Nishie Tomomi, Komaru Aika, Shiroguchi Syota, Yamaizumi Takako, Ono Yuriko, Motomochi Atsuko, Tooyama Ikuo, Fujioka Yasuhiro, Sakai Noriyoshi, Higaki Shogo, Takada Tatsuyuki
Laboratory of Cell Engineering, Department of Pharmaceutical Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan.
Molecular Neuroscience Research Center and Medical Innovation Research Center, Shiga University of Medical Science, Shiga 520-2192, Japan.
Toxicol In Vitro. 2023 Jun;89:105565. doi: 10.1016/j.tiv.2023.105565. Epub 2023 Feb 4.
Nonylphenol (NP), an endocrine disrupting chemical, is widely used in industrial and agricultural processes, causing NP influx into aquatic environments. NP induces hormonal imbalance, and male feminization, and reduces germ cell production during spermatogenesis; however, the mechanism by which it affects spermatogenesis remains unknown. Here, we investigated the effect of NP on spermatogenesis in honmoroko (Gnathopogon caerulescens), an endangered fish endemic to Lake Biwa, Japan, using an in vitro differentiation system. We collected spermatogonia from the testes of non-spawning G. caerulescens and subjected them to suspension culture. The spermatogonia differentiated into flagellated spermatozoa in 3 weeks, regardless of the presence of NP. NP concentrations as low as 1 nM caused a decrease in the number of germ cells in a dose-dependent manner, whereas the number of somatic cells decreased only at a high concentration of 1 μM. Flow cytometric analysis revealed that the decrease in germ cell number was attributed to haploids (spermatids and spermatozoa); the number of spermatogonia and spermatocytes was not affected by NP treatment. This result is consistent with the hypothesis that NP might repress the second meiosis or induce apoptosis in haploids. This study demonstrated that the combination of in vitro germ cell differentiation and flow cytometric analysis is useful for evaluating the direct effects of NP on germ cell differentiation in endangered endemic fish.
壬基酚（NP）是一种内分泌干扰化学物质，广泛应用于工农业生产过程中，导致NP流入水生环境。NP会导致激素失衡和雄性雌性化，并减少精子发生过程中的生殖细胞产生；然而，其影响精子发生的机制尚不清楚。在此，我们使用体外分化系统研究了NP对日本琵琶湖特有的濒危鱼类日本颌须鮈（Gnathopogon caerulescens）精子发生的影响。我们从未产卵的日本颌须鮈的睾丸中收集精原细胞，并对其进行悬浮培养。无论是否存在NP，精原细胞在3周内都会分化为有鞭毛的精子。低至1 nM的NP浓度会导致生殖细胞数量呈剂量依赖性减少，而体细胞数量仅在1 μM的高浓度下才会减少。流式细胞术分析表明，生殖细胞数量的减少归因于单倍体（精子细胞和精子）；精原细胞和精母细胞的数量不受NP处理的影响。这一结果与NP可能抑制第二次减数分裂或诱导单倍体细胞凋亡的假设一致。本研究表明，体外生殖细胞分化和流式细胞术分析相结合，有助于评估NP对濒危特有鱼类生殖细胞分化的直接影响。
