General and Comparative Endocrinology 158 (2008) 178–182

Contents lists available at ScienceDirect

General and Comparative Endocrinology

journal homepage: www.elsevier.com/ locate /ygcen

Effects of Castration castration and Androgen-Treatment androgen-treatment on the Expression expression of FSH- b FSH-b

and LH- b LH-b in the Threespine three-spine stickleback, Gasterosteus aculeatus – Feedback Dif ferences Mediating gasterosteus aculeatus—Feedback

differences mediating the Photoperiodic Maturation Response?photoperiodic maturation response?q

Anna Hellqvist a,*, Monika Schmitz b, Bertil Borg a

a Department of Zool­ogy, Stoc­k­hol­m Univers­ity, S-106 91 Stoc­k­hol­m, Swedenb Department of Biol­ogy, Karl­s­tad Univers­ity, S-65188 Karl­s­tad, Sweden

a r t i c l e i n f o

Artic­l­e his­tory:

Received 3 March 2008

Revised 7 July 2008

Accepted 9 July 2008

Available online 18 July 2008

Keywords­:

Stickleback

Castration

Androgen treatmentAndrogen-treatment

FSH

LH

mRNA expression

Photoperiod

0016-6480/$ - see front matter © 2008 Elsevier Inc. A

doi:10.1016/j.ygcen.2008.07.004

q Supported by the Swedish Natural Science Researc

Swedish Forestry and Agriculture Research Council (to

mous reviewers for valuable comments.

* Corresponding author. Fax: +46 08 164095.

E-mail­ addres­s­: hellqvist.anna@gmail.com (A. Hell

a b s t r a c t

In many animals, including the threespine three-spine stickleback (Gas­teros­teus­ ac­ul­eatus­), photoperiod strongly

influences reproduction. The aim of this study was to investigate if feedback mechanisms on the brain-pituitary-gonadal brain–

pituitary–gonadal axis play a role in mediating the photoperiodic response in the stickleback. To that

end, stickleback males, exposed to either non-stimulatory short photoperiod (Light:Dark (light/dark 8:16) or under

stimulatory long photoperiod (LD 16:8), were subjected to either sham-operation, castration, castration

combined with treatment with the androgens 11-ketoandrostenedione (11KA) and testosterone (T), and

the effects on levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) b -mRNA (FSH)-b mRNA were

analyzed. During breeding season the kidney of the stickleback male hypertrophies and produces a glue

used for building nests. Kidney weight and expression of both LH- b LH-b and FSH- b FSH-b were higher in sham-

operated fish kept under long than under short photoperiod. Under both photoperiods, LH- b LH-b mRNA

levels were lower in castrated males compared to sham-operated males and treatment with 11KA and T

increased expression, indicating a positive feedback. A positive feedback was also found on FSH- b FSH-b expres-

sion under long photoperiod, where castration decreased, and androgen replacement restored FSH- b FSH-b

mRNA expression. On the contrary, castration under short photoperiod instead increased FSH- b FSH-b levels

whereas treatment with 11KA and T decreased FSH- b FSH-b expression, indicating a negative feedback on FSH- b FSH-

b under these conditions. The positive feedback on FSH- b FSH-b expression under stimulatory photoperiod

may accelerate maturation, whereas the negative feedback under inhibitory photoperiod may suppress

maturation. This could be part of the mechanisms by which photoperiod controls maturation.

© 2008 Elsevier Inc. All rights reserved.

This has been observed in several studies on birds, e.g. in Japanese

quail, Coturnix japonic­a, where both plasma LH and FSH levels were

higher in castrated birds kept under stimulatory long photoperi-

ods than under shorter photoperiods (Urbanski and Follett, 1982).

These two ways in which photoperiods may control the BPG axis BPG-axis

are not mutually exclusive, both mechanisms were found to be

present in tree sparrows, Spiz­el­l­a arborea (Wilson, 1985).

