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Background

Antagonistic analogues of GnRH for the treatment of prostate cancer may be used clinically in persons for whom return to fertility after such treatment is important or desirable. The purpose of this study was, therefore, to evaluate the effects of a long term treatment with orntide, a GnRH antagonist, on testosterone levels and fertility in male rats.

Methods

Two groups of male rats received either 120-day orntide microspheres (8.8 mg orntide/kg/120 days) or vehicle alone (control group). Serum orntide and testosterone levels in both groups were monitored at certain intervals for 9 months from the initiation of treatment. After recovery of normal serum testosterone levels in the treated animals, each rat was housed with two proven breeder, but drug-naive, females.

Results

All mates of treated rats achieved pregnancy as rapidly as the mates of control rats although two of the control rats did not sire a litter with either female and one sired only one litter. The mean size of the litters of treated (12.3 offspring per litter) and control (10.6 offspring per litter) were similar. All offspring were grossly normal morphologically and behaviorally during the time to weaning.

Conclusions

These results suggest that lack of fertility due to testosterone suppression is reversible after cessation of treatment with this GnRH antagonist.

Orntide acetate, a novel luteinizing hormone-releasing hormone (LHRH) antagonist, was prepared and evaluated in vivo in 30-day and 120-day sustained delivery formulations using a rat animal model. Orntide poly(d,l- lactide-co-glycolide) (PLGA) and poly(d,l- lactide) (PLA) microspheres were prepared by a dispersion method and administered subcutaneously in a liquid vehicle to rats at 2.2 mg Orntide/kg of body weight (30-day forms) or 8.8 mg Orntide/kg (120-day forms). Serum levels of Orntide and testosterone were monitored by radioimmunoassays, and a dose-response study at 4 closes (3, 2.25, 1.5, and 1.75 mg Orntide/kg) was conducted to determine the effective dose of Orntide. Microspheres with diameters between 3.9 and 14 μ were prepared. The onset and duration of testosterone suppression varied for different microsphere formulations and were influenced both by polymer properties and by microsphere characteristics. Microspheres prepared with 50∶50 and 75∶25 copolymers effectively sustained peptide release for 14 to 28 days, whereas an 85∶15 copolymer and the PLA microspheres extended the pharmacological response for more than 120 days. Increase in drug load generally accelerated peptide release from the microspheres, resulting in higher initial serum levels of Orntide and shorter duration of the release: In general, apparent release was faster in vivo than under in vitro conditions. Orntide microspheres effectively suppressed testosterone in rats, providing rapid onset of release and extended periods of chemical castration. Testosterone suppression occurred immediately after microsphere administration without the initial elevation seen with LHRH superagonists.

AIM: To evaluate the long-term effects of gonadotropin-releasing hormone (GnRH)-based vaccine on levels of GnRH antibody and testosterone, and vaccine-induced immunocastration on sexual behavior of male rats.

METHODS: The rats were treated with GnRH-PE40 intraperitoneally every other day for 12 wk. GnRH antibody and testosterone level in rat blood were determined by ELISA and radioimmunoassay, respectively. Morphological changes in testes and sexual behavior of rats were evaluated.

RESULTS: GnRH-PE40 induced a high production in GnRH antibody, decreased the serum testosterone level, testis atrophy and sexual function in rats.

CONCLUSION: Intraperitoneal administration of GnRH-PE40 produces structural and functional castration of male rat reproductive system by inducing anti-GnRH antibody.

Testosterone replacement is the choice of treatment in androgen-deficient osteoporosis. However, long-term use of testosterone is potentially carcinogenic. Eurycoma longifolia (EL) has been reported to enhance testosterone level and prevent bone calcium loss but there is a paucity of research regarding its effect on the bone structural parameters. This study was conducted to explore the bone structural changes following EL treatment in normal and androgen-deficient osteoporosis rat model. Thirty-six male Sprague-Dawley rats aged 12 months were divided into normal control, normal rat supplemented with EL, sham-operated, orchidectomised-control, orchidectomised with testosterone replacement, and orchidectomised with EL supplementation groups. Testosterone serum was measured both before and after the completion of the treatment. After 6 weeks of the treatment, the femora were processed for bone histomorphometry. Testosterone replacement was able to raise the testosterone level and restore the bone volume of orchidectomised rats. EL supplementation failed to emulate both these testosterone actions. The inability of EL to do so may be related to the absence of testes in the androgen deficient osteoporosis model for EL to stimulate testosterone production.

