Human Granulosa-luteal Cells Research Articles

Tumor necrosis factor-alpha activates nuclear factor-kappaB but does not regulate progesterone production in cultured human granulosa luteal cells

Background. The role of tumor necrosis factor-α (TNF-α) in granulosa luteal cell function and steroidogenesis is still controversial. Our aim was to examine the steroidogenic response, together with the simultaneous expression and activation of nuclear factor-κB (NF-κB), in cultured human granulosa luteal cells (GLCs) following administration of TNF-α.Materials and methods. This prospective controlled study was conducted in the Human Reproduction Division at the Institute of Maternal and Child Research, Faculty of Medicine, University of Chile and the San Borja Arriarán Hospital, National Health Service, Santiago, Chile. GLCs were obtained from aspirates of follicles from women undergoing in vitro fertilization (IVF). Thirty-two women undergoing IVF for tubal-factor and/or male-factor infertility participated in this study. Protein levels of NF-κB, the NF-κB inhibitor IκBα and steroidogenic acute regulatory protein (StAR) were determined by Western blot and localization of NF-κB was studied by indirect immunofluorescence. Progesterone production was determined by radioimmunoassay.Results. TNF-α did not affect the expression of StAR protein or the synthesis of progesterone. NF-κB was expressed in the GLCs and activated by TNF-α, resulting in degradation of IκBα and mobilization of the p65 NF-κB subunit into the nucleus.Conclusions. These results indicate that TNF-α did not modulate steroidogenesis in cultured human GLCs. However, NF-κB was activated by TNF-α. Therefore the activation of NF-κB via the TNF-α pathway is likely associated with other preovulatory granulosa cell processes important for human ovarian function.

Expression of inhibin-activin subunits, follistatin and smads in granulosa-luteal cells collected at oocyte retrieval

To examine the expression of inhibin-activin subunits, follistatin, and Smads 2, 3 and 4 in granulosa-luteal cells at the time of oocyte retrieval. Quantitative reverse transcriptase-polymerase chain reaction was performed to quantify the mRNA expression of the investigated genes: inhibin alpha-subunit, inhibin-activin betaA- and betaB-subunits, follistatin, and Smads 2, 3 and 4. alpha-, betaA- and betaB-subunits of inhibin-activin, and follistatin were all well expressed in granulosa-luteal cells obtained from either dominant or cohort follicles. alpha-subunits strongly correlated with Smad2 (p < 0.001). betaA-subunit significantly correlated with Smad4, p < 0.001. betaB-subunit significantly correlated with Smad2, p = 0.002, and follistatin expression strongly correlated with the expression of Smads 2 and 4 (p < 0.001 and = 0.007 respectively). We found, for the first time that inhibin-activin betaB-subunit mRNA was well expressed in human granulosa-luteal cells obtained from either dominant or cohort follicles. alpha-, betaA- and betaB-subunits and follistatin correlated with Smads to varied degrees, suggesting the active roles of the above genes at the time of oocyte retrieval.

Phytoestrogens and their low dose combinations inhibit mRNA expression and activity of aromatase in human granulosa-luteal cells

There is evidence that certain phytoestrogens inhibit aromatase, the enzyme that converts androgens to oestrogens. Kinetic studies in cell-free preparations show that they may inhibit aromatase by competitive binding to the enzyme, but there is a paucity of studies investigating longer-term effects of phytoestrogens on the expression of steroidogenic enzymes. This study tested the hypothesis that phytoestrogens could reduce aromatase activity by down-regulation of its expression. Experiments were carried out on primary cultures of human granulosa-luteal (GL) cells after they had been exposed to phytoestrogens for 48 h. Aromatase activity was measured by the ability of cells to convert testosterone to estradiol over a 4 h period and aromatase mRNA expression (mRNA arom) was subsequently measured from the same cells using quantitative real-time PCR. The compounds investigated were the flavones, apigenin and quercetin, and the isoflavones, genistein, biochanin A and daidzein at doses of 10 μM and 100 nM. Combinations of these compounds at the lower dose were also investigated. All compounds tested dose-dependently reduced mean mRNA arom compared with controls. Apigenin was the most potent inhibitor with significant inhibition of mRNA arom seen at both 10 μM and 100 nM, whilst other flavonoids (except biochanin A) only induced significant inhibition ( p ≤ 0.05) at the higher dose. Low dose (100 nM) mixtures of the compounds were ineffective except for a combination of the three isoflavones that induced a significant inhibition of mRNA arom. The changes in aromatase activity paralleled the mRNA arom results and additional studies showed that the reduction in aromatase activity was significantly delayed in time compared with the reduction in mRNA arom. This is the first study to compare the action of various phytoestrogens, either singly or in low-dose combination, on the expression and activity of aromatase in human cells and it suggests that chronic dietary exposure and tissue accumulation of low-dose mixtures of phytoestrogens could have important consequences for aromatase activity and the production of oestrogens.

