This study aimed to investigate the role of microRNA (miRNA) in heat stress-induced spermatogenic impairment. Testes from 15 adult ICR mice subjected to testicular hyperthermia at 43°C for 30 min and from 15 control mice were collected and pooled into 3 samples. Isolated RNA from these samples was subjected to small RNA high-throughput sequencing, and differentially expressed miRNAs were identified and validated using RT-PCR. The identified miRNAs were further subjected to Gene Ontology and KEGG analyses, which revealed significant enrichment for pathways potentially involved in heat stress-induced spermatogenic impairment. Additionally, a correlation analysis of the relative levels of validated miRNAs with germ cell apoptosis was performed. Of the 11 miRNAs identified as differentially expressed, 8 were validated as consistent with sequencing data. Further analyses suggested that the target genes of those miRNAs were involved in various pathways (e.g., ribosomal, HIF-1, MAPK) that may be critical to heat stress-induced testicular damage. Some identified miRNAs, including miR-449a-3p, miR-92a-1-5p, miR-423-3p, and miR-128-3p, correlated closely with germ cell apoptosis. The study results reveal a detailed miRNA profile of heat stress-induced testicular damage and highlight new and potentially important candidate targets in the process of male infertility.
Cold-inducible RNA-binding protein (CIRBP) is reduced by scrotal hyperthermia in cryptorchidism, varicocoele and heat treatment, but there is no direct evidence clarifying the relationship between CIRBP and spermatogenesis. The aim of this study was to investigate the expression of CIRBP in GC2-spd cells (a mouse spermatocyte cell line) before and after heat treatment, and to determine the effects of the downregulation or overexpression of CIRBP on spermatocyte cell proliferation and apoptosis. GC2-spd cells overexpressing CIRBP and GC2-spd cells in CIRBP was knocked down were constructed to investigate the function of CIRBP in cell proliferation and apoptosis using a cell counting kit-8 and flow cytometry respectively. In addition, proliferation and apoptosis were evaluated in GC2-spd cells that had been heated for 30 or 60min, and were analysed 12, 24, and 48h after heat treatment. Heat treatment clearly suppressed the proliferation of GC2-spd cells, and upregulation of CIRBP expression in GC2-spd cells promoted cell proliferation and decreased apoptosis before and after heat stress; in contrast, downregulation of CIRBP expression inhibited cell proliferation and increased apoptosis. These findings suggest that CIRBP exerts a protective effect against spermatogenic injury caused by heat stress.
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