The Molecular Mechanisms Of MiR-27a Suppresses The Cysteine-rich Secretory Protein 2 In Asthenoteratozoospermia

There are about 15% couples infertility in our world, which male-factor infertility accounted for 50%. Clinical studies have shown that asthenozoospermia and asthenoteratozoospermia are the common factor leading to male infertility. First, we achieved the gene expression profiles of asthenozoospermia and normal male sperm by the human whole genome microarray. SEMG1 gene may be one of the candidate genes of asthenozoospermia by by bioinformatic screening. CRISP2 also confirmed low expression in asthenozoospermia, and the protein which was structural protein of sperm participated in acrosome reaction and gamete in sperm egg fusion in other studies. Secondly, we further evaluated the CRISP2 gene/protein expression and clinical significance in asthenoteratozoospermia, and found the CRISP2 protein expression was significantly low, but the CRISP2 gene expression was no difference in Q-PCR results. Suggesting that the expression of CRISP2 protein is post-transcription level regulation. Therefore, the study intends to further evaluate the molecular mechanism of low expression of CRISP2 in asthenoteratozoospermia. We know that the abnormal expression of genes often cause by genetic mechanisms and epigenetic mechanism. Epigenetics is known as the sequence of DNA does not change, but the gene expression has undergone a heritable changes. This change is genetic material changes in addition to the genetic information in cells, and these changes can stably transfer in process development and cell proliferation. Epigenetic mechanisms include DNA methylation, histone modification, chromosome remodeling, RNA interference (MICRNA, LNCRNA).We studied the CRISP2 gene methylation by methylation specific PCR and Bisulfite sequencing method. We did not found the existence of CRISP2 gene promoter CpG island methylation in 10 cases of normal control group and 10 cases of asthenoteratozoospermias. Results indicated no significant difference of CRISP2 gene by Q-PCR, and significant down-regulation expression of CRISP2 protein in 20 cases of normal control group and 20 cases of asthenoteratozoospermias. Combined with clinical data, found that the down regulation expression of CRISP2 is positively associated with the sperm morphology, sperm vitality and male infertility.CRISP2 protein expression was significantly low in asthenoteratozoospermia,but CRISP2 gene promoter CpG island methylation was not existence. Suggesting that the expression of CRISP2 protein is post-transcription level regulation. Therefore, we will study the microRNA targeting CRISP2 gene/protein.Using miRDB, miRWalk, miRTargetscan bioinformatics software to predict microRNA that can target the CRISP2 and choose the highest rated, common intersection of microRNA. We found miR-27a, miR-27b, miR-340, found in miR-502-3p, miR-510, miR-640 and miR-767-5p can specifically bind to CRISP2 3 ’-UTR region and regulating its expression.Results indicated miR-27a was up-regulation expression by Q-PCR in 20 cases of normal control group and 20 cases of asthenoteratozoospermias. Combined with clinical data, found that the up-regulation expression of miR-27a is negatively associated with the sperm morphology, sperm vitality and male infertility.To this end, we further investigate whether the miR-27a leads to the occurrence of asthenoteratozoospermia through inhibiting the expression of CRISP2. To screen miR-27a targeted to CRISP in 293 cells by luciferase report gene. Contained CRISP2 3’-UTR (clone) pEZX-MT05 plasmid and miR-27a were transfected in the 293 cells, and the protein of supernatant was mensurated through fluorescent assay. The results indicated that the miR-27a resulted in asthenoteratozoospermia through a combination of CRISP2 3’-UTR. The following are the research methods, contents and conclusions of this study.Part 1 Study on the molecular markers of adult male asthenozoospermia by combining the microarray and bioinformatics techniquesObjectivewe obtained gene expression profiles of spermatozoa from asthenozoospermic and normal controls by genome wide microarrays, screened differentially expressed genes in asthenozoospermic semen samples by bioinformatics techniques, found new genes related with sperm motility and explored their value and significance.Methods1. We collected 12 adult asthenozoospermic semens nd 12 normal control group. Using Percoll density gradient centrifugation to purificate the sperm, we extracted the total RNA of sperm, cRNA synthesis in vitro transcription and marked cRNA. After purification and hybridization with Agilent 4110B chip, the scanned images of microarrays were analysed using Feature Extraction