Expression And Function Verification Of CDC25C Gene In Ovarian Granulosa Cells Of Adult Goats And Young Goats

Follicular granulosa cell is an important part of follicle which is surrounded by oocytes.With the development of follicles,follicular granulosa cell will change in the proliferation of a variety of hormones and growth factors,which are important to oocytes' growth and maturation.In this study,it was found that the maturation rates of oocytes were significantly different in the process of in vitro maturation between ewes and lambs,and the ratio of ewes' oocytes developed to four cells after maturation and fertilization were also significantly lower than lambs'.We also found that ewes and lambs' follicular granulosa cells were very different in quantity and the form of surrounding oocytes.Another finding is that the expression of cell division cycle 25 homologous protein C gene(CDC25C)in oocytes of ewes and lambs was significantly different.CDC25C can regulate cell division cycle and eukaryotic cell mitosis,and it is one of the key factors in cell cycle's entry into M phase.Based on the above two findings,we decided to verify the function of CDC25C from follicular granulosa cells and try to find out the reason of difference in oocytes' maturation rate at different ages.In this experiment,the white goat of Nantong,Jiangsu Province was used as the research object.The ovaries of 1.5-year-old lambs and 2-month-old ewes were collected and transported to the laboratory with 0.01M PBS.The follicular granules were cultured in vitro and the expression of CDC25C,CDK1,WEE1,estrogen,progesterone and cell cycle were studied after transfection in the follicular granulosa cells of ewes and lambs.The results showed that:1.Cloning of goat CDC25C geneAccording to the CDC25C gene sequence provided by NCBI,the CDS region of the CDC25C gene was designed to clone,and followed by construction of the gene pCMV-HA-CDC25C vector and small molecule interference vector CDC25C.The sequencing results showed that pCMV-HA-CDC25C vector was constructed correctly,At the same time,the small molecule interference vector siCDC25C was confirmed to be effective in the pre-experiment and could be used for subsequent experiments.2.Identification of primary isolated cellsAccording to the principle of enzyme-linked immunosorbent assay,there are specific FSH receptors on the surface of goat granulosa cells,Rabbit Anti-FSHR polyclonal antibody as primary antibody,SA1074-rabbit IgG as secondary antibody,and after enzyme-linked immunosorbent assay,the cells isolated from this experiment were mostly follicular granules needed to continue the follow-up test.3.Effects of CDC25C gene on follicular granulosa cellsConstruction of pCMV-HA-CDC25C vector and small molecule interference vector siCDC25C,after transfection of adult goat and young goat follicular granulosa cells,the relative expression of CDC25C,CDK1 and WEE1 genes was detected by real-time fluorescence quantitative PCR,the secretion of PROG and E2 was detected by enzyme-linked immunosorbent assay(ELISA),Using the technique of slicing to compare the difference between adult goat and young goat ovaries,and cell cycle detection was used to compare the cell cycle differences between two kinds of goat follicular granule cells transfected with recombinant vector and interfering vector.(1)After transfection of pCMV-HA-CDC25C vector,The relative expression of CDC25C,CDK1 and WEE1 genes in follicular granulocytes of adult goats has been significantly improved;The secretion of both PROG and E2 hormones also increased,and the difference was significant compared with the blank group;Adult goat ovarian follicles less,but better development;The proportion of cells in the divisional phase was significantly increased.The relative expression of CDC25C gene in follicular granulosa cells of young goat was increased after transfection of pCMV-HA-CDC25C vector,but the relative expression of CDK1 and WEE1 genes decreased;PROG and E2 both hormone secretion also increased;The follicles on the ovaries of young goats are more,but the degree of development is not good;The proportion of cells in the divisional period is significantly increased.(2)After transfection of small molecule interference vector siCDC25C,the relative expression of CDC25C,CDK1 and WEE1 genes in follicular granulocytes of adult goats decreased;PROG and E2 secretion of two kinds of hormones have increased;Cell division in the proportion of intermittent significant decline.The relative expression levels of CDC25C,CDK1 and WEE1 genes in follicular granulosa cells of young goats were decreased;The secretion of PROG and E2 had a certain increase;And the proportion of cells in the divisional stage was significantly decreased.