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Luteinizing hormone/human chorionic gonadotropin stimulates progesterone biosynthesis in the corpus luteum by activating cyclic adenosine monophosphate/protein kinase A cascade. Recent studies have shown that cyclic adenosine monophosphate-mediated activation of protein kinase A interacts with the mammalian target of rapamycin signaling pathways. Furthermore, the use of mammalian target of rapamycin inhibitors for immunosuppression in transplant patients has shown adverse effects in reproductive functions. This study examined whether the mammalian target of rapamycin pathway plays any role in luteinizing hormone-mediated regulation of progesterone production. Human granulosa lutein cells were isolated from follicular aspirates of women undergoing in vitro fertilization. Cells were cultured for 72 h and treated with human chorionic gonadotropin (50 ng/ml) for different time periods with or without pretreatment with mammalian target of rapamycin complex 1 inhibitor, rapamycin, (20 nM) for 1 h. Expression of steroidogenic enzymes, including steroidogenic acute regulatory protein, cholesterol side chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase type 1 messenger RNA, were examined by real-time polymerase chain reaction after 6 h of human chorionic gonadotropin treatment. Expressions of phospho-ribosomal protein S6 kinase and cholesterol side chain cleavage enzyme were analyzed after 15 min and 24 h of human chorionic gonadotropin treatment, respectively. Progesterone production was analyzed by an enzyme immunoassay kit after human chorionic gonadotropin (50 ng/ml) or forskolin (10 μM) treatment for 24 h. Treatment with human chorionic gonadotropin increased the expression of downstream targets of mammalian target of rapamycin complex 1, as well as cholesterol side chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase type 1 and steroidogenic acute regulatory protein messenger RNAs. These increases were inhibited by rapamycin pretreatment. Increased progesterone production in response to treatment with human chorionic gonadotropin or forskolin was also blocked by rapamycin pretreatment. Our findings support a role for mammalian target of rapamycin complex 1 in regulating steroidogenesis in human granulosa lutein cells.

A specific luteinizing hormone receptor (LHR) mRNA binding protein (LRBP) has been identified and purified. This LH receptor mRNA binding protein selectively binds to the polypyrimidine rich bipartite sequence in the coding region of the LHR mRNA and accelerates its degradation. In response to preovulatory LH surge, the LH receptor expression in the ovary undergoes downregulation by accelerated degradation of LH receptor mRNA through the involvement of this RNA binding protein. Here we describe the intracellular mechanism triggered by LH/hCG (human chorionic gonadotropin) that leads to the regulated degradation of LH receptor mRNA. Downregulation of LH receptor mRNA was induced by treatment of cultured human granulosa cells with 10 IU of hCG. Activation of downstream target, extracellular signal-regulated protein kinase 1 and 2 (ERK 1/2) showed an increase within five min and sustained up to 1 h. Confocal analysis showed that ERK1/2 translocates to the nucleus after 15 min of hCG treatment. This leads to an increase in LRBP expression which then causes downregulation of LH receptor mRNA by accelerating its degradation. Treatment with UO126 or transfection with ERK specific siRNA (small interfering RNA) resulted in the abolishment of ERK activation as well as LHR mRNA downregulation. RNA electrophoretic mobility gel shift assay of the cytosolic fractions showed that hCG-induced increase in the LH receptor mRNA binding activity was also abrogated by these treatments. These results show that LH/hCG-induced LH receptor mRNA downregulation is initiated by the activation of ERK1/2 pathway by regulating the expression and activity of LH receptor mRNA binding activity.

LH triggers the biosynthesis of androgens in the theca-interstitial (T-I) cells of ovary through the activation of a cAMP-dependent pathway. We have previously shown that LH/human chorionic gonadotropin (hCG) activates mammalian target of rapamycin complex 1 (mTORC1) signaling network, leading to cell proliferation. In the present study, we provide evidence that the LH/hCG-mediated activation of the mTORC1 signaling cascade is involved in the regulation of steroidogenic enzymes in androgen biosynthesis. Treatment with LH/hCG increased the expression of downstream targets of mTORC1, ribosomal protein S6 kinase 1, and eukaryotic initiation factor 4E as well as steroidogenic enzymes. LH/hCG-mediated stimulation of the steroidogenic enzyme mRNA was blocked by the mTORC1 inhibitor, rapamycin. This inhibitory effect was selective because rapamycin failed to block hCG-mediated increase in the expression of Star mRNA levels. Furthermore, pharmacological targeting of mTORC1 with rapamycin also blocked LH/hCG- or forskolin-induced expression of cAMP response element-binding protein (CREB) and steroidogenic enzymes (P450 side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase type 1, and 17α-hydroxylase/17,20 lyase) but produced no effect on steroidogenic acute regulatory protein levels. These results were further confirmed by demonstrating that the knockdown of mTOR using small interfering RNA selectively abrogated the LH/hCG-induced increase in steroidogenic enzyme expression, without affecting steroidogenic acute regulatory protein expression. LH/hCG-stimulated androgen production was also blocked by rapamycin. Furthermore, the pharmacological inhibition of mTORC1 or ribosomal protein S6 kinase 1 signaling prevented the LH/hCG-induced phosphorylation of CREB. Chromatin immunoprecipitation assays revealed the association of CREB with the proximal promoter of the Cyp17a1 gene in response to hCG, and this association was reduced by rapamycin treatment. Taken together, our findings show for the first time that LH/hCG-mediated activation of androgen biosynthesis is regulated by the mTORC1 signaling pathway in T-I cells.

Luteinizing hormone receptor and follicle stimulating hormone receptor play a crucial role in female and male reproduction. Significant new information has emerged about the structure, mechanism of activation, and regulation of expression of these receptors. Here we provide an overview of the current information on those aspects with an in-depth discussion of the recent developments in the post-transcriptional mechanism of LH receptor expression mediated by a specific LH receptor mRNA binding protein, designated as LRBP. LRBP was identified by electrophoretic gel mobility shift assay using cytosolic fractions from ovaries in the down regulated state. LRBP was purified, its binding site on LH receptor mRNA was identified and characterized. During ligand-induced down regulation, LRBP expression is increased through the cAMP/PKA and ERK signaling pathway, is translocated to translating ribosomes, binds LH receptor mRNA and forms an untranslatable ribonucleoprotein complex. This complex is then routed to the mRNA degradation machinery resulting in diminished levels of both LHR mRNA and cell surface expression of LH receptor. The studies leading to these conclusions are presented.

We have identified a specific LHR mRNA binding protein that selectively binds to the polypyrimidine-rich bipartite sequence in the coding region of the LHR mRNA and accelerates its degradation. This process has been shown to be one of the mechanisms that is responsible for the loss of the steady-state levels of LHR mRNA following the preovulatory LH surge or the down regulation of the receptor in response to the administration of a pharmacological dose of LH or hCG. The trans factor, designated as the LHR mRNA binding protein (LRBP), was purified and its identity was established as being mevalonate kinase, an enzyme involved in cholesterol biosynthesis. When mevalonate kinase expression was abolished by treating cultured luteal cells with 25-hydroxycholesterol, the ability to undergo LH-induced down regulation of LHR mRNA was completely abrogated. Examination of the crystal structure of mevalonate kinase coupled with mutagenesis of the critical residues in the catalytic site revealed that the catalytic site is in close proximity to the LHR mRNA binding site. Further studies revealed that mevalonate kinase causes LHR mRNA degradation by acting as a translational suppressor by forming an untranslatable ribonucleoprotein (RNP) complex which is then targeted for degradation. These studies show that LHR expression in the ovary is regulated by a post-transcriptional mechanism mediated by mevalonate kinase thereby linking LHR expression with cholesterol metabolism.