The purpose of this study was to develop and evaluate a dialysisin vitro release technique for peptide-containing poly(d, 1-lactide-coglycolide) (PLGA) microspheres (ms) that would correlate within vitro data. Using a luteinizing hormone- releasing hormone analogue (LHRH), Orntide acetate, solubility and stability were determined in 0.1 M phosphate buffer (PB), pH 7.4, and in 0.1 M acetate buffer (AB), pH 4.0, with highperformance liquid chromotography (HPLC), and peptide permeability through a dialysis membrane (molecular weight cut-off 300,000) was determined. Orntide ms were prepared by a dispersion/solvent extraction/evaporation method and characterized for drug content (HPLC), particle size distribution (laser diffraction method), and surface morphology (scanning electron microscopy).In vitro release was studied in PB using a conventional extraction method and with a new dialysis method in AB. Gravimetric analyses of polymer mass loss and matrix hydration, and peptide adsorption to blank PLGA ms (50∶50, Mw 28 022) were carried out in PB and AB upon incubation at 37°C. Serum Orntide and testosterone levels in rats after administration of Orntide ms were determined by radioimmunoassay. Orntide acetate solubility was influenced by pH; approximately 2.3 mg/mL dissolved in PB and >18 mg/mL in AB. Stability was pH- and temperature-dependent. The peptide was very stable at pH 4.0, 4°C, but degraded rapidly at pH 7.4,37°C. Peptide permeability through the dialysis membrane was accelerated by agitation and>95% equilibrium was reached within 48 hours. The overall release rate was higher with the dialysis method. Mass loss of the Orntide ms was faster in AB (50% loss in 3 weeks: 95% in 35 days) than in PB (65% in 35 days). In contrast, hydration after 35 days was 4-fold higher in PB. The nonspecific adsorption to blank ms was greater in PB (128 μ g Orntide/10 mg PLGA) compared with AB (<5 μ g Orntide/10 mg PLGA). Administration of 30-day Orntide PLGA ms to rats resulted in an initial serum Orntide level of 21 ng/mL after 6 hours and a Cmax of 87 ng/mL after 6 days. Testesterone levels were suppressed immediately after ms administration (3 mg Orntide/Kg) from 5.2 ng/mL to 0.3 ng/mL (after 24 hours) and remained suppressed for 38 days. Orntide acetate solubility and degradation kinetics were markedly influenced by pH of the buffer systems and mass loss; matrix hydration, as well as the nonspecific adsorption to blank ms, was pH-dependent. Thein vitro release profile obtained with the dialysis method in AB correlated well with thein vivo data, therepy providing a more reliable prediction ofin vivo performance.

D17DT consists of the GnRH decapeptide linked to diphtheria toxoid. The aim of this pilot study was to assess the tolerance of D17DT and the production of anti-GnRH antibodies from two doses, 30 and 100 μg, in patients with locally advanced prostate cancer. Twelve patients with histologically proven prostate cancer in whom hormonal therapy was indicated were recruited. Patients received either 30 or 100 μg given intramuscularly on three separate occasions over six weeks. Patients were followed up and blood was taken for estimation of serum testosterone, PSA and anti-GnRH antibody titre. Overall the drug was well tolerated. In 5 patients a significant reduction in serum testosterone and PSA was seen. Castrate levels of testosterone were achieved in 4 and maintained for up to 9 months. Patients with the highest antibody titre had the best response in terms of testosterone suppression. This study shows that it is possible to immunize a patient with prostate cancer against GnRH to induce castrate levels of testosterone. This state appears to be reversible. This novel form of immunotherapy may have advantages over conventional forms of hormonal therapy and further studies are warranted in order to try and increase the proportion of responders. © 2000 Cancer Research Campaign

The influence of testosterone on gonadotropin-releasing hormone (GnRH) secretion was assessed indirectly by altering the serum testosterone concentration of male rats and measuring GnRH release from their incubated hypothalami 1 wk later.

GnRH release from hypothalami of castrated rats was 13.4±1.2 (SE) pg/h, compared to 35.3±3.8 pg/h from hypothalami of intact rats (P < 0.001). GnRH release from the hypothalami of castrated rats treated with testosterone propionate, 100 or 500 μg daily, was 25.0±3.4 pg/h and 27.9±3.6 pg/h, which is significantly greater (P < 0.05 and P < 0.01, respectively) than that from hypothalami of castrated rats treated only with sesame oil.

A similar decrease in GnRH release from hypothalami of hypophysectomized rats and prevention of this decrease by treating the hypophysectomized rats with testosterone propionate is evidence that the observed effects of testosterone are not mediated via luteinizing hormone and(or) follicle-stimulating hormone secretion. Treatment of castrated rats with either dihydrotestosterone propionate or estradiol benzoate also prevented the decrease in GnRH release from the hypothalami of castrated rats.

