Development of an acute ovine model of polycystic ovaries to assess the effect of ovarian denervation.

Introduction: Polycystic ovary syndrome (PCOS) seems to be associated with increased ovarian sympathetic nerve activity and in rodent models of PCOS reducing the sympathetic drive to the ovary, through denervation or neuromodulation, improves ovulation rate. We hypothesised that sympathetic nerves work with gonadotropins to promote development and survival of small antral follicles to develop a polycystic ovary phenotype. Methods: Using a clinically realistic ovine model we showed a rich sympathetic innervation to the normal ovary and reinnervation after ovarian transplantation. Using needlepoint diathermy to the nerve plexus in the ovarian vascular pedicle we were able to denervate the ovary resulting in reduced intraovarian noradrenaline and tyrosine hydroxylase immunostained sympathetic nerves. We developed an acute polycystic ovary (PCO) model using gonadotrophin releasing hormone (GnRH) agonist followed infusion of follicle stimulating hormone (FSH) with increased pulsatile luteinising hormone (LH). This resulted in increased numbers of smaller antral follicles in the ovary when compared to FSH infusion suggesting a polycystic ovary. Results: Denervation had no effect of the survival or numbers of follicles in the acute PCO model and did not impact on ovulation, follicular and luteal hormone profiles in a normal cycle. Discussion: Although the ovary is richly inervated we did not find evidence for a role of sympathetic nerves in ovarian function or small follicle growth and survival.

Insights into Manipulating Postprandial Energy Expenditure to Manage Weight Gain in Polycystic Ovary Syndrome.

Women with polycystic ovary syndrome (PCOS) are more likely to be obese and have difficulty in losing weight. They demonstrate an obesity-independent deficit in adaptive energy expenditure. We used a clinically realistic preclinical model to investigate the molecular basis for the reduced postprandial thermogenesis (PPT) and develop a therapeutic strategy to normalize this deficit. Sheep exposed to increased androgens before birth develop the clinical features of PCOS. In adulthood they develop obesity and demonstrate an obesity-independent reduction in PPT. This is associated with reduced adipose tissue uncoupling protein expression and adipose tissue noradrenaline concentrations. These sheep are insulin resistant with reduced insulin signaling in the brain. Increasing brain insulin concentrations using intranasal insulin administration increased PPT in PCOS sheep without any effects on blood glucose concentrations. Intranasal insulin administration with food is a potential novel strategy to improve adaptive energy expenditure and normalize the responses to weight loss strategies in women with PCOS.

Ovine trophoblasts express cathelicidin host defence peptide in response to infection.

Cationic host defence peptides (CHDP; also known as antimicrobial peptides) are key components of the immune response in the female reproductive tract. The role of the placental trophoblast in ovine host defence remains poorly understood. This study characterises expression of genes for cathelicidin and defensin peptides in primary ovine placental tissues, the ovine trophoblast cell line (AH-1) and in response to the TLR-4 ligand LPS, the abortifacient organism Waddlia chondrophila and 1α,25-dihydroxyvitamin D3. Using RT-PCR, expression of the CHDP SMAP-29, sBD-1 and sBD-2 was assessed in the AH-1 cell line in response to LPS, 1α,25-dihydroxyvitamin D3 exposure (a known stimulator of cathelicidin gene expression), or W. chondrophila infection. Expression of cathelicidin in the trophoblast compartment of the ovine placenta and in the ovine trophoblast cell line (AH-1) was also established. AH-1 cells did not upregulate expression of CHDP in response to LPS, but sBD-1 and sBD-2 expression was significantly increased in response to W. chondrophila infection. SMAP-29 expression was not altered by in vitro exposure to 1α,25-dihydroxyvitamin D3. This study demonstrates that the ovine trophoblast expresses cathelicidins, but does not upregulate expression of CHDP in response to LPS. Ovine trophoblasts are shown to differentially regulate expression of CHDP and lack a demonstrable vitamin D-mediated cathelicidin response.

