雄激素通过PKC通路调控缝隙连接蛋白的表达及功能促进卵母细胞发育的研究

Poor ovarian response (POR) is one of the most challenging tasks in assisted reproductive technology. In view of the importance of androgen to follicle recruitment, growth and development, domestic and foreign reproductive centers try to add androgen as an adjuvant to in-vitro fertilization (IVF) treatment protocols in POR patients, in order to improve ovarian response. The mechanism of action of androgen is unclear. Our previous studies found that the expression of connexin (Cx) in cumulus granulosa cells was closely related to the ovarian response, the quality of oocytes and the outcome of clinical pregnancy. In addition, adding a certain amount of androgen in mouse cumulus oocyte complex can increase the expression of connexin which was located the surface of oocytes and somatic cells in ovary. In order to explore the mechanism of action, we investigated the expression of PKC pathway related protein, phosphorylation of Cx43, Cx43 localization and gap junction intercellular communication (GJIC) function after androgen pretreatment in three aspects: 1. clinical study: collect the basic data and granulosa cells before and after androgen pretreatment in POR patients; 2. in vitro cell experiment: add different concentrations of androgen in the physiological level to the mouse granulosa cells; 3. animal model: construct mouse model at different concentrations of androgen stimulation. The objective of this study is to clarify that androgen can activate the PKC pathway, then PKC phosphorylates the Cx43 at serine 262 and 368, and the phosphorylation of Cx43 further regulates GJIC function, which finally affects follicular growth and oocyte quality. Through the in-depth discussion of the mechanism of androgen promoting follicular development, we not only can profoundly understand the physiological role of androgen, but also clarify its target of action more clearly. The results of this study might provide the theoretical basis for performing androgen pretreatment in POR patients.

卵巢低反应（POR）是辅助生殖领域最具有挑战性的难题之一，临床上可用雄激素预处理提高卵巢反应性，但雄激素作用机制尚不清楚。我们的前期研究发现颗粒细胞缝隙连接蛋白（Cx）的表达与卵巢反应性、卵母细胞质量密切相关；在小鼠卵丘卵母细胞复合物中添加雄激素可提高卵母细胞与周围体细胞之间Cx的表达。为阐明雄激素作用机制拟进行：1.临床研究：收集POR患者进行雄激素预处理前后的临床资料及颗粒细胞；2.体外细胞实验：小鼠颗粒细胞添加生理水平范围内不同浓度的雄激素刺激；3.动物模型：构建不同浓度雄激素刺激下的动物模型三部分研究来观察雄激素处理后PKC通路相关蛋白、Cx43磷酸化水平、Cx43定位以及GJIC功能等指标的变化，从而阐明雄激素可通过激活PKC通路，调控Cx43丝氨酸262和368号位点的磷酸化，进一步调节GJIC功能，从而影响卵泡生长发育及卵母细胞质量，为POR患者进行雄激素预处理提供理论依据。

卵巢低反应（POR）是辅助生殖领域最具有挑战性的难题之一。随着对生殖生理各个环节的深入研究，人们已认识到卵巢除受促性腺激素的调节外，卵巢微环境内的一些类固醇激素（如雌激素、孕激素、雄激素等）也共同参与了卵泡的生长发育过程。鉴于雄激素在卵泡生长发育中的重要作用，全球大约1/4的生殖中心将雄激素用于POR患者的治疗中，来提高患者的获卵数及临床妊娠率。为了阐明雄激素的作用机制，本课题从以下三个方面进行了研究：1、从临床水平比较DHEA预处理组和对照组的临床妊娠结局以及颗粒细胞中PKC、Cx43、磷酸化Cx43的表达量，结果发现DHEA预处理可通过提高不孕症患者的基础睾酮水平，一方面增加缝隙连接蛋白的表达，另一方面通过PKC信号通路增加磷酸化Cx43的表达，从而改善卵巢反应性，提高获卵数、受精率及优质胚胎率，最终提高临床妊娠率。2、通过体外细胞实验，我们用生理水平范围内不同浓度的睾酮、雄激素受体拮抗剂以及PKC抑制剂处理卵巢颗粒细胞，结果发现生理水平范围内的雄激素可通过激活PKC信号通路，从而导致Cx43磷酸化水平升高，进一步增强细胞通讯功能，影响卵泡的生长发育及卵母细胞质量。3、通过构建不同浓度雄激素刺激下的动物模型，检测各组小鼠颗粒细胞中PKC、磷酸化Cx43的水平及获卵数，进一步验证了雄激素通过PKC通路调控缝隙连接蛋白的表达及功能促进卵母细胞发育的研究假说。综合上述结果，本课题阐明了雄激素的作用机制：雄激素可通过激活PKC通路，调控Cx43丝氨酸262和368号位点的磷酸化，进一步调节细胞通讯功能，从而影响卵泡生长发育及卵母细胞质量。通过本课题研究，不仅深刻认识了雄激素的生理作用，而且也更清楚地明确其作用靶点，为临床上卵巢低反应患者进行雄激素预处理提供理论依据。