Figura 2. Electron micrographs of canine preantral follicles from control
and frozen-thawed using 1.5 M EG or GLY. 93
Capítulo IV
Figura 1. Histological features of canine ovarian fragments before and
after freezing-thawing with 1.5 M DMSO or PROH. 108
Figura 2. Percentages of morphologically normal preantral follicles in
ovarian tissue before (fresh control) and after exposure and
freezing-thawing tests in medium containing 1.5 M DMSO or
PROH or without cryoprotectants (exposure and freezing
controls). 109
Figura 3. Electron micrographs of canine preantral follicles from control
and frozen-thawed using 1.5 M DMSO or PROH. 111
Figura 4. Viability assessment of canine preantral follicles using
fluorescent probes. 113
Figura 5. Percentages of viable canine preantral follicles in fresh ovaries
and after exposure and freezing-thawing using 1.5 M DMSO as
assessed by trypan blue dye exclusion test and labeling with
calcein-AM and ethidium homodimer. 114
Capítulo V
Figura 1. Percentages of viable canine preantral follicles before (fresh
control time 0 h) and after in vitro culture for 24 h with in
medium containing no serum or 10% pre-immune serum
(control) or anti-pZP. 128
Figura 2. Viability evaluation of canine preantral follicles using
fluorescent probes. 129
Figura 3. Assessment of anti-pZP antibodies ability to inhibit canine
oocyte-sperm interaction. 130
Figura 4. Assessment of the contraceptive efficiency of anti-porcine zona
pellucida (pZP) antibodies in dogs using an in vitro test for
canine sperm binding to homologous ZP. 131
LISTA DE TABELAS
Capítulo II
Tabela 1. Percentages (viable/total) of viable canine preantral follicles in
control and after storage. 74
Capítulo III
Tabela 1. Degenerated preantral follicles (%) in control and after exposure
and freezing-thawing tests. 91
Capítulo IV
Tabela 1. Percentages of viable follicles in the fresh control and after
exposure to cryoprotectants and slow freezing-thawing as
assessed using trypan blue. 112
LISTA DE ABREVIATURAS E SIGLAS
A Antro
ANOVA Analysis of variance (Análise de variância)
ATP Adenosina trifosfato
bm basement membrane (membrana basal)
BrdU Bromo-deoxiuridina
BSA Bovine serum albumin (Albumina sérica bovina)
CAPES Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
CCZ Centro de Controle de Zoonoses
CG Células da granulosa
CGP Células germinativas primordiais
CNPq Conselho Nacional de Desenvolvimento Científico e Tecnológico
CT Células da teca
DMSO Dimetilsulfóxido
DNA Deoxyribonucleic acid (Ácido desoxirribonucléico)
EG Etilenoglicol
er endoplasmic reticulum (retículo endoplasmático)
FOPA Folículo ovariano pré-antral
FSH Follicle stimulating hormone (Hormônio folículo estimulante)
GC Granulosa cells (Células da granulosa)
GLY Glycerol (glicerol)
GnRH Gonadotrophin releasing hormone (Hormônio liberador de gonadotrofinas)
h Horas
HC Histologia clássica
HE Hematoxilina-eosina
ICC Instituto Central de Ciências
IVM in vitro maturation (maturação in vitro)
IZW Institut for Zoo and Wildlife Reseach
l Lipid droplet (gota lipídica)
L Litro
LAMOFOPA Laboratório de Manipulação de Oócitos e Folículos Ovarianos Pré-
Antrais
LFCR Laboratório de Fisiologia e Controle da Reprodução
M Molar
MEM Meio essencial mínimo
mL Mililitro
mM Milimolar
MNPF Morphologically normal preantral follicles (Folículos pré-antrais
morfologicamente normais)
MOIFOPA Manipulação de oócitos inclusos em folículos pré-antrais
mRNA Ácido ribonucléico mensageiro
n número de amostras
NUBIS Núcleo de Biotecnologia de Sobral
PAS Periodic Acid Schiff (Ácido periódico de Schiff)
PBS Phosphate-buffered saline (Salina tamponada com fosfato)
PROH 1,2-Propanediol (1,2-Propanodiol)
pZP Zona pelúcida porcina
OMS Organização Mundial da Saúde
RT Room temperature (temperatura ambiente)
sc stromal cells (células do estroma)
SEM Standard error of the mean (Erro padrão da média)
SNK Student-Newman-Keuls
TB Trypan Blue (Azul de Tripan)
TEM Transmission Electron Microscopy (Microscopia Eletrônica de
Transmissão)
UECE Universidade Estadual do Ceará
UFC Universidade Federal do Ceará
UnB Universidade de Brasília
v Vesicles (vesículas)
ZP Zona pellucida (Zona pelúcida)
μg Micrograma
μL Microlitro
μm Micrômetro
μM Micromolar
ºC Graus Celsius
SUMÁRIO
1. INTRODUÇÃO 16
2. REVISÃO DE LITERATURA
2.1 A superpopulação de cães 18
2.2 Contracepção para cadelas 19
2.3 Imunocontracepção 20
2.4 Ovário mamífero 23
2.5 Oogênese e foliculogênese 23
2.6 População folicular ovariana 24 2.7 A biotécnica de MOIFOPA 25 2.8 Preservação de FOPA durante o transporte de ovários 26
2.9 Criopreservação 27 2.10 Cultivo in vitro de folículos ovarianos 32
3. JUSTIFICATIVA 35 4. HIPÓTESES CIENTÍFICAS 37 5. OBJETIVOS 38 6. CAPÍTULO I
39
Imunocontracepção em mamíferos com ênfase no controle populacional de cães
7. CAPÍTULO II 58Preservação de folículos pré-antrais caninos por períodos curtos: efeitos da temperatura, meio e tempo
8. CAPÍTULO III 83Criopreservação de folículos pré-antrais caninos utilizando-se etilenoglicol e glicerol
9. CAPÍTULO IV 99Criopreservação bem-sucedida de folículos pré-antrais caninos utilizando-se dimetil sulfóxido e 1,3-propanodiol
10. CAPÍTULO V 121Potencial de anticorpos anti-zona pelúcida para a imunoesterilização de cães: efeitos sobre folículos pré-antrais in vitro
11. CONCLUSÕES 138 12. PERSPECTIVAS 139 13. REFERÊNCIAS BIBLIOGRÁFICAS 140
16
1 INTRODUÇÃO
A superpopulação de cães constitui um grave problema em diversas cidades do
mundo, caracterizado pela existência de grandes conjuntos de animais sem
responsáveis, que podem compor um importante reservatório de zoonoses. Neste
contexto, as autoridades sanitárias realizam a eliminação sistemática destes animais
como estratégia de controle populacional. Todos os anos, esta situação resulta na
eutanásia de milhões de animais sadios, além do dispêndio de um elevado volume de
recursos econômicos estimados na ordem de bilhões de dólares (Frank e Carlisle-Frank,
2007). Esta abordagem, no entanto, é estritamente paliativa, por não atuar sobre a
origem do problema, que consiste em elevadas taxas de natalidade dos cães. Além
disso, a eliminação dos animais sem responsáveis, associada à sua má qualidade de
vida, faz emergirem questões éticas de bem-estar animal, que definem a necessidade de
novas estratégias de ação. Assim, um sistema de controle populacional para cães, para
ser efetivo, racional e humanitário, deve basear-se no controle de natalidade.
Apesar dos esforços despendidos nos últimos anos para o desenvolvimento de
métodos farmacológicos e químicos para a esterilização cães, as técnicas cirúrgicas têm
se mantido como os principais procedimentos para este propósito (Howe, 2006).
Entretanto, estes métodos demandam muito tempo e possuem alto custo financeiro para
aplicação em escala populacional (Kutzler e Wood, 2006), sendo assim logística e
economicamente inviáveis para uso em populações caninas numerosas, como no caso
das metrópoles.
Uma nova abordagem para o desenvolvimento de métodos contraceptivos,
denominada imunocontracepção, surge como uma importante perspectiva para o
controle reprodutivo canino em larga escala. Esta modalidade contraceptiva tem como
princípio induzir o sistema imunológico à síntese de anticorpos específicos para
moléculas bioativas do aparelho reprodutor que, uma vez revestidas por tais anticorpos,
têm sua função cessada, interrompendo-se assim o processo reprodutivo. Em
determinadas situações, pode ocorrer a eliminação da população de células germinativas
e conseqüente esterilidade, denominando-se este processo imunoesterilização.
Estudos têm demonstrado que a vacinação com proteínas da zona pelúcida (ZP),
estrutura que circunda os óocitos, resulta em infertilidade prolongada em diversas
espécies de mamíferos. Sugere-se que este efeito decorre do bloqueio da ZP à ligação de
17
espermatozóides pelos anticorpos produzidos. A resposta imunológica aos antígenos da
ZP pode ainda causar a eliminação dos oócitos (Ringleb et al., 2004).
Nesta tese, foi avaliado o potencial de anticorpos anti-ZP para a
imunoesterilização de cadelas, através da análise da capacidade de um antisoro
produzido por imunização de coelhos com ZP porcina (pZP) para promover a atresia de
folículos pré-antrais caninos. Para este propósito, a biotécnica de manipulação de
oócitos inclusos em folículos pré-antrais (MOIFOPA) foi utilizada para se estabelecer
um modelo experimental in vitro. Uma vez que os trabalhos relacionados ao uso desta
técnica em cães são bastante escassos e limitados à avaliação de métodos de maturação,
avaliaram-se neste estudo protocolos de preservação de folículos pré-antrais caninos
durante o período compreendido entre a coleta dos ovários e o uso no laboratório, além
da criopreservação utilizando-se diferentes crioprotetores para o armazenamento de
folículos a longo prazo. Por último, os folículos preservados pelos métodos
estabelecidos neste trabalho foram cultivados in vitro para a avaliação dos efeitos dos
anticorpos anti-ZP.
Para uma melhor compreensão dos estudos realizados nesta tese, segue-se uma
revisão de literatura abordando diversos temas relacionados à superpopulação de cães, à
imunocontracepção e à biotécnica de MOIFOPA.
18
2 REVISÃO DE LITERATURA
2.1 A superpopulação de cães
A domesticação do cão ocorreu, segundo estudos arqueológicos, na Eurásia, há
cerca de 14.000 anos. Canídeos selvagens passaram a adentrar a área dos acampamentos
humanos, para se alimentarem com sobras de comida, o que fez com que
estabelecessem, paulatinamente, um convívio harmonioso com o homem. Em
contrapartida, esses animais passaram a auxiliar na defesa dessas comunidades e na
caça. Este vínculo de lealdade fora rompido unilateralmente pelo homem quando, tendo
adotado o padrão urbano de assentamento, passou a permitir a reprodução deliberada de
seus cães, não assumindo a responsabilidade pela vida dos animais das numerosas
ninhadas, que são abandonados nas ruas (World Society for the Protection of the
Animals, 1999).
2.1.1 Dinâmica da população de cães
O conjunto de cães sem responsáveis que vivem nos espaços públicos constitui
um importante reservatório de zoonoses, o que leva as autoridades de saúde a realizarem
a eliminação sistemática destes animais. Entretanto, de acordo com a Organização
Mundial da Saúde (OMS), não existe nenhuma prova de que a eliminação de cães possa
gerar um impacto significativo sobre as densidades populacionais desses animais
(World Health Organization, 1992). Assim, os sistemas de controle populacional de
cães baseados em apreensão sistemática e eutanásia em grande escala têm sido adotados
em várias partes do mundo erroneamente, em função do desconhecimento sobre a
composição e a dinâmica da população canina (World Health Organization, 1990).
Qualquer redução no tamanho da população canina, resultante do aumento da
taxa de mortalidade, é rapidamente compensada pelo aumento da taxa de sobrevivência
das frações remanescentes, que terão melhor acesso aos recursos ambientais disponíveis
(Wandeler et al., 1988). Além disso, a taxa de reposição de animais sobrepõe facilmente
a taxa de eliminação, sendo que, em relação a esta, a mais elevada registrada até hoje é
de aproximadamente 15% da população canina (World Health Organization, 1992).
As populações de cães errantes são mantidas a partir dos filhotes em excesso dos
animais domiciliados (que possuem elevadas taxas de sobrevivência, por receberem
abrigo e alimento antes de serem abandonados às ruas), e não pelas proles dos próprios
19
animais errantes, que apresentam taxas de sobrevivência muito reduzidas (Wandeler et
al., 1988). Desta forma, um sistema de controle populacional de cães, para ser efetivo,
deve basear-se na contenção da natalidade, através de programas de controle
reprodutivo.
Modelos matemáticos de dinâmica populacional demonstram que, para espécies
que se reproduzem através de sistemas poligâmicos de acasalamento, como a canina e a
felina, o controle da reprodução de 65% das fêmeas pode permitir a estabilização de
uma determinada população. Por outro lado, a contracepção direcionada aos machos
deve abranger mais de 95% desses animais para que se obtenha o mesmo efeito, o que é
inviável em termos práticos (Jewgenow et al., 2006). Neste contexto, sistemas de
manejo demográfico para cães devem ter como foco o controle reprodutivo das fêmeas.
2.2 Contracepção para cadelas
Os métodos contraceptivos atualmente disponíveis para o controle reprodutivo
canino não são adequados para utilização em escala populacional por razões
econômicas, operacionais, culturais e/ou de segurança.
O confinamento de cadelas durante o estro é considerado um método bastante
satisfatório, por sua simplicidade e por não envolver custo algum. Contudo, em várias
localidades, sua aplicação é dificultada por questões culturais, não se podendo contar
com a cooperação efetiva das comunidades, sobretudo as de baixa renda (World Health
Organization, 1992). Durante a execução de um programa de controle populacional de
cães e gatos realizado em vilas rurais do noroeste do Estado do Paraná, observou-se a
existência de um contigente substancial de pessoas que demonstraram pouca
preocupação com o controle reprodutivo de seus animais. Cerca de um terço dos
proprietários de animais de companhia dessas localidades não aderiu ao projeto, o que
sugere que a consciência acerca da importância da posse responsável depende do nível
educacional da comunidade (Molento et al., 2005).
O controle reprodutivo de cadelas pode ser realizado também através de métodos
farmacológicos. O uso de progestágenos, dentre os quais se destacam o megestrol, a
medroxiprogesterona e a proligestona, é o recurso mais utilizado. Estes compostos
consistem em esteróides sintéticos, que atuam através de retroalimentação negativa
sobre a secreção do hormônio liberador de gonadotrofinas (GnRH) e controle da
liberação das gonadotrofinas pela hipófise, com supressão da esteroidogênese e da
20
gametogênese (Rodrigues e Rodrigues, 2005). Diversos efeitos adversos têm sido
associados à utilização prolongada de progestágenos. A ocorrência de neoplasias
mamárias, alterações clínico-patológicas características de diabetes mellitus e letargia
foi reportada em cadelas após o uso de megestrol. Por sua vez, uma elevada incidência
de hiperplasia endometrial cística e supressão adrenocortical têm sido relatada em
conseqüência do tratamento com medroxiprogesterona (Kutzler e Wood, 2006). Selman
et al. (1995) descreveram alterações histopatológicas em diversos órgãos de cadelas
tratadas com proligestona, sendo notáveis a atrofia do córtex da glândula adrenal,
tumores mamários e vacuolização das células das ilhotas de Langerhans. Além disso,
Selman et al. (1996) demonstraram a afinidade da proligestona com receptores de
glicocorticóides, o que pode resultar em supressão do eixo hipotalâmico-hiposifário-
adrenocortical e resistência à insulina.
A esterilização cirúrgica de cadelas, comumente realizada através de
ovariohisterectomia, possui um custo financeiro elevado e demanda muito tempo, sendo
assim inadequada em termos operacionais para a aplicação em populações caninas
muito numerosas (Kutzler e Wood, 2006). Ademais, complicações pós-operatórias
como hemorragia, piometra e/ou granuloma do coto, fistulações, adesões peritoniais e
incontinência urinária têm sido relatadas, embora sejam facilmente prevenidas por uma
técnica cirúrgica realizada com rigor (Howe, 2006). Apesar de seu limitado potencial
para a redução das taxas de natalidade caninas, este método ainda se constitui na melhor
alternativa para o controle da reprodução de cães.
Neste contexto, faz-se urgente o desenvolvimento de agentes esterilizantes
inócuos injetáveis, para que seja possível o controle reprodutivo de cães de modo
efetivo (World Health Organization, 1992).
2.3 Imunocontracepção
A imunocontracepção consiste em induzir o sistema imunológico a neutralizar
elementos de importância estrutural e funcional envolvidos na reprodução. Antígenos
presentes na superfície dos espermatozóides, as proteínas da ZP, as gonadotrofinas
(FSH e LH), seus receptores específicos e seu hormônio liberador (GnRH) têm sido
utilizados como alvos para a elaboração de vacinas contraceptivas para carnívoros.
Atualmente, os melhores resultados têm sido obtidos através de imunização com ZP,
21
que tem se mostrado capaz de promover contracepção por longos períodos em diversas
espécies (Jewgenow et al., 2006).
A ZP consiste em uma matriz glicoprotéica extracelular que circunda os oócitos
de mamíferos e desempenha funções fundamentais no processo de fecundação através
da regulação da ligação de espermatozóides, indução da reação acrossômica e do
bloqueio à polispermia (Wassarman, 2008). Ao microscópio eletrônico, a ZP é vista
como uma rede entrelaçada com fenestrações, apresentando uma camada interna
compacta com pequenos poros, e uma camada externa frouxamente arranjada com
grandes poros (Greve e Wassarman, 1989).
A vacinação ativa com proteínas da ZP suprime a fertilidade em diversas
espécies por interferência na interação oócito-espermatozóides durante a fecundação,
além de afetar a população de folículos ovarianos em crescimento (Ringleb et al., 2004).
Diversos estudos têm demonstrado a ocorrência de alterações patológicas em folículos
após a vacinação com antígenos da ZP, sendo encontradas evidências que indicam o
envolvimento da resposta imunológica celular nesses processos (Millar et al., 1989;
Rhim et al.,1992; Lou and Tung, 1993; Jackson et al., 1998; Lou et al. 2000). Millar et
al. (1989), utilizando um anticorpo monoclonal para a ZP3 murina (mZP3) com
reconhecida capacidade de bloquear a fecundação, identificou uma seqüência de sete
aminoácidos na referida proteína, que consistiria de um epítopo para linfócitos B, e o
testou como antígeno imunocontraceptivo. Um peptídeo de 16 aminoácidos
incorporando a referida seqüência foi sintetizado e utilizado para imunizar ativamente
fêmeas de camundongo. Os anticorpos produzidos determinaram contracepção
duradoura na ausência de histopatologia do ovário e citotoxicidade celular. Estes autores
sugeriram que tais resultados deviam-se à ausência de epítopos para linfócitos T na
referida seqüência.
A redução do número de folículos primordiais após imunização ativa contra uma
das glicoproteínas zonárias (ZP3) é uma consequência comum (Aitken, 2002). Apesar
dos diversos indícios de mecanismos imunológicos celulares para a patogênese das
alterações de folículos ovarianos, em nenhum dos estudos em que foi verificada
depleção de folículos primordiais, observou-se a infiltração de linfócitos T (Kerr et al.,
1998). Uma explicação para esse fenômeno consiste no recrutamento acelerado de tais
folículos para compor a população de folículos em crescimento que é eliminada devido
à ação de anticorpos anti-ZP (Skinner et al., 1984). Alternativamente, sugere-se que
uma proporção significativa de folículos primordiais expressa a proteína ZP3, sendo
22
destruídos por anticorpos anti-ZP por fixação de complemento (Grootenhuis et al.,
1996). Gwatkin et al. (1977) localizaram anticorpos e complemento na ZP de oócitos de
camundongo inférteis imunizados com ZP de hamster. Outro possível mecanismo pode
consistir da interrupção da comunicação entre oócitos e células da granulosa via junções
do tipo ‘gap’ obstruídas por grandes quantidades de anticorpos anti-ZP aderidos ( Mahi-
Brown et al., 1988).
Barber et al. (2001) demonstraram através de imunohistoquímica ultra-estrutural
que anticorpos anti-ZP são capazes de se ligar a proteínas zonárias presentes no
aparelho de golgi de oócitos caninos em folículos pré-antrais. Além disso, a expressão
da proteína codificada pelo gene ZPA foi detectada no citoplasma de oócitos caninos
inclusos em folículos primordiais, enquanto em folículos primários e secundários foi
observada a transcrição de mRNA a partir dos genes ZPA, ZPB e ZPC (Blackmore et
al., 2004). Um padrão semelhante de expressão de proteínas da ZP foi descrito por
Jewgenow e Fickel (1999) em felinos. Assim, nestas duas espécies de carnívoros, os
mecanismos descritos acima para eliminação de folículos primordiais podem se
processar, estabelecendo-se assim o potencial de anticorpos anti-ZP para a
imunoesterilização desses animais.
Na maioria dos estudos sobre imunocontracepção baseada na ZP, as
glicoproteínas da pZP têm sido utilizadas devido à sua alta disponibilidade em
matadouros e ao elevado grau de homologia com as proteínas da ZP de diversas
espécies mamíferas, o que garante a reatividade cruzada dos anticorpos produzidas com
a ZP nativa (Niu et al., 2006).
Até o presente, o estudo da ação de anticorpos anti-ZP sobre oócitos tem sido
realizado utilizando-se apenas modelos experimentais in vivo. Entretanto, o uso de um
sistema de cultivo in vitro de folículos ovarianos pode proporcionar uma análise mais
precisa do processo de eliminação dos referidos gametas. Neste contexto, a biotécnica
MOIFOPA pode prover os métodos necessários ao desenvolvimento de um modelo
experimental in vitro para este propósito.
No capítulo I, constituído por um artigo de revisão de literatura sobre métodos
imunocontraceptivos, mais informações detalhadas acerca desta estratégia de
contracepção podem ser obtidas.
23
2.4 Ovário mamífero
O ovário mamífero é composto por muitos tipos de células diferenciadas, que
trabalham em conjunto promovendo um ambiente ideal para o desempenho de suas
funções endócrinas e gametogênica (Bristol-Gould e Woodruff, 2006). O ovário é
constituído por duas regiões: cortical e medular. O córtex ovariano, localizado na parte
mais externa, é circundado pelo epitélio germinal e corresponde à região funcional do
órgão, sendo formado por tecido conectivo (fibroblastos, colágeno e fibras reticulares),
folículos ovarianos e corpos lúteos em vários estádios de desenvolvimento ou em
regressão (Liu et al., 2006). A região medular, localizada mais internamente, é
constituída por tecido conjuntivo, células musculares lisas, nervos, vasos sanguíneos e
linfáticos responsáveis pela nutrição e estruturação do ovário (Hafez, 1996).
2.5 Oogênese e foliculogênese
A oogênese consiste na formação e diferenciação do gameta feminino que se
desenvolve em várias fases e tem início na vida fetal com as células germinativas
primordiais (CGP) e culmina com a formação do oócito haplóide fecundado (Bristol-
Gould e Woodruff, 2006). Em mamíferos, nos estádios iniciais do desenvolvimento
ovariano, ocorre a migração das células germinativas primordiais (CGP) do saco
vitelínico para a gônada primitiva com sua posterior colonização (Eppig et al., 2004).
Imediatamente após a diferenciação das gônadas, ocorre a transformação das CGP em
oogônias mitoticamente ativas e, então, em oócitos primários (Suh et al., 2002). Em
seguida, uma camada de células somáticas planas conhecidas como células da pré-
granulosa, originárias do epitélio celômico, circundam os oócitos primários formando
os folículos primordiais (Magoffin, 2005), iniciando assim a foliculogênese. Após a
formação dos folículos primordiais, as células da pré-granulosa param de se multiplicar
e entram num período de quiescência. Os oócitos primários inclusos nesses folículos
encontram-se na fase de prófase I da meiose. A progressão da divisão meiótica ocorre
somente na puberdade, com a liberação do pico pré-ovulatório de FSH e LH, formação
dos oócitos secundários e outra parada da meiose na fase de metáfase II (Gordon, 1994).
A meiose será retomada novamente somente após a fecundação do oócito pelo
espermatozóide, originando o oócito haplóide fecundado e marcando o fim da oogênese
(Figueiredo et al., 2008).
24
Desta forma, aos processos de formação, crescimento e maturação folicular dá-
se o nome de foliculogênese, que é iniciado com a formação do folículo primordial
culminando com o estádio de folículo pré-ovulatório (van den Hurk e Zhao, 2005). O
folículo ovariano é considerado a unidade funcional do ovário mamífero, cuja principal
função é proporcionar um ambiente ideal para o crescimento e maturação do oócito
(Cortvrindt e Smitz, 2001). Durante a foliculogênese, a morfologia folicular é alterada,
uma vez que o oócito cresce e as células circundantes se diferenciam (Bristol-Gould e
Woodruff, 2006). De acordo com o grau de evolução, os folículos podem ser
classificados em folículos pré-antrais (primordiais, em transição, primários e
secundários) e antrais (terciários e pré-ovulatórios) (Silva et al., 2004). Vale ressaltar
que os folículos pré-antrais (FOPA) representam 90% da população folicular e são
responsáveis pela renovação contínua de folículos antrais no ovário (Katska-
Ksiazkiewicz, 2006).
2.6 População folicular ovariana
Em muitos mamíferos, a população folicular ovariana é estabelecida ainda na
vida intra-uterina (primatas e ruminantes – Knight e Glister, 2006) ou em um curto
período de tempo após o nascimento (roedores – Ojeda et al., 2000). Por outro lado,
recentes trabalhos têm demonstrado mecanismos envolvidos na formação de novas
células germinativas e folículos em mulheres (Bukovsky et al., 2004) e camundongas
adultas (Johnson et al., 2004). Esses autores demonstraram indícios da continuidade da
oogênese e foliculogênese no período pós-natal, pela atuação de células-tronco. Para
Bukovsky et al. (2004), a renovação folicular pós-natal ocorre regularmente em
mulheres, mas a origem das células-tronco para tal não seria a medula óssea, e sim
células germinativas presentes na superfície do epitélio ovariano. No entanto, devido
aos resultados controversos até o momento, mais trabalhos sobre esse assunto são
necessários para esclarecer esse fenômeno.
