The characteristics of the sex-differentiating action of sex steroids on the androgen receptors of the rat liver

A study was made of the specificities of polydifferentiating action of androgens (A) and estrogens (E) on the level of androgen receptors (AR) of the rat liver cytosol. Androgen receptors were shown to be detected in the neonatal period of ontogenesis, their level was progressively on the increase with the body development. Sex differences in their content were revealed in mature animals only. The AR level is a reversibly controlled, but unprogrammed sex-related sign. The effects of sex steroids are dose-related and modulated reciprocally by the action of endogenous A and E. The action of physiological doses of A and E is characterized by slow development of the effect. Correlation between the level of AR and the efficacy of A action on the content of these proteins was revealed.     

Role of various hormones of total metabolic action in the expression of sex differentiation of the rat liver by a specific estrogen-binding protein

The effect of some hormones of total metabolic action on a degree of sex differentiation of the liver by a specific estrogen-binding protein (SEBP) was investigated. Insulin administration did not change the SEBP level of the male rat liver. Adrenalectomy and dexamethasone administration caused no significant changes of sex differentiation of the SEBP level in the male and female rat liver. Neither did thyroidectomy cause significant changes in the SEBP level. Dose-related T3 administration decreased reversibly the SEBP level in the liver of adult male rats. The effect of T3 on the SEBP level was preserved after castration or hypophysectomy of males. T3 administration decreased a degree of determination of a raised level of SEBP in the female rat liver by androgens. STH injections were effective mainly in hypophysectomized male rats resulting, on the one hand, in an increase in the level of SEBP and creating conditions, on the other hand, for a decrease in its level caused by estradiol. It has been concluded that thyroid hormones and STH are able to be modulators of adaptive changes of sex differences of the liver by SEBP.     

Various mechanisms of the participation of the hypophysis and somatotropin in the hormonal regulation of the level of specific estrogen-binding protein in the rat liver

A study was made of the role of the hypophysis and STH in the regulation of the level of specific estrogen-binding protein (SEBP) and estrogen receptors (ER) in the rat liver, in the expression of the SEPB-determining effect of androgens (A) and the regulating action of estrogens (E) on the SEPB level. The SEPB and ER content was determined by quantitative methods permitting the differential testing of one E-binding protein from another. Hypophysectomy of males caused a decrease in the SEPB and ER levels. STH administration increased the SEPB and ER levels in the liver of the hypophysectomized males but did not influence the level of these proteins in intact animals. Hypophysectomy of intact and ovariectomized females blocked the SEPB-determining action of A even at early time after operation. Estrogens (E) were capable of producing an inhibitory effect on the SEPB level at early time after hypophysectomy of males. The efficacy of E action after hypophysectomy decreased in parallel with a decrease in the ER content in the liver. The administration of STH to hypophysectomized males resulted in an increase in the ER level and a subsequent reduction of SEPB negative reaction to E. It was concluded that hypophysial factors could produce a direct, sensitizing and permissive for sex steroids action on the SEPB level.     

Endocrinotropic actions of the benzamide sultopride (author's transl)]

The influence on the pituitary hypothalamic system of Sultopride are different of those of a chemically related compound, Sulpiride. In the mammary gland the development of the acini is associated with an increase of the connective tissue. The processus of secretion, lactogenesis is quiet not stimulated. The changes on the anterior lobe of the pituitary involve the carmino and eosinophilic cells. This modification seems to reflect a stimulation of the secretion of LT and an inhibition of the release of STH. In the juvenile rat, Sultopride determines an inhibition of the somatic growth.     

