Castrated Animals Research Articles

Testosterone replacement increases fat-free mass and muscle size in hypogonadal men.

Testosterone-induced nitrogen retention in castrated male animals and sex-related differences in the size of the muscles in male and female animals have been cited as evidence that testosterone has anabolic effects. However, the effects of testosterone on body composition and muscle size have not been rigorously studied. The objective of this study was to determine the effects of replacement doses of testosterone on fat-free mass and muscle size in healthy hypogonadal men in the setting of controlled nutritional intake and exercise level. Seven hypogonadal men, 19-47 yr of age, after at least a 12-week washout from previous androgen therapy, were treated for 10 weeks with testosterone enanthate (100 mg/week) by im injections. Body weight, fat-free mass measured by underwater weighing and deuterated water dilution, and muscle size measured by magnetic resonance imaging were assessed before and after treatment. Energy and protein intake were standardized at 35 Cal/kg.day and 1.5 g/kg.day, respectively. Body weight increased significantly from 79.2 +/- 5.6 to 83.7 +/- 5.7 kg after 10 weeks of testosterone replacement therapy (weight gain, 4.5 +/- 0.6 kg; P = 0.0064). Fat-free mass, measured by underwater weighing, increased from 56.0 +/- 2.5 to 60.9 +/- 2.2 kg (change, +5.0 +/- 0.7 kg; P = 0.0004), but percent fat did not significantly change. Similar increases in fat-free mass were observed with the deuterated water method. The cross-sectional area of the triceps arm muscle increased from 2421 +/- 317 to 2721 +/- 239 mm2 (P = 0.045), and that of the quadriceps leg muscle increased from 7173 +/- 464 to 7720 +/- 454 mm2 (P = 0.0427), measured by magnetic resonance imaging. Muscle strength, assessed by one repetition maximum of weight-lifting exercises increased significantly after testosterone treatment. L-[1-13C]Leucine turnover, leucine oxidation, and nonoxidative disappearance of leucine did not significantly change after 10 weeks of treatment. There was no significant change in hemoglobin, hematocrit, creatinine, and transaminase levels. Replacement doses of testosterone increase fat-free mass and muscle size and strength in hypogonadal men. Whether androgen replacement in wasting states characterized by low testosterone levels will have similar anabolic effects remains to be studied.

M-Calpain Activation/Depletion is Associated With Androgen-Induced Reduction of Protein Kinase C and Proliferation of Male Accessory Sex Organ Smooth Muscles Cells

In the guinea pig seminal vesicle smooth muscle (SVM), androgen-dependent proliferation and terminal differentiation appear to be coupled to protein kinase C (PKC). This is based on the observations that both the soluble (cytosolic) enzyme and the Triton X-100 solubilizable form of the particulate enzyme were reduced during proliferation but were androgen-resistant in the amitotic state of adults. The purpose of the present investigation was to determine if the reduction in PKC activity was linked to the translocation of the activated enzyme to acceptor sites in the Triton X-100 insoluble fraction of the cell or reflected enzyme depletion due to proteolysis by androgen-dependent activation of the u- and/or m-calpains. SVM was harvested from treated animals, homogenized and separated into soluble and particulate components. The particulate material was further fractionated into Triton X-100 soluble and insoluble fractions. PKC activity was determined in all fractions using radioactive ATP. Cultures of pure smooth muscle cells from both human prostate and SVM were also employed to assess the role of calpain in smooth muscle growth. During androgen-induced proliferation, instead of a translocation of PKC activity to the Triton X-100 insoluble particulate fraction of the cell, PKC activity in this fraction was significantly reduced. The m-calpain was the only isoform detected in SVM. At the peak of androgen-induced DNA synthesis in pre-pubertal castrate animals, m-calpain decreased 45% whereas in proliferative resistant SVM of adult castrates the protease was not significantly affected by androgen treatment. In pure smooth muscle cultures from the SVM as well as human prostate glands calpeptin the cell permeable inhibitor of calpains produced a concentration-dependent inhibition (IC50 approximately equal to 35 microM) of cellular proliferation. Given that biochemical assays of calpain quantify the residual proenzyme and that upon activation calpain is rapidly degraded, our findings indicate that m-calpain activation occurs in association with androgen-induced degradation of PKC and SMC proliferation. Thus in vitro inhibition of m-calpain activation is antiproliferative. The relative resistance of m-calpain in adult SVM may be an important component of the terminal differentiation process in normal smooth muscle.

