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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: 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]

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]

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]

Abstract: Calcium ATPase (Ca2+-ATPase) is a key enzyme that participates in the translocation of calcium in the uterus of oviparous amniotes during eggshell formation. We used Western blot and indirect immunofluorescence microscopy to determine expression and localisation of uterine Ca2+-ATPase during the reproductive cycle of king quail and zebra finch. The pattern of Ca2+-ATPase expression and localisation during the reproductive cycle was similar for both species. Immunoblots of uterine extracts from quail and finch indicated that Ca 2+-ATPase expression is reduced in non-reproductive compared to reproductive females. Similarly, in non-reproductive females, weak apical immunofluorescent staining of Ca2+-ATPase is localised to epithelial cells in a small number of uterine tubular glands. A large increase in apical immunofluorescent staining of tubular gland epithelia occurs in both vitellogenic and reproductive females. The presence of Ca2+-ATPase on the apical surface of tubular gland epithelial cells suggests that the enzyme is involved in the translocation of calcium out of the tubular gland epithelia and into the concentrated fluid of the uterine lumen. Presence of Ca2+-ATPase in vitellogenic females indicates that the enzyme is expressed prior to the time of ovulation and eggshell calcification. [Copyright &y& Elsevier]

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]

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]

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]

The eggshell of lizards is a complex structure composed of organic and inorganic molecules secreted by the oviduct, which protects the embryo by providing a barrier to the external environment and also allows the exchange of respiratory gases and water for life support. Calcium deposited on the surface of the eggshell provides an important nutrient source for the embryo. Variation in physical conditions encountered by eggs results in a tradeoff among these functions and influences eggshell structure. Evolution of prolonged uterine egg retention results in a significant change in the incubation environment, notably reduction in efficiency of gas exchange, and selection should favor a concomitant reduction in eggshell thickness. This model is supported by studies that demonstrate an inverse correlation between eggshell thickness and length of uterine egg retention. One mechanism leading to thinning of the eggshell is reduction in size of uterine shell glands. Saiphos equalis is an Australian scincid lizard with an unusual pattern of geographic variation in reproductive mode. All populations retain eggs in the uterus beyond the embryonic stage at oviposition typical for lizards, and some are viviparous. We compared structure and histochemistry of the uterus and eggshell of two populations of S. equalis, prolonged egg retention, and viviparous to test the hypotheses: 1) eggshell thickness is inversely correlated with length of egg retention and 2) eggshell thickness is positively correlated with size of shell glands. We found support for the first hypothesis but also found that eggshells of both populations are surprisingly thick compared with other lizards. Our histochemical data support prior conclusions that uterine shell glands are the source of protein fiber matrix of the eggshell, but we did not find a correlation between size of shell glands and eggshell thickness. Eggshell thickness is likely determined by density of uterine shell glands in this species.

Calcium ATPase (Ca2+-ATPase) is a key enzyme that participates in the translocation of calcium in the uterus of oviparous amniotes during eggshell formation. We used Western blot and indirect immunofluorescence microscopy to determine expression and localisation of uterine Ca2+-ATPase during the reproductive cycle of king quail and zebra finch. The pattern of Ca2+-ATPase expression and localisation during the reproductive cycle was similar for both species. Immunoblots of uterine extracts from quail and finch indicated that Ca2+-ATPase expression is reduced in non-reproductive compared to reproductive females. Similarly, in non-reproductive females, weak apical immunofluorescent staining of Ca2+-ATPase is localised to epithelial cells in a small number of uterine tubular glands. A large increase in apical immunofluorescent staining of tubular gland epithelia occurs in both vitellogenic and reproductive females. The presence of Ca2+-ATPase on the apical surface of tubular gland epithelial cells suggests that the enzyme is involved in the translocation of calcium out of the tubular gland epithelia and into the concentrated fluid of the uterine lumen. Presence of Ca2+-ATPase in vitellogenic females indicates that the enzyme is expressed prior to the time of ovulation and eggshell calcification.

Placental nutrient provision has evolved in multiple lineages of squamate reptiles and although possible structural specializations for placentotrophy have been described in a variety of species, neither the pathways nor the mechanisms of placental transfer are known. Lizards of the Australian genus Pseudemoia are placentotrophic and have elaborate placental structures that are thought to enhance nutrient transfer. The chorioallantoic placenta, which occupies the embryonic hemisphere of the egg, is regionally diversified into a large area with low epithelial height and a smaller placentome with cuboidal or columnar epithelia. Both regions are underlain by an extensive vascular bed. The abembryonic hemisphere of the egg is covered by an omphaloplacenta, which is similar to the placentome in having cuboidal or columnar epithelia but with a different embryonic vascular supply. We tested the hypothesis that embryonic epithelial cells of the placentome and the omphaloplacenta of Pseudemoia entrecasteauxii are each capable of endocytosis. Embryos (stages 33-39) with intact extraembryonic membranes were surgically removed from the uterus and incubated in a solution containing fluorescein isothiocyanate-dextran (77,000 MW). The fluorescent label was detected in the cytoplasm of scattered populations of epithelial cells in both placental regions of all embryonic stages. We conclude that both the placentome and the omphaloplacenta of P. entrecasteauxii are sites of histotrophic nutrient transport. However, there are histological and cytological differences in the embryonic epithelia of these two placental regions. The histological differences reflect differences in the evolutionary precursors of each tissue. The cytological differences likely portray different functional specializations., (2006 Wiley-Liss, Inc.)