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.

Decreased myo-inositol to chiro-inositol (M/C) ratios and increased M/C epimerase activity in PCOS theca cells demonstrate increased insulin sensitivity compared to controls.

Previous studies from our and other labs have shown that insulin resistance is associated with an inositol imbalance of excess myo-inositol and deficient chiro-inositol together with a deficiency of myo-inositol to chiro-inositol epimerase in vivo and in vitro. In this report, we utilized well characterized theca cells from normal cycling women, with normal insulin sensitivity, and theca cells from women with polycystic ovary syndrome (PCOS), with increased insulin sensitivity to examine the myo-inositol to chiro-inisitol (M/C) ratio and the myo-inositol to chiro-inositol epimerase activity. PCOS theca cells with increased insulin sensitivity were specifically used to investigate whether the inositol imbalance and myo-inositol to chiro-inositol epimerase are regulated in a similar or the opposite direction than that observed in insulin resistant cells. The results of these studies are the first to demonstrate that in insulin sensitive PCOS theca cells the inositol imbalance goes in the opposite direction to that observed in insulin resistant cells, and there is a decreased M/C ratio and an increased myo-inositol to chiro-inositol epimerase activity. Further biochemical and genetic studies will probe the mechanisms involved.

The bovine genome contains three differentially methylated paralogous copies of the P450c17 encoding gene (CYP17A1).

CYP17A1 encodes the key enzyme of androgen biosynthesis, P450c17. The gene is expressed in a number of steroidogenic tissues among them testis, ovary, placenta and adrenal gland. The proper analysis of CYP17A1 expression and of epigenetic parameters however, is hampered by the presence of more than one copy of the gene within the bovine genome. Therefore, as a prerequisite for future studies we characterized these copies and analyzed their promoter methylation and expression profiles in different tissues. DNA methylation levels were determined by bisulfite modification, amplification, cloning and sequencing. Transcription was analyzed by RT-PCR. From bovine genomic DNA three different CYP17A1 promoter sequences could be amplified with a sequence similarity of 94.8%, 95.6% and 98.7%. Based on these sequences we could reconstruct, by in silico analysis, the promoter regions and eight potentially coding exons of two loci, CYP17A1a and CYP17A1b, and the promoter region and truncated first exon of a third locus, CYP17A1x. By using locus-specific primers, only transcripts of CYP17A1a, but not of CYP17A1b could be detected in testis, epididymis, theca, corpus luteum, placental cotyledons, adrenal gland and preoptic brain area. Methylation analysis revealed that only the CYP17A1a promoter was hypo-methylated in the tested P450c17 active tissues, whereas both other copies showed higher levels of methylation. From these data we conclude that the bovine genome contains three paralogous copies of the CYP17A1 gene, of which two (CYP17A1b and CYP17A1x) might be silenced by epigenetic modification (promoter methylation).

Gonadotropins up-regulate the expression of enolase 2, but not enolase 1, in the rat ovary.

It has been demonstrated that the glycolytic enzymes, enolase 1 (ENO1) and enolase 2 (ENO2), are expressed in the rat ovary. In the present study, we found that mRNA levels of ovarian ENO2 but not ENO1 in normal cycling adult female rats changed significantly during the estrous cycle: ovarian ENO2 mRNA levels at metestrus were lower than those at estrus. Single injection of human CG (hCG) or equine CG (eCG) into immature (3 week old) rats up-regulated ovarian expression of ENO2. hCG mainly increased ENO2 expression in oocytes and theca cells of preantral and antral follicles, and eCG did in theca cells of these follicles. In contrast, hCG and eCG did not affect the expression of ENO1, which was mainly expressed in granulosa cells. These results suggest that endogenous gonadotropins up-regulate expression of ENO2 in oocytes and theca cells of preantral and antral follicles, which would activate glycolysis in these cells. It is also suggested that the activated glycolysis is necessary for ovarian functions such as follicle growth and maturation, and hormone production.

DNA methylation is not involved in preovulatory down-regulation of CYP11A1, HSD3B1, and CYP19A1 in bovine follicles but may have a role in permanent silencing of CYP19A1 in large granulosa lutein cells.