Vascular endothelial growth factor A (VEGF-A) is one of the most important regulators of ovarian angiogenesis. In this study, we examined the temporal relationship between VEGF-A and luteinizing hormone receptor (LHR) mRNA expression during ligand-induced down-regulation of LHR. Immature female rats were treated with pregnant mare's serum gonadotropin followed by 25 IU hCG 56 h later (day 0). On day 5, treatment with hCG (50 IU) to down-regulate LHR showed a temporal decrease in VEGF-A mRNA and protein levels in parallel with decreasing LHR mRNA. This effect was specific since the expression of CYP11A1 mRNA showed no decline. Examination of VEGF-A mRNA expression, using in situ hybridization histochemistry with 35S-labeled antisense VEGF-A mRNA probe, showed intense signal in the corpora lutea on day 5. Treatment with 50 IU hCG to down-regulate LHR mRNA showed a decline in the intensity of VEGF-A mRNA in the corpora lutea. VEGF-A mRNA expression returned to control level 53 h later when the expression of LHR mRNA also recovered. These results show that the transient down-regulation of VEGF-A mRNA and protein closely parallels the ligand-induced down-regulation of LHR mRNA. The present study establishes a close association between VEGF-A and LHR mRNA expression, suggesting the possibility that VEGF-A-induced vascularization of the ovary is dictated by the expression of LHR and this might play a regulatory role in ovarian physiology.

Elevated levels of 5α-reduced androgens have been shown to be associated with hyperandrogenism and hyperinsulinemia, the leading causes of ovulatory dysfunction in women. 5α-Dihydrotestosterone reduces ovarian granulosa cell proliferation by inhibiting FSH-mediated mitogenic signaling pathways. The present study examined the effect of insulin on 5α-reductase, the enzyme that catalyses the conversion of androgens to their 5α-derivatives. Granulosa cells isolated from immature rat ovaries were cultured in serum-free, phenol red-free DMEM-F12 media and treated with different doses of insulin (0, 0.1, 1.0, and 10.0 μg/ml) for different time intervals up to 12 h. The expression of 5α-reductase type 1 mRNA, the predominant isoform found in granulosa cells, showed a significant (P < 0.05) increase in response to the insulin treatment up to 12 h compared with control. The catalytic activity of 5α-reductase enzyme was also stimulated in a dose-depended manner (P < 0.05). Inhibiting the Akt-dependent signaling pathway abolished the insulin-mediated increase in 5α-reductase mRNA expression, whereas inhibition of the ERK-dependent pathway had no effect. The dose-dependent increase in 5α-reductase mRNA expression as well as catalytic activity seen in response to insulin treatment was also demonstrated in the human granulosa cell line (KGN). In addition to increased mRNA expression, a dose-dependent increase in 5α-reductase protein expression in response to insulin was also seen in KGN cells, which corroborated well with that of mRNA expression. These results suggest that elevated levels of 5α-reduced androgens seen in hyperinsulinemic conditions might be explained on the basis of a stimulatory effect of insulin on 5α-reductase in granulosa cells. The elevated levels of these metabolites, in turn, might adversely affect growth and proliferation of granulosa cells, thereby impairing follicle growth and ovulation.

Luteinizing hormone receptor (LHR) mRNA is post-transcriptionally regulated during ligand-induced downregulation. This process involves interaction of LHR mRNA with a specific mRNA-binding protein (LRBP), identified as mevalonate kinase (MVK), resulting in inhibition of translation followed by targeting the ribonucleoprotein complex to accelerated degradation. The present study investigated the endogenous association of LRBP with the translational machinery and its interaction with LHR mRNA during LH/hCG-induced downregulation. Ovaries were collected from rats that were injected with the ligand, hCG, to induce downregulation of LHR mRNA expression. Western blot analysis showed significantly higher levels of LRBP in polysomes from downregulated ovaries compared to controls. Western blot analysis of ribosome-rich fractions from FPLC-assisted gel filtration of post-mitochondrial supernatants confirmed the presence of LRBP in translating ribosomes isolated from the downregulated state but not from controls. The association of LRBP with LHR mRNA in the downregulated polysomes was demonstrated by immunoprecipitation with LRBP antibody followed by qPCR analysis of the associated RNA. Increased association of LHR mRNA with LRBP during downregulation was also demonstrated by subjecting the polysome-associated RNAs to oligo(dT) cellulose chromatography followed by immunoprecipitation and qPCR analysis. Additionally, analysis of in vitro translation of LHR mRNA showed increased inhibition of translation by polysomes from downregulated ovaries compared to control. This study provides strong in vivo and in vitro evidence to show that during ligand-induced downregulation, LRBP translocates to ribosomes and associates with LHR mRNA to form an untranslatable ribonucleoprotein complex and inhibits LHR mRNA translation, paving the way to its degradation.

Theca-interstitial (T-I) cells of the ovary synthesize androgens in response to luteinizing hormone (LH). In pathological conditions such as polycystic ovarian syndrome, T-I cells are hyperactive in androgen production in response to LH and insulin. Because cholesterol is an essential substrate for androgen production, we examined the effect of human chorionic gonadotropin (hCG) and insulin on signaling pathways that are known to increase cholesterol accumulation in steroidogenic cells. Specifically, the effect of hCG and insulin on sterol regulatory element-binding transcription factor 1a (SREBF1a) required for cholesterol biosynthesis and uptake was examined. Primary cultures of T-I cells isolated from 25-day-old rat ovaries responded to hCG and insulin to increase the active/processed form of SREBF1a. The hCG and insulin significantly reduced insulin-induced gene 1 (INSIG1) protein, a negative regulator of SREBF processing. Furthermore, an increase in the expression of selected SREBF target genes, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr) and mevalonate kinase (Mvk), was also observed. Protein kinase A (PRKA) inhibitor completely abolished the hCG-induced increase in SREBF1a, while increasing INSIG1. Although the hCG-induced depletion of total and free cholesterol was abolished by aminoglutethimide, the stimulatory effect on SREBF1a was not totally suppressed. Treatment with 25-hydroxycholesterol abrogated the effect of hCG on SREBF1a. Inhibition of the phosphatidylinositol 3-kinase pathway did not block the insulin-induced increase in SREBF1a, whereas mitogen-activated protein kinase inhibition reduced the insulin response. These results suggest that the increased androgen biosynthesis by T-I cells in response to hCG and insulin is regulated, at least in part, by increasing the expression of sterol response element-responsive genes by increasing SREBF1a.

The processing of Luteinizing hormone receptor (LHR) shows marked differences in different species. While the human LHR is predominantly expressed as the mature, 90 kDa species, rat LHR exists mostly in the 70 kDa precursor form. Since the extracellular domain of the LHR is unusually large in comparison with other G protein coupled receptors, the present studies examined the role of extracellular domain in its processing. FLAG-tagged chimeric LH receptors were constructed by substituting the extracellular domain of the human receptor in rat LHR (hrr) and the extracellular domain of the rat receptor in human LHR (rhh). The intracellular processing, ligand binding and recycling of the chimeric receptors were compared with that of the wild type receptors in 293 T cells. The results showed that the human and rat LHR were expressed predominantly as 90 kDa and 70 kDa species, respectively, as expected. The introduction of the rat extracellular domain into the human LHR (rhh) decreased the abundance of the mature form with an increase in the precursor form. Conversely, substitution of the extracellular domain of the rat LHR by the extracellular domain of the human LHR (hrr) led to an increase in the mature form with a corresponding decrease in the precursor form. Changes were also observed in the ligand binding and recycling of the wild type and chimeric receptors. These results suggest that the extracellular domain of the LHR is one of the determinants that confer its ability for proper maturation and cell surface expression.

We have shown that hormone-induced downregulation of luteinizing hormone receptor (LHR) in the ovary is post-transcriptionally regulated by an mRNA binding protein. This protein, later identified as mevalonate kinase (MVK), binds to the coding region of LHR mRNA, suppresses its translation, and the resulting ribonucleoprotein complex is targeted for degradation. Mutagenesis and crystallographic studies of rat MVK have established Ser146, Glu193, Asp204 and Lys13 as being crucial for its catalytic function. The present study examined the structural aspects of MVK required for LHR mRNA recognition and translational suppression. Single MVK mutants (S146A, E193Q, D204N and K13A) were overexpressed in 293T cells. Cytosolic fractions were examined for LHR mRNA binding activities by RNA electrophoretic mobility shift analysis. All the single MVK mutants showed decreased LHR mRNA binding activity compared with the wild-type MVK. Double mutants (S146A & E193Q, E193Q & D204N and E193Q & K13A) of MVK also showed a significant decrease in binding to LHR mRNA, suggesting that the residues required for catalytic function are also involved in LHR mRNA recognition. Mutation of the residues outside the catalytic site (D316A and S314A) did not cause any change in LHR mRNA binding activity of MVK when compared with wild-type MVK. To examine the biological effects of these mutants on LHR mRNA expression, a full-length capped rat LHR mRNA was synthesized and translated using a rabbit reticulocyte lysate system in the presence or absence of the MVK mutant proteins. The results showed that mutations of the active site residues of MVK abrogated the inhibitory effect on LHR mRNA translation. Therefore, these data indicate that an intact active site of MVK is required for its binding to rat LHR mRNA and for its translational suppressor function.

