HORMONE AND DRUG STIMULATION OF TESTOSTERONE SECRETION IN THE RAT LEYDIG CELL

Abstract

The steroidogenic response of rat Leydig cell to gonadotrophin and prolactin was used to modify an in vitro experimental model for studies an the effect of some medicinal extracts on testosterone secretion. The crude methanol extract of Quassia amara -quassinoid, and its purified compounds (quassin and 2-methoxycanthin-6-one) on Leydig cell steroidogenesis were studied. The results showed that quassinoid, quassia and methoxycanthin-6-one did not produce lethal effect at the wide range of doses (1-2000 mgKg⁻¹ B.W for quassinoid and 1-2000 μgKg⁻¹ B.W for quassin and 2-methoxycanthin-6-one) tested in rats. Quassinoid (100 mgKg⁻¹ B.W) and quassin (100 μgKg⁻¹ B.W) significantly reduced (P<0.05) the weights of the testis, epididymis and seminal vesicle but, significantly increased (p< 0.05) the weights of the anterior pituitary gland and liver. Sperm counts were also significantly reduced (P<0.05). However, 2-methoxycanthin-6-one did not produce any noticeable change in these variables. Moreover, all the three drugs did not affect the total body weight. The serum glutamic-oxaloacetic transaminase (SGOT) levels were slightly increased (P<.05) while the serum LH, FSH and testosterone levels were significantly reduced (P<0.01) in quassinoid and quassin but not 2-methoxycanthin-6-one treated rats. To investigate further if the drugs exert a direct effect on steroidogenesis, the reactivity of Leydig cells was determined: (a) after addition of gonadotrophin and prolactin in vitro in the presence or absence of 10 mM theophylline; (b) after 2 months daily treatment of rats with drugs in vivo; (c) after addition of the drugs in vitro; and (d) after addition of the drugs in vitro in the presence or absence of ovine luteinizing hormone (oLH) (50ng/ml) and or theophylline (10mM). In (a) - (d), Leydig cells from the rat testis were prepared by collagenase digestion and gentle dispersions. The digestion with collagenase was carried out in Dulbecco's modified Eagle's medium containing bovine serum albumin and trypsin inhibitor (DME). About 100,000 Leydig cells/ml were incubated (37⁰C, 3 hrs) separately with phosphate buffered saline (PBS) pH.7.4, graded doses of gonadotrophin, prolactin and quassinoid, quassin and 2-methoxycanthin-6-one. The effects of ovine luteinizing hormone and theophylline on testosterone secretion by quassinoid, quassin and 2-methoxycanthin -6-one were similarly investigated. Leydig cell count was performed by the trypan blue dye exclusion test. Hormone and drug solutions were prepared in PBS which served as the control. Each point was run in quintuplicate. The reaction was terminated by the addition of cold DME (4⁰C), and testosterone secretion in suitable aliquots of samples was estimated by a validated radioimmunoassay (RIA) technique. Extraction and purification of quassinoid was done as described by Grandolini et al, (1987). The average viability of Leydig cells before treatments was 97.80 ± 0.14%. The mean basal testosterone secretion was 0.15 ± 0.05 nmol/10⁵ cells/3hrs. Human luteinizing hormone (hLH) and human chorionic gonadotrophin (hCG) produced a similar dose-dependent increase in testosterone secretion. However, oLH-induced testosterone secretion was higher (P<0.05) than that by hLH, hCG, or follicle stimulating hormone (FSH) at the same doses. The effects of oLH, hLH, hCG and FSH (1-100ng/ml) were potentiated by 10mM theophylline. Prolactin (1.25-10.0 iu/L) which inhibited testosterone secretion, had no effect at lower doses (0.31-1.0 iu/L). The Leydig cell viability after hormone treatments was 96.80±0.12%. Furthermore the isolated cells stained positively for 3ß-HSD, and the percentage that stained for 3ß-HSD activity was more than 90% indicating that they were pure Leydig cells. Quassinoid and quassin caused an inhibition of basal and stimulated testosterone production, all the same if they were applied in vivo or in vitro. However, 2-methoxycanthin-6-one did not produce any change in testosterone production when applied either in vivo or in vitro. The addition of 50ng/ml oLH or 10mM theophylline had no effect on the inhibitory action of quassinoid and quassin on testosterone secretion. Washing of Leydig cells after incubation with drugs restored their steroidogenic ability. The Leydig cell viabilities after quassinold and quassin treatments were respectively 93.95±1.4% and 93.20±0.98%. These cells were responsive to oLH stimulation alter washing in DME. The mean Leydig cell viability was 91.0±0.70% after treatment with 2-methoxycanthin-6-one. The intra and inter-assay variations of testosterone RIA were 9.5±0.95% and 10.20±1.50% respectively. The in vitro experimental model in this study seems suitable for studies on the effect of drugs on steriodogenesis. Quassinoid, quassin and 2-methoxycanthin-6-one were not detrimental to the Leydig cell and quassin appears to possess anti-fertility effects.