In vivo administration of a GnRH antagonist to male mice: effects on pituitary gonadotropin secretion in vitro.

The potent luteinizing hormone-releasing hormone antagonist [N-Ac-D-p-Cl-Phe1,2,D-Trp3,D-Arg6,D-Ala10]GnRH (4 mg/kg) was administered sc once or daily for 21 days to immune-deficient (nude) and normal immune-competent (NIC) male mice derived from the same genetic background. Effects of in vivo pretreatment with the antagonist on gonadotropin secretion from hemipituitary glands from both types of mice were studied in vitro in the presence or absence of synthetic GnRH. Treatment with the GnRH antagonist caused differential effects on release of FSH and LH from and amounts of FSH and LH in hemipituitary glands. Pituitary FSH secretion was effectively inhibited, whereas effects on pituitary LH were less evident or nonsignificant under these experimental conditions. Long-term treatment with the antagonist caused larger effects on pituitary secretion and content of FSH, when compared with short-term treatment. No significant effects of duration of treatment on secretion or pituitary content of LH were detected. Addition of synthetic GnRH to the incubation medium caused stimulation of gonadotropin release. Therefore, it was concluded that the high doses of this GnRH antagonist were not able to block GnRH receptors effectively in the pituitary glands of nude and NIC male mice. The incomplete suppression of LH secretion by this high dose of the GnRH antagonist may partly explain the inability of the antagonist to suppress plasma testosterone levels and the growth of androgen-dependent tumours in male mice.

Effects of treatment of neonatal rats with highly purified FSH alone and in combination with LH on testicular function and endogenous hormone levels at various ages.

Factors which play a role in the regulation of testicular size in rats were investigated using neonatal animals treated with exogenous gonadotrophins for 2 or 3 weeks, starting on the day after birth. Effects on testis weight and various aspects of the pituitary-testicular axis were evaluated up to the age of 9 weeks. Daily treatment with human FSH (Metrodin; 0.15 U/g body wt) for 2 or 3 weeks, starting on the first day or 1 week after birth, resulted in enlargement of the testes, increased testicular content of inhibin and a suppression of pituitary and plasma FSH. The relative increase of testis weight decreased after cessation of treatment. Injections of human FSH combined with administration of human LH (Pergonal) for 3 weeks, starting on the first day after birth, resulted in larger testes immediately after treatment. In addition, an increased amount of interstitial tissue was observed in these animals. Pituitary and plasma FSH and LH were suppressed after this treatment, while the growth of the accessory sex organs was significantly stimulated. In animals treated with human FSH during the first 2 or 3 weeks of life, levels of rat FSH in blood samples collected at weekly intervals were significantly suppressed until the animals were killed at the age of 9 weeks. In the animals treated with human FSH and human LH, both FSH and testosterone concentrations were significantly lower than those in control animals between the ages of 4 and 9 weeks. At the age of 9 weeks testicular weights were still higher than those in control animals after these treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibin-like activity in Sertoli cell culture media and testicular homogenates from rats of various ages.

The influence of age on testicular inhibin in untreated, neonatally hemicastrated and prenatally irradiated rats was studied using in-vivo and in-vitro experiments. In testicular cytosols prepared from 1-, 7-, 14-, 21-, 42- and 63-day-old rats concentrations of testicular inhibin could be measured with an in-vitro bioassay method using dispersed pituitary cells. Preparations of testicular cytosols caused a dose-dependent suppression of pituitary FSH secretion, whereas no effects were found on LH secretion. Testicular content of inhibin increased gradually with age, while after 14 days of age a relatively large increase of peripheral FSH concentrations occurred in all experimental groups. Neonatal hemicastration or prenatal irradiation resulted in decreased inhibin content of the testis and increased plasma FSH levels. The production of inhibin activity by Sertoli cells obtained from 7-, 14-, 21-, 42- and 63-day-old normal rats was measured during a 24-h incubation period on the third day of culture. The inhibin production per 10(6) plated Sertoli cells decreased rapidly after 14 days of age and the lowest production of inhibin was found in Sertoli cells from rats of 63 days of age. After pre-incubation with ovine FSH significantly larger amounts of inhibin activity were detected in spent media from 21-day-old rat testes. In contrast, suppression of inhibin production was found after pre-culture in the presence of testosterone at most of the ages studied. These data from in-vivo and in-vitro experiments indicate that a reciprocal relationship exists between pituitary FSH secretion and inhibin production before the age of 21 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration of an LHRH-antagonist to male mice: effects on in vivo secretion of hormones and on the growth of a transplantable human prostatic carcinoma.

