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Squamate reptiles (lizards and snakes) offer a unique model system for testing hypotheses about the evolutionary transition from oviparity (egg-laying) to viviparity (live-bearing) in amniote vertebrates. The evolution of squamate viviparity has occurred remarkably frequently (>108 times) and has resulted in major changes in reproductive physiology. Such frequent changes in reproductive strategy pose two questions: (1) what are the molecular mechanisms responsible for the evolution of squamate viviparity? (2) Are these molecular mechanisms the same for separate origins of viviparity? Molecular approaches, such as RT-PCR, in situ hybridisation, Western blotting and immunofluorescence, have been invaluable for identifying genes and proteins that are involved in squamate placental development, materno-foetal immunotolerance, placental transport, placental angiogenesis, hormone synthesis and hormone receptor expression. However, the candidate-gene or -protein approach that has been used until now does not allow for de novo gene/protein discovery; results to date suggest that the reproductive physiologies of mammals and squamate reptiles are very similar, but this conclusion may simply be due to a limited capacity to study the subset of genes and proteins that are unique to reptiles. Progress has also been slowed by the lack of appropriate molecular and genomic resources for squamate reptiles. The advent of next-generation sequencing provides a relatively inexpensive way to conduct rapid high-throughput sequencing of genomes and transcriptomes. We discuss the potential use of next-generation sequencing technologies to analyse differences in gene expression between oviparous and viviparous squamates, provide important sequence information for reptiles, and generate testable hypotheses for the evolution of viviparity. [ABSTRACT FROM AUTHOR]

Abstract: Regardless of the mode of reproduction, three things must occur at birth or hatching in amniote vertebrates: initiation of breathing, pulmonary fluid elimination and reabsorption, and adequate perfusion of pulmonary circulation. Although data on these events are few, there appears to be no fundamental difference in them that can be associated with the oviparity to viviparity transition. There are, however, differences in the timing of these events in oviparous and viviparous amniotes. The transition to neonatal respiration tends to be very quick in viviparous species because the vascular support for oxygen uptake provided by the mother is rapidly disassociated from the mechanism for uptake by the embryo. By contrast, hatching often is a slow process, taking 24 h or more in some species, as chorioallantoic blood flow slowly gives way to clearing of the lungs and pulmonary gas exchange. Little is known of the mechanisms of pulmonary fluid elimination and reabsorption or lung inflation in reptiles, but the cellular structures and surfactant systems are similar in all amniote vertebrates. Nevertheless, there are differences, particularly of timing and maturation of various systems, but there has been no exploration of the functional (or phylogenetic) bases of these differences. Perfusion of the neonatal pulmonary system to support respiration in reptiles remains to be investigated. In mammals and birds, closure of the ductus arteriosus is important, but the role played by the ductus arterioisus in reptilian hatching or birth is not known. [Copyright &y& Elsevier]

Occludin, an integral protein associated with the mammalian tight junction, has for the first time been identified in the uterus of squamate reptiles. The tight junction is made up of anastamosing strands and forms a selective barrier that regulates paracellular diffusion of solutes across uterine epithelium. Occludin exclusively labels tight junctional strands and is an excellent marker for tight junction permeability. Using western blotting and immunohistochemistry, occludin expression was examined in the uterine epithelium of five species of Australian skinks at different stages of gestation. More occludin was detected during late stage pregnancy/gravidity compared to the lower levels of occludin detected in vitellogenic and post-parturient females in three of the five species. We conclude that the paracellular permeability of the squamate uterine epithelium decreases as gestation progresses. As placental transport of ions and solutes to the embryo is highest during the last third of pregnancy in viviparous squamates, it is likely that a decrease in paracellular permeability is compensated by an upregulation of other transporting mechanisms such as histotrophy. [ABSTRACT FROM AUTHOR]

Abstract: Lampropholis guichenoti is an oviparous lizard that lays eggs with a calcareous outer shell. We used immunofluorescence microscopy to describe the occurrence and distribution of Ca2+ ATPase pumps in the uterus of L. guichenoti at different stages of the reproductive and egg-shelling cycles. Ca2+ ATPase pumps were not demonstrated by immunofluorescent techniques in any uterine tissue until egg-shelling had commenced and at least partly calcified eggs were in the uterus. During egg-shelling, Ca2+ ATPase pumps occur on the apical and baso-lateral surfaces of uterine epithelial cells, and those of associated shell glands in the stroma of the uterus. We conclude that Ca2+ ATPase pumps provide a major mechanism for deposition of the calcareous eggshell of L. guichenoti and that the pumps are up-regulated when required in the reproductive cycle. Furthermore, it is likely that specific calcium glands in the stroma of the uterus are involved in the rapid transport required for egg-shelling, but the differential contribution of luminal and glandular epithelial cells is not known. [Copyright &y& Elsevier]

