Prostaglandin f2alpha treatment in vivo, but not in vitro, stimulates protein kinase C-activated superoxide production by nonsteroidogenic cells of the rat corpus luteum.

Luteal regression is associated with the generation of reactive oxygen species (ROS). To determine the nature of the ROS generator, cells isolated from luteinized rat ovaries were examined for ROS production using luminol-amplified chemiluminescence (LCL). Cells cultured for 2-48 h exhibited minimal LCL, but there was a significant (30- to 50-fold), rapid (maximum at 3-5 min), and dose-dependent increase in LCL in response to phorbol ester (phorbol 12-myristate 13-acetate; TPA; ED50 = 0.03 microM) and diacylglycerol (1,2-dioctanoyl-glycerol; ED50 = 30 microM). The TPA-induced response was cell number dependent and was virtually abolished by superoxide dismutase, freezing, or heating (95 degrees C for 5 min). Zymosan, known to induce a phagocytic response in leukocytes, stimulated a superoxide (O2-.) response with a slow onset (maximum at 40 to 60 min) and a maximum about one third of that observed for TPA. The response to TPA and zymosan was inhibited by the NADPH/NADH-oxidase inhibitor, diphenylene iodonium (ID50 = 5 microM for TPA), but not by the mitochondrial inhibitors, potassium cyanide, rotenone, or sodium azide. Fractionation of cells by centrifugal elutriation showed that TPA-stimulated O2-. production coeluted with the nonsteroidogenic cells and that little, if any, O2-. generation coeluted with the steroidogenic cells. Cells isolated 1, 2, and 4 h after in vivo treatment with a luteolytic dose of prostaglandin F2alpha (PGF2alpha) showed a significant increase in TPA-stimulated O2-. production at 2 h, whereas luteal cells or corpora lutea incubated directly with 1 microM PGF2alpha did not show any increase in response. Corpora lutea isolated from naturally regressed ovaries (18 days after ovulation) showed a significantly elevated level of TPA-stimulated O2-. production. In conclusion, there is a superoxide generator in luteinized ovaries that is activated through a protein kinase C pathway, localized in nonsteroidogenic cells, transiently increased during PGF2alpha-induced luteolysis in vivo, and elevated during natural luteal regression.

Accumulation of ascorbate by endocrine-regulated and glucose-sensitive transport of dehydroascorbic acid in luteinized rat ovarian cells.

The corpus luteum is notable for very high levels of ascorbic acid. In luteal cells, ascorbic acid depletion occurs as a result of consumption during radical scavenging, inhibition of ascorbic acid uptake, and stimulation of its secretion. Oxidation of ascorbic acid generates dehydroascorbic acid (DHAA). Although levels of DHAA in blood are much lower than those of ascorbic acid, DHAA serves as the major transportable form of ascorbate for certain cell types. The aim of the present studies was to investigate whether DHAA transport is a potential mechanism for conserving ascorbic acid in the corpus luteum. DHAA uptake by rat luteal cells precultured for 24 h was linear for up to 30 min. Kinetics studies showed that uptake of DHAA was a concentration-dependent and saturable process with an estimated Michaelis constant (Km) of 830 microM and a maximum velocity (Vmax) of 700 pmol/min per 10(6) cells, a rate 50 times that of ascorbate transport. More than 90% of DHAA was reduced to ascorbic acid within 2 h of cellular uptake. DHAA uptake was energy- and microfilament-dependent, as transport was inhibited by 2,4-dinitrophenol (1 mM) and cytochalasin B (10 microM). Menadione (50 microM), an intracellular generator of reactive oxygen species, also markedly reduced DHAA uptake. In contrast to ascorbic acid transport, DHAA uptake was potently inhibited by glucose and phloretin, an inhibitor of glucose transporters, with IC50s of approximately 5 mM and 10 microM, respectively. DHAA uptake appears to occur via an insulin-insensitive transporter, as insulin (10 nM) had no effect on uptake. However, 24-h preincubation with insulin-like growth factor (IGF)-I dose-dependently (10-100 ng/ml) stimulated DHAA uptake; similar concentrations of IGF-II had no effect. The secretion of radioactivity by cells preloaded with radiolabeled DHAA was significantly increased by prostaglandin F2alpha (1 microM). The ability of luteal cells to transport DHAA in a regulated manner may serve to maintain vital levels of ascorbic acid within the corpus luteum.

