Effects of cannabinoids on testosterone and protein synthesis in rat testis Leydig cells in vitro.

Various water-insoluble cannabinoids as well as SP-111A, the water-soluble derivative of delta 9-tetrahydrocannabinol (delta 9-THC), reduced hCG and dibutyryl-cAMP stimulated testosterone production by rat testicular Leydig cell preparations. With 0.15 microM (0.05 micrograms/ml) 8-beta-OH-delta 9-THC the inhibition was about 50% of stimulated testosterone synthesis. Dose-related inhibitions were apparent with other cannabinoids and their order of potency in inhibiting stimulated steroidogenesis by the interstitial cells in vitro was found to be: 8-beta-OH-delta 9-THC greater than or equal to 11-OH-delta 9-THC greater than CBN = CBD = CBG greater than or equal delta 9-THC = delta 8-THC. The non-stimulated, basal, steroidogenesis was not affected even with 15 microM cannabinoids. The incorporation of L-[U-14C]leucine into the protein of Leydig cells was markedly reduced by 15 microM cannabinoids under both basal and stimulated conditions. The inhibition of steroidogenesis as well as protein synthesis in rat testicular Leydig cell preparations by various cannabinoids cannot be correlated with their psychoactivity. The present data suggest that cannabinoids at very low concentrations may interfere directly in Leydig cells with both protein and testosterone synthesis, and thus with their function.     

Early effects of hCG on human testicular cyclic AMP content, protein kinase activity, in-vitro progesterone conversion and the serum concentrations of testosterone and oestradiol

Ten infertile men underwent testicular biopsy. Cyclic AMP concentration and cAMP-dependent protein kinase activity were determined in biopsies obtained before, and at 3, 10, 20 and 30 min after an intravenous injection of hCG (1500-5000 IU). The in-vitro conversion of progesterone by testicular tissue, and the serum concentrations of testosterone and oestradiol were then studied before and at 30 min after hCG injection. Intravenous injection of hCG induced a rapid increase in cAMP concentration and in the activity of cAMP-dependent protein kinase. The kinetics of this response indicated that cAMP and cAMP-dependent protein kinase mediate hCG effects on the human testis, presumably via effects on the Leydig cells. No stimulatory effect on steroid conversion in vitro or on the serum concentrations of testosterone and oestradiol were seen after 30 min.     

Treatment with an LHRH agonist or hCG increases interstitial fluid volume and permeability to Evans blue in the mouse testis

Treatment of adult mice with 1 microgram of an LHRH-agonist or with 5 i.u. hCG, subcutaneously, resulted in an increase in the permeability to intravenously injected Evans blue into the testicular interstitial space and in the volume of testicular interstitial fluid. These changes are probably related to an increase in vascular permeability but, in contrast to the situation in rats, this was accompanied neither by an accumulation of polymorphonuclear leucocytes in testicular blood vessels nor by the formation of large inter-endothelial cell gaps in postcapillary venules. The mechanisms mediating the gonadotrophin-induced increase in vascular permeability in the mouse testis thus remain unknown.     

The importance of the Leydig cells in the vascular response to hCG in the rat testis

The increase in permeability of the testicular blood vessels following an injection of hCG into rats is abolished completely if the animals are treated 3 days earlier with ethane dimethane sulphonate (EDS), a compound that effectively eliminates Leydig cells from the testes. As there is other evidence that androgens or prostaglandins are not involved in this vascular response, further studies will be necessary to determine whether these data mean that another vasoactive substance is secreted by the Leydig cells or whether the EDS also eliminates other cells besides the Leydig cells, for example the mast cells found in the vicinity of the testicular artery.     

Hormone binding modified endogenous proteolysis of LH/hCG receptors in rat ovarian plasma membranes

Purified rat ovarian plasma membranes were subjected to incubation under conditions where luteinizing hormone receptors were either free or bound to hCG. When receptor proteolysis was followed by labeling the receptor with tritiated borohydride or [125I]hCG, occupied receptors were found to be more accessible to endogenous proteinases than unoccupied receptors. They exhibited greater rates of degradation and also produced an additional degradation product upon proteolysis. Degradation of other plasma membrane (glyco)polypeptides, however, was not affected by hormone binding. These results indicate that hCG binding induces a conformational or a structural change in its receptor, thereby increasing its susceptibility to endogenous plasma membrane proteinases.     

Male Hypogonadism Associated with Elevated LH, Normal FSH and Low Testosterone Possibly Due to an Abnormal LH Molecule

A male patient of 20 years of age with clinical features of hypogonadism presented high LH, low T and normal FSH blood level. Although the LH data suggested a primary gonadal defect, he responded normally to hCG administration, with a rise of plasma testosterone. During therapeutical administration of hCG he also improved clinically. Thus, the patient's Leydig cells responded to exogenous hCG but not to endogenous LH. This syndrome is likely to be attributable to the presence of an immunologically active but biologically inactive LH molecule.     

Autoregulation of LH/hCG Receptors and Catabolism of hCG in Rat Testis

The number of free and occupied LH/hCG-receptors in adult rat testes after subcutaneous injections of hCG was studied. When 3–10 IU of hCG was injected, there was initially (between 6 and 16 h) a significant increase in free binding sites, without detectable occupancy of receptors. Higher doses (100 and 1000 IU) induced a drop in the amount of free receptors which was explained by simultaneous receptor occupancy. Later (between 12 and 48 h) there was a pronounced net loss of both free and occupied binding sites. Gel filtration of radioactivity extracted from testicular tissue demonstrated that following the initial uptake [¹²⁵I]hCG undergoes a cleavage to subunits and hydrolysis to peptide fragments and amino acids. The autoradiographic results suggest that the degradative processes take place in Leydig cells. The degradation of [¹²⁵I]hCG was markedly reduced by simultaneous injection of an excess of unlabelled hormone, supporting the autoradiographic evidence of the degradation site of [¹²⁵I]hCG.     

SYNTHESIS OF CARBOXYL TERMINAL SEGMENTS OF BETA-SUBUNIT OF HUMAN CHORIONIC GONADOTROPIN

The synthesis in solution of carboxyl terminal peptide segments of the beta-subunit of human chorionic gonadotropin is described. The protected segments include sequences 119–131, 132–137, and 138–145. The syntheses were based on a standardized liquid-liquid extraction program for routine purification of intermediates (two-phase method). Condensation of terminally deblocked segments afforded protected peptides 132–145, 126–145, 120–145 and 119–145. Protected peptides 126–145 and 120–145 were deprotected in liquid hydrogen fluoride and used in conjugated form for immunization of rabbits. Data on the specificity of the antibody response are reported.