【作者】 阿依木古丽；

【导师】 崔燕；

【作者基本信息】 甘肃农业大学 ， 基础兽医学， 2011， 博士

【摘要】 目的意义:牦牛是青藏高原特有牛种,生活在高寒、高海拔、低氧、冰封期长的严酷自然条件下。既可用于农耕,又可作为运输工具,还能提供一定量的乳、肉、毛绒等产品。牦牛较其它牛种性成熟晚,是一种典型的季节性发情动物。在牧草丰富的暖季发情,繁殖力低,通常两年一胎或三年两胎,一生产仔四到五头。由于受自然条件限制,相关研究较少,目前还未见到牦牛子宫中生殖激素受体相关的研究报道。为了深入研究牦牛生殖生理特性,探讨雌激素、孕激素、促卵泡激素和黄体生成素对牦牛发情周期子宫功能的调控机制,为进一步进行发情周期调控,提高其繁殖能力奠定理论基础。本实验进行了以下研究:实验方法:(1)分别取处于发情期、发情后期、间情期和发情前期的健康的母牦牛各4头,经颈部放血致死,肉眼检查内脏器官正常,尤其是生殖器官无异常后,迅速取出子宫,剪取子宫角标记后直接投入液氮中带回实验室进行mRNA的定量分析;另取子宫角用生理盐水冲洗3遍,40 g/L多聚甲醛磷酸缓冲液(pH=7.4)固定,带回实验室后置4℃固定24 h- 48 h。采用实时荧光定量PCR法和免疫组织化学SP法,分别检测发情周期不同阶段牦牛子宫中雌激素受体α(ERα)、孕激素受体(PR)、促卵泡素受体(FSHR)和黄体生成素受体(LHR)mRNA和蛋白质的表达情况。(2)选取健康、发育良好的性成熟未孕牦牛,颈部放血致死,迅速取出子宫,结扎两端,用冰冷无菌的不含钙、镁离子的PBS液(添加100 IU/mL青霉素,100μg/mL链霉素)保存,并置于冰盒中,3 h内带回实验室,采用I型胶原酶消化,离心分离和差速消化纯化的方法分离纯化上皮细胞和基质细胞,对其进行免疫细胞化学染色鉴定,绘制生长曲线,最后用MTT法测定不同浓度17β-雌二醇和孕酮对细胞增殖的影响。实验结果:(1)ERα蛋白免疫阳性产物主要定位于子宫腔上皮细胞、腺上皮细胞、基质细胞、血管内皮细胞及子宫肌层平滑肌细胞的核中,细胞质有少量表达;ERα在发情期牦牛子宫中表达都强,其中腺上皮表达最强;发情后期普遍下降,间情期表达最低,发情前期有所回升;牦牛子宫内膜和子宫肌层中ERαmRNA的表达都在发情前期最高,间情期最低;子宫肌层中ERαmRNA的表达普遍高于内膜中的表达。(2)PR蛋白免疫阳性产物主要定位于子宫腔上皮细胞、腺上皮细胞、基质细胞、血管内皮细胞及子宫肌层平滑肌细胞的细胞核中,少量存在于胞质;PR在发情期牦牛子宫中表达较强,其中腺上皮细胞和血管内皮细胞中表达较强,腔上皮和基质细胞中表达较弱;发情后期普遍下降,间情期表达最低,发情前期有所回升;牦牛子宫内膜中PR mRNA的表达在发情后期最高,间情期最低;肌层PR mRNA的表达则在发情前期最高,间情期最低;但整体而言,内膜中PR mRNA的表达显著低于肌层中的表达。(3)发情周期不同阶段牦牛子宫中均有FSHR蛋白和mRNA的表达,FSHR免疫阳性产物定位于子宫腔上皮细胞、腺上皮细胞、基质细胞、血管内皮细胞和子宫肌层平滑肌细胞的细胞质中;子宫中FSHR蛋白在发情期表达强度最强,发情后期下降,间情期表达显著降低,发情前期回升;而FSHR mRNA则在发情前期和发情期表达最强,发情后期下降,间情期表达显著降低并达最低值。(4)发情周期不同阶段牦牛子宫内膜及肌层平滑肌中均有LHR蛋白和LHR mRNA的表达;LHR蛋白免疫阳性产物定位于牦牛子宫内膜腺上皮细胞、基质细胞、血管内皮细胞、血管平滑肌细胞和肌层平滑肌细胞的细胞质中;腺上皮细胞、基质细胞和肌层平滑肌细胞中发情前期和发情期表达最弱,发情后期表达增加,间情期表达最强;子宫内膜血管平滑肌细胞中LHR的表达在发情期最强,间情期最弱;血管内皮中LHR在发情期和发情前期表达很强,发情后期和间情期显著下降。牦牛子宫内膜中LHR mRNA在发情前期表达最低,间情期表达显著升高并达最高值;子宫肌层平滑肌中,LHR mRNA在发情期表达最低,间情期表达最高。(5)1g/L胶原酶消化1 h,500 r/min离心10 min,经两代差速消化纯化获得的上皮细胞纯度为96 %,基质细胞纯度为93 %;各浓度17β-雌二醇均能促进上皮细胞和基质细胞的增殖,孕酮促进基质细胞增殖,但对上皮细胞的增殖有抑制作用,不同浓度的17β-雌二醇和孕酮共同作用均能显著促进基质细胞增殖,而对上皮细胞增殖的影响随混合液中孕酮浓度的增加而由促进转为抑制。结论:(1)发情周期不同阶段牦牛子宫中ERα、PR、FSHR和LHR mRNA和蛋白质都出现规律性的变化。表明,牦牛子宫功能的变化与ERα、PR、FSHR和LHR转率和翻译水平的调控有关。(2)本研究获得了高纯度的牦牛子宫内膜上皮细胞和基质细胞,雌激素能促进两种细胞的增殖,孕激素促进基质细胞增殖但抑制上皮细胞增殖。更多还原

【Abstract】 Objective: Yak (Bos grunniens) is one of the most important and special breed living in high mountain grassland at harsh climate with extremely cold temperature and low oxygen content. They are excellent pack animals, and as they also produce milk and meat, they are of vital importance to the people living in high altitude areas. Yak is typical animal with seasonal estrous, has low reproductive efficiency, most yak cows calve only once every 2 years. There is, however, no information available on the expression of reproductive hormone receptors in yak. Present study was undertaken to investigate the different expression of estrogen receptorα(ERα)、Progestogerone receptor(PR)、follicle-stimulating hormone receptor (FSHR)and luteinizing hormone receptor (LHR) in yak Uterus