Research On The Expression Of CRISP2 Gene And Protein In Asthenospermia

Infertility in humans has had a high profile around the world. In recent years, the incidence of infertility is growing step by step. Research has shown that infertility in couples caused by male factor has risen to 50%. And one recently study shown the male factor has increased to 2/3 in the couples who were treated for infertility.Clinical studies have shown the low sperm motility, is the major cause of infertility in humans, it also has a scientific name:asthenospermia. There are so many causes can lead to asthenospermia, for example, the genitourinary infections, abnormal semen liquefaction, immunity factors, endocrine factors, Kartagener's syndrome, chromosome abnormity, varicocele, absence of trace element and unhealthy life styles et al. Because of the etiological factor were so complicated and the pathogenesis of asthenospermia hasn't been study clearly. There weren't unable to take a targeted treatment, and the treatment effect wasn't satisfactory. The only way for the infertility couples were assisted reproduction. Recent research have found a few mRNAs in human sperm have shown a significant expressed differently in low motile sperm compared to high motile sperm isolated from the same semen sample of normospermic patients. And some genes have been verified associated with asthenospermia such as Tektin-2, DNAI1, DNAH5, DNAH11.Those studies tell us that the difference in levels of sperm mRNA expression are closely associated with the occurrence and development in asthenospermia. Previously we obtained the gene expression profiles of spermatozoa from asthenospermia and normal controls by microarrays, and found some genes differently expressed in asthenospermia. By applying the threshold value of 2 folds and p<0.05 (Student's t-test), the previous microarray screening data has been analyzed by expression profile analysis software GeneSpring10.0. Totally we found 1265 genes differently expressed in asthenospermia of which 307 up regulated,958 down regulated(Log2Ratio≤1 or Log2Ratio≥1 P<0.05), by Agilent human 4x44K slides containing 41000 single-spotted human cDNA. However, microarray technique which was an efficient and large-scale access to biological information, it was bound to produce massive amounts of data exist in some false positives. Bioinformatics was an emerging disciplines which comprehensive utilized biology, computer science and information technology, it provides an effective mean to eliminate the false and retain the true from massive data by data mining. Therefore, we planned to analyze the genes which differently expressed in asthenospermia obtained by microarray screening, find the genes which associated with asthenospermia and explore the relationship with occurrence and development in asthenospermia by bioinformatics methods. The following are the research methods, contents and conclusions of this study.Part 1 Research on the differently expressed genes in asthenospermia based onthe bioinformatics methodsObjective:We analyze the differently expressed genes in asthenospermia(Totally we found 1265 genes differently expressed in asthenospermia of which 307 up regulated,958 down regulated) which obtained by previous microarray screening, from which find the genes closely associated with asthenospermia. For better understanding the molecular characteristics and explore the relationship among occurrence and development in asthenospermia by bio informatics tools depth analysis.Methods:1. The previous microarray screening data has been analyzed by expression profile analysis software GeneSpring10.0. Totally we found 1265 genes differently expressed in asthenospermia of which 307 up regulated,958 down regulated(Log2Ratio<1 or Log2Ratio>1 P<0.05). And uploaded the data to online bio informatics tools such as GATHER, PANTHER and ToppGene.2. Analyzed the differently expressed genes in asthenospermia, and studied their function and pathways.Results:1. GATHER analysis Uploaded the differently expressed genes to the Gather tools, there were 1205 genes can be recognized by GATHER. GO classification analysis shown those genes associated with cellular physiological process and metabolic process, such as G protein-coupled signal pathway, cellular protein and macro molecular metabolism:2. PATHER analysis There were 932 genes recognized by PANTHER in those differently expressed genes. Pathway analysis shown the Integrin signaling pathway, Interleukin signaling pathway, Oxidative stress response, TGF-beta signaling pathway and Apoptosis signaling pathway were up-regulated. But in Adrenaline and noradrenaline biosynthesis,5-Hydroxytryptamine biosynthesis, Transcription regulation by bZIP transcription factor and Histamine synthesis were down-regulated.3. ToppGene analysis Totally 234 genes recognized in up-regulated genes and 633 genes recognized in down-regulated genes by ToppGene. Biological process analyzed of shown that the up-regulated genes mainly related to cellular immune responses, antigen recognition and presentation, cell death, apoptosis and mediates programmed cell death. The down-regulated genes mainly related to protein hydrolysis, cell protein macro molecule metabolism, protein modified and macro molecules metabolism, protein K48 ubiquitination and structure of microtubule and cytoskeleton.Conclusions:1. The bio info rmatic analysis shown that the motility of sperm was declining meantime the basic life activities were, and prone to apoptosis and death which form the asthenospermia patients.2. Such genes as KIF3B, MY015A, KIF6, KIF26B, KIF3A, DNHD2, DMN, DYNC2H1, STARD9, MYOHD1, TPM1, etc. Involved in cytoskeletal structure, micro tubules movement, cell movement, maybe associated with asthenospermia.3. Literature mining shown the expression of CRISP2 gene and protein may be related closely to asthenospermia. And it's worth for further depth study.Part 2 Tentative research on the expression of CRISP2 gene and protein in AsthenospermiaObjective:Combined with previous microarray screening, bioinformatic tools analysis and literature mining, we found the expression of CRISP2 gene and protein closely related to asthenospermia. We research the expression level of Cysteine-rich secretory protein 2 (CRISP2) gene and its protein product in Asthenospermia, and explore its relationship with sperm motility and the related molecular mechanism.Methods:1. We have collected 77 semen samples from adult male with asthenospermia and 67 samples from healthy volunteers as control groups. Purified the spermatozoa through 50% one layer Percoll gradient centrifugation, and stored at -80℃.2. Sperm samples after serparation were stored in -80℃refrigerator. After thawing in 37℃water box, then every four spermatozoal pellets from asthenospermia or healthy donors were pooled. RNA was isolated from these solutions using an RNeasy Mini kit (Qiagen, Germany), according to the producers' manuals. Detect the relative expression of CRISP2 mRNA and protein between the two groups by RT-PCR, SYBR Green real-time PCR.3. After thawing, total protein was isolated using Total Protein Extraction Kit (Bestbio, China), according to the producers'manuals. Detect the relative expression of CRISP2 protein between the two groups by Western Blot.4. STRING analysis of CRISP2 protein. Upload CRISP2 protein to the STRING database website (http://string-db.org). To analyze the proteins this associated with CRISP2 protein, and the interactions between those proteins and CRISP2 protein.5. Further research on CRISP2 gene and its protein product by literature mining.Results:1. The expression of CRISP2 mRNA in asthenospermia is significantly lower than in the normal control group, do wn-regulation to 4.3-fold.2. Western Blot showed the expression of CRISP2 protein in asthenospermia was 1.71 times lower than the normal. The expression levels between the two groups are statistically significant difference (P<0.05).3. STRING analysis showed that CRISP2 protein interacted with GRSF1, GGN, TPX2, SPATA22, MAP3K11, NSUN4, PGK2, SPZ1, LDHC, LUZP4 proteins.Conclusions:1. CRISP2 gene regulated sperm development by Wnt pathway, and affected the sperm cell Ca2+ channel activity by RyR receptor signal pathway. CRISP2 protein directly played an important role in the sperm tail cytoskeleton structures.2. CRISP2 gene and protein expression level is closely related to sperm motility. The down-regulated expression of CRISP2 mRNA and protein may closely relate with the poor sperm motility in asthenospermia.3. It suggested CRISP2 gene and protein may be a potential diagnostic marker for asthenospermia. And further functional analysis of this gene may help us expound the relevant patho genesis of asthenospermia.