A população folicular difere entre as espécies, além de ser observada uma
grande variação individual (Katska-Ksiazkiewicz, 2006), sendo de aproximadamente
1.500 na camundonga (Shaw et al., 2000); 35.000 na cabra (Lucci et al., 1999); 114.000
na gata (Lima, 2006); 160.000 na ovelha (Amorim et al., 2000); 235.000 na vaca
(Betteridge et al., 1989) e aproximadamente 2.000.000 na mulher (Erickson, 1986). Ao
longo da vida do animal, ocorre uma redução ordenada do número de FOPA (Shaw et
25
al., 2000). Essa redução deve-se a dois fenômenos que ocorrem naturalmente no ovário,
a ovulação e a atresia ou morte folicular. Somente uma pequena parte (0,1%) dos
folículos primordiais chega à ovulação (Nuttinck et al., 1993), enquanto os demais
tornam-se atrésicos durante as fases de crescimento e maturação oocitária (Otala et al.,
2002).
2.7 A biotécnica de MOIFOPA
A disponibilidade de oócitos é um fator limitante no desenvolvimento de novas
técnicas reprodutivas (Smitz e Cortvrindt, 2002). Os métodos atuais para a produção in
vitro de embriões dependem de uma oferta escassa de oócitos competentes de grandes
folículos antrais ou pré-ovulatórios, que estão presentes no ovário em número
relativamente reduzido (Telfer, 1998). Sabe-se que os FOPA representam cerca de 90%
de toda a população folicular, armazenando assim a grande maioria dos oócitos
presentes em ovários mamíferos (Silva et al., 2003). Dessa forma, a possibilidade de
desenvolver sistemas in vitro que explorem o grande número de oócitos provenientes de
folículos imaturos de ovários mamíferos deve ser considerada, destacando-se portanto a
biotécnica de MOIFOPA.
A MOIFOPA é uma biotécnica da reprodução que vem sendo aprimorada nos
últimos tempos e consiste em uma das principais ferramentas utilizadas atualmente para
a elucidação da foliculogênese inicial. Tal biotécnica consiste no isolamento,
conservação (resfriamento e criopreservação) e/ou cultivo in vitro de FOPA, visando a
estocagem, ativação, crescimento e maturação in vitro do folículo primordial até o
estádio pré-ovulatório (Figueiredo et al., 2008), prevenindo-se, assim, a ocorrência da
atresia. Assim, no futuro, será possível obter, de um único ovário, milhares de FOPA
que, cultivados e submetidos a outras biotécnicas da reprodução como a fecundação in
vitro e a clonagem, viabilizarão a produção in vitro de um grande número de embriões.
Destes, um número de crias saudáveis significativamente maior do que aquele obtido
através da reprodução natural também poderá ser alcançado. Na medicina veterinária, o
principal objetivo é aumentar a produtividade de animais de alto valor genético, ou
mesmo a preservação de espécies ameaçadas de extinção.
A biotécnica de MOIFOPA também representa uma excelente alternativa para
incrementar e auxiliar no desenvolvimento de pesquisas relacionadas à indústria
26
farmacêutica (Figueiredo et al., 2008) e à imunoesterilização utilizando-se anticorpos
anti-ZP.
2.8 Preservação de FOPA durante o transporte de ovários
A preservação da viabilidade e da capacidade de desenvolvimento dos FOPA
durante o período compreendido entre a coleta dos ovários e seu uso no laboratório é
um fator crítico em decorrência da condição de isquemia. Para que isto seja possível,
mesmo após longos períodos de transporte, como ocorre nos casos em que os animais
doadores se encontram em regiões bastante afastadas dos laboratórios de manipulação,
alguns fatores essenciais para a sobrevivência das células devem ser levados em
consideração, tais como o meio, a temperatura e o tempo de preservação (Santos et al.,
2004).
Diferentes meios têm sido utilizados para a preservação de FOPA, podendo ser
citados: TCM 199 (Costa et al., 2005), solução salina 0,9% (Matos et al., 2004;
Celestino et al., 2008), solução salina tamponada com fosfato (PBS) - (Santos et al.,
2002), solução Braun-Collins (Carvalho et al., 2001), solução à base de água de coco
(Lucci et al., 2004a) e Meio Essencial Mínimo (MEM) – (Chaves et al., 2008). Métodos
para armazenamento de ovários por curtos períodos já foram desenvolvidos para
caprinos (Silva et al., 2000), ovinos (Andrade et al., 2002a,b; Matos et al., 2004) e
bovinos (Lucci et al., 2004a; Celestino et al., 2007). Nesses estudos, as temperaturas de
4, 20 e 39°C foram testadas para a preservação de FOPA. Em geral, a temperatura mais
eficiente é a de 4°C, permitindo a preservação da morfologia e capacidade de FOPA
suínos de crescer in vitro após períodos de armazenamento de até 18 h, enquanto a
20°C, isto foi possível por apenas 6 h (Lucci et al., 2007). Em caprinos, apenas
temperatura de 4ºC permitiu preservar, após 4 h de armazenamento, a qualidade de
FOPA inclusos em fragmentos de córtex ovariano, que, após cultivo in vitro por sete
dias, apresentaram percentuais de folículos morfologicamente normais similares ao
controle fresco (Chaves et al., 2008).
Durante o transporte dos ovários para o laboratório, a privação de oxigênio do
tecido resulta em uma mudança do metabolismo aeróbico para anaeróbico, sendo o
produto principal, o ácido láctico, acumulado dentro da célula causando redução do pH
(Wongsrikeao et al., 2005). Com a intenção de se minimizarem os efeitos deletérios da
isquemia, o estabelecimento de hipotermia provoca uma redução do metabolismo
27
celular e, assim, dos requerimentos de energia e nutrientes, aumentando a resistência de
folículos durante a preservação in vitro (Silva et al., 2003). A preservação de FOPA por
períodos indeterminados pode ser obtida, por sua vez, através da utilização de técnicas
eficientes de criopreservação (Santos, 2005).
2.9 Criopreservação
2.9.1 Princípios básicos
A criopreservação consiste em um método de preservação de material biológico
a baixas temperaturas, geralmente em nitrogênio líquido a –196°C, ou em sua fase de
vapor a -150°C. Os únicos estados físicos existentes abaixo de aproximadamente
-130°C são o cristalino e o vítreo e, em ambos, a viscosidade é muito elevada, a difusão
é considerada insignificante (dependendo do tempo de armazenamento), a energia
cinética molecular é muito baixa e reações metabólicas impulsionadas por energia
térmica ocorrem muito lentamente ou são paralisadas completamente (Kartha, 1985).
Portanto, à temperatura do nitrogênio líquido, a viabilidade durante o armazenamento
pode ser estendida por longos períodos de tempo, com manutenção da estabilidade do
material genético (Stushnoff e Seufferheld, 1995). Para a criopreservação, faz-se
necessária a utilização de um ou mais componentes que confiram proteção às células
durante a congelação, conhecidos como agentes crioprotetores (Mullen, 2007). A
capacidade do material biológico de sobreviver ao processo de criopreservação depende
de sua tolerância aos agentes crioprotetores, à desidratação, ao resfriamento e ao re-
aquecimento.
2.9.2 Agentes crioprotetores
Os agentes crioprotetores são componentes utilizados durante a congelação para
proteger as células contra os efeitos deletérios da desidratação, resfriamento e da
redução extrema de temperatura. O modo de ação dos agentes crioprotetores na célula
não é plenamente conhecido e isso se dá provavelmente devido aos inúmeros efeitos
deles na célula. Em geral, esses agentes podem agir (i) penetrando nas células
(crioprotetores intracelulares) e substituindo as moléculas de água, (ii) reduzindo o
ponto de congelação, (iii) protegendo membranas celulares por meio da sua ligação às
28
cabeças dos grupos fosfolipídicos, (iv) aumentando a viscosidade do meio, e/ou (v)
diminuindo a concentração de eletrólitos durante a criopreservação, e, assim, o risco de
danos osmóticos. Contudo, agentes crioprotetores podem ser tóxicos, bem como podem
facilitar a entrada de agentes tóxicos nas células (Mullen, 2007; Santos, 2007c).
Os agentes crioprotetores podem ser alcoóis, açúcares, amidas e grandes
polímeros, que atuam por diferentes mecanismos (Fuller, 2004; Acker, 2007), sendo
classificados como intra ou extracelulares. Crioprotetores intracelulares ou penetrantes
possuem baixo peso molecular e a capacidade de substituir a água intracelular. Como
exemplos, podem ser citados o glicerol (GLI), etilenoglicol (EG), dimetilsulfóxido
(DMSO) e propanodiol (PROH). Quando os crioprotetores são adicionados, a água
intracelular (solvente) deixa a célula e os crioprotetores penetrantes (soluto) a penetram,
interagindo com a membrana celular, estabilizando as proteínas intracelulares e
reduzindo o ponto de congelação. Estudos recentes demonstraram que o GLI, DMSO,
PROH e EG nas concentrações de 1,5 e 3,0 M foram utilizados com sucesso para a
criopreservação de tecido ovariano de caprinos (Rodrigues et al., 2004a,b) e ovinos
(Santos et al., 2006) previamente exposto a esses compostos a uma temperatura de 20°C
por um período de 20 minutos (período de equilíbrio).
O GLI tem sido usado largamente para a congelação de embriões bovinos devido
à sua baixa citotoxicidade. Entretanto, o GLI induz a severos danos no citoplasma
devido à baixa permeabilidade da membrana plasmática a esta substância (Széll et al.,
1986). Oócitos de camundongos isolados são menos permeáveis ao glicerol que ao
DMSO, PROH e EG (Paynter et al., 1997). A baixa permeabilidade das células ao GLI
aumenta o risco do estresse osmótico durante a descongelação e diluição, pois a entrada
de água na célula ocorre mais rapidamente do que a saída do GLI. Isso explica as baixas
taxas de sobrevivência e de maturação de oócitos criopreservados em GLI (Wani et al.,
2004).
Crioprotetores extracelulares ou não-penetrantes, entre eles os açúcares como a
sacarose e a trealose, e polissacarídeos como o ficol, agem otimizando o efeito dos
crioprotetores penetrantes. Utilizada com freqüência, a sacarose age como um tampão
osmótico contra o estresse causado durante a remoção dos crioprotetores intracelulares
(Mandelbaum et al., 1988). A sacarose pode ser utilizada com sucesso como
crioprotetor extracelular para folículos pré-antrais murinos, especialmente quando
combinada com EG (Salehnia et al., 2002). Ainda, a sacarose (0,1 M) associada ao
DMSO ou PROH mostrou-se eficiente para criopreservar o tecido ovariano bovino, fato
29
demonstrado pela integridade ultra-estrutural dos folículos pré-antrais criopreservados
(Lucci et al., 2004b).
Crioprotetores extracelulares, também conhecidos como agentes de alto peso
molecular, aumentam a viscosidade da solução (por exemplo, o polivinil álcool) ou se
ligam às cabeças dos grupos fosfolipídicos (açúcares, tais como sacarose e trealose),
protegendo as membranas celulares contra as injúrias do frio. Outra substância
comumente adicionada para melhorar a eficiência do meio de criopreservação consiste
no soro fetal bovino. Contudo, a importância do soro na criopreservação não é
cientificamente comprovada, além do risco de conter agentes infecciosos que não são
destruídos durante os procedimentos de criopreservação (Santos, 2007c).
2.9.3 Criopreservação: métodos e danos
O processo de criopreservação envolve basicamente as seguintes etapas: (1)
adição de agente crioprotetor (período de equilíbrio); (2) resfriamento e indução da
formação de gelo, seguidos por congelação ou vitrificação; (3) estocagem em nitrogênio
líquido; (4) descongelação ou aquecimento e (5) remoção ou diluição do agente
crioprotetor. A criopreservação pode ser realizada por dois métodos básicos: congelação
convencional (lenta) e vitrificação.
A congelação lenta é caracterizada pela exposição das células ou tecidos a baixas
concentrações de agente crioprotetor (∼1,5 M) - (Paynter et al., 2000), por um período
que pode variar de 20 (Rodrigues et al., 2004a,b) a 60 minutos (Candy et al., 1997).
Nesse método, é utilizado um freezer programável, em que o material é resfriado
lentamente a uma velocidade de 2ºC/min até –4 a –9ºC, mantendo-se esta temperatura
por um curto período (10 a 15 min) para a estabilização térmica e indução da
cristalização da solução crioprotetora (seeding) através do contato de um elemento
metálico pré-resfriado em nitrogênio líquido com a parede do recipiente que contém o
as amostras. Este procedimento visa prevenir a ocorrência da cristalização do meio em
temperaturas inferiores ao seu ponto de solidificação, que resulta na liberação de
energia sob forma de calor e elevação da temperatura da solução, seguida de redução
brusca durante o equilíbrio térmico com o freezer (Reichenbach et al., 2008). Em
seguida, o sistema continua sendo resfriado lentamente a uma velocidade de 0,3ºC/min.
Uma vez que a desidratação celular é suficientemente atingida (entre –30 a –140ºC), o
material é estocado em nitrogênio líquido (-196°C). Este é o método mais utilizado para
30
a criopreservação de tecido ovariano (Cox et al., 1996; Candy et al., 1997; Newton et
al., 1998; Liu et al., 2008; Qi et al., 2008; Tsuribe et al., 2008). Utilizando este método,
já foi possível a restauração da fertilidade após transplante em ovinos (Salle et al., 1999;
Almodim et al., 2004) e murinos (Liu et al., 2008), bem como já foram obtidos
nascimentos a partir de tecido ovariano criopreservado de murino (Guenasena et al.,
1997), ovino (Gosden et al., 1994; Salle et al., 2003) e humano (Oktay, 2001; Donnez et
al., 2004; Meirow et al., 2005).
O método de vitrificação consiste em utilizar uma taxa de congelação
extremamente rápida, que leva à formação de um estado vítreo do material
criopreservado, protegendo-se as células da formação de cristais de gelo (Huang et al.,
2008). A vitrificação foi idealizada por Luyet em 1937, e, depois de quase 50 anos, Rall
e Fahy descreveram este método como uma alternativa ao processo de congelação lenta
(Rall e Fahy, 1985). Ao contrário da congelação lenta, a vitrificação envolve a
exposição do material biológico a altas concentrações de agente crioprotetor
(geralmente entre 4 e 6 M) por um curto período de tempo (25 segundos a 5 minutos),
geralmente à temperatura ambiente, seguido de um resfriamento ultra-rápido em
nitrogênio líquido, não sendo necessária a utilização de equipamentos sofisticados e de
alto custo. De acordo com Stachecki e Cohen (2004), a vitrificação possui dois aspectos
básicos a serem levados em consideração. O primeiro consiste no fato de que as altas
concentrações de agentes crioprotetores utilizadas na exposição aumentam os efeitos
tóxicos e, em segundo lugar, apesar desse efeito durante o período de equilíbrio, a
vitrificação, por ser uma congelação altamente rápida, aumenta as taxas de
sobrevivência. Este método de congelação já foi reportado para oócitos maturos (Chian
et al., 2004), embriões (Isachenko et al., 2005) e tecido ovariano de diferentes espécies
como camundongas (Kagabu e Umezu, 2000), ratas (Sugimoto et al., 2000) e ovelhas
(Al-aghbari e Menino, 2002). Assim como na congelação lenta, resultados satisfatórios
com nascimentos foram obtidos após vitrificação de ovários inteiros ou FOPA de
camundongas (Liu et al., 2001; de la Peña et al., 2002; Migishima et al., 2003),
entretanto os resultados, principalmente em outras espécies, ainda são controversos.
Existem dois fatores que podem levar à morte celular durante o processo de
congelação/descongelação: a formação de gelo intracelular e o choque osmótico. Esses
efeitos negativos podem ser reduzidos ou evitados com a utilização de agentes
crioprotetores (de la Vega e Wilde, 1991), bem como modificando-se o processo de
criopreservação. De acordo com Stachecki e Cohen (2004), os efeitos do gelo
31
intracelular e do choque osmótico são fatores letais se as células não são tratadas
apropriadamente.
A prevenção da formação de gelo intracelular é considerada um dos fatores mais
importantes quando se deseja realizar um eficiente protocolo de criopreservação. Na
congelação clássica (lenta), essa formação pode ser evitada com um resfriamento celular
lento, permitindo a progressiva desidratação celular (Mazur et al., 2005). Existem duas
hipóteses para a formação de gelo intracelular. A primeira, postulada por Muldrey e
McGann (1990), sugere que o gelo intracelular seja formado como conseqüência de
danos ou defeitos na membrana plasmática, que permitiriam a passagem do gelo
extracelular através da membrana (teoria do fluxo osmótico). Com o super-resfriamento
celular durante a congelação, a força de efluxo da água para o meio extracelular
atingiria um valor crítico e, consequentemente, causaria danos à membrana, permitindo
a entrada do gelo extracelular para o meio intracelular. Uma outra hipótese é a de que o
gelo extracelular em contato (direto ou indireto) com a membrana plasmática, causaria a
formação de gelo intracelular, levando injúria ao conteúdo celular. Há duas versões para
essa segunda hipótese. Na primeira, Toner et al. (1990) sugerem que o gelo extracelular
provocaria uma mudança conformacional na membrana, que se transformaria em um
nucleador heterogêneo dos conteúdos celulares (contato indireto). Na segunda,
postulada por Mazur (2004), o gelo extracelular cresceria através de poros preexistentes
na membrana, onde tal contato direto levaria à formação de gelo intracelular.
Outra causa de morte celular consiste no efeito solução, que envolve alterações
citoplasmáticas como resultado da desidratação, aumento da concentração e
precipitação de solutos e alterações de pH (Mazur et al., 1984). Por muito tempo,
acreditou-se que a morte celular poderia ser causada pelo aumento da concentração de
soluto fora da célula devido ao resfriamento e a congelação da água extracelular
(Lovelock, 1953). Contudo, estudos mais recentes têm reportado uma alta tolerância
celular ao estresse osmótico. Agca et al. (2000) expuseram oócitos bovinos a crescentes
concentrações de cloreto de sódio para elevar a osmolaridade a concentrações
supravitais (até 4800 mOsm) e observaram que até 2400 mOsm, posteriormente os
oócitos foram capazes de ser fecundados e foram obtidos blastocistos. Outros
pesquisadores expuseram oócitos humanos e murinos a concentrações variáveis de
agente crioprotetor ou açúcares sem resfriamento e observaram considerável tolerância
celular às condições osmóticas impostas (Oda et al., 1992; Hotamisligil et al., 1996).
Os danos supracitados não são observados exclusivamente durante o
32
resfriamento, mas também durante o processo de descongelação/re-aquecimento, uma
vez que as células recuperam seu metabolismo na presença de substâncias tóxicas como
os agentes crioprotetores. Assim, El-Naggar et al. (2006) sugerem que as células ou
tecidos criopreservados devem ser descongelados em uma velocidade alta. Agentes
crioprotetores podem ser removidos em uma (Leibo, 1984), três (Rodrigues et al.,
2004a,b) ou mesmo em seis lavagens (Shelton, 1992). Apesar de uma lavagem ser
suficiente para embriões descongelados, folículos isolados e tecido ovariano devem ser
lavados em três passos para retirada de resquícios de agentes crioprotetores (Lima et al.,
2006; Sadeu et al., 2006; Santos et al., 2006).
2.9.4 Métodos de análise de FOPA criopreservados
Para a aplicação de um protocolo de criopreservação, é necessário determinar
o(s) agente(s) crioprotetore(s) mais indicado(s), sua concentração, tempo de exposição e
método de remoção. Um método prático para se verificar a qualidade folicular após a
congelação/descongelação é a histologia clássica. Contudo, esta análise morfológica não
é suficiente para se avaliar o processo de criopreservação (Schotanus et al., 1997; van
den Hurk et al., 1998; Martinez-Madrid et al., 2004), por permitir apenas a identificação
dos sinais primários da atresia (picnose nuclear, danos citoplasmáticos, desconexão
entre as células da granulosa e o oócito, bem como irregularidades na membrana basal)
- (Jorio et al., 1991; Hulshof et al., 1995; Demirci et al., 2002). A morfologia celular
observada por histologia não está sempre correlacionada com a integridade ultra-
estrutural, que requer o uso da microscopia eletrônica de transmissão para sua análise
(Santos et al., 2006). Além das organelas celulares, a verificação da integridade da
membrana plasmática também é fundamental para a avaliação da viabilidade celular, e
tem sido realizada utilizando-se o corante vital azul de trypan (Santos et al., 2007a,b) ou
o marcador fluorescente etídio homodímero (Schotanus et al., 1997; van den Hurk et al.,
1998). Outro parâmetro utilizado para a análise da viabilidade consiste na atividade
enzimática no citoplasma, detectável nas células foliculares através do composto
calceína-AM (Schotanus et al., 1997; van den Hurk et al., 1998), que é clivado por
enzimas esterase em células vivas, resultando em um produto fluorescente (De Clerck et
al., 1994).
33
2.10 Cultivo in vitro de folículos ovarianos
O objetivo principal do cultivo in vitro de FOPA é permitir o desenvolvimento
folicular assegurando o crescimento e a maturação oocitária, bem como a multiplicação
e posterior diferenciação das células da granulosa inclusas nesses folículos (Figueiredo
et al., 2008). O cultivo de ovários tem sido utilizado com diferentes propósitos como
avaliar a importância da vascularização, apoptose, fatores de crescimento e hormônios
para o desenvolvimento de FOPA dentre outros (Fortune et al., 2000; Erickson, 2001;
Flaws et al., 2001; O`Brien et al., 2003; Matos et al., 2007). Dentre os hormônios
testados e já comumente adicionados ao meio de base para o cultivo de folículos
ovarianos, destaca-se o FSH, sendo demonstrada sua importância para a manutenção da
viabilidade e proliferação das células da granulosa de FOPA (Matos et al., 2007; Choi et
al., 2008)
Em roedores, a pequena dimensão dos ovários possibilita o cultivo do órgão
inteiro, o que tem sido bastante útil para o estudo da foliculogênese inicial em pequenos
mamíferos (Fortune, 2003). Em animais domésticos de médio e grande porte, devido às
grandes dimensões dos ovários, não é possível utilizar este modelo. Para estes animais,
o cultivo de pequenos fragmentos de córtex ovariano, rico em folículos primordiais, tem
sido realizado para o estudo da ativação e crescimento de folículos primordiais caprinos
(Silva et al., 2004), bovinos (Braw-Tal e Yossefi, 1997) e humanos (Scott et al., 2004).
Esse tipo de cultivo in vitro tem a vantagem de manter a integridade estrutural folicular
e as interações entre as células foliculares e células do estroma, facilitando a perfusão
do meio para o tecido ovariano (Telfer, 1996). Já o segundo sistema de cultivo folicular
envolve o isolamento, mecânico ou enzimático, dos FOPA (Abir et al., 2001b),
permitindo o monitoramento diário do crescimento folicular, bem como o efeito in vitro
de hormônios e fatores de crescimento sobre cada classe folicular (Abir et al., 2001a).
Apesar dos métodos enzimáticos de isolamento serem mais práticos, os mecânicos têm
sido mais comumente adotados para o isolamento de FOPA de ovários bovinos (Itoh et
al., 2002), caprinos (Arunakumari et al., 2007), ovinos (Amorim et al., 2000), ratas
(Zhao et al., 2000) e camundongas (Lenie et al., 2004). Tais métodos têm
proporcionado a recuperação de um grande número de FOPA, sendo mais utilizados a
microdissecção para folículos com diâmetro igual ou superior a 150 μm (Lee et al.,
2007) e o isolamento pelo tissue chopper para folículos menores (Martinez-Madrid et
al., 2004) ou ainda a associação de ambos os métodos (Gupta et al., 2008). Além disso,
34
o isolamento mecânico mantém a integridade da estrutura folicular, a membrana basal
permanece intacta após o procedimento e as interações oócito-células da granulosa-teca
são mantidas (Demeestere et al., 2002).
Em relação aos avanços obtidos através da MOIFOPA, em gatas (Jewgenow e
Stolte, 1996), gambás (Butcher e Ullman, 1996) e macacas (Fortune et al., 1998) foi
observado o crescimento de FOPA após cultivo in vitro, porém sem a formação de
antro. Nas espécies bovina (Itoh et al., 2002), caprina (Huanmin e Yong, 2000) e
humana (Roy e Treacy, 1993), os FOPA isolados foram cultivados in vitro e
desenvolveram-se até o estádio antral. Em ovinos, folículos secundários isolados e
cultivados por 6 dias atingiram a maturação nuclear (Tamilmani et al., 2005). Em suínos
e bubalinos, folículos secundários crescidos in vitro chegaram até a ovulação, tiveram
seus oócitos fecundados in vitro, com desenvolvimento até estádio de blastocisto (Wu e
Tian, 2007; Gupta et al., 2008). No entanto, os maiores avanços do cultivo folicular
foram alcançados em camundongos, com o nascimento de crias viáveis a partir da
maturação de oócitos provenientes de FOPA cultivados in situ e isolados, em que o
oócito adquiriu competência meiótica para ser fecundado e completar o
desenvolvimento embrionário (O’Brien et al., 2003). Provavelmente, essa estratégia de
associar o cultivo de folículos in situ e isolados seja necessária para promover o
crescimento de folículos primordiais de espécies domésticas e primatas (Silva et al.,
2006).
Para a espécie canina, poucos estudos com FOPA têm sido realizados,
restringindo-se todos à avaliação de sistemas de maturação (Bolamba et al., 1998, 2002,
2006). Nenhum trabalho foi realizado com o objetivo de estabelecer métodos de
preservação ou de cultivo in vitro.
35
3 JUSTIFICATIVA
A superpopulação de cães nas cidades resulta na eutanásia de milhões de
animais sadios, além do dispêndio de um elevado volume de recursos econômicos
estimados na ordem de bilhões de dólares anualmente (Frank e Carlisle-Frank, 2007).
Nos países em desenvolvimento, estes recursos são utilizados, principalmente, para a
execução de programas de controle da raiva urbana, que se baseiam na eliminação de
cães errantes, na vacinação da população animal e no tratamento de pessoas
potencialmente expostas ao vírus rábico (Wandeler et al., 1988).
O tratamento profilático pós-exposição para pessoas agredidas por cães de rua,
instituído obrigatoriamente nestes casos por não ser possível a observação do animal
agressor, onera bastante os cofres públicos. Isto porque o custo médio de um esquema
completo de soro-vacinação pode exceder a quantia de U$ 4.000, quando se utilizam
vacinas produzidas em cultivo celular e imunoglobulinas anti-rábicas homólogas
(Dhankhar et al., 2008), e a freqüência desses eventos de agressão em cidades de países
em desenvolvimento, a exemplo do município de Fortaleza (Ceará, Brasil), é bastante
elevada (Lopes et al., 2001).
