Acupuncture regulates the autophagy of ovarian granulosa cells in polycystic ovarian syndrome ovulation disorder by inhibiting the PI3K/AKT/mTOR pathway through LncMEG3.

The main features of polycystic ovary syndrome (PCOS) are abnormal follicular development and ovulation dysfunction, which are caused by the excessive autophagy of ovarian granulosa cells. Acupuncture has been shown to improve ovulation dysfunction and abnormal follicular development in PCOS patients, but its mechanism is unclear. This study hypothesized that the beneficial effects of acupuncture are the result of LncMEG3-mediated effects on the PI3K/AKT/mTOR pathway. Acupuncture (CV-4, RN-3, CV-6, SP-6 and EX-CA 1) was used to treat a rat model of polycystic ovary syndrome. Hematoxylin-eosin staining was used to observe ovarian morphology and enzyme-linked immunosorbent assay, western blotting, immunohistochemistry and real-time PCR were used to detect LH, E2, FSH, T, AMH, LncMEG3, PI3K, AKT, mTOR, P62 and LC3II/I expression. The ovarian morphology of 90% of the rats in the acupuncture treatment group was significantly improved after 11 consecutive days of therapy. Acupuncture also resulted in a significant decrease in serum LH, FSH, T and AMH levels and a significant increase in E2 level (P<0.01). LncMEG3, PI3K, AKT, mTOR, P62 and LC3II/I expression was decreased in ovarian granulosa cells after acupuncture compared with PCOS and lentiviral Intervention Group (P<0.05), while the expression of follicle stimulating hormone receptor was increased (P<0.05). These results indicate that acupuncture can down-regulate the expression of LncMEG3 and thereby inhibit the PI3K/AKT/mTOR pathway, reducing granulosa cell autophagy and normalizing their proliferation. These factors ultimately remedy abnormal follicular development. These findings suggest that acupuncture has clinical potential as a safe treatment for PCOS ovulatory dysfunction.

In Search of New Therapeutics-Molecular Aspects of the PCOS Pathophysiology: Genetics, Hormones, Metabolism and Beyond.

In a healthy female reproductive system, a subtle hormonal and metabolic dance leads to repetitive cyclic changes in the ovaries and uterus, which make an effective ovulation and potential implantation of an embryo possible. However, that is not so in the case of polycystic ovary syndrome (PCOS), in which case the central mechanism responsible for entraining hormonal and metabolic rhythms during the menstrual cycle is notably disrupted. In this review we provide a detailed description of the possible scenario of PCOS pathogenesis. We begin from the analysis of how a set of genetic disorders related to PCOS leads to particular malfunctions at a molecular level (e.g., increased enzyme activities of cytochrome P450 (CYP) type 17A1 (17α-hydroxylase), 3ß-HSD type II and CYP type 11A1 (side-chain cleavage enzyme) in theca cells, or changes in the expression of aquaporins in granulosa cells) and discuss further cellular- and tissue-level consequences (e.g., anovulation, elevated levels of the advanced glycation end products in ovaries), which in turn lead to the observed subsequent systemic symptoms. Since gene-editing therapy is currently out of reach, herein special emphasis is placed on discussing what kinds of drug targets and which potentially active substances seem promising for an effective medication, acting on the primary causes of PCOS on a molecular level.

FSH receptor binding inhibitor influences estrogen production, receptor expression and signal pathway during in vitro maturation of sheep COCs.