Posttranscriptional mechanism plays a crucial role in regulating LH receptor (LHR) expression in the ovary. We have identified a novel trans-factor, LHR mRNA binding protein (LRBP), which binds to a polypyrimidine-rich bipartite sequence of the coding region of LHR mRNA, and its identity was established as mevalonate kinase (Mvk). Although an inverse relation between LHR mRNA expression and RNA binding activity of LRBP has been established, its intermediary role in LHR mRNA expression has not been demonstrated. The present study examined the direct role of Mvk in regulating LHR expression by using primary cultures of human granulosa cells as a model system. A marked decrease in LHR mRNA stability and an increase in Mvk expression were seen when cultured granulosa cells were treated with human chorionic gonadotropin (hCG) in vitro. This treatment also resulted in an increase in LHR mRNA binding activity in the cytosolic fractions prepared from hCGtreated cells compared with the control. Because Mvk expression is regulated by sterol response element-binding protein-1, which is sensitive to the cellular concentration of 25-hydroxycholesterol (25-OHC), cultured granulosa cells were treated with this oxysterol, and the expression of Mvk gene was examined. As expected, treatment with 25OHC inhibited the Mvk (LRBP) expression, as well as the LHR mRNA binding activity of LRBP. To determine the role of Mvk in ligand-mediated down-regulation of LHR mRNA, cells were additionally treated with 25-OHC when treated with hCG. The results showed that the decrease in Mvk expression by oxysterol treatment abrogated ligand-induced down-regulation of LHR mRNA. These results therefore establish a direct participation of Mvk in regulating LHR expression and suggest a novel relationship between cholesterol metabolism and LHR expression in the ovary. (Molecular Endocrinology 21: 2233–2241, 2007)

FSH-mediated regulation of mammalian target of rapamycin (mTOR) signaling in proliferating granulosa cells and the effect of dihydrotestosterone (DHT) on this pathway were examined. Inhibiting mTOR activation using rapamycin significantly reduced the FSH-mediated increase in cyclin D2 mRNA expression, suggesting that mTOR plays a role in the FSH-mediated increase in granulosa cell proliferation. FSH treatment of granulosa cells showed a 2-fold increase in phosphorylation of p70S6 kinase (p70S6K), the downstream target of mTOR. The increase in p70S6K phosphorylation by FSH treatment was abolished by prior exposure to DHT, suggesting that DHT inhibits FSH-mediated activation of mTOR signaling in cultured granulosa cells. The effect of FSH and DHT treatment on tuberin (TSC2), the upstream regulator of mTOR, was then examined. FSH treatment increased TSC2 phosphorylation, and pretreatment with DHT for 24 h reduced this stimulation. These results indicate that reduced p70S6K phosphorylation observed in DHT-treated cells might be the result of reduced TSC2 phosphorylation. Because Akt is the upstream activator of TSC2 phosphorylation, the effect of Akt inhibition was examined to test whether FSH-mediated TSC2 phosphorylation proceeds through an Akt-dependent pathway. Our results show that inhibiting Akt phosphorylation did not block FSH-stimulated TSC2 phosphorylation, whereas ERK inhibition reduced FSH-mediated stimulation. These results demonstrate the involvement of ERK rather than Akt in FSH-mediated TSC2 phosphorylation in granulosa cells. Based on these observations, we conclude that in granulosa cells, FSH uses a protein kinase A-/ERK-dependent pathway to stimulate TSC2 phosphorylation and mTOR signaling, and DHT treatment significantly reduces this response.

Context: In normally cycling women, LH receptor mRNA expression undergoes transient down-regulation after the LH surge. The same phenomenon is also seen during a hormonally induced ovarian cycle where the LH receptor mRNA expression is down-regulated in response to the administration of human chorionic gonadotropin (hCG). Although the granulosa cells isolated from the follicular aspirates at this stage show a decline in the expression of LH receptor mRNA, this diminished receptor expression returns to control levels upon culturing in serum-containing medium. Objective: To understand the mechanism of hCG-induced loss of LH receptor mRNA expression, a cytosolic fraction (S100) was isolated from the granulosa cells, and its ability to bind LH receptor mRNA was assayed by performing RNA electrophoretic mobility gel shift analysis. Results: The results showed that the mRNA binding activity of the S100 fraction was induced in the freshly isolated granulosa cells (d 1) from the follicular aspirates collected from women who had been injected with hCG to induce ovulation. The LH receptor mRNA expression in granulosa cells on d 1, as assessed by real-time PCR and Northern blot analysis, was significantly suppressed. Both the expression of LH receptor mRNA and RNA binding activity in the S100 fractionwerethenassessedafterculturinggranulosacellsfor4d.The results showed that the LH receptor mRNA expression was significantly higher on d 4 compared with that seen on d 1. However, the RNA binding activity of the S100 fraction was significantly decreased on d 4 compared with that seen on d 1. These results show an increased association of RNA binding protein during LH receptor mRNA down-regulation. Conclusion:The present results support the notion that LH receptor mRNA expression in the human ovaries is regulated by an RNA binding protein. (J Clin Endocrinol Metab 91: 2239–2243, 2006)

The effect of 5α-dihydrotestosterone (DHT) on insulin-stimulated granulosa cell proliferation was examined using cyclin D2 mRNA as a marker. Granulosa cells from 3-d estradiol-treated immature rats showed a concentration-dependent increase in cyclin D2 mRNA expression in response to insulin. Exposure to DHT reduced the insulin-stimulated cyclin D2 mRNA expression. Inhibition of the two insulin-signaling pathways, ERK and phosphatidylinositol 3 kinase (PI3 kinase), by using specific inhibitors, also reduced this insulin-stimulated response. These results suggest that both ERK and PI3 kinase signaling are involved in insulin stimulated granulosa cell proliferation. DHT exposure resulted in reduced insulin-stimulated ERK phosphorylation. DHT treatment also reduced the insulin mediated insulin receptor substrate-1 and Raf-1 phosphorylation, the upstream molecules of ERK in insulin signaling pathway. Additionally, inhibition of insulin stimulated PI3 kinase activation reduced ERK phosphorylation. The present study therefore shows that the inhibitory effect of DHT on insulin-stimulated granulosa cell proliferation occurs early in the signaling pathway at the level of insulin receptor substrate-1 phosphorylation, leading to reduced ERK phosphorylation and subsequent inhibition of cyclin D2 mRNA expression.

In the ovary, lutropin (LH) stimulates the selective uptake and transport of cholesterol for steroid biosynthesis from HDL particles via the scavenger receptor class B type I (SR-BI). Furthermore the expression of SR-BI mRNA in the ovary is stimulated by LH and cyclic AMP (cAMP). Since the promoter of the rat SR-BI gene is devoid of consensus cyclic AMP response element (CRE) sequences, this study examined if cAMP response element binding protein (CREB) plays a role in the transactivation of SR-BI promoter (SR-BIpr). The transactivation of SR-BIpr was examined in transfected 293T cells and human granulosa SVOG-4o cells, and in primary cultures of rat theca-interstitial cells infected with adenoviral constructs containing the SR-BIpr and a luciferase reporter gene. Dose-related increases in SR-BRpr activity ranging from 2- to 4-fold was induces by 293T cells co-transfected with the catalytic subunit of protein kinase A (cPKA). Co-transfections with CREB and cPKA produced a concentration-dependent increase ranging from 6- to 32-fold. The cAMP-mediated transactivation was significantly attenuated by co-transfection with CREB M1, a non-phosphorylatable, dominant-negative form of CREB. An increase in transactivation of SR-BIpr activity was also seen in SVOG-4o cells co-transfected with CREB. In primary cultures of rat theca-interstitial (T-I) cells infected with an adenoviral construct of SR-BIpr, forskolin produced a marked increase in promoter activity. These data indicate that stimulation of the cAMP-PKA-CREB pathway enhances rat SR-BIpr activity and substantiate the role of CREB as an intermediary in this process. The absence of canonical CRE sequences in the rat SR-BIpr suggests that the activation of SR-BI by CREB may occur either through non-canonical CRE sequences or through additional transcription factors that cooperate with CREB in the activation of SR-BI promoter activity.

