The Molecular Mechanism Of BMP15 Regulates The Function Of Follicular Granulosa Cells Through BMPR1B In Shanbei White Cashmere Goat

Shanbei White Cashmere Goat is a cashmere and mutton breed formed by crossbreeding of Liaoning Cashmere Goat and Shanbei Black Goat,with the features of high cashmere yield,good cashmere quality,strong roughage resistance and stress tolerance.As one of the important economic traits,reproductive trait directly affects the production and economic benefits of Shanbei White Cashmere Goat.The study on the biological mechanism of cashmere goat litter size is of significant practical value of cultivation income improved,also theoretical value of reproductive performance regulatory mechanism research.Fecundity of goats is a complicated processes regulated by multi-gene.As a candidate gene of goat litter size,the expression regulation of bone morphogenetic protein receptor1B(BMPR1B),the regulation mechanism and the biological mechanism of BMPR1B during follicular development were still unclear.The ovaries,ovarian follicles and follicular granulosa cells of Shanbei White Cashmere Goat were used to determine the regulation of BMPR1B gene expression by immunohistochemical,real-time fluorescent quantitative PCR,western blot,5-ethynyl-2'deoxyuridine incorporation,enzyme linked immunosorbent assay and flow cytometry.And the molecular regulatory mechanism of bone morphogenetic protein 15(BMP15)regulate cells prolifeation,apoptosis and steroid secretion through receptor BMPR1B by gene overexpression and co-expression.The main results are as follows:1.The study cloned and analyzed BMPR1B gene CDS region of Shanbei White Cashmere Goat.The CDS region of BMPR1B gene with 1509 bp in length was amplified.Sequence analysis showed that BMPR1B is a membrane protein,it has specific Gly/Ser kinase domain of the type I receptor of TGF?family.2.The expression pattern and characteristics of BMPR1B gene of reproductive system on Shanbei White Cashmere Goat were analyzed.The m RNA of BMPR1B gene were detected in uterus,ovaries and fallopian tubes of Shanbei White Cashmere Goat.The expression level in ovaries was the highest among them,followed by fallopian tubes,and uterus.The m RNA and protein of BMPR1B gene were detected in all developmental stages of goat ovaries,the m RNA abundance level was gradually increasing with the development of the follicle with highest expression level in the large antral follicles.The m RNA of BMPR1B gene were both detected in granulosa cells and oocytes,the former expression level was higher than the latter.The expression level of BMPR1B gene in the ovaries of multi-lamb goat was significantly higher than that of single-lamb goat.3.The regulation of BMPR1B expression by BMP15 from the cellular level was analyzed.The expression of its type II receptor BMPR2 and type I receptor BMPR1B in cultured goat follicular granulosa cells were up-regulated by exogenous BMP15.BMP15up-regulated BMPR1B by suppressing the expression of mi R-125b.4.Studies showed that BMP15 activated the MAPK ERK signaling pathway to regulate proliferation and apoptosis of follicular granulosa cells through BMPR1B.BMP15promoted cell proliferation by increasing the phosphorylation level of ERK protein through BMPR1B,up-regulating the expression of Cyclin E and cyclin-dependent kinase CDK2,thus increased the proportion of cells in the S phase of cell cycle.BMP15 inhibited cell apoptosis by increasing the phosphorylation level of ERK protein through BMPR1B,up-regulating the expression of Bcl2 meanwhile Inhibiting the expression of Bax and Caspase-3,thus interrupted the process of apoptosis.5.Studies showed that BMP15 activates the Smad signaling pathway to regulate the steroid secretion of follicular granulosa cells through BMPR1B.BMP15 increased the secretion of E₂ and P₄ in follicular granulosa cells by increasing the phosphorylation level of Smad1/5/8 protein through BMPR1B,then up-regulating the expression level of Smad4protein,and FSHR,inducing the expression level of St AR,CYP11A1 and CYP19A1,thus promoted the steroid secretion of follicular granulosa cells.In summary,this project established cell model by culturing follicular granulosa cells in vitro,constructed a regulatory network of BMP15 for cells prolifeation,apoptosis and steroid secretion through receptor BMPR1B,revealed the molecular mechanism of follicular development and atresia by BMP15 regulation,providing novel experimental evidence and a good theoretical and technical guidance for improving the litter size of cashmere goats.