Allantoplacental Ultraestructure Mabuya

Allantoplacental Ultrastructure of an Andean Populationof Mabuya (Squamata, Scincidae)Martha Patricia Ramırez-Pinilla,1* Gloria De Perez,2 and J. Fernando Carreno-Escobar1

1Laboratorio de Biologıa Reproductiva de Vertebrados, Escuela de Biologıa, Universidad Industrial de Santander,Bucaramanga, Colombia2Laboratorio de Microscopıa Electronica, Departamento de Biologıa, Universidad Nacional de Colombia,Bogota, Colombia

ABSTRACT Mabuya species are highly matrotrophicviviparous lizards with Type IV epitheliochorial allan-toplacenta. The allantoplacenta of an Andean popula-tion of this genus, currently assigned to Mabuya sp.,possesses specializations related to histotrophic nutri-tion at the embryonic hemisphere (placentome, parapla-centome, and chorionic areolas), while at the abembry-onic hemisphere it has a mixed function: histotrophictransfer (absorptive plaques) and hemotrophic nutrition(gas exchange in respiratory segments). These placentalspecializations were studied using high-resolution lightmicroscopy and transmission electron microscopy, andwere compared with those found in other squamatereptiles and eutherian mammals. Cytological featuresof the placentome suggest that this is an importantregion for nutritional provision; the paraplacentomealso shows characteristics for nutrient transfer, espe-cially lipids. Chorionic areolas allow the absorption ofglandular products, as well as uterine and chorioniccellular debris produced by lysis of some cells of bothepithelia during areola formation. In the absorptiveplaques both uterine and chorionic epithelia are firmlyattached and their cellular apices exhibit electron-densegranules that could be related to autocrine and para-crine functions. The short interhemal distance found inthe respiratory segments confirms their role in gas ex-change. A common feature of all regional specializationsin the Mabuya sp. allantoplacenta is the presence oflipids in the interacting chorionic and uterine epithelia,suggesting that lipids are transferred throughout theentire embryonic chamber; placental transfer of lipidsmay be the principal fetal energy and lipid source in thisspecies. In spite of this feature, each one of the special-ized areas of the allantoplacenta has different featuressuggesting particular functions in the transfer of nutri-ents (as ions, lipids, proteins, amino acids, sugar, water,and gases), and in the possible synthesis of hormonesand proteins. The placental complexity observed in thisspecies of Mabuya is greater than in any other reptile,and resembles that of eutherian mammals: Each one ofthese specializations of the placental membranes inMabuya sp. is similar to those found among differenteutherian mammals, indicating a very impressive evo-lutionary convergence at the histological and cytologicallevels between both clades. However, no eutherianmammal species simultaneously displays all of thesespecializations in the embryonic chamber as doesMabuya sp. J. Morphol. 267:1227–1247, 2006.© 2006 Wiley-Liss, Inc.

KEY WORDS: fetal membranes; Mabuya; matrotrophy;placenta; reptiles; viviparity

The allantoplacenta or chorioallantoic placenta isthe term used for placental structures in which thefetal contribution consists of the chorion, allantois,and sometimes amnion, but does not include anypart of the yolk-sac endoderm (Yaron, 1985). Weekes(1935) described three types of chorioallantoic pla-centas in lizards based on cytological and histologi-cal features; moreover, the presence of specializedstructures for maternal–fetal nutrient and gasexchange in the Neotropical Mabuya heathi led tothe recognition of a new placental morphotype, the“Type IV” allantoplacenta (Blackburn and Vitt,2002).

The lizard family Scincidae (Squamata) has thegreatest diversity in placental morphologies amongamniotes (Stewart and Thompson, 2000). Amongthem, Mabuya species have the most complex andspecialized squamate allantoplacentae, the Type IVallantoplacenta. This clade shows evolutionary con-vergence with the eutherian mammals for fetal nu-trition (Blackburn et al., 1984; Blackburn and Vitt,2002), and other reproductive specializations suchas a long gestation period, ovulation of tiny ova(microlecithic), and placental provision of virtuallyall of the nutrients for development (Blackburn etal., 1984). Flemming and Blackburn (2003) sug-gested that these features are widespread, if notuniversal in New World Mabuya, including M. agi-lis, M. brachypoda, M. caissara, M. frenata, M.

Contract grant sponsor: COLCIENCIAS – UIS; Contract grantnumber: 1102-05-13556.

*Correspondence to: Martha Patricia Ramırez-Pinilla, Laboratoriode Biologıa Reproductiva de Vertebrados, Universidad Industrial deSantander, A.A. 678, Bucaramanga, Colombia.E-mail: [email protected]

Published online 18 July 2006 inWiley InterScience (www.interscience.wiley.com)DOI: 10.1002/jmor.10471

JOURNAL OF MORPHOLOGY 267:1227–1247 (2006)

© 2006 WILEY-LISS, INC.

heathi, M. nigropunctata, M. macrorhyncha, and M.mabouya; however, placental morphology has beenstudied for only three of these species (M. heathi:Blackburn et al., 1984; Blackburn and Vitt, 2002; M.nigropunctata: Vitt and Blackburn, 1991; and anAndean population temporarily assigned to M.mabouya: Jerez and Ramırez-Pinilla, 2001, 2003).Recently, Mausfeld et al. (2002) divided Mabuyainto four different genera, keeping the nameMabuya only for this American clade, which seemsto constitute a monophyletic group (Greer et al.,1999; Mausfeld et al., 2002; Carranza and Arnold,2003). Interestingly, the African skinks Eumecia an-chietae and Trachylepis ivensii share reproductivespecializations with New World Mabuya such asovulation of microlecithic ova, intimate apposition offetal and maternal tissues after breakdown of theegg shell, development of an absorptive chorioallan-tois, and development of an unusual yolk sac thatlacks an isolated yolk mass (Flemming and Branch,2001; Flemming and Blackburn, 2003).

The studies of placental morphology in viviparousreptiles have been based almost exclusively on lightmicroscopy. Ultrastructural information is neces-sary to solve the anatomical and developmental am-biguities that complicate attempts to recognizestructural correlates of functional attributes (Black-burn et al., 2002; Stewart and Brasch, 2003). Theultrastructure of allantoplacentae in Squamata hasbeen studied in species with relatively simple allan-toplacentas (Thamnophys sirtalis: Hoffman, 1970;Virginia striatula: Stewart and Brasch, 2003; Eu-lamprus tympanum: Hosie et al., 2003; Thamnophisordinoides, T. sirtalis, T. radix: Blackburn et al.,2002; Blackburn and Lorenz, 2003a) and complexallantoplacentas (Pseudemoia entrecasteauxii: Ad-ams et al., 2005); but not in placentotrophic Mabuya,in which are found different and highly specializedstructures for placental exchange.

In the allantoplacenta of an Andean population ofMabuya sp., Jerez and Ramırez-Pinilla (2001) de-scribed several placental specializations related tohemotrophic and histotrophic transport in both themesometrial hemisphere (placentome, paraplacen-tome, and chorionic areolas) and at the antimesome-trial hemisphere (absorptive plaques and respira-tory segments). The present study describes, usinghigh-resolution light microscopy and transmissionelectron microscopy (TEM), the cytological featuresof these specializations in late pregnant femalesfrom the same population of Mabuya. These featuresare compared with those known for other squamatereptiles and eutherian mammals.

MATERIALS AND METHODS

In past studies, the Andean populations of Mabuya from Co-lombia were temporarily identified as Mabuya mabouya (Jerezand Ramırez-Pinilla, 2001, 2003; Ramırez-Pinilla et al., 2002;Gomez and Ramırez-Pinilla, 2004). However, according to

Miralles (2005) M. mabouya Lacepede (1788) is a species re-stricted to the Lesser Antilles. Also, in their revision of the Ven-ezuelan Mabuya, Miralles et al. (2005a,b) concluded that M.mabouya is not present in the Andean regions and that Andeanspecies of Mabuya cannot be identified to any known species.Given that the external morphology is so conservative in thegenus, it is necessary that a study based on detailed morpholog-ical analyses and molecular data be performed to resolve thetaxonomic status of these populations. We refer to our studiedpopulation as Mabuya sp.

Mabuya sp. pregnant females were obtained by manual collec-tion in the Inspeccion de Policıa de Guadualito, Municipio deYacopı, Departamento de Cundinamarca, Colombia (N: 05° 37�,W: 74° 18�, 840 m altitude). The females were the same used inpast studies (Jerez and Ramırez-Pinilla, 2001; Ramırez-Pinilla etal., 2002). Seven pregnant females with embryos of Stages 39 and40 (following the table of Dufaure and Hubert, 1961) were stud-ied. For the study of absorptive plaques we also included embry-onic chambers of one female in embryonic Stage 31. The incuba-tory chambers were dissected to obtain small pieces of placentaltissues that were fixed in 2.5% glutaraldehyde in Millonig buffer(pH 7.3) and postfixed in osmium tetroxide (1%). The tissues wereembedded in Epon-Araldite and sectioned at 0.1–2 �m for semi-thin sections and 60 nm for ultrathin section observation in aLKB 4801 ultramicrotome. Semithin sections were stained withToluidine blue; the high-resolution photomicrographs were takenusing a Nikon microscope. Ultrathin sections were contrastedwith uranyl acetate and lead citrate. All the allantoplacentalstructures (placentome, paraplacentome, chorionic areolas, ab-sorptive plaques, and respiratory segments) were observed, de-scribed, and photographed using a Jeol JEM transmission elec-tron microscope.