Follicle-stimulating hormone (FSH) promotes secretion of follicle fluid and follicle development. FSH acts via cognate FSH receptor (FSHR). It remains unknown whether the supplement of FSH-receptor binding inhibitor (FRBI) into the in vitro maturation (IVM)medium influence the estrogen receptor expression and signal pathway of oocytes in sheep. The present study aimed to investigate FRBI effects on inositol trisphosphate (IP3) of oocytes and protein kinase A (PKA) of sheep granulosa cells, further to elucidate the signal pathway of FRBI effects. Cumulus-oocyte complexes (COCs) were recovered from antral follicles. COCs were cultured for 24 h in the IVM medium supplemented with varying concentrations of FRBI (0, 10, 20, 30 and 40 µg/mL) and FSH (10IU/mL). ELISA was used to measure the concentrations of estradiol (E2) and IP3 in the IVM medium. Western blotting was utilized to detect protein expression of ERß of COCs and protein kinase A (PKA) of granulosa cells. The results showed IP3 concentrations of FRBI-3 and FRBI-4 groups were less than that of CG and FSH groups at 22 h and 24 h (P < 0.05). PKA levels of FRBI-3 and FRBI-4 groups were significantly less than that of CG and FSH group (P < 0.05 or P < 0.01). Expression levels of ERß mRNA and protein of FRBI-treated groups were gradually decreased in comparison to CG and FSH group. The minimum value was detected in the FRBI-4 group. ERß protein level of the FRBI-4 group was significantly less than that of FSH group (P < 0.05). E2 concentrations of FRBI-treated groups were elevated as compared to CG, with the highest increment of FRBI-2 group (P < 0.05). Our results revealed a higher dose of FRBI reduced IP3 production. FRBI could suppress slightly expression levels of ERß mRNA and protein of COCs and PKA of granulosa cells, additionally increased E2 production of sheep COCs.

Modulation of steroidogenesis by vitamin D3 in granulosa cells of the mouse model of polycystic ovarian syndrome.

Polycystic ovarian syndrome (PCOS) is the most common endocrine disorder of women of reproductive age characterized by polycystic ovarian morphology, anovulation or oligomenorrhea, and hyperandrogenism. It is shown that disruption in the steroidogenesis pathway caused by excess androgen in PCOS is a critical element of abnormal folliculogenesis and failure in dominant follicle selection. Vitamin D plays an important role in the regulation of ovulatory dysfunction and can influence genes involved in steroidogenesis in granulosa cells. In the present study, we investigated the effects of vitamin D3 on steroidogenic enzyme expression and activities in granulosa cell using a PCOS mouse model. In our study, the PCOS mouse model was developed by the injection of dehydroepiandrosterone (DHEA) for 20 days. The mRNA and protein expression levels of genes involved in steroidogenesis in granulosa cells were compared between polycystic and normal ovaries using real-time PCR and Western blotting assays. Granulosa cells of DHEA-induced PCOS mice were then cultured with and without vitamin D3 and mRNA and protein expression levels of steroidogenic enzymes and serum 17beta-estradiol and progesterone levels were investigated using qRT-PCR, western blot, and radioimmunoassay, respectively. Steroidogenic enzymes including Cyp11a1, StAR, Cyp19a1, and 3ß-HSD were upregulated in granulosa cells of PCOS mice when compared to normal mice. Treatment with vitamin D3 decreased mRNA and protein expression levels of steroidogenic enzymes in cultured granulosa cells. Vitamin D3 also decreased aromatase and 3ß-HSD activity that leads to decreased 17beta-estradiol and progesterone release. This study suggests that vitamin D3 could modulate the steroidogenesis pathway in granulosa cells of PCOS mice that may lead to improving follicular development and maturation. This is a step towards a possible conceivable treatment for PCOS. ABBREVIATIONS: AMHR-II: anti-müllerian hormone receptor-II; 3ß-HSD: 3ß-hydroxysteroid dehydrogenase; Cyp11a1: Cytochrome P450 Family 11 Subfamily A Member 1; Cyp19a1: cytochrome P450 aromatase; DHEA: dehydroepiandrosterone; FSH: follicle stimulating hormone; FSHR: follicle stimulating hormone receptor; IVF: in vitro fertilization; 25OHD: 25-hydroxy vitamin D; OHSS: ovarian hyperstimulation syndrome; PCOS: polycystic ovarian syndrome; P450scc: P450 side-chain cleavage enzyme; StAR: steroidogenic acute regulatory protein; VDRs: vitamin D receptors.

Coenzyme Q-dependent mitochondrial respiratory chain activity in granulosa cells is reduced with aging.

