Kavarthapu Raghuveer, Dufau Maria L
Section on Molecular Endocrinology, Program on Developmental Endocrinology and Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Mol Endocrinol. 2015 Dec;29(12):1792-804. doi: 10.1210/me.2015-1198. Epub 2015 Oct 20.
Gonadotropin-regulated testicular RNA helicase (GRTH) (GRTH/DDX25), is a testis-specific protein essential for completion of spermatogenesis. Transgenic mice carrying 5'-flanking regions of the GRTH gene/green fluorescence protein (GFP) reporter revealed a region (-6.4/-3.6 kb) which directs its expression in germ cells (GCs) via androgen action. This study identifies a functional cis-binding element on the GRTH gene for GC nuclear factor (GCNF) (GCNF/RTR) required to regulate GRTH gene expression in postmeiotic testis GCs and explore the action of androgen on GCNF and GRTH transcription/expression. GCNF expression decreased in mice testis upon flutamide (androgen receptor antagonist) treatment, indicating the presence of an androgen/GCNF network to direct GRTH expression in GC. Binding studies and chromatin immunoprecipitation demonstrated specific association of GCNF to a consensus half-site (-5270/-5252) of the GRTH gene in both round spermatids and spermatocytes, which was abolished by flutamide treatment in round spermatids. Moreover, flutamide treatment of wild-type mice caused selective reduction of GCNF and GRTH in round spermatids. GCNF knock-down in seminiferous tubules from GRTH-transgenic mice (dark zone, round spermatid rich) caused decreased GFP expression. Exposure of tubules to flutamide caused decrease in GCNF and GFP expression, whereas androgen exposure induced significant increase. Our studies provide evidence for actions of androgen on GCNF cell-specific regulation of GRTH expression in GC. GRTH associates with GCNF mRNA, its absence caused increase on GCNF expression and mRNA stability indicative of a negative autocrine regulation of GCNF by GRTH. These in vivo/in vitro models link androgen actions to GC through GCNF, as regulated transfactor that controls transcription/expression of GRTH.
促性腺激素调节的睾丸RNA解旋酶（GRTH）（GRTH/DDX25）是一种睾丸特异性蛋白，对精子发生的完成至关重要。携带GRTH基因5'-侧翼区域/绿色荧光蛋白（GFP）报告基因的转基因小鼠显示出一个区域（-6.4/-3.6 kb），该区域通过雄激素作用指导其在生殖细胞（GCs）中的表达。本研究确定了GRTH基因上一个功能性顺式结合元件，该元件是减数分裂后睾丸生殖细胞中调节GRTH基因表达所需的生殖细胞核因子（GCNF）（GCNF/RTR），并探讨了雄激素对GCNF和GRTH转录/表达的作用。氟他胺（雄激素受体拮抗剂）处理后，小鼠睾丸中GCNF表达降低，表明存在雄激素/GCNF网络以指导生殖细胞中GRTH的表达。结合研究和染色质免疫沉淀表明，GCNF在圆形精子细胞和精母细胞中均与GRTH基因的一个共有半位点（-5270/-5252）特异性结合，在圆形精子细胞中氟他胺处理可消除这种结合。此外，氟他胺处理野生型小鼠导致圆形精子细胞中GCNF和GRTH选择性减少。GRTH转基因小鼠（富含圆形精子细胞的暗区）的生精小管中GCNF敲低导致GFP表达降低。生精小管暴露于氟他胺会导致GCNF和GFP表达降低，而雄激素暴露则诱导显著增加。我们的研究为雄激素对生殖细胞中GRTH表达的GCNF细胞特异性调节作用提供了证据。GRTH与GCNF mRNA相关，其缺失导致GCNF表达和mRNA稳定性增加，表明GRTH对GCNF存在负自分泌调节。这些体内/体外模型通过GCNF将雄激素作用与生殖细胞联系起来，GCNF作为调节转录因子控制GRTH的转录/表达。
