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 Ca 2ϩ concentrations. Binding experiments were performed using progesterone-11␣-glucuronide-[ 125 I]iodotyramine as tracer. Computer analysis of competition curves using different steroids as competitors

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... indicated the presence of two distinct binding sites for P. The high affinity site (K d in the nanomolar range) appears to be specific for P, whereas the low affinity one (K d in the micromolar range) binds with equal affinity 11␤-hydroxyprogesterone (11␤OHP) and 17␣-hydroxyprogesterone (17␣OHP). A significant correlation exists among affinity constants (as determined by binding studies) and EC 50 values for the effects of P, 11␤OHP, and 17␣OHP on intracellular Ca 2ϩ 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 EC 50 in the nanomolar range and another in the micromolar range. Conversely, curves for 11␤OHP and 17␣OHP were monophasic, with an EC 50 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 ␣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 ␣c262 on P-induced acrosome reaction of capacitated human spermatozoa. (J Clin Endocrinol