Masculinization of growth hormone (GH) secretory pattern by dihydrotestosterone is associated with augmentation of hypothalamic somatostatin and GH-releasing hormone mRNA levels in ovariectomized adult rats.

The role of androgen in the sexual dimorphism in hypothalamic growth hormone (GH)-releasing hormone (GHRH) and somatostatin (SS) gene expression was examined in rats. In the first study, the SS and GHRH mRNA levels were measured in both male and female rats at 4, 6, 8, and 10 weeks of age. A significant sex-related difference in the SS and GHRH mRNA levels was observed after 8 weeks of age, when sexual maturation is fully attained. Male rats had higher SS and GHRH mRNA levels than the female rats. In the second study, adult ovariectomized rats received daily injection of dihydrotestosterone (DHT), nonaromatizable testosterone, at a dose of 2 mg/rat for 21 days. The DHT treatment masculinized the GH secretory pattern, which was indistinguishable from that of intact male rats, and simultaneously augmented the SS and GHRH mRNA levels. The DHT treatment of ovariectomized rats after hypophysectomy significantly raised the level of SS mRNA, but not that of GHRH mRNA compared to the control animals. These findings suggest that the activation of the SS gene expression through androgen receptor plays an important role in the maintenance of sexual dimorphism in GH secretion in rats.     

Estrogen and androgen elicit growth hormone release via dissimilar patterns of hypothalamic neuropeptide secretion

The dimorphic pattern of growth hormone (GH) secretion and somatic growth in male and female mammals is attributable to the gonadal steroids. Whether these hormones mediate their effects solely on hypothalamic neurons, on somatotropes or on both to evoke the gender-specific GH secretory patterns has not been fully elucidated. The purpose of this study was to determine the effects of 17beta-estradiol, testosterone and its metabolites on release of GH, GH-releasing hormone (GHRH) and somatostatin (SRIF) from bovine anterior pituitary cells and hypothalamic slices in an in vitro perifusion system. Physiological concentrations of testosterone and estradiol perifused directly to anterior pituitary cells did not affect GH releases; whereas, dihydrotestosterone and 5alpha-androstane-3alpha, 17beta-diol increased GH. Perifusion of testosterone at a pulsatile rate, and its metabolites and estradiol at a constant rate to hypothalamic slices in series with anterior pituitary cells increased GH release. The androgenic hormones increased GHRH and SRIF release from hypothalamus; whereas, estradiol increased GHRH but decreased SRIF release. Our data show that estradiol and the androgens generated distinctly different patterns of GHRH and SRIF release, which in turn established gender-specific GH patterns.     

Single exposure to testosterone in adulthood rapidly induces regularity in the growth hormone release process

The neonatal gonadal steroid milieu is known to be important in imprinting the striking sexual dimorphism of growth hormone (GH) secretion; however, the influence of the sex steroids on GH control in adult life and their mechanism/site of action are largely unknown. In the present study, we tested the hypothesis that testosterone (T) subserves the gender-specific regularity of the GH release process in adulthood. The approximate entropy statistic (ApEn) was used to quantify the degree of regularity of GH release patterns over time. Eighteen hours after a single subcutaneous injection of 1 mg T, both sham-operated and ovariectomized (OVX) female adult rats displayed plasma GH profiles that were strikingly similar to the regular male-like ultradian rhythm of GH secretion. The highest ApEn values, denoting greater disorderliness of GH secretion, were observed in the ovary-intact group, and T injection significantly (P < 0.001) reduced this irregularity whether or not the ovaries were present. Serial intravenous injections of GH-releasing hormone (GHRH) caused a similar increase in plasma GH levels in sham-operated females independently of time of administration. In contrast, female rats administered T exhibited a male-like intermittent pattern of GH responsiveness to GHRH, the latter known to be due to the cyclic release of endogenous somatostatin. These results demonstrate that acute exposure to T during adult life can rapidly and profoundly "masculinize" GH pulse-generating circuits in the female rat. Our findings suggest that the enhanced orderliness characteristic of the GH release process in males, compared with females, is regulated by T. We postulate that this T-induced regularity is mediated at the level of the hypothalamus by inducing regularity in somatostatin secretion, which in turn governs overall GH periodicity.     

