Scanning electron microscopy of the fetal membranes of an oviparous squamate,the corn snake Pituophis guttatus (Colubridae)

Although the fetal membranes of viviparous squamates have received much study, morphology of their homologues among oviparous reptiles is poorly understood. The scarcity of information about these membranes in egg‐laying reptiles hampers attempts to distinguish specializations for viviparity from ancestral oviparous features. We used scanning electron microscopy to examine fetal membranes of an oviparous snake (Pituophis guttatus) throughout the developmental period from oviposition to hatching. The external surface of the chorion contains broad, flattened cells that lack surface features; these cells form a continuous layer over the allantoic capillaries and offer a minimal barrier to respiratory exchange. In contrast, the surface epithelium of the omphalopleure bears elaborate surface ridges suggestive of absorptive capabilities. These ridges are prominent in the first few weeks after oviposition, but diminish thereafter. During development, the isolated yolk mass (IYM) of the omphalopleure becomes depleted, and the tissue becomes heavily vascularized by allantoic vessels. Surface features of the omphalopleure progressively take on the appearance of the chorioallantois, but the changes are not synchronous with loss of the IYM or membrane vascularization. Previous studies on viviparous snakes suggest that the chorioallantois and omphalopleure are respectively specialized for gas exchange and absorption in the intrauterine environment. Our studies of fetal membranes in P. guttatus offer evidence that cytological specializations for these functions originated under oviparous conditions, reflecting functional capacities that predate viviparity. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Ultrastructure of the fetal membranes of the oviparous kingsnake,Lampropeltis getula (Colubridae) as revealed by scanning electron microscopy

In reptilian sauropsids, fetal (extraembryonic) membranes that line the eggshell sustain developing embryos by providing for gas exchange and uptake of water and eggshell calcium. However, a scarcity of morphological studies hinders an understanding of functional specializations and their evolution. In kingsnakes (Lampropeltis getula), scanning electron microscopy reveals two major fetal membranes: the chorioallantois and yolk sac omphalopleure. In early development, the chorioallantois contains tall chorionic epithelial cells, avascular connective tissue, and enlarged allantoic epithelial cells. During its maturation, the chorionic and allantoic epithelia thin dramatically and become underlain by a rich network of allantoic capillaries, yielding a membrane ideally suited for respiratory gas exchange. Yolk sac development initially is like that of typical lizards and snakes, forming an avascular omphalopleure, isolated yolk mass (IYM), and yolk cleft. However, unlike the situation in most squamates studied, the omphalopleure becomes transformed into a “secondary chorioallantois” via three asynchronous events: flattening of the epithelium, regression of the IYM, and vascularization by the allantois. Progressive expansion of chorioallantois parallels growing embryonic needs for gas exchange. In early through mid‐development, external surfaces of both the chorionic and omphalopleure epithelium show an abundance of irregular surface protrusions that possibly increase surface area for water absorption. We postulate that the hypertrophied allantoic epithelial cells produce allantoic fluid, a viscous substance that facilitates water uptake and storage. Our findings are consistent with a previous study on the corn snake Pantherophis guttatus, but include new observations and novel functional hypotheses relevant to a reconstruction of basal squamate patterns. J. Morphol. 276:1467–1481, 2015. © 2015 Wiley Periodicals, Inc.     

Placentation in the lizard Gerrhonotus coeruleus with a comparison to the extraembryonic membranes of the oviparous Gerrhonotus multicarinatus (Sauria,Anguidae)