We conclude that testosterone, dihydrotestosterone, and estradiol all prevent the decrease in GnRH release from hypothalami of castrated rats treated with these steroids. The possibility exists that these steroids may also maintain GnRH secretion in vivo.

Reproductive aging in males is characterized by a diminution in sexual behavior beginning in middle age. We investigated the relationships among testosterone, androgen receptor (AR) and estrogen receptor alpha (ERα) cell numbers in the hypothalamus, and their relationship to sexual performance in male rats. Young (3 months) and middle-aged (12 months) rats were given sexual behavior tests, then castrated and implanted with vehicle or testosterone capsules. Rats were tested again for sexual behavior. Numbers of AR and ERα immunoreactive cells were counted in the anteroventral periventricular nucleus and the medial preoptic nucleus, and serum hormones were measured. Middle-aged intact rats had significant impairments of all sexual behavior measures compared to young males. After castration and testosterone implantation, sexual behaviors in middle-aged males were largely comparable to those in the young males. In the hypothalamus, AR cell density was significantly (5-fold) higher, and ERα cell density significantly (6-fold) lower, in testosterone- than vehicle-treated males, with no age differences. Thus, restoration of serum testosterone to comparable levels in young and middle-aged rats resulted in similar preoptic AR and ERα cell density concomitant with a reinstatement of most behaviors. These data suggest that age-related differences in sexual behavior cannot be due to absolute levels of testosterone, and further, the middle-aged brain retains the capacity to respond to exogenous testosterone with changes in hypothalamic AR and ERα expression. Our finding that testosterone replacement in aging males has profound effects on hypothalamic receptors and behavior has potential medical implications for the treatment of age-related hypogonadism in men.

The effects of a long-acting gonadotropin-releasing hormone (GnRH) agonist, [D-Trp6]-GnRH (GnRH-A) on developmental profiles of plasma luteinizing hormone (LH), follicle stimulation hormone (FSH) and testosterone (T), and pituitary responsiveness to exogenous GnRH were studied in male Dutch Landrace x Large White crossbred pigs from 1 to 30 wk of age. Group 1 control animals (control; n = 12) were injected subcutaneously in the neck with vehicle at 1 and 16 wk of age. Group 2 animals (early treatment; n = 10) were injected with 600 micrograms [D-Trp6]-GnRH at 1 wk and with vehicle at 16 wk. Group 3 animals (late treatment; n = 8) were injected with vehicle and 3 mg GnRH-A at 1 and 16 wk, respectively. Group 4 animals (early plus late treatment; n = 9) were injected at both 1 and 16 wk with GnRH-A. Blood was collected by brachiocephalic puncture at weekly or biweekly intervals, and through brachiocephalic cannulae, to determine longitudinal profiles of LH, FSH and T, and plasma gonadotropin responses to intravenous injection of GnRH (0.1 microgram/kg), respectively. In control animals, LH and FSH declined over the first 5 wk of postnatal life and peaked again at 10-14 wk. Levels of both hormones were basal from 18 to 30 wk. Plasma T was high in the first week, declined progressively over the next few weeks and remained low until 24 wk when a transient increment was noted. The LH and FSH responses to acute GnRH stimulation were similar at 7 and 14 wk and declined significantly at 23 wk of age.(ABSTRACT TRUNCATED AT 250 WORDS)

The in vivo antiandrogenicity of Casodex has been confirmed and characterised. Androgen receptor (AR) binding assays of rat ventral prostate gland cytosols revealed a relative binding affinity (RBA) for the AR of 0.267 and a k1 of 1.25 x 10(-7) M for Casodex. In addition, the peripheral selectivity of Casodex relative to other non-steroidal antiandrogens was confirmed in that daily treatment of non-castrated rats with Casodex (25 mg kg-1) did not elicit any changes in serum LH and testosterone concentrations relative to vehicle-treated controls, whereas elevated serum LH and testosterone were observed in rats treated with flutamide (25 mg kg-1). The peripheral selectivity of Casodex in the intact male rat was related to the distribution of radiolabelled antiandrogen following intravenous injection. All tissues with the exception of the hypothalamus and cerebral cortex (CC) sequestered radioactivity such that the tissue:serum ratio (TSR) for the drug was greater than unity. In the testis, the TSR was less than unity 1 h after injection but approached unity 5 h after injection and was greater than unity 10 h after injection. This may be explained by the presence of a blood-testis barrier for the drug, resulting in delayed equilibration between the blood and testis tissue. By comparison, an order of magnitude lower amounts of radioactivity in the hypothalamus and CC were maintained for the 10 h period after injection. These data, together with known physicochemical properties of Casodex suggest that a blood-brain barrier exists for the drug which results in exclusion of this antiandrogen from central sites of androgen negative feedback and that this accounts for its peripherally selective antihormonal profile.

