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Interleukin-1 beta (IL-1 beta) may be active in the ovary at ovulation and may be produced by immune and non-immune cells. This study evaluates the production of IL-1 beta by granulosa cells and macrophages from the human ovulatory follicle. The concentrations of IL-1 beta and IL-1 beta mRNA were measured in follicular aspirates taken from patients undergoing in-vitro fertilization and in cultures of cells from aspirates. Macrophages were detected by immunocytochemistry and by the reverse transcriptase polymerase chain reaction (RT-PCR). The macrophages were removed from granulosa cell preparations immediately or after the cells have been cultured for 24 h. IL-1 beta was detected by radioimmunoassay and transcripts were detected by RT-PCR and by in-situ hybridization on cytospun preparations using a [35S]IL-1 beta riboprobe. IL-1 beta and IL-1 beta mRNA were found in follicular aspirates, in granulosa luteal cell preparations containing macrophages and in highly purified granulosa cell preparations after removal of macrophages by all the methods used. Both macrophages and granulosa cells contained high concentrations of IL-1 beta transcripts. Moreover, the number of IL-1 beta reactive cells in granulosa cell preparations cultured for 24 h with 10-15% macrophages before removal was twice that of granulosa cells cultured without macrophages. Thus, both granulosa cells and macrophages are actively involved in the ovarian production of IL-1 beta at ovulation and the ability of granulosa cells to produce IL-1 beta may require ovarian macrophages. [ABSTRACT FROM AUTHOR]

Abstract Background CXCL12 has been widely reported to play a biologically relevant role in tumor growth and spread. In epithelial ovarian cancer (EOC), CXCL12 enhances tumor angiogenesis and contributes to the immunosuppressive network. However, its prognostic significance remains unclear. We thus compared CXCL12 status in healthy and malignant ovaries, to assess its prognostic value. Methods Immunohistochemistry was used to analyze CXCL12 expression in the reproductive tracts, including the ovaries and fallopian tubes, of healthy women, in benign and borderline epithelial tumors, and in a series of 183 tumor specimens from patients with advanced primary EOC enrolled in a multicenter prospective clinical trial of paclitaxel/carboplatin/gemcitabine-based chemotherapy (GINECO study). Univariate COX model analysis was performed to assess the prognostic value of clinical and biological variables. Kaplan-Meier methods were used to generate progression-free and overall survival curves. Results Epithelial cells from the surface of the ovary and the fallopian tubes stained positive for CXCL12, whereas the follicles within the ovary did not. Epithelial cells in benign, borderline and malignant tumors also expressed CXCL12. In EOC specimens, CXCL12 immunoreactivity was observed mostly in epithelial tumor cells. The intensity of the signal obtained ranged from strong in 86 cases (47%) to absent in 18 cases ( Conclusion Our findings highlight the previously unappreciated constitutive expression of CXCL12 on healthy epithelia of the ovary surface and fallopian tubes, indicating that EOC may originate from either of these epithelia. We reveal that CXCL12 production by malignant epithelial cells precedes tumorigenesis and we confirm in a large cohort of patients with advanced EOC that CXCL12 expression level in EOC is not a valuable prognostic factor in itself. Trial Registration ClinicalTrials.gov: NCT00052468

