Secretion Of Hypothalamus GnRH And The Expression Of GnRHR In The Pituitary And Ovary During The Estrus Cycle Of Ganjia Tibetan Sheep

The reproductive activity of female animals was regulated by the(hypothalamuspituitary-ovary axis HPOA),gonadotropin-releasing hormone(GnRH)was key information molecule of hypothalamus-pituitary-ovary axis,it can directly affect the secretion of gonadotrophin(GTH)and ovary steroid hormones and then it regulated the reproductive process of estrus and ovulation.Regulation of reproductive function by GnRH must be mediated by gonadotropin releasing hormone receptor(GnRHR).Therefore,the dynamic secretion of hypothalamus GnRH and the distribution of its receptors in the pituitary and ovary and the dynamic changes of gene mRNA expression in the estrus cycle of Ganjia Tibetan sheep were studied.This study provided theoretical basis for studied reproductive hormones regulated the reproductive physiological activities of Ganjia Tibetan sheep.In order to elucidate the roles of hypothalamic gonadotropin-releasing hormone(GnRH)and its receptor(GnRHR)in reproductive regulation in the estrus cycle of Ganjia Tibetan sheep.In this study,the secretion of GnRH in plasma and the distribution of GnRHR in pituitary and ovarian tissues and the dynamic changes of GnRHR mRNA expression in different stages of estrus cycle were studied.In the breeding season,48 male Ganjia Tibetan sheep aged 2.5 to 3.5 years were selected and developed normally,According to the four-point method of the estrus cycle,the Ganja Tibetan sheep were divided into four groups: proestrus,oestrus,metestrus,dioestrum.The plasma,pituitary and ovarian tissues of Tibetan sheep were collected at various stages of the estrus cycle.(1)The content of GnRH in plasma of Ganjia Tibetan sheep was determined by enzyme-linked immunosorbent assay(ELISA),and the dynamic changes in the estrus cycle were analyzed.(2)The dynamic expression of GnRHR gene in the pituitary and ovarian tissue of Ganjia Tibetan sheep was studied by real-time RT-PCR(RT-PCR).(3)The structure of pituitary and ovarian tissues was observed by HE staining;(4)Immunohistochemical SP method was used to analyze the localization of GnRHR positive cells in the pituitary and ovarian tissues of Ganjia Tibetan sheep in proestrus,oestrus,metestrus,dioestrum and combining with computer iage analysis software(Image pro-plus),the integrated optical density of GnRHR immunoreactive products in pituitary and ovarian tissues of Ganjia Tibetan sheep was analyzed.The results of the study were as follows:1.The secretion of hypothalamus GnRH in plasma had certain regularity in the estrus cycle of Ganjia Tibetan: the level of GnRH in plasma was the lowest in the dioestrum(55.687 pg/ml),after which secretion increased,in the proestrus(57.409 pg/ml),the estrus increased to maximum(73.389 pg/ml),and the secretion in the metestrus(62.058 pg/ml)decreased.The secretion volume of GnRH in estrus was the highest and was different from the other three periods(P<0.05),the secretion of GnRH was pulsatile at the whole estrous cycle,the same time,the diestrus and proestrus was volatility.2.The relative expression of GnRHR mRNA in pituitary during the estrous cycle of Ganjia Tibetan sheep in the proestrus,estrus,metestrus and diestrus were respectively 0.8246±0.1739,1.5100±0.4187,0.2923±0.0202 and 1.0000±0.2100.The relative expression of GnRHR mRNA in estrus was the highest and was very significantly different from that in metestrus(P<0.01),and was different from that in proestrus and diestrus(P<0.05).In the ovaries,the relative expression of GnRHR mRNA were respectively 1.0910± 0.7665,0.5420±0.0763,1.3799±0.7452 and 1.0000±1.1221.The relative expression of GnRHR mRNA in metestrus was the highest and was very significantly different from that in estrus(P<0.01),and was different from that in proestrus and diestrus(P<0.05).3.HE staining shows that(1)According to the different cytoplasm staining of the pituitary,could distinguish three types of cells,the cytoplasm of pituitary cells were stained red for eosinophils with the large volume and a round shape;the nucleus were stained purple for basophil with the largest volume,round or oval;the cytoplasmics were stained light color,the smallest and irregularly shaped.The increased number of basophilic cells from proestrus,estrus,metestrus,dioestrum of Ganjia Tibetan sheep,reduced during the dioestrum;The eosinophils were less in proestrus and estrus,the number of metestrus and dioestrum were more;The chromosome cells do not have complete cell pellets and were more in proestrus and dioestrum.