The potent luteinizing hormone releasing hormone (LHRH) antagonist [N-Ac-D-p-Cl-Phe1,2,D-Trp3,D-Arg6,D-Ala10]-LHRH was chronically administered to male nude mice bearing the transplantable human hormone-dependent prostatic adenocarcinoma PC-82. Treatment of tumor-bearing male mice with a daily dose of 100 micrograms (4 mg/kg b w.) for 21 days did not significantly affect the growth of the PC-82 tumor tissue, or the weights of ventral prostate, seminal vesicles and testes. At 24 hours after the last dose of the antagonist the mean plasma-testosterone (T) value in these animals was not different from the control level. Administration of similar doses of the antagonist to intact normal immunocompetent male mice significantly reduced plasma LH concentrations and suppressed plasma-T to near-castrate levels, when blood was taken 2 hours after the last injection. At 24 hours after the last dose, however, plasma concentrations of LH and T had returned to control levels. This time-dependent pattern of T suppression by the antagonist was confirmed by a time-course experiment in animals receiving a single dose of the compound. These data demonstrate that a daily high dose of this antagonist cannot effectively suppress plasma-T in male mice. Therefore, the mouse may not be a suitable model for the investigation of the "castration-like" effect of LHRH-antagonists on androgen-dependent prostate xenografts.

In-vitro secretion of inhibin-like activity by Sertoli cells from normal and prenatally irradiated immature rats.

The influence of in-vitro conditions on the production of inhibin by Sertoli cells from 21-day-old normal and prenatally irradiated rat testes was studied by measuring inhibin activity in culture media, using the suppression of the release of FSH from cultured rat pituitary cells. Sertoli cells secreted inhibin-like activity during at least 21 days of culture, and cells cultured at 37 degrees C produced significantly more inhibin than those cultured at 32 degrees C. The presence of fetal calf serum had no significant effect on inhibin production at 32 degrees C, while at 37 degrees C the production was decreased. The presence of ovine FSH stimulated inhibin secretion, while inhibin concentrations in Sertoli cell culture media were decreased after the addition of testosterone. Testosterone, added together with ovine FSH, suppressed inhibin secretion when compared with the levels found in the presence of FSH alone. The presence of spermatogenic cells decreased the release of inhibin. From these results it was concluded that both Sertoli cells isolated from normal immature rat testes and those from testes without spermatogenic cells can secrete inhibin-like activity in culture. A number of discrepancies with in-vivo observations was observed. Therefore, it is likely that the in-vivo situation is too complicated for direct study of the regulation of inhibin production, because of mutual interactions between the testicular compartments.

Cell-cell interaction between rat Sertoli cells and mouse germ cells in vitro.

An antiserum against rat germ cell membranes was prepared, and after absorption with protein extracts of rat liver and kidney and mouse testis, this antiserum reacted only with rat germ cell membranes and juxtanuclear vesicles in rat Sertoli cells. Germ cell-free rat Sertoli cell monolayers were cultured in vitro. Freshly isolated mouse germ cells adhered to these monolayers within 1 h. After a minimum of 3 days of such a co-culture, immunofluorescence and immunoblotting revealed that the mouse germ cells had obtained rat-antigenic determinants in their membranes. Our results indicate that this appearance of rat-specific antigens on mouse germ cells is specific and inducible.

Influence of neonatal hemicastration on in-vitro secretion of inhibin, gonadotrophins and testicular steroids in male rats.

Pituitary secretion of FSH in male animals is regulated, at least partly, by a protein hormone, inhibin, which is produced by Sertoli cells in the testes. To establish at which age the role of testicular inhibin in the regulation of FSH secretion becomes apparent, groups of male rats were hemicastrated or sham-operated on day 1 of life and pituitary and testicular function were investigated in vitro at 21, 42 or 63 days of age. Testis weights were increased in hemicastrated rats at all ages studied. Peripheral concentrations of gonadotrophins generally showed a good correlation with the concentrations of FSH and LH measured in the medium of hemipituitary glands which were incubated in vitro at 37 degrees C in the absence or presence of LH-releasing hormone. Peripheral testosterone concentrations in hemicastrated animals were not significantly different from those in sham-operated rats at all ages studied. Steroid production by Leydig cells in vitro was not significantly influenced by hemicastration. The secretion of inhibin by Sertoli cells from 21-day-old hemicastrated rats was decreased while Sertoli cells from 42- and 63-day-old hemicastrated animals secreted slightly but not significantly more inhibin than Sertoli cells from sham-operated rats. It is concluded that although compensatory increases of testosterone and inhibin production at later ages make it difficult to draw conclusions about the relative importance of inhibin in the feedback regulation of FSH secretion at different ages, it is likely that inhibin plays a role in the feedback of FSH in immature, rather than in mature male rats.