钙促蛋鸭卵泡成熟机制研究

Calcium is the macromineral that supports formation of egg shell and maintenance of bone growth for egg-laying poultry. However, calcium was found in our lab to affect the number of post-ovulatory follicle, which is probably involved in the meiotic maturation process of follicle. Still the mechanism involved in the regulatory function of calcium in the maturation of follicle remains unclear. Based on the recent report on the mechanism involved in maturation of follicle, we hypothesized that calcium might decrease the level of cAMP, inhibitor of meiotic oocyte maturation, through regulating the activities of calcium-sensitive adenylyl cyclase and phosphodiesterase family members, herein promoting meiotic maturation of oocyte. To test the hypothesis, we are to evaluate the effect of dietary calcium level on the meiotic maturation of follicle in egg-laying ducks by employing calcium-deficient diet; and to confirm the regulatory role of calcium in affecting the maturation of oocyte through in vitro oocyte culture. Then, we plan to confirm the regulatory function in oocyte in vitro. Subsequently, we plan to do treatment on the isolated oocyte, granulosa cell, oocyte and granulosa complex with activator of L-type calcium channel/calcium chelator, activator/inhibitor of adenylyl cyclase and phosphodiesterase, fllowing which the oocyte under meiotic maturation will be recorded according to the germinal vesicle breakdown. Additinoally, the cAMP content as well as the signaling pathway involved in oocyte meiotic maturation are to be detected based on the experiment above, aiming at: 1) evaluating the regulatory role of calcium in affecting the activity of adenylyl cyclase and phosphodiesterase family members; 2) estimating the potential role of theses enzyme in mediating oocyte meiotic maturation. The results of this study will explore the potential mechanism of calcium that is involved in the meiotic maturation of oocyte in avian species, thereby providing a theoretical basis for the biological function of calcium.

钙是参与蛋禽蛋壳形成和骨骼生长的重要矿物元素，但近年来本小组研究发现日粮钙影响到蛋鸭的排卵数量，其原因可能与卵泡发育成熟有关，而相应机理尚不明确。结合国内外研究，我们假设认为：钙通过调节卵泡颗粒细胞与卵母细胞中钙敏感性腺苷酸环化酶（AC）（cAMP合成酶）和磷酸二酯酶（PDE）（cAMP降解酶），影响环腺苷酸（cAMP）（卵泡成熟抑制因子）的生成，从而促进卵泡成熟。基于此假设，本项目拟开展钙缺乏日粮饲养试验，探讨钙对蛋鸭卵泡发育的调控作用，并结合体外卵泡培养进行验证。在此基础上，通过钙通道激动剂/钙螯合剂、AC与PDE激动剂/抑制剂处理颗粒细胞与卵母细胞，检测cAMP水平和促成熟信号分子，观测卵母细胞成熟发生，研究：1）钙对AC和PDE家族成员的调节；2）两种酶对卵泡成熟的介导作用，从而对前述假说进行论证。本研究将可能揭示钙参与家禽卵泡成熟的调控机制，并为丰富钙新的生物学功能提供理论基础。

本项目针对钙参与蛋鸭卵泡发育调控这一假设进行了科学研究，首先对蛋鸭进行了钙缺乏和钙恢复两阶段饲喂试验，结果发现，钙缺乏显著降低了蛋鸭优势卵泡数量，且提高了卵巢中卵泡成熟抑制因子cAMP的产生，同时对cAMP合成（腺苷酸环化酶）与分解酶（磷酸二酯酶）家族基因及卵巢中缝隙连接蛋白基因均产生调节影响，并诱导PKA信号的激活；而在钙恢复期以上指标均恢复到正常水平，从而在体内证实了蛋鸭卵泡发育对饲粮中钙的依赖性。通过体外细胞培养发现，钙螯合剂及性激素并未对卵泡减数分裂产生显著影响，暗示着家禽卵泡成熟可能不同于哺乳动物。在颗粒细胞中发现，钙参与cAMP的正调节，且通过促进缝隙连接蛋白的表达提高细胞间互作关系。同时，通过钙通道抑制剂（verapamil）及螯合剂（EGTA）诱导的颗粒细胞钙缺乏能诱导cAMP降解酶PDE1A基因上调，表明，钙很可能通过抑制磷酸二酯酶基因表达来提高颗粒细胞中cAMP水平。此外，本项目研究还发现饲粮钙可调节壳腺组织中钙转运、蛋壳基质蛋白、碳酸酐酶等蛋壳形成过程，从而影响蛋壳超微结构和蛋壳质量。本研究结果进一步在cAMP途径及细胞学上明确了钙参与的蛋鸭卵泡发育的可能机制，为蛋鸭卵泡成熟调控提供了理论基础。