| According to the statistics of the World Health Organization,about 10%-15%of the couples of childbearing age in the world have fertility problems,and infertility has become the focus of attention around the world.In these cases,infertility caused by male factors accounts for about 50%[1,2,26].Awfully,the sperm density and sperm activity decrease yearly at a certain rate,which may be associated with the increasing environmental pollution[4].Asthenozoospermia(AZS)is characterized by reduced sperm motility or absent sperm motility,with normal sperm morphology in the fresh ejaculate,based on the World Health Organization(WHO)guidelines(5th ed.).AZS may be the result of urogenital infections,abnormal varicocele,semen liquefaction,abnormal hormones and or other causes[5,27].However,of these AZS,approximately 50%of the causes can't be found,which are referred to as idiopathic male infertility[2].The pathogenesis of idiopathic AZS remains largely unknown,which hinders the progress of diagnosis and treatment of AZS.To date,some research have shown that certain genes involved in the asthenozoospermia.Some genes,including cilia and flagella associated protein 69(CFAP69)and cysteine-rich secretory protein 2(CRISP2),have been reported to be associated with AZS in mice or human[8,28].Furthermore,our previous study have identified semenogelin 1(SEMG1)as a potential marker for idiopathic AZS[9].SEMG1,the major secreted protein in male semen,originates from seminal vesicles as a formation of gel matrix that encases ejaculated spermatozoa.After ejaculation,prostate-specific antigen(PSA)degraded it into smaller peptides,which allows the spermatozoa to get motile ability and move quickly[10].Martinez-Heredia et al.used two-dimensional proteomic analysis to identify proteins which are differently expressed in AZS semen samples,and found that SEMG1 protein was highly expressedc[11,12],Consistently,our previous study revealed over-expression of SEMG1 in AZS patients through gene expression profile analyses of AZS patients and volunteers spermatozoa,which suggest the important role of SEMGI in AZS[9].However,the molecular mechanism underlying over-expression of SEMG1 in patients with AZS remains unclear.In the current study,we detected SEMG1 and miR-525-3p expression levels in the ejaculated spermatozoa from patients with AZS and normozoospermic volunteers.Furthermore,we identified whether miR-525-3p has the regulatory effects on the expression of SEMG1 in AZS.This may provide the opportunity to further illustrate the molecular mechanisms of AZS.Meanwhile,a follow-up study of reproductive history indicated that low miR-525-3p expression and high SEMG1 expression were correlated with abnormal morphology,low progressive sperm motility and infertility respectively.Our findings demonstrate that miR-525-3p deletion contributes to aberrant expression of SEMG1,clinically involving in AZS and male infertility,which provides a possible target for the treatment of male sterility or male contraception.Methods and Results:1.Comparison of basic parameters of semen in AZS patients and normozoosper mic volunteersWe compared the semen routine parameters of 30 AZS patients and 30 age-matched normal volunteers.No difference was observed between patients with AZS and normozoospermic volunteers in terms of age,sperm volume,pH,and sperm concentration.Notably,compared with those in normozoospermia control group,sperm progressive motility and normal morphology in AZS patients was lower(p<0.001).2.SEMG1 mRNA and protein expression in the ejaculated spermatozoa of AZS patients and normozoospermic volunteersSEMG1 mRNA and protein expression levels were analyzed in the spermatozoa of AZS patients and normozoospermic volunteers.The results revealed that the levels of SEMG1 mRNA were significantly higher in AZS patients when compared with those in normozoospermic volunteers(1.40±0.35 vs.0.46±0.23,P<0.001).Similarly,the Western Blot analysis of the SEMG1 protein also showed that the expression of SEMG1 protein was significantly higher in the spermatozoa of AZS patients than that in normozoospermic controls(AZS vs.Norm,p<0.001)3.miR-525-3p was lower expression in the ejaculated spermatozoa of AZS,and directly suppressed SEMG1 expression through binding to its 3'-UTRWe used miRWalk2.0 database to predict miRNAs potentially targeting SEMG1 gene.Consequently,six candidate miRNAs(miR-525-3p,miR-524-3p,miR-133b,miR-671-5p,miR-133a-3p and miR-130a-5p)were selected.We detected the expression levels of these selected miRNAs in the spermatozoa by qRT-PCR.Interestingly,we found that the expression level of miR-525-3p was uniquely low in the ejaculated spermatozoa of AZS patients than normozoospermic controls(1.22±1.00 vs.0.64±0.55,P= 0.007).The results suggested that there may be a potential association between miR-525-3p and SEMG1 in AZS.To further confirm whether miR-525-3p could specifically regulate SEMG1 expression,we performed luciferase activity assays.When co-transfected 293T cells with miR-525-3p mimic,a clear suppression of luciferase activity was observed for both the wild-type and the mutant construct.Our results confirmed that miR-525-3p could directly regulate SEMG1 by binding to its 3'-UTR..4.Correlation of miR-525-3p and SEMG1 mRNA expression with clinical sperm parametersThe correlations between the expression of miR-525-3p or SEMG1 mRNA and clinical sperm parameters were carried out.The results illustrated that miR-525-3p expression was positively associated with sperm progressive motility and normal morphology(r = 0.393,p = 0.002;r = 0.382,P= 0.003,Fig 5b,respectively).On the contrary,SEMGI mRNA expression was negatively associated with sperm progressive motility and normal morphology(r =-0.685,p<0.001;r =-0.711,,P<0.001,respectively).Furthermore,the relative expression of miR-525-3p or SEMG1 mRNA has no correlation with other sperm parameters,such as semen volume and sperm concentration(p>0.05).5.Correlation of miR-525-3p and SEMG1 