Testosterone has been shown to increase the volume of steroid-sensitive brain nuclei in adulthood in several vertebrate species. In male Japanese quail the volume of the male-biased sexually dimorphic medial preoptic nucleus (POM), a key brain area for the control of male sexual behavior, is markedly increased by testosterone. Previous studies assessed this effect after a period of 8–14 days but the exact time-course of these effects is unknown. We asked here whether testosterone-dependent POM plasticity could be observed at shorter latencies. Brains from castrated male quail were collected after 1, 2, 7 and 14 days of T treatment (CX+T) and compared to brains of untreated castrates (CX) collected after 1 or 14 days. POM volumes defined either by Nissl staining or by aromatase immunohistochemistry increased in a time-dependent fashion in CX+T subjects and almost doubled after 14 days of treatment with testosterone while no change was observed in CX birds. A significant increase in the average POM volume was detected after only one day of testosterone treatment. The optical density of Nissl and aromatase staining were also increased after one or two days of testosterone treatment. Activation of male copulatory behavior followed these morphological changes with a latency of approximately one day. This rapid neurochemical and neuroanatomical plasticity observed in the quail POM thus seems to limit the activation of male sexual behavior and offers an excellent model to analyze features of steroid-regulated brain structure and function that determine behavior expression.

Objective

To elucidate the physiologic mechanism responsible for the supraphysiologic gonadotropin release from the pituitary induced by GnRH-agonist in GnRH-antagonist-primed female rats.

Design

Controlled experimental intervention.

Setting

Government research facility.

Intervention

Forty oophrectomized rats were randomized into 4 groups of 10 and treated with: Group A) control vehicles; Group B) GnRH-a (leuprolide acetate; 1.7μg/kg BID) on day 4; Group C) GnRH-ant (Nal-Lys; 3mg/kg QD) days 1–4; or D) GnRH-ant (Nal-Lys; 3mg/kg QD) days 1–4 and GnRH-a (1.7μg/kg BID) on day 4.

Main Outcome Measure(s)

Immunohistochemical methods, Northern, and in situ hybridization to quantitate pituitary FSH-β, LH-β and GnRH-R mRNA and receptor protein levels in all treatment groups.

Results

Treatment with GnRH-ant was associated with increased storage of gonadotropin in the pituitary for FSH-β and LH-β, but mRNA levels were unchanged. GnRH-R mRNA decreased following GnRH-a treatment but remained stable in the GnRH-ant-treated groups. Levels of GnRH-R were decreased following GnRH-ant treatment.

Conclusions

These data indicate that the in vivo mechanism responsible for the exaggerated release of gonadotropins in the GnRH-ant primed, GnRH-a treated rat was an increase in releasable gonadotropin pools coupled with a reduction in GnRH-R, but receptor function was preserved.

Background

Testosterone (T) and the sympathetic nervous system each contribute to the pathology of hypertension. Altered blood vessel reactivity is also associated with the pathology of high blood pressure. The purpose of this study was to examine the effects of T manipulation in the regulation of resistance-sized blood vessel reactivity.

Methods

Adult spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) male rats at 8 weeks of age were used. The rats were divided into groups consisting of gonadally intact controls (CONT), castrate with sham implant (CAST) and castrate with T implant (CAST + T) (n = 6 to 12 per group). Following a short-term period of T treatment (approximately 4 weeks), plasma norepinephrine (NE) and plasma T were assessed by performing high-performance liquid chromatography and RIA, respectively. Resistance-sized mesenteric artery reactivity was assessed on a pressurized arteriograph for myogenic reactivity (MYO), phenylephrine (PE) responsiveness and passive structural mechanics.

Results

SHR and WKY males exhibited similar physiological trends in T manipulation, with castration significantly lowering plasma T and NE and T replacement significantly increasing plasma T and NE. T manipulation in general resulted in significant alterations in MYO of second-order mesenteric arteries, with T replacement decreasing MYO in SHR (P < 0.05) compared to CONT, T replacement increasing MYO, and CAST decreasing MYO in WKY rats (P < 0.001) compared to CONT rats. Additionally, PE-induced constriction was significantly altered in both strains following T treatment, with the effective concentration of PE to constrict the vessel to 50% of the total diameter significantly increased in the CAST + T SHR compared to CONT (P < 0.05). Comparisons of passive structural mechanics between SHR and WKY treatment groups indicated in SHR a significantly increased wall-to-lumen ratio and decreased circumferential wall stress compared to WKY treatment groups.