Abstract Background Little is known about the molecules that contribute to tumor progression of epithelial ovarian cancer (EOC), currently a leading cause of mortality from gynecological malignancies. Glucocorticoid-Induced Leucine Zipper (GILZ), an intracellular protein widely expressed in immune tissues, has been reported in epithelial tissues and controls some of key signaling pathways involved in tumorigenesis. However, there has been no report on GILZ in EOC up to now. The objectives of the current study were to examine the expression of GILZ in EOC and its effect on tumor cell proliferation. Results GILZ expression was measured by immunohistochemical staining in tissue sections from 3 normal ovaries, 7 benign EOC and 50 invasive EOC. GILZ was not detected on the surface epithelium of normal ovaries and benign tumors. In contrast, it was expressed in the cytoplasm of tumor cells in 80% EOC specimens. GILZ immunostaining scores correlated positively to the proliferation marker Ki-67 (Spearman test in univariate analysis, P < 0.00001, r = 0.56). They were also higher in tumor cells containing large amounts of phosphorylated protein kinase B (p-AKT) (unpaired t test, P < 0.0001). To assess the effect of GILZ on proliferation and AKT activation, we used the BG-1 cell line derived from ovarian tumor cells as a cellular model. GILZ expression was either enhanced by stable transfection or decreased by the use of small interfering (si) RNA targeting GILZ. We found that GILZ increased cell proliferation, phospho-AKT cellular content and AKT kinase activity. Further, GILZ upregulated cyclin D1 and phosphorylated retinoblastoma (p-Rb), downregulated cyclin-dependent kinase inhibitor p21, and promoted the entry into S phase of cell cycle. Conclusion The present study is the first to identify GILZ as a molecule produced by ovarian cancer cells that promotes cell cycle progression and proliferation. Our findings clearly indicate that GILZ activates AKT, a crucial signaling molecule in tumorigenesis. GILZ thus appears as a potential key molecule in EOC.

Recent studies have highlighted the importance of understanding the molecular determinants of CXCL12-mediated effects in cancers. Once previously thought to interact exclusively with CXCR4, CXCL12 also binds with high affinity to CXCR7 (recently renamed ACKR3), which belongs to an atypical chemokine receptor family whose members fail to activate Gαi proteins but interact with β-arrestins. In addition to its capacity to control CXCL12 bioavailability, ACKR3 can either enhance or dampen CXCR4-mediated signaling and activity. In light of the most recent findings, we have examined the role of ACKR3 in cancer, including a subset of virus-related cancers., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Ovarian cancer is the gynecological cancer exhibiting the highest morbidity and improvement of treatments is still required. Previous studies have shown that Estrogen-receptor beta (ERβ) levels decreased along with ovarian carcinogenesis. Here, we present evidence that reintroduction of ERβ in BG-1 epithelial ovarian cancer cells, which express ERα, leads in vitro to a decrease of basal and estradiol-promoted cell proliferation. ERβ reduced the frequency of cells in S phase and increased the one of cells in G2/M phase. At the molecular level, we found that ERβ downregulated total retinoblastoma (Rb), phosphorylated Rb and phospho-AKT cellular content as well as cyclins D1 and A2. In addition, ERβ had a direct effect on ERα, by strongly inhibiting its expression and activity, which could explain part of the anti-proliferative action of ERβ. By developing a novel preclinical model of ovarian cancer based on a luminescent orthotopic xenograft in athymic Nude mice, we further revealed that ERβ expression reduces tumor growth and the presence of tumor cells in sites of metastasis, hence resulting in improved survival of mice. Altogether, these findings unveil a potential tumor-suppressor role of ERβ in ovarian carcinogenesis, which could be of potential clinical relevance for the selection of the most appropriate treatment for patients.

Background: CXCL12 has been widely reported to play a biologically relevant role in tumor growth and spread. In epithelial ovarian cancer (EOC), CXCL12 enhances tumor angiogenesis and contributes to the immunosuppressive network. However, its prognostic significance remains unclear. We thus compared CXCL12 status in healthy and malignant ovaries, to assess its prognostic value., Methods: Immunohistochemistry was used to analyze CXCL12 expression in the reproductive tracts, including the ovaries and fallopian tubes, of healthy women, in benign and borderline epithelial tumors, and in a series of 183 tumor specimens from patients with advanced primary EOC enrolled in a multicenter prospective clinical trial of paclitaxel/carboplatin/gemcitabine-based chemotherapy (GINECO study). Univariate COX model analysis was performed to assess the prognostic value of clinical and biological variables. Kaplan-Meier methods were used to generate progression-free and overall survival curves., Results: Epithelial cells from the surface of the ovary and the fallopian tubes stained positive for CXCL12, whereas the follicles within the ovary did not. Epithelial cells in benign, borderline and malignant tumors also expressed CXCL12. In EOC specimens, CXCL12 immunoreactivity was observed mostly in epithelial tumor cells. The intensity of the signal obtained ranged from strong in 86 cases (47%) to absent in 18 cases (<10%). This uneven distribution of CXCL12 did not reflect the morphological heterogeneity of EOC. CXCL12 expression levels were not correlated with any of the clinical parameters currently used to determine EOC prognosis or with HER2 status. They also had no impact on progression-free or overall survival., Conclusion: Our findings highlight the previously unappreciated constitutive expression of CXCL12 on healthy epithelia of the ovary surface and fallopian tubes, indicating that EOC may originate from either of these epithelia. We reveal that CXCL12 production by malignant epithelial cells precedes tumorigenesis and we confirm in a large cohort of patients with advanced EOC that CXCL12 expression level in EOC is not a valuable prognostic factor in itself., Trial Registration: ClinicalTrials.gov: NCT00052468.

