山羊卵泡选择中miRNAs的参与及其调控机制研究

The number of ovulations at each ovarian cycle is determined by a tightly controlled follicle selection process. The mechanism underlying follicle selection take place on different scales and different levels, how precisely the follicular selection is controlled remains a critical and important question in reproductive biology. It is a supplement and improvement of the follicle selection mechanism by studying the involvement and regulation of miRNAs in goat follicular selection , it also provides important values to goat ovulation research and follicular resources development. In this study, we employ miRNA microarray using miRNAs expressed in goat ovaries to evaluate the differently expressed miRNAs between pre-selected growing antral follicle, dominant follicle and subordinate one. Some of these differentially expressed miRNAs are validated using quantitative PCR assay. Top significant GO categories, biological functions and different canonical pathways are analyzed for the specific targets of differentially expressed miRNAs. Using luciferase reporter system to validate target genes. Inducing granulosa cell proliferation by FSH to identify the regulation between FSH and miRNAs. Using miRNA inhibitors or locked nucleic acid oligonucleotides complementary to specific miRNAs to identify the function of miRNAs on granulosa cell proliferation, hormone production and follicular wave dynamics. Our aim is to elucidate the potential role of miRNAs in regulating follicle selection.

卵泡选择（follicle selection）是决定排卵数量的重要步骤，属于多因素参与调节的复杂生命过程，其机制尚未明确。探索miRNAs参与卵泡选择调控的分子机制是对卵泡选择调控机理的有益补充和完善，也对山羊排卵机理研究及卵泡储备资源开发具有重要的意义。本项目以山羊卵泡为研究材料，利用山羊卵巢表达的miRNAs（前期基础）制定miRNA芯片寻找募集卵泡、优势卵泡及从属卵泡中差异表达的miRNAs，通过生物信息学手段预测miRNA的靶基因，进一步利用荧光素酶报告系统明确miRNA与靶基因的调控关系；在此基础上，利用FSH诱导颗粒细胞增殖研究FSH与miRNAs的调控关系,利用miRNA抑制剂（和类似物）体内外干扰途径研究miRNA及其靶基因调控颗粒细胞增殖、激素合成及对卵泡发育波动力学的影响，阐明miRNAs参与卵泡选择的调控机制。

优势卵泡选择是决定排卵数量的重要步骤，属于多因素参与调控的复杂生命过程，其机制尚未明确。探索 miRNAs 参与优势卵泡选择调控的分子机制是对卵泡发育与选择调控机制的有益补充和完善，也对山羊排卵机理研究及卵泡储备资源开发具有重要的意义。本项目的研究结果如下：1）将卵泡期山羊卵泡按直径大小分组，通过小 RNA 测序从优势卵泡组和从属卵泡组中初步筛选出了 chi-miR-582-5p 等 9 个差异极显著的 miRNAs（p<0.01）；另外有 novel-344 等 4 个 miRNAs 在不同大小卵泡中呈现共同差异表达；2）对小 RNA 测序的同批次卵泡样品进行 mRNA 转录本测序，分析转录本与miRNA 预测靶基因的汇交信息。在优势卵泡组和从属卵泡组间差异极显著的 chi-miR-130b-5p 及 chi-miR-214-3p 的预测靶基因富集在 Ovarian Steroidogenesis 通路中，其中 GDF9 转录本与 chi-miR-214-3p 的表达模式是相反的，qPCR 结果也验证了这一点。在大小卵泡中共同差异表达的 novel-344 与其预测靶基因 FSHR 转录本的表达模式也是相反的；3）在大小卵泡中差异表达的 chi-miR-449a-5p 与 VEGF 基因有靶向结合位点。用 VEGF、activin A 和 VEGF+activin A 处理体外培养的初级卵泡时显著提高卵泡生长率（p<0.05）。本研究中筛选出的 miRNAs 可能参与卵泡的发育、优势卵泡的选择等生命过程需要后续研究中进一步研究。