鹅就巢期HSP70调控卵泡颗粒细胞自噬的机制研究

Broodiness, specific behavior of poultry, affects egg-laying capability and economic value, becoming a development bottleneck of poultry farming. Geese have strongly broody behavior limiting their reproductive ability. Follicular degeneration and resorption at the broody stage in goose have been descripted, but its regulatory mechanism is not reported. We are committed to study the regulation mechanism of broodiness in goose for long. We found that the interaction between granulosa cells and internal environment of follicle was considered to be a key determinant of follicular development; enhanced expressions of HSP70 (70kDa heat-shock protein) gene and autophagy-related genes, including LC3 and Beclin1, in follicle, and increased level of autophagy in granulosa cells were occurred in broody goose follicles. Therefore, we suppose that HSP70 expression level, the stability of follicular internal environment, and follicular degeneration induced by autophagy are close linked with broodiness in goose. To prove this hypothesis, Zhedong white goose, strongly broody species, is used as material. We will study the regular pattern of autophagy in hierarchical follicles during egg-laying and broody stages in goose, and explore the relationships between autophagy and the expression of HSP70. Furthermore, to uncover the function of HSP70 in regulating the autophagy, we knock down HSP70 expression using siRNA method and elevate its expression by virus-mediated method in the follicular granulosa cells in vitro. Moreover, the HSP70 regulating or binding autophagy-related proteins are detected by Western blot and Co-IP (Co-immunoprecipitation) methods. The autophagosomes in the follicular granulosa cells are observed using SEM (Scanning electron microscope). The results will illustrate the molecular mechanism of granulosa cell autophagy regulated by HSP70 affecting goose broody behavior. This project has guiding significance for poultry breeding and molecular breeding, and will enhance the egg-laying ability and promote the development of goose farming.

就巢性影响禽类繁殖力和产蛋性能，是制约禽类养殖业发展的瓶颈。鹅为就巢行为较强的禽类，就巢期鹅卵泡退化和重吸收，但其调控机制未见报道。本项目组致力于鹅就巢行为的调控机制研究，前期初步研究发现鹅就巢期卵泡内环境紊乱，激素和氧化水平明显变化，HSP70和自噬相关基因表达上升；且卵泡颗粒细胞层自噬体数目和自噬标记蛋白LC3显著增加。因此，推测鹅就巢期HSP70与调控卵泡颗粒细胞自噬密切相关，但HSP70在此方面的功能研究未见报道。本项目以就巢性强的浙东白鹅为材料，主要解决：鹅就巢期卵泡颗粒细胞自噬发生的规律、卵泡HSP70表达与自噬发生之间的联系；体外培养颗粒细胞，通过siRNA干涉和过表达HSP70，结合电镜、Western blot和Co-IP等技术研究HSP70对鹅卵泡颗粒细胞自噬的调控机制。本项目的完成将解析鹅就巢期卵泡退化的分子机制，为提高鹅产蛋性能提供理论基础。

就巢严重影响禽类的产蛋性能。鹅的就巢行为强，就巢期鹅卵泡退化和重吸收。研究发现，卵泡的发育与闭锁是卵泡内环境和颗粒细胞相互作用的结果，但关于鹅就巢期卵泡退化的分子机制尚不清楚。我们前期发现，HSP70和自噬相关基因的表达存在相关性，因此，推测鹅就巢期HSP70参与调控卵泡颗粒细胞自噬，并调控鹅卵泡发育。为了验证此假设，我们首先分析鹅就巢前后不同发育阶段卵泡内生理生化水平和分子水平变化；再探索就巢期鹅卵泡颗粒细胞发生自噬的规律；接下来，明确HSP70表达规律及对卵泡颗粒细胞自噬的影响；最后，解析HSP70调控颗粒细胞自噬的分子机制。根据项目的设计，我们基本上完成了研究目标。. 我们研究发现，就巢抑制鹅大白卵泡到小黄卵泡的发育。就巢期与产蛋期卵泡相比较，催乳素、黄体酮和雌二醇等激素含量显著提高，SOD和LDH酶活性也明显提升，但MDA的含量却降低。电镜和Western Blot结果显示，就巢期卵泡颗粒细胞自噬水平显著增加，且自噬主要发生在大白卵泡和小黄卵泡两个阶段。转录组结果表明，激素通路、氧化还原通路和自噬调控通路在调控鹅就巢中起重要作用，与表型以及生理生化指标结果相印证。H2O2添加实验证明，ROS正向调控鹅卵泡颗粒细胞自噬。HSP70家族基因表达结果表明，大部分HSP70家族基因在就巢期大白卵泡中显著上调，而且与自噬的发生密切相关，提示HSP70家族基因在调控颗粒细胞自噬中的重要作用。我们构建了HSP70基因过表达和沉默载体并转化卵泡颗粒细胞，验证HSP70互作蛋白工作正在有序开展。此外，我们还发现鹅就巢早期，卵泡颗粒细胞线粒体会发生形态变化和功能缺陷。本项目揭示了鹅就巢期卵泡退化的分子机制，为提高鹅产蛋性能提供了理论基础。