The role of sexual steroids in the modulation of growth hormone (GH) secretion in humans

Sex steroids contribute to modulate GH secretion in man. However, both the exact locus and mechanism by which their actions are exerted still remain not clearly understood. We undertook a number of studies designed to ascertain: (1) whether or not sudden or chronic changes in circulating gonadal steroids may affect GH secretion in normal adults; and (2) the reason(s) for gender-related dimorphic pattern of GH release. The pituitary reserve of GH, as evaluated by means of a GHRH challenge, was similar in women with anorexia nervosa and in normally menstruating women. Estrogenic receptor blockade with tamoxifen (TMX) did not significantly change GHRH-induced GH response in these normal women. Therefore, acute or chronic hypoestrogenism apparently had no important effects at level of somatotrophs. In another group of normal women we tested the possibility that changes in circulating estrogens might induce changes in the hypothalamic-somatotroph rhythm (HSR). GHRH challenges were performed throughout a menstrual cycle, and again after having achieved functional ovarian blockade with a GnRH agonist treatment. Short-term ovarian blockade did not significantly affect the parameters of GH response to GHRH, although it was accompanied by an increase in the number of women in a refractory HSR phase at testing. This suggested a low potentiating effect on the basic pattern of somatostatin (SS) release occurring as a consequence of the decrease in circulating estrogens. In normal men, neither the GH response to GHRH nor the HSR were affected by functional testicular blockade (after GnRH agonist treatment). However, the administration of testosterone enanthate (250 mg) to another group of men increased both the GHRH-induced GH release and the number of subjects in a spontaneous secretory HSR phase at testing; these were reversed by estrogenic receptor blockade with TMS. In another group of normal men, the fraction of GH secreted in pulses (FGHP) during a nocturnal sampling period was significantly decreased by testicular blockade. Other parameters of GH secretion,such as the number of GH pulses and their mean amplitude (A), and the mean plasma GH concentration (MCGH), showed a slight, although not significant, decrease following the lack of androgens. The administration of testosterone enanthate (500 mg) reversed these parameters to values similar to those in the basal study. Interestingly, when tamoxifen was given after testosterone enanthate, A, MCGH and FGHP increased to values significantly higher than in any other experimental condition in that study.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sexual dimorphic expression of ADH in rat liver: importance of the hypothalamic-pituitary-liver axis

Hepatic alcohol dehydrogenase (ADH) activity is higher in female than in male rats. Although sex steroids, thyroid, and growth hormone (GH) have been shown to regulate hepatic ADH, the mechanism(s) for sexual dimorphic expression is unclear. We tested the possibility that the GH secretory pattern determined differential expression of ADH. Gonadectomized and hypophysectomized male and female rats were examined. Hepatic ADH activity was 2.1-fold greater in females. Because protein and mRNA content were also 1.7- and 2.4-fold greater, results indicated that activity differences were due to pretranslational mechanisms. Estradiol increased ADH selectively in males, and testosterone selectively decreased activity and mRNA levels in females. Effect of sex steroids on ADH was lost after hypophysectomy; infusion of GH in males increased ADH to basal female levels, supporting a role of the pituitary-liver axis. However, GH and L-thyroxine (T4) replacements alone in hypophysectomized rats did not restore dimorphic differences for either ADH activity or mRNA levels. On the other hand, T4 in combination with intermittent administration of GH reduced ADH activity and mRNA to basal male values, whereas T4 plus GH infusion replicated female levels. These results indicate that the intermittent male pattern of GH secretion combined with T4 is the principal determinant of low ADH activity in male liver.     

Sex differences in fatty acid composition of rat liver phosphatidylcholine are regulated by the plasma pattern of growth hormone

The effects of continuous and intermittent (at 12 h intervals) administration of growth hormone (GH), and the effects of gonadal steroids on the regulation of the fatty acid composition of liver phosphatidylcholine were studied in gonadectomized and hypophysectomized adult female Sprague-Dawley rats. Gonadal steroids have been shown to influence the fatty acid composition of liver phosphatidylcholine in the rat. It is shown in the present study that neither testosterone nor estradiol had any effects on liver phosphatidylcholine in hypophysectomized rats. There was a 'masculinizing' effect of hypophysectomy of female rats on the fatty acid composition of liver phosphatidylcholine (i.e., an increase in the proportion of palmitic, oleic and linoleic acids and a decrease in the proportion of stearic and arachidonic acids). Continuous infusion of human GH and bovine GH partly reversed the 'masculinizing' effect of hypophysectomy. In contrast, there were no effects of intermittent administration of human GH. Also, there was no effect of prolactin infusion. It is concluded that the sexually dimorphic secretory pattern of GH may be involved in the regulation of the sexual differentiation of the fatty acid composition of liver phosphatidylcholine in the rat.     