Neonatal exposure to diethylstilbestrol permanently alters the basal and 17 β-estradiol induced expression of c- fos proto-oncogene in mouse urethroprostatic complex

Perinatal estrogen exposure induces permanent structural and functional changes in the male reproductive tract. We have studied the effect of neonatal estrogenization on the estrogen-responsive c- fos proto-oncogene expression in mouse prostate. Fos is involved in growth and differentiation, and may play a central role in regulating diverse estrogen-related cellular differentiation. In adult control mouse prostate, basal c- fos mRNA expression is very low. Neonatal treatment with diethylstilbestrol on days 1–3 (neoDES) results in permanently increased fos expression in the prostatic urethra and all prostatic lobes. In adult castrated animals, estradiol induces a rapid transient increase in c- fos expression in the prostatic urethra, with maximum induction being higher in neoDES animals. In situ hybridization and immunohistochemistry show that in neoDES mice fos transcripts and protein are localized primarily in the epithelium of posterior periurethral prostatic collecting ducts. These are the sites previously reported to show the most pronounced morphological changes after estrogen treatment. Our results indicate that neonatal estrogenization affects both basal and estrogen stimulated c- fos mRNA levels in the prostate of mature mice, which supports the hypothesis that estrogen-induced morphological changes in mouse prostate may involve altered c- fos expression.

Androgens modulate the alpha-adrenergic responsiveness of vascular smooth muscle in the corpus cavernosum.

Rat penile erection is an androgen-dependent process with castration leading to a loss of potency. The present study was designed to determine if one of the mechanisms by which androgens maintain the erectile response is the regulation of the alpha-adrenergic responsiveness of cavernosal smooth muscle. Electrical stimulation of the major pelvic ganglion (MPG) was used to elicit erection in untreated, castrated rats (CASTRATE) or castrated rats given testosterone replacement (TESTO). The effects of phenylephrine (an alpha 1-adrenergic agonist) and prazosin (an alpha 1-adrenergic antagonist) on the erectile response were investigated. Phenylephrine, when administered to both TESTO and CASTRATE animals during erection, resulted in a dose-dependent decrease in the intracavernosal pressure (CCP) with an ED50 value of 1.8 +/- 0.48 micrograms/kg BW for TESTO rats; in the CASTRATE animals, the ED50 was significantly reduced to 0.29 +/- 0.08 microgram/kg BW. The increases in mean arterial pressure (MAP) resulting from phenylephrine injection in TESTO and CASTRATE animals were of similar magnitude and were not significantly different. Prazosin administration resulted in an enhancement of the erectile response in CASTRATE but not in TESTO animals. Taken together these results demonstrate that the cavernosal vasculature in CASTRATE animals possesses increased reactivity to alpha-adrenergic stimulation as compared to the sensitivity in TESTO rats. Based on these findings, we conclude that one of the mechanisms by which androgens maintain erectile function is by regulating the alpha 1-adrenergic responsiveness of the cavernosal smooth muscle.

Effect of castration on the VIPergic innervation and 125I-labelled vasoactive intestinal peptide (VIP) binding sites in the hamster seminal vesicle a quantitative immunohistochemical and receptor autoradiographic study

In the present work we have investigated the effects of medium-(15 days) and long-term (2 months) castration on vasoactive intestinal peptide (VIP)-immunoreactive nerve fibres and 125I-labelled VIP binding sites in the adult hamster seminal vesicle. The density of VIP- and synaptophysin (general neuronal marker)-containing nerve fibres was determined in immunofluorescently stained cryostat sections using a computerised image analysis system. The morphological analysis of 125I-VIP binding sites in seminal vesicle cryostat sections was performed by quantitative receptor autoradiography. Our results show that the densities of the overall (synaptophysin immunoreactive) and VIPergic innervation increase in both medium and long-term castrated animals. In absolute terms, the quantity of VIP- and synaptophysin- containing nerves is not altered in medium-term castrates, but decreases for synaptophysin in long-term castrates. Medium-term castration does not affect the density of 125I-VIP binding sites in the gland muscular coat, but a significant decrease is observed after long-term castration. In conclusion, our results indicate that whereas VIP nerves are apparently unaffected by castration. 125I-VIP binding sites in the muscular coat of hamster seminal vesicle are sensitive to androgen levels.

Androgen and pituitary control of penile nitric oxide synthase and erectile function in the rat.