software.2. The acquired data analysis was performed using GeneSpringlO and BRB-Array Tools respectively, two different AZS-related gene expression profiles were obtained and genes of the intersection of both profiles were considered as the molecular signatures of asthenozoospermia.3. We use such as NextBio,EDLINE and FACTA software tools for further screening differentially expressed genes and verify the chip results by fluorescence quantitative PCR.Results1. We obtained 1265 differentially expressed transcripts between two groups through the analysis of GeneSpring 10. We also used BRB-ArrayTools to analyse these two gene expression profiles,262 transcripts were found to be differentially expressed. Of the transcripts overlapping the analysis of GeneSpring and BRB-ArrayTools,71 transcripts were found to be differentially expressed between groups, the transcripts list, termed molecular signatures of asthenozoospermia, comprising 10 elevated and 61 decreased in asthenozoospermia spermatozoa.2. we next compared our gene list with expression profiles of non-obstructive azoospermia, and teratozoospermia performed by NextBio. We further excluded all the common genes we have obtained from our gene list, and using this filter strategy, we finally refined our gene list to 21 genes which comprised 7 up-regulated and 14 down-regulated genes.3. We next matched these 21 genes against the MEDLINE database using the FACTA software tool. This analysis identified 2 genes (PGAP1, SEMG1) that were linked to male-fertility-related articles, with PGAP1 gene was not linked to sperm motility.4. Real-time PCR data of SEMG1 gene was consistent with the result of microarray analysis. SEMG1 may be the candidate gene for idiopathic asthenozoospermiaConclusions1. We obtained gene expression profiles of spermatozoa from asthenozoospermic and normal donors and screened differentially expressed genes. The results suggest that differences in gene expression between the asthenozoospermic samples and normal controls might be responsible for the decline of sperm motility.2. These genes discovered by this endeavour could serve as potential diagnostic biomarkers.SEMG1 gene may be one of the candidate genes of asthenozoospermia by by bioinformatic screening. CRISP2 also confirmed low expression in asthenozoospermia and was close to sperm motility. Further study of these genes might help us to explain the molecular mechanisms involved in asthenozoospermia.Part 2 Expression level of cysteine-rich secretory protein 2 (CRISP2) in asthenoteratozoospermia Patients and its clinical significanceObjectiveTo discuss the expression level of cysteine-rich secretory protein 2 (CRISP2) gene and its protein product in asthenoteratozoospermia and study the relationship between CRISP2 gene/protein and asthenoteratozoospermia. To study the clinical significance of CRISP2 expression level.Methods1. We have collected 20 semen samples from adult male asthenoteratozoospermia and 20 cases from normal as control groups. To purify the spermatozoa through Percoll gradient centrifugation and extract the sperm total RNA and total protein. Using SYBR Green real-time PCR and Western Blot to detect the relative expression of CRISP2 mRNA and protein between the two groups.2. We investigated whether the couples of asthenoteratozoospermia and normal control group was reproductive for 1 year through Retrospective investigation. Spearman correlation analysis was calculated to assess the correlation between the expression level of CRISP2 mRNA and protein and Male infertility. All data were analysed by GraphPad Prism 5 software.Results1. The expression of CRISP2 mRNA in asthenoteratozoospermia is not different than in the normal control group (P=0.8580).2. The expression of CRISP2 protein in asthenoteratozoospermia is differently down-regulation than in the normal control group (P=0.0156).3. Correlation analysis showed that CRISP2 protein expression was positively correlated with progressive motility (r=0.6197,P=0.0316) and normal sperm morphology (r=0.5933, P=0.0420) in asthenoteratozoospermia. There was no correlation between CRISP2 protein expression and age, semen volume, sperm concentration or pH value.4. The sterile rate (73%) in asthenoteratozoospermia was significantly higher than that in normal control group (24%, P=0.0188); Sterile rate (67%) in low expression of CRISP2 protein group was significantly higher than that of CRISP2 protein with a high expression group (17%, P=0.0361).Conclusions1. The expression level of CRISP2 protein is positively correlated with normal sperm morphology, progressive motility and Male infertility in asthenoterat-ozoospermia.2. CRISP2 could be as a novel molecular target for studying the pathogenesis of asthenoteratozoospermia. It