Testosterone maintains pituitary and serum FSH and spermatogenesis in gonadotrophin-releasing hormone antagonist-suppressed rats

Groups of adult male rats were treated continuously for 30 days with either vehicle or the potent gonadotrophin-releasing hormone (GnRH) antagonist. (N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Trp3,D-hArg(Et2)6,D-Ala10 )- GnRH (RS 68439; 35 micrograms/day). In addition, groups of vehicle- and antagonist-treated rats received s.c. testosterone implants sufficient to maintain serum testosterone concentrations 3.5- to 5-fold higher than those of vehicle-treated control rats. After 30 days of antagonist treatment serum LH, FSH and testosterone concentrations were at or below the detection limits of their respective assays and pituitary FSH content and GnRH receptor binding were reduced, relative to control animals, by 77 and 98% respectively. Testis weight in antagonist-treated rats was reduced by 75% and spermatogenesis was suppressed to an extent comparable to that observed in hypophysectomized rats. Testosterone, which caused a 40% reduction in serum FSH relative to control animals, prevented the antagonist-induced fall in both serum and pituitary FSH, but not GnRH receptors, below that observed in the vehicle plus testosterone-treated group. Furthermore, spermatogenesis in the antagonist plus testosterone-treated group was indistinguishable from that observed in control animals. It is concluded that testosterone is capable of maintaining serum and pituitary FSH levels in vivo, under conditions which presumably render the pituitary insensitive to hypothalamic GnRH.     

Gonadotropin gene expression and secretion in gonadotropin-releasing hormone antagonist-treated male rats: effect of sex steroid replacement.

The possibility of direct pituitary effects of sex steroids on gonadotropin gene expression and synthesis was studied in male rats. The animals were treated with a potent GnRH antagonist, Ac-D-pClPhe-D-pClPhe-D-Trp-Ser-Tyr-D-Arg-Leu-Arg-Pro-D-Ala-+ ++NH2CH3COOH (Org 30276; 0.5 mg/kg BW, sc, twice daily) for 10 days. Groups of the antagonist-treated rats were implanted at the beginning of the injections with Silastic capsules containing testosterone (T), 5 alpha-dihydrotestosterone (DHT), or diethylstilbestrol (DES). Groups treated with the antagonist alone or vehicle served as controls. The antagonist treatment decreased unoccupied pituitary receptors of GnRH by 93% (P less than 0.001), serum LH by 34% (P less than 0.01), and serum FSH by 30% (P less than 0.05), and serum T became undetectable (less than 0.10 nmol/liter). Compared to antagonist treatment alone, no further effects on serum or pituitary LH levels found after steroid replacements. In contrast, the antagonist-induced decreases in serum and pituitary FSH (30% and 70%, respectively; P less than 0.05-0.01) were totally reversed by the T and DHT implants, but not by DES. Pituitary levels of the LH beta-subunit mRNA were decreased by 60% (P less than 0.01) after antagonist treatment. Combination treatment with androgens had no further effect on this mRNA, whereas DES partially reversed this suppression (P less than 0.05). In contrast, the pituitary mRNA level of the FSH beta-subunit, which decreased with antagonist treatment by 90% (P less than 0.01), returned to the control level with T and DHT replacements, but only partially with DES. The pituitary mRNA level of the common alpha-subunit was significantly suppressed only by combined antagonist plus DHT treatment (P less than 0.01). However, combination of DES with the antagonist increased alpha-subunit mRNA levels 2.4-fold (P less than 0.05) compared to antagonist treatment alone. It is concluded that the suppression of gonadotropin secretion by GnRH antagonist treatment is accompanied in male rats by a parallel reduction in mRNA levels of the gonadotropin beta-subunits. Sex steroid replacement of the antagonist-treated animals selectively reverses some of the mRNA changes. Androgens (T and DHT) increase the mRNA of FSH beta-subunit, but have no effect on the LH beta-subunit. Estrogen increases the mRNA levels of common alpha- and LH beta-subunits and slightly increases that of FSH beta.(ABSTRACT TRUNCATED AT 400 WORDS)     

Gonadotropin-releasing hormone regulates follicle-stimulating hormone beta-subunit gene expression in the male rat

