Effect of desialylated human chorionic gonadotropin (hCG) on the bioactivity of rat Leydig cells.

Human chorionic gonadotropin is a glycoprotein hormone that, like LH, stimulates steroidogenesis in gonadal cells. Using a desialylation process. 95 per cent of the sialic acid residues from an intact standard hCG molecule were eliminated and then the electrophoretic properties and the bioactivity of the desialylated hCG were determined. Using rat Leydig cells as a biological model, the binding affinity to LH receptors of Leydig cell membranes, steroidogenic activity and second messenger production were studied. The results indicate that the loss of sialic acid from the hCG molecule slightly increases the binding activity to LH receptors and results in steroidogenic activity with an increased ED50. Cyclic AMP production was significantly reduced however and arachidonic acid release was not observed. Several possible mechanisms that could explain these results are discussed.

Melatonin and testicular function: characterization of binding sites for 2-[125I]-iodomelatonin in immature rat testes.

Melatonin-binding sites in membrane preparation immature rat testes were demonstrated by utilizing 2-[125I]-iodomelatonin as a radioligand. Binding at these sites was found to be reversible, saturable, specific and of, high affinity. Scatchard analysis of the specific binding revealed an equilibrium binding constant (kd) of 215 +/- 23 pmol/L and a total number of binding sites (Bmax) of 0.94 +/- 0.1 fmol/mg protein. The Hill coefficient of 1.0 suggests a single class of 2-[125I]-iodomelatonin-binding site in the rat testes. The Kd value determined from kinetic analysis was 179 pmol/L, which is in close agreement with the value determined from equilibrium studies. In competition studies, the order of pharmacological affinity for 2-[125I]-iodomelatonin binding sites in the rat membrane testes was: melatonin > 6-hydroxymelatonin > N-acetylserotonin > 5-hydroxyindole-3-acetic acid > 5-hydroxytryptamine > 5-hydroxy-L-tryptophan > tryptamine > > 5-methoxytryptamine, 5-methoxyl-DL-tryptophan, D-L-tryptophan. The 2-[125I]-iodomelatonin binding was markedly reduced by guanine nucleotides; treatment with nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) caused a 10-fold decrease in receptor affinity. In this paper, we report evidence indicating the presence of binding sites in immature rat tests, suggesting a possible direct role of melatonin on testicular steroidogenesis.

Melatonin in the rat testis: evidence for local synthesis.

The vertebrate pineal gland rhythmically produces melatonin, a hormone involved in regulation of several physiological and behavioral processes. Melatonin is synthesized from serotonin essentially by two enzymatic steps involving N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase activities. We have previously demonstrated the presence of melatonin binding sites in the rat testes, and an inhibitory effect of melatonin on testicular gonadotrophin-stimulated androgen production. It is unknown whether these effects are mediated by melatonin synthesized locally or by melatonin from pineal origin. To assess the potential capacity of melatonin production by the testis, we used radiolabeled precursors to measure the activities of N-acetyltransferase and hydroxyindole-O-methyltransferase. The production of N-acetylserotonin was time-dependent during over 10 min of incubation. Melatonin had a linear increase throughout the 30 min incubation period with S-adenosyl-L-[methyl-14C]methionine. Identities of melatonin and N-acetylserotonin were confirmed by thin-layer chromatography. The ability of the testis to synthesize melatonin during sexual maturation was also analyzed. When activity of NAT was expressed per mg of protein, the maximal activity was observed on day 40. In contrast, when activity of NAT is expressed by the testis, the amount of NAT increased to peak on day 40 and remained elevated through day 70. We determined that both activities were predominantly localized in interstitial cells. NAT activity in seminiferous tubules was substantially decreased, representing 6.4% of NAT activity in interstitial cells. We concluded that rat testes are capable of synthesizing melatonin due to the presence of the enzymes necessary for the transformation of serotonin to melatonin.

Melatonin binding sites in interstitial cells from immature rat testes.

Melatonin binding sites in rat testes interstitial cells were identified using 2-[125I]-iodomelatonin. Saturation studies of cells revealed a single class of high affinity binding sites, with an apparent equilibrium dissociation constant (Kd) of 100 +/- 20 pM, and a total binding capacity (B max) of 3.0 +/- 0.2 x 10(3) melatonin molecules per cell. Binding was reversible and inhibited by non radioactive melatonin. These results suggest that interstitial cells from immature rat testes have specific receptors for melatonin.

Melatonin binding sites in interstitial cells from immature rat testes

Melatonin binding sites in rat testes interstitial cells were identified using 2-[125I]-iodomelatonin. Saturation studies of cells revealed a single class of high affinity binding sites, with an apparent equilibrium dissociation constant (Kd) of 100 +/- 20 pM, and a total binding capacity (B max) of 3.0 +/- 0.2 x 10(3) melatonin molecules per cell. Binding was reversible and inhibited by non radioactive melatonin. These results suggest that interstitial cells from immature rat testes have specific receptors for melatonin