Viviparity (live-bearing) has evolved from oviparity (egg-laying) more than 100 times within snakes and lizards, and thermal factors are thought to have driven this shift. However, other major features of reptilian reproduction may reflect selective pressures related to hydric rather than thermal exchanges between the egg and its incubation environment. Notably, why are intermediate stages of prolonged egg retention so rarely seen? Embryonic stages at oviposition in squamates are largely dichotomous: most oviparous species lay eggs with embryos about one-third developed, whereas viviparous taxa retain the eggs until development is complete. Why don't more species oviposit with embryos at either earlier or later stages? We suggest that the scarcity of squamates that lay eggs soon after ovulation (and thus, with very early-stage embryos) may reflect the need to delay oviposition until embryos have developed sufficient physiological control over water influx and efflux to survive in the challenging hydric environment of the nest. The scarcity of retention to later stages (intermediate between typical oviparity and viviparity), and the apparent lack of reversals from viviparity back to oviparity, may be due to a conflict between adaptations for water versus gas exchange; retention of larger embryos in utero requires eggshell thinning to allow gas exchange, but a thinner shell precludes effective hydric control after oviposition. Thus, although the transition from oviparity to viviparity in squamates has been driven largely by thermal advantages, the clustering of species at two main positions along the oviparity-viviparity continuum may be due to challenges of controlling embryonic water balance. [ABSTRACT FROM AUTHOR]

We compared the uterine component of the placentae of each placental region of two species of lizards, Eulamprus tympanum and Pseudemoia entrecasteauxii, to identify potential routes and mechanisms of nutrient transport. We focused on the uterine epithelium because nutrients must pass though it to reach the embryo. At this point, we have only been able to study two species in detail, but each has a placenta of different complexity and quantitatively different placentotrophy. The uterine epithelium of the chorioallantoic placenta has a different morphology from the omphaloplacenta, and the chorioallantoic placenta of P. entrecasteauxii, the most placentotrophic of the two species, is further differentiated in placentomal and paraplacentomal regions. The chorioallantoic region of E. tympanum, and the paraplacentomal region of P. entrecasteauxii have morphologies indicative of gas exchange epithelia. The blood-uterine lumen distance in P. entrecasteauxii, however, is about one tenth that in E. tympanum, suggesting a greater diffusive capacity for gases in P. entrecasteauxii. The omphaloplacenta of both species has a similar structure, with hypertrophied cells, electron-dense granules in the apical cytoplasm and vesicles budding off into the uterine lumen. This result is particularly interesting because E. tympanum exhibits little net nutrient transfer to the embryo during pregnancy. Nutrient provision across the omphaloplacenta in both species occurs by histotrophy. The placentome of P. entrecasteauxii also has a morphology suggestive of nutrient provision, but by some non-histotrophic mechanism and may be very important in the evolution of substantial placentotrophy. [ABSTRACT FROM AUTHOR]

The aim of this review is to collate data relevant to understanding the evolution of viviparity in general, and complex placentae in particular. The wide range of reproductive modes exhibited by lizards provides a solid model system for investigating the evolution of viviparity. Within the lizards are oviparous species, viviparous species that have a very simple placenta and little nutrient uptake from the mother during pregnancy (lecithotrophic viviparity), through a range of species that have intermediate placental complexities and placental nutrient provision, to species that lay microlecithal eggs and most nutrients are provided across the placenta during development (obligate placentotrophy). In its commonest form, lecithotrophic viviparity, some uptake of water, inorganic ions and oxygen occurs from the mother to the embryo during pregnancy. In contrast, the evolution of complex placentae is rare, but has evolved at least five times. Where there is still predominantly a reliance on egg yolk, the omphaloplacenta seems to be paramount in the provision of nutrition to the embryo via histotrophy, whereas the chorioallantoic placenta is more likely involved in gas exchange. Reliance on provision of substantial organic nutrient is correlated with the regional specialisation of the chorioallantoic placenta to form a placentome for nutrient uptake, particularly lipids, and the further development of the gas exchange capabilities of the other parts of the chorioallantois. [ABSTRACT FROM AUTHOR]

Abstract: Frequent evolution of viviparity in squamate reptiles provides excellent models for study of the transition from oviparity to viviparity, and the evolution of complex placentae. Embryonic reptiles have a chorioallantoic placenta at the embryonic pole of the egg, and an omphaloplacenta at the abembryonic pole. The uterus of viviparous reptiles and mammals exhibits common plasma membrane transformation including uterodome formation associated with pregnancy. The transition from oviparity to viviparity requires few changes: (1) reduction in an eggshell, (2) delay of expulsion of oviposition/birth, (3) increased vascularisation of the uterus for gas exchange. We used immunohistochemistry to address the question of eggshell thinning in a range of species, and SEM and TEM to infer the functions of different placental regions in one species with a complex placenta. We conclude that: (1) changes in the calcium channels among species accompany the evolution of viviparity; (2) the omphaloplacenta has a role in nutrient transport via apocrine secretion and histotrophy; (3) the placentome has a role in nutrient transport, but not via apocrine secretion; (4) the paraplacentome of the chorioallantoic placenta has a major role in gas exchange. [Copyright &y& Elsevier]