Conclusions

These data suggest that T and NE are involved in a complex interaction with both myogenic reactivity and structural alterations of resistance-sized blood vessels and that these factors likely contribute to the development and maintenance of hypertension.

A link exists between stress during pregnancy and altered hypothalamic–pituitary–adrenal (HPA) activity and behaviour in children. In the guinea pig, male offspring born to mothers that were exposed to stress during pregnancy demonstrated increased anxiety, basal cortisol levels and decreased testosterone concentrations. Testosterone is known to inhibit HPA function and anxiety behaviours. Therefore, we hypothesized that restoring plasma testosterone would ameliorate the differences observed in HPA function and behaviour. Pregnant guinea pigs were exposed to a stressor during the period of rapid fetal brain growth (prenatal stress, PS) or left undisturbed (control, C). Behaviour in an open-field and prepulse inhibition (PPI) of the acoustic startle reflex (ASR) was assessed in juvenile offspring. In adulthood, male offspring were divided into four groups: Control + sham gonadectomy (GDX), control + GDX + testosterone replacement, PS + sham GDX and PS + GDX + testosterone. Male offspring were retested in the open-field and PPI. Basal HPA activity was also assessed. As juveniles, PS males exhibited significantly lower ASR (P < 0.05) and elevated PPI. In adulthood, PS male offspring exhibited significantly decreased PPI (P < 0.02) and this was reversed by administration of testosterone. We also found that adult PS offspring exhibited significantly less activity in the open-field (P < 0.05) and administration of testosterone increased ambulatory activity in PS animals. Basal plasma adrenocorticotrophin hormone (ACTH) levels were significantly greater in PS animals and there was a trend towards reversal by administration of testosterone in PS males. In conclusion, prenatal stress results in male guinea pig offspring that exhibit age-dependent differences in ambulatory activity, sensorimotor gating and HPA activity. In adulthood, the behavioural changes are reversed by replacement of plasma testosterone.

A link exists between stress during pregnancy and altered hypothalamic–pituitary–adrenal (HPA) activity and behaviour in children. In the guinea pig, male offspring born to mothers that were exposed to stress during pregnancy demonstrated increased anxiety, basal cortisol levels and decreased testosterone concentrations. Testosterone is known to inhibit HPA function and anxiety behaviours. Therefore, we hypothesized that restoring plasma testosterone would ameliorate the differences observed in HPA function and behaviour. Pregnant guinea pigs were exposed to a stressor during the period of rapid fetal brain growth (prenatal stress, PS) or left undisturbed (control, C). Behaviour in an open-field and prepulse inhibition (PPI) of the acoustic startle reflex (ASR) was assessed in juvenile offspring. In adulthood, male offspring were divided into four groups: Control + sham gonadectomy (GDX), control + GDX + testosterone replacement, PS + sham GDX and PS + GDX + testosterone. Male offspring were retested in the open-field and PPI. Basal HPA activity was also assessed. As juveniles, PS males exhibited significantly lower ASR (P < 0.05) and elevated PPI. In adulthood, PS male offspring exhibited significantly decreased PPI (P < 0.02) and this was reversed by administration of testosterone. We also found that adult PS offspring exhibited significantly less activity in the open-field (P < 0.05) and administration of testosterone increased ambulatory activity in PS animals. Basal plasma adrenocorticotrophin hormone (ACTH) levels were significantly greater in PS animals and there was a trend towards reversal by administration of testosterone in PS males. In conclusion, prenatal stress results in male guinea pig offspring that exhibit age-dependent differences in ambulatory activity, sensorimotor gating and HPA activity. In adulthood, the behavioural changes are reversed by replacement of plasma testosterone.

To assess the correlation between the remaining serum testosterone and bone mineral density(BMD), and to determine the effect of exogenous testosterone on BMD in subjects with male hypogonadism, we evaluated the serum testosterone levels and BMDs of the femur neck, Ward's triangle and the spine(L1-4) in 20 subjects with Klinefelter's syndrome and 7 with hypogonadotropic hypogonadism before and after testosterone replacement. BMDs of the femur neck, Ward's triangle and the spine were below the age-matched normal mean at 77.8%(21/20), 74.1%(20/27) and 88.9%(24/27), respectively. There were significant differences in serum testosterone levels and the spinal BMD between the two groups and the BMD of the spine closely correlated with the serum testosterone level (R = 0.63, p < 0.001). Following a mean 11.8 +/- 4.9 months of testosterone replacement, the BMD at all sites increased significantly and the pretreatment difference in spinal BMD between the two groups disappeared. We conclude that, although testosterone may increases the bone density, it has a site-specific effect of maintaining and increasing the bone mass especially at the spine in male hypogonadism.

