Binding characteristics, hormonal regulation and identity of the sperm membrane progestin receptor in Atlantic croaker

Recently a novel cDNA was discovered in spotted seatrout ovaries encoding a protein with seven transmembrane domains that has the characteristics of the membrane progestin receptor (mPR) mediating maturation-inducing steroid (MIS) induction of oocyte maturation in this species. Preliminary results suggested the MIS also activates an mPR on the spermatozoa of spotted seatrout and a closely related species, Atlantic croaker, to stimulate sperm motility. We show here that plasma membranes of croaker sperm demonstrate high affinity (Kd ∼ 20 nM), limited capacity (Bmax 0.08 nM), specific and displaceable binding for progestins that is characteristic of mPRs. The MIS (17,20β,21-trihydroxy-4-pregnen-3-one, 20β-S) displayed the greatest binding affinity for the sperm mPR among the steroids tested. Treatment of croaker testicular tissue in vitro with gonadotropin caused a several-fold increase in sperm mPR receptor concentrations that was partially blocked in the presence of cyanoketone, which suggests this action of gonadotropin is partially mediated by stimulation of steroidogenesis. Protein bands of the predicted sizes for the mPR and its dimer (40 and 80 kDa) were detected by Western blotting of croaker sperm membranes using a specific antibody to the novel seatrout mPR (mPRα). Immunocytochemistry of whole croaker spermatozoa with the mPRα antibody showed that staining was primarily localized on the midpiece, consistent with a role of the mPRα in mediating MIS stimulation of sperm motility. The results suggest that the mechanism of progestin action on fish sperm involving mPRs is basically similar to that in mammals and has been evolutionarily conserved amongst vertebrates.