The expression of cysteine-rich secretory protein 2 (CRISP2) and its

BOR Papers in Press. Published on December 29, 2014 as DOI:10.1095/biolreprod.114.124487
The expression of cysteine‐rich secretory protein 2 (CRISP2) and its specific regulator miR‐27b in the spermatozoa of patients with asthenozoospermia1 Jun‐Hao Zhou,4,5 Qi‐Zhao Zhou,4,5 Xiao‐Ming Lyu,4,6,7 Ting Zhu,7 Zi‐Jian Chen,5 Ming‐Kun Chen,5 Hui Xia,5 Chun‐Yan Wang,8 Tao Qi,8 Xin Li,3,5,7 and Cun‐Dong Liu2,5 5
Department of Urology, The Third Affiliated Hospital of Southern Medical University, Guangzhou, Guangdong, China 6
Medical Laboratory Centre, The Third Affiliated Hospital of Southern Medical University, Guangzhou, Guangdong, China 7
Cancer Research Institute, Southern Medical University, Guangzhou, Guangdong, China 8
Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China 1
Supported by the Guangdong Provincial Natural Science Foundation of China (No. S2012010009126), and Guangdong Provincial Science and Technology Program (No. 2013B021800317). 2
Correspondence: Cundong Liu, Department of Urology, The Third Affiliated Hospital of Southern Medical University, Guangzhou, 510630. E‐mail: [email protected] 3
Correspondence:Xin Li, Department of Urology, The Third Affiliated Hospital of Southern Medical University, Guangzhou, 510630, China or Cancer Research Institute, Southern Medical University, Guangzhou, 510515, Guangdong, China. E‐mail: [email protected] 4
These authors contributed equally to this work. Running head: MiR‐27b & CRISP2 in asthenozoospermia Summary: MiR‐27b contributes to the reduction of CRISP2 protein in asthenozoospermia. Both high miR‐27b expression and low CRISP2 protein expression were significantly associated with low sperm progressive motility, abnormal morphology, and infertility. ABSTRACT Cysteine‐rich secretory protein 2 (CRISP2) is an important sperm protein and plays roles in spermatogenesis, modulating the flagellar motility, acrosome reaction and gamete fusion. Clinical evidence shows a reduced CRISP2 expression in spermatozoa from asthenozoospermic patients, but the molecular mechanism underlying its reduction remains unknown. Herein, we carried out a study focusing on the CRISP2 reduction and its roles in asthenozoospermia. Initially, through analyzing CRISP2 expression and methylation on CRISP2 promoter activity in sperm, we observed a decreased expression of CRISP2 protein rather than its mRNA in the ejaculated spermatozoa from asthenozoospermic patients and no methylation in CRISP2 promoter, suggesting CRISP2 expression may be regulated in sperm at the post‐transcriptional level. Subsequently, we found that microRNA 27b (miR‐27b), predicted as a candidate regulator of CRISP2 using bioinformatics, was highly expressed in the ejaculated spermatozoa from asthenozoospermic patients. Luciferase reporter assay and transfection experiments disclosed Copyright 2014 by The Society for the Study of Reproduction.