In an Ovine Model of Polycystic Ovary Syndrome (PCOS) Prenatal Androgens Suppress Female Fetal Renal Gluconeogenesis.

Increased maternal androgen exposure during pregnancy programmes a polycystic ovary syndrome (PCOS)-like condition, with metabolic dysfunction, in adult female offspring. Other in utero exposures associated with the development of insulin resistance, such as intrauterine growth restriction and exposure to prenatal glucocorticoids, are associated with altered fetal gluconeogenesis. We therefore aimed to assess the effect of maternal androgenisation on the expression of PEPCK and G6PC in the ovine fetus. Pregnant Scottish Greyface sheep were treated with twice weekly testosterone propionate (TP; 100mg) or vehicle control from day 62 to day 102 of gestation. At day 90 and day 112 fetal plasma and liver and kidney tissue was collected for analysis. PEPCK and G6PC expression were analysed by quantitative RT-PCR, immunohistochemistry and western blotting. PEPCK and G6PC were localised to fetal hepatocytes but maternal androgens had no effect on female or male fetuses. PEPCK and G6PC were also localised to the renal tubules and renal PEPCK (P<0.01) and G6PC (P = 0.057) were lower in females after prenatal androgenisation with no change in male fetuses. These tissue and sex specific observations could not be explained by alterations in fetal insulin or cortisol. The sexual dimorphism may be related to the increase in circulating estrogen (P<0.01) and testosterone (P<0.001) in females but not males. The tissue specific effects may be related to the increased expression of ESR1 (P<0.01) and AR (P<0.05) in the kidney when compared to the fetal liver. After discontinuation of maternal androgenisation female fetal kidney PEPCK expression normalised. These data further highlight the fetal and sexual dimorphic effects of maternal androgenisation, an antecedent to adult disease and the plasticity of fetal development.

The local effects of ovarian diathermy in an ovine model of polycystic ovary syndrome.

In order to develop a medical alternative to surgical ovarian diathermy (OD) in polycystic ovary syndrome (PCOS) more mechanistic information is required about OD. We therefore studied the cellular, molecular and vascular effects of diathermy on the ovary using an established ovine model of PCOS. Pregnant sheep were treated twice weekly with testosterone propionate (100 mg) from day 30-100 gestation. Their female offspring (n = 12) were studied during their second breeding season when the PCOS-like phenotype, with anovulation, is fully manifest. In one group (n = 4) one ovary underwent diathermy and it was collected and compared to the contralateral ovary after 24 hours. In another group a treatment PCOS cohort underwent diathermy (n = 4) and the ovaries were collected and compared to the control PCOS cohort (n = 4) after 5 weeks. Ovarian vascular indices were measured using contrast-enhanced ultrasound and colour Doppler before, immediately after, 24 hours and five weeks after diathermy. Antral follicles were assessed by immunohistochemistry and ovarian stromal gene expression by quantitative RT-PCR 24 hours and 5 weeks after diathermy. Diathermy increased follicular atresia (P<0.05) and reduced antral follicle numbers after 5 weeks (P<0.05). There was an increase in stromal CCL2 expression 24 hours after diathermy (P<0.01) but no alteration in inflammatory indices at 5 weeks. Immediately after diathermy there was increased microbubble transit time in the ovarian microvasculature (P = 0.05) but this was not seen at 24 hours. However 24 hours after diathermy there was a reduction in the stromal Doppler blood flow signal (P<0.05) and an increased ovarian resistance index (P<0.05) both of which persisted at 5 weeks (P<0.01; P<0.05). In the ovine model of PCOS, OD causes a sustained reduction in ovarian stromal blood flow with an increased ovarian artery resistance index associated with atresia of antral follicles.

Excess androgens in utero alters fetal testis development.

