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G. P. Risbridger and D. M. de Kretser

Summary. The characteristics of the fetal and adult populations of Leydig cells from postnatal rat testes were compared by Percoll gradient centrifugation. A single peak of hCG binding, due to the presence of fetal Leydig cells, was obtained after purification of intertubular cells from 8-day-old animals. Two peaks of specific hCG binding were obtained after purification of intertubular cells from 15-day-old rats: it was confirmed by autoradiographic techniques that the hCG was bound by adult-like Leydig cells in one peak and fetal Leydig cells in the other. Similarly, intertubular cell preparations from 21- and 25-day-old rats resolved into two peaks of hCG binding; adult-like Leydig cells were observed in the first peak, but fetal Leydig cells were rarely observed in the second of these peaks. These results demonstrate the separation of two Leydig cell populations from intertubular cells obtained from animals aged up to 15 days. Thereafter the pattern of the hCG binding profile is similar but is not due to the presence of the same cell types. Therefore these results emphasize the necessity for morphological identification of cell types to permit the correct interpretation of the corresponding biochemical data.

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G. P. Risbridger, G. Jenkin, and D. M. de Kretser

Summary. Rat testicular interstitial fluid and hydroxycholesterol both stimulated testosterone production by isolated Leydig cells in vitro in a dose-dependent manner, but the dose—response lines were not parallel. The addition of cycloheximide blocked the stimulation by interstitial fluid but not that of hydroxycholesterol. Use of the compounds SU 10603 and cyanoketone (which inhibit 3β-hydroxysteroid dehydrogenase and 17α-hydroxylase respectively) or aminoglutethimide (which acts on the cholesterol side-chain cleavage enzyme) showed that the stimulatory factor(s) in interstitial fluid stimulated steroidogenesis at the cholesterol side-chain cleavage enzyme, before the conversion of pregnenolone. This enzyme is rate-limiting in the synthesis of testosterone by Leydig cells and a site of action of LH; therefore, these results support the view that an interstitial fluid factor may be involved in the paracrine regulation of testicular steroidogenesis.

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A. E. Drummond, G. P. Risbridger, P. C. O'Leary, and D. M. de Kretser

Summary. A single dose of EDS was given to mature male rats and interstitial fluid (IF) was collected to determine the potency of mitogenic and steroidogenic activities therein. The potency of the factor stimulating testosterone secretion in vitro by Percoll-purified Leydig cells was significantly elevated 2 weeks after EDS, whilst the potency of mitogenic activities (stimulation of DNA synthesis by BALB/c 3T3 cells) was not elevated until 4 weeks after EDS treatment.

This study suggests that two separate factors, one with mitogenic and the other steroidogenic activity, may be involved in the response of Leydig cells after EDS administration. The mitogenic factor may play a role in Leydig cell regeneration whereas the testosterone-stimulating factor may be involved in testicular testosterone homeostasis.

Keywords: mitogenesis; interstitial fluid; steroidogenesis; Leydig cell

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Y. T. Sun, D. M. Robertson, G. Gonzales, G. P. Risbridger, and D. M. de Kretser

Summary. Intact and hypophysectomized rats were treated with graded doses of testosterone via subcutaneous Silastic implants over a 13-week period. Serum inhibin concentrations fell 50% (P < 0·001) after 2 weeks of hypophysectomy, remaining suppressed at this level for 13 weeks. The administration of testosterone to hypophysectomized rats (serum testosterone values 2–12 ng/ml; control values 5·5 ng/ml) was without effect on serum inhibin values. In contrast, administration of testosterone to intact animals for 7 weeks resulted in an initial fall (P < 0·05) in inhibin levels to 50–70% of controls then increasing to reach control levels at higher doses. Serum FSH concentrations were similarly biphasic with increasing dose of testosterone and values for these two hormones were significantly correlated (r = 0·44, P < 0·01). Segments of seminiferous tubules in culture from rats after various times of hypophysectomy showed a partly suppressed secretion of inhibin. The administration of testosterone did not modify inhibin production although inhibin production was sensitive to FSH.

It is concluded that (1) serum inhibin concentrations are partly suppressed after hypophysectomy and testosterone has no effect on serum inhibin values; and (2) the suppression of serum inhibin in intact rats treated with increasing doses of testosterone is attributable to the concomitant fall in serum FSH concentrations.

Keywords: inhibin; testosterone; hypophysectomy; rat

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D. C. Irby, J. B. Kerr, G. P. Risbridger, and D. M. de Kretser

Summary. Serum concentrations of LH, FSH and testosterone were measured monthly throughout the year in male bush rats. Testicular size and ultrastructure, LH/hCG, FSH and oestradiol receptors and the response of the pituitary to LHRH were also recorded.

LH and FSH rose in parallel with an increase in testicular size after the winter solstice with peak gonadotrophin levels in the spring (September). The subsequent fall in LH and FSH levels was associated with a rise in serum testosterone which reached peak levels during summer (December and January). In February serum testosterone levels and testicular size declined in parallel, while the pituitary response to an LHRH injection was maximal during late summer. The number of LH/hCG, FSH and oestradiol receptors per testis were all greatly reduced in the regressed testes when compared to active testes.

In a controlled environment of decreased lighting (shortened photoperiod), temperature and food quality, the testes of sexually active adult males regressed at any time of the year, the resultant testicular morphology and endocrine status being identical to that of wild rats in the non-breeding season. Full testicular regression was achieved only when the photoperiod, temperature and food quality were changed: experiments in which only one or two of these factors were altered failed to produce complete sexual regression.

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G. P. Risbridger, J. B. Kerr, R. Peake, K. A. Rich, and D. M. de Kretser

Summary. Adult rats were made bilaterally cryptorchid and studied at intervals of 3, 7, 14 or 21 days to study temporal changes in Leydig cell function. Serum FSH and LH levels were measured and the cross-sectional area of the Leydig cells assessed by morphometry. The function of the Leydig cells was judged by the binding of 125I-labelled hCG to testicular tissue in vitro and the testosterone response of the testis to hCG stimulation in vitro. By 3 days after cryptorchidism, the binding of labelled hCG to testicular tissue was significantly decreased compared to that of controls, but the testes were able to respond to hCG stimulation in vitro. At 7, 14 and 21 days after cryptorchidism, an enhanced testosterone response was observed and the size of the Leydig cells was significantly greater than that of the controls, which indicated increased secretory activity by the cryptorchid testis. Although serum FSH levels were significantly elevated after 3 days of cryptorchidism, serum LH levels did not rise until 7 days, thereby suggesting that the loss of receptors is unlikely to result from down-regulation by LH. The reduced testosterone response of the cryptorchid testis in vivo to low doses of hCG and the enhanced response at high doses are probably related to the reduced blood flow to the cryptorchid testis and the decreased sensitivity of the Leydig cells induced by LH/hCG receptor loss.

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G. P. Risbridger, A. E. Drummond, J. B. Kerr, and D. M. de Kretser

Summary. There was a significant (P < 0·05) and consistent increase in the potency of steroidogenic stimulatory activity (testosterone production by purified Leydig cells in vitro) in testicular interstitial fluid of the cryptorchid compared to the scrotal testis from 1 to 4 weeks after the induction of unilateral cryptorchidism. In contrast, the level of mitogenic activity ([3H]thymidine incorporation into 3T3 cells) was not significantly different between interstitial fluid from cryptorchid and scrotal testes for up to 4 weeks after surgery. These results indicate that the steroidogenic activity and the mitogenic activity are due to different, as yet unidentified, factors in testicular interstitial fluid.