The Feature And Mirna Regulation Of Follicular Atresia In Poecine

In female mammalian ovary, only a limited number of follicles develop to ovulation, most of them (70%-99%) undergo degeneration and disappear at any stage of growth and development. This process is called "follicular atresia". Follicular atresia, which is a normal physiological process, is the end of the fate for most follicles. Many researches have been done about follicular atresia for a long time, but the exact molecular mechanism of follicular atresia regulation has not yet clarified so far, it still needs further exploration. It is of great significance to clarify the molecular mechanism of this physiological process for rich theory of reproduction, improving animal fertility, promoting development of embryo engineering and the Treatment of human reproductive diseases.Recent studies have found that a miRNA named (or microRNA) family of small RNA involved in the various activities of organisms, such as embryonic development, metabolism, signal transduction, tumorigenesis, cell proliferation, differentiation and apoptosis. It is becoming a hot topic in current researches for its variety and wide range functions.Pig was selected as experimental animal in this research, and the main research topics are as follows:(1) observe the characteristics of porcine follicular atresia; analyze the relationship of three methods for atresia judgment that are apparent observation, granulosa cell density in follicular fluid and P/E ratio in follicular fluid.(2) detect the apoptosis pathway of cell aopotosis during follicular atresia between3-5mm in diameter; explore the molecular regulation mechanism of important cell death ligand and receptor during apoptosis pathway.(3) establish miRNA expression profile in the process of follicular atresia, and screen differential expression of miRNA during follicular atresia, especially in the startup process, predict target genes and functions of the differential expression of miRNA by bioinformatics.(4) miR-26b was chosen from differential expression miRNA for further research, verify miR-26b functions such as granulosa cell apoptosis promoting in porcine, explore molecular mechanism of miR-26b promote cell apoptosis. Through all the studies, we can provide a theoretical basis for further elucidate the molecular mechanism of follicular atresia, especially in miRNA regulation of follicle atresia.We can draw the following conclusions throughout comprehensive results of this study:(1) The characteristics and determination of follicular atresiaThe essence of pig follicular atresia is granulosa cell apoptosis. Granulosa cells near the follicle antral tend to apoptosis first, and then gradually develop to the base membrane during follicular atresia in porcine. Mass of apoptotic granulosa cells shed into the follicular fluid, leading to sharp increase of granulosa cell density in follicular fluid. Meanwhile, the P/E ratio in follicular fluid was significantly increased during the process of follicular atresia. According to our experimental results, granulosa cell density in follicular fluid of health follicle, early atretic follicle, advanced atretic follicle were<10,10-30and>30respectively; P/E ratios of follicular fluid were<5,5-10,>10respectively. Generally, the three indicators to judge the degree of follicular atresia are in line, we suggested to determine the extent of follicular atresia with two indicators.(2) Apoptosis pathway of3-5mm follicular atresia in pig ovariesFollicular atresia of3-5mm in pig ovaries went primarily via TRAIL and Fas L pathway. Caspase-8acted as an important intermediate molecular in apoptotic signal delivering. After apoptosis signal deliver from extracellular into intracellular, apoptosis is further induced by Caspase-8.Anti-apoptosis gene XIAP plays an important role in the anti-apoptotic process of granulosa cells during3-5mm follicular atresia. The transcription factor Fox01may be involved in transcription regulation of cell death ligand and receptor genes during follicular atresia in porcine.(3) Expression of miRNA during follicular atresia in pig ovaries1251mature miRNA expression was examined in the process of follicular atresia in pig ovaries, and329,369,435species of miRNA were respectively expressed in healthy follicles, early atretic follicles and progressed atresia follicles. It is found that about200species miRNA differentially express in the process of follicular atresia, we take special attention on23miRNA which differentially express in the start-up phase of follicular atresia, and we found miRNA such as let-7a, miR-26b maybe play a important regulatory role in cell apoptosis pathway after target gene, function, pathway analysis of the23miRNA. (4) the mechanism of miR-26b regulate porcine granulosa cell apoptosisIt has been discovered that miR-26b was up regulated during porcine follicular atresia. miR-26b can induce porcine granulosa cells apoptosis in vitro, and it is proved that ATM is a direct target genes of miR-26b in pig granulosa cells, DNA damage in the granulosa cells increased significantly when miR-26b overexpressed, it suggested that miR-26b affect DNA damage repair, increase DNA damage and then promote the apoptosis of granulosa cell.A preliminary study of the regulating role of miRNA in follicular atresia was performed in this paper, which expands miRNA function, and contributes to clarify the molecular mechanisms of follicular atresia. It is a preliminary attempt that applied epigenetics theory in animal reproduction study.