The Molecular Mechanism Underling The Down-expression Of CRISP2 Protein In The Ejaculated Spermatozoa From Patient With Asthenozoospermia And Its Clinical Significance

Infertility is becoming a global health problem affecting 15% of couples worldwide. Male infertility is responsible for 50% of these couples’ inability to conceive. Acquired and congenital sperm abnormalities are a critical factor in male infertility. Asthenozoospermia is the most common sperm quality abnormality and one of major causes of male infertility. It is defined as less than 32% of progressive motility (PR) spermatozoa according to the World Health Organization (WHO) guidelines (5th Ed.). The etiology and pathophysiology of asthenozoospermia seem complicated and multifactorial. Many hypothetical etiologies have been put forward, such as congenital or acquired urogenital abnormalities, endocrine disturbances, increased scrotal temperature (varicocele), urogenital infection, testicular injury or pathology, and antisperm antibodies or genetic abnormalities, but the exact causal factors underlying asthenozoospermia still remain largely unknown.Recently, several studies have paid attention to the molecular mechanism of asthenozoospermia. Some differentially expressed genes including Tekt2 (tektin-t), DNAI1, DNAH5, DNAH11, Akap4, Sept4 and CRISP2 (cysteine-rich secretory protein 2, previously known as TPX1) are supposedly related to asthenozoospermia. CRISP2, as a member of the CAP cysteine-rich secretory proteins (CRISPS), antigen 5 (Ag5), and pathogenesis-related 1 (Pr-1) super-family of proteins, arouses broader interests because it is the only CRISP family member expressed within the testis in an androgen-independent manner, and eventually located at sperm acrosome and the outer dense fibres of sperm tail. Functionally, this protein has been reported to be a regulator of calcium influx through ryanodine receptors, which modulates the abilities of sperm flagellar motility. It can be also released from the acrosomal vesicle for the subsequent acrosome reaction or the re-association with the equatorial segment of acrosome-reacted human sperm, involving in sperm-egg fusion.In particular, a recent gene expression profiling study discloses a close association between CRISP2 and human spermatogenesis and infertility. This protein is evidently down-expressed in the ejaculated spermatozoa of patients with asthenozoospermia .Decreased CRISP2 expression in the ejaculated spermatozoa is correlated with low pregnancy rate in cattle. These previous results suggest that CRISP2 plays roles in asthenozoospermia. Therefore, the molecular mechanism underlying CRISP2 down-expression in asthenozoospermia deserves investigation.Gene expression is often regulated by either genetic or epigenetic mechanisms .DNA methylation and microRNA (miRNA) regulation are two major epigenetic regulatory mechanisms involved in multiple biological processes. Some studies reveal that abnormal sperm DNA methylation patterns are able to be associated with the reduced sperm count and function and others report that miRNA-mediated gene control plays a role in the maintenance of undifferentiated state or the induction of differentiation of spermatogonia. Expression profiling studies have identified a number of miRNAs that are enriched in the mammalian testis, mouse male germ cells, and human ejaculated spermatozoa,suggesting that miRNAs are more likely involved in modulating various stages of spermatogenesis. However, it is still unclear whether these two epigenetic mechanisms are involved in the down-expression of CRISP2 in asthenozoospermia.In the present study, we analyzed CRISP2 expression, methylation status of CRISP2 promoter, and microRNA 27b (miR-27b) expression in the ejaculated spermatozoa from patients with asthenozoospermia, and further investigated the regulatory effects of miR-27b on the CRISP2 down-expression in asthenozoospermia. This may provide a novel insight into a portion of mechanism underling asthenozoo spermia.Methods and Results:1. Low expression of CRISP2 protein rather than its mRNA in the ejaculated spermatozoa from patients with asthenozoospermia.The mRNA and protein expression levels of CRISP2 gene were examined by qRT-PCR and western blot respectively in the ejaculated spermatozoa samples from 48 asthenozoospermic patients and 42 normozoospermic volunteers. Notably, no significant difference of CRISP2 mRNA expression was found between two groups (P=0.3854, Fig.1-3), but CRISP2 protein expression was obviously reduced in the ejaculated spermatozoa from asthenozoospermic patients relative to normozoospermic controls (Fig.1-4).2. No methylation in the promoter of CRISP2 CpG islands in the ejaculated spermatozoa from asthenozoospermic patients.The ejaculated spermatozoa can maintain the epigenetic signature of previous spermatogenic stages and it is feasible to using ejaculated spermatozoa to assess the germline DNA methylation profile of spermatogenesis genes. The methylation of the CpG island in the CRISP2 promoter region was examined by both MSP and BSP in the semen samples from 42 normozoospermic and 48 asthenozoospermic males, and then confirmed by DNA sequencing. Notably, no methylation appeared in the CpG islands of CRISP2 promoter in the ejaculated spermatozoa from asthenozoospermic patients and normozoospermic