Heat shock protein-70 induction mediates luteal regression in the rat.

Induction of heat shock protein 70 (HSP-70) is associated with inhibition of hormone-sensitive steroidogenesis and interruption of cholesterol translocation to or into the mitochondria. A number of pharmacological and physiological inhibitors of luteal cell function stimulate HSP-70 synthesis via activation of the heat shock transcription factor (HSF). In the present study we address the following questions: 1) is HSP-70 synthesis increased during prostaglandin F2 alpha (PGF2 alpha)-induced luteolysis or natural luteal regression? 2) Does blocking HSP-70 synthesis reverse the inhibition of hormone-sensitive steroidogenesis induced by heat stress and PGF2 alpha? Gel-retardation assays demonstrated HSF activation within 7.5 min after PGF2 alpha (400 micrograms) administered in vivo. Western blotting revealed HSP-70 synthesis by 1 h with higher levels seen at 2 h. The stress response was similarly activated during natural regression of the corpus luteum in rats. Gel retardation assays demonstrated maximal HSF activation 10 days after ovulation. Western blotting showed that HSP-70 levels increased dramatically on this same day and were sustained for several days after the initiation of luteal regression. Inhibition of HSP-70 synthesis was investigated in isolated luteal cells using a cholesteryl-conjugated phosphorothioate antisense oligodeoxynucleotide. The control was an oligodeoxynucleotide with the same base composition, but with scrambled base sequence. Incubation with 3 microM antisense oligodeoxynucleotide for 2 h before a 42 C heat shock prevented synthesis of HSP-70 while up to 4.5 microM control oligodeoxynucleotide had no effect on HSP-70 synthesis in response to heat shock. Preincubation with antisense oligodeoxynucleotide partially reversed heat stress-induced inhibition of LH-stimulated steroidogenesis. More importantly, preincubation with antisense oligodeoxynucleotide also significantly reversed inhibition of cAMP-stimulated steroidogenesis induced by PGF2 alpha. Treatment with control oligodeoxynucleotide did not reverse heat shock or PGF2 alpha inhibition of hormone-dependent steroidogenesis. The findings that the synthesis of HSP-70 coincides with the loss of luteal function and that blocking its synthesis reverses inhibition of hormone-dependent steroidogenesis strongly suggest a role for HSPs as physiological mediators of luteal regression.

Evidence that hydrogen peroxide blocks hormone-sensitive cholesterol transport into mitochondria of rat luteal cells.

In luteal and granulosa cells, hydrogen peroxide abruptly inhibits activation of adenylate cyclase by receptor-bound gonadotropin and blocks steroidogenesis. In the present studies a post-cAMP site of peroxide action on inhibition of steroidogenesis was investigated. Steroidogenesis, stimulated by dibutyryl or 8-bromo-cAMP, was inhibited by hydrogen peroxide. Yet, cAMP-dependent protein kinase activation in cytosol or intact cells was unaffected by peroxide treatment. Hydrogen peroxide also did not inhibit the activity of cholesterol esterase and acyl coenzyme-A:acyltransferase. Progesterone synthesis was maximally increased 5- to 50-fold with 25- and 22-hydroxycholesterol, respectively. Unlike that seen with cAMP analogs and LH, however, progestin synthesis stimulated by these cell- and mitochondria-permeant cholesterol analogs was not inhibited by hydrogen peroxide. Treatment of animals with amino-glutethimide produces a marked accumulation of steroidogenic cholesterol substrate and a large increase in hormone-independent steroidogenesis in subsequently isolated and washed luteal tissue. In this paradigm, hydrogen peroxide did not inhibit elevated basal progesterone synthesis in luteal cells produced by in vivo aminoglutethimide treatment, yet LH-stimulated steroidogenesis was blocked. However, treatment of luteal cells with hydrogen peroxide inhibited pregnenolone synthesis in isolated mitochondria, an effect partially reversed by the addition of luteal cell cytosol. In summary, while peroxide inhibited cAMP-dependent steroidogenesis, it did not appear to inhibit protein kinase activation or mobilization of cholesterol from intracellular esterified stores. Although peroxide inhibited pregnenolone synthesis, it had no effect on steroidogenesis when substrate was made available by either addition of cholesterol analogs or prior treatment with aminoglutethimide in vivo. We conclude, therefore, that hydrogen peroxide inhibits steroidogenesis by blocking intracellular transport of cholesterol to mitochondria or translocation of cholesterol across the outer mitochondrial membrane.

Inhibition of gonadotropin action and progesterone synthesis by xanthine oxidase in rat luteal cells.