Outra consideração importante acerca desses tratamentos é o risco de reações
adversas severas quando se utilizam vacinas produzidas por cultivo em tecido nervoso
de animais, que constituem o único tipo disponível em alguns países (Rupprecht et al.,
2002). Por exemplo, reações de desmielinização central e/ou periférica, algumas das
quais fatais, têm sido associadas ao uso da vacina Fuenzalida & Palácios, obtida a partir
de cultivo em cérebros de camundongos lactentes, sendo reportadas incidências de até
um caso para cada 1.250 tratamentos (Meltzer e Rupprecht, 1998).
O Estado do Ceará possui uma das maiores incidências de raiva canina e raiva
humana transmitida por cães no Brasil. No período de 2002 a 2005, confirmaram-se em
laboratório 175 casos de raiva canina, o que indica uma elevada circulação da doença
em cães (Secretaria de Saúde do Estado do Ceará, 2006). Devido ao fato de que o
número de casos de raiva animal e humana tende a ser maior em áreas com elevada
densidade de cães errantes, onde os contatos entre homens e animais são mais
freqüentes (Haddad et al., 1988; Osman e Haddad, 1988), as autoridades sanitárias do
referido, bem como de outros estados da federação, têm realizado a apreensão
sistemática e eutanásia de cães de rua. Contudo, estudos realizados em diversos países
demonstram que a a eliminação de cães errantes é ineficaz e apresenta maior custo que
36
o investimento no controle da reprodução animal e em programas educativos para a
população (World Health Organization, 1990).
Mais que um dilema de ordem econômica e sanitária, a superpopulação canina e
a forma como é controlada compõem um complexo dilema ético. A qualidade de vida
de cães abandonados é extremamente baixa, sendo marcantes a fome, doenças,
desconforto por condições climáticas adversas, atropelamentos, crueldades, enfim, uma
situação multifatorial de sofrimento intenso. Neste contexto, faz-se fundamental e
urgente a realização de programas de controle da reprodução de cães, bem como o
desenvolvimento de métodos contraceptivos eficientes, seguros e de custo
suficientemente baixo para permitir sua utilização em massa. Assim, será possível
intervir de maneira racional, efetiva e humanitária sobre o problema da superpopulação
de cães, obtendo-se, conseqüentemente, uma economia expressiva de recursos públicos,
além de promoção da saúde pública e do bem-estar animal. O potencial da técnica de
imunoesterilização por anticorpos anti-ZP de reunir todas as características supracitadas
representa uma importante perspectiva para a solução desta questão.
Considerando-se a importância da realização de estudos in vitro preliminares aos
testes in vivo, com o intuito de se estabelecer uma base sólida e precisa de
conhecimentos, minimizando-se a experimentação com animais, utilizou-se no presente
trabalho a biotécnica de MOIFOPA como modelo experimental. Entretanto, uma vez
que ainda não há informações relativas à aplicação desta tecnologia para cães, estudos
foram conduzidos para a definição de métodos de preservação e cultivo in vitro de
folículos pré-antrais caninos. Neste contexto, realizou-se a avaliação de protocolos de
resfriamento e criopreservação utilizados para outras espécies, sendo ainda empregado
um sistema de cultivo em desenvolvimento para possibilitar a análise dos efeitos de
anticorpos anti-ZP sobre os referidos folículos.
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4 HIPÓTESES CIENTÍFICAS
Folículos ovarianos pré-antrais caninos podem ser preservados por meio de
armazenamento hipotérmico durante o período de isquemia compreendido entre a coleta
dos ovários e seu uso no laboratório.
Folículos pré-antrais caninos podem ainda ser criopreservados através de
congelação lenta utilizando-se agentes crioprotetores, mantendo-se a viabilidade e a
integridade ultra-estrutural.
Folículos pré-antrais caninos frescos ou criopreservados podem ser cultivados in
vitro mantendo sua viabilidade. Esta técnica pode ser utilizada como modelo
experimental para o teste de agentes esterilizantes.
Anticorpos anti-ZP são capazes de se ligar especificamente à ZP de oócitos de
caninos, causando bloqueio da ligação de espermatozóides ou atresia de folículos pré-
antrais.
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5 OBJETIVOS
5.1 Objetivo Geral
Avaliar o potencial de anticorpos anti-ZP para a imunoesterilização de cadelas.
5.2 Objetivos Específicos
- Definir um método de preservação de FOPA caninos por períodos curtos,
avaliando-se os parâmetros meio (solução de NaCl a 0,9% - salina fisiológica – ou Meio
Essencial Mínimo – MEM), temperatura (4, 20 ou 38°C) e tempo de conservação
(2,6,12 ou 24 h);
- estabelecer um método de criopreservação de FOPA caninos, avaliando-se a
eficiência dos agentes crioprotetores dimetilsulfóxido, etilenoglicol, glicerol e 1,3-
propanodiol durante a execução da técnica de congelação lenta;
- cultivar in vitro FOPA caninos frescos ou criopreservados de modo a manter-
se sua viabilidade;
- analisar o efeito de anticorpos policlonais anti-pZP sobre FOPA caninos
cultivados in vitro.
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6 CAPÍTULO I
Imunocontracepção em mamíferos com ênfase no controle populacional de cães
Immunocontraception in mammals emphasizing dog population control
Revista Brasileira de Reprodução Animal
(Publicado, v.29, n.3/4, p.159-166, 2005)
40
Imunocontracepção em mamíferos com ênfase no controle populacional de cães
(Immunocontraception in mammals emphasizing dog population control)
Cláudio Afonso Pinho Lopes*, Diana Célia Sousa Nunes-Pinheiro, José Ricardo de
Figueiredo
Faculdade de Veterinária, Universidade Estadual do Ceará, CEP 60740-000, Fortaleza,
CE, Brasil.
* Autor para correspondência: [email protected]
Resumo
A superpopulação de cães constitui um sério problema em diversas cidades do
mundo, sendo necessário o desenvolvimento de sistemas de contenção de natalidade
baseados no controle da reprodução. Os métodos contraceptivos atualmente disponíveis
são inadequados para uso em escala populacional. Neste contexto, a imunocontracepção
surge como uma importante perspectiva para a solução desta questão. No presente
trabalho, são abordados diferentes métodos de imunocontracepção em mamíferos,
baseados no uso de hormônios reprodutivos, antígenos do embrião, da zona pelúcida e
do espermatozóide, apresentando seus princípios e os avanços obtidos em seu
desenvolvimento.
Palavras-chave: imunocontracepção, imunoesterilização, mamíferos, cães.
Abstract
Dog overpopulation is a serious problem in many cities throughout the world,
which requires the development of birth control systems based upon reproduction
blocking. Contraception methods available nowadays are not suitable for use in a
population scale. In this context, immunocontraception is an important perspective for
filling this gap. In this article, immunocontraceptive methods for mammals based on use
of reproductive hormones and antigens from embryo, zona pellucida and spermatozoon
are reviewed, focusing on their principles and development advances.
Keywords: Immunocontraception, immunosterilization, mammals, dogs.
41
Introdução
A superpopulação de cães constitui um sério problema em diversas cidades do
mundo, caracterizado pela existência de uma grande quantidade de animais sem
responsáveis que vivem nos espaços públicos. Esses animais compõem um reservatório
de zoonoses, e, assim, um risco de agravo à saúde pública, o que implica o dispêndio
expressivo de recursos. Neste contexto, as autoridades sanitárias realizam a eliminação
sistemática desses animais como estratégia de controle populacional.
Esta abordagem, no entanto, é estritamente paliativa, por não atuar sobre a
origem do problema, que consiste das elevadas taxas de natalidade de cães. Além disso,
a eliminação dos animais sem responsáveis faz emergirem questões éticas que definem
a necessidade de novas estratégias de ação. Assim, um sistema de controle populacional
para cães, para ser efetivo, racional e humanitário, deve basear-se no controle de
natalidade.
As técnicas contraceptivas atualmente disponíveis para cães não são adequadas
para uso em escala populacional. Os métodos cirúrgicos, além do custo elevado e dos
riscos inerentes a qualquer procedimento cirúrgico, são logisticamente inviáveis para
populações grandes. Os métodos farmacológicos, baseados no uso de hormônios, têm
apresentado efeitos adversos. Um novo conceito em métodos contraceptivos, a
imunocontracepção, surge como uma importante perspectiva para o desenvolvimento de
métodos aplicáveis ao controle da reprodução de cães em larga escala.
Além de cães, outras espécies de mamíferos também encontram-se em
superpopulação, como os felinos domésticos em diversas regiões do mundo (Slater,
2001) e, em particular, na Austrália (Bradley et al., 1999), o elefante em alguns países
do sul da África (Stout e Colenbrander, 2004) e o cervo de cauda branca (Rutberg et al.,
2004) na América do Norte. Problemas de animais em excesso também ocorrem em
parques zoológicos, em virtude da reprodução dos animais cativos (Kirkpatrick e
Rutberg, 2001). Assim, o uso da imunocontracepção pode ser útil também no controle
das populações dessas espécies. Neste contexto, no presente trabalho, serão abordados
diferentes métodos de imunocontracepção em mamíferos, baseados no uso de
hormônios reprodutivos e de antígenos do embrião, da zona pelúcida e do
espermatozóide, apresentando-se seus princípios e os avanços obtidos em seu
desenvolvimento.
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A imunocontracepção
A imunocontracepção consiste da ligação de anticorpos a moléculas bioativas do
sistema reprodutor responsáveis por interações fundamentais ao processo reprodutivo,
que, desta forma, é interrompido. Por exemplo, a ligação de anticorpos à superfície da
zona pelúcida (ZP) de oócitos torna estes inacessíveis a espermatozóides, impedindo,
assim, as interações moleculares necessárias ao processo de fertilização. O efeito
contraceptivo é reversível, mantendo-se até que as concentrações (títulos) dos
anticorpos específicos, que gradativamente vão decaindo a níveis abaixo de um limiar
contraceptivo, ou seja, tornem-se insuficientes para o bloqueio da reprodução. A
duração deste efeito varia em função do título inicial de anticorpos contraceptivos,
sendo observados períodos de até cinco anos de infertilidade (Brown et al., 1997).
A imunocontracepção tende a ser destituída de efeitos colaterais adversos, uma
vez que as moléculas a serem bloqueadas devem desempenhar funções limitadas ao
processo reprodutivo. Além disso, a verificação criteriosa do potencial de reatividade
cruzada de anticorpos contraceptivos com outros elementos do organismo, em
decorrência de algum grau de homologia, deve sempre ser procedida.
Em alguns casos, os anticorpos produzidos e/ou células citotóxicas do sistema
imunológico ativadas nas vacinações causam a destruição de elementos não-
regeneráveis do sistema reprodutivo, caracterizando-se, assim, um processo de
esterilização imunológica, também denominado imunoesterilização.
Diversos antígenos do sistema reprodutor são passíveis de ligação por
anticorpos, sendo, portanto, moléculas-alvo em potencial para o desenvolvimento de
métodos de imunocontracepção ou imunoesterilização (Fig. 1). O hormônio liberador de
gonadotrofinas (GnRH), secretado pelo hipotálamo, controla a liberação dos hormônios
folículo estimulante (FSH) e luteinizante (LH), que, por sua vez, controlam a
gametogênese em machos e fêmeas. Assim, a ligação de anticorpos às moléculas desses
hormônios ou aos seus receptores resulta na interrupção da ovulação ou da produção de
espermatozóides. As glicoproteínas da ZP e determinados antígenos da superfície do
espermatozóide, responsáveis pela cascata de reações que constituem o processo de
fertilização, também são passíveis de bloqueio por anticorpos. As gonadotrofinas
coriônicas (GC), responsáveis pela manutenção da gestação até o desenvolvimento
completo da placenta, podem também constituir moléculas-alvo para a
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imunocontracepção. O bloqueio por anticorpos de proteínas bioativas do embrião pode
também ser utilizado como método para interrupção da gestação em seu início.
Imunização ativa
Os anticorpos contraceptivos podem ser produzidos por meio de imunização
ativa (vacinação), utilizando-se como imunógeno a molécula-alvo a ser bloqueada.
Entretanto, em populações com grande diversidade genética, uma parcela significativa
dos indivíduos pode não ter capacidade de resposta imunológica a determinados
antígenos, o que resulta na redução da eficácia do método.
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Imunização passiva
Uma técnica alternativa à vacinação contraceptiva, com potencial de reunir
maiores eficácia, segurança e praticidade, consiste da administração de anticorpos
contraceptivos pré-formados em outros animais, ou seja, a imunização passiva
(soroterapia). A maior eficácia decorre do fato de este método não depender da
capacidade de cada indivíduo de responder imunologicamente ao elemento-alvo a ser
neutralizado. Esta técnica é ainda mais segura que a vacinação, pois é possível analisar a
especificidade dos anticorpos produzidos antes de serem administrados (Talwar, 1999).
Outra vantagem deste método é sua praticidade, uma vez que uma única aplicação de
anticorpos pode resultar em títulos adequados, enquanto os procedimentos de
vacinação, geralmente, requerem diversas aplicações de imunógeno. Os anticorpos de
interesse podem ser produzidos pela hiperimunização de plantéis de animais
selecionados para a produção de soro, do qual se procede a purificação e a concentração
das imunoglobulinas específicas para o antígeno a ser neutralizado, a exemplo dos soros
anti-ofídicos produzidos em eqüinos.
Imunocontracepção baseada em hormônios reprodutivos
GnRH
A molécula de GnRH consiste de um decapeptídeo incapaz de desencadear uma
resposta imunológica quando utilizado como vacina. Elementos com estruturas
pequenas não são capazes de estimular as células do sistema imunológico, ou seja, não
são imunogênicos, sendo denominados haptenos (Janeway et al., 2001). Para que uma
resposta imunológica seja estabelecida para haptenos, é necessário que estes sejam
ligados a proteínas de grandes dimensões, denominadas proteínas carreadoras. Assim, o
GnRH tem sido quimicamente conjugado a diversas proteínas, como os toxóides
tetânico (TT) e diftérico (TD), para seu uso como agente imunocontraceptivo.
Ladd et al. (1994) vacinaram cães com GnRH conjugado ao toxóide tetânico,
obtendo uma redução dos níveis séricos de testosterona a limiares observados em
animais castrados, o que resultou em azoospermia. Miller et al. (2000) analisaram a
eficácia de uma vacina constituída por GnRH para o controle reprodutivo de um grupo
de cervos machos e fêmeas de cauda branca. Após um esquema de vacinação composto
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por duas aplicações apenas de imunógeno, observou-se uma redução de 88% da
fertilidade das fêmeas, além da interrupção da produção de testosterona pelos machos,
com conseqüente perda da libido. Estes efeitos mantiveram-se por até dois anos, sem a
necessidade de vacinações de reforço. Robbins et al. (2004) vacinaram felinos
domésticos machos e fêmeas com GnRH conjugado à leucotoxina A e observaram a
produção de anticorpos em títulos imunoneutralizantes que se mantiveram por mais de
20 meses. Neste período, os machos apresentaram supressão da produção de
testosterona e da espermatogênese, verificando-se atrofia das células de Sertoli e
ausência de espermátides. As fêmeas mantiveram-se em anestro durante o mesmo
período, não sendo observada a ocorrência de folículos terciários ao exame histológico
dos ovários.
Uma única injeção de anticorpos monoclonais anti-GnRH ao início do proestro
suprimiu a ovulação subseqüente em fêmeas de rato. Quando administrados a cadelas,
estes anticorpos também interromperam o estro, conforme verificado pela avaliação do
comportamento, citologia vaginal e perfis de hormônios sexuais (Talwar et al., 1985).
FSH
Nos machos, a ausência de estimulação por FSH afeta significativamente a
proliferação de espermatogônias e, assim, a produção quantitativa de espermatócitos
primários (Suresh et al., 1995). Além disso, evidências sugerem que a privação de FSH
também afeta o processo de espermiogênese, levando à produção de espermatozóides
de baixa qualidade ou imaturos, resultando em infertilidade. Dessa forma, a
neutralização do FSH por anticorpos pode ser utilizada como técnica
imunocontraceptiva (Moudgal et al., 1997a).
Macacos vacinados com FSH ovino (oFSH) apresentaram oligozoospermia e
infertilidade (Srivastav e Das, 1992). A administração a macacos, de anticorpos para o
FSH ou para o receptor deste hormônio (FSH-R), causou uma inibição da transformação
de espermatogônias em espermatócitos primários, bem como do processo de
espermiogênese (Aravindan et al., 1993; Suresh et al., 1995). Não houve alteração da
produção de testosterona, observando-se apenas oligozoospermia, mas a qualidade dos
espermatozóides foi afetada de forma suficiente ao estabelecimento de infertilidade.
Aravindan et al. (1993) observaram que os títulos de anticorpos anti-FSH em
animais vacinados com o FSH ovino não duram mais de 90-100 dias, pois estes são
46
rapidamente usados para a bioneutralização do FSH, produzido de forma contínua pela
hipófise. Assim, uma alternativa de técnica imunocontraceptiva para se afetar a
espermatogênese consiste do uso de anticorpos para o receptor de FSH. Macacos
vacinados com um fragmento recombinante do receptor do FSH apresentaram títulos
efetivos de anticorpos para o antígeno utilizado, que se mantiveram por mais de 300
dias após apenas duas a três injeções do imunógeno. Estes anticorpos ligaram-se
efetivamente a moléculas do receptor do FSH nos testículos, impedindo a ligação do
FSH, o que resultou em inibição da transformação de espermatogônias em
espermatócitos primários. Os animais vacinados, ao serem submetidos ao teste de
cópula, apresentaram-se inférteis. Não houve qualquer alteração nas concentrações
séricas de testosterona (Moudgal et al., 1997b).
LH
A eficácia de vacinas baseadas em LH ovino (oLH) para a interrupção da função
testicular foi avaliada em coelhos (Jeyakumar e Moudgal, 1996) e macacos (Suresh et
al., 1995). Os anticorpos produzidos foram capazes de se ligar ao LH, resultando em
uma redução intensa (~90%) das concentrações séricas de testosterona. A quantificação
da população de células germinais por citometria de fluxo mostrou que, 15-18 semanas
após a vacinação, a população de espermátides foi reduzida em mais de 90%, e a de
espermatócitos primários em mais de 50%. Os autores sugeriram que a azoospermia
resultante decorreu da interrupção da meiose, que é controlada pela testosterona
testicular. Remy et al. (1996) observaram que a vacinação de ratos machos com
receptor de LH (LH-R) suprimiu a fertilidade de forma proporcional aos títulos de
anticorpos produzidos contra os receptores de LH.
Coelhas vacinadas com receptores de LH bovino produziram anticorpos para
este receptor e apresentaram falha de ovulação após cópula, inseminação artificial ou
injeção de gonadotrofina coriônica (Singh et al., 1995). Essas coelhas mantiveram-se
inférteis por 10 meses após a vacinação, mesmo após repetidas cópulas. Entretanto, a
ovulação foi reestabelecida com o declínio dos anticorpos. De modo semelhante,
babuínos e macacos rhesus vacinados com receptores de LH tornaram-se inférteis, mas,
com o declínio dos anticorpos, retornaram à fertilidade normal (Shukla et al., 1991; Pal
et al., 1992).
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Cadelas vacinadas com receptores de LH bovino apresentaram redução dos
níveis de progesterona, sugerindo-se falha de ovulação e de função do corpo lúteo e não
manifestaram sinais de estro. Com o declínio dos anticorpos anti-receptores de LH cerca
de 500 dias após a vacinação, os perfis hormonais, a citologia vaginal e os sinais de
estro retomaram o padrão normal. Assim, comprovou-se que a vacinação de cadelas
com receptores de LH leva a um estado reversível de infertilidade (Saxena et al., 2002).
Imunocontracepção baseada em proteínas embrionárias
Outra modalidade de técnica imunocontraceptiva consiste da ligação de
anticorpos a moléculas bioativas do embrião, como a proteína transportadora de
riboflavina (RCP). A proteína transportadora de riboflavina é o principal mediador do
suprimento de vitamina do embrião em desenvolvimento. A vacinação de fêmeas de
primatas com a proteína transportadora de riboflavina de galinha resultou na produção
de anticorpos que interromperam a gestação, provavelmente durante o estágio peri-
implantação do embrião no útero (Adiga et al., 1997). A demonstração por
imunohistoquímica da presença da proteína transportadora de riboflavina em oócitos
ovulados e embriões em clivagem de fêmeas de rato sugere que estes podem constituir
um alvo para a citotoxicidade mediada por anticorpos e processos degenerativos.
Imunocontracepção baseada na Zona Pelúcida
A zona pelúcida é uma estrutura extracelular que circunda e protege oócitos e,
subseqüentemente, o embrião (Wolgemuth et al., 1984). A zona pelúcida é composta
por três glicoproteínas denominadas ZP1, ZP2 e ZP3. As duas últimas se unem para
formar filamentos, que são interligados por ZP1 (Greve e Wassarman, 1985). A zona
pelúcida desempenha funções fundamentais no processo de fertilização, que é iniciado
pela ligação dos espermatozóides à sua superfície (Gupta et al., 1997). Evidências
sugerem que, em camundongos, hamsters e no homem, a ZP3 constitui o receptor
primário ao qual se ligam os espermatozóides (Moller et al., 1990; Van-Duin et al.,
1994). Em suínos, sugere-se que a ZP3 e a ZP1 associadas participem do processo de
ligação dos espermatozóides (Yurewicz e Sacco, 1996).
A ligação da ZP3 ao receptor cognato do espermatozóide induz uma cascata de
transdução de sinal que determina a reação acrossômica (Gupta et al., 1997). A reação
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acrossômica resulta na liberação de enzimas proteolíticas necessárias para que os
espermatozóides penetrem na estrutura da zona pelúcida e na remodelação da superfície
dessas células para manutenção da adesão à zona pelúcida e para a fusão com a
membrana do oócito (Yanagimachi, 1994). Após a reação acrossômica, a ZP2 passa a
desempenhar a função de receptor (secundário) para os espermatozóides, mantendo-os
aderidos ao oócito (Gupta et al., 1997).
A ZP2 também desempenha uma função importante no mecanismo de bloqueio
à polispermia. Após a fusão de um espermatozóide ao oócito, ocorre a clivagem da ZP2
(Moller e Wassarman, 1989). Os fragmentos resultantes permanecem não-
covalentemente ligados à zona pelúcida, tornando essa zona resistente à proteólise e,
portanto, prevenindo a penetração espermática.
Assim, devido às funções fundamentais das glicoproteínas da zona pelúcida no
processo de fertilização, estas tornam-se moléculas-alvo em potencial para a
constituição de técnicas imunocontraceptivas. A viabilidade do uso de glicoproteínas da
zona pelúcida para a imunocontracepção foi demonstrada pela primeira vez pela
vacinação de fêmeas de camundongo com zona pelúcida de hamster, que resultou na
produção de anticorpos capazes de se ligar à zona pelúcida endógena, induzindo
infertilidade (Gwatkin et al., 1977). Antisoro de hamsters imunizados com ZPA humana
recombinante foi administrado a fêmeas de hamster, observando-se uma redução
significativa da fecundidade (Koyama e Hasegawa, 2004). Anticorpos produzidos
contra um peptídeo sintético correspondente a um segmento da zona pelúcida felina
(fZP) foram capazes de bloquear a ligação de espermatozóides a oócitos de gata e a
fertilização in vitro (Ringleb et al., 2004).
Estudos iniciais de caracterização imunológica revelaram que os anticorpos
produzidos para as glicoproteínas da zona pelúcida de uma espécie são capazes de se
ligar às glicoproteínas de várias outras espécies (Sacco et al., 1981). Esta reatividade
imunológica cruzada deve-se a graus variáveis de homologia das seqüências protéicas, o
que estabelece a possibilidade de vacinação heteróloga. Devido à grande
disponibilidade de ovários suínos em abatedouros, as preparações de zona pelúcida
suína (pZP) tornaram-se os antígenos de escolha (Gupta et al., 2004). A vacinação com
pZP mostrou-se efetiva para o controle populacional de cavalos selvagens (Kirkpatrick
et al., 1990), várias espécies de animais cativos em zoológicos (Kirkpatrick et al.,
1996), cervos de cauda branca (Rutberg et al., 2004) e elefantes-africanos (Fayrer-
Hosken et al., 1999).
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A vacinação de coelhas com pZP resultou em infertilidade (Wood et al., 1981).
O monitoramento subseqüente dos animais imunizados mostrou que o efeito foi
irreversível. A histologia dos ovários destes animais revelou uma destruição de oócitos
em todos os folículos em crescimento e uma depleção severa da população de folículos
quiescentes (Skinner et al., 1984). A vacinação de primatas não-humanos com pZP
também resultou em infertilidade irreversível associada a alterações de folículos
ovarianos (Gulyas et al., 1983). Estes e outros estudos, empregando modelos animais
diferentes, demonstraram que a vacinação com pZP resulta em infertilidade mais
provavelmente por distrofia do ovário que por um bloqueio da fertilização (Gupta et al.,
2004).
Diversos estudos têm acumulado evidências que indicaram o envolvimento da
resposta imunológica celular na histopatologia dos ovários após a vacinação com
antígenos da zona pelúcida (Naz et al., 1995). Rhim et al. (1992) demonstraram ser
possível transferir a ooforite de fêmeas de camundongo vacinadas com peptídeos que
continham segmentos da mZP3, para animais normais, através da transferência de
linfócitos T CD4+, sem a detecção de anticorpos anti-mZP3 nos receptores. Gu et al.
(2004), ao infectarem coelhas com um vírus myxoma recombinante que expressa a ZP2
de coelho (MV-ZPB), observaram a produção de anticorpos específicos para esta
proteína, que se ligaram à zona pelúcida nativa. Houve uma redução significativa das
quantidades de folículos ovarianos terciários e pré-ovulatórios das fêmeas infectadas,
sendo observada a infiltração de linfócitos T nos ovários de alguns animais.
Apesar dos diversos indícios de mecanismos imunológicos celulares para a
patogênese da redução de folículos ovarianos, em nenhum dos estudos em que foi
verificada depleção de folículos primordiais após vacinação com zona pelúcida,
observou-se a infiltração de linfócitos T (Aitken, 2002). De fato, não se conhece uma
associação entre a ooforite observada após a vacinação com zona pelúcida e a depleção
de folículos primordiais (Kerr et al., 1998).