controls (Fig.2-2,2-3 and 2-4). These data further confirmed that CRISP2 reduction was not attributable to DNA methylation and might be regulated at a posttranscrip-tional level in asthenozoospermia.3. MiR-27b was highly expressed in the ejaculated spermatozoa from patients with asthenozoospermia. MiR-27b directly regulated CRISP2 by binding to its 3’-UTR.MiRNAs are usually required for post-transcriptionally silencing of gene expression, so we next determined the possible regulatory roles of some miRNAs in CRISP2 down-expression. A comprehensive database miRWalk, including miRDR, miRWalk and Targetscan, was applied to predict miRNAs potentially regulating CRISP2 gene. As a result, several candidate miRNAs (miR-27a, miR-27b, miR-502-3p, miR-510, miR-640 and miR-767-5p) were extracted (Fig.3-3). Interestingly, of these miRNAs, miR-27b was uniquely confirmed by qRT-PCR to be highly expressed in the ejaculated spermatozoa from patients with asthenozoospermia relative to normal controls (P=0.0071, Fig 3-4A), which was further validated in a larger set of clinical samples (P=0.0013, Fig.3-5), suggesting a potential association of miR-27b with CRISP2 gene in asthenozoospermia.To determine whether miR-27b could specifically regulate CRISP2 expression, we generated a CRISP2 3’-UTR pEZX-MT05 plasmid, and then performed luciferase reporter assay by the co-transfection of this plasmid with miR-27b mimic into HEK 293T cells. Notably, our results revealed that miR-27b significantly suppressed the luciferase activity of reporter genes containing 3’-UTR of CRISP2 compared with the controls, indicating that CRISP2 was a miR-27b target and miR-27b might involve in asthenozoospermia by directly suppressing CR1SP2.To validate our results, we next transfected miR-27b mimic, inhibitor, negative control or miR-27b mimic+CRISP2 plasmid into 293T cells, respectively. As shown in Fig.3-7, CRISP2 protein expression was indeed decreased upon the transfection with miR-27b mimic, whereas it was increased after treatment with miR-27b inhibitor. Moreover, CRISP2 protein level was increased upon the re-expression of CRISP2 in 293T cells treated with miR-27b mimic.Furthermore, we evaluated the relationship of miR-27b with CRISP2 protein expression in clinical samples by Spearman’s correlation analysis, which revealed that the expression levels of miR-27b and CRISP2 protein were negatively correlated in the ejaculated spermatozoa (r=-0.5039, F=0.0121; Fig.3-8). However, no evidence was still found that miR-27b expression was correlated with CRISP2 mRNA expression (P>0.05, Fig.3-9), consistent with the findings of no difference in CRISP2 mRNA expression in the ejaculated spermatozoa between asthenozoospermic and normozoospermic males.Collectively, these results suggested that miR-27b could directly regulate CRISP2 expression.4. Clinical correlations of miR-27b and CRISP2 protein expression with sperm progressive motility, normal morphology and infertility.To explore the clinical relevance of miR-27b and CRISP2 protein expression to asthenozoospermia, we further conducted a correlation analysis of miR-27b or CRISP2 protein expression with low sperm progressive motility and normal morphology in clinical samples. Notably, miR-27b expression level was negatively correlated with sperm progressive motility (r=-0.2745,P=0.0088; Fig.4-1) and normal morphology (r=-0.3397, P=0.0012; Fig.4-2) in 90 semen samples (from 48 asthenozoospermic patients and 42 normozoospermic volunteers). In addition, as shown in Fig.4-3 and 4-4, the study subjects with relatively low CRISP2 protein expression tended to have lower sperm progressive motility and normal morphology.Finally, we carried out a retrospective follow-up study of the reproductive history in all study subjects with 1 year. All 63 included subjects were divided into relatively high and low expression groups based on their expression levels of miR-27b or CRISP2 protein. The infertility rate was calculated for each group respectively and the significant differences of infertility rate between different groups were determined by Chi-square or Fisher’s exact test (Fig.4-5). As expected, we observed a higher infertility rate (69%) in asthenozoospermic group than in normal group (32%, P=0.0038, Table 4-3). Of note, a higher infertility rate appeared in the group with relatively high miR-27b expression (P=0.0376, Table 4-4) or in the group with relatively low CRISP2 protein expression (P=0.0335, Table 4-5). These data further supported the assumption that low CRISP2 protein and high miR-27b expression were associated with infertility.MiRNAs, as a family of short (20-23 nucleotides), single-stranded noncoding RN A molecules, are generally required for post-transcriptionally silencing of multiple target genes by base-pair binding to their 3’-UTR, thereby inducing mRNA degradation or translational repression. Sperm miRNAs have been shown to modulate various stages of spermatogenesis. Some altered sperm miRNAs have been identified by microarray-based approaches in the ejaculated spermatozoa of patients with different spermatogenic impairments. Impressively, the expression of miR-15a and its target gene HSPA1B has been recently reported in the ejaculated spermatozoa from patients with varicocele. These miRNAs identified in the ejaculated spermatozoa could be remnants of untranslated