Effect Of NYD-SP27 Gene On Sperm Quality And Sperm Signal Pathway In Ovine

The development of mammalian sperm is mainly the testis spermatogenic cell develops into spermatogonial cell,and then a complex series of biological changes,finally formation of sperm.Spermatogenesis was regulated by a complex series of programmed expression,including changes in the chromosome structure,a number of endogenous regulatory factors,and a series of complex procedural expressional regulation of genes.NYDSP27,as an inhibitor of PLC,is a regulator of sperm formation and plays an important role in the regulation of sperm production.[Objective] To explore NYD-SP27 gene effects on ovine sperm motility,deformity rate,changes of reproductive hormones in serum and expression levels of key enzymes in the cAMP of sperm by injected up-regulated and down-regulated plasmid vectors of NYD-SP27 gene in testis.It provides scientific basis for the molecular mechanism of NYD-SP27 gene in the process of sperm formation.[Methods](1)In this study,primers were designed based on the NYD-SP27 gene sequence in Genbank to clone NYD-SP27 gene,and made it connected to a eukaryotic vector named pmcherry-n1.Recombinant plasmids were obtained by coexpression of the target gene and the red fluorescent protein which were connected with P2 A.(2)The up-regulated and down-regulated plasmid vectors of NYD-SP27 gene were injected into the testicles,collectioned semen by electrical stimulation,and the effect of the gene on the sperm motility and deformity rate of the sheep was examined;semen used to made frozen semen particlesthen,which after was thawed and detected its effect on changes of actin and myosin and expression levels.(3)The effect of NYD-SP27 gene on the expression of related enzymes in the cAMP pathway in sperm was detected by ELISA.(4)After injecting the up-regulated and down-regulated plasmid vectors of NYD-SP27 gene into ovine testicles,collected blood and detected the changes in reproductive hormones in serum.[Results](1)The recombinant expression vector pmcherry-Flag-NYDSP-P2 A was successfully constructed;(2)There was no significant difference that the sperm survival rate of between control group and FNP group in the sheep(P>0.05),but control group and Sh group have significant difference(P<0.05);there was no significant difference that the sperm deformity rate of between control group and test group in the sheep(P>0.05);there was no significant difference that the actin expression of between control and FNP group in the sheep(P>0.05),but the control group and Sh group have extremely significant difference(P<0.01);(3)There was significant difference that the sperm survival rate of between control group and Sh group in the sheep(P<0.05),but control group and FNP group have extremely significant difference(P<0.01);There was no significant difference that the protein kinases expression of control group and Sh group in the sheep(P>0.05),but control group and FNP group have significant difference(P<0.05);(4)Down-regulation of the NYD-SP27 gene promotes the secretion of testosterone levels in sheep.[Conclusion](1)The P2 A plays a shearing role in the protein translation process of NYD-SP27 gene expression and red fluorescent;(2)Down-regulation of NYD-SP27 gene do not only promotes the improvement of sperm motility in sheep,but also regulates actin in sperm;(3)Down-regulation of the NYD-SP27 gene regulates the expression of cAMP and PK in the sperm cAMP signaling pathway;(4)Down-regulation of the NYD-SP27 gene regulates the secretion of testosterone levels in sheep.