To describe the histology and ultrastructure features of theallantoplacenta of this population of Mabuya, we followed theterminology employed by Mossman (1987), Blackburn (Blackburn1993, 2000), Stewart and Thompson (2000), and Jerez andRamırez-Pinilla (Jerez and Ramırez-Pinilla 2001, 2003).

RESULTSPlacentome

The placentome is an area located dorsal to theembryo. Its distinctive feature is the very stronginterdigitation between the villous folds of the uter-ine endometrium and chorioallantois. The interact-ing epithelia consist of a uterine syncytium and ahypertrophied chorionic epithelium with giant andinterstitial cells. The interdigitation between theuterine villous folds and chorioallantoic invagina-tions follows a radial pattern emerging from theembryonic pole.

The chorionic and uterine epithelia form a widemicrovillar band (�7 �m) (Fig. 1a) that consists ofstraight, long microvilli protruding from both epi-thelial surfaces. They are closely interdigitated,with no significant gaps. The uterine epithelial syn-cytium has a palely staining cytoplasm with finereticular material, small lipid droplets, and smallclear vesicles; the nuclei are spherical, with onenucleolus and without heterochromatic material.The chorion has a basal layer of squamous cells andan external layer consisting of two cellular types:very thin cells (interstitial cells), and large broadbinuclear cells (giant cells) (Fig. 1b). The giant cellsexhibit a homogeneous darkly stained cytoplasmthat contains small vesicles, abundant lysosomes,

1228 M.P. RAMIREZ-PINILLA ET AL.

Journal of Morphology DOI 10.1002/jmor

and lipid droplets in the lateral and basal regions;their nuclei are ovoid, with one or two prominentnucleoli. Occasionally, thin cells with dense cyto-

plasm are found inserted into the apex of the uterinesyncytium. Their ultrastructure is similar to that ofthe interstitial cells and they can extend as far asthe uterine epithelial basement membrane (Fig. 1a).

At the ultrastructural level, the chorionic–uterineinterface is occupied by a broad microvillar bandconsisting of microvilli from the uterine epitheliuminterdigitated closely with microvilli from giant cellsof the chorionic epithelium. Between the bases of theuterine microvilli are inpocketings of the plasma-lemma that bud off coated and uncoated vesicles intothe uterine epithelial cytoplasm (Fig. 2). On thechorionic side, in contrast, there are narrow tubularextensions of the plasmalemma into the apical cyto-plasm (Fig. 3). At lower magnification, the chorioniccytoplasm is stained homogeneously and its moder-ate electron density allows the observation of nu-merous small mitochondria and lysosomal bodieswith heterogeneous content, some of which may besecondary lysosomes (Fig. 4). Higher magnificationreveals an extensive rough endoplasmic reticulum(RER) with dilated cisternae that contain storedproducts (Fig. 5). Giant and interstitial cells havehighly folded basolateral membranes with multipleprojections that define irregular spaces loosely in-terlocked with each other and close to lymphatic andblood vessels. Interstitial cells have a very slendershape, with RER surrounding the nuclei, mitochon-dria, and some lysosomes. The nuclei are ellipsoid,heterochromatic, with prominent nucleoli (Fig. 4).

The ultrastructural investigation confirmed theexistence of a uterine syncytium, since no intercel-lular membranes with tight junctions around theirapices were found (Fig. 6a). The cytoplasm is rich insmall mitochondria (0.19 � 0.04 �m equator width),polyribosomes, and lysosomes. The cytoplasm con-tains cisternae of rough and smooth endoplasmicreticula around the nucleus. At the basal region,RER profiles (Fig. 6b) and empty vesicles predomi-nate; some large lipid droplets are also present inthe cytoplasm. The uterine epithelium is underlainby a basement membrane below, which is a thinlayer of connective tissue in which capillaries andother blood vessels are abundant and very close tothe epithelium. Indications of localized invasion ofthe uterine syncytium by chorionic cells were ob-served occasionally in the ultrathin sections (Fig.6c). The invader chorionic cell has a small nucleusand a cytoplasm with RER cisternae and electron-dense granules (Fig. 6d).

Paraplacentome

The paraplacentome is a narrow zone, peripheralto the placentome, at the dorsal hemisphere of theincubatory chamber; it is seen macroscopically as anarrow yellow band ventral to the placentome. Theuterine endometrium and chorioallantois are notfolded in this region. Also, there are no apical uter-ine or chorionic microvilli, like those observed in the

Fig. 1. Mabuya sp. Placentome. Toluidine blue. Stage 40. a: Theapical surfaces of the chorionic and the uterine epithelia havevery thin and long microvilli (mv) that interdigitate. The uterineepithelium is a syncytium (us) with large euchromatic nuclei (n).A chorionic invasive cell (ic) extends into the syncytium. Giant(gi) and interstitial (i) cells constitute the chorionic epithelium. lp,a muscular band underlie the lamina propria. b: Section throughthe base of a villous fold of the chorionic epithelium. The very thininterstitial cells (i) surround the giant cells (gi). Note the well-developed allantoic capillaries, which vascularize the chorionicepithelium (v).

1229PLACENTAL ULTRASTRUCTURE OF MABUYA


Fig. 2. Apical surface of the uterine epithelium in Mabuya sp. placentome. TEM. Stage 40. a: The uterine syncytium (us) exhibitsa microvillar surface (mv) with very long, straight microvilli. ce, chorionic epithelium; n, nucleus. b: Detail of the apical surface of theuterine syncytium. At the bottom of the microvilli are coated vesicles (arrows) and membrane dilations (arrowhead). L, lysosomes; mi,mitochondria; mv, microvilli.

Fig. 3. Apical surfaces of chorionic epithelium (ce) and uterine epithelial syncytium (us) of the Mabuya sp. placentome. TEM. Stage40. a: The microvillar band (mv) in transverse section. Note the electron-dense cytoplasm of the chorionic epithelium in contrast withthe electron-lucent cytoplasm of the uterine syncytium, and the lateral processes between chorionic cells. b: Detail of the chorionicepithelial apex with long microvilli (mv); note the canaliculus in the interdigitation with uterine microvilli (arrowhead). L, lysosome.



placentome. Furthermore, the uterine syncytium isreplaced abruptly by a columnar uterine epitheliumwith well-defined tight junctions at cell apices (Fig.7). The cytoplasm of uterine epithelial cells is palerthan those of the chorion; it contains numerous lipiddroplets that almost fill the whole cell volume andfuse to form large droplets (Fig. 8). Alveolar uterineglands can be found in the lamina propria; glandducts or secretion granules could not be seen, al-though glands contain densely stained material attheir lumen. The external layer of the chorionic ep-ithelium is similar to that of the placentome, com-posed of giant and interstitial cells.

At the ultrastructural level, the paraplacentomaluterine and chorionic epithelia adjacent to the pla-centome display irregular apices with interdigitat-ing convoluted cytoplasm projections (Fig. 9); at theparaplacentomal region these cytoplasmic projec-tions disappear and the apical surfaces of both uter-ine and chorionic epithelia do not interdigitate. Thechorionic–uterine interface is very thin and no ma-terial was observed between the epithelia. The uter-ine epithelial cells have well-defined basolateral

membranes and nuclei with heterochromatin that iseither dispersed or aggregated close to the nuclearenvelope. Numerous small mitochondria and vesi-cles of heterogeneous sizes with dispersed fine ma-terial are present in the cytoplasm and short cister-nae of RER are also common (Fig. 10).

The cytoplasm of chorionic cells has numerousvesicles surrounded by membranes that correspondto lysosomal bodies that contain residual material;some of them are completely filled with this material(Fig. 9). At the basal region, the lysosomal bodies arefewer than in the apex, and the cytoplasm showsdilated cisterns of RER (Fig. 11). Lipid droplets areabundant in the basolateral areas and there arehighly convoluted membrane projections betweencells. Also in this region, allantoic lymphatic anduterine blood vessels are in close proximity to oneanother (Fig. 12).

Chorionic Areolas

In the studied population of Mabuya sp. the cho-rionic areolas are found at the dorsal hemisphere,

Fig. 4. Chorionic epithelium of the Mabuya sp. placentome. TEM. Stage 40. The electron-dense chorionic epithelium shows highlyfolded basolateral membranes that form extended cytoplasmic processes (p), and large intercellular spaces between giant cells (gi) andinterstitial cells. n, nucleus of interstitial cell; nu, nucleolus of interstitial cell; pL, primary lysosomes; sL, secondary lysosomes.