Feedback effects on the BPG-axis play an important role in con-

trolling reproduction in teleost fishes. Both positive and negative

feedback effects on the BPG-axis have been reported (Crim and

Evans, 1979; Billard et al., 1977). Aromatization, the conversion of

androgens to estrogens, has been found to be involved in both the

positive and negative feedback effects in fishes (Crim et al., 1981;

de Leeuw et al., 1986). It is, however, not known whether changes

1. Intro­duc­tio­n

The photoperiod is an important factor in the control of sea-

sonal reproduction. Under stimulatory photoperiods, an increased

secretion of gonadotropins (GTHs) from the pituitary activate ver-

tebrate gonads. Previous studies have shown that photoperiod can

alter the sensitivity of steroid feedback on the secretion of GTHs

in the brain-pituitary-gonadal brain–pituitary–gonadal (BPG) axis in mammals (Turek,

1977; Rosa and Bryant, 2003). For instance, higher doses of testos-

terone (T) were needed to suppress LH and FSH plasma levels in

castrated golden hamsters, Mes­oc­ric­etus­ auratus­, under a stimula-

tory long photoperiod than under a non-stimulatory short photo-

period (Turek, 1977). However, GTH secretion patterns may also

change independently of changes in feedbacks on the BPG-axis.

ll rights reserved.

in feedback mechanisms play a role in the photoperiodic control of

reproduction in fish.

The threespine three-spine stickleback (Gas­teros­teus­ ac­ul­eatus­) has a

marked seasonal reproductive cycle in which spermatogenesis

and the development of androgen-dependent secondary sexual

characters in the male are separated in time (e.g. Borg, 1982).

h Council (to BB) B.B.) and by the

MS). M.S.). We thank two anony-

qvist).

A. Hel­l­q­vis­t et al­. / General­ and Comparative Endoc­rinol­ogy 158 (2008) 178–182 179

During the breeding season, the stickleback displays distinctive

secondary sexual characters such as the red breeding colours and

a hypertrophied kidney that secrets a glue, consisting of the andro-

gen induced protein spiggin (Jakobsson et al., 1999; Jones et al.,

2001), used for nest building. Sexual maturation in the stickleback

is strongly dependent on photoperiod. In winter, long photoperi-

ods induce maturation, while shorter photoperiods do not, even if

temperatures are high (e.g. Baggerman, 1957; Borg et al., 1987).

The aim of the present study was to investigate whether feed-

back mechanisms play a role in mediating the photoperiodic

response in the stickleback. To that end, males exposed to either

short or long photoperiod were subjected to either sham-operation

castration, castration combined with androgen replacement with

the non-aromatisable androgen 11-ketoandrostenedione (11KA)

or the aromatisable androgen testosterone (T), and the effects on

expression of LH- b LH-b and FSH- b FSH-b mRNA were analyzed.

2. Mate­rial & Me­th­o­dsMate­rials and me­th­o­ds

2.1. The animal­s­ and experimental­ protoc­ol­

The study was performed with the permission of the Stockholm

Northern Animal Experiment Ethical Board (no. (No. N184/00). Adult

non-breeding sticklebacks were caught in the Öresund on the 15-16 15–

16 December 2001. The fish were kept at 7 °C and under short day

photoperiod (LD 8:16) until the onset of the experiment.

The castration/implant experiment was started on the 4-6 4–6 Janu-

ary 2002. The fish were anaesthetized with 0.1% 2-phenoxyethanol,

c. ca. 1.5 mm long incisions were made into the abdominal cavity on

each side and the testes were excised using fine forceps. This type

of surgery drastically reduces circulating levels of T and 11-ketotes-

tosterone (11KT) in breeding sticklebacks (Mayer et al., 1990).

Sham-operated fish were treated similarly except that the testes

were mot removed. The castrated control and sham-operated fish

were implanted with empty Silastic capsules (length 5 mm, 1.2 mm

outer diameter) sealed with silicone glue. The other two castrated

groups were implanted with capsules, described as above, contain-

ing either crystalline T (KEBO) or 11KA (Sigma), which is converted

to 11KT in blood cells of sticklebacks (Mayer et al., 1990). The fish

were divided between two 700 l aquaria kept under long photope-

riod (16:8 Light:Dark) light/dark) or under short photoperiod (8:16 LD). In the

castration/replacement experiment, 16 sham-operated, 18 control

castrated, 17 11KA treated and 17 T treated fish were placed under

each regime.