The luteinizing hormone-induced morphological and physiological reorganization of the bovine follicle is preceded by a profound and well-orchestrated modulation of gene expression. In the present study, the cell type-specific methylation profiles of CYP11A1, HSD3B1, and CYP19A1, genes that encode key enzymes of steroid hormone biosynthesis, were analyzed to elucidate whether epigenetic parameters such as DNA methylation might be involved in gene regulation during luteinization. Transcript abundance and DNA methylation levels were determined in granulosa and theca of large dominant and late preovulatory follicles and in large granulosa lutein cells isolated from corpora lutea cyclica and graviditatis. Levels of the steroid hormones progesterone and estradiol-17beta were monitored to assess the physiological status of individual follicles. From our results, we conclude that (1) individual, even closely neighboring, CpG dinucleotides can show very different methylation levels; (2) proximal (<300 base pair [bp] from the respective transcription start sites) but not distal CpGs show cell type-specific methylation levels; (3) higher methylation levels suggestively preclude high levels of gene expression; (4) DNA methylation is not involved in the transient (HSD3B1 and CYP11A1) respectively permanent (CYP19A1) down-regulation of gene expression in late preovulatory follicles; and (5) DNA methylation may have a role in the permanent shutdown of promoter 2-directed CYP19A1 expression in large (granulosa derived) lutein cells.

Evidence that high variation in antral follicle count during follicular waves is linked to alterations in ovarian androgen production in cattle.

Androgens have an important role in ovarian follicular growth and function, but circulating androgen concentrations are also associated with ovarian dysfunction, cardiovascular disease, and metabolic disorders in women. The extent and causes of the variation in androgen production in individuals, however, are unknown. Because thecal cells of follicles synthesize androstenedione and testosterone, variation in production of these androgens is hypothesized to be directly related to the inherently high variation in number of healthy growing follicles in ovaries of individuals. To test this hypothesis, we determined whether thecal CYP17A1 mRNA (codes for a cytochrome P450 enzyme involved in androgen synthesis), LH-induced thecal androstenedione production, androstenedione concentrations in follicular fluid, and circulating testosterone concentrations were lower in cattle with relatively low versus high number of follicles growing during follicular waves and whether ovariectomy reduced serum testosterone concentrations. Results demonstrated that cattle with a low follicle number had lower (P<0.05) abundance of CYP17A1 mRNA in thecal cells, reduced (P<0.01) capacity of thecal cells to produce androstenedione in response to LH, lower (P<0.01) androstenedione concentrations in ovulatory follicles, and lower (P<0.02) circulating testosterone concentrations during estrous cycles compared with animals with high follicle number. Also, serum testosterone in cattle with low or high follicle number was reduced by 63 and 70%, respectively, following ovariectomy. In conclusion, circulating androgen concentrations are lower in cattle with low versus high number of follicles growing during follicular waves, possibly because of a reduced responsiveness of thecal cells to LH.

Two distinct types of theca cells in the medaka gonad: germ cell-dependent maintenance of cyp19a1-expressing theca cells.

Aromatase is a steroidogenic enzyme catalyzing the production of estrogens and is important for the proper development and function of the reproductive system. The lineage of cyp19a1 (ovarian-type aromatase)-expressing cells in the developing gonad was analyzed using a transgenic medaka (Oryzias latipes) that recapitulates endogenous cyp19a1 expression with EGFP fluorescence. Our results show that cyp19a1-expressing cells arise in the ventral stromal cells of the developing female gonad, then expand anteriorly as the gonadal region extends anteriorly. These cells become located close to the developing follicles, and are distinguishable from the P450c17-I-expressing theca cells. In the adult ovary, the expression of P450c17-I and cyp19a1 are mutually exclusive in the outer theca-cell layer. Cyp19a1 expression in the granulosa cells is found only in the population of large follicles. These observations demonstrate two types of theca cells in the medaka ovary. We also show that the maintenance of cyp19a1-expressing cells depends on germ cells.

Localization of nuclear subunits of cyclic AMP-dependent protein kinase by the immunocolloidal gold method.