Castration of adult male rats reduces by half the penile erectile response to electrical field stimulation (EFS) of the cavernosal nerve, and the activity of penile nitric oxide synthase (NOS). Both changes are prevented by androgen administration. We have now investigated whether other strategies of androgen ablation or competition may act as stronger inhibitors, and, if so, whether the stronger inhibition is due to the depletion of penile NOS content. Rats were castrated or left intact and were treated daily as follows: 1) intact, with the antiandrogen flutamide (25 mg/kg/day, i.p.); 2) castrated, with similar treatment; 3) castrated, with 17 beta-estradiol 3-benzoate (estradiol; via silastic tubing, s.c.). Additional groups of intact rats received injections of a GnRH antagonist (GnRHA, 1.25 mg/kg, s.c.), or were hypophysectomized and left untreated. Controls were untreated intact and castrated animals. After 7 days, rats were subjected to EFS, and the ratios between maximal intracavernosal pressure (MIP) and mean arterial pressure (MAP) were measured. Penile NOS activity and the contents of neuronal NOS (nNOS) and endothelial NOS (eNOS) were determined. Castration reduced the MIP:MAP ratio and penile NOS activity. Androgen receptor blockade with flutamide induced a similar response in intact rats. When flutamide treatment was combined with castration, the erectile response was nearly abolished, but NOS activity was not decreased below the values in castrated rats. Estradiol given to castrated rats and hypophysectomy or GnRHA treatment in intact rats diminished the erectile response below the level in castrated animals. In hypophysectomized rats, penile NOS activity fell below levels in castrated animals. contents of nNOS and eNOS were not significantly reduced by any treatment. These data suggest that penile erection in the rat is completely dependent on androgens, presumably because of their role in the maintenance of penile NOS activity and of other ancillary factors. However, only the complete blockade of residual androgen effects at the tissue level or a total androgen depletion can abolish the erectile response.

Endocrine and metabolite responses to porcine growth hormone administered by sustained release implant for different lengths of time in male pigs.

The effects of long term administration of GH on serum concentrations of hormones and metabolites was investigated in intact and castrate male swine. At 10 weeks of age, male swine were assigned to six treatments (n = 10/group): nonimplanted intact and castrate males; intact males implanted for 6 weeks, from 22-28 weeks of age; intact males implanted for 12 weeks, from 16-28 weeks of age; and intact and castrate males implanted for 18 weeks, from 10-28 weeks of age. Recombinant porcine GH was administered with sustained release implants designed to deliver a dose of 4 mg/day for 6 weeks. Throughout the study, blood samples were collected, and serum was harvested to quantitate circulating concentrations of glucose, urea nitrogen, GH, insulin, insulin-like growth factor I (IGF-I), IGF-II, and PRL. The pattern of administered GH in the serum suggests that the presence of testes and prior treatment with GH influence GH clearance. Somatotropin treatment elevated serum concentrations of GH and increased serum levels of glucose, insulin, IGF-I, and IGF-II in both intact and castrate animals. However, during the prepubertal period of 10-16 weeks, GH-treated intact males were resistant to the diabetogenic actions of GH, whereas significantly increased serum levels of glucose and insulin occurred in GH-treated castrates during this period. Changes in serum levels of IGF-I throughout the study and in insulin after the first 6 weeks followed the pattern of circulating GH concentrations in the treated animals. Serum concentrations of IGF-II were increased after GH administration, but, in contrast to the IGF-I response, IGF-II levels remained elevated as GH concentrations waned in the latter portion of the implant period. The maintenance of higher serum levels of IGF-II may be less dependent upon GH than are insulin and IGF-I. Administration of GH to intact males is more efficacious in altering metabolites and hormones, with the exception of IGF-I, during the peripubertal and postpubertal periods than during the prepubertal period.

Endocrine and metabolite responses to porcine growth hormone administered by sustained release implant for different lengths of time in male pigs

The effects of long term administration of GH on serum concentrations of hormones and metabolites was investigated in intact and castrate male swine. At 10 weeks of age, male swine were assigned to six treatments (n = 10/group): nonimplanted intact and castrate males; intact males implanted for 6 weeks, from 22-28 weeks of age; intact males implanted for 12 weeks, from 16-28 weeks of age; and intact and castrate males implanted for 18 weeks, from 10-28 weeks of age. Recombinant porcine GH was administered with sustained release implants designed to deliver a dose of 4 mg/day for 6 weeks. Throughout the study, blood samples were collected, and serum was harvested to quantitate circulating concentrations of glucose, urea nitrogen, GH, insulin, insulin-like growth factor I (IGF-I), IGF-II, and PRL. The pattern of administered GH in the serum suggests that the presence of testes and prior treatment with GH influence GH clearance. Somatotropin treatment elevated serum concentrations of GH and increased serum levels of glucose, insulin, IGF-I, and IGF-II in both intact and castrate animals. However, during the prepubertal period of 10-16 weeks, GH-treated intact males were resistant to the diabetogenic actions of GH, whereas significantly increased serum levels of glucose and insulin occurred in GH-treated castrates during this period. Changes in serum levels of IGF-I throughout the study and in insulin after the first 6 weeks followed the pattern of circulating GH concentrations in the treated animals. Serum concentrations of IGF-II were increased after GH administration, but, in contrast to the IGF-I response, IGF-II levels remained elevated as GH concentrations waned in the latter portion of the implant period. The maintenance of higher serum levels of IGF-II may be less dependent upon GH than are insulin and IGF-I. Administration of GH to intact males is more efficacious in altering metabolites and hormones, with the exception of IGF-I, during the peripubertal and postpubertal periods than during the prepubertal period.

