Terpenes, hormones and life: isoprene rule revisited.

The year 2019 marks the 80th anniversary of the 1939 Nobel Prize in Chemistry awarded to Leopold Ruzicka (1887-1976) for work on higher terpene molecular structures, including the first chemical synthesis of male sex hormones. Arguably his crowning achievement was the 'biogenetic isoprene rule', which helped to unravel the complexities of terpenoid biosynthesis. The rule declares terpenoids to be enzymatically cyclized products of substrate alkene chains containing a characteristic number of linear, head-to-tail condensed, C5 isoprene units. The number of repeat isoprene units dictates the type of terpene produced (i.e., 2, monoterpene; 3, sesquiterpene; 4, diterpene, etc.). In the case of triterpenes, six C5 isoprene units combine into C30 squalene, which is cyclized into one of the signature carbon skeletons from which myriad downstream triterpenoid structures are derived, including sterols and steroids. Ruzicka also had a keen interest in the origin of life, but the pivotal role of terpenoids has generally been overshadowed by nucleobases, amino acids, and sugars. To redress the balance, we provide a historical and evolutionary perspective. We address the potential abiotic generation of isoprene, the crucial role that polyprene terpenoids played in early membranes and cellular life, and emphasize that endocrinology from microbes to plants and vertebrates is firmly grounded on Ruzicka's pivotal insights into the structure and function of terpenes. A harmonizing feature is that all known lifeforms (including bacteria) biosynthesize triterpenoid substances that are essential for cellular membrane formation and function, from which signaling molecules such as steroid hormones and cognate receptors are likely to have evolved.

Targeting lysyl oxidase reduces peritoneal fibrosis.

BACKGROUND: Abdominal surgery and disease cause persistent abdominal adhesions, pelvic pain, infertility and occasionally, bowel obstruction. Current treatments are ineffective and the aetiology is unclear, although excessive collagen deposition is a consistent feature. Lysyl oxidase (Lox) is a key enzyme required for crosslinking and deposition of insoluble collagen, so we investigated whether targeting Lox might be an approach to reduce abdominal adhesions. METHODS: Female C57Bl/6 mice were treated intraperitoneally with multiwalled carbon nanotubes (NT) to induce fibrosis, together with chemical (ß-aminoproprionitrile-BAPN) or miRNA Lox inhibitors, progesterone or dexamethasone. Fibrotic lesions on the diaphragm, and expression of fibrosis-related genes in abdominal wall peritoneal mesothelial cells (PMC) were measured. Effects of BAPN and dexamethasone on collagen fibre alignment were observed by TEM. Isolated PMC were cultured with interleukin-1 alpha (IL-1α) and progesterone to determine effects on Lox mRNA in vitro. RESULTS: NT-induced fibrosis and collagen deposition on the diaphragm was ameliorated by BAPN, Lox miRNA, or steroids. BAPN and dexamethasone disrupted collagen fibres. NT increased PMC Lox, Col1a1, Col3a1 and Bmp1 mRNA, which was inhibited by steroids. Progesterone significantly inhibited IL-1α induced Lox expression by PMC in vitro. CONCLUSION: Our results provide proof-of-concept that targeting peritoneal Lox could be an effective approach in ameliorating fibrosis and adhesion development.

On gonads and gadflies: the estrus angle.

The first sex steroid to be crystallized was the vertebrate ovarian hormone, estrone - a less potent metabolite of 17ß-estradiol, which in mammals stimulates the female urge to mate (estrus). The gadfly (Greek oistros) lent its name to the process of estrus, as an insect that bites and torments in classical Greek mythology. With the purification and crystallization of a moult-inducing steroid (ecdysone) from insects, an interesting parallel emerged between mating and moulting in lower mammals and arthropods. Ecdysterone (potent ecdysone metabolite) has anabolic effects in mammalian muscle cells that can be blocked by selective estrogen receptor antagonists. Insects utilize ecdysteroids in similar ways that vertebrates use estrogens, including stimulation of oocyte growth and maturation. Ecdysteroids also modify precopulatory insect mating behaviour, further reinforcing the gonad-gadfly/mate-moult analogy.

Local estrogen metabolism in epithelial ovarian cancer suggests novel targets for therapy.