An immunocolloidal gold electron microscopy method is described allowing the ultrastructural localization and quantitation of the regulatory subunits RI and RII and the catalytic subunit C of cAMP-dependent protein kinase. Using a postembedding indirect immunogold labeling procedure that employs specific antisera, the catalytic and regulatory subunits were localized in electron-dense regions of the nucleus and in cytoplasmic areas with a minimum of nonspecific staining. Antigenic domains were localized in regions of the heterochromatin, nucleolus, interchromatin granules, and in the endoplasmic reticulum of different cell types, such as rat hepatocytes, ovarian granulosa cells, and spermatogonia, as well as cultured H4IIE hepatoma cells. Morphometric quantitation of the relative staining density of nuclear antigens indicated a marked modulation of the number of subunits per unit area under various physiologic conditions. For instance, following partial hepatectomy in rats, the staining density of the nuclear RI and C subunits was markedly increased 16 h after surgery. Glucagon treatment of rats increased the staining density of only the nuclear catalytic subunit. Dibutyryl cAMP treatment of H4IIE hepatoma cells led to a marked increase in the nuclear staining density of all three subunits of cAMP-dependent protein kinase. These studies demonstrate that specific antisera against cAMP-dependent protein kinase subunits may be used in combination with immunogold electron microscopy to identify the ultrastructural location of the subunits and to provide a semi-quantitative estimate of their relative cellular density.

Plasminogen activators in tissue remodeling and invasion: mRNA localization in mouse ovaries and implanting embryos.

To assess in vivo the postulated participation of urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators in processes involving tissue remodeling and cell migration, we have studied the cellular distribution of u-PA and t-PA mRNAs during mouse oogenesis and embryo implantation. By in situ hybridizations, we detected t-PA mRNA in oocytes and u-PA mRNA in granulosa and thecal cells from preovulatory follicles. These findings are compatible with a role for plasminogen activators in oogenesis and follicular disruption. We demonstrated the presence of u-PA mRNA in the invasive and migrating trophoblast cells of 5.5- and 6.5-d-old embryos. At 7.5 days, u-PA mRNA was predominantly localized to trophoblast cells that had reached the deep layers of the uterine wall, while the peripheral trophoblast cells surrounding the presomite stage embryo were devoid of specific signal. In 8.5-d-old embryos abundant u-PA mRNA expression resumed transiently in the giant trophoblast cells at the periphery of the embryo and in the trophoblast cells of the ectoplacental cone, to become undetectable in 10.5-d-old embryos. These observations establish the in vivo expression of the u-PA gene by invading and migrating trophoblast cells in a biphasic time pattern; they are in agreement with the proposed involvement of the enzyme in the extracellular proteolysis accompanying embryo implantation.

Differential expression of signal transduction factors in ovarian follicle development: a functional role for betaglycan and FIBP in granulosa cells in cattle.

Ovarian follicles develop in groups yet individual follicles follow different growth trajectories. This growth and development are regulated by endocrine and locally produced growth factors that use a myriad of receptors and signal transduction pathways to exert their effects on theca and granulosa cells. We hypothesize that differential growth may be due to differences in hormonal responsiveness that is partially mediated by differences in expression of genes involved in signal transduction. We used the bovine dominant follicle model, microarrays, quantitative real-time PCR and RNA interference to examine this. We identified 83 genes coding for signal transduction molecules and validated a subset of them associated with different stages of the follicle wave. We suggest important roles for CAM kinase-1 and EphA4 in theca cells and BCAR1 in granulosa cells for the development of dominant follicles and for betaglycan and FIBP in granulosa cells of regressing subordinate follicles. Inhibition of genes for betaglycan and FIBP in granulosa cells in vitro suggests that they inhibit estradiol production in regressing subordinate follicles.

Role of adiponectin in regulating ovarian theca and granulosa cell function.