Palmitoylation is a well-conserved posttranslational modification among members of the G protein-coupled receptor superfamily. The present study examined the role of palmitoylation in endocytosis and postendocytic trafficking of the human LH receptor (LHR). Palmitoylation of the LHR was determined by incorporation of [3H]palmitic acid into wild-type (WT) or mutant receptor in which the potential palmitoylation sites, C643 and C644, were mutated to glycine residues. The WT receptor showed incorporation of [3H]palmitic acid into the mature 90-kDa form of the receptor whereas mutation of the two Cys residues abrogated this incorporation, indicating that Cys 643 and C644 are the sites of palmitoylation. The role of palmitoylation on endocytosis and postendocytic processing was examined by testing the ability of the WT and mutant receptor to undergo internalization, recycling, and lysosomal degradation. Compared with the WT receptor, the mutant receptor showed increased internalization and decreased recycling, suggesting that retention of palmitic acid residues at Cys 643 and 644 promotes LHR recycling. The role of palmitoylation on receptor recycling was substantiated by demonstrating that a different mutant, D578H LHR, which is deficient in palmitoylation, also recycled less efficiently. Furthermore, the data show that palmitoylation, not the rate of internalization, determines the efficiency of recycling. The present study shows that palmitoylation of cysteine residues 643 and 644 of the human LHR is a determinant of recycling.

The LH/hCG receptor, a member of the G protein coupled receptor family mediates the cellular actions of LH in the ovary. A considerable amount of information regarding its structure, mechanism of activation, and regulation of expression has emerged in recent years. Here we provide a brief overview of the current information on the structural organization of the receptor and the mechanism of receptor mediated signaling as well as an in-depth discussion on recent developments pertaining to the regulation of receptor expression. Specifically, we describe studies from our laboratory showing that the posttranscriptional regulation of the receptor involves an LH/hCG receptor mRNA-binding protein. We also propose a model to explain the loss of steady-state LH/hCG receptor mRNA levels during receptor down-regulation.

Granulosa cell mitogenesis is critical for the development of normal ovarian follicles. FSH and other mitogenic stimuli play a crucial role in this process. We have shown that exposing granulosa cells to 5alpha-dihydrotestosterone (DHT) reduces forskolin-stimulated cyclin D2 mRNA expression, which leads to cell cycle arrest resulting in reduced cell proliferation. The present study investigated the signaling molecules upstream of cyclin D2 in FSH-mediated, cAMP-dependent signaling pathway that may be negatively affected by DHT, leading to inhibition of cell cycle progression. Because ERK is an important molecule in mitogenic signaling, the possible effect of DHT on its phosphorylation was examined. Granulosa cells from 3-d estradiol-primed immature rats were treated with DHT (90 ng/ml) for 24 h and subsequently stimulated with forskolin. DHT treatment reduced forskolin stimulation of ERK phosphorylation. Although DHT exposure did not affect cellular cAMP production in response to forskolin, treating the cells with DHT for 24 h significantly reduced protein kinase A activity. DHT also caused a reduction in ERK-2 phosphorylation in response to FSH similar to that seen with forskolin. Furthermore, blocking ERK phosphorylation as well as DHT treatment resulted in a reduction in FSH-stimulated cyclin D2 mRNA expression. From these results, we conclude that DHT treatment reduces the FSH-mediated ERK phosphorylation in granulosa cells, leading to reduced cyclin D2 mRNA expression that culminates in cell cycle arrest.

Post-transcriptional mechanisms play a major role in regulating luteinizing hormone (LH) receptor mRNA expression in the ovary. An ovarian cytosolic protein that we have identified in rats and humans, which binds to a polypyrimidine-rich bipartitate sequence in the coding region of LHR mRNA, acts as a trans-acting factor in this process. In the present study, we isolated and characterized this LH receptor mRNA-binding protein (LRBP) from rat ovary. LRBP was purified to homogeneity by cation exchange chromatography followed by Northwestern analysis and subsequent elution of the single protein band from SDS-polyacrylamide gel. Purified LRBP was subjected to N-terminal microsequencing followed by homology search, which revealed its identity as mevalonate kinase. Purified rat mevalonate kinase antibody recognized the gel-purified LRBP on Western blots performed with one- and two-dimensional SDS-polyacrylamide gels. When recombinant mevalonate kinase produced in human embryonic kidney cells (293 cells) was tested, it showed all of the characteristics of LRBP with respect to specificity of LHR mRNA binding sequence, as examined by gel mobility shift analysis. Inhibition of LHR mRNA binding activity of mevalonate kinase in the presence of ATP and mevalonate indicates that the RNA recognition site of mevalonate kinase might involve the ATP/mevalonate binding region of the protein. Treatment of 293 cells with mevastatin to deplete cellular mevalonate resulted in an increase in LHR mRNA binding activity of mevalonate kinase. Collectively, the data support the novel function of rat mevalonate kinase as a LHR mRNA-binding protein in the post-transcriptional regulation of LH receptor expression in the ovary.

The theca-interstitial cells take up plasma high-density lipoprotein (HDL)- and low-density-lipoprotein-derived cholesterol to convert into steroid hormones. The uptake of HDL-derived cholesterol is mediated by the scavenger receptor, class B, type I (SR-BI). In nonsteroidogenic cells, HDL-stimulated efflux of cholesterol has been shown to be mediated by the ATP-binding cassette A1 (ABCA1) transporter. Its expression has not been documented in steroidogenic cells. The goal of the present study was to determine: 1) the role of SR-BI in theca-interstitial cell androgen production; 2) whether theca-interstitial cells express ABCA1 transporter mRNA; and 3) the relative roles of SR-BI and ABCA1 transporter in androgen production. The ABCA1 transporter mRNA expression in rat theca-interstitial cells was shown using RT-PCR and Northern blot analyses. The role of SR-BI and ABCA1 in androstenedione production was also examined by treating cells with anti-SR-BI and 2-hydroxypropyl-beta-cyclodextrin in the presence and absence of human chorionic gonadotropin and/or human HDL(3). The treatment of theca-interstitial cells with anti-SR-BI antibody blocked more than 90% of HDL plus human chorionic gonadotropin-stimulated androstenedione production, and selective HDL-CE uptake. On the other hand, the use of inhibitors of ABCA1 transporter function had no discernible effect on HDL-supported androgen production. These data demonstrate that, although theca-interstitial cells express both SR-BI and ABCA1 transporter mRNA, the SR-BI pathway supplies the majority of the cholesterol required for androgen production. Furthermore, the present study presents evidence for a crucial role for SR-BI in HDL-mediated androgen production.

Luteinizing hormone (LH) receptor mRNA is post-transcriptionally regulated. An ovarian cytosolic LH receptor mRNA-binding protein (LRBP) identified in our laboratory binds to a polypyrimidine-rich bipartite sequence in the coding region of LH receptor mRNA. The present studies show a role for LRBP in the regulation of LH receptor mRNA. We demonstrated that increased LH receptor mRNA degradation occurs during hormone-induced LH receptor down-regulation. Furthermore, increased degradation of LH receptor mRNA was seen when partially purified LRBP was included in an in vitro mRNA decay reaction. The LH receptor mRNA binding activity of LRBP measured by RNA electrophoretic mobility shift analysis showed an inverse relationship to LH receptor mRNA levels during different physiological states. These results suggest that LRBP is a physiological regulator of LHR mRNA expression in the ovary and provides a novel mechanism for the regulation of LH receptor expression in the ovary.