Background: Little is known about the molecules that contribute to the growth of epithelial ovarian carcinomas (EOC), which remain the most lethal gynecological cancer in women. The chemokine Fractalkine/CX(3)CL1 has been widely reported to play a biologically relevant role in tumor growth and spread. We report here the first investigation of the expression and role of CX(3)CL1 in EOC., Results: Epithelial cells from the surface of the ovary and the Fallopian tubes and from benign, borderline and malignant tumors all stained positive for CX(3)CL1. In tumor specimens from 54 women who underwent surgical treatment for EOC diagnosis, CX(3)CL1 immunoreactivity was unevenly distributed in epithelial tumor cells, and ranged from strong (33%) to absent (17%). This uneven distribution of CX(3)CL1 did not reflect the morphological heterogeneity of EOC. It was positively correlated with the proliferation index Ki-67 and with GILZ (glucocorticoid-induced leucine zipper), previously identified as an activator of the proliferation of malignant EOC cells. Hierarchical clustering analysis, including age at diagnosis, tumor grade, FIGO stage, Ki-67 index, CX(3)CL1, SDF-1/CXCL12 and GILZ immunostaining scores, distinguished two major clusters corresponding to low and high levels of proliferation and differing in terms of GILZ and CX(3)CL1 expression. GILZ overexpression in the carcinoma-derived BG1 cell line resulted in parallel changes in CX(3)CL1 products. Conversely, CX(3)CL1 promoted through its binding to CX(3)CR1 AKT activation and proliferation in BG1 cells. In a mouse subcutaneous xenograft model, the overexpression of GILZ was associated with higher expression of CX(3)CL1 and faster tumor growth., Conclusion: Our findings highlight the previously unappreciated constitutive expression of CX(3)CL1 preceding tumorigenesis in ovarian epithelial cells. Together with GILZ, this chemokine emerges as a regulator of cell proliferation, which may be of potential clinical relevance for the selection of the most appropriate treatment for EOC patients.

Background: Little is known about the molecules that contribute to tumor progression of epithelial ovarian cancer (EOC), currently a leading cause of mortality from gynecological malignancies. Glucocorticoid-Induced Leucine Zipper (GILZ), an intracellular protein widely expressed in immune tissues, has been reported in epithelial tissues and controls some of key signaling pathways involved in tumorigenesis. However, there has been no report on GILZ in EOC up to now. The objectives of the current study were to examine the expression of GILZ in EOC and its effect on tumor cell proliferation., Results: GILZ expression was measured by immunohistochemical staining in tissue sections from 3 normal ovaries, 7 benign EOC and 50 invasive EOC. GILZ was not detected on the surface epithelium of normal ovaries and benign tumors. In contrast, it was expressed in the cytoplasm of tumor cells in 80% EOC specimens. GILZ immunostaining scores correlated positively to the proliferation marker Ki-67 (Spearman test in univariate analysis, P < 0.00001, r = 0.56). They were also higher in tumor cells containing large amounts of phosphorylated protein kinase B (p-AKT) (unpaired t test, P < 0.0001). To assess the effect of GILZ on proliferation and AKT activation, we used the BG-1 cell line derived from ovarian tumor cells as a cellular model. GILZ expression was either enhanced by stable transfection or decreased by the use of small interfering (si) RNA targeting GILZ. We found that GILZ increased cell proliferation, phospho-AKT cellular content and AKT kinase activity. Further, GILZ upregulated cyclin D1 and phosphorylated retinoblastoma (p-Rb), downregulated cyclin-dependent kinase inhibitor p21, and promoted the entry into S phase of cell cycle., Conclusion: The present study is the first to identify GILZ as a molecule produced by ovarian cancer cells that promotes cell cycle progression and proliferation. Our findings clearly indicate that GILZ activates AKT, a crucial signaling molecule in tumorigenesis. GILZ thus appears as a potential key molecule in EOC.