Uma explicação alternativa para a patologia do ovário após a vacinação com
zona pelúcida é que a perda de folículos primordiais reflete seu recrutamento acelerado
para compor a população de folículos em crescimento que é eliminada devido à ação de
anticorpos anti-zona pelúcida (Aitken, 2002). Outra possibilidade é que uma proporção
significativa de folículos primordiais expressem níveis baixos de ZP3, sendo destruídos
por anticorpos antizona pelúcida por fixação de complemento (Grootenhuis et al.,
1996). Em coelhos, foi demonstrado que a resposta imunológica humoral (de
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anticorpos) pode causar perdas foliculares, provavelmente por meio da ligação de
anticorpos aos oócitos, que bloqueia a comunicação entre estas células e as da granulosa
(Skinner et al., 1984).
Em cadelas, o potencial do uso de técnicas imunocontraceptivas que têm as
glicoproteínas da zona pelúcida como moléculas-alvo tem sido avaliado visando ao
controle populacional de cães nas cidades. Mahi e Yanagimachi (1979) demonstraram
que anticorpos produzidos contra extratos de ovários de cadela são capazes de inibir a
fertilização de oócitos caninos in vitro. Em outro estudo, cadelas vacinadas com zona
pelúcida de suínos produziram altos títulos de anticorpos capazes de se ligar
cruzadamente à zona pelúcida canina (dZP) e apresentaram-se inférteis após
acasalamento (Mahi-Brown et al., 1982). A vacinação de cadelas com ZP3 canina
recombinante conjugada ao toxóide diftérico também resultou em infertilidade
(Srivastava et al., 2002). A análise histológica dos ovários dos animais imunizados
revelou um aumento da taxa de atresia folicular, não sendo observada infiltração por
linfócitos. Nosso grupo está avaliando o potencial da imunização passiva com
anticorpos antizona pelúcida canina para a imunoesterilização de cadelas.
Imunocontracepção baseada em antígenos espermáticos
Outra estratégia para o desenvolvimento de técnicas imunocontraceptivas que
bloqueiam a fertilização consiste da utilização de anticorpos capazes de se ligar às
moléculas bioativas da superfície do espermatozóide responsáveis pelas interações que
constituem este processo. Alternativamente, estes anticorpos, se presentes no muco
cervical, podem causar a aglutinação dos espermatozóides, impedindo-os de acessar o
trato reprodutivo superior feminino (Delves et al., 2002). Casos de ocorrência natural de
anticorpos aglutinadores de espermatozóides em mulheres de casais inférteis indicam
que estas estratégias podem ser efetivas (Van Voorhis e Stovall, 1997) e seguras, pois
não se observam quaisquer outros efeitos, além da infertilidade, devido à presença
destes anticorpos (Aitken, 2002). Potencialmente, estas técnicas podem ser aplicadas a
machos, mas o número de espermatozóides a serem neutralizados (~108-109) torna isso
um desafio maior que fazê-lo sobre um número limitado (dezenas a centenas) destas
células no trato reprodutivo superior feminino (Delves et al., 2002).
A vacinação de cangurus machos com espermatozóides homólogos resultou em
produção de anticorpos capazes de penetrar o trato reprodutivo e de se ligar ao
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acrossoma e à peça intermediária dos espermatozóides, resultando em redução das taxas
de fertilização (Asquith et al., 2006).
Dentre os antígenos espermáticos com potencial para servir como moléculas-
alvo em técnicas imunocontraceptivas, destacam-se as enzimas hialuronidase (PH-20),
lactato desidrogenase C4 (LDH-C4) e o antígeno associado ao espermatozóide-9
(SPAG9). A PH-20 está presente na superfície externa da membrana plasmática que
reveste o acrossoma, possibilitando ao espermatozóide penetrar a massa de células do
cumulus rica em ácido hialurônico (Sabeur et al., 1997). Homólogos desta molécula têm
sido encontrados em uma ampla variedade de espécies e sua eficácia contraceptiva
como imunógeno tem sido demonstrada em porcos-da-índia machos. A LDH-C4 é uma
enzima específica do espermatozóide de várias espécies (Goldberg, 1986). A vacinação
de fêmeas de babuíno com a LDH-C4 resultou em uma redução do índice de fertilidade
de 77% (controle) para 29% (Goldberg e Herr, 1999). O SPAG9 é uma molécula
presente no acrossoma em espermatozóides humanos e de outras espécies de primatas,
que parece participar da adesão destas células a oócitos. Anticorpos produzidos contra
SPAG9 recombinante humano (rhSPAG9) mostraram-se capazes de bloquear in vitro a
ligação entre espermatozóides e oócitos humanos (Jagadish et al., 2005).
Conclusão
A viabilidade do desenvolvimento de técnicas imunocontraceptivas eficazes,
demonstrada pelos estudos compilados neste trabalho, constitui uma importante
perspectiva para a solução do problema de superpopulação de cães. O controle da
reprodução de fêmeas é preferível ao de machos, uma vez que, em programas de
contenção de natalidade, poucos indivíduos machos não-controlados são capazes de
manter os índices de fertilidade de uma população conforme já foi verificado em alguns
casos. Neste contexto, para cadelas, destacam-se as técnicas imunocontraceptivas
baseadas no GnRH, receptor de LH, proteína transportadora de riboflavina, zona
pelúcida e antígenos espermáticos. Dentre estas, o uso da zona pelúcida para a
imunoesterilização através da eliminação dos folículos primordiais é uma técnica
bastante promissora.
52
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58
7 CAPÍTULO II
Preservação de folículos pré-antrais caninos por períodos curtos: efeitos da
temperatura, meio e tempo
Short-term preservation of canine preantral follicles: effects of temperature, medium
and time
Animal Reproduction Science
(Aceito para publicação, 2008)
59
Short-term preservation of canine preantral follicles: effects of temperature, medium and time
Cláudio Afonso Pinho Lopesa,b,*, Regiane Rodrigues dos Santosc, Juliana Jales de
Hollanda Celestinoa, Mônica Aline Parente Meloa, Roberta Nogueira Chavesa, Claudio
Cabral Campelloa, José Roberto Viana Silvad, Sônia Nair Báoe, Katarina Jewgenowb,
José Ricardo de Figueiredoa.
a. Faculty of Veterinary, PPGCV, LAMOFOPA, State University of Ceará, Fortaleza, Brazil.
Postal address: Av. Paranjana 1700, Campus do Itaperi, CEP 60.740-903, Fortaleza, CE,
Brazil.
b. Institute for Zoo and Wildlife Research, Berlin, Germany.
Postal address: Postfach 601103, D-10252, Berlin, Germany.
c. Department of Farm Animal Health, Utrecht University, The Netherlands.
Postal address: Yalelaan 7, 3584 CL Utrecht, Netherlands.
d. Biotechnology Nucleus of Sobral (NUBIS), Federal University of Ceará, Sobral, Brazil.
Postal address: Rua Gerardo Rangel s/n, Campus do Derby, CEP 62042-280, Sobral, CE,
Brazil.
e. Laboratory of Electron Microscopy, Institute of Biology, University of Brasília, Brazil.
Postal address: Universidade de Brasília, Campus Universitário Darcy Ribeiro (Asa Norte),
Instituto Central de Ciências (ICC Sul), CEP 70910, Brasília, DF, Brazil.
* Corresponding author. Tel.: +49 176 2671 8956. Fax +49 30 5126 104. E-mail address:
[email protected] (C.A.P. Lopes).
Correspondence address: Institut für Zoo- und Wildtierforschung (IZW). Postfach 601103,
D-10252, Berlin, Germany.
Resumo
O uso da vasta população de folículos pré-antrais é uma alternativa promissora para a
obtenção de elevados números de oócitos fertilizáveis para a biotecnologia
reprodutiva. Esta questão é particularmente importante para canídeos, uma vez que a
taxas atuais de successo das técnicas in vitro que utilizam oócitos são muito limitadas,
e muitas espécies desta família encontram-se ameaçadas pela extinção. O objetivo
deste estudo foi avaliar os efeitos da temperatura, meio e tempo sobre a morfologia e
viabilidade de folículos pré-antrais caninos durante a preservação por curtos períodos.
Ovários caninos foram divididos em fragmentos, que foram incubados em solução de
NaCl a 0,9% ou em Meio Essencial Mínimo (MEM) a 4, 20 ou 38°C por 2, 6, 12 or 24
h. Os folículos pré-antrais inclusos nos fragmentos foram então analisados por
60
histologia, microscopia eletrônica de transmissão e teste de viabilidade utilizando-se
Azul de Tripan, calceína-AM e etídio homodímero-1. As percentagens de folículos
morfologicamente normais e viáveis foram mantidas similares ao controle (tempo 0 h)
após a incubação em salina a 4ºC ou 20ºC por até 6 h, e a 38°C por 2 h. Utilizando-se
MEM, tal preservação foi possível por 12 h a 4°C ou 20ºC, e por 6h a 38ºC. Estes
resultados indicam que a preservação de folículos pré-antrais caninos é realizada com
maior eficiência através do armazenamento hipotérmico (4 ou 20°C) em MEM, que
possibilita a manutenção da morfologia e viabilidade por até 12 h.
Palavras-Chave: Preservação; Hipotermia; Morfologia; Viabilidade; Folículos pré-antrais;
Canino.
Abstract
The use of the large pool of preantral follicles is a promising alternative to
provide high numbers of fertilizable oocytes to reproductive biotechnology. This issue is
particularly important to canids, since current rates of success of in vitro techniques
using oocytes are very limited, and many species within this family are threatened by
extinction. The aim of this study was to evaluate effects of temperature, medium and
time on morphology and viability of canine preantral follicles during short-term
preservation. Canine ovaries were cut into fragments which were incubated in 0.9%
NaCl solution or in Minimum Essential Medium (MEM) at 4, 20 or 38°C for 2, 6, 12 or
24 h. Afterwards, preantral follicles were analyzed by histology, transmission electron
microscopy and viability testing using trypan blue, calcein-AM and ethidium
homodimer-1. Percentages of morphological normal and viable follicles were
maintained similar to control (time 0 h) after incubation in 0.9% NaCl at 4ºC or 20ºC for
up to 6 h and at 38°C for 2 h. Using MEM, such preservation was possible for 12 h at
4°C or 20ºC, and for 6h at 38ºC. These results indicate that preservation of canine
preantral follicles might be better accomplished through hypothermic (4 or 20°C)
storage in MEM, which ensures maintenance of morphology and viability for up to 12
h.
Keywords: Preservation; Hypothermia; Morphology; Viability; Preantral follicle; Canine.
61
1. Introduction
Reproductive biotechnology has a great potential to contribute for the
conservation of endangered wildlife species. However, a limiting factor for the
development and efficiency of reproductive techniques is the lack of abundant numbers
of fertilizable oocytes. This problem could be addressed by using the large source of
oocytes enclosed in preantral follicles (Telfer, 2001), which exist in ovaries in numbers
of thousands to millions depending on the species. In vitro culture systems capable to
promote growth and maturation of these follicles have been developed for several
species, and birth of healthy offspring after embryo transfer of in vitro fertilized oocytes
derived from primordial follicles has, so far, been achieved in mice (Eppig and
Schroeder, 1989).
Due to the fact that time from ovary collection to the outset of in vitro culture can
be very long, especially when the donor animal is far from the reproductive laboratory,
as is the case with free-ranging individuals in the wild, preservation of viability is a
critical issue since cells are under ischemia. In this context, studies have assessed
short-term preservation of preantral follicles combining different temperatures and
media in several species such as caprine (Silva et al. 2000, 2001; Carvalho et al. 2001;
Ferreira et al. 2001), ovine (Andrade et al. 2001, 2002a,b; Matos et al. 2004), bovine
(Lucci et al. 2004) and swine (Lucci et al. 2007).
Conversely, to our knowledge, there is not to date a study on short term
preservation of canine preantral follicles. Few works have addressed the storage of
canine ovaries, but only effects on in vitro maturation (IVM) of oocytes from antral
follicles have been evaluated and findings are controversial. Taş et al. (2006)
demonstrated that bitch oocytes maintained at 4°C in physiologic saline had higher
maturation rates than those kept at 35-38°C. In contrast, Lee et al. (2006) observed
that viability of canine antral oocytes after in vitro maturation was significantly
decreased when ovaries were stored at 4°C in relation to 38°C.
Thus, methods for preservation of canine oocytes are still to be established, and
more studies are needed to gather consistent data. Once established, in vitro embryo
production in dogs based upon the vast pool of preantral follicles could be adapted to
endangered wild canids, which would contribute enormously to the multiplication of
individuals of such species. Nine canid species are under risk of extinction, which has
already occurred to the Falklands Wolf, Dusicyon australis (IUCN, 2007).
In this context, the aim of this study was to assess the preservation of canine
preantral follicles maintained in physiologic saline solution or in Minimum Essential
Medium (MEM) at different temperatures and times of storage. To this end, evaluation
62
of morphology through histological and ultrastructural analyses, as well as viability
assays based on trypan blue dye exclusion and the fluorescent probes calcein-AM and
ethidium homodimer were performed.
2. Materials and methods 2.1. Source and preparation of canine ovaries
Pairs of ovaries (n=15) were aseptically collected during ovariectomy of healthy
mixed-breed bitches (Canis familiaris) whose reproductive history and age were
unknown, but it was estimated that most were 12-24 months old and believed
nulliparous. After removal from the bursa ovarica, ovaries were rinsed once with 70%
ethanol for 10 seconds, twice in sterile phosphate-buffered saline (PBS) and eventual
corpora lutea were excised using a scalpel.
2.2. Experimental design
2.2.1. Experiment I: morphological evaluation of stored canine preantral follicles
Immediately after collection and preparation, pairs of ovaries (n=5) were divided
each into 25 fragments, from which one was randomly selected as control (time 0 h) and fixed for histology and ultrastructural analysis. The remaining fragments were
placed into 15 ml tubes (Corning Glass Works, Corning, NY, USA) containing 2 ml of
sterile saline solution (0.9% NaCl solution, osmolarity 300 mOsmol/l, pH 7.2) or
HEPES-buffered Minimum Essential Medium (MEM, osmolarity 280 mOsmol/l, pH7.2;
Sigma, St. Louis, MO, USA) at 4, 20 or 38ºC and stored for 2, 6, 12, or 24 h, and
subsequently fixed for morphological analysis as the non-stored control sample (Figure
1). Thermoflasks filled with water were used for maintaining temperatures, which were
monitored throughout the experiment. Each treatment was repeated five times.
63
Figure 1. Design of experiment I: pairs of canine ovaries were divided into 25
fragments, being one selected as non-stored control (time 0 h), whilst the others were
incubated in saline solution or in Minimum Essential Medium (MEM) at 4, 20 or 38ºC
for 2, 6, 12, or 24 h. Afterwards, fragments were submitted to morphological analysis.
2.2.2. Experiment II: assessment of viability of stored canine preantral follicles
In this study, effects of storage on canine preantral follicles were further
analyzed through assessment of viability. Five pairs of canine ovaries were cut each
into 25 fragments, and one was randomly selected and immediately submitted to
follicle isolation followed by trypan blue dye exclusion test as described later. The other
fragments were maintained in saline solution or in MEM at 4, 20 or 38°C for 2, 6, 12 or
24 h, and afterwards evaluated similarly as for the control.
Based on results of experiment I and trypan blue test, a second viability trial
was performed. Pairs of ovaries (n=5) were cut into three portions, from which one was
immediately processed for follicle isolation and testing using trypan blue as well as
calcein-AM and ethidium homodimer, which was employed to provide an additional and
more precise method of viability assessment. The remaining fractions were stored in
MEM at 4°C for 12 and 24 h, and afterwards analyzed similarly as for the control.
64
2.3. Histological analysis
In order to assess the morphology of canine preantral follicles submitted to the
different treatments, fragments of ovarian tissue were fixed in Carnoy for 12 h,
dehydrated in a graded series of ethanol, clarified with xylene, embedded in paraffin
wax and serially sectioned at 7 µm. Every fifth section was mounted on glass slides,
stained with periodic acid Schiff (PAS)-hematoxylin and evaluated by light microscopy
at a 400x magnification (Zeiss, Germany). Preantral follicles were defined as an oocyte
surrounded either by one layer of flattened or cuboidal granulosa cells, or several
layers of cuboidal granulosa cells with no antrum. Follicular morphology was evaluated
based on the integrity of the oocyte, granulosa cells and basement membrane.
Preantral follicles were classified and counted as (i) morphologically normal, when
containing an oocyte with regular shape and uniform cytoplasm, and organized layers
of granulosa cells, or (ii) degenerated, when the oocyte exhibited pycnotic nucleus
and/or ooplasma shrinkage, and occasionally granulosa cells layers became
disorganized, detached from the basement membrane and/or included enlarged cells.
To avoid evaluating and counting a follicle more than once, preantral follicles were
analyzed only in the sections where oocyte nucleus was observed.
2.4. Ultrastructure analysis
In order to better examine follicular morphology, transmission electron
microscopy (TEM) was performed to analyze ultrastructure of preantral follicles from
the control, as well as from treatments that did not differ statistically from control in
histology. A portion with a maximum dimension of 1 mm3 was cut from each fragment
of ovarian tissue and fixed in Karnovsky solution (2% paraformaldeyde and 2%
glutaraldeyde in 0.1 M sodium cacodylate buffer pH 7.2) for 3 h at room temperature
(RT, approximately 25°C). After three washes in sodium cacodylate buffer, specimens
were post-fixed in 1% osmium tetroxide, 0.8% potassium ferricyanide and 5 mM
calcium chloride in 0.1 M sodium cacodylate buffer for 1 h at RT. The samples were
then dehydrated through a gradient of acetone solutions and thereafter embedded in
Spurr’s epoxy resin. Afterwards, semi-thin sections (3 µm) were cut, stained with
toluidine blue and analyzed by light microscopy at a 400x magnification. Ultra-thin
sections (60–70 nm) were obtained from preantral follicles classified as morphologically
normal in semi-thin sections, according to the criteria adopted in histology.
Subsequently, ultra-thin sections were contrasted with uranyl acetate and lead citrate,
65
and examined under a Jeol 1011 (Jeol, Tokyo) transmission electron microscope
operating at 80 kV.
2.5. Assessment of preantral follicles viability
Canine preantral follicles were isolated from control and stored ovarian
fragments using the mechanical method described by Figueiredo et al. (1993). Briefly,
using a tissue chopper (The Mickle Laboratory Engineering Co., Gomshal, Surrey, UK)
adjusted to a sectioning interval of 87.5 µm, samples were cut into small pieces, which
were placed in MEM and suspended 40 times using a large Pasteur pipette (diameter
of about 1600 µm) and subsequently 40 times with a smaller Pasteur pipette (diameter
of approximately 600 µm) to dissociate preantral follicles from stroma. The obtained
material was passed through 500- and 100-μm nylon mesh filters, resulting in a
suspension containing preantral follicles smaller than 100 μm in diameter. This
procedure was carried out within 10 min at RT.
Thereafter, viability of preantral follicles was assessed through trypan blue dye
exclusion test (Jewgenow et al., 1998). Briefly, 10 μl of 0.4% trypan blue (Sigma,
Deisenhofen, Germany) were added to 90 μl of the suspension of isolated preantral
follicles, which were examined using an inverted microscope after incubation for 5 min
at RT. Follicles were classified as viable if the oocyte and <10% granulosa cells
remained unstained, or as non-viable if uptake of the dye by the oocyte and/or ≥10%
granulosa cells occurred.
Preantral follicles were also analyzed using a two-color fluorescence cell
viability assay based on the simultaneous determination of live and dead cells by
calcein-AM and ethidium homodimer-1, respectively. Whilst the first probe detected
intracellular esterase activity of viable cells, the later labeled nucleic acids of non-viable
cells with plasma membrane disruption. Since it is difficult to distinguish the outline of
live cells labeled with calcein-AM within follicle structure, determination of the
percentage of viable granulosa cells was achieved through using Hoechst 33343 to
detect all nuclei, enabling counting of the total number of cells. The test was performed
by adding 4 μM calcein-AM, 2 μM ethidium homodimer-1 (Molecular Probes,
Invitrogen, Karlsruhe, Germany) and 10 μM Hoechst 33342 (Sigma, Deisenhofen,
Germany) to the suspension of isolated follicles, followed by incubation at 37°C for 10
min. After being labeled, follicles were washed three times by centrifugation at 100xg
for 5 min and resuspension in MEM, mounted on a glass microscope slide in 5 μl
antifading medium (DABCO, Sigma, Deisenhofen, Germany) to prevent
photobleaching, and finally examined using an a DMLB fluorescence microscope
66
(Leica, Germany). The emitted fluorescent signals of Hoechst, calcein-AM, and
ethidium homodimer were collected at 350, 488, and 568 nm, respectively. Oocytes
and granulosa cells were considered live if the cytoplasm was stained positively with
calcein-AM (green) and chromatin was not labelled with ethidium homodimer (red).
Preantral follicles were classified as viable when the oocyte and <10 % of granulosa
cells were live, and as non-viable, when the oocyte and/or ≥10 % granulosa cells were
dead.
2.6. Statistical analysis
All experiments were replicated five times. Analysis of variance (ANOVA) of the
data was performed using the GLM procedure of the software SAS (SAS Institute Inc.,
Cary, NC, USA) with a factorial design (medium, temperature and time of storage).
Differences of percentages of morphologically normal preantral follicles (MNPF) and
viable follicles between control and treatments (combinations of medium, temperature
and time of storage) were determined by Duncan’s multiple range test. Differences
were considered statistically significant when P < 0.05.
3. Results
3.1. Experiment 1: Morphological evaluation of stored canine preantral follicles
3.1.1. Histological analysis
A total of 4,045 preantral follicles were examined in histology, with a range of
146 to 179 in each treatment. Proportions of preantral follicles at each developmental
stage were similar among fragments obtained from a pair of ovaries and thus follicular
diameters were homogenous between treatments. Most of the studied follicles were at
the primordial or resting stage and had a diameter of 25-30 µm. Primary or activated
follicles (30-40 µm) and secondary follicles (40-100) were also analyzed in lower
numbers equally found in all experimental groups. MNPF in control as well as after
storage exhibited a spherical or elliptical oocyte with a large central or eccentric
nucleus and uniform cytoplasm. Granulosa cells without pycnotic nuclei were well-
organized in layers surrounding the oocyte and a distinguishable intact basement
membrane could be observed (Fig. 2A-C). Degenerated follicles showed a retraced
oocyte with or without a pycnotic nucleus, and occasionally a strongly eosinophilic
cytoplasm (Fig. 2D). Layers of granulosa cells remained unaltered or became
67
disorganized into a low density mass of cells which were many times swollen and/or
detached from basement membrane. Occurrence of pycnotic bodies in granulosa cells
or rupture of the basement membrane were not observed.
Fig. 2. Histological features of stored canine ovarian fragments. Morphologically normal
primordial (A) and primary (B) follicles comprised an oocyte (o) displaying a large
nucleus (nu) and homogenous cytoplasm surrounded by one layer of flattened or
cuboidal granulosa cells (gc) respectively, whilst in secondary follicles two or more
layers were present without antrum (C). Degenerated preantral follicles often displayed
oocyte retraction (D, arrow) and disorganization of granulosa cell layers. The
arrowhead indicates an intact basement membrane. Scale bars represent 6 µm (A) and
10 µm (B,C and D). PAS-hematoxilin.
68
The effects of medium, temperature and time of storage on the percentages of
MNPF are shown in Fig. 3. Maintenance in saline solution at 4°C for up to 24 h, at 20°C
for up to 12 h or at 38°C for 6 h kept proportions of MNPF similar (P > 0.05) to control
values (time zero). A decrease (P < 0.01) of the percentages of MNPF in relation to the
control was observed after holding in saline solution at 20°C for 24 h and at 38°C for 12
and 24 h. With respect to MEM, similar results were obtained, with exception of the
maintenance of follicles at 20°C for 24 h and at 38°C for 12 h, for which percentages of
MNPF were also similar (P > 0.05) to control values.
Fig. 3. Effects of medium, temperature and time of storage on the percentages of
morphologically normal canine preantral follicles. (*) Differs significantly from the
control (P < 0.05); (a, b, c) different letters for the same medium and time of incubation
denotes significant differences between temperatures (P < 0.05); (A, B, C) different
letters for a given combination of medium and temperature indicates significant
differences among times of incubation (P < 0,05); (X,Y) different letters represent
significant differences between media for fixed time and temperature of incubation.
The effect of storage time on the percentages of MNPF at each temperature
and for each solution was analyzed. At 4°C, percentages of MNPF were not affected by
the incubation time, for both solutions. Similar results were obtained after preservation
in MEM at 20°C. Otherwise, when ovarian fragments were kept at 20°C in saline
solution, there was a decrease (P < 0.01) in the percentages of MNPF after 24 h
compared to 2, 6 or 12 h. A reduction (P < 0.05) in the proportions of MNPF occurred
69
at 38°C with the increase of incubation time to 12 h in saline solution and to 24 h in
MEM.
Regarding the effect of temperature on percentages of MNPF for a defined
period of storage, a decrease (P < 0.05) in percentages of MNPF was observed when
ovarian fragments were maintained in MEM for 24 h at 38ºC in comparison to 4ºC.
The comparison between saline solution and MEM at a same temperature and
storage period showed that at 38°C after 12 h, percentages of MNPF were higher when
ovarian fragments were maintained in MEM.
3.1.2. Ultrastructure analysis
Transmission electron microscopy of stored preantral follicles was performed for
assessment of ultrastructure in comparison to normal follicles from the control group. At
least five follicles per group were analyzed. Normal follicles contained an oocyte
displaying a very homogenous cytoplasm plenty of round shaped mitochondria with
continuous membranes, few peripheral cristae and electron-dense granules. Some
elongated forms with parallel cristae could also be seen. Small Golgi apparatus
cisternae were rarely observed. Smooth and rough endoplasmic reticulum were
present, either as isolated aggregations or as complex associations with mitochondria.
The nuclei of oocytes were large and usually round, well delimited by the nuclear
envelope, the chromatin was uncondensed and a nucleolus could often be identified.
Granulosa cells were small and presented a high nucleus-to-cytoplasm ratio. Their
irregularly shaped nuclei contained loose chromatin in the inner part and small
peripheral aggregates of condensed chromatin. The cytoplasm exhibited a great
number of mitochondria and well-developed smooth and rough endoplasmic reticulum.
