IDENTIFICATION AND CHARACTERIZATION OF FUNCTIONAL NONGENOMIC PROGESTERONE RECEPTORS ON HUMAN SPERM MEMBRANE

The presence of functional nongenomic progesterone (P) receptors in hu man spermatozoa has been investigated by equilibrium binding studies i n intact spermatozoa, ligand blot and Western blot analysis of sperm l ysates, as well as determination of the effects of the steroid on sper m intracellular Ca2+ concentrations. Binding experiments were performe d using progesterone-11 alpha-glucuronide-[I-125]iodotyramine as trace r. Computer analysis of competition curves using different steroids as competitors indicated the presence of two distinct binding sites for P. The high affinity site (K-d in the nanomolar range) appears to be s pecific for P, whereas the low affinity one (K-d in the micromolar ran ge) binds with equal affinity 11 beta-hydroxyprogesterone (11 beta OHP ) and 17 alpha-hydroxyprogesterone (17 alpha OHP). A significant corre lation exists among affinity constants (as determined by binding studi es) and EC50 values for the effects of P, 11 beta OHP, and 17 alpha OH P on intracellular Ca2+ in fura-2-loaded spermatozoa, strongly indicat ing the involvement of P-binding sites in the biological effect of the steroid. In particular, dose-response curves for P were biphasic, wit h an EC50 in the nanomolar range and another in the micromolar range. Conversely, curves for 11 beta OHP and 17 alpha OHP mere monophasic, w ith an EC50 just in the micromolar range; Ligand blot analysis of sper m total lysates performed with peroxidase-conjugated P revealed the pr esence of two binding proteins of 54 and 57 kDa that were specific for P. Indeed, peroxidase-conjugated P binding was blocked by the simulta neous presence of the unconjugated steroid. Using alpha c262 antibody, which is directed against the P-binding domain of genomic receptor, w e detected two proteins of similar molecular mass (54 and 57 kDa), whe reas 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 memb ranes and virtually absent in the cytoplasm. The involvement of these proteins in the biological effects of P is indicated by the strong inh ibitory effect of alpha c262 on P-induced acrosome reaction of capacit ated human spermatozoa.