Extra‐pituitary function of GnRH‐I and GnRH‐II in human reproduction

Recent studies have indicated that both the classical form of mammalian GnRH (GnRH-I) and its novel isoform (GnRH-II) are potent autocrine regulators in several non-pituitary tissues, including the ovary, endometrium and placenta. In human granulosa-luteal cells, we have demonstrated the expression of GnRH-I and GnRH-II, as well as GnRH receptor (GnRH-R). GnRH-II, like GnRH-I, directly inhibits progesterone production in human granulosa-luteal cells. In normal ovarian surface epithelium (OSE) and in 80% of human ovarian epithelial tumors, GnRHR is expressed. A direct anti-proliferative effect has been observed following treatment with GnRH-I or GnRH-II in different ovarian cancer cell lines, presumably via a common GnRHR (the type I GnRH-R). In human endometrial stromal cells and in the first-trimester trophoblast, GnRH-I and GnRH-II are expressed. These peptides differentially regulate the balance between the urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor (PAI-1) levels in the human decidua. GnRH-I and GnRH-II are potent regulators of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) that are important for the overall proteolytic activity of trophoblasts during human implantation. Together, these findings strongly support a multi-faceted role of the GnRH/GnRH-R system in the control of reproduction. [This research was supported by the Canadian Institutes of Health Research].

Dose–response effects of phytoestrogens on the activity and expression of 3β-hydroxysteroid dehydrogenase and aromatase in human granulosa-luteal cells

There is evidence that certain phytoestrogens can inhibit key steroidogenic enzymes although most studies have been carried out on microsomal or purified enzyme preparations, some using cell lines. This study was designed to test the hypothesis that low doses of phytoestrogens, at concentrations that would be attained through the diet, could inhibit 3β-hydroxysteroid dehydrogenase (HSD) and/or aromatase in primary cultures of human granulosa-luteal (GL) cells and that this effect was due to a decrease in the expression of these proteins. Based on published evidence, eight compounds were selected for investigation and these included the flavones apigenin and quercetin, the isoflavones genistein, biochanin A and daidzein, the lignans, enterodiol and enterolactone, and the mycotoxin zearalenone. Human GL cells were cultured for 48 h in the presence of these phytoestrogens at concentrations ranging from 0.01 to 100 μM and after addition of fresh media the conversion of pregnenolone to progesterone or androstenedione to oestradiol over a 4 h period was measured. Biochanin A was the only phytoestrogen that displayed any dose-dependent inhibition of 3β-HSD, others showing inhibition at doses ≥10 μM. Apigenin and quercetin only inhibited aromatase/17β-HSD at high doses as did genistein, biochanin A and daidzein. The lignans had weak inhibitory effects on aromatase/17β-HSD, whilst zearalenone showed potent inhibition at 0.1 μM. Phytoestrogens did not exert any significant effects on protein expression of 3β-HSD or aromatase as determined by Western blots. It is concluded that steroidogenic enzymes are inhibited by phytoestrogens in primary cultures of human GL cells but these cells are less sensitive to the effects of phytoestrogens than cell-free systems. This may be due to poor lipid solubility or cellular metabolism. We have also shown for the first time that phytoestrogens do not act by inhibiting the cellular concentration of 3β-HSD and aromatase even though exposure time would have allowed for changes in gene expression.

