Hormonal status modifies renin-angiotensin system-regulating aminopeptidases and vasopressin-degrading activity in the hypothalamus-pituitary-adrenal axis of male mice

Local renin-angiotensin systems (RAS) have been postulated in brain, pituitary and adrenal glands. These local RAS have been implicated, respectively, in the central regulation of the cardiovascular system and body water balance, the secretion of pituitary hormones and the secretion of aldosterone by adrenal glands. By other hand, it is known that the hypothalamus-pituitary-adrenal (HPA) axis is involved in blood pressure regulation, and is affected by sex hormones. The aim of the present work is to analyze the influence of testosterone on RAS-regulating aminopeptidase A, B and M activities and vasopressin-degrading activity in the HPA axis, measuring these activities in their soluble and membrane-bound forms in the hypothalamus, pituitary and adrenal glands of orchidectomized males and orchidectomized males treated subcutaneously with several doses of testosterone. The present data suggest that in male mice, testosterone influences the RAS- and vasopressin-degrading activities at all levels of the HPA axis.     

The pattern of testosterone replacement influences the recovery of the stimulatory effect of clonidine on growth hormone (GH) secretion in orchidectomized rats

It has previously been described that the growth hormone (GH) releasing effect of clonidine (CLO), an agonist of ₂-adrenoreceptors, disappears after orchidectomy and is restored by testosterone replacement when started immediately after orchidectomy. In the present experiments, the effects of CLO on GH release was analysed in long-term (LTO; 12 weeks) and short-term (STO; 2 weeks) orchidectomized rats. In the first experiment, LTO males were implanted with silastic capsules containing testosterone 10 weeks after orchidectomy and killed 2 weeks later, 15 min after injection of CLO (150 μg/kg) or vehicle. In the second experiment, adult males were implanted with testosterone at the moment of orchidectomy and decapitated 2 or 12 weeks later, 15 min after vehicle or CLO administration. In addition, in order to evaluate the effects of orchidectomy and androgen replacement on ₂ agonists GH release further, prepubertal males (21-days-old) implanted with testosterone or 5--androstane-3-, 17β diol (-diol) at the moment of orchidectomy were killed 2 weeks later, 15 min after ketamine-xylazine (an ₂ agonist) administration. Finally, 10-day-old males (orchidectomized 72 h before) were decapitated 15 min after CLO or vehicle administration. Our results show that: (a) LTO and STO abolished the stimulatory effect of clonidine on GH secretion; (b) orchidectomy also abolished the stimulatory effect of clonidine in neonatal rats and that of xylazine in prepubertal males; (c) testosterone implanted at the moment of orchidectomy prevented the loss of the CLO effect in LTO and STO, but testosterone-delayed administration in LTO was unable to restore the effectiveness of CLO inducing GH release. We conclude that orchidectomy at all ages tested abolishes GH secretion induced by ₂ agonists, which suggests that the functionality of -adrenergic receptors involved in the control of GH secretion is critically dependent on a permanent exposure to testosterone in males.     

Compensatory testosterone secretion in unilaterally orchidectomized rats

Blood and testis samples were taken from rats 3 weeks after unilateral (sinistral) orchidectomy or sham operation to study the regulation of circulating testosterone. Although plasma testosterone concentrations did not differ, the concentration of testicular testosterone was twofold greater in orchidectomized rats than in sham operated controls. At autopsy, weights of right testes as well as Leydig cell number of orchidectomized and control rats were similar. These observations indicate that, after unilateral orchidectomy, compensatory hypersecretion is not related to compensatory testicular hypertrophy.     

Selective contributions of the medial preoptic nucleus to testosterone-dependant regulation of the paraventricular nucleus of the hypothalamus and the HPA axis