Brain and Pituitary Melatonin Receptors in Male Rat during Post‐Natal and Pubertal Development and the Effect of Pinealectomy and Testosterone Manipulation

Using quantitative autoradiography, melatonin receptors have been studied during post-natal and pubertal development of the rat in 2 brain and 2 pituitary structures. In the pars distalis of anterior pituitary, melatonin receptors decrease gradually in density after birth and disappear in 30 day-old animals. In contrast melatonin binding is only expressed in the paraventricular nuclei of the thalamus at the age of 21-23 days and is always present in adult animals. In the suprachiasmatic nuclei and in the pars tuberalis of the pituitary, melatonin receptor density decreases after birth, remains stable for approximately 1 month and increases again at puberty to reach the birth values in the adult. This increase was absent in pinealectomized and in castrated animals but present in castrated animals receiving testosterone suggesting that it depends upon circulating testosterone and melatonin levels. These results show that melatonin receptors are differentially regulated during post-natal development in each of the 4 structures studied, and that melatonin and testosterone are 2 factors which could be involved in the regulation of melatonin receptor density in the suprachiasmatic nuclei and pars tuberalis.

Immunisation of goat bucks against GnRH to prevent seasonal reproductive and agonistic behaviour

The study was designed to test the efficacy of a commercially available vaccine, Vaxstrate ® (Arthur Webster Pty Ltd, Castle Hill, N.S.W.), to immunise adult male goats against GnRH. The general hypothesis tested was that immunisation of goats against GnRH using Vaxstrate ® would suppress gonadotrophin secretion, steroidogenesis and gametogenesis in the testis, production of male odour and agonistic behaviour between males. We also compared intervals of 2 and 4 weeks between primary and booster immunisations and monitored recovery from immunisation for approximately 1 year. Twenty bucks of feral origin obtained from a commercial feedlotter were allocated to one of four treatment groups ( n = 5): entire control (intact non-immunised bucks); Vaxstrate 2 (immunised with 1 ml of Vaxstrate ® on Days 0 and 14); Vaxstrate 4 (immunised with 1 ml of Vaxstrate ® on Days 0 and 28); and castrate control (surgical castration on Day 0). The level of immunity against GnRH was significantly elevated ( P < 0.01) 14 days following the booster immunisation compared with pre-treatment levels in both Vaxstrate 2 and Vaxstrate 4 treatments. Immunisation prevented the seasonal increase in LH and testosterone concentration seen in entire bucks, and resulted in lower concentrations of these hormones and FSH in vaccinated bucks from Day 28 ( P < 0.001). Scrotal circumference of immunised bucks was smaller than entire bucks from Day 56 ( P < 0.05). Bucks in the Vaxstrate 2 and Vaxtrate 4 treatments had lower ( P < 0.01) odour scores than entire controls from Day 42 and 56 respectively. Numbers of spermatozoa ml −1 were lower in immunised than entire bucks at Days 175 and 329 ( P < 0.05 and P < 0.01 respectively). Agonist confrontations recorded at Day 64 in vaccinated bucks appeared to be intermediate between that of surgically castrate animals and entire bucks. The results have shown immunisation against GnRH in goats is effective in suppressing the increase in testes size, male odour and agonistic behaviour that is associated with the seasonal breeding season. Immunisation against GnRH was successful in all goats and in 90% of goats the testes remained small for more than a year following primary immunisation. In this experiment, a 2 week interval was as effective as a 4 week interval between primary and booster immunisations.