(2)The primordial follicles were mainly located in the superficial cortex,small volume,numerous in number and concentrated together,and the follicle cell boundaries were not obvious.The primary follicle follicle cells had distinct boundaries surrounding the primary oocyte.The secondary follicles were located in the deeper of ovarian cortex,the volum of follicles were increased,and the follicle cells increased to multiple layers surrounding the secondary oocytes.The tertiary follicles were located in the medulla,and the follicle cells divided into more granulosa cells.The cell boundaries were clear and the follicular cavity appeared,filled with follicular fluid.The mature follicles located on the surface of the ovary and had the largest volume.The follicular cavity grows to the maximum and fills with the follicular fluid.At this time,the zona pellucida and radiating crown were more pronounced.Occlusion of follicular oocytes was atrophic and irregular.Luteal cells were larger,the boundaries between the cells and the surrounding tissue were clear,and the cytoplasm was darker.4.Immunohistochemistry showed that:(1)GnRHR was mainly distributed in the pituitary gland in the distal part of the pituitar;Gn RHR positive cells were distributed in the highest density in the estrus,and the cell shape was mostly round.The number of positive cells in the metestrus and diestrus was small and the cells were ovoid;the positive cells in the proestrus were relatively numerous and irregular in shape.Using computer image analysis software(IPP),the integrated optical density of GnRHR immunoreactive cells in the pituitary during the estrus cycle of Ganjia Tibetan sheep in the proestrus,estrus,metestrus,dioestrum were respectively: 9126874.8624±39582.6795,9628562.0168±49365.2516,7486923.0574±52684.8536,7395086.6842±49572.8143,the expression of GnRHR protein in the estrus was significantly higher than that in the metestrus and the diestrus(P<0.01).There was a significant difference between the proestrus and the metestrus and diestrus(P<0.01).(2)GnRHR immunoreactive cells were distributed in all stages of follicular oocytes,follicles,follicles,follicles,corpus luteum,and corpus luteum in all phases of the estrus cycle of Tibetan Gandhi Tibetan goats.There were also immunopositive reactions in the perifollicular stromal cells.According to the analysis of IPP software,the integrated optical density of GnRHR immunoreactive cells in the proestrus,estrus,metestrus,dioestrum respectively: 869625.19 54±6921.2894,895264.6931±6137.9752,973841.3840±5941.3698,897316.2547±5723.46 18,GnRHR protein expression was most in metestrus and was significantly different from that in estrus,proestrus,and diestrus(P<0.01).There was no significant difference between estrus,proestrus and diestrus(P>0.05).Conclusion: The content of GnRH in plasma during the estrus cycle of Ganjia Tibetan sheep was the lowest in the estrus and the highest in the estrus.The relative expression of GnRHR mRNA in estrus was highest in pituitary,and the relative expression of GnRHR mRNA in ovaries was highest in metestrus.The GnRHR immunoreactive cells in the pituitary were mainly distributed in the distal part,and the distribution density was highest in the estrus.The GnRHR-positive cells were distributed in the follicle oocytes,follicle cells,follicle granular layer,follicle membrane,and corpus luteum.The integrated optical density of GnRHR immunopositive products was highest in metestrus.The above results could provide the theory basis of including hormone,molecular and cell levels for the further studied the influence of hypothalamic reproductive hormones for the pituitary,follicles and reproductive performance in different development stages.