Sexual dimorphism and growth hormone regulation of a hybrid gene in transgenic mice.

The sexually dimorphic expression of the urinary protein genes of mice (Mup genes) in the liver is mediated by the different male and female temporal patterns of circulating GH. Normal females were induced to male levels when GH was administered by injection to mimic the male GH pattern, showing that expression at the male level does not require a male sex steroid status in addition to intermittent GH. Two Mup-alpha 2u-globulin hybrid transgenes with different Mup gene promoters showed sexually dimorphic expression, and their expression in females increased to male levels upon testosterone treatment. GH-deficient (lit/lit) mice did not express these transgenes, and GH-deficient females did not respond to testosterone treatment, showing that GH was required for induction. Both normal and GH-deficient females were induced to male levels when GH was administered by injection. This is the first report of a transgene responsive to GH. A transgene consisting of a Mup promoter fused to a Herpes simplex virus thymidine kinase reporter sequence also showed sexual dimorphism, although to a lesser degree. It was expressed at the same level in normal females and GH-deficient mice of both sexes and was induced when GH-deficient mice were treated with GH. We propose that this transgene has a basal constitutive expression, possibly due to the absence of any rodent DNA downstream of the promoter. Since expression of the transgene was significantly induced by GH, the GH response is due at least in part to sequences in the promoter region.     

A construct of interactive feedback control of the GH axis in the male

Growth hormone (GH) secretion is controlled by GH-releasing hormone (GHRH), the GH release-inhibiting hormone somatostatin (SRIF), and autofeedback connections. The ensemble network produces sexually dimorphic patterns of GH secretion. In an effort to formalize this system, we implemented a deterministically based autonomous feedback-driven construct of five principal dose-responsive regulatory interactions: GHRH drive of GH pituitary release, competitive inhibition of GH release by SRIF, GH autofeedback via SRIF with a time delay, delayed GH autonegative feedback on GHRH, and SRIF inhibition of GHRH secretion. This formulation engenders a malelike pattern of successive GH volleys due jointly to positive time-delayed feedback of GH on SRIF and negative feedback of SRIF on GH and GHRH. The multipeak volley is explicated as arising from a reciprocal interaction between GH and GHRH during periods of low SRIF secretion. The applicability of this formalism to neuroendocrine control is explored by initial parameter sensitivity analysis and is illustrated for selected feedback-dependent experimental paradigms. The present construct is not overparameterized and does not require an ad hoc pulse generator to achieve pulsatile GH output. Further evolution of interactive constructs could aid in exploring more complex feedback postulates that confer the vivid sexual dimorphism of female GH profiles.     

Growth hormone-releasing hormone neurons in the anestrus cat do not express progesterone receptors

Ovarian steroids have been implicated in the regulation of growth hormone (GH) secretion in several species and increased progesterone secretion has been associated with elevated circulating GH levels in the cat. These high GH concentrations may be due, at least in part, to a direct action of progesterone on growth hormone-releasing hormone (GHRH) neurons. Using standard immunocytochemical methods coupled to high-temperature antigen retrieval, the objective of this study was to determine whether progesterone receptors were colocalized in GHRH neurons of the anestrus cat. GHRH perikarya were restricted to the infundibular nucleus and the ventral ventromedial nucleus and although frequently surrounded by numerous progesterone receptor-immunoreactive cells, none was colocalized. This study, therefore, provides evidence that, in the adult anestrus female cat, GHRH neurons do not express nuclear progesterone receptors.     

Hypothalamic-pituitary somatotropic function in prepubertal hypothyroid rats: effect of growth hormone replacement therapy.

