Regulation Mechanism Of Goat Reproductive Hormone Secretion By Illumination Signal Tachykinin And Its Receptor Genes

Tachykinins(TKs)belong to the kinin family.Studies have shown that tachykinins affect the release of gonadotropin-releasing hormone(GnRH)through hypothalamic neurons,thus affecting a series of reproductions such as estrus,ovulation and pregnancy process in animals.At present,it is believed that there are two main pathways that regulate mammal reproduction,one is that the pineal body secrets melatonin,which directly acts on the isthmus of the pituitary gland and regulates thyrotropin,under the influence of external light.Thyrotropin then feeds information back to the hypothalamus to regulate the reproductive function of mammals.Another pathway is that the pituitary regulates the seasonal reproduction of mammals by regulating the rhythmic release of prolactin(PRL)and the important signaling substances in this pathway may include a series of low-molecular-weight peptides including tachykinins.Therefore,we studied the relationship between the expression of tachykinin and its receptor gene in hypothalamus,pituitary and ovary of goats and the length of illumination.The purpose of this study is to provide a theoretical basis for its involvement in the regulation of hypothalamic-pituitary-gonadal axis in goat reproductive process.In this experiment,20 Henan Huai goats were raised in the pasture of Zhoushan campus of Henan University of Science and Technology.The average age of the ewes was one year old and the average weight was 37.75 kg.The goats were divided into short illumination period(8 h light: 16 h dark)and long illumination period(16 h light: 8 h darkness).The short illumination period was six weeks and the long light period was four weeks.The cDNA sequences of TAC1,TACR1,TACR2 and TACR3 genes of Huai goat were successfully cloned and sequenced by using primer premier 6.0primer design software to design the specific primers of the target gene.The first strand of cDNA was used as the template to clone and sequence the cDNA sequences of the TAC1,TACR1,TACR2 and TACR3 genes of Huai goat.The genes of tachykinin and its receptor were analyzed deeply by bioinformatics analysis software.The results showed that both TAC1,TACR1 and TACR2 genes had no transmembrane domain,encoding 130,407 and 384 amino acid residues respectively,and TACR3 gene had seven transmembrane domains encoding 539 amino acid residues.The transcriptional group sequencing and bioinformatics analysis were used to screen the signal pathways and candidate genes related to goat reproduction.The results showed that a total of 440 million reads,samples were obtained from RNA-Seq,and the average reads number per sample was 52.49 million,and a total of 241 signaling pathways,including 448 differentially expressed genes(DEGs)were found in the two groups.The results of Real-time PCR analysis showed that the sequencing results of transcription group were reliable,and the differentially expressed gene(DEGs)was significantly enriched in calcium signaling pathway(chx04020),and three candidate genes,TACR1,TACR2 and TACR3 were screened out.By means of immunofluorescence,immunohistochemistry,Western blot and Real-time PCR,enzyme-linked immunosorbent assay,the relative expression of TAC1,TACR1,TACR2 and TACR3 genes in the organs of each group was studied under long and short light conditions and the changes of PRL,LH and FSH in peripheral blood.The results showed that when animals were exposed to short light for 24 hours,the levels of serum hormones such as prl,lh and fsh were higher than those of animals under long illumination.The expression levels of tac1,tacr1 and tacr3 genes in all tissues and organs decreased after short light exposure to long light exposure(tacr2 gene showed an upward trend).It was found that the expression of tachykinin gene in pituitary cells could be inhibited or promoted by the addition of exogenous tachykinin and its receptor gene expression inhibitors or agonists,and the secretion of prolactin by pituitary cells could be influenced to a certain extent by adding exogenous tachykinin and its receptor gene expression inhibitors or agonists.The content of prolactin increased with the increase of tachykinin gene expression and decreased on the contrary.Conclusion: the long and short illumination conditions and the gene of tachykinin and its receptor are closely related to the seasonal reproduction and the secretion of prolactin and other reproductive hormones in goats.Light may co-regulate the function of hypothalamus-pituitary-gonad axis and other organs in goats with tachykinin gene.In this study,the effects of light-mediated tachykinin and its receptor genes on reproductive regulation or neuroendocrine activity in goats were investigated.It was found that goats,as seasonal breeding animals under short light,were affected by light signals.The levels of reproduction-related tachykinin and its receptor genes also changed in the body,which resulted in a series of regulatory effects on the secretion of prolactin and other reproductive hormones in goats.The results showed that the gene level of tachykinin and its receptor decreased in pituitary and reproductive organs after the goat changed from short light period to long illumination period,and the gene level of tachykinin and its receptor gene decreased.The content of prolactin and other reproductive hormones in goat body was decreased by the regulation of tachykinin and its receptor gene.This study not only provides a theoretical reference for further elucidating the regulation of mammalian reproductive physiology by tachykinin gene,but also provides relevant bioinformatics data for the biological research of goat tachykinin and its receptor genes in the future.