Binding of glucocorticoid to the androgen receptor of mouse submandibular glands

An increase in esteroprotease activity, a known cytodifferentiating response to androgen in the submandibular gland, occurred after cortisol acetate, dexamethasone or methyltrienolone (R1881) treatment in castrated genetically normal male (X/Y-castrated) mice, but not in normal male (X/Y) and testicular feminized male (Tfm/Y) mice. A peak with specific binding for [3H]cortisol appeared in sucrose density gradient patterns of extracts from X/Y-castrated mice and in almost the same fraction number as that for [3H]R1881 binding. However, peaks specific for neither [3H]cortisol nor [3H]R1881 binding were observed in Tfm/Y mice. The peak binding [3H]cortisol in extracts from X/Y-castrated mice, as well as the one binding [3H]R1881, were inhibited by unlabelled R1881 and cyproterone acetate, an antiandrogen; the peak was not, however, affected by unlabelled oestradiol-17 beta. The binding capacity of [3H]cortisol determined by Scatchard analysis was similar to that of [3H]R1881 (103 and 106 fmol/mg protein respectively). The Kd value of [3H]cortisol, however, was about 13.6-fold higher than that of R1881. These results suggest that cortisol has the ability to promote androgenic cytodifferentiating action in the mouse submandibular gland by binding to its androgen receptors, if androgens are absent or deficient.     

Induction of androgen-dependent protease and serous-like granules by tri-iodothyronine in the submandibular gland of mice with testicular feminization.

Esteroprotease, an androgen-dependent enzyme of the mouse submandibular gland, was increased by injection of tri-iodothyronine (T3) in mice with testicular feminization (Tfm) which are genetically deficient in androgen receptors. Histochemical and electron microscopic studies also demonstrated increases of RNA and serous-like granules in cells of the convoluted tubules of the gland. These findings suggest that the esteroprotease gene in Tfm mice is normal and that T3 can induced both esteroprotease and serous-like granules independently of androgen.     

Testicular steroidogenesis in the testicular feminized (Tfm) mouse: loss of 17 alpha-hydroxylase activity.

Testicular feminized (Tfm) mice are totally insensitive to androgen and may be used to study the role of the androgen receptor in normal development and function. We have examined testicular and Leydig cell steroidogenesis in Tfm mice. Serum bioactive LH was high in Tfm mice but serum testosterone was low and this was associated with a severe reduction in testicular testosterone production in vitro. Examination of [3H]pregnenolone metabolism by testes of Tfm mice indicated that progesterone, rather than testosterone, was the major steroid produced. Leydig cells were isolated from normal and Tfm mice and from normal mice in which testicular descent was surgically prevented before puberty. As in whole testes, androgen production in response to human chorionic gonadotrophin was severely reduced in Leydig cells from testes of Tfm mice compared with normal or cryptorchid groups. In contrast, progesterone production by Leydig cells from testes of Tfm mice was markedly increased in comparison with other groups. Total steroid production (progesterone plus androstenedione plus testosterone), however, was only 24% of normal in Leydig cells from Tfm mice. The pattern of steroid production by Leydig cells from cryptorchid testes was similar to control, although total steroid production was reduced to about 50% (this was significantly higher than the Tfm group, P less than 0.05). The high progesterone/androgen ratio in testes from Tfm mice suggested that 17 alpha-hydroxylase was depleted in these animals. To confirm this, activity of the four major steroidogenic enzymes associated with the smooth endoplasmic reticulum was measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Promotion of murine hepatocarcinogenesis by testosterone is androgen receptor-dependent but not cell autonomous.