Fig. 5. Chorionic epithelium of the Mabuya sp. placentome. Stage 40. The cytoplasm of the giant chorionic cells (gi) has an extensivesystem of rough endoplasmic reticulum (rer). Note the intercellular spaces (s) formed by the interdigitation of cytoplasmic processes(p) between giant cells and the adjacent interstitial cells (i). n, nucleus of interstitial cell.



out of the paraplacentomal region; few of them reachthe equatorial region of the incubatory chamber.They can be recognized only at the light-microscopiclevel; macroscopically, they do not differ from ab-sorptive plaques. Areola formation is initiated atStage 39 of development. The chorion proliferatestoward the allantois, forming a pluristratified sac-cular structure and the uterine epithelium exhibitsa slight evagination (Fig. 13). Lipids accumulateinside chorionic cells, forming large lipid dropletsthat make the observation of cell borders difficult.The fold of the uterine epithelium is progressivelyextended toward the chorion (Fig. 14a), and the are-

olar cavity is formed by gland secretion and cellulardegeneration of apical cells of the chorionic epithe-lium (Fig. 14b). The areolar cavity is invaded byglandular secretions and cytoplasmic materials(vacuoles, granules, and membranous debris) re-leased from the endometrial and chorionic cells. En-dometrial glands open via short ducts to the cavity ofthe areola, and although a mixed secretion is ob-served in the glandular lumen of sections stainedwith Toluidine blue (small granules and condensedsubstance), intracellular granules cannot be seen(Fig. 15a). The glandular content stored in the lu-men is released in the areolar concavity (Fig. 15b).

Fig. 6. Uterine epithelium of Mabuya sp. placentome. TEM. Stage 40. a: The uterine epithelium forms a syncytium. Two adjacentnuclei (n) are present in a clear vesicular cytoplasm with abundant mitochondria (m). b: The basal region of the uterine syncytiumexhibits well-developed rough endoplasmic reticulum (rer), and is in close apposition to uterine blood vessels (v). n, nucleus ofendothelial cell. c: Part of a chorionic invasive cell (ic) penetrating the uterine syncytium (us). The cytoplasm of the chorionic invasivecell exhibits numerous electron-dense organelles (*), and rough endoplasmic reticulum (rer) running parallel to its apical region. Thecytoplasm of the uterine syncytium is highly vesicular (L) with several mitochondria. n, nucleus. d: Detail of the chorionic invasive cell(ic).



Abundant lymphatic and blood vessels underlieuterine and chorionic epithelia.

Ultrastructurally, some luminal uterine epithelialcells have an intact apical membrane. However, inmost of them the apex is broken and massively ex-pels cytoplasmic material in dissolution and lipiddroplets of all sizes (Fig. 16). This material dispersesinto the areolar cavity, in front of the chorionic epi-thelium. Uterine epithelial cells are joined by junc-tional complexes. The apical portion of the cyto-plasm exhibits small, smooth vesicles and RERcisternae. Abundant lipid droplets are distributedthroughout the cytoplasm. Plasma membranes ofneighboring cells are very close and follow a straighttrajectory. Blood vessels are in tight contact to theepithelial basal lamina. The chorionic epitheliumconsists mainly of giant cells with irregular apicesthat exhibit short, thin projections to which is ad-hered abundant interface material. The nucleus iseuchromatic, with small heterochromatin aggre-gates (Fig. 17). The apical region of the cell containslarge lipid droplets, abundant spherical and irregu-lar vesicles with granular fine content, and mem-brane debris. Loosely interlocked intercellular con-voluted membranes define the neighboring cellularspaces.

Uterine glands have columnar cells with shortapical microvilli (Fig. 15c). A few electron-densegranules of heterogeneous form and size are distrib-uted in the apical region. Some isolated granules arealso present in the basal region. Some cells releasesecretory granules into the lumen. Because both thegranules and stored secretion products in the lumenhave similar electron density, a process of granulefusion apparently occurs in the glandular lumen. Atlow magnification, conspicuous junctional complexesare observed and cytoplasmic membranes run par-allel without an appreciable space between them. Insome cells, basolateral sinuous membranes sur-round vesicles located laterally. These vesicles haveirregular form and size, and a finely granular dis-persed and membranous content. The cytoplasm dis-plays elongated mitochondria and abundant RER,especially in the basal region. The uterine lumen atthe areolar concavity is filled by aggregations ofcytoplasmic granular material, cellular debris asmembranous vesicles, mitochondria, lysosomes,lipid droplets, and glandular secretion products (Fig.18).

Absorptive Plaques

The absorptive plaques are located at the abem-bryonic hemisphere of the incubation chamber, ven-tral to the chorionic areolas area. Approximately50–60 absorptive plaques were estimated per incu-batory chamber. Hypertrophied chorionic and uter-ine epithelia are in close association (Figs. 19, 20).The chorion has two layers. Both giant and intersti-tial cells occur in the very tall external layer. The

Fig. 7. The placentome–paraplacentome transition is abrupt(arrow). In the paraplacentome the microvillar band (mv) is lostand the uterine epithelium consists of columnar secretory cells(ue), while the chorionic epithelium (ce) is similar to that found inthe placentome. Note the considerable endometrial vasculariza-tion (v), and especially the blood vessels that penetrate the basalregion of the uterine syncytium (us). A, allantois; n, nucleus; um,thin band of uterine myometrium. Toluidine blue. Stage 40.

Fig. 8. Mabuya sp. paraplacentome. Toluidine blue. Stage40.Chorionic (ce) and uterine epithelia (ue) show numerous lipiddroplets (li). The allantois (A) is vascularized and wide. lv, lym-phatic vessel; um, uterine muscle, v, allantoic blood vessel.



cytoplasm above the nuclei of chorionic cells stainsdeeply. One noteworthy feature of the giant cells isthe presence of one to three large lipid dropletsbeneath the nucleus. Lipid droplets inside chorioniccells in other cell regions are numerous and small.The luminal uterine epithelial cells have basal nu-clei, some of which are binucleated. Small lipid drop-lets practically fill the cytoplasm. Some are fused,forming great lipid lagoons.

At the ultrastructural level, the chorionic anduterine epithelial apices are closely adherent (Fig.

21a), displaying different junctional complexes. Incertain areas the cells appear to be joined by gapjunctions, while others may exhibit junctional com-plexes similar to zonula adherentes and desmo-somes (Fig. 21b). A very close association is presentbetween cytoplasmic short interdigitating projec-tions of both epithelia because focal joint points be-tween both apical membranes are found. The uter-ine apical cytoplasm is rich in lipid droplets andorganelles (Fig. 22). It contains small electron-densegranules that are difficult to observe in semithin

Fig. 9. Mabuya sp. apical surfacesof chorionic and uterine epithelia ofthe placentomal–paraplacentomallimit. The contact surface betweenuterine (ue) and chorionic epithelia(ce) is strongly attached by convo-luted cytoplasmic processes that in-terdigitate (p). L, lysosomes; n, nu-clei of uterine epithelial cells. TEM.Stage 40.

Fig. 10. Paraplacentomal uter-ine epithelium cells of Mabuya sp.Stage 40. TEM. The cells have evi-dent intercellular membranes thatfollow a parallel course and displaytight junctional complexes betweenthem (arrows). The apical region ofthe cells is characterized by the pres-ence of numerous electron-densegranules (*). The basal region ex-hibits rough endoplasmic reticulum(rer). The nuclei (n) are mostly eu-chromatic with some heterochro-matic regions.



sections (see Fig. 21b). As in other regions, neigh-boring uterine cells are joined by complex junctionalstructures in apical, lateral, and basolateral regions.Adjacent membranes run parallel without visible

spaces between them. The apical cytoplasm of giantchorionic cells contains lysosomes, electron-densegranules, dilated cisternae of RER, and abundantsmooth ER (Fig. 23). The lateral region has shortprojections that interlock with neighboring cells. De-pending on the level, the adjacent cells can be giantor interstitial, because interstitial cells are shorterand do not reach the apical portion of the chorionicepithelium. Nuclei of the giant cells are located cen-trally. Interstitial cells are very thin and elongatedand their cytoplasm displays lysosomes, mitochon-dria, and basal lipid droplets. The euchromatic nu-clei have an elongated shape. Squamous cells of thechorionic basal layer contain numerous small lipiddroplets and are associated with other cells throughcytoplasmic projections. Abundant allantoic capil-laries are adjacent to this layer of chorionic cells(Fig. 24).

Respiratory Segments

The respiratory segments of the allantoplacentaoccur outside the polar placentomal–paraplacentomalregion of the incubatory chamber. A profuse subep-ithelial vascularization in the intimately associatedchorionic and uterine epithelia characterizes the re-spiratory segments (Fig. 25). The height of the cho-rionic and uterine epithelia decreases progressivelyduring the last stages of development. This causesthe uterine and chorionic blood vessels to approachso closely that the interhemal distance is only �6–9�m (Fig. 26). The allantois is vascularized by nu-merous capillaries that contact the basal lamina ofthe chorionic squamous cells. Lymphatic vessels arepresent in the external portion of the allantois orbetween blood vessels. In the uterus lymphatic ves-sels underlie the epithelium and exhibit numerouslymphocytes and neutrophils. Active uterine glandsare also present; however, gland ducts and openingswere not observed.