Adiponectin is an adipokine that has been implicated in insulin resistance, a condition associated with polycystic ovarian syndrome in humans, but whether adiponectin can directly affect ovarian theca or granulosa cell function is unknown. Therefore, to determine the effects of adiponectin on proliferation, steroidogenesis and gene expression of large-follicle theca and granulosa cells, experiments were conducted using bovine ovarian cell cultures. RT-PCR was used to elucidate the effects of adiponectin on gene expression of CYP11A1 and LH receptor (LHR) in large-follicle theca and granulosa cells, as well as expression of CYP17A1 in theca cells and CYP19A1 in granulosa cells. Adiponectin decreased (P<0.05) insulin-induced progesterone and androstenedione production as well as attenuated IGF-I-induced LHR, CYP11A1, and CYP17A1 gene expression in theca cells. In contrast, adiponectin decreased (P<0.05) LHR mRNA abundance in granulosa cells but did not affect steroidogenic enzyme gene expression in granulosa cells. Adiponectin had no effect (P>0.10) on proliferation of large-follicle theca cells. RT-PCR also revealed that abundance of mRNA for the adiponectin receptor (ADIPOR2) was greater (P<0.05) in large-follicle than in small-follicle theca cells and did not significantly differ between small- and large-follicle granulosa cells. In cultured theca cells, LH increased (P<0.05) and IGF-I decreased (P<0.05) ADIPOR2 mRNA abundance. These results indicate that the inhibitory effects of adiponectin on steroidogenesis are primarily localized to theca cells and that the response of theca cells to adiponectin (i.e., ADIPOR2) may be regulated by LH and IGF-I.

Effect of bisphenol A on steroid hormone production in rat ovarian theca-interstitial and granulosa cells.

Many studies have shown that 2,2-bis 4-hydroxyphenyl propane (BPA), an estrogenic chemical, affects the reproductive health of wildlife and possibly of humans. In this study, we investigated the effects of BPA on steroid hormone production in rat ovarian theca-interstitial cells (T-I cells) and granulosa cells. In T-I cells, BPA increased testosterone synthesis and mRNA expression of 17-alpha hydroxylase (P450c17), cholesterol side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory protein (StAR) at concentrations of 10(-7) to 10(-4)M after a 72 h incubation period. Treatment of granulosa cells with BPA at concentrations of 10(-7) to 10(-5)M caused an increase in progesterone levels and P450scc mRNA expression, with an unexpected decrease at 10(-4)M. BPA (10(-7) to 10(-5)M) tended to elevate the expression of StAR mRNA with a significant increase at 10(-4)M concentration. A significant concentration-dependent inhibitory effect of BPA (10(-6) to 10(-4)M) on estradiol levels and the expression of P450arom mRNA was observed. These results suggest that BPA may interrupt ovarian steroidogenesis by altering the steroidogenic enzymes.

IGF-1 receptor signaling pathways and effects of AMPK activation on IGF-1-induced progesterone secretion in hen granulosa cells.

IGF-1 plays a key role in the proliferation and differentiation of granulosa cells. However, the molecular mechanism of IGF-1 action in avian granulosa cells during follicle maturation is unclear. Here, we first studied IGF-1 receptor (IGF-1R) expression, IGF-1-induced progesterone production and some IGF-1R signaling pathways in granulosa cells from different follicles. IGF-1R (mRNA and protein) was higher in fresh or cultured granulosa cells from the largest follicles (F1 or F2) than in those from smaller follicles (F3 or F4). In vitro, IGF-1 treatment (10(-8)M, 36h) increased progesterone secretion by four-fold in mixed F3 and F4 (F3/4) granulosa cells and by 1.5-fold in F1 granulosa cells. IGF-1 (10(-8)M, 30min)-induced increases in tyrosine phosphorylation of IGF-1R beta subunit and phosphorylation of ERK were higher in F1 than in F3/4 granulosa cells. Interestingly, IGF-1 stimulation (10(-8)M, 10min) decreased the level of AMPK Thr172 phosphorylation in F1 and F3/4 granulosa cells. We have recently showed that AMPK (AMP-activated protein kinase) is a protein kinase involved in the steroidogenesis in chicken granulosa cells. We then studied the effects of AMPK activation by AICAR (5-aminoimidazole-4-carboxamide ribonucleoside), an activator of AMPK, on IGF-1-induced progesterone secretion by F3/4 and F1 granulosa cells. AICAR treatment (1mM, 36h) increased IGF-1-induced progesterone secretion, StAR protein levels and decreased ERK phosphorylation in F1 granulosa cells. Opposite data were observed in F3/4 granulosa cells. Adenovirus-mediated expression of dominant negative AMPK totally reversed the effects of AICAR on IGF-1-induced progesterone secretion, StAR protein production and ERK phosphorylation in both F3/4 and F1 granulosa cells. Thus, a variation of energy metabolism through AMPK activation could modulate differently IGF-1-induced progesterone production in F1 and F3/4 granulosa cells.

