James P. Preslock

A review of in vitro testicular steroidogenesis in rodents, monkeys and humans

Abstract This review covers pertinent literature regarding the synthesis of androgens by the testis of the rodent, the subhuman primate and man. By increasing the activity of the cholesterol side-chain cleavage mitochondrial enzyme complex, LH is rate-limiting for the synthesis of testicular androgens by regulating the conversion of cholesterol to pregnenolone. In the rat, available information suggests that pregnenolone is converted to testosterone primarily through the 4-ene pathway. Testosterone is the major metabolite of pregnenolone and progesterone in the newborn and adult rat, while in the immature rat 5α-reduced androgens are predominant. In subhuman primates, the 4-ene pathway also appears to be predominant for testosterone synthesis in more primitive species, such as the marmoset and the baboon. However, in higher primates, such as the orang-utan, both the 4-ene and the 5-ene pathways appear to be utilized for testosterone synthesis. In man, the 5-ene pathway is the major pathway for the metabolism of pregnenolone to testosterone, and this pathway may involve the conversion of sulfated intermediates to testosterone. There is apparently little 5α-reductase activity in the prepubertal human testis, but the activity of this enzyme increases with age after puberty. The reduction in 20α-hydroxysteroid dehydrogenase activity prior to puberty may invoke the onset of puberty in man.

Lesions of the sexually dimorphic nucleus of the preoptic area: Effects upon LH, FSH and prolactin in rats

Bilateral lesions were placed in the sexually dimorphic nucleus of the preoptic area (SDN-POA) in castrated adult male rats in an attempt to determine a physiologic role for this nucleus. These lesions significantly attenuated the increase in plasma FSH and LH due to simultaneous castration at 24 hr, and at 7 and 14 days following surgery, and significantly decreased the levels of plasma prolactin on comparison with pre-operative values and those of castrated controls. When rats were castrated and lesions placed at 14 days following castration, plasma levels of FSH, LH and prolactin were significantly decreased at 24 hr and at 7 days following surgery. Lesions which were placed lateral or caudal to the SDN-POA simulated the effects of lesions placed within the SDN-POA upon plasma LH, FSH and prolactin, with lateral lesions being most effective. However, lesions which were placed dorsal or rostral to the SDN-POA had no effect. The results of these studies suggest that the SDN-POA may be involved in the regulation of LH, FSH, and prolactin release.

Gonadal steroid regulation of pineal melatonin synthesis.

Abstract The regulation of gonadal function by the vertebrate pineal, primarily through the biological actions of melatonin has received much attention from investigators during the past decade (1–5). Melatonin is synthesized from serotonin through the activities of the pineal enzymes N-acetyl transferase (NAT) and hydroxyindole-O-methyl transferase (HIOMT). NAT converts serotonin to N-acetyl serotonin by coupling of the acetyl function from Acetyl Coenzyme A to serotonin. HIOMT transfers the methyl group from S-adenosylmethionine to N-acetyl serotonin, to form melatonin.

Pathway of testosterone biosynthesis in the testis of the marmoset Saguinus oedipus

These studies were undertaken to determine the principal pathway of androgen biosynthesis by the testis of the marmoset Saguinus oedipus. Testicular fragments (25 mg) were incubated at 37 degrees C in Krebs-Ringer bicarbonate buffer, pH 7.4, containing pregnenolone-7-3H (3beta-hydroxy-5-pregnen-20-one) or progesterone-7-3H. Duplicate fragments were incubated with each substrate for 30 min, one hr, three hr, or five hr, for a total of 16 separate incubations. Metabolites were separated by paper and thin-layer chromatography, with identity established by recrystallization to constant specific activities and 3H/14C ratios. Pregnenolone was readily metabolized to progesterone, 17alpha-hydroxyprogesterone, androstenedione (4-androstene-3, 17-dione) and testosterone. Progesterone was converted to 17alpha-hydroxyprogesterone, androstenedione and testosterone. 17alpha-hydroxyprogesterone was the predominant metabolite obtained from both substrates at one, three and five hrs of incubation. Neither 17alpha-hydroxypregnenolone (3beta-17-dihydroxy-5-pregnen-20-one) nor dehydroepiandrosterone (3beta-hydroxy-5-androsten-17-one) was detected in the incubates. These data suggest a predominant delta4 pathway with accumulation of 17alpha-hydroxyprogesterone in the testis of this primate specie.

Androgen biosynthesis by marmoset testes in vitro

These studies were undertaken to determine the major steroid metabolites formed from selected androgen precursors by the testis of the marmoset, Saguinus oedipus, a New World primate of the family Callitricadae. Testicular fragments (50 mg) were incubated for 3.0 hr with pregnenolone-7-3H or with progesterone-7-3H. The major metabolites formed from pregnenolone were 17α-hydroxyprogesterone (42.7%), testosterone (20.5%), androstenedione (11.4%) and progesterone (9.2%). Nonmetabolized substrate was 6.8% of radioactivity. For porgesterone incubations, 17α-hydroxyprogesterone was the major matabolite (49.0%), with testosterone (21.2%) and androstenedione (10.7%) as lesser metabolites. Unreacted progesterone accounted for 14.9% of all radioactivity. The unusually high levels of 17α-hydroxyprogesterone in marmosets is in contrast to that observed in other mammalian species.

