Jacquie F. Herbert

CALCIUM TRANSPORT ACROSS THE UTERINE EPITHELIUM OF PREGNANT LIZARDS

ABSTRACT 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.

Uterine and eggshell structure and histochemistry in a lizard with prolonged uterine egg retention (Lacertilia, Scincidae, Saiphos)

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. J. Morphol., 2010.

Maternal provision and embryonic uptake of calcium in an oviparous and a placentotrophic viviparous Australian lizard (Lacertilia: Scincidae)

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.

Placental calcium provision in a lizard with prolonged oviductal egg retention

A prominent scenario for the evolution of viviparity and placentation in reptiles predicts a step-wise pattern with an initial phase of prolonged oviductal egg retention accompanied by progressive reduction in eggshell thickness culminating in viviparity; calcium placentotrophy evolves secondarily to viviparity. Saiphos equalis is an Australian scincid lizard with a reproductive mode that is uncommon for squamates because eggs are retained in the oviduct until late developmental stages, and the embryonic stage at oviposition varies geographically. We studied calcium mobilization by embryos in two populations with different oviductal egg retention patterns to test the hypothesis that the pattern of nutritional provision of calcium is independent of the embryonic stage at oviposition. Females from one population are viviparous and oviposit eggs containing fully formed embryos, whereas embryos in oviposited eggs of the second population are morphologically less mature, and these eggs hatch several days later. The reproductive mode of this population is denoted as prolonged oviductal egg retention. Yolk provided the highest proportion of calcium to hatchlings in both populations. Eggs of both populations were enclosed in calcified eggshells, but shells of the population with prolonged egg retention had twice the calcium content of the viviparous population and embryos recovered calcium from these eggshells. Placental transfer accounted for a substantial amount of calcium in hatchlings in both populations. Hatchling calcium concentration was higher in the population with prolonged egg retention because these embryos mobilized calcium from yolk, the eggshell and the placenta. This pattern of embryonic calcium provision in which both a calcified eggshell and placentotrophy contribute to embryonic nutrition is novel. The reproductive pattern of S. equalis illustrates that calcified eggshells are compatible with prolonged oviductal egg retention and that viviparity is not requisite to calcium placentotrophy.

Comparison of the fatty-acid compositions of prey items and yolks of Australian insectivorous scincid lizards.

The yolk fatty-acid profiles of a range of species of insectivorous scincid lizards generally conform to a common pattern, typified by high proportions of linoleic acid (13.5–18.5% of total fatty acids), substantial proportions of α-linolenic acid (2.4–8.2%), and significant amounts of the long-chain polyunsaturated fatty acids, arachidonic (1.6–3.3%), eicosapentaenoic (0.7–1.2%) and docosahexaenoic (0.7–1.6%) acids. We characterised the fatty-acid compositions of ten prey taxa that are eaten by female skinks during vitellogenesis. Linoleic acid is the major polyunsaturated fatty acid in all prey, excepting Orthoptera where α-linolenic acid predominates. To varying extents, α-linolenic acid is present in all the prey items. Arachidonic acid forms over 1% of total fatty acids for six of the prey items. Four of the prey items contain eicosapentaenoic acid at over 1%. Most notably, docosahexaenoic acid is essentially absent from all the prey items. There is a general similarity between the fatty-acid profiles of prey and yolk, suggesting that the linoleic, α-linolenic, arachidonic and eicosapentaenoic acids required for egg formation can be supplied directly from the maternal diet. However, the docosahexaenoic acid of the egg lipids cannot derive from the diet and must, therefore, be formed by biosynthesis in the maternal liver, using dietary α-linolenic and eicosapentaenoic acids as precursors.

Placental development and expression of calcium transporting proteins in the extraembryonic membranes of a placentotrophic lizard

Pseudemoia pagenstecheri is a viviparous Australian scincid lizard in which the maternal–embryonic placental interface is differentiated into structurally distinct regions. The chorioallantoic placenta contains an elliptical‐shaped region, the placentome, characterized by hypertrophied uterine and embryonic epithelial cells supported by dense vascular networks. The remainder of the chorioallantoic placenta, the paraplacentome, is also highly vascularized but uterine and chorionic epithelia are thin. An omphaloplacenta with hypertrophied epithelia is located in the abembryonic hemisphere of the egg. There is extensive placental transport of organic and inorganic nutrients, e.g., 85–90% of neonatal calcium is received via placental transfer. Calcium uptake by extraembryonic membranes of squamates correlates with expression of the intracellular calcium binding protein, calbindin‐D28K, and plasma membrane calcium ATPase (PMCA) is a marker for active calcium transport. We estimated expression of calbindin‐D28K and PMCA in the chorioallantoic membrane in a developmental series of embryos using immunoblotting and used immunohistochemistry to define the cellular localization of calbindin‐D28K to test the hypotheses that 1) expression of calcium transporting proteins is coincident with placental transport of calcium and 2) the placenta is functionally specialized for calcium transport in regions of structural differentiation. Calbindin‐D28K and PMCA were detected at low levels in early stages of development and increased significantly prior to birth, when embryonic calcium uptake peaks. These data support the hypothesis that placental calcium secretion occurs over an extended interval of gestation, with increasing activity as embryonic demand escalates in late development. In addition, calbindin‐D28K expression is localized in chorionic epithelial cells of the placentome and in the epithelium of the omphalopleure of the omphaloplacenta, which supports the hypothesis that regional structural differentiation in the placenta reflects functional specializations for calcium transport. J. Morphol. 2012.