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孕激素及其受体在大菱鲆精子发生过程中的作用研究
丰程程
学位类型博士
导师李军
2017-11
学位授予单位中国科学院大学
学位授予地点北京
学位专业水产养殖
关键词生长期+繁殖期+孕激素+孕激素受体+大菱鲆
摘要硬骨鱼类繁殖受到内分泌系统调控,其中孕激素在雄鱼精子发生过程中起重要作用,促进精液排放,提高精子活力。所有养殖鱼类几乎都存在繁殖机能障碍,人工养殖的大菱鲆精液排放量少,精子存活时间短严重制约人工养殖的发展。因此研究孕激素在大菱鲆精子发生过程中的作用既对阐明硬骨鱼类繁殖内分泌机制具有重要意义,又能针对大菱鲆人工养殖技术提供理论基础。本论文以人工养殖的从生长期至繁殖期的大菱鲆为研究对象,运用组织学,激素放射免疫测定和酶联免疫吸附法,分子生物学方法,荧光定量,原位杂交和精子参数分析方法,将大菱鲆孕激素合成关键酶的表达,孕激素水平变化及孕激素受体的表达模式与整个精巢发育过程中精巢结构和生殖细胞类型的变化相结合进行研究,分析孕激素及其受体对大菱鲆精子发生过程中的作用,主要研究结果如下:
1.      组织学观察表明大菱鲆精巢属于小叶型,精原细胞主要但不局限分布于生殖上皮边缘区域。生长期大菱鲆精巢在6-14月龄为精原细胞增殖期,精小叶体积小,生殖上皮形成的隔间厚,雄性分化标记基因amh的表达量显著升高;16-22月龄为减数分裂期,初级精母细胞标记基因sycp3表达量和生殖细胞标记基因dazl的表达量显著增加并持续高表达。大菱鲆进入繁殖期后,根据生殖上皮形态和占主导地位的生殖细胞类型将大菱鲆整个繁殖周期分为III-VI期四个阶段。III期精巢中精小叶之间排列紧密,由完整的生殖上皮相分隔,小叶中主要出现的是初级精母细胞第一次减数分裂形成次级精母细胞;IV期有少量生殖上皮出现破裂,输精管部分的面积扩大,靠近输精管位置的精小叶中的部分精子细胞变态形成成熟精子;V期大量生殖上皮变薄断裂形成巨大的小叶腔,与输精管相连,成熟精子汇入小叶腔流向输精管排出体外;VI期精巢退化,精巢边缘生殖上皮重新生长分隔形成新的精小叶,细胞类型主要为精母细胞和少量精原细胞,精巢中央残余少量成熟精子。
2.      在生长期大菱鲆血清中,孕激素P4和DHP,雄激素T和11-KT的含量都在精原细胞增殖期较低,在精母细胞减数分裂起始阶段开始显著升高,在减数分裂结束主要细胞类型为精子细胞时显著下降。另外,P4向DHP和雄激素方向转化的关键酶 P450c17-IP450c17-II在精原细胞增殖期显著高表达,为在减数分裂起始阶段合成高水平的DHP,T和11-KT提供条件。
3.      孕激素核受体pgr在生长期大菱鲆精原细胞增殖期表达量显著增加,在减数分裂期持续高表达,与DHP的水平变化一致。而孕激素膜受体mPRαpgrmc1pgrmc2在减数分裂期后期才开始显著增加。推测在大菱鲆中,高水平的DHP通过调节pgr的转录水平启动并维持减数分裂的正常进行,而mPRαpgrmc1pgrmc2在减数分裂后期才开始发挥作用。
4.      繁殖周期中,P4,DHP和T在血清中的含量随精巢发育显著升高,精巢退化时显著下降,P450c17-IP450c17-II 的表达量与DHP含量具有相同的表达时序。表明在整个繁殖周期中,不断有胆固醇通过一系列转化合成P4,再通过P450c17-IP450c17-II转化成DHP和T促进繁殖周期的进行。另外,P4和DHP含量高的雄鱼,其精子存活率也高,进一步证明高浓度P4和DHP促进精子成熟。
排精期,pgr在精巢中高表达,定位在精子细胞上,表明在繁殖期大菱鲆中,pgr的主要作用是通过基因组作用介导DHP促进减数分裂和精子细胞变态成熟,在成熟的精子中不发挥作用。mPRαpgrmc2 在成熟精子中的表达量显著高于在精巢中的表达量,且在质量高的精子组中的表达量显著高于质量低的精子组,推测这两种膜受体通过高表达介导孕激素非基因组作用影响精子质量。
其他摘要In teleost, sex steroid hormones are critical to reproductive endocrine, especially progestin is well known to promote spermiation. Almost all fish reared in captivity exhibit some form of reproductive dysfunction. In males turbot (Scophthalmus maximus), low quality and quantity of milt production seriously restricts the development of artificial breeding industry. To further understand the functions of progestin via its receptors during spermatogenesis in male turbot, we observed testicular development, quantified several sex steroid hormones, detected the expression of progestin receptors and the enzymes of progestin synthesis, and measured various sperm parameters from the onset of puberty to reproductive cycle. The purpose is to study the function of progestin via its functionally distinct progestin receptors on male germ cells development. Main contents and results are as follows:
1.        In this study, turbot testicular structure was characterized by the lobular type and represented a cystic type of spermatogenesis. The spermatagonial cells proliferation stage was from 6-14 months. The expression of amh of male differentiation marker gene increased significantly. The meiosis stage was from 16 to 22 mah. The expression of primary spermatocyte marker gene sycp3 and germ cell marker gene dazl increased significantly. The reproductive cycle of turbot was divided into four stages based on the proportion of different germ cells and the shape of germinal epithelium in testes. From stages III to VI, the dominant germ cells types were spermatocytes, spermatids, spermatozoa, and spermatocytes, respectively. At stage V, almost all the seminiferous lobules were filled with spermatozoa and spermatids, and a large number of discontinuous germinal epithelium appeared. At stage VI, germ cells regressed to spermatocytes. Only some seminiferous lobules still contained a small amount of spermatozoa. The number of sertoli cells increased markedly and the cyst wall had thickened.
2.        In growing period, the results showed that the levels of progesterone (P4), 17α, 20β-dihydroxy-4-pregnen-3-one (DHP), testosterone (T) and 11-ketotestostrone (11-KT) were high at the initial of meiosis, and dropped significantly at spermiogenesis. In addition, the expression of enzymes of progestin and androgen synthesis P450c17 were high during the spermatagonial cells proliferation, which provides conditions for DHP, T and 11-KT synthesis.
3.        The expression of nuclear progesterone receptor (pgr) was high from spermatogonial proliferation to meiosis, which was consistent with the level of DHP. While the expression of three membrane progestin receptors (mPRαpgrmc1 and pgrmc2) did not increase significantly until late meiosis period. Hence, we speculated that DHP might play an essential role in the initiation of meiosis through pgr. And mPRαpgrmc1 and pgrmc2 play an important role at meiosis II and spermiogenesis.
4.        During reproductive cycle, serum T, P4 and DHP levels presented higher values from stage IV to V than other stages. Furthermore, the expression of P450c17-I and P450c17-II were also high from stage IV to V. The expression profiles indicated that the cholesterol transformed into P4 continuously, and then continued to transform into DHP and T via P450c17-I and P450c17-II to promote the development of reproductive cycle. In addition, the males with higher motility sperm showed higher levels of P4 and DHP comparing with those with lower motility sperm. These results indicated that P4 and DHP might induce spermiogenesis by seasonal changes.
The expression of pgr reached the peak at stage V and located in spermatid. While the expression of mPRα and pgrmc2 were higher in sperm than in testes. Futhermore, the expression of mPRα and pgrmc2 were higher in high quality sperm group than in low quality sperm group. These results indicated that progestin might exert a direct pgr-mediated effect on spermatogenesis by genomic actions and improve sperm quality depending on the abundance of membrane progestin receptors in sperm by nongenomic actions.
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/154368
专题实验海洋生物学重点实验室
推荐引用方式
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丰程程. 孕激素及其受体在大菱鲆精子发生过程中的作用研究[D]. 北京. 中国科学院大学,2017.
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