Tfm (testicular feminization) mutant mice lack functional androgen receptors. By studying liver tumor development in Tfm mice, we have shown that the greater susceptibility of male mice relative to female mice for liver tumor induction by N,N-diethylnitrosamine is androgen receptor-dependent. C57BL/6J normal and Tfm mutant mice were injected at 12 days of age with N,N-diethylnitrosamine (0.2 mumol/g, i.p.), and liver tumors were enumerated in 50-week-old animals. Normal males averaged 20 liver tumors per animal; Tfm males, 0.7; normal females, 0.6; and Tfm/+ heterozygous females, 1.5. The androgen receptor gene and the Tfm mutation are X chromosome linked. Because of random X chromosome inactivation, hepatocytes from Tfm/+ heterozygous female mice are mosaic with respect to the expression of mutant or wild-type receptors. To determine if testosterone acts directly as a liver tumor promoter, through the androgen receptor in preneoplastic hepatocytes, or by an indirect mechanism, we chronically treated these mosaic female mice with testosterone and measured the androgen receptor content of the resulting tumors. B6C3F1 Tfm/+ mosaic and +/+ wild-type female mice were injected i.p. at 12 days of age with N,N-diethylnitrosamine (0.1 mumol/g) and ovariectomized at 8 weeks of age. Half of the mice of each group subsequently received biweekly s.c. injections of testosterone (0.15 mg per mouse) for 30 weeks. Tumor multiplicity was the same for wild-type and Tfm/+ mosaic females treated with testosterone (31-32 tumors per animal at 38 weeks of age) and was increased relative to females not treated with testosterone (13-17 tumors per animal at 50 weeks of age). Testosterone treatment did not significantly increase the percentage of androgen receptor-positive tumors in Tfm/+ mosaic females: 58% of the tumors from Tfm/+ mosaic females treated with testosterone were receptor positive compared to 48% in Tfm/+ females not treated with testosterone and 92% in wild-type females treated with testosterone. Finally, the number of androgen receptors in the majority of liver tumors examined was greatly decreased relative to the surrounding normal liver tissue. We conclude that liver tumor promotion by testosterone requires a functional androgen receptor in the intact animal. However, this promotion is not cell autonomous; that is, the response of the preneoplastic hepatocyte is not dependent on the expression of functional receptor in the target cell.     

Expression of the epidermal growth factor gene in mouse lachrymal gland: comparison with that in the submandibular gland and kidney

Expression of the epidermal growth factor (EGF) gene in mouse lachrymal gland was studied. The lachrymal gland of adult male and female mice contained immunoreactive EGF at a concentration of 0.013 +/- 0.002 (S.E.M.) and 0.014 +/- 0.003 ng/mg wet tissue respectively. Northern blot analysis of RNA from the lachrymal gland revealed the presence of 4.1-4.3 kb preproEGF mRNA, which was smaller in size than the 4.7-4.8 kb preproEGF mRNA in the submandibular gland and kidney. There was no sex difference in the mRNA level in the lachrymal gland, whereas the mRNA levels in the submandibular gland and kidney of adult male mice were 42-fold higher and twofold lower respectively than those of female animals. Administration of testosterone propionate or L-thyroxine to female mice increased submandibular gland levels of preproEGF mRNA by 35-fold or 12-fold respectively, but caused no change in lachrymal gland levels of preproEGF mRNA. The level of mRNA in the kidney was decreased by administration of androgen to one-third of that in control animals. The mRNA in the lachrymal gland was detected as early as 2 weeks of age and thereafter it remained at a constant level throughout adulthood, while that in the submandibular gland increased greatly during the pubertal stage. These results indicate differential hormonal and developmental regulation of EGF gene expression in the lachrymal gland, submandibular gland and kidney.     

Androgen receptors in thymic epithelium modulate thymus size and thymocyte development

Castration of normal male rodents results in significant enlargement of the thymus, and androgen replacement reverses these changes. Androgen-resistant testicular feminization (Tfm) mice also show significant thymus enlargement, which suggests that these changes are mediated by the androgen receptor (AR). The cellular targets of androgen action in the thymus are not known, but may include the lymphoid cells (thymocytes) as well as nonlymphoid epithelial cells, both of which have been believed to express AR. In the present study immunohistochemical analysis and hormone binding assays were used to demonstrate the presence of AR in thymic epithelial cells. The physiological significance of this epithelial cell AR expression was defined by further studies performed in vivo using chimeric mice, produced by bone marrow transplantation, in which AR expression was limited to either lymphoid or epithelial components of the thymus. Chimeric C57 mice engrafted with Tfm bone marrow cells (AR(+) epithelium and AR(-) thymocytes) had thymuses of normal size and showed the normal involutional response to androgens, whereas chimeric Tfm mice engrafted with C57 bone marrow cells (AR(-) epithelium and AR(+) thymocytes) showed thymus enlargement and androgen insensitivity. Furthermore, phenotypic analyses of lymphocytes in mice with AR(-) thymic epithelium showed abrogation of the normal responses to androgens. These data suggest that AR expressed by thymic epithelium are important modulators of thymocyte development.     