Since the role of GnRH in the control of FSH release and synthesis is controversial, we have examined the effect of elimination of GnRH action on gonadotropes on FSH beta gene expression, FSH release, and synthesis. GnRH stimulation of the pituitary was abolished by continuous infusion of either a GnRH antagonist or a GnRH antiserum. We also examined the effects of gonadotrope desensitization, using a continuous infusion of GnRH or GnRH agonist analog. FSH beta mRNA levels were determined by dot blot hybridization using rat FSH beta cDNA, and changes were related to pituitary and serum FSH concentrations. FSH beta mRNA levels increased after orchidectomy and correlated well with serum FSH concentrations. Overall FSH synthesis was increased after castration, as judged by elevated serum FSH and unchanged pituitary FSH content. In orchidectomized rats, continuous GnRH antagonist infusion prevented the postcastration rise in FSH beta mRNA levels and serum FSH. Pituitary FSH content was reduced at 7 days, but not at 14 days. In intact rats, GnRH antagonist infusion for 7 days had no effect on FSH beta mRNA levels, but after 14 days, there was a 33% reduction, and serum FSH was suppressed. Pituitary FSH content was decreased after GnRH antagonist treatment for 7 or 14 days. Daily injection of GnRH antiserum for 6 days abolished the increases in FSH beta mRNA levels and serum FSH in orchidectomized rats, but pituitary FSH content was unaffected. In intact rats, GnRH antiserum treatment reduced FSH beta mRNA levels by 38%, suppressed serum FSH, and decreased pituitary FSH content. When gonadotropes were desensitized by a continuous infusion of GnRH for 14 days or GnRH agonist analog for 28 days, FSH beta mRNA levels and pituitary FSH content were markedly reduced, and serum FSH was suppressed to undetectable levels. We concluded that 1) endogenous GnRH is required for the maintenance of FSH beta mRNA levels in both intact and orchidectomized rats; 2) FSH beta mRNA levels are coupled to the level of FSH biosynthesis, indicating the physiological importance of this pretranslational regulation; 3) desensitization is more effective at inhibiting FSH beta gene expression and FSH synthesis than preventing gonadotrope stimulation using the GnRH antagonist or antiserum; and 4) the actions of GnRH on FSH beta mRNA levels are paralleled by its effects on LH beta mRNA levels, suggesting that GnRH provides a common primary stimulus for the induction of both beta-subunit genes in vivo. These data provide further evidence for the crucial stimulatory role of GnRH in the control of FSH synthesis.     

The role of androgen in the development of sexual differences in pituitary responsiveness to gonadotropin releasing hormone (GnRH) agonist in the bullfrog, Rana catesbeiana

Sexual differences in pituitary responsiveness to acute injection of gonadotropin-releasing hormone (GnRH) agonist, as measured by increments in plasma FSH and LH, were examined throughout development in the bullfrog, Rana catesbeiana. Untreated tadpoles, in various stages of metamorphosis, were unresponsive to GnRH agonist. Postmetamorphic males showed a progressive increase in the magnitude of pituitary response with age, whereas females remained relatively insensitive until after sexual maturation; males were always more responsive than females. Chronic (1–2.5 week) Silastic implants containing 5α-dihydrotestosterone (DHT) significantly augmented the pituitary response (for both gonadotropins) in intact postmetamorphic females at all ages; a similar, though less pronounced, action of testosterone in subadult females may have been due to its conversion to DHT. (Silastic implants of comparable size always produced higher circulating levels of DHT in females than in males as was observed in previous studies with gonadectomized frogs.) DHT enhanced the responsiveness of intact tadpoles (sexes undetermined); only the treated tadpoles responded to the GnRH agonist. Supplemental DHT did not enhance pituitary response in intact males; in fact, it attentuated the response in FSH. GnRH responsiveness paralleled changes in pituitary gonadotropin content; pituitary content of FSH and LH was higher in males than females; it showed a marked increase with age from tadpole to adult; and it was increased by DHT treatment. The potentiating effect of DHT on GnRH responsiveness and the significantly higher levels of DHT observed in males of all ages suggest that the nonaromatizable androgen DHT may be responsible for the early establishment and maintenance of sexual dimorphism in pituitary GnRH responsiveness in the bullfrog.     

Effects of androgens on bioactivity and immunoreactivity of pituitary FSH in GnRH antagonist-treated male rats