Temperature was raised gradually to 20 °C over the first week to

allow adaptation. All aquaria contained brackish water (0.5% salin-

ity), which was constantly filtered and aerated, and the bottoms

were covered with sand. The fish were fed daily with frozen red

midge larvae. The fish in the castration/replacement experiment

were sampled on the 14-15 14–15 February 2002. The fish were anaesthe-

tized and weighed. Pituitaries were collected and frozen in liquid

nitrogen, and kidneys were excised and weighed (± 1(±1 mg). Visual

inspection under a dissecting microscope confirmed that the pres-

ence of capsules and the completeness of castration (absence of

testes remnants) in all fish.

2.2. Dot-bl­ot anal­ys­is­ of LH¡ b LH-b and FSH- b FSH-b mRNA expres­s­ion

Total RNA was extracted from single pituitaries using Trizol

reagent (Invitrogen) following the manufacture’s instructions, and

was then denatured and blotted on Hybond-N+, Nylon membrane

(Amersham Pharmacia Biotech) through a Hybri-blot manifold

(Life Technologies). The membranes were allowed to dry and were

then UV- crosslinked.UV-crosslinked.

The membranes were hybridized using a cRNA probe. The ca.

300 bp long antisense probes, based on stickleback LH- b LH-b and FSH- b FSH-

b (Hellqvist et al., 2004, accession nr. Accession Nos. AJ534969 and AJ534871,

respectively) were synthesized by in vitro transcription using SP6

or T7 RNA polymerase, respectively, and labelled with (a-32P) UTP

according to the Strip-EZ

TM Strip-EZ™ RNA kit (Ambion). The blots were

prehybridized for 1hour h at 65 °C, with ULTRAhyb hybridization solu-

tion (Ambion) and then incubated over night with the radioactive

probe (1x10(1 £ 10 6 cpm/ml hybridisation solution). The blots were

washed twice in 1 x 1£ SSC, 0.1% SDS at 65 °C for 15minutes min each and

then once for 15 min at 65 °C and once for 30 min at 68 °C in 0.1 x 0.1£

SSC, 0.1% SDS. The hybridization signals of the blots were counted

and analysed analyzed using a biophosphoimager (BioRad). The membranes

were stripped according to the Strip-EZ

TM Strip-EZ™ protocols and subse-

quently hybridized with a 32P-labeled stickleback 18S rRNA probe

to correct for unequal RNA loadings.

2.3. Statis­tic­s­

The data were analyzed with STATISTICA ’99 edition (StatSoft

Inc.), using two-way ANOVA for multiple comparisons and Tukey

honest significant difference (post hoc) test for comparisons

between pair of groups. Prior to analysis, the data were log trans-

formed.

3. Re­sults

3.1. Kidney s­omatic­ index

Sham-operated males displayed significantly higher KSI under

long photoperiod than under short photoperiod (p(p < 0.001, Table

1). Under long photoperiod, castrated males had significantly lower

kidney somatic index (KSI) compared to sham-operated males

(p(p < 0.001), whereas under short photoperiod, where KSI was low

in both sham-operated and castrated males, there was no signifi-

cant difference in KSI. Treatment with 11KA significantly increased

KSI in castrated males under both photoperiods (p(p < 0.001 in each

comparison), whereas treatment with T did not. There was no

effect of photoperiod on KSI in the groups treated with 11KA, T or

in castrated controls.

3.2. FSH- b FSH-b and LH- b LH-b mRNA expres­s­ion

In both experiments, expressions of both FSH- b FSH-b and LH- b LH-b in

sham-operated males were significantly higher under long photo-

period than under short photoperiod (P(p < 0.001, Figs. 1 and 2).