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.

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.

Cytokine induction of heat shock protein in human granulosa-luteal cells.

The infiltration of leukocytes is a characteristic feature of luteolysis in humans. Leukocytes are known to generate physiological inducers of cell stress such as cytokines which have been implicated as mediators of functional luteal regression. In cells exposed to stress, a response characterized by an increase in heat shock protein (HSP) synthesis occurs. Recently, the induction of HSP-70 in rat luteal cells has been shown to inhibit luteinizing hormone (LH) and cAMP-sensitive progesterone production, possibly by interfering with the translocation of cholesterol to the mitochondrial cytochrome P450scc. We therefore investigated whether HSP-70 is induced in human granulosa-luteal cells and its relationship to steroidogenesis. [35S]Methionine labelling showed an increase in a 70 kDa protein after heat treatment which was demonstrated to be HSP-70 by Western analysis using monoclonal antibodies against the constitutive and inducible forms of HSP-70. Induction of HSP-70 in human granulosa-luteal cells was also seen with interferon (IFN) gamma (10 ng/ml), tumour necrosis factor (TNF)-alpha (100 ng/ml) and a combination of IFN gamma/TNF-alpha (10/50 ng/ml). Interleukin-1 beta (IL-1 beta) (30 ng/ml) showed minimal induction of HSP-70 above control values. An increase in activated heat shock factor, which binds to the heat shock transcriptional control element, was detected after heat shock, IFN/TNF, and IFN treatment. Coincident with the induction of HSP-70 by heat shock was the inhibition of progesterone production compared with non-shocked granulosa-luteal cells. Heat shock inhibition of progesterone synthesis was partially reversed by the cell- and mitochondria-permeant cholesterol analogue, 22R-hydroxycholesterol. Cell viability was unaffected by heat treatment. White blood cell-depleted granulosa-luteal cell cultures treated with IFN demonstrated a significant reduction in progesterone production. Treatment with IFN/TNF, TNF, and IL-1 also decreased progesterone secretion, although statistical significance was not achieved. These findings provide evidence that a stress response occurs in human granulosa-luteal cells in response to heat and cytokines. The inhibition of gonadotrophin-sensitive steroidogenesis coincident with the induction of HSP-70 synthesis by physiological agents which are present in the corpus luteum implicates HSP-70 as a potential mediator of luteolysis in the human.

Endocrine regulation of ascorbic acid transport and secretion in luteal cells.

Luteal ascorbic acid depletion by LH and prostaglandin (PG) F2 alpha is well known, but how such depletion occurs is not. We therefore investigated the nature and regulation of ascorbic acid uptake and depletion in the rat CL and luteal cells. In vivo studies showed that blockade of steroidogenesis by aminoglutethimide prevented ascorbate depletion by LH, but not PGF2 alpha. Also, the time course for half-maximal depletion of ascorbic acid in vivo in response to PGF2 alpha was extremely rapid (2-3 min) compared to that known for LH (60 min). Thus, ascorbate depletion by LH and PGF2 alpha appears to occur by different mechanisms. In luteal cells, ascorbate uptake was energy-, sodium-, and microfilament-dependent with a Michaelis constant (Km) of 33 microM, similar to that reported for other cells. In contrast to findings for other cells, PGF2 alpha was found to be a potent and rapid inhibitor of ascorbate uptake with a half-maximal inhibition (IC50) of about 5 nM in luteal cells. Ascorbate uptake was unaffected by LH, PGE2, glucose, bromo-cAMP, progesterone, phorbol ester, ionomycin, hydrogen peroxide (H2O2), or aminoglutethimide. Also novel was the finding that luteal cell secretion of ascorbic acid was rapidly and potently stimulated by PGF2 alpha (IC50 about 5 nM), an effect mimicked by LH, H2O2, generators of reactive oxygen, calcium ionophore, and cytochalasin B. Basal release of ascorbic acid was energy-dependent, as secretion was blocked by a mitochondrial uncoupler and lowered temperature. Phorbol ester, bromo-cAMP, progesterone, aminoglutethimide, and ouabain had no effect on ascorbic acid secretion in luteal cells. These findings indicate that the secretion of ascorbic acid induced by PGF2 alpha, and possibly LH, may be mediated by calcium, reactive oxygen, and cytoskeletal changes. The ability of PGF2 alpha to inhibit ascorbate transport and to stimulate secretion implicates these processes as the basis for the rapid depletion of ascorbic acid in the CL. Ascorbate depletion by LH is associated with stimulation of steroidogenesis and an increase in ascorbic acid secretion.