Adenosine Triphosphate Induces Activation of Caspase-3 in Apoptosis of Human Granulosa-luteal Cells

Adenosine triphosphate (ATP) has been shown to induce programmed cell death in various systems. However, little is known about the effect of ATP on human granulosa-luteal cells (hGLCs). The present study was designed to examine the effect of ATP on the activation of the caspase signaling pathway and its role in inducing programmed cell death. Human GLCs were collected from patients undergoing in vitro fertilization programs, and then were cultured in FBS-supplemented DMEM for 3 days prior to our studies. To examine the dose-response relationship, hGLCs were treated with increasing concentrations of ATP (10 microM, 100 microM, 1 mM or 10 mM) for 24 hours. For time-course experiments, hGLCs were treated with 10 mM ATP for 6, 12, or 24 hours. Western blot analysis was performed using antibodies against the pro- and active forms of caspase-3, -9, or PARP. To quantify the induction of apoptosis, DNA fragmentation was measured using the cell death detection enzyme-linked immunosorbent assay. To examine the effect of human chorionic gonadotropin (hCG) in protecting cells from apoptosis, hGLCs were treated with 10 IU hCG in the presence of 10 mM ATP for 12 hours. It was demonstrated that ATP was capable of inducing DNA fragmentation in a dose- and time-dependent manner. Furthermore, Western blot analysis, which detected the pro- and active forms of caspase-3, or PARP, demonstrated that ATP activated the caspase-signaling pathway, leading to the proteolytic conversion of pro-caspase-3 to active caspase-3, and the subsequent cleavage of the caspase substrate PARP. Based on our observation, caspase-9 was not triggered by ATP. Interestingly, hCG attenuated the effect of ATP in activating the caspase signaling pathway. To our knowledge, this is the first demonstration of the ATP-induced activation of the caspase signaling pathway in the human ovary. These results support the notion that the caspase-signaling pathway is involved in mediating ATP actions in the human ovary.

Adenoviral Gene Transfer Allows Smad-Responsive Gene Promoter Analyses and Delineation of Type I Receptor Usage of Transforming Growth Factor-β Family Ligands in Cultured Human Granulosa Luteal Cells

In the human ovary, cell growth and differentiation are regulated by members of the TGF-beta superfamily, including growth differentiation factor-9 (GDF9), TGF-beta, and activin. TGF-beta and activin are known to signal via Smad3 activation, and we have recently shown the involvement of Smad3 in cellular responses to GDF9. Recent studies with Smad3-deficient mice have also indicated a key role for this signaling mediator in ovarian folliculogenesis. We now demonstrate the use of a Smad3 reporter (CAGA-luciferase) adenovirus in primary cultures of human granulosa-luteal (hGL) cells to detect GDF9, TGF-beta, and activin responses. In rodent granulosa cells, TGF-beta and GDF9 signal through the TGF-beta type I receptor or activin receptor-like kinase 5 (Alk5), whereas the effect of activin is mediated though the activin type IB receptor, also known as Alk4. We now show that the GDF9 response in hGL cells is markedly potentiated upon overexpression of Alk5 by adenoviral gene transduction, as measured by the CAGA-luciferase reporter activity. A similar response to Alk5 overexpression was observed for TGF-beta, but not for activin. Adenoviral overexpression of the activin type IB receptor Alk4 in hGL cells specifically potentiated activin signaling, but not GDF9 or TGF-beta signaling. Alk5 overexpression in hGL cells also potentiated the GDF9 response when inhibin B production was used as the read-out. These results indicate that the CAGA-luciferase adenovirus can be used to study Smad3 signaling in primary cultures of human cells, and that adenoviral overexpression of wild-type receptors of the TGF-beta superfamily can be used to amplify the cellular response to ligands such as GDF9, TGF-beta, and activin. Furthermore, these studies indicate the involvement of Alk5 in GDF9 signaling in human cells and therefore, along with other recent studies, highlight how a limited number of type I and II receptors cooperate to generate specificity of action within the TGF-beta superfamily.

Adenosine 5'-triphosphate activates nuclear translocation of mitogen-activated protein kinases leading to the induction of early growth response 1 and raf expression in human granulosa-luteal cells.