There was no difference in LH- b LH-b levels between the two pho-

toperiods in castrated control males and in castrated males treated

with either 11KA or T (Fig. 2). Under long photoperiod, LH- b LH-b mRNA

levels were significantly reduced in castrated males compared to

Table­ 1

Kidneysomatic indices (KSI) in sham-operated males, castrated controls and

castrated males treated with 11-ketoandrostenedion (11KA) or testosterone (T)

implants and kept under different photoperiods (16 h light/8 h dark, 8 h light/16 h

dark)*

Treatment KSI

16:8 sham 2.67 ± 0.17b

16:8 castrated 0.78 ± 0.06

16:8 11KA 2.72 ± 0.25b

16:8 T 1.21 ± 0.15

8:16 sham 0.94 ± 0.17a

8:16 castrated 0.74 ± 0.09

8:16 11KA 2.65 ± 0.18b

8:16 T 1.17 ± 0.19

* Values are mean ± SEM, n = 10–13 fish per group. a p < 0.001 compared to sham-operated fish under LD 16:8. b p < 0.001 compared to castrated to castrated controls under the same photope-

riod.

180 A. Hel­l­q­vis­t et al­. / General­ and Comparative Endoc­rinol­ogy 158 (2008) 178–182

sham-operated males (p(p < 0.001, Fig. 2), and treatment with 11KA

and T significantly increased the LH- b LH-b expression in castrated

males (P(p < 0.001 for both treatments), with T being more effective

than 11KA. Under short photoperiod there was no significant differ-

ence in expression of LH- b LH-b between sham-operated and castrated

males, but treatment with 11KA and T significantly increased the LH- b

LH-b expression in castrated males compared to the castrated con-

trols (P(p < 0.01 for 11KA treatment and Pp < 0.001 for T treatment). T

had a tendency to be more effective than 11KA.

Under long photoperiod FSH- b FSH-b expression in castrated males

was significantly suppressed compared to sham-operated ones,

and treatment with 11KA and T both significantly raised FSH- b FSH-b

levels in castrated fish (P(p < 0.001 for 11KA treatment and p < 0.01

for T treatment, Fig. 1). In contrast, under short photoperiod, cas-

trated males instead showed a dramatic rise in FSH- b FSH-b expression

compared to the sham-operated males, and FSH- b FSH-b levels were sig-

nificantly suppressed in castrated males treated with 11KA and T

(P(p < 0.001).

4. Disc­ussio­n

KSI and mRNA expression of both LH- b LH-b and FSH- b FSH-b was higher

in sham-operated fish kept under long than under short photope-

riod in both experiments. This is consistent with previous stud-

ies on effects of photoperiod in the stickleback (Borg et al., 1987;

Hellqvist et al. 2004). KSI and breeding colours were always sup-

pressed in castrated controls, this is in accordance with previous

studies (e.g. Borg et al., 1989; Mayer et al., 1990), and also demon-

strates the effectiveness of the operations.

Castrated males kept under long photoperiod had lower levels

of LH- b LH-b mRNA compared to sham-operated males, whereas under

short photoperiod levels were similarly low in both groups. The

effect under long photoperiod is consistent with reports showing

that breeding sticklebacks castrated in summer and kept under

long photoperiod and high temperature showed a drastic decrease

in LH- b LH-b levels compared to sham-operated males (Hellqvist et al.,

2003). Also, castration of the male African catfish (Cl­arias­ gariepi-

nus­) resulted in suppressed LH- b LH-b mRNA levels (Rebers et al., 1996).

Positive effects of sex steroids on LH production have also been

found in a number of previous studies on fishes, see Yaron et al.

(2003) for review. In some cases, effects appear to be largely medi-

ated via estrogen receptors. In salmonids, for instance, LH pituitary

levels are stimulated by estrogens and aromatisable androgens,

e.g. T (Crim et al., 1981), whereas the non-aromatisable 11KT is far

less effective. Treatment with T was more than a hundred times

more effective than 11KA in stimulating LH pituitary levels in cas-

trated Atlantic salmon (Borg et al., 1998). In other studies, effects

have been found that appear rather to be mediated via androgen

receptors. Treatment of intact adult male African catfish with 11KT

resulted in an increase in LH- b LH-b mRNA levels (Rebers et al. (1996).