Previous data have consistently demonstrated an inhibitory effect of androgens on stress-induced hypothalamic-pituitary-adrenal (HPA) responses. Several brain regions may influence androgen-mediated inhibition of the HPA axis, including the medial preoptic area. To test the role of the medial preoptic nucleus (MPN) specifically, we examined in high- and low-testosterone-replaced gonadectomized rats bearing discrete bilateral lesions of the MPN basal and stress-induced indexes of HPA function, and the relative levels of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) mRNA in the amygdala. High testosterone replacement decreased plasma adrenocorticotropin hormone (ACTH) and paraventricular nucleus (PVN) Fos responses to restraint exposure in sham- but not in MPN-lesioned animals. AVP-, but not CRH-immunoreactivity staining in the external zone of the median eminence was increased by testosterone in sham animals, and MPN lesions blocked this increment in AVP. A similar interaction between MPN lesions and testosterone occurred on AVP mRNA levels in the medial nucleus of the amygdala. These findings support an involvement of MPN projections in mediating the AVP response to testosterone in both the medial parvocellular PVN and medial amygdala. We conclude that the MPN forms part of an integral circuit that mediates the central effects of gonadal status on neuroendocrine and central stress responses.     

Testosterone deficiency impairs glucose oxidation through defective insulin and its receptor gene expression in target tissues of adult male rats

Testosterone and insulin interact in their actions on target tissues. Most of the studies that address this issue have focused on the physiological concentration of testosterone, which maintains normal insulin sensitivity but has deleterious effects on the same when the concentration of testosterone is out of this range. However, molecular basis of the action of testosterone in the early step of insulin action is not known. The present study has been designed to assess the impact of testosterone on insulin receptor gene expression and glucose oxidation in target tissues of adult male rat. Adult male albino rats were orchidectomized and supplemented with testosterone (100 microg/100 g b. wt., twice daily) for 15 days from the 11th day of post orchidectomy. On the day after the last treatment, animals were euthanized and blood was collected for the assay of plasma glucose, serum testosterone and insulin. Skeletal muscles, such as gracilis and quadriceps, liver and adipose tissue were dissected out and used for the assay of various parameters such as insulin receptor concentration, insulin receptor mRNA level and glucose oxidation. Testosterone deprivation due to orchidectomy decreased serum insulin concentration. In addition to this, insulin receptor number and its mRNA level and glucose oxidation in target tissues were significantly decreased (p<0.05) when compared to control. However, testosterone replacement in orchidectomized rats restored all these parameters to control level. It is concluded from this study that testosterone deficiency-induced defective glucose oxidation in skeletal muscles, liver and adipose tissue is mediated through impaired expression of insulin and its receptor gene.     

Testosterone & lipid metabolism in rabbits.

Testosterone and lipid metabolism was studied in rabbits. The effect of orchidectomy in rabbits fed normal diets and of testosterone propionate administration to these animals on total cholesterol, phospholipids, and triglycerides of serum, liver, aortic arch, thoracic aorta, and abdominal aorta as well as the activity of lipoprotein lipase in the aortic segments and heart was investigated. With a few exceptions, total cholesterol,phospholipids, and triglycerides increased in these tissues in orchidectomized animals and testosterone counteracted this increase. 3 segments of the aorta revealed variations in response to lipids in the orchidectomized animals as well as in the testosterone administered. Lipoprotein lipase activity decreased in the heart and the 3 aortic segments on orchidectomy, and testosterone administration caused increased enzyme activity.     

Orchidectomy attenuates impaired endothelial effects of a high-salt diet in Sprague-Dawley rats

The effect of sex hormones on vascular reactivity is considered one of the underlying factors contributing to gender differences in cardiovascular functions and diseases. Experiments were designed to investigate the role of androgens in salt-induced hypertension by assessing the relaxation response of isolated aortic rings to acetylcholine and sodium nitroprusside in the presence or absence of l-nitroarginine methyl ester in Sprague-Dawley rats. The rats were either orchidectomized or sham-operated, with or without testosterone replacement, and were placed on a normal or high-salt diet for 6?weeks. The results indicate a significant increase (p?< 0.001) in the mean arterial blood pressure of rats on the high-salt diet, when compared with control or orchidectomized rats. Orchidectomy elicited a reduction in mean arterial blood pressure (p?< 0.01), while testosterone replacement normalized mean arterial blood pressure to values seen in intact rats on the high-salt diet. The high-salt diet reduced the relaxation response to acetylcholine both in the presence and absence of inhibition of endothelial nitric oxide synthase with l-nitroarginine methyl ester. Bilateral orchidectomy attenuated the impaired endothelial function induced by the high-salt diet in rats, but this was reversed by concomitant administration of testosterone, suggesting a role for androgens in enhancing long-term vascular smooth muscle tone and hence maintenance of high blood pressure in salt-induced hypertension.     