In-vitro evidence for the autoregulation of prolactin secretion at the level of the pituitary gland in the rat

The autoregulation of rat prolactin secretion at the level of the pituitary gland was investigated, using a static incubation system. The rate of prolactin secretion from the female anterior pituitary gland in vitro was found to be constant when the medium was changed at 20-min intervals. However, when the medium was left unchanged and secretory products were allowed to accumulate, prolactin secretion began to decline within 60 min. This effect was not observed with the male tissue, where the level of accumulated prolactin did not reach that at which the inhibition occurred using female tissue. The nature of the putative secretory product causing the inhibition of prolactin secretion was investigated. Exogenous bovine prolactin (1-4 mg/l) caused an inhibition of endogenous rat prolactin secretion. Inclusion of monoamine oxidase in unchanged medium, to prevent dopamine accumulation in the medium (a possible consequence of co-storage and co-secretion with prolactin granules), did not prevent the inhibition observed in the control incubation. We therefore conclude that in-vitro autoregulation of prolactin secretion can occur at the level of the pituitary gland, probably due to the accumulated prolactin having a feedback action on the lactotroph. This might be of physiological significance if localized concentrations of the hormone within the gland are high.     

Immunohistochemical localization of the androgen receptor in rat and human tissues.

Immunohistochemical localization of the androgen receptor (AR) was performed in reproductive tissues, submaxillary gland, pituitary, and brain of the rat and in human prostate. AR was visualized using either of two polyclonal antibodies raised against peptides with sequences derived from rat and human AR. Tissue sections of 6-8 microns, frozen in isopentane and fixed in paraformaldehyde, were stained using immunoglobulin G fractions of immune, preimmune, and peptide-adsorbed immune sera in the avidin-biotin peroxidase procedure. AR was prominent in nuclei of acinar epithelial cells of epididymis, ventral prostate, seminal vesicle, and ductus deferens from the intact rat. Androgen withdrawal, 3 days after castration, resulted in the loss of receptor immunostaining, which was restored within 15 min of androgen administration. Stromal cell staining was absent or weak in the ventral prostate of intact rats, but was more evident in the epididymis. AR was confined to nuclei of cells within and bordering the interstitial compartment of the testis, including Sertoli cells, peritubular myoid cells, and interstitial cells, and was undetectable in germ cells. Submaxillary gland epithelial cells and a population of rat anterior pituitary cells showed strong nuclear staining of AR. In rat brain, AR was present in the medial preoptic, arcurate, and ventromedial nuclei of the hypothalamus, the medial nucleus of the amygdala, the CA-1 hippocampus, and the cortex. AR was prominent in acinar epithelial cells in human benign prostatic hyperplasia and was also present in stroma of fibromuscular benign hyperplasia. Heterogeneous staining was observed in stromal and epithelial cells of prostatic adenocarcinoma. The results of these studies indicate that AR can be detected immunohistochemically in a variety of tissues and cell types using antipeptide polyclonal antibodies. The presence of AR in tissues correlated with their known androgen responsiveness.     

Tissue-specific actions of the Ept1, Ept2, Ept6, and Ept9 genetic determinants of responsiveness to estrogens in the female rat

Ept1, Ept2, Ept6, and Ept9 are quantitative trait loci mapped in crosses between the ACI and Copenhagen (COP) rat strains as genetic determinants of responsiveness of the pituitary gland to estrogens. We have developed four congenic rat strains, each of which carries, on the genetic background of the ACI rat strain, alleles from the COP rat strain that span one of these quantitative trait loci. Relative to the female ACI rats, female ACI.COP-Ept1 rats exhibited reduced responsiveness to 17beta-estradiol (E2) in the pituitary gland, as evidenced by quantification of pituitary mass and circulating prolactin, and in the mammary gland, as evidenced by reduced susceptibility to E2-induced mammary cancer. The ACI.COP-Ept2 rat strain exhibited reduced responsiveness to E2 in the pituitary gland but did not differ from the ACI strain in regard to susceptibility to E2-induced mammary cancer. Interestingly, female Ept2 congenic rats exhibited increased responsiveness to E2 in the thymus, as evidenced by enhanced thymic atrophy. The ACI.COP-Ept6 rat strain exhibited increased responsiveness to E2 in the pituitary gland, which was associated with a qualitative phenotype suggestive of enhanced pituitary vascularization. The ACI.COP-Ept9 rat strain exhibited reduced responsiveness to E2 in the anterior pituitary gland, relative to the ACI rat strain. Neither Ept6 nor Ept9 impacted responsiveness to E2 in the mammary gland or thymus. These data indicate that each of these Ept genetic determinants of estrogen action is unique in regard to the tissues in which it exerts its effects and/or the direction of its effect on estrogen responsiveness.     