Hydrogen peroxide produces marked antigonadotropic and lytic actions in luteal cells, but the effects of superoxide, the archetypal oxygen radical, are unknown. Xanthine oxidase generates superoxide, and the activity of this enzyme, and purine substrate, are increased under ischemia, such as that seen at luteal regression. We therefore examined the actions of xanthine oxidase on luteal cells to assess the effects of this enzyme and the superoxide anion on luteal function. Xanthine oxidase, in the presence of hypoxanthine (50 microM), produced marked inhibition of LH-sensitive cAMP and progesterone production with complete inhibition at 25 mU/ml and half-maximal inhibition at about 5 mU/ml. These antigonadotropic actions of xanthine oxidase were rapid with maximal effects within 5 min, followed several minutes later by substantial depletion of ATP. Heat, superoxide dismutase, and catalase or catalase alone abolished the actions of xanthine oxidase. While depletion of ATP by xanthine oxidase was prevented by 3-amino-benzamide, an inhibitor of DNA repair, inhibition of cAMP and progesterone production was still evident. Xanthine oxidase also inhibited progesterone synthesis stimulated by 8-bromo-cAMP. Isobutylmethylxanthine, a cAMP phosphodiesterase inhibitor, did not reverse the inhibition of cAMP accumulation by xanthine oxidase, and the enzyme had no effect on LH receptor binding activity. Since catalase reversed the effects of xanthine oxidase, we conclude that superoxide was rapidly dismuted to hydrogen peroxide and mediated the antigonadotropic and antisteroidogenic actions of xanthine oxidase in luteal cells. The sensitivity of luteal cells to xanthine oxidase raises the possibility that this enzyme may serve as a significant source of hydrogen peroxide in the corpus luteum.

The antigonadotropic actions of prostaglandin F2 alpha and phorbol ester are mediated by separate processes in rat luteal cells.

Protein kinase-C (PKC) has been suggested as a possible mediator of the antigonadotropic action of prostaglandin F2 alpha (PGF2 alpha) in luteal cells. To examine this possibility, we evaluated the effects of phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] in relation to those of PGF2 alpha on cAMP accumulation and ATP levels as well as on the subcellular distribution of PKC activity in rat luteal cell cultures. Treatment of luteal cells for 1 h with TPA or PGF2 alpha produced a dose-dependent inhibition of LH-stimulated cAMP accumulation. Maximal inhibition produced by PGF2 alpha was about 35% greater than that produced by TPA. Moreover, PGF2 alpha produced a further inhibition of LH action when the cells were maximally inhibited by TPA. Staurosporine, a PKC inhibitor, reversed inhibition of LH-dependent cAMP accumulation produced by TPA, but had no effect on the response to PGF2 alpha. Furthermore, cells in which PKC was persistently activated by prolonged TPA treatment lost their responsiveness to additional TPA, but continued to show inhibition of cAMP accumulation by PGF2 alpha. TPA also produced a dose-dependent decrease in cell levels of ATP in contrast to PGF2 alpha. Finally, TPA produced a rapid redistribution of PKC activity from the cytosolic to the particulate fraction, whereas PGF2 alpha produced only a slight redistribution. We conclude that the acute antigonadotropic action of PGF2 alpha in rat luteal cells occurs via mechanisms other than phorbol ester-sensitive PKC activation.

Inhibition of protein synthesis and hormone-sensitive steroidogenesis in response to hydrogen peroxide in rat luteal cells.

Hydrogen peroxide (H2O2) is generated in the corpus luteum at functional luteal regression and produces rapid antigonadotropic effects in rat luteal cells. However, the mechanism by which peroxide interrupts LH- and cAMP-sensitive progesterone synthesis is unknown. The post-cAMP site of H2O2 action is due to the reduced cholesterol availability in mitochondria, and this process is well known to be dependent on protein synthesis. Therefore, we examined whether H2O2 may interfere with protein and RNA synthesis, and whether such responses may be associated with inhibition of steroidogenesis. Incorporation of radiolabeled amino acids into luteal proteins was inhibited in response to H2O2 in a time- and dose-dependent manner, and these doses are similar to those that inhibit progesterone synthesis, shown earlier in the identical paradigm. The inhibitory effect of H2O2 on amino acid incorporation was not due to increased protein degradation, impaired transport of amino acids, or depletion of cellular ATP levels. H2O2 also inhibited RNA synthesis, increased RNA degradation, and impaired the efficiency of mRNA as a translation template. The time course for the inhibitory effect of H2O2 on protein and RNA synthesis was very rapid and coincident with inhibition of steroidogenesis. Inhibition of protein and RNA synthesis and steroidogenesis were reversed by preincubation of cells with the cell-permeable metal chelator o-phenanthroline, which implicates metal-dependent radical generation as the probable mediator of these actions of H2O2. We conclude that the target of the post-cAMP site of peroxide-induced inhibition of cAMP-dependent steroidogenesis is the inhibition of rapidly inducible proteins that are known to mediate translocation of cholesterol within mitochondria, where it is used as a substrate for pregnenolone synthesis.