Effect of orchidectomy and testosterone substitution on enzyme activities and DNA content in rat liver and epididymal fat

Orchidectomy of rats resulted in increased concentration and whole organ amount of DNA both in the epididymal fat pad and liver. Liver hexokinase (HK) and phosphofructokinase (PFK) activities were raised after orchidectomy, but were normalized by testosterone substitution. Several glycolytic enzymes, and fumarase and aspartate aminotransferase were increased by orchidectomy in epididymal fat. Most of the enzyme changes tended to normalize after testosterone administration. Activities of NADPH generating enzymes were increased after orchidectomy both in liver and epididymal fat. When related to DNA, several enzyme activities in both tissues fell following castration. However, liver HK, PFK and NADPH generating enzymes, as well as epididymal fat HK and isocitrate dehydrogenase were elevated after castration also when related to DNA. The results suggest that the influence of testosterone on cell proliferation is organ-specific. The observed enzyme alterations after orchidectomy might partly explain fat accumulation and hyperlipoproteinemia encountered in castrates.     

Prepuberal reproductive defects in neonatal estrogenized male rats

Intact Wistar male rats injected on Day 1 with 500 micrograms of estradiol benzoate or olive oil were decapitated on Days 15 and 22 or maintained until adulthood to analyze the balanopreputial separation. Other oil or estradiol-treated rats were orchidectomized on Day 15 and decapitated on Day 22. The neonatal estrogenization produced the following reproductive changes prior to puberty: testis, adrenal, and ventral prostate atrophy; increase in the weights of seminal vesicles and epididymis; decrease in testosterone plasma levels; delayed balanopreputial separation; abolition of luteinizing hormone response to orchidectomy; transient increase in prolactin plasma levels; and blockade in seminal and prostate response to orchidectomy.     

Transient sex-related changes in the mice hypothalamo-pituitary-adrenal (HPA) axis during the acute phase of the inflammatory process

The potential role of endogenous sex hormones in regulating hypothalamo-pituitary-adrenal (HPA) axis function was investigated after a single injection of endotoxin in adult (8 week old) BALB/c mice of both sexes. The effect of LPS on plasma ACTH, corticosterone (B), testosterone and oestradiol (E) levels and on anterior pituitary (AP) ACTH and adrenal B contents at different times after treatment was studied. The results indicate that: (a) basal B but not ACTH plasma levels were significantly higher in female than in male mice; (b) LPS significantly increased both ACTH and B plasma levels over the baseline 2 h after injection, both hormone levels being higher in female than in male mice; (c) although plasma ACTH concentrations recovered the basal value at 72 h after LPS in animals of both sexes, plasma B levels returned to the baseline only at 120 h after treatment; (d) E plasma levels significantly increased 2 h after LPS and returned to the baseline at 72 h post-treatment, in both sexes; (e) at 2 h after LPS, testosterone plasma levels significantly decreased in male mice and increased in female mice, recovering the baseline level at 120 and 72 h after LPS, respectively; (f) AP ACTH content was similar in both sexes in basal condition and it was significantly diminished 72 h post-treatment without sex difference; whereas AP ACTH returned to basal content 120 h after LPS in males, it remained significantly decreased in females; (g) basal adrenal B content was higher in female than in male mice, and it significantly increased in both sexes 2 h post-LPS, maintaining this sex difference. Whereas adrenal B returned to basal content 72 h after treatment in male mice, it remained significantly enhanced up to 120 h post-LPS in female animals. The data demonstrate the existence of a clear sexual dimorphism in basal condition and during the acute phase response as well as in the recovery of the HPA axis function shortly after infection.     