The involvement of ovarian factors in maintaining the pituitary glands of female rats in a state of low LH responsiveness to LHRH

The effects of discontinuation and restoration of ovarian influences on the pituitary LH response to LHRH in vitro were investigated. When female rat pituitary glands taken on day 2 of dioestrus were incubated with LHRH the release of LH was low during the first hour (lag phase response) and afterwards a progressive, protein synthesis-dependent increase took place (second phase response), this being the self-priming action of LHRH. Short-term discontinuation (less than 1 day) of ovarian influences on the rat pituitary gland in vivo (ovariectomy) or in vitro (incubation in medium only) resulted in an increased LHRH-induced LH response during the lag phase. The biphasic LH response or the self-priming action of LHRH disappeared completely after long-term discontinuation of ovarian influences on the pituitary gland, LH release being at its maximum from the start of the incubation. The biphasic response was reinstated when ovaries were implanted under the kidney capsules of ovariectomized rats. Auto-implantation of an ovary into the spleen immediately after bilateral ovariectomy did not, however, prevent the disappearance of the LHRH self-priming action. Ovarian activity responsible for the presence of the low LH response during the lag phase was thus effectively removed by the liver, but inhibin-like activity suppressing serum FSH levels remained present. Silicone elastomer implants (s.c.) containing oestradiol-17 beta, implanted for 4 weeks, did not reverse the loss of the biphasic LH response to LHRH. It is concluded that liver-labile factors released by the ovaries keep the pituitary gland in a state of low responsiveness to LHRH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estrogen action in the regulation of cell proliferation, cell survival, and tumorigenesis in the rat anterior pituitary gland

Estrogens act as important regulators of cell proliferation, cell survival, and differentiation in a variety of organ systems and tissues and have been implicated in the etiology of a variety of malignant cancers and benign tumors. The anterior pituitary gland of the rat provides an excellent model for the study of estrogen action in the regulation of cell proliferation and survival. Estrogens stimulate proliferation of the prolactin (PRL)-producing lactotroph and enhance lactotroph survival. Through these actions on lactotroph proliferation and survival, estrogens induce or contribute to the development of PRL-producing pituitary tumors in several rat strains. Data from our laboratory and others indicate that estrogen-induced pituitary growth is rat strain specific and segregates as a quantitative genetic trait in crosses between different rat strains. The purpose of this review is to summarize current know ledge pertaining to estrogen action in the regulation of cell proliferation, cell survival, and tumorigenesis in the anterior pituitary gland of the rat species, Rattus norvegicus, and to illustrate the advantages of the rat pituitary gland as a model for elucidating the mechanisms through which estrogens regulate these processes.     

Demonstration of a sex differentiation in the content of androgen receptors in the liver cytosol of rats

The paper is concerned with differential quantification of the level of androgen receptors (AR) in the liver cytosol of male and female rats as well as in the prostatic cytosol. AR affinity to synthetic androgen R1881 was shown to be similar in all investigated tissues. In male rats the AR content in the liver cytosol was 20.5 +/- 2.1 fmol per 1 mg of protein, lower (p less than 0.05) than in the prostatic cytosol (30.8 +/- 2.5 fmol per 1 mg of protein). Sex differentiation of the liver AR content with 3-fold predominance in males was determined. Gonadectomy of mature rats caused leveling down of sex differences in the liver AR content as a result of a decrease in AR concentration in castrated males and its increase in ovariectomized females. Sex differentiation of the liver AR content could be an additional factor enhancing the multiple differentiating action of androgens on the male rat liver.     