Testosterone facilitates aggression by modulating vasopressin receptors in the hypothalamus

In many species, testosterone treatment facilitates offensive aggression tested in resident-intruder models. As the mechanisms of action of testosterone remain unclear, we hypothesized that testosterone interacts with neurotransmitter systems involved in the regulation of offensive aggression. We tested this hypothesis with the vasopressinergic system in golden hamsters in three separate experiments. First, we compared the density of V 1 vasopressin (AVP) receptor binding between castrated animals treated with testosterone and their untreated controls. The most noticeable difference was found within the ventrolateral hypothalamus (VLH), a site involved in the control of aggression in several species of mammals. Within this area, V 1 AVP receptor binding disappeared after castration, while being maintained by testosterone-treatment. Second, we tested behavioral effects of AVP within the VLH. Microinjections of ANT (100 nl, 1 or 100 μM) within the VLH accelerated the onset of offensive aggression in testosterone-treated animals. However, AVP-injected animals did not bite more than their vehicle-injected controls. Third, microinjections of AVP failed to activate offensive aggression in animals deprived of testosterone. As AVP receptors appeared to overlay previously described distributions of androgen and estrogen receptors in golden hamsters, we propose that testosterone facilitates the onset of offensive aggression, at least partly, through an activation of AVP receptors within the VLH.

Paradoxical effect of an aromatase inhibitor, CGS 20267, on aromatase activity in guinea pig brain.

To determine the effect of in vivo treatment of guinea pigs with a non-steroidal aromatase inhibitor (CGS 20267; letrozole), we treated subjects with subcutaneous Silastic implants containing crystalline letrozole. We studied four treatment groups: intact, intact letrozole-treated, castrate and castrate letrozole-treated. After treatment for 1 week, brain tissues (preoptic area, septum, medial basal hypothalamus, amygdala and parietal cortex) were removed, and microsomal aromatase activity (AA) was determined by an in vitro 3H2O assay using 1beta-3H-androstenedione as substrate. Kinetic experiments were performed to determine the competitive nature of letrozole and an approximate Ki was calculated. Letrozole appears to be a reversible, competitive inhibitor of aromatase activity with an apparent Ki of 1.2 nM. Aromatase activity in intact letrozole-treated animals was elevated compared to untreated controls in all brain areas tested (P< 0.05). Letrozole also stimulated AA in the brains of letrozole-treated castrated guinea pigs compared to untreated castrated animals (P< 0.05). These data indicate that letrozole administered in vivo causes an increase in AA. Possible mechanisms include an autoregulatory mechanism which is interrupted by enzyme inhibition, or an effect of the inhibitor on turnover rates of P450 aromatase.

Androgens modulate glucocorticoid receptor mRNA, but not mineralocorticoid receptor mRNA levels, in the rat hippocampus.

Androgen, mineralocorticoid and glucocorticoid receptors (AR, MR and GR, respectively) are ligand-activated transcription factors that alter gene expression and have a wide variety of effects in the central nervous system. High levels of AR, MR and GR mRNA have been found in the CA1 pyramidal cell region of the rat hippocampus and all 3 of these proteins bind to a similar hormone response element in DNA suggesting the possibility of common receptor function or cross-talk between these receptors at the level of transcription. To begin to investigate this hypothesis, we examined the regulation of AR, MR and GR mRNA expression in the rat hippocampus following treatment with androgens in combination with gonadectomy and/or adrenalectomy. Three-month-old male Sprague-Dawley rats were either castrated for 3 weeks, castrated and immediately implanted with 2 Silastic capsules filled with the non-aromatizable androgen, dihydrotestosterone, or left gonadally intact. Four days prior to sacrifice, these animals were either adrenalectomized or sham operated. GR, MR and AR mRNA were measured in the hippocampal subfields using in situ hybridization. In the CA1 region, dihydrotestosterone treatment of castrates decreased GR mRNA levels to 69 percent of levels found in gonadally intact rats and prevented the adrenalectomy-induced increases in GR mRNA observed in the gonadally intact and castrated animals. No changes in GR mRNA were observed in the CA3 region or dentate gyrus, where AR expression is low or absent. There was no effect of androgen treatment on MR mRNA levels nor did gonadectomy or androgen replacement alter the increases in MR mRNA following adrenalectomy. AR mRNA levels in the CA1 region were unchanged across all treatment groups. In vitro binding studies revealed almost complete nuclear occupancy of hippocampal AR in dihydrotestosterone-treated castrates. No appreciable in vitro binding of dihydrotestosterone to hippocampal MR or GR (Ki approximately 1500 nM) was observed which suggests that androgen regulation of GR mRNA in the hippocampus is occurring through AR binding. These data demonstrate a functional similarity of androgens and glucocorticoids in the regulation of GR mRNA levels in an area where AR and GR are colocalized. Androgen-mediated downregulation of GR expression may prove to be an important event in the adaptive responses of CA1 pyramidal cells to hormonal stimuli.