OBJECTIVE: To examine coenzyme Q10 (CoQ10)-dependent mitochondrial respiratory chain (MRC) activity in granulosa cells (GC) with aging and examine the effect of in vitro CoQ supplementation. DESIGN: Experimental study. SETTING: Hospital laboratory. PATIENT(S): Ten younger (<32 years) and 10 older (>39 years) patients undergoing in vitro fertilization (IVF) treatment. INTERVENTION(S): Measurement of succinate-cytochrome c reductase (MRC complex II + III) activity in the presence and absence of CoQ1 (a soluble CoQ analog). MAIN OUTCOME MEASURE(S): MRC enzymatic activity in human GC via complex II + III measured in GC homogenate by spectrophotometry and compared with CoQ in dependent MRC complex II and citrate synthase (CS). RESULT(S): Complex II + III activity was 1.9 times higher in young patients compared with older patients (18.3 ± 5.8 and 9.6 ± 3 nmol/min/mg, respectively) whereas II and CS were not statistically significantly different. Increased II + III activity in the presence CoQ1 was observed in both groups but was statistically significantly higher in the older patients, reaching similar levels. Compared with baseline (II + III + Q/II + III), the increase was 2.47 times higher in older patients compared with young patients (6.5 ± 2.0 and 2.62 ± 0.83, respectively). CONCLUSION(S): Coenzyme Q10-dependent MRC activity in GC reduces with aging. This reduction is diminished upon in vitro CoQ1 supplementation, indicating that CoQ10 deficit is the underlying cause for the mitochondrial dysfunction. The results show that functional CoQ10 status can be assessed by measuring complex II + III activity in GC and might provide a useful monitoring tool for future clinical studies of oral CoQ10 supplementation to older patients undergoing IVF treatment.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and phytoestrogen genistein on the activity and the presence of steroidogenic enzyme proteins in cultured granulosa cells of pigs.

Environmental estrogens such as dioxins (e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) and phytoestrogens (e.g. genistein; G) are known to influence endocrine and reproductive processes in humans and animals. Because living organisms are usually exposed to small, non toxic, doses of dioxins and phytoestrogens, the aims of the study were to determine the effects of small, environmentally relevant doses of TCDD (100pM) and/or genistein (500nM) on: (1) the activity of steroidogenic enzymes (cholesterol side-chain cleavage enzyme, P450scc; 3ß-hydroxysteroid dehydrogenase, 3ß-HSD and aromatase, P450arom) and (2) amount of protein of the enzymes in granulosa cells isolated from medium and large ovarian follicles of pigs. To determine the activity of the enzymes, the incubation medium was supplemented with specific steroid substrates (25-hydroxycholesterol; pregnenolone; testosterone) of particular steroidogenic enzymes (P450scc, 3ß-HSD and P450arom, respectively). Subsequently, the production of progesterone (P450scc and 3ß-HSD) or estradiol (P450arom) was compared in the presence and absence of the appropriate steroid precursor. Neither genistein nor genistein combined with TCDD affected activity of P450arom and relative amounts of steroidogenic enzyme proteins in the examined granulosa cells of pigs. In contrast, genistein alone and in combination with TCDD decreased P450scc and 3ß-HSD activity as well as progesterone production in granulosa cells isolated from medium and large follicles of pigs. Because TCDD alone did not affect steroid hormone production or enzyme activity, the above effects should be ascribed solely to genistein. It appears that the effects of the examined doses of TCDD and genistein on granulosal cell functions were not additive.

Survival role of superoxide dismutase 1 on human granulosa luteinized cells in vitro.

