Tsuruta J K, O'Brien D A, Griswold M D
Laboratories for Reproductive Biology, University of North Carolina at Chapel Hill 27599-7500.
Biol Reprod. 1993 Nov;49(5):1045-54. doi: 10.1095/biolreprod49.5.1045.
Sertoli cells were shown to synthesize and secrete cystatin C, a potent inhibitor of cysteine proteases. The evidence for this observation was obtained from protein sequencing. Western analysis using antiserum specific to cystatin C, and immunoprecipitation of 35S protein secreted by cultured cells. The Western analysis with an antiserum to human cystatin C showed that cultured Sertoli cells secrete three previously reported immunoreactive forms of cystatin C: a predominant pair of proteins at 13-14 kDa and a less abundant 20-kDa protein. Immunohistochemical localization of cystatin C in sections of rat testes showed intense staining in Sertoli cells; no immunoreactivity was observed in spermatogonia or spermatocytes. A cDNA fragment for rat cystatin C was obtained by use of the polymerase chain reaction and was used as a probe in Northern analyses to examine the steady-state levels of cystatin C mRNA in intact testes and in Sertoli and spermatogenic cells. Sertoli cells contained a 700-nucleotide cystatin C transcript, and a mixed population of spermatids and spermatocytes contained a 550-nucleotide transcript. Analysis of RNA from purified spermatogenic cells revealed that round and condensing spermatids contained the 550-nucleotide transcript, while pachytene spermatocytes contained a smaller 500-nucleotide transcript. The 700-nucleotide transcript was present in testes isolated from rats of 5-79 days of age, the 500-nucleotide transcript was detected initially in testes from 24-day-old rats, and the 550-nucleotide transcript was detected initially at 35 days of age. Both the 500- and 550-nucleotide transcripts increased in abundance until 50 days of age. RNA from stage-synchronized testes showed that steady-state levels of both the 550- and 700-nucleotide transcripts were lowest in stages VI-VII of the cycle. These data suggest that the role of cystatin C in the testis may be to inhibit the proteolytic activity of the cysteine protease cathepsin L in all stages except stages VI-VII.
研究表明，支持细胞能够合成并分泌胱抑素C，它是一种有效的半胱氨酸蛋白酶抑制剂。这一观察结果的证据来自蛋白质测序、使用针对胱抑素C的抗血清进行的蛋白质免疫印迹分析，以及对培养细胞分泌的35S蛋白进行的免疫沉淀。用抗人胱抑素C的抗血清进行的蛋白质免疫印迹分析表明，培养的支持细胞分泌三种先前报道的胱抑素C免疫反应形式：一对主要的13 - 14 kDa蛋白质和一种含量较少的20 kDa蛋白质。胱抑素C在大鼠睾丸切片中的免疫组织化学定位显示，支持细胞中有强烈染色；精原细胞或精母细胞中未观察到免疫反应性。通过聚合酶链反应获得了大鼠胱抑素C的cDNA片段，并将其用作探针进行Northern分析，以检测完整睾丸以及支持细胞和生精细胞中胱抑素C mRNA的稳态水平。支持细胞含有一个700个核苷酸的胱抑素C转录本，混合的精子细胞和精母细胞群体含有一个550个核苷酸的转录本。对纯化的生精细胞RNA的分析表明，圆形和浓缩的精子细胞含有550个核苷酸的转录本，而粗线期精母细胞含有一个较小的500个核苷酸的转录本。700个核苷酸的转录本存在于从5 - 79日龄大鼠分离的睾丸中，500个核苷酸的转录本最初在24日龄大鼠的睾丸中检测到，550个核苷酸的转录本最初在35日龄时检测到。500和550个核苷酸的转录本丰度均在50日龄前增加。来自阶段同步睾丸的RNA表明，550和700个核苷酸转录本的稳态水平在周期的VI - VII阶段最低。这些数据表明，胱抑素C在睾丸中的作用可能是在除VI - VII阶段之外的所有阶段抑制半胱氨酸蛋白酶组织蛋白酶L的蛋白水解活性。