that this miRNA could target CRISP2 by specifically binding its 3’‐UTR and suppressed CRISP2 expression. The extended clinical observation further confirmed a highly expressed miR‐27b and its obviously negative correlation with CRISP2 protein expression in ejaculated spermatozoa samples from asthenozoospermic patients. Finally, we conducted a retrospective follow‐up study to support that either high miR‐27b expression or low CRISP2 protein expression was significantly associated with low sperm progressive motility, abnormal morphology and infertility. Thus, this study provide the first preliminary insight into the mechanism leading to the reduced CRISP2 expression in asthenozoospermia, offering a potential therapeutic target for treating male infertility or for male contraception. Keywords: Cysteine‐rich secretory protein 2 (CRISP2), asthenozoospermia, miR‐27b, male infertility INTRODUCTION Infertility is becoming a global health problem affecting 15% of couples worldwide [1, 2]. Male infertility is responsible for 50% of these couples’ inability to conceive. Acquired and congenital sperm abnormalities are a critical factor in male infertility [3]. Asthenozoospermia is the most common sperm quality abnormality [3] and one of major causes of male infertility [4]. It is defined as less than 32% of progressive motility (PR) spermatozoa according to the World Health Organization (WHO) guidelines (5th Ed.) [5]. 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 [6‐10], but the exact causal factors underlying asthenozoospermia still remain largely unknown [11]. Recently, several studies have paid attention to the molecular mechanism of asthenozoospermia. Some differentially expressed genes including Tekt2 (tektin‐t) [12], DNAI1, DNAH5, DNAH11 [13], Akap4 [14], Sept4 [15] 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 [16], arouses broader interests because it is the only CRISP family member expressed within the testis [17, 18] in an androgen‐independent manner [19], and eventually located at sperm acrosome and the outer dense fibres of sperm tail [20‐24]. Functionally, this protein has been reported to be a regulator of calcium influx through ryanodine receptors [25], which modulates the abilities of sperm flagellar motility [26, 27]. 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 [28], involving in sperm‐egg fusion [29]. In particular, a recent gene expression profiling study discloses a close association between CRISP2 and human spermatogenesis and infertility [30]. This protein is evidently down‐expressed in the ejaculated spermatozoa of patients with asthenozoospermia [31, 32]. Decreased CRISP2 expression in the ejaculated spermatozoa is correlated with low pregnancy rate in cattle [33]. 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 [34]. DNA methylation and microRNA (miRNA) regulation are two major epigenetic regulatory mechanisms involved in multiple biological processes [35, 36]. Some studies reveal that abnormal sperm DNA methylation patterns are able to be associated with the reduced sperm count and function [37‐41] and others report that miRNA‐mediated gene control plays a role in the maintenance of undifferentiated state or the induction of differentiation of spermatogonia [42‐45]. Expression profiling studies have identified a number of miRNAs that are enriched in the mammalian testis [46, 47], mouse male germ cells (including spermatogonia, pachytene spermatocytes, spermatids, and spermatozoa) [48], and human ejaculated spermatozoa [49], suggesting that miRNAs are more likely involved in modulating various stages of spermatogenesis [50, 51]. 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 asthenozoospermia. MATERIALS AND METHODS Ethics Statement The present study was approved by the bioethics committees of Nanfang Hospital and The Third Affiliated Hospital of Southern Medical University, Guangzhou, China. The written informed consent was signed by study subjects. The experimental protocol was established according to the associated national guidelines from Ministry of Science and Technology of China. Subjects and semen samples Totally 90 study subjects including 48 asthenozoospermic patients and 42 normozoospermic volunteers were enrolled in the study in Department of Laboratory Medicine of Nanfang Hospital, Southern Medical University from January 2013 to June 2013, after excluding those with varicocele, teratozoospermia and leukocytospermia. Additional exclusion criterions included abnormal semen liquefaction, reproduction tract infection, testicular injury or pathology, history of cryptorchidism, orchitis or epididymitis and some systemic diseases (diabetes