OBJECTIVE: Deleterious effects of free radicals do not only result from the amount of free radicals produced but also are related to the efficiency and to the activities of enzymatic antioxidant systems. We investigated the effect of exogenous superoxide dismutase (SOD1) or Cu-chelating agent diethyldithiocarbamate (DDC) on the apoptosis (caspase-3 activity) of human granulosa luteinized cells (hGLC) in vitro. METHODS: The effects of SOD1 and DDC were studied using in vitro culture system, caspase-3 and the total SOD activity in hGLCs were measured using AcDEVD-PNA substrate and Beaushap and Fridovich methods, respectively, after 48 h of the culture period. RESULTS: The activity of SOD1 was the lowest in GLCs treated with 100 µM DDC as compared to control cells and to the cells supplemented with Cu, Zn-SOD or DDC (10 µM). The effect of DDC was associated with elevated caspase-3 activity as compared to control cells. CONCLUSIONS: It was demonstrated for the first time that the supplementation of cultured hGLCs with Cu2+, Zn2+-SOD (200 U/ml) maintains the viability of hGLCs via caspase-3 suppression.

Rosiglitazone and pioglitazone inhibit estrogen synthesis in human granulosa cells by interfering with androgen binding to aromatase.

The effects of rosiglitazone or pioglitazone (thiazolidinediones, TZDs) on estrogen production and aromatase activity in human ovarian cells were examined. Human granulosa cells were incubated in the tissue culture medium supplemented with androstenedione or testosterone, with or without insulin, TZDs, or type 1 17ß-hydroxysteroid-dehydrogenase (17ß-HSD) inhibitor. Estrogen concentrations in the conditioned medium, aromatase mRNA and protein expression in the cells and androgen substrate binding to aromatase were measured. With androstenedione as substrate, rosiglitazone or pioglitazone inhibited estrone production by up to 22% (p<0.012) while type 1 17ß-HSD inhibitor enhanced this effect of rosiglitazone or pioglitazone by 37% (p<0.001) and by 67% (p<0.001), respectively. With testosterone as substrate, rosiglitazone or pioglitazone inhibited estradiol production by 32% (p<0.001). With (3)H-testosterone as substrate, rosiglitazone or pioglitazone inhibited the (3)H-tritiated water release by the cultured cells by 45% and 35%, respectively, thus directly demonstrating inhibition of aromatase. Rosiglitazone or pioglitazone, however, had no significant effect on aromatase mRNA or protein expression. Rosiglitazone or pioglitazone inhibited (125)I-androstenedione and (125)I-testosterone binding to aromatase by 38% (p<0.001). It was concluded that rosiglitazone or pioglitazone inhibit estrogen synthesis in human granulosa cells by interfering with androgen binding to aromatase.

Biological effects on granulosa cells of hydroxylated and methylated resveratrol analogues.

Several resveratrol analogues have been designed to improve bioactivity: among these polymethoxystilbenes appear to be particularly promising. The present study was set up to investigate the biological functions of polymethoxystilbenes 2 and 3, recently found in our lab as antiangiogenic agents, on a well-defined swine granulosa cell model. Proliferative activity and effects on steroidogenesis were evaluated, as well as the effect on granulosa cell vascular endothelial growth factor (VEGF) production, since these cells in basic conditions synthesize the main proangiogenic peptide. Moreover, we considered the effect of these two resveratrol analogues on granulosa cell redox status. Analogue 3 inhibited granulosa cell growth, while it stimulated steroidogenesis. A similar effect was displayed by 2 on estradiol 17beta production and cell proliferation at the highest concentration tested. On the other hand, at the same dosage 2 decreased progesterone levels. Both analogues inhibited VEGF output. Granulosa cell redox status was unaffected by resveratrol analogue 2 while the highest concentration of 3 stimulated free radicals generation and scavenging enzyme activities. The overall results indicate that analogue 3 is the more powerful compound, thus suggesting that a slight modification in the structure markedly increases effectiveness. These data could be useful to develop more active resveratrol analogues for therapeutic use.

Effects of unsaturated fatty acids on progesterone secretion and selected protein kinases in goat granulosa cells.