Growth and inhibition of metamorphosis in the newt Taricha torosa by mammalian hypophysial and placental hormones

Three experiments were undertaken with late larval California newts, Taricha torosa, to determine the effects of pituitary prolactin (PL) and growth hormone (STH) and human placental lactogen (HPL) on growth and metamorphosis in this urodele amphibian. Mammalian PL and STH had distinctive actions in the larva before metamorphosis: STH promoted growth (body weight gain and trunk and tail elongation), whereas PL was ineffective in this regard. However, PL, in contrast to STH, was highly effective in blocking metamorphosis. PL-treated newts, even in the presence of thyroxine, were able to continue normal larval growth. HPL exhibited an action that was similar to that of pituitary PL in that it also inhibited metamorphosis and allowed larval growth to continue.     

Dynamics and mechanics of corticosteroid feedback at the hypothalamus and anterior pituitary gland.

Corticosteroid feedback mechanisms were investigated at the hypothalamic level using the rat hypothalamus in vitro and the pituitary level using basal hypthalamic-lesioned rats. Both fast and delayed corticosteroid feedback effects were demonstrated at the level of the hypothalamus and pituitary gland with doses of corticosteroids within or near the physiological range. These two phases of feedback were separated temporally by a 'silent period' during which no feedback was apparent. Studies on the mechanism of action of corticosteroids at the hypothalamic level showed that the fast feedback mechanism acts by inhibition of release whilst the delayed feedback mechanism acts by inhibition of both synthesis and release. The fast feedback action of corticosterone does not appear to act by excitation of neuroinhibitory pathways since neither picrotoxin nor phentolamine prevented the feedback action of corticosteroids in vitro. Corticosterone inhibition of corticotrophin releasing factor release was overcome by depolarization of the membrane with K+ suggesting that the mechanism of action of the fast feedback of corticosteroids is by membrane stabilization.     

Androgen receptors in gonadotrophs in pituitary cultures from adult male monkeys and rats

There is substantial evidence demonstrating that the principal feedback action of androgens to decrease LH secretion in male primates, including man, is to slow the GnRH pulse generator, whereas in male rats androgens not only decrease GnRH but also suppress LH synthesis and secretion through a direct pituitary effect. Previous experiments in our laboratory revealed that testosterone (T) suppresses LH secretion and decreases alpha-subunit mRNA levels in male rat pituitary cell cultures perifused with pulses of GnRH but not in pituitary cells from adult male monkeys. In the present study, we sought to determine whether the lack of responsiveness of gonadotrophs to androgens in the primate is androgen receptor (AR) related. Primary cultures were prepared from the anterior pituitary glands of adult male monkeys and rats. Cells were identified as gonadotrophs if they were immunoreactive for LH-beta or FSH-beta. Of these cells in the monkey, 80% contained both gonadotropins, 17% contained only LH-beta, and 3% contained only FSH-beta. AR immunoreactivity (IR) was nuclear in 22% and 15%, respectively, of monkey and rat FSH-beta-positive cells in the absence of T. Following T treatment, nuclear AR IR was identified in 79% of monkey and 81% of rat gonadotrophs. T treatment similarly intensified AR IR in mouse gonadotroph alphaT3-1 and LbetaT2 cells and in monkey and rat fibroblasts. Single-cell RT-PCR confirmed coexpression of LH-beta and AR mRNA as well as LH-beta and GH mRNA in monkey gonadotrophs. Our data reveal that most monkey, as well as rat, gonadotrophs are AR-positive with nuclear localization in the presence of T. GH expression is not required for AR expression in gonadotrophs. We conclude that the failure of T to inhibit LH secretion and decrease alpha-subunit mRNA expression in the male primate is not due a disturbance in AR nuclear shuttling.     

