Regulation of Blood-Testis Barrier (BTB) proteins in Sertoli-germ cell nanotube formation in EF-treated spermatogenesis

Abstract

Abstract Objective: The aim of this study was to analyze visual data analysis of nanotubes formation and actin expression between Sertoli germ cells, gene silencing with the use of FAK siRNA and EF application, confirmation with mRNA levels, cell viability test and immunofluorescent staining associated ezrin, Fascin 1, FAK and N-cadherin. Expressions of blood-testicular barrier (BTB) proteins were evaluated. Materials and Method: As the experimental group of the research; control group (CG), in which intercellular nanotubes and cargo proteins were followed under normal culture conditions; Sertoli and germ cells co-culture; co-culture of testosterone (T) group, Sertoli and germ cells in which intercellular nanotubes and cargo proteins are tracked; The group in which actin organization and intercellular nanotubes and cargo proteins are monitored, and the group in which the focal adhesion kinase is suppressed with siRNA (FAK RNAi) and the co-culture of Sertoli and germ cells, the electromagnetic field applied group (EF), in which intercellular nanotubes and cargo proteins are followed under normal culture conditions, were used. Results:In the control groups, nanotubes formations started at the 6th hour during the culture and increased at the 40th hour, while the number of nanotubes formation and disappearance was 52 in the Control group; 58 in the EF group; 44, 12 in the FAK RNAi group and 5 in the EF+ FAK RNAi group. It was shown that actin associated nanotubes formations were significantly decreased in FAK RNAi and EF+ FAK RNAi groups compared to control. Stable nanotubes formation rate but low disappearance rate was detected in the EF applied group. It was observed that there was a decrease in ezrin and Fascin 1 expressions in nanotubes formation regions, except for control and testosterone groups, and there was no significant difference in N-cadherin expression levels. It was determined that FAK, Ezrin and Fascin 1 cargo passage were significantly retained in the cytoplasm in the FAK RNAi groups. Conclusions:With the results we obtained; It has been shown that the FAK molecule has an important role in the germ cell development process in vitro. It has been shown that in Sertoli-germ cell co-culture in which FAK gene is silenced and FAK RNAi and EF applied together, vesicle contents cannot be released by endocytosis and these molecules affect nanotubes formation due to decreasing the ratios of FAK, ezrin and Fascin 1 proteins. Based on our results, a research pattern and culture model were proposed for the detection of intercellular signaling due to the passage of regulatory proteins and nanotubes formation.