mellitus, hypertension, hypercholesterolaemia and hypoandrogenism). Each patient with asthenozoospermia was confirmed by the routine semen analysis of his three ejaculated semen samples collected at different time points. All volunteers were also proven to have normal semen parameters. The routine semen analysis conducted on SCA (Sperm Class Analyzer, MicroPtic, Spain) covered such factors as the liquefaction time, volume, pH, viscosity, agglutination, motility, viability, density, and morphology of sperm. Then semen samples were stained using Diff‐Quik staining for evaluating sperm morphology. All semen samples were collected from study subjects by masturbation after 3 days of sexual abstinence, and then allowed to liquefy at 37°C for 30 minutes. Liquefied semen samples were loaded on to 50% discontinuous Percoll gradient (Pharmacia, USA) and centrifuged at 2000g for 15 minutes at room temperature [52, 53]. The spermatozoa pellet was washed twice with phosphate buffered saline (PBS), and stored at ‐80°C for use. From 90 semen samples, 48 semen samples (derived from 24 asthenozoospermic patients and 24 normal volunteers) were randomly selected for preliminarily detecting the expression levels of six candidate miRNAs. Additionally, 24 semen samples (from 12 asthenozoospermic patients and 12 normal volunteers) were randomly selected for detecting both miR‐27b and CRISP2 protein expression levels, which were further used for evaluating the correlation of miR‐27b with CRISP2 protein expression, and the clinical correlations of their expression levels with sperm progressive motility, normal morphology and infertility. Prediction of CpG islands CpG islands in CRISP2 promoter region were predicted by Methyl Primer Express software (version 1.0). A CpG island was defined as a DNA fragment with a length of at least 300 base pairs (bp), a GC content of more than 55 %, and a ratio of more than 0.65 between the observed and the expected CpGs. DNA extraction and sodium bisulfite modification of genomic DNA Genome DNA was extracted from semen samples using Sperm DNA Purification Kit (Simgen, Hangzhou, China), and then treated with sodium bisulfite modification by DNA Modification Kit (CWBio, Beijing, China) to transform unmethylated cytosines into uridines and keep methylated cytosines unchanged. The modified DNA samples were stored at −20°C for further use. All procedures were performed according to the manufacturer’s recommendations. Methylation‐specific PCR The methylation of CpG islands in CRISP2 promoter region was detected by methylation‐specific polymerase chain reaction (MSP) as in previous studies[54]. The PCR conditions and procedures included predenaturation at 95°C for 1 minute, denaturtion for 35 cycles, (each cycle including denatution at 95°C for 10 seconds, 60 °C for 30 seconds and 72°C for 30 seconds), and an extension at 72 °C for 10 minutes. The expected PCR product at the size of 139 bp was separated on 2% agarose gels with ethidium bromide, and then visualized under ultraviolet light. When a sample was amplified only by M‐primers or U‐primers, it was defined as full methylation or full unmethylation and if a sample was amplified by both M‐primers and U‐primers, it was considered as partial methylation [55]. Bisulfite‐sequencing PCR The methylation status of CpG island of CRISP2 in spermatozoa samples was also detected by bisulfite‐sequencing polymerase chain reaction (BSP) as described previously [56]. The PCR conditions and procedures were consistent with those in our previous descriptions. The expected PCR product size was 262 bp. The PCR products were electrophoresed on 2% agarose gels visualized under ultraviolet light, and then sent to Life Technologies Company for sequencing. The primers used for detecting DNA methylation and CRISP2 gene were shown in Supplemental Table S1 (Supplemental Data are available online at www.biolreprod.org). Extraction of total RNA from semen samples Total RNA, including miRNAs, was extracted from each spermatozoa pellet using TRIzol reagent (Invitrogen, Carlsbad, USA) in line with the manufacture’s instruction. The quantity and quality of extracted RNA were measured by E‐Spect (Malcon, Japan). Reverse Transcription and Quantitative Real‐Time PCR 200 ng of total RNA was reverse‐transcribed respectively by PrimerScriptTM RT Kit (TaKaRa) for mRNA and by SYBR PrimeScript miRNA RT‐PCR Kit (TaKaRa) for miRNA. The quantitative Real‐Time PCR for mRNA was performed in a 96‐well plate format using SYBR Premix Ex Taq Real Time PCR Kit (TaKaRa). Amplification reactions were carried out in a final volume of 20 µl and were performed on an Mx3005P Stratagene on the thermal