Aims—To examine the effects of oestradiol and testosterone on the early carcinogenic changes expressed in rat liver from the diethylnitrosamine (DEN), 2-acetylaminofluorene (AAF), partial hepatectomy (PH) model of hepatocarcinogenesis. 
Methods—Preneoplastic liver lesions were evaluated using immunohistochemical analysis of glutathione-S-transferase placental form (GST-P) expression; oestrogen and androgen receptor levels were measured by radioimmunoassay.
Results—Oestradiol administration to non-castrated DEN-AAF-PH treated males resulted in a decrease in the area of GST-P positive foci, while testosterone increased the serum oestradiol level and reduced the area. In males, castration alone and castration with oestradiol replacement significantly reduced the GST-P positive area, and increased the hepatic oestrogen receptor level. In DEN-AAF-PH treated females, castration with testosterone replacement was associated with a significant increase in the GST-P positive area and the hepatic androgen receptor level. 
Conclusion—These findings suggest that exogenous and endogenous oestradiol can suppress chemical hepatocarcinogenesis. It appears that oestrogen receptors may be involved in the inhibition of malignant transformation of preneoplastic liver cells, while androgens and androgen receptors are involved in hepatocarcinogenesis. 



Keywords: hormone receptor; testosterone; diethylnitrosamine; glutathione-S-transferase placental form; antioxidant

Background

In this study, we tested the hypothesis that 17β-estradiol contributes to testosterone-mediated restoration of baroreflex-mediated bradycardia in short-term (3 weeks) castrated rats. Further, a reported increase in serum testosterone after long-term (6 weeks) castration constituted a basis for testing the hypothesis that a spontaneous increase in serum testosterone or androstenedione in this model causes a commensurate increase in baroreflex-mediated bradycardia.

Results

Testosterone (1 week) replacement enhanced baroreflex-mediated bradycardia in short-term castrated rats without changing 17β-estradiol level. A spontaneous recovery of baroreflex-mediated bradycardia occurred following long-term castration, although circulating testosterone and androstenedione remained suppressed.

Conclusion

The data suggest: 1) 17β-Estradiol does not contribute to testosterone restoration of the baroreflex-mediated bradycardia in short-term castrated rats. 2) The long-term modulation of baroreflex-mediated bradycardia occurs independent of androgens, or the baroreflex mechanism may become adapted to low levels of circulating androgens.

The pattern of the gonadotropin-releasing hormone (GnRH) stimulus is critically important in the regulation of pituitary gonadotropin secretion and continuous infusions down-regulate secretion while intermittent pulses maintain luteinizing hormone (LH) and follicle-stimulating hormone (FSH) responsiveness. We examined the effects of pulsatile GnRH administration on pituitary GnRH receptors (GnRH-R) and gonadotropin secretion in the presence of physiological concentrations of testosterone (T) to elucidate the mechanisms and sites of action of GnRH and T on the pituitary gonadotroph. Castrate male rats received one, two, or four testosterone (T) implants (serum T concentrations of 1.1, 2.4, and 5.2 ng/ml, respectively) to suppress endogenous GnRH secretion. Subsequently, intracarotid pulse injections of GnRH (5-250 ng/pulse) or saline in controls were given every 30 min for 48 h, after which gonadotropin responses and pituitary GnRH-R were measured. In control rats, the T implants prevented the rise in GnRH-R that was seen in castrates (empty implant--600 fmol/mg protein) and maintained receptors at the level that was present in intact animals (300 fmol/mg). Pulsatile GnRH administration increased GnRH-R in castrate T-implanted rats, but the response was dependent on the serum T concentration. With one T implant, increasing GnRH doses per pulse stimulated GnRH-R in a linear manner and the maximum receptor concentration (703 +/- 99 fmol/mg) was seen after the 250 ng GnRH dose. In the presence of two T implants, GnRH-R was maximal (705 +/- 45 fmol/mg) after the 25-ng dose and higher doses did not increase receptors above control values. With four T implants, GnRH doses of 5 ng induced a maximum response, 17-50 ng/pulse did not increase GnRH-R, but receptors were again increased by the 250-ng dose (633 +/- 86 fmol/mg). After 48 h of pulsatile GnRH administration there was no correlation between the number of GnRH-R and LH responses to GnRH. In rats with one or two T implants, LH responses were absent after all but the 250-ng doses. In contrast, LH responsiveness was not impaired in the presence of four implants. Thus, low dose GnRH pulses down-regulate LH secretion by an action at a post GnRH-R site, and this effect is regulated by testosterone. The results show that GnRH, given in a pulsatile manner, regulates its own receptor, and physiological increases in serum T produce a 50-fold increase in the sensitivity of GnRH-R stimulation by GnRH.

