Evolution of Placental Calcium Transport in Reptiles

陆生脊椎动物表现出两种基本的繁殖模式：胎生和卵产，其中胚胎发育发生在雌性身体之外。除单孔动物外，所有哺乳动物都是胎生的。海龟、鳄鱼和鸟类完全是卵生的。相比之下，鳞状爬行动物(蛇和蜥蜴)表现出多样化的繁殖模式，大约20%是胎生的，至少有三种是双峰繁殖的。鳞类中的胎盘已经独立进化了100多次，胎盘的进化与胚胎营养模式的转变有关，从加强卵子中的能量储存到胎盘营养交换。所有脊椎动物胚胎都需要钙才能成功发育。卵生物种向胚胎提供钙的来源和方式与胎生物种不同。卵生物种的胚胎从卵黄和蛋壳中获得钙，这些来源是在卵黄发生和产蛋前的脱壳过程中积累下来的。胎生胚胎从卵黄中获得一些钙，但也依赖于发育后期的胎盘转移。钙转运到胚胎的时间、来源和机制是否影响导致胎生的进化创新的潜力？该项目将探索在陆地脊椎动物中表现出最高生殖模式多样性的一个谱系(鳞状爬行动物)中，胚胎钙供应模式与生殖模式(产卵或活产)之间的关系。我们将比较选定的卵生和胎生鳞状动物胚胎的钙供应模式和机制，以开发一个胎盘钙运输进化的模型。该项目将比较子宫分泌钙的时间和机制以及胚胎动员钙的时间和机制：1)最近进化出胎盘的两种物种，2)胎盘钙转运较少的胎生物种，以及3)严重依赖胎盘钙的胎生物种。亲缘关系密切的产卵物种将被研究以进行比较。原子吸收光谱将被用来定义胚胎吸收钙的模式。通过免疫印迹和免疫组织化学方法确定钙转运蛋白Calbindin-D28K、Ca~(2+)-ATPase的发育表达，研究子宫和胚胎膜钙转运的时间和机制。这项研究将有助于通过研究高度保守的向胚胎输送营养的机制来更好地了解陆生脊椎动物的胚胎发育。该项目将通过在博士授予系的研究环境中为以本科生为主的本科生提供机会，将研究和教育结合起来。将通过与校园罗纳德·E·麦克奈尔学者计划的管理人员合作，积极招募来自社会代表性不足部分的本科生。这些努力将针对任何院校的任何符合条件的本科生。这些学生将接受培训，目的是提高他们未来在研究生项目中取得成功的潜力。

Terrestrial vertebrates exhibit two basic modes of reproduction; viviparity, or the birth of live young, and oviparity, or the laying of eggs in which embryonic development occurs outside the body of the female. All mammals, with the exception of monotremes, are viviparous. Turtles, crocodilians and birds are exclusively oviparous. In contrast, squamate reptiles (snakes and lizards) show a diversity of reproductive patterns with approximately 20% being viviparous and at least three species being reproductively bimodal. Viviparity in squamates has evolved independently more than 100 times and the evolution of viviparity is associated with a transition in the pattern of embryonic nutrition from fortification of energy stores in the egg to placental nutrient exchange. All vertebrate embryos require calcium for successful development. The source and pattern of calcium provision to embryos of oviparous species differs from that of viviparous species. Embryos of oviparous species derive calcium from yolk and eggshell, sources laid down during vitellogenesis and shelling prior to egglaying. Viviparous embryos derive some calcium from yolk but depend also on placental transfer late in development. Does the timing, source and mechanism of calcium transport to embryos influence the potential for evolutionary innovations leading to viviparity? This project will explore the relationship between the pattern of embryonic calcium provision and reproductive mode (egg-laying or live-bearing) in a lineage (squamate reptiles) that exhibits the highest diversity of reproductive patterns among terrestrial vertebrates. The pattern and mechanism of calcium provision to embryos of selected oviparous and viviparous squamates will be compared to develop a model for the evolution of placental calcium transport. The project will compare the timing and mechanism of calcium secretion by the uterus and the timing and mechanism of calcium mobilization by embryos of: 1) two species that have recently evolved viviparity, 2) a viviparous species with little placental calcium transport, and 3) a viviparous species that is heavily dependent on placental calcium. Egg-laying species that are closely related will be studied for comparison. Atomic absorption spectroscopy will be used to define the pattern of calcium uptake by embryos. The timing and mechanism of calcium transport in the uterus and embryonic membranes will be studied by defining the developmental expression of the calcium transporting proteins, calbindin-D28K, Ca2+-ATPase, using immunoblotting and immunohistochemistry. This research will contribute a greater understanding of embryonic development of terrestrial vertebrates through study of a highly conserved mechanism for delivery of nutrients to embryos. The project will integrate research and education by providing opportunities for undergraduate students from predominantly undergraduate departments in the research environment of a Ph.D. granting department. Undergraduate students from underrepresented segments of society will be actively recruited through collaboration with administrators of the Ronald E. McNair Scholars Program on campus. These efforts will target any eligible undergraduate from any institution. These students will receive training with an aim to increase their potential for future success in graduate programs.