ABSTRACT

Inhibin and testosterone were measured in the serum of young and old men with proven fertility before and after stimulation with human chorionic gonadotrophin (hCG) in order to characterize endocrinological changes in senescence further. While there was a significant increase of both hormones in all young men, there was a decreased response of serum testosterone and an insignificant increase in inhibin in the older men. Although basal hormone levels and ejaculate parameters were not different, hCG stimulation revealed that there were decreased secretory capacities of Leydig as well as of Sertoli cells in old age.

In hypogonadal male Djungarian hamsters FSH alone can induce normal spermatogenesis. However, for the induction of mating behavior, supplementation with testosterone is necessary. We have here investigated, by in vitro fertilization, whether sperm produced by photoinhibited hamsters treated with FSH alone can fertilize without testosterone. Photoinhibited hypogonadal male Djungarian hamsters were injected daily with human FSH (10 IU; Fertinorm) for 5-7 weeks. The hormone stimulated regrowth of the testes. Neither body weight nor the weights of the androgen-dependent organs-epididymides, prostates, accessory glands-showed significant differences from photoinhibited controls; furthermore FSH treatment did not raise intratesticular or serum testosterone levels. In eleven out of the twelve FSH-treated photoinhibited hamsters, elongated spermatids were found in the testes; in five out of nine epididymides, sperm was found by histological examination. In two out of the twelve FSH-treated hamsters, the amount of sperm in the caudal part of the epididymis was sufficient for in vitro fertilization with oocytes collected from superstimulated females. These spermatozoa fertilized 16-29% of the oocytes. Spermatozoa from photostimulated controls produced similar levels of fertilization.

Abstract

Follicle-stimulating hormone is known to be highly heterogeneous in serum and in the pituitary. In the present study, we have partially separated different molecular species of human pituitary FSH and characterized their immunoreactivity and in vitro bioactivity. Pooled extracts of male (n=15) and female (n=9) human pituitary glands were chromatographed on a column of Sephacryl S-200 and FSH-containing fractions were fractionated by chromatofocusing in the pH range 4–6. FSH was measured in the individual fractions by an in vitro bioassay, based on the FSH-dependent aromatase activity of immature rat Sertoli cells, and by the following methods based on commercial kits: radioimmunoassay (RIA), immunofluorimetric assay (IFMA), immunoradiometric assay (IRMA), immunoenzymometric assay (IEMA). In each assay, the kit standard, calibrated against the 2nd International Reference Preparation (IRP) 78/549, and the International Standard (IS) 83/575 were run in parallel. The relative potencies of the kit standards in terms of IS 83/575 were: IFMA 3·08, IRMA 1·62, RIA 2·42, IEMA 1·45 and bioassay 1·14. After chromatofocusing, pituitary FSH eluted mostly in fractions with pH≈4·5, without sex-related differences. In both sexes ≈25% of bioactive material showed a pI<4 and eluted with 1 m NaCl. Although the same IS 83/575 was used in the various assays, the profiles of immunoreactive FSH were significantly different. The highest intermethod variability was observed in the case of male pituitary FSH. The relative biopotency of the different molecular species of FSH did not appear to change according to their pI but, rather, varied with the assay method and the standard. In terms of 2nd IRP 78/549 the activity profiles were similar but not identical to those obtained in terms of IS 83/575 and the ratios between the two values (IS:IRP ratios) were significantly different among the methods. No significant correlation was found between IS:IRP ratios and FSH concentrations or pH. These data suggest that: (1) all the methods have different affinities for standard and unknown and/or for different molecular isoforms of FSH; (2) overall, they perform more accurately when assaying female pituitary FSH, perhaps because of a closer resemblance between standard and unknown; (3) the variability of the IS:IRP ratios indicates a different affinity of the antibodies for IS 83/575 and the kit standard, highlighting the importance of the molecular composition of the reference preparations for the final result; and (4) the results do not support the commonly accepted concept of a higher in vitro biopotency of less acidic species of pituitary FSH.

