Human granulosa-luteal cells initiate an innate immune response to pathogen-associated molecules.

The microenvironment of the ovarian follicle is key to the developmental success of the oocyte. Minor changes within the follicular microenvironment can significantly disrupt oocyte development, compromising the formation of competent embryos and reducing fertility. Previously described as a sterile environment, the ovarian follicle of women has been shown to contain colonizing bacterial strains, whereas in domestic species, pathogen-associated molecules are concentrated in the follicular fluid of animals with uterine infection. The aim of this study is to determine whether human granulosa-luteal cells mount an innate immune response to pathogen-associated molecules, potentially disrupting the microenvironment of the ovarian follicle. Human granulosa-luteal cells were collected from patients undergoing assisted reproduction. Cells were cultured in the presence of pathogen-associated molecules (LPS, FSL-1 and Pam3CSK4) for 24h. Supernatants and total RNA were collected for assessment by PCR and ELISA. Granulosa-luteal cells were shown to express the molecular machinery required to respond to a range of pathogen-associated molecules. Expression of TLR4 varied up to 15-fold between individual patients. Granulosa-luteal cells increased the expression of the inflammatory mediators IL1B, IL6 and CXCL8 in the presence of the TLR4 agonist E. coli LPS. Similarly, the TLR2/6 ligand, FSL-1, increased the expression of IL6 and CXCL8. Although no detectable changes in CYP19A1 or STAR expression were observed in granulosa-luteal cells following challenge, a significant reduction in progesterone secretion was measured after treatment with FSL-1. These findings demonstrate the ability of human granulosa-luteal cells to respond to pathogen-associated molecules and generate an innate immune response.

Cytokines tumor necrosis factor-α and interferon-γ participate in modulation of the equine corpus luteum as autocrine and paracrine factors.

Knowledge on the regulation of corpus luteum (CL) function in the mare is scarce. In this study, the presence of cytokines tumor necrosis factor alpha (TNF) and interferon gamma (IFNG), and their receptors (TNFRI, TNFRII and IFNRI), was investigated in equine CL throughout the luteal phase. The effects of TNF and IFNG on secretory function and viability of luteal cells were defined in vitro. Cytokine ligands and receptors were present in steroidogenic and endothelial cells. Protein expression for TNF was greater in mid-phase and regressing CL, while TNFRI was increased in regressing CL and TNFRII did not change. IFNG and IFNRI showed the highest expression in regressing CL. Transcription of mRNA for TNF increased from mid-phase to regressing CL and both TNFRI and TNFRII decreased from early to regressing CL. Transcription of mRNA for IFNG was lower in CL from early phase than in mid or regressing luteal phase, while IFNRI expression was not changed. In the early CL, TNF acted to increase P(4) and PGE(2) but decrease PGF(2α) secretion. In the mid luteal phase, TNF increased PGF(2α) secretion and TNF+IFNG decreased PGE(2) secretion. In the regressing luteal phase, TNF, IFNG and TNF+IFNG decreased P(4) and PGE(2) secretion, but TNF and TNF+IFNG increased PGF(2α) secretion by luteal cells. Cell viability was reduced by TNF+IFNG in regressing CL. These data show the presence of cytokines TNF and IFNG, and their receptors, in the equine CL and indicate their potential involvement in regulation of luteal function.

Signal mechanisms of vascular endothelial growth factor and interleukin-8 in ovarian hyperstimulation syndrome: dopamine targets their common pathways.

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is a serious complication of ovarian stimulation with massive ascites, pleural effusion and hemoconcentration. The pathophysiological signal mechanisms of OHSS are still unclear and merit further investigation. METHODS: Various angiogenic cytokines of follicular fluid and ascites of patients with risk of OHSS were measured, and examined for inducing endothelial permeability. These include vascular endothelial growth factor (VEGF), interleukin (IL)-6, IL-8, basic fibroblast growth factor, tumor necrosis factor-alpha, IL-1alpha, IL-1beta and platelet-derived growth factor. We explore the molecular signal pathways of major contributing cytokines in granulosa-lutein cells and endothelial cells possibly involved in OHSS. RESULTS: Neutralizing antibodies of VEGF or IL-8 significantly decreased follicular fluid- and ascites-induced endothelial permeability. Human chorionic gonadotrophin induced VEGF secretion of granulosa-lutein cells through the Sp1 and CREB dependent pathways. IL-8 activated CXCR1/2 of endothelial cells leading to VEGF receptor (VEGFR)-2 transactivation. Both VEGF and IL-8 of follicular fluid enhanced endothelial permeability via VEGFR-2-mediated Rho/Rock activation, actin polymerization and phosphorylations of VE-cadherin and occludin, resulting in opening of adherens junctions and tight junctions. Dopamine (2 microM) inhibited follicular fluid-induced VEGFR-2 signals and endothelial permeability, without diminishing migration and tube formation. CONCLUSIONS: Our results suggest that VEGF and IL-8 secreted from corpora luteae may play major roles in OHSS. Delineation of signal pathways would be helpful for treatment. Dopamine may block VEGF- and IL-8-induced endothelial permeability by inhibiting common VEGFR-2 dependent signals.