Paraffin sections of an ontogenetic series of embryos of the viviparous lizard Gerrhonotus coeruleus and the oviparous congener G. multicarinatus reveal that although general features of the development of the chorioallantoic and yolk sac membranes are similar, differences are evident in the distribution of the chorioallantoic membrane in late stage embryos. An acellular shell membrane surrounds the egg throughout gestation in both species although the thickness of this structure is much reduced in G. coeruleus over that of G. multicarinatus. The initial vascular membrane to contact the shell membrane in both species is a trilaminar omphalopleure (choriovitelline membrane) composed of ectoderm, mesoderm of the area vasculosa, and endoderm. This transitory membrane is replaced by the vascularized chorioallantois as the allantois expands to contact the inner surface of the chorion. Prior to the establishment of the chorioallantois at the embryonic pole, a membrane begins to form within the yolk ventral to the sinus terminalis. This membrane, which becomes vascularized, extends across the entire width of the abembryonic region and isolates a mass of yolk ventral to the yolk mass proper. The outer membrane of the yolk pole is a nonvascular bilaminar omphalopleure (chorionic ectoderm and yolk endoderm). In G. multicarinatus the bilaminar omphalopleure is supported internally by the vascularized allantoic membrane, whereas in G. coeruleus the allantois does not extend beyond the margin of the isolated yolk mass and the bilaminar omphalopleure is supported by the vascularized intravitelline membrane. Both the chorioallantoic placenta (uterine epithelium, chorionic ectoderm and mesoderm, and allantoic mesoderm and endoderm) and the yolk sac placenta at the abembryonic pole (uterine epithelium, chorionic ectoderm, and yolk sac endoderm) persist to the end of gestation in G. coeruleus.     

Development of yolk sac and chorioallantoic membranes in the Lord Howe Island skink,Oligosoma lichenigerum

Development of the yolk sac of squamate reptiles (lizards and snakes) differs from other amniote lineages in the pattern of growth of extraembryonic mesoderm, which produces a cavity, the yolk cleft, within the yolk. The structure of the yolk cleft and the accompanying isolated yolk mass influence development of the allantois and chorioallantoic membrane. The yolk cleft of viviparous species of the Eugongylus group of scincid lizards is the foundation for an elaborate yolk sac placenta; development of the yolk cleft of oviparous species has not been studied. We used light microscopy to describe the yolk sac and chorioallantoic membrane in a developmental series of an oviparous member of this species group, Oligosoma lichenigerum. Topology of the extraembryonic membranes of late stage embryos differs from viviparous species as a result of differences in development of the yolk sac. The chorioallantoic membrane encircles the egg of O. lichenigerum but is confined to the embryonic hemisphere of the egg in viviparous species. Early development of the yolk cleft is similar for both modes of parity, but in contrast to viviparous species, the yolk cleft of O. lichenigerum is transformed into a tube‐like structure, which fills with cells. The yolk cleft originates as extraembryonic mesoderm is diverted from the periphery of the egg into the yolk sac cavity. As a result, a bilaminar omphalopleure persists over the abembryonic surface of the yolk. The bilaminar omphalopleure is ultimately displaced by intrusion of allantoic mesoderm between ectodermal and endodermal layers. The resulting chorioallantoic membrane has a similar structure but different developmental history to the chorioallantoic membrane of the embryonic hemisphere of the egg. J. Morphol. 2012. © 2012 Wiley Periodicals, Inc.     

Ontogeny of the extraembryonic membranes of the oviparous lizard, Eumeces fasciatus (Squamata: scincidae)

Oviposited eggs of Eumeces fasciatus contain embryos in the limb bud stage. Amniogenesis is complete and two yolk sac membranes, vascular trilaminar omphalopleure (choriovitelline membrane) and bilaminar omphalopleure, enclose the yolk vesicle. A small allantoic vesicle contacts the chorion. The choriovitelline membrane is the primary vascular system. Blood islands, sites of hematopoiesis, are associated with omphalomesenteric vessels of the choriovitelline membrane. The bilaminar omphalopleure, which contacts the eggshell over the abembryonic hemisphere of the egg, lies external to an isolated yolk mass and yolk cleft and is not vascularized. The definitive yolk sac (splanchnopleure) is formed when the extraembryonic coelom and allantoic vesicle intrude into the choriovitelline membrane. Omphalomesenteric vessels are retained with the yolk sac splanchnopleure and the associated hematopoietic sites are present throughout incubation. The chorioallantoic membrane reaches the equator of the egg, entirely supplanting the choriovitelline membrane, after 25% of incubation is completed. Further growth of the allantois is stalled until 65% of incubation is completed when rapid expansion of the allantoic vesicle, in conjunction with resorption of the isolated yolk mass, supplants the bilaminar omphalopleure. As a result, the chorioallantoic membrane completely envelopes the egg for the final 35% of incubation. This developmental event is coincident with published reports for the timing of increased growth and metabolism of embryos. As the isolated yolk mass regresses, intravitelline cells associated with the yolk cleft invade and resorb the yolk to form a large cavity. The wall of this cavity is a germinal epithelium that produces cells that fill the cavity. This structure appears to be a site of hematopoiesis previously undescribed in vertebrates.     