Epithelial ovarian cancer (EOC) accounts for about 90% of malignant ovarian tumors, and estrogen is often implicated in disease progression. We therefore compared the potential for gating of estrogen action via pre-receptor metabolism in normal human ovarian surface epithelium (OSE), EOC and selected EOC cell lines (SKOV3 and PEO1). Steroid sulphatase (STS), estrogen sulfotransferase (EST), 17ß-hydroxysteroid dehydrogenases 2 (17BHSD2) and 5 (17BHSD5) mRNAs, proteins and enzymatic activities were all detectable in primary cell cultures of OSE and EOC, whereas aromatase and 17BHSD1 expression was negligible. qRT-PCR assay on total mRNA revealed significantly higher EST mRNA expression in OSE compared to EOC (P<0.05). Radioenzymatic measurements confirmed reduced sulfoconjugation (neutralization) of free estrogen in EOC relative to OSE. OSE cells were more effective at converting free [(3)H]-E1 to [(3)H]-E1S or [(3)H]-E2S, while EOC cell lines mainly converted [(3)H]-E1 to [(3)H]-E2 with minimal formation of [(3)H]-E1S or [(3)H]-E2S. IL1α treatment suppressed EST (P<0.01) and 17BHSD2 (P<0.001) mRNA levels in OSE and stimulated STS mRNA levels (P<0.001) in cancer (SKOV3) cells. These results show that estrogen is differentially metabolized in OSE and EOC cells, with E2 'activation' from conjugated estrogen predominating in EOC. Inflammatory cytokines may further augment the local production of E2 by stimulating STS and suppressing EST. We conclude that local estrogen metabolism may be a target for EOC treatment.

CYP3A variation, premenopausal estrone levels, and breast cancer risk.

BACKGROUND: Epidemiological studies have provided strong evidence for a role of endogenous sex steroids in the etiology of breast cancer. Our aim was to identify common variants in genes involved in sex steroid synthesis or metabolism that are associated with hormone levels and the risk of breast cancer in premenopausal women. METHODS: We measured urinary levels of estrone glucuronide (E1G) using a protocol specifically developed to account for cyclic variation in hormone levels during the menstrual cycle in 729 healthy premenopausal women. We genotyped 642 single-nucleotide polymorphisms (SNPs) in these women; a single SNP, rs10273424, was further tested for association with the risk of breast cancer using data from 10 551 breast cancer case patients and 17 535 control subjects. All statistical tests were two-sided. RESULTS: rs10273424, which maps approximately 50 kb centromeric to the cytochrome P450 3A (CYP3A) gene cluster at chromosome 7q22.1, was associated with a 21.8% reduction in E1G levels (95% confidence interval [CI] = 27.8% to 15.3% reduction; P = 2.7 × 10(-9)) and a modest reduction in the risk of breast cancer in case patients who were diagnosed at or before age 50 years (odds ratio [OR] = 0.91, 95% CI = 0.83 to 0.99; P = .03) but not in those diagnosed after age 50 years (OR = 1.01, 95% CI = 0.93 to 1.10; P = .82). CONCLUSIONS: Genetic variation in noncoding sequences flanking the CYP3A locus contributes to variance in premenopausal E1G levels and is associated with the risk of breast cancer in younger patients. This association may have wider implications given that the most predominantly expressed CYP3A gene, CYP3A4, is responsible for metabolism of endogenous and exogenous hormones and hormonal agents used in the treatment of breast cancer.

Glucocorticoid regulation of SLIT/ROBO tumour suppressor genes in the ovarian surface epithelium and ovarian cancer cells.

The three SLIT ligands and their four ROBO receptors have fundamental roles in mammalian development by promoting apoptosis and repulsing aberrant cell migration. SLITs and ROBOs have emerged as candidate tumour suppressor genes whose expression is inhibited in a variety of epithelial tumours. We demonstrated that their expression could be negatively regulated by cortisol in normal ovarian luteal cells. We hypothesised that after ovulation the locally produced cortisol would inhibit SLIT/ROBO expression in the ovarian surface epithelium (OSE) to facilitate its repair and that this regulatory pathway was still present, and could be manipulated, in ovarian epithelial cancer cells. Here we examined the expression and regulation of the SLIT/ROBO pathway in OSE, ovarian cancer epithelial cells and ovarian tumour cell lines. Basal SLIT2, SLIT3, ROBO1, ROBO2 and ROBO4 expression was lower in primary cultures of ovarian cancer epithelial cells when compared to normal OSE (P<0.05) and in poorly differentiated SKOV-3 cells compared to the more differentiated PEO-14 cells (P<0.05). Cortisol reduced the expression of certain SLITs and ROBOs in normal OSE and PEO-14 cells (P<0.05). Furthermore blocking SLIT/ROBO activity reduced apoptosis in both PEO-14 and SKOV-3 tumour cells (P<0.05). Interestingly SLIT/ROBO expression could be increased by reducing the expression of the glucocorticoid receptor using siRNA (P<0.05). Overall our findings indicate that in the post-ovulatory phase one role of cortisol may be to temporarily inhibit SLIT/ROBO expression to facilitate regeneration of the OSE. Therefore this pathway may be a target to develop strategies to manipulate the SLIT/ROBO system in ovarian cancer.

