The Development Of Spermatogenic Cells And The Analysis Of LncRNA Differential Expression In Goats

For male animals,sexual maturation and semen quality are determined by their testicular development status,and the reproductive performance of males is ultimately affected.Spermatogenesis originates in the self-renewal and differentiation of spermatogonial stem cell(SSCs),and the distribution of SSCs in testis tissue reflects the degree of testis development.The histological analysis can be directly used to observe the changes of testicular structure and the distribution of spermatogenic cells.lncRNAs(long noncoding RNAs)are transcripts of more than 200 nucleotides in length with little or no potential for translation.They are expressed only at specific stages of cell development and differentiation,and have obvious expression specificity.Increasing evidence points toward a role of lncRNAs as determinants of stem cell fate;specifically,as regulators of self-renewal and differentiation.However,the information on the regulation of lncRNAs on proliferation and differentiation of SSCs is scanty.In order to explore the difference of lncRNA expression in testis tissue of Shaanbei white cashmere goats at different developmental stages,the testes of Shaanbei white cashmere goats(one-,three-,six-month-old and adult goats)were collected and cut into pieces for staining using hematoxylin eosin.The morphology of seminiferous tubules and the number of germ cells were observed and calculated under a microscope to analyze testis developmental degree.In addition,the immunostaining was also performed.To analyze the relative abundance of SSCs of Shaanbei write cashmere goats at different developmental stages,the relative expression of SSC marker genes(CD49f,PLZF,Thy1,ETV5,VASA,Oct4,Nanog,Nanos,CyclinA)in testis was detected and compared using q-PCR.Finally,the lncRNA expression levels of one-,three-month-old and adult goats testis were detected and compared using lncRNA sequencing.The main results of this research were as following:1.The diameter of goat seminiferous tubules increased significantly with age(P < 0.05).The difference in diameter between 3-month-old and adult goats was not significant,representing 150.72 μm and 161.94 μm,respectively(P > 0.05).The cell layer of goats also increased with age,the cell layer of 6-month-old goat(7.70 layers)was significantly higher than that of one-and three-month-old goats,respectively(P< 0.05).Moreover,the proportion of spermatogonia of 3-month-old goats(46.40%)was significantly higher than that of one-,six-month-old and adult goats,just about 33.10%,9.10% and 7.60%,respectively(P< 0.05).The total number of cross-sectional cells in seminiferous tubules both of six-month-old and adult goats was strikingly different compared to those of 1-month-old and 3-month-old cashmere goats(P< 0.05).The results showed that the cashmere goats reached sexual maturity in 6 month old,and the most abundant spermatogonia were in 3-month-old testis.2.The CD49 f,CD90,GFRa1,ETV5 and PLZF were highly expressed in the testis of 3-month-old goats(about 41.67%,40.20%,21.92%,25.36%,respectively)than those of 6-month-old and adult goats(P< 0.05);The positive rates of CD49 f,CD90,GFRa1,ETV5 and PLZF of 6-month-old goats were slightly higher than those of adult goat(P> 0.05).The expression of PLZF in testis decreased significantly with age(P< 0.05).Marker genes of SSCs,such as CD49 f,PLZF,Thy1,ETV5,VASA,Oct4,Nanog and Nanos,were highly expressed in the testis of 3-month-old goats,and except for the CD49 f gene,expression levels of other genes were significantly higher than those of other groups(P< 0.05).In addition,the expression of CyclinA in 6-month-old goat testis was significantly higher than that of other groups(P< 0.05).The results demonstrated that compared with other groups,SSCs accounted for the highest proportion in 3-month-old goat testis(P< 0.05),and that 3-month-old goat testis was the ideal material for isolation and enrichment of SSCs in vitro.3.Sequencing results showed that17 lncRNAs were expressed differentially in testis between one-and three-month-old goats,of which 13 were up-regulated and 4 down-regulated expression.By comparing 1-month-old goat and adult cashmere goat,we found that the expression levels of 247 lncRNAs were different,of which 239 were up-regulated and 8 down-regulated expression.Meanwhile,there were also differences between three-month-old and adult goats with 201 lncRNAs,of which 174 expression of 27 genes down-regulatedexpression.These differentially expressed genes and lncRNA may be involved in the regulation of spermatogenic cell development.The analysis of these differentially expressed lncRNAshowed that these lncRNA played a role in various pathways,which laid the foundation for the study of the effects of lncRNA on spermatogenesis.This study shows that the proportion of SSCs in 3-month-old goat testis was the highest,which was suitable for SSCs enrichment.6-month-old goat has reached sexual maturity with a large number of sperm cells in the testes.lncRNA sequencing results showed differential expression of lncRNA existed in testes at different developmental stages.These differential expressed lncRNAs may be related to the regulation of spermatogenic cell development.