Ultrastructurally, the chorionic epithelium is lowand resembles the endothelium of blood vessels. Thechorionic apices display very small projections, leav-ing a very small lumen between the epithelia (Fig.25). The uterine epithelial apex has no cytoplasmicprojections. Adjacent lateral and basolateral mem-branes are in close contact, and have a parallel andsome what sinuous course. The apical region showsjunctional complexes. The large nuclei of the uterineepithelial cells have an irregular shape and smallnucleoli. The cytoplasm exhibits short cisternae ofrough and smooth endoplasmic reticulum, dispersedsmall mitochondria, and small lipid droplets. At lateStage 40, the uterine epithelium is cuboidal to flat.Nuclei with small nucleoli occupy a large portion ofcytoplasm volume and RER is present. Lipid drop-lets are fewer than in previous stages. In the basalregion the uterine epithelial cells broaden, increas-ing the surface between them and the endothelium

Fig. 11. Basal region of chorionic epithelium at the parapla-centome of Mabuya sp. Stage 40. TEM. Dilated cisterns of roughendoplasmic reticulum (rer) present in the giant cells (gi) aresimilar to those found in the placentome. Also, as in the placen-tome, the chorionic epithelium exhibits highly folded basolateralmembranes forming extended cytoplasmic processes (p). i, inter-stitial cells.

Fig. 12. Basal region of chorionic epithelium at the parapla-centome of Mabuya sp. Stage 40. TEM. Giant (gi) and interstitialcells are close to lymphatic vessels (lv). li, lipid droplets in thegiant cell cytoplasm. Lipids have been extracted by the dehydra-tion procedure. n, nuclei of interstitial cells; p, cytoplasmic pro-cess between adjacent chorionic cells.



of blood vessels. However, there are always basallaminae between epithelial and endothelial cells.

DISCUSSION

The descriptions of allantoplacentas for other spe-cies of Mabuya have been restricted to the embry-onic hemisphere, where placentome, paraplacen-tome, and chorionic areolas are located (Vitt andBlackburn, 1991; Blackburn and Vitt, 1992; Black-burn, 1993; M. heathi: Blackburn and Vitt, 2002; M.macrorhyncha and M. nigropunctata: Flemming andBlackburn, 2003). These studies have not reportedchorionic specializations for histotrophic exchangein the antimesometrial hemisphere. However, theabsorptive plaques are present in our studied popu-lation, in other Colombian and Venezuelan popula-tions (Jerez and Ramırez-Pinilla, 2001; pers. obs.),and in Central American M. brachypoda (Villagran-Santacruz et al., 1994). Therefore, it is possible thatthey are present in all species with Type IV allan-toplacentas, suggesting that these characters aresynapomorphies for the American lineage. Thus, thecombination of all the allantoplacental specializa-tions (placentome, paraplacentome, chorionic areo-lae, absorptive plaques, and respiratory segments)distinguishes this clade from all viviparous squa-mates. Moreover, these morphological specializa-tions that allow nutrient exchange in the Mabuyaallantoplacenta are similar to those present amongplacentas of eutherian mammals.

The placentome has been described in ruminantmammals (Hoffman and Wooding, 1993; Wooding etal., 1996, 1997). The chorionic areolas are present incamelids (Lama pacos: Olivera et al., 2003a; Cam-elus dromedarius: Abd-Elnaeim et al., 2003), inequids (Wooding et al., 2000, 2001), and in pigs(Bielanska-Osuchowska and Kunska, 1995). Ab-sorptive plaques are structures associated withmammal groups that possess villous epitheliochorialand endotheliochorial labyrinthine placentas (Moss-man, 1987). The respiratory segments of Mabuya sp.

Fig. 13. Chorionic areola at Stage 39 of gestation in Mabuyasp. During the process of formation of the chorionic areolas,concave pluristratified saccular structures are formed in the cho-rionic epithelium (ec). Abundant lipid droplets (li) accumulate inthe cytoplasm and among chorionic cells. A, allantois; n, nucleus;v, blood vessel. Toluidine blue.

Fig. 14. Typical dome-shaped areolas in Mabuya sp. with thecavity filled by areolar material (am). Stage 40. Toluidine blue.a: A uterine fold is projected to the chorionic areola. The areolarconcavity is formed by degeneration of external layers of thechorionic epithelium (ce) and uterine epithelial cells of the pro-jecting fold (ue). In the lamina propria (lp) note a uterine gland(gl) with a large duct (arrow) oriented to the areolar cavity. b: Thebasal layers of the chorionic epithelium (ce) in the areolar cavitycontact the areolar interface material (am) formed by glandularand cytoplasmic materials. ue, uterine epithelium; v, allantoicblood vessels.



Fig. 15. Mabuya sp. areolar glands. Stage 40. a: The active uterine glands (gl) exhibit a finely granular and amorphous material(gs), which is secreted into the areolar cavity. am, areolar interface material; ue, uterine epithelium. Toluidine blue. b: Glandularmaterial is secreted into the glandular lumen (arrow). A, allantois; ce, chorionic epithelium; gl, uterine gland; ue, uterine epithelium;v, allantoic blood vessels. Toluidine blue. c: A uterine gland that secretes to the areola. TEM. Glandular cells release theirelectron-dense granular contents (arrows) by exocytosis into the glandular lumen. The cytoplasm of glandular cells has a well-developed rough endoplasmic reticulum (rer), several mitochondria (mi), and apical electron-dense secretory granules. The nuclei (n)are euchromatic with small heterochromatin aggregates.



correspond to gas exchange areas widespread inmammal groups (Enders and Blakenship, 1999).However, the variety of combined specializations ob-

served in the Mabuya sp. incubatory chamber hasnot been described in any mammal species. Sincethis is a highly matrotrophic species, we suggest

Fig. 16. Uterine epithelium (ue)in front of the areola concavity ofMabuya sp. Stage 40. TEM. The lu-minal epithelium consists of colum-nar secretory cells with numerouslipid droplets (li) and lysosomes (L).Some cells lose their apical surfacesand partially expulse their cyto-plasm into the areolar cavity formingthe areolar interface material (am).n, nucleus; v, uterine blood vessel.

Fig. 17. Basal layer of chorionicepithelium in the Mabuya sp. chori-onic areola. Stage 40. TEM. The api-ces of giant cells (a) contain abun-dant lipid droplets (li) and electron-dense granules (arrows) and are incontact with the areolar materials(am); cytoplasmic processes (p) arepresent in the wide lacunar spacesbetween adjacent cells. gi, giant cell;n, nucleus.

Fig. 18. Apical region of the uter-ine epithelium (ue) in front of theinterface material in the cavity ofthe chorionic areola. Stage 40. TEM.Abundant remnants of cytoplasmicmaterial, including membranousvesicles, lipids (li), electron-densegranules (arrows) and fibers composethe interface material. ue, uterineepithelium.



that each of these allantoplacental specializationsshould have specific functions in nutrient exchange.

Placentome

The observed morphological features in theMabuya sp. placentome suggest that this is a veryimportant region for nutrient transfer. This special-ized region is observed from earlier developmentalstages (gastrula stages) with a morphology similarto the absorptive plaques (Jerez and Ramırez-Pinilla, 2003), suggesting an active and early nutri-ent transfer of molecules. However, its highest spe-

cialized features, such as the interdigitant uterineand chorionic mucosal folds bearing long and thinmicrovilli, syncytial uterine epithelium, invasivechorionic cells in the uterine syncytium, and fea-tures of high metabolic activity, are present onlyduring the final stages of development (fetal growthstages).

The placentome of this population of Mabuya wasconsidered synepitheliochorial due to the formationof a uterine epithelial syncytium in front of thechorionic epithelium (Jerez and Ramırez-Pinilla,2001). Ruminant mammals exhibit a cotyledonaryepithelichorial placenta, which shows five or eightplacentomes in the embryonic chamber (Mossman,1987); each placentome is conformed by a syncytium(Wooding et al., 1997). This syncytium originatesthrough the fusion of endometrial and binucleatetrophoblastic cells, forming a hybrid syncytium. Be-cause of its hybrid nature, this type of ruminantplacenta is termed synepitheliochorial (Hoffmanand Wooding, 1993; Wooding et al., 1997). The pres-ence in the placentome of Mabuya sp. of some inva-sive chorionic cells protruding into the uterine syn-cytium could suggest a chorionic invasion thatresults in close proximity to the endothelial endome-trium. However, cellular fusion between chorionicand syncytial endometrial cells was not observed,and thus a hybrid nature in Mabuya sp. syncytiumsuch as occurs in ruminant mammals is not sug-gested. Chorionic invasion of the uterine epitheliummight be a common feature in highly matrotrophicsquamates because the allantoplacenta of the highlymatrotrophic African lizard Trachylepis ivensii con-tains giant chorionic cells that directly contact uter-ine endothelial cells to form the only known exampleof endothelial placentation in reptiles (Flemmingand Blackburn, 2005). However, both observationsfor squamates must be better studied and analyzed.