Regulation of Thyrotropin Releasing Hormone Degrading Enzymes in Rat Brain and Pituitary by L- 3,5,3''-Triiodothyronine

The effect of treatment with L-3,5,3'-triiodothyronine (T3) on the levels of pyroglutamyl peptidase I and pyroglutamyl peptidase II in rat brain regions, pituitary, and serum was studied. Pyroglutamyl peptidase I cleaves pyroglutamyl peptides such as thyrotropin releasing hormone (TRH), luteinizing hormone releasing hormone, neurotensin, and bombesin, whereas pyroglutamyl peptidase II appears to be specific for TRH. Acute administration of T3 did not affect pyroglutamyl peptidase I in any of the regions studied, whereas pyroglutamyl peptidase II was significantly elevated in frontal cortex and pituitary. Treatment with T3 for 10 or 14 days significantly elevated pyroglutamyl peptidase I in pituitary, hypothalamus, olfactory bulb, hippocampus, and thalamus. Chronic T3 treatment elevated pyroglutamyl peptidase II in frontal cortex and in serum. These studies demonstrate regulation of neuropeptide degrading enzymes by thyroid hormones in vivo. This regulation may play a role in the negative feedback control of thyroid status by T3.     

Pyrrolidon carboxypeptidase activities in the hypothalamus-pituitary-thyroid and hypothalamus-pituitary-ovary axes of rats with mammary gland cancer induced by N-methyl nitrosourea.

Pyrrolidon carboxypeptidase is an omega-peptidase that hydrolyses N-terminal pyroglutamyl residues from biologically active peptides such as gonadotropin-releasing and thyrotrophin-releasing hormones. We previously described a decrease in both rat and human pyrrolidon carboxypeptidase activity with breast cancer, suggesting that gonadotropin-releasing hormone may be an important local intracrine, autocrine and/or paracrine hormonal factor in the pathogenesis of breast cancer while playing a role in the tumoral process. However, the other susceptible substrate of pyrrolidon carboxypeptidase, thyrotrophin-releasing hormone, may also be modified with breast cancer, supporting an association between breast cancer and thyroid disorders. The present work analyses soluble and membrane-bound pyrrolidon carboxypeptidase activities in the hypothalamus-pituitary-thyroid and hypothalamus-pituitary-ovary axes in N-methyl nitrosourea-induced breast cancer in rats. Our aim was to determine the possible relationship between gonadotropin-releasing hormone and thyrotrophin-releasing hormone regulation through pyrrolidon carboxypeptidase activity. We propose that pyrrolidon carboxypeptidase activity dysregulation at various local and systemic levels may participate in the initiation, promotion and progression of breast cancer induced in rat by N-methyl nitrosourea through the increase in gonadotropin-releasing hormone. Since pyrrolidon carboxypeptidase activity also acts on thyrotrophin-releasing hormone, the dysregulation of this enzyme's activity could indirectly affect hypothalamus-pituitary-thyroid axis function, and thus potentially represent a link between the diseases of thyroid and breast cancer.     

Chronic orchidectomy does not influence the sensitivity of the pituitary somatotropes to varying doses of GHRH administered intravenously to the adult male rhesus monkey

In the present study, the pituitary growth hormone (GH) response to graded doses of GH-releasing hormone (GHRH) was determined in intact (n = 3) and chronically orchidectomized (n = 3) adult rhesus monkeys (Mucaca mulatta). GHRH in doses of 0, 6.25, 12.5 and 25 microg/kg BW was infused through a teflon cannula implanted in the saphenous vein. Blood samples were collected 60 min before and 90 min after the injection of the neurohormone at 15 min intervals. All bleedings were carried out under ketamine hydrochloride anesthesia. The plasma levels of GH were determined by using AutoDELFIA time-resolved flouroimmunoassay, whereas plasma levels of testosterone and estradiol were determined using specific radioimmunoassay systems. The GH responses to GHRH were not significantly different between intact and chronically orchidectomized monkeys at any of the dose levels tested (p > 0.05). The administration of GHRH resulted in a significant (p < 0.05) stimulation of GH secretion at all the doses tested and in both the groups studied. In both intact and orchidectomized animals, the greatest response was observed at 6.25 microg/kg and no further increase was noted with the higher doses of GHRH. In conclusion, the present study suggests that chronic orchidectomy does not influence the sensitivity of the pituitary somatotropes to GHRH stimulation implying that the responsiveness of the pituitary somatotropes to GHRH is independent of testicular steroid modulation.     

