The presence of 17α,20β-dihydroxy-4-pregnen-3-one receptor activity in the ovary of the brook trout,Salvelinus fontinalis, during terminal stages of oocyte maturation

Abstract

The presence of 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DHP) oocyte receptor activity has been demonstrated in brook troutSalvelinus fontinalis. Scatchard analyses of the cytosol fraction during various terminal stages of oocyte maturation gave a high equilibrium association constant (K_a) value of 1.394±0.669 10⁸M⁻¹ (n=7) and low maximum binding capacities (N_max). The association kinetics of the receptor was second order k₊₁=2.292×10⁶M⁻¹ sec⁻¹. The dissociation rate constant k_a was 1.502×10⁻² sec⁻¹ for the first order dissociation reaction. The K_a=1.526×10⁸M⁻¹, when it was determined from k₊₁/k₋₁ a value close to that found from the Scatchard analysis. Competition studies showed the following binding affinities testosterone > 17α-HP > 17α,20β-DHP > Promegestone > progesterone > estradiol > pregnenolone; cortisol showed no competitive inhibition. Cytosolic extracts when pre-equilibrated with various labelled steroids and eluted from a Sephacryl S-300 column gave multiple specific binding peaks. On sucrose density gradient centrifugation specific binding was observed at 3.05 S in cytosol containing 0.15M sodium chloride buffer. The receptor lost binding activity when incubated with various proteases, but DNase and RNase had no effect. Blood plasma without heparin at (1∶10) dilution also bound [³H]17α,20β-DHP, Ka was 8.04×10⁷ M⁻¹. The nuclear pellet extract (750×g) gave very little specific binding activity even at high radiolabelled steroid concentrations and a linear Scatchard plot was not obtained. Nevertheless the nuclear extract, after dextran-charcoal treatment, pre-equilibrated with [³H]17α,20β-DHP, bound specifically to DNA cellulose, and cytosol from the same oocytes also bound to DNA cellulose under similar conditions. Although specific binding to DNA cellulose was obtained the salt concentrations at which the steroid-receptor complex elution took place was not reproducible in both nuclear extracts and cytosol samples. Also binding activity was extremely small compared to the total cytosolic binding. The nuclear extract when pre-equilibrated with high concentrations (20 nM) of the labelled steroid and then chromatographed on Sephacryl S-300 column gave a specific binding peak which was similar to that of the cytosolic preparation. The receptor levels in cytosol decreased progressively during final maturation (Stages 1–7). There is preliminary evidence for the presence of 17α,20β-DHP receptor activity in cytosol of landlocked Atlantic salmonSalmo salar ouananiche, and rainbow troutSalmo gairdneri. The zona radiata fraction from late stages oocyes 5, 6, and 7 in brook and rainbow trout oocytes were isolated by ultracentrifugation; from this fraction a protein was characterized which covalently bound [³H]R5020 after photoaffinity labelling. The same protein also bound [³H]17α,20β-DHP after solubilization in Brig 35 buffer. The SDS gel electrophoresis subunit composition of the above protein was similar to the cytosol counterpart binding [³H]17α,20β-DHP, although the molecular weights were different. The blood sample [³H]R5020 binding component subunit composition was different from that of the membrane extracted protein. These results demonstrate the presence of 17α,20β-DHP receptor activity in the cytosol and zona radiata membranes of the oocytes during final maturation.