Mabuya sp. giant and interstitial placentomalchorionic cells are binucleated, similar to those ob-served in the mammalian trophoblast, where thesecells have diploid nuclei and play an important rolein the production of lactogen and steroids (Hoffmanand Wooding, 1993). In viviparous reptiles such asChalcides chalcides (with Type III allantoplacenta),progesterone production was detected in the mater-nal component of the placentome, after corpus lu-teum degeneration in late pregnancy (Guarino et al.,1998). The role of uterine and chorionic cells in hor-mone production must be evaluated in viviparouslizards, and particularly in species with Type IVallantoplacentation because it has been observedthat corpora lutea degenerate very early in gestationin this population of Mabuya (Gomez and Ramırez-Pinilla, 2004).

The Mabuya sp. placentome exhibits a consider-able increase in the exchange surface betweenmother and fetus. Two features increase theMabuya sp. placentomal surface area: 1) the radialdevelopment of branching endometrial folds from

Fig. 19. Absorptive plaque at 40 stage of development inMabuya sp. The very tall cells of the chorionic epithelium (ce) andthe hypertrophied uterine epithelium (ue) are in close associa-tion. Abundant lipid droplets are present in the uterine epithe-lium, whereas in the chorionic epithelium they accumulate aslarge lipid droplets in the basal portion of the cells (li) bellow thenuclei (n). A, allantois; v, allantoic blood vessels. Toluidine blue.

Fig. 20. Absorptive plaque at Stage 31 of Mabuya sp. At thisstage the epithelia are not as hypertrophied as in Stage 40, andthe lipids in the uterine epithelium are not so abundant. How-ever, in the chorion the lipids accumulate in the basal portion,forming large lipid droplets (li). A, allantois; ce, chorionic epithe-lium, ue, uterine epithelium; v, allantoic blood vessels. Toluidineblue.



Fig. 21. Apical surfaces of the chorionic and uterine epithelia of the absorptive plaque of Mabuya sp. TEM. Stage 31. a: The apicesof the two epithelia are apposed in such a way that there is no space between them. Both have thin, irregular projections thatinterdigitate and are reinforced by junction points (f). Electron-dense granules (arrows) of different sizes are present in the uterine (ue)and chorionic (ce) epithelial apices. The uterine epithelium contains abundant lipid droplets (li). b: Detail of the interdigitationbetween uterine (ue) and chorionic (ce) cytoplasmic apical projections. Both epithelia are tightly joined by focal junction points (f). L,lysosomes; li, lipids.



the incubatory embryonic pole that contact corre-sponding chorionic folds, and 2) the presence of long,thin apical microvilli at the surfaces of both epithe-lia, forming a tightly joined band of interlocked mi-crovilli (interdigitant brush border) between mater-nal and fetal tissues. Chorionic and uterine

epithelial interdigitation is also present in the dif-fuse epitheliochorial placentas of Suiformes, Tragu-lidae, and Camelidae eutherian groups (Mossman,1987). Also, it was described in the placentome oflizards such as Pseudemoia entrecasteauxii (with aType III allantoplacenta; Adams et al., 2005), and

Fig. 22. Uterine epithelium (ue) of the Mabuya sp. absorptiveplaque. Stage 31. TEM. The cytoplasm contains numerous lipiddroplets (li). n, nucleoli; nu, nucleus.

Fig. 23. Chorionic epithelium of the Mabuya sp. absorptiveplaque. Stage 31. TEM. The external layer of the chorionic epi-thelium is composed by giant cells (gi) and interstitial cells (i)that are very tall. The apical region of the giant cells has abun-dant smooth ER and small electron-dense granules (arrows),whereas in the basal portion they contain large lipid droplets (li)bellow the nucleus (n). nu, nucleolus; p, laterobasal interdigita-tion projection between adjacent giant and interstitial cells.

Fig. 24. Basal region of the chorionic epithelium in theMabuya sp. absorptive plaque. Stage 31. Stage 40. TEM. Thechorionic epithelium is composed by two layers: the externallayer has interstitial cells (i) and giant cells, and the basal layer(b) consists of squamous cells close to the allantoic blood vessels(v). Large lipid droplets are present in giant cells, whereas verysmall droplets (li) are in the basal chorionic cells. mi, mitochon-dria.



other Mabuya species, as M. heathi (Blackburn andVitt, 2002), M. macrorhyncha, and M. nigropunctata(Flemming and Blackburn, 2003).

In the Mabuya sp. allantoplacenta the microvillarsurfaces are observed from gastrula stages in theentire extraembryonic ectoderm and uterine epithe-

lial surfaces. However, they are especially developedin the epithelia of the dorsal absorptive plaque thatwill constitute the placentome during allantoplacen-tal development (Jerez and Ramırez-Pinilla, 2003).In the mature allantoplacenta, placentomal mi-crovillar distribution is apparently similar in both

Fig. 25. Respiratory segment of Mabuya sp. Toluidine blue. a: Both uterine (ue) and chorionic epithelia (ce) are very low, andallantoic and uterine blood vessels are very close to each other. A, allantois; gl, gland; v, blood vessel. b: Active endometrial glands (gl)are present in the respiratory regions of the allantoplacenta. They seem to have the same ultrastructure as glands depicted in Figure15. A, allantois; c, chorion; u, uterus.

Fig. 26. Respiratory segment of Mabuya sp. Stage 40. TEM. Both chorionic (ce) and uterine epithelia (ue) are very low. Small lipiddroplets (li) are still present in the attenuated uterine epithelium. The interhemal distance is �8 �m. um, uterine muscle; v, bloodvessels.



epithelia, and the interface is completely filled bythe microvillar interdigitation, with no included ma-terial component. This type of interdigitant brushborder provides an extensive platform for nutrienttransport and is commonly observed between epi-thelia with active transfer of materials (i.e., the zonaradiata of ovarian follicles), such as in placentas ofmammals (i.e., the brush border membrane of thesyncytiotrophoblast of the human placenta is recog-nized as the major site of synthetic and transportactivity; Whitsett, 1980) and squamates (brush bor-der cells of the bilaminar omphalopleure in theomphalantoic placenta of the snakes Thamnophisradix and T. sirtalis; Blackburn and Lorenz, 2003a).In mammals, the interdigitated microvillar zone be-tween maternal and fetal epithelial apices accommo-dates the transport molecules of glucose and aminoacids (Wooding et al., 2003). Also, the brush borderand the mitochondrial richness of the apical cyto-plasm in the chorionic and uterine epithelia of theMabuya sp. placentome suggest an active transportof molecules and ions. The uterine syncytium of theplacentome contains small mitochondria (0.19 �0.04 �m equator width) and abundant RER in thebasal region, completely euchromatic nuclei, andabundant vesicles and lipids, suggesting a high levelof metabolic activity. Additionally, the close proxim-ity of subjacent blood vessels to the syncytial epithe-lium suggests considerable transfer of moleculesand ions from maternal blood. For mammals, Mat-subara et al. (2001) observed that mitochondria andRER morphology in the villous syncytiotrophoblasts,cytotrophoblasts, and villous cytotrophoblast pla-centas is related to metabolic activity, suggestingthat features such as small mitochondria (0.22 �0.04 �m equator width) and abundant RER are re-lated to high cellular metabolism.

In the placentomal chorion of Mabuya sp., giantcells exhibit primary and secondary lysosomes andabundant RER, features that suggest high levels ofprotein synthetic activity and an important capacityfor macromolecule digestion. The morphology ofthese cells is similar in appearance to the early stagetrophoblast of Lama pacos, where protein syntheticactivity, ion transport, and high phagocytic activityoccur (Olivera et al., 2003a). Immunohistochemicallocalization revealed the important synthetic activ-ity of ruminant placentomal trophoblast and equinetrophoblast girdle and cup cells, which synthesizeplacental lactogen hormones, glycoproteins (Wood-ing et al., 1997), and chorionic gonadotropin (Wood-ing et al., 2001). Mabuya sp. giant and interstitialcells develop lateral interdigitant processes that areextensively elaborated and entwined. These pro-cesses form channels and structures like gap junc-tions that suggest the chorionic transcellular trans-fer of substances from the uterine syncytium. Afterthe molecules have been pumped into the chorion,their subsequent transfer into the channels couldenhance transport to the fetal circulation. Also, in

the viviparous snake Virginia striatula a wide meshof membranous channels and cytoplasmic exten-sions that interdigitate with adjacent chorionic cellsoccurs (Stewart and Brasch, 2003). The absence ofimmunocytochemical studies in the chorion of theMabuya species hampers a detailed comparisonwith mammals because the chorion morphology sug-gests absorption, synthesis, and transference ofmacromolecules; however, their nature has not beendescribed.

Paraplacentome

The Mabuya sp. paraplacentome was described asa region with histologically distinct characteristicsthat surrounds and limits the placentome, locatednear the embryonic pole (Jerez and Ramırez-Pinilla,2001). It is different from those described with thesame nomination in other scincids (Chalcides chal-cides: Blackburn and Callard, 1997; M. heathi:Blackburn and Vitt, 2002; Pseudemoia entrecas-teauxii: Adams et al., 2005). Due to the apparentlylesser complexity of the allantoplacenta in these spe-cies, the paraplacentome is considered a generalizedregion of the embryonic chamber, adjacent to theplacentome, which plays an important role in gasexchange.

