Preliminary Research On DNA Methylation Of Paternal And Maternal Imprinted Gene In Sperm Of Infertile Males

Male infertility refers to the inability of a male to conceive after at least one year of unprotected intercourse. Male infertility is affected by genetic, environmental and other multiple factors, No causal factor is found in 60%-75% of infertile male (idiopathic male infertility), but the cause is not fully understood. Genomic imprinting is a mechanism that regulates gene expression in a parental origin-dependent way, leading to monoallelic gene expression. This process is mediated by the methylation of DNA. During normal spermatogenesis, the erasure of methylation marks of the maternally imprinted gene SNRPN and the resetting of the paternally imprinted gene H19 have been reported to be completed before germ cells enter meiosis. The paternal methylation imprints are then maintained through meiosis and during the subsequent male germ cell differentiation. Imprint erasure, resetting and maintenance errors can lead to disorder of imprinted genes. The present research is to study the DNA methylation of paternal imprinted gene H19 and maternal imprinted gene SNRPN in sperm of infertile male by bisulfite sodium sequencing, and some CpG sites were analysis with restriction enzymes.To explore the epigenetic causes and pathogenesis of male infertility and the impact of epigenetic factors on the security of assisted reproductive technology. The present study indicated that most of H19 and SNRPN are showed correct imprinting status in each group, there is a small number of CpG sites with scattered imprinting change. In clinic, ART may be a safety and efficient method to treat severe oligospermia , severe asthenospermia and severe teratospermia, but we should note the potential risk of imprinted disease in offspring and treat according to ART indications. Interestingly, one clone of H19 of severe asthenospermia group and SNRPN of severe teratospermia group presents critical abnormal methylation. Severe oligospermia and severe asthenospermia group are more prone to have abnormal imprinting in CpG 9 of H19,CTCF- binding site 6 and CpG 16 of SNRPN than normospermia group and severe teratospermia group. This may be one of reasons of poor semen quality and infertility of severe oligospermia, asthenospermia and teratospermia.Partâ… Sperm preparation and the establishment of DNA methylation detection technologyObjective:1. To establish bisulfite sodium sequencing technology for DNA methylation detection of imprinted gene in sperm of infertile male.2. To optimize experimental conditions and select the appropriate sequencing method. Facilitate the following experiment.Methods:1. Routine semen analysis and morphological analysis were performed; Sperm were purified by density gradient centrifugation; DNA was extracted by a standard extraction method.2. Genomic DNA was bisulfite treated. Then a hemi-nest PCR was performed. The first round PCR products should be purified before the second round of reaction.3. The purified PCR products were cloned into pCR?2.1-TOPO? vector and Proceed to Chemical Transformation. Positive clones were analyzed by PCR. 4. For each sample, PCR products and 15 positive clones were selected for sequencing analysis. Sequence was analyzed with Vector NTI Suite 8 software.Results:1. DNA OD 260/280 value of all samples is between 1.7 and 1.9, indicating that DNA is pure, no protein and RNA contamination occurs.2. The first round PCR products without purification as a template of the second round of PCR, electrophoresis showed non-specific band. And the purified PCR products as template, electrophoresis showed a single and specific band.3. In direct sequencing of PCR products, the front of the sequence was lost about 40bp, methylation information of CpG 1 was lost. There is only one sequence result in each sample, and there will present partial methylation in CpG sites.4. In Clone sequencing, no loss of sequence, methylation information of all CpG sites was complete. There is different methylation in 15 clones and could show the average methylation of this sample.5. Clone sequencing of PCR product is an appropriate method for detection of DNA methylation status in infertile male.Conclusion:1. The first round PCR products were purified with Exonuclease I and FastAP? Thermosensitive Alkaline Phosphatase could remove unincorporated primers and degrades unincorporated nucleotides, No non-specific band presents in he second round of PCR, the band is single and specific. Facilitate the subsequent cloning operations.2. The purified PCR products were successfully cloned into pCR?2.1-TOPO? vector and Proceed to Chemical Transformation. Most of the clones are positive. We successfully detected the methylation in sperm by bisulfite sodium sequencing technology.3. In direct sequencing of PCR products, partial sequence and methylation information of CpG sites were lost. But clone sequencing did not lose any sequence, the methylation information of CpG sites were complete. 