Antigonadotropic and antisteroidogenic actions of peroxide in rat granulosa cells.

Reactive oxygen species are produced in the ovary. In luteal cells, peroxide abruptly inhibits LH-sensitive cAMP and progesterone production, and may serve a role as a mediator of luteolysis by such mechanisms. The objective of the present studies was to evaluate the acute actions of peroxide in rat granulosa cells. Peroxide at concentrations in the low micromolar range produced a marked and dose-dependent inhibition of FSH-sensitive cAMP accumulation and progesterone production, and depleted cell levels of ATP within 1 min. Longer treatment with peroxide (60 min) caused complete abrogation of the actions of FSH. Peroxide-induced depletion of ATP was prevented by 3-aminobenzamide, an inhibitor of DNA repair, but maintenance of cell levels of ATP did not prevent the anti-FSH effects of peroxide. Peroxide also abrogated cAMP accumulation and progesterone production in response to LH in granulosa cells. Unlike that seen with LH, inhibition of FSH-sensitive cyclic AMP accumulation by peroxide was partially reversed with isobutylmethyl xanthine, an inhibitor of cyclic AMP phosphodiesterase. Although peroxide inhibited cAMP accumulation in response to cholera toxin, it did not inhibit this same response to forskolin, which indicates that peroxide may interfere with G-protein-dependent activation of adenylate cyclase. Peroxide inhibited steroidogenesis in response to cholera toxin, forskolin, and 8-bromo-cAMP. The marked inhibitory actions of peroxide on gonadotropic hormone action and steroidogenesis in granulosa cells raise the possibility that peroxide may mediate events associated with loss of follicular function.

Physiological and pharmacological inhibitors of luteinizing hormone-dependent steroidogenesis induce heat shock protein-70 in rat luteal cells.

Heat shock protein (HSP) synthesis increases in cells with a broad range of stress conditions. We recently showed that induction of HSP-70 is associated with inhibition of hormone-sensitive steroidogenesis, but not hormone-sensitive cAMP accumulation, in rat luteal cells by a mechanism associated with interruption of cholesterol translocation in mitochondria. As HSP induction may be an early mediator of luteal regression, we investigated whether physiological and pharmacological inhibitors of luteal function would induce HSP-70 in rat luteal cells. Both [35S]methionine labeling and Western blotting with antibodies against the inducible form of HSP-70 revealed HSP induction in rat luteal cells by 1 microM prostaglandin F2 alpha (PGF2 alpha) coincident with inhibition of progesterone synthesis. In contrast, PGE2 (1 microM) failed to increase HSP-70 synthesis. Phorbol 12-myristate 13 acetate (3 microM), tumor necrosis factor-alpha (100 ng/ml), and ionomycin (1 microM) also induced HSP synthesis. Induction of HSP-70 was preceded by the rapid activation of heat shock transcription factor, which binds to the heat shock transcriptional control element. Gel retardation assays demonstrated heat shock transcription factor activation within 15 min of PGF2 alpha treatment. Northern analysis with an oligonucleotide probe specific for inducible HSP-70 showed induction at the transcriptional level by the above agents within 30 min. As functional luteal regression is known to display elements of a stress response, the finding that a number of factors that inhibit hormone-sensitive progesterone synthesis rapidly activate the heat shock response further implicates HSPs as possible mediators of luteolysis.

Coordinate induction and activation of metalloproteinase and ascorbate depletion in structural luteolysis.

