Binding characteristics and regulation of the 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) receptor on testicular and sperm plasma membranes of spotted seatrout (Cynoscion nebulosus)

Abstract

The binding characteristics of 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) to plasma membranes prepared from the testes and sperm of spotted seatrout (Cynoscion nebulosus) were investigated using a filtration method to retain the bound 20β-S. A single class of high affinity (Kd = 17.9 nM), low capacity (Bmax = 0.072 nM g-1 testes) binding sites was identified by saturation and Scatchard analyses on testicular membranes of spermiating spotted seatrout. A corresponding receptor (Kd = 22.17 nM, Bmax = 0.00261 nM ml-1 milt) was also detected in spermatozoan membrane preparations. The rates of 20β-S association and dissociation were rapid, both had Thalfs of less than 1 min. Competition studies indicated that the receptor was highly specific for 20β-S. 17,20β-dihydroxy-4-pregnen-3-one, which had the highest affinity of the other steroids tested, had a relative binding affinity (RBA) of 14.3%. Progesterone, 11-deoxycortisol and testosterone competed with an order of magnitude less affinity (RBA's of 7.4, 1.8 and 1.1%, respectively). Estradiol displayed low affinity for the receptor (RBA = 0.4%) and cortisol did not cause any displacement at 1000-fold excess concentration. Specific 20β-S receptor binding was detected in plasma membranes from testes of both spermiating and non-spermiating seatrout and on spermatozoa. Prolonged incubation of testicular fragments from a spermiating fish with gonadotropin (15 IU ml-1 human chorionic gonadotropin) or forskolin (10 µM) caused a 2–3 fold increase in membrane receptor binding. Previous studies have shown that gonadotropin-induced upregulation of the 20β-S plasma membrane receptor in seatrout ovaries is required for the oocytes to become responsive to 20β-S and undergo final maturation. The existence of a 20β-S membrane receptor on sperm and its upregulation in the testes by gonadotropin raises the possibility that final maturation of spermatozoa in male seatrout may be regulated by a similar mechanism.