Raymond F. Aten

Estrogen receptors and androgen receptors in the mammalian liver

An estrogen receptor and an androgen receptor are present in the mammalian liver. In the liver of the rat, the estrogen receptor concentration increases markedly at puberty and this change correlates with enhanced estrogen stimulation of plasma renin substrate synthesis. High doses of estrogen are required for nuclear binding in liver when compared to doses for the uterus. The high dose requirement appears to be predominantly due to extensive metabolism in the hepatocyte of the estrogen to inactive derivatives. Furthermore, estradiol is much weaker than ethinyl estradiol for promoting nuclear binding in the liver. This is due to extremely rapid and extensive metabolism of estradiol. In human liver the concentration of estrogen receptor is low. An androgen receptor is present in high concentration in rabbit liver and is located predominantly in the nucleus after androgen administration. High concentrations of a putative androgen receptor are also present in human liver cytosol. Preliminary studies indicate that synthetic progestins can attach to the human liver androgen receptor. To date, a progesterone receptor has not been found in the mammalian liver. Thus, it appears that extensive steroid metabolism in liver preferentially diminishes sex steroid interaction with liver receptors and that androgen receptors may mediate progestin effects in liver. These observations provide a scientific basis for improved safety of oral contraceptives. Lowering the estrogen and progestin doses in oral contraceptives will decrease the major side-effects, which are liver mediated, and still maintain the desired effects at the hypothalamic-pituitary axis and uterus. Furthermore, it is likely that by selecting which estrogen, progestin or androgen is administered as well as by utilizing a parenteral route of administration that sex steroid effects on the liver could be minimized.

Specific macromolecular binding of estradiol in the mammalian liver supernatant

Abstract The liver supernatant of adult female rats contains proteins with a high specificity for binding radioactive estradiol and other potent estrogens. Several properties of the liver macromolecules are similar to those of the presumed estrogen receptor found in the uterus. Specific estrogen binding is also present in the liver supernatant of the rabbit and green monkey. Estrogens are known to influence liver composition and function in mammals. The estrogen binding protein observed in the mammalian liver may be an estrogen receptor.

Corpus luteum function and regression

Several conclusions can be drawn from a review of the formation, function and regression of the corpus luteum. Ovulation and luteinization encompass degenerative and growth changes. Inflammatory conditions associated with ovulation lead to the breakdown of the follicle wall and the membrana granulosa, along with initial damage to theca and granulosa cells. The early corpus luteum is, therefore, a tissue in stress. Thus, one view of the corpus luteum is that it, like the phoenix, rises from the inflammatory ashes of the postovulatory follicle to exist briefly and to be consumed by a similar process at regression. The luteinization process is associated with parenchymal cell hypertrophy and matrix remodelling, which appear to be regulated by IGFs and androgens, and with angiogenesis, which is induced mostly by bFGF. High levels of functional activity of the corpus luteum are regulated by control at the level of the LH receptor, whose activation leads to the translocation of cholesterol into the cell and mitochondria for conversion to steroids. Functional luteal regression can be considered as another inflammatory-like condition with apparent activation of the immune system, along with cytokine, reactive oxygen, and eicosanoid production. Structural luteolysis is subsequently invoked that leads to matrix dissolution and cellular degeneration. It is perhaps not surprising that the invocation of immune activation, which causes the production of DNA-damaging reactive oxygen species and cytotoxic cytokines each cycle, may increase the risk of pathologies. One example may be ovarian cancer which appears to be associated with the use of fertility-enhancing drugs and associated with the number of ovulations in a womans lifetime.

Oral contraceptives--possible mediation of side effects via an estrogen receptor in liver.

