大鼠睾丸内皮型一氧化氮合酶表达的增龄变化

Expression of eNOS in rat testis from infancy to maturity

为研究雄性大鼠睾丸在发生、发育和衰老过程中内皮型一氧化氮合酶 (eNOS)在生精功能中的作用及其变化规律 ,本实验采用了免疫组织化学染色及体视学图像分析等方法 ,对生后 1d至生后 2 4月龄大鼠睾丸eNOS表达的变化进行了系统研究 ,并统计测量了阳性血管内皮细胞及间质细胞面密度的变化。结果表明 :生后1d至 2周龄eNOS阳性表达极少 ;3周龄血管内皮细胞、间质细胞及精子细胞均出现了阳性表达 ;1月龄至 18月龄生精小管靠近管腔的精子细胞也呈阳性 ,阳性血管内皮细胞、间质细胞数目差异显著 ;2 4月龄血管内皮细胞、间质细胞eNOS大量表达 ,部分生精细胞也有表达。本结果提示一氧化氮参与精子的生成及睾酮的分泌过程 ,衰老时eNOS阳性表达显著增加 ,这种变化可能会抑制睾酮的分泌 ,最终会影响睾丸的生精功能.

In order to investigate change of endothelium nitric oxide synthase (eNOS) in rat spermatogenesis during development, immunohistochemistry and morphometric analyses were used to examine the testes of postnatal day 1 to postnatal 24 months old rats. Testicular tissue was removed from decapitated male Sprague-Dawley rats in 10 age groups: postnatal 1 day, postnatal 1 week, postnatal 2 weeks, postnatal 3 weeks, postnatal 1 month, postnatal 2 months, postnatal 3 months, postnatal 12 months, postnatal 18 months and postnatal 24 months, and then placed immediately in a fixative consisting of 4% paraformaldehyde and 0.1 M phosphate buffer (PB, pH 7.4). Fixed testes were rinsed for at least 12 h at 4℃ in 0.1 M PB (pH 7.2) containing 30% sucrose, and then cryostat serial sections were cut at 4 μm thickness and mounted onto glass slides covered with 1% gelatinum. Rabbit anti-rat eNOS antibody and SP kit were purchased from the Beijing Zhongshan Biotechnical Company (product of ZYMED in America). Immunohistochemical staining for eNOS was performed using the SP technique with immunohistochemistry. The eNOS content of postive blood vessel endothelium as well as Leydig cells were measured with Beihang analytic software. The results indicate that the cytoplasm of postive cells appeared homogeneously yellow and the nuclear counterstaining of hematoxylin were iodes. Specimens from postnatal day 1 to postnatal 2 weeks showed little immunoreactivity for eNOS. In contrast, blood vessel endothelium and Leydig cells from postnatal 3 weeks testes were strongly expressed eNOS immunoreactivity, while spermatids were immunostained appearing “cresccentiform”. Postnatal 1st month testes had strong positive substances in the blood vessel endothelium and Leydig cells, spermatids were heterogeneously stained appearing “cresccentiform” and “long-bar-shaped”. Immunoreactivity was found in blood vessel endothelium, Leydig cells and spermatids in postnatal 2nd month testes and the number of positive cells conspicuously increased. In postnatal 3rd month testes, the number of postive cells persistently increased. In postnatal 12th month testes the number of postive Leydig cells persistently increased, whereas the number of blood vessels with positive endothelium decreased slightly; In postnatal 18th month testes only the number of postive Leydig cells decreased slightly; blood vessel endothelium and Leydig cells usually expressed eNOS in 24th month testes, and some spermatocytes also displayed strong eNOS immunoreactivity. In this age group, the testes were old so few spermatids showed immunoreactivity. Sertoli cells, spermatogenous cells and myoid cells in each group did not stain for eNOS. The above results suggests that: eNOS positive cells are distributed in the blood vessel endothelium, Leydig cells and spermatids; positive cells were found in spermatids during different stages of spermatogenesis, which suggests that NO might accelerate the process of spermatogenesis. The number of blood vessels with positive endothelium was increased sharply, which might relate to testicular aging and the reduction of spermatogenesis. NO possibly plays a role in the relaxation of seminiferous tubules and blood vessels, to modulate sperm transport and testicular blood flow respectively. The regulation of NO in blood vessels requires further study. The number of positive Leydig cells were increased with aging, and Leydig cells in 24-month old testes appeared strongly positive. As a result, NO produced by Leydig cells might directly influence the secretion of testosterone. NO produced by Leydig cells may act as a messenger to mediate the action of numerous intracellular and extracellular neuroactive substances and growth factors, to regulate local blood flow and permeability, and to influence the contraction of peritubular myoblasts and the permeability of the lamina propria. We first observed spermatids that appeared to have positive reactivity to eNOS which were stained as “crescentiform” in the testes of post 3 week old rats. eNOS was strongly expr