Down-regulation of LH receptor (LHR) in the ovary by its ligand is mediated by a specific RNA-binding protein, designated LH receptor mRNA-binding protein (LRBP), through translational suppression and mRNA degradation. Using yeast 2-hybrid screens, we previously identified eukaryotic initiation factor 5A (eIF5A) as one of the proteins that interacts with LRBP during LHR mRNA down-regulation. The present study examined the role of eIF5A and its hypusination in the context of LHR mRNA down-regulation. The association of eIF5A with LRBP or LHR mRNA was determined using immunoprecipitation and RNA immunoprecipitation assays. The results showed that the association of eIF5A with the LHR mRNA-LRBP complex increased significantly during down-regulation. Furthermore, gel fractionation and the hypusination activity assay both showed increased hypusination of eIF5A during LHR mRNA down-regulation. Abolishment of hypusination by pretreatment with the chemical inhibitor GC7 prevented the association of eIF5A with LHR mRNA and LRBP. Inhibition of hypusination also reduced the extent of ligand-induced down-regulation of LHR mRNA as well as the expression of functional LHRs assessed by real-time PCR and (125)I-human chorionic gonadotropin (hCG) binding assays, respectively. The loss of human chorionic gonadotropin-mediated downstream signaling during LHR down-regulation was also restored by inhibition of hypusination of eIF5A. Thus, the present study, for the first time, reveals the crucial role of eIF5A and its hypusination in the regulation of LHR expression in the ovary.

The luteinizing hormone/human chorionic gonadotropin receptor (LH/hCGR) undergoes palmitoylation at cysteine residues 621 and 622 located in the carboxyl terminal tail of the receptor. This study examined the biological function of palmitoylation with respect to its effect on receptor internalization. Coexpression of wild-type (WT) or C621/622G mutant receptors with arrestin-2 increased receptor internalization in 293T cells. Furthermore, measurements of rate enhancement upon overexpression of arrestin indicate that the palmitoylation deficient mutant receptor is more prone to utilizing the arrestin mediated internalization pathway than the WT receptor. Coexpression of G-protein-coupled receptor kinase 4 (GRK4) with wild type receptor resulted in an increase in internalization, while coexpression with the mutant receptor did not result in further enhancement of internalization. Additionally, 293T cells expressing mutant receptor were responsive to hCG with respect to production of inositol phosphates. Taken together, these results suggest that the palmitoylation state of the receptor governs internalization by regulating the accessibility of the receptor to the arrestin-mediated internalization pathway.

In the first step of asparagine-linked oligosaccharide chain maturation, terminal glucose residues are removed from the high mannose oligosaccharide core by glucosidases I and II. The role that glucose residues play in trafficking the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor from the endoplasmic reticulum to the cell surface was investigated. Glucosidases I and II were inhibited by incubating 293 T cells transiently transfected with LH/hCG receptor cDNA with 5 mM 1-deoxynojirimycin (DNJ). DNJ treatment resulted in a marked reduction in cell surface [(125)I]hCG binding. Similar results were obtained from glucosidase I-deficient Lec 23 Chinese hamster ovarian (CHO) cells and wild-type CHO cells that were transiently transfected with LH/hCG receptor cDNA. Immunoprecipitation followed by Western blotting of transfected 293 T cells incubated in the presence or absence of 5 mM DNJ revealed that there is substantially less receptor in DNJ-treated cells than in control cells. These results show that the removal of glucose residues is necessary for trafficking the LH/hCG receptor to the cell surface.

The synthesis of androgens by theca-interstitial cells is stimulated by LH and the insulin/insulin-like growth factor I (IGF-I) system. An essential element of the steroidogenesis is the uptake of plasma cholesterol and transformation to steroid hormones. In the rat, the uptake of cholesterol by the theca-interstitial cells is mediated by the high density lipoprotein receptor. The goal of the present study was to examine whether insulin has any effect on cholesterol delivery into theca-interstitial cells. The effects of insulin and hCG on the expression of the high density lipoprotein receptor (SR-BI) messenger RNA (mRNA) and intracellular cholesterol levels were examined in rat theca-interstitial cells under in vivo and in vitro conditions. Twenty-five-day-old rats were treated with insulin, hCG, or insulin followed by hCG. The expression of SR-BI mRNA was then examined in ovaries enriched in theca-interstitial cell population by Northern blot analysis. Treatment with insulin increased the expression of ...

We investigated the physiological basis for the trophic effect of glucocorticoids in rat corpora lutea in the absence of pituitary gonadotropins. Immature (Day 29) Sprague-Dawley rats were given eCG and hCG to induce the development of corpora lutea and were hypophysectomized on Day 32. Beginning on Day 40, rats received twice-daily s.c. injections of either dexamethasone (dex; 200 microg/rat/day) or vehicle (controls) and then were killed on Day 44. Plasma 20alpha-dihydroprogesterone, a major steroid produced by the corpora lutea, was higher (p 2-fold of plasma 20alpha-dihydroprogesterone concentration compared to controls. Glucocorticoid receptor protein (about 92 kDa) was detected in both luteal and nonluteal ovarian tissues in this animal model. These effects of glucocorticoids and the presence of the glucocorticoid receptor raise the possibility of a physiological role for glucocorticoids in the rat corpus luteum.

The luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor, which belongs to the family of G-protein coupled receptors, plays an important role in gonadal steroidogenesis. Substitution of aspartic acid 556 of the LH/hCG receptor with glycine (D556G) creates a constitutively active receptor that activates adenylyl cyclase in the absence of hormone. To examine receptor internalization, human embryonic kidney cells (293 T) expressing wild type (WT) or D556G mutant receptors were incubated with [125I]hCG and subsequently analyzed for cell surface bound and internalized radioactivity. Comparison of the rate constants of internalization of the D556G mutant and WT receptors revealed that the rate of internalization of the D556G mutant was five times greater than that of the WT receptor. Although the D556G receptor internalizes [125I]hCG rapidly, a corresponding increase in [125I]hCG degradation was not seen. The internalization of another constitutively active LH/hCG receptor (aspartic acid 556 to tyrosine) was also greater than that of the WT receptor. Internalization of receptor bound [125I]hCG was inhibited by a hypertonic sucrose solution, confirming that the ligand enters the cell by receptor-mediated endocytosis. Furthermore, the constitutively active D556G and D556Y LH/hCG receptors utilize the arrestin dependent internalization pathway. These results suggest that the active state conformation of the constitutively active receptor is conducive to rapid internalization.

To elucidate the molecular events associated with the regulation of luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor mRNA stability during hCG-induced receptor down-regulation, we have identified an LH/hCG receptor-specific mRNA binding protein. Proteins were isolated from control and down-regulated rat ovary and were incubated with in vitro transcribed RNAs corresponding to the full-length LH/hCG receptor, as well as 5'- and 3'-truncated receptor forms. Resultant ribonucleoprotein complexes were analyzed by RNA gel mobility shift. A prominent Mr 50,000 ribonucleoprotein complex was identified with the following characteristics: 1) specificity for LH/hCG receptor open reading frame sequences located between nucleotides 102 and 282; 2) lack of competition by nonspecific RNAs; 3) a 3-fold increase in RNA binding activity during hCG-induced receptor down-regulation; and 4) limited tissue expression. This report describes the first evidence of an LH/hCG receptor mRNA binding protein, which we term LRBP-1, for luteinizing hormone receptor RNA binding protein-1. This protein is a candidate for a trans-acting factor involved in the hormonal regulation of LH/hCG receptor mRNA stability in rat ovary.

Objective To assess, in the human endometrial cell line HEC-1-A, the presence of protein tyrosine phosphatase 1 (PTP1D) and the possible regulation of its mRNA expression by mitogens such as forskolin (an agent that increases intracellular cyclic adenosine monophosphate [cAMP] levels), epidemial growth factor (EGF), and insulin-like growth factor-I (IGF-1).

