The role of microRNAs in regulation of bovine ovarian follicular development

卵巢卵泡生理学涉及复杂和协调的过程以及不同细胞类型的相互作用，已知其依赖于多种基因的严格调控的表达和相互作用。在本研究中，microRNAs参与卵巢卵泡发育相关基因的调控，将通过我们以前的研究中发现的miRNAs的系统表征和功能分析进行研究。首先，研究了24种候选miRNAs在第3、7和19天（分别对应于选择期、优势期和排卵前期）的卵泡膜、颗粒细胞、卵丘-卵母细胞复合体中的表达。在此基础上，我们将研究一些选定的预测目标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，将使用体外完整腔前卵泡培养物分析最多7种候选miRNA参与卵泡生长的情况。此外，相同的候选人将在体外培养的颗粒细胞和卵泡膜细胞中进行功能研究。此外，将通过将5种选定的miRNA的抑制剂或模拟物注射到卵巢中，然后研究卵泡发育的改变以及miRNA、mRNA和相关蛋白质表达的变化来进行体内功能研究。这项研究将揭示牛卵泡更新的miRNAs依赖性调节，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.