The effects of androgens on the bioactivity and molecular composition of pituitary FSH were examined in intact and GnRH antagonist-suppressed male rats. Eight groups of adult Sprague-Dawley rats were subjected to the following treatments: antagonist (75 micrograms/day by osmotic minipumps; sc), testosterone-filled Silastic implants (3 X 5 cm, sc), dihydrotestosterone-filled Silastic implants (3 X 5 cm, sc), E2 benzoate (15 micrograms/day, sc), and combined administration of antagonist with either steroid for 3 weeks. At the end of the treatment period, pituitaries were dissected out and homogenised. FSH content was determined in the pituitary extracts by an in vitro bioassay and a radioimmunoassay. Individual pituitary extracts from rats treated with vehicle, testosterone and testosterone + antagonist were subjected to isoelectric-focusing on sucrose density gradients performed in the pH range from 3.5 to 7.0. Individual isoelectric-focusing fractions (100-120) were analysed for bioactive and immunoreactive FSH. Treatment with antagonist, E2 or antagonist + E2 caused a significant decrease in pituitary FSH, whereas testosterone and dihydrotestosterone alone or in combination with antagonist prevented the decrease in pituitary FSH. The effects of all treatments on both bioactive and immunoreactive FSH were similar. Testosterone treatment not only maintained FSH synthesis but also altered the molecular composition of pituitary FSH. Following treatment with testosterone there was a shift of maximal FSH bioactivity to the more acidic pH range. On the other hand, less bioactivity was recovered than corresponding immunoreactivity in the higher pH region, resulting in significantly reduced ratios of bioactivity to immunoreactivity of FSH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exogenously administered testosterone maintains spermatogenesis quantitatively in adult rats actively immunized against gonadotropin-releasing hormone.

The administration of testosterone via Silastic capsules has been shown previously to maintain advanced spermatid number quantitatively in intact rats in which LH but not FSH was suppressed, but not in hypophysectomized rats, indicating that pituitary factors in addition to LH are required for the quantitative maintenance of spermatogenesis in the rat. The objective of the present study was to examine whether testosterone is capable of maintaining quantitatively normal spermatogenesis in rats in which both LH and FSH are suppressed. Intact adult male rats were actively immunized against GnRH by intradermal injection of GnRH conjugated to human serum globulin; control rats received intradermal injections of saline and adjuvant. Four weeks after the primary immunization, GnRH-immunized rats received the first booster injection and, at the same time, received testosterone-filled polydimethylsiloxane (PDS) implants of 4, 8, 12, or 24 cm or empty implants. Booster injections were repeated every 2 weeks for 8 weeks. At that time, rats were killed, and serum levels of LH, FSH, and testosterone, testicular advanced spermatid number, and seminiferous tubule fluid testosterone concentrations were determined. Four weeks after the initial administration of GnRH immunogen, i.e. before the first booster injection, serum levels of testosterone, LH, and FSH and the number of advanced spermatids per testis were not different from those in controls. Eight weeks after the first booster injection, serum LH and FSH and advanced spermatids were undetectable in all GnRH-immunized rats. The administration of testosterone-filled PDS implants of 4 and 8 cm to GnRH-immunized rats for 8 weeks resulted in the maintenance of 105 +/- 6 and 161 +/- 5 x 10(6) advanced spermatid/testis, respectively, significantly less than the control value (237 +/- 19 x 10(6)). In GnRH-immunized rats that received testosterone-filled PDS implants of 12 or 24 cm, the advanced spermatid numbers per testis (228 +/- 4 and 229 +/- 8 x 10(6), respectively) were not significantly different from those in controls. These results indicate that testosterone is capable of maintaining spermatogenesis quantitatively in the adult rats testis, in the absence of both radioimmunoassayable LH and FSH.     

Effect of dihydrotestosterone on pituitary follicle-stimulating hormone isoforms in adult male rats treated with a gonadotropin-releasing hormone antagonist

The effect of androgens on the oligosaccharide structure of follicle-stimulating hormone (FSH) isoforms was studied in adult male rats treated with a potent gonadotropin-releasing hormone (GnRH) antagonist. Animals were castrated (Cx), and 24 h later were treated with physiological doses of either testosterone propionate (T) or dihydrotestosterone propionate (DHT) (0.25 mg/rat in corn oil, daily, s.c.) for 7 days. The antiandrogen flutamide (F; 5 mg/day/rat, twice a day, s.c.) was administered to either Cx-T or Cx-DHT rats to block androgen action. The GnRH antagonist Org 30276 (Ant; 1 mg/kg/day, s.c.) was injected to both Cx- and Cx-DHT treated rats. FSH serum levels reached normal values in Cx rats treated with either T or DHT, whereas those treated with F retained Cx conditions. Both Cx-Ant and Cx-Ant-DHT treated animals presented normal serum FSH levels. Concanavalin A affinity chromatography was used to isolate pituitary FSH isoforms according to their carbohydrate inner structure. An increased proportion of FSH isoforms bearing complex-type oligosaccharides was observed in Cx rats treated with T or DHT, whereas the proportion of these isoforms was reduced in Cx and Cx-T-F or Cx-DHT-F rats. These results demonstrate that functional androgens are needed to complete the oligosaccharide processing of FSH. In addition, the proportion of the different pituitary FSH isoforms after DHT replacement was equivalent to that found when GnRH action was blocked, indicating that androgens are involved in the regulation of carbohydrate incorporation to the FSH molecule by acting mainly at the pituitary level, independently of GnRH action.     