The cellular membranes of oocyte and surrounding granulosa cells were closely
justaposed, and sometimes few short microvilli could be observed. A distinct
continuous basement membrane surrounded follicles and was tightly attached to
ovarian stroma (Fig. 4).
70
Fig. 4. Electron micrographs of normal follicles from control group (A) and stored in
MEM at 4°C for 12 h (B). O, oocyte; GC, granulosa cells; ne, nuclear envelope; m,
mitochondria; ser, smooth endoplasmic reticulum; bm, basement membrane; sc,
stromal cells. (A, 5000x, scale bar=5 µm; B, 10000x, scale bar = 2 µm).
The ultrastructural pattern described above was observed in normal follicles
from control as well as after storage in MEM at 4°C or 20°C for up to 12 h and at 38°C
for up to 6 h; in saline solution at 4°C or 20°C for up to 6 h. When ovarian fragments
were incubated at 4°C in MEM or in saline solution for 24 h and 12 h respectively, and
at 38ºC in saline solution for 2 h, discreet changes in ultrastructure suggesting onset of
degeneration could be observed in follicles evaluated as morphological normal in semi-
thin sections by light microscopy. Some mitochondria both in the oocyte and in
granulosa cells were very enlarged, displayed fewer cristae and lower electron density
of the matrix, which may indicate swelling (fig. 5).
71
Fig. 5. Ultrastructure of follicles stored in MEM at 4°C for 24 h. Mitochondria displaying
substantial enlargement, loss of peripheral cristae and an electron-lucent matrix
(arrows) were observed both in oocytes (A) and granulosa cells (B). O, oocyte; GC,
granulosa cells; nu, nucleolus; m, mitochondria; bm, basement membrane. (A, 6000x,
scale bar=5 µm; B, 10000x, scale bar = 2 µm).
More severe alterations of ultrastructure were noticed after storage in saline
solution at 4ºC for 24 h, at 20ºC for 12 h and at 38ºC for 6 h; in MEM at 20ºC for 24 h
and at 38ºC for 12 h. Follicles contained high numbers of vacuoles spread throughout
the cytoplasm of all cells, which often fused creating large vacated areas. Furthermore,
signs of proliferation of the endoplasmic reticulum and damage to mitochondrial
membranes and cristae were observed. Granulosa cells had a swollen aspect and
presented a very low density of organelles. Some of these cells completely
disappeared, resulting in a empty space. In oocytes, swelling of the nuclear cisterna
and disruption of the nuclear envelope were observed along some segments of the
nuclear outline (fig. 6). In some follicles kept in MEM at 20ºC for 24 h, the cytoplasm of
oocyte and granulosa cells seemed to be clotted, possibly due to nuclear content leak.
72
Fig. 6. Electron micrographs of follicles stored in saline solution at 4°C for 24 h. A
primordial follicle containing several vacuoles spread throughout the ooplasma as
indicated by arrows (A, 5000x, scale bar=5 µm); the insert is displayed at a higher
magnification (B, 12000x, scale bar=2 µm), note the occurrence of nuclear cisterna
swelling (arrowhead). Another primordial follicle exhibiting widespread vacuolization
and a granulosa cell with a very large vacated area indicated by an asterisk (C, 8000x,
scale bar=2 µm); a higher magnification of the same follicle (D, 15000x, scale bar=2
µm), observe the discontinuity of the nuclear envelope (thick arrow). O, oocyte; GC,
granulosa cells; nu, nucleolus; ne, nuclear envelope; m, mitochondria; bm, basement
membrane.
73
3.2. Experiment II: assessment of viability of stored canine preantral follicles
Preantral follicles were mechanically isolated from control and stored canine
ovarian fragments and viability was assessed by trypan blue dye exclusion test (fig. 7).
A total of 6,037 follicles were examined, with a range of 171 to 281 in each treatment.
Fig. 7. Viability assessment of canine preantral follicles using trypan blue (TB) dye
exclusion test. Isolated preantral follicles were incubated with 0.04% TB for five
minutes and then classified as viable if the oocyte and <10% granulosa cells remained
unstained (A), or as non-viable if oocyte and/or ≥10% granulosa cells were stained (B).
Scale bars represent 10 µm.
The effects of medium, temperature and time of storage on the percentages of
viable preantral follicles are shown in Table 1. Maintenance in saline solution at 4°C or
20ºC for up to 6 h kept proportions of viable follicles similar (P > 0.05) to control values
(time zero). A decrease (P < 0.01) of the percentages of viable follicles in relation to the
control was observed after holding in saline solution at 4ºC or 20°C for 12 and 24 h,
and at 38°C after all incubation times. With respect to MEM, storage of follicles at 4ºC
or 20ºC for up to 12 h, and at 38°C for up to 6 h preserved viable follicles, whose
percentages were similar (P > 0.05) to control values.
74
Table 1.
Percentages (viable/total) of viable canine preantral follicles in control and after storage.
Medium Temperature
(°C)
Time (h)
2 6 12 24
Control 67 (186/279)
Saline 4º 55% abA
(151/276)
52% abA
(132/255)
41% *abA
(107/259)
37% *abA
(87/234)
20º 54% abA
(123/229)
52% abAB
(145/281)
39% *abB
(97/246)
20% *bC
(35/171)
38º 43% *bA
(96/222)
43% *bA
(115/265)
30% *bB
(69/231)
23% *bB
(45/195)
MEM 4º 67% aA
(172/258)
61% aA
(159/259)
64% cA
(176/275)
48% *aB
(96/200)
20º 65% aA
(171/264)
58% abAB
(160/277)
60% cAB
(162/272)
32% *abB
(59/185)
38º 62% aA
(138/222)
58% abAB
(148/255)
47% *aB
(111/238)
22% *bC
(42/189)
(*) Differs significantly from control (P < 0.05); (a, b, c) different letters denotes
significant difference between values within a column (P < 0.05); (A, B, C) different letters
indicates significant difference between values of a row (P < 0,05).
The effect of storage time on the percentages of viable follicles at each
temperature and for each solution was analyzed. At 4°C, percentages of viable follicles
in saline solution were not affected by the incubation time, but a significant reduction (P
< 0.05) was observed in MEM from 12 to 24 h of holding. When ovarian fragments
were kept at 20°C in saline solution, there was a decrease (P < 0.01) of the
percentages of viable follicles after 12 h, and a further reduction from 12 h to 24 h; in
MEM, this reduction was observed after 24 h only. A decrease (P < 0.05) in the
proportions of viable follicles occurred with the increase of incubation time to 12 h in
both solutions at 38°C. An additional decrease (P < 0.01) was observed in MEM at
38°C from 12 to 24 h.
75
Regarding the effect of temperature on percentages of viable follicles for a
defined period of storage, a reduction (P < 0.05) in the viability of follicles was
observed when ovarian fragments were maintained in MEM at 38°C for 12 h compared
to 4ºC and 20ºC, and for 24 h, in relation to 4ºC only.
The comparison between saline solution and MEM at a same temperature and
incubation period showed that after storage at 38ºC for 2 h, and at all temperatures for
12 h, percentages of viable follicles were higher when ovarian fragments were
maintained in MEM.
Based on the results of morphological evaluation (experiment I) and viability
assessment with trypan blue, which evidenced that storage in MEM at 4ºC is able to
maintain viability of canine preantral follicles for the longer time (12 h) and to preserve
ultrastructure more efficiently, as verified after 24 h of incubation, a second viability trial
using this protocol was performed with five replicates. In addition to trypan blue testing,
a fluorescence cell viability assay based on labeling of live and dead cells by calcein-
AM and ethidium homodimer-1, respectively, was employed. Hoechst 33343 was
integrated to the test to allow counting of total cell number for calculation of the
percentage of viable granulosa cells within each follicle (fig. 8).
76
Fig. 8. Viability assessment of canine preantral follicles using fluorescent probes. (A)
An isolated preantral follicle classified as viable (B) since cells were labeled by calcein-
AM (green fluorescence). (D) A secondary follicle considered non-viable (E) as cells
were marked with ethidium homodimer-1 (red fluorescence). (C, F) The same follicles
in A and D respectively, stained with Hoechst 33342, which was also used in the assay
to allow counting of the total number of cells for calculation of the percentage of dead
cells. Scale bars represent 20 µm.
After storing ovarian fragments in MEM at 4ºC for 12 h, percentages of viable
follicles remained unaltered in comparison to control, as assessed by both trypan blue
and calcein-AM/ethidium homodimer assays, whose values did not differ from each
other (P < 0.01). However, a significant decrease (P < 0.05) in percentages of viable
follicles was observed with the increase of incubation to 24 h according to the two
viability tests, which once more retrieved similar results (fig.9).
77
Fig. 9. Percentages of viable canine preantral follicles in fresh ovaries and after storage
in MEM at 4ºC for 12 h or 24 h as assessed by trypan blue dye exclusion test and
labeling with calcein-AM and ethidium homodimer. (*) Differs significantly from control
(P < 0.05); (a, b) different letters for values of viability assays within the same time of
incubation denotes significant differences (P < 0.05); (A, B) different letters for results
of a same test indicates significant differences between times of incubation (P < 0,05).
4. Discussion
The present study evaluated for the first time the short term preservation of
canine preantral follicles, which can be successfully accomplished using either 0.9%
NaCl solution or MEM, but efficiency of each medium depends on temperature and
time of holding.
Percentages of MNPF remained unaltered, as observed by histological
analysis, after incubation at 4ºC in both media for up to 24 h, and at 20ºC in saline
solution and MEM for up to 12 h and 24 h respectively. Conversely, holding at
physiologic temperature (38ºC) maintained percentages of MNPF for only 6 h in saline
solution and 12 h in MEM. Therefore, hypothermia during storage of canine ovaries
provides more efficient preservation of morphology of preantral follicles. This may be
explained by a reduction of metabolism, followed by a decrease in requirements of
oxygen and nutrients as well as metabolites production, which is critically important
since cells are under ischemia. Indeed, hypothermia has proven to be an exceptional
means of protection of any organ, with metabolic rate falling exponentially by about
78
50% per 6°C drop in organ temperature (Kirklin & Barrat-Boyes, 1993). Our results are
in accordance with other works, which reported that maintenance at 4ºC and 20ºC
preserves morphology of bovine (Lucci et al., 2004), caprine (Carvalho et al., 2001;
Ferreira et al., 2001; Silva et al., 2000, 2001) and ovine (Andrade et al., 2001; Matos et
al., 2004) preantral follicles for longer times than at physiological temperature. In
addition, Wood et al. (1997) showed that maintenance in saline at 4ºC inhibited
degeneration of cat follicles for 48 h after excision. Transmission electron microscopy revealed that, after storage in saline solution
at 4ºC and 20ºC for 12 h, at 38°C for 2 h, in MEM at 4ºC and 20ºC for 24 h and at 38ºC
for 12 h, preantral follicles considered morphologically normal in histology had
alterations in ultrastructure indicative of degeneration. Therefore, electron microscopy
proved to be a valuable tool to detect early morphological modifications due to atresia,
which can be observed only at the ultrastructural level before becoming pronounced
more grossly to be identified through light microscopy. Ultrastructure of ovine (Matos et
al., 2004) and caprine (Silva et al., 2000) preantral follicles could be preserved for up to
24 h at 4ºC. The shorter periods of preservation of ultrastructure observed in the
present study may be due to a higher sensibility of canine preantral follicles to ischemia
and/or to cold storage in relation to follicles of small ruminants.
Follicles in ovarian fragments maintained at 4ºC in MEM and saline solution for
24 h and 12 h respectively, and at 38ºC in saline solution for 2 h displayed the least
degree of damaging, containing some enlarged mitochondria with reduced cristae and
electron-lucent matrix. Silva et al. (2001) observed that damage to mitochondria is one
of the first signs of degeneration in goat preantral follicles during storage in vitro.
Ischemia impairs cellular energetic metabolism which decreases the activity of the
Na+/K+-ATPase pump with a gain in sodium (Bonz et al., 1998). During mild ischemia,
mitochondrial matrix swells moderately due to uptake of sodium (Garlid, 1996).
Incubation in MEM at 20ºC for 24 h and at 38ºC for 12 h, and in saline solution
at 4ºC for 24 h and at 20ºC for 12 h led to more severe changes characterized mainly
by high numbers of vacuoles spread throughout the cytoplasm of both oocyte and
granulosa cells. Silva et al. (2001) demonstrated that structural changes due to
degeneration progress with the increase of preservation temperature and incubation
time. Atresia in vivo is also characterized by cytoplasmic vacuoles in oocytes (van den
Hurk et al., 1998) and granulosa cells (Hay et al., 1976), which may be originated from
endoplasmic reticulum swelling. On other hand, these vacuoles may be derived from
altered mitochondria, as observed in cryopreserved bovine oocytes by Fuku et al.
(1995).
79
Notwithstanding previous studies on preservation of preantral follicles provided
important knowledge on this issue, approaches were limited to analysis of morphology,
which is not always correlated with the viability of follicles (Santos et al., 2007).
Therefore, in the present work, viability assessment using the trypan blue dye
exclusion test was employed. It was observed that storage in MEM at 4ºC and 20ºC
maintained percentages of viable preantral follicles for up to 12 h, whilst at 38ºC such
preservation was possible for up to 6 h. In saline, percentages of viable follicles were
kept at 4ºC and 20ºC for up to 6 h, and at 38ºC, a significant decrease of viability was
observed after only 2 h of incubation. Percentages of viable follicles were much lower
than those of morphologically normal follicles in every treatment. This proves that a
more precise determination of the proportions of follicles with adequate quality for
subsequent applications such as in vitro culture may rely on viability assessment.
We further analyzed viability of canine preantral follicles stored in MEM at 4ºC,
the protocol which maintained proportions of viable follicles for the longer time (12 h)
while preserving ultrastructure more efficiently after 24 h, using a more accurate
method based on fluorescent probes. For this purpose, labeling of non-viable cells with
disruption of plasma membrane was performed again by using ethidium homodimer-1,
which enabled a better individualized visualization of dead cells through fluorescent
staining of nuclei. Concomitantly, identification of viable cells was performed through
detection of esterase activity with calcein-AM. Hoechst 33342 was used to mark all
nuclei in each follicle for total cell number counting and calculation of percentages of
live cells. Results of this assay were similar to values observed with trypan blue testing,
which thus proved to be a reliable, practical and quick method for viability assessment
of preantral follicles. Accordingly, Poeschmann et al. (2008) observed a significant
correlation between both methods while analyzing viability of feline preantral follicles.
In this study, MEM was more efficient than saline solution to preserve viability of
follicles after 12 h of incubation at any tested temperature. In addition, at 38ºC, MEM
was able to maintain viability similar to control for 6 h, whilst, in saline solution,
percentages of viable follicles were decreased after only 2 h of incubation.
Furthermore, after 24 h at 4ºC, ultrastructure of follicles was altered to a lesser extent
in ovarian fragments maintained in MEM. Therefore, composition of medium is critical
for short-term storage of canine preantral follicles. We infer that endogenous nutrient
resources in these follicles are very limited, thus it is necessary to provide
supplementary nutritional compounds in preservation solutions. Although hypothermia
may have reduced metabolic rates, these were possibly high enough to have depleted
own energetic sources of follicles kept in saline at 4ºC and 20ºC after 6 h, leading to
80
degeneration, whereas MEM, which comprises sugars, aminoacids, vitamins and
inorganic salts, supported survival for up to 12 h.
In conclusion, preservation of canine preantral follicles during storage is
achieved more efficiently through hypothermia in a nutritive medium. Maintenance of
viability and ultrastructural integrity can be accomplished at 4°C and 20ºC in saline
solution for 6 h or in MEM for 12 h, being the later solution also adequate at 38ºC for 6
h. We suggest the use of MEM at 4ºC for up to 12 h in order to provide optimal
preservation of quality of canine preantral follicles for subsequent applications such as
in vitro culture.
Acknowledgements
During the period of this work, Claudio A. P. Lopes received financial support
from the National Council for Scientific and Technological Development (CNPq;
doctoral scholarship) and the Coordination for Improvement of Graduate Personnel
(CAPES; fellowship for studies abroad, PDEE, grant 5212/06-5), both institutions of the
Brazilian Government. José Ricardo de Figueiredo was also recipient of a grant from
CNPq. We would like to express our appreciation to the Center for Zoonosis Control
(CCZ) of Fortaleza Municipality (Brazil) and to the veterinary clinics of the animal
shelter Tierheim-Berlin and Tierklinik-Biesdorf (Germany), which gently provided the
dog ovaries. We also thank Dr. Vicente J. F. Freitas (LFCR, UECE) for logistical
support.
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Morphological and ultrastructural analysis of sheep primordial follicles preserved in
0.9% saline solution and TCM 199. Theriogenology 62, 65–80.
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Veterinary and Human Reproductive Medicine. Reprod. Domest. Anim. 43 (Suppl.
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Hurk, R., 2007. Vitrification of goat preantral follicles enclosed in ovarian tissue by
using conventional and solid-surface vitrification methods. Cell Tissue Res. 327,
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Silva, J.R.V., Lucci, C.M., Carvalho, F.C.A., Báo, S.N., Costa, S.H.F., Santos, R.R.,
Figueiredo, J.R., 2000. Effect of coconut water and Braun–Collins solutions at
different temperatures and incubation times on the morphology of goat preantral
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Figueiredo, J.R., 2001. Morphological and ultrastructural changes occuring during
degeneration of goat preantral follicles preserved in vitro. Anim. Reprod. Sci. 66,
209–23.
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Telfer, E.E., 2001. In vitro development of pig preantral follicles. Reprod. Suppl. 58,
81–90.
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taphonomy in cold-stored domestic cat ovaries. Mol. Reprod. Dev. 46, 190–200.
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8 CAPÍTULO III
Criopreservação de folículos pré-antrais caninos utilizando-se etilenoglicol e
glicerol
Cryopreservation of canine preantral follicles using ethylene glycol and glycerol
Brazilian Journal of Veterinary Research
(Submetido, 2008)
84
Cryopreservation of canine preantral follicles using ethylene glycol and glycerol C.A.P. Lopes1,3, M.C.A. Luque2, S. B. Vasconcelos2, G.M. Silva1, S.N. Báo2, J.R. Figueiredo1 1 LAMOFOPA, PPGCV, Faculty of Veterinary, State University of Ceará, Brazil. 2 Laboratory of Electron Microscopy, Institute of Biology, University of Brasília, Brazil. 3 Corresponding author. Email: [email protected]
Resumo
O objetivo do presente estudo foi avaliar os efeitos da congelação lenta-
descongelação rápida sobre a morfologia de folículos pré-antrais caninos utilizando-se
glicerol e etilenoglicol. Fragmentos de tecido ovariano canino foram equilibrados por
20 min a 20ºC em Meio Essencial Mínimo contendo um dos crioprotetores
mencionados à concentração de 1,5 M, sendo então congelados, estocados em
nitrogênio líquido (-196ºC) por uma semana e descongelados. A morfologia dos
folículos foi analisada através de histologia e microscopia eletrônica de transmissão.
As percentagens de folículos pré-antrais morfologicamente normais (FPMN) foram
significativamente reduzidas após a exposição ao glicerol em relação ao controle, o
que não foi observado com o etilenoglicol. Após a congelação/descongelação, as
percentagens de FPMN apresentaram-se significativamente inferiores em comparação
ao controle utilizando-se ambos crioprotetores, que não diferiram estatisticamente
entre si. Entretanto, a microscopia eletrônica de transmissão revelou que apenas os
folículos congelados com etilenoglicol possuíam ultra-estrutura normal. Como
conclusão, este estudo demonstrou que a morfologia de folículos pré-antrais caninos
pode ser preservada através de congelação lenta seguida de descongelação rápida
utilizando-se etilenoglicol a 1,5 M. Abstract
The aim of the present study was to evaluate the effects of slow freezing-rapid
thawing using glycerol and ethylene glycol on morphology of canine preantral follicles.
Slices of canine ovarian tissue were equilibrated for 20 min at 20ºC in minimum
essential medium containing either cryoprotectants at 1.5 M, and then frozen, stored in
liquid nitrogen (-196ºC) for a week and thawed. Morphology of follicles was analyzed
through histology and transmission electron microscopy. The results showed that the
percentages of morphologically normal preantral follicles (MNPF) were significantly
85
reduced after exposure to glycerol, but not to ethylene glycol in comparison to the
control. After freezing/thawing, the percentages of MNPF were significantly decreased
as compared to the control using either cryoprotectants, which were statistically similar
in relation to each other. However, transmission electron microscopy revealed that only
follicles frozen with ethylene glycol had normal ultrastructure. In conclusion, this study
demonstrated that morphology of canine preantral follicles can be successfully
preserved through slow freezing-rapid thawing using 1.5 M ethylene glycol.
Keywords: cryopreservation, slow freezing, preantral follicles, canine.
Introduction
Cryopreservation of gametes is potentially an important tool for the conservation
of endangered species and biodiversity maintenance. Significant advances in this field
has been obtained through genome resource banking which has been allowed by the
relative success achieved with semen cryopreservation, in which widely implemented
protocols are used. Conversely, almost all mammalian species oocytes studied have
proven very difficult to successfully cryopreserve (Woods et al., 2004).
The process of cryopreservation can cause degeneration of oocytes,
disassembly of cell microtubules resulting in the disruption of the spindle apparatus
followed by aneuploidy, and zona pellucida (ZP) hardening which thus may impair the
subsequent fertilization process. These effects can be a direct consequence of
intracellular ice-crystal formation, rapid or massive osmotic and temperature change
and the relative toxicity of cryoprotectant under certain conditions (Ko et al., 2008). In
order to overcome these problems, the cryopreservation of preantral ovarian follicles
emerges as a promising alternative for female gamete cryobanking. Immature oocytes
in preantral follicles still have not metaphase spindle fibers and lack a ZP and cortical
granules. Moreover, such oocytes are far smaller and less differentiated than
Metaphase II oocytes. All these characteristics are potentially beneficial for freeze
preservation (Oktay et al., 1998). Together with these qualitative advantages, this
strategy for female gamete banking can also potentially provide a much higher quantity
of fertilizable oocytes, because thousands of these follicles can be obtained from an
ovary (Rutherford & Gosden, 1999).
Cryopreservation of isolated primordial follicles through slow freezing was
successfully achieved in the mouse (Carroll and Gosden, 1993). Follicles were
transplanted to sterilized hosts which could afterwards produce normal offspring after
mating. Live young were also obtained after in vitro culture, maturation and fertilization
86
of mouse oocytes within frozen-thawed secondary follicles (Smitz and Cortvrindt,
1998). More recently, live births after orthotopic autotransplantation of cryopreserved
ovarian tissue were described in a woman (Donnez et al., 2004), as well as in ewes
(Bordes et al., 2005).
Although cryopreservation of preantral follicles from carnivores would be
specially important since many wild species of these are currently threatened by
extinction, few studies have been published on this topic. Jewgenow et al. (1998)
demonstrated that small preantral follicles isolated from cat ovaries survive
cryopreservation, remaining structurally intact and physiologically active after thawing.
In another work, Bosch et al. (2004) reported the survival of cat ovarian tissue
subjected to slow freezing-thawing and transplantation to an immunocompromised
host, which was followed by development of follicles from early up to the antral stage.
With respect to canids, to our knowledge, only one attempt to cryopreserve preantral
follicles is reported. Ishijima et al. (2006) vitrified canine ovarian cortex and
xenotransplanted to immunodeficient mice. Although antral follicle formation did not
occur, grafted tissue was morphological normal and proliferating cell nuclear antigen
immunoreactivity was detected in primary follicles. Nonetheless, detailed morphological
and quantitative analyses on cryopreservation of canine preantral follicles are still to be
performed, and knowledge gathered in this species could be applied to endangered
canids.
The aim of this study was to evaluate the cryopreservation of canine preantral
follicles through slow freezing of ovarian tissue using 1.5 M glycerol (GLY) or ethylene
glycol (EG). For this purpose, morphology of follicles after exposure to the
cryoprotectants and freezing-thawing was analyzed through histology and transmission
electron microscopy (TEM).
Materials and Methods
Collection and preservation of canine ovaries
Ovarian pairs (n=5) were aseptically collected at local veterinary clinics during
ovariohysterectomy of healthy mixed-breed bitches (Canis lupus familiaris), whose
reproductive history and age were unknown, but it was estimated that they were 12-24
months old. After removal from the bursa ovarica and excision of eventual corpora
lutea using a scalpel, ovaries were placed into 50 ml tubes (Corning Glass Works,
Corning, NY, USA) containing 15 ml of HEPES-buffered Minimum Essential Medium
(H-MEM, osmolarity 280 mOsmol/l, pH7.2; Sigma, St. Louis, MO, USA). Then,
87
transportation to the laboratory was carried out at 4°C within one hour in thermoflasks
filled with water. All procedures were performed aseptically.
Exposure test
At the laboratory, five fragments of cortex (approximately 3 x 3 x 1 mm) were
cut from each pair of ovaries, and one of these pieces was randomly selected as
control and fixed for histology and ultrastructural analysis. The remaining samples were
placed into cryovials containing 1.8 ml of freezing medium which consisted of Minimum
Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 1.5 M
GLY or EG, prepared immediately prior to use under sterile conditions. After incubation
at 20ºC for 20 min (equilibration period), half of the fragments was subjected to
cryoprotectant removal according to the method described by Candy et al. (1997).
Briefly, three washes of 5 min each in MEM with 10% FBS were performed at room
temperature (RT, approximately 25°C). Samples were subsequently fixed for histology
and TEM in order to evaluate effects of exposure to cryoprotectants on preantral
follicles.
Freezing-thawing
The cryovials with the remaining fragments were transferred to a biological
programmable freezer (Freeze Control, CryoLogic Pty Ltd.,Waverley, Australia) pre-set
at 20 °C. The following program was used to freeze the tissue samples: cooling from
20 to -7°C at a rate of 2°C/min; holding at -7°C for 15 min (after 5 min, ice crystal
formation - seeding - was induced manually by touching the vials with a forceps pre-
chilled in liquid nitrogen); temperature was then lowered 0.3°C/min to -30°C, and
further reduced by 0.15°C/min to -33°C; finally, the vials were plunged into liquid
nitrogen (-196°C) and stored for 7 days. Thereafter, ovarian tissue pieces were thawed
through rapid warming by exposing the cryovials to air at RT for 1 min, followed by
immersion in a water bath at 38ºC for 3 min. Cryoprotectant removal was carried as
describe for the exposure test, and pieces of ovarian cortex were fixed for histological
and ultrastructural analyses. This experiment was repeated five times.