during estrous cycle, and study the effects of different concentration combination of 17β-estrodiol and progesterone on yak endometrial epithelial and stromal cells proliferation.Methods: (1) Healthy yak at different estrus including estrus, metestrus, diestrus and proestrus were collected from Tian Zhu county of Gan Su province, bleeding at the local abattoir. Animals were eviscerated, then the uterine well were taken and put into liquid nitrogen for fluorescent quantitative RT-PCR; additional uterine well were flushed by physiological saline three times and immersed in 4% neutral buffered formalin at 4℃for 24 h-48 h. Method of fluorescent quantitative RT-PCR and streptavidin-perosidase (SP) immunohistochemistry were used to determine the levels of mRNAs and expression of proteins for ERα, PR, FSHR and LHR.(2) Healthy and Sexual matured yak without pregnant were collected from Tian Zhu county of Gan Su province, bleeding at the local abattoir. Animals were eviscerated, the uterus were taken immediately to laboratory at low temperature in 3 hours. Collagenase1enzymolysis, centrifugal and different-time degesting purification were used to isolate and purify the endometrial epithelial and stromal cells of yak. Purified cells passaged, immunocytochemical stained, growth curve were analyzed, then endometrial epithelial cells and stromal cells were cultured respectively in different concentration combination of 17β-estrodiol and progesterone added-media, the proliferative capability of the cells in vitro was determined indirectly by MTT.Results: (1) ERαimmunoreactivity expressed in surface epithelium cells, gland epithelium cells, stromal cells, endometrial blood vessel and myometrial smooth muscle cells, most at nuclei and some at cytoplasm. ERαexhibited stronger immunoreactive at estrus, especially at gland epithelium cells; ERαdecreased at metestrus, and to the lowest at diestrus, then re-increased at proestrus. ERαmRNA was expressed in both endometrium and myometrium of yak uterus. In endometrium, high expression of ERαmRNA were observed at metestrus, significantly decreased at diestrus, then re-increased at proestrus; In myometrium, it were highly expressed at estrus, significantly decreased at metestrus, lowest at diestrus, and re-increased to its highest at proestrus. And the ERαmRNA in myometrium was higher than that in endometrium.(2) PR immunoreactivity expressed in surface epithelium cells, gland epithelium cells, stromal cells, endometrial blood vessel and myometrial smooth muscle cells, most at nuclei and a few at cytoplasm. PR exhibited stronger immunoreactive at estrus, and expressed higher at gland epithelium and endometrial blood vessel cells than that at epithelium and stromal cells; PR decreased at metestrus, and to the lowest at diestrus, then re-increased at proestrus. PR mRNA was expressed in both endometrium and myometrium of yak uterus. In endometrium, expression of PR mRNA observed the highest at metestrus and the lowest at diestrus, then re-increased at proestrus; In myometrium, expression of PR mRNA observed the highest at proestrus and the lowest at diestrus. And the PR mRNA in myometrium was higher than that in endometrium.