Most reptiles are oviparous, with the developing embryos relying on the contents of the yolk to sustain development until hatching (lecithotrophy). The yolk is composed primarily of lipid and protein, which act as an energy source and the essential components to build embryonic tissue. Nevertheless, yolk and the resulting embryos contain many other nutrients, including inorganic ions, vitamins, carotenoids, water and hormones. Apart from water and oxygen, which may be taken up by eggs, and some inorganic ions that can come from the eggshell or even from outside the egg, everything required by the embryo must be in the egg when it is laid. Approximately 20% of squamate reptiles are viviparous, exhibiting a variety of placental complexities. Species with complex placentae have reduced yolk volumes, with the mother augmenting embryonic nutrition by provision across the placenta (placentotrophy). Despite assumed advantages of placentotrophy, only 5 out of approximately 100 lineages of viviparous squamates exhibit substantial placentotrophy. This paper reviews available and recent information on the yolk contents of a variety of squamate reptiles to ask the question, how are nutrients transported from the yolk to the embryo or across the placenta? Although, current available data suggest that, in broad terms, yolk is taken up by embryos without discrimination of the nutrients, there are some apparent exceptions, including the very long chain polyunsaturated fatty acids. In addition, fundamental differences in the patterns of energy utilisation in lizards and snakes suggest fundamental differences in lipid profiles in these taxa, which appear to reflect the differences between placentotrophic and lecithotrophic viviparous lizards. [Copyright &y& Elsevier]

Historically, Australia has been important in the study of, and the development of hypotheses aimed at understanding, the evolution of viviparity in amniote vertebrates. Part of the importance of Australia in the field results from a rich fauna of skinks, including one of the broadest ranges of diversity of placental structures within one geographic region. During the last decade, we have focussed our studies on one lineage, the Eugongylus group of skinks of the subfamily Lygosominae because it contains oviparous species and some that exhibit complex placentae. Our specific objective has been to attempt to understand the fundamental steps required when viviparity, and ultimately complex placentae, evolve from oviparous ancestors. We have taken a three-prong approach: (1) detailed study of the morphology and ontogeny of the placentae of key species at the light microscope level; (2) study of changes in the uterus associated with pregnancy, or the plasma membrane transformation; and (3) measures of the net exchange of nutrients across the placenta or eggshell of key species. In turn, we have found that: (1) details of the morphology and ontogeny of placentae are more complex that originally envisaged, and that the early conclusions about a sequence in the evolution of complex placentae was naı¨ve; (2) a plasma membrane transformation occurs in viviparous, but not oviparous lizards, and thus may be a fundamental feature of the evolution of viviparity in amniotes; and (3) species with more complex chorioallantoic placentae tend to transport more nutrients across the placenta during pregnancy than those with simpler chorioallantoic placentae but, because the correlation is not tight, the importance of the omphaloplacenta in transporting nutrients may have been overlooked. Also, the composition of yolk of highly matrotrophic species is broadly similar, but not identical, to the yolk of oviparous species. Some of the interpretation of our data within the context of our specific objective is not yet possible, pending the publication of a robust phylogeny of Eugongylus group skinks. Once such a phylogeny is available, we are in a position to propose specific hypotheses about the evolution of viviparity that can be tested using another lineage of amniotes, possibly Mabuya group skinks. [Copyright &y& Elsevier]

Focuses on a study which measured the ion, energy, lipid, nitrogen and fat-soluble vitamin contents of freshly ovulated eggs and neonates of the viviparous lizard to quantify uptake of nutrients across the placenta. Similarity of the triacylglycerol fatty acid profiles of eggs and neonates; Description of phosphatidylcholine in the eggs; Amount of total lipid-soluble components; Conclusion.

Shark placentae are derived from modifications to the fetal yolk sac and the maternal uterine mucosa. In almost all placental sharks, embryonic development occurs in an egg capsule that remains intact for the entire pregnancy, separating the fetal tissues from the maternal tissues at the placental interface. Here, we investigate the structure and permeability of the egg capsules that surround developing embryos of the placental Australian sharpnose shark (Rhizoprionodon taylori) during late pregnancy. The egg capsule is an acellular fibrous structure that is 0.42 ± 0.04 μm thick at the placental interface between the yolk sac and uterine tissues, and 0.67 ± 0.08 μm thick in the paraplacental regions. This is the thinnest egg capsule of any placental shark measured so far, which may increase the diffusion rate of respiratory gases, fetal wastes, water and nutrients between maternal and fetal tissues. Molecules smaller than or equal to ~ 1000 Da can diffuse through the egg capsule, but larger proteins (~ 3000–26,000 Da) cannot. Similar permeability characteristics between the egg capsule of R. taylori and other placental sharks suggest that molecular size is an important determinant of the molecules that can be exchanged between the mother and her embryos during pregnancy. [ABSTRACT FROM AUTHOR]

The article focuses on the Fifth World Congress of Herpetology held at the University of Stellenbosch in South Africa in 2005. It cites papers presented at the conference, including a review of the utility of reptiles to a model of the evolution of viviparity. Also stated is that the rapid development that has been made in understanding the evolution of viviparity was highlighted in the event. It adds that one main advantage of the symposium was in establishing potential future collaborations.