Structural luteolysis was found decades ago to be induced by PRL in the hypophysectomized rat, but the mechanisms of this process are unknown. To gain information on mechanisms of luteal involution, we developed an animal model that circumvented complex surgery and provided ample tissue for analyses. Gonadotropin-synchronized ovulation and luteinization were induced in immature rats, followed by treatment with ergot alkaloid and PRL. PRL-induced structural luteolysis, as shown by loss of luteal weight, protein, and DNA after pretreatment with ergot alkaloid, was evident after 36 h. Ascorbic acid depletion was rapid, severe, and lasting in luteal tissue during structural luteolysis, but lipid peroxidation or depletion of vitamin E was not evident. PRL treatment of animals with functional corpora lutea did not induce luteal involution. Significantly, after natural functional luteolysis occurred, PRL was highly effective in inducing structural luteolysis. Thus, either natural or ergot-induced functional luteolysis permitted the luteolytic expression of PRL. A greater depletion of protein than DNA was seen during PRL-induced structural luteolysis and was associated with a significant increase in neutral caseinase activity in luteal extracts. Caseinase activity was markedly reduced by calcium chelators and profoundly inhibited by the chelator orthophenanthroline; only slightly reduced activity was seen with serine, aspartate, or cysteine proteinase inhibitors. These findings implicate metalloproteinase (MMP) as the relevant caseinase that was increased during structural luteolysis. The major proteinase identified by zymography had apparent sizes of 72 and 66 kilodaltons (kDa), and slight but detectable activity was also seen at 92 and 84 kDa. Organomercurial treatment caused a major shift of the 72-kDa band to 66 kDa and the 92-kDa band to 84 kDa, confirming MMP-2 and MMP-9 by activation of latent activity of each MMP, respectively. Structural luteolysis caused a significant increase in the activated 66-kDa form and the latent 72-kDa form of MMP-2, which occurred before a loss of luteal weight or protein. As MMP-2 degrades collagen (type IV) in basement membranes, we conclude that an early event in PRL-induced structural luteolysis is the degradation of extracellular matrix. This conclusion is further emphasized by the marked and lasting depletion of ascorbic acid, a vitamin long known to serve an essential role in collagen synthesis.

Lipid hydroperoxides evoke antigonadotropic and antisteroidogenic activity in rat luteal cells.

At functional luteolysis, the rat corpus luteum generates hydrogen peroxide (H2O2), which is known to rapidly inhibit gonadotropin-sensitive cAMP and progesterone production in isolated luteal cells. Lipid peroxides also increase markedly in the rat corpus luteum with the onset of functional luteolysis, and H2O2 is a potent inducer of lipid peroxidation. However, the actions of lipid peroxides on cell function are unknown. The objective of this study was to investigate the impact of typical lipid peroxides, cumene hydroperoxide (CuOOH) and 15(S)-hydroperoxyeicosatetraenoic acid, on rat luteal cells. CuOOH inhibited both LH-sensitive cAMP accumulation (ED50, 25 microM) and progesterone production (ED50, 20 microM). 15(S)-hydroperoxyeicosatetraenoic acid also dose dependently inhibited steroidogenesis. A significant reduction of LH-stimulated progesterone production was evident within 5 min of treatment with CuOOH, whereas inhibition of cAMP accumulation was not evident until 60 min. 8-Bromo-cAMP and 22-hydroxycholesterol caused partial and complete reversal of CuOOH-inhibited progesterone secretion, respectively. Preincubation of cells with o-phenanthroline completely reversed the inhibitory effects of CuOOH on cAMP accumulation and partially reversed its effects on progesterone production. Incorporation of radiolabeled amino acids into luteal proteins was significantly inhibited by CuOOH (25 microM) within 2 min of treatment and was reduced to 40 +/- 14% of control levels at 60 min. CuOOH (25 microM) maximally stimulated PGE2 production within 30 min of treatment (180 +/- 30% of control), a response that was completely blocked by aristolochic acid (100 microM), a phospholipase-A2 inhibitor, and indomethacin (1 microgram/ml), a prostaglandin (PG) synthesis inhibitor. The present results suggest that the acute inhibitory action of lipid peroxides on LH-stimulated progesterone production occurs down-stream of cAMP synthesis and appears to be due to impaired cholesterol utilization for steroidogenesis, possibly through inhibition of protein synthesis. The stimulation of PGE2 production by CuOOH appears to involve the activation of phospholipase-A2, which is a rate-limiting step in PG synthesis. Lipid peroxides as well as H2O2 may serve as mediators of functional luteolysis.

Ovarian vitamin E accumulation: evidence for a role of lipoproteins.