Scanning electron microscopy of the placental interface in the viviparous lizard Sceloporus jarrovi (Squamata: Phrynosomatidae)

Placental membranes mediate maternal‐fetal exchange in all viviparous reptilian sauropsids. We used scanning electron microscopy to examine the placental interface in the mountain spiny lizard, Sceloporus jarrovi (Phrynosomatidae). From the late limb bud stage until birth, the conceptus is surrounded by placental membranes formed from the chorioallantois and yolk sac omphalopleure. The chorioallantois lies directly apposed to the uterine lining with no intervening shell membrane. Both fetal and maternal sides of the chorioallantoic placenta are lined by continuous layers of flattened epithelial cells that overlie dense capillary networks. The chorioallantoic placenta shows specializations that enhance respiratory exchange, as well as ultrastructural evidence of maternal secretion and fetal absorption. The yolk sac placenta contains enlarged fetal and maternal epithelia with specializations for histotrophic nutrient transfer. This placenta lacks intrinsic vascularity, although the vascular allantois lies against its inner face, contributing to an omphallantoic placenta. In a specialized region at the abembryonic pole, uterine and fetal tissues are separated by a compact mass of shed shell membrane, yolk droplets, and cellular debris. The omphalopleure in this region develops elongate folds that may contribute to sequestration and absorption of this material. Fetal membrane morphogenesis and composition in S. jarrovi are consistent with those of typical squamates. However, this species exhibits unusual placental specializations characteristic of highly placentotrophic lizards. J. Morphol., 2011. © 2011 Wiley‐Liss, Inc.     

Morphology, development, and evolution of fetal membranes and placentation in squamate reptiles

Current studies on fetal membranes of reptiles are providing insight into three major historical transformations: evolution of the amniote egg, evolution of viviparity, and evolution of placentotrophy. Squamates (lizards and snakes) are ideal for such studies because their fetal membranes sustain embryos in oviparous species and contribute to placentas in viviparous species. Ultrastructure of the fetal membranes in oviparous corn snakes (Pituophis guttatus) shows that the chorioallantois is specialized for gas exchange and the omphalopleure, for water absorption. Transmission and scanning electron microscopic studies of viviparous thamnophine snakes (Thamnophis, Storeria) have revealed morphological specializations for gas exchange and absorption in the intra-uterine environment that represent modifications of features found in oviparous species. Thus, fetal membranes in oviparous species show morphological differentiation for distinct functions that have been recruited and enhanced under viviparous conditions. The ultimate in specialization of fetal membranes is found in viviparous skinks of South America (Mabuya) and Africa (Trachylepis, Eumecia), in which placentotrophy accounts for nearly all of the nutrients for development. Ongoing research on these lizards has revealed morphological specializations of the chorioallantoic placenta through which nutrient transfer is accomplished. In addition, African Trachylepis show an invasive form of implantation, in which uterine epithelium is replaced by invading chorionic cells. Ongoing analysis of these lizards shows how integration of multiple lines of evidence can provide insight into the evolution of developmental and reproductive specializations once thought to be confined to eutherian mammals.     