Histological analysis
In order to assess the morphology of canine preantral follicles submitted to the
different treatments, fragments of ovarian tissue were fixed in Carnoy for 12 h,
88
dehydrated in a graded series of ethanol, clarified with xylene, embedded in paraffin
wax and serially sectioned at 7 μm. Every fifth section was mounted on glass slides,
stained with periodic acid Schiff (PAS)-hematoxylin and evaluated by light microscopy
at a 400x magnification (Zeiss, Germany). Preantral follicles were defined as an oocyte
surrounded either by one layer of flattened or cuboidal granulosa cells, or several
layers of cuboidal granulosa cells with no antrum. Follicular morphology was evaluated
based on the integrity of the oocyte, granulosa cells and basement membrane.
Preantral follicles were classified and counted as (i) morphologically normal, when
containing an oocyte with regular shape and uniform cytoplasm, and organized layers
of granulosa cells; (ii) degenerated grade 1, when the oocyte exhibited pycnotic
nucleus and/or ooplasma shrinkage; (iii) degenerated grade 2, when in addition to
oocyte damage, granulosa cells layers became disorganized, detached from the
basement membrane and/or included enlarged cells. To avoid evaluating and counting
a follicle more than once, preantral follicles were analyzed only in the sections where
oocyte nucleus was observed. All the slides were coded to prevent observer bias.
Ultrastructural analysis
In order to better examine follicular morphology, TEM was performed to analyze
ultrastructure of preantral follicles from the control as well as from treatments that did
not differ statistically from control in histology. A portion with a maximum dimension of
1 mm3 was cut from each fragment of ovarian tissue and fixed in modified Karnovsky
solution (2% paraformaldeyde and 2% glutaraldeyde in 0.1 M sodium cacodylate buffer
pH 7.2) for 3 h at room temperature (RT, approximately 25°C). After three washes in
sodium cacodylate buffer, specimens were post-fixed in 1% osmium tetroxide, 0.8%
potassium ferricyanide and 5 mM calcium chloride in 0.1 M sodium cacodylate buffer
for 1 h at RT. The samples were then dehydrated through a gradient of acetone
solutions and thereafter embedded in Spurr’s epoxy resin. Afterwards, semi-thin
sections (3 μm) were cut, stained with toluidine blue and analyzed by light microscopy
at a 400x magnification. Ultra-thin sections (60–70 nm) were obtained from preantral
follicles classified as morphologically normal in semi-thin sections, according to the
criteria adopted in histology. Subsequently, ultra-thin sections were contrasted with
uranyl acetate and lead citrate, and examined under a Jeol 1011 (Jeol, Tokyo)
transmission electron microscope operating at 80 kV.
89
Statistical analysis
All experiments were replicated five times. Analysis of variance (ANOVA) of the
data was performed using the GLM procedure of the software SAS (SAS Institute Inc.,
Cary, NC, USA). Differences of percentages of morphologically normal preantral
follicles (MNPF) and degenerated follicles between control and treatments were
determined by t-test. Differences were considered statistically significant when
P < 0.05.
Results
Histological analysis of ovarian fragments before and after exposure to cryoprotectants
and freezing-thawing
Thirty preantral follicles were examined per treatment in each replicate through
histology, resulting in a total of 750 analyzed follicles. MNPF in control as well as after
exposure to cryoprotectants and freezing-thawing exhibited a spherical or elliptical
oocyte with a large central or eccentric nucleus and uniform cytoplasm. Granulosa cells
without pycnotic nuclei were well-organized in layers surrounding the oocyte and a
distinguishable intact basement membrane could be observed.
Degenerated grade 1 follicles showed a retraced oocyte with or without a
pycnotic nucleus, and occasionally a strongly eosinophilic cytoplasm, whilst layers of
granulosa cells remained unaltered. In degenerated grade 2 follicles, along with the
described oocyte alterations, granulosa cells layers became disorganized into a low
density mass of cells which were many times swollen and/or detached from basement
membrane. Occurrence of pycnotic bodies in granulosa cells or rupture of the
basement membrane was not observed.
The percentages of MNPF observed in the control and after the exposure and
freezing-thawing tests are shown in figure 1. Immersion of ovarian fragments into the
solution containing 1.5 M EG had no effect on the percentages of normal follicles as
compared with the control (P>0.05). Conversely, exposure to 1.5 M GLY reduced
significantly (P <0.05) the percentages of MNPF in comparison to the control and to
immersion in 1.5 M EG. After freezing-thawing, the percentages of normal follicles were
significantly decreased as compared to the control (P <0.05) using either
cryoprotectant, which were statistically similar in relation to each other (P >0.05). For
both cryoprotectants, it was observed that post-thawing percentages of MNPF were
90
significantly reduced in relation to the values obtained after the exposure test (P
<0.05).
Fig. 1. Percentages of morphologically normal preantral follicles in ovarian tissue
before (control) and after exposure and freezing-thawing tests in medium containing
1.5 M EG or GLY. (*) Differs significantly from the control (P <0.05); (a, b) different
letters for the same test (exposure or freezing-thawing) denote significant differences
between media (P <0.05); (A, B) different letters for a given cryoprotectant indicates
significant differences among exposure and post-thawing values (P <0.05).
Regarding to the distribution of follicular degeneration among the different
experimental groups, table 2 shows the percentages of grade 1 and grade 2
degenerated preantral follicles in ovarian fragments from the control, exposure and
freezing-thawing tests. A significant (P <0.05) predominance of degenerated grade 1
follicles over those of grade 2 was observed in all treatments. In the exposure test,
percentages of degenerated grade 1 follicles increased significantly as compared to the
control (P <0.05) when GLY was used, but remained unchanged in the presence of EG
(P >0.05). Percentages of degenerated grade 2 were similar to control using either
GLY or EG (P >0.05).
91
Table 2. Degenerated preantral follicles (%) in control and after exposure and freezing-
thawing tests.
Exposure test Freezing-thawing Control
GLY EG GLY EG
Grade 1 35*aAd 16bAd 40*aAd 47*aBd 13d
Grade 2 6aAe 5aAe 17*aBe 12*aBe 4e
*P < 0:05, significantly differs from control.
Different superscripts (a, b) differ significantly between cryoprotectants at a same test.
Different superscripts (A, B) differ significantly between exposure test and
cryopreservation.
Different superscripts (d, e) differ significantly between Grades 1 and 2 of
degeneration.
After freezing-thawing, significantly (P <0.05) higher values of degenerated
grade 1 and 2 follicles in comparison to the control were observed with both
cryoprotectants, which were similar to each other (P >0.05). Percentages of
degenerated grade 1 follicles were significantly higher after cryopreservation as
compared to the exposure test when EG was used (P <0.05), but not with GLY (P
>0.05). Regarding percentages of degenerated grade 2 follicles, a significant (P <0.05)
rise was observed after freezing-thawing relative to exposure to either cryoprotectants.
Ultrastructural analysis
TEM of frozen-thawed preantral follicles was performed in order to assess the
ultrastructure of follicles classified as normal at the histological level. At least five
follicles per group were analyzed. Normal follicles contained an oocyte displaying a
very homogenous cytoplasm plenty of round shaped mitochondria with continuous
membranes, few peripheral cristae and electron-dense granules. Some elongated
forms with parallel cristae could also be seen. Small Golgi apparatus cisternae were
rarely observed. Smooth and rough endoplasmic reticulum were present, either as
isolated aggregations or as complex associations with mitochondria. The nuclei of
oocytes were large and usually round, well delimited by the nuclear envelope. The
chromatin was uncondensed and a nucleolus could often be identified. A few vacuoles
were also observed. Granulosa cells were small and presented a high nucleus-to-
cytoplasm ratio. Their irregularly shaped nuclei contained loose chromatin in the inner
part and small peripheral aggregates of condensed chromatin. The cytoplasm exhibited
92
a great number of mitochondria and well-developed smooth and rough endoplasmic
reticulum. The cellular membranes of oocyte and surrounding granulosa cells were
closely juxtaposed, and sometimes few short microvilli could be observed. A distinct
continuous basement membrane surrounded follicles and was tightly attached to
ovarian stroma (Fig. 2A-C).
The ultrastructural pattern described above was observed in normal follicles
from control and freezing-thawing with EG. When GLY was used for freezing, despite
some follicles displayed normal morphology, changes could be detected in follicles
evaluated as normal in semi-thin sections by light microscopy. The ooplasm presented
a reduced electron density and lower numbers of organelles, heterogeneously
distributed in small clusters. Some large vacuoles could also be seen. In granulosa
cells, loss of cytoplasm content was noticed, leading to the existence of very large
empty spaces in some cases (Fig. 2D).
Discussion
The present study evaluated for the first time the cryopreservation of canine
preantral follicles through slow freezing, employing GLY and EG as cryoprotectants. It
was demonstrated that ultrastructural integrity of such follicles can be successfully
maintained with the use of 1.5 M EG.
Exposure of canine ovarian tissue to 1.5 M GLY at 20ºC during 20 minutes
resulted in a significant reduction of MNPF percentages. An osmotic stress due to the
presence of cryoprotectants in the freezing medium is one of the primary theories of
injury during cryopreservation, whilst the inherent toxicity of these compounds is also
an important consideration (Mullen et al., 2008). GLY induces severe osmotic damage
to the cytoplasm due to its low membrane permeability (Szell et al., 1986). In
accordance to our results, it has been demonstrated that exposure of ovarian tissue to
1.5 GLY results in a substantial reduction of the percentage of normal follicles (caprine:
Rodrigues et al., 2004; ovine: Santos et al., 2006). Therefore, we suggest that the
current method for exposure to GLY is inadequate and some protocol adjustments
such as gradual addition/removal and use of an osmotic buffer like sucrose (Adams et
al., 2003) should be done to minimize osmotic damage.
93
Fig. 2. Electron micrographs of canine preantral follicles from control (a) and frozen-
thawed using 1.5 M EG (b, c) or GLY (d). In Fig. 2a (2,500x, scale bar= 10 µm) and
Fig. 2b (3,000x, scale bar= 10 µm), normal follicles containing an oocyte with
homogenous cytoplasm and a large nucleus well delimited by the nuclear envelope are
displayed. A nucleolus could often be identified (nu). In Fig. 2c (8,000x, scale bar= 2
µm), the same follicle shown in Fig. 2b is displayed at a higher magnification. Note the
great number of intact mitochondria (m) and smooth endoplasmic reticulum (ser) in the
cytoplasm of the oocyte and surrounding granulosa cells cryopreserved with EG. In
Fig. 2d (10,000x, scale bar= 2 µm), observe the reduced electron density of the
ooplasm, the lower numbers of organelles and a large empty space (asterisk) after
freezing-thawing with GLY. O, oocyte; GC, granulosa cells; l, lipid droplet.
On other hand, 1.5 M EG, which has a molecular weight of 62.07 compared
with 92.10 for GLY (Massip, 2001), did not exert any effect on preantral follicles after
94
exposure of ovarian tissue under the same conditions of GLY addition. Amorim et al.
(2003) exposed isolated ovine primordial follicles to EG at different concentrations and
observed that viability was maintained in concentrations up to 2 M. Sommerfeld and
Niemann (1999) showed that bovine embryos are able to survive exposure to
concentrations of 3.6 M EG without appreciable loss of developmental capacity.
Conversely, Lucci et al. (2004) observed that exposure of zebu bovine ovarian tissue to
1.5 M EG resulted in a high decrease of MNPF, which was greater than that observed
after exposure to GLY under the same conditions. This discrepancy of EG cytotoxicity
might reflect a higher sensitivity of bovine preantral follicles to chemical effects caused
by this cryoprotectant compared to canine follicles. This is in accordance with the fact
that cells from different species can respond very differently to a cryoprotectant
(Schellander et al., 1994; Cocero et al., 1996).
After freezing and thawing, the percentages of MNPF were significantly reduced
from values observed after exposure to both cryoprotectants, which showed similar
preservation efficiency. Therefore, the freezing and/or thawing processes resulted in
damage to follicles, probably due to intracellular ice formation (IIF). We hypothesize
that the concentration of cryoprotectants used (1.5 M) was not enough to protect cells
against crioinjury. Permeating cryoprotectants such as GLY and EG are small
molecules that permeate the membranes of cells, form hydrogen bonds with water
molecules and prevent ice crystallization. At high enough concentrations, they inhibit
the formation of the characteristic ice crystal and lead to the development of a solid,
glasslike, so-called vitrified state in which water is solidified, but not expanded (Jain
and Paulson, 2006). Rodrigues et al. (2004) demonstrated that a rise in concentration
of EG from 1.5 to 3.0 M was able to significantly increase percentages of normal
preantral follicles during cryopreservation of caprine ovarian tissue.
In the present study, since follicles proved to not be affected by exposure to EG
at 1.5 M, we suggest that concentration of this cryoprotectant may be increased up to a
threshold on which cytotoxic and/or osmotic effects are still inexistent and protection
against cryoinjuries is maximum. Regarding to freezing with GLY, we also have to
consider that the protocol of cryoprotectant addition might have not been adequate for
complete permeation of cells, which resulted in intracellular concentrations not
reaching sufficient levels for cryoprotection. Candy et al. (1997) showed that the
survival rate of mice primordial follicles after freezing increased in function of exposure
time to GLY, which suggests that the extent of follicular survival is at least in part
determined by the final tissue concentration achieved.
In all treatments, percentages of grade 1 degenerated follicles were higher than
that for grade 2 degenerated follicles, which characterizes that oocytes are more
95
susceptible than granulosa cells to degeneration in vivo and after exposure to
cryoprotectants and freezing-thawing. The increase of grade 1 degenerated follicles
after freezing and thawing observed in this work was also found in studies with non-
human primate (Candy et al., 1995), human (Hovatta et al., 1996), bovine (Lucci et al.,
2004), caprine (Rodrigues et al., 2004a,b) and ovine (Santos et al., 2006) ovarian
tissue. We supposed that oocytes are less resistant to exposure to cryoprotectants and
cryopreservation than granulosa cells because of its lower surface area-to-volume
ratio, which reduces permeability to both water and ACPs (Wood et al., 2004).
TEM performed on frozen-thawed follicles classified as normal in histology
revealed that only those frozen with EG had preserved ultrastructure, whilst follicles
frozen with GLY showed severe ultrastructural damages. Therefore, TEM proved to be
an essential tool to detect damage due to the freezing-thawing process which can be
observed only at the ultrastructural level but not through light microscopy. The most
severe damage observed was made up of large empty spaces not surrounded by
membranes. This feature suggests the occurence of crystallization, and was also
observed in glycerol-frozen ovine embryos (Cocero et al., 2002).
Once established, knowledge on cryopreservation of canine preantral follicles
could be applied to endangered canids in order to lengthen the genetic lifespan of each
rare female. To this end, development of in vitro culture systems capable to promote
growth and maturation of this vast pool of oocytes has to be accomplished. However,
at present, such systems still do not lead to normal full follicle development in non-
rodent species, for which oocyte growth is a long-term, coordinated and complex
process, and the time required to reach the mature stage is thought to be up to several
months. Currently, xenotransplantation into immunodeficient recipients seems to be
one the most promising ways for obtaining oocytes from ovaries for further exploration
of assisted reproduction techniques (Jewgenow and Paris, 2006). Fassbender et al.
(2007) demonstrated the survival of cat ovarian cortex tissue transplanted under the
kidney capsule of athymic nude rats. Follicular development was monitored through
high-resolution ultrasonography, and cumulus oocytes complexes could be collected
and in vitro matured.
In conclusion, preservation of morphology of canine preantral follicles can be
accomplished through slow freezing and rapid thawing using 1.5 M EG. Nevertheless,
further studies are necessary to evaluate developmental capacity of such follicles,
which demands the establishment of in vitro culture systems or use of transplantation
approaches.
96
Acknowledgements
CAP Lopes and JR Figueiredo are recipients of grants from CNPq (Brazil). We
would like to express our appreciation to the Center for Zoonosis Control (CCZ) of
Fortaleza Municipality (Brazil), which gently provided the canine ovaries.
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9 CAPÍTULO IV
Criopreservação bem-sucedida de folículos pré-antrais caninos utilizando-se
dimetilsulfóxido e 1,3-propanodiol
Successful cryopreservation of canine preantral follicles using dimethyl sulfoxide and
1,3-propanediol
Reproduction, Fertility and Development
(Submetido, 2008)
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Successful cryopreservation of canine preantral follicles using dimethyl sulfoxide and 1,3-propanediol C. A. P. LopesA,B,E, A. K. F. LimaA, R. R. SantosC, M. H. T. MatosA, A. P. R. RodriguesA,
S. N. BáoD, K. JewgenowB, J. R. FigueiredoA
ALAMOFOPA, PPGCV, Faculty of Veterinary, State University of Ceará, Brazil. BLeibniz-Institute for Zoo and Wildlife Research Berlin, Germany. CDepartment of Equine Sciences, Pharmaceuticals, Pharmacology and Toxicology
Division, Faculty of Veterinary Medicine, Utrecht University, The Netherlands. DLaboratory of Electron Microscopy, Institute of Biology, University of Brasília, Brazil. ECorresponding author. Email: [email protected]
Resumo
A criopreservação de folículos pré-antrais é uma abordagem potencialmente
importante para a conservação de espécies ameaçadas pela extinção, uma vez que
técnicas têm sido desenvolvidas para a obtenção de oócitos fertilizáveis a partir desta
vasta fonte de gametas. O objetivo deste estudo foi avaliar os efeitos da congelação
lenta-descongelação rápida utilizando-se dimetilsulfóxido (DMSO) ou 1,3-propanodiol
(PROH) sobre a morfologia e viabilidade de folículos pré-antrais caninos. Fragmentos
de tecido ovariano canino foram equilibrados por 20 min a 20ºC em Meio Essencial
Mínimo contendo um dos crioprotetores mencionados à concentração de 1,5 M, sendo
então congelados e descongelados. A morfologia dos folículos foi analisada através de
histologia e microscopia eletrônica de transmissão (MET), e a viabilidade avaliada
utilizando-se Azul de Tripan e marcadores fluorescentes. Os resultados demonstraram
que a exposição de tecido ovariano canino a ambos crioprotetores não apresentou
efeito sobre a morfologia e viabilidade foliculares. Após a congelação e
descongelação, os folículos mantiveram-se histologicamente normais, mas a MET
revelou danos à ultra-estrutura dos folículos congelados em PROH. A viabilidade foi
significativamente reduzida com ambos crioprotetores. Entretanto, altos percentuais de
folículos viáveis (65%) foram matidos com DMSO. Como conclusão, este estudo
demonstrou que a criopreservação de folículos pré-antrais caninos viáveis com ultra-
estrutura intacta pode ser realizada com sucesso através de congelação lenta e
descongelação rápida utilizando-se DMSO a 1,5 M.
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Palavras-chave: criopreservação, folículos pré-antrais, canino, congelação lenta,
dimetilsulfóxido, propanodiol. Abstract
Cryopreservation of preantral follicles is potentially an important approach for
the conservation of endangered species in such way that techniques have been
developed in order to retrieve fertilizable oocytes from this large pool of gametes. The
aim of this study was to evaluate the effects of slow freezing-rapid thawing using
dimethyl sulfoxide (DMSO) or 1,3-propanediol (PROH) on morphology and viability of
canine preantral follicles. Slices of canine ovarian tissue were equilibrated for 20 min at
20ºC in minimum essential medium (MEM) containing either cryoprotectants at 1.5 M,
and then frozen-thawed. Morphology of follicles was analyzed through histology and
transmission electron microscopy (TEM), and viability assessed using trypan blue and
fluorescent probes. The results showed that exposure of canine ovarian tissue to both
cryoprotectants had no effect on follicular morphology and viability. After freezing-
thawing, follicles remained histologically normal, but TEM revealed damage to the
ultrastructure of those frozen in PROH. Viability was significantly reduced when either
compound was used. However, high percentages of viable follicles (65%) were
maintained with DMSO. In conclusion, this study demonstrated that cryopreservation of
viable canine preantral follicles with intact ultrastructure can be successfully
accomplished through slow freezing and rapid thawing using 1.5 M DMSO.
Additional keywords: cryopreservation, preantral follicles, canine, slow freezing,
dimethyl sulfoxide, propanediol.
Introduction
Along with the global warming, extinction of species is an increasing major
concern worldwide, since biodiversity is a central component of Earth’s life supporting
systems (Khuroo et al., 2007), and the removal of single species can affect the
functioning of global ecosystems (Myers et al., 2000). In this context, in addition to the
crucial protection of habitats, reproductive biotechnology has a great potential to
contribute for the conservation of endangered wild animal species.
Nevertheless, a limiting factor for the development of reproductive techniques,
and also for their efficiency, is the lack of abundant numbers of fertilizable oocytes.
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This problem could be addressed by using the large source of oocytes enclosed in
preantral follicles (Telfer, 2001), which exist in ovaries in number of thousands to
millions depending on the species. This category comprises around 90 to 95% of entire
follicular population, storing a vast majority of the oocytes in mammalian ovaries
(Figueiredo et al., 2006).
In vitro culture systems capable to promote growth and maturation of preantral
follicles have been developed for several species. Currently, birth of healthy offspring
after embryo transfer of in vitro fertilized oocytes derived from primordial follicles has
been accomplished only in mice (Eppig and Schroeder, 1989). The oocyte growth
period in this species is about 3 weeks, whereas, in non-rodents, the time required to
reach the mature stage is thought to be up to several months (Jewgenow and Paris,
2006). Therefore, the creation of such systems is a challenging task still at an early
stage of development, and long-term preservation of preantral follicles until the
establishment of routine culture is an important issue. It could permit to lengthen the
genetic lifespan of each rare female of an endangered species after death through the
storage of its large pool of gametes.
Cryopreservation of isolated primordial follicles through slow freezing was
successfully achieved in the mouse (Carroll and Gosden, 1993). Follicles were
transplanted to sterilized hosts which could afterwards produce normal offspring after
mating. Live young were also obtained after in vitro culture, maturation and fertilization
of mouse oocytes within frozen-thawed secondary follicles (Smitz and Cortvrindt,
1998). More recently, live births after orthotopic autotransplantation of cryopreserved
ovarian tissue were described in a woman (Donnez et al., 2004), as well as in ewes
(Bordes et al., 2005).
Although cryopreservation of preantral follicles from carnivores would be
specially important since many wild species of these are currently threatened by
extinction, few studies have been published on this topic. Jewgenow et al. (1998)
demonstrated that small preantral follicles isolated from cat ovaries survive
cryopreservation, remaining structurally intact and physiologically active after thawing.
In addition, Bosch et al. (2004) reported the survival of cat ovarian tissue subjected to
slow freezing-thawing and transplantation to an immunocompromised host, which was
followed by development of follicles from early up to the antral stage. With respect to
canids, to our knowledge, only one attempt to cryopreserve preantral follicles was
reported. Ishijima et al. (2006) vitrified canine ovarian cortex and xenotransplanted to
immunodeficient mice. Although antral follicle formation did not occur, grafted tissue
was morphological normal and proliferating cell nuclear antigen immunoreactivity was
detected in primary follicles. Nonetheless, detailed morphological and quantitative
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analyses on cryopreservation of canine preantral follicles are still to be performed, and
knowledge gathered in this species could be applied to endangered canids.
The aim of this study was to evaluate the cryopreservation of canine preantral
follicles through slow freezing of ovarian tissue using 1.5 M dimethyl sulfoxide (DMSO)
or 1.5 M 1,3-propanediol (PROH). For this purpose, morphology of follicles after
exposure to the cryoprotectants and freezing-thawing was analyzed through histology
and transmission electron microscopy (TEM), and viability was assessed through the
trypan blue dye exclusion test as well as using the fluorescent probes calcein-AM and
ethidium homodimer.
Materials and methods Collection and preservation of canine ovaries
Ovarian pairs were collected during ovariectomy of healthy 12-24 months old
bitches (Canis lupus familiaris) at local veterinary clinics. After removal from the bursa
ovarica and excision of eventual corpora lutea using a scalpel, ovaries were placed into
50 ml tubes (Corning Glass Works, Corning, NY, USA) containing 15 ml of HEPES-
buffered Minimum Essential Medium (H-MEM, osmolarity 280 mOsmol/l, pH7.2; Sigma,
St. Louis, MO, USA). Then, transportation to the laboratory was carried out at 4°C
within one hour in thermoflasks filled with water. All procedures were performed
aseptically.
Experiment 1: Morphological evaluation of follicles after exposure to cryoprotectants
and freezing-thawing
At the laboratory, seven fragments of cortex (approximately 3 x 3 x 1 mm) were
cut from each pair of ovaries (n=5), and one of these pieces was randomly selected as
fresh control and fixed for histology and ultrastructural analysis. The remaining samples
were placed into cryovials containing 1.8 ml of freezing medium which consisted of
Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS)
and 1.5 M DMSO or 1.5 M PROH or without cryoprotectant (freezing control), prepared
immediately prior to use under sterile conditions. After incubation at 20ºC for 20 min
(equilibration period), half of the fragments was subjected to cryoprotectant removal
according to the method described by Candy et al. (1997). Briefly, three washes of 5
min each in MEM with 10% FBS were performed at room temperature (RT,
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approximately 25°C). Samples were subsequently fixed for histology and TEM in order
to evaluate effects of exposure to cryoprotectants on preantral follicles.
The cryovials with the remaining fragments were transferred to a biological
programmable freezer (Freeze Control, CryoLogic Pty Ltd.,Waverley, Australia) pre-set
at 20 °C. The following program was used to freeze the tissue samples: cooling from
20 to -7°C at a rate of 2°C/min; holding at -7°C for 15 min (after 5 min, ice crystal
formation - seeding - was induced manually by touching the vials with a forceps pre-
chilled in liquid nitrogen); temperature was then lowered 0.3°C/min to -30°C, and
further reduced by 0.15°C/min to -33°C; finally, the vials were plunged into liquid
nitrogen (-196°C) and stored for 7 days. Thereafter, ovarian tissue pieces were thawed
through rapid warming by exposing the cryovials to air at RT for 1 min, followed by
immersion in a water bath at 38ºC for 3 min. Cryoprotectant removal was carried out as
describe for the exposure test, and pieces of ovarian cortex were fixed for histological
and ultrastructural analyses. This experiment was repeated five times.