Reactive oxygen species, such as superoxide, hydrogen peroxide, and lipid peroxides, impair luteal function. Vitamin E, a lipophilic antioxidant vitamin, provides a major avenue of protection by scavenging free radicals and terminating lipid peroxidation. We previously showed that ovarian vitamin E levels increase after functional regression (loss of progesterone production) of the corpus luteum in the pseudopregnant rat and the objective of the present studies was to determine the mechanism(s) that resulted in such increased levels of vitamin E. Luteal vitamin E levels were significantly elevated after function regression and remained elevated. Luteal cholesterol ester levels, in contrast, decreased in parallel with the decrease in plasma progesterone levels, whereas plasma vitamin E, cholesterol, and cholesterol ester levels did not change. Because vitamin E is transported in blood by chylomicrons and lipoproteins, ovarian vitamin E levels were determined after treatments known to modify ovarian lipoprotein receptor content and serum lipoproteins. Acute treatment with aminoglutethimide during the mid-luteal phase decreased serum progesterone levels and increased luteal vitamin E and cholesterol ester levels. Daily treatment with 4-amino-pyrazolo-(3,4-d)pyrimidine reduced serum vitamin E and cholesterol ester levels, diminished the accumulation of vitamin E associated with luteal regression, significantly reduced luteal cholesterol esters levels, and increased luteal high density lipoprotein-binding sites. Analysis of the distribution of vitamin E between a membrane/particulate pellet and a lipid droplet/granule cytosol before and after luteal regression revealed no changes. Vitamin E levels were divided 60:40 between a crude particulate/membrane fraction and a cytosol/lipid droplet fraction, although functional regression produced a 2.5-fold increase in total luteal vitamin E levels. In conclusion, the uptake of vitamin E by the corpus luteum appears to be mediated by lipoprotein receptors and the increase in vitamin E that follows functional regression, we suggest, may be due to a diminished consumption of vitamin E by oxidative radicals, most likely generated during steroidogenesis.

Ovarian gonadotropin-releasing hormone-like protein(s): demonstration and characterization.

Both GnRH and prostaglandin F2 alpha inhibit LH-stimulated cAMP accumulation and progesterone secretion in isolated luteal cells. Moreover, since ovarian GnRH receptors have been demonstrated, this study was conducted to determine if GnRH-like substances were present in the luteinized rat ovary. Detection of GnRH-like activity was based on a sensitive and specific ovarian membrane GnRH radioreceptor assay and an immunoassay specific for GnRH. Ovaries were extracted with an aqueous medium containing formic acid, HCl, trifluoroacetic acid, and NaCl. Material present in the supernatant fraction which adsorbed to Waters C18 Sep-Paks was subsequently eluted with acetonitrile and lyophilized. The redissolved ovarian extract showed substantial radioreceptor activity, but very little immunoassayable activity. The GnRH-like activity of the ovary was sensitive to proteolytic enzyme digestion and to incubations at 50 C for as little as 5 min, had an apparent mol wt greater than 1,000 but less than 10,000, and was not soluble in ether. Extracts of plasma did not exhibit radioreceptor or immunoreactive activity, whereas hypothalamic extracts exhibited both radioreceptor and immunoreactive activities. Liver and kidney extracts showed less radioreceptor activity than ovarian extracts and very little immunoreactive activity. Two peaks of radioreceptor activity appeared when the ovarian extract was further fractionated by reverse phase HPLC. The two peaks of GnRH-like activity were clearly separated from GnRH or [D-Ala6, des-Gly10] GnRH ethylamide, an analog used in this study, when these were included in ovarian extracts. It is concluded that rat ovaries contain a GnRH-like protein(s) with membrane binding properties similar to those of GnRH but with other characteristics distinctly different from those of GnRH. The ovarian GnRH-like protein is immunologically different from GnRH, sensitive to elevated temperatures which do not effect GnRH, and chromatographically different from GnRH during reverse phase HPLC.

A cell adhesion receptor antiserum abolishes, whereas laminin and fibronectin glycoprotein components of extracellular matrix promote, luteinization of cultured rat granulosa cells.

We recently showed that structural regression is marked by an endocrine-induced increase in matrix metalloproteinase activity specific for basement membrane, which suggests that extracellular matrix (ECM) may play an important role in sustaining luteal cell function. Such a role for ECM has been demonstrated for cultured mammary epithelial cells, hepatocytes, and keratinocytes. To test this hypothesis, granulosa cells from preovulatory follicles that were induced to luteinize by gonadotropin stimulation in vivo were examined. Initial studies established that cells cultured on plastic in medium supplemented with 1% fetal bovine serum, LH (100 pg/ml), PRL (1 microgram/ml), and insulin-like growth factor-I (5 ng/ml) showed a time-dependent increase in the secretion of progesterone (P4) and total progestin (P4 plus 20 alpha-dihydroprogesterone) for at least 10 days and that replacement of fetal bovine serum with 0.1% BSA stimulated P4 secretion and reduced the 20 alpha-dihydroprogesterone to P4 ratio from 10:1 to as low as 3:1. The inclusion of an anticell adhesion receptor subunit sera (Lenny IV, against the integrin beta 1-subunit) in the culture medium for the first 2 days resulted in an irreversible loss of progestin secretion by the cultured granulosa cells, but the inclusion of a bacterial collagenase (form III) had no effect. Granulosa cells from preovulatory follicles cultured on ECM (Matrigel matrix) formed cell aggregates and projected cellular sprouts, but secreted less P4 than those cultured on plastic. The inclusion of laminin in the culture medium or laminin coating the culture wells stimulated P4 secretion by granulosa cells and promoted the enlargement of steroidogenic cells (3 beta-hydroxysteroid dehydrogenase). Fibronectin-coated, but not collagen-I-coated, wells similarly promoted P4 secretion. These results suggest that a cell adhesion receptor (an integrin), and laminin and fibronectin, major glycoprotein components of ECM, play important roles in the differentiation of granulosa cells to luteal cells.

