Conditioned medium and secretome from epididymal epithelial cell cultures improve sperm kinetics and capacitation.

Background and Aim: Sperm maturation occurs in the epididymis through interactions with existing molecules inside the lumen. However, the mechanism of epididymis molecular transfer is currently unclear. This study was aimed to determine the necessity of the epididymal epithelial cells (EECs) in the process of sperm maturation in terms of sperm kinetics and tyrosine phosphorylation. Materials and Methods: A true experimental research design was used in this study. The medium tested was a primary culture of mice caput epididymal cells (cells and culture medium), conditioned medium (CM) (supernatant of EECs), and secretome (CM filtered at 0.22 µm). Sperm was cocultured in EEC culture, CM, and secretome for 1, 2, 3, or 4 h. The original culture medium was used as the control. Sperm kinetic analysis was performed after the indicated times using computer-assisted sperm analysis, and tyrosine phosphorylation was detected using the Western blot technique. Results: A primary culture of caput EECs was successfully generated. The results showed increased sperm motility and progressive movement after 3 h of incubation (p < 0.05). There was a significant decrease in the average path velocity (VAP) values after 4 h of incubation (p < 0.05), but there was no significant change in the 1, 2, and 3 h incubation groups. The EEC culture-CM and secretome groups showed a significant increased progressivity and VAP percentage values compared with the control medium (p < 0.05). In terms of percentage motility, the culture and CM groups were significantly different from the control medium, but the secretome group was not. Conclusion: The sperm kinetics of sperm cultured in CM, secretome, and EEC were significantly increased after 3 h of incubation, suggesting that CM and secretome can be used to replace EECs, especially when analyzing molecules secreted by the epididymal epithelium during sperm maturation. The results of this study highlight the potential of CM and secretome as therapy mediums for sperm kinetic abnormalities.

Various gene modification techniques to discover molecular targets for nonhormonal male contraceptives: A review.

The identification and characterization of relevant targets are necessary for developing nonhormonal male contraceptives. The molecules must demonstrate that they are necessary for reproduction. As a result, a sophisticated technique is required to identify the molecular targets for nonhormonal male contraceptives. Genetic modification (GM) techniques are one method that can be applied. This technique has been widely used to study gene function that effected male fertility and has resulted in the discovery of numerous nonhormonal male contraceptive target molecules. We examined GM techniques and approaches used to investigate genes involved in male fertility as potential targets for nonhormonal contraceptives. The discovery of nonhormonal contraceptive candidate molecules was increased by using GM techniques, especially the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 method. The discovery of candidate nonhormonal contraceptive molecules can be a wide-open research for the development of nonhormonal male contraceptives. Therefore, we are believing that one day nonhormonal male contraceptives will be released.

In silico docking analysis of beta-defensin 20 against cation channel sperm-associated protein 1-4 to predict its role in the sperm maturation.

Beta-defensin 20 (DEFB20) is widely expressed in the epididymis with gene features involved in epididymal sperm maturation. However, the action mechanism and function of DEFB20 in sperm maturation are still unclear. One of the important roles of beta-defensin is the ion channel activity. The cation channel sperm-associated protein (CatSper) alpha is an ion channel protein found on the sperm surface. This study aimed to investigate the interaction between DEFB20 and CatSper1-4 protein in relation to the sperm maturation process. Protein sequences were obtained from the National Center for Biotechnology Information (NCBI). Protein modeling and validation were carried out by using the Robetta modeling server and the Ramachandran plot method. Rosetta web server was used for the docking analysis. The results revealed a natural interaction between DEFB20 and CatSper1-4. The interaction occurred at the cation channel (close to the casein kinase II), ion transport protein, and kinase c phosphorylation of the CatSper1-4 active site. The DEFB20 region interacting with CatSper2-4 was the beta-defensin domain, while with CatSper1 was the non-beta-defensin domain. Based on the analysis, DEFB20 may interact with CatSper α subunits, particularly CatsSper1, to affect ion channel activity during sperm maturation.

Association of Variants in COMT, RASSF1 and GPM6A with the Risk of Paranoid Schizophrenia Patients in Prof HB Saanin Psychiatric Hospital, West Sumatra, Indonesia.

