scRNA-seq reveals that origin recognition complex subunit 6 regulates mouse spermatogonial cell proliferation and apoptosis via activation of Wnt/ß-catenin signaling.

ABSTRACT: The regulation of spermatogonial proliferation and apoptosis is of great significance for maintaining spermatogenesis. The single-cell RNA sequencing (scRNA-seq) analysis of the testis was performed to identify genes upregulated in spermatogonia. Using scRNA-seq analysis, we identified the spermatogonia upregulated gene origin recognition complex subunit 6 (Orc6), which is involved in DNA replication and cell cycle regulation; its protein expression in the human and mouse testis was detected by western blot and immunofluorescence. To explore the potential function of Orc6 in spermatogonia, the C18-4 cell line was transfected with control or Orc6 siRNA. Subsequently, 5-ethynyl-2-deoxyuridine (EdU) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, flow cytometry, and western blot were used to evaluate its effects on proliferation and apoptosis. It was revealed that ORC6 could promote proliferation and inhibit apoptosis of C18-4 cells. Bulk RNA sequencing and bioinformatics analysis indicated that Orc6 was involved in the activation of wingless/integrated (Wnt)/ ß-catenin signaling. Western blot revealed that the expression of ß-catenin protein and its phosphorylation (Ser675) were significantly decreased when silencing the expression of ORC6. Our findings indicated that Orc6 was upregulated in spermatogonia, whereby it regulated proliferation and apoptosis by activating Wnt/ß-catenin signaling.

Seminal plasma miR-210-3p induces spermatogenic cell apoptosis by activating caspase-3 in patients with varicocele.

The aim of this study was to investigate the role of seminal plasma miR-210-3p in the impairment of semen quality caused by varicocele. This study included 102 patients whose semen quality was normal when they were diagnosed with varicocele. A 2-year follow-up for included patients was performed, and they were divided into Group A (semen quality became abnormal) and Group B (semen quality remained normal) according to the results of semen analysis during the follow-up. Semen parameters and seminal plasma miR-210-3p expression were investigated by semen analysis and quantitative real-time polymerase chain reaction, respectively. In vitro experiments with GC-2 cells were performed to explore the role of miR-210-3p in spermatogenic cells. The results of quantitative real-time polymerase chain reaction showed that the level of seminal plasma miR-210-3p in Group A was higher than that in Group B both after 2-year follow-up and when they were diagnosed with varicocele (both P < 0.01). Apoptosis and proliferation assays showed that miR-210-3p induces apoptosis of spermatogenic cells by promoting caspase-3 activation. In conclusion, our study indicated that seminal plasma miR-210-3p induces spermatogenic cell apoptosis by activating caspase-3 in patients with varicocele. Seminal plasma miR-210-3p may be a potential biomarker for predicting impaired semen quality caused by varicocele.