| Spermatogenesis and testicular development in mammals is regulated by many genes.Spermatogenesis and testicular development are directly related to the quality of semen,and semen quality directly affects the reproductive performance of male animals.It is reported that luteinizing hormone receptor(LHR)and prolactin receptor(PRLR)genes play an important role in mammalian reproductive development and exist in various tissues.GH and IGF-1 genes are the key genes on the growth axis of animals.Therefore,a large number of studies focus on improving the growth rate and meat production performance of animals,while relatively few studies on reproductive performance.However,LHR,PRLR,GH and IGF-1 genes play important roles in spermatogenesis,testicular development and functional maintenance in male animals,while studies on the different stages of testicular development in yak,a species endemic to the qinghai-tibet plateau,are still in a blank stage.Therefore,it is of great significance to explore the regulatory role of LHR,PRLR,GH and IGF-1 genes in the development of yak testis,and to reveal the molecular regulatory mechanism of spermatogenesis and maintenance of testis function in male yak.In this study,different tissue of yak such as testis,kidney,liver,lung,heart,spleen,skeletal muscle,subcutaneous fat were collected from different growing stages prepubertal stage(6 months),first estrus(24 months),sexual maturity(36 months),and adult(72 months)of male yak.The LHR,PRLR,GH,IGF-1 genes were detected by qRT-PCR and immunohistochemistry for mRNA detection and protein localization.The main findings are as follows:1.The expression level of LHR gene increased gradually from prepubertal stage to sexual maturity,while,reached its peak in sexual maturity,and slightly decreased in adulthood.Among them,the expression level in the sexual maturity stage was significantly higher than that in the primary and juvenile stages(P<0.01),with no significant difference from that in the adult stage.In addition to that LHR mRNA was evaluated in 7 different tissues of yak.Expression level of LHR gene was the highest in testis(P<0.05),followed by kidney and skeletal muscle,and the lowest in liver(P<0.05).While,immunohistochemistry results showed that the expression of LHR protein was mainly localized in mesenchymal cells and spermatogenic cells.2.The expression level of PRLR gene increased gradually from infancy to adulthood.Among them,there was no significant difference between the adult stage and the sexual maturity stage,but both were significantly higher than the primary and juvenile stages(P<0.01).The expression level of PRLR gene was highest in kidney followed by skeletal muscle,testis and spleen(P<0.05),and lowest in liver.Furthermore,immunohistochemistry findings illustrated that PRLR protein was expressed in the supporting cells,mesenchymal cells,primary spermatocytes and secondary spermatocytes of the testis during prepubertal,sexually mature and adult stages.The expression level of PRLR protein increased rapidly with advanced age,and the expression level of PRLR protein in the early stage was significantly higher than that in the early stage(P<0.05),and the expression level in the sexual maturity stage was higher than that in the adult stage(P<0.05).3.The expression level of GH gene reached its peak in the period of prebubertal and sexual maturity,while slightly decreased in the adult period.Among them,the expression level was higher in the sexual maturity and prepubertal stage than that of adult.qPCR findings showed that the relative expression of GH gene was higher(P>0.05)in the spleen followed by testis,heart,liver,kidney,subcutaneous fat and skeletal muscle(P<0.05).The expression level of skeletal muscle was the lowest,while that of heart was slightly higher.There was also a significant difference between skeletal muscle and lung(P<0.05).Western blot results showed that GH protein expression was significantly higher in the early and sexual maturation stages than that of other developmental stages.Immunohistochemical results showed that GH protein signals were present in the cytoplasm of some testicular mesenchymal cells in the infancy,and GH positive reactions were found in spermatogonial cells,spermatogonial cells and spermatogonial cells in the sexual maturity and adult stages.GH integral optical density value was significantly higher in sexual maturity than in adulthood,and adult stage is significantly higher than the prepubertal stage(P<0.05).4.The expression level of IGF-1 gene increased gradually from infancy to sexual maturity,and decreased slightly in adulthood.Among them,the expression level at the sexual maturity stage was significantly higher than that at the adult stage(P<0.01),while the expression level at the adult stage was significantly higher than that at the first estrus,and the expression level at the first estrus was significantly higher than that at the prepubertal stage(P<0.05).IGF-1 mRNA was expressed in 7 tissue samples.The relative expression level in liver was 12.97,with the highest expression abundance,followed by spleen,testis,subcutaneous fat,lung,heart and skeletal muscle(P<0.05),among which there was no significant difference between heart and skeletal muscle and kidney(P>0.05).Western blot results showed that the expression level of IGF-1 protein in sexual maturity was significantly higher than that in other developmental stages,and the expression trend was basically consistent with mRNA.Immunohistochemical results show: in the juvenile stage,IGF 1 protein signal exists in part of the cytoplasm of leydig cells,maturation stage and adult stage in testicle spermatocyte,spermatagonial cells,sperm cells were positive reaction of IGF-1,suggests that IGF 1 protein may through autocrine/paracrine manner to adjust the development of testicles,no positive signals and supporting cells.IGF-1 integral optical density value was significantly higher in sexual maturity than in adulthood,and adult stage is significantly higher than the prepubertal stage(P<0.05).IGF-1 gene expression level in sexual maturation period is significantly higher than other three periods. |
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