Placentation in watersnakes II: Placental ultrastructure in Nerodia erythrogaster (Colubridae: Natricinae)

Ultrastructure of the placental tissues from redbelly watersnakes (Nerodia erythrogaster ) was analyzed during late pregnancy to provide insight into placental development and function. Examination of the chorioallantoic placenta with transmission electron microscopy reveals that chorionic and uterine epithelia are extremely attenuated but intact and that the eggshell membrane is vestigial and lacks a calcareous layer. These features minimize the interhemal diffusion distance across the placenta. Scanning electron microscopy reveals that fetal and maternal components of the placentas are richly vascularized by dense networks of capillaries. Although the yolk sac omphalopleure has largely been replaced by chorioallantois by late gestation, it retains patches of yolk droplets and regions of absorptive cells with microvilli and abundant mitochondria. Transmission electron microscopy reveals that yolk material is taken up for digestion by endodermal cells. As yolk is removed, allantoic capillaries invade to occupy positions just beneath the epithelium, forming regions of chorioallantoic placentation. Ultrastructural features indicate that the chorioallantoic placenta is specialized for gas exchange, while the omphalallantoic (“yolk sac”) placenta shows evidence of functions in yolk digestion and maternal‐fetal nutrient transfer. Placental features of this species are consistent with those of other thamnophines, and are evolutionarily convergent on snakes of other viviparous clades.     

Extraembryonic membrane development in a reproductively bimodal lizard, Lacerta (Zootoca) vivipara

Reproductive mode has been remarkably labile among squamate reptiles and the evolutionary transition from oviparity to viviparity commonly has been accompanied by a shift in the pattern of embryonic nutrition. Structural specializations for placental transfer of nutrients during intrauterine gestation are highly diverse and many features of the extraembryonic membranes of viviparous species differ markedly from those of oviparous species. However, because of a high degree of evolutionary divergence between the species used for comparisons it is likely that the observed differences arose secondarily to the evolution of viviparity. We studied development of the extraembryonic membranes and placentation in the reproductively bimodal lizard Lacerta vivipara because the influence of reproductive mode on the structural/functional relationship between mothers and embryos can best be understood by studying the most recent evolutionary events. Lecithotrophic viviparity has evolved recently within this species and, although populations with different reproductive modes are allopatric, oviparous and viviparous forms interbreed in the laboratory and share many life history characteristics. In contrast to prior comparisons between oviparous and viviparous species, we found no differences in ontogeny or structure of the extraembryonic membranes between populations with different reproductive modes within L. vivipara. However, we did confirm conclusions from previous studies that the tertiary envelope of the egg, the eggshell, is much reduced in the viviparous population. These conclusions support a widely accepted model for the evolution of squamate placentation. We also found support for work published nearly 80 years ago that the pattern of development of the yolk sac of L. vivipara is unusual and that a function of a unique structure of squamate development, the yolk cleft, is hematopoiesis. The structure of the yolk sac splanchnopleure of L. vivipara is inconsistent with a commonly accepted model for amniote yolk sac function and we suggest that a long standing hypothesis that cells from the yolk cleft participate in yolk digestion requires further study.     

Expression of calbindin-D28K by yolk sac and chorioallantoic membranes of the corn snake, Elaphe guttata

The yolk splanchnopleure and chorioallantoic membrane of oviparous reptiles transport calcium from the yolk and eggshell to the developing embryo. Among oviparous amniotes, the mechanism of calcium mobilization to embryos has been studied only in domestic fowl, in which the mechanism of calcium transport of the yolk splanchnopleure differs from the chorioallantoic membrane. Transport of calcium is facilitated by calbindin-D(28K) in endodermal cells of the yolk splanchnopleure of chickens but the chorioallantoic membrane does not express calbindin-D(28K). We used immunoblotting to assay for calbindin-D(28K) expression in yolk splanchnopleure and chorioallantoic membrane of the corn snake, Elaphe guttata, to test the hypothesis that the mechanism of calcium transport by extraembryonic membranes of snakes is similar to birds. High calbindin-D(28K) expression was detected in samples of yolk splanchnopleure and chorioallantoic membrane during late embryonic stages. We conclude that calbindin-D(28K) is expressed in these extraembryonic membranes to facilitate transport of calcium and that the mechanism of calcium transport of the chorioallantoic membrane of the corn snake differs from that of the chicken. Further, we conclude that calbindin-D(28K) expression is developmentally regulated and increases during later embryonic stages in the corn snake.     