Most of the side effects of oral contraceptives may be caused by direct estrogen interaction in the liver resulting in alteration in liver function, including the synthesis of critical plasma proteins affecting the cardiovascular system. This study has succeeded in demonstrating an estrogen receptor in the mammalian liver. The liver receptors were found to consist, at least partly, of protein, and bound estrogens with high specificity. A strong binding affinity was indicated. The steroid hormones first bind to cytoplasmic receptors, after which the cytoplasmic receptor-steroid complex translocates from the cytoplasm to the nucleus and attaches to chromatin. Estrogen binding and translocation were shown in rat liver in vitro and in vivo. Minimizing the liver interaction thus might reduce the risk of side effects, but the estrogen-receptor function in the hypothalmic-pituitary axis and in the endometrium would have to be maintained. Possible differences in receptor characteristics of the 2 organs might be exploited to reduce liver interaction and, at the same time, possibly improve therapeutic benefits. Studies of receptor characteristics in rats indicate that a higher dose of estrogen is required in the liver than in the uterus and other organs to induce at least some direct effects. There seems to be a possibility that the newer combined oral contraceptives, which contain lower amounts of estrogen, ethinyl estradiol (reduced from 50 ug to 30 ug), and progestin, might produce smaller changes in estrogen-related plasma protein and at the same time achieve contraceptive effectiveness and regular menstrual bleeding patterns. Another possibility would be the future development or selection of another and safer estrogen.

Developmental correlation of higher levels of estrogen binding by macromolecules in rat liver supernatant and of increases in plasma renin substrate levels after estrogen administration

Developmental correlation of higher levels of estrogen binding by macromolecules in rat liver supernatant and of increases in plasma renin substrate levels after estrogen administration is reported. Gel filtration columns were used to separate bound from free radioactivity in studying binding of radioactive estradiol to tissue supernatants. The liver of the prepubescent female rat has less estrogen-specific binding macromolecules than the adult (p less than .01). This difference in quantity was maintained when binding activities were partially purified by precipitation with ammonium sulfate at 30% saturation. After administration in vivo of 100 mcg of ethinyl estradiol (sc), plasma renin substrate (PRS) levels increased 167% above control in the adult female rat (p less than .05). The corresponding increase was only 15% in the prepubescent rat. In contrast, renin substrate levels were significantly increased in both the prepubescent and adult by administration of 4 mg/kg of dexamethasone (p less than .05). The marked increase in the amount of estrogen binding and PRS responsiveness to estrogen administration with sexual maturation indicates that the estrogen-binding protein may be an estrogen receptor involved in modulating synthesis of at least 1 plasma protein.

Heat shock protein induction blocks hormone-sensitive steroidogenesis in rat luteal cells

A variety of agents induce heat shock proteins (HSPs) in addition to heat shock. The heat shock response and its effects on luteal function have not been investigated, but provocatively, many of the agents known to induce HSPs impair progesterone synthesis in luteal cells. We therefore investigated whether HSP induction might influence luteal function. Rat luteal cells exposed to a commonly used heat shock paradigm (45 degrees C; 10 min) were shown to induce HSP of 70 kDa (HSP-70). Heat shock also caused a complete abrogation of LH-sensitive progesterone and 20 alpha-dihydroprogesterone secretion, and blocked steroidogenesis in response to 8-bromo-cAMP and forskolin. In contrast, heat shock had no effect on cAMP accumulation in response to LH or forskolin, or on basal progestin secretion. Heat shock inhibition of steroidogenesis was fully reversed by 22R-hydroxycholesterol (22-OH cholesterol), a cell- and mitochondria-permeant cholesterol analog. Inhibition of transcription with actinomycin D blocked HSP-70 induction and significantly reversed the inhibition of steroidogenesis by heat shock treatment. The antisteroidogenic response of heat shock was coincident with induction of HPSs and both events were transcription dependent. These findings provide strong evidence that HSP induction inhibits steroidogenesis. The mechanism of the antisteroidogenic action of HSP induction appears to be due to interference with translocation of cholesterol to mitochondrial cytochrome P450scc, a conclusion based on reversal of inhibition by 22-OH cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)

Female and male green monkey liver estrogen receptor.

Abstract Adult female and male green monkey liver cytosol contains tritiated 17β-estradiol ([ 3 H]E 2 ) binding sites. The binding of [ 3 H]E 2 to these sites is reduced substantially by diethylstilbestrol. Unlike adult male rate liver cytosol, neither male nor female green monkey liver cytosol contains moderate affinity high capacity [ 3 H]E 2 binding sites. The green monkey liver cytosol [ 3 H]E 2 sites are precipitated by ammonium sulfate at 30% of saturation. The properties of the redissolved ammonium sulfate precipitated female and male [ 3 H]E 2 binding sites are similar: they have a high [ 3 H]E 2 affinity, a low [ 3 H]E 2 capacity, are estrogen specific, and are sensitive to sulfhydryl inactivation and protease digestion. Although the female and male sites are similar, there appears to be a 2- to 3-fold sex difference in the affinity (female K d , 0.8 to 1.3 × 10 −10 M; male K d , 0.4 × 10 −10 M) and capacity (female, 2.3 to 3.7 fmoles/mg of liver; male, 1.2 fmoles/mg of liver)of the [ 3 H]E 2 binding sites. Accordingly, adult female and male green monkey liver cytosol contains putative estradiol receptors.