Cooperative expression of monocyte chemoattractant protein 1 within the bovine corpus luteum: evidence of immune cell-endothelial cell interactions in a coculture system.

Endothelial cells (EC) of the bovine corpus luteum (CL) are a known source of proinflammatory mediators, including monocyte chemoattractant protein 1 (CCL2) and endothelin 1 (EDN1). Here, a coculture system was devised to determine if immune cells and PGF 2alpha together affect CCL2 and EDN1 secretion by EC. Luteal EC were cultured either alone or together with peripheral blood mononuclear cells (PBMC), and treated without or with PGF 2alpha for 48 h (n = 6 experiments). Coculture of EC with PBMC increased CCL2 secretion an average of 5-fold higher compared with either cell type alone (P < 0.05). Basal secretion of EDN1 by EC was substantial (approximately 2 ng/ml), but was not affected by coculture with PBMC (P > 0.05). EC cocultured with concanavalin A-activated PBMC (ActPBMC) increased CCL2 secretion an average of 12-fold higher compared with controls (P < 0.05), but again, EDN1 secretion was unchanged (P > 0.05). Interestingly, PGF 2alpha did not alter either CCL2 or EDN1 secretion, regardless of culture conditions (P > 0.05). In a second series of experiments (n = 3 experiments), mixed luteal cells (MLC) were cultured alone or with PBMC as described above. Secretion of CCL2 and EDN1 was not affected by coculture or by PGF 2alpha (P > 0.05), but MLC produced less progesterone in the presence of ActPBMC (P < 0.05). Collectively, these results suggest that immune cells and EC can interact cooperatively to increase CCL2 secretion in the CL, but this interaction does not affect EDN1 secretion nor is it influenced by PGF 2alpha. Additionally, activated immune cells appear to produce a factor that impairs progesterone production by luteal steroidogenic cells.

Luteinizing hormone up-regulates the expression of interleukin-1 beta mRNA in human granulosa-luteal cells.

PROBLEM: Previously, we observed that follicular fluid obtained from patients with premature luteinization contained elevated interleukin-1 beta (IL-1 beta) levels. In this study. we aimed to examine the effects of luteinizing hormone (LH) on IL-1 beta expression and IL-1 beta on steroidogenesis in human granulosa-luteal cells. METHOD OF STUDY: Human granulosa-luteal cells were obtained during oocyte retrieval. The cells were treated with either LH or IL-1 beta and subsequently were examined for the level of IL-1 beta transcript. The conditioned media were examined for IL-1 beta protein and steroid hormone levels. RESULTS: LH (250-500 mIU/mL) up-regulated the expression of IL-1 beta mRNA (up to a 4-fold increase over control; P<0.05) in the granulosa-luteal cells. IL-1 beta (5-50 ng/mL) increased the basal, but not LH-dependent, progesterone production from these cells in a dose-dependent manner after 96 and 144 hr of culture (P<0.05). However, an inhibitory effect of IL-1 beta on LH-dependent estradiol production was observed (up to 20% decrease, P<0.05). CONCLUSIONS: LH is capable of stimulating IL-1 beta transcript expression in human granulosa-luteal cells and may regulate ovarian steroidogenesis, at least partly through the activation of IL-1 beta.

Co-expression of integrin-associated protein (IAP/CD47) and its ligand thrombospondin-1 on human granulosa and large luteal cells.

In the present study, we have raised a monoclonal antibody (mAb) designated OG-4 against human granulosa cells (GC). By immunohistochemistry, the expression of OG-4 antigen was observed on human GC and large luteal cells, but not on thecal and small luteal cells. A complementary DNA (cDNA) clone encoding OG-4 antigen was screened and isolated by a panning method using OG-4 mAb from a human corpus luteum (CL) cDNA library that was expressed transiently in COS-7 cells. Nucleotide sequencing revealed that OG-4 antigen was identical to integrin-associated protein (IAP)/CD47 antigen. Subsequent reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the isoform 2 mRNA of IAP is predominantly expressed in human GC and CL. The expression of thrombospondin-1 (TSP-1), which is a ligand for IAP, was also observed in human GC by immunocytochemistry and RT-PCR. Co-expression of IAP and TSP-1 on human GC may suggest that TSP-1 has a physiological role in GC function possibly via IAP in an autocrine fashion.

Immunodetection of cotinine protein in granulosa-lutein cells of women exposed to cigarette smoke.