Effect of melatonin in vivo upon FSH and LH release from hamster pituitary glands.

The effect of chronic daily afternoon injections of melatonin upon basal and melatonin-modulated release of FSH and LH was investigated in superfused hamster anterior pituitary glands. The basal release rate of both FSH and LH began to decline following the beginning of melatonin injections, and reached a nadir after six weeks. Basal release rate of FSH and LH then began to spontaneously increase and reached a plateau at 13 weeks of injections. The inhibition by melatonin upon FSH and LH release in vitro gradually declined during the period of melatonin injections. After six weeks of melatonin injections the release rate of FSH was no longer suppressed by melatonin superfusion, while the release rate of LH became refractory to melatonin suppression in vitro after nine weeks of melatonin injections. These results demonstrate a change in the release rates of both basal and melatonin-inhibited gonadotropin release during melatonin-induced testicular regression and recrudescence in hamsters.

Regulation of Pineal Enzymes by Photoperiod, Gonadal Hormones and Melatonin in Coturnix Quail

The photoperiodic and hormonal regulation of melatonin-synthesizing enzymes was determined in pineals of Coturnix quail. N-Acetyl transferase (NAT) and hydroxyindole-O-methyl transferase (HIOMT) were twofold higher in pineals of female and male Coturnix quail during exposure to darkness (16L:8D). Castration decreased pineal HIOMT activity in both female and male Coturnix, while selective gonadal steroids restored activity. NAT was not affected by castration or gonadal steroids. Implantation of melatonin into female Coturnix decreased both HIOMT and NAT activities. These results suggest that NAT is regulated primarily by photoperiodicity, while HIOMT activity is a consequence of the external perceptive environment and the internal hormonal milieu, with both enzymic activities modulated by the feedback inhibitory influence of endogenous melatonin.

Photoperiodic and gonadal steroid regulation of pineal hydroxyindole-O-methyl transferase and N-acetyl transferase in Coturnix quail.

Abstract The activities of the melatonin-synthesizing enzymes were determined in pineals of Coturnix quail in response to photoperiodicity and gonadal hormones. Both hydroxyindole-O-methyl transferase (HIOMT) and N-acetyl transferase (NAT) were two-fold higher during exposure to darkness in female and male Coturnix maintained in a gonad-stimulating photoperiod (16L:8D). Castration decreased HIOMT activity in both female and male Coturnix. Administration of diethylstilbestrol, estradiol benzoate and progesterone into castrated females, and testosterone propionate and androstenedione into castrated males, restored HIOMT activity similar to that of intact controls. NAT was not affected by castration or gonadal steroids. These results suggest that the activity of pineal NAT is regulated primarily by photoperiodicity, while HIOMT activity is a consequence of photoperiodic and gonadal steroid regulation.

Testicular steroidogenesis in the mature and immature baboon Papio anubis

Abstract The following studies were undertaken to compare testicular steroidogenesis in the mature and immature baboon. Testicular fragments (50 mg) were incubated for 3 hr with [7- 3 H]pregnenolone, or with [7- 3 H]progesterone. The mature testis formed more testosterone (4.6%), androstenedione (1.6%), and progesterone (28.5%) from pregnenolone than did the immature testis (0.6, 0.5, and 26.1%). The immature testis formed more 17α-hydroxyprogesterone (34.7%) and 20α-dihydroprogesterone (23.2%) from pregnenolone than did the mature testis. Similar conversions were obtained in progesterone incubates. 5α-Androstanediol was identified only in mature incubates. These results suggest that the mature baboon testis has greater C 17 -C 20 lyase, 17β-hydroxysteroid dehydrogenase, and 5α-reductase activities than the immature testis, while the immature testis has greater 20α-reductase activity.

Melatonin's differential inhibition of FSH and LH release from hamster pituitary glands

Melatonin initially inhibited FSH secretion from superfused adult male hamster anterior pituitary glands obtained at 8:30 a.m. However, there was a gradual rebound during melatonin superfusion followed by an overshoot above baseline when melatonin was discontinued (morning response). Melatonin continuously inhibited FSH secretion during superfusion of hamster anterior pituitary glands obtained at 3:30 p.m., with a rebound evident only when melatonin was withdrawn (afternoon response). Melatonin continuously inhibited LH secretion from these superfused pituitaries, with a return to baseline levels only upon melatonin withdrawal. Prior pinealectomy or constant light could reverse the FSH morning response to an afternoon response, and late-morning melatonin injections could reverse the FSH afternoon response to a morning response. Neither prior pinealectomy, constant light nor melatonin injections affected melatonin inhibition of LH secretion from superfused pituitary glands. These results suggest that melatonin differentially inhibits FSH and LH secretion from isolated superfused hamster anterior pituitary glands.