Molecular Cloning Of BMP4 Gene And Its Effect On Apoptosis Of Granulosa Cells In Goose

BMP4 as a member of BMPs, affectting the production of steroids hormone that seretes by granulosa cells and theca cells and the differentiation of granulosa cells. In some species, the proliferation or apoptosis of granulosa cells can be regulated by BMP4. Granulosa cell as one of the most important cell type in the ovarian follicle can not only produce steroids hormones and molecules that are essential for follicle growth but also cause follicular atresia by an apoptotic process. Therefore, studying the mechanism of granulosa cell apoptosis can be helpful to explore follicular atresia.Thus, this study aimed to obtain the coding sequence of BMP gene and explore its mRNA expression in granulosa cells and theca cells by using qRT-PCR. In order to exmine its effect on granulosa cells proliferation and apoptosis, a eukaryotic expression vector was constructed. Then granulosa cells were transfected with BMP4 gene in vitro by using liposomal transfection method. And the results are as follows:(1) The longth of goose BMP4 coding sequence was 1212bp. The nucletide sequence of this gene shared 96.3% similarity with zebra finch BMP4 gene while 98.3% at amino acid level.(2) In generally speaking, the relative expression of goose BMP4 mRNA in granulosa cells was higher than in theca cells in different developmental stages of ovarian follicle. BMP4 mRNA level of granulosa cells was low in small follicle but rise up when follicle reached 8～10mm in diameter.-The highest BMP4 mRAN expression level of granulosa cells is in the granulosa cells of F5. And its level drop quickly after that. In theca cells, BMP4 mRNA expression was lower than that in granulosa cells at many developmental stages except for follicles in 6～8mm diameter. (3) Liposomal transfection method was used to transfect granulosa cells with pEGFP-N1-BMP4.36h after transfection, OD450 value from the well of transfected group was significant high than that of contral group. No significant difference was found between transfected group and contral group at other transfection time. This indicate that the number of living granulosa cells were high in transfection time than in contral group at 36h after transfection (P<0.05).(4) We detected the proliferative granulosa cells and the apoptotic granulosa cells at 36h after transfection. The number of proliferative granulosa cells in transfected group has no significant difference with contral group. But there are more apoptotic granulosa cells in contral group than in transfected group (P<0.05).(5)BCL2 mRNA and caspase-3 mRNA as apoptosis related gene were detected in granulosa cells of transfection group and contral group by using qRT-PCR. There is a descent of caspase-3 mRNA in transfection group (P<0.05).