mRNA expression with fertilityTo explore the clinical relevance of miR-525-3p or SEMGl mRNA expression to AZS,we conducted a retrospective follow-up study of the reproductive history of all participants.In the present study,12 subjects were excluded from our study.Excluded subjects included lost contact or refused to cooperate(n=9),unmarried(n=1),and female partner infertility(n=2).Overall,21 AZS patients and 27 normozoospermic volunteers were included in this study.Subsequently,we grouped the included subjects into relative high and low expression groups according to the expression levels of miR-525-3p or SEMG1 mRNA in their spermatozoa.As shown in Fig 6,the fertility rate was higher in normozoospermic volunteers than those in AZS patient(70.37%vs.19.05%,p<0.001).Of note,a higher fertility rate was observed in relatively low SEMG1 mRNA expression group(75.00%vs.20.83%,P<0.001)and relatively high miR-525-3p expression group(66.67%vs.29.16%5,p<0.001).SEMG1 is a major protein of semen coagulum that has been shown to inhibit human sperm capacitation[29].Clinical evidence showed that SEMG1 plays roles in modulating semen liquefaction,sperm capacitation,and regulation of sperm cell membrane permeability[12,29,30].In this study,we found that the mRNA and protein of SEMG1 were elevated in the spermatozoa of AZS patients.MiR-525-3p was identified as a specific suppressor of SEMG1 by directly targeting its 3'-UTR,clinically involving in AZS and male infertility.MiRNAs are the class of small endogenous RNAs that function as important gene expression regulators by base-pair binding to their 3'-UTR,thus eliciting mRNA degradation or translational repression[26].There have been extensive studies reported the miRNA expression profiles of testis in humans or animals,suggesting the important roles of miRNAs in the process of testis development and spermatogenesis[31,32]Currently,there are many studies on the regulation of miRNA on primordial germ cells to spermatogonia,but few from primary spermatocytes to mature spermatids[33].Nevertheless,research on spermatozoaL RNA has made considerable progress.Ma J found that testosterone-dependent miR-26a-5p and let-7g-5p act as signaling mediators to regulate sperm apoptosis via targeting PTEN and PMAIP1[34]Furthermore,microRNAs control mRNA fate by compartmentalization based on 3'-UTR length in male germ cells[35].Nowadays,it is generally believed that spermatozoa are translationally silent,whereas other researchers[36,37]reveal that in fact protein translation does occur in mammalian spermatozoa prior to fertilization.Brohi RD also showed that posttranslational modifications in spermatozoa have an effect on sperm viability and male fertility[38]Recent study found that the expression of miR-15a was significantly decreased in the ejaculated spermatozoa of varicocele patients and miR-15a repressed the expression of HSPA1B through directly binding its 3'-UTR[39]Consistently,our previous researches also observed that miR-27b and miR-27a could target the 3'UTR of CRISP2 and down-regulate its expression in AZS or asthenoteratozoospermia respectively[40,41]In our research,bioinformatics predicted that miR-525-3p could be a specific regulator of SEMG1.Dual luciferase activity assay further verified that miR-525-3p could directly bind to the 3'-UTR of SEMG1 and suppress its expression.Taken together,we proposed that one of the mechanisms underlying SEMG1 over-expression in AZS is due to the deletion of miR-525-3p,though there may be more regulators that probably contribute to SEMG1 over-expression,which deserves further investigation.Study show that miR-525-3p acted as an oncogenic miRNA in liver tumor,which enhances the migration and invasion of liver cancer cells[42].The expression level of miR-525-3p also increases in cisplatin resistant germ cell tumor cell lines[43],suggesting miR-525-3p may positively associated with the malignancy of germ cell tumor cells.Nevertheless,there is no report on the function and mechanism of miR-525-3p in AZS.As mentioned in the literature,reduced expression of miR-525-3p in semen of infertile men compared with normal semen[44].Consistent with our current observations,relatively high expression of SEMG1 mRNA or low expression of miR-525-3p in the spermatozoa were correlation with low progressive sperm motility and abnormal morphology respectively.SEMG1 is a major protein of semen coagulum that has been shown to inhibit human sperm capacitation.In our research,there were higher spermatozoa abnormal morphology rate with relatively high expression of mRNA SEMG1 or low expression of miR-525-3p.More research is needed to confirm how SEMG1 influences sperm morphology.A strong relationship between high SEMG1 mRNA or low miR-525-3p and male infertility has been reported in our follow-up study of reproductive history.It is interesting to note that the infertility rate in AZS patients was higher and high infertility rate was closely related to high SEMG1 or low miR-525-3p expression.Though there may be more unidentified regulatory mechanism that may contributes to AZS and sterility,our research first demonstrates that miR-525-3p deletion contributes to aberrant expression of SEMG1,clinically involving in AZS and male infertility likely by affecting sperm motility and morphology.Conclusions:Our findings demonstrated that miR-525-3p expression in ejaculatory spermatozoa from AZS patients was lower,and SEMG1 expression was higher compared with normal spermatozoa.Furthermore,miR-525-3p deletion contributes to aberrant expression of SEMGlL Clinically,elevated SEMG1 and reduced miR-525-3p levels are associated with AZS and male infertility,likely through influencing sperm motility and morphology.Our research brings valuable suggestions for analysis of AZS and male infertility,which may help to provide a potential diagnostic marker and target for the treatment of male infertility or male contraception. |
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