is worth further study of its function and regulation mechanism.Part 3 Expression level of Mir-27a in asthenoteratozoospermia Patients and its clinical significanceObjectiveTo discuss the expression level of mir-27a gene in asthenoteratozoospermia and study the relationship between mir-27a gene and asthenoteratozoospermia. To study the clinical significance of mir-27a expression level.Methods1. We have collected 20 semen samples from adult male asthenoteratozoospermia and 20 cases from normal as control groups. To purify the spermatozoa through Percoll gradient centrifugation and extract the sperm total RNA and total protein. Using SYBR Green real-time PCR to detect the relative expression of mir-27a mRNA between the two groups.2. We investigated whether the couples of asthenoteratozoospermia and normal control group was reproductive for 1 yeas through Retrospective investigation. To know the correlation between the expression level of mir-27a mRNA and Male infertility.Results1. The expression of mir-27a mRNA in asthenoteratozoospermia is differently up-regulation than in the normal control group (P=0.0179)2. Correlation analysis showed that mir-27a mRNA expression was negatively correlated with progressive motility (r=-0.4800, P=0.0322) and normal sperm morphology (r=-0.5415, P=0.0137) in asthenoteratozoospermia. There was no correlation between mir-27a mRNA expression and age, semen volume, sperm concentration or pH value.3. The sterile rate (25%) in low expression of mir-27a mRNA group was significantly lower than that of mir-27a mRNA with a high expression group (69%, P=0.0320). These data suggest that Mir-27a is closely related with Male infertility.Conclusions1. The expression level of mir-27a mRNA is negatively correlated with normal sperm morphology, progressive motility and Male infertility in asthenoterat-ozoospermia.2. Mir-27a could be as a novel molecular target for studying the pathogenesis of asthenoteratozoospermia. It is worth further study of its function and regulation mechanism.Part 4 The molecular mechanisms of MiR-27a suppresses the cysteine-rich secretory protein 2 in asthenoteratozoospermiaObjectiveTo investigate the molecular mechanism of down-regulated expression of CRISP2 in asthenoteratozoospermia, determine whether Mir-27a is related to CRISP2, and whether Mir-27a regulate the expression of CRISP2.Methods1. We have collected 10 semen samples from adult male asthenoteratozoospermia and 10 cases from normal as control groups. To purify the spermatozoa through Percoll gradient centrifugation and extract the sperm DNA. The target fragment was amplified by PCR and BSP method, and sequenced for detecting CpG island methylation of CRISP2 gene.2. Spearman correlation analysis was calculated to assess the correlation between the expression level of CRISP2 gene/protein and mir-27a mRNA in asthenoteratozoospermia.3. we further investigate whether the miR-27a leads to the occurrence of asthenoteratozoospermia through inhibiting the expression of CRISP2 by luciferase report gene. Contained CRISP2 3’-UTR (clone) pEZX-MT05 plasmid and miR-27a were transfected in the 293 cells, and the protein of supernatant was mensurated through fluorescent assay.Results1. We did not found the existence of CRISP2 gene promoter CpG island methylation in 10 cases of normal control group and 10 cases of asthenoteratozoospermias.2. Correlation analysis showed that mir-27a mRNA expression was negatively correlated with expression level of CRISP2 protein (r=-0.4330, P=0.0345) There were no correlation between mir-27a mRNA expression and expression level of CRISP2 gene (P>0.05)3. We discovered that fluorescence activity of containing CRISP2 3’UTR plasmid group was lower when miR-27a put in this group. The results indicated that the miR-27a resulted in asthenoteratozoospermia through a combination of CRISP2 3’-UTR.Conclusions1. The expression of CRISP2 mRNA in asthenoteratozoospermia is not different than in the normal control group. We did not found the existence of CRISP2 gene promoter CpG island methylation in asthenoteratozoospermias and normal control group.The expression of CRISP2 protein in asthenoteratozoospermia is differently down-regulation than in the normal control group.2. The expression of mir-27a mRNA in asthenoteratozoospermia is differently up-regulation than in the normal control group. Correlation analysis showed that mir-27a mRNA expression was negatively correlated with expression level of CRISP2 protein.3. The results indicated that the miR-27a suppresses the cysteine-rich secretory protein 2 in asthenoteratozoospermia through a combination of CRISP2 3’-UTR. This provides a new basis for further research on CRISP2 and miR-27a function and CRISP2 signal pathway.