T stimulated LH- b LH-b subunit production in European eel (Anguil­l­a

anguil­l­a) pituitaries (Huang et al., 1997). The latter positive effect

was also induced by the non-aromatisable androgens androstane-

diol and dihydrotestosterone, but not by estradiol (E2).

In the present study, treatment with 11KA and T in castrated

males increased LH- b LH-b expression under both long and short pho-

toperiod. Treatment with aromatisable T was more effective than

non-aromatisable 11KA, though the effects were in the same order

of magnitude. Thus, it appears that the positive steroid effects on LH- b

LH-b expression in the stickleback are largely mediated via andro-

gen receptors. The present investigation does not indicate where

the feedback effects are exerted; whether directly on the pituitary

level, via the GnRH system and/or via dopamine or some other sys-

tem. In some studies, positive feedback effects of steroids on LH

have been found to be exerted on the pituitary level in other fishes;

e.g. in salmonids (e.g. Gielen and Goos, 1983) and eel (Huang et al.,

1997). It is possible that this is also the case in the stickleback.

In the present study, castration suppressed FSH- b FSH-b mRNA

expression under long photoperiod, whereas androgens stimu-

lated it, indicating a positive feedback. Using quantitative electron

microscopy, Borg et al. (1989) observed that castration reduced

the amounts of dilated granulated endoplasmic reticulum in the

GTH cells in sticklebacks exposed to long photoperiod in winter,

indicating a suppressed activity of GTH cells and a physiologi-

cal positive feedback. Although effects on LH and FSH could not

be distinguished in that study, the positive feedback reported is

consistent with the positive feedbacks on the expression of both

GTHs observed under long photoperiod in the present study. Borg

et al. (1989) did not observe an increase of GTH cell granules in

Fig. 1. FSH-b mRNA expression under different photoperiods (light/dark 16:8 or LD

8:16) in sham-operated and castrated control males, and in castrated fish treated

with 11-ketoandrostenedione (11KA) or testosterone (T). Mean ± SEM shown. Let-

ters on top of bars indicate significant differences. Groups different from the simi-

larly treated group under the other photoperiod; (a) p < 0.005, (b) p < 0.001. Effects

of treatment: Groups different from castrated controls under the same photope-

riod; (c) p < 0.01; (d) p < 0.001.

Fig. 2. LH-b mRNA expression under different photoperiods (light/dark 16:8 or LD

8:16) in sham-operated and castrated males, and in castrated fish treated with 11-

ketoandrostenedione (11KA) or testosterone (T). Mean ± SEM shown. Letters on top

of bars indicate significant differences. Groups different from the similarly treated

group under the other photoperiod; (a) p < 0.001. Effects of treatment: (b) p < 0.05

compared to 11KA under the same photoperiod; (c) p < 0.01 compared to castrated

controls under the same photoperiod; (d) p < 0.001 compared to castrated controls

under the same photoperiod.

A. Hel­l­q­vis­t et al­. / General­ and Comparative Endoc­rinol­ogy 158 (2008) 178–182 181

sham-operated fish, but instead a decrease compared to castrated

fish. This indicates that the increased amounts of GTHs that the

former produced were released, rather than stored. Unfortunately,

it is at present not possible to measure circulating levels of LH and

FSH in the stickleback.

Contrarily, castrated males under short photoperiod showed

a dramatic rise in FSH- b FSH-b expression compared to the sham-oper-

ated males, indicating a negative feedback instead under this con-

dition. This is also consistent with androgens suppressing FSH- b FSH-b

mRNA expression under the short photoperiod. It cannot be said

at which level(s) the feedback effects on FSH- b FSH-b are exerted. The

complexity of the response could be consistent with more than

one level being involved.

