Association Of Kiss1/GPR54 Gene With Testicular Development And Its Polymorphism In Hu Sheep

Kiss1 and its receptor(Kiss1/GPR54)are closely related to the reproductive system.The purpose of this study was to explore the relationship between Kiss1/GPR54 gene and testicular development and its polymorphism in sheep,and to provide theoretical basis for further study on the function of Kiss1/GPR54 gene and its relationship with reproductive traits.In this study,three male Hu sheep of newborn,30,60,90,120,150 and 180 days were selected,and their body weight,body size,scrotal circumference,testicular weight and testicular size(length,width and thickness)were measured,and testicular index was calculated.The correlation between testicular weight and body weight was analyzed,and their Logistic growth curve fitting model were analyzed,and the inflexion age was calculated.HE staining was performed on testicular tissue sections.The number of spermatogenic cells at all levels and cell layers in seminiferous tubules were counted.The diameter of seminiferous tubules was measured and the correlation coefficient between the measured values was calculated.The expression and distribution of Kiss1/GPR54 in testis were observed by immunohistochemistry.The relative expression of Kiss1/GPR54 gene in testis was determined by Real-time quantitative PCR,and the correlation between the relative expression of Kiss1/GPR54 gene and testicular measurements was analyzed.Hu sheep with average number of lambing at two tails in normal distribution were selected and divided into high-fertility group and low-fertility group.Single nucleotide polymorphism loci of Kiss1 gene and GPR54 gene in Hu sheep population were detected by DNA mixing pool sequencing.Chi-square test was used to analyze the difference of allele frequency and genotype frequency of SNP locus of Kiss1/GPR54 gene between the two groups.The main results are as follows:1.Testicular weight was significantly correlated with body weight,and the correlation coefficient was 0.895(P < 0.05).Logistic model fitting results showed that the inflexion age of body weight and testicular weight were 98 days and 113 days,respectively.There was a significant difference in testicular index between 90-days and 120-days(P < 0.05).2.There were spermatogonia and Sertoli cells in the seminiferous tubules of Hu sheep at birth.At 90-day-old,a small number of primary spermatocytes appeared in seminiferous tubules.At 120-day-old,the number of cell layers in seminiferous tubules increased,and spermatogonia,spermatocyte,early spermatocyte and a small number of spermatozoa appeared with lumen.The number of spermatogenic cells in seminiferous tubules increased at 150 and 180-day-old.The diameter of seminiferous tubules increased from birth to 180-days-old,and there was a significant difference between 90-days-old and 120-day-old(P < 0.05).The diameter of seminiferous tubules was significantly correlated with the number of cell layers,and the correlation coefficient was 0.970(P < 0.05).3.Kiss1 and GPR54 positive products were detected in spermatogonia,spermatocytes,spermatid and leydig cell of testis.The relative expression of Kiss1/GPR54 gene in the testis of Hu sheep from birth to 180-day-old showed the same trend.The relative expression of the two genes was the highest at birth.The relative expression of the two genes decreased from birth to 60-day-old,increased from 60 to 120-day-old and decreased from 120 to 180-day-old.4.During the rapid growth period of testicular weight,testicular volume and seminiferous tubule diameter: from 60 days to 120 days,the relative expression of Kiss1 and GPR54 was significantly correlated with testicular weight and testicular volume(P < 0.05),and extremely significant correlated with seminiferous tubule diameter(P < 0.01).5.Seventeen SNPs were detected in the Kiss1 gene,g.37C>T,g.38G>A,g.89G>T in the 5'UTR region of exon 1,g.3556G>A is located in the coding region of exon 2,resulting in the conversion of valine to methionine(Val>Met),five SNPs in intron 1 and eight SNPs in intron 2.Genotype distribution of g.5117T>C locus was significantly different between high-fecundity groups and low-fecundity groups(P<0.05).The allele frequencies of g.37C>T,g.38G>A,g.89G>A,g.4783C>G,g.5117T>C were significantly different between high-fertility group and low-fertility group(P<0.05).6.Five SNP loci were detected in the GPR54 gene,all located in the 5'UTR region of exon 1.Three loci,g.2606 T > C,g.2625 C > A and g.2721 A > C,formed a linkage block,which constituted two haplotypes: TCA and CAC.Both haplotypes existed in the high-fertility group and only one haplotype in the low-fertility group.In conclusion,Kiss1/GPR54 plays an important role in inducing the onset of puberty,testicular development and spermatogenesis of Hu sheep.The g.37C>T,g.38G>A,g.89G>A,g.4783C>G,g.5117T>C loci of Kiss1 gene and the g.2606T>C,g.2625C>A,g.2721A>C loci of GPR54 gene may be related to the high fertility of Hu sheep.