Regulation of the androgen and glucocorticoid receptors in rat and mouse skeletal muscle cytosol

Using a charcoal technique, we determined the relative binding affinity of some anabolic compounds for the androgen and glucocorticoid receptors in cytosol from rat skeletal muscle. Only a few of the compounds analyzed competed for the receptor-binding sites. The androgen and glucocorticoid receptors were analyzed in rat and mouse skeletal muscle cytosols by Scatchard analysis. In rats grouped according to sex and age, the cytosolic protein content was about the same in all groups, but the DNA content decreased with increased weight of the animal regardless of sex (male, female, or castrated male). The glucocorticoid receptor did not differ in concentration (2-3 pmol/g tissue) or ligand affinity (Kd, 10-40 nM) among the groups, but the androgen receptor concentration decreased with increased weight and age of the animals, more in the case of males than in the case of females or castrates. The Kd for the androgen receptor increased with age in males but was constantly about 0.2 nM for castrates or females. In adult intact rats, the androgen and glucocorticoid receptor concentrations in muscle cytosol from females were about 100 and 3000 fmol/g tissue, respectively, the corresponding values for males being about 50 and 2000 fmol/g tissue, respectively. Short term castration or adrenalectomy increased the concentration of and ligand affinity for the androgen and glucocorticoid receptors, respectively. After long term castration of male rats, the concentration of both receptors increased during 5 weeks to about the female level, only to decrease later. Neonatally castrated male rats had about the same androgen receptor concentrations and Kd values as female rats. Female mice had higher androgen receptor concentrations (approximately 700 fmol/g tissue) than rats. Intact male mice had about 200 fmol androgen receptor-binding sites/g tissue, and the same amount was found in mice bearing the testicular feminization (Tfm) mutant gene. In summary, the concentrations of androgen and glucocorticoid receptors in rat skeletal muscle are regulated at least by the testes. The presence of androgen receptors in skeletal muscle from Tfm mice is surprising and may motivate a reinvestigation of the regulation of androgen receptors in Tfm animals.     

Growth hormone (GH) response to GH-releasing hormone by perifused pituitary cells from male, female, and testicular feminized rats

To determine whether a normal complement of androgen receptors is required to permit full expression of sex-related differences in pituitary GH secretion, we compared the GHRH-stimulated GH secretory responses of continuously perifused anterior pituitary cells from normal male, normal female, and androgen-resistant testicular feminized (Tfm) rats. In each experimental replicate, acutely dispersed pituitary cells were exposed to GHRH (0.03-100 nM) administered as 2.5-min pulses in random order at 30-min intervals. The eluate was collected in 5-min fractions for GH determination by RIA. Basal unstimulated secretion of GH by cells from male rats was greater than that by cells from female (P = 0.007) and Tfm (P = 0.03) rats; basal secretion by the other two groups was similar (P = 0.55). Linear concentration-response relationships between GHRH and GH release were defined for cells from male (P = 0.0002), female (P = 0.0001), and Tfm (P = 0.0002) rats. Overall GHRH-stimulated GH secretion by cells from male rats was greater (P less than 0.0001) than that by cells from female rats. Overall secretion by cells from Tfm rats was less (P less than 0.001) than that by cells from male rats but greater (P less than 0.001) than that by cells from female rats. For all experimental groups, body weight was strongly correlated with both basal (r2 = 0.42; P = 0.001) and GHRH-stimulated (r2 = 0.53; P = 0.0001) GH secretion by the dispersed pituitary cells. These data suggest that a deficiency of androgen receptors results in a diminution of the in vitro GH secretory capability of anterior pituitary cells to a level below that by cells from normal males, but not to the level in normal females. The intermediate position of cells from the Tfm rat may represent a partial masculinization or defeminization within this generally female phenotype.     

