Changes in ovarian maturation-inducing steroid (MIS; 17,20 beta, 21-trihydroxy-4-pregnen-3-one [20 beta-S]) membrane receptor concentrations during the reproductive cycle were investigated in spotted seatrout (Cynoscion nebulosus) captured at their spawning grounds. Ovarian receptor concentrations increased gradually during ovarian recrudescence and subsequently increased rapidly during oocyte maturation, reaching 3.5-fold the prematuration values by the beginning of ovulation. The significant elevation of receptor concentrations by the germinal vesicle migration stage of oocyte maturation was accompanied by increases in circulating levels of gonadotropin (LH, GTH II) and MIS (20 beta-S). The regulation and physiological significance of the increase in ovarian MIS membrane receptor concentrations were investigated in a double in vitro incubation system. Incubation of fully grown, follicle-enclosed oocytes with hCG (10 IU/ml) for 6 h caused a two- to fourfold increase in oocyte and ovarian MIS receptor concentrations and the development of oocyte maturational competence (OMC; ability to complete oocyte maturation in vitro in response to exogenous 20 beta-S in a second incubation). Both upregulation of the MIS receptor and development of OMC in response to gonadotropin were blocked by coincubation with actinomycin D or cycloheximide, which are inhibitors of mRNA and protein synthesis, respectively, but not by cyanoketone, which is an inhibitor of 3 beta-hydroxysteroid dehydrogenase-dependent steroid synthesis. Incubation with a variety of steroids, including 20 beta-S, failed to increase receptor concentrations or to induce OMC, further supporting a steroid-independent mechanism of gonadotropin action. In contrast, insulin-like growth factor I (IGF-I) mimicked the actions of gonadotropin, which suggests IGF-I may be a component of the hormone signaling pathway. A close correlation was found between the relative increase in MIS receptor concentrations and the percentage of oocytes that became maturationally competent after treatment with different concentrations of gonadotropins and drugs that elevate cAMP levels. The finding that upregulation of the MIS receptor in response to gonadotropin and other treatments is invariably associated with the development of OMC indicates that these two processes are intimately related, and it suggests that the increase in MIS receptor concentrations is a critical regulatory step in the hormonal control of oocyte maturation.

Incubation of mature, hydrated, follicle-enclosed oocytes of the spotted seatrout, Cynoscion nebulosus, with the maturation-inducing steroid (MIS), 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S), for 9–12 h resulted in the appearance of ovulated oocytes in the culture media. The ovulation response was concentration-dependent and steroid-specific. The other teleost MIS, 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P), was also a potent inducer of ovulation, whereas progesterone and 11-deoxycorticosterone did not stimulate ovulation above control levels and partially antagonized the action of 20β-S. The agonist and antagonist activities of these steroids on ovulation are consistent with their relative binding affinities for the ovarian nuclear progestogen receptor previously characterized in this species. Both the RNA synthesis inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide blocked MIS-induced ovulation. This suggests that induction of ovulation by the MIS is through a genomic mechanism of action, and potentially involves the previously characterized nuclear progestogen receptor. Gonadotropin (hCG)-induced ovulation was blocked by addition of the steroid synthesis inhibitor cyanoketone, which was overcome by the addition of 20β-S, but not pregnenolone. Thus, the most likely mechanism of gonadotropin-induced ovulation is an increase in the synthesis of the MIS. It is concluded that the processes of final oocyte maturation and ovulation are both regulated by the MIS. Whereas final oocyte maturation is mediated by the 20β-S membrane receptor (P. Thomas and S. Das, 1997, Biol. Reprod. 57, 999–1007), ovulation is regulated by a genomic mechanism and is potentially mediated by the previously characterized nuclear progestogen receptor.

A nuclear progestogen receptor has previously been characterized in the ovary of the spotted seatrout. The steroid specificity of this receptor was further defined in the present study by determining the binding affinity of a wide variety of progestin and corticosteroid agonists and antagonists. The addition of a hydroxyl or keto group to the 11 position resulted in a 10–100-fold decrease in relative binding affinity (RBA). The significance of the 17, 20, and 21 positions in determining the RBA of closely related steroids was investigated in detail. Modification of the 17α-hydroxyl to an acetyl or carbyne group resulted in a 10-fold decrease in RBA. The substitution of a ketone group with a hydroxyl group at the 20 position increased binding, whereas the addition of a 21-hydroxyl group consistently decreased RBA by 40–60%. The effect of the 17α-hydroxyl group on RBA was dependent on what functional group was present at the 20 position. The addition of a 17α-hydroxyl decreased affinity by one- to 10-fold if a ketone group was present at position 20. However, the RBA increased five- to 10-fold upon addition of the 17α-hydroxyl group if a hydroxyl was present at the 20 position. The effects of the different substitutions at the 17, 20 and 21 positions explain why the two teleost maturation-inducing steroids 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-P) and 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-S) have higher affinities than progesterone for this receptor. It is concluded that the seatrout progestogen receptor demonstrates steroid specificity different from progesterone receptors in other vertebrates.

A nuclear progestogen receptor was identified in the ovary of the spotted seatrout, Cynoscion nebulosus. A single class of high-affinity, low-capacity cytoplasmic binding sites for 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-P) was characterized by saturation and Scatchard analyses (KD = 1.89 +/- 0.61 nM, Bmax = 1.80 +/- 0.63 pmol/g ovary, n = 4), as well as by one-point assay (Bmax = 1.41 +/- 0.26 pmol/g ovary, n = 12). Analysis of the binding kinetics indicated a fairly rapid association rate (T1/2 = 72 +/- 10.2 min) and a slightly slower dissociation rate (T1/2 = 99 +/- 9.4 min). Competition studies revealed that several steroids exhibited the same range of affinity (17 alpha, 20 beta-P > 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one (20 beta-S) > 11-deoxycorticosterone > progesterone) while others displayed an order of magnitude less affinity (17 alpha-hydroxy-4-pregnen-3-one > pregnenolone > 11-deoxycortisol > testosterone). No displacement was found with 1000-fold excess estradiol-17 beta or cortisol. Binding activity was also present within the testis, but not in the brain, gill, muscle, or plasma. Nuclear binding was detected by DNA-cellulose column chromatography and was inhibited by the addition of molybdate, a characteristic of nuclear steroid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)