Growth factor regulation of normal and cancerous cell proliferation has been well-documented and may be mediated by proto-oncogene activity. The purpose of this study was to assess changes in proliferation and mitogen-induced c-fos mRNA expression of an endometrial carcinoma cell line, HEC-1-A, in response to TGF-beta, a potent growth-inhibitory peptide. HEC-1-A cells were incubated in the presence or absence of TGF-beta. Mitogen-stimulated cells were additionally treated with epidermal growth factor (EGF). Changes in proliferation were measured by [3H]thymidine uptake assays. Alterations in EGF-induced c-fos expression following TGF-beta pretreatment were assessed by Northern blot using a 32P-labeled human c-fos probe. Finally, chloramphenicol acetyltransferase assays were performed to evaluate c-fos promoter activity in response to treatment conditions. Basal and EGF-stimulated proliferation was inhibited by TGF-beta in a dose- and time-dependent manner. TGF-beta also reversibly decreased EGF-induced c-fos mRNA expression in a dose- and time-dependent manner. Sequences in the c-fos promoter that were stimulated by EGF showed suppressed activity when preincubated with TGF-beta. These results show that TGF-beta negatively modulates EGF-induced c-fos expression, which may be related to the observed inhibition of carcinoma cell proliferation.

The conserved cysteine residues 621 and 622 of luteinizing hormone/human chorionic gonadotropin receptors were converted to serine (C621S, C622S, C621/622S) and glycine residues (C621/C622G) by site directed mutagenesis. The wild type and mutant receptor cDNAs were cloned into the mammalian expression vector (PCMV4) and human embryonic kidney cells (293 cells) were transiently transfected with these constructs. Equilibrium binding studies with [(125)I]hCG (human chorionic gonadotropin) showed that the mutant and wild type receptors expressed on the cell surface exhibited similar K(d). The effect of mutation of the conserved cysteine residues on the ability of the receptors to undergo ligand-induced down-regulation was then tested. In vitro exposure of cells expressing the wild type receptor to a saturating concentration of human chorionic gonadotropin (100 ng/ml) for 24 h resulted in modest down-regulation of receptors. The palmitoylation deficient mutants, C621S, C622S, C621/622S and C621/622G, showed increased down-regulation compared with the wild type receptor. The extent of down-regulation of the mutant receptors correlated with increased internalization of the receptor. Additionally, the G protein coupling efficiency of the palmitoylation deficient mutants was not different from the wild type since the EC(50)s for cyclic AMP (cAMP) production were identical in both groups. These studies demonstrate that palmitoylation deficient mutants are more prone to ligand-induced receptor down-regulation. Furthermore, abrogation of palmitoylation by mutagenesis showed no effect on the efficiency of the palmitoylation deficient mutants to couple to Gs protein.

Point mutations in the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor have been shown to cause constitutive activation which results in precocious puberty in affected males. We introduced one of these mutations, Asp-556 → Gly, into the rat LH/hCG receptor and demonstrated that the mutant receptor constitutively activated adenylate cyclase in transfected 293 T cells. The cell surface expression of the mutant receptor was lower than that of the wild type receptor. Pulse-chase studies showed that the 73-kDa precursor of both the mutant and wild type receptors was synthesized at comparable efficiencies. However, post-translational processing of the mutant receptor to the mature 92-kDa form, which has N-linked complex type oligosaccharide chains, was impaired. Sensitivity of the mutant receptor to peptide-N-glycanase F and endoglycosidase H, and insensitivity to sialidase indicated that the 73-kDa species represents the high mannose form that has not yet been trafficked through the medial and trans Golgi. Additionally, although the wild type receptor was palmitoylated, the mutant receptor was not. Although the high mannose 73-kDa species is capable of binding LH/hCG, our results show that post-translational processing in the Golgi is required for the mature 92-kDa receptor to reach the cell surface.

LH receptor (LHR) expression in the ovary is regulated by the RNA binding protein, (LHR mRNA binding protein [LRBP]), which has been identified as being mevalonate kinase. This study examined the role of microRNA miR-122 in LRBP-mediated LHR mRNA expression. Real-time PCR analysis of ovaries from pregnant mare serum gonadotropin/human chorionic gonadotropin (hCG)-primed female rats treated with hCG to down-regulate LHR expression showed that an increase in miR-122 expression preceded LHR mRNA down-regulation. The expression of miR-122 and its regulation was confirmed using fluorescent in situ hybridization of the frozen ovary sections using 5′-fluorescein isothiocyanate-labeled miR-122 locked nucleic acid probe. The increased expression of miR-122 preceded increased expression of LRBP mRNA and protein, and these increases were followed by LHR mRNA down-regulation. Inhibition of protein kinase A (PKA) and ERK1/2 signaling pathways by H89 and UO126, respectively, attenuated the hCG-mediated up-regulation of miR-122 levels. This was also confirmed in vitro using human granulosa cells. These results suggest the possibility that hCG-mediated miR-122 expression is mediated by the activation of cAMP/PKA/ERK signaling pathways. Inhibition of miR-122 by injection of the locked nucleic acid-conjugated antagomir of miR-122 abrogated the hCG-mediated increases in LRBP protein expression. Because it has been previously shown that miR-122 regulates sterol regulatory element-binding proteins (SREBPs) and SREBPs, in turn, regulate LRBP expression, the role of SREBPs in miR-122-mediated increase in LRBP expression was then examined. The levels of active forms of both SREBP-1a and SREBP-2 were increased in response to hCG treatment, and the stimulatory effect was sustained up to 4 hours. Taken together, our results suggest that hCG-induced down-regulation of LHR mRNA expression is mediated by activation of cAMP/PKA/ERK pathways to increase miR-122 expression, which then increases LRBP expression through the activation of SREBPs.

The present study examined the effect of insulin-mediated activation of the mammalian target of rapamycin complex 1 (MTORC1) signaling network on the proliferation of primary culture of theca-interstitial (T-I) cells. Our results show that insulin treatment increased proliferation of the T-I cells through the MTORC1-dependent signaling pathway by increasing cell cycle regulatory proteins. Inhibition of ERK1/2 signaling caused partial reduction of insulin-induced phosphorylation of RPS6KB1 and RPS6 whereas inhibition of PI3-kinase signaling completely blocked the insulin response. Pharmacological inhibition of MTORC1 with rapamycin abrogated the insulin-induced phosphorylation of EIF4EBP1, RPS6KB1 and its downstream effector, RPS6. These results were further confirmed by demonstrating that knockdown of Mtor using siRNA reduced the insulin-stimulated MTORC1 signaling. Furthermore, insulin-stimulated T-I cell proliferation and the expression of cell cycle regulatory proteins CDK4, CCND3 and PCNA were also blocked by rapamycin. Taken together, the present studies show that insulin stimulates cell proliferation and cell cycle regulatory proteins in T-I cells via activation of the MTORC1 signaling pathway.

Luteinizing hormone receptor undergoes downregulation during preovulatory Luteinizing hormone surge through a post-transcriptional mechanism involving an RNA binding protein designated as LRBP. The present study examined the mechanism by which LRBP induces the degradation of Luteinizing hormone receptor mRNA, specifically the role of decapping of Luteinizing hormone receptor mRNA and the translocation of LRBP-bound Luteinizing hormone receptor mRNA to degradative machinery. Immunoprecipitation of the complex with the 5′cap structure antibody followed by real time PCR analysis showed progressive loss of capped Luteinizing hormone receptor mRNA during downregulation suggesting that Luteinizing hormone receptor mRNA undergoes decapping prior to degradation. RNA immunoprecipitation analysis confirmed dissociation of eukaryotic initiation factor 4E from the cap structure, a step required for decapping. Furthermore, RNA immunoprecipitation analysis using antibody against the p body marker protein, DCP1A showed that Luteinizing hormone receptor mRNA was associated with the p bodies, the cytoplasmic foci that contain RNA degradative enzymes and decapping complex. Immunohistochemical studies using antibodies against LRBP and DCP1A followed by confocal analysis showed colocalization of LRBP with DCP1A during downregulation. This was further confirmed by co-immunoprecipitation of LRBP with DCP1A. The association of LRBP and Luteinizing hormone receptor mRNA in the p bodies during downregulation was further confirmed by examining the association of a second p body component, rck/p54, using immunoprecipitation and RNA immunoprecipitation respectively. These data suggest that the association of LRBP with Luteinizing hormone receptor mRNA results in the translocation of the messenger ribonucleoprotein complex to the p bodies leading to decapping and degradation.

