| The KISS-1/GPR54 system is composed of the melanoma metastasis suppressor gene(KISS-1)and its expression product kisspeptin and its specific G protein-coupled receptor 54(G protein coupled receptor 54,GPR54).kisspeptin mainly through regulation of hypothalamic gonadotropin-releasing hormone(GnRH),which in turn affects the secretion of follicle-stimulating hormone(FSH)and luteinizing hormone(LH),plays an important role in regulating reproductive activities such as priming,estrus cycle,ovulation and so on in mammals.Ganjia Tibetan sheep live in alpine areas above 3000 m above sea level,belong to typical seasonal estrus animals,low reproduction rate,one child per year,one lamb per child.At Current,there is no reports have been reported at home and abroad on whether the KISS-1/GPR54 system can directly affect the reproductive activities such as seasonal reproduction,estrus cycle and molecular regulation mechanism of Ganjia Tibetan sheep.In order to elucidate the role of KISS-1/GPR54 in reproductive activity of Ganjia Tibetan sheep,this study for the first time systematically investigated the dynamic changes and expression and distribution of KISS-1/GPR54 gene and protein of hypothalamicpituitary-ovarian axis(HPOA)of Ganjia Tibetan sheep in different stages of estrus cycle.Thirty-two Ganjia Tibetan sheep with healthy body and not pregnant were selected and were divided into four groups: estrus,postestrus,diestrus and proestrus.Each group of 8 was tested.After determining estrus,the hypothalamus,pituitary and ovarian tissues were taken in each period of estrus cycle.(1)Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the changes of KISS-1/GPR54 mRNA expression in HPOA during each period of estrus cycle;(2)Using immunohistochemical(IHC),combined with computer image analysis software(Image pro-plus),the number,distribution and integral optical density of kisspeptin/GPR54 immunoreactive cells in the estrous cycle of Ganjia Tibetan sheep were studied.(3)Western blotting(WB)was used to quantitatively detect and analyze the expression of kisspeptin/GPR54 proteins in the hypothalamus,pituitary and ovarian tissues of Ganjia Tibetan sheep during the estrus cycle.The findings are as follows:1.qRT-PCR results show that there were KISS-1 and GPR54 mRNA expression in the hypothalamus,pituitary and ovary tissues of Ganjia Tibetan sheep during estrus cycle,and the expression was different :(1)The relative expression of KISS-1 andGPR54 mRNA in hypothalamus was the highest in preestrus and estrus,which was significantly higher than other estrus periods(p<0.05);(2)The relative expression of KISS-1 and GPR54 mRNA in pituitary was the highest in preestrus,significantly higher than other periods(p<0.05);(3)the relative expression of KISS-1 and GPR54 mRNA in ovary was the highest in diestrus and postestrus,which was significantly higher than other periods(p<0.05).Indicating that the KISS-1/GPR54 mRNA system has a strong regulatory effect on hypothalamic and pituitary functional activity from preestrus to estrus stage in Tibetan sheep,and the regulatory effect on ovary is mainly focused on late estrus and interestrus stage.2.The results of immunohistochemical staining showed: kisspeptin and GPR54 immunoreactive expression was found in the hypothalamus,pituitary and ovarian tissues of Ganjia Tibetan sheep during estrus cycle.(1)kisspeptin in the hypothalamus are mainly expressed in the cytoplasm of nerve cells and pyramidal cells,and some dendrites of nerve cells also have positive reactions,GPR54 in addition to the above reaction areas,positive reactions are also detected on the nuclear membrane of some granulosa cells.(2)The kisspeptin and GPR54 in pituitary tissue are mainly expressed in the cytoplasm of basophils.(3)The kisspeptin and GPR54 in the ovary are mainly expressed in follicular cells in the preestrus stage,the follicular membrane has a small amount of expression,the estrus stage is mainly high in the granulosa layer and the inner layer of the follicular membrane,and enters the postestrus and dieestrus stage,mainly in the granulosa layer,follicular intima and extrafollicular membrane,especially in the increased expression of the extrafollicular membrane.The average integral optical density of kisspeptin immune positive products in hypothalamus and pituitary tissues was the highest in preestrus,significantly higher than other estrus periods(p<0.05),GPR54 the highest in estrus and preestrus;the highest expression of kisspeptin/GPR54 in ovary in preestrus and estrus,and the significant difference in expression between estrus(p<0.05).3.Western blot analysis: The kisspeptin and GPR54 in hypothalamus,pituitary and ovary were expressed in estrus cycle of Ganjia Tibetan sheep,and the expression of different estrus periods was different.(1)The relative expression of kisspeptin and GPR54 in hypothalamus and pituitary in preestrus were significantly higher than those in other estrus periods(p<0.05);(2)The relative expression of kisspeptin and GPR54 in ovary tissue in postestrus and in preestrues were the highest,significantly higher than those in other estrus periods(p<0.05).Suggesting that kisspeptin/GPR54 functional regulation of hypothalamic and pituitary tissues is mainly dependent on high kisspeptin expression.kisspeptin mainly in the meestrus on ovarian function regulation.Conclusion:(1)there are kisspeptin/GPR54 immune positive cells and KISS-1/GPR54 mRNA and their protein distribution expression in the HPOA tissues at all stages of the estrus cycle,which show that kisspeptin/GPR54 plays an important role in regulating HPOA function of Ganjia Tibetan sheep.(2)the regulation of HPOA functional activities mainly depends on the high expression of kisspeptin,and the regulation of hypothalamus and pituitary functional activities is strong in the preestrus to estrus period of Ganjia Tibetan sheep,and the regulation of ovary is mainly in the estrus period and metestrus period.KISS-1/GPR54 system regulates the estrus cycle HPOA functional activities of Ganjia Tibetan sheep. |
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