Altered circulating hormone levels,endothelial function and vascular reactivity in the testicular feminised mouse

OBJECTIVE: Testicular feminised (Tfm) mice express a non-functional androgen receptor, and also have reduced levels of circulating testosterone. Recent studies support a cardio-protective role for testosterone since it elicits systemic and pulmonary vasodilatation. The aim of the present study was to determine whether androgen insensitivity and hypotestosteronaemia in the Tfm mouse are associated with abnormal vascular reactivity or hormone status. METHODS: Adult male Tfm and littermate control mice were killed and the blood collected. Femoral (diameter range = 183-508 microm) and pulmonary (diameter range = 320-816 microm) arteries were dissected and loaded in either a wire or pressure myograph, at 100 mmHg or 17.5 mmHg respectively. Pharmacological assessment of the vasoreactivity to potassium chloride (KCl, 80 mmol/l) and either noradrenaline (NA, 1 nmol/l-100 micromol/l) and acetylcholine (ACh, 0.1-100 micromol/l) or testosterone (1 nmol/l-100 micromol/l) was then made. RESULTS: Tfm mice had reduced levels of testosterone (1.8+/-0.3 nmol/l) compared with controls (9.3+/-2.0 nmol/l, P<0.001) and elevated levels of cholesterol (3.6+0.1 mmol/l) compared with controls (3.2+0.1 mmol/l, P<0.05). Femoral arteries from Tfm mice exhibited reduced vasoconstriction to 80 mmol/l KCl (3.27+/-0.23 mN/mm) compared with vessels from controls (4.44+/-0.41 mN/mm, P<0.05), and reduced endothelial-dependent vasodilatation to 0.1-100 micromol/l ACh (23.3+/-3.6% relaxation) compared with vessels from controls (41.6+/-5.4% relaxation, P<0.05). Vasoconstriction to NA (1 nmol/l-100 micromol/l) and vasodilatation to testosterone were unaffected. CONCLUSIONS: Androgen receptor deficiency and hypotestosteronaemia in the Tfm mouse reduced endothelial function and impaired voltage-operated calcium channel activity, which may pre-dispose to cardiovascular disease. Testosterone-induced vasodilatation was unaffected, demonstrating no involvement of the androgen receptor in this response.     

Androgen effects on hippocampal CA1 spine synapse numbers are retained in Tfm male rats with defective androgen receptors

The effects of estradiol benzoate (EB), dihydrotestosterone (DHT), or the antiandrogen hydroxyflutamide on CA1 pyramidal cell dendritic spine synapses were investigated in adult male rats. To elucidate the contribution of the androgen receptor to the hormone-induced increase in hippocampal CA1 synapses, wild-type males were compared with males expressing the Tfm mutation, which results in synthesis of defective androgen receptors. Orchidectomized rats were treated with EB (10 microg/rat.d), DHT (500 mug/rat.d), hydroxyflutamide (5 mg/rat.d), or the sesame oil vehicle sc daily for 2 d and examined using quantitative electron microscopic stereological techniques, 48 h after the second injection. In wild-type males, DHT and hydroxyflutamide both induced increases in the number of spine synapses in the CA1 stratum radiatum, whereas EB had no effect. DHT almost doubled the number of synaptic contacts observed, whereas hydroxyflutamide increased synapse density by approximately 50%, compared with the vehicle-injected controls. Surprisingly, in Tfm males, the effects of EB, DHT, and hydroxyflutamide were all indistinguishable from those observed in wild-type animals. These observations demonstrate that Tfm male rats resemble normal males in having no detectable hippocampal synaptic response to a dose of EB that is highly effective in females. Despite the reduction in androgen sensitivity as a result of the Tfm mutation, hippocampal synaptic responses to both DHT and a mixed androgen agonist/antagonist (hydroxyflutamide) remain intact in Tfm males. These data are consistent with previous results suggesting that androgen effects on hippocampal spine synapses may involve novel androgen response mechanisms.     

Gonadotropin-releasing hormone-stimulated luteinizing hormone secretion by perifused pituitary cells from normal, gonadectomized, and testicular feminized rats

