Gilmont R R, Coulter G H, Sylvester S R, Griswold M D
Genetics and Cell Biology Program, Washington State University, Pullman 99164.
Biol Reprod. 1990 Jul;43(1):139-50. doi: 10.1095/biolreprod43.1.139.
Techniques were developed for generating enriched cultures of bovine Sertoli cells and indifferent supporting cells (immature Sertoli cells). The [35S]methionine and [35S]sulfate-labeled proteins secreted by cultured cells were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. The electrophoretic pattern of the major Sertoli cell-secreted proteins was distinct from that of the major proteins secreted by cultured peritubular cells (the predominant contaminating cell type). Five major polypeptides ranging in molecular mass from 22 kDa to 77 kDa were resolved by 2D-PAGE in reducing conditions and were assigned numbers for reference purposes. Polypeptides 1 and 2 appeared to be analogous to two rat Sertoli cell-secreted proteins, sulfated glycoprotein-1 and sulfated glycoprotein-2, because of similar molecular mass, isoelectric point, subunit composition, sulfation, and sialation characteristics. Transferrin was detected in conditioned medium by immunoprecipitation using an antibody to bovine serum transferrin. Cultured Sertoli cells isolated from prepubertal bulls secreted higher levels of transferrin than did cells isolated from infant bulls. An 850 bp cDNA corresponding to the 3' portion of bovine transferrin mRNA was cloned and sequenced. Transferrin message was shown to be present in testicular tissue isolated from infant and prepubertal bulls and it increased as bulls matured. Levels of testicular transferrin mRNA were subsequently shown to correlate with daily sperm production in yearling beef bulls.
已开发出用于培养牛支持细胞和未分化支持细胞（未成熟支持细胞）富集培养物的技术。通过二维聚丙烯酰胺凝胶电泳（2D-PAGE）和荧光自显影分析培养细胞分泌的[35S]甲硫氨酸和[35S]硫酸盐标记的蛋白质。支持细胞分泌的主要蛋白质的电泳图谱与培养的睾丸周细胞（主要的污染细胞类型）分泌的主要蛋白质的电泳图谱不同。在还原条件下，通过2D-PAGE分离出5种分子量从22 kDa到77 kDa的主要多肽，并为便于参考为其编号。由于分子量、等电点、亚基组成、硫酸化和唾液酸化特征相似，多肽1和2似乎类似于两种大鼠支持细胞分泌的蛋白质，硫酸化糖蛋白-1和硫酸化糖蛋白-2。使用抗牛血清转铁蛋白抗体通过免疫沉淀在条件培养基中检测到转铁蛋白。从青春期前公牛分离的培养支持细胞比从幼年公牛分离的细胞分泌更高水平的转铁蛋白。克隆并测序了与牛转铁蛋白mRNA 3'部分相对应的850 bp cDNA。已证明转铁蛋白信息存在于从幼年和青春期前公牛分离的睾丸组织中，并且随着公牛成熟而增加。随后表明，一岁肉牛睾丸中转铁蛋白mRNA的水平与每日精子产量相关。