Castration of sticklebacks kept under long photoperiod and

high temperature in summer had no effect on FSH- b FSH-b expression

(Hellqvist et al., 2003). There are few other studies on the effects of

castration on expression of FSH- b FSH-b mRNA in fishes. In coho salmon

(Onc­orhync­hus­ k­is­utc­h), castration resulted in elevated plasma

levels of FSH (Larsen and Swanson, 1997), and castration of Atlan-

tic salmon could have both positive or negative effects on plasma

and pituitary FSH levels depending on season (Borg et al., 1998;

Antonopoulou et al., 1999a, b). In the present study, FSH- b FSH-b

mRNA levels under long photoperiod were restored with 11KA

and T treatment which increased the expression in castrated

fish. In contrast, under short photoperiod, FSH- b FSH-b levels were

suppressed in castrated males treated with 11KA and T. Since

both the non-aromatisable 11KA and the aromatisable T lowered

the FSH- b FSH-b expression under short photoperiod and increased

it under long photoperiod, both the inhibitory and the stimula-

tory effects are probably exerted via an androgen receptor. In

contrast, treatment of intact coho salmon with the aromatisable

androgen T or E resulted in a reduction of transcript levels of

FSH- b FSH-b (Dickey and Swanson, 1998) indicating an effect via an

estrogen receptor. In Atlantic salmon, T had both negative and

positive effects on pituitary FSH in salmon, depending on dose

and season (Antonopoulou et al., 1999b). Atlantic salmon treated

with 11KA showed increased plasma and pituitary FSH levels

(Borg et al., 1998). Unlike the feedback control of LH which is

relatively consistent, the feedback control of FSH is complex and

changes markedly between seasons/photoperiods in both the

stickleback and salmonids. There are very few previous studies

on feedback effects under different photoperiods in fishes. Early

maturing male masu salmon, Onc­orhync­hus­ mas­ou, reached full

maturity (spermiation) earlier under LD 8:16 than under LD 16:8

in autumn (Amano et al. 2001). Castration had similar effects on

LH (GTH II) pituitary content under both photoperiods; levels

were much lower than in controls. Castration increased pituitary

FSH (GTH I) content under the non-stimulatory long, but not

under stimulatory short photoperiod after 30 days (Amano et al.

2001). It is dif­fi­cult to interpret what role, if any, this could have

in the control of maturation as there was no significant effect

of photoperiod on FSH pituitary levels in controls. T implants

increased LH pituitary levels similarly in immature salmon kept

under LD 8:16 and 16:8, but had no effect on FSH levels (Amano

et al. 2001).

In the present study, there are no indications for a feedback-

independent increase of GTH secretion by the stimulatory

photoperiods as found in birds (Urbanski & Follett). The expres-

sion of LH- b LH-b was not influenced by photoperiod in the castrated

groups, whereas the expression of FSH- b FSH-b in castrated controls was

much higher under LD 8:16 than under LD 16:8, which is the oppo-

site to what one should expect with a feedback -independent feedback-independent stim-

ulation. The possible biological significance of this is unknown.

Feedback effects on LH- b LH-b were similar under both photoperiods.

A positive feedback was also found on FSH- b FSH-b mRNA expression

under long photoperiod, where castration drastically decreased,

and androgen replacement restored, expression. In contrast, cas-

tration under short photoperiod increased FSH- b FSH-b transcript levels

drastically and treatment with 11KA and T decreased FSH- b FSH-b expres-

sion, revealing a negative feedback on FSH- b. FSH-b. Thus, the feedback

effects on FSH are very different under long and short photoperiod.

The shift between negative and positive feedback under different

photoperiods is not found in mammals, where there are instead

changes in effectiveness of negative feedbacks (Turek, 1977; Rosa

and Bryant, 2003).

FSH levels peak earlier in the season than LH levels in most

studied fishes (Suzuki et al. 1988). Also in the stickleback FSH- b FSH-b

expression reaches a peak earlier in the season than LH- b LH-b and FSH

is therefore more likely than LH to control the onset of maturation

(Hellqvist et al., 2006). The positive feedback under long photope-

riod is likely to accelerate maturation whereas the negative feed-

back under short photoperiod may suppress maturation, which

could be part of the mechanisms by which photoperiod controls

maturation in the stickleback.

Appe­ndix A. Supple­me­ntary data

Supplementary data associated with this article can be found,

in the online version, at doi:10.1016/j.ygcen.2008.07.004.

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