Previous studies in cattle have shown influences of dietary unsaturated fatty acid (UFA) supplementation on ovarian function. However, it is unclear whether these UFA exert direct or indirect effects on ovarian steroid production or their mechanisms of action. We have recently shown that 5'AMP-activated protein kinase (AMPK) regulates progesterone secretion through mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MAPK ERK1/2) in rodent granulosa cells. Here, we investigated the effects of 3 UFAs, oleic acid (OA), linoleic acid (LA), and alpha-linolenic acid (ALA) on progesterone secretion in goat granulosa cells. Finally, we examined the effects of UFAs on MAPK ERK1/2 and AMPK phosphorylation in these granulosa cells. Oleic acid and LA (10 microM each), but not ALA (100 microM), increased progesterone secretion (P<0.05) in the presence or absence of insulin-like growth factor (IGF)-1 (10(-8) M) or FSH (5 x 10(-8)M). The different AMPK subunits, except for gamma3, are present in the goat ovary. Treatment with metformin (10mM), an activator of AMPK, increased AMPK phosphorylation (P<0.05) and reduced progesterone secretion by 50% (P<0.05) in the basal state and in response to IGF-1 or FSH in goat granulosa cells. Oleic acid and LA had no effect on AMPK phosphorylation, whereas they rapidly increased MAPK ERK1/2 phosphorylation (P<0.05). Finally, U0126, a MAPK ERK1/2 inhibitor, decreased OA- and LA-induced progesterone secretion (P<0.05), suggesting that these UFAs could stimulate progesterone secretion partly through MAPK ERK1/2 in the absence of IGF-1 and FSH in goat granulosa cells. The involvement of AMPK in this process remains to be demonstrated. Taken together, some fatty acids could improve ovarian steroidogenesis through the MAPK ERK1/2 signaling pathway and, consequently, have beneficial effects on goat fertility.

Molecular mechanism for repression of 17alpha-hydroxylase expression and androstenedione production in granulosa cells.

CONTEXT: According to the traditional two-cell two-gonadotropin model of follicular steroidogenesis, androgen production arises exclusively from theca cells. The granulosa cells, in turn, utilize androstenedione and testosterone, which are aromatized into estrone and estradiol, respectively. Differential expression of the activator protein-1 (AP-1) transcription factor, c-fos, has been postulated to result in distinct patterns of steroidogenesis in the theca and granulosa cell compartments. We hypothesize that c-fos functions to inhibit the production of 17alpha-hydroxylase 17,20 lyase (CYP17) in granulosa cells, thereby suppressing androgen synthesis. OBJECTIVE: Our objective was to define the role of c-fos in the regulation of CYP17 production in granulosa cells. DESIGN AND METHODS: Human luteinized granulosa (HGL5) cells were utilized for all experiments. The following techniques were used: mRNA extraction, steroid quantification, small interfering RNA silencing, microarray analysis, and immunohistochemistry. RESULTS: Immunohistochemistry studies demonstrated significant staining of c-fos in the granulosa cell layer, but absent staining for CYP17. Conversely, the theca cell layer did not stain for c-fos, but staining was evident for CYP17. Treatment of HGL5 cells with the MAPK kinase inhibitor PD98059 resulted in an 11-fold increase in CYP17 mRNA levels. In c-fos gene silenced cells, CYP17 mRNA levels increased 8-fold. Androstenedione production was increased 13-fold after treatment with PD98059. CONCLUSIONS: These results suggest that the AP-1 transcription factor, c-fos, may be one of the factors responsible for CYP17 repression and hence suppression of androstenedione production in granulosa cells. This may provide an explanation for the lack of CYP17 in granulosa cells.

Molecular cloning and characterization of cDNA encoding buffalo (Bubalus bubalis) cytochrome P450 aromatase in the ovary.

One of the predominant causes of poor reproduction in buffaloes is low levels of ovarian estrogens. A rate limiting enzyme in estrogen biosynthesis is cytochrome P450 aromatase (P450 AROM), the product of CYP19 gene. In the present study CYP19 cDNA was cloned and its 5'UTR was characterized by 5'RACE in granulosa cells of large follicles. CYP19 transcripts with four different 5'UTRs (206, 114, 90 and 3 bases) were found in buffalo granulosa cells of large ovarian follicles. Interestingly, a predominant aromatase transcript with short 5'UTR (3 nucleotides) was found. Further studies are required to understand the relevance of these transcripts and their translational efficiency in granulosa cells of large follicles during folliculogenesis of buffalo ovary.