OBJECTIVE: To detect immunoreactivity to cotinine protein, a major metabolite of nicotine, in granulosa-lutein cells from patients exposed to cigarette smoke, as measured by levels of cotinine in follicular fluid (FF) samples. DESIGN: Controlled immunocytochemical study. SETTING: Hospital IVF-ET program treating infertile patients. PATIENT(S): Twenty-eight women classified by self-reported smoking habits: active smokers (n = 17), passive smokers (n = 4), and nonsmokers (n = 7). INTERVENTION(S): Ovarian hyperstimulation. MAIN OUTCOME MEASURE(S): Grades of immunostaining intensity were assessed in granulosa-lutein cells. Patient scores of cell immunostaining were calculated and regressed on levels of FF cotinine. RESULT(S): Cotinine levels in FF were higher in active smokers than in passive smokers or nonsmokers. Cotinine immunostaining was visualized in the nucleus and cytoplasm of granulosa-lutein cells. Mean grades and mean scores of immunostaining intensity were higher in active smokers than in passive smokers or nonsmokers. There was a strong positive correlation between scores of cell immunostaining and FF cotinine levels. CONCLUSION(S): The association between cotinine expression in granulosa-lutein cells and FF cotinine provides reliable evidence for a dose-related effect. This constituent of cigarette smoke appears to interact directly with and incorporate into these ovarian cells. Our approach seems useful for monitoring ovarian exposure to environmental toxins.

Monocyte chemotactic protein-1 expression in human preovulatory follicles and ovarian cells.

There is a considerable population of macrophages (5-15% of the cells) within the human ovarian follicle at the time of ovulation. Macrophages are also present within the ovarian stroma, mostly near perifollicular capillaries. We hypothesized that macrophage migration in and around the preovulatory follicle is hormonally regulated and that regulation of macrophage migration occurs through local modulation of monocyte chemotactic protein-1 (MCP-1) that chemoattracts and activates monocytes/macrophages. In this regard, we investigated the expression and regulation of MCP-1 in human follicular fluid and in ovarian stromal and granulosa-lutein cell cultures. The concentration of MCP-1 in follicular fluid samples obtained from women prior to the administration of hCG was (n = 4) 90 +/- 27 (mean +/- S.E.) pg/ml; in samples obtained 12 h after the hCG administration it was (n = 3) 135 +/- 23 pg/mL; in follicular fluids obtained 34 h after the hCG administration it was (n = 126) 322 +/- 46 pg/mL (P = 0.007 vs. pre-hCG). The mean ratio of follicular fluid/serum MCP-1 levels was 4.18. There was a correlation between follicular fluid MCP-1 levels and follicular fluid or serum progesterone levels (r = 0.21, P = 0.02; r = 0.29, P = 0.03, respectively). MCP-1 mRNA and the protein were expressed in ovarian stromal and granulosalutein cells in culture and were increased by interleukin-1 alpha and tumor necrosis factor-alpha in a time- and concentration-dependent manner. LH/hCG induced higher levels of MCP-1 mRNA expression and protein production in both cell cultures. We propose that regulation of MCP-1 in ovarian stromal and granulosa-lutein cells by cytokines may play a role in the physiology of periovulatory events.

Interleukin-1 alpha and -beta modulation of luteinized human granulosa cell oestrogen and progesterone biosynthesis.

This study was designed to test the hypothesis that interleukin-1 alpha (IL-1 alpha) and beta directly affect progesterone, and oestradiol production in cultures of purified human granulosa cells. Luteinized granulosa cells were obtained from women during in-vitro fertilization cycles. Granulosa cells with and without associated white blood cells were cultured in the presence of IL-1 alpha and IL-1 beta (0.5-50 ng/ml) for 48 h. Media were changed at 24 h intervals and assayed for progesterone and oestradiol. In separate experiments, granulosa cell viability was assessed with the tetrazolium salt reduction assay, haemocytometer cell counts, and Trypan blue dye exclusion. Our results indicate that progesterone synthesis by basal and human chorionic gonadotrophin (HCG)-stimulated granulosa cells co-cultured with white blood cells was inhibited by 5.0 ng/ml of IL-1 alpha and IL-1 beta at 48 h of culture. In the presence of white blood cells, granulosa cell oestradiol synthesis was inhibited by IL-1 beta but not IL-1 alpha. Oestradiol was inhibited after both 24 and 48 h of culture and was maximally affected by 5.0 ng/ml of IL-1 beta. In contrast, basal and HCG-stimulated oestradiol production by granulosa cells cultured free of white blood cells was inhibited only by IL-1 alpha. IL-1 alpha at 5.0 ng/ml produced maximal inhibition of basal oestradiol (57%) and HCG-stimulated oestradiol (41%) production at 48 h of culture. Gonadal steroid inhibition by IL-1 alpha and IL-1 beta was not mediated through cytotoxic or antiproliferative effects on granulosa cells. Specificity of the granulosa cell response to IL-1 alpha and IL-1 beta was demonstrated by abrogation of steroid inhibition with anti-IL-1 alpha and IL-1 beta neutralizing antibodies. In conclusion, IL-1 alpha directly inhibited the production of oestradiol by human ovarian granulosa cells. IL-1 alpha and IL-1 beta also exerted indirect effects on steroid production via white blood cells that are usually present in granulosa cell cultures if steps are not taken to remove them. These data support the hypothesis that cytokines play an important role in intra-ovarian regulation of steroid biosynthesis.