Cites the occurrence of temperature-dependent sex determination in viviparous lizard. Effect of gestation temperature on the sex of viviparous lizard offspring; Other factors that influence thermoregulation in viviparous lizard mothers.

How innovations such as vision, flight and pregnancy evolve is a central question in evolutionary biology. Examination of transitional (intermediate) forms of these traits can help address this question, but these intermediate phenotypes are very rare in extant species. Here we explore the biology and evolution of transitional forms of pregnancy that are midway between the ancestral state of oviparity (egg‐laying) and the derived state, viviparity (live birth). Transitional forms of pregnancy occur in only three vertebrates, all of which are lizard species that also display intraspecific variation in reproductive phenotype. In these lizards (Lerista bougainvillii, Saiphos equalis, and Zootoca vivipara), geographic variation of three reproductive forms occurs within a single species: oviparity, viviparity, and a transitional form of pregnancy. This phenomenon offers the valuable prospect of watching 'evolution in action'. In these species, it is possible to conduct comparative research using different reproductive forms that are not confounded by speciation, and are of relatively recent origin. We identify major proximate and ultimate questions that can be addressed in these species, and the genetic and genomic tools that can help us understand how transitional forms of pregnancy are produced, despite predicted fitness costs. We argue that these taxa represent an excellent prospect for understanding the major evolutionary shift between egg‐laying and live birth, which is a fundamental innovation in the history of animals. [ABSTRACT FROM AUTHOR]

Mechanisms of reproductive allocation are major determinants of fitness because embryos cannot complete development without receiving sufficient nutrition from their parents. The nourishment of offspring via placentas (placentotrophy) has evolved repeatedly in vertebrates, including multiple times in squamate reptiles (lizards and snakes). Placentotrophy has been suggested to evolve only if food is sufficiently abundant throughout gestation to allow successful embryogenesis. If scarcity of food prevents successful embryogenesis, females should recoup nutrients allocated to embryos via abortion, reabsorption, and/or cannibalism. We tested these hypotheses in the placentotrophic southern grass skink Pseudemoia entrecasteauxii. We fed females one of four diets (high constant, high variable, low constant, and low variable) during gestation and tested the effects of both food amount and schedule of feeding on developmental success, cannibalism rate, placental nutrient transport, offspring size, and maternal growth and body condition. Low food availability reduced developmental success, placental nutrient transport, offspring size, and maternal growth and body condition. Cannibalism of offspring also increased when food was scarce. Schedule of feeding did not affect offspring or mothers. We suggest that high food abundance and ability to abort and cannibalize poor-quality offspring are permissive factors necessary for placentotrophy to be a viable strategy of reproductive allocation. [ABSTRACT FROM AUTHOR]

Abstract: Plastic changes occur in the morphology of the uterus at various stages of the reproductive cycle in both oviparous and viviparous lizards and these may be influenced by estrogen. Estrogen driven phosphorylation of effector proteins on tyrosine residues plays a major role in the plastic modulation of uterine anatomy and physiology in vertebrates. We used electrophoresis and Western blotting to characterize the phosphotyrosine protein profiles at various stages of the reproductive pathway in an oviparous lizard Lampropholis guichenoti and a viviparous lizard Eulamprus tympanum. L. guichenoti displayed major bands in the 200–35 kDa range and a triplet of bands of molecular masses 61 kDa, 52 kDa and 48 kDa in 50% of specimens and a 38 kDa band in all specimens. In contrast, E. tympanum samples all displayed a single major band at 40 kDa, which was significantly elevated at the early pregnancy stage. Somewhat paradoxically, the viviparous species, which has the more complex uterine epithelial changes during pregnancy, has the fewest phosphotyrosine bands, so how tyrosine phosphorylation is affected during the evolution of viviparity is not clear. [Copyright &y& Elsevier]