IL1α and IL4 signalling in human ovarian surface epithelial cells.

The human ovarian surface epithelium (hOSE) is a mesothelial layer that surrounds the ovary and undergoes injury and repair cycles after ovulation-associated inflammation. We previously showed that IL4 is a key regulator of progesterone bioavailability during post-ovulatory hOSE repair as it differentially up-regulated 3ß-HSD1 and 3ß-HSD2 mRNA transcripts and total 3ß-hydroxysteroid dehydrogenase activity whereas it inhibited androgen receptor (AR) expression. We now show that the pro-inflammatory effect of IL1α on 3ß-HSD1 expression is mediated by nuclear factor-κB (NF-κB), whereas its anti-inflammatory action on 3ß-HSD2 expression is exerted via p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K) and NF-κB signalling pathways. The anti-inflammatory IL4 effects on 3ß-HSD1 and 3ß-HSD2 mRNA expression are mediated through STAT6 and PI3K signalling networks. IL4 effects on AR and 3ß-HSD2 expression involve the p38 MAPK pathway. We also document that IL4 up-regulates lysyl oxidase (LOX) mRNA transcripts, a key gene for extracellular matrix (ECM) deposition and inhibits IL1α-induced expression of cyclooxygenase-2 (COX-2) mRNA, a gene involved in breakdown of ECM, showing a further role in post-ovulatory wound healing. We conclude that IL1α and IL4 actions in the post-ovulatory wound healing of hOSE cells are mediated by different signalling transduction pathways. The p38 MAPK signalling pathway may have possible therapeutic benefit in inflammation-associated disorders of the ovary, including cancer.

Thrombospondin-1 inhibits angiogenesis and promotes follicular atresia in a novel in vitro angiogenesis assay.

Thrombospondin-1 (TSP-1) is a putative antiangiogenic factor, but its role in regulating physiological angiogenesis is unclear. We have developed a novel in vitro angiogenesis assay to study the effect of TSP-1 on follicular angiogenesis and development. Intact preantral/early antral follicles dissected from 21-d-old rat ovaries were cultured for 6 d in the presence or absence of TSP-1. At the end of the culture period, angiogenic sprouting from the follicles was quantified using image analysis. Follicles were fixed and sectioned, and follicular apoptosis was assessed by immunohistochemistry for activated caspase-3 in granulosa cells. The results showed that TSP-1 inhibited follicular angiogenesis (P < 0.01) and promoted follicular apoptosis (P < 0.001) in a dose-dependent manner. To determine whether the proapoptotic activity of TSP-1 is mediated by direct effects on granulosa cells, isolated granulosa cells were cultured with TSP-1 (0, 10, 100, and 1000 ng/ml) for 48 h. Apoptosis was quantified using a luminescent caspase-3/7 assay. TSP-1 promoted apoptosis of granulosa cells in a dose-dependent manner (P < 0.05), suggesting that TSP-1 can act independently of the angiogenesis pathway to promote follicular apoptosis. These results show that TSP-1 can both inhibit follicular angiogenesis and directly induce apoptosis of granulosa cells. As such, it may have potential as a therapeutic for abnormal ovarian angiogenesis and could facilitate the destruction of abnormal follicles observed in polycystic ovary syndrome.

Premenopausal mammographic density in relation to cyclic variations in endogenous sex hormone levels, prolactin, and insulin-like growth factors.

Mammographic density is strongly associated with breast cancer risk, and endogenous hormones, which are risk factors for breast cancer, may be involved in the mechanism. This cross-sectional study of 494 premenopausal women is the first to account for cyclic variations in estrogen levels, by measuring urinary estrone glucuronide (E1G) in the periovulatory and luteal phases of the menstrual cycle, and to assess the role of androgens. Computer-assisted density readings were obtained from digitized mammograms. Mean ovulatory E1G level and daily E1G load were both positively associated with percent density before adjustment for body mass index (BMI), with women in the top fourth having 10.2% (95% CI: 2.9%, 18.1%) and 8.9% (1.7%, 16.7%), respectively, higher density than those in the bottom fourth (Ptrend before/after BMI adjustment=0.006/0.11 and 0.01/0.13, respectively). Neither the peak nor luteal E1G levels were predictive of density after adjustment for E1G levels at other points in the cycle. The plasma androgens testosterone, androstenedione, and dehydroepiandrosterone sulfate were negatively associated with density. In mutually adjusted analyses, density was positively associated with insulin-like growth factor (IGF)-I and negatively with IGF-II (Ptrend=0.006 for both) but not with IGF binding protein-3. There was also weak evidence of a positive association of prolactin with density. The study supports the hypothesis that endogenous hormones affect density in premenopausal women; in particular, it shows a positive association between estrogen levels and density and suggests that the mean level throughout the cycle is the most biologically relevant measure. Most of these hormone-density associations were attenuated with further adjustment for BMI.

