Han I S, Sylvester S R, Kim K H, Schelling M E, Venkateswaran S, Blanckaert V D, McGuinness M P, Griswold M D
Department of Biochemistry and Biophysics, Washington State University, Pullman 99164.
Mol Endocrinol. 1993 Jul;7(7):889-97. doi: 10.1210/mend.7.7.8413313.
Previously, a proteinacious factor secreted by a mixture of rat testicular spermatocytes and round spermatids was shown to stimulate transferrin mRNA and protein levels in Sertoli cells. To identify the germ cell-secreted proteins which affect Sertoli cell functions, concentrated germ cell-conditioned medium was fractionated by reverse-phase HPLC. The fraction which eluted at 35% acetonitrile increased transferrin secretion in Sertoli cell cultures 2.4-fold above the basal level. Both the active fraction and a protein extract from cultured germ cells were positive for basic fibroblast growth factor (bFGF) as determined by Western blot analysis and immunoprecipitation. The apparent molecular sizes of the immunoreactive proteins were 30, 27, and 24 kilodaltons (kDa). By immunohistochemistry, bFGF was shown to be present in pachytene spermatocytes and Leydig cells. The bFGF receptor was also examined by immunohistochemistry and found to be present in Leydig cells, round and elongated spermatids, and Sertoli cells. The presence of receptors was more pronounced in stages I-VIII. Western blot analysis confirmed that the receptors were expressed in isolated round spermatids, elongated spermatids, and Sertoli cells. Two major receptor species with apparent molecular sizes of 120 and 145 kDa were detected in the rat testis. Germ cells contained both of these receptors, but Sertoli cells possessed only the 120-kDa receptor. From these experiments, it is evident that bFGF is a germ cell product which may regulate Sertoli cell function. The expression of bFGF and its receptor may be an important component of germ cell-Sertoli cell and/or germ cell-germ cell communication during spermatogenesis.
先前的研究表明，大鼠睾丸精母细胞和圆形精子细胞混合物分泌的一种蛋白质因子可刺激支持细胞中转铁蛋白mRNA和蛋白质水平。为了鉴定影响支持细胞功能的生殖细胞分泌蛋白，通过反相高效液相色谱法对浓缩的生殖细胞条件培养基进行了分级分离。在35%乙腈处洗脱的级分使支持细胞培养物中的转铁蛋白分泌量比基础水平增加了2.4倍。通过蛋白质印迹分析和免疫沉淀法测定，活性级分和培养的生殖细胞蛋白提取物中的碱性成纤维细胞生长因子（bFGF）均呈阳性。免疫反应性蛋白的表观分子大小分别为30、27和24千道尔顿（kDa）。通过免疫组织化学方法显示，bFGF存在于粗线期精母细胞和睾丸间质细胞中。还通过免疫组织化学方法检测了bFGF受体，发现其存在于睾丸间质细胞、圆形和长形精子细胞以及支持细胞中。在I-VIII期，受体的存在更为明显。蛋白质印迹分析证实，这些受体在分离的圆形精子细胞、长形精子细胞和支持细胞中均有表达。在大鼠睾丸中检测到两种主要的受体类型，表观分子大小分别为120和145 kDa。生殖细胞同时含有这两种受体，但支持细胞仅拥有120 kDa的受体。从这些实验可以明显看出，bFGF是一种生殖细胞产物，可能调节支持细胞的功能。bFGF及其受体的表达可能是精子发生过程中生殖细胞-支持细胞和/或生殖细胞-生殖细胞通讯的重要组成部分。
