THE DOUBLE-STRAND BREAK PROTEINS NBS1, KU70, KU80, RAD51 AND BRCA1 EXHIBIT SIGNIFICANT ALTERATIONS IN NON-OBSTRUCTIVE AZOOSPERMIC INFERTILE MEN HAVING PRIMARY SPERMATOCYTE AND ROUND SPERMATID ARREST

Abstract

Objective

Azoospermia, characterized by the absence of sperm in ejaculate, affects about 1% of general population. Non-obstructive azoospermia (NOA), which constitutes 60% of azoospermic infertility cases, remains poorly understood at the molecular level. DNA double-strand breaks (DSBs) being formed for different reasons such as DNA replication, DNA lesions, crossing-over and genotoxic agents are repaired timely and correctly to maintain genome integrity. In response to sensing DSBs, the histone variant H2AX is phosphorylated at serine 139 by ATM kinase to form γH2AX. Thus, the main DSB repair pathways such as homologous recombination (HR) and canonical non-homologous end joining (cNHEJ) are activated. While the KU70 and KU80 proteins play key roles in cNHEJ repair, the NBS1, RAD51, and BRCA1 proteins contribute HR-based repair. Importantly, absence or expressional changes in these proteins is found to be associated with male infertility development. Our study aims to elucidate the role of γH2AX, KU70, KU80, NBS1, RAD51, and BRCA1 proteins in losing fertility in NOA patients.

Materials and Methods

After taking ethical committee approval (protocol no. 13.05.2020/KAEK-345), we utilized previously collected testicular sperm extraction (TESE) samples in this study. We categorized the TESE samples according their histopathological status into four groups: hypospermatogenesis (n=6), primary spermatocyte arrest (n=6), round spermatid arrest (n=6), and Sertoli cell-only syndrome (n=6). Additionally, a control group having normal spermatogenesis (n=6) was created. With immunohistochemistry, we stained all the aforementioned proteins and analyzed their expression patterns with the ImageJ software. The resulting data were then evaluated using the non-parametric Mann-Whitney U test.

Results

The relative levels of γH2AX were significantly higher in primary spermatocyte and round spermatid arrest groups when compared to the control group (P<0.01). The NBS1 protein was mainly localized in the nuclei of the spermatogenic cells, and its levels significantly reduced in patients with primary spermatocyte and round spermatid arrest groups (P<0.05). Additionally, significant reductions in the KU70 (P<0.01), KU80 (P<0.01) and RAD51 (P<0.05) proteins were observed in the total and seminiferous tubules of the primary spermatocyte and round spermatid arrest groups when compared with the control group. In contrast, BRCA1 protein levels significantly increased in the hypospermatogenesis, primary spermatocyte arrest and round spermatid arrest groups compared to the control group (P<0.01).

Conclusions

The significant reductions in NBS1, KU70, KU80, and RAD51 proteins, along with increasing BRCA1 levels may be associated with developing spermatogenic arrest in NOA patients. Further studies are required to elucidate the mechanistic effects of changed expression of these proteins in cNHEJ and HR repairs in primary spermatocytes and round spermatids.