During gestation in the viviparous lizard Pseudemoia entrecasteauxii, the fetus obtains nutrients from two sources: uptake of yolk components from the retained egg (lecithotrophy) and transfer of nutrients from the maternal circulation via the placenta (placentotrophy). Although net placentotrophy in this species is indicated by the observation that the neonate contains 1.7 times more dry matter than the egg, the placental transfer of lipid has not been previously demonstrated. Lipid analysis was performed on newly ovulated eggs and on neonates. The weight of total lipid per neonate (8.2±0.5 mg) is significantly (P=0.049) greater than that in the egg (6.8±0.4 mg), indicating that the placenta must contribute some lipid to the fetus. On the assumption that 50% of the lipid delivered to the fetus from either source is oxidized for energy, it is calculated that the placenta accounts for 58.5% of the fetal lipid requirements, with the remaining 41.5% being derived from the egg. The fatty acid compositions of the triacylglycerol and phospholipid recovered in the neonatal tissue differ substantially from those of the egg. In particular, the proportions of 18:2n−6 and 18:3n−3 are far lower in the neonatal lipids compared with the egg lipids. On the other hand, the proportion of 22:6n−3 in the phospholipid of the neonate is six times higher than in the phospholipid of the egg. The absolute amount (mg) of 22:6n−3 recovered in the total lipid of the neonate is 3.8 times greater than the amount initially present in the egg. By comparison, the amount of total fatty acid in neonatal lipid is 1.2 times greater than the amount in the egg. Thus, there is a preferential use of 22:6n−3 for tissue phospholipid synthesis during development. We conclude that there is net transfer of fatty acids across the placenta to the fetus of P. entrecasteauxii and a high degree of selectivity in the use of the various fatty acids for fetal tissue lipid synthesis. [Copyright &y& Elsevier]

Viviparity has evolved from oviparity at least 150 independent times in vertebrates. More than 80% of these transitions have occurred in squamate reptiles, where both reproductive modes are rarely seen in different populations of the same species. This condition (bimodal reproduction) is ideal for studying the physiological and morphological changes underpinning the evolution of reproductive mode, and their genetic determinants. Here we analysed the genomes of Zootoca vivipara populations with either oviparous or viviparous reproduction using a RAD sequencing approach. No signature of interbreeding between oviparous and viviparous individuals was found. We conservatively identified 22 annotated coding sequences in genes potentially associated with parity mode differences. Six of these genes are transcription regulators that are also expressed in reproductive tissues of mammals and reptiles, suggesting that changes in gene expression are important for the evolution of viviparity. Using a more inclusive approach based on contigs mapping in either coding or non-coding regions, 45 genes were identified. Twelve of these candidate genes are transcription regulators and four encode protease enzymes. We propose that the evolution of proteases may support morphological changes to the uterus during pregnancy. This study provides the foundation for further experimental studies of the genetic basis of parity mode in Z. vivipara. [ABSTRACT FROM AUTHOR]

In oviparous amniotes (reptiles, birds, and mammals) the chorioallantoic membrane (CAM) lines the inside of the egg and acts as the living point of contact between the embryo and the outside world. In livebearing (viviparous) amniotes, communication during embryonic development occurs across placental tissues, which form between the uterine tissue of the mother and the CAM of the embryo. In both oviparous and viviparous taxa, the CAM is at the interface of the embryo and the external environment and can transfer signals from there to the embryo proper. To understand the evolution of placental hormone production in amniotes, we examined the expression of genes involved in hormone synthesis, metabolism, and hormone receptivity in the CAM of species across the amniote phylogeny. We collected transcriptome data for the chorioallantoic membranes of the chicken (oviparous), the lizards Lerista bougainvillii (both oviparous and viviparous populations) and Pseudemoia entrecasteauxii (viviparous), and the horse Equus caballus (viviparous). The viviparous taxa differ in their mechanisms of nutrient provisioning: L. bougainvillii is lecithotrophic (embryonic nourishment is provided via the yolk only), but P. entrecasteauxii and the horse are placentotrophic (embryos are nourished via placental transport). Of the 423 hormone-related genes that we examined, 91 genes are expressed in all studied species, suggesting that the chorioallantoic membrane ancestrally had an endocrine function. Therefore, the chorioallantoic membrane appears to be a highly hormonally active organ in all amniotes. No genes are expressed only in viviparous species, suggesting that the evolution of viviparity has not required the recruitment of any specific hormone-related genes. Our data suggest that the endocrine function of the CAM as a placental tissue evolved in part through co-option of ancestral gene expression patterns. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor A is a major mediator of angiogenesis, a critically important process in vertebrate growth and development as well as pregnancy. Here we report for the first time the expression of a rare and unusually potent splice variant, VEGF , in vivo in mammals. This variant has previously only been found in mammals in cultured human cells exposed to genotoxic agents. Our discovery of VEGF in the uterus of both a eutherian (rat) and a marsupial (fat-tailed dunnart) suggests that the splice variant may be common to all mammals. As VEGF is also expressed in the uterus of at least one lineage of lizards, the expression of this splice variant may be a widespread amniote phenomenon. We measured expression of VEGF and two major VEGF-A splice variants in the uterus of pregnant rats, showing that the three variants show different expression patterns across pregnancy. Our results suggest that viviparous mammals possess a precisely regulated milieu of VEGF isoforms producing the angiogenesis required for successful pregnancy. The discovery of VEGF in rat uterus paves the way for the development of in vivo models of VEGF activity in a highly tractable laboratory animal, and is particularly significant in the context of early pregnancy loss and cancer research. [ABSTRACT FROM AUTHOR]