Differential regulation of anterior pituitary prodynorphin and gonadotropin-subunit gene expression by steroid hormones.

Prodynorphin is expressed by neurons of the hypothalamus and gonadotrophs of the anterior pituitary gland (AP) and plays a role in the negative feedback regulation of the reproductive neuroendocrine axis. The present study examined whether gonadal steroid hormones are capable of modulating pituitary prodynorphin expression in immature, female rats. Steroids were administered via subcutaneous Silastic implants and rats were killed at 29 days of age. Northern blot analysis was used to measure AP prodynorphin, luteinizing hormone-beta (LH beta), follicle-stimulating hormone-beta (FSH beta), and common alpha-subunit mRNA levels (normalized to 18S ribosomal RNA). Treatment groups (n = 5-6) consisted of control (CNT; empty implants), estradiol (E2; 4 days), E2 + progesterone (E2 + P4; 8 days and 4 days, respectively), and dihydrotestosterone (DHT; 4 days). Pituitary prodynorphin mRNA was significantly suppressed in only the DHT-treated animals (26 +/- 10% of CNT, p < 0.01). LH beta mRNA was suppressed by all steroid treatments (p < 0.01), FSH beta was lower in only the E2 group, and alpha-subunit was reduced in both the E2 + P4 and DHT groups (p < 0.01). Serum LH was suppressed by all steroid treatments but FSH was reduced in only the E2 and E2 + P4 groups (p < 0.01). Treatment of prepubescent rats with continuous high levels of gonadal steroids is known to severely reduce endogenous hypothalamic gonadotropin releasing hormone (GnRH) release and this is supported by our observation of reduced gonadotropin-subunit gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression and recovery of pituitary gonadotrophin secretion in intact and orchidectomized rats treated neonatally with a gonadotrophin-releasing hormone antagonist

Suppression of neonatal rat pituitary-testis function by gonadotrophin-releasing hormone (GnRH) antagonists results in delayed sexual maturation and infertility. Since the mechanism is not understood, the acute effects of a GnRH antagonist on gonadotrophin secretion in neonatal male rats has been studied in more detail. Treatment with a GnRH antagonist analogue, N-Ac-D-Nal(2)1,D-p-Cl-Phe2,D-Trp3,D-hArg(Et2)6,D-Ala10 -GnRH (2 mg/kg per day) on days 1-10 of life had prolonged effects on gonadotrophin secretion; serum LH and FSH recovered in 1 week, but the pituitary content took 2 weeks to recover. Likewise, LH and FSH responses to acute in-vivo stimulation with a GnRH agonist were still suppressed 1 week after the treatment. Interestingly, a rebound (86% increase) in basal serum FSH was found 16 days after treatment with the antagonist. Whether testis factors influence gonadotrophin secretion during treatment with the GnRH antagonist and/or in the subsequent recovery period was also assessed. Neonatal rats were castrated on days 1, 5 or 10 of the 10-day period of antagonist treatment. Orchidectomy on days 1 and 5 only marginally affected gonadotrophin secretion. When orchidectomy was performed at the beginning of the recovery period, no effects on pituitary recovery were seen within 1 week of castration. After 16 days, serum LH and FSH in the antagonist-treated and control castrated rats were equally increased but the pituitary contents of the antagonist-treated rats were still suppressed. Finally, the effect of testosterone treatment on the recovery of gonadotrophin secretion after antagonist suppression was studied in intact and orchidectomized animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pituitary gonadotropin-releasing hormone (GnRH) receptor responses to GnRH in hypothalamus-lesioned rats: inhibition of responses by hyperprolactinemia and evidence that testosterone and estradiol modulate gonadotropin secretion at postreceptor sites