Expression of calcium transport proteins in the extraembryonic membranes of a viviparous snake, Virginia striatula

Yolk is the primary source of calcium for embryonic growth and development for most squamates, irrespective of mode of parity. The calcified eggshell is a secondary source for embryonic calcium in all oviparous eggs, but this structure is lost in viviparous lineages. Virginia striatula is a viviparous snake in which embryos obtain calcium from both yolk and placental transport of uterine calcium secretions. The developmental pattern of embryonic calcium acquisition in V. striatula is similar to that for oviparous snakes. Calbindin-D(28K) is a marker for epithelial calcium transport activity and plasma membrane Ca(2+)-ATPase (PMCA) provides the energy to catalyze the final step in calcium transport. Expression of calbindin-D(28K) and PMCA was measured by immunoblotting in yolk sac splanchnopleure and chorioallantois of a developmental series of V. striatula to test the hypothesis that these proteins mediate calcium transport to embryos. In addition, we compared the expression of calbindin-D(28K) in extraembryonic membranes of V. striatula throughout development to a previously published expression pattern in an oviparous snake to test the hypothesis that the ontogeny of calcium transport function is independent of reproductive mode. Expression of calbindin-D(28K) increased in yolk sac splanchnopleure and chorioallantois coincident with calcium mobilization from yolk and uterine sources and with embryonic growth. The amount of PMCA in the chorioallantois did not change through development suggesting its expression is not rate limiting for calcium transport. The pattern of expression of calbindin-D(28K) and PMCA confirms our initial hypothesis that these proteins mediate embryonic calcium uptake. In addition, the developmental pattern of calbindin-D(28K) expression in V. striatula is similar to that of an oviparous snake, which suggests that calcium transport mechanisms and their regulation are independent of reproductive mode.     

Evolution of reptilian placentation: Development of extraembryonic membranes of the Australian scincid lizards,Bassiana duperreyi (Oviparous) and Pseudemoia entrecasteauxii (Viviparous)

A prominent model for the evolution of placentation among Reptilia is based on placental structure among species in the Eugongylus group of Australian lygosomatine skinks. We studied the development of the extraembryonic membranes of an oviparous species, Bassiana duperreyi, and a viviparous species, Pseudemoia entrecasteauxii, within this taxonomic group. We observed differences in the timing of development of shared features and in the structure of extraembryonic membrane epithelia in the two species. In the viviparous species, there is earlier vascularization of the yolk sâc and increased vascular support for the abembryonic yolk sac splanchnopleure. Structural differences between species result in partitioning of the egg into two distinct hemispheres and produce epithelia which appear functionally histotrophic in both the chorioallantoic membrane and the bilaminar omphalopleure of the viviparous species. We propose that the evolution of placentation in P. entrecasteauxii involved a combination of heterochrony and structural innovation. Further, because our interpretation of placental structure of this species provides new information relevant to placental function, we propose a revision of a classic model for the evolution of placentation among Reptilia. This model predicts specific relationships among reproductive characteristics and thus is testable by comparative analysis among other species within the Eugongylus group of Australian skinks. © 1996 Wiley-Liss, Inc.     