The evolution of new organs is difficult to study because most vertebrate organs evolved only once, more than 500 million years ago. An ideal model for understanding complex organ evolution is the placenta, a structure that is present in live bearing reptiles and mammals (amniotes), which has evolved independently more than 115 times. Using transcriptomics, we characterized the uterine gene expression patterns through the reproductive cycle of a viviparous skink lizard, Pseudemoia entrecasteauxii. Then we compare these patterns with the patterns of gene expression from two oviparous skinks Lampropholis guichenoti and Lerista bougainvillii. While thousands of genes are differentially expressed between pregnant and non-pregnant uterine tissue in the viviparous skink, few differentially expressed genes were identified between gravid and non-gravid oviparous skinks. This finding suggests that in P. entrecasteauxii, a pregnant-specific gene expression profile has evolved, allowing for the evolution of pregnancy-specific innovations in the uterus. We find substantial gene expression differences between the uterus of the chorioallantoic and the yolk sac placenta in P. entrecasteauxii, suggesting these placental regions are specialized for different placental functions. In particular, the chorioallantoic placenta is likely a major site of nutrient transport by membrane-bound transport proteins, while the yolk sac placenta also likely transports nutrients but via apocrine secretions. We discuss how the evolution of transcription factor networks is likely to underpin the evolution of the new transcriptional states in the uterine tissue of viviparous reptiles. [ABSTRACT FROM AUTHOR]

Genomic imprinting is a process that results in the differential expression of genes depending on their parent of origin. It occurs in both plants and live-bearing mammals, with imprinted genes typically regulating the ability of an embryo to manipulate the maternal provision of nutrients. Genomic imprinting increases the potential for selection to act separately on paternally and maternally expressed genes, which increases the number of opportunities that selection can facilitate embryonic control over maternal nutrient provision. By looking for imprinting in an independent matrotrophic lineage, the viviparous lizard Pseudemoia entrecasteauxii (Scincidae), we test the hypothesis that genomic imprinting facilitates the evolution of substantial placental nutrient transport to embryos (matrotrophy). We sequenced transcriptomes from the embryonic component of lizard placentae to determine whether there are parent-of-origin differences in expression of genes that are imprinted in mammals. Of these genes, 19 had sufficiently high expression in the lizard to identify polymorphisms in transcribed sequences. We identified bi-allelic expression in 17 genes (including insulin- like growth factor 2), indicating that neither allele was imprinted. These data suggest that either genomic imprinting has not evolved in this matrotrophic skink or, if it has, it has evolved in different genes to mammals. We outline how these hypotheses can be tested. This study highlights important differences between mammalian and reptile pregnancy and the absence of any shared imprinting genes reflects fundamental differences in the way that pregnancy has evolved in these two lineages. [ABSTRACT FROM AUTHOR]

Although the morphological and physiological changes involved in pregnancy in live-bearing reptiles are well studied, the genetic mechanisms that underlie these changes are not known. We used the viviparous African Ocellated Skink, Chalcides ocellatus, as a model to identify a near complete gene expression profile associated with pregnancy using RNA-Seq analyses of uterine transcriptomes. Pregnancy in C. ocellatus is associated with upregulation of uterine genes involved with metabolism, cell proliferation and death, and cellular transport. Moreover, there are clear parallels between the genetic processes associated with pregnancy in mammals and Chalcides in expression of genes related to tissue remodeling, angiogenesis, immune system regulation, and nutrient provisioning to the embryo. In particular, the pregnant uterine transcriptome is dominated by expression of proteolytic enzymes that we speculate are involved both with remodeling the chorioallantoic placenta and histotrophy in the omphaloplacenta. Elements of the maternal innate immune system are downregulated in the pregnant uterus, indicating a potential mechanism to avoid rejection of the embryo. We found a downregulation of major histocompatability complex loci and estrogen and progesterone receptors in the pregnant uterus. This pattern is similar to mammals but cannot be explained by the mammalian model. The latter finding provides evidence that pregnancy is controlled by different endocrinological mechanisms in mammals and reptiles. Finally, 88% of the identified genes are expressed in both the pregnant and the nonpregnant uterus, and thus, morphological and physiological changes associated with C. ocellatus pregnancy are likely a result of regulation of genes continually expressed in the uterus rather than the initiation of expression of unique genes. [ABSTRACT FROM AUTHOR]

