高原甘加型藏羊垂体-卵巢轴GTH细胞和受体表达及生殖激素变化规律研究

Ganjia Tibetan sheep is a better kind of district species, which are herded by the natural grassland of Qinghai-Tibetan Plateau ，2800-3500 meters above sea level, but the reproduction hormone endocrine mechanism regulated by the FSH(Follicle stimulating hormone)，LH (Luteinizing hormone)，E2 (Estrogens) and P (Progesterone) has not been studied up to now. The project selects the Tibetan sheep as experimental samples, combining RIA (radioimmunoassay) method with the Immunohistochemistry technology and electronic microscope analysis and RT-PCR technique, and the program aims to carry out the following research experiments， 1.the endocrine mechanism of the FSH，LH，E2 and P during the oestrus cycle. 2.the GTH（gonadotropic hormone）cell types and secretory characteristics of pituitary gland with FSH and LH contend dynamics in jugular vein blood samples during different estrus period，and the reproduction regulation relation of the morphology changes of GTH（gonadotropic hormone）cell microstructure and endocrine particle of pituitary gland during seasonal empathema period. 3. the periodicity changes of FSHR(Follicle stimulating hormone receptor) and LHR(Luteinizing hormone receptor) numbers and its distribution as well as mRNA gene expression level in the ovary during the oestrus and anoestrus, and the regulation mechanism between GTH and the ovum development in different developing periods. The project explores firstly the reproduction hormone endocrine mechanism from GTH cell morphology changes and secretion dynamic regularity of reproduction hormone in blood of Tibetan sheep. We are mainly focus on the function changes of GTH cell in the pituitary-gonadal axis, endocrine mechanism of reproduction hormones, molecular expression of GTH receptor, and physiological regulation mechanism in Tibetan sheep. These kinds of study will be validated their great importance and far-reaching meanings to understand the physiological regulation mechanism of reproduction and to master the breeding performance of Tibetan sheep.

高原型藏羊是我国三大原始绵羊品种之一，生长在海拔2800-3500ｍ青藏高原，迄今对藏羊垂体-卵巢轴促性腺激素细胞功能和受体表达及生殖激素变化规律等研究国内外尚无报道。本课题以青藏高原甘加型藏羊为研究对象，采用放射免疫分析、免疫组织化学、电镜和RT-PCR等研究方法，主要研究:①发情期藏羊FSH、LH、E2和P分泌规律；②发情期藏羊垂体促性腺激素细胞形态、超微结构、分泌颗粒变化规律及其与血浆FSH和LH水平变化调控的相互关系；③发情期卵巢中FSHR和LHR的分布、mRNA基因表达水平变化规律，探讨GTH对卵巢功能调控的作用机理。本课题首次从GTH细胞、受体功能变化结合血浆生殖激素水平变化，探讨藏羊生殖激素分泌调控规律，拟揭示藏羊垂体-卵巢轴GTH细胞功能、受体分子表达、生殖激素变化规律及其相互作用关系和调控机理，对掌握藏羊生殖生理调控机制和提高繁殖性能等具有重要的科学意义。

为了揭示甘加藏羊发情周期垂体GTH细胞功能、卵巢GTH受体和与血浆生殖激素分泌变化及其调控机理,本项目连续三年定时定点在甘加藏羊发情周期进行了跟踪观察实验，采集了64只发情周期藏羊血浆、下丘脑、垂体、卵巢等组织，应用ELISA、免疫组化、PCR等技术首次对藏羊发情周期生殖激素分泌特点、垂体GTH细胞及其受体分子表达与调控机理进行了研究。获得如下研究成果：.1.通过阴道涂片法探究了藏羊发情周期各期阴道细胞种类变化规律。发情期角化上皮细胞数显著高于其它三个时期；发情前期表层上皮细胞数显著高于其它三个时期；间情期和发情后期白细胞数显著高于发情前期和发情期。此法可科学准确的鉴定藏羊发情周期。.2.揭示了藏羊发情周期血浆FSH、LH、E、P、GnRH等生殖激素含量的动态变化规律。 FSH和LH在发情期极显著高于发情后期、间情期和发情前期；E在发情后期极显著高于其它三个时期。P在发情前期极显著高于其它三个时期。GnRH在发情期显著高于其它三个时期。.3.在分子水平上揭示了藏羊垂体和卵巢组织中FSHR、LHR和GnRHR基因表达。卵巢组织中FSHRmRNA在发情期表达量最高，间情期最低；LHRmRNA在发情后期表达量最高，间情期最低；GnRHR mRNA在发情后期最高。垂体组织中FSH mRNA在间情期表达量最高；LH mRNA在发情后期最高；GnRHR mRNA在发情期最高。.4.在细胞水平上揭示了藏羊发情期垂体和卵巢GTH细胞及其受体变化规律。FSH和LH细胞分布在垂体的远侧部，FSH阳性细胞数在发情前期表达最多；LH阳性细胞数在间情期表达最多。发情周期卵巢组织中FSHR和LHR阳性细胞分布于卵巢的原始、初级和成熟卵泡，FSHR阳性细胞在发情期表达最多，LHR在发情后期表达最多。.5.探明了腺垂体GTH细胞超微结构变化规律。发情前期和发情期腺垂体远侧部GTH分泌颗粒数量多，之后逐渐减少，部分大型分泌颗粒物向外分泌空泡形成增多。.本项目首次综合研究了藏羊在发情周期垂体GTH细胞、卵巢GTH受体和血浆中生殖激素分泌的变化对藏羊繁殖的调控机理，填补了空白，为藏羊生殖生理调控提供了科学依据，具有重要的科学意义。