Mast cells proliferate in vivo in areas of active fibrosis, during parasite infestations, in response to repeated immediate hypersensitivity reactions and in patients with mastocytosis. We investigated how progesterone reduces the proliferation of HMC-1(560) mast cells that proliferate spontaneously in culture. Cells were incubated with 1 microM to 1 nM progesterone for 24-48 h. Progesterone (1 microM) reduced the spontaneous proliferation of HMC-1(560) mast cells to half that of cells cultured without hormone. [(3)H] thymidine incorporation was only 50% of control; there were fewer cells in G2/M and more cells in G0/G1. The amounts of phospho-Raf-1 (Tyr 340-341) and phospho-p42/p44 MAPK proteins were also reduced. In contrast progesterone had no effect on MAP kinase-phosphatase-1. The Raf/MAPK pathway, which depends on Src kinase activity, is implicated in the control of cell proliferation. HMC-1(560) cells incubated with the tyrosine kinase inhibitor PP1 proliferated more slowly than controls and had less phospho-Raf-1 (Tyr 340-341) and phospho-p42/p44 MAPK. The Csk homologous kinase (CHK), an endogenous inhibitor of Src protein tyrosine kinases, was also enhanced in progesterone-treated cells. In contrast, progesterone had no effect on the growth of cells transfected with siRNA CHK. We conclude that progesterone increases the amount of csk homologous kinase, which in turn reduces HMC-1(560) mast cell proliferation. This effect parallels decreases in the phosphorylated forms of Raf-1 and p42/44 MAPK, as their production depends on Src kinase activity., ((c) 2007 Wiley-Liss, Inc.)

Mast cell recruitment is implicated in many physiological functions and several diseases. It depends on microenvironmental factors, including hormones. We have investigated the effect of progesterone on the migration of HMC-1(560) mast cells toward CXCL12, a chemokine that controls the migration of mast cells into tissues. HMC-1(560) mast cells were incubated with 1 nM to 1 microM progesterone for 24 h. Controls were run without progesterone. Cell migration toward CXCL12 was monitored with an in vitro assay, and statistical analysis of repeated experiments revealed that progesterone significantly reduced cell migration without increasing the number of apoptotic cells (P = 0.0084, n = 7). Differences between progesterone-treated and untreated cells were significant at 1 microM (P < 0.01, n = 7). Cells incubated with 1 microM progesterone showed no rearrangment of actin filaments in response to CXCL12. Progesterone also reduced the calcium response to CXCL12 and Akt phosphorylation. Cells incubated with progesterone had one-half the control concentrations of CXCR4 (mRNA, total protein, and membrane-bound protein). Progesterone also inhibited the migration of HMC-1(560) cells transfected with hPR-B-pSG5 plasmid, which contained 2.5 times as much PR-B as the control. These transfected cells responded differently (P < 0.05, n = 5) from untreated cells to 1 nM progesterone. We conclude that progesterone reduces mast cell migration toward CXCL12 and that CXCR4 may be a progesterone target in mast cells.

Granulosa-cumulus cells are located around the oocyte and may be involved in competence acquisition and in the resumption of oocyte meiosis. We assessed Bax expression in granulosa-cumulus cells and its correlation to in vitro resumption of oocyte meiosis in women presenting PCOS (polycystic ovarian syndrome), as well as the role of the chemokine SDF1-CXCL12 in human preovulatory follicle in the follow-up of granulosa cell survival and embryo quality in IVF.