Effects of transferrin on aromatase activity in porcine granulosa cells in vitro.

Proliferating cells have an absolute requirement for iron, which is delivered by transferrin with subsequent intracellular transport via the transferrin receptor. Recent studies have reported that transferrin plays a crucial role in the local regulation of ovarian function, apart from its iron-binding characteristic. Therefore, the present study was undertaken to explore the possible role of transferrin in porcine granulosa cells function by examining its influence on aromatase activity, the most important indicator of follicular cell differentiation. In the first series of studies, pig granulosa cells isolated from small, immature follicles were cultured in the presence of transferrin alone (10 microg/ml or 100 microg/ml) or with the addition of FSH (100ng/ml). The second series of studies was undertaken to determine transferrin-stimulated granulosa cells ability to aromatize exogenous testosterone (1x10(-7)M). One hour after the establishment of cultures an aromatase inhibitor CGS16949A was added to test its influence on estradiol production. After 48 hours, cultures were terminated and cells were processed for immunocytochemical staining of aromatase. Media were frozen for further estradiol level analysis. Positive immunostaining for aromatase was found in all granulosa cell cultures. The intensity of immunostaining was always stronger in cultures supplemented with FSH whereas the addition of transferrin had no effect. Granulosa cells in vitro synthesized the highest amount of estradiol after the addition of FSH and exogenous testosterone as measured radioimmunologically. Concomitant treatment with FSH and transferrin caused an inhibition of FSH-stimulated aromatase activity. The production of estradiol also declined in the presence of FSH, testosterone and transferrin. This study demonstrates that transferrin had a dose-dependent inhibitory effect on FSH-stimulated aromatase activity, which was confirmed by radioimmunoassay. Our results indicate that transferrin may be an important factor in the regulation of granulosa cell diferentiation.

Effects of adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) hull extracts on the secretion of progesterone and estradiol in vivo and in vitro.

Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) has been used as a traditional Chinese medicine for dysfunction of the endocrine system. However, there have been few studies on the effects of adlay seed on the endocrine system. In the present study, both the in vivo and in vitro effects of methanolic extracts of adlay hull (AHM) on progesterone synthesis were studied. AHM was partitioned with four different solvents: water, 1-butanol, ethyl acetate, and n-hexane. Four fractions, namely, AHM-Wa (water fraction), AHM-Bu (1-butanol fraction), AHM-EA (ethyl acetate fraction), and AHM-Hex (n-hexane fraction), were respectively obtained. Granulosa cells (GCs) were prepared from pregnant mare serum gonadotropin-primed immature female rats and were challenged with different reagents, including human chorionic gonadotropin (hCG; 0.5 IU/ml), 8-bromo-adenosine-3',5'-cyclic monophosphate (8-Br-cAMP; 0.1 mM), forskolin (10 microM), 25-OH-cholesterol (10 microM), and pregnenolone (10 microM), in the presence or absence of AHM (100 microg/ml). The functions of steroidogenic enzymes, including protein expression of the steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage enzyme (P450scc), protein kinase A (PKA), and aromatase activity, were investigated. The expression of StAR mRNA was also explored by using real-time reverse transcription-polymerase chain reaction. In the in vivo study, AHM decreased plasma progesterone and estradiol levels after an intravenous injection of AHM (2 mg/ ml/kg). In the in vitro studies, AHM decreased progesterone and estradiol via inhibition of (i) the cAMP-PKA signal transduction pathway, (ii) cAMP accumulation, (iii) P450scc and 3beta-HSD enzyme activities, (iv) PKA, P450scc and StAR protein expressions and StAR mRNA expression, and (v) aromatase activity in rat GCs. These results suggest that AHM decreased the production of progesterone via mechanisms involving the inhibition of the cAMP pathway, enzyme activities, and the protein expressions of P450scc and StAR in rat GCs.

Activation of protein kinase Czeta mediates luteinizing hormone- or forskolin-induced NGFI-B expression in preovulatory granulosa cells of rat ovary.