Interaction of androgen-receptor complexes in rat liver cytosol with cellular nuclei and DNA

A study was made of the ability of androgen-receptor complexes (ARC) of male rat liver cytosol to interact with hepatic cell nuclei and DNA in a model free-cell system and after administration of androgens to animals. In a free-cell system androgen receptors (AR) within a partially purified cytosol preparation were capable of binding strongly with hepatic cell nuclei and DNA of cellulose-conjugated cow spleen. The administration of testosterone propionate for 3 days resulted in the redistribution of liver AR between cytoplasm and nuclei during testing 1 h after the last injection. The observed regularities were unrelated to changes in AR content caused by the regulatory effect of administered androgen. Similar changes in AR content in the studied subcellular fractions were revealed 1 h after single administration of R1881 but not testosterone propionate. The revealed regularities were indicative of a possibility of AR involvement in androgenic action on the liver at the nuclear level.     

Daily rhythms of liver cAMP, total liver lipids, prolactin-like hormone and growth hormone cell activities in Sarotherodon mossambicus acclimated to different photoperiod regimes

Liver cAMP, total liver lipids, and pituitary prolactin-like (PRL) hormone and growth hormone (STH) cell activities were determined eight times over a 24-hr period in Sarotherodon mossambicus maintained under long and short photoperiod regimes. Diurnal variations were observed in all parameters monitored. The data illustrate that the photoperiod and the time of sacrifice are important considerations in the study of metabolic hormonal parameters in fishes. A significant correspondence between the PRL cell cycle and the total liver lipid levels cycle, and between the STH cell cycle and the liver cAMP level cycle, was also observed. Possible explanations of these facts are discussed.     

Effect of estrogen on androgen receptor dynamics in female rat pituitary

We examined the role of estrogen in regulating the number of androgen receptors (AR) in the pituitary gland of the female rat. Female Sprague-Dawley rats (240-280 g) that were ovariectomized for 3 days were used in this study. AR numbers were determined in pituitary cytosol and nuclear fractions by binding and exchange assays using the synthetic ligand 17 alpha-methyl-3H-trienolone. The administration of a single dose of estradiol benzoate [(EB) 10 micrograms/100 g BW] to ovariectomized female rats resulted in a 60% increase in cytosolic AR which was significantly (P less than 0.01) elevated above that of oil-treated controls by 12 h post EB. Cytosolic AR levels remained elevated for as long as 48 h post EB (213% of controls). Saturation analysis of pituitary cytosolic AR revealed a single, high affinity binding site for 17 alpha-methyl-3H-trienolone exhibiting an apparent dissociation constant (Kd) of 0.5 X 10(-10) M in both EB- and oil-treated animals. The administration of cycloheximide (1 mg/kg BW) before EB administration prevented the EB-induced rise in AR when measured 8 h post EB. When dihydrotestosterone (1.5 mg) was injected 24 h after EB or oil, there was a rapid increase in nuclear AR accompanied by a rapid decrease in cytosolic AR. The increase in nuclear AR was significantly greater (P less than 0.05) in EB-pretreated animals vs. oil-treated controls. These observations show that a potential synergism exists between androgen and estrogen in the female rat pituitary and suggest that androgens may play an important role in regulating cyclic pituitary hormone release.     

Localization of oestrogen receptor alpha, oestrogen receptor beta and androgen receptors in the rat reproductive organs