cycling conditions (denaturation at 95°C for 30 seconds [1×], followed by 40 cycles of denaturation [95°C, 30s], annealing [60°C, 10s] and extension [72°C, 15s]). Human actin beta (ACTB) was used as an endogenous reference. The expression of miRNAs was quantified by SYBR PrimeScript miRNA RT‐PCR Kit (TaKaRa) on Mx3005P Stratagene. Amplification reaction was performed in a final volume of 20 µl for 40 cycles (95°C, 10s; 60°C, 10s, 72°C, 15s). The primers used for detecting the expression of miRNAs were showed in Supplemental Table S2. U6b small nuclear RNA was used as the endogenous reference for normalization. The comparative cycle threshold method was performed for relative quantification. Prediction of miRNAs targeting CRISP2 gene The miRNA binding sites in the 3’ untranslated region (3’‐UTR) of CRISP2 were predicted using a miRWalk database (http://www.umm.uni‐heidelberg.de/apps/zmf/ mirwalk/predictedmirnagene.html), a comprehensive database on miRNAs, which hosts predicted or validated miRNA binding sites as well as the information of all known genes of human, rat and mouse [57]. Plasmid construction, Cell culture and Transfection The 732 bp ORF (open reading frame) sequence of CRISP2 was cloned into an EX‐G0250‐M02 plasmid (GeneCopoeia, Guangzhou, China) with CMV promoter for transfection experiment. HEK 293T cells were cultured in DMEM supplemented with 10% fetal bovine serum in 5% CO2 at 37°C, and then plated into 6‐well plates until 80% confluence. The cells were transfected respectively with 100 nM of miR‐27b mimic, inhibitor, negative control (GeneCopoeia, Guangzhou, China) or miR‐27b mimic+CRISP2 plasmid using Lipofectamine 2000 (Invitrogen), and finally harvested 72h after transfection for protein extraction and Western Blot. Western blotting Spermatozoa pellets were purified by Percoll gradient centrifugation and washed with cold PBS. About 2×109 spermatozoa were lysed with 100μl of lysis buffer and 1μl of protease inhibitors on ice for 30 minutes. HEK 293T cells were transfected with mimic or inhibitor and lysed as well. Subsequently, proteins were extracted from them by using Total Protein Extraction Kit (BestBio, Shanghai, China). 30 µg of the total protein was electrophoresed on 12% SDS‐PAGE gels and transferred to PVDF membranes (BioTrace, Mexico). The blots were probed respectively with antibodies against CRISP2 (19066‐1‐AP, Proteintech, USA) [58] (Supplemental Figure S2) or ACTB (60008‐1‐Ig, Proteintech, USA) at 4°C overnight. The horseradish peroxidase‐conjugated secondary antibodies were added respectively. The signals were detected by enhanced chemiluminescence (Pierce, Rockford, IL). The gray value of each protein band was analyzed by Image Lab software. Dual luciferase reporter assay The target sequence of CRISP2 3’‐UTR was cloned into a luciferase reporter vector containing Gaussia Luciferase (GLuc) and Secreted Alkaline Phosphatase (SeAP) dual‐luciferase reporter genes (GeneCopoeia, Guangzhou, China). HEK 293T cells were plated into 24‐well plates until 80% confluence before transfection. 350ng of CRISP2 3’‐UTR pEZX‐MT05 plasmid (3’‐UTR vector, GeneCopoeia, Guangzhou, China) or negative control pEZX‐MT05 plasmid (NC vector, GeneCopoeia, Guangzhou, China) was transiently cotransfected with 50 nM miR‐27b mimic (GenePharma, Shanghai, China) or negative control mimic (NC mimic) into HEK 293T cells. Cells culture mediums were harvested 72h after transfection, and then the activities of Gluc and SeAP were measured using Secrete‐Pair Dual Luminescence Assay Kit (GeneCopoeia, Guangzhou, China) on a Berthold AutoLumat LB9507 rack luminometer. Gluc activities were normalized to SeAP luciferase activities to control for transfection efficiency. Each experiment was performed in duplicated and repeated at least three times. Follow‐up study of reproductive history A retrospective follow‐up study of reproductive history was carried out in 90 study subjects. According to their miR‐27b expression levels relatively separated by a broken line in Fig. 4A and B, the qualified study subjects (Supplemental Table S3B) were divided into the relatively high and low expression groups, which were followed up for the reproductive history. In addition, randomly selected 24 study subjects (12 asthenozoospermic patients and 12 normal volunteers, Supplemental Table S3C) were also divided according to their CRISP2 protein expression levels relatively separated by a broken line in Fig. 4C and D, and then followed up. Male infertility is defined as the inability of a couple to conceive a child due to male factors after one year of unprotected sexual intercourse with a normal female partner or spouse [11].