Effects of sex hormones on protein and collagen content of the temporomandibular joint disc of the rat

Purpose: The effect of sex hormones on the protein and collagen content of the temporomandibular joint (TMJ) disc of adult male and female rats. Materials and Methods: One hundred forty-four Wistar rats were assigned to 14 groups of 12 each. Two groups, one female and one male, served as a control and received no treatment, and two other groups (one female and one male) received a sham gonadectomy and placebo hormone. The remaining 10 groups (five males and five females) received either orchiectomy or ovariectomy, followed by administration of estrogen, progesterone, combined estrogen and progesterone, or testosterone. The total protein and collagen content of the TMJ disc were determined using the calorimetric hydroxyproline method. Results: The collagen content of TMJ discs of control males was statistically greater than the collagen content of the control female rats. This difference disappeared after ovariectomy of females and orchiectomy of males. Also, there was a general trend for a decrease in collagen and protein content to be produced by estrogen, progesterone, and by estrogen combined with progesterone in castrated male and female rats, and by orchiectomy of male rats. There was also a trend toward an increase in collagen and protein content after ovariectomy in female rats and administration of testosterone to castrated male and female rats. However, the only statistically significant effect of the drugs tested was that of estrogen combined with progesterone in ovariectomized female rats (a lowering effect on the total protein) and of estrogen alone in orchiectomized male rats (a lowering effect on the collagen content). Conclusion: Steroid sex hormones have an effect on the collagen and protein content of the TMJ disc of the rat as indicated by the difference in the values between control males and females and by the disappearance of this difference on castration of both male and female animals. This was also manifested by the significant effect of estradiol on collagen content of castrated males, by the effect of estrogen combined with progesterone on the protein content of castrated females.

Hormonal regulation of skeletal muscle hypertrophy in rats: the testosterone to cortisol ratio.

This study determined the influence that the catabolic hormone, corticosterone (C), and the anabolic hormone, testosterone (T), had in regulating skeletal muscle hypertrophy using the rat hind limb ablation model. Specifically, the ratio of T:C (TCR) was manipulated via hormone implants and injections and concentrations measured to evaluate the relative contribution of each hormone to skeletal muscle protein balance. Skeletal muscle growth was measured 16 days after gastrocnemius muscle ablation. Elevations in plasma concentrations of C (via daily C injections, 50 mg kg*kg(-1) body mass) resulted in TCR of 0.007 that was less than the control group TCR of 0.249. In this C-injected group, whole body and skeletal muscle atrophy was elicited-this being greater in the fast-twitch plantaris muscle than in the slow-twitch soleus muscle. The overloaded leg resisted the C-induced atrophy. Castration of animals (TCR 0.024) resulted in less whole body and skeletal muscle growth. However, elevations in plasma concentrations of T (two groups, with TCR of 1.35 and 1.64) did not result in significantly greater muscle growth. Furthermore, T was also ineffective in antagonizing the C-induced atrophy in a group that received both T implants and C injections. This group had a TCR of 0.175 that was similar to the control group ratio of 0.249 that received no manipulations. We concluded that glucocorticoids were able to induce pronounced atrophy, but at the same time overloaded muscles were able to over-ride the glucocorticoid signal. Plasma concentrations of C were a better predictor of muscle growth/atrophy than T and/or the TCR. In addition, it is suggested that the volume of contractile activity of the muscle is perhaps an important determinant of C-induced atrophy, because less atrophy occurs in the more active slow twitch muscles.

The effect of castration on steady state levels of luteinizing hormone-releasing hormone (LHRH) mRNA and proLHRH processing: time course study utilizing semi-quantitative reverse transcription/polymerase chain reaction.