With the stimulation of many types of cell surface receptors, MAPKs are activated. We have previously demonstrated an effect of extracellular ATP on ERKs and gonadotropin- induced progesterone secretion, implicating the significance of ATP in the regulation of ovarian function. However, little is known about the specific role of ATP in the subsequent MAPK-induced signaling cascade in human granulosa-luteal cells (hGLCs). The present study was designed to examine the effect of ATP on the activation of the MAPK signaling pathway, including nuclear translocation and the expression of the immediate early genes in hGLCs. Western blot analysis, using a monoclonal antibody, which detected the total and phosphorylated forms of ERK1 and ERK2 (p42(mapk) and p44 (mapk), respectively), demonstrated that exogenous ATP evoked ERKs in a dose- and time-dependent manner. In contrast, p38 and JNK were not significantly activated after ATP treatment. To examine the translocation of activated ERKs, fluorescein isothiocyanate-conjugated secondary antibody was used to detect the distribution of total and phosphorylated ERKs. Immunofluorescent staining revealed that phosphorylated ERKs were translocated from cytoplasm into nucleus subsequent to 10 microm ATP treatment. To study the gene(s) induced by exogenous ATP, mRNA was extracted from hGLCs in the presence or absence of 10 microm ATP. Gene array for 23 genes associated with members of the mitogenic pathway cascade and immediate early genes revealed that the expression of early growth response 1 and c-raf-1 was increased. To our knowledge, this is the first demonstration of the ATP-induced nuclear translocation of MAPKs in the human ovary. These results suggest that the MAPK signaling pathway plays a role in mediating ATP actions in the human ovary.

ATP activates nuclear translocation of mitogen-activated protein kinases in human granulosa-luteal cells

To investigate the specific role of adenosine triphosphate (ATP) in the subsequent MAPK-induced signaling cascade in human granulosa-luteal cells (hGLCs). We have previously demonstrated an effect of extracellular adenosine triphosphate (ATP) on ERKs and gonadotropin-induced progesterone secretion, implicating the significance of ATP in the regulation of ovarian function. The present study was designed to examine the effect of ATP on the activation of the MAPK signaling pathway, including nuclear translocation in hGLCs. Human GLCs were collected from patients undergoing in vitro fertilization programs. Human GLCs were cultured in FBS-supplemented DMEM for 3 days prior to our studies. Cells were starved in serum-free medium for 4h prior to subsequent treatments. To examine the dose-response relationship, hGLCs were treated with increasing concentrations of ATP (100nM, 1μ, 10μ, 100μ) for 5 min. For time-course experiments, hGLCs were treated with 10μM ATP for 1, 5, 10, 20 min. To determine the translocation of MAPKs, hGLCs were fixed 5 min after 10μM ATP exposure. Western blot analysis was performed using antibodies against the phosphorylated forms of ERK1 and ERK2 (P-MAPK, p42mapk and p44mapk, respectively), p42/p44 MAPK (total MAPK, T- MAPK), phospho JNK and activated P38. To examine the translocation of activated ERKs, FITC-conjugated secondary antibody was used to detect the distribution of total and phosphorylated ERKs. Western blot analysis, which detected the total and phosphorylated forms of ERK1 and ERK2, demonstrated that exogenous ATP evoked ERKs in a dose- and time-dependent manner. In contrast, p38 and JNK were not significantly activated following ATP treatment. Immunofluorescent staining revealed that phosphorylated ERKs were translocated from cytoplasm into nucleus subsequent to 10μ ATP treatment. To our knowledge, this is the first demonstration of the ATP-induced nuclear translocation of MAPKs in the human ovary. These results suggest that the MAPKs signaling pathway plays a role in mediating ATP actions in the human ovary.

Mechanism of apoptosis of human granulosa luteal cells by GnRH -agonist

Mechanism of apoptosis of human granulosa luteal cells by GnRH -agonist

Direct effect of gonadotropin-releasing hormone agonist and antagonist on the growth, apoptosis and steroidogenesis in human granulosa cells from women undergoing in vitro fertilization

To evaluate the direct effect of a GnRH-a (leuprolide acetate, LA) and a GnRH-ant (antide, ANT) on the steroidogenesis, growth and programmed cell death (apoptosis) of human granulosa-luteal cells (GLC) obtained from patients undergoing ART.