Evolutionary transformations of the fetal membranes of viviparous reptiles: a case study of two lineages

The reptilian placenta is a composite structure formed by a functional interaction between extraembryonic membranes and the maternal uterus. Study of placental structure of squamate reptiles over the past century has established that each of the multiple independent origins of placentation, which characterize the reproductive diversity of squamates, has resulted from the evolutionary transformation of these homologous structures. Because each evolutionary transformation is an independent novel relationship between maternal and embryonic tissues, the resulting placentae are not homologous, even though the individual components may be. The evolution of reptilian placentation should reveal much about evolutionary patterns and mechanisms because similar structural-functional systems have been transformed along parallel trajectories on multiple occasions. We compared extraembryonic membrane and placental development and pattern of embryonic nutrition in thamnophiine snakes and Pseudemoia lizards in the context of recent hypotheses of phylogenetic relationships. Two primary types of placentation, chorioallantoic and yolk sac, evolved in each lineage. Smooth, highly vascular regions of chorioallantoic placentation are indistinguishable homoplasies that evolved in parallel, likely to facilitate respiratory exchange. The yolk sac placenta of each lineage is specialized for histotrophic nutrient transfer, yet composition of these structures differs because of variation in the ancestral snakes and lizards. In addition, the omphalopleure that contributes to yolk sac placentation persists to later embryonic stages compared to oviparous outgroups, but the two lineages have evolved different structures that prevent replacement of the omphalopleure by the allantois. Each lineage has also evolved unique structural specializations of the chorioallantoic placenta.     

Placentation in garter snakes. III. Transmission EM of the omphalallantoic placenta of Thamnophis radix and T. sirtalis

The omphalallantoic placenta is a complex organ that is unique to viviparous squamates. Using transmission EM and light microscopy, we examined this placenta in garter snakes in order to understand its structural organization and functional capabilities. The omphalallantoic placenta is formed from the uterine lining and the bilaminar omphalopleure, the latter of which is associated with the isolated yolk mass and allantois. A thin shell membrane separates the fetal and maternal tissues throughout gestation. The uterine epithelium contains cuboidal cells with large droplets or granules and appears to be secretory. Epithelium of the omphalopleure is specialized for absorption and contains cells with prominent microvilli and others with large cytoplasmic droplets or granules. The brush-border cells are rich in mitochondria and Golgi bodies and interdigitate extensively with adjacent cells, forming elaborate intercellular canaliculi. Their morphology is consistent with their proposed role in sodium-coupled water movement. During development, the isolated yolk mass becomes depleted as yolk droplets are digested by cells of the omphalopleure and allantois. However, the allantois does not fuse to or vascularize the inner face of the omphalopleure. Consequently, the distance between fetal and maternal circulatory systems remains large (about 250-300 microm), precluding efficient gas exchange and hemotrophic transfer. The morphology of the omphalallantoic placenta strongly suggests that it functions in nutrient transfer through uterine secretion and fetal absorption.     

Histology and ultrastructure of the placental membranes of the viviparous brown snake,Storeria dekayi (Colubridae: Natricinae)

The placental membranes of the viviparous brown snake Storeria dekayi were examined following mid‐gestation by means of light microscopy, scanning electron microscopy, and transmission electron microscopy to reveal their structural organization and cytological composition. By Zehr stage 32, the chorioallantoic placenta (allantoplacenta) is established around much of the egg, and a well‐developed omphalallantoic placenta occurs in the abembryonic hemisphere. The allantoplacenta exhibits multiple features that enhance interhemal exchange: the uterus and allantois are well vascularized, the chorionic and uterine epithelia are attenuated, and the shell membrane is vestigial and has begun to degenerate. In the omphalallantoic placenta, the uterine epithelium is enlarged and appears to be secretory. The omphalopleure contains two distinct populations of cells, and shows cytological evidence for absorption. In intermediate areas, regions of omphalallantoic placenta are being transformed into allantoplacenta, through depletion of the isolated yolk mass and reduction in epithelial height of both uterus and omphalopleure. Morphological evidence suggests that the allantoplacenta is specialized for gas exchange, and the omphalallantoic placenta, for maternal secretion and fetal absorption. On the basis of the available evidence, we postulate that this pattern is characteristic of the thamnophine radiation of snakes. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.     