We investigated the production and the role of the chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) in pre-ovulatory follicles of women undergoing in vitro fertilization. We detected CXCL12 and its receptor CXCR4 by flow cytometry, western blotting and RT-PCR. We tested cell migration in Transwell experiments. We measured apoptosis using delta psi m-sensitive fluorescent probe DiOC6(3) and we screened apoptosis-related gene expression with macro-arrays. Granulosa cells from follicular aspirates produce CXCL12 that contributes to T lymphocytes recruitment. CXCL12 reduces early apoptosis of granulosa cells. This effect is accompanied by a shift of bcl2/bax ratio, and decreased expression of p53-targeted genes (pig7, pig8, p21, gadd45). Removal of lymphocytes disables CXCL12-mediated anti-apoptotic effect on granulosa cells. Anti-apoptotic activity of CXCL12 is positively correlated to high quality of embryos. In conclusion, CXCL12 is locally produced by luteinizing granulosa cells. It specifically contributes to T lymphocytes recruitment and coordinates with local lymphocytes to increase granulosa cell survival and embryo quality.

Ovarian carcinomas have a poor prognosis, often associated with multifocal i.p. dissemination accompanied by intense neovascularization. To examine tumor angiogenesis in the tumor microenvironment, we studied malignant ascites and tumors of patients with untreated ovarian carcinoma. We observed that malignant ascites fluid induced potent in vivo neovascularization in Matrigel assay. We detected a sizable amount of vascular endothelial cell growth factor (VEGF) in malignant ascites. However, pathologic concentration of VEGF is insufficient to induce in vivo angiogenesis. We show that ovarian tumors strongly express CXC chemokine stromal-derived factor (SDF-1/CXCL12). High concentration of CXCL12, but not the pathologic concentration of CXCL12 induces in vivo angiogenesis. Strikingly, pathologic concentrations of VEGF and CXCL12 efficiently and synergistically induce in vivo angiogenesis. Migration, expansion, and survival of vascular endothelial cells (VEC) form the essential functional network of angiogenesis. We further provide a mechanistic basis for explaining the interaction between CXCL12 and VEGF. We show that VEGF up-regulates the receptor for CXCL12, CXCR4 expression on VECs, and synergizes CXCL12-mediated VEC migration. CXCL12 synergizes VEGF-mediated VEC expansion and synergistically protects VECs from sera starvation-induced apoptosis with VEGF. Finally, we show that hypoxia synchronously induces tumor CXCL12 and VEGF production. Therefore, hypoxia triggered tumor CXCL12 and VEGF form a synergistic angiogenic axis in vivo. Hypoxia-induced signals would be the important factor for initiating and maintaining an active synergistic angiogeneic pathway mediated by CXCL12 and VEGF. Thus, interrupting this synergistic axis, rather than VEGF alone, will be a novel efficient antiangiogenesis strategy to treat cancer.

Exposure to lead causes decreased fertility in women. In the present study, we examined the in vitro effects of lead on cytochrome p450 aromatase (p450 arom) and on estrogen receptor beta (ERbeta), two key proteins for the human ovary. Aromatase is required for the bioconversion of androgen to estradiol; ERbeta mediates estrogen effects in granulosa cells. Granulosa cells were collected from women undergoing in vitro fertilization and then cultured with 10 microM lead acetate. Using atomic absorption spectrometry, we showed that lead accumulated in cells. Aromatase activity as measured by a tritiated water production assay was significantly reduced. Using semiquantitative reverse transcription-polymerase chain reaction and Western blotting procedures, we showed that p450 arom and ERbeta mRNA and protein content were both significantly reduced. Adding 10 microg/ml of cycloheximide, a protein inhibitor, did not eliminate the effects of lead. The present results support the hypothesis that the action of lead on fertility in women may result, in part, from the down-regulation of p450 arom and ERbeta gene transcription in ovarian granulosa.