Follicle-stimulating hormone (FSH) stimulates the expression of Pin1, a peptidyl-prolyl isomerase, in the bovine granulosa cells.

A peptidyl-prolyl isomerase, Pin 1, has been shown to play a role in the regulation of cell cycle progression, both in vitro and in vivo. However, the involvement of Pin 1 during follicular development is not well understood. The aim of this study was first to investigate the expression of Pin 1 mRNA in the granulosa and theca cells of the follicle at different developmental stages of follicles in the bovine ovary, and second, to examine the effects of follicle-stimulating hormone (FSH) and estradiol (E2) on the expression of Pin 1 in the cultured bovine granulosa cells. Follicles were classified into four groups based on the diameter (dominant follicles >8.5mm in diameter, subordinate follicles <8.5mm in diameter) and the relative levels of E2 and progesterone (P4) (E2:P4>1, estrogen active; E2:P4<1, estrogen inactive): i.e. preovulatory dominant follicles (POFs); E2 active dominant follicles (EADs); E2 inactive dominant follicles (EIDs); small follicles (SFs). The expression of the Pin 1 gene was significantly increased in the granulosa cells of EADs as compared with those of other follicles, whereas its expression in theca cells did not differ among follicles at different developmental stages. The concentration of 5 ng/ml FSH alone and the combination of 1 ng/ml E2 and 5 ng/ml FSH stimulated the expression of the Pin 1 gene in bovine granulosa cells. Our data provide the first evidence that Pin 1 expression in the granulosa cells but not the theca cells changes during follicular development, and that FSH stimulate the expression of the Pin 1 gene. These results suggest that Pin 1 regulates the timing of cell proliferation and may act as an intracellular signal responder in the granulosa cells during bovine follicle development.

Gossypol inhibits LH-induced steroidogenesis in bovine theca cells.

Gossypol, a polyphenolic aldehyde found in cottonseed, has been shown to perturb steroidogenesis in granulosa and luteal cells of rats, pigs and cattle. However, little is known about the direct effect of gossypol on theca cell functions in any species. The present study was conducted to investigate the effect of gossypol on the steroidogenesis and the expression of genes involved in it in cultured bovine theca cells. Theca cells were isolated from healthy preovulatory follicles and were cultured in the presence of luteinizing hormone (LH) for up to 7 days. During the culture period, main steroid products of the theca cells shifted from androstenedione (A4) at day 1 to progesterone (P4) from day 2 onward. At days 1 and 7, theca cells were treated with gossypol (0-25 µg/mL) for 24 h. Gossypol inhibited LH-stimulated theca cell A4 and P4 production in a dose-dependent manner at both occasions. The viability of theca cells was not affected by gossypol at any doses used. Gossypol down-regulated expressions of steroidogenic enzymes CYP11A1, HSD3B1 and CYP17A1, but not that of LHR. These results indicate that gossypol inhibits thecal steroidogenesis through down-regulating gene expressions of steroidogenic enzymes but without affecting cell viability in cattle.

Human fetal ovary development involves the spatiotemporal expression of p450c17 protein.