Frequent evolutionary changes in reproductive mode have produced a wide range of placental structures in viviparous squamate reptiles. Closely related species with different placental structures and resolved phylogenetic relationships are particularly useful for reconstructing how placentae might have transformed during the evolutionary process. We used light microscopy to study placental morphology in mid- to late stage embryos of four closely related species of Eulamprus, a genus of viviparous scincid lizards that we had reason to suspect may display significant interspecific variation in placental morphology. Embryos from all four species possess a chorioallantoic placenta, an omphaloplacenta and an interomphalopleuric membrane, characteristics present in other viviparous skinks. However, unlike other viviparous skinks but characteristic of oviparous skinks, the allantois expands to surround the yolk sac in each species, supplanting the omphalopleure with a larger area of chorioallantois until a chorioallantoic placenta surrounds the entire egg in one specimen that is only a few days from birth. All four Eulamprus species share the same extraembryonic membrane morphology, but the cellular morphology of the uterine epithelium in the chorioallantoic placenta and omphaloplacenta varies between species. We determined that the interomphalopleuric membrane is a shared derived character of the Eulamprus quoyii species group. New phylogenetic information indicates that variation in the chorioallantoic placenta is a result of two independent transitions, but that variation in the omphaloplacenta can be explained using a single change within the species studied. Our results indicate that E. quoyii group skinks are a valuable model for investigating the evolution of viviparity, as extraembryonic membrane development in these species shows features characteristic of both oviparous and viviparous skinks. [ABSTRACT FROM AUTHOR]

Although the morphological and physiological changes involved in pregnancy in live-bearing reptiles are well studied, the genetic mechanisms that underlie these changes are not known. We used the viviparous African Ocellated Skink, Chalcides ocellatus, as a model to identify a near complete gene expression profile associated with pregnancy using RNA-Seq analyses of uterine transcriptomes. Pregnancy in C. ocellatus is associated with upregulation of uterine genes involved with metabolism, cell proliferation and death, and cellular transport. Moreover, there are clear parallels between the genetic processes associated with pregnancy in mammals and Chalcides in expression of genes related to tissue remodeling, angiogenesis, immune system regulation, and nutrient provisioning to the embryo. In particular, the pregnant uterine transcriptome is dominated by expression of proteolytic enzymes that we speculate are involved both with remodeling the chorioallantoic placenta and histotrophy in the omphaloplacenta. Elements of the maternal innate immune system are downregulated in the pregnant uterus, indicating a potential mechanism to avoid rejection of the embryo. We found a downregulation of major histocompatability complex loci and estrogen and progesterone receptors in the pregnant uterus. This pattern is similar to mammals but cannot be explained by the mammalian model. The latter finding provides evidence that pregnancy is controlled by different endocrinological mechanisms in mammals and reptiles. Finally, 88% of the identified genes are expressed in both the pregnant and the nonpregnant uterus, and thus, morphological and physiological changes associated with C. ocellatus pregnancy are likely a result of regulation of genes continually expressed in the uterus rather than the initiation of expression of unique genes. [ABSTRACT FROM AUTHOR]

The eastern water skink ( Eulamprus quoyii) has lecithotrophic embryos and was previously described as having a simple Type I chorioallantoic placenta. Indeed, it was the species upon which the definition of a Type I placenta was thought to be based, although we had cause to question that assumption. Hence we have described the morphology of the uterus of E. quoyii and found it to be more complex than previously supposed. The mesometrial pole of the uterus in E. quoyii displays a vessel-dense elliptical structure (the VDE) with columnar uterine epithelial cells. As pregnancy proceeds, the uterine epithelium near the mesometrial pole becomes folded and glands become hypertrophied, so that the morphology of VDE resembles that of a placentome, characteristic of Type III placentae. Unlike species with a Type III placenta, the apposing chorioallantoic membrane of E. quoyii is lined with squamous cells and interdigitates with the folded uterine epithelium. The remainder of the uterus is thin with a squamous uterine epithelium throughout pregnancy. Immunohistochemical localisation of blood vessels reveals a dense network of small capillaries directly beneath the folded epithelium of the VDE, while blood vessels are larger and sparser at the abembryonic pole of the uterus. Alkaline phosphatase (AP) activity is present in the uterine epithelium and sub-epithelial blood vessels in newly ovulated females. AP activity disappears from the epithelium between stages 27 and 29 of embryonic development and from the blood vessels after stage 34, but appears in the uterine glands at stage 35, where it remains until the end of pregnancy. Although the VDE is structurally similar to the placentomes found in other viviparous lizards, different distributions of AP activity in the uterus of E. quoyii and Pseudemoia spenceri suggest that the VDE may be functionally different from the placentome of the latter species. Our description of uterine morphology in E. quoyii provides evidence that, at least in some lineages, the evolution of a placentome may not occur in concert with the evolution of microlecithal eggs and obligate placentotrophy. [ABSTRACT FROM AUTHOR]