Background: Schizophrenia is a multifactorial disease in which genetic factors play a greater role than other factors. The genes of importance in schizophrenia patients are the genes that encode for neurotransmitters associated with low minor allele frequency (MAF) scores. This study was aimed to determine the association of genetic variations in catechol-O-methyl transferase (COMT), Ras association domain family member 1 (RASSF1) and glycoprotein M6A (GPM6A) with the risk of paranoid schizophrenia (PS) in patients admitted to Prof HB Saanin Psychiatric Hospital, West Sumatra, Indonesia. Methods: Genotyping analysis through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and PCR-amplification refractory mutation system (ARMS) was performed in 100 PS patients and 100 healthy controls. Chi-square and Fisher's exact tests were used to compare the frequencies of genotype and allotype between the PS and control groups. Odds ratio (OR) with 95% confidence interval (95% CI) were calculated to determine the relative risk of PS with respect to genetic variations. Results: Polymorphism rs13142920 in GPM6A was associated with significantly elevated risk of PS (P = 0.020; OR = 1.60 [95% CI: 1.08, 2.39]). However, COMT rs4680 and RASSF1 rs2073499 polymorphisms were not significantly associated with PS. Conclusion: The GPM6A rs13142920 polymorphism holds great potential as a genetic marker in PS patients.

Epstein-Barr virus nuclear antigen-1 is useful as therapeutic efficacy marker in serum but not in saliva of nasopharyngeal cancer patients who underwent radiotherapy.

INTRODUCTION: Nasopharyngeal carcinoma (NPC) is a multifactorial disease with genetic, viral, environmental and lifestyle-related risk factors. Epstein-Barr virus (EBV) can promote the oncogenic transformation of an infected cell into malignant. EBV encodes many stimulating products including Epstein-Barr virus nuclear antigen-1 (EBNA-1) which plays a key role in the regulation of gene expression and replication of the genome in the latent period of infection. EBNA-1 in serum and tumour tissue of NPC patients correlates with NPC prognosis. Moreover, the presence of EBV DNA in serum samples from NPC patients' blood circulation can be used as an early marker in the diagnosis of NPC. OBJECTIVE: The objective of this study was to find effective methods for monitoring the progress of NPC patients undergoing radiotherapy and therapeutic efficacy by observing the changes in EBV DNA in serum and saliva. METHODOLOGY: The pre-experimental design compared blood and saliva taken from a pre-test and post-test group of NPC patients before and after radiation therapy. The concentration of EBV DNA was measured in the serum and saliva after amplification using quantitative polymerase chain reaction (qPCR) with compatible primers for the EBNA-1 gene. The data were statistically analysed by paired T-test. RESULTS: Highly significant (p = 0.0001) increase in cycle threshold qPCR and decrease in the mean concentration of EBV DNA (p = 0.0001) were observed in serum samples, but no significant changes were observed in saliva. CONCLUSIONS: The results suggest that EBV DNA in serum can be used as the gold standard and a marker for monitoring the response to radiation therapy in NPC patients, whereas the examination of EBV DNA from saliva samples is not accurate and thus, not appropriate.

Decreased Expression of CDC25A in Azoospermia as the Etiology of Spermatogenesis Failure.

BACKGROUND: Spermatogenesis is a tightly regulated developmental process of male germ cells. The stages in spermatogenesis are mitosis, meiosis and spermiogenesis. One of the genes playing a role in meiosis is Cell Division Cycle 25A (CDC25A). Decreased expression of CDC25A is associated with failure of spermatogenesis and sperm retrieval. Infertility examination for azoospermia has been limited on histological examination. Hence, molecular research to find marker genes for infertility will improve the examination of testis biopsies. METHODS: This research is a cross sectional study of 50 testicular biopsies with Johnsen scoring categories from scoring 2 to 8. Analysis of mRNA expression used qPCR and protein expression using immunohistochemistry. Statistical analysis with Spearman correlation was considered significant at p<0.05. RESULTS: The result showed that transcript level and protein expression of CDC25A decreased in score 5 of Johnsen scoring categories. Moderate Spearman rho correlation (r=0.546) between mRNA relative expression and protein expression of CDC25A was significant at p<0.01. CONCLUSION: Decreased expression of CDC25A is associated with meiotic arrest as the etiology of spermatogenic failure in many azoospermic men.