stores during spermatogenesis, presenting a window into molecular events in spermatogenesis or a record of the requirement of haploid gene expression and post-meiotic equilibration. Alternatively, these spermatozoal miRNAs could be delivered into the oocyte following fertilization for further functions. In the present study, we discovered that miR-27b might be a putative miRNA regulating CRISP2 expression. Based on several lines of evidence (including bioinformatics prediction, clinical samples validation, luciferase reporter assays, miRNAs transfection experiments in 293T cell and correlation analysis between miR-27b and CRISP2), we firstly confirmed that miR-27b could directly suppress CRISP2 protein by binding to its 3’-UTR.MiR-27b has been identified as a tumor suppressor in several cancers. It also inhibits the inflammatory response,angiogenesis as well as impairs adipogenesis and mitochondrial function. Interestingly, miR-27b has been observed to be expressed in testis, ovary and zygote by miRNAs profile.In particular, it was highly expressed in the ejaculated spermatozoa from asthenozoospermic patients relative to normozoospermic men. These findings suggest that miR-27b may be an important regulator in multiple processes including different stages of spermatogenesis. CRISP2 displays a stage-specific expression pattern during spermatogenesis, playing essential roles in various stages of spermatogenesis and the post-testicular maturation of spermatozoa. Broad research indicates that CRISP2 is involved in germ cell-Sertoli cell adhesion within the testis , specially expressed in testis, and then incorporated into the growing acrosome and sperm tail and even implicated in fertilization via the acrosome reaction or the re-association with the equatorial segment of acrosome-reacted human sperm. Particularly, its aberrant expression has been reported to be closely associated with spermatogenesis arrested and defects in sperm function. More interestingly, although it is widely accepted that spermatozoa are translationally silent, some studies (eg. by Dr. Yael Gur and Haim Breitbart) showed that in fact protein translation does take place in mammalian spermatozoa prior to fertilization. Consistently, the present study observed a reduced CRISP2 protein rather than its mRNA expression in the ejaculated spermatozoa from patients with asthenozoospermia. All these findings make it suggestive that CRISP2 and its regulator miR-27b may play roles in the whole processes of spermatogenesis prior to fertilization though further studies using other human experimental models are encouraged to provide supporting evidence in the future.Asthenozoospermia is frequently characterized by low sperm progressive motility, clinically accompanied by a low total sperm count or increased numbers of spermatozoa with abnormal morphology. Low sperm motility is considered to be more likely reason for male infertility and sperm motility pattern is closely related with natural pregnancy rate. Low pregnancy rate has been reported to be due to low CRISP2 expression level in the ejaculated spermatozoa in cattle. To further support the roles of miR-27b and CRISP2 in asthenozoospermia and even infertility, we carried out a clinical correlation analysis and a retrospective follow-up study of the reproductive history in all study subjects. Consistently, our clinical data revealed that either high miR-27b or low CRISP2 protein expression tended to be correlated with lower sperm progressive motility and normal morphology. The follow-up study further observed a higher infertility rate in asthenozoospermic patients and a close relationship between high infertility rate and low CRISP2 protein expression or high miR-27b expression. Although there may be more unidentified miR-27b targets that likely contribute to asthenozoospermia and infertility, these data demonstrates that miR-27b suppresses CRISP2 protein expression during spermatogenesis prior to fertilization, clinically involving in asthenozoospermia and male infertility probably via influencing sperm motility and morphology.This study conclusively provide a novel insight into a portion of mechanism leading to the reduced CRISP2 expression in asthenozoospermia, offering a potential therapeutic target for treating male infertility or for male contraception.Conclusions:1. CRISP2 protein rather than its mRNA was down-expressed in the ejaculated spermatozoa from patients with asthenozoospermia. This suggested that there was no transcriptional regulation of CRISP2 in sperm and the reduced expression of CRISP2 protein rather than its mRNA was associated with asthenozoospermia.2. No methylation appeared in the CpG islands of CRISP2 promoter in the ejaculated spermatozoa from normozoospermic and asthenozoospermic males. These data further reflected that CRISP2 reduction was not attributed to DNA methylation and might be regulated at a post-transcriptionally level in asthenozoospermia.3. MiR-27b, was highly expressed in the ejaculated spermatozoa from patients with asthenozoospermia and it could directly suppress CRISP2 expression by binding to its 3’-UTR.4. MiR-27b suppresses CRISP2 protein expression during spermatogenesis prior to fertilization, clinically involving in asthenozoospermia and male infertility probably via influencing sperm motility and morphology.