MicroRNA-665靶向HPGDS基因调控绵羊黄体退化机制研究

Corpora lutea (CL) are ephemeral endocrine glands occurring in all most mammals. Cyclical changes of CL can directly affect the estrus cycle and production efficiency of the farm livestock, which plays a vital role in the efficient breeding of sheep. Currently, PGF2α in the prostaglandin family member is considered to be the most critical factor causing the degeneration of the corpus luteum, but PGD2 belongings to the family member has not yet been studied in depth. Given that HPGDS is the target of miRNA-665, and is also a key synthesizing enzyme invovles promoting PGD2 synthesis. Thus, based on the miRNA-665-HPGDS pair, miRNA-665 mimic and inhibitor and CRISPR-Cas knockout system were transfected into luteal cells, and then HPGDS expression level and its protein level, apoptosis level, inflammatory factors and immunofluorescence in the varies groups were detected to find out the functional regulation of PGD2 in luteal cells. Meanwhile, ewes were treated with using PGD2and PGF2α, and finally blood samples, corpus luteum tissues are collected to conduct qRT-PCR, WB, Elisa and Immunofluorescence assay and to explore whether there is the interaction between PGD2 and PGF2α, both of which ultimately lays the foundation for comprehensive understanding and analysis of the mechanism of regulation of corpus luteum degradation, providing a theoretical reference for further optimization of the efficient breeding techniques.

黄体在哺乳动物中属于暂时性内分泌腺，其周期性变化直接影响动物的发情周期及生产效率，对绵羊高效繁殖发挥着重要作用。目前，前列腺素家族成员PGF2α被认为是引起黄体退化最关键因素，但该家族PGD2仍尚未受到重视。鉴于miRNA-665靶标HPGDS，是促进PGD2关键合成酶，本项目以miRNA-665-HPGDS为着力点，首先通过靶细胞转染miRNA-665 mimic和inhibitor及CRISPR-Cas敲除HPGDS系统试验，检测靶标HPGDS在黄体细胞中表达和蛋白含量变化，结合细胞凋亡、炎症因子分泌变化及免疫荧光细胞检测，探究PGD2在黄体细胞中参与功能性调节作用；选取母羊个体分别进行PGD2和PGF2α子宫肌内注射，采集血液、卵巢黄体组织通过qRT-PCR、WB、Elisa及免疫荧光组化检测，探析PGD2和PGF2α间是否存在互作影响，从而为全面认识和探析黄体退化调控机制奠定基础。

黄体退化在哺乳动物的生殖周期中起着重要的“关卡”作用，对于重启母畜的生殖周期至关重要。目前，已知前列腺素家族PGF2α成员是引起黄体退化的关键因素，但其家族中PGD2成员的研究却鲜有报道。本研究基于PGD2是miR-665靶向HPGDS合成的最终产物，主要开展了两方面的研究工作：（1）基于细胞层面，开展miR-665与其靶标基因HPGDS参与的黄体退化分子调控机制研究；（2）基于个体层面，开展靶标基因HPGDS合成产物（PGD2）参与黄体退化的调控机制研究。最终，我们研究发现（1）在黄体细胞中过表达miR-665和下调HPGDS后，miR-665/HPGDS能够显著地影响黄体细胞的凋亡过程（即结构退化），即导致黄体细胞的凋亡率明显下调，分泌孕酮水平显著上调及其凋亡相关基因BCL-2的表达增加、凋亡关键执行蛋白Caspase-3的表达下降；提示miR-665靶向HPGDS对黄体细胞的作用主要表现为明显的保护效应，而不是破坏效应。（2）在母体子宫肌内单独或联合注射PGD2和PGF2α后，miR-665靶向HPGDS的合成产物PGD2能够直接或间接影响母体的黄体退化过程，即PGD2可以通过促进PGF2α关键合成酶PGFS显著上调，与其受体DP1和FP表达的增加，促进内源性PGF2α向黄体的转运、促进黄体退化；而且，当其与PGF2α联合应用时，又可通过促进CRTH2的表达，启动黄体组织的凋亡过程、进一步加速黄体退化；提示miR-665靶向HPGDS的合成产物PGD2无论单独使用还是结合PGF2α使用，均能够促进黄体的退化，尤其是二者结合使用时更是呈现明显地协同效应。这为全面认识哺乳动物黄体退化的调控机制奠定了基础，也为进一步优化高效繁殖技术（尤其是PGs方案）提供了新的思路。