Objective To determine whether metformin has direct effects on ovarian theca-interstitial (T-I) cell proliferation through activation of adenosine monophosphate–activated protein kinase (AMPK). Design In vitro experimental study. Setting Academic medical center laboratory. Animal(s) Immature Sprague-Dawley female rats. Intervention(s) Ovarian T-I cells were isolated, purified, and cultured in the absence (control) or presence of insulin (1 μg/mL) with or without metformin or other activators/inhibitors of AMPK (AICAR, compound C). Main Outcome Measure(s) Proliferation assessed by determination of expression levels of proteins involved in cell cycle progression, cyclin D3, and cyclin-dependent kinase 4 (CDK4) with Western blot analysis, and determination of DNA synthesis with bromodeoxyuridine (BrdU) incorporation assay; activation of AMPK, Erk1/2, and S6K1 determined by Western blot analysis with the use of antibodies specific for the phosphorylated (activated) forms. Result(s) Metformin inhibited insulin-induced ovarian T-I cell proliferation and the up-regulation of the cell cycle regulatory proteins, cyclin D3 and CDK4. Metformin independently activated AMPK in a dose-dependent manner. Treatment with metformin inhibited insulin-induced activation of Erk1/2 and S6K1. This effect was reversed with the addition of compound C, a known AMPK inhibitor. Conclusion(s) Metformin directly inhibits proliferation of ovarian T-I cells via an AMPK-dependent mechanism. These findings further validate the potential benefits of metformin in the treatment of conditions associated with hyperinsulinemia and excessive growth of ovarian T-I cells (such as polycystic ovary syndrome).

We have examined whether the two cysteine residues (621 and 622) of the carboxyl-terminal cytoplasmic domain of the rat luteinizing hormone (LH/hCG) receptor are potential sites for palmitoylation. The full-length LH/hCG receptor cDNA was cloned into an expression vector (hCGR-pCMV4). A human embryonic kidney cell line expressing large T antigen (293T cells) was transiently transfected with hCGR-pCMV4 by the calcium phosphate precipitation technique. The functional expression of the receptor was confirmed by 35S-labeled cysteine incorporation into the receptor as well as by 125I-human chorionic gonadotropin (hCG) binding. The transfected cells were then labeled with [3H]palmitic acid, and the labeled receptors purified on hCG-Affi-Gel matrix and subjected to SDS-polyacrylamide gel electrophoresis and autoradiography. The hCGR-pCMV4-transfected cells incorporated [3H]palmitic acid into a 92-kDa band corresponding to the mature form of the LH/hCG receptor; this band was absent in cells transfected with vector alone. Site-directed mutagenesis of either cysteine 621 or 622 to serine residue was partially inhibitory, whereas mutation of both cysteine residues (621 and 622) completely abolished palmitoylation. Scatchard analyses revealed that the mutant and wild type receptors have similar affinities for 125I-hCG. The biological function of palmitoylation was then examined in the transfected cells. The results showed that although the intracellular trafficking of the receptor and the ability to stimulate cyclic AMP production were unaffected, the rate of ligand-induced internalization of the receptor was higher in palmitoylation-deficient mutants compared to the wild type receptors. The first order rate constants of internalization of the mutant receptors were over 2-fold higher than the wild type. Intracellular degradation of the receptor-bound ligand was also higher in the mutants. These studies suggest that the native LH/hCG receptor is palmitoylated at cysteine residues 621 and 622, creating a membrane-anchoring site at the putative cytoplasmic domain. This palmitic acid-mediated anchoring decreases the ligand-induced receptor internalization thereby prolonging the retention of the ligand-bound receptor on the cell surface.

Previous studies have demonstrated that ligand-induced down-regulation of the LH/hCG receptor in rat corpus luteum results in a loss of ligand-binding activity and a parallel decline in steady state levels of receptor mRNA. The purpose of the present study was to determine whether this loss of receptor mRNA during receptor down-regulation is due to inhibition of transcription or increased degradation of the receptor mRNA. To differentiate between these two possibilities, nuclear run-off assays were performed to study transcription rates of nuclei isolated from control and hCG down-regulated rat ovaries. A 750-mer LH/hCG receptor cDNA probe, spanning the carboxy-terminal region of the peptide and the 3'-untranslated region of the receptor cDNA, was constructed by polymerase chain reaction. RNA transcripts were synthesized from existing nuclear RNA from control and down-regulated nuclei and hybridized to the receptor cDNA probe immobilized on nylon membranes and subjected to autoradiography. Hybridization to actin cDNA was also run alongside as a control. The results of run-off transcription analysis indicated that during down-regulation, the transcription rates of LH/hCG receptor mRNA are not decreased compared to those in saline-treated controls. Although no decrease in the transcription rates of the receptor mRNA was seen in the down-regulated state, the steady state levels of the receptor mRNA showed a decline when assayed by either solution hybridization or Northern blot analysis. Furthermore, hCG induced down-regulation appears to increase the transcriptional activity of the nuclei. Estimates of steady state LH/hCG receptor mRNA turnover rates indicate that the half-life of the message in down-regulated ovaries is markedly reduced compared to that in the control. It is concluded that the loss of steady state LH/hCG receptor mRNA is not due to a decrease in transcription, but probably represents an increased degradation of the receptor mRNA.

The ligand-induced down-regulation of LH receptor (LHR) expression in the ovaries, at least in part, is regulated by a posttranscriptional process mediated by a specific LH receptor mRNA binding protein (LRBP). The LH-mediated signaling pathways involved in this process were examined in primary cultures of human granulosa cells. Treatment with 10 IU human chorionic gonadotropin (hCG) for 12 h resulted in the down-regulation of LHR mRNA expression while producing an increase in LHR mRNA binding to LRBP as well as a 2-fold increase in LRBP levels. The activation of ERK1/2 pathway in LH-mediated LHR mRNA down-regulation was also established by demonstrating the translocation of ERK1/2 from the cytosol to the nucleus using confocal microcopy. Inhibition of protein kinase A using H-89 or ERK1/2 by U0126 abolished the LH-induced LHR mRNA down-regulation. These treatments also abrogated both the increases in LRBP levels as well as the LHR mRNA binding activity. The abolishment of the hCG-induced increase in LRBP levels and LHR mRNA binding activity was further confirmed by transfecting granulosa cells with ERK1/2 specific small interfering RNA. This treatment also reversed the hCG-induced down-regulation of LHR mRNA. These data show that LH-regulated ERK1/2 signaling is required for the LRBP-mediated down-regulation of LHR mRNA.

This study examined the role of Rab5a GTPase in regulating hCG-induced internalization and trafficking of the hCG-LH receptor complex in transfected 293T cells. Coexpression of wild-type Rab5a (WT) or constitutively active Rab5a (Q79L) with LHR significantly increased hCG-induced LHR internalization. Conversely, coexpression of dominant negative Rab5a (S34N) with LHR reduced internalization. Confocal microscopy showed LHR colocalizing with Rab5a (WT) and Rab5a (Q79L) in punctuate structures. Coexpression of Rab5a (WT) and Rab5a (Q79L) with LHR significantly increased colocalization of LHR in early endosomes. Conversely, dominant negative Rab5a (S34N) decreased this colocalization. While Rab5a stimulated internalization of LHR, it significantly decreased LHR recycling to the cell surface and increased degradation. Dominant negative Rab5a (S34N) increased LHR recycling and decreased degradation. These results suggest that Rab5a plays a role in LHR trafficking by facilitating internalization and fusion to early endosomes, increasing the degradation of internalized receptor resulting in a reduction in LHR recycling.