OBJECTIVE: The purpose of this research was to characterize the spatiotemporal expression of P450c17 in the human fetal ovary. DESIGN: P450c17 protein was visualized in sections of control and anencephalic ovaries using immunohistochemistry. SUBJECTS: Subjects included control (nonanencephalic) and anencephalic human fetal ovaries during the second and third trimesters. RESULTS: In second-trimester control ovaries, P450c17 was highly expressed in primary interstitial cells (PIC) located between the ovigerous cords near the cortical-medullary border where meiosis and primordial follicle formation were occurring. Morphometric analysis revealed a progressive decrease in the number of PIC during the second trimester, suggesting that PIC might have a finite lifetime. Between 25 and 32 wk, relatively few cells stained positive for P450c17; however, after 33 wk, P450c17 was strongly expressed in theca interstitial cells (TIC) bordering developing follicles. Surprisingly, the TIC appeared remarkably early during folliculogenesis, e.g. as early as the primary-to-secondary transition, and exhibited notable hyperplasia throughout preantral and early antral follicle growth. Owing to large numbers of developing preantral follicles, the third trimester was characterized by an increased abundance of P450c17-positive TIC. During this time period, P450c17 was strongly expressed in the hilus interstitial cells juxtaposed to the rete ovarii. Studies of ovaries of anencephalic fetuses revealed a similar spatiotemporal pattern of P450c17 expression in the PIC, TIC, and hilus interstitial cells, consistent with the possibility that pituitary hormones may not be involved in P450c17 expression in fetal ovaries. CONCLUSION: We identified three different classes of P450c17-expressing interstitial cells in the human fetal ovary, each having a different spatiotemporal pattern of P450c17 expression and, presumably, a different set of physiological functions.

Induction of cytochromes P450, caspase-3 and DNA damage by PCB3 and its hydroxylated metabolites in porcine ovary.

Polychlorinated biphenyl (PCBs) levels of tens and hundreds of pg/ml for individual congeners are measured in human follicular fluid. PCB3 (4-chlorobiphenyl), caused a significant increase in estradiol secretion in porcine granulose-theca cell co-cultures and its two metabolites, 4-OH-PCB3 and 3,4-diOH-PCB3, were even more potent than PCB3 itself [Ptak, A., Ludewig, G., Lehmler, H.J., Wojtowicz, A.K., Robertson, L.W., Gregoraszczuk, E.L. 2005. Comparison of the actions of 4-chlorobiphenyl and its hydroxylated metabolites on estradiol secretion by ovarian follicles in primary cells in culture. Reprod. Toxicol. 20, 57-64]. The question is whether these follicle cells are potentially able to metabolize PCB3 to hydroxylated and genotoxic or cytotoxic intermediates. We report here that granulose-theca co-cultures express xenobiotic-metabolizing cytochrome P450 activities, with CYP1A1>CYP2B>>CYP1A2. A significant increase in CYP1A1 and 2B, but not CYP1A2, activity was seen in cells that were exposed to 6 ng/ml PCB3 or 20 nM 17-beta-estradiol. An increase in caspase-3 activity, indicative for apoptosis, was only observed in PCB3-exposed cells after 24 h exposure. Genotoxicity, determined with the Comet assay, was initially reduced after 24 h exposure to PCB3 and both metabolites compared to untreated controls, followed by a significant transient increase in Comets at the 4 and 24 h time point with PCB3 and 4-OH-PCB3. 3,4-diOH-PCB3 induced a significant increase only after 72 h of recovery. We hypothesize that these biphasic damage kinetics may be due to cross-links caused by adduct formation. These results show for the first time that granulose-theca cells in co-culture express CYP1A1, 2B and 1A2 activities and that PCBs at concentrations that are reached in the environment induce genotoxicity in granulosa cells.

Patterns of expression of steroidogenic enzymes during the first wave of the ovine estrous cycle as compared to the preovulatory follicle.

The expression patterns of steroidogenic enzymes in ovarian antral follicles at various stages of growth in a follicular wave have not been reported for sheep. Ovaries were collected from ewes (n=4-5 per group) when the largest follicle(s) of the first wave of the cycle, as determined by ultrasonography, reached (i) 3 mm, (ii) 4 mm, (iii) > or =5 mm in diameter or when there was a single (iv) preovulatory follicle in the last wave of the cycle, 12h after estrus detection. The expression pattern of steroidogenic enzymes was quantified using immunohistochemistry and grey-scale densitometry. The expression of CYP19 in the granulosa and 3beta-HSD and CYP17 in the theca increased (P<0.01) progressively from 3 to > or =5 mm follicles in the first wave of the cycle and was lower (P<0.01) in the preovulatory follicle compared to > or =5 mm follicles. However, the expression of 3beta-HSD in the granulosa increased (P<0.05) from 3 to > or =5 mm follicles and was maintained (P<0.05) at a high level in the preovulatory follicles. The amount of CYP19 in the granulosa of the growing follicles correlated positively (r=0.5; P<0.03) with the concurrent serum estradiol concentrations. We concluded that the expression pattern of steroidogenic enzymes in theca and granulosa of follicles growing in each wave in the ewe, paralleled with serum estradiol concentrations, with the exception that concentrations of 3beta-HSD in granulosa increased continuously from follicles 3mm in diameter to the preovulatory follicle.