An unusual sex steroid-binding protein in mature male rat liver cytosol.

Mature male rat liver cytosol contains a moderate affinity and capacity estrogen-binding protein in at least a 200-fold higher level than mature female or immature male rat liver cytosol. Binding of estradiol to this protein is very rapid, is stabilized by EDTA, and is inhibited by divalent cations. This is the major binding protein for [3H]estradiol ([3H]E2) in mature male rat liver cytosol, and it has properties clearly distinguishing it from putative liver or uterine estrogen receptors. In addition to binding [3H]E2, this protein seems to rapidly bind a [3H]5alpha-dihydrotestosterone ([3H]DHT) metabolite at the same binding site. The binding of this androgen metabolite is stabilized by EDTA and is inhibited by divalent cations. The binding properties of the [3H]DHT metabolite suggest that these binding sites are not classical androgen receptors. Cytosol binding levels of both the [3H]E2 and the [3H]DHT metabolites change in a similar direction in resonse to endocrine manipulation. The putative liver estrogen receptor level, determined after partial purification (in a redissolved 30% ammonium sulfate-precipitated fraction), seems to change in an opposite direction in response to these same endocrine manipulations.

Estrogen receptor in rat liver: translocation to the nucleus in vivo.

Following in vivo ethinyl estradiol (EE2) administration (100 microgram sc) to adult female rats, the estradiol-specific binding sites (ESBS) of liver cytosol were markedly reduced at 30 and 60 min. The reduction at 30 min was to one-quarter of that found in rats treated with vehicle alone. Coincident with this reduction, nuclear ESBS were increased. The ESBS of partially purified cytosol and of dense sucrose-purified nuclei were determined by gel filtration after incubations with tritiated estradiol using exchange assay conditions. An elevated temperature during the exchange assay incubations was necessary to demonstrate most of the ESBS in purified nuclei of EE2-treated rats and suggested that estrogens are attached at these sites. Following administration of 5 microgram EE2, the decrease in cytosol ESBS and the increase in nuclear ESBS were smaller. In contrast, 5 microgram EE2 was as effective as 100 microgram EE2 in substantially increasing the ESBS observed in uterine nuclear fractions. The low level of ESBS found in whole brain purified nuclei was unchanged by 100 microgram EE2 administration. The steroid specificity and proteolytic enzyme sensitivity of the purified nuclear ESBS of treated rats were similar to that of the partially purified cytosol ESBS of rats treated with vehicle alone. The data are consistent with the ESBS being estrogen receptor proteins which translocate from the liver cytosol to the nucleus after estrogen administration in vivo.

Estrogen receptor in adult male rat liver.

A 30% ammonium sulfate precipitation has previously been utilized to partially purify the estrogen receptor(s) of female rat liver cytosol. This procedure has now been used to fractionate the estradiol-binding sites of adult male rat liver cytosol. The 30% ammonium sulfate precipitation partially purifies a group of estradiol-binding sites which have properties quite distinct from the large number of sites present in male liver cytosol. The partially purified male sites seem to have the same properties as the partially purified female estradiol-binding sites. They seem to be proteins that are estrogen specific and have a high estradiol affinity (Kd = 1 X 10(-10) M) and a low estrogen capacity (2.3 fmol/mg liver). The estradiol-binding sites of prepubescent male rat liver cytosol have also been fractionated by 30% ammonium sulfate precipitation. The redissolved ammonium sulfate precipitates from prepubescent male rat liver cytosol contain fewer estradiol-binding sites then those from adult male or female rats. It seems that adult male as well as adult female rat liver contains estrogen receptors.

Presence of gonadotropin-releasing hormone-like proteins in bovine and ovine ovaries.

