Lee K O, Oh Y, Giudice L C, Cohen P, Peehl D M, Rosenfeld R G
Department of Medicine, National University of Singapore.
J Clin Endocrinol Metab. 1994 Nov;79(5):1367-72. doi: 10.1210/jcem.79.5.7525634.
Previous studies have demonstrated that insulin-like growth factor (IGF) peptides, IGF-binding proteins (IGFBPs), and IGFBP-3 proteolytic activity, are present in human seminal plasma (SP). In this study, we have further characterized the IGFBPs in SP using immunoprecipitation and Western ligand blotting, Western immunoblotting, affinity cross-linking and immunoprecipitation, and RIA of IGFBP-3 using two different assays and have identified additional proteolytic activities for IGFBP-4 and IGFBP-5 in SP. Immunoprecipitation with antibodies to IGFBP-2, IGFBP-3, and IGFBP-4, before and after affinity cross-linking, demonstrated that intact IGFBP-2 and IGFBP-4 are present in SP, but intact IGFBP-3 is absent. Low mol wt fragments of IGFBP-3, which did not bind to IGF-I or IGF-II on Western ligand blot and did not cross-link to IGF-II, were demonstrated on Western immunoblot and were measurable by two different RIAs. Proteolytic activities for IGFBP-4 and IGFBP-5 were demonstrated in SP by incubation with the respective iodinated IGFBPs. On comparing the proteolytic activity for IGFBP-4 by purified prostate-specific antigen (PSA; a known IGFBP-3 protease in SP) or by SP with measured equivalent concentrations of PSA, the dose response and fragment patterns were identical. With IGFBP-5, however, proteolysis by purified PSA was different from that by SP with measured equivalent concentrations of PSA: 1) proteolysis by pure PSA was less efficient than matched concentrations of SP; 2) the pattern of fragments after proteolysis by pure PSA was different from that after proteolysis by matched concentrations of SP; and 3) proteolysis by purified PSA was significantly inhibited by phenylmethylsulfonylfluoride and aprotinin, but proteolysis by SP was not. We conclude that human SP contains intact IGFBP-2 and IGFBP-4, but has only IGFBP-3 fragments with low affinity for IGF peptides; that PSA is able to proteolyze IGFBP-4 and IGFBP-5 (as well as IGFBP-3); and that an additional IGFBP-5 protease is probably present in SP. There was no significant difference in any of these findings in SP from normal volunteers, vasectomized patients, or patients with idiopathic azoospermia. The roles of IGFBPs and IGFBP proteases in the male reproductive system and male infertility remain to be further elucidated.
以往研究表明，胰岛素样生长因子（IGF）肽、IGF结合蛋白（IGFBP）以及IGFBP-3蛋白水解活性存在于人类精液中。在本研究中，我们利用免疫沉淀和Western配体印迹法、Western免疫印迹法、亲和交联和免疫沉淀法，以及采用两种不同检测方法的IGFBP-3放射免疫分析法，进一步对精液中的IGFBP进行了特性分析，并确定了精液中IGFBP-4和IGFBP-5的其他蛋白水解活性。用针对IGFBP-2、IGFBP-3和IGFBP-4的抗体进行免疫沉淀，在亲和交联前后，结果表明精液中存在完整的IGFBP-2和IGFBP-4，但不存在完整的IGFBP-3。在Western免疫印迹上显示出IGFBP-3的低分子量片段，这些片段在Western配体印迹上不与IGF-I或IGF-II结合，也不与IGF-II交联，并且可用两种不同的放射免疫分析法进行检测。通过与各自的碘化IGFBP一起孵育，在精液中证实了IGFBP-4和IGFBP-5的蛋白水解活性。在比较纯化的前列腺特异性抗原（PSA；精液中一种已知的IGFBP-3蛋白酶）或精液对IGFBP-4的蛋白水解活性时，在测量的等效PSA浓度下，剂量反应和片段模式是相同的。然而，对于IGFBP-5，纯化的PSA的蛋白水解作用与在测量的等效PSA浓度下精液的蛋白水解作用不同：1）纯化的PSA的蛋白水解效率低于匹配浓度的精液；2）纯化的PSA蛋白水解后的片段模式与匹配浓度的精液蛋白水解后的片段模式不同；3）纯化的PSA的蛋白水解作用被苯甲基磺酰氟和抑肽酶显著抑制，但精液的蛋白水解作用不受影响。我们得出结论，人类精液中含有完整的IGFBP-2和IGFBP-4，但只有对IGF肽具有低亲和力的IGFBP-3片段；PSA能够蛋白水解IGFBP-4和IGFBP-5（以及IGFBP-3）；并且精液中可能存在另一种IGFBP-5蛋白酶。在正常志愿者、输精管结扎患者或特发性无精子症患者的精液中，这些发现均无显著差异。IGFBP和IGFBP蛋白酶在男性生殖系统和男性不育中的作用仍有待进一步阐明。