The percentage was used to describe the infertility rate in each group and the high infertility rate was a relative rate. For example, when comparing the infertility rate between two groups, if the number of subjects with infertility in the group A is significantly more than that of the group B, we can determine that group A has a high infertility rate relative to group B. Statistical analysis Continuous variables were expressed as the mean values ± standard deviation (SD). A two‐tailed Student’s t test was performed to determine the differences of CRISP2 mRNA and miR‐27b expression in the ejaculated spermatozoa between asthenozoospermic and normozoospermic males. One‐way ANOVA and Bonferroni’s test were used to determine significant differences in luciferase activity. A Spearman correlation coefficient (r) was calculated to assess the significance of association between clinical features of semen samples and the expression level of miR‐27b. Chi‐square or Fisher's exact test based on fourfold table was performed to determine significant differences of infertility rate between different groups. All statistical analyses were performed using SPSS13.0 for Windows. P<0.05 was considered to be statistically significant. RESULTS Low expression of CRISP2 protein rather than its mRNA in the ejaculated spermatozoa from patients with asthenozoospermia We initially compared the baseline sperm characteristics between 48 asthenozoospermic patients and 42 normozoospermic volunteers. As a result, no significant differences in age, ejaculate volume, pH‐value and sperm concentration were observed between the two groups except sperm morphology and progressive motility (Table 1). Subsequently, the mRNA and protein expression levels of CRISP2 gene were examined by qRT‐PCR and western blot respectively in the ejaculated spermatozoa samples from the two groups. Notably, no significant difference of CRISP2 mRNA expression was found between two groups (P=0.3854, Fig. 1A), but CRISP2 protein expression was obviously reduced in the semen samples from asthenozoospermic patients relative to normozoospermic controls (Fig. 1B). 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. No methylation in the promoter of CRISP2 CpG islands 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 [40]. To support that CRISP2 was not transcriptionally down‐regulated in asthenozoospermia, we also carried out an analysis of methylation status of CRISP2 gene. A CpG island, containing 35 CpG sites, was identified in the 5’‐flanking of CRISP2 gene, covering the region from −301 to +383, relative to the transcription start site. The methylation of these CpG sites 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 all samples (Fig. 2A, B and C). These data further reflected that CRISP2 reduction was not attributed to DNA methylation and might be regulated at a post‐transcriptionally level in asthenozoospermia. MiR‐27b, a candidate regulator of CRISP2, was highly expressed in the ejaculated spermatozoa from patients with asthenozoospermia 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. 3A). 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 (Supplemental Fig. S1), which was further validated in a larger set of clinical samples (P=0.0013, Fig. 3B), suggesting a potential association of miR‐27b with CRISP2 gene in asthenozoospermia. MiR‐27b directly regulated CRISP2 by binding to its 3’‐UTR To determine whether miR‐27b could specifically regulate CRISP2 expression, we generated a CRISP2 3’‐UTR pEZX‐MT05 plasmid that contained 414‐nucleotide 3’‐UTR of CRISP2 downstream of the luciferase gene (Fig. 3C), 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 (Fig. 3C), indicating that CRISP2 was a miR‐27b target and miR‐27b might involve in asthenozoospermia by directly suppressing CRISP2. 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. 3D, 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, P=0.0121; Fig. 3E). However, no evidence was still found that miR‐27b expression was correlated with CRISP2 mRNA expression (P>0.05, Fig. 3F), 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. Clinical correlations of miR‐27b and CRISP2 protein expression with sperm progressive motility and normal morphology