Recently, the rat ovary was shown to contain significant levels of a GnRH-like protein, but no detectable GnRH. In the present studies, extracts of bovine ovaries and ovine corpora lutea were examined for their content of both GnRH-like protein and GnRH. The GnRH-like proteins were detected with a rat ovarian membrane radioreceptor assay, and GnRH was detected with a specific GnRH RIA. The biological activity of the GnRH-like protein was evaluated with a rat luteal cell assay. GnRH-like activity, but not GnRH, was clearly present in extracts of the entire bovine ovary, bovine corpus luteum, bovine granulosa cells, and ovine corpus luteum. The highest levels of GnRH-like activity were present in granulosa cells. Neither GnRH-like activity nor GnRH was detected in extracts of bovine follicular fluid or bovine jugular plasma. Fractionation of the bovine and ovine GnRH-like proteins by reverse phase HPLC resulted in retention times similar to that of the rat ovarian GnRH-like protein, but distinctly different from that of authentic GnRH. The bovine and ovine GnRH-like fractions, like those in the rat, were sensitive to protease and heat. The bovine GnRH-like protein, obtained by preparative reverse phase HPLC, evoked a dose-dependent inhibition of LH-stimulated cAMP accumulation in rat luteal cells similar to that caused by GnRH. Based on these results, we suggest that the bovine ovary, granulosa cells, and corpus luteum and the ovine corpus luteum contain an antigonadotropic GnRH-like protein similar to the GnRH-like protein of the rat ovary. The presence of a similar GnRH-like protein (but the absence of GnRH) in ovaries of domestic species and the rat raises the possibility that this substance may play a paracrine antigonadotropic role in the ovary of diverse species.

Hormone induction of ascorbic acid transport in immature granulosa cells.

Ascorbic acid serves a vital role as an antioxidant, and like FSH, it inhibits apoptosis of granulosa cells in cultured follicles. In contrast, reactive oxygen species block the action of FSH and induce DNA damage in these cells. As the uptake of ascorbic acid by granulosa cells may be a site for regulation, we examined the nature of this process and whether uptake is under hormone control. Granulosa cells were isolated from immature rats pretreated with estradiol or diethylstilbestrol for 3-4 days and placed in culture. Culture of the cells with either FSH (50 ng/ml) or insulin-like growth factor I (IGF-I; 30 ng/ml) for 48 h increased ascorbic acid uptake by 2.7- and 1.9-fold (P < 0.05), respectively, and the response to FSH plus IGF-I was additive (4.5-fold; P < 0.05). The interval for maximum induction of ascorbic acid transport by FSH was between 4-8 h, whereas a significant response to IGF-I was not seen until 48 h. GnRH (1 microM), phorbol ester (phorbol 12-myristate 13-acetate; 1 microM), and 8-bromo-cAMP (8Br-cAMP; 1 mM) also induced ascorbic acid transport by 1.7-, 1.9-, and 2.3-fold (P < 0.05) within 24 h, and the response to maximal levels of phorbol ester and 8Br-cAMP was synergistic (4.8-fold; P < 0.05). Kinetic analysis showed a similar Michaelis constant (K(m); 50.8 +/- 5.3 microM) and maximum velocity (3.3 +/- 0.4 pmol/10(6) cells.min) for ascorbic acid transport in FSH-, 8Br-cAMP-, or phorbol ester-treated cells. Ouabain (100 microM) or removal of extracellular Na+ significantly inhibited ascorbic acid uptake, as did dinitrophenol (1 mM), an inhibitor of mitochondrial production of ATP. The induction of ascorbic acid transport by FSH, IGF-I, or GnRH was abolished by simultaneous incubation with tyrphostin (AG-18; 80 microM), a specific tyrosine kinase inhibitor, whereas induction was unaffected by an inactive, but chemically similar, compound (A-1; 80 microM). From these results we conclude that ascorbic acid uptake is energy and Na+ dependent and that the induction of ascorbic acid transporters in granulosa cells occurs through multiple hormones that ultimately influence tyrosine-specific protein kinases. The hormone-dependent induction of ascorbic acid accumulation in granulosa cells appears to be an essential process for the development and maintenance of a viable follicle.

Androgen receptor in the rabbit liver and apparent translocation to the nucleus in vivo.

The present study demonstrates that the liver of a mammal, the rabbit, contains an androgen receptor. Rabbit liver cytosol or purified nuclei were incubated with the radioactive androgen R-1881 (methyltrienolone). The cytosol of adult female rabbit liver contained androgen-binding sites of high affinity, Kd 0.9 nM, and a capacity of 7000 fmol/g liver or 79 fmol/mg cytosol protein. After partial purification by 35% ammonium sulfate precipitation (AS cytosol), the binding specificity pattern was consistent with that of androgen receptor. Apparent translocation from cytosol to nucleus was examined by administering 100 micrograms nonradioactive R-1881 in vivo. One hour later, almost all of the receptor was detected in liver nuclei. The receptor concentration in purified nuclei, as determined by an exchange procedure, was 2100 fmol/g liver, which is an increase of 6-fold relative to the low levels in nuclei of untreated rabbits. The binding affinity, specificity pattern, and protease sensitivity for the sites in the nucleus after in vivo androgen in general resembled those as AS cytosol binding in untreated liver. Androgen receptors were also present in AS cytosol of the immature female rabbit liver and, in lower concentration, of the intact adult male rabbit. The properties of liver androgen binding are quite different from those of testosterone binding protein present in serum. Accordingly, an androgen binding protein with high affinity and specificity and capable of translocation to the nucleus in vivo has been detected in mammalian liver. An androgen receptor in the mammalian liver may mediate androgen effects on liver function, including modulation of synthesis of selective plasma proteins.