ATP-induced apoptosis of human granulosa luteal cells cultured in vitro

Objective: To investigate the role of extracellular adenosine triphosphatase (ATP) as an inducer of apoptotic cell death in human granulosa cells and to elucidate its underlying mechanism. Design: Prospective study. Setting: Gynecologic clinic and human reproduction research laboratory. Patient(s): Twenty-five patients undergoing IVF or IVF-ET. Intervention(s): ATP treatment of granulosa luteal cells subjected to primary culture. Main outcome measure(s): Apoptosis was assessed by the annexin V binding assay and the terminal deoxynucleotidyl transferase–mediated dUTP–digoxigenin nick-end labeling assay. The receptor type that binds ATP, thus mediating ATP-induced apoptosis, was determined by using the calcium imaging and patch-clamp techniques. Mitochondrial depolarization was assessed by staining with the membrane potential-sensitive dye 5,5′,6,6′-tetrachloro-1,1′,3,3′- tetraethylbenzimidazolyl carbocyanide iodide (JC-1). Result(s): ATP elevated [Ca 2+] i by mobilizing intracellularly stored Ca 2+. An ionic membrane current evoked by ATP was mediated by the Ca 2+-activated K + channel. ATP induced a mitochondrial depolarization with a concomitant increase in cellular apoptosis. Treatment with hCG eliminated both ATP-induced mitochondrial depolarization and apoptosis. Conclusion(s): Extracellular ATP recognized by P 2Y type purinoceptor triggers apoptosis in human granulosa luteal cells, and the downstream apoptotic cascade may act at least in part through mitochondria. The antiapoptotic effect of hCG has possible clinical implications in the treatment of disorders involving granulosa cell degeneration (such as follicular atresia).

Expression of betaglycan, an inhibin coreceptor, in normal human ovaries and ovarian sex cord-stromal tumors and its regulation in cultured human granulosa-luteal cells.

Activins and inhibins are often antagonistic in the regulation of ovarian function. TGFbeta type III receptor, betaglycan, has been identified as a coreceptor to enhance the binding of inhibins to activin type II receptor and thus to prevent the binding of activins to their receptor. In this study we characterized the expression and regulation pattern of betaglycan gene in normal ovaries and sex cord-stromal tumors and in cultured human granulosa-luteal cells from women undergoing in vitro fertilization. Expression of betaglycan mRNA was detected by RT-PCR or Northern blotting in normal ovarian granulosa, thecal, and stroma cells as well as in granulosa-luteal cells. Immunohistochemical analysis revealed positive staining for betaglycan in antral and preovulatory follicular granulosa and thecal cells and in corpora lutea of normal ovaries. Furthermore, betaglycan expression was detected in the vast majority of granulosa cell tumors, thecomas, and fibromas, with weaker staining in granulosa cell tumors compared with fibrothecomas. In cultured granulosa-luteal cells, FSH and LH treatment increased dose-dependently the accumulation of betaglycan mRNA, as did the protein kinase A activator dibutyryl cAMP and the protein kinase C inhibitor staurosporine. In contrast, the protein kinase C activator 12-O-tetradecanoyl phorbol 13-acetate had no significant effect on betaglycan mRNA levels. Treatment with prostaglandin E(2) and with its receptor EP2 subtype agonist butaprost increased betaglycan mRNA accumulation and progesterone secretion dose- and time-dependently. In summary, betaglycan gene is expressed in normal human ovarian steroidogenic cells and sex cord-stromal ovarian tumors. The accumulation of its mRNA in cultured granulosa-luteal cells is up-regulated by gonadotropins and prostaglandin E(2), probably via the protein kinase A pathway. The specific expression and regulation pattern of betaglycan gene may be related to the functional antagonism of inhibins to activin signal transduction in human ovaries.

