In vitro bioactive and immunoactive inhibin concentrations in bovine fetal ovaries and testes throughout gestation.

This study investigates the concentrations of inhibin in the bovine fetal ovary and testis throughout gestation (days 40 to 270/term) as determined by inhibin in vitro bioassay and RIA techniques. In addition, the expression of the inhibin alpha- and beta-subunits (beta A and beta B) in these tissues was evaluated by Northern blot analysis and in situ hybridization. Testicular concentrations of inhibin bioactivity and immunoactivity increased 2.5-fold (103 +/- 24.5 to 256 +/- 11.7 U/g wet weight; mean +/- SE) and 10.4-fold (139 +/- 56 to 1430 +/- 172) respectively, between 90 days and term. The corresponding ratio of inhibin biological: immunological activities (B/I ratio) decreased 8.3-fold (1.5 +/- 0.7 to 0.18 +/- 0.01). The concentration of ovarian bioactive inhibin increased significantly (P less than 0.05) 4.6-fold between 90 days and term (73.6 +/- 14.7 to 340 +/- 11.1 U/g wet weight), whereas the immunoactive inhibin concentration increased 10.3-fold between 120 and 210 days of gestation (7.2 +/- 1.9 to 40.0 +/- 8.7). The corresponding B/I ratio remained unchanged throughout gestation (8.3 +/- 2.4 to 12.5 +/- 4.0). Although the levels of alpha subunit mRNA in the testis and ovary increased over gestation, the levels of testicular beta A subunit mRNA remained low and unchanged. Ovarian levels of beta A subunit mRNA were also low but variable. Furthermore, no beta B subunit mRNA could be detected in gonadal tissue throughout gestation. alpha-Subunit mRNA was detected by in situ hybridization in the sex cords of the fetal testis and the granulosa cells of the fetal ovary while beta A subunit mRNA was detected only in the granulosa cells of the fetal ovary. It is concluded that inhibin is produced by the fetal testis and ovary and these tissue levels increase throughout gestation. The location of alpha- and beta A-subunit mRNA to the sex cords of the testis and granulosa cells of the ovary indicate that these cells are the primary source of inhibin production. The rapid fall in inhibin B/I ratio in testicular extracts over gestation is attributed to the production of an inhibin-related protein with limited or negligible biological activity.

Cellular localization of inhibin mRNA in the bovine ovary by in-situ hybridization.

Hybridization histochemistry has been used to detect the presence of mRNA for the alpha and beta A subunit of inhibin in tissue sections of the ovary of cows. 32P-labelled cDNAs, complementary to the bovine alpha or beta A subunit of inhibin or to a control segment of plasmid DNA (pBR 322), were used. The alpha subunit mRNA was located in the granulosa layer of antral follicles greater than 0.36 mm in diameter while the alpha and beta A subunit mRNA were both present in follicles of greater than 0.8 mm. In these latter follicles, the thecal layer hybridized with only the alpha subunit mRNA. No hybridization of the alpha or beta A subunit probe was found in the cells of the corpus luteum. Hybridization of both probes was abolished when the tissue sections were pretreated with ribonuclease (RNAse). The plasmid cDNA did not hybridize to any of the tissue sections. This study demonstrates that mRNA for the alpha inhibin subunit can be detected in granulosa and theca cells whereas the beta A inhibin subunit mRNA is restricted to the granulosa cells. These results provide evidence for an independent regulation of expression for the two subunits of inhibin.

Testosterone response of cryptorchid and hypophysectomized rats to human chorionic gonadotrophin (hCG) stimulation.