The effect of thyroid hormone deficiency and growth hormone (GH) treatment on hypothalamic GH-releasing hormone (GHRH)/somatostatin (SS) concentrations, GHRH/SS mRNA levels, and plasma GH and somatomedin-C (IGF-I) concentrations were studied in 28- and 35-day-old rats made hypothyroid by giving dams propylthiouracil in the drinking water since the day of parturition. Hypothyroid rats, at both 28 and 35 days of life, had decreased hypothalamic GHRH content and increased GHRH mRNA levels, unaltered SS content and SS mRNA levels, and reduced plasma GH and IGF-I concentrations. Treatment of hypothyroid rats with GH for 14 days completely restored hypothalamic GHRH content and reversed the increase in GHRH mRNA, but did not alter plasma IGF-I concentrations. These data indicate that, in hypothyroid rats, the changes in hypothalamic GHRH content and gene expression are due to the GH deficiency ensuing from the hypothyroid state. Failure of the GH treatment to increase plasma IGF-I indicates that the feedback regulation on GHRH neurons is operated by circulating GH and/or perhaps tissue but not plasma IGF-I concentrations. Presence of low plasma IGF-I concentrations would be directly related to thyroid hormone deficiency.     

Estradiol increases growth hormone secretion in rats exposed to swimming stress or reserpine treatment

The secretory pattern of growth hormone (GH) in female rats differs from that in males with respect to e.g. the inter-peak baseline levels being higher in females. In the present study the influence of sex steroids on plasma GH levels was investigated in male rats under various conditions. Administration of estradiol, but not testosterone, was found to increase GH release in rats with suppressed levels induced by exposure to swimming stress or by treatment with the monoamine depleting agent reserpine. In line with previous studies, administration of estradiol was found to increase also inter-peak GH levels in adult male rats; i.e. to cause a feminization of the secretory pattern. In stressed and in reserpinized animals as well as in normal male rats, the effect of estradiol is similar to that earlier demonstrated for somatostatin antiserum, and hence it is suggested that estradiol may act antagonistic to the GH inhibiting factor.     

Effects of GH and/or sex steroids on circulating IGF-I and IGFBPs in healthy, aged women and men

Circulating GH, IGF-I, IGFBP-3, and sex steroid concentrations decrease with age. GH or sex steroid treatment increases IGFBP-3, but little is known regarding the effects of these hormones on other IGFBPs. We assessed the effects of 26 wk of administration of GH, sex steroids, or GH + sex steroids on AM levels of IGF-I, IGFBPs 1-5, insulin, glucose, and osteocalcin and 2-h urinary excretion of deoxypyridinolline (DPD) cross-links in 53 women and 71 men aged 65-88 yr. Before treatment, in women and men, IGF-I was directly related to IGFBP-3 (P < 0.001 and P < 0.0001) and IGFBP-1 to IGFBP-2 (P = 0.0001). In women, IGFBP-1 was inversely related to insulin (P < 0.0005) and glucose (P < 0.005) and IGFBP-4 to osteocalcin (P < 0.01). IGFBP-4 and IGFBP-5 were not significantly related to DPD cross-links. GH and/or sex steroid increased IGF-I levels in both sexes, with higher concentrations in men (P < 0.001). In women, the IGF-I increment after GH was attenuated by hormone replacement therapy (HRT) coadministration (P < 0.05). Hormone administration also increased IGFBP-3. IGFBP-1 was unaffected by GH + sex steroids, whereas GH decreased IGFBP-2 by 15% in men (P < 0.05). Hormone administration did not change IGFBP-4, whereas in men IGFBP-5 increased by 20% after GH (P < 0.05) and 56% after GH + testosterone (P = 0.0003). These data demonstrate sexually dimorphic IGFBP responses to GH. Additionally, HRT attenuated or prevented GH-mediated increases in IGF-I and IGFBP-3. Whether GH and/or sex steroid administration alters local tissue production of IGFBPs and whether the latter influence autocrine or paracrine actions of IGF-I remain to be determined.     

Anthropometric analysis of homosexuals and heterosexuals: implications for early hormone exposure

Early exposure to sex steroids is thought to be important in mediating the differentiation of male-typical sexual orientation. Bone morphology is a marker of childhood sex steroid exposure, because estrogens and androgens control sexual dimorphism in skeletal size. Anthropometric analysis of heterosexuals and homosexuals indicates that those bones, which become sexually dimorphic in childhood, but not those which become sexually dimorphic after puberty, are different in length in homosexuals and heterosexuals. Persons with a sexual preference for males have less long bone growth in the arms, legs and hands, than those with sexual preference for females. The data support the hypothesis that male homosexuals have had less steroid exposure during development than male heterosexuals and that female homosexuals have had greater steroid exposure during development than their heterosexual counterparts.     