Brain-derived neurotrophin factor activates mitogen-activated protein kinase in human granulosa-luteal cells

Objective: Brain-derived neurotrophin factor (BDNF) was produced by human cumulus granulosa-luteal cells (hCGLCs) in the preovulatory follicle. Our data showed that BDNF regulated steroidogenesis in human mural granulosa-luteal cells (hMGLCs) (unpublished). The present studies were to investigate the effect of BDNF on activation of Mitogen-Activated Protein Kinase (MAPK) signaling pathway in hMGLCs. Further, the role of BDNF-induced MARK activation in steroidogenesis was investigated. Design: The effect of BDNF in activating MAPK signaling pathway was determined in cultured hMGLCs. The effect of MARK inhibition on BDNF-induced influence on estrodial and progestone production was studied. Materials and Methods: hMGLCs were obtained from women undergoing in vitro fertilization-embryo transfer. For time-course experiments, hMGLCs were treated with BDNF at 100 ng/ml for 10, 20, 30 min. For dose-course experiments, hMGLCs were treated with BDNF at 0, 50, 100, 200 ng/ml for 20 min. Phosphorylated forms of p38 MAPK and p44 MAPK were examined by Western Blot. To determine the intracellular signaling pathway subsequent to BDNF treatment and the role of MARK in steroidogenesis, hMGLCs were treated with BDNF at 100 ng/ml for 24h with or without pre-treatment of SB203580 (an inhibitor for p38) at 10 μM or PD098059 (an inhibitor for p44) at 50 μM for 30 min. Enzyme imunoassay was used to determine the level of the estradiol and progesterone in culture medium of hMGLCs. Data were analyzed by Student-Newman-keuls tests. Results: BDNF up-regulated p38 MAPK activity and down-regulated p44 MAPK activity in a dose- and time- dependent manner in hMGLCs. Treatment with BDNF at 0 ng/ml or 100 ng/ml for 24 hours, E2 in culture medium of hMGLCs was 15142.33±2152.46 pg/ml, 18861.63±3765.16 pg/ml and P4 was 163.15±29.78ng/ml, 116.70±23.29 ng/ml, respectively. Treatment of BDNF at 100ng/ml with pre-treatment of SB203580 or PD098059, E2 in culture medium of hMGLCs was 16609.07±3020.17 pg/ml, 20118.90±3125.36pg/ml and P4 was 149.02±29.03 ng/ml, 106.78±17.78 ng/ml, respectively. Treatment of hMGLCs with p38 inhibitor significantly inhibited the E2 production (P0.05). Conclusion: Our data demonstrated that BDNF activated p38 MAPK signaling pathway in hMGLCs. And this activation was associated with changes in estradiol and progesterone production. It is suggested MARK signaling pathway is involved in mediating BDNF effect in the human ovary.

Phytoestrogens inhibit aromatase but not 17beta-hydroxysteroid dehydrogenase (HSD) type 1 in human granulosa-luteal cells: evidence for FSH induction of 17beta-HSD.

Studies using purified enzyme preparations, placental microsomes or cell lines have shown that certain phytoestrogens can inhibit the enzymes that convert androgens to estrogens, namely aromatase and 17beta-hydroxysteroid dehydrogenase (HSD) type 1 and type 5. The study aim was to investigate the effects of selected phytoestrogens on aromatase and 17beta-HSD type 1 activity in primary cultures of human granulosa-luteal (GL) cells. GL cells, cultured for 48 h in medium containing 5% fetal calf serum and for a further 24 h in serum-free medium with or without hFSH or hCG, were exposed to steroid substrates during the last 1-4 h of the experiment. The production of progesterone in the presence of pregnenolone or estradiol synthesis from androstenedione, estrone or testosterone showed dose- and time-dependent increases. Whilst hCG priming had no effect on progesterone production, FSH priming induced mean 68 and 56% increases in the production of estradiol from androstenedione (A-dione) and estrone respectively, but had no significant effect on the metabolism of testosterone to estradiol. None of the phytoestrogens investigated had any acute effects on enzyme activity. In contrast, when GL cells were exposed to the compounds for 24 h prior to exposure to steroid substrates for 4 h, 10 micro mol/l apigenin and zearalenone significantly inhibited aromatase activity, whilst biochanin A and quercetin had no effect. None of the phytoestrogens inhibited FSH-induced 17beta-HSD type 1 activity, and only quercetin significantly inhibited progesterone production. The inability of phytoestrogens to acutely inhibit steroidogenic enzymes in human GL cells (as has been shown in cell-free models) suggests that they are either rapidly metabolized to relatively inactive compounds or that the high enzyme activity in human GL cells masks any inhibitory effects of the compounds at the concentration tested.