Ascorbic acid-dependent cytoprotection of ovarian cells by leukocyte and nonleukocyte peroxidases.

Luteal cells contain high levels of ascorbic acid that is secreted by stimulation with agents like luteinizing hormone (LH) and prostaglandin F2 alpha (PGF2 alpha). One role for interstitial ascorbic acid, we propose, may be the detoxification of H2O2 by regeneration of catalytically active peroxidase. By serving as a preferred secondary substrate, ascorbic acid regenerates the catalytically active peroxidase that is inhibited irreversibly by H2O2 alone. To test this hypothesis, luteal cells were incubated in the absence and presence of peroxidases and H2O2, and the maximal cyclic AMP and steroidogenic response to LH was examined. In luteal cells, H2O2 is known to severely inhibit LH-sensitive cyclic AMP accumulation and steroidogenesis, and the addition of lactoperoxidase, myeloperoxidase, eosinophil peroxidase, or ascorbic acid (1 mM) alone had no effect on these responses to H2O2. However, co-incubation of ascorbic acid and the peroxidases completely reversed the inhibition of cyclic AMP accumulation and steroidogenesis produced by H2O2. These findings and the results that show direct oxidation of ascorbic acid in the presence of peroxidase and H2O2, but not with H2O2 alone, support the conclusion that ascorbic acid released from cells may detoxify H2O2 by regenerating the catalytically active state of peroxidases.

Differential modulation of thymosin genes in the immature rat ovary by gonadotropins

A cloned thymosin beta-10 cDNA and a synthetic oligonucleotide specific for the thymosin beta-4 gene were used to study the in vivo expression of these two genes in the immature rat ovary in response to exogenously administered gonadotropins. Despite the fact that both genes were co-expressed in the rat ovary, it became evident that they exhibit distinctly unique differential responses to in vivo hormonal challenge. Administration of pregnant mares serum gonadotropin (PMSG) to immature rats provoked a pronounced stimulation of ovarian thymosin beta-10 expression, the maximal effect (2- to 4-fold) of which coincided with the time at which folliculogenesis was also maximally enhanced. In contrast, the transcriptional status of the thymosin beta-4 gene varied little in response to the PMSG. Administration of human chorionic gonadotropin (hCG) to PMSG-primed rats inhibited ovarian thymosin beta-10 but stimulated thymosin beta-4 gene expression. These findings suggest that despite a mutually high degree of homology, these two proteins may, under the influence of gonadotropins, play independent roles in normal ovarian function.

Preliminary evidence that GnRH has the properties of a growth hormone-releasing factor in vivo in the rat.

Objective: We investigated whether GnRH has the properties of a growth hormone-releasing factor in vivo in a mammalian species. Methods: Sexually mature female Sprague-Dawley rats were prepared for in vivo dynamic hormone testing. The rats were assigned to one of four groups: I) GnRH, II) apomorphine and GnRH, III) somatostatin and GnRH, or IV) saline control. Blood samples were drawn at 0, 30, 60, 90, and 120 minutes after treatment. Growth hormone (GH) and LH were measured by radioimmunoassay. Results were analyzed using repeated-measures analysis of variance, Student t test, and comparisons of the area under the curve where appropriate. Results: Gonadotropin-releasing hormone caused a marked rise in both GH and LH levels as a function of time. Apomorphine caused a significant blunting of the LH response to GnRH but did not blunt the GnRH-induced rise in GH. Somatostatin blocked the GnRH-induced rise in GH but did not block the increase in LH. In saline-treated controls, neither the GH nor LH levels exhibited a statistically significant variance over the 2-hour test period. Conclusions: Gonadotropin-releasing hormone appears to have the properties of a growth hormone-releasing factor.