Whitcomb R W, Schneyer A L, Roser J F, Hughes J P
Department of Medicine, Massachusetts General Hospital, Boston 02114.
Endocrinology. 1991 May;128(5):2497-502. doi: 10.1210/endo-128-5-2497.
Using a LH radioligand receptor assay (RRA) previously validated for use in serum and an equine monoclonal RIA, we have distinguished a subset of subfertile stallions with an elevated RRA/RIA ratio. After purification of the active moiety by anion exchange chromatography and immunoprecipitation with the equine LH (eLH) monoclonal antibody, RRA activity remained in the supernatant. This activity was also recognized by a polyclonal LH antibody (GDN 15) with wide cross-species recognition. This active fraction was further purified by gel filtration chromatography and shown to displace labeled eLH in a dose-dependent fashion in the RRA with an inhibition slope of 2.8 compared with a slope of 1.1 for native eLH. This fraction also inhibited the LH-stimulated steroidogenesis of Leydig cells in vitro in a dose-dependent fashion, but had no effect on basal (minus LH) steroid production. Polyacrylamide gel electrophoresis and electroelution of this material demonstrated RRA activity in a fraction with a mol wt between 45-66 kDa. We conclude that this substance 1) competitively inhibited binding of eLH and hCG to the LH receptor, 2) antagonized LH-stimulated steroidogenesis in vitro, and 3) may represent a LH isoform found in association with infertility in these animals.
使用先前已验证可用于血清的促黄体生成素（LH）放射性配体受体分析（RRA）和马单克隆放射免疫分析（RIA），我们鉴别出了一部分RRA/RIA比值升高的亚生育力种马。通过阴离子交换色谱法纯化活性部分并用马促黄体生成素（eLH）单克隆抗体进行免疫沉淀后，RRA活性仍存在于上清液中。该活性也被具有广泛跨物种识别能力的多克隆促黄体生成素抗体（GDN 15）所识别。通过凝胶过滤色谱法进一步纯化该活性部分，结果显示其在RRA中能以剂量依赖性方式取代标记的eLH，抑制斜率为2.8，而天然eLH的斜率为1.1。该部分还能以剂量依赖性方式在体外抑制促黄体生成素刺激的睾丸间质细胞类固醇生成，但对基础（无促黄体生成素）类固醇产生没有影响。对该物质进行聚丙烯酰胺凝胶电泳并电洗脱后，在分子量介于45 - 66 kDa的部分显示出RRA活性。我们得出结论，该物质1）竞争性抑制eLH和人绒毛膜促性腺激素（hCG）与促黄体生成素受体的结合，2）在体外拮抗促黄体生成素刺激的类固醇生成，3）可能代表在这些动物中与不育相关的一种促黄体生成素同工型。