Experiment II: Viability assessment of follicles exposed to cryoprotectants and frozen-
thawed
A second study using a similar experimental protocol as described above in the
morphological investigation was performed with the objective of analyzing effects of
exposure to cryoprotectants and freezing-thawing on viability of preantral follicles. Five
ovaries were used, and fragments from the fresh control and testing groups undergone
follicle isolation followed by trypan blue dye exclusion test as described later. Based on
the results of this assay and experiment I, viability of follicles cryopreserved with the
substance which provided the best outcome was further analyzed using a more
accurate method of assessment based on fluorescent probes. Additional pairs of
ovaries (n=5) were cut into fragments, from which one was immediately processed for
follicle isolation and analyzed using trypan blue as well as calcein-AM and ethidium
homodimer. Remaining fractions were subjected to exposure to the selected
cryoprotectant, frozen-thawed and analyzed after each procedure similarly as for the
control. All samples were evaluated in duplicate.
Histological analysis
In order to assess the morphology of canine preantral follicles submitted to the
different treatments, fragments of ovarian tissue were fixed in Carnoy for 12 h,
dehydrated in a graded series of ethanol, clarified with xylene, embedded in paraffin
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wax and serially sectioned at 7 μm. Every fifth section was mounted on glass slides,
stained with periodic acid Schiff (PAS)-hematoxylin and evaluated by light microscopy
at a 400x magnification (Zeiss, Germany). Preantral follicles were defined as an oocyte
surrounded either by one layer of flattened or cuboidal granulosa cells, or several
layers of cuboidal granulosa cells with no antrum. Follicular morphology was evaluated
based on the integrity of the oocyte, granulosa cells and basement membrane.
Preantral follicles were classified and counted as (i) morphologically normal, when
containing an oocyte with regular shape and uniform cytoplasm, and organized layers
of granulosa cells, or (ii) degenerated, when the oocyte exhibited pycnotic nucleus
and/or ooplasma shrinkage, and occasionally granulosa cells layers became
disorganized, detached from the basement membrane and/or included enlarged cells.
To avoid evaluating and counting a follicle more than once, preantral follicles were
analyzed only in the sections where oocyte nucleus was observed.
Ultrastructural analysis
In order to better examine follicular morphology, TEM was performed to analyze
ultrastructure of preantral follicles from the control as well as from treatments that did
not differ statistically from control in histology. A portion with a maximum dimension of
1 mm3 was cut from each fragment of ovarian tissue and fixed in modified Karnovsky
solution (2% paraformaldeyde and 2% glutaraldeyde in 0.1 M sodium cacodylate buffer
pH 7.2) for 3 h at room temperature (RT, approximately 25°C). After three washes in
sodium cacodylate buffer, specimens were post-fixed in 1% osmium tetroxide, 0.8%
potassium ferricyanide and 5 mM calcium chloride in 0.1 M sodium cacodylate buffer
for 1 h at RT. The samples were then dehydrated through a gradient of acetone
solutions and thereafter embedded in Spurr’s epoxy resin. Afterwards, semi-thin
sections (3 μm) were cut, stained with toluidine blue and analyzed by light microscopy
at a 400x magnification. Ultra-thin sections (60–70 nm) were obtained from preantral
follicles classified as morphologically normal in semi-thin sections, according to the
criteria adopted in histology. Subsequently, ultra-thin sections were contrasted with
uranyl acetate and lead citrate, and examined under a Jeol 1011 (Jeol, Tokyo)
transmission electron microscope operating at 80 kV.
Assessment of preantral follicles viability
Canine preantral follicles were isolated from ovarian fragments using the
mechanical method described by Figueiredo et al. (1993). Briefly, using a tissue
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chopper (The Mickle Laboratory Engineering Co., Gomshal, Surrey, UK) adjusted to a
sectioning interval of 87.5 μm, samples were cut into small pieces, which were placed
in MEM and suspended 40 times using a large Pasteur pipette (diameter of about 1600
μm) and subsequently 40 times with a smaller Pasteur pipette (diameter of
approximately 600 μm) to dissociate preantral follicles from stroma. The obtained
material was passed through 500- and 100-μm nylon mesh filters, resulting in a
suspension containing preantral follicles smaller than 100 μm in diameter. This
procedure was carried out within 10 min at RT.
Thereafter, viability of preantral follicles was assessed through trypan blue dye
exclusion test (Jewgenow et al., 1998). Briefly, 10 μl of 0.4% trypan blue (Sigma,
Deisenhofen, Germany) were added to 90 μl of the suspension of isolated preantral
follicles, which were examined using an inverted microscope after incubation for 5 min
at RT. Follicles were classified as viable if the oocyte and <10% granulosa cells
remained unstained, or as non-viable if uptake of the dye by the oocyte and/or ≥10%
granulosa cells occurred.
Preantral follicles were also analyzed using a two-color fluorescence cell
viability assay based on the simultaneous determination of live and dead cells by
calcein-AM and ethidium homodimer-1, respectively. Whilst the first probe detected
intracellular esterase activity of viable cells, the later labeled nucleic acids of non-viable
cells with plasma membrane disruption. The test was performed by adding 4 μM
calcein-AM and 2 μM ethidium homodimer-1 (Molecular Probes, Invitrogen, Karlsruhe,
Germany) to the suspension of isolated follicles, followed by incubation at 37°C for 10
min. After being labeled, follicles were washed three times in MEM and mounted on a
glass microscope slide in 5 μl antifading medium (DABCO, Sigma, Deisenhofen,
Germany) to prevent photobleaching, and finally examined using an a DMLB
fluorescence microscope (Leica, Germany). The emitted fluorescent signals of calcein-
AM and ethidium homodimer were collected at 488 and 568 nm respectively. Oocytes
and granulosa cells were considered live if the cytoplasm was stained positively with
calcein-AM (green) and chromatin was not labelled with ethidium homodimer (red).
Statistical analysis
All experiments were replicated five times. Kolmogorov-Smirnov and Bartlett’s
tests were applied to verify normal distribution of data and homogeneity of variances
respectively. Afterwards, analysis of variance (ANOVA) was performed using the GLM
procedure of the software SAS (SAS Institute Inc., Cary, NC, USA). Differences of
percentages of morphologically normal preantral follicles (MNPF) and viable follicles
107
between control and treatments were determined by Dunnett’s test. Student Newman
Keuls’ (SNK) test was used to compare results among testing groups. Data were
expressed as means ± standard error of means (SEM). Differences were considered
statistically significant when P <0.05.
Results
Experiment 1: Morphological evaluation of follicles after exposure to cryoprotectants
and freezing-thawing
A total of 1079 preantral follicles were examined in histology, with a range of
145 to 171 in each treatment. Morphologically normal preantral follicles (MNPF) in
control as well as after exposure to cryoprotectants and freezing-thawing exhibited a
spherical or elliptical oocyte with a large central or eccentric nucleus and uniform
cytoplasm. Granulosa cells without pycnotic nuclei were well-organized in layers
surrounding the oocyte and a distinguishable intact basement membrane could be
observed (Fig. 1a-c). Degenerated follicles showed a retraced oocyte with or without a
pycnotic nucleus, and occasionally a strongly eosinophilic cytoplasm (Fig. 1d). Layers
of granulosa cells remained unaltered or became disorganized into a low density mass
of cells which were many times swollen and/or detached from basement membrane.
Occurrence of pycnotic bodies in granulosa cells or rupture of the basement membrane
was not observed.
The percentages of MNPF observed in the control and after the exposure and
freezing-thawing tests are shown in figure 2. Immersion of ovarian fragments into the
cryopreservation medium, which contained either DMSO or PROH at 1.5 M had no
effect on the percentages of normal follicles as compared with the fresh control
(P>0.05). Similarly, freezing-thawing with either cryoprotectant maintained the
percentages of MNPF (P >0.05). Nevertheless, the absence of cryoprotectants in the
freezing medium (freezing control) led to a severe decrease of the percentages of
normal follicles (22%) as compared with the fresh control (90%) and freezing with
DMSO (84%) or PROH (80%) (P <0.05). For both cryoprotectants, it was observed that
percentages of MNPF after freezing-thawing were similar to the values obtained after
the exposure test (P <0.05).
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Fig. 1. Histological features of canine ovarian fragments before (a) and after freezing-
thawing with 1.5 M DMSO (b) or PROH (c, d). Morphologically normal preantral follicles
comprised an oocyte displaying a large nucleus (nu) and homogenous cytoplasm
surrounded by one or more layers of flattened or cuboidal granulosa cells (gc) and
without antrum (a, b and c). Degenerated preantral follicles often displayed oocyte
retraction (d, arrow) and disorganization of granulosa cell layers. Scale bars represent
10 µm (a, b and c) or 20 µm (d). PAS-hematoxilin.
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Fig. 2. Percentages of morphologically normal preantral follicles in ovarian tissue
before (fresh control) and after exposure and freezing-thawing tests in medium
containing 1.5 M DMSO or PROH or without cryoprotectants (exposure and freezing
controls). (*) Differs significantly from the control (P <0.05); (a, b) different letters for the
same test denote significant differences between media (P <0.05); (A, B) different
letters for a given medium indicates significant differences among exposure and post-
thawing values (P <0.05).
Ultrastructural analysis
TEM of frozen-thawed preantral follicles was performed in order to assess the
ultrastructure of follicles classified as normal at the histological level. At least five
follicles per group were analyzed. Normal follicles contained an oocyte displaying a
very homogenous cytoplasm plenty of round shaped mitochondria with continuous
membranes, few peripheral cristae and electron-dense granules. Some elongated
forms with parallel cristae could also be seen. Small Golgi apparatus cisternae were
rarely observed. Smooth and rough endoplasmic reticulum were present, either as
isolated aggregations or as complex associations with mitochondria. The nuclei of
oocytes were large and usually round, well delimited by the nuclear envelope. The
chromatin was uncondensed and a nucleolus could often be identified. A few vacuoles
were also observed. Granulosa cells were small and presented a high nucleus-to-
cytoplasm ratio. Their irregularly shaped nuclei contained loose chromatin in the inner
part and small peripheral aggregates of condensed chromatin. The cytoplasm exhibited
a great number of mitochondria and well-developed smooth and rough endoplasmic
110
reticulum. The cellular membranes of oocyte and surrounding granulosa cells were
closely juxtaposed, and sometimes few short microvilli could be observed. A distinct
continuous basement membrane surrounded follicles and was tightly attached to
ovarian stroma (Fig. 3a-c).
The ultrastructural pattern described above was observed in normal follicles
from control, after exposure to either cryoprotectants and freezing-thawing with DMSO.
When PROH was used for freezing, despite some follicles displayed normal
morphology, changes could be detected in follicles evaluated as normal in semi-thin
sections by light microscopy. The ooplasm presented a reduced electron density and
lower numbers of organelles, heterogeneously distributed in small clusters. Some large
vacuoles could also be seen. In granulosa cells, loss of cytoplasm content was noticed,
leading to the existence of very large empty spaces in some cases (Fig 3c).
Experiment II: Viability assessment of follicles exposed to cryoprotectants and frozen-
thawed
Preantral follicles from ovarian fragments of the control and testing groups were
mechanically isolated and viability was assessed by trypan blue dye exclusion test. A
total of 1,289 follicles were examined, with a range of 152 to 200 in each treatment.
The percentages of viable follicles observed in each treatment are presented in table 1.
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Fig. 3. Electron micrographs of canine preantral follicles from control (a) and frozen-
thawed using 1.5 M DMSO (b, d) or PROH (c). In Fig. 3a (2500x, scale bar= 10 µm)
and 3b (6000x, scale bar= 5 µm), normal follicles containing an oocyte with
homogenous cytoplasm and a large nucleus well delimited by the nuclear envelope are
displayed. A nucleolus could often be identified (nu), as well as a continuous basement
membrane (bm). In Fig. 3c (2500x, scale bar= 10 µm), observe the large vacuoles
(arrowheads) and a large empty space (asterisk) after freezing-thawing with PROH. In
Fig. 3d (6000x, scale bar= 5 µm), note the great number of intact mitochondria (m) and
smooth endoplasmic reticulum (ser) in the cytoplasm of an oocyte and surrounding
granulosa cells cryopreserved using DMSO. O, oocyte; GC, granulosa cells; v,
vacuole.
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Table 1. Percentages of viable follicles in the fresh control and after exposure to
cryoprotectants and slow freezing-thawing as assessed using trypan blue.
Treatments Exposure Freezing-thawing
Fresh control 77.68±2.89
No cryoprotectant 75.82±2.62aA 5.94±2.82*cB
1.5 M PROH 73.24±3.35aA 48.74±4.55*bB
1.5 M DMSO 73.76±2.63aA 65.68±1.66*aB
(*) Differs significantly from control (P <0.05); (a, b, c) different letters denotes
significant difference between values within a column (P <0.05); (A, B) different letters
indicate significant difference between values of a row (P <0,05).
Immersion of ovarian fragments into 1.5 M DMSO or 1.5 M PROH had no effect
on the percentages of viable follicles as compared with the fresh and test control
(medium without cryoprotectant) (P >0.05). Nevertheless, freezing in either 1.5 M
DMSO or 1.5 M PROH and rapid thawing resulted in a significant reduction of the
percentages of viable follicles in relation to the fresh control (P <0.05), which were
higher on the use of DMSO than when PROH was employed (P <0.05). The absence
of cryoprotectants in the freezing medium (cryopreservation control) led to a severe
and significant decrease of the percentages of viable follicles in comparison with the
fresh control and with freezing with DMSO or PROH. For both cryoprotectants, it was
observed that percentages of viable follicles after freezing-thawing were significantly
decreased as compared with the values obtained after the exposure test.
Based on the results of morphological evaluation (experiment I) and viability
assessment with trypan blue, which evidenced that freezing-thawing using 1.5 M
DMSO provides preservation of ultrastructure and higher percentages of viable follicles
than PROH, a second viability trial using the former cryoprotectant was performed. In
addition to trypan blue testing, a fluorescence cell viability assay based on labelling of
live and dead cells by calcein-AM and ethidium homodimer-1, respectively, was
employed (Fig. 4).
113
Fig. 4. Viability assessment of canine preantral follicles using fluorescent probes. (a)
An isolated preantral follicle classified as viable after slow freezing- rapid thawing with
1.5M DMSO, since cells were labeled by calcein-AM (b) (green fluorescence). (c)
Another follicle from the same treatment considered non-viable as cells were marked
with ethidium homodimer-1 (d) (red fluorescence). Scale bars represent 10 µm.
Percentages of viable follicles remained unaltered after exposure of ovarian
cortex fragments to 1.5 M DMSO in comparison to the control (P >0.05) as assessed
by trypan blue and calcein-AM/ethidium homodimer assays, whose values did not differ
from each other (P >0.05). However, a significant reduction in the percentages of
viable follicles relative to the fresh control and exposed fragments was observed after
freezing-thawing according to the two viability tests, which once more retrieved similar
results (Fig.5).
114
Fig. 5. Percentages of viable canine preantral follicles in fresh ovaries and after
exposure and freezing-thawing using 1.5 M DMSO as assessed by trypan blue dye
exclusion test and labeling with calcein-AM and ethidium homodimer. (*) Differs
significantly from control (P <0.05); (a, b) different letters within the same test
(exposure or freezing-thawing) denote significant differences between values of the
viability assays (P <0.05); (A, B) different letters for a same viability assay indicates
significant differences between tests (P <0.05).
Discussion
The present study evaluated for the first time the cryopreservation of canine
preantral follicles through slow freezing, employing DMSO and PROH as
cryoprotectants. It was demonstrated that viability and ultrastructural integrity of such
follicles can be successfully maintained with the use of 1.5 M DMSO.
Exposure of ovarian fragments to either DMSO or PROH at 1.5 M did not cause
reduction of the percentages of MNPF follicles as evaluated in histology, and
ultrastructural analysis through TEM confirmed the integrity of such follicles.
Accordingly, Lucci et al. (2004) also observed no effect of such cryoprotectants at the
same concentrations on bovine preantral follicles. On other hand, caprine (Rodrigues
et al., 2004) and ovine (Santos et al., 2006) ovarian tissue exposed to 1.5 M DMSO or
PROH at similar conditions (temperature and time) showed a significant reduction of
the percentage of normal preantral follicles. Indeed, an osmotic stress due to the
presence of cryoprotectants in the freezing medium is one of the primary theories of
115
injury during cryopreservation, whilst the inherent toxicity of these compounds is also
an important consideration (Mullen et al., 2008). We hypothesize that these differences
reflect a species-specific resistance of preantral follicles enclosed in ovarian cortex to
exposure to DMSO and PROH at 1.5 M.
After freezing and thawing with either cryoprotectants, percentages of MNPF
also remained unchanged as evaluated by histology. However, TEM revealed
alterations in the ultrastructure of follicles frozen with PROH which were previously
evaluated as normal in histology. Therefore, TEM proved to be an essential tool to
detect damage due to the freezing-thawing process which can be observed only at the
ultrastructural level but not through light microscopy. The most severe damage
observed was made up of large empty spaces not surrounded by membranes. This
feature suggests the occurence of crystallization, and was also observed in glycerol-
frozen ovine embryos (Cocero et al., 2002). This relative inefficiency of PROH to
protect canine preantral follicles during freezing-thawing may be due to the fact that its
ability to prevent ice-crystal formation is limited at 1.5 M (Jain and Paulson, 2006).
Similarly, integrity of caprine (Rodrigues et al., 2004) and bovine (Lucci et al., 2004)
preantral follicles cryopreserved in 1.5 M DMSO was confirmed, whilst similar
modifications were found in follicles frozen-thawed with 1.5 M PROH.
Notwithstanding previous studies on cryopreservation of preantral follicles
provided important knowledge on this issue, most approaches were limited to analysis
of morphology, which is not always correlated with the viability of follicles (Santos et al.,
2007). Therefore, in the present work, we further analyzed the quality of follicles
through assessment of viability using the trypan blue dye exclusion test. After exposure
to either DMSO or PROH at 1.5 M, percentages of viable follicles remained similar to
those of the controls. Accordingly, Jewgenow et al. (1998) demonstrated that adding
these cryoprotectants at the same concentrations to isolated feline small preantral
follicles did not result in decrease of viability after in vitro culture in comparison to the
control. Amorim et al. (2004) observed that only exposure to DMSO or PROH at
concentrations higher than 2 M and 1.5 M respectively reduced the numbers of viable
preantral follicles isolated from ovine ovaries. Thus, our results show that addition of
DMSO or PROH at up to 1.5 M to canine ovarian tissue affects neither the integrity nor
the viability of preantral follicles as a consequence of osmotic and toxic effects of the
freezing solution.
When post-thaw viability of follicles was examined, a significant decrease was
found with the use of both cryoprotectants in comparison to the fresh control. However,
a high percentage (65%) of viable follicles was maintained through the employment of
1.5 M DMSO, whilst PROH did not show a comparable efficiency for follicular
116
preservation, which might be due to the ultrastructural damages detected in the
morphological analysis. In accordance, Jewgenow et al. (1998) showed that viability of
isolated feline preantral follicles was also reduced after freezing-thawing with either 1.5
M DMSO or PROH. Conversely, Rodrigues et al. (2005) observed that
cryopreservation using 1.5 M DMSO or PROH had no effect on viable caprine preantral
follicles, and this outcome was also achieved with ovine follicles after using 1.5 M
DMSO (Amorim et al., 2007). Hence, we suggest that carnivore preantral follicles are
less resistant to freezing-thawing with these cryoprotectants at 1.5 M than their
counterparts from small ruminants.
Percentages of viable follicles were much lower than those of morphologically
normal follicles in every treatment. This proves that a more precise determination of the
proportions of follicles with adequate quality for subsequent applications such as in
vitro culture may rely on viability assessment. In this context, we have further analyzed
follicles cryopreserved in DMSO using a more accurate method. For this purpose,
labeling of non-viable cells with disruption of plasma membrane was performed again
by using ethidium homodimer-1, which enabled a better visualization of dead cells
through fluorescent staining of nuclei. Concomitantly, identification of viable cells was
performed through detection of esterase activity with calcein-AM. These fluorescent
probes have been successfully used to assess viability of bovine early-staged follicles
(Schotanus et al.,1997; Van den Hurk et al., 1998) and after cryopreservation of
caprine preantral follicles (Santos et al., 2006, 2007). In the present study, results of
this assay were similar to values observed with trypan blue testing, which thus proved
to be a reliable, practical and quick method for preliminary viability assessment of
preantral follicles. Accordingly, Poeschmann et al. (2008) observed a significant
correlation between both methods while analyzing viability of feline preantral follicles.
Despite a significant decrease of viable follicles was observed after freezing-
thawing in the present study, we consider that preservation rates with the use of DMSO
to freeze canine preantral follicles are by this time high and can be further improved.
Since these follicles proved to not be affected by exposure to DMSO at 1.5 M at the
described conditions, concentration of this cryoprotectant could be increased up to a
threshold on which cytotoxic and/or osmotic effects are still inexistent and protection
against cryoinjuries is maximum. It must also be considered for such adjustment that
during cell dehydration, a delicate balance must be maintained between the removal of
free water that can form ice crystals and the removal of bound water, because
excessive loss of the latter results in loss of structural support to proteins and lipids
(Wright et al., 2004). The use of isolated follicles could also enhance results, since
117
mammalian cells frozen in situ are more susceptible to damage caused by this process
than cells in suspension (Armitage et al., 1995; Armitage and Juss, 1996).
Once established, knowledge on cryopreservation of canine preantral follicles
could be applied to endangered canids in order to lengthen the genetic lifespan of each
rare female. To this end, development of in vitro culture systems capable to promote
growth and maturation of this vast pool of oocytes has to be accomplished. However,
at present, such systems still do not lead to normal full follicle development in non-
rodent species, for which oocyte growth is a long-term, coordinated and complex
process, and the time required to reach the mature stage is thought to be up to several
months. Currently, xenotransplantation into immunodeficient recipients seems to be
one the most promising ways for obtaining oocytes from ovaries for further exploration
of assisted reproduction techniques (Jewgenow and Paris, 2006). Ishijima et al. (2006)
transplanted canine ovarian tissue into non-obese diabetic severe combined
immunodeficient mice and observed a significant shift from primordial to primary
follicles, in which the proliferating cell nuclear antigen could be detected. In addition,
Fassbender et al. (2007) demonstrated the survival of cat ovarian cortex tissue
transplanted under the kidney capsule of athymic nude rats. Follicular development
was monitored through high-resolution ultrasonography, and cumulus oocytes
complexes could be collected and in vitro matured.
In conclusion, cryopreservation of canine preantral follicles can be
accomplished through slow freezing and rapid thawing using 1.5 M DMSO or PROH.
However, only DMSO provided maintenance of high percentages of viable follicles with
intact ultrastructure. Further studies are necessary to evaluate developmental capacity
of such follicles, which demands the establishment of in vitro culture systems or use of
transplantation approaches.
Acknowledgements
During the period of this work, Claudio A. P. Lopes received financial support
from the National Council for Scientific and Technological Development (CNPq;
doctoral scholarship) and the Coordination for Improvement of Graduate Personnel
(CAPES; fellowship for studies abroad, PDEE, grant 5212/06-5), both institutions of the
Brazilian Government. José Ricardo de Figueiredo was also recipient of a grant from
CNPq. We would like to express our appreciation to the Center for Zoonosis Control
(CCZ) of Fortaleza Municipality (Brazil) and to the veterinary clinics of the animal
shelter Tierheim-Berlin and Tierklinik-Biesdorf (Germany), which gently provided the
canine ovaries.
118
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10 CAPÍTULO V
Potencial de anticorpos anti-zona pelúcida para a imunoesterilização de cães:
efeitos sobre folículos pré-antrais in vitro
Potential of anti-zona pellucida antibodies for the immunosterilization of dogs: effects
on preantral follicles in vitro
Journal of Reproduction and Development
(Submetido, 2008)
122
Potential of anti-zona pellucida antibodies for the immunosterilization of dogs: effects on preantral follicles in vitro
Lopes CAPABD, Niu YC, Alves AMCVA, Chaves RNA, Campello CCA, Jewgenow KB, Figueiredo JRA
A Faculty of Veterinary, PPGCV, LAMOFOPA, State University of Ceará, Fortaleza, Brazil.
Postal address: Av. Paranjana 1700, Campus do Itaperi, CEP 60.740-903, Fortaleza, CE, Brazil.
B Institute for Zoo and Wildlife Research, Berlin, Germany.
Postal address: Postfach 601103, D-10252, Berlin, Germany.
CKunming Primate Research Center and Kunming Institute of Zoology, Chinese Academy of Sciences,
China.
Postal address: 32 Jiao Chang Dong Lu, Kunming, Yunnan 650223, China.
DCorresponding author. Email: [email protected]
Resumo
A superpopulação de cães constitui um grave problema em diversas cidades do
mundo, que tem sido controlado através da eliminação sistemática de animais sem
responsáveis, que podem atuar como reservatórios de zoonoses. A esterilização de cadelas
através de imunização contra as proteínas da zona pelúcida (ZP), estrutura que reveste os
oócitos, é uma alternativa promissora para o controle reprodutivo desta espécie. O objetivo
deste estudo foi avaliar in vitro o potencial de um antisoro contra ZP suína (pZP) de induzir
degeneração de folículos pré-antrais caninos e/ou de bloquear a ligação espermática à ZP.
Folículos pré-antrais foram isolados de ovários caninos e cultivados in vitro com soro anti-
pZP. Oócitos obtidos de folículos antrais foram tratados com o mesmo soro e co-incubados
com sêmen canino de modo a se analisar a ligação espermática. A avaliação da viabilidade
após o cultivo revelou que o soro anti-pZP foi efetivo para induzir degeneração de folículos
pré-antrais maiores que 100 µm, conforme comparação com o controle (soro pré-imune),
mas não teve efeito sobre folículos menores. Este processo parece envolver a ativação do
sistema complemento sérico. Anticorpos anti-pZP também se mostraram capazes de
bloquear a ligação espermática. Diante do exposto, concluímos que anticorpos anti-pZP
podem promover a eliminação de folículos pré-antrais caninos e inibir a ligação espermática,
constituindo-se, assim, uma importante perspectiva para o controle efetivo da reprodução
em cães.
Palavras-chave: Imunocontracepção, anticorpos, canino, zona pelúcida, folículos pré-antrais.