In the boundary between placentome and parapla-centome there is a strong interdigitation betweenboth epithelia, mediated by convoluted processesvery different from the placentomal brush border.These processes can be observed only in this transi-tional region with the placentome, and could func-tion as seal mechanisms, similar to the border sealsobserved in Camelus dromedarius areolas that pre-vent the dissipation of stored secretory products intothe areolar cavity (Abd-Elnaeim et al., 2003).

Cellular and subcellular chorionic characteristicsof the paraplacentome are similar to those describedin the placentome, suggesting that extensive molec-ular transfer and metabolic activity is maintained inthe paraplacentomal chorion. Although lipid drop-lets occur in both epithelia of the entire exchangeareas of the Mabuya sp. embryonic chamber, onedistinguishing paraplacentomal feature is the pres-ence of abundant uterine and chorionic lipid drop-lets. Significant intra- and transcellular transport oflipids toward allantoic blood and lymphatic capillar-ies is suggested by the proximity between the chori-onic cells and allantoic blood vessels and by theabundant lysosomes and stored lipid droplets closeto the lateral and basolateral regions of giant chori-onic cells. This suggests that the paraplacentome isa site of much maternal–fetal lipid transfer. Othermaterials can also be transferred by the paraplacen-tome. The electron-dense granules in the supranu-clear region of uterine epithelial cells (eosinophilicin optical microscopy; Jerez and Ramırez-Pinilla,2001) seem to be secreted and absorbed by the cho-



rionic apical region; however, the role and nature ofthis secretion are unknown.

Chorionic Areolas

The process of formation of the chorionic areolasin Mabuya sp. is similar to that observed in theareola of Camelus dromedarius. Abd-Elnaeim et al.(2003) described an initial proliferation of tropho-blast (mitosis) followed by cellular degeneration,creating the areolar cavity and producing materialfor later absorption by the nondegenerated tropho-blast. Nonetheless, the formation process of chori-onic areolas in C. dromedarius, does not show thedevelopment of the large lipid droplets found in theareolas of Mabuya sp. before and after the degener-ation stage.

The Mabuya sp. chorionic areolas exhibit threetypes of secretory products that seem to be absorbedby the chorion: 1) products from luminal uterinecells; 2) products from chorionic cell lysis during theformation of the areolar cavity; and 3) products fromendometrial glands. The first type is characterizedby an amorphous secretion of eosinophilic nature(Jerez and Ramırez-Pinilla, 2001) produced by anapocrine mechanism, suggesting the extrusion ofsoluble and membrane-associated proteins and cy-toplasm remnants. The second type of secretoryproduct is characterized by the presence of largelipid droplets and chorionic cellular debris producedby degeneration of the apical chorionic cells (holo-crine products). These secretory materials are ab-sorbed by the basal chorionic cells of the areolarconcavity. The third type has ultrastructural fea-tures that seem to be enzymatic in nature and itsfunction is unknown. This glandular secretion couldmediate in the formation of the chorionic cavity ofthe areola, in the extrusion and lysis of the apocrineand holocrine secretory products, or in the digestionand absorption of chorionic and uterine cellularproducts. The areolar glands of Mabuya sp. havecharacteristics that differ from those described inthe areola of mammals (in Lama pacos, Olivera etal., 2003a; and in Camelus dromedaries, Abd-Elnaeim et al., 2003), particularly in the appearanceof glandular secretions and the basolateral mem-brane arrangement.

In mammals, most placental types use histotro-phic production from uterine glands to chorionic are-olae. Iron deposits and acid phosphatase activity areobserved in alpaca areolar cavities (Olivera et al.,2003a) and equid areolar cells allow iron and cal-cium transfer (Wooding et al., 2001). In pigs, twotypes of chorionic areolas are described: 1) irregular,with sulfomucin vacuoles characterized by resis-tance to trypsin digestion, and 2) regular, with PAS-positive glycoproteins, associated with uteroferrineabsorption (Bielanska-Osuchowska and Kunska,1995). It is possible that in Mabuya sp. some of thesemolecules and ions are transferred via chorionic are-

olas. Transplacental movements of Ca�� in Mabuyasp. increase dramatically during the last stages of ges-tation, when fetal skeletal mineralization reaches itsmaximum and when the placentome is mature andchorionic areolas occur (Ramırez-Pinilla et al., 2005).Morphological evidence suggests lipid transfer and ex-change of other molecules such as proteins and othernutrients; histochemical studies would identify thesesubstances.

Absorptive Plaques

The original descriptions of Type IV allantopla-centas, restricted to the embryonic hemisphere andon formalin-fixed material, indicated the presence ofa highly specialized placentome and chorionic areo-las related to the histotrophic transfer of nutrients(Vitt and Blackburn, 1991; Blackburn and Vitt,1992, 2002; Blackburn, 1993; Flemming and Black-burn, 2003). However, for this population of AndeanMabuya Jerez and Ramırez-Pinilla (2001) describedfor the first time the presence of absorptive plaques.These flat, round areas located at the antimesome-trial hemisphere, immediately ventral to the zone ofchorionic areolas, have histological characteristicsthat correspond to specialized structures for trans-fer of nutrients. Thus, the antimesometrial hemi-sphere has two placental functions (gas exchangeand nutrient transfer), whereas the mesometrialhemisphere functions predominantly in histotrophictransfer.

The origin and nature of transferred products inMabuya sp. absorptive plaques are different fromthose in mammals. In eutherian mammals the ab-sorptive plaques occur near one or more endometrialglands and allow the transfer of maternal productsby phagocytosis (Mossman, 1987). In Mabuya sp.absorptive plaques, the endometrial glands are notspecially developed and their ducts do not open tothe interface of the absorptive plaque; thus, a sig-nificant histotroph uptake by the chorion cannot besuggested. The absorptive plaques have closely ap-posed chorionic and uterine epithelia without mi-crovillar surfaces between them, and thus, mem-brane transport of ions and molecules would not beespecially enhanced. However, absorptive plaquesexhibit a complex system of interwoven apical mem-brane projections. Lama pacos also has points ofattachment (Olivera et al., 2003b) morphologicallysimilar to these observed in Mabuya sp. absorptiveplaques. These are glycan–glycan interactions atthe maternal–fetal interface that improve the adhe-sion of the placenta (Olivera et al., 2003a). ForMabuya sp. these adhesion areas can also functionas a mechanism to prevent the dispersion of mate-rials to respiratory segments, and to improve theinteraction between the two epithelia.

Similar to the other specializations described inthe embryonic pole of the Mabuya sp. allantopla-centa, in the absorptive plaques the numerous lipid



droplets in the uterine epithelium and in the chori-onic cytoplasm, and in the intercellular and basalspaces between chorionic cells, suggest the transferof lipids from uterus to chorioallantois. Moreover,the presence of small lipid droplets near the endo-thelium of allantoic blood capillaries suggests lipidtransfer from uterus to allantoic blood vessels. Giantcells of absorptive plaques are very tall and haveconsiderable smooth ER in the chorionic apical re-gion. Giant multinucleate cells in the camel producesteroids (Wooding et al., 2003) and the giant cells inMabuya sp. may have a similar function. Also, in theabsorptive plaques numerous electron-dense gran-ules are present in the chorionic apex and their sizesuggests an autocrine or paracrine function. In thehuman placenta the chorion plays an important rolein implantation, fetal development, and parturitionthrough peptides that function like endocrine andparacrine markers (Petraglia et al., 1996). However,to determine the exact nature of these materials andspeculate more accurately on their function inMabuya sp. absorptive plaques, further immunocy-tochemical studies are required.

Respiratory Segments

In the respiratory segments of the Mabuya sp.allantoplacenta, the uterine and chorionic epitheliaare closely apposed and the diffusion distance be-tween uterine and allantoic vessels is highly dimin-ished, because both epithelia are thin and epithelialprojections or microvilli do not occur. The respira-tory segments alternate with absorptive plaques inthe subparaplacentomal area of the embryonicchamber exhibiting a small interhemal distance. Asmall thickness and a wide surface area facilitateeasy transfer of oxygen, carbon monoxide, and othersubstances (Schroder, 1995).

The Type IV allantoplacenta of Mabuya heathialso exhibits attenuated chorionic and uterine epi-thelia (Blackburn and Vitt, 2002). In the allantopla-centa of the snakes Virginia striatula (Stewart andBrasch, 2003), Thamnophis sirtalis, and T. radix(Blackburn and Lorenz, 2003a) uterine epithelia arethinned by overlying blood vessels that create atightly folded area, caused by the increasing pres-sure of expanding blood vessels. This displaces thecytoplasm, attenuating the overlying epithelialcells. In the paraplacentome of Pseudemoia entrecas-teauxii, the extremely attenuated cytoplasm and theloss of cellular organelles suggest a variant of epi-theliochorial placenta, termed cyto-epitheliochorial(Adams et al., 2005). In contrast, in mature placen-tas from eutherian mammals, such as guinea pigs(Enders and Blakenship, 1999), the interhemal dis-tance is reduced by the loss of trophoblast layers. Inthe Mabuya sp. respiratory segments, loss of chori-onic layers was not observed and the epithelial at-tenuation is less than that observed in Pseudemoiaentrecasteauxii.