To elucidate further the manner in which gonadal steroids influence the secretion of LH, we examined the effects of gonadectomy and the absence of functional androgen receptors on GnRH-induced LH release from dispersed rat anterior pituitary cells. Intact and gonadectomized (GNX) normal rats and androgen-resistant, testicular feminized (Tfm) animals from the King x Holtzman strain (a mutant strain that possesses defective androgen receptors) were used. Dispersed pituitary cells were perifused with Medium 199 during a 4-h equilibration period and then subjected to eight 2.5-min pulses of GnRH introduced at 30-min intervals at concentrations ranging from 0.03-100 nM. Basal LH secretion by cells from intact male and female rats was indistinguishable (P = 0.79) and was substantially lower (P less than 0.0001) than that by cells from GNX male and female animals. Basal LH secretion by cells from Tfm rats was significantly higher (P less than 0.01) than that by cells from intact animals, but lower (P less than 0.005) than that by cells from GNX animals. In response to GnRH, perifused pituitary cells from animals representing all experimental groups demonstrated concentration-dependent LH release. Pituitary cells from intact female rats showed an overall greater (P less than 0.05) response to GnRH than cells from intact male rats. Pituitary cells from Tfm rats demonstrated a greater GnRH-stimulated LH mean response than cells from intact male (P less than 0.0001) or intact female (P less than 0.0001) rats. Gonadectomy of male rats resulted in an overall GnRH-stimulated LH release similar to that exhibited by cells from gonadectomized female rats (P = 0.61). Cells from Tfm animals released more LH in response to GnRH than those from gonadectomized male and female rats (P less than 0.001). These data demonstrate that the release of LH in response to GnRH by pituitary cells from intact male rats (i.e. in the presence of androgen and functional androgen receptors) is less than that seen by cells from intact females rats. Since circulating levels of testosterone and estradiol are known to be elevated in the testicular feminized rat, the heightened GnRH-stimulated LH release by cells from such animals may reflect either the long term lack of androgenic influence and/or the combined effects of androgen resistance and elevated levels of circulating estrogens.     

The regulation by thyroid hormones and androgen of epidermal growth factor synthesis in the submandibular gland and its plasma concentrations in mice

The effects of androgen and thyroid hormones on epidermal growth factor (EGF) synthesis in the submandibular gland and on plasma EGF concentrations in mice were examined. Testosterone propionate was administered alone or in combination with L-thyroxine (T4) to female mice for 2 weeks. The submandibular EGF concentrations were increased by the administration of testosterone propionate in a dose-dependent fashion; the maximal increase, 20-fold, being produced by a dose of 2 mg every other day. The EGF levels were increased sevenfold by T4, which also enhanced the stimulatory effect of suboptimal doses of testosterone propionate. Cyproterone acetate, an anti-androgen, inhibited the testosterone propionate-induced increase, but not the T4-induced increase. Plasma EGF concentrations were raised by testosterone propionate but not by T4. Both hormones stimulated the accumulation of 4.7 kb preproEGF mRNA in the submandibular gland, which occurred almost in a parallel manner with the increase in submandibular EGF concentrations. These results suggest that EGF synthesis in the submandibular gland is regulated by alterations in the level of its mRNA by thyroid hormones and androgen, and that the rise in plasma EGF concentrations is under the influence of androgen but not of thyroid hormones.     

Altered expression of genes involved in regulation of vitamin A metabolism, solute transportation, and cytoskeletal function in the androgen-insensitive tfm mouse testis

Androgens are essential for the development and maintenance of spermatogenesis, but the underlying mechanisms of androgen action in the testis remain unclear. To help clarify these mechanisms, gene expression was measured in testes of pubertal (20 d old), androgen-insensitive, testicular feminized (Tfm) mice and in normal controls. Using microarrays (Affymetrix chips 430A and 430B), initial data identified a large number of genes down-regulated in the Tfm testis (>4700). These genes were largely of germ cell origin, reflecting the arrest of spermatogenesis that is apparent in the 20-d-old Tfm testis. Subsequent screening in vitro and in silico of this gene set identified 20 genes of a somatic tubular origin that were significantly down-regulated in the Tfm testis and six genes that were significantly up-regulated. Altered expression of these genes was confirmed by real-time PCR, and genes down-regulated in the Tfm testis were shown to be up-regulated in testes of hypogonadal (hpg) mice treated with androgen. In a developmental study using real-time PCR most of the regulated genes showed normal expression during fetal and neonatal development and deviated from control only between 10 and 20 d. In all cases, expression was also reduced in the adult, although interpretation is more complex because of the inherent cryptorchidism in the adult Tfm mouse. Of the total number of somatic genes showing differential expression in the Tfm testis, 50% were associated with three separate groups of genes involved in regulation of vitamin A metabolism, solute transportation, and cytoskeletal function. Thus, effects of androgens on tubular function and spermatogenesis may be mediated in part through regulation of the tubular environment and control of retinoic acid concentrations.     