Prenatal androgenization induces a polycystic ovary syndrome-like phenotype in adult female offspring, which is associated with alterations that can be detected in the fetal ovary, suggesting gestational origins of this condition. We therefore investigated whether increased prenatal androgen exposure also altered testicular development using ovine animal models. Biweekly maternal testosterone propionate (TP; 100 mg) from day 62 to day 70/day 90 of gestation altered male developmental trajectory. In male fetuses serum LH was decreased (P < .01), and testicular STAR, CYP11, and CYP17 abundance were reduced. Coincident with this, basal testicular T synthesis was decreased in vitro (P < .001). Leydig cell distribution was severely perturbed in all testes prenatally exposed to TP (P < .001). To examine the contribution of estrogens, fetuses were injected with TP (20 mg), the potent estrogen agonist, diethylstilbestrol (DES; 20 mg), or vehicle control at day 62 and day 82 and assessed at day 90. The effects of fetal (direct) TP treatment, but not DES, paralleled maternal (indirect) TP exposure, supporting a direct androgen effect. Cessation of maternal androgenization at day 102 returned Leydig cell distribution to normal but increased basal T output, at day 112, demonstrating Leydig cell developmental plasticity. Earlier maternal androgen exposure from day 30 similarly influenced Leydig cell development at day 90 but additionally affected the expression of Sertoli and germ cell markers. We show in this study that increased prenatal androgen exposure alters development and function of Leydig cells at a time when androgen production is paramount for male development. This supports the concept that gestational antecedents associated with polycystic ovary syndrome may have effects on the male fetus.

Maternal undernutrition does not alter Sertoli cell numbers or the expression of key developmental markers in the mid-gestation ovine fetal testis.

BACKGROUND: The aim of this study was to determine the effects of maternal undernutrition on ovine fetal testis morphology and expression of relevant histological indicators. Maternal undernutrition, in sheep, has been reported, previously, to alter fetal ovary development, as indicated by delayed folliculogenesis and the altered expression of ovarian apoptosis-regulating gene products, at day 110 of gestation. It is not known whether or not maternal undernutrition alters the same gene products in the day 110 fetal testis. DESIGN AND METHODS: Mature Scottish Blackface ewes were fed either 100% (Control; C) or 50% (low; L) of estimated metabolisable energy requirements of a pregnant ewe, from mating to day 110 of gestation. All pregnant ewes were euthanized at day 110 and a sub-set of male fetuses was randomly selected (6 C and 9 L) for histology studies designed to address the effect of nutritional state on several indices of testis development. Sertoli cell numbers were measured using a stereological method and Ki67 (cell proliferation index), Bax (pro-apoptosis), Mcl-1 (anti-apoptosis), SCF and c-kit ligand (development and apoptosis) gene expression was measured in Bouins-fixed fetal testis using immunohistochemistry. RESULTS: No significant differences were observed in numbers of Sertoli cells or testicular Ki67 positive cells. The latter were localised to the testicular cords and interstitium. Bax and Mcl-1 were localised specifically to the germ cells whereas c-kit was localised to both the cords and interstitium. SCF staining was very sparse. No treatment effects were observed for any of the markers examined. CONCLUSIONS: These data suggest that, unlike in the fetal ovary, maternal undernutrition for the first 110 days of gestation affects neither the morphology of the fetal testis nor the expression of gene products which regulate apoptosis. It is postulated that the effects of fetal undernutrition on testis function may be expressed through hypothalamic-pituitary changes.

Glucocorticoid receptor-mediated regulation of MMP9 gene expression in human ovarian surface epithelial cells.