We have previously demonstrated that luteinizing hormone (LH) induces a rapid and transient expression of NGFI-B in the ovary. In this report, we investigated the signaling pathway for LH- and forskolin-induced NGFI-B expression in cultured rat granulosa cells of preovulatory follicles. LH- or forskolin-induced NGFI-B expression was suppressed by high dose of protein kinase C (PKC) inhibitor RO 31-8220 (10 microM), but not by low doses RO 31-8220 (0.1-1.0 microM) or adenylate cyclase inhibitor MDL-12,300A, implicating the involvement of atypical PKCs. Kinase assay revealed that LH treatment of granulosa cells resulted in a rapid stimulation of atypical PKCzeta activity. Interestingly, like LH, forskolin was also able to activate PKCzeta. Treatment with the cell-permeable PKCzeta-specific inhibitor pseudosubstrate peptide inhibited LH-or forskolin-induced NGFI-B expression, indicating the essential role of PKCzeta. Consistent with this promise, in granulosa cells depleted of diacylglycerol sensitive PKCs by prolonged treatment with tetradecanoylphobol-13-acetate, LH or forskolin could still induce NGFI-B expression, and RO 31-8220 or the PKCzeta pseudosubstrate peptide inhibited LH- or forskolin-induced NGFI-B expression. Furthermore, overexpression of dominant-negative PKCzeta in primary granulosa cells using a replication-defective adenovirus vector resulted in the suppression of LH- or forskolin-induced NGFI-B expression. Our findings demonstrate that PKCzeta, which is activated by LH or forskolin, contributes to the induction of NGFI-B in granulosa cells of preovulatory follicles.

Cloning of the bovine beta-carotene-15,15'-oxygenase and expression in gonadal tissues.

Beta-carotene-15,15'-oxygenase (betaCO), found mainly in intestinal mucosa and liver, is the enzyme responsible for cleaving beta-carotene into retinal, which can be used or stored at these sites or carried by the bloodstream to different target cells within the body. We isolated the cDNA for bovine betaCO and demonstrated its expression in gonadal tissues. A cDNA of 2130 base pairs (bp) was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), using degenerate oligonucleotides; the deduced protein shared an identity of 75% with its homologues from other mammalian species. In order to evaluate the expression of this enzyme, we performed RT-PCR and in situ hybridizations in the ovary and testis of bovines. RT-PCR showed the expression of betaCO in testis, ovary, and cultured granulosa cells. In situ hybridization of complete ovary and testis revealed expression in granulosa cells and the corpus luteum in the ovary and in germinal and interstitial cells in the testis. These results suggest that beta-carotene could act as a local source of retinoids, which have been shown to be important during proliferation, differentiation, and maturation of both female and male germinal cells.

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

BACKGROUND: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Cyclooxygenase (COX)-2 and granulosa cell apoptosis in vitro.

PURPOSE: C-myc was studied in cyclooxygenase (COX)-2 associated granulosa cell apoptosis, METHODS: Granulosa cells (N = 5 cases) were incubated for 24 h in either 1 or 50 microM COX-2 inhibitor, 1 or 50 microM COX-1/COX-2 inhibitor, negative or positive controls Single primer polymerase chain reaction of c-myc exon 1 were performed. Bisbenzimide-stained control single-stranded (ssDNA) were hybridized to SYBR Gold-stained ssDNA and fluorescent images analyzed. RESULTS: C-myc was disrupted by the high-dose COX-2 inhibitor. Cell viability decreased with COX-1 and COX-2 inhibition. However, cell viability was similar for the positive control and at low-dose COX-2 inhibition. CONCLUSIONS: Inhibition of both COX-1 and COX-2 initiated apoptosis without disrupting c-myc suggesting a protective effect on c-myc. The low dosage of the COX-2 inhibitor did not disrupt c-myc and cell viability. C-myc sensitization was not part of apoptosis.

Transcriptional regulation of the cyclooxygenase-2 gene changes from protein kinase (PK) A- to PKC-dependence after luteinization of granulosa cells.