Increased hypothalamic GnRH secretion appears to influence positively the number of pituitary GnRH receptors (GnRH-R). GnRH-R increase after castration in male rats, and this rise can be prevented by testosterone (T), anti-GnRH sera, or hypothalamic lesions. GnRH also increases serum LH and GnRH-R in hypothalamus-lesioned rats, and these animals injected with exogenous GnRH are, therefore, a good model in which to study the site of steroid feedback at the pituitary level. Adult male and female rats were gonadectomized, and radiofrequency lesions were placed in the hypothalamus. Males received T implants, and females received estradiol implants at the time of surgery. Empty capsules were placed in the control animals. Beginning 3-5 days later, animals in each group were injected every 8 h with vehicle (BSA) or GnRH (0.002-200 micrograms/day) for 2 days. After these GnRH injections, all rats received 6.6 micrograms GnRH, sc, 1 h before decapitation to determine acute LH and FSH responses. GnRH-R were determined by saturation analysis using 125I-D-Ala6-GnRH ethylamide as ligand. In males, GnRH injections increased GnRH-R. T inhibited acute LH and FSH responses to GnRH in all groups, but had little effect on GnRH-R, indicating that T inhibits gonadotropin secretion at a post-GnRH receptor site. In females, the GnRH-R response to GnRH was less marked, and only the 200 micrograms/day dose of GnRH increased GnRH-R, indicating that the positive feedback effects of estradiol at the pituitary level are also exerted at a site distal to the GnRH receptor. There was no positive correlation between the number of GnRH-R and GnRH-stimulated gonadotropin release in males or females. Female rats with hypothalamic lesions had markedly elevated serum PRL levels (greater than 300 ng/ml). Suppression of PRL secretion by bromocryptine resulted in augmented GnRH-R responses to GnRH, and GnRH-R concentrations rose to the same values induced in males. This suggests that hyperprolactinemia inhibits GnRH-R responses to GnRH in females by a direct action on the pituitary gonadotroph.     

Suppression of pituitary-testis function in rats treated neonatally with a gonadotrophin-releasing hormone agonist and antagonist: acute and long-term effects

The acute and long-term effects of pituitary-testis suppression with a gonadotrophin-releasing hormone (GnRH) agonist, D-Ser(Bu(t))6des-Gly10-GnRH N-ethylamide (buserelin; 0.02, 0.1, 1.0 or 10 mg/kg body weight per day s.c.) or antagonist, N-Ac-D-Nal(2)1,D-p-Cl-Phe2,D-Trp3,D-hArg(Et2)6,D-Ala10 -GnRH (RS 68439; 2 mg/kg body weight per day s.c.) were studied in male rats treated on days 1-15 of life. The animals were killed on day 16 (acute effects) or as adults (130-160 days; long-term effects). Acutely, the lowest dose of the agonist decreased pituitary FSH content and testicular LH receptors, but with increasing doses pituitary and serum LH concentrations, intratesticular testosterone content and weights of testes were also suppressed (P less than 0.05-0.01). No decrease was found in serum FSH or in weights of accessory sex organs even with the highest dose of the agonist, the latter finding indicating continuing secretion of androgens. The GnRH antagonist treatment suppressed pituitary LH and FSH contents and serum LH (P less than 0.05-0.01) but, as with the agonist, serum FSH remained unaltered. Testicular testosterone and testis weights were decreased (P less than 0.01) but testicular LH receptors remained unchanged. Moreover, the seminal vesicle and ventral prostate weights were reduced, in contrast to the effects of the agonists. Pituitary LH and FSH contents had recovered in all adult rats treated neonatally with agonist and there was no effect on serum LH and testosterone concentrations or on fertility.(ABSTRACT TRUNCATED AT 250 WORDS)     

Testicular receptors for luteinizing hormone after immunoneutralization of gonadotrophin releasing hormone in the male rat.

The effects of immunoneutralization of endogenous gonadotrophin releasing hormone (GnRH) on the serum concentrations of testosterone and gonadotrophins and the binding of 125I-labelled human chorionic gonadotrophin (HCG) to testicular membrane fractions were studied in adult male rats. Four days after the administration of 1 ml anti-GnRH serum, the level of testosterone in the serum decreased to 44% of the concentration before the injection, whereas administration of normal rabbit serum had no effect. Multiple injections of anti-GnRH serum for 4 days dramatically suppressed the secretion of gonadotrophins in rats orchidectomized 2 months earlier. In intact male rats treated identically, immunoneutralization of GnRH decreased the level of serum testosterone to 32% of the concentration present in saline-treated controls, but did not decrease the number of testicular binding sites for HCG (LH). Administration of testosterone or oestradiol for 3 or 6 days caused a marked reduction in the concentration of serum gonadotrophins but did not decrease the number of LH receptors. This study provides further support for the concept that one releasing hormone governs secretion of both FSH and LH. In addition, these studies indicate that selective reduction of gonadotrophins for 3-6 days has no effect on the number of testicular LH receptors. This suggests that pituitary hormones other than gonadotrophins may be important in the maintenance of testicular receptors for LH.     