Journal of Endocrinology (1994) 141, 359–367

ABSTRACT

Serum FSH levels in fertile and infertile men were determined by applying the Sertoli cell in-vitro bioassay and six different immunoassays. Bioassay and immunoassay estimates were significantly correlated (r ranging from 0·78 to 0·86; P<0·01). On average, all immunoassays measured lower FSH concentrations in samples with low FSH levels and higher FSH concentrations in those with high FSH levels compared with the bioassay. Ratios of bioactivity to immunoreactivity (B/I) were highest in fertile men and lowest in men with severe disturbances of testicular function. Depending on which immunoassay was used these differences were either significant or only marginal. Dose–response characteristics for WHO FSH standard preparation 78/549, used in the bioassay as well as in the immunoassays, were different between immunoassays and the bioassay, suggesting that decreasing B/I ratios with increasing FSH serum levels were method-related and reflected different slopes of the dose–response characteristics of the assays, rather than being true changes in the molecular composition of FSH. The present investigation underlines the necessity of choosing the immunoassay used for comparison with the bioassay carefully and of validating the system in regard to parallelism between dose–response characteristics. B/I ratios must be interpreted with great caution and previous studies which report changing B/I ratios in various endocrine situations may have to be reevaluated.

Journal of Endocrinology (1990) 127, 523–532

ABSTRACT

We reported previously that after a single injection of a gonadotrophin-releasing hormone (GnRH) antagonist to male monkeys, exogenous GnRH stimulated LH secretion in a time- and dose-dependent manner, indicating that GnRH antagonist-induced blockade of LH secretion resulted from pituitary GnRH receptor occupancy. The present study was performed to investigate whether GnRH can also restore a blockade of LH and testosterone secretion during chronic GnRH antagonist administration. Four adult male cynomolgus monkeys (Macacafascicularis) received daily s.c. injections of the GnRH antagonist [N-Ac-d-pCl-Phe^1,2,d-TRP³,d-Arg⁶,d-Ala¹⁰]-GnRH (ORG 30276) at a dose of 1400–1600 μg/kg for 8 weeks. Before the GnRH antagonist was given and during weeks 3 and 8 of treatment, pituitary stimulation tests were performed with 0·5, 5, 50 and 500 μg synthetic GnRH, administered in increasing order at intervals of 24 h. At 8 weeks, a dose of 1000 μg GnRH was also given. All doses of GnRH significantly (P < 0·05) stimulated serum concentrations of bioactive LH (3- to 8-fold) and testosterone (2·6- to 3·8-fold) before the initiation of GnRH antagonist treatment. After 3 weeks of GnRH antagonist treatment, only 50 and 500 μg GnRH doses were able to increase LH and testosterone secretion. Release of LH was significantly (P < 0·05) more elevated with 500 μg compared with 50 μg GnRH. After 8 weeks, only the highest dose of 1000 μg elicited a significant (P < 0·05) rise in LH secretion. Basal hormone levels just before the bolus injection of GnRH were similar (P > 0·10–0·80). This finding eliminated the possibility that the increasing doses of GnRH had primed the pituitary thereby resulting in higher stimulatory effects of the larger doses of GnRH.

In conclusion, the present data indicate that, even after prolonged exposure to a GnRH antagonist, the pituitary retains some degree of responsiveness to GnRH. This observation supports the view that the inhibitory effects of chronic GnRH antagonist treatment are also mediated, at least in part, by occupancy of the pituitary GnRH receptor rather than by receptor down-regulation.