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. 4A) and normal morphology (r= ‐0.3397, P=0.0012; Fig. 4B) in 90 semen samples (from 48 asthenozoospermic patients and 42 normozoospermic volunteers), but not associated with other clinical features such as age, sperm concentration and semen volume (P>0.05; Supplemental Table S4). In addition, as shown in Fig. 4C and D, the study subjects with relatively low CRISP2 protein expression tended to have lower sperm progressive motility and normal morphology. These data suggested that miR‐27b and CRISP2 protein were clinically involved in asthenozoospermia probably via influencing sperm morphology and progressive motility, although this deserves to be replicated in a larger set of clinical samples. Clinical correlation of miR‐27b and CRISP2 protein expression with infertility Finally, we carried out a retrospective follow‐up study of the reproductive history in all study subjects. Of 90 subjects, 27 were excluded for individual reasons (Table 2). All 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 (Supplemental Table S3). As expected, we observed a higher infertility rate (69%) in asthenozoospermic group than in normal group (32%, P=0.0038, Fig. 4E, Supplemental Table S3A). Of note, a higher infertility rate appeared in the group with relatively high miR‐27b expression (P=0.0376, Supplemental Table S3B) or in the group with relatively low CRISP2 protein expression (P=0.0335, Supplemental Table S3C). These data further supported the assumption that low CRISP2 protein and high miR‐27b expression were associated with infertility. DISCUSSION MiRNAs, as a family of short (20‐23 nucleotides), single‐stranded noncoding RNA 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 [59, 60]. Sperm miRNAs have been shown to modulate various stages of spermatogenesis [50, 51, 61]. Some altered sperm miRNAs have been identified by microarray‐based approaches in the ejaculated spermatozoa of patients with different spermatogenic impairments [62‐64]. Impressively, the expression of miR‐15a and its target gene HSPA1B has been recently reported in the ejaculated spermatozoa from patients with varicocele [65]. These miRNAs identified in the ejaculated spermatozoa could be remnants of untranslated stores during spermatogenesis [66], presenting a window into molecular events in spermatogenesis or a record of the requirement of haploid gene expression and post‐meiotic equilibration [67]. Alternatively, these spermatozoal miRNAs could be delivered into the oocyte following fertilization for further functions [68, 69]. In the present study, we discovered that miR‐27b might be a putative miRNA regulating CRISP2 expression. To explore its putative mechanism, we firstly confirmed that miR‐27b was highly expressed in the ejaculated spermatozoa from patients with asthenozoospermia, and then carried out a serial of experiments. Our results revealed that miR‐27b indeed directly regulated CRISP2 protein expression, based on several lines of evidence. First, bioinformatics prediction and luciferase reporter assays indicated that miR‐27b could directly suppress CRISP2. Second, CRISP2 protein expression was decreased in 293T cells after transfection with miR‐27b mimic, whereas CRISP2 protein expression was increased upon the transfection with miR‐27b inhibitor. Third, CRISP2 protein expression was obviously decreased while miR‐27b was highly expressed in the ejaculated spermatozoa of patients with asthenozoospermia. The expression level of CRISP2 protein was negatively correlated with miR‐27b expression. MiR‐27b has been identified as a tumor suppressor in several cancers [70‐72]. It also inhibits the inflammatory response [73, 74], angiogenesis [75] as well as impairs adipogenesis [76] and mitochondrial function [77]. Interestingly, miR‐27b has been observed to be expressed in testis, ovary and zygote [78]. In particular, it was highly expressed in the ejaculated spermatozoa from asthenozoospermic patients relative to normozoospermic men [63]. These findings suggest that miR‐27b may be an important regulator in multiple processes including different stages of spermatogenesis. Of note, within mammals, male germ cells also encounter CRISPs at virtually every phase of development and maturation [79]. 