Regulation of pituitary gonadotropin-releasing hormone receptors by androgens in the male rabbit

The regulation of pituitary GnRH receptors was studied in adult male rabbits after castration and androgen replacement with testosterone (T) or 7 alpha-methyl-19-nortestosterone acetate (U-15,614; T analog) supplied by Silastic capsules implanted sc. Castration increased pituitary GnRH receptors significantly, from 99.3 to 329.5 fmol/mg protein within 4 weeks, without a change in the equilibrium association constant. Serum LH concentrations increased from 0.45 to maximum levels of 2.6 ng/ml by day 8 after orchiectomy; these levels persisted throughout the 4 weeks of study. Serum FSH reached maximum levels of 33.6 ng/ml 5 days after castration. T replacement with 250, 500, and 1000 micrograms/kg X day, prevented a postcastration rise in both pituitary GnRH receptor concentrations and gonadotropin secretion, while 100 micrograms/kg X day prevented an increase in GnRH receptors, but did not completely inhibit hypersecretion of gonadotropins. Administration of T analog at doses of 6.25 and 12.5 micrograms/kg X day partially suppressed the castration-induced increase in pituitary GnRH receptor concentrations, while 25, 50, and 100 micrograms/kg X day suppressed GnRH-binding sites to the levels found in intact controls in 15 of 16 rabbits. By contrast, none of the T analog doses was able to prevent completely LH and FSH hypersecretion. The fact that both T and T analog induced dose-dependent stimulation of prostate and seminal vesicle weights indicates that there are tissue-specific differences in the sensitivity to androgens. We conclude that in the male rabbit 1) pituitary GnRH receptors significantly increase after castration; 2) this increase may partially mediate the postcastration hypersecretion of LH and FSH; 3) castration-induced effects can be prevented by androgen replacement. These results are similar to those obtained in rats, where castration increases LHRH receptors, but contrast with results in mice and hamsters, where castration either reduces or does not change receptor levels. This indicates significant species differences in the response of pituitary GnRH receptor concentrations to elimination of the negative feedback effects of androgens.     

Testosterone stimulates pituitary and serum FSH in GnRH antagonist-suppressed rats

Thirty days of continuous treatment of adult male rats with 35 micrograms/day of the potent GnRH antagonist, (N-Ac-D-Nal (2)1, D-pCl-Phe2, D-Trp3, D-hArg (Et2)6, D-Ala10)-GnRH (RS-68439) reduced serum FSH to values below the limit of detection of the assay. Testosterone supplementation in the form of subcutaneous testosterone-filled silastic capsule implants present during an additional 30 days of GnRH antagonist administration restored serum FSH to values comparable to those observed after vehicle treatment. Pituitary FSH content, which was substantially reduced after GnRH antagonist treatment, was completely restored after concurrent testosterone supplementation. These results show that, under conditions of GnRH receptor blockade, testosterone is capable of stimulating pituitary and serum FSH in adult male rats.     

Daily administration of gonadotrophin-releasing hormone increases pituitary gonadotroph number and pituitary gonadotrophin content, but not serum gonadotrophin levels, in female rats on day 1 of dioestrus.

Gonadotrophin-releasing hormone (GnRH) has been shown to regulate the synthesis and release of gonadotrophins acutely, yet few studies have investigated the chronic effects of this agent on pituitary gonadotrophins. In the present study we determined the effect of chronic administration of GnRH on the female rat pituitary gland. Rats of 8 weeks of age were injected s.c. with various doses of GnRH daily for 30 days. After completion of the GnRH treatment, treated rats and age-matched controls were killed by decapitation at 09.00 h on the first day of dioestrus, as determined from vaginal smears. Treatment with 10 ng-10 micrograms GnRH/day increased pituitary contents of FSH and LH in a dose-dependent manner. The change in FSH content was much greater than that of LH content. The pituitary FSH content of rats treated with 40 micrograms GnRH was significantly less than that of rats treated with 10 micrograms GnRH. There was a marked increase in the number of cells which stained positively for FSH (266%) and LH (28%) in the anterior pituitary of rats given 10 micrograms GnRH, but there was no demonstrable change in the areas of single cells stained positively for FSH and LH. Serum levels of LH, FSH and oestradiol were not affected by the GnRH treatment. These data indicate that chronic administration of GnRH is capable of increasing the pituitary gonadotrophin content and numbers of FSH and/or LH-stained cells and that FSH cells are affected more than LH cells by the GnRH treatment. The increase in pituitary gonadotrophin content, however, does not necessarily produce an increase in circulating levels of gonadotrophins.     