The role of protein kinase A and cyclin-dependent (CDC2) kinase in the control of basal and IGF-II-induced proliferation and secretory activity of chicken ovarian cells.

The aim of these experiments was to study the role of protein kinase A (PKA), cyclin-dependent kinase 2 (CDC2) and insulin-like growth factor II (IGF-II) in the control of ovarian function in domestic fowl, as well as the role of PKA and CDC2 in mediating the effects of IGF-II on the ovary. For this purpose, we studied the influence of an inhibitor of PKA (KT5720; 50 ng/ml), a CDC2 blocker (olomoucine; 1 microg/ml), IGF-II (0, 1, 10 or 100 ng/ml) and their combinations on cultured fragments of chicken ovarian follicular wall. Accumulation of PKA and CDC2 and secretion of progesterone (P4), testosterone (T), estradiol (E2) and arginine-vasotocin (AVT) were evaluated by using SDS-PAGE-Western blotting and RIA/EIA. IGF-II addition to culture medium stimulated T, E2 and AVT secretion and inhibited P4 secretion. These changes were associated with an increase in PKA and a decrease in CDC2 accumulation. The PKA blocker KT5720, when given alone, increased accumulation of PKA and secretion of T and E2, but not AVT and inhibited P4 secretion. The PKA blocker also prevented and even reversed the effects of IGF-II on PKA and steroid hormones secretion, but enhanced the action of IGF-II on AVT. The inhibitor of CDC2, olomoucine, when given alone, suppressed the expression of CDC2 and the secretion of P4 and AVT (but not T and E2). When given together with IGF-II, it augmented IGF-II-induced suppression of CDC2 and reversed the effects of IGF-II on P4 (but not on T, E2 or AVT). These observations demonstrate the involvement of PKA, CDC2 and IGF-II in regulating the secretory activity of avian ovarian cells. Our data also suggest the involvement of PKA in the mediation of IGF-II effects on P4, T, E2 and AVT secretion. CDC2 can mediate the effects of IGF-II on ovarian P4 secretion but not on other hormones.

Resistin stimulation of 17alpha-hydroxylase activity in ovarian theca cells in vitro: relevance to polycystic ovary syndrome.

CONTEXT: A newly discovered hormone resistin has been shown to be increased in women with polycystic ovary syndrome (PCOS). OBJECTIVE: The purpose of this study was to confirm increased resistin concentrations in women with PCOS and to test the direct effect of resistin on human theca cell androgen production. DESIGN: Resistin was measured in fasting serum samples by RIA. To test the direct effects of resistin on ovarian androgen biosynthesis, human theca cells were cultured with resistin for 3 d in the presence and absence of forskolin and insulin. PATIENTS: Fasting serum samples were obtained from 45 women with PCOS and 74 regularly cycling premenopausal control women in the follicular phase of their menstrual cycles, and ovarian theca cell cultures were established from two control women. RESULTS: The mean serum resistin concentration was increased (40%) in women with PCOS. Serum resistin concentrations correlated positively with body mass index and testosterone in PCOS women but not in controls. There were no significant correlations between resistin and fasting insulin or indicators of insulin resistance when corrected for body mass index. In cultured human theca cells, basal 17alpha-hydroxylase activity was unchanged by resistin alone, but resistin enhanced 17alpha-hydroxylase activity in the presence of forskolin or a combination of forskolin plus insulin. Resistin (> or =1 ng/ml) augmented forskolin and forskolin plus insulin stimulation of CYP17 mRNA expression in a concentration-dependent manner. CONCLUSION: These data indicate that abnormal resistin secretion in PCOS may play a role in causing ovarian hyperandrogenism.