Regulation of Microcystin-LR-Induced Toxicity in Mouse Spermatogonia by miR-96

Abstract

Microcystin (MC)-LR is a cyclic heptapeptide that acts as a potent reproductive system toxin, especially by decreasing sperm quality through affecting spermatogonia. However, the molecular mechanisms of MC-induced spermatogonial cytotoxicity still remain unclear. The present study was designed to investigate changes in microRNA (miRNA) profiles and their potential functions in spermatogonia (GC-1 cell line) following treatment with MC-LR. With microarray analysis, 101 miRNAs were identified to be significantly altered in GC-1 cells treated with MC-LR. Among the 25 miRNAs associated with spermatogenesis, miR-96 was down-regulated most dramatically and thus selected for further functional analysis. Deleted-in azoospermia-associated protein 2 (DAZAP2) was predicted to have a binding sequence for miR-96 within its 3'-untranslated region. Fluorescent reporter assay confirmed that DAZAP2 was the target gene of miR-96. The expression of DAZAP2 decreased significantly when miR-96 was up-regulated. Consistently, down-regulation of miR-96 significantly increased the level of DAZAP2. Up-regulation of miR-96 promoted cell viability in GC-1 cells as a result of exposure to MC-LR. Our study suggested a crucial role for miR-96 in the regulation of cytotoxic effects of MC-LR in spermatogonia, which provides new perspectives in the diagnosis and treatment strategies for MC-induced male infertility.

Roles of miRNAs in microcystin-LR-induced Sertoli cell toxicity

“…Total protein was isolated from cells following various treatments. Western blot was applied according to a previous study (Zhou et al, 2014). Briefly, cells were washed three times with PBS and lysed in ice-cold extraction buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP, 0.1% SDS, and a 1× protease inhibitor cocktail).…”

A review of reproductive toxicity of microcystins

“…The expression of 5 multispecific organic anion-transporting polypeptides (OATPs, -1a5, -3a1, -6b1, -6c1, and -6d1) was affected by MC-LR [68], especially OATP3a1, and Oatps may transport MC-LR into spermatogonia, resulting in cytotoxicity to the male reproductive system. With microarray analysis [69], 101 miRNAs were identified to be significantly altered in spermatogonia (GC-1 cells) treated with MC-LR, revealing an important link between miRNA and MC-LR in inducing reproductive toxicity. Inhibition of miR-96 may be one of the mechanisms for MC-induced sperm abnormality through regulating the expression of deleted-in azoospermia-associated protein 2 (DAZAP2).…”

“…These studies revealed that numerous genes and metabolic pathways involved in cytoskeleton assembly, oxidative stress, cell cycle regulation, and energetic metabolism were differentially regulated. Whilst most of these studies focused on the hepatotoxicity of MCs, only a few proteomic and microarray studies investigated the male reproductive toxicity in zebrafish testis [32], mouse spermatogonia [69] and Sertoli cells [74], respectively. Future work could better clarify the reproductive toxicological mechanism involved in MCs by the powerful 'omics' approaches.…”

Novel Role of ER Stress and Autophagy in Microcystin-LR Induced Apoptosis in Chinese Hamster Ovary Cells

“…Many studies have demonstrated the lethality of MC-LR on various cells including the human liver 7702 cells, rat testicular Sertoli cells and mouse spermatogonia (Chen et al, 2013; Sun et al, 2014; Zhou et al, 2014). Here, we found that exposure to 1~30 μM of MC-LR brought about inhibition of CHO cells growth.…”