(3) FSHR immunoreactivity localized in surface epithelium cells, gland epithelium cells, stromal cells, endometrial blood vessel and myometrial smooth muscle cells, FSHR exhibited stronger immunoreactive at estrus compared to that at diestrus, and re-increased at proestrus. FSHR mRNA were highly expressed at proestrus and estrus, decreased at metestrus, significantly decreased and to lowest at diestrus, and increased to its highest at proestrus(4) LHR protein and LHR mRNA were expressed in both endometrium and myometrium; LHR immunoreactivity localized in gland epithelium cells, stromal cells, blood vessel endothelium, vascular smooth muscle and myometrial smooth muscle cells; in gland epithelium cells, stromal cells and myometrial smooth muscle cells, the immunoreactivity of LHR were weakly exhibited at proestrus and estrus, increased at metestrus, and strongly exhibited at diestrus; in vascular smooth muscle cells LHR were strongly exhibited at estrus, significantly decreased at metestrus and most weakly exhibited at diestrus; but in blood vessel endothelium, strongly exhibited at proestrus and estrus, significantly decreased at metestrus and diestrus. In endometrium, LHR mRNA were lowest expressed at proestrus and increased significantly at diestrus; In myometrium, LHR mRNA were lowest expressed at estrus and increased significantly at diestrus.(5) High purity epithelial cells and stromal cells were isolated digested by 1g/L collagenase1 for 1 h and centrifuged at 500 r/min for 10 min, cell purification of epithelial and stromal cells respectively were 96 % and 93 %. Different concentration of 17β-estrodiol all could stimulate proliferation of epithelial cells and stromal cells; different concentration of progesterone could stimulate proliferation of stromal cells, but inhibit that of epithelial cells; combination of 17β-estrodiol and progesterone could stimulate proliferation of stromal cells, and inhibit epithelial cells when progesterone dominated. Conclusion: (1) mRNAs and proteins for ERα、PR、FSHR and LHR in yak uterus were differed at different stages of the estrous cycle. The differences suggested that ERα、PR、FSHR and LHR played important roles in yak uterus during the estrous cycle.(2) High purity yak endometrial epithelial cells and stromal cells were isolated, 17β-estrodiol stimulate proliferation of both endometrial cells, progesterone stimulate proliferation of stromal cells while inhibit that of epithelial cells.更多还原