Effects of androgens on aromatase activity and 11C‐vorozole binding in granulosa cells in vitro

Locally produced androgens and estrogens are important in the hormonal regulation of follicular development. The present study aimed to further elucidate the mechanism through which androgens exert their ambivalent effects on aromatization. Non-cultured human granulosa-luteal cells (GC) were treated with different concentrations of androstenedione (A4), testosterone (T), 5alpha-androstane-3,17-dione (5alpha-A) and dihydrotestosterone (DHT). The effects on aromatase activity were evaluated in a tritiated water assay (incubation time 2 h) and the availability of aromatase active sites was measured in a radiotracer-binding assay using the non-steroidal competitive aromatase inhibitor [11C]-vorozole (incubation time 15 min). A4, T and 5alpha-A caused dose-dependent inhibition of both aromatase activity and [11C]-vorozole binding; IC50-values for both inhibition processes were calculated for these three steroids, revealing A4 as the most potent inhibitor and T and 5alpha-A as moderate inhibitors. At low concentrations (0.01 and 0.1 micro M), DHT stimulated aromatase activity but did not affect [11C]-vorozole binding. At the higher concentrations tested (1 and 10 micro M) DHT suppressed both processes thus weakly binding the aromatase active site. Because the incubation time in the tritiated water assay was short, the stimulation by DHT at low concentrations might therefore most likely include mechanisms other than new synthesis of aromatase protein such as allosteric action of DHT upon aromatase or liganded androgen receptor-aromatase interaction.

Differential regulation of gonadotropin-releasing hormone (GnRH)I and GnRHII messenger ribonucleic acid by gonadal steroids in human granulosa luteal cells.

In humans, reproduction was generally believed to be controlled by only one form of GnRH (called mammalian GnRH or GnRHI). However, recently, a second form of GnRH, analogous to chicken GnRHII, was discovered in several tissues, including the human ovary. The regulation and function of GnRHI in the hypothalamus has been well studied. However, the function and regulation of GnRHI, and particularly GnRHII in the ovary, is less well understood. Because gonadal sex steroids are one of the main regulators of reproduction, we investigated, in the present study, the regulation of GnRHI and GnRHII mRNA expression by 17beta-estradiol (E2) and RU486 (a progesterone antagonist) in human granulosa luteal cells (hGLCs). The levels of the mRNA transcripts encoding the two GnRH forms were examined using semiquantitative RT-PCR followed by Southern blot analysis. With time in culture, GnRHI and GnRHII mRNA levels significantly increased, by 120% and 210%, at d 8 and d 1, respectively. The levels remained elevated until the termination of these experiments at d 10. A 24-h treatment of hGLCs with E2 (10(-9) to 10(-7) M) resulted in a dose-dependent decrease and increase in mRNA expression of GnRHI and GnRHII, respectively. E2 (10(-9) M) significantly decreased GnRHI mRNA levels (by 55%) and increased GnRHII mRNA levels (by 294%). Time-course studies demonstrated that E2 (10(-9) M) significantly decreased GnRHI mRNA levels in a time-dependent manner, with maximal inhibition of 77% at 48 h. In contrast, GnRHII mRNA levels significantly increased in a time-dependent fashion, reaching a maximum level of 280% at 24 h. Cotreatment of hGLCs with E2 and tamoxifen (an E2 antagonist) reversed the inhibitory and stimulatory effects of E2 on the mRNA expression of GnRHI and GnRHII, respectively. Time- and dose-dependent treatment with RU486 did not affect GnRHI mRNA levels in hGLCs. In contrast, RU486 treatment significantly increased GnRHII mRNA levels in hGLCs in a time- and dose-dependent fashion, with a maximum increase being observed at 24 h (with 10(-5)M RU486). In summary, the present study demonstrated that the expression of GnRHI and GnRHII at the transcriptional level is differently regulated by E2 and P4 in hGLCs.

Growth differentiation factor-9 induces Smad2 activation and inhibin B production in cultured human granulosa-luteal cells.