Lipids found in the chorionic and endometrialcells of the respiratory segments are remnants oftransfer in early developmental stages (at gastrulaand neurula stages; Jerez and Ramırez-Pinilla,2003) when oxygen transfer is not highly required.Because oxygen demand is highest during thepreparturition stages, as is observed in other squa-mates (i.e., Eulamprus tympanum; Robert andThompson, 2000) and in eutherian mammals (i.e.,humans; Burton et al., 2001), some regions amongthe absorptive plaques, mainly in the abembryonichemisphere, constitute the respiratory segmentsspecialized for gas exchange.

A common feature of all structural specializationsof the allantoplacenta of Mabuya sp. is the transferof lipids, which supply the metabolic and energeticnecessities for embryo development because the eggis microlecithal. In eutherian mammals, glucose isthe principal fetal and placental energy source(Smith et al., 1992; Takata et al., 1997; Burton et al.,2001; Ward et al., 2003; Fuchs and Ellinger, 2004;Wooding et al., 2005), while lipids and proteins arethe main energy sources in reptiles (Stewart andThompson, 1993; Speake and Thompson, 1999;Thompson et al., 1999a–c). Significant lipid transferoccurs in the Mabuya sp. embryonic chamber fromthe earliest stages of embryo development (Jerezand Ramırez-Pinilla, 2003) and throughout allanto-placentation. In Mabuya sp. the necessity of lipidsfor embryonic development must be supplied by pla-cental transfer because of the almost complete sup-pression of vitellogenesis (Gomez and Ramırez-Pinilla, 2004). However, in the lipid transfer fromuterus to chorion, each lipid must be metabolized toits constituent molecules, and each type of molecule(i.e., fatty acids, cholesteryl esters) must have theirown membrane transport systems. Lipid dropletsmust be re-formed in the fetus with a different com-position, as has been found in other placentotrophiclizards (Speake et al., 2004). Studies by Speake et al.(2004) revealed that the evolution of placentotrophyin Pseudemoia entrecasteauxii requires the expres-sion of molecular mechanisms to accomplish thetransfer of lipids from the maternal circulation tothe fetus during gestation. This assertion suggeststhat in the highly matrotrophic Type IV allantopla-centas, these transport systems and molecularmechanisms must be highly developed.

In this Andean population of Mabuya, each spe-cialized area of the allantoplacenta has differenthistological and cellular features related to: 1) thematernal–fetal apical interactions between epithe-lia, 2) the morphology of endometrial and chorial-lantoic tissues and cells, and 3) the material stored,secreted, and absorbed in uterine and chorioniccells. These characteristics suggest that each spe-cialization of Mabuya sp. allantoplacenta has par-ticular functions in the transfer of nutrients as ions,lipids, proteins, amino acids, sugar, water, and gasexchange, and in the possible synthesis of hormones



and proteins. Different studies show that the pla-centa of eutherian mammals also has regional func-tional specializations, such as the cotyledonary andintercotyledonary regions in ruminants, which showdifferent synthetic capacity (Wooding et al., 1996),or areolas and microcotyledons in the equid pla-centa, which allow the transfer of different sub-stances (Wooding et al., 2000). It is possible that thecomplex regionalization of the Mabuya sp. allanto-placenta follows a similar eutherian mammal pat-tern in such a way that the molecular transfer andsynthesis of particular molecules can be regionallyspecialized. The nature of the molecules and thecellular and molecular mechanisms that allow thefunction of each specialization should be deter-mined.

The Mabuya sp. allantoplacenta exhibits greatmorphological complexity related to its high level ofplacentotrophy. This complexity is observed not onlyin the remarkable regionalization of specializationsfor nutrient transfer, but also in the histological andcytological features of interacting chorionic and ma-ternal tissues in the incubatory chamber. These al-lantoplacental features and other characteristics ofextraembryonic membrane development (Jerez andRamırez-Pinilla, 2003), such as the fact that the yolksac does not develop according to the standard squa-mate pattern, reveal strong differences from thegeneralized condition of other viviparous squa-mates, including placentotrophic species. The mor-phological differences observed between M. heathiand our population of Mabuya suggests that moredetailed morphological and physiological studies onextraembryonic membranes and in their develop-ment must be done in this lineage of Mabuya spe-cies, and also in the highly placentotrophic Africanskinks Eumecia anchietae and Trachylepis ivensii.On the other hand, several of the described featuresare very similar to those found in the placentas ofeutherian mammals, suggesting clear evidence ofevolutionary convergence at the histological and cy-tological level between two clades, and from whichevolved the highest levels of placentotrophy amongvertebrates and developed a complex chorioallantoicplacenta that provides all the nutrients for fetaldevelopment.

ACKNOWLEDGMENTS

We thank the authorities and the people of theInspeccion de Policıa de Guadalito for hospitalityduring our collection trips, especially E. de Al-varez and her family, the students and teachers atthe elementary school of Guadualito, and R. Al-varez and his family; M.L. Calderon, M.I. Chacon,A. Jerez, J.P. Ramırez, and R.M. Torres for sup-port and helpful suggestions reviewing the article,and especially P. Wooding and two anonymous

reviewers for helpful commentaries and criticalreading of the article.

LITERATURE CITED

Abd-Elnaeim M, Hassan A, Abou-Elmagd H, Klisch K, Jones C,Leisser R. 2003. Development of the areola in the early placentaof the one-humped camel (Camelus dromedarius): a light, scan-ning and transmission electron microspical study. Anat HistolEmbryol 32:326–34.

Adams SM, Biazik JM, Thompson MB, Murphy CR. 2005. Cyto-epitheliochorial placenta of the viviparous lizard Pseudemoiaentrecasteauxii: a new placental morphotype. J Morphol 264:264–276.

Bielanska-Osuchowska Z, Kunska A. 1995. A new approach to theareolar structures in the pig placenta: histochemistry and de-velopment of mucous areolae. Folia Histochem Cytobiol 33:95–101.

Blackburn DG. 1993. Chorioallantoic placentation in squamatereptiles: structure, function, development, and evolution. J ExpBiol 266:414–430.

Blackburn DG. 2000. Reptilian viviparity: past research, futuredirections, and appropriate models. Comp Biochem PhysiolPart A 127:391–409.

Blackburn DG, Callard I. 1997. Morphogenesis of placental mem-branes in the viviparous, placentotrophic lizard Chalcides chal-cides (Squamata: Scincidae). J Morphol 232:35–55.

Blackburn DG, Lorenz RL. 2003a. Placentation in garter snakes.II. Transmission EM of the corioallantoic placenta of Thamno-phis radix and T. sirtalis. J Morphol 256:171–186.

Blackburn DG, Lorenz RL. 2003b. Placentation in garter snakes.III. Transmission EM of the omphalallantoic placenta of Tham-nophis radix and T. sirtalis. J Morphol 256:187–204.

Blackburn DG, Vitt LJ. 1992. Reproduction in viviparous SouthAmerican lizards of the genus Mabuya. In: Hamlett W, editor.Reproduction in South American vertebrates: aquatic and ter-restrial. New York: Springer. p 150–164.

Blackburn DG, Vitt LJ. 2002. Specializations of the cho-rioallantoic placenta in the Brazilian scincid lizard, Mabuyaheathi: a new placental morphotype for reptiles. J Morphol254:121–131.

Blackburn DG, Vitt L, Beuchat C. 1984. Eutherian-like reproduc-tive specializations in a viviparous reptile. Proc Natl Acad Sci US A 81:4860–4863.

Blackburn DG, Stewart JR, Baxter DC, Hoffman LH. 2002. Pla-centation in garter snakes: scanning EM of the placental mem-branes of Thamnophis ordinoides and T. sirtalis. J Morphol252:263–275.

Burton GJ, Hempstock J, Jauniaux E. 2001. Nutrition of thehuman fetus during the first trimester—a review. Placenta22:S70–S77.

Carranza S, Arnold EN. 2003. Investigating the origin of trans-oceanic distributions: mtDNA shows Mabuya lizards (Reptilia,Scincidae) crossed the Atlantic twice. Syst Biodivers 1:275–282.

Dufaure JP, Hubert J. 1961. Table de developpment de lezardvivipare: Lacerta (Zootoca) vivipara. Jacquin. Arch Anat Mi-crosc Morphol Exp 50:309–328.

Enders C, Blakenship T. 1999. Comparative placental structure.Adv Drug Deliv Rev 38:3–15.

Flemming AF, Blackburn DG. 2003. Evolution of placental spe-cializations in viviparous African and South America lizards. JExp Zool 299A:33–47.

Flemming AF, Blackburn DG. 2005. Close contacts of the giantkind: on placental interfaces in matrotrophic lizards. FifthWorld Congress of Herpetology. Abstracts and programme. p44.

