The role of microRNAs in regulation of bovine ovarian follicular development

卵巢卵泡生理涉及复杂而协调的过程和不同细胞类型的相互作用，已知依赖于多种基因的严格调控表达和相互作用。在本研究中，我们将通过对先前研究中发现的microrna进行系统表征和功能分析，来研究microrna在卵巢卵泡发育相关基因调控中的作用。首先，24个候选mirna将在第3天、第7天和第19天分别对应于选择期、优势期和排卵前阶段，在卵泡膜、颗粒细胞、卵丘-卵母细胞复合物中的表达情况进行研究。基于这一结果，我们将研究筛选出的一些预测靶mrna的差异表达模式，并与相关mirna的表达谱进行比较，以找出参与卵泡发育的miRNA-mRNA相互作用的调控网络。随后，最多7个候选mirna将通过体外全腔前卵泡培养分析其与卵泡生长的关系。此外，同样的候选细胞将在体外培养的颗粒和卵泡细胞中进行功能研究。此外，将通过将5种选定的miRNAs的抑制剂或模拟物注射到卵巢进行体内功能研究，然后研究卵泡发育的改变以及miRNAs、mrna和相关蛋白的表达变化。该研究将揭示牛卵巢卵泡转换的miRNAs依赖性调控，并可用于人类辅助生殖的诊断和干预程序。

Ovarian follicular physiology, which involves complex and coordinated processes and the interactions of different cell types, is known to be dependent on tightly regulated expression and interaction of a multitude of genes. In the present study the involvement of microRNAs in the regulation of genes involved in ovarian follicular development will be investigated through a systematic characterization and functional analysis of miRNAs identified in our previous study. Initially, 24 candidate miRNAs will be investigated for their expression in the follicular theca, granulosa cells, cumulus-oocyte complexes from the leading and subordinated follicles at days 3,7 and 19, whichs correspond to the selection, dominance and preovulatory stages, respectively. Based on this result, the differential expression pattern of some selected predicted target mRNAs will be investigated and compared to the expression profile of related miRNAs to find out the regulatory network of miRNA-mRNA interactions involved in follicular development. Subsequently, a maximum of 7 candidate miRNAs will be analysed for their involvement in follicular growth using a whole preantral follicle culture in vitro. Moreover, the same candidates will functionally studied in in vitro cultured granulosa and theca cells. Further, in vivo functional studies will be conducted by injection of inhibitor or mimics of 5 selected miRNAs into the ovary followed by investigation of alteration in follicular development as well as changes on the expression of miRNAs, mRNAs and related proteins. The proposed study will uncover the miRNAs dependent regulation of ovarian follicle turnover in bovine and could be modified for diagnostic and interventional procedures in human assisted reproduction.