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.