The TGF beta family member growth differentiation factor-9 (GDF-9) is an oocyte-derived factor that is essential for mammalian ovarian folliculogenesis. GDF-9 mRNAs have been shown to be expressed in the human ovarian follicle from the primary follicle stage onward, and recombinant GDF-9 has been shown to promote human ovarian follicle growth in vitro. In this study with primary cultures of human granulosa-luteal (hGL) cells, we investigated whether recombinant GDF-9 activates components of the Smad signaling pathways known to be differentially activated by TGF beta and the bone morphogenetic proteins (BMPs). As with TGF beta, GDF-9 treatment caused the phosphorylation of endogenous 53-kDa proteins detected in Western blots with antiphospho-Smad2 antibodies (alpha PS2). However, unlike BMP-2, GDF-9 did not activate the phosphorylation of antiphospho-Smad1 antibody (alphaPS1)-immunoreactive proteins in hGL cells. Infection of hGL cells with an adenovirus expressing Smad2 (Ad-Smad2) confirmed that GDF-9 activates specifically phosphorylation of the Smad2 protein. Infection of hGL cells with Ad-Smad7, which expresses the inhibitory Smad7 protein, suppressed the levels of both GDF-9-induced endogenous and adenoviral alpha PS2-reactive proteins. Furthermore, GDF-9 increased the steady state levels of inhibin beta(B)-subunit mRNAs in hGL cells and strongly stimulated the secretion of dimeric inhibin B. Again, Ad-Smad7 blocked GDF-9-stimulated inhibin B production in a concentration-dependent manner. We identify here for the first time distinct molecular components of the GDF-9 signaling pathway in the human ovary. Our data suggest that GDF-9 mediates its effect through the pathway commonly activated by TGF beta and activin, but not that activated by many BMPs. The results are also consistent with the suggestion that in addition to endocrine control of inhibin production by gonadotropins, a local paracrine control of inhibin production is likely to occur via oocyte-derived factors in the human ovary.

Regulation of follistatin-related gene (FLRG) expression by protein kinase C and prostaglandin E(2) in cultured granulosa-luteal cells.

Activin and its binding protein follistatin may act as local regulators of cell growth and steroidogenesis in the human ovary. The recently identified follistatin-related gene (FLRG) is expressed abundantly in the human ovary, has high affinity for activin, and is able to inhibit activin-induced transcriptional responses. However, little is known about the regulation of FLRG expression in specific cell types in the ovary, while it is known that gonadotrophins induce follistatin gene expression in human granulosa-luteal cells. In this study, we investigated the expression of FLRG mRNA in granulosa-luteal cells of preovulatory follicles obtained from women undergoing IVF. FLRG mRNA was detected by RT-PCR in fresh and cultured granulosa-luteal cells, as well as in normal ovarian stroma, theca and granulosa cells. Northern blot analysis revealed a 2.5 kb transcript of the FLRG in cultured granulosa-luteal cells. The protein kinase C activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA, 160 nmol/l), and prostaglandin E(2) (PGE(2), 1 micromol/l) increased FLRG mRNA accumulation up to 3-8 fold over the control level after 24 h of treatment, and these stimulatory effects were dose-dependent. Co-treatment with the protein kinase C inhibitor, Ro-31-8220 (3 micromol/l), blocked the stimulatory effect of TPA. Although short term treatment with the protein kinase A activator, (Bu)(2)cAMP (1 mmol/l), slightly reduced FLRG mRNA expression in most experiments, long term treatment with FSH (100 IU/l), LH (100 IU/l), or (Bu)(2)cAMP had no significant effect on the FLRG mRNA levels. As expected, gonadotrophins, protein kinase A and C activators and PGE(2) increased granulosa-luteal cell progesterone secretion into the culture media. Taken together, previous and our present data suggest that protein kinase C and A signal transduction pathways differently regulate the expression of FLRG and follistatin genes in human ovarian granulosa-luteal cells.

The role of LH receptor in human IVF-ET program

Objective: To study the role of LH receptor on folliculogenesis, we examined the expression levels of LH receptor (LH-R) mRNA in human granulosa luteal cell (GLC)s and compared these expression patterns with pregnancy rates, number of retrieved oocytes, and quality of oocytes and embryos.