Testosterone affects hormone-sensitive lipase (HSL) activity and lipid metabolism in the left ventricle

Fatty acids, which are the major cardiac fuel, are derived from lipid droplets stored in cardiomyocytes, among other sources. The heart expresses hormone-sensitive lipase (HSL), which regulates triglycerides (TG) breakdown, and the enzyme is under hormonal control. Evidence obtained from adipose tissue suggests that testosterone regulates HSL activity. To test whether this is also true in the heart, we measured HSL activity in the left ventricle of sedentary male rats that had been treated with testosterone supplementation or orchidectomy with or without testosterone substitution. Left ventricle HSL activity against TG was significantly elevated in intact rats supplemented with testosterone. HSL activity against both TG and diacylglyceride was reduced by orchidectomy, whereas testosterone replacement fully reversed this effect. Moreover, testosterone increased left ventricle free fatty acid levels, caused an inhibitory effect on carbohydrate metabolism in the heart, and elevated left ventricular phosphocreatine and ATP levels as compared to control rats. These data indicate that testosterone is involved in cardiac HSL activity regulation which, in turn, may affect cardiac lipid and carbohydrate metabolism.     

Effects of different sex hormones on male rabbit urodynamics: an experimental study

BACKGROUND: The effects of different sex hormones on urodynamics in female rabbits have been investigated previously. Estrogen induces an increase in bladder capacity and compliance, whereas testosterone and progesterone reduced bladder capacity and compliance. OBJECTIVE: The aim of this study was to investigate the effects of sex hormones on bladder urodynamics in male rabbits. METHODS: 5 groups were set up for the study: group I, low midline laparotomy (LML) + 0.9% NaCl; group II, LML + testosterone; group III, LML + bilateral orchiectomy (BO) + testosterone; group IV, LML + BO + progesterone, and group V, LML + BO + estrogen. Baseline urodynamic records and blood sex hormone levels were measured. In the follow-up all rabbits from each group underwent urodynamics 5, 10 and 30 days after injection. Estrogen, progesterone and testosterone levels were also measured during the follow-up period. For statistical verification Mann-Whitney U and Kruskal-Wallis chi(2) tests were used. RESULTS: Hormone levels: Testosterone levels were found to be increased in groups II and III 5 and 10 days after the injection. Testosterone declined thereafter and returned to baseline levels on day 30. In groups IV and V progesterone and estrogen levels increased after the injection and returned to baseline levels on day 30. Urodynamics: In groups II and III testosterone increased the bladder capacity and compliance on days 5 and 10. In these groups, capacity and compliance decreased thereafter and returned to the baseline levels on day 30. These urodynamic findings correlated with the alterations in blood testosterone levels. In groups I and IV no changes were observed in bladder capacity and compliance. In group V capacity and compliance were found to be increased on day 5 after the injection and returned to baseline levels on day 30. The changes in bladder capacity were found to be statistically significant in groups II, III and V. CONCLUSION: In this study, after the injection of testosterone, bladder capacity and compliance increased with high blood testosterone levels in male rabbits. The most interesting finding was observed in the estrogen group, questioning the role of estrogens in males. These findings allow us to reconsider the role of sex hormones in bladder functions.     

Testosterone control of brain and anterior pituitary beta-adrenergic receptors

Orchidectomy of adult albino rats resulted in a quick, (approximately 70%) increase in the density of beta-adrenergic receptors in the anterior pituitary gland within the first day. There was a concurrent rapid increase in plasma levels of pituitary gonadotropins. The beta-receptor density continued to increase slowly for at least 16 days after castration, but it could be lowered significantly to the levels of sham-operated animals by treatment with testosterone (3 mg/kg/day) beginning on the fourth day after castration and continuing for 4 days. This treatment also completely reversed the elevation in plasma levels of luteinizing hormone (LH), and significantly reduced the circulating follicle-stimulating hormone (FSH) levels. Prolactin levels were not significantly altered by the treatments used in these studies. Most of the beta-adrenergic receptors induced by orchidectomy in the anterior pituitary were shown, using a beta 1-selective antagonist, practolol, or a beta 2-selective antagonist, IPS-339, to be of the beta 2-subtype. The density of the beta-adrenergic receptors in the cerebral cortex also increased significantly (10-24%) after castration, and returned to the levels of sham-operated animals following treatment with testosterone. No significant change in the density of the beta-adrenergic receptors in the hypothalamus resulted from either castration or testosterone replacement.     