The androgen receptor of the testicular-feminized (Tfm) mutant mouse is smaller than the wild-type receptor

The physicochemical and immunological properties of androgen receptors from kidney and brain of testicular-feminized (Tfm) mutant mice and wild-type mice were compared. Analysis by gel filtration and sucrose density gradients revealed that the mol wt of the mutant receptor was 66K (38A; 3.8S) which was significantly smaller than the 110K (53A; 4.6S) size of the wild-type androgen receptor (P less than 0.05). Mixing experiments failed to demonstrate any role for differential proteolysis in the size differences between these receptors. Interaction of the mutant androgen receptor with specific polyclonal antiandrogen receptor antibodies produced significantly smaller immune complexes than that formed with wild-type receptor (12S vs. 17S; P less than 0.01). This confirmed the smaller size of the Tfm mutant androgen receptor and suggested that it contained fewer epitopes. The Tfm kidney cytosols also demonstrated a decreased concentration of androgen receptor-binding activity relative to that of the wild type. Together, these results suggest that the androgen insensitivity associated with the Tfm phenotype is due to a deficiency of androgen receptor in target tissues and a qualitative defect in the androgen receptor protein itself.     

Androgens alter brain catecholamine content and blood pressure in the testicular feminized male rat

Androgens interact with catecholamines in the central nervous system (CNS) to regulate many physiological processes including blood pressure (BP). To test the hypothesis that testosterone (T) and 5a-dihydrotestosterone (DHT) modulate CNS catecholamines and BP through androgen receptor (AR)-dependent and independent mechanisms, we used the testicular feminized male (Tfm) rat. Females that carry the AR mutation (Tfm mutation) on the X chromosome were bred with spontaneously hypertensive rat (SHR) males. The normal AR male and Tfm offspring were divided into groups: control, castrated, castrated, and T or (DHT) replacement. In both AR normal and Tfm males, BP was reduced by castration, but T restored BP in both groups. In the amygdale, castration decreased dopamine (DA) in both strains and both T and DHT restored it. In the bed nucleus of the stria terminalis castration increased DA which was further increased by DHT and reduced to normal by T in both strains. In the frontal cortex, castration reduced DA content in both strains but only T restored it to normal in SHR but not in Tfm. Brain norepinephrine (NE) content showed a significant strain effect for the preoptic area (POA), but no treatment effect. Although castration did not change NE in the amygdala or POA in either strain, both T and DHT increased NE in the Tfm castrates. Blood pressure was influenced by T manipulation and correlated most significantly with DA content in the amygdala, frontal cortex, and stria terminalis. These data demonstrate an action of androgen on brain catecholamines and BP, which is independent of the classic androgen receptor.     

Effects of androgens and estradiol on spine synapse formation in the prefrontal cortex of normal and testicular feminization mutant male rats

Recent studies suggest that, in female monkeys and rats, estrogens elicit dendritic spine synapse formation in the prefrontal cortex, an area that, similar to the hippocampus, plays a critical role in cognition. However, whether gonadal hormones induce synaptic remodeling in the male prefrontal cortex remains unknown. Here we report that gonadectomy reduced, whereas administration of 5alpha-dihydrotestosterone or estradiol-benzoate to castrated male rats increased, the number of medial prefrontal cortical (mPFC) spine synapses, with estradiol-benzoate being less effective than 5alpha-dihydrotestosterone. To investigate whether the androgen receptor contributes to the mediation of these changes, we compared the response of testicular feminization mutant (Tfm) male rats to that of wild-type animals. The number of mPFC spine synapses in gonadally intact Tfm rats and 5alpha-dihydrotestosterone-treated castrated Tfm males was considerably reduced compared to intact wild-type animals, whereas the synaptogenic effect of estradiol-benzoate was surprisingly enhanced in Tfm rats. These data are consistent with the hypothesis that remodeling of spine synapses in the prefrontal cortex may contribute to the cognitive effect of gonadal steroids. Our findings in Tfm animals indicate that androgen receptors may mediate a large part of the synaptogenic action of androgens in the mPFC of adult males. However, because this effect of 5alpha-dihydrotestosterone is not completely lost in Tfm rats, additional mechanisms may also be involved.     