4. Direct sequencing could only show the overall methylation of the sample, and there will present partial methylation in CpG sites. Each sample selected 15 clones for sequencing could show the average methylation of the sample.Partâ…¡DNA methylation of paternal and maternal imprinted gene in sperm of infertile maleObjective:1. To investigate DNA methylation of paternal imprinted gene H19 and maternal imprinted gene SNRPN in sperm of infertile male.2. To explore the epigenetic causes and pathogenesis of male infertility and the impact of epigenetic factors on the security of assisted reproductive technology.Methods:1. Routine semen analysis and morphological analysis were performed; Sperm were purified by density gradient centrifugation; DNA was extracted by a standard extraction method.2. Genomic DNA was bisulfite treated. Then a hemi-nest PCR or a conventional PCR was performed.3. The purified PCR products were cloned into pCR?2.1-TOPO? vector and Proceed to Chemical Transformation. Positive clones were analyzed by PCR.4. 15-20 positive clones of each sample were selected for sequencing. Sequence was analyzed with Vector NTI Suite 8 software.5. According to the sequencing results, the methylation and unmethylation clone of CpG 7, CpG 9 of H19 gene and CpG 16 of SNRPN gene were selected. Plasmid DNA was extracted, Then performed PCR reaction.6. CpG 7 of H19 gene and CpG 16 of SNRPN gene was digested with Hhaâ… , CpG 9 of H19 gene was digested with Taqâ… . The results were analysis by electrophoresis.7. Statistical analysis was performed using SPSS 16.0 software.Results:1. most of H19 gene are showed methylation status in each group, there is a small number of CpG sites with scattered imprinting change, but hypomethylation and unmethylation did not show in CpG sites. one clone of severe asthenospermia group presents 8 CpG sites unmethylation, but it could not reach the hypomethylation criterion(>9 CpG sites present unmethylation).2. most of SNRPN gene are showed ummethylation status in each group, there is a small number of CpG sites with scattered imprinting change. one clone of normospermia group presents 20 CpG sites methylation(>11 CpG sites present methylation ), but hypermethylation and methylation did not show in other clone, one clone of severe teratospermia group presents 9 CpG sites methylation, but it could not reach the hypermethylation criterion.3. The percent of H19 gene CpG sites'complete methylation in severe teratospermia group was significantly lower than severe oligozoospermia and severe asthenospermia group.4. The percent of H19 gene CpG 7 site'unmethylation in severe oligozoospermia was significantly lower than normospermia and severe teratospermia group.5. The percent of H19 gene CpG 9 site's unmethylation, CTCF-binding site 6 three and more than three CpG sites'unmethylation, and SNRPN gene CpG 16 site's methylation in severe oligozoospermia and severe asthenospermia group were higher than normospermia and severe teratospermia group, but the differences are not significant.6. Restriction enzyme analysis is consistent with the clone sequencing result.Plasmid PCR products showed undigested unmethylation bands and digested methylation bands. PCR products of sample presented digested methylation bands in CpG 7 and CpG 9 in H19 gene, and presented undigested unmethylation bands in CpG 16 in SNRPN gene.7. the unimprinted gene LINE-1 showed similar hypermethylation in each group, indicating that the globe DNA methylation is normal in these samples.Conclusion:1. Most of H19 and SNRPN are showed correct imprinting status in each group, there is a small number of CpG sites with scattered imprinting change.2. One clone of normospermia group presents hypermethylation status, indicated that abnormal methylation of imprinted gene can be presented in normospermia male.3. One clone of H19 of severe asthenospermia group and SNRPN of severe teratospermia group presents critical abnormal methylation. This may be one of reasons of infertility of severe asthenospermia and teratospermia.4. Most of CpG sites and CpG 7 present normal methylation in severe oligospermia group.5. In clinic, ART may be a safety and efficient method to treat severe oligospermia , severe asthenospermia and severe teratospermia, but we should note the potential risk of imprinted disease in offspring and treat according to ART indications.6. Severe oligospermia and severe asthenospermia group are more prone to have abnormal imprinting in CpG 9 of H19,CTCF- binding site 6 and CpG 16 of SNRPN than normospermia group and severe teratospermia group. This may be one of reasons of poor semen quality and infertility of severe asthenospermia and teratospermia, indicated that we should do further research for the imprinting status of gamete and its effect on embryonic imprinting.7. The unimprinted gene LINE-1 showed similar hypermethylation in each group, indicating that the globe DNA methylation is normal in these samples. methylation errors occurring during spermatogenesis are specific for imprinted genes and associate with abnormal sperm qaulity.