| Mammalian spermatogenesis is a cascade process controlled by multiple gene,which is carried out smoothly to directly determine semen quality and have great influence on reproductive performance for male.The research reports show that the Dmrt1,Dmrt7,Boule and Dazl gene are closely involved in the regulation of testicular function maintenance and spermatogenesis in male gonads.However,most reports on the Dmrt1,Dmrt7,Boule and Dazl were human and mouse,and the regulation mechaisms are not exactly same in broad variety of animals.No reports on expression patterns and function of the Dmrt1,Dmrt7,Boule and Dazl gene in sheep at present.Accordingly,it is significant to elucidating the mechanisms of ram spermatogenesis by investigating regulatory role of the Dmrt1,Dmrt7,Boule and Dazl gene in the ovine testicular development.In this study,testicular tissues of sheep from five crucial development stages,including 0-month-old(neonate),2-month-old(weaning),5~6-month-old(puberty),12-month-old(after sexual maturity)and 24-month-old(adult),and heart,liver,spleen,lung,kidney and muscle tissues of 12-month-old sheep,was taken as the experimental materials.The expression levels of Dmrt1,Dmrt7,Boule and Dazl gene were measured on the level of both transcription and translation by qRT-PCR and Western blot,respectively.Meanwhile,the expression levels of Dmrt1,Boule and Dazl mRNA were detected in various tissues of 12-month-old sheep by qRT-PCR.Moverover,the positive signals for Dmrt1,Dmrt7,Boule and Daz protein were examined in sheep testes from different development stages using the immunohistochemical staining.The purpose of this study was to preliminarily evaluate and explore the role of Dmrt1,Dmrt7,Boule and Dazl gene in the regulation of ovine testicular development and spermatogenesis.The main findings are as follows:1.The expression levels of Dmrt1 mRNA and protein in 12 and 24-month-old sheep testes were extremely significantly higher than those in 0 and 2-month-old sheep testes(P<0.01).The qRT-PCR results in various tissues(heart,liver,spleen,lung,kidney,muscle and testis)of sheep at the age of 12-month-old showed that Dmrt1 mRNA was predominantly expressed in testis,while it was almost undetectable in other tissues.Immunohistochemical staining in testes at different developmental stages showed that the positive signals for Dmrt1 protein were mainly localized in Sertoli cells and gonocytes in 0-,2-and 5~6-month-old sheep testes,while the signals were localized in spermatocytes,sperm cells,and some spermatogonia and Sertoli cells in 12-and 24-month-old.These results suggested the Dmrt1 gene plays a vital role during ovine spermatogenesis,perhaps by regulating the maturation and functional maintenance of Sertoli cells,the proliferation and differentiation of gonocytes in prepubertal ram testes,and the mitosis and meiosis of germ cells in adult ram.2.The Dmrt7 mRNA and protein were expressed in sheep testes throughout development,and their change patterns were similar.However,there were differences concerning the expression level of Dmrt7 for the different month groups.Dmrt7 mRNA and protein were lowly expressed,and immunoreactive responses of Dmrt7 protein were localized to minority of spermatogonia,in 0,2 and 5~6-month-old sheep testes.The expression levels of Dmrt7 mRNA and protein were extremely significantly higher than those in 0,2 and 5~6-month-old sheep testis tissues(P<0.01),and immunoreactive responses of Dmrt7 protein were mainly localized to secondary spermatocytes,and partial primary spermatocytes and spermatids,in 12,24-month-old sheep testes.The results demonstrated that Dmrt7 gene plays an essential regulation role in the division processes of various spermatogenic cells in testicular seminiferous tubule,especially meiotic division from primary spermatocytes to spermatids.3.Boule mRNA was specifically expressed in sheep testis,while no expression in other tissues such as heart,liver,spleen,lung,kidney and muscle.The qRT-PCR and Western blot results in testes at different developmental stages showed that the expression levels of Dazl mRNA and protein were extremely significantly lower than those in 12-and 24-month-old sheep testes(P<0.01),and the positive signals of Boule protein were localized to seminiferous tubules,in pre-pubertal sheep(0-,2-and 5~6-month-old)testes.The expression levels of Boule mRNA and protein were significantly elevated in 12-month-old sheep testis,peaked in 24-month-old sheep testis.The positive signals for Boule protein were localized to primary spermatocytes,secondary spermatocytes and sperm cells.These results showed the Boule gene might be involved in regulating meiosis during ovine spermatogenesis.4.The qRT-PCR and Western blot results showed that the mRNA and protein of Dazl gene were expressed in sheep testes throughout development,and their expression patterns were almost the same.The Dazl mRNA and protein were at a lower expression level in pre-pubertal sheep testes(0-,2-and 5-month-old),and then their expression levels in 12-month-old were significantly higher than those in 0-,2-and 5~6-month-old testes(P<0.01),whereas their expression levels were decreased in 24-month-old sheep testis.The qRT-PCR results in multiple sheep tissues at 12 months of age,including testis,heart,liver,spleen,lung,kidney and muscle tissues,showed that the Dazl mRNA was predominantly expressed in testis,and low-expressed in heart,whereas no expression in other tissues.The immunohistochemical staining in testes at different developmental stages showed that Dazl protein was localized to Leydig cells in pre-pubertal sheep testes,while it was localized to Leydig cells,primary spermatocytes,secondary spermatocytes and sperm cells in 12-and 24-month-old sheep testes.These results suggested that the Dazl gene was highly expressed in testes to play vital roles in the proliferation of Leydig cells in various age of sheep testes,spermatocytes and sperm cells in post-pubertal sheep.It is inferred that the Dazl gene is involved in the regulation of postnatal ovine spermatogenesis via direct effect(modulating the maturation and meiosis of spermatocytes)and indirect effect(modulating the proliferation of Leydig cells). |
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