Images

In the rat, neonatal administration of testosterone propionate to a castrated male causes masculinization of behavior. However, if an intact male is treated neonatally with testosterone (hyper-androgen condition), male sexual behavior in adulthood is disrupted. There is a possibility that the hyper-androgen treatment is suppressing male sexual behavior by altering the male’s partner preference and thereby reducing his motivation to approach the female. If so, this would suggest that exposure to supra-physiological levels of androgen during development may result in the development of male-oriented partner preference in the male. To test this idea, male rats were treated either postnatally or prenatally with testosterone, and partner preference and sexual behavior were examined in adulthood. The principal finding of this study was that increased levels of testosterone during early postnatal life, but not prenatal, decreased male sexual behavior and increased the amount of time a male spent with a stimulus male, without affecting the amount of time spent with a stimulus female during partner preference tests. Thus, the reduction in male sexual behavior produced by early exposure to high levels of testosterone is not likely due to a reduction in the male’s motivation to approach a receptive female.

The lumbar spinal cord of rats contains the sexually dimorphic, steroid-sensitive spinal nucleus of the bulbocavernosus (SNB). Androgens are necessary for the development of the SNB neuromuscular system, and in adulthood, continue to influence the morphology and function of the motoneurons and their target musculature. However, estrogens are also involved in the development of the SNB system, and are capable of maintaining function in adulthood. In this experiment we assessed the ability of testosterone metabolites, estrogens and non-aromatizable androgens, to maintain neuromuscular morphology in adulthood. Motoneuron and muscle morphology was assessed in adult normal males, sham-castrated males, castrated males treated with testosterone, dihydrotestosterone, estradiol, or left untreated, and gonadally intact males treated with the 5α-reductase inhibitor finasteride or the aromatase inhibitor fadrozole. After 6 weeks of treatment, SNB motoneurons were retrogradely labeled with cholera toxin-HRP and reconstructed in three dimensions. Castration resulted in reductions in SNB target muscle size, soma size, and dendritic morphology. Testosterone treatment after castration maintained SNB soma size, dendritic morphology, and elevated target muscle size; dihydrotestosterone treatment also maintained SNB dendritic length, but was less effective than testosterone in maintaining both SNB soma size and target muscle weight. Treatment of intact males with finasteride or fadrozole did not alter the morphology of SNB motoneurons or their target muscles. In contrast, estradiol treatment was completely ineffective in preventing castration-induced atrophy of the SNB neuromuscular system. Together, these results suggest that the maintenance of adult motoneuron or muscle morphology is strictly mediated by androgens.

Spironolactone and potassium canrenoate are diuretics that are used widely for management of cirrhotic ascites. The administration of spironolactone frequently leads to feminization, which has been noted less frequently with the use of potassium canrenoate, a salt of the active metabolite of spironolactone. The use of these two drugs has been associated with decreases in serum testosterone levels and spironolactone with a reduction in androgen receptor (AR) activity. This decrease in AR has been cited as the cause of the anti androgen effect of these drugs. We therefore assessed the effect of both drugs on levels of androgen and estrogen receptors (ER) in the liver, a tissue that is responsive to sex steroids. Three groups of male rats (n = 12 rats each) were studied. Group 1 (control) received vehicle only; group 2 received spironolactone (5 mg/day); group 3 received potassium canrenoate (5 mg/day). After 21 days of treatment, the animals of all groups were killed and liver tissue was assayed for nuclear and cytosolic AR and ER, and for male specific estrogen binder (MEB), an androgen-responsive protein. Both drugs drastically decreased the nuclear AR content, as compared with the control group, but only spironolactone decreased cytosolic AR. When the total hepatic content of AR is considered, a highly significant decrease is observed only in rats treated with spironolactone. This reduction in hepatic AR content suggested loss of androgen responsiveness of liver. We confirmed this by assessing levels of MEB, and found that livers from group 2 animals had no detectable MEB activity, whereas livers from both group 1 and 3 had normal MEB activity. No changes were observed in nuclear ER and cytosolic ER of group 3 as compared with group 1. Nuclear estrogen receptor decreased and cytosolic ER increased in group 2, but with no change in total ER content. These results indicate that (a) only spironolactone appears to act as an antiandrogen in liver, resulting in a decrease in both AR and male specific estrogen binder content, and (b) neither drug results in elevated hepatic ER content, although spironolactone-treated animals show an altered subcellular localization.