Contributions to the study of oviparity–viviparity transition: Placentary structures of Liolaemus elongatus (Squamata: Liolaemidae)

Liolaemus elongatus (Liolaemidae) is a viviparous, mainly lecithotrophic species with placental structures specialized for uptake of oxygen and inorganic nutrient transport. An allantoplacenta and an omphaloplacenta are present during early embryonic stages (25–28) and there is a moderate distension of the uterine wall and major glandular activity in the uterine mucosa and submucosa compared with nonpregnant females. The uterine epithelium increases in height, first as a growth in the height of some dispersed cells localized in all regions of the placenta, and later as groups of cells localized in the periembryonic and central‐abembryonic regions. At embryonic stage 39, the allantoplacenta reaches its maximum extension around the yolk sac. Omphaloplacenta is restricted to the abembryonic zone, the yolk cleft limiting the newly formed isolated vitelline mass. At more advanced embryonic stages (39–42), the blood supply to the allantoplacenta's periembryonic zone increases, matching the profuse allantoic vascularization. At embryonic stage 42, a secondary cleft opens in the main vitelline mass, above the first yolk cleft, and allantoic blood vessels enter into this secondary cleft. This secondary cleft subdivides the vitelline mass into a large embryonic region connected to a much smaller abembryonic region. In L. elongatus most nutritional resources seem to be provided by the yolk that remains attached to the newborn for 2 or 3 days as an external supply. The embryo's wet weight doubles the weight of the decrease observed in vitelline mass. But the dry wet diminishes, evidencing the importance of the exchange of water and inorganic nutrients. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Placentation in garter snakes: scanning EM of the placental membranes of Thamnophis ordinoides and T. sirtalis

Surface topography and cross-sections of the placental membranes were examined by scanning electron microscopy in two species of Thamnophis. The chorionic epithelium of the chorioallantoic placenta consists of broad, squamous cells that lack surface specializations. The apposed uterine epithelium contains ciliated cells and larger, nonciliated cells. Neither the epithelium of the chorion nor that of the uterus is eroded; thus, underlying capillaries are not exposed to the luminal surface. In both the omphaloplacenta and the omphalallantoic placenta, epithelium of the omphalopleure consists of brush-border cells bearing prominent microvilli, interspersed with cells bearing minuscule microvilli. These surface epithelial cells are joined at their apices and their lateral surfaces are extensively sculpted by intercellular channels, presenting the appearance of an epithelium specialized for absorption. Deep to the epithelium lie the yolk spheres of the isolated yolk mass, interspersed with endodermal cells. Surface topography of the uterine epithelia of the omphaloplacenta and omphalallantoic placenta is relatively unspecialized. The acellular shell membrane separates maternal and fetal tissues in each of the three placental types. Marked differences in surface features of the chorioallantois and omphalopleure probably reflect different roles of these membranes in gas exchange and transfer of water and nutrients.     

Uterine morphology during the annual cycle in Chalcides ocellatus tiligugu (Gmelin) (Squamata: scincidae)

The uterus of the viviparous skink Chalcides ocellatus tiligugu was studied by SEM and LM during the annual cycle. Three functional phases were identified: preovulatory (spring), gestatory (summer), and quiescent (autumn-winter), characterized by changes in the uterine wall (mainly the endometrial layer). In the preovulatory phase, the uterine wall increases in thickness; its luminal epithelium has ciliated cells and two types of unciliated secretory cells. The first type secretes sulfated glycosaminoglycans (GAGs), which form the amorphous inner layer of the eggshell membrane; the second type secretes acidic glycoproteins that form the intrafibrillar matrix of the outer layer of the eggshell membrane. The lamina propria contains simple alveolar glands that secrete the collagen fibers of the eggshell membrane. During the gestatory phase, the glycoproteins produced by secretory cells of the second type have histotrophic activity for the developing embryo. The uterus widens to form incubation chambers with two hemispheres, one embryonic and the other abembryonic. Both a chorioallantoic placenta and an omphaloplacenta with histotrophic activity are present in late gestation. The chorioallantoic placenta, with aspects of a Weekes (1935) Type III placenta, develops in the embryonic hemisphere. The omphaloplacenta forms at the vegetative pole of the egg and shows cellular hypertrophy of the bilaminar omphalopleure and uterus. During the quiescent phase, the uterus gradually decreases in thickness and activity; its luminal epithelium does not show secretory activity. The annual variations in the myometrial layer involved the inner circular and the outer longitudinal muscle layers.     