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.

Modulation of cholesteryl ester hydrolase messenger ribonucleic acid levels, protein levels, and activity in the rat corpus luteum.

Previous studies have shown that the induction of functional luteolysis (loss of progesterone production) with either prostaglandin F2 alpha (PGF2 alpha) treatment or hypophysectomy (APX) diminished neutral cholesteryl ester hydrolase (CEH) activity in the corpus luteum (CL) and that prolactin (PRL) replacement of APX animals prevented luteolysis and maintained CEH activity at control levels. More recent studies have shown that CEH is the same protein as hormone-sensitive lipase (HSL) and that CEH/HSL activity may be regulated by phosphorylation. However, the possibility that CEH/HSL activity may be under transcriptional and/or translation control has not been excluded. Therefore, in the present study we examined whether PGF2 alpha treatment, APX, or inhibition of PRL secretion by bromocryptine (BrC) treatment modulated CEH/HSL mRNA and/or protein levels in a coordinate fashion with CEH activity. Furthermore, we examined whether CEH/HSL mRNA and/or protein levels changed after luteinization of the ovary and after natural functional regression. PGF2 alpha treatment and APX significantly reduced CEH activity; and PGF2 alpha treatment, APX, and BrC treatment significantly reduced CEH/HSL protein and mRNA levels. PRL replacement after APX substantially blocked the reductions in CEH activity, CEH/HSL protein, and CEH/HSL mRNA levels. PRL replacement during BrC treatment significantly inhibited the reductions in CEH/HSL protein and mRNA levels. CEH/HSL mRNA levels increased twofold after luteinization. Whereas CEH/HSL mRNA levels remained elevated after natural luteal regression, CEH/HSL protein significantly decreased. In summary, the luteolytic actions of PGF2 alpha, APX, and BrC resulted in coordinate reductions in luteal CEH activity, protein levels, and mRNA levels; PRL replacement significantly reversed the luteolytic effects of APX and BrC; natural luteal regression resulted in a reduction in CEH/HSL protein without a concomitant reduction in CEH/HSL mRNA. These results suggest that ovarian CEH activity is controlled at the level of both transcription and translation, and that PRL is important for continued CEH/HSL mRNA transcription in the CL.

Peroxisomes and sterol carrier protein-2 in luteal cell steroidogenesis: a possible role in cholesterol transport from lipid droplets to mitochondria.

In the present investigation, we have studied peroxisomes and sterol carrier protein-2 (SCP2) in control and luteinizing hormone stimulated rat luteal cells. Superovulated immature rats in mid-luteal phase (8 days after ovulation) were divided into two groups (n = 4/group) and treated with vehicle (0.2 ml saline), or luteinizing hormone (LH, 20 micrograms/rat). In this animal model, LH acutely stimulates steroidogenesis. Thirty minutes later, corpora lutea were fixed by whole body perfusion and processed for (1) electron microscopic immunocytochemistry to localize SCP2 via the protein A gold immunolabeling technique, and for (2) electron microscopic histochemistry to stain peroxisomal catalase via the alkaline 3,3'-diaminobenzidine tetrahydrochloride method. In the steroidogenic, mid-phase luteal cells of vehicle treated rats (controls), SCP2 was highly concentrated in peroxisomes and sparsely scattered on mitochondria, but no labeling was observed in lipid droplets. In the luteal cells of rats acutely stimulated with LH, peroxisomes immunolabeled for SCP2 were observed within the luteal cell lipid droplets and mitochondria, and in union with lipid droplets and mitochondria. Moreover, in contrast to control luteal cells, significant immunolabeling for SCP2 was detected within the lipid droplets and mitochondria in luteal cells of LH-treated rats. As SCP2 binds cholesterol to 1:1 molar ratio and is known to be involved in the intracellular movement of cholesterol, these findings suggest that peroxisomes and SCP2 may possibly be involved in delivering cholesterol from lipid droplets to the mitochondria when luteal cell steroidogenesis is acutely stimulated by LH.

