The eggshell of oviparous lizards is a significant source of calcium for embryos, whereas the eggshell of viviparous lizards, when present, contains little calcium. In view of the potential cost to embryonic nutrition occasioned by the loss of eggshell calcium, the large number of independent origins of viviparity among lizards is surprising. Concomitant evolution of viviparity and calcium placentotrophy would ameliorate the loss of eggshell calcium, but a mechanism linking these events has yet to be discovered. Zootoca vivipara, a lizard with geographic variation in its mode of parity, is an excellent model for studying mechanisms of calcium transport to oviparous and viviparous embryos because each is highly dependent on calcium secreted by the uterus (eggshell or placenta) and ontogenetic patterns of embryonic calcium mobilization are similar. We compared developmental expression of the calcium transport protein calbindin-D 28K in yolk splanchnopleure and chorioallantoic membranes of oviparous and viviparous embryos to test the hypothesis that the mechanism of calcium transport does not differ between modes of parity. We found that the ontogenetic pattern of protein expression is similar between reproductive modes and is correlated with calcium uptake from yolk and either eggshell or placenta. Calbindin-D 28K is localized in the chorionic epithelium of embryos of both reproductive modes. These findings suggest that the embryonic calcium transport machinery is conserved in the transition between reproductive modes and that an adaptation of oviparous embryos for calcium uptake from eggshells functions similarly to transport calcium directly from uterine secretions.

The chorioallantoic membrane resides adjacent to either the inner surface of the egg shell or uterine epithelium in oviparous and viviparous reptiles, respectively. Chorionic cells face the shell or uterine epithelium and transport calcium to underlying embryonic capillaries. Calcium transport activity of the chorioallantois increases in the final stages of development coincident with rapid embryonic growth and skeletal ossification. We excised embryos from viviparous Zootoca vivipara females at a stage prior to significant calcium accumulation and incubated them ex utero with and without calcium to test the hypothesis that chorioallantois calcium transport activity depends on developmental stage and not calcium availability. We measured calcium uptake by monitoring incubation media calcium content and chorioallantois expression of calbindin-D28K, a marker for transcellular calcium transport. The pattern of calcium flux to the media differed by incubation condition. Eggs in 0 mM calcium exhibited little variation in calcium gain or loss. For eggs in 2 mM calcium, calcium flux to the media was highly variable and was directed inward during the last 3 days of the experiment such that embryos gained calcium. Calbindin-D28K expression increased under both incubation conditions but was significantly higher in embryos incubated with 2 mM calcium. We conclude that embryos respond to calcium availability, yet significant calcium accumulation is developmental stage dependent. These observations suggest the chorioallantois exhibits a degree of functional plasticity that facilitates response to metabolic or environmental fluctuations.

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-D 28K, and plasma membrane calcium ATPase (PMCA) is a marker for active calcium transport. We estimated expression of calbindin-D 28K and PMCA in the chorioallantoic membrane in a developmental series of embryos using immunoblotting and used immunohistochemistry to define the cellular localization of calbindin-D 28K 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-D 28K 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-D 28K 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.

Embryos of oviparous squamate reptiles obtain all organic and most inorganic nutrients from yolk; yolk provides 19-86% of hatchling calcium content. The remaining calcium is extracted from the eggshell. Yolk calcium provision to viviparous embryos also is variable and includes three patterns. The contribution of yolk to embryonic development for most viviparous squamates is similar to oviparous species, but the attenuated eggshell of viviparous species is a poor source of calcium because it lacks an outer layer of calcium carbonate, and embryos supplement yolk calcium via placental transfer. In a second pattern, yolk provides all organic nutrients and calcium. The final pattern occurs in viviparous species that are substantially placentotrophic and placental transfer accounts for most organic and inorganic nutrients, including calcium. The many independent evolutionary transitions to viviparity among squamates have inspired interest in a possible link to patterns of embryonic calcium nutrition. A prominent model predicts that the pattern of maternal provision and embryonic uptake of calcium unique to squamates facilitates the evolution of viviparity. A primary assumption of the model is that the evolution of viviparity precedes the evolution of calcium placentotrophy. An alternative model predicts that viviparity and placentotrophy evolve concurrently because mechanisms for nutrient provision and mobilization are not dependent on reproductive mode. These hypotheses have not been tested directly but review of the literature indicates that neither fully explains the diversity of squamate embryonic calcium nutrition. Viviparous species differ from oviparous species primarily in the timing of uterine calcium secretion and structure of eggshell calcium. Future studies should focus on the mechanisms that promote these differences.

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