The effects of castration and hormone treatment on cognitive performance were evaluated in male rats. Castrated animals received either testosterone or estradiol and were compared with gonadally intact animals and with castrated controls. Results revealed a dissociation between the effects of testosterone and estradiol on cognitive performance in male rats. Specifically, estradiol enhanced acquisition of a delayed matching-to-position spatial task, similar to previously published observations in females. In contrast, neither castration nor testosterone treatment had any significant effect on acquisition of the delayed matching-to-position task, but did appear to affect delay-dependent working memory. None of the treatments had any significant effect on acquisition of a configural association negative patterning task, suggesting that effects on the delayed matching-to-position task were not due to effects on motivational factors. These data demonstrate that, as in females, gonadal hormones influence cognitive performance in males and suggest that estradiol and testosterone affect distinct cognitive domains.

Isoflurane preconditioning neuroprotection in experimental stroke is male-specific. The role of androgens in the ischemic sensitivity of isoflurane preconditioned male brain and whether androgen effects are androgen receptor dependent were assessed. Male C57BL/6 mice were implanted with flutamide (androgen receptor antagonist), or castrated and implanted with testosterone, dihydrotestosterone, flutamide, letrozole (aromatase inhibitor), or vehicle 7–13 days before preconditioning. P450 estrogen aromatase wild-type and knockout mice were also evaluated. All mice were preconditioned for 4 h with 0% (sham preconditioning) or 1% isoflurane (isoflurane preconditioning) and recovered for 24 h. Mice then underwent 2 h of middle cerebral artery occlusion and were evaluated 22 h later for infarct volume. For neurobehavioral outcomes, sham and isoflurane preconditioned castrated male±dihydrotestosterone groups underwent 1 h of middle cerebral artery occlusion followed by 9 days of reperfusion. Isoflurane preconditioning neuroprotection relative to infarct volume outcomes were testosterone and dihydrotestosterone dose-specific and androgen receptor-dependent. Relative to long-term neurobehavioral outcomes, front paw sensorimotor function improved in isoflurane preconditioned mice regardless of androgen status while androgen replacement independently improved sensorimotor function. In contrast, isoflurane preconditioning improved cognitive function in castrates lacking endogenous androgens, but this improvement was absent in androgen replaced mice. Our findings suggest that androgen availability during isoflurane preconditioning may influence infarct volume and neurobehavioral outcomes in male mice following experimental stroke.

The effects of decreasing the frequency of pulsatile gonadotropin-releasing hormone (GnRH) stimulation on pituitary responsiveness were studied in (a) men with isolated GnRH deficiency who had achieved normal sex steroid levels during prior long-term pulsatile GnRH replacement and (b) perifused dispersed pituitary cells from male rats in the absence of sex steroids. In three groups of four GnRH-deficient men, the frequency of GnRH stimulation was decreased at weekly intervals from (a) every 2-3-4 h (group I), (b) every 2-8 h without testosterone replacement (group II), or (c) every 2-8 h with testosterone replacement (group III). In three groups of three columns of perifused dispersed pituitary cells, pulses of GnRH were administered every 2, 4, or 8 h. In groups I and II, mean area under the luteinizing hormone (LH) curve increased (P less than 0.025) and serum testosterone levels fell (P less than 0.035) as the frequency of GnRH stimulation was decreased. In group III, the area under the LH curve also increased (P less than 0.01) although serum testosterone levels were constant, thereby demonstrating that the increase in pituitary responsiveness to slow frequencies of GnRH stimulation occurs independently of changes in the sex steroid hormonal milieu. The area under the LH curve also increased in the perifused dispersed rat pituitary cells when the frequency of GnRH administration was decreased to every 8 h (P less than 0.05), thus demonstrating that the enhanced pituitary responsiveness to slow frequencies of GnRH stimulation is maintained even in the complete absence of gonadal steroids. Nadir LH levels fell in all three groups (P less than 0.01) as the frequency of GnRH stimulation was decreased. In contrast, mean peak LH levels, the rate of LH rise, and the rate of endogenous LH decay were constant as the frequency of GnRH stimulation was decreased. Finally, as the GnRH interpulse interval increased, mean LH levels fell, and mean follicle-stimulating hormone levels were stable or fell. These results indicate that (a) pituitary responsiveness to GnRH increases at slower frequencies of GnRH stimulation in models both in vivo and in vitro, (b) these changes in pituitary responsiveness occur independently of changes in gonadal steroid secretion, and (c) the increases in LH pulse amplitude and area under the curve at slow frequencies of GnRH stimulation are due to decreases in nadir, but not peak, LH levels. Slowing of the frequency of GnRH secretion may be an important independent variable in the control of pituitary gonadotropin secretion.

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