Intracerebral Sex Differences in the Vasotocin System in Birds: Possible Implication in Behavioral and Autonomic Functions

The brain vasotocinergic system demonstrates clear sexual dimorphism in birds investigated so far. This paper examines the evidence obtained in studies on gallinaceous (domestic fowl, Japanese quail) and passerine (canary, junco, zebra finch) birds. Vasotocin (VT)-immunoreactive parvocellular neurons are present in the nucleus of stria terminalis of males, but they are less abundant or absent in the corresponding structure of females. A similar difference has been observed in the dorsal paraventricular area of domestic fowl. Sex-related differences in VT-gene expression have been confirmed byin situhybridization. Moreover, overall brain content of VT mRNA in cockerels is about twice that of hens, suggesting that VT synthesis may also be sexually dimorphic in other brain areas where morphological sex differences have not yet been revealed. The vasotocinergic system in birds is implicated in body fluid homeostasis, and during ontogeny it starts to respond to osmotic challenges in a sexually dimorphic way. Photoperiod, aging, or castration—all associated with changes in circulating testosterone levels—affect sexually dimorphic VT pathways and cell clusters. Sexually dimorphic vasotocinergic circuits are distributed in regions containing steroid-concentrating cells and are closely intermingled with aromatase-containing neurons that may mediate activational effects of gonadal steroids on this peptidergic system. However, it remains undetermined whether the observed neuroanatomical sex differences are related to sexually dimorphic autonomic and behavioral effects induced by VT. Most likely, VT in birds has a modulatory rather than a specific regulatory function in control of male sexual behavior and vocalization.     

Complex regulation of GH autofeedback under dual-peptide drive: studies under a pharmacological GH and sex steroid clamp

To test the postulate that sex difference, sex steroids, and peptidyl secretagogues control GH autofeedback, 11 healthy postmenopausal women and 14 older men were each given 1) a single iv pulse of GH to enforce negative feedback and 2) continuous iv infusion of saline vs. combined GHRH/GHRP-2 to drive feedback escape during pharmacological estradiol (E(2); women) or testosterone (T; men) supplementation vs. placebo in a double-blind, prospectively randomized crossover design. By three-way ANCOVA, sex difference, sex hormone treatment, peptide stimulation, and placebo/saline responses (covariate) controlled total (integrated) GH recovery during feedback (each P < 0.001). Both sex steroid milieu (P = 0.019) and dual-peptide stimulation (P < 0.001) determined nadir (maximally feedback-suppressed) GH concentrations. E(2)/T exposure elevated nadir GH concentrations during saline infusion (P = 0.003), whereas dual-peptide infusion did so independently of T/E(2) and sex difference (P = 0.001). All three of sex difference (P = 0.001), sex steroid treatment (P = 0.005), and double-peptide stimulation (P < 0.001) augmented recovery of peak (maximally feedback-escaped) GH concentrations. Peak GH responses to dual-peptidyl agonists were greater in women than in men (P = 0.016). E(2)/T augmented peak GH recovery during saline infusion (P < 0.001). Approximate entropy analysis corroborated independent effects of sex steroid treatment (P = 0.012) and peptide infusion (P < 0.001) on GH regularity. In summary, sex difference, sex steroid supplementation, and combined peptide drive influence nadir, peak, and entropic measurements of GH release under controlled negative feedback. To the degree that the pharmacological sex steroid, GH, and dual-peptide clamps provide prephysiological regulatory insights, these outcomes suggest major determinants of pulsatile GH secretion in the feedback domain.     