OBJECTIVE: To obtain proof-of-concept that locally produced anti-inflammatory steroids suppress ovulation-associated extracellular matrix proteases in human ovarian surface epithelial (OSE) cells. DESIGN: Primary OSE cell cultures treated with interleukin-1alpha (IL-1alpha) (500 pg/mL) as proxy for inflammation, with/without anti-inflammatory steroid (cortisol or progesterone [P], 0.01-1.0 microM). SETTING: Academic medical center. PATIENT(S): Sixteen premenopausal women (29-46 years) undergoing surgery for nonmalignant gynecological conditions. MAIN OUTCOME MEASURE(S): Semiquantitative extracellular matrix protease gene expression profiling with verification by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and gelatinase zymography. RESULT(S): Treatment with IL-1alpha stimulated messenger RNA (mRNA) expression of several ovulation-associated matrix metalloproteinase genes by OSE cell cultures, including gelatinase B (MMP9) but not gelatinase A (MMP2). The IL-1alpha-stimulated MMP9 mRNA production was suppressed by cortisol but not P. Cortisol but not P also dose-dependently suppressed IL-1alpha-stimulated MMP9 gelatinase activity and this effect was blocked by the glucocorticoid receptor antagonist RU486. CONCLUSION(S): In human OSE cells, stimulation of MMP9 gene expression and proteolytic activity by IL-1alpha is suppressed by anti-inflammatory cortisol through a glucocorticoid receptor-mediated mechanism. Because IL-1alpha also generates cortisol formation in OSE by stimulating cortisone reductase activity, these results support a role for intracrine cortisol in minimizing proteolytic damage to the OSE at ovulation.

Oestrogen formation and connective tissue growth factor expression in rat granulosa cells.

Ovarian follicular development involves continual remodelling of the extracellular matrix (ECM) forming the basement membrane and intercellular framework that support granulosa cell (GC) growth and differentiation. Insight into the molecular regulation of ovarian ECM remodelling is potentially translatable to tissue remodelling elsewhere in the body. We therefore studied the link between a gene marker of ECM remodelling (connective tissue growth factor (CTGF)) and oestrogen biosynthesis (cytochrome P450(aromatase) (P450(arom))) in rat granulosa cells. To determine if a cause-effect interaction exists, we used semi-quantitative in situ hybridisation to analyse patterns of CTGF and P450(arom) mRNA expression and immunohistochemistry to detect CTGF protein localisation throughout follicular development, and tested the actions of CTGF on oestrogen biosynthesis and oestradiol on CTGF mRNA expression in isolated GC in vitro. CTGF mRNA levels in GC rose gradually through small preantral (SP) and small antral (SA) stages of development to a maximum (fivefold higher) in large antral (LA) follicles. In preovulatory (PO) follicles, the CTGF mRNA level fell to 30% of that in SP follicles. P450(arom) mRNA also increased (threefold in LA relative to SP) throughout antral development follicles, but in contrast to CTGF continued to increase (12-fold) in PO follicles. In the cumulus oophorus of PO follicles, the residual GC CTGF mRNA expression increased with proximity to the oocyte, being inversely related to P450(arom). Elsewhere in the follicle wall, there was a mural-to-antral gradient of CTGF mRNA expression, again inversely related to P450(arom). Immunohistochemistry showed CTGF protein localisation broadly followed mRNA expression during follicular development, although the protein was also present in the theca interna and ovarian surface epithelium. Gradients in CTGF expression across the cumulus oophorus and follicle wall were similar to those observed for mRNA with CTGF protein expression being greatest in proximity to the oocyte. Treatment of isolated GC from preantral and SA follicles with recombinant human CTGF (1-100 ng/ml) did not affect basal or FSH-stimulated GC aromatase activity. However, in the absence of FSH, oestradiol (10(-7)-10(-5) M) stimulated CTGF mRNA expression up to twofold. Conversely, FSH (10 ng/ml) alone reduced CTGF mRNA expression by 40% and combined treatment with FSH and oestradiol further suppressed CTGF to 10% of the control value. The oestrogen receptor (ER) antagonist, ICI 182 780 blocked the stimulatory and inhibitory effects of oestradiol, suggesting a specific ER-mediated mode of action on CTGF. Therefore, CTGF gene expression in GC is under local control by oestrogen whose effect (positive or negative) is modulated by FSH. This helps explain why gene expression of CTGF and P450(arom) diverge in FSH-induced PO follicles and has implications for oestrogenic regulation of CTGF formation elsewhere in the body.