Regulation of 3beta-hydroxysteroid dehydrogenase type 1 and type 2 gene expression and function in the human ovarian surface epithelium by cytokines.

The human ovarian surface epithelium (hOSE) is a squamous-to-cuboidal layer that surrounds the ovary. hOSE undergoes injury and repair cycles as a result of ovulation-induced inflammation, an event relevant to the development of epithelial ovarian cancer (EOC). Locally produced steroids mediate the response to inflammation. 3beta-Hydroxysteroid dehydrogenase (3beta-HSD) drives the intracrine generation of progestogens and androgens that potentially affect cell survival and proliferation. We therefore investigated the regulation of 3beta-HSD along with downstream steroid signalling in hOSE. Double immunofluorescence of cultured primary hOSE cells confirmed the expression of 3beta-HSD protein Interleukin (IL). IL-1alpha treatment of primary cells to mimic ovulation-associated inflammation suppressed 3beta-HSD1 expression and stimulated 3beta-HSD2 mRNA (P < 0.001), without affecting total 3beta-HSD protein and activity or androgen or progesterone receptor (PR) mRNA levels. Conversely, IL-4 as a proxy for a post-ovulatory healing cytokine increased both 3beta-HSD transcripts, total protein and activity (P < 0.01). IL-4 also suppressed androgen receptor expression (P < 0.01) without affecting that of the PR, thereby potentially sustaining both progesterone biosynthesis and its underlying signalling in the ovarian surface. 3beta-HSD protein was immunodetectable in primary ascites of women who were diagnosed with EOC but both mRNA transcripts were diminished relative to normal cells (P < 0.05). Notably, this difference was countered by IL-4 treatment (P < 0.01). We conclude that stimulation by IL-4 could be physiologically relevant to post-ovulatory ovarian healing and suggest a novel therapeutic strategy for the activation of progesterone-associated apoptosis in ovarian cancer. Also, our results suggest an attenuation of 3beta-HSD expression in EOC although further studies are required for confirmation.

Cortisol inactivation by 11beta-hydroxysteroid dehydrogenase-2 may enhance endometrial angiogenesis via reduced thrombospondin-1 in heavy menstruation.

CONTEXT: Heavy menstrual bleeding (HMB; menorrhagia) impairs quality of life for women and requires medication or surgery. Because glucocorticoids inhibit angiogenesis in other organs, we hypothesized that endometrium of women with HMB is subject to decreased local glucocorticoid exposure and enhanced angiogenesis, thereby increasing menstrual bleeding. DESIGN: Endometrium was collected from 29 women with menstrual complaints. Menstrual blood loss was measured by alkaline-hematin assay (n = 12, > 80 ml (HMB); n = 17, < 80 ml). Quantitative RT-PCR for thrombospondin-1 (TSP-1) and glucocorticoid-metabolizing enzymes, 11beta-hydroxysteroid dehydrogenases-1 and -2 (11betaHSD1,2) was performed. Glucocorticoid effects on endometrial stromal cells and uterine endothelial cells (UECs) were determined. RNA interference studies in UECs examined the effect of TSP-1 ablation on cortisol action. RESULTS: Secretory phase endometrium mRNA levels for the cortisol inactivating enzyme 11betaHSD2 were higher [3.78 +/- 1.29 vs. 1.40 +/- 0.6 (arbitrary units), P < 0.05], whereas TSP-1 mRNA was lower [0.40 +/- 0.13 vs. 1.66 +/- 1.02 (arbitrary units), P < 0.05] in women with HMB. In cultured endometrial stromal cells and UECs, cortisol increased TSP-1 expression. Both cortisol and TSP-1 inhibited new vessel formation in endometrial explants embedded in Matrigel. In UECs cortisol inhibition of tube-like structure formation was blocked by small interfering RNA (siRNA) against TSP-1 (25 +/- 2.5% cortisol inhibition with scrambled siRNA vs. 0% cortisol inhibition with TSP-1 siRNA inactivation, P<0.01). CONCLUSIONS: Enhanced inactivation of cortisol by 11betaHSD2 in endometrium from women with HMB may explain reduced TSP-1 levels and hence endothelial cell dysfunction and abnormal angiogenesis. Inhibition of 11betaHSD2 may be a rational novel therapy for heavy menstrual bleeding.

