Effect of Nitric Oxide on NGF-Receptor of seminiferous Epithelial Cells

我们研究了一氧化氮（NO）对神经营养因子受体的影响本研究通过对大鼠睾丸生精上皮细胞（Sertoli）的神经生长因子受体（NGF-R）和酪氨酸激酶（TRKs）的研究，探讨了NO在睾丸内生殖细胞与上皮细胞间通讯中的作用。神经营养因子受体的基因产物如NGF-R（p75）和Trks（A和B）定位于睾丸支持细胞。NGF-R，TrkA和TrkB在培养的Sertoli细胞中的定位显示，受体随机分布在整个细胞表面。弥散免疫荧光染色代表质膜受体的特征性免疫染色，这些受体未预先聚集在被膜小凹中。此外，某些Sertoli细胞也显示这些受体的免疫荧光染色定位于细胞的聚集区域。 关于我们 用RT-PCR方法检测支持细胞总RNA中神经营养因子受体mRNA的表达;用Western blot方法检测支持细胞提取物中神经营养因子受体（NGF-R）、TrkA和TrkB的免疫反应条带。用NO供体NOC 18（0.4mM）处理Sertoli细胞24 h后，其NGF-R、TrkA和TrkB的蛋白和mRNA表达均明显下降。用NO供体处理也导致细胞核上区NGF-R、TrkA和TrkB的下降。这表明，包括受体在内的包被小凹的内吞作用被添加到培养物中的支持细胞的外源性NO消除。另一方面，用神经营养因子如BDNF（100 ng/ml）和NGF（100 ng/ml）培养的Sertoli细胞显示Trk（146 kDa）的上调，Trk-pan抗体检测到，并且神经营养因子和NO供体的联合处理消除了由NO供体处理引起的Trk（146 kDa）的降低。这些结果表明，睾丸支持细胞表达神经营养因子受体NGF-R、TrkA和TrkB，可能通过旁分泌调节机制调节生殖细胞的增殖和分化。此外，在急性或慢性病理生理条件下，睾丸内过量产生的NO干扰神经营养因子受体的表达和定位，从而干扰精子发生的信号转导，而这些受体的配体如BDNF和NGF可能从过量NO诱导的抑制中拯救受体，维持正常的精子发生调节。少

We investigated the effect of nitric oxide (NO) on neurotrophic factor receptors (NGF-R and TRKs) of rat seminiferous epithelial (Sertoli) cells to elucidate some aspects of the role of NO in intratesticular communication between germ cells and epithelial cells.Immunoreactivity with a specific antibody against NGF gene product localized to germ cells (pachytene spermatocytes and round spermatids) within testis, while gene products of neurotrophic factor receptors such as NGF-R (p75) and Trks (A and B) localized to Sertoli cells. Localization of the NGF-R, TrkA and TrkB in cultured Sertoli cells revealed that the receptors were distributed randomly throughout the cell surface. The diffused immunofluorescence staining represented characteristic immunostaining of plasma membrane receptors that were not pre-clustered in coated pits. In addition, a certain Sertoli cells exhibited that the immunofluorescence staining of these receptors also localized to be in clustered areas of the cells.Exp … More ression of mRNA of these neurotrophic factor receptors was detected by RT-PCR in total RNA samples obtained from the cultured Sertoli cells, and immunoreactive bands of NGF-R (76kDa), TrkA (140kDa) and TrkB (145kDa) were identified in the extracts of cultured Sertoli cells by the Western blot analyses. Expressions of both proteins and mRNAs of the NGF-R, TrkA and TrkB were apparently decreased in the Sertoli cells treated with NOC18 (an NO donor, 0.4mM) for 24h. Treatment with NO donor also resulted in the decline of the NGF-R, TrkA and TrkB at the supranuclear areas of the cells. This indicates that endocytosis of coated pits including the receptors was eliminated by exogenous NO added to the Sertoli cells in the culture. On the other hand, the culture of Sertoli cells with neurotrophic factors such as BDNF (100ng/ml) and NGF (100ng/ml) revealed the up-regulation of Trk (146kDa) detected the Trk-pan antibody, and the combination-treatment of neurotrophic factor and NO donor eliminated the decrease of Trk (146kDa) induced by the NO donor treatment. These results suggest that Sertoli cells express the neurotrophic factor receptors such as NGF-R, TrkA and TrkB and may regulate the proliferation and differentiation of germ cells through the paracrine regulation mechanism within testis. Furthermore, NO produced excessively within testis during acute or chronic pathophysiological conditions perturbs the expression and localization of neurotrophic factor receptors and in turn the signal transduction for spermatogenesis, whereas the ligands of these receptors such as BDNF and NGF might rescue the receptors from the repression induced by the excessive NO and maintain the regulation of normal spermatogenesis. Less