Steroid signalling in the ovarian surface epithelium.

Human ovarian surface epithelium (HOSE) undergoes serial injury-repair with each ovulation, which is probably why most ovarian epithelial cancers arise there. Considering the proposed inflammatory aetiology of ovarian cancer, anti-inflammatory steroid signalling might be vital for HOSE regulation. HOSE cells express hydroxysteroid dehydrogenase (HSD) enzymes that undertake prereceptor metabolism of bioinert steroidogenic precursors formed elsewhere in the body. Ovulation-associated cytokines activate anti-inflammatory cortisol from precursor cortisone in HOSE cells owing to up-regulation of the gene encoding 11betaHSD type 1 (HSD11B1) in vitro. Cortisol further enhances its own formation and action through augmentation of cytokine-induced HSD11B1 and glucocorticoid receptor gene expression. Understanding this feed-forward signalling process has implications for the improved diagnosis and treatment of inflammation-associated reproductive disease states such as ovarian cancer.

Antiinflammatory steroid action in human ovarian surface epithelial cells.

The human ovarian surface epithelium (OSE) is subject to serial injury and repair during ovulation, which is a natural inflammatory event. We asked whether there is a compensatory antiinflammatory component to this process, involving steroid hormones produced locally at the time of ovulation. Quantitative RT-PCR analysis of total RNA from cultured human OSE cell monolayers showed that exposure to proinflammatory IL1alpha (500 pg/ml) increased mRNA levels of cyclooxygenase-2 (COX-2) (P < 0.01) at 48 h. The COX-2 mRNA response to IL1alpha was associated with an approximate 18-fold (P < 0.01) increase in mRNA levels of 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1), encoding the steroid dehydrogenase that reversibly reduces cortisone to antiinflammatory cortisol. Addition of cortisol to OSE cell culture medium dose-dependently suppressed the COX-2 mRNA response to IL1alpha (P < 0.01) but reciprocally enhanced the 11betaHSD1 mRNA response (P < 0.05), with both effects strongest at 1 microm cortisol. Presence of glucocorticoid receptor-alpha mRNA and protein was established in OSE cell monolayers and treatment with IL1alpha shown to significantly up-regulate the glucocorticoid receptor-alpha mRNA level (P < 0.05). Glucocorticoid receptor antagonist (RU486, 10 microm) fully reversed the inhibitory effect of 1 microm cortisol on IL1alpha-stimulated COX-2 mRNA expression. Progesterone also suppressed IL1alpha-induced COX-2 mRNA expression but had no significant effect on IL1alpha-stimulated 11betaHSD1 expression. These data provide direct evidence for antiinflammatory actions of cortisol and progesterone in human OSE cells.

Glucocorticoid metabolism and reproduction: a tale of two enzymes.

Within potential target cells, the actions of physiological glucocorticoids (cortisol and corticosterone) are modulated by isoforms of the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD). To date, two isoforms of 11 beta HSD have been cloned: 11 beta HSD1 acts predominantly as an NADP(H)-dependent reductase to generate active cortisol or corticosterone, and 11 beta HSD2 is a high affinity NAD(+)-dependent enzyme that catalyses the enzymatic inactivation of glucocorticoids. Whereas the regeneration of active glucocorticoids by 11 beta HSD1 has been implicated in the cellular mechanisms of pituitary function, ovulation and parturition, the enzymatic inactivation of cortisol and corticosterone by 11 beta HSD enzymes appears to be central to the protection of gonadal steroidogenesis, prevention of intra-uterine growth retardation, and lactation. Recent evidence indicates that follicular fluid contains endogenous modulators of cortisol metabolism by 11 beta HSD1, the concentrations of which are associated with the clinical outcome of assisted conception cycles and are altered in cystic ovarian disease. In conclusion, the two cloned isoforms of 11 beta HSD fulfil diverse roles in a wide range of reproductive processes from conception to lactation.