This study was designed to elucidate the molecular mechanism(s) mediating cyclooxygenase-2 (Cox-2) regulation during differentiation of the granulosa cell. The 5' flanking sequence of the Cox-2 gene was linked to a vector with a luciferase reporter gene, and this vector was transfected into freshly isolated bovine granulosa cells or granulosa cells after culture with or without forskolin to induce luteinization in vitro. The Cox-2 promoter was inducible by 8-bromo cAMP but not by phorbol esters in fresh granulosa cells, and maximal expression by cAMP was delayed until 48 h after treatment. In contrast, after luteinization of granulosa cells by 8-day treatment with forskolin, the Cox-2 promoter was immediately inducible by phorbol esters but not by cAMP. In granulosa cells cultured for 8 days without forskolin, the Cox-2 promoter continued to be inducible only by cAMP and not by phorbol esters. Unexpectedly, no delay was observed in the induction of Cox-2 by cAMP in granulosa cells that were cultured without forskolin, compared with an approximately 1 day delay in Cox-2 induction by cAMP in fresh granulosa cells. Myristoylated protein kinase (PK) A and PKC inhibitory peptides were utilized to further confirm the PKA- or PKC-dependence of Cox-2 induction. Time-course experiments showed that only 2 days of forskolin treatment could induce PKC-responsiveness of the Cox-2 promoter, although maximal responsiveness was not observed until 10 days of luteinization. Promoter activity was also analyzed in a series of deletion mutants as well as site-directed mutants of C/EBP, CRE, and E-box. A 282-base pair sequence in the Cox-2 5' flanking region maintained full inducibility by PKA in granulosa cells and by PKC in luteinized granulosa cells. The E-box element was found to be the critical regulatory element for Cox-2 induction by either PKA in granulosa cells or by PKC in luteinized granulosa cells. Electrophoretic mobility shift assays were performed on nuclear extracts from fresh or luteinized granulosa cells. Upstream stimulatory factor (USF)-1 and USF-2 bound to the E-box of the Cox-2 gene, and binding was similar for nuclear extracts from fresh, cultured, or luteinized granulosa cells. Thus, although luteinization changes transcriptional regulation of Cox-2 from PKA- to PKC-dependence, the crucial role of the E-box element in this transcriptional activation is conserved.

Presence of a 5-HT7 receptor positively coupled to adenylate cyclase activation in human granulosa-lutein cells.

Although serotonin (5-HT) has been shown to stimulate progesterone production by human granulosa-lutein cells (hGLC), the receptor type and associated signaling pathway remain uncharacterized. We report here that 5-HT receptors in these cells are positively coupled to adenylate cyclase activity. Formation of cAMP was stimulated by 5-HT and its agonists in a dose- and time-dependent manner. Mianserin, amoxapine, and loxapine were equipotent in antagonizing 5-HT-induced cAMP formation. For both cAMP formation in cells and adenylate cyclase assay using membrane fractions, the rank order of potency for agonists of 5-HT were: 5-carboxy-aminotryptamine >5-HT> or =5-methoxytryptamine, consistent with a typical pharmacological profile of human 5-ht7 (h5-ht7) receptor. Sequence data of amplified complementary DNA fragments reverse transcribed from hGLC RNA revealed complete identity with published sequence of h5-ht7 receptor complementary DNA. Northern analysis showed the presence of 2.8-kb h5-ht7 transcripts in hGLC. The three variants h5-ht7A, h5-ht7B, and h5-ht7D were also detected in hGLC. Preincubation of hGLC with 5-HT (10(-8)-10(-6) M) resulted in a marked reduction in the cAMP response when the cells were subsequently stimulated with gonadotropin, and this heterologous desensitization could be reversed by 5-ht7 receptor antagonist clozapine. These data demonstrate that h5-ht7 receptor is present and stimulate cAMP formation in hGLC. In addition, the h5-ht7 receptor seems to be implicated in the heterologous down-regulation hCG-stimulated cAMP response in hGLC, with a possible ramification for luteal insufficiency.