Independent regulatory influence of growth hormone on the hepatic synthesis of alpha 2u-globulin

Administration of GH through sc injections to hypophysectomized male rats induces the hepatic mRNA for alpha 2u-globulin (a male-specific urinary protein) from an undetectable level to 43.4% of the normal male level. The same treatment administered to hypophysectomized-gonadectomized rats and androgen-insensitive Tfm rats induces alpha 2u-globulin mRNA to a level of only 5-10% of that in the normal male. However, none of these types of animals shows an appreciable response when GH is administered continuously through osmotic minipumps. Perfusion of the livers derived from hypophysectomized male rats with the blood from hypothyroid rabbits (also deficient in GH) was used to examine in vitro effects of GH on alpha 2u-globulin synthesis. Supplementation of the perfusion medium with GH and T4 failed to induce alpha 2u-globulin within the perfusion period of 120 min. These results show that GH can influence alpha 2u-globulin synthesis independent of the androgen and that the mode of administration of GH plays an important role in its biological response.     

Multiple Sites of Regulation of Mouse Renin Expression in Ontogeny

How local renin expression is regulated in many tissues has yet to be defined. In the present studies the ontogeny of renin in submandibular gland (SMG) and kidney of CD-1 mice was examined in order to determine whether renal and extrarenal renin are similarly expressed. In males, submandibular gland (SMG) renin and renin secretory rate increase at puberty as androgen rises. The ratio of secreted forms (1-chain renin cf. 2-chain) seen on Western blots shows predominance of 1-chain prior to puberty and 2-chain thereafter. This androgen influence on renin processing and secretion was supported by reversion to prepubertal patterns with castration of adult males and by conversion to male pattern in androgen treated females which otherwise have low renin levels. In contrast, renal renin remains unchanged throughout development. The influence of ontogeny on renal and SMG renin mRNA was examined by Northern analysis using renin cDNA: SMG renin mRNA increases from near zero to high levels at puberty while renal renin mRNA level is high throughout. Taken together, these data suggest that SMG renin is influenced by androgens, whereas renal renin is not. Apparent differences in tissue renin regulation may have important implications for local function of this enzyme.     

Growth hormone secretion by individual somatotropes of the testicular feminized rat

To investigate the cellular mechanisms underlying the unique GH secretory apparatus of the androgen-resistant testicular feminized (Tfm) rat we employed a reverse hemolytic plaque assay to assess GH secretion by individual cells from normal male, normal female, and Tfm rats. Acutely dispersed pituitary cells were incubated for 90 min with GH anti-serum in the presence of medium alone, 0.01, 0.1, 1, 10, or 100 nM GHRH, or 3 microM forskolin after which hemolytic plaques were developed over an additional 30 min. Body weights of the Tfm rats [318 +/- 7 g (mean +/- SEM)] were intermediate between intact males (372 +/- 18 g) and females (218 +/- 7 g). The total number of cells recovered from dispersion of Tfm rat pituitaries [3.20 +/- 0.42 X 10(6) (mean +/- SEM)] was greater than that from males (1.43 +/- 0.12 X 10(6); P = 0.001), but not distinguishable from that from females (2.31 +/- 0.30 X 10(6); P = 0.06). However, the absolute population of recovered somatotropes from the Tfm animals (1.24 +/- 0.22 X 10(6) exceeded both male (0.56 +/- 0.10 X 10(6); P = 0.002) and female (0.80 +/- 0.14 X 10(6); P = 0.046) values. Mean basal and maximal GH plaque areas were greater for cells from male rats than for those from either female or Tfm rats (P less than 0.05) regardless of whether GHRH or forskolin was used as the secretagogue. Plaque areas from female and Tfm cells were indistinguishable under all study conditions. These data suggest that a deficiency of androgen receptors prevents establishment of the greater GH secretory capacity of individual somatotropes characteristic of the adult male rat. This androgen receptor-dependent modulation of GH secretory capacity appears to occur at a step distal to the GHRH receptor. The data also suggest that an increase in the absolute population of somatotropes is an additional consequence of androgen receptor deficiency. This combination of individual somatotropes, each possessing a GH secretory capacity similar to that of cells from normal females, but present in greater absolute numbers, may explain the intermediate values found during previous studies of the Tfm rat GH axis which were based on assessment of large mixed populations of pituitary cells.