In addition to playing a cardinal role in androgen production, LH also regulates growth and proliferation of theca-interstitial (T-I) cells. Here, we show for the first time that LH/human chorionic gonadotropin (hCG) regulates T-I cell proliferation via the mammalian target of rapamycin complex 1 (mTORC1) signaling network. LH/hCG treatment showed a time-dependent stimulation of T-I cell proliferation and phosphorylation of protein kinase B (AKT), ERK1/2, and ribosomal protein (rp)S6 kinase 1 (S6K1), and its downstream effector, rpS6. Pharmacological inhibition of ERK1/2 signaling did not block the hCG-induced phosphorylation of tuberin, the upstream regulator of mTORC1 or S6K1, the downstream target of mTORC1. However, inhibition of AKT signaling completely blocked the hCG response. Furthermore, the AKT-specific inhibitor abolished forskolin (FSK)-stimulated phosphorylation of AKT, tuberin, S6K1, and rpS6. Human CG and FSK-mediated phosphorylation of AKT and downstream targets of mTORC1 were attenuated by inhibition of adenylyl cyclase. Pharmacologic targeting of mTORC1 with rapamycin also abrogated hCG or FSK-induced phosphorylation of S6K1, rpS6, and eukaryotic initiation factor 4E binding protein 1. In addition, hCG or FSK-mediated up-regulation of the cell cycle regulatory proteins cyclin-dependent kinase 4, cyclin D3, and proliferating cell nuclear antigen was blocked by rapamycin. These results were further confirmed by demonstrating that knockdown of mTORC1 using small interfering RNA abolished hCG-mediated increases in cell proliferation and the expression of cyclin D3 and proliferating cell nuclear antigen. Taken together, the present studies show a novel intracellular signaling pathway for T-I cell proliferation involving LH/hCG-mediated activation of the AKT/mTORC1 signaling cascade.

Rat luteal cells preferentially utilize cholesterol derived from high density lipoproteins (HDL) as a substrate for steroid hormone synthesis. The uptake of cholesterol from HDL by these cells is in contrast to nonsteroidogenic cells, which export cholesterol to HDL. A previous study demonstrated that HDL binding to luteal cell membranes was increased in conjunction with in vivo cholesterol depletion or cholesterol loading of the ovary induced by pharmacological agents. These results suggest a biphasic regulation of the HDL receptor in luteinized rat ovaries. In the present studies, the in vitro regulation of HDL binding in rat luteal cells by increased intracellular cholesterol was examined. Cultured luteal cells were incubated with increasing doses of low density lipoproteins (LDL) for 2 days after which the cellular sterol content and the effects on progesterone production and HDL binding were measured. As expected, the LDL treatment increased total cellular sterol content in a dose-dependent manner, resulting in a 2.1-fold increase over control at a dose of 1 mg LDL/ml. Increased cellular cholesterol was accompanied by a comparable increase in progesterone secretion. These results suggest that exogenous cholesterol was utilized by these cells. The LDL treatment also increased the binding of HDL to the cells in a dose-dependent manner to a maximum of 2.2-fold over control. The effect of increased cellular sterol on HDL binding was also examined using a more polar cholesterol derivative, 25-hydroxycholesterol. Cells were cultured for 2 days in media containing 0.3-40 micrograms/ml 25-hydroxycholesterol in the presence of 100 micrograms/ml aminoglutethimide, an inhibitor of cholesterol metabolism. The HDL binding to luteal cells exhibited dose-dependent up-regulation by 25-hydroxycholesterol with a 5.8-fold increase in binding at the maximum dose tested. Equilibrium binding studies using cells treated with 10 micrograms/ml 25-hydroxycholesterol revealed a 2.1-fold increase in the number of HDL binding sites on the luteal cells without affecting the binding affinity. From the results of this study, it is concluded that HDL binding in rat luteal cells is up-regulated by an increase in the intracellular cholesterol level.

Luteinizing hormone receptor (LHR) mRNA binding protein (LRBP), identified as mevalonate kinase, has been shown to be a trans factor mediating the post-transcriptional regulation of LHR mRNA expression in ovaries. LRBP binds to the coding region of LHR mRNA and accelerates its degradation. Our previous studies in an in vitro system showed that LRBP represses the translation of LHR mRNA by forming an untranslatable ribonucleoprotein (mRNP) complex, further suggesting that the untranslatable mRNP complex is directed to the mRNA repression/decay machinery for subsequent mRNA turnover. In the present studies, we used yeast two-hybrid system to screen a cDNA library which was constructed from LHR down-regulated ovaries. Two proteins were identified interacting with LRBP: ribosomal protein S20 (RP S20) and ubiquitin conjugating enzyme 2i (UBCE2i). Their interactions with LRBP were confirmed by the mating assay, co-immunoprecipitation analyses and in vitro sumoylation assays. Furthermore, we show that LRBP is a target for modification by SUMO2/3 but not by SUMO1, at K256 and/or K345. Mutation of both lysine residues is sufficient to abrogate the sumoylation of LRBP. These findings suggest that the direct interaction of LRBP with the translation machinery, through RP S20, may be responsible for the transition of LHR mRNA to an untranslatable complex, and that sumoylation of LRBP may play a role in targeting the untranslatable mRNP complex to the mRNA decay machinery in specific cytoplasmic foci.

Objective To determine the effect of thiazolidenedione derivatives (TZDs) on vascular endothelial growth factor (VEGF) production by human luteinized granulosa cells and the morphologic development of murine embryos. Design Prospective, experimental, in vitro and in vivo study. Setting Research laboratory. Patient(s) Follicular aspirates from 10 women undergoing oocyte retrieval. Intervention(s) Isolated human granulosa cells were treated with a dimethyl sulfoxide (DMSO) control or ciglitazone, in the presence and absence of an hCG stimulus. Embryos extracted from superovulated B6C3F1 female mice were cultured in the presence of DMSO or pioglitazone. Main Outcome Measure(s) Vascular endothelial growth factor concentrations at 24 and 48 hours. Morphologic development of murine embryos at 96 hours. Result(s) Following an hCG stimulus, treatment with 20 μM or 40 μM ciglitazone decreased VEGF production in a statistically significant manner at both time intervals. Blastocyst development at 96 hours did not significantly differ between untreated zygotes and those treated with pioglitazone. Conclusion(s) Ciglitazone significantly decreased VEGF production by human granulosa cells in an in vitro model. Pioglitazone did not adversely impact the development of cultured murine embryos. Although mechanistic evidence is not provided, the pivotal role of VEGF in ovarian hyperstimulation syndrome prompts investigation of TZDs as a novel treatment for this condition.

FSH, acting through multiple signaling pathways, regulates the proliferation and growth of granulosa cells, which are critical for ovulation. The present study investigated whether AMP-activated protein kinase (AMPK), which controls the energy balance of the cell, plays a role in FSH-mediated increase in granulosa cell proliferation. Cells isolated from immature rat ovaries were grown in serum-free, phenol red free DMEM-F12 and were treated with FSH (50 ng/ml) for 0, 5, and 15 min. Western blot analysis showed a significant reduction in AMPK activation as observed by a reduction of phosphorylation at thr 172 in response to FSH treatment at all time points tested. FSH also reduced AMPK phosphorylation in a dose-dependent manner with maximum inhibition at 100 ng/ml. The chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, 0.5 mm) increased the cell cycle inhibitor p27 kip expression significantly, whereas the AMPK inhibitor (compound C, 20 μm) and FSH reduced p27kip expression significantly compared with control. FSH treatment resulted in an increase in the phosphorylation of AMPK at ser 485/491 and a reduction in thr 172 phosphorylation. Inhibition of Akt phosphorylation using Akt inhibitor VIII reversed the inhibitory effect of FSH on thr 172 phosphorylation of AMPK, whereas ERK inhibitor U0126 had no effect. These results show that FSH, through an Akt-dependent pathway, phosphorylates AMPK at ser 481/495 and inhibits its activation by reducing thr 172 phosphorylation. AMPK activation by 5-amino-imidazole-4-carboxamide-1-β-d-ribofuranoside treatment resulted in a reduction of cell cycle regulatory protein cyclin D2 mRNA expression, whereas FSH increased the expression by 2-fold. These results suggest that FSH promotes granulosa cell proliferation by increasing cyclin D2 mRNA expression and by reducing p27 kip expression by inhibiting AMPK activation through an Akt-dependent pathway. FSH stimulates granulosa cell proliferation by reducing cell cycle inhibitor p27 kip through AMP kinase inhibition.