There is now evidence that oestrogens and androgens can influence male and female reproductive systems. In order to accurately identify the sites of action of oestrogens and androgens, we have proceeded to the histological localization of the two oestrogen receptor (ER) subtypes, ERalpha and ERbeta, and the androgen receptor (AR) in the reproductive tissues of adult rats of both sexes. AR was detected by immunocytochemistry, while ERalpha and ERbeta were localized by both immunocytochemistry and in situ hybridization. In the pituitary gland of animals of both sexes, ERalpha was found in the majority of nuclei of secretory cells in the anterior pituitary. The intermediate and posterior lobes did not show any staining. ERbeta was not found to be expressed in any of the pituitary lobes. Using AR antibodies, nuclear staining was detected in about 50% of secretory cells of the anterior lobe, the intermediate and posterior lobes being completely unstained. In the testis, ERalpha was localized in nuclei of Leydig cells as well as in round spermatocytes and spermatids, while ERbeta could only be detected in Sertoli cell nuclei. AR immunoreactivity was found in nuclei of Sertoli, peritubular myoid and Leydig cells. In the prostate, ERbeta was observed in epithelial cells of tubulo-alveoli, while the stroma was unlabelled. ERalpha was not found to be expressed in any prostate cells. In the prostate, AR was detected in nuclei of epithelial, stromal and endothelial cells. In seminal vesicles, staining of ERalpha was found in nuclei of epithelial and stromal cells. Similar findings were observed using AR antibodies. While ERbeta mRNA could not be detected by in situ hybridization, weak staining for ERbeta was localized in epithelial cells of seminal vesicles. In the ovary, both ERalpha and ERbeta were found to be expressed. ERbeta mRNA was found in granulosa cells of growing follicles, while ERalpha was present in theca cells, interstitial gland cells and germinal epithelium. AR immunoreactivity was detected in granulosa cell nuclei in growing follicles and also in scattered interstitial cells. In the oviduct and uterus, ERalpha was observed in nuclei of epithelial cells as well as of stromal and muscle cells. Similarly, AR immunoreactivity was present in nuclei of epithelial cells, stromal and muscle cells in both the oviduct and uterus. ERbeta was not detected in the oviduct and uterus. The present findings indicate a cell-specific localization of ERalpha, ERbeta and AR in reproductive tissues in rats of both sexes. By establishing the precise sites of action of oestrogens and androgens they contribute to a better understanding of the respective role of these steroids in reproduction function.     

Role of hormones in maintaining the sex-differentiated level of estrogen receptors in rat liver cytosol

A study was made of the endocrine mechanisms of the formation and maintenance of a sex-differentiated level of estrogen receptors (ER) in rat liver cytosol. The administration of testosterone-propionate (TP) at a dose of 3 mg for 3 days was shown to cause a significant decrease in the concentration of ER in the liver of gonadectomized animals to the level in intact male rats. In a week after the discontinuation of TP, a complete restoration of the basal level of receptors was observed. Neonatal and prepubertal administration of TP to gonadectomized male rats at early stages of ontogenesis made no effect on the level of ER in the liver cytosol of these animals at the age of 12-14 weeks. The removal of the adrenal and thyroid glands produced no changes in the level of ER in the liver of rats of both sexes. Hypophysectomy in rats resulted even on the 1st day in a decrease in ER concentration in the liver of male and female animals to the same basal level which later on remained unchanged. Ectopic transplantation of a homologous hypophysis and human STH administration led to a significant rise of the level of ER in hypophysectomized animals. TP inhibited a stimulating effect of STH in rats with the removed hypophysis.     

Novel ligand specificity of pituitary vasopressin receptors in the rat

The ligand specificities of sites binding tritium-labeled arginine vasopressin (3H-AVP) were investigated in membrane preparations of rat anterior pituitary gland and liver. Labeled AVP interacted with high-affinity, low-capacity binding sites in liver as well as in pituitary membrane fractions. Binding displacement studied showed that AVP was a potent ligand for liver (KI = 0.23 nM) and pituitary (KI = 0.4 nM) receptors. Oxytocin and the antidiuretic (V2) agonist dDAVP had low affinities (KI greater than 10 nM) both for liver and pituitary binding sites. The pressor (V1) antagonist d(CH2)5Tyr(Me)AVP was equipotent with AVP in liver membranes, but was 1,000-fold less potent on pituitary receptors. Another V1 antagonist, dPenTyr(Me)AVP, displaced 3H-AVP with KI values of 3 and 8 nM from pituitary and liver receptors, respectively. These data are in reasonable agreement with recent studies of the in vitro biological activities of the analogs tested, and suggest that the ligand specificity of rat pituitary AVP receptors is distinct from those of previously characterized V1, V2 and oxytocin binding sites in the rat.