Many studies have consistently shown that castration induces a prompt increase in serum levels and pituitary content of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), as well as a concomitant rise in steady state levels of the messenger RNAs directing their synthesis. The reports of effects of castration on the overall physiology of hypothalamic luteinizing hormone-releasing hormone (LHRH)--steady state levels of LHRH mRNA, post-translational processing and secretion--have, however, not been consistent. The goal of the studies reported here was to provide the first analysis of the effect of castration, at multiple postoperative time points, on steady state levels of LHRH mRNA and on the levels of hypothalamic proLHRH. All these data are correlated with hypothalamic levels of the mature LHRH decapeptide and with serum and pituitary levels of immunoreactive LH and FSH. Adult male rats were either castrated or sham-castrated (controls) and then sacrificed at 1, 3, 5, 7, 14, 21 or 28 days postoperatively. As expected, there was a prompt and sustained rise in serum immunoreactive LH and FSH in castrates compared with sham-operated animals. Intra-pituitary LH levels rose above levels in the sham-operated animals by 14 days post castration. Intra-pituitary FSH showed a biphasic response, first falling significantly below control levels, then rising above control levels at 21 days. Steady state levels of LHRH mRNA in castrates, measured by reverse transcription/polymerase chain reaction, were increased about 2-fold above control levels by 1 day postoperatively, but were virtually identical to control levels at each of the other time points despite marked changes in the gonadotropins. ProLHRH content in castrates was 1.8-times that seen in controls at 1 day post castration (P<0.05), concomitant with the rise in steady state levels of LHRH mRNA at that time point. However, proLHRH content in castrates was no different from that seen in controls at each of the later time points examined. LHRH content was unchanged through 7 days after castration, but then fell significantly to 57% of control levels in hypothalami from animals gonadectomized 14 to 21 days previously (P<0.001 vs control), and to 54% of sham-operated levels at 28 days postoperatively (P<0.001). We conclude that: (1) changes in steady state levels of LHRH mRNA after castration are small and transient and (2) increased proLHRH coupled with unchanged LHRH levels at 1 day post castration, and castrate animal proLHRH at control levels coupled with falling LHRH at later post-castration time points indicate that the effect of gonadectomy on post-translational processing of proLHRH to LHRH is, likewise, small and transient. In aggregate our data suggest that most of the increase in serum LH and FSH seen in male rats after castration is not mediated at the hypothalamic level.

CGP 53153: a new potent inhibitor of 5α-reductase

CGP 53153 ( N-2-(cyano-2-propyl)-3-oxo-4-aza-5α-androst-1-ene-17β-carboxamide) is a steroidal inhibitor of 5α-reductase, the enzyme which effects the conversion of testosterone (T) to 5α-dihydrotestosterone (DHT). In vitro, CGP 53153 competitively inhibited rat microsomal 5α-reductase from prostate by 50% (IC 50) at a concentration of 36 nM compared to the reference compound finasteride which inhibited 5α-reductase with an IC 50 of 11 nM in the same system. In vivo, inhibition of 5α-reductase activity was characterized in three different test systems. Inhibition of 5α-reductase activity was first assessed in a standard test designed to compare directly the potency of different 5α-reductase inhibitors. This test assesses potency through the inhibition of prostate growth in juvenile castrate male rats treated with a standard dose of T-propionate (1 mg/kg, s.c.) and a 5α-reductase inhibitor administered orally at various doses for 4 days. CGP 53153 and finasteride significantly reduced T-propionate-mediated prostate growth by about 25% (ED 25) compared to T-propionate-treated controls at oral doses of 0.01 and 0.1 mg/kg, respectively. Second, the effects on prostate weight were studied in normal adult male rats treated orally once daily for 14 days with 1, 3 and 10 mg/kg CGP 53153 and with 10 mg/kg finasteride. CGP 53153 significantly reduced prostate weight at 3 and 10 mg/kg by 31% and 37%, respectively, compared to vehicle-treated controls, whereas the dose of 10 mg/kg finasteride did not significantly reduce prostate weight. Third, the effects on prostate volume were studied in normal 6–9-year-old male dogs treated orally once daily with 5 mg/kg CGP 53153 and with 5 mg/kg finasteride for 12 weeks. Prostate volume was monitored with magnetic resonance imaging every 2 weeks beginning 6 weeks before start of the treatment with 5α-reductase inhibitors and ending after a recovery period of 8 weeks after termination of treatment. Treatment for 12 weeks with both CGP 53153 and finasteride was equally effective in reducing prostate volume by more than 70% in individual dogs. Anti-androgenic potency of CGP 53153 and finasteride was assessed in juvenile castrate male rats treated with DHT-propionate (1 mg/kg, s.c.) and a 5α-reductase inhibitor (p.o.) for 4 days. Neither CGP 53153 nor finasteride given at a dose of 10 mg/kg had any significant effect on DHT-propionate-mediated prostate growth, whereas the reference anti-androgen flutamide given at a dose of 10 mg/kg reduced prostate weight to levels comparable to those seen in untreated castrate animals. For CGP 53153, the dose of 10 mg/kg is 1000-fold higher than the ED 25 for 5α-reductase inhibition in vivo. In conclusion, both CGP 53153 and finasteride are potent inhibitors of the rat 5α-reductase enzyme system in vitro without showing any anti-androgenic effects in vivo. Both CGP 53153 and finasteride were equally potent in reducing prostate volume in aged male dogs, whereas in rats, CGP 53153 is up to 10 times more potent than finasteride in reducing prostate weight as shown in two different rat models.