The testosterone responses to a single injection of hCG (100 i.u.) in hypophysectomized (hypox.), cryptorchid or sham-operated rats were followed over a 5-day period. In sham-operated rats, hCG induced a biphasic rise in serum testosterone, peaks being observed at 2 and 72 h. Reduced testis weights, elevated FSH and LH levels and reduced serum testosterone levels were found after 4 weeks of cryptorchidism, but hCG stimulation resulted in a normal 2 h peak in serum testosterone. However, the secondary rise at 72 h in cryptorchid rats was significantly lower than sham-operated rats. Reduced testis weight and undetectable serum FSH and LH levels together with decreased testosterone levels were found 4 weeks after hypophysectomy. Serum testosterone levels rose 2 h after hCG in comparison to hypox. controls but this peak was significantly reduced compared with sham-operated rats. The second rise in serum testosterone began on day 2, peaking on day 4 at levels comparable to that seen in sham-operated rats after hCG. The in vitro basal and hCG stimulated secretion of testosterone by cryptorchid testes was greater than that secreted by normal rat testes (518.0 +/- 45.9 and 3337.6 +/- 304.1 pmol per testis per 4 h compared with 223.6 +/- 24.9 and 1312.9 +/- 141.4 pmol per testis per 4 h for normal rat testes). In cryptorchid animals a single injection of 100 i.u. hCG resulted in a pattern of in vitro refractoriness similar to normal rats, lasting from 12 h to 2 days, during which testosterone secretion was reduced to near basal levels. The in vitro basal and hCG-stimulated secretion of testosterone by hypox. rat testes was severely diminished compared with normal rat testes. The temporal pattern of in vitro secretion of testosterone from hypox. rat testes mimicked the in vivo serum testosterone pattern seen in these animals. This study demonstrates important differences in the in vivo and in vitro testosterone response to hCG after testicular damage.

The temporal response of rat testes to hCG stimulation during sexual maturation.

Serum testosterone responses to a single sc injection of hCG (25 IU/100 g body weight) were monitored for 5 days in rats throughout sexual maturation (22-70 days). Two hours after hCG injection serum testosterone levels rose in 22, 37 and 53 day-old animals and remained elevated for 2 days, returning to control levels on day 3. This response differed markedly from the biphasic secretion of testosterone reported for adult animals. In 70 day-old animals the serum testosterone response approached that seen in adult animals. Testosterone levels were elevated 2 h after hCG injection (25.4 +/- 2.5 ng/ml) and declined significantly at 12 and 24 h to 17.1 +/- 1.0 and 16.1 +/- 3.4 ng/ml, respectively. Testosterone levels tended to increase again on days 2 and 3 (19.9 +/- 2.8 and 21.1 +/- 3.5 ng/ml, respectively) but the increase was not statistically significant. This response differed markedly to the biphasic secretion of testosterone reported for adult animals. In vitro patterns of basal and hCG-stimulated testosterone secretion by decapsulated testes following a single hCG injection also changed during sexual maturation. In 22 day-old animals the testes exhibited refractoriness to in vitro hCG stimulation at 12 h, but testes from 37 day old rats were refractory from 2 to 24 h. In vitro testosterone responses of testes from 53 and 70 day-old rats were similar to that reported for adult rats with a period of refractoriness from 12 h to 2 days. This study demonstrates that during sexual maturation in the rat alterations occur in the temporal patterns of testosterone secretion in vivo and in vitro following hCG stimulation.

Acute responses of Leydig cells to hCG: evidence for early hypertrophy of Leydig cells.

The cross-sectional area of Leydig cells has been studied in rats following a single injection of 100 IU hCG. Leydig cell size was not changed within 2 h of injection (89.73 +/- 3.02 micron 2) but had increased significantly by 12 h (114.76 +/- 3.57 micron 2). Leydig cell size continued to increase until 24 h when a maximum cross-sectional area of 186.30 +/- 4.72 micron 2 was measured. This area was significantly greater than that recorded after 1 week of daily hCG treatment (143.18 +/- 6.25 micron 2, P less than 0.05). At 48 and 72 h Leydig cell size decreased slightly and was not significantly different from that seen after chronic hCG treatment. A single injection of 100 IU hCG induced a biphasic serum testosterone response with peaks in serum testosterone at 2 h and 72 h, the intervening nadir suggesting a period of in vivo refractoriness. A corresponding period of Leydig cell refractoriness in vitro was also demonstrated 12 h following a single injection of 100 IU hCG and persisted for 48 h. Further injections of hCG on day 2 or 3 did not induce a second biphasic serum testosterone pattern or the accompanying period of refractoriness, indicating that Leydig cell refractoriness is a temporary phenomenon which cannot be maintained during persistent stimulation with hCG. The absence of further refractory periods to hCG during recurrent stimulation may be related to the trophic action of the hormone on Leydig cells.

