Luconi M, Bonaccorsi L, Maggi M, Pecchioli P, Krausz C, Forti G, Baldi E
Dipartimento di Fisiopatologia Clinica, Unità di Andrologia, Università di Firenze, Italy.
J Clin Endocrinol Metab. 1998 Mar;83(3):877-85. doi: 10.1210/jcem.83.3.4672.
The presence of functional nongenomic progesterone (P) receptors in human spermatozoa has been investigated by equilibrium binding studies in intact spermatozoa, ligand blot and Western blot analysis of sperm lysates, as well as determination of the effects of the steroid on sperm intracellular Ca2+ concentrations. Binding experiments were performed using progesterone-11alpha-glucuronide-[125I]iodotyramine as tracer. Computer analysis of competition curves using different steroids as competitors indicated the presence of two distinct binding sites for P. The high affinity site (Kd in the nanomolar range) appears to be specific for P, whereas the low affinity one (Kd in the micromolar range) binds with equal affinity 11beta-hydroxyprogesterone (11betaOHP) and 17alpha-hydroxyprogesterone (17alphaOHP). A significant correlation exists among affinity constants (as determined by binding studies) and EC50 values for the effects of P, 11betaOHP, and 17alphaOHP on intracellular Ca2+ in fura-2-loaded spermatozoa, strongly indicating the involvement of P-binding sites in the biological effect of the steroid. In particular, dose-response curves for P were biphasic, with an EC50 in the nanomolar range and another in the micromolar range. Conversely, curves for 11betaOHP and 17alphaOHP were monophasic, with an EC50 just in the micromolar range. Ligand blot analysis of sperm total lysates performed with peroxidase-conjugated P revealed the presence of two binding proteins of 54 and 57 kDa that were specific for P. Indeed, peroxidase-conjugated P binding was blocked by the simultaneous presence of the unconjugated steroid. Using alpha c262 antibody, which is directed against the P-binding domain of genomic receptor, we detected two proteins of similar molecular mass (54 and 57 kDa), whereas using antibodies directed against the DNA-binding and N-terminal domains of the genomic P receptors, the two proteins were not detected. In addition, p54 and p57 appear to be mostly localized in sperm membranes and virtually absent in the cytoplasm. The involvement of these proteins in the biological effects of P is indicated by the strong inhibitory effect of alpha c262 on P-induced acrosome reaction of capacitated human spermatozoa.
通过对完整精子进行平衡结合研究、对精子裂解物进行配体印迹和蛋白质免疫印迹分析以及测定该类固醇对精子细胞内钙离子浓度的影响，对人类精子中功能性非基因组孕酮（P）受体的存在情况进行了研究。使用孕酮-11α-葡萄糖醛酸-[125I]碘酪胺作为示踪剂进行结合实验。使用不同类固醇作为竞争者对竞争曲线进行计算机分析，结果表明存在两个不同的P结合位点。高亲和力位点（解离常数Kd在纳摩尔范围内）似乎对P具有特异性，而低亲和力位点（Kd在微摩尔范围内）对11β-羟基孕酮（11βOHP）和17α-羟基孕酮（17αOHP）具有同等亲和力。亲和力常数（通过结合研究确定）与P、11βOHP和17αOHP对用fura-2加载的精子细胞内钙离子影响的半数有效浓度（EC50）值之间存在显著相关性，这有力地表明P结合位点参与了该类固醇的生物学效应。特别是，P的剂量反应曲线呈双相，一个EC50在纳摩尔范围内，另一个在微摩尔范围内。相反，11βOHP和17αOHP的曲线呈单相，EC50仅在微摩尔范围内。用与过氧化物酶结合的P对精子总裂解物进行配体印迹分析，发现存在两种分子量分别为54和57 kDa的结合蛋白，它们对P具有特异性。实际上，未结合的类固醇同时存在时会阻断与过氧化物酶结合的P的结合。使用针对基因组受体P结合域的α c262抗体，我们检测到两种分子量相似的蛋白质（54和57 kDa），而使用针对基因组P受体DNA结合域和N末端域的抗体时，未检测到这两种蛋白质。此外，p54和p57似乎主要定位于精子膜上，而在细胞质中几乎不存在。α c262对获能人类精子的P诱导顶体反应具有强烈抑制作用，这表明这些蛋白质参与了P的生物学效应。