Hodgkin's disease appearing as, or associated with, fibrous thyroiditis has only been described rarely. We report the observation of a patient presenting with a goitre, fibrosis of the thyroid and adjacent structures, and hypothyroidism. The histological examination was compatible with fibrosclerotic thyroiditis. This diagnosis was reviewed 6 months later when the biopsy of a supraclavicular nodule that had subsequently appeared led to the diagnosis of a nodular-sclerosis type of Hodgkin's disease. The plasmatic levels of interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-alpha) were very high compared to the levels in healthy subjects (12 and 40 IU/l vs. 0.05 and 2.0 IU/l, respectively). These cytokine levels decreased when the initial illness was treated, and their normalization was associated with the disappearance of the cervical and thyroidal fibroses. A parallel in vitro study of these cytokines and of TNF-alpha receptors and IL-13 was performed. The results suggest a possible cause-and-effect relationship between IL-6 and IL-13 produced locally by the tumoral tissue and the development of cervical fibrosis.

Dendritic-cell (DC) trafficking and function in tumors is poorly characterized, with studies confined to myeloid DCs (DC1s). Tumors inhibit DC1 migration and function, likely hindering specific immunity. The role of plasmacytoid DCs (DC2s) in tumor immunity is unknown. We show here that malignant human ovarian epithelial tumor cells express very high levels of stromal-derived factor-1, which induces DC2 precursor (preDC2) chemotaxis and adhesion/transmigration, upregulates preDC2 very late antigen (VLA)-5, and protects preDC2s from tumor macrophage interleukin-10-induced apoptosis, all through CXC chemokine receptor-4. The VLA-5 ligand vascular-cell adhesion molecule-1 mediated preDC2 adhesion/transmigration. Tumor preDC2s induced significant T-cell interleukin-10 unrelated to preDC2 differentiation or activation state, and this contributed to poor T-cell activation. Myeloid precursor DCs (preDC1s) were not detected. Tumors may weaken immunity by attracting preDC2s and protecting them from the harsh microenvironment, and by altering preDC1 distribution.

We examined the production of regulated on activation normal T expressed and secreted (RANTES) chemokine, which may contribute to the recruitment and local accumulation of leukocytes in human preovulatory follicles. Cells were obtained from follicular aspirates collected from in vitro fertilization patients, then cultured. RANTES production in culture was measured by immunoenzymatic assay, RANTES-producing cells were measured by flow cytometry, and messenger ribonucleic acid as measured by RT-PCR and in situ hybridization. RANTES was detected in follicular fluids and culture supernatants; RANTES protein and messenger ribonucleic acid were expressed in granulosa cells. RANTES production was stimulated by tumor necrosis factor-alpha (TNFalpha) and was inhibited in cultures containing a neutralizing anti-TNFa antibody. p55 TNF receptors were detected by RT-PCR and visualized on granulosa cells by flow cytometry. RANTES production was increased by phorbol 12-myristate 13-acetate, but not by 8-bromo-cAMP. RANTES was produced by granulosa cells from human preovulatory follicles. This production was activated by TNFalpha, probably through TNF receptor p55. This suggests that RANTES may play an active role in ovarian processes involving the local accumulation of immune cells.

Cells were obtained from patients undergoing in vitro fertilization. They were cultured and those producing vascular endothelium growth factor (VEGF) were detected by flow cytometry; relative amounts of mRNA were detected by RT-PCR and measured by PCR Elisa after RT-PCR products were biotinylated. Most of the granulosa cells produced VEGF. This production was maintained over 5 days in culture without adding hCG. The two diffusible forms, VEGF 121 and 165, were the most abundant. VEGF 145, which is specific to the reproductive system, was less abundant. VEGF 189, which is not freely secreted, was not produced by granulosa cells; small amounts were only detected in preparations containing leukocytes. TNF-alpha decreased VEGF production; the effect of TNF-alpha was neutralized by 10 nM staurosporine. Thus, the VEGF in human preovulatory follicles is mostly in the granulosa cells. These cells are therefore a major source of VEGF at ovulation and may play a key role in physiological and pathological processes which involve changes in vascular permeability and/or angiogenesis. The data also suggest that TNF-alpha via protein kinase C modulates the production of VEGF.