Hydrogen peroxide evokes antisteroidogenic and antigonadotropic actions in human granulosa luteal cells.

The nature of the luteolysin in humans is unknown. Hydrogen peroxide (H2O2), notably released by activated leukocytes, is generated in the rat corpus luteum at luteolysis and evokes luteolytic-like effects in rat luteal cells. We, therefore, evaluated the actions of H2O2 in human luteinized granulosa cells. After 2 days of preculture with low levels of hCG, human granulosa luteal cells were placed in suspension culture for 1 h in the presence of isobutylmethylxanthine (100 microM). A 60-min challenge with hCG evoked dose-dependent stimulation of cAMP and progesterone production. H2O2 dose-dependently inhibited progesterone production (ED50, 50-100 microM) in the absence or presence of hCG and blocked hCG-stimulated cAMP accumulation. Inhibition of progesterone synthesis by H2O2 was near maximal within 5 min, whereas inhibition of cAMP accumulation was not evident until 60 min. Cell viability was unaffected by H2O2, and inhibition of cAMP was reversible, but inhibition of steroidogenesis was long-lasting. Progesterone production stimulated by 8-bromo-cAMP, 22-hydroxycholesterol, and pregnenolone was inhibited by H2O2 as was androstenedione-dependent estradiol production. These findings indicate that H2O2 blocked progesterone synthesis by inhibition of cholesterol side-chain cleavage cytochrome P450, 3 beta-hydroxysteroid dehydrogenase, aromatase, and/or 17 beta-hydroxysteroid dehydrogenase. While H2O2 blocked stimulation of cAMP accumulation in response to hCG and cholera toxin, this same response produced by forskolin or aluminum fluoride was unaffected by H2O2. Thus, H2O2 appears to uncouple LH (hCG) receptors by interruption of G-protein-dependent activation of adenylate cyclase. In summary, H2O2 evokes effects in isolated human granulosa luteal cells that are associated with luteal regression, which raises the interesting possibility that H2O2 may serve a role as a mediator of this process like that in the rat.

A gonadotropin-releasing hormone (GnRH)-like protein in human ovaries: similarity to the GnRH-like ovarian protein of the rat.

The rat ovary contains significant amounts of a GnRH-like protein, but does not contain readily detectable GnRH. We, therefore, determined whether human ovaries contain a similar GnRH-like protein. Extracts of human ovaries as well as rat ovaries, rat hypothalami, and rat plasma were examined for their content of both GnRH-like protein and GnRH. GnRH-like activity was detected by inhibition of binding of GnRH to rat ovarian membrane receptors [radioreceptor assay (RRA)]. GnRH was detected with a specific GnRH RIA. Equivalent amounts of GnRH-like activity were found in both human and rat ovaries, whereas GnRH was not detected. The human and rat ovarian GnRH-like materials had identical retention times after reverse phase high pressure liquid chromatography, and these were clearly different from that of authentic GnRH. Furthermore, the ovarian GnRH-like activity was clearly separated by HPLC from GnRH and GnRH-like activities extracted from rat hypothalami. Plasma contained neither GnRH-like nor GnRh activity. The ovarian GnRH-like activity was protease and heat sensitive, whereas hypothalamic GnRH and GnRH-like activity were protease sensitive, but not heat sensitive. The ovarian GnRH-like protein did not inhibit the specific binding of prostaglandin F2 alpha to rat ovarian membranes under conditions of assay similar to the GnRH RRA. The ovarian GnRH-like protein did not have GnRH peptidase activity, its inhibition of GnRH binding in the RRA was reversible, and bacitracin (protease inhibitor) had no effect on assay of the ovarian GnRH-like protein by RRA. We conclude that the human ovary contains a GnRH-like protein similar, if not identical, to the GnRH-like protein of the rat ovary, which is different from GnRH and the GnRH-like protein of the hypothalamus. The presence of the same GnRH-like material in human and rat ovary raises the possibility that this protein may mediate responses in the human ovary similar to the antigonadotropic actions of GnRH in the rat ovary.