3beta-Hydroxysteroid dehydrogenases and pre-receptor steroid metabolism in the human ovarian surface epithelium.

Ovulation-associated inflammation with accompanied cytokines and reproductive hormones impact upon the human ovarian surface epithelium (hOSE) and probably have a role in the aetiology of ovarian cancer. Progesterone and progestin-related events, i.e. pregnancy and oral contraception, protect from the disease. We have investigated the pre-receptor metabolism of progesterone in primary hOSE cells and an immortalised hOSE cell line, OSE-C2, focusing on transcriptional regulation of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) by inflammatory, anti-inflammatory and apoptotic factors. In hOSE cells, we show that anti-inflammatory effects of IL-1alpha and IL-4 on 3beta-HSD2 mRNA involve a p38 MAPK signalling pathway, whereas pro-inflammatory response of IL-1alpha to 3beta-HSD1 mRNA involves a NF-kappaB inflammatory pathway. In OSE-C2 cells, retinoic acid and transforming growth factor-beta1 massively induce 3beta-HSD1 mRNA levels. In conclusion, we elaborate several mechanisms for intracrine formation of progesterone in hOSE that could contribute in the development of novel strategies to prevent, diagnose and/or treat ovarian cancer.

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.

Follicular growth and oocyte competence in the in vitro cultured mouse follicle: effects of gonadotrophins and steroids.

Although there have been extensive studies on the effects of gonadotrophins and steroids on follicular development, less is known as to the effects these hormones have on the acquisition of oocyte developmental competence. This study investigates the effect of altering the gonadotrophin or steroidal environment on follicular development and on oocyte viability and DNA methylation. Oocytes were obtained from pre-ovulatory follicles after individual follicle culture from the pre-antral stage; gonadotrophin or steroid levels were manipulated during the culture period. Oocytes obtained from follicles grown in gonadotrophin free conditions were able to fertilize and develop to the blastocyst stage despite their impaired follicle development. There was no effect of luteinizing hormone or steroids on follicular growth. Altering the steroidal environment did, however, affect oocyte development. The oocytes of follicles exposed to high estrogen levels had lower fertilization rates, regardless of the presence or absence of high androgen levels. The combined presence of high levels of both steroids altered the level of global methylation. This study demonstrates that gonadotrophins and steroids influence the acquisition of developmental competence of the oocyte and suggests that optimal steroid exposure during follicle development is required for the oocyte to mature correctly.

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.

Connective tissue growth factor expression in the human corpus luteum: paracrine regulation by human chorionic gonadotropin.

CONTEXT: The molecular mechanisms of luteolysis and its inhibition during maternal recognition of pregnancy remain unclear. OBJECTIVE: The objective of this study was to investigate the differential regulation of connective tissue growth factor (CTGF) expression in human corpora lutea using in vivo and in vitro models. DESIGN: Corpora lutea from different stages of the luteal phase and after luteal rescue with human chorionic gonadotropin (hCG) were studied. Primary cultures and cocultures of luteinized granulosa cells and luteal fibroblast-like cells were performed. SETTING: This study was performed at the research center of a university teaching hospital. PATIENTS: Women with regular cycles having hysterectomy for nonmalignant conditions and women undergoing oocyte collection for assisted conception were studied. INTERVENTIONS: CTGF localization was determined by in situ hybridization, and expression by quantitative RT-PCR. OUTCOMES: The outcome measures were the effect of hCG on the expression and localization of CTGF mRNA in human corpora lutea and the effect of hCG on CTGF expression in primary cultures of luteinized granulosa cells and luteal fibroblast-like cells. RESULTS: Luteal rescue reduced CTGF expression compared with that in the late luteal phase (P < 0.05). CTGF expression was localized to fibroblast-like cells and endothelial cells of larger blood vessels, not to steroidogenic cells. The expression of CTGF by fibroblast-like cells in vitro was not regulated by hCG. When cocultured with luteinized granulosa cells, fibroblast-like cell CTGF expression was inhibited by hCG (P < 0.001). This effect was independent of stimulated progesterone concentrations and was not blocked by follistatin or indomethacin. Both IL-1alpha (P < 0.05) and cAMP (P < 0.001) inhibited CTGF expression in fibroblast-like cells. CONCLUSIONS: These results provide evidence for negative regulation of CTGF by hCG during luteal rescue mediated by paracrine signals.

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.