Voluntary running, skeletal muscle gene expression, and signaling inversely regulated by orchidectomy and testosterone replacement

Declines in skeletal muscle size and strength, often seen with chronic wasting diseases, prolonged or high-dose glucocorticoid therapy, and the natural aging process in mammals, are usually associated with reduced physical activity and testosterone levels. However, it is not clear whether the decline in testosterone and activity are causally related. Using a mouse model, we found that removal of endogenous testosterone by orchidectomy results in an almost complete cessation in voluntary wheel running but only a small decline in muscle mass. Testosterone replacement restored running behavior and muscle mass to normal levels. Orchidectomy also suppressed the IGF-I/Akt pathway, activated the atrophy-inducing E3 ligases MuRF1 and MAFBx, and suppressed several energy metabolism pathways, and all of these effects were reversed by testosterone replacement. The study also delineated a distinct, previously unidentified set of genes that is inversely regulated by orchidectomy and testosterone treatment. These data demonstrate the necessity of testosterone for both speed and endurance of voluntary wheel running in mice and suggest a potential mechanism for declined activity in humans where androgens are deficient.     

Time-Dependent Effect of Orchidectomy on Vascular Nitric Oxide and Thromboxane A2 Release. Functional Implications to Control Cell Proliferation through Activation of the Epidermal Growth Factor Receptor

This study analyzes whether the release of nitric oxide (NO) and thromboxane A₂ (TXA₂) depends on the time lapsed since gonadal function is lost, and their correlation with the proliferation of vascular smooth muscle cells (VSMC) mediated by the epidermal growth factor receptor (EGFR). For this purpose, aortic and mesenteric artery segments from control and 6-weeks or 5-months orchidectomized rats were used to measure NO and TXA₂ release. The results showed that the basal and acetylcholine (ACh)-induced NO release were decreased 6 weeks post-orchidectomy both in aorta and mesenteric artery, but were recovered 5 months thereafter up to levels similar to those found in arteries from control rats. The basal and ACh-induced TXA₂ release increased in aorta and mesenteric artery 6 weeks post-orchidectomy, and was maintained at high levels 5 months thereafter. Since we previously observed that orchidectomy, which decreased testosterone level, enlarged the muscular layer of mesenteric arteries, the effect of testosterone on VSMC proliferation was analyzed. The results showed that treatment of cultured VSMC with testosterone downregulated mitogenic signaling pathways initiated by the ligand-dependent activation of the EGFR. In contrast, the EGFR pathways were constitutively active in mesenteric arteries of long-term orchidectomized rats. Thus, the exposure of mesenteric arteries from control rats to epidermal growth factor (EGF) induced the activation of EGFR signaling pathways. However, the addition of EGF to arteries from orchidectomized rats failed to induce a further activation of these pathways. In conclusion, this study shows that the release of NO depends on the time lapsed since the gonadal function is lost, while the release of TXA₂ is already increased after short periods post-orchidectomy. The alterations in these signaling molecules could contribute to the constitutive activation of the EGFR and its downstream signaling pathways after long period post-orchidectomy enhancing the proliferation of the vascular muscular layer.     

A decrease in thymus-mediated immune responses as a result of treatment of neonatal rats with glutamate

We investigated whether administration of monosodium l-glutamate (MSG) to neonatal rats would disrupt immune responses in intact and orchidectomized adult male rats. Neonatal male rats were treated with saline or MSG which causes severe endocrine abnormalities. Half of each group of animals were orchidectomized as adults and killed one week later along with intact rats. MSG treatment resulted in suppressed serum LH levels in intact rats. Thymus weight and spleen cellularity in intact animals were not affected by MSG treatment, but thymus weight increased within one week after orchidectomy in both saline- and MSG-treated groups. In intact rats, lymphocyte stimulation by the T cell specific mitogens (concanavalin A or phytohemagglutinin) or the B cell specific mitogen (lipopolysaccharide) was unaffected by prior treatment with MSG. However, MSG treatment blocked the decrease attributable to orchidectomy in concanavalin A and phytohemagglutinin stimulation of lymphocyte blastogenesis. The results suggest that administration of MSG to neonatal male rats can alter some immune responses in the adult animal.