Journal of Endocrinology (1992) 133, 439–445

ABSTRACT

The aim of the present investigation was to investigate the effects of testosterone on basal and gonadotrophin-releasing hormone (GnRH)-stimulated gonadotrophin secretion in the presence and absence of a GnRH antagonist in a non-human primate model (Macaca fascicularis). Orchidectomized animals were used in order to avoid interference by testicular products other than testosterone involved in gonadotrophin feedback. Concomitant and delayed administration of testosterone at doses that provided serum levels either within the intact range (study 1) or markedly above that range (study 2) did not influence the suppression of basal gonadotrophin release induced by the GnRH antagonist during a 15-day period. To assess the possible effects of testosterone treatment at the pituitary level (study 3) GnRH stimulation tests (500 μg) were performed before and on days 8 and 15 of treatment with high-dose testosterone and GnRH antagonist alone or in combination. Testosterone alone abolished the gonadotrophin responses to exogenous GnRH observed under pretreatment conditions. With GnRH antagonist alone, an increased responsiveness (P <0·05) to GnRH was seen on day 8 and a similar response compared with pretreatment on day 15. Following combined treatment with GnRH antagonist and testosterone, GnRH-induced gonadotrophin secretion was consistently lower compared with that after GnRH antagonist alone (P <0·05), but was increased compared with that after testosterone alone (P<0·05). Thus, in the presence of a GnRH antagonist the feedback action of testosterone on LH and FSH was diminished. The present work in GnRH antagonist-treated orchidectomized monkeys demonstrates that (I) unlike in rats, testosterone fails to stimulate FSH secretion selectively, (II) the negative feedback action of testosterone on GnRH-stimulated LH and FSH secretion is altered in the presence of a GnRH antagonist and (III) GnRH antagonists induce a transient period of increased responsiveness of gonadotrophic hormone release to exogenous GnRH. The observation that a GnRH antagonist reduced the feedback effects of testosterone suggests that testosterone action on pituitary gonadotrophin release, at least in part, is mediated via hypothalamic GnRH.

Journal of Endocrinology (1991) 129, 363–370

We have investigated the antigonadotropic and antispermatogenic effects of exposure to a long-acting testosterone ester in the cynomolgus monkey model. Groups of five adult animals were exposed either to vehicle or to 10 mg/kg or 20 mg/kg testosterone buciclate (TB) over a 26-week period with injections given in weeks 0, 11 and 18. In week 26, testicular biopsy tissue was collected. Serum testosterone levels were in the upper normal range with 10 mg/kg TB and were approximately twofold higher with 20 mg/kg TB. The estradiol pattern followed that of testosterone and body weights increased in a testosterone-dependent manner. TB completely abolished serum LH bioactivity. Serum concentrations of FSH and inhibin-alpha were suppressed in a TB dose-dependent manner. During weeks 4-8 after the first injection, a rebound of FSH and inhibin but not bioactive LH secretion occurred. This rebound was followed immediately by a restimulation of testis size and sperm numbers. After the next TB injections these parameters were once again suppressed. Nadir testis size was 30-40% of baseline and animals were severely oligozoospermic or transiently azoospermic. Consistent azoospermia was not achieved. Quantitation of serum inhibin B, proliferating cell-nuclear antigen staining and flow cytometric analysis of germ cell populations revealed pronounced suppression of spermatogenesis in both TB-treated groups whereas androgen receptor expression remained unchanged. Testicular androgens levels, determined in week 26, did not differ among all three groups and did not correlate with sperm numbers, histological and immunocytochemical findings. All suppressive effects were fully reversed during the recovery period. We have concluded that pronounced suppression of primate spermatogenesis seemingly requires inhibition of FSH rather than testicular androgen levels, at least in this preclinical non-human primate model. For the purpose of male contraception, FSH inhibition appears mandatory.

The effects of bovine inhibin, testosterone and GnRH on gonadotrophin secretion by primate pituitary cells were characterized in vitro using pituitaries from six male rhesus monkeys and one male cynomolgus monkey. The effect of inhibin on basal secretion of FSH and LH was investigated. Dose-response curves in monkeys and rats were compared. GnRH dose-response curves in the presence and absence of testosterone were also examined in monkeys. In monkey pituitary cells, testosterone at a concentration of 10(-7) M had no effect on LH or FSH secretion. Inhibin suppressed FSH secretion to 50.8% of that of controls with no effect on LH. In rats, FSH secretion was suppressed to 45.0% of that of controls with a median effective dose (ED50, 95% range) of 1.298 (1.064-1.584) U/ml, compared with 1.024 (0.7204-1.455) U/ml in monkeys. In monkey pituitary cells, LH release was stimulated 9.9-fold and FSH 3.3-fold by GnRH. Testosterone had no effect on basal or GnRH-stimulated gonadotrophin release. These results support the view that the pituitary is not the target organ for the negative feedback action of testosterone in the male. In vitro, inhibin is the major regulator of FSH secretion at the pituitary level.