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 [80, 81], expressed in germ cells, and then incorporated into the growing acrosome and sperm tail [20] and even implicated in fertilization [29, 82] via the acrosome reaction or the re‐association with the equatorial segment of acrosome‐reacted human sperm [28]. Particularly, its aberrant expression has been reported to be closely associated with spermatogenesis arrested and defects in sperm function [83]. More interestingly, although it is widely accepted that spermatozoa are translationally silent, some studies (eg. by Dr. Yael Gur and Haim Breitbart [84‐86] ) 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 [3]. Low sperm motility is considered to be more likely reason for male infertility [87] and sperm motility pattern is closely related with natural pregnancy rate [88, 89]. Low pregnancy rate has been reported to be due to low CRISP2 expression level in the ejaculated spermatozoa in cattle [33]. 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 first 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. Conclusively, this study 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. ACKNOWLEDGMENT The authors thank Prof. Rangke Wu for his contributions to the manuscript revision. REFERENCES
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FIGURE LEGENDS FIG. 1. Low expression of CRISP2 protein rather than its mRNA in the ejaculated spermatozoa of patients with asthenozoospermia. A) The expression level of CRISP2 mRNA was detected by qRT‐PCR in the ejaculated spermatozoa between asthenozoospermic patients and normozoospermic volunteers. B) The CRISP2 protein expression was detected by Western Blot in the ejaculated spermatozoa, and then the relative gray value of each protein band was analyzed by Image Lab software. Data were shown as mean ± SEM, ***P<0.001. Norm, normozoospermic volunteers group; Asth, asthenozoospermic patients group. FIG. 2. No methylation in the promoter of CRISP2 CpG islands in the ejaculated spermatozoa of patients with asthenozoospermia. A) Methylation status of CRISP2 promoter by MSP. B) Methylation status of CRISP2 promoter by BSP. C) DNA sequencing of bisulfite‐sequencing PCR results. M, methylated products; U, unmethylated products; Norm, normozoospermic volunteers group; Asth, asthenozoospermic patients group; NC; negative control of PCR system. FIG. 3. miR‐27b directly regulated CRISP2 by binding to its 3’‐UTR. A) A Venn diagram showed the overlap of candidate miRNAs that were predicted by miRWalk, and the CRISP2 3’‐UTR with a potential binding‐site for miR‐27b was displayed. B) miR‐27b was detected by qRT‐PCR to be highly expressed in the ejaculated spermatozoa of patients with asthenozoospermia relative to normal controls. Data were shown as mean ± SEM, **P<0.01. C) Schematic diagram of luciferase reporter construct, and results of luciferase reporter assay in HEK 293T cells with co‐transfection of 3’‐UTR vector or negative control vector. The bar graph showed the mean ± SD in the three independent experiments. *P<0.05, ***P<0.001. D) The expression of CRISP2 protein was detected by Western Blot in 293T cells transfected with miR‐27b mimic, inhibitor, negative control and miR‐27b mimic+CRISP2 plasmid, respectively. E) Spearman correlation coefficient (r) was calculated to assess the significance of association between miR‐27b and CRISP2 protein expression levels. F) Spearman correlation coefficient was calculated to assess the significance of association between miR‐27b and CRISP2 mRNA expression levels. Norm, normozoospermic volunteers group; Asth, asthenozoospermic patients group; NC, negative control. FIG. 4. miR‐27b expression level