Flemming AF, Branch WR. 2001. Extraordinary case of matrot-rophy in the African skink Eumecia anchietae. J Morphol 247:264–287.



Fuchs R, Ellinger I. 2004. Endocytic and transcytotic process invillous syncytiotrophoblast: role in nutrient transport to thehuman fetus. Traffic 5:725–738.

Gomez D, Ramırez-Pinilla MP. 2004. Ovarian histology of theplacentotrophic Mabuya mabouya (Squamata, Scincidae). JMorphol 259:90–105.

Greer AE, Arnold CJP, Arnold EN. 1999. The systematic signif-icance of the number of presacral vertebrae in the scincid lizardgenus Mabuya. Amphibia-Reptilia 21:121–126.

Guarino F, Paulesu L, Cardone A, Bellini L, Ghiara G, Angelini F.1998. Endocrine activity of the corpus luteum and placentaduring pregnancy in Chalcides chalcides (Reptilia, Squamata).Gen Comp Endocrinol 111:261–270.

Hoffman LH. 1970. Placentation in the garter snake, Thamno-phis sirtalis. J Morphol 131:57–88.

Hoffman L, Wooding FBP. 1993. Giant and binucleate tropho-blast cells of mammals. J Exp Biol 266:559–557.

Hosie JM, Adams MS, Thompson MB, Murphy CR. 2003. Vivip-arous lizard, Eulamprus tympanum, shows changes in the uter-ine surface epithelium during early pregnancy that are similarto the plasma membrane transformation of mammals. J Mor-phol 258:346–357.

Jerez A, Ramıez-Pinilla MP. 2001. The allantoplacenta ofMabuya mabouya (Sauria, Scincidae). J Morphol 249:132–146.

Jerez A, Ramırez-Pinilla MP. 2003. Morphogenesis of extraem-bryonic membranes and placentation in Mabuya mabouya(Squamata, Scincidae). J Morphol 258:15–178.

Matsubara S, Takayama T, Iwasaki R, Minakami H, Takizawa T,Sato I. 2001. Morphology of the mitochondria and endoplasmicreticula of the chorion leave cytotrophoblasts: their resem-blance to villous syncytiotrophoblasts rather than villous cy-totrophoblasts. Histochem Cell Biol 116:9–15.

Mausfeld P, Schmitz A, Bohme W, Misof B, Vrcibradic D, DuarteRCF. 2002. Phylogenetic affinities of Mabuya atlanticaSchmidt, 1945, endemic to the Atlantic Ocean Archipelago ofFernando de Noronha (Brazil): necessity of partitioning thegenus Mabuya Fitzinger, 1826 (Scincidae: Lygosominae). ZoolAnz 241:281–293.

Miralles A. 2005. The identity of Lacertus mabouya Lacepede,1788, with description of a neotype: an approach toward thetaxonomy of new world Mabuya. Herpetologica 61:46–53.

Miralles A, Rivas Fuenmayor G, Barrio-Amoros CL. 2005a. Tax-onomy of the genus Mabuya (Reptilia, Squamata, Scincidae) inVenezuela. Zoosystema 27:825–837.

Miralles A, Rivas Fuenmayor G, Schargel WE. 2005b. A newspecies of Mabuya (Squamata, Scincidae) from the VenezuelanAndes. Zootaxa 895:1–11.

Mossman H. 1987. Vertebrate fetal membranes. New Brunswick,NJ: Rutgers University Press.

Olivera L, Zago D, Leiser R, Jones C, Bevilacqua E. 2003a. Pla-centation in the alpaca Lama pacos. Anat Embriol 207:45–62.

Olivera L, Zago D, Leiser R, Jones C, Bevilacqua E. 2003b. De-velopmental changes at the maternal-embryonic interface inearly pregnancy of the alpaca, Lamos pacos. Anat Embryol207:317–331.

Petraglia F, Florio P, Nappi C, Genazzani A. 1996. Peptide sig-naling in human placenta and membranes: autocrine, para-crine, and endocrine mechanisms. Endocr Rev 17:156–186.

Ramırez-Pinilla MP, Serrano VH, Galeano JC. 2002. Annual re-productive activity of Mabuya mabouya (Squamata, Scincidae).J Herpetol 36:667–677.

Ramırez-Pinilla MP, De Perez GR, Stashenko E, Carreno F. 2005.Placental transference of nutrients during gestation and andultrastructure of the placentae in Mabuya mabouya. FifthWorld Congress of Herpetology. Abstracts and programme. p84.

Robert KA, Thompson MB. 2000. Energy consumption by em-bryos of viviparous lizard, Eulamprus tympanum, during de-velopment. Comp Biochem Physiol A 127:481–486.

Schroder HJ. 1995. Comparative aspects of placental exchangefunctions. Eur J Obstet Gynecol Reprod Biol 63:81–90.

Smith CH, Moe AJ, Ganapathy V. 1992. Nutrient transport path-ways across the epithelium of the placenta. Annu Rev Nutr12:138–206.

Speake BK, Thompson MB. 1999. Comparative aspects of yolklipid utilization in birds and reptiles. Poult Avian Biol Rev10:181–211.

Speake BK, Hebert JF, Thompson MB. 2004. Evidence for pla-cental transfer of lipids during gestation in the viviparous liz-ard, Pseudemoia entrecasteauxii. Comp Biochem Physiol A 139:213–220.

Stewart JR, Brasch K. 2003. Ultrastructure of the placentae ofthe natricine snake, Virginia striatula (Reptilia: Squamata). JMorphol 255:177–201.

Stewart JR, Thompson MB. 1993. A novel pattern of embryonicnutrition in a viviparous reptile. J Exp Biol 174:97–108.

Stewart JR, Thompson MB. 2000. Evolution of placentationamong squamate reptiles: recent research and future direc-tions. Comp Biochem Physiol A 127:411–431.

Takata K, Fujikura K, Shin B. 1997. Ultrastructure of the rodentplacental labyrinth: a site of barrier and transport. J ReprodDev 43:13–24.

Thompson MB, Speake BK, Stewart JR, Rusell KJ, McCartneyRJ, Surai PF. 1999a. Placental nutrition in the viviparous liz-ard Niveoscincus metallicus: the influence of placental type. JExp Biol 202:2985–2992.

Thompson MB, Stewart JR, Speake BK, Rusell KJ, McCartneyRJ, Surai PF. 1999b. Placental nutrition in a viviparous lizard(Pseudemoia pagenstecheri) with a complex placenta. J ZoolLond 248:295–305.

Thompson MB, Stewart JR, Speake BK, Rusell KJ, McCartneyRJ. 1999c. Placental transfer of nutrients during gestation inthe viviparous lizard, Pseudemoia spenceri. J Comp Physiol B169:319–328.

Villagran-Santacruz M, Gillette JL, Mendez-de la Cruz FR. 1994.Placentacion en el Scincido Mabuya. Resumenes de la III Re-union Nacional de Herpetologıa. San Cristobal de las Casas,Chiapas, Mexico.

Vitt LJ, Blackburn DG. 1991. Ecology and life history of theviviparous lizard Mabuya bistriata (Scincidae) in the BrazilianAmazonia. Copeia 1991:916–927.

Ward JW, Wooding FBP, Fowden AL. 2003. Ovine feto-placentalmetabolism. J Physiol 554:529–541.

Weekes HC. 1935. A review of placentation among reptiles, withparticular regard to the function and evolution of the placenta.Proc Zool Soc Lond 2:625–645.

Whitsett JA. 1980. Specialization in plasma membranes of thehuman placenta. J Pediatr 96:600–603.

Wooding FBP, Morgan G, Monaghan S, Hamon M, Heap RB.1996. Functional specialization in the ruminant placenta: evi-dence for two populations of fetal binucleate cells of differentselective synthetic capacity. Placenta 17:75–86.

Wooding FBP, Morgan G, Adam CL. 1997. Structure and functionin the synepitheliochorial placenta: cental role of the tropho-blast binucleate cell in deer. Microsc Res Tech 38:88–99.

Wooding FBP, Morgan G, Fowden AL, Allen WR. 2000. Separatesites and mechanisms for placental transport of calcium, ironand glucose in the equine placenta. Placenta 21:635–645.

Wooding FBP, Morgan G, Fowden AL, Allen WR. 2001. A struc-tural and immunological study of chorionic gonadotrophin pro-duction by equine trophoblast girdle and cup cells. Placenta22:749–767.

Wooding FBP, Ozturk M, Skidmore JA, Allen WR. 2003. Devel-opmental changes in localization of steroid synthesis enzymesin camelid placentas. Reproduction 126:239–247.

Wooding FBP, Stewart F, Mathias S, Allen WR. 2005. Placentationin the African elephant, Loxodonta africanus. III. Ultrastructuraland functional features of the placenta. Placenta 26:449–470.

Yaron Z. 1985. Reptilian placentation and gestation: structure,function, and endocrine control. In: Gans C, Billet F, editors.Biology of the Reptilia, vol. 15. Development B. New York: JohnWiley & Sons. p 527–603.



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Allantoplacental Ultraestructure Mabuya