Different Expression Of Reproductive Hormone Receptors In Yak Uterus During Estrous Cycle

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.