123
Abstract
Overpopulation of dogs in urban areas is a critical issue worldwide, which has been
controlled by means of systematic euthanasia of unowned animals that can act as zoonoses
reservoirs. Sterilization of bitches through immunization against proteins of the zona
pellucida (ZP), the egg coat, is a promising alternative for the reproduction control in this
species. The aim of this work was to evaluate in vitro the potential of an antiserum against
porcine ZP (pZP) to cause degeneration of canine preantral follicles and/or blocking of sperm
binding. Preantral follicles were isolated from bitch ovaries and in vitro cultured with anti-pZP
serum. Oocytes from antral follicles were also obtained, treated with the same serum and co-
incubated with dog semen in order to analyze sperm binding. Viability assessment after
culture showed that anti-pZP serum was effective to induce degeneration of preantral follicles
larger than 100 µm as compared to the control (pre-immune serum), but had no effect on
smaller follicles. This process might involve activation of the serum complement system.
Anti-pZP antibodies also proved capable of blocking sperm binding. In conclusion, anti-pZP
antibodies can promote the elimination of canine preantral follicle and inhibit sperm binding,
which is an important prospect for effective control of reproduction in dogs.
Keywords: Immunocontraception, antibodies, canine, zona pellucida, preantral follicles.
Introduction
Overpopulation of dogs is a critical problem is many cities around the world, which is
characterized by the occurrence of large groups of surplus animals without a responsible
owner. Since these individuals can act as zoonoses reservoirs, health authorities perform
systematic euthanasia in order to control this subpopulation. Each year, this situation results
in millions of deaths at shelters and spending in the billions of dollars (Frank e Carlisle-Frank,
2007).
Despite efforts in recent years to identify reliable methods of pharmacologic and
chemical sterilization for dogs, surgical methods have remained the mainstay. Although
these procedures are often viewed as ‘‘routine’’ surgeries, complications may result from
inappropriate techniques, and efforts should be made to follow good surgical and aseptic
standards (Howe, 2006). In addition, such methods can be too time consuming and
expensive to be performed on a large-scale (Kutzler e Wood, 2006), and hence are not
logistically and economically feasible to be applied to large canine populations, as is the case
in metropolis.
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In this context, immunocontraception arises as a promising perspective for the control
of reproduction in dogs. This approach consists of directing the body’s immune response
towards functionally and structurally important molecules that are involved in mammalian
reproduction. Sperm surface antigens, the zona pellucida (ZP), gonadotropins (LH and FSH)
or their receptors, and the gonadotropin releasing hormone (GnRH) have been identified and
are used as targets for immunocontraceptive vaccines in carnivores. At present, the best
contraceptive vaccines are zona pellucida based, which has been shown to cause long-
lasting contraception in several wildlife species (Jewgenow et al., 2006).
The ZP consists of an extracellular glycoprotein matrix which surrounds the
mammalian oocytes and plays fundamental roles in the fertilization process through the
regulation of sperm binding, induction of the acrossome reaction and of the block to
polyspermy (Wassarman, 2008). Active immunization against ZP proteins inhibits fertility in
many species by interfering with sperm–egg interaction during fertilization, and affecting the
growing pool of ovarian follicles. The synthesis of ZP takes place during oocyte growth.
Therefore, a immune reaction against ZP can induce destruction of early ovarian oocytes,
leading sometimes to irreversible infertility (Ringleb et al., 2004). Loss of primordial follicles is
a common outcome of the immunization against one ZP glycoprotein (ZP3), which might
occur due to the elimination of growing follicles by anti-ZP antibodies followed by accelerated
recruitment of the quiescent follicles (Aitken, 2002).
The aim of the present study was to evaluate in vitro the potential of serum containing
anti-pZP antibodies to cause degeneration of canine preantral follicles. It was also tested the
capacity of these antibodies to block canine sperm binding to homologous ZP.
Material e Methods
All chemicals were obtained from Sigma-Aldrich (Sigma Chemie GmbH, Deisenhofen,
Germany) unless stated otherwise and were of the highest purity available.
Animals and gonad collection
Ovaries and testes were obtained from dogs at the veterinary clinic of the animal
shelter of the City of Berlin (Tierheim Berlin - Tierschutzverein für Berlin und Umgebung
Corporation e.V.). Following excision, ovaries were placed into 15 mL of HEPES-buffered
Minimum Essential Medium (MEM, osmolarity 280 mOsmol/l) at 4ºC. Testes were stored at
125
the same temperature without any medium. Gonads were transported to the laboratory in
thermoflasks within 4 h and then immediately processed.
Collection of oocytes, preantral follicles and spermatozoa
Ovaries were dissected in M199 containing Earle’s salts, supplemented with 3 mg/ml
BSA, 0.6 mg/ml sodium lactate, 1.4 mg/ml Hepes, 0.25 mg/ml sodium pyruvate, 0.1 mg/ml
cysteine and 0.055 mg/ml gentamicin. Grade 1 oocytes displaying an even dark ooplasm
surrounded by a multilayered cumulus cell mass were selected, vortexed for 3 min to detach
cumulus cells and then washed three times.
Canine preantral follicles were isolated using the mechanical method described by
Figueiredo et al. (1993). Briefly, using a tissue chopper (The Mickle Laboratory Engineering
Co., Gomshal, Surrey, UK) adjusted to a sectioning interval of 100 µm, ovarian cortex was
cut into small pieces, which were placed in MEM and suspended 40 times using a large
Pasteur pipette (diameter of about 1600 µm) and subsequently 40 times with a smaller
Pasteur pipette (diameter of approximately 600 µm) to dissociate preantral follicles from the
stroma. The obtained material was passed through 500- and 100-μm nylon mesh filters,
resulting in a suspension containing preantral follicles smaller than 100 μm in diameter. This
procedure was carried out within 10 min at room temperature (RT, approximately 25°C).
Preantral follicles with a diameter of 100 to 200 μm were isolated from canine ovaries
as described by Mao et al. (2004). Briefly, ovarian cortical tissue (<1 mm thickness) was
sliced from the ovarian surface, placed in HEPES-buffered α-MEM and visualized under a
dissecting microscope (SMZ 645 Nikon, Tokyo, Japan) equipped with an ocular micrometer.
Follicles were then manually isolated with a 28-gauge needle and surgical blade. Those
displaying normal morphology, no sign of oocyte retraction and well-organized layers
granulosa cells adhered to an intact basement membrane were selected.
Canine spermatozoa were obtained from caudae epididymides by mincing them in
1ml TALP medium (Tyrode’s salt solution containing 6 mg/ml BSA, 1.2 mg/ml Hepes, 1.1
mg/ml sodium lactate, 0.11 mg/ml sodium pyruvate). Sperm cells were centrifuged at 500 g
for 7 min and resuspended in 100–200ml TALP medium. After estimation of sperm
concentration and motility, a final concentration of 1 x 105 motile sperm cells/ml was
established by addition of TALP medium.
126
In vitro culture of canine preantral follicles with anti-pZP serum
Serum containing anti-pZP polyclonal antibodies was produced through immunization
of rabbits as previously described by (Jewgenow et al. 2000). Canine preantral follicles
(n=2,560) were isolated as described previously and divided into two groups according to the
diameter (<100 µm or ≥100 µm) and in vitro cultured with 10% pre-immune serum (control)
or anti-pZP serum. The used medium consisted of α-MEM (osmolarity: 300 mOsm/L, pH:
7.2) supplemented with 1% ITS (6.25 µg/ml insulin, 6.25 µg/ml transferrin and 6.25 ng/ml
selenium), glutamine (2 mM), hypoxanthine (2 mM) and bovine serum albumin (BSA,1.25
mg/ml).
Assessment of preantral follicles viability
Viability of canine preantral follicles was assessed before and after in vitro cultured
through a test based on the simultaneous determination of live and dead cells by calcein-AM
and ethidium homodimer-1, respectively. Whilst the former compound detected intracellular
esterase activity of viable cells, the latter marked nucleic acids in non-viable cells with
damaged plasma membrane. The test was performed by adding by adding 4 μM calcein-AM
and 2 μM ethidium homodimer-1 (Molecular Probes, Invitrogen, Karlsruhe, Germany) to
isolated follicles, followed by incubation at 37°C for 10 min. After being labeled, follicles were
washed three times in MEM, mounted on a glass microscope slide in 5 μl antifading medium
(DABCO, Sigma, Deisenhofen, Germany) to prevent photobleaching, and finally examined
using an a DMLB fluorescence microscope (Leica, Germany). The emitted fluorescent
signals calcein-AM, and ethidium homodimer were collected at 488 and 568 nm,
respectively. Oocytes and granulosa cells were considered live if the cytoplasm was stained
positively with calcein-AM (green) and chromatin was not labelled with ethidium homodimer
(red).
In vitro test for sperm binding test
Denuded oocytes were incubated for 1 h at 37.5°C with pre-immune serum (control),
anti-pZP serum (undiluted or diluted 1:10 or 1:20) or in HEPES-buffered MEM (positive
control). Afterwards, oocytes were transferred to separate insemination drops containing 400
ml of IVF medium (TALP solution containing 10mg/ml heparin) and 1 x 105 motile
spermatozoa/ml. After 18–20 h of co-incubation at 39°C in an atmosphere of 5% CO2,
oocytes were washed three times in DPBS with 3 mg/ml BSA to remove loosely attached
127
spermatozoa and finally placed into DPBS supplemented with 4% (w/v) paraformaldehyde
and 0.02% (v/v) Triton-X100 for 45–60 min at 39 8C. After washing twice again, oocytes
were stained with 10mg/ml Hoechst 33258 for 45 min, mounted on slides and the number of
attached sperm heads was counted under fluorescence excitation using a DMLB Leica
Microscope (Wetzlar, Germany).
Statistical analysis
All experiments were replicated five times. Kolmogorov-Smirnov and Bartlett’s tests
were applied to verify normal distribution of data and homogeneity of variances respectively.
Afterwards, analysis of variance (ANOVA) was performed using the GLM procedure of the
software SAS (SAS Institute Inc., Cary, NC, USA). Differences of percentages of viable
preantral follicles between treatments were determined by Student Newman Keuls’ (SNK)
test. For the sperm binding experiments, differences between groups were assessed by the
non-parametric Mann–Whitney test. Data were expressed as means ± standard error of the
mean (SEM) and differences were considered statistically significant when P <0.05.
Results
Effect of anti-pZP serum on viability of canine preantral follicles
Viability of canine preantral follicles was assessed before and after in vitro culture
through a test based on the simultaneous determination of live and dead cells by calcein-AM
and ethidium homodimer-1, respectively. Percentages of viable follicles observed in each
treatment are showed in Figure 1.
128
Figure 1. Percentages of viable canine preantral follicles before (fresh control time 0 h) and
after in vitro culture for 24 h with in medium containing no serum or 10% pre-immune serum
(control) or anti-pZP. (a, b) different letters indicate significant differences (P <0.05) between
values within a follicular category (<100 µm or ≥100 µm).
After in vitro culture for 24 h in medium without serum, with 10% pre-immune serum
(control) or anti-pZP serum, it was observed a significant reduction (P <0,05) of the
percentages of viable follicles with diameter smaller than 100 µm in comparison to the fresh
control (time 0h). No significant difference (P >0,05) was found among the different culture
groups (P >0,05).
With respect to the group of follicles with diameter of 100-200 µm, a significant
decrease (P <0,05) of the percentages of viable follicles was also seen after in vitro culture in
medium without serum or with either sera in comparison to the fresh control. Moreover, it
was noticed that the presence of anti-pZP serum led to a massive reduction of the
percentages of viable follicles, which were significantly lower (P <0,05) than those obtained
with the addition of pre-immune serum (control) or with culture medium alone. It is
noteworthy that along with the oocyte, no single follicular cell could survive, which is an
important outcome considering the potential of remaining granulosa cells to develop cysts
(Mahi-Brown et al., 1998). In figure 2, viable and non-viable follicles analyzed using
fluorescent labels are presented.
a
129
Fig. 2. Viability evaluation of canine preantral follicles using fluorescent probes. (A) A follicle
in vitro cultured for 24 h with pre-immune serum (control) and classified as viable since cells
were labeled by calcein-AM (green fluorescence). The arrow indicates a thick zona pellucida
surrounding the oocyte. (B) A follicle cultured with anti-pZP serum and considered non-viable
as cells were marked with ethidium homodimer-1 (red fluorescence). Scale bars represent 20
µm.
Effect of anti-pZP antibodies on canine sperm binding to homologous oocytes
The capacity of anti-pZP antibodies to block binding of dog spermatozoa to bitch
oocytes was assessed through an in vitro test which consisted of co-incubation of oocytes
treated with pre-immune (control) or hyperimmune serum with sperm followed by counting of
Hoechst 33258 stained sperm heads bound to oocytes under a fluorescence microscope (fig.
3).
A B
130
Fig. 3. Assessment of anti-pZP antibodies ability to inhibit canine oocyte-sperm interaction.
(A) Oocyte incubated in MEM (positive control) displaying several bound spermatozoa; (B)
oocyte treated with pre-immune serum (control); (C) oocyte treated with anti-pZP serum
showing no bound spermatozoon; (D) oocyte treated with anti-pZP diluted 1:10. ZP: zona
pellucida.
A B
C D
ZP
ZP
ZP ZP
131
The mean numbers of bound spermatozoa bound per oocyte for each treatment are
presented in figure 4. Incubation of oocytes with pre-immune serum led to a significant
decrease (P <0,05) of the sperm binding as compared to the positive control (incubation in
MEM). Treatment of oocytes with anti-pZP serum reduced significantly the numbers of bound
sperm (P <0,05) in comparison to the test control (pre-immune serum) and to the positive
control. Conversely, anti-pZP serum diluted at 1:10 lowered (P <0,05) sperm binding in
relation to the positive control, but no significant difference (P >0,05) was found in relation to
treatment with pre-immune serum. Dilution of anti-pZP at 1:20 had no effect on numbers of
bound sperm as compared to the controls.
Fig. 4. Assessment of the contraceptive efficiency of anti-porcine zona pellucida (pZP)
antibodies in dogs using an in vitro test for canine sperm binding to homologous ZP. Bitch
oocytes were incubated with pre-immune serum (control), anti-pZP serum (undiluted or
diluted 1:10 or 1:20) or in HEPES-buffered MEM (positive control). Subsequently, oocytes
were transferred to IVF drops and co-incubated with 1 x 105 motile spermatozoa/ml for 18-20
h. After staining with Hoechst 33258, the number of attached sperm heads on each oocyte
was counted under a fluorescence microscope. (a, b, c) Different letters indicate significant
differences (P <0.05).
a
b
c
b
ab
132
Discussion
The present study demonstrates the ability of anti-ZP antibodies to cause atresia of
ovarian preantral follicles, and thus their potential to be used for the immunosterilization of
dogs. In most of the works on immunological contraception targeting ZP, including this one,
pZP glycoproteins have been used due to its high availability in abattoirs and the high degree
of homology with the ZP proteins of many mammalian species, which ensures the cross-
reactivity of produced antibodies to native zonae (Niu et al., 2006). We have used a serum
produced against purified pZP, which contained antibodies with high specificity for native
zona proteins as shown by ELISA, immunoelectrophoresis and immunohistology (Jewgenow
et al., 2000).
In order to analyze the effects of anti-pZP serum on viability of canine preantral
follicles, we used as experimental model an efficient in vitro culture system which has been
developed by our group to promote growth of preantral follicles from some mammalian
species, but it had not been employed hitherto in canine. The maintenance of high
percentages of viable follicles after culture observed in this study indicates that this system is
also suitable for canine preantral follicles. Moreover, since it enabled observations on
isolated follicles, it was possible to assess directly and exclusively the action of anti-ZP
antibodies on these structures, contributing for the elucidation of the pathogenesis of follicle
damage showed previously in studies in vivo. It has been postulated by many authors that
cytotoxic T-cell responses due to ZP immunization would be a major mechanism (Millar et
al., 1989; Rhim et al.,1992; Lou and Tung, 1993; Jackson et al., 1998; Lou et al. 2000). The
absence of immunological cells in our system indicates that antibodies can act solely for
follicle elimination. Another important aspect of the model used in this work is the possibility
of experimentation without use of animals.
Addition of anti-pZP serum to the culture medium had no effect on viability of follicles
with diameter smaller than 100 µm as compared to the use of medium alone or with 10%
pre-immune serum. However, pZP antiserum caused extensive atresia of preantral follicles
measuring 100-200 µm. Since such effect was not observed when follicles of this category
were cultured in medium containing pre-immune serum (control), polyclonal anti-ZP
antibodies previously confirmed in the hyperimmune serum, and not other components, may
have accounted for this outcome. We suggest that massive binding of antibodies to the ZP
led to the activation of the cascade of serum complement proteins, which in turn resulted in
the formation of the membrane-attack complexes (Janeway et al., 2001) and subsequent
degeneration of follicular cells. This hypothesis is supported by the study of Gwatkin et al.
(1977), who localized antibodies and complement in the zonae of oocytes from infertile mice
133
immunized with hamster zonae. Jackson et al. (1998) postulated that in mice, antibodies
binding to the developing ZP of growing eggs may kill oocytes via antibody-dependent cell-
mediated cytotoxicity or complement lysis. Another possible mechanism for this effect could
be the interruption of communication between oocytes and granulosa via gap junctions by
large amounts of bound antibodies as postulated by Mahi-Brown et al. (1988). However,
despite these authors observed deposit of electron-dense material at the surface of the ZP in
bitches immunized with pZP, no alterations of cell junctions were found.
The absence of effect of the anti-pZP serum on follicles smaller than 100 µm might be
explained by a lower frequency of antibody binding due to lesser amounts of ZP
glycoproteins within these follicles. The zona proteins are the products of three gene families,
named ZPA, ZPB, and ZPC (Harris et al., 1994). A vast majority of follicles in this size
category comprised primordial follicles (data not presented), for which only expression of
ZPA could be detected in the cytoplasm of oocytes (Blackmore et al., 2004). Furthermore,
Barber et al. (2001) showed through ultrastructural immunohistochemistry that anti-pZP
antibodies react with zona proteins present in the golgi apparatus of canine of oocytes within
developing follicles but not when they are at the primordial stage. Conversely, follicles of the
other group (100-200 µm) corresponded to mid and late secondary follicles, which present
peak mRNA transcription for ZPC, in addition to ZPA and ZPB expression (Blackmore et al.,
2004). A comparable sequential ZP expression pattern was also described by Jewgenow
and Fickel (1999) in cats, for which ZPB gene transcripts were firstly present in primary
follicles, whilst ZPA and ZPC were detectable only from transition to secondary stage
onwards.
We also suggest as a reason for the inability of anti-pZP serum to cause
degeneration to smaller follicles that the affinity of the present antibodies may be higher or
even specific to the ZP glycoproteins synthesized by late secondary follicles. Blackmore et
al. (2004) showed through probing canine ovarian tissue with a panel of lectins that patterns
of glycosylation of zona proteins occurs in a developmentally specific manner. Residues of α-
mannose were weakly labeled in granulosa cells of primary follicles and strongly labeled in
the ZP and in surrounding granulosa cells of secondary follicles. On other hand, using the
lectin obtained from Erythrina cristagalli (ECL), which binds D-galactose and N-acetyl
glucosamine, labeling was identified in the ZP and adjacent granulosa cells of secondary
follicles only. Therefore, in order to achieve immunosterilization targeting directly the pool of
oocytes within the quiescent follicles, a promising strategy could be the characterization of
the ZPA proteins already expressed in canine primordial follicles to be subsequently used in
immunogens. Grootenhuis et al. (1996) observed the binding of anti-ZP antibodies to oocytes
and granulosa cells of primordial follicles in several species, suggesting that complement
134
fixation following antibody binding may be the mechanism of depletion of these follicles
observed in studies in vivo.
Despite primordial follicles were not affected by anti-pZP serum in the present study,
the observed degeneration of late secondary follicles is also an important prospect for the
immunosterilization of bitches. Skinner et al. (1984) demonstrated that immunization of
rabbits with pZP resulted in a complete disappearance of growing follicles within 30 weeks
with associated reduction of the number of primordial follicles. These authors postulated that
developing follicles provide some signal that prevents the activation of primordial follicles,
and since the former are destroyed by anti-ZP antibodies, the latter start a suicidal growth
until ZP proteins are secreted. These progressive changes persist whilst ovaries are under
influence of such antibodies. Thus, the anti-ZP antibodies studied here could promote a
similar process in vivo leading to an immunological castration.
Mahi-Brown et al. (1988) observed that immunization of bitches with pZP caused
extensive oocyte destruction. These authors suggested that this consequence could
probably lead to permanent sterility. In some animals, follicles failed to survive beyond the
primordial stage. However, it was also noticed the occurrence of follicular cysts and
abnormal estrous cycles in which prolonged elevated 17 β-estradiol was recorded in most of
the treated females. Despite bitches were rendered infertile, this side effect is undesirable,
since some sequela such as bone marrow hypoplasia and/or pyometra can develop (Meyers-
Wallen, 2007). In the present study, complete follicular degeneration was achieved, i.e. no
single granulosa cell could survive along with the dead oocyte. Therefore, potential of cyst
formation would be unlikely if in vitro conditions can be established in vivo. We hypothesize
that levels of antibodies that could interact with follicles in our work were higher than those
attained within ovaries in the study of Mahi-Brown et al. (1988). Anti-pZP titres of the sera
used in the current study ranged from 1:100 000 to 1:160 000, whilst in active immunized
animals in the mentioned previous work, titers ranged from 1:1000 to 1:20 000. Hence, levels
of anti-pZP antibodies in the former study could possibly had not reached a threshold to
cause massive follicle degeneration, and remaining live granulosa cells may have developed
to the observed cysts. In accordance to this theory, Srivastava et al. (2002) reported an
increase in the number of atretic follicles associated with the antibody titres in bitches
immunized with recombinant canine ZP3.
Capacity of the used anti-ZP antibodies to block binding of spermatozoa to the canine
ZP was also evaluated in this study. We could observe a significant inhibition of sperm
binding when oocytes were incubated for 1 h with undiluted serum, but dilution (1:10 and
1:20) suppressed this effect. Nevertheless, bitches do not need to have extremely high anti-
135
ZP titers in order for their eggs to be impenetrable. Since oocytes in vivo are exposed
continuously to circulating immunoglobulins, there may be more thorough coating of their
zonae pellucidae than after in vitro incubation (Mahi-Brown et al., 1985). Oocytes recovered
from the ovary of one bitch immunized with pZP were not penetrable by spermatozoa,
whereas antiserum from this same bitch did not prevent sperm penetration (Mahi-Brown et
al., 1982). Hence, the anti-ZP antibodies tested in the present work have also potential to be
used for the immunocontraception of dogs through fertilization blocking.
In conclusion, anti-pZP antibodies are capable to cause atresia of canine preantral
follicles as the secondary stage of development is reached. This process might involve the
serum complement system. Therefore, immunosterilization of bitches can be potentially
accomplished through vaccination against pZP proteins or passive immunization with anti-
pZP antibodies. Contraception through blocking of sperm binding can possibly be achieved
by using these antibodies as well. Once established, such techniques could enable an
effective control of dog populations in many urban areas.
Acknowledgements
During the period of this work, Claudio A. P. Lopes received financial support from the
National Council for Scientific and Technological Development (CNPq; doctoral scholarship)
and the Coordination for Improvement of Graduate Personnel (CAPES; fellowship for studies
abroad, PDEE, grant 5212/06-5), both institutions of the Brazilian Government. José Ricardo
de Figueiredo was also recipient of a grant from CNPq. We would like to express our
appreciation to the veterinary clinics of the animal shelter Tierheim-Berlin and Tierklinik-
Biesdorf (Germany), which gently provided the canine ovaries.
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11 CONCLUSÕES
A preservação de folículos pré-antrais caninos durante o armazenamento é realizada
com maior eficiência através de hipotermia em um meio nutritivo. A manutenção da
viabilidade e integridade ultra-estrutural pode ser obtida a 4ºC e 20ºC em solução salina por 6
h ou em MEM por 12 h, sendo esta última solução também adequada a 38ºC por 6 h. Sugere-
se o uso de MEM a 4ºC por até 12 horas para a preservação ótima da qualidade de folículos
pré-antrais caninos destinados a aplicações como o cultivo in vitro.
A criopreservação de folículos pré-antrais caninos pode ser realizada através de
congelação lenta e descongelação rápida utilizando-se DMSO a 1,5 M. Este crioprotetor
proporciona a manutenção de altas percentagens de folículos viáveis com ultra-estrutura
intacta. Índices elevados de preservação da ultra-estrutura folicular podem também ser
obtidos com EG a 1,5 M. Entretanto, faz-se necessário avaliar a viabilidade dos folículos
criopreservados utilizando-se este composto.
Folículos pré-antrais caninos frescos ou criopreservados podem ser cultivados in vitro
mantendo sua viabilidade. Assim, pode-se utilizar este sistema como modelo experimental
para o teste de agentes esterilizantes.
Anticorpos anti-ZP são capazes de se ligar à ZP de oócitos caninos, causando bloqueio
da ligação de espermatozóides ou atresia de folículos pré-antrais.
139
12 PERSPECTIVAS
Os conhecimentos acerca da preservação de folículos pré-antrais caninos obtidos neste
estudo poderão servir como base para o desenvolvimento pleno desta técnica, que, uma vez
estabelecida, poderá ser aplicada a canídeos em risco de extinção de forma a se preservar o
patrimônio genético de cada fêmea rara. Ainda, o uso do método de congelação lenta e
descongelação rápida utilizando-se DMSO a 1,5 M já possibilita a manutenção de elevados
percentuais de folículos viáveis com ultra-estrutura intacta. Entretanto, é preciso avaliar a
capacidade de desenvolvimento de tais folículos, sendo necessário o estabelecimento de
sistemas de cultivo in vitro ou in vivo, sendo as técnicas de xenotransplante ou alotransplantes
recursos promissores. O método de cultivo in vitro utilizado neste trabalho também
demonstrou potencial para este propósito.
A alta eficiência da congelação utilizando-se EG a 1,5 M para a manutenção da ultra-
estrutura folicular indica o potencial deste composto para a criopreservação de folículos pré-
antrais caninos. No entanto, a viabilidade, bem como a capacidade de desenvolvimento
folicular, devem ser avaliados, sendo importante também a comparação direta da eficiência
deste crioprotetor àquela observada para o DMSO, que proporcionou os melhores resultados.
A demonstração da capacidade de anticorpos anti-ZP de causar atresia em folículos
pré-antrais caninos consolida a importante perspectiva do desenvolvimento de um método de
imunoesterilização de cães, que poderá se estabelecer através da composição de uma vacina
ou de um soro hiperimune. Isto poderá permitir o controle reprodutivo de cães nas cidades e
estabelecer uma abordagem racional, efetiva e humanitária para o controle populacional
destes animais.
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