不同繁殖模式石龙子的胚胎钙供应及其分子机制

Viviparity is a reproductive mode evolving from oviparity through gradual increases in the length of intrauterine development until parturition. This transition requires dramatic changes to reproductive morphology and physiology, and is among the most fundamental evolutionary transitions. The primary pattern of embryonic nutrition for squamates is lecithotrophy. The evolution of viviparity presents an opportunity for an additional source of embryonic nutrition through delivery of uterine secretions. The assumptions that nutrients for oviparous embryos are mobilized from yolk, and that this source is not disrupted in the transition to viviparity, are supported for most nutrients. In contrast, calcium, an essential nutrient for embryonic development, is mobilized from both yolk and eggshell by oviparous embryos and reduction of eggshell calcium is correlated with viviparity. If embryonic fitness is compromised by disruption of a primary source of calcium, selection may not favor evolution of viviparity, yet viviparity has arisen independently in many squamate lineages. Studies of fetal nutrition in reproductively bimodal species suggest a resolution to this paradox. If uterine calcium secretion occurs during egg retention, calcium placentotrophy evolves prior to viviparity as a replacement for eggshell calcium and embryonic nutrition will not be compromised. This hypothesis is integrated into the current model for evolution of viviparity to address the unique attributes of calcium nutrition. The sequence of events requires a shift in timing of uterine calcium secretion and the embryonic mechanism of calcium retrieval to be responsive to calcium availability. Regulation of uterine calcium secretion and the mechanism of embryonic uptake of calcium are important elements to understanding evolution of viviparity. Although much is known about the morphological and physiological changes involved in reptilian pregnancy, there has been little examination of the underlying genetic mechanism. Nowadays, advances in next-generation transcriptome sequencing allow detailed analysis of gene expression in non-model organisms, which provides the snapshots of nearly all the genes expressed in a tissue and allows direct comparisons of molecular function amongst squamate lineages. Here we will use three pairs of congeneric skinks with different reproductive modes to study calcium dynamics in eggshell and yolk at different embryonic stages. We will compare the transcriptomic data of the uterus tissues from non-pregnant, pregnant and postpartum females to elucidate the molecular mechanisms of calcium provision during pregnancy. This study aims to address questions of regulation of uterine calcium secretion and the mechanism of embryonic uptake of calcium which are important elements to understanding evolution of viviparity, and data generated from this study will provide the clue for the gradual evolutionary transition from oviparity to viviparity in squamate reptiles.

繁殖模式由卵生到卵胎生的进化转变是受自然选择驱动的，直接影响物种的繁殖、形态、生理和行为等方面，要求动物在形态和生理上发生相应的变化。因此有鳞类卵生进化研究一直是学术界关注的热点问题。尽管该研究领域已在形态和生理方面开展了大量研究，但这种变化背后潜在的分子机制尚不清楚，迫切需要选取合适的动物模型开展深入研究。本项目拟以三组同属但不同繁殖模式的石龙子为材料，通过实验操纵缩短或延长卵滞留时间，检测各胚胎时期新生卵胚胎时期与卵壳或胚胎膜以及卵黄内钙含量关系，比较同属不同繁殖模式石龙子新生卵胚胎和幼体的干重比，在转录组水平分别比较三组同属不同繁殖模式石龙子在未怀卵、怀卵和产后状态输卵管组织的基因表达差异，筛选与石龙子胚胎钙供应相关的调控基因并进一步比较其在不同时期的表达量变化。研究结果旨在探讨不同繁殖模式石龙子在胚胎不同发育时期钙供应差异及其内在的分子机制，为阐述卵胎生的渐进式进化提供实验证据。

繁殖模式由卵生到卵胎生的进化转变是受自然选择驱动的，直接影响物种的繁殖、形态、生理和行为等方面。因此有鳞类卵生进化研究一直是学术界关注的热点问题。至今，繁殖模式进化背后潜在的分子机制尚不清楚，迫切需要选取合适的动物模型开展深入研究。本项目以三组同属但不同繁殖模式的石龙子为材料，检测各胚胎时期新生卵胚胎时期与卵壳或胚胎膜以及卵黄内钙含量关系，在转录组水平分别比较三组同属不同繁殖模式石龙子在未怀卵、怀卵和产后状态输卵管组织的基因表达差异，筛选与石龙子胚胎钙供应相关的调控基因并进一步比较其在不同时期的表达量变化。本项目通过RACE技术对CaBP-D28k和PTGS2两个基因进行全长扩增，测定这两个基因在不同繁殖模式不同历期输卵管组织中的表达情况，以研究这两个与钙离子调控相关的基因在不同繁殖模式下促进母体产卵的具体过程和作用机制。本项目完成南滑蜥，宁波滑蜥，印度蜓蜥，股鳞蜓蜥四个物种处于不同繁殖阶段的共100个雌体输卵管组织样品的转录组测序，基于输卵管组织的转录组数据发现石龙子卵胎生进化过程主要表现在卵壳腺退化、气体交换、粘附和胚胎发育这三个方面。两种卵生物种中与卵壳腺形成相关的基因eEF在产前都呈现出相同高表达趋势，两种卵胎生物种中与胎盘粘附相关的基因BMP在怀卵中期都呈现出高表达的趋势；基因调控过程揭示两种繁殖模式进化出了不同的气体交换机制。这三个方面的证据都揭示了卵生向卵胎生进化过程中具有趋同性的特点。CaBP-D28k和PTGS2基因在相同繁殖模式物种的不同胚胎发育时期的输卵管组织中表达量在四个物种中具有高度一致性，主要表现在这两个基因在卵生临产30-31期表达量最高，在卵胎生石龙子30-31期胚胎之后表达下调，在临产期40期表达量上升，与国内外同行已报道的研究结果相一致。本项目研究结果揭示了这两个基因在石龙子卵胎生进化过程中起到非常重要的作用，为阐述卵胎生的渐进式进化提供新的实验证据。