Sexual dimorphism and sex steroid modulation of glial fibrillary acidic protein messenger RNA and immunoreactivity levels in the rat hypothalamus

By using the techniques of in situ hybridization histochemistry and immunocytochemistry, we have found that both glial fibrillary acidic protein messenger RNA levels and glial fibrillary acidic protein immunoreactive surface density in the arcuate nucleus and median eminence are modulated by both the neonatal and adult sex steroid environments. No effect was seen on the number of immunoreactive glia. Intact adult males had significantly higher glial fibrillary acidic protein messenger RNA levels and glial fibrillary acidic protein immunoreactive surface density than females. Both adult and neonatal castration of male animals significantly reduced glial fibrillary acidic protein messenger RNA levels and glial fibrillary acidic protein immunoreactive surface density. Neonatal and adult testosterone treatment increased both of these parameters in both sexes; however, there was no additive effect of the steroid treatments. Glial cells are involved in the proliferation, survival, migration and maturation of neurons, as well as in the modulation of synaptic connectivity, and therefore it follows that hormonal modulation of glia may mediate some of the known steroid effects on neurons. The data reported here show that astroglia are significantly influenced by both the neonatal and adult sex steroid environments and suggest that some of the steroid effects on neurons during both of these developmental periods may be mediated, at least in part, through modulation of glial cells.

Effects of Dehydroepiandrosterone (DHEA) on Pituitary Prolactin and Arcuate Nucleus Neuron Tyrosine Hydroxylase mRNA Levels in the Rat

It is well documented that dehydroepiandrosterone (DHEA), an adrenal androgen, is converted into potent androgens and/or estrogens in peripheral tissues. Since sex steroids are involved in the regulation of prolactin (PRL) secretion, we have studied the effect of DHEA administration on PRL mRNA levels in both adult male and female rats. Since tuberoinfundibular dopaminergic (TIDA) neurons are involved in the negative regulation of PRL, we have also evaluated the effects of DHEA on the genetic expression of tyrosine hydroxylase (TH), the limiting enzyme in catecholamine biosynthesis in TIDA neurons. Sham-operated and castrated animals of both sexes received during 2 days DHEA at the dose of 6 mg/kg/day, starting on the first day after castration. PRL and TH mRNA levels were measured by quantitative in situ hybridization. In the male rat, orchiectomy performed 3 days earlier did not modify PRL mRNA levels. DHEA administration increased the hybridization signal in both sham-operated and orchiectomized animals. In the female, ovariectomy decreased PRL mRNA levels and, as observed in the male, DHEA treatment induced an increase in the hybridization signal in both control and ovariectomized rats. In TIDA neurons, castration increased TH mRNA levels as evaluated by number of grains over labelled neurons and the number of TH-labelled cells per section in both male and female animals. In both sham-operated male rats and orchiectomized animals, DHEA decreased the hybridization signal. In the female, DHEA administration completely prevented the increase in TH mRNA levels due to ovariectomy. In sham-operated female rats, the treatment had no effect. These data clearly indicate that in both male and female rats DHEA exerts an estrogenic influence on both PRL and TH gene expression. Although these in vivo experiments do not allow to establish whether the stimulation of PRL gene expression is due to an action of the steroid on the pituitary or at the hypothalamic level or alternatively at both sites, it is likely that one of the mechanisms of action of DHEA might be related to a decrease in dopamine release following a depression of TIDA neuron activity.

Changes in LHβ-gene and FSHβ-gene expression in the ram pars tuberalis according to season and castration

Luteinizing hormone beta (LHbeta) and follicle stimulating hormone beta (FSHbeta) subunits and their mRNAs were studied in the ram pars tuberalis following different seasonal (winter vs summer) and experimental (intact vs castrated animals) conditions. Hormone-containing cells were identified by immunohistochemistry, using homologous double-stranded 35S-cDNAs. The labelling was quantified by image analysis. Immunohistochemical staining showed that cells containing LHbeta and FSHbeta were localized mainly in the ventral part of the pars tuberalis but that, in the summer, additional LHbeta containing cells were present in the dorsal part in intact rams. On the other hand, LHbeta-mRNA labelling was found in the whole pars tuberalis in wethers but only in the ventral part in intact rams. The magnitude of LHbeta-mRNA labelling was significantly greater in summer than in winter rams, and in castrated than in intact animals (P<0.001). However, the number of labelled cells was found to be the greatest in the winter (P<0.001) and was not affected by castration. FSHbeta-mRNA expression was similar to that of LHbeta-mRNA except that the level and extent were considerably lower. Thus, our results show an increase in the magnitude of gonadotropin beta subunit-mRNA in the summer and following castration; this increase appears to involve the entire pars tuberalis.