was negatively correlated with sperm progressive motility and normal morphology. The study subjects with relatively low CRISP2 protein expression tended to have lower sperm progressive motility and normal morphology. Low CRISP2 protein and high miR‐27b expression were associated with infertility. A) Spearman correlation analysis showed the significance of association between miR‐27b expression level and sperm progressive motility in 90 samples. B) Spearman correlation analysis showed the significance of association between miR‐27b expression level and sperm normal morphology in 90 samples. C) Spearman correlation analysis showed the significance of association between CRISP2 protein expression level and sperm progressive motility in 24 samples. D) Spearman correlation analysis showed the significance of association between CRISP2 protein expression level and sperm normal morphology in 24 samples. E) The infertility rates were displayed in the indicated groups. Chi‐square or Fisher's exact test was performed to determine significant differences of infertility rate between different groups. *P<0.05, ** P<0.01. Abbreviations: Norm: normozoospermic volunteers group. Asth: asthenozoospermic patients group. TABLE 1. Demographic and semen characteristics. a
Characteristics Norm (n = 42)a,b Asth (n = 48)a,c Age (yr) 29.00 ± 4.804 30.13 ± 5.841 Volume (ml) 3.32 ± 1.520 3.18 ± 1.345 pH 7.53 ± 0.113 7.51 ± 0.130 Count (106/ml) 74.53 ± 60.193 85.80 ± 80.298 Motility (% motile) 61.51 ± 11.665* 17.98 ± 9.124 Morphology (%) 22.90 ± 6.630* 6.721 ± 1.567 Data presented as mean±SD. Norm, normozoospermia control group. c
Asth, asthenozoospermia group. *Student t‐test, P < 0.05 versus the asthenozoospermia group. b
TABLE 2. The 63 study subjects included and 27 study subjects excluded. Participants
Status Descriptions 19 Excluded Lost contact or refused to cooperate
4 Excluded
Female partner infertility
4 Excluded
Unmarried
31 Included The included normozoospermic volunteers
32 Included The included asthenozoospermic patients 90 ‐ Total study subjects Figure 1
B
2.5
Asth
Norm
CRISP2
P=0.3854
2.0
ACTB
1.5
1.0
0.5
0.0
Norm
Asth
(n=42)
(n=48)
Relative gray values
(CRISP2 protein / ACTB)
Relative expression
(CRISP2 mRNA / ACTB)
A
***
2.0
1.5
1.0
0.5
0.0
Norm
Asth
(n=12)
(n=12)
Figure 2
M
A
Norm
Maker
U
Asth
Norm
Asth
NC
Maker
MSP
B
Maker
NC
Norm
Maker
Asth
Maker
BSP
C
U: GTGGTGATGAAAGTATAAAGT T TAGTGATGT TGT TTATATGTGGTGT T TGGTAAGTGAATAATTGTGGTGAGAGGGGTGTG TTGTAG T T T TTTAATG TTGTAA TGTGTT
M: GCGGCGATGAAAGTATAAAGTT TAGTGACGTCGT TTATACGTGGCGT TTGGTAAGTGAATAATCGCGGTGAGAGGGGCGCG TCGTAGT T T TTTAACGTCGTAA CGCGTC
Figure 3
miRDB
A
miRWalk
Has-miR-27b-3p
0
6
0
miR-27b
position 266
miR-27a
CRISP2 3’-UTR
5' -… A
G
AG
C …-3'
miR-502-3p
UAGGAUUUA GUCACU
GA
miR-510
GUCUUGAAU CGGUGA
CU
miR-640
miR-27b
3' -
C
CA
U
-5'
Targetscan
**
1.0
miR-27b
0.8
0.6
0.4
CRISP2 3’-UTR
0.2
Norm
CRISP2
ACTB
*
***
1.0
0.8
0.6
0.4
0.2
0.0
3’-UTR vector
NC vector
miR-27b
miR-NC
Asth
(n=48)
E
F
2.0
Relative expression
(CRISP2 mRNA / ACTB)
0.0
(n=42)
D
C
Relative gray values
(CRISP2 protein / ACTB)
Relative expression
(miR-27b / U6b)
B
R e la t iv e lu c if e ra s e ra t io
miR-767-5p
P = 0.0121
1.5
r = －0.5039
1.0
0.5
0.0
0.0
0.2
0.4
0.6
miR-27b / U6b
(n=24)
0.8
＋
－
＋
－
2.5
＋
－
－
＋
－
＋
＋
－
P > 0.05
2.0
1.5
1.0
0.5
0.0
0.0
0.2
0.4
0.6
miR-27b / U6b
(n=90)
0.8
1.0
Figure 4
A
P = 0.0088
r = －0.2745
100
50
0
0.0
0.2
0.4
0.6
0.8
1.0
50
P = 0.0012
r = －0.3397
40
30
20
10
0
0.0
0.2
0.4
20
P = 0.0024
r = 0.5911
10
5
1.0
1.5
Relativ e gray values
(CRISP2 protein / ACTB)
(n=24)
2.0
P = 0.0003
r = 0.6697
40
20
0
0.0
0.5
40
Fertility
Infertility
**
n=31 n=32
30
69%
20
42%
10
0
32%
*
n=20
70%
1.0
1.5
Relative gray values
(CRISP2 protein / ACTB)
(n=24)
n=43
Numbers of study subjects
Normal morphology (%)
25
60
*
E
0.5
1.0
(n=90)
D
0
0.0
0.8
0.6
Re lativ e expre ssion
(miR-27b / U6b)
Relative expression
(miR-27b / U6b)
(n=90)
15
Progressive motility(%)
150
C
Normal morphology (%)
Progressive motility(%)
B
n=16
63%
n=8
13%
2.0

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