Identification And Expression Analysis Of Sex Related Genes In Yellow Catfish And Function Study Of Mir-34a In Zebrafish Sperm Motility

Reproductive development and its regulatory mechanism of fish has always been one of the most important issues in the life science research,which not only has important theoretical significance but also has extensive application prospect on guidance of fish species improvement.Sperm motility,an important index of male reproductive ability,has direct impacts on the fertilization rate and quality of offspring.Improvement of male sperm motility is one of the important direction of fish species improvement.We identified genes related to sex and analyzed their expression levels in healthy XX females,XY males,YY super males of yellow catfish(Pelteobagrus fulvidraco).In addition,we found a miRNA which has certain regulatory fuction on sperm motility in zebrafish(Danio rerio).To investigate the molecular mechanism of sex differentiation and gonad development in yellow catfish,we screened out 13 related genes from the transcriptome data of gonad.The expression profile of these 13 genes indicated that the male sex-related genes such as piwi-1 and amhr2 had higher expression in XY and YY testsis than in XX ovary,and the expression of amh in XY testis was higher than in XX ovary and YY testis.There were almost no expression of cyp19 a and foxl2 in XY and YY testis,whereas their expressions in XX ovary were detected.We chose amh and amhr2 genes for further research.RT-PCR results indicated that amh and amhr2 were highly expressed in gonad among the hypothalamus-pituitary-gonad axis and liver.The expressions of amh in male gonad and pituitary were significantly higher than in females,and its expression in female liver was higher than in male.There was no significant difference of amhr2 expression between testis and ovary,whereas its expressions in male hypothalamus,pituitary and liver were higher than in female.For the hormone treatment experiment,healthy XX female individuals were injected with 17α-methyltestosterone(MT)and XY male individuals with 17α-ethinylestradiol(EE2).EE2 treatment resulted in upregulation of amh and amhr2 at first and subsequent downregulation in males.After MT treatment in females,the relative expression of amh in ovary was lower than normal level at 48 hours and 72 hours post treatment(hpt)but return to normal at 120 hpt.The amhr2 expression was significantly reduced at 48 hpt and was about 50% of the control at 72 and 120 hpt.The expression changes of these genes might be caused by stress response of yellow catfish.This study identified and analyzed the expression of sex determination and differentiation related genes,providing a reference for further study of sex determination and differentiation mechanism in yellow catfish.Sperm motility is influenced by multiple factors both in vivo and in vitro.Meanwhile,each process in spermatogenesis will affect the final sperm motility.The functions of miR-34 in gonad development and spermatogenesis have been reported in previous studies.To investigate the function of mi R-34 a in zebrafish,miR-34a-/-zebrafish mutant was created by CRISPR/Cas9 system through deleting the pre-miR-34 a.miR-34a-/-male zebrafish has higher sperm motility compared to WT zebrafish,with significant differences in VAP(average path velocity),VSL(straight line velocity),VCL(curvilinear velocity)of motile sperm(P<0.05).Searching all the published data,we know that gene mutations or deletion of Gsk3 a or Wt1 have some levels of damage on spermatogenesis or sperm motility in mammals,these findings suggest the positive effects of above-mentioned genes on sperm motility.We find that all these above-mentioned genes have complementary sequence with seed sequence of miR-34a(5’-...GGCAGU...-3’)in 3’ UTR.Such complementarity indicates that miR-34 a may direct target these genes and then has some effect on sperm motility in zebrafish.We detected an upregulated expression of these predicted targeted genes of miR-34 a in liver,testis,brain of mutant zebrafish compared with wildtype.Our study revealed the negative regulation effect of miR-34 a on sperm motility with the reduced expression of these predicted targeted genes.mi R-34 a and its predicted targeted genes may play important roles in gene regulatory network of sperm motility.