We compared the membrane effects of estradiol, progesterone, and androstenedione in a single experimental model, the ovarian granulosa cells collected from immature Large White sows. We measured changes in cytosolic free calcium concentration ([Ca2+]i) in confluent Fura-2 loaded cells. We used pharmacological tools and polyclonal phospholipase C-beta (PLC-beta) antibodies. Each steroid (0.1 pM to 1 nM) transiently increased intracellular calcium concentration ([Ca2+]i) within 5 sec. They mobilized Ca2+ from the endoplasmic reticulum as shown by using two phospholipase C inhibitors, neomycin and U-73122. Ca2+ mobilization involved PLC-beta1 for progesterone, PLC-beta2 for estradiol and PLC-beta4 for androstenedione. A pertussis toxin-insensitive G protein was involved in the effects of progesterone on Ca2+ mobilization whereas estradiol and androstenedione effects were mediated via a pertussis toxin-sensitive G-protein. Ca2+ influx from the extracellular milieu was involved in the increase in [Ca2+]i induced by progesterone and estradiol, but not by androstenedione. Influx of Ca2+ was independent of Ca2+ mobilization from calcium stores, and it was suggested that L-type Ca2+ channels for estradiol and T-type Ca2+ channels for progesterone were involved. The three steroids had no effect on cAMP. Rapid effects of progesterone, estradiol, and androstenedione involved a direct action on cell membrane elements such as PLC-beta, G-proteins, and calcium channels, and these mechanisms were hormone-specific.

Cytokines, originally identified as products of immune cells, are synthesized throughout the female reproductive tract. Evidence has accumulated supporting the role of cytokines in reproduction, including gamete and follicle development and steroidogenesis. In these processes, cytokines act either through a paracrine or autocrine mechanism. The present article focuses on the role of cytokines during ovulation, which shares many of the features of the inflammatory reaction. The intraovarian production of cytokines, as well as its regulation by sex steroid and peptide hormones, is considered. The role of cytokines in follicle rupture and remodelling, leukocyte infiltration, angiogenesis, steroid hormone production and oocyte maturation is also described.

We investigated the early effects (5-60 s) of progesterone (1 pM-0.1 microM) on cytosolic free calcium concentration ([Ca2+]i) and inositol 1,4,5-trisphosphate (InsP3) formation in nonluteinized and in vitro luteinized porcine granulosa cells (pGCs). Progesterone increased [Ca2+]i and InsP3 formation within 5 s in both cell types. Progesterone induced calcium mobilization from the endoplasmic reticulum via the activation of a phospholipase C linked to a pertussis-insensitive G-protein. This process was controlled by protein kinases C and A. In contrast, only nonluteinized pGCs showed a Ca2+ influx via dihydropyridine-insensitive calcium channel. In both cell types, the nuclear progesterone receptor antagonist RU-38486 did not inhibit the progesterone-induced increase in [Ca2+]i, progesterone immobilized on bovine serum albumin, which did not enter the cell, increased [Ca2+]i within 5 s and was a full agonist, but less potent than the free progesterone; pertussis toxin did not inhibit progesterone effect on InsP3. In conclusion, progesterone may interact with membrane unconventional receptors that belong to the class of membrane receptors coupled to a phospholipase C via a pertussis toxin-insensitive G-protein. The source of the Ca2+ for the progesterone-induced increase in [Ca2+]i also depends on the stage of cell luteinization.

We explored the potential relevance of interleukin-6 (IL6) to ovarian function in cynomolgus monkey females which were treated to induce multiple follicular growth. Present work gave evidences that IL6 was produced in the ovulatory follicle after hCG administration by using anti-IL6 monoclonal antibody and immunohistochemical techniques. IL6 was produced by granulosa cells in cultures. However, this production was not stimulated by adding to cultures FSH or Bt2cAMP, known to induce IL6 production in various cell types. IL6 was shown to antagonize luteinizing FSH effects. Inhibitory IL6 effect was probably mediated at a site distal to cAMP generation since it had no effect on progesterone production induced by dibutyryl cAMP treatment. The presence of IL6 in the ovulatory follicle show that inflammatory cytokines may play a role at ovulation. On the other hand, its inhibitory effect on progesterone production by granulosa cells could not likely affect the process of luteinization which is controlled by LH/hCG.

