VD3调控AMH信号提高鹅小黄卵泡FSH敏感性的研究

VD3对于家禽产蛋性能的维持至关重要。申请人前期研究发现，AMH能够降低鹅小黄卵泡的FSH敏感性，体外添加VD3可降低鹅小黄卵泡颗粒细胞AMH基因表达量，提高FSH敏感性。但是，VD3调控AMH基因表达的分子机制及其在鹅小黄卵泡发育中的作用尚不清楚。本项目拟首先通过体内饲喂方法观察VD3对产蛋鹅卵巢被选入等级发育的卵泡数量和小黄卵泡颗粒细胞AMH信号通路和FSHR基因表达的影响；其次，以体外培养的鹅小黄卵泡颗粒细胞为细胞模型，通过过表达和RNA干扰VDR基因方法，观察VD3对AMHR2-Smad1/5/9和FSHR-cAMP信号通路相关基因和蛋白表达以及孕酮分泌的影响；然后，通过荧光素酶报告基因系统以及ChIP和EMSA方法进一步验证VD3抑制AMH基因的转录。通过以上研究可阐明VD3通过抑制AMH信号提高鹅小黄卵泡FSH敏感性的分子机制，也可为种鹅日粮中添加VD3增加产蛋量提供理论依据。

Vitamin D3 (VD3) plays a vital role in maintaining the egg-laying performance in poultry. Our previous studies have found that anti-mullerian hormone (AMH) played a role in reducing the FSH sensitivity of ovarian small yellow follicles in geese, and in vitro VD3 decreased the mRNA expression of AMH gene in the granulosa cells of small yellow follicles and promoted the FSH sensitivity. However, the molecular mechanism by which VD3 regulates the mRNA expression of AMH gene and its role in the development of ovarian small yellow follicles in geese are still unclear. In this study, we will first investigate that the effect of VD3 on the number of recruited small yellow follicles and the expression of genes of AMH signal pathway and FSHR gene by in vivo feeding method in laying geese. Secondly, using the granulosa cells of goose small yellow follicles cultured in vitro as the cell model, it will be observed that VD3 alters the expression of genes and proteins related to AMHR2-Smad1/5/9 and FSHR-cAMP signal pathways, and the secretion of progesterone under the condition of over-expression and interfering with the VDR gene. Thirdly, VD3 inhibits the transcription activity of goose AMH gene, which will be determined by the luciferase reporter gene assay，ChIP and EMSA. The above results are expected to elucidate the molecular mechanism of VD3 promoting the FSH sensitivity of small yellow follicles by inhibiting AMH signal, and also provide the theoretical basis for the application of dietary VD3 supplementation to increase egg production in geese.