Effect of oestradiol and tamoxifen on the testosterone response in male rats to a single injection of hCG.

A single s.c. injection of hCG (100 i.u.) produced a biphasic serum testosterone response in adult male rats, peaks being noted at 2 h (24 ng/ml) and 3 days (16 ng/ml). The levels fell to control during the intervening interval (8 ng/ml), although there were elevated levels of serum hCG. Maintenance of high oestradiol levels by a s.c. injection of 50 micrograms oestradiol benzoate given on Day 2 after the initial hCG injection failed to prolong the refractory period and the secondary peak of testosterone (16 ng/ml) occurred on Day 3. Administration of the antioestrogen, tamoxifen (2 mg or 3 micrograms), 24 h before or simultaneously with hCG did not prevent testicular refractoriness in vivo because serum testosterone levels still declined after 2 h to reach a nadir at 2 days. The basal in-vitro testosterone production by decapsulated testes from animals injected with hCG was enhanced at 2 h. Stimulation by hCG increased the amount of testosterone produced (X 1.5 that in controls). By 12 h basal production decreased and there was no further increment in testosterone in the presence of hCG. This refractoriness to further hCG stimulation prevailed until Day 3, but the total production of testosterone fell so that at 24 h and 2 days testes were producing basal amounts of testosterone. Testes recovered from refractoriness at 4 and 5 days, when basal and stimulated testosterone production were greater than in controls. Injection of 50 micrograms oestradiol benzoate at 2 days did not prolong the in-vitro refractory period and 2 mg or 3 micrograms tamoxifen had no effect on the in-vitro steroidogenic activity, since testes were still refractory to further hCG stimulation from 12 h to 3 days. The results of the present study do not support the hypothesis that oestradiol is involved in the hCG-induced refractoriness of the Leydig cell. The nadir between the peaks of serum testosterone in vivo corresponds to the period during which the testis is refractory to in-vitro stimulation by hCG.

Serum Testosterone response to single injection of hCG ovine-LH and LHRH in male rats.

A biphasic pattern of testosterone secretion in response to a single injection of 100 IU hCG has been observed in the rat. Serum testosterone increased from basal levels of 8.7 +/- 3.1 ng/ml (mean +/- SEM) to 23.0 +/- 1.4 ng/ml within 2 h of hCG-stimulation and returned to control levels by 2 days. A second, delayed, but significant increase in serum testosterone occurred, reaching a peak of 24.6 +/- 4.0 ng/ml at 3 days and declining to basal values at 5 days. To study this response further, lower doses of hCG were tried. Administration of 10 IU hCG produced a single peak of testosterone, which did not occur until 24 h. Differences in the serum testosterone response were related to the concentration of hCG measured in the serum after injection, as injection of 1 IU, which failed to increase serum hCG levels above detection, was also inadequate to increase serum testosterone. The response after stimulation with 500 micrograms ovine-LH or 0.1-10.0 micrograms LHRH was also evaluated. Injection of 500 micrograms ovine-LH produced a significant rise in serum testosterone reaching a peak at 2 h of 25.2 +/- 2.6 ng/ml and subsequently declining over the next 48 h to control levels where it remained for 5 days. Stimulation with doses of 0.1 - 10.0 micrograms LHRH produced rapid and short increase in serum LH concentration which induced peaks of testosterone up to 48.8 +/- 14.1 ng/ml 1 h post injection. No secondary peak of testosterone followed. Failure of ovine-LH and LHRH to produce a second testosterone peak suggests that this response may be due to a re-stimulation of the Leydig cell by elevated levels of hCG which persist until the fourth day after injection.