The influence of chronic morphine treatment on the negative feedback regulation of gonadotropin secretion by gonadal steroids

The influence of continuous stimulation of opiate receptors with morphine (M) on the negative feedback effects of testosterone (T), 5 alpha-dihydrotestosterone (DHT), and 17 beta-estradiol (E2) on LH and FSH secretion was studied in rats that had been castrated 2 weeks previously. In the absence of gonadal steroids, 4 days of continuous M exposure did not alter LH or FSH levels. Similarly, Silastic capsules containing crystalline T (5 mm) or E2 [5 mm long (75 micrograms E2/ml) to 7.5 mm long (300 micrograms E2/ml)] alone had little effect on LH or FSH release. However, in M-exposed rats, T reduced serum LH by greater than 90%, and E2 reduced LH by more than 75%. Among the doses of DHT evaluated, only the highest dose (7.5-mm Silastic capsules packed with crystalline DHT) reduced LH secretion, and M exposure only slightly enhanced this suppression. M or gonadal steroids alone produced little change in FSH levels in castrated rats. However, the combination of M plus E2 or DHT further reduced FSH levels. Evaluation of pituitary responses to LHRH revealed that when administered alone, T did not alter, DHT reduced, and E2 enhanced the LH response to the decapeptide. Neither M treatment alone nor M plus T or DHT altered the pituitary LH response to LHRH. On the other hand, M appeared to enhance the stimulatory effects of E2 on pituitary responsiveness to LHRH. These findings suggest that the interaction of M and gonadal steroids at the level of the pituitary could not explain the observed marked suppression of gonadotropin secretion by suboptimal T or E2 during opiate receptor stimulation with M. Collectively, these observations are in accord with the view that endogenous opioid peptides may play a role in modulating the sensitivity of the hypothalamus to the negative feedback effects of gonadal steroids.     

Pituitary gonadotropin-releasing hormone receptor regulation in mice. I: Males

The regulation of pituitary GnRH receptors (GnRH-R) has been examined in male mice (C3H/HeH/101H F1 hybrid) after castration and testosterone replacement. GnRH-R were quantified in individual mouse pituitaries by equilibration with 125I(D-Ser(tBut)6) des Gly10 GnRH N ethylamide and compared with serum and pituitary LH and FSH concentrations. The equilibrium association constant was 2.7 X 10(9) M-1 for both intact and castrated male mouse pituitary GnRH-R. Six hours after orchidectomy there was a transient 50% reduction in GnRH-R; 13.6 +/- 3.8 fmol/pituitary (castrate) vs. 25.4 +/- 2.5 (intact). A subsequent partial return of binding sites began at 12 h, reaching a peak value of 18.2 +/- 1.5 fmol/pituitary (33% increase vs. 6 h) at 24-h post orchidectomy. This was followed by a gradual decrease in GnRH-R, reaching a plateau by 72 h. The decrease in GnRH-R was associated with a rapid (6-12 h) increase in serum LH and serum FSH. The pituitary GnRH-R concentration remained 45% below intact control values for up to 3 months and was accompanied by a persistent 5-fold rise in serum LH values. Treatment of male mice with testosterone propionate (TP), 25 micrograms/day, completely prevented the GnRH-R fall and the serum and pituitary LH responses to castration, whereas 12.5 micrograms/day TP produced variable results and 5 micrograms/day TP were ineffective. In another strain of mouse (BALB/c white). GnRH-R values also fell by 66% at 7 days post orchidectomy, with no change in the receptor affinity. In mice with androgen resistance from birth due to absence of androgen receptors (Tfm mice), GnRH-R were 14.45 +/- 0.49 vs. 19.8 +/- 1.67 fmol/pituitary in normal male littermates, and serum LH was 472 +/- 78 ng/ml compared with 52.5 +/- 11.7 ng/ml in normals. These findings are qualitatively similar to those in orchidectomized normal adult mice. Thus, in contrast to reports in rats, pituitary GnRH-R content falls after orchidectomy in mice. Possible explanations for this consistent finding include: persistent receptor occupancy by increased endogenous GnRH secretion, endogenous GnRH-induced receptor loss (down-regulation), or a species difference in the pituitary GnRH-R response to removal of negative steroid feedback, unrelated to changes in endogenous GnRH secretion.     

Cortisol regulates secretion and pituitary content of the two gonadotropins differentially in female rats: effects of gonadotropin-releasing hormone antagonist.