Previous studies in vivo provide evidence that extra-ovarian factors, currently unknown but nevertheless present and associated with pregnancy, directly prevent the growth of follicles beyond a diameter of 2 mm during the last trimester of pregnancy in the ewe. In the present study, the effect of charcoal-treated extract from sheep placenta on total aromatase activity, as determined by the conversion of [3H] androstenedione to estradiol and measurement of [3H] water, was investigated using primary culture of human granulosa cells in serum-free medium as a model. Addition of different doses (50, 150, 300 and 600 micrograms protein) of cotyledons extract of day 110 of pregnancy produced a dose-dependent diminution of granulosa cell aromatase activity in the absence of FSH. The maximal inhibition of aromatase activity occurred at a dose of 600 micrograms. These results showed that the cotyledons of late pregnancy contain a non-steroidal factor that inhibits basal aromatase activity in granulosa cells. Extracts prepared from cotyledons of days 90 and 110 of pregnancy but not extracts of days 50 and 70 significantly reduced basal aromatase activity in a dose-dependent manner. These results suggest that the production of the aromatase inhibiting factor increases with the advance of pregnancy. The aromatase inhibiting activity was lost after heating (80 degrees C, 30 min) or after treatment with trypsin (1 mg/ml) of cotyledons extract of day 110 of pregnancy, demonstrating the protein nature of the aromatase inhibiting factor. In conclusion, these experiments demonstrate that ovine placenta contains a heat- and trypsin-sensitive factor likely to be a protein which inhibits granulosa cell aromatase activity.

Gonadotropin regulation of granulosa cell (GC) differentiation can be modulated by non-steroidal factors, including cytokines. Interleukin-6 (IL-6), a broad spectrum cytokine, has been previously demonstrated to be produced by GCs and to directly influence follicle stimulating hormone (FSH) differentiated functions of ovarian GCs. In the present study, primary cultures of GCs were prepared from prepubertal sow ovaries. No significant amount of biological active IL-6 was detected in these cultures using the B9 cell growth bioassay. Although our findings suggest that GCs are not source of IL-6 in the porcine ovary, this cytokine may be released by leukocytes present in the ovary and modulate ovarian functions by acting on GCs. Here, adding recombinant human (rh)IL-6 to GC cultures inhibited differentiated functions induced by FSH such as aromatase activity, LH receptor (LHr) expression measured by specific 125I-hCG binding and progesterone (P) production. On the opposite, rhIL-6 did not modulate stimulatory human chorionic hormone (hCG) effects on P release by GCs and did not prevent hCG binding to LHr. These preliminary results clearly showed that IL-6 acted differently on FSH and hCG induced functions although these gonadotropins act primarily through the same transduction pathway involving generation of cyclic AMP. We suggest that IL-6 might act more likely by reducing FSH binding capacity than by modulating transduction pathways. Inhibitory IL-6 effects on FSH-induced functions were not neutralized by adding to culture media a monoclonal antibody against the human IL-6 signal transducer gp130, previously reported to inhibit IL-6 mediated effects in human cell lines.

Objective: To explore the conditions of a suitable luteal phase in human stimulated cycles, progesterone (P) production by cultured granulosa cells from preovulatory follicles was related to preovulatory serum estradiol (E2) and number of oocytes., Design: Progesterone production was measured in the presence or absence of human chorionic gonadotropin (hCG) using radioimmunoassay; data were compared using Student's t-test; correlations used linear regression., Setting: In vitro fertilization and embryo transfer (IVF-ET) for infertility treatment at hospital Antoine Beclère, Clamart, France; scientific studies at Institut National de la Santé et de la Recherche Médicale, Unit 187, Clamart, France., Patients, Participants: Nineteen women, 33 +/- 4 years old, undergoing IVF-ET for nonovarian causes., Main Outcome Measures: High preovulatory E2 usually correlates with high luteal P level. Atretic follicle has reduced follicular E2 production combined with a loss of responsiveness to gonadotropins., Results: Granulosa-luteal cell P production correlated with E2 level (P less than 0.0002). Six cycles, with 14 oocytes recovered per cycle on average, showed reduced plasma E2 per oocyte (P less than 0.001) combined with reduced responsiveness to hCG by granulosa-luteal cells (P less than 0.02)., Conclusion: Recovery of numerous oocytes might be associated with follicular atresia and deficient luteal phase.