We have discovered a modification of the uterus that appears to facilitate maternal-fetal communication during pregnancy in the scincid lizard Eulamprus quoyii. A vessel-dense elliptical area (VDE) on the mesometrial side of the uterus expands as the embryo grows, providing a large vascular area for physiological exchange between mother and embryo. The VDE is already developed in females with newly ovulated eggs, and is situated directly adjacent to the chorioallantois of the embryo when it develops. It is likely that signals from the early developing embryo determine the position of the VDE, as the VDE is off-centre in cases where the embryo sits obliquely in the uterus. The VDE is not a modification of the uterus over the entire chorioallantoic placenta, as the VDE is smaller than the chorioallantois after embryonic stage 33, but expansion of the VDE and growth of the chorioallantois during pregnancy are strongly correlated. The expansion of the VDE is also strongly correlated with embryonic growth and increasing embryonic oxygen demand (VO2). We propose that angiogenic stimuli are exchanged between the VDE and the chorioallantois in E. quoyii, allowing the simultaneous growth of both tissues. [ABSTRACT FROM AUTHOR]

Abstract: Embryos of oviparous lizards have two sources of calcium for embryonic development: 1) calcium that accumulates in yolk during vitellogenesis, and 2) calcium carbonate deposited in the eggshell from oviductal secretions. Eggs of viviparous lizards lack a calcified eggshell and calcium secreted by the uterus is delivered to the embryo across a placenta. Whereas oviparous lizard embryos recover calcium from the eggshell during late developmental growth stages, viviparous embryos have a lengthy intimate association with the uterus and the potential for an extended interval of placental calcium transfer. We compared the pattern of calcium mobilization of embryos of the viviparous, placentotrophic scincid lizard, Pseudemoia pagenstecheri, to that of a closely related oviparous species, Saproscincus mustelinus, to determine if the timing of uterine calcium secretion was influenced by reproductive mode. Embryos of both species receive a substantial amount of calcium from either the eggshell or placenta (54% and 85% respectively). The ontogeny of calcium uptake by embryos of P. pagenstecheri reveals that the onset of embryonic acquisition of calcium occurs earlier relative to embryonic stage but the timing of peak uterine secretion of calcium is delayed, compared to S. mustelinus. [Copyright &y& Elsevier]

Calcium is a major requirement for developing embryos, but an insufficient amount is present in egg-yolk to sustain embryonic development in most reptiles. Consequently, the eggshell acts as an extra-lectithal source of calcium in oviparous amniote vertebrates, including squamates. A number of anatomical and physiological changes must accompany the evolutionary transitions from oviparity to viviparity, including the reduction or elimination of the eggshell. In our quest to understand the evolution of viviparity, we have used indirect immunofluorescence to characterize Ca2+ATPase pumps in the uterus of a targeted range of species of Eugongylus group skinks and bird species at various stages of the reproductive cycle, including pregnancy. During egg-shelling, apical localisation of Ca2+ATPase pumps occurs in the glandular epithelium of the uterus in the oviparous lizard Lampropholis guichenoti and the King quail Coturnix chinensis, presumably to allow movement of calcium against its electrochemical gradient into the lumen of the glandular duct and ultimately to the uterine lumen and developing embryo. Interestingly, structurally similar shell glands containing Ca2+ATPase pumps occur in the uterine lamina propria (stroma) of viviparous species, although we have so far seen them only in early vitellogenic N. ocellatus and non-pregnant female Pseudemoia spenceri. Our findings suggest that the down regulation of Ca2+ATPase in the shell glands may be an important contributor to the evolution of viviparity through the loss of an eggshell. [ABSTRACT FROM AUTHOR]

Abstract: The gene HoxA10 and its protein product are essential for the formation of the extensions of the plasma membrane called uterodomes or pinopods in mammalian uterine epithelia. In mice, the presence of the HoxA10 protein and uterodomes is needed for uterine receptivity to blastocyst implantation. The viviparous lizard Eulamprus tympanum displays uterodomes whereas the oviparous lizard Lampropholis guichenoti does not. To explore the theory that HoxA10 is involved in the formation of uterodomes we investigated whether HoxA10 immunoreactive proteins were present in both species during their reproductive cycles. Oviduct proteins from vitellogenic, gravid or non-reproductive L. guichenoti (n =19) and E. tympanum (n =28) were separated by electrophoresis and analysed by Western blot and specific antibodies to HoxA10. E. tympanum displayed HoxA10 immunoreactive bands at 59 and 63 kDa in 20 out of the 28 samples. All of the L. guichenoti samples displayed HoxA10 immunoreactive bands, 18 had bands at 59 and 64 kDa and 1 animal had a single band at 59 kDa. There were no significant differences in the level of HoxA10 immunoreactivity between the different stages of reproductive cycle in either species. The different molecular mass of the larger band in L. guichenoti (64 kDa) compared to E. tympanum (63 kDa) indicates that the two lizards express different isoforms of the HoxA10-like proteins and it will be interesting in future studies to determine whether there are differences in the biological activity of the proteins that regulate different physiological functions in the uterus of viviparous and oviparous lizards. [Copyright &y& Elsevier]