Changes in the hypothalamic-pituitary somatotropic function of infant hypothyroid rats

The effects of the perturbation of the pituitary-thyroid axis induced during development on the functional activity of the growth hormone (GH) regulatory neuronal systems, GH-releasing hormone (GHRH), and somatostatin (SS) were studied in 14- and 21-day-old rats made hypothyroid by giving dams propylthiouracil in the drinking water since the day of parturition. Infant hypothyroid rats, both at 14 and 21 days of life, had elevated plasma thyroid-stimulating hormone levels and decreased pituitary and plasma GH levels. Simultaneous determination of hypothalamic GHRH/SS-like immunoreactivity (LI) and GHRH/SS mRNA levels did not reveal any difference in 14-day-old hypothyroid rats when compared with age-matched controls. In contrast, 21-day-old hypothyroid rats had decreased GHRH-LI content and a striking rise in GHRH mRNA levels, whereas SS-LI content and SS gene expression remained unaltered. These data indicate that in infant hypothyroid rats, changes in the functional activity of the GHRH neuronal system occur later than changes in GH secretion and are probably dependent on the GH deficiency. The functional activity of SS neurons was apparently unaltered in these hypothyroid rats, pointing to a lesser sensitivity of this system to the perturbation of the pituitary-thyroid axis.     

Regulation of somatostatin and growth hormone-releasing factor by gonadal steroids in fetal rat hypothalamic cells in culture.

The mechanism underlying the sexually dimorphic pattern of growth hormone (GH) secretion in the rat has not been clearly elucidated. In the present study, we assayed the possible direct effect of gonadal steroids on both somatostatin (SS) and growth hormone-releasing factor (GRF) in fetal rat hypothalamic cells in culture. Hypothalamic cells, obtained by mechanical dispersion, were maintained as monolayer cultures in serum-supplemented medium. After 20 days in culture, cells were incubated with serum free medium containing testosterone (T, 10, 20, 40 ng/dl) or estradiol (E, 0.1, 1, 10 ng/dl) for 48 h. At the end of the experiments, immunoreactive SS (IR-SS) and immunoreactive GRF (IR-GRF) were measured by specific radioimmunoassays (RIAs) in media and cell extracts. After 48 h of incubation with testosterone, somatostatin in both media and cells was significantly reduced. On the contrary, this treatment lead to a dose-dependent increase in media and cell GRF content. When cells were incubated with estradiol for 48 h, a significant inhibition in medium SS release was observed, whereas intracellular SS slightly increased at the highest concentration of 10 ng/dl. Estradiol treatment resulted in an inconsistent decrease in media and cells IR-GRF. Our results indicate that both SS and GRF are under the influence of testosterone and estradiol acting at the hypothalamic level, and furthermore suggest that at this stage of brain development, gonadal steroids may regulate GH secretion through their ability to modulate hypothalamic SS and GRF.     

Growth hormone-releasing hormone and corticotropin-releasing hormone enhance non-rapid-eye-movement sleep after sleep deprivation

The neuropeptides growth hormone (GH)-releasing hormone (GHRH) and corticotropin-releasing hormone (CRH) regulate sleep and nocturnal hormone secretion in a reciprocal fashion, at least in males. GHRH promotes sleep and GH and inhibits hypothalamo-pituitary-adrenocortical (HPA) hormones. CRH exerts opposite effects. In women, a sexual dimorphism was found because GHRH impairs sleep and stimulates HPA hormones. Sleep deprivation (SD) is the most powerful stimulus for inducing sleep. Studies in rodents show a key role of GHRH in sleep promotion after SD. The effects of GHRH and CRH on sleep-endocrine activity during the recovery night after SD are unknown. We compared sleep EEG, GH, and cortisol secretion between nights before and after 40 h of SD in 48 normal women and men aged 19-67 yr. During the recovery night, GHRH, CRH, or placebo were injected repetitively. After placebo during the recovery night, non-rapid-eye-movement sleep (NREMS) and rapid-eye-movement sleep (REMS) increased and wakefulness decreased compared with the baseline night. After GHRH, the increase of NREMS and the decrease of wakefulness were more distinct than after placebo. Also, after CRH, NREMS increased higher than after placebo, and a positive correlation was found between age and the baseline-related increase of slow-wave sleep. REMS increased after placebo and after GHRH, but not after CRH. EEG spectral analysis showed increases in the lower frequencies and decreases in the higher frequencies during NREMS after each of the treatments. Cortisol and GH did not differ between baseline and recovery nights after placebo. After GHRH, GH increased and cortisol decreased. Cortisol increased after CRH. No sex differences were found in these changes. Our data suggest that GHRH and CRH augment NREMS promotion after SD. Marked differences appear to exist in peptidergic sleep regulation between spontaneous and recovery sleep.