Universitätsfrauenklinik, 74 Tübingen, and *II. Medizinische Universitätsklinik, Düsseldorf, Germany

(Received 9 May 1975)

We have reported previously changes of plasma dehydroepiandrosterone (DHA) and plasma dehydroepiandrosterone sulphate (DHAS) before and during pregnancy (Nieschlag, Walk & Schindler, 1974). There are strong indications that labour represents a stressful stimulus to the endocrine system. This is reflected in a rise of maternal plasma corticotrophin (ACTH), corticosteroids, free fatty acids, insulin, androgens and oestrogens (Migneon, Kenny & Taylor, 1968; Rivarola, Forest & Migneon, 1968; Lefebvre, Chapedelaine & Bolté, 1970; Nieschlag, Wombacher, Kremer & Martin, 1970; Kuwabara, Kihara, Arai & Sakamoto, 1971; Kauppila, Tuimala & Haapalahti, 1974). Steroid changes in cord blood during labour have also been noticed (Arai, Kuwabara, Kihara, Okinaga & Sakamoto, 1972). Determinations of DHA and DHAS concentrations in maternal blood during the course of labour are rare and do show a variety of results (Lefebvre et al. 1970; Gandy, 1971). Therefore, we

Meiosis constitutes a crucial phase of spermatogenesis since the recombination of genetic information and production of haploid round spermatids need to be achieved. Although it is well established that gonadotrophic hormones are required for completion of the spermatogenic process, little is known about the dynamic and kinetic aspects of development of spermatocytes into spermatids and its endocrine control in the primate. In this study, S-phase germ cells were labelled using 5-bromodeoxyuridine (BrdU) incorporation and were then followed throughout meiosis under normal conditions and following GnRH antagonist (ANT)-induced gonadotrophin withdrawal in a nonhuman primate model, the cynomolgus monkey (Macaca fascicularis). Adult animals received either vehicle (VEH, n = 4) or the ANT cetrorelix (n = 5) throughout 25 days. On day 7 all animals received a bolus injection of BrdU. A biopsy was performed after 3 h, one testis was removed 9 days later (day 16 of treatment) and the other testis after 18 days (day 25 of treatment). Serum testosterone and inhibin levels, and testis weight were reduced (P < 0.05) by ANT treatment. BrdU localized to pachytene spermatocytes 9 days after BrdU and to round spermatids 18 days after BrdU in both groups, demonstrating that BrdU-labelled pachytene spermatocytes had undergone meiosis. Flow cytometric analysis revealed that the relative number and number per testis of BrdU-tagged 2C and 4C cells were reduced significantly (P < 0.05) within 16 days of ANT treatment. Numbers of 1C cells were lowered by day 25. The cell ratio for 1C:4C was similar with VEH and ANT (P > 0.05). These findings indicate that ANT reduced the number of cells available for meiosis but did not alter the rate of transition into round spermatids. Unexpectedly, however, the stage-dependent progression of BrdU-tagged round spermatids was significantly (P < 0.05) retarded under ANT as seen from the frequency of tubules containing BrdU-labelled round spermatids. The average duration of spermatogenic cycle was slightly prolonged (9.8 days in the VEH group and 10.8 days in the ANT group (P = 0.09)). Since no atypical germ cell associations could be found, it remains unclear whether this slight prolongation is entirely due to altered spermatid progression or whether earlier phases are affected. We conclude for the nonhuman primate that (1) BrdU-labelling of premeiotic germ cells is suitable for tracing their meiotic transition into postmeiotic cells, (2) unlike in the rat, gonadotrophin suppression initially affects premeiotic cell proliferation and thus the number of cells available for meiosis, (3) the meiotic process continues quantitatively despite gonadotrophin deficiency and (4) prolonged gonadotrophin deficiency might alter the timing of germ cell development.