Elucidating the mechanisms and mitigation strategies for six-phthalate-induced toxicity in male germ cells.

Phthalate esters (PAEs) are primary plasticizers and endocrine-disrupting chemicals (EDCs) that are extensively used in numerous everyday consumer products. Although the adverse effects of single PAEs have been studied, our understanding of the effect of multiple phthalate exposure on male germ cell vitality remains limited. Therefore, this study aimed to investigate the collective effects of a mixture of PAEs (MP) comprising diethyl-, bis (2-ethylhexyl)-, dibutyl-, diisononyl-, diisobutyl-, and benzyl butyl-phthalates in the proportions of 35, 21, 15, 15, 8, and 5%, respectively, on differentiated male germ cells using GC-1 spermatogonia (spg) cells. As a mixture, MP substantially hindered GC-1 spg cell proliferation at 3.13 µg/mL, with a half-maximal inhibitory concentration of 16.9 µg/mL. Treatment with 25 µg/mL MP significantly induced reactive oxygen species generation and promoted apoptosis. Furthermore, MP activated autophagy and suppressed phosphorylation of phosphoinositide 3-kinase, protein kinase B, and mammalian target of rapamycin (mTOR). The triple inhibitor combination treatment comprising parthenolide, N-acetylcysteine, and 3-methyladenine effectively reversed MP-induced GC-1 spg cell proliferation inhibition, mitigated apoptosis and autophagy, and restored mTOR phosphorylation. This study is the first to elucidate the mechanism underlying MP-induced male germ cell toxicity and the restoration of male germ cell proliferation mediated by chemical inhibitors. Therefore, it provides valuable insights into the existing literature by proposing a combinatorial toxicity mitigation strategy to counteract male germ cell toxicity induced by various EDCs exposure.

Restoration of PM2.5-induced spermatogonia GC-1 cellular damage by parthenolide via suppression of autophagy and inflammation: An in vitro study.

Particulate matter (PM) generated by environmental and air pollution is known to have detrimental effects on human health. Among these, PM2.5 particles (diameter < 2.5 µm) can breach the alveolar-capillary barrier and disseminate to other organs, posing significant health risks. Numerous studies have shown that PMs can harm various organs, including the reproductive system. Therefore, this study aimed to investigate the harmful effects of PM2.5 on mouse GC-1 spermatogonia cells (GC-1 spg cells) and to verify the ameliorative effects of parthenolide (PTL) treatment on damaged GC-1 spg cells. We observed a significant dose-dependent reduction in cell proliferation after PM2.5 concentration of 2.5 µg/cm2. Additionally, treatment with 20 µg/cm2 PM2.5 concentration significantly increased the expression of autophagy-related proteins ATG7, the ratio of LC3-II/LC3-I, and decreased phosphorylation of PI3K and AKT. Furthermore, PM2.5 exposure augmented inflammation mediator gene expressions, the phosphorylation of the inflammation-related transcription factor NF-κB p65 at Ser536, and ubiquitination. Treatment of PM2.5-exposed GC-1 spg cells with PTL significantly reduced NF-κB p65 phosphorylation and the expression of autophagy-related proteins ATG7 and LC3-II, leading to a statistically significant recovery in cell proliferation. Together, our findings elucidated the detrimental effects of PM2.5 exposure on male germ cells, and the restorative properties of PTL against air pollutants.

The inflammatory mediators TNFα and nitric oxide arrest spermatogonia GC-1 cell cycle.

During an inflammatory process of the testis, the network of somatic, immune, and germ cell interactions is altered leading to organ dysfunction. In testicular biopsies of infertile men, spermatogenesis impairment is associated with reduced spermatogonia proliferation, increased number of immune cells, and content of pro-inflammatory cytokines. TNFα-TNFR and nitric oxide (NO)-NO synthase systems are up-regulated in models of testicular damage and in human testis with maturation arrest. The purpose of this study was to test the hypothesis that TNFα-TNFR system and NO alter the function of spermatogonia in the inflamed testis. We studied the effect of TNFα and NO on GC-1 spermatogonia cell cycle progression and death by flow cytometry. GC-1 cells expressed TNFR1 and TNFR2 (immunofluorescence). TNFα (10 and 50 ng/ml) and DETA-Nonoate (0.5 and 2 mM), a NO releaser, increased the percentage of cells in S-phase of the cell cycle and reduced the percentage in G1, inducing also cell apoptosis. TNFα effect was not mediated by oxidative stress unlike NO, since the presence of N-acetyl-l-cysteine (2.5 and 5.0 mM) prevented NO induced cell cycle arrest and death. GC-1 spermatogonia overpass NO induced cell cycle arrest but no TNFα, since after removal of NO, spermatogonia progressed through the cell cycle. We propose TNFα and NO might contribute to impairment of spermatogenesis by preventing adequate functioning of the spermatogonia population. Our results showed that TNFα and NO impaired spermatogonia cell cycle, inducing GC-1 arrest in the S phase.