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.

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.

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.

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.

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)

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.

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.

Mechanisms for the antigonadotropic action of the ovarian gonadotropin-releasing hormone-binding inhibitor protein/histone H2A on ovarian cells.

A GnRH-binding inhibitor (GnRH-BI) was recently purified from bovine ovaries. On the basis of amino acid composition and partial sequence analysis this antigonadotropic GnRH-BI was identified as histone H2A. In the present study the mechanism for the antigonadotropic action of histone H2A was examined and compared to that of GnRH and poly-L-lysine. The potential sites examined were the receptor-coupled pathway of second message synthesis including receptor binding of hormone, G protein activation, and adenylyl cyclase activation. Histone H2A inhibited (ID50 = 2 microM) the binding of hCG by membrane receptors from luteinized rat ovaries in a noncompetitive and dose-dependent manner. The binding of FSH by membrane receptors from immature rat ovaries was not inhibited by histone H2A. Binding of GnRH by pituitary membrane receptors was inhibited by histone H2A, and the ID50 of 8 microM was similar to that previously observed for GnRH binding sites in rat ovarian membranes. No high-affinity binding of histone H2A by rat ovarian membranes was detected. Near-maximal doses of histone H2A (7 microM), poly-L-lysine (10 microM), and GnRH (1 microM) inhibited LH-stimulated cAMP production in isolated rat luteal cells. Inhibition by H2A and poly-L-lysine was larger than by GnRH. Furthermore, histone H2A and poly-L-lysine inhibited cholera toxin (CT)-stimulated cAMP production, but GnRH did not. Like GnRH, neither histone H2A nor poly-L-lysine inhibited forskolin (FK)-stimulated cAMP production. In isolated rat granulosa cells, histone H2A and poly-L-lysine inhibited FSH-, CT-, and FK-stimulated cAMP production.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of ovarian antioxidant vitamins, reduced glutathione, and lipid peroxidation by luteinizing hormone and prostaglandin F2 alpha.

Reactive oxygen species are generated by the rat ovary, and they evoke marked antigonadotropic responses in ovarian cells. Protection against reactive oxygen species is provided by antioxidants such as vitamins C, E, and A, and reduced glutathione (GSH). Our objectives were to establish the ovarian levels of these antioxidants during development and regression of the corpus luteum of the pseudopregnant rat and to determine whether these levels were changed by an acute treatment with either a luteotropic (LH) or luteolytic (prostaglandin [PG] F2 alpha) agent. In addition, we evaluated the extent of oxidative activity in the ovary by determining the level of lipid peroxidation. Follicular development was associated with a significant increase in ovarian levels of vitamin A and GSH, whereas levels of vitamins E and C were unchanged. During the luteal phase, vitamin E levels tended to increase, whereas vitamin A and GSH levels decreased. Luteal regression was associated with a marked increase in ovarian levels of vitamins E and A, whereas GSH levels increased only transiently. Acute treatment with LH in the midluteal phase produced a transient decrease in vitamin C levels that was maximal at 4 h. Luteal vitamin E levels were markedly increased 24 h after LH treatment, whereas vitamin A levels were unchanged, and no evidence of lipid peroxidation was seen. Acute treatment with PGF2 alpha produced a transient decrease in luteal vitamin C levels coincident with transient lipid peroxidation and a sustained fall in serum progesterone levels. Ovarian vitamin A levels were elevated 24 h after PGF2 alpha treatment.(ABSTRACT TRUNCATED AT 250 WORDS)