To determine if LH and FSH respond to cortisol exposure the same way in females as they do in males, metestrous females were implanted with cholesterol or cortisol (F) subcutaneously, and either ovariectomized or left intact 4 days later. Tail vein injections of 1000 ng of GnRH in saline, or saline alone, were given 4.5, 23.5, or 47.5 h after the time of ovariectomy. Animals were killed 30 min after the injections at 5, 24, and 48 h after surgery. F attenuated the postovariectomy increase in serum LH at 48 h. F also suppressed GnRH-stimulated LH release 24 and 48 h after surgery in ovariectomized animals and in intact animals at 48 h. Pituitary content of LH was increased moderately by F at 5 h. These effects of F are similar to those seen in males. In contrast to LH, F increased serum FSH in intact females and suppressed levels in ovariectomized animals at 24 and 48 h, while inducing a remarkable increase in pituitary FSH content at all three times. These divergent effects of F on serum FSH (suppression in gonadectomized and stimulation in intact groups) were not seen in males, and the increase in pituitary FSH as a result of exposure to F was much more profound and reliable in females than in males. To determine if the F-induced increase in pituitary FSH was dependent on endogenous secretion of GnRH, intact metestrous females were implanted with either cholesterol or F pellets. Each implant group received sc injections of 100 micrograms GnRH antagonist or control injections every 48 h beginning at the time of steroid implantation. Animals were killed 5 days after implantation. The antagonist suppressed both serum and pituitary LH. F also suppressed serum LH levels, but had no effect on pituitary content of LH. Neither the antagonist nor F affected serum FSH. F greatly increased pituitary content of FSH in the presence or absence of GnRH antagonist. These data suggest that 1) LH responds to F treatment in a similar way in females and males; 2) pituitary FSH content is more sensitive to the enhancing effect of F in females than in males; 3) the ability of F to increase pituitary FSH in females is not dependent on GnRH.     

Differential suppression of follicle-stimulating hormone and luteinizing hormone secretion in vivo by a gonadotropin-releasing hormone antagonist

Because of some indication that FSH secretion is less dependent than LH secretion on GnRH in vivo, we performed experiments to examine the effects of a GnRH antagonist (antag) on LH and FSH secretion. We first showed that pituitary cells superfused with GnRH showed a similar pattern of suppressed secretion of both LH and FSH in response to addition of antag. In contrast, antag administration to ovariectomized rats had differing effects on LH and FSH secretion. Serum LH was suppressed in a dose-dependent fashion by 2 h (20-50% of control values). Recovery from the lower doses of antag was seen by 12 h, but the two highest doses maintained serum LH levels at 10% of control values for 72 h. In contrast, the effect on serum FSH was not manifested until 12 h. FSH was maximally decreased only to 40-60% of control values. The two highest doses maintained this effect for 72 h. These results reinforce previous suggestions that FSH secretion in vivo may occur independently of acute changes in GnRH secretion, and may have an GnRH-independent component.     

Interdependence of oestradiol and 5 alpha-dihydrotestosterone in modulating LH and FSH secretion in rats

The effects of oestradiol, 5 alpha-dihydrotestosterone (DHT) and oestradiol plus DHT on pituitary responsiveness to LHRH were studied. Rats ovariectomized for 2 weeks were infused s.c. (by osmotic minipump) with LHRH at 250 ng/h for 6 days. Control rats received a sham s.c. pump. On day 3, silicone elastomer implants containing oestradiol or DHT were implanted s.c. and on day 6 the effects of these in-vivo treatments on pituitary LH and FSH content and on in-vitro (perifusion) LH and FSH secretion following maximal LHRH stimulation (1 microgram/ml perifusion medium) were assessed. Luteinizing hormone-releasing hormone alone decreased pituitary LH/FSH content and, in response to acute LHRH challenge in vitro, the absolute rate of LH/FSH release, but not LH/FSH release expressed as a fraction of pituitary content. Oestradiol alone increased pituitary LH/FSH content and LHRH-induced LH/FSH release in vitro, both absolutely and as a fraction of pituitary LH/FSH. Oestradiol exacerbated the decrease in pituitary LH/FSH caused by LHRH pretreatment in vivo, and decreased the absolute rate of LHRH-stimulated LH/FSH release in vitro, but increased this rate when it was expressed as a fraction of pituitary LH/FSH. In both LHRH-treated and control rats, DHT increased pituitary LH/FSH content, did not change the absolute rate of LH/FSH release in response to acute LHRH challenge in vitro, but decreased the rate of LH/FSH release expressed as a fraction of pituitary LH/FSH content.(ABSTRACT TRUNCATED AT 250 WORDS)