扬子鳄性激素水平及其受体基因表达对卵泡发育的调控机制

Chinese alligator is an endemic species in China.We have successfully captived this species and hatched the eggs artificially.But the number of eggs was fluctuated in different years. The reproductive conditions are difficult to be make an accurate assessment in both wild and captive alligator population, ect. So. We hope to make a comprehensive knowledge in reproductive mechanism of Chinese alligators. In this project, we shall test the hormone level including Estrogen(E2), Testosterone(T), Progesterone(P), Luteinizing hormone(LH), Follicle stimulating hormone(FSH) and Gonadotropin releaseing hormone(GnRH) in blood plasma once a month, analyse their receptors（ERα，ERβ,PR,TR,LHR,FSHR and GnRHR）gene expression in different organs by Q-PCR, and exame the distribution and location of their receptors in different organs by immunofluorescene histochemistry, and study the structure of follicular development by microscope, submicroscope and ultrasonography. our targets are: (1) to analyse the correlations of the concentration of sex hormone and the structure of follicular development by measuring the sex hormone concentration throughout the reproductive recycle and Ultrasonography monitoring, (2) to understand the gene expression of sex hormone receptors in different organs in reproductive recycle, (3) to exame the distribution and Localization of sex hormone receptors in alligator,(4) to elucidate the regulation mechanism of sex hormone receptors gene expression in follicular development, (5) to evaluate the feasibility of monitoring reproductive condition of Chinese alligators using Ultrasonography. The result of this project will let us obtain more clear apperceive in reproductive biology of Chinese alligators, and also set up a availability method to evaluate the reproductive condition in both wild and captive population of Chinese alligators. These results will also help to promote the artifical breeding and to improve the reproductive rate of Chinese alligator.

虽然人工孵化问题已得到解决，但扬子鳄产卵量存在不确定性，年度间波动大，深入研究其卵巢发育及卵子发生、揭示雌性扬子鳄的繁殖机制具有重要理论和实际意义。本项目通过放射免疫荧光测定、免疫荧光组织化学、超声波扫描、Q-PCR等方法，测定扬子鳄血液中性激素（雌激素、孕激素和雄激素）、促性腺激素（促黄体生成激素、促卵泡成熟激素）及促性腺激素释放激素含量的年变化，定量分析扬子鳄性激素受体在不同器官的分布和定位及其基因表达。研究目标(1)查明血液中性激素水平的年变化与卵泡发育的关系；(2)揭示性激素受体的基因表达与卵泡发育的关系；(3)阐明性激素受体在不同器官中的分布和定位；（4）探讨卵泡发育过程中，性激素受体基因的调控机制；（5）探索利用超声波扫描检测卵巢发育状况与性激素水平的相关性。通过研究，期望弄清扬子鳄的繁殖的激素调控机理，为扬子鳄繁殖状态评估、开展人工辅助繁殖、提高种群的繁育率等提供科学依据。

虽然人工孵化问题已得到解决，但扬子鳄生殖机理的研究很少，深入研究其卵巢发育及卵子发生、揭示雌性扬子鳄的繁殖机制具有重要理论和实际意义。本项目运用组织学方法、电镜技术对扬子鳄不同时期的卵巢及卵泡发育周期进行了分析，将卵巢发育过程分为五个时期，且发现每个月份中均存在不同发育阶段的卵母细胞，说明了卵细胞成熟呈非同步性。利用B超技术成功地对扬子鳄进行产前卵巢发育检测，B超这种低损伤方法增加了对扬子鳄检测的有效性，提高了卵泡发育状况评估的准确性以及繁殖鳄筛选的正确率。运用放射免疫分析法定量检测雌雄扬子鳄血浆中17β-雌二醇、睾丸酮和孕激素三种生殖激素的浓度，结果表明其生殖活动可能始于秋季；扬子鳄血浆生殖激素水平呈明显的周期性，能够调控其求偶交配等生殖活动，且激素水平与环境温度的变化有关。此外，我们利用荧光定量PCR以及免疫组织化学等技术对扬子鳄雌激素受体α和β（ESRα、ESRβ）、雄激素受体（AR）、孕激素受体（PR）、卵泡刺激素（FSHβ）及其受体（FSHR）等基因cDNA序列进行了克隆及定量表达等相关分析，探讨了各激素对卵泡发育的影响，结果表明ESRα、AR、FSH 和FSHR在繁殖期具有相对较高的mRNA水平，对卵泡的发育起着正调控作用；ESRβ在繁殖期表达量较低，PR在成熟的卵泡中表达量低，而在未成熟的卵泡中表达量较高，表明ESRβ和PR对卵泡发育起着负调控作用。通过研究，基本了解了扬子鳄雌性生殖的激素调控机理，分析扬子鳄繁殖状态、开展人工辅助繁殖、提高种群的繁育率等具有重要的指导意义。