Extraordinary case of matrotrophy in the African skink Eumecia anchietae

The viviparous African skink, Eumecia anchietae, exhibits a matrotrophic fetal nutritional pattern. Until well after the limb bud stage, extravitelline nutritional provision is in the form of holocrine secretion originating from the stratified uterine epithelium of the uterine incubation chambers. Uterine secretions are absorbed by a specialized yolk sac ectoderm and chorioallantois through histotrophy. The yolk sac is not in close contact with the uterine lining from the limb bud stage onwards. The yolk sac ectoderm forms invaginations filled with uterine secretion and consists of a single layer of vacuolated hypertrophied cells bearing microvilli. The chorioallantois at the limb bud stage is extensive, well-vascularized, and not intimately associated with the uterine epithelium. Where the uterus is folded, the chorioallantois may interdigitate loosely. Chorionic cells are low to high columnar, clearly vacuolated, and bear microvilli. The allantoic layer consists primarily of squamous cells exhibiting villous projections. By the time embryos have well-defined digits, the specialized yolk sac ectoderm has regressed and the yolk sac lumen has been invaded by vitelline cells. The chorioallantois is very extensive and in areas greatly folded. Where it contacts the uterine epithelium, a proper chorioallantoic placenta is formed. Cell layers of the chorioallantois and uterine epithelium are thin and cuboidal to squamous in appearance. The chorioallantoic placenta is simple in structure, occurs throughout the incubation chamber, and is epitheliochorial in arrangement. It is unknown whether the placentome observed in other highly matrotrophic scincids is formed in late stage embryos of this species.     

Variation in eggshell characteristics and in intrauterine egg retention between two oviparous clades of the lizard Lacerta vivipara: insight into the oviparity-viviparity continuum in squamates

The concept of the oviparity-viviparity continuum refers to the wide range in the length of intrauterine egg retention and, hence, in the stage of embryonic development at oviposition existing in squamates. The evolutionary process underlying this continuum may involve not only a lengthening of egg retention in utero, but also a marked reduction in the thickness of the eggshell. The idea that there may exist a negative correlation between the developmental stage reached by the embryo at oviposition and the eggshell thickness within squamates, although supported by the comparison of oviparous vs. viviparous species, has seldom been evaluated by comparing eggshell thickness of oviparous forms with different lengths of intrauterine egg retention. Eggs of two distinct oviparous clades of the lizard Lacerta vivipara were compared. The eggs laid by females from Slovenian and Italian populations have thicker eggshells, contain embryos on average less developed at the time of oviposition, and require a longer incubation period before hatching than the eggs laid by females from French oviparous populations. Our data and several other examples available from the literature support the idea that the lengthening of intrauterine retention of eggs and the shortening of the subsequent external incubation of eggs are associated with reduction in the thickness of the eggshell, at least in some lineages of oviparous squamates. The current hypotheses that may account for this correlation are presented and a few restrictions and refinements to those hypotheses are discussed. In particular, other changes, such as increased vascularization of the oviduct and of the extraembryonic membranes, may play the same role as the decrease of eggshell thickness in facilitating prolonged intrauterine egg retention in squamates. Future studies should also consider the hypothesis that the length of intrauterine retention might directly depend on the extent of maternal-fetal chemical communication through the eggshell barrier.