Oocyte in-vitro maturation: BCL2 mRNA content in cumulus cells reflects oocyte competency.

BCL2-associated X protein (BAX) and B-cell leukaemia/lymphoma gene-2 (BCL2), which are, respectively, pro- and anti-apoptotic proteins of the BCL2 gene family, participate in the mitochondria-dependent apoptosis pathway. A correlation between low incidence of apoptosis in cumulus cells and oocyte maturation has previously been suggested in ovarian stimulation. However, little is known in unprimed ovaries. These authors have investigated whether BAX and BCL2 expression in cumulus cells affects the competency of in-vitro matured oocytes. We have studied 100 cumulus-oocyte-complexes (COC) recovered from unprimed ovaries of 13 women diagnosed with polycystic ovary syndrome (PCOS) and undergoing in-vitro maturation (IVM) with their informed consent. COC were matured for 24 h in a specific maturation medium and the cumulus was stripped from the oocyte. BAX and BCL2 mRNA content was measured in each COC using real-time polymerase chain reaction. We found that BCL2 mRNA expression was significantly higher in cumulus cells associated with mature oocytes than those associated with immature oocytes while BAX mRNA concentrations did not vary in cumulus cells. Regarding fertilization, higher BCL2 mRNA content was found in cumulus cells enclosing fertilized oocytes (0.140 versus 0.075; P = 0.03). These results suggest that BCL2 expression is strongly associated with the ability of oocytes to complete nuclear maturation and to be fertilized.

[Granulosa cells as indicators of oocyte quality]. / Cellules de la granulosa et indicateur de la qualité ovocytaire.

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.

Stromal-derived factor-1 in human tumors recruits and alters the function of plasmacytoid precursor dendritic cells.

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.

Regulated on activation normal T expressed and secreted chemokine is induced by tumor necrosis factor-alpha in granulosa cells from human preovulatory follicle.

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.

Cellular distribution and relative amounts of vascular endothelium growth factor mRNA in granulosa cells from human preovulatory follicles.

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.

Phospholipase C-beta and ovarian sex steroids in pig granulosa cells.

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.

Nongenomic effects of androstenedione on human granulosa luteinizing cells.

This study examines rapid (5-60 s) effects of androgens on the cytosolic free Ca2+ concentration ([Ca2+]i) in human granulosa lutenizing cells. Cells were obtained from human preovulatory follicles, and [Ca2+]i was measured with the use of the Ca(2+)-responsive fluorescent dye fluo-3. Molar concentrations between 100 pmol/L and 1 mumol/L androstenedione increased [Ca2+]i within 5 s after addition to cells. This [Ca2+]i increase resulted from both Ca2+ influx, as shown by the effects of ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and the voltage-dependent Ca2+ channel blocker verapamil, and Ca2+ mobilization from the endoplasmic reticulum, as shown by the effects of thapsigargin. Treatment with pertussis toxin and U-73,122, a specific inhibitor of phospholipase C, abolished the effects of androstenedione on [Ca2+]i. Flutamide, a nuclear androgen receptor antagonist, did not block the increase in [Ca2+]i induced by androstenedione. Testosterone (100 pmol/L to 1 mumol/L) had no effect. This is the first report showing that androstenedione increases [Ca2+]i in granulosa cells. These data provide evidence for the presence in granulosa cells of a novel, short term mechanism of androstenedione action involving voltage-dependent Ca2+ channels in the plasma membrane and phospholipase C activation via a pertussis toxin-sensitive G protein.

Production of ovarian cytokines and their role in ovulation in the mammalian ovary.

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.

Tumor necrosis factor-alpha induces interleukin-6 mRNA and protein in human granulosa luteinizing cells via protein tyrosine kinase without involving ceramide.

This study examines how interleukin-6 (IL-6) expression by human luteinizing granulosa cells is regulated. IL-6 was assayed in culture supernatants, mRNA in cells by in situ hybridization and by a competitive reverse-transcriptase polymerase chain reaction (RT-PCR). TNF alpha (100 pg-1 ng/ml) induced IL-6 mRNA and protein. Phorbol 12-myristate 13-acetate (PMA) (50 nM) mimicked this effect. DibutyrylcAMP (1 mM) and 10 microM forskolin. C2-, C6- and C8-ceramide (15 microM), all had no effect. The inhibitor of protein tyrosine kinase (PTK), genistein (100 micrograms/ml) reduced tumor necrosis factor (TNF) effects. The inhibitors of protein kinase C (PKC) (staurosporine, 10 nM), of phospholipase C (U73122, 2 microM), of phospholipase A2 (PLA2), (indomethacin 30 microM, mepacrin 50 microM, nordihydroguaiaretic acid 10 microM, ONO-RS-082 3,5 microM), none prevented it. Hence, IL-6 is induced by TNF alpha via activation of PTK. Protein kinase A, phosphoinositide and conventional PKC, sphingomyelin and PLA2 pathways are not implicated.

Progesterone triggers rapid transmembrane calcium influx and/or calcium mobilization from endoplasmic reticulum, via a pertussis-insensitive G-protein in granulosa cells in relation to luteinization process.

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.

Ovarian production of IL6 and its potential inhibitory effect on progesterone secretion in Cynomolgus fascicularis.

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.

Macrophage and granulosa interleukin-1 beta mRNA in human ovulatory follicles.

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.

Evidence that a non-steroidal factor from ovine placenta inhibits aromatase activity of granulosa cells in vitro.

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.

Interleukin-6 biosynthesis in human preovulatory follicles: some of its potential roles at ovulation.

In the present work we explored cellular sites of interleukin-6 (IL-6) biosynthesis in human follicular aspirates from patients undergoing in vitro fertilization therapy and the effects of this cytokine on oocyte fertilization and granulosa cell (GC) steroidogenesis. Biological IL-6 activity from 20-40 IU/mL was present in follicular fluids from 22 patients; it was also detected in 10 of 22 supernatants of cultured oocyte-cumulus complexes and in cumulus cell and GC cultures. Biological IL-6 activity in oocyte-cumulus complex cultures was not related to fertilization rates. Total ribonucleic acid was isolated from follicular aspirates and GC-enriched preparations. After reverse transcriptase and polymerase chain reaction cycles using oligonucleotide primers corresponding to known cDNA sequences for IL-6, a 126-basepair band characterized the amplification product of IL-6 transcripts on gel electrophoresis. To localize IL-6 messenger ribonucleic acid, in situ hybridization analysis was performed using a [35S]IL-6 riboprobe. The distribution of transcripts was more dense (15% vs. 3% stained cells) in GC-enriched preparations, which contained more than 95% GCs, than in original follicular preparations, which contained 20-40% viable GCs; it was not significantly modified by the presence of macrophage contaminants. The expression of IL-6 protein was assessed by positive immunohistological stainings. Biological IL-6 activity was higher, and in situ hybridization signals were more dense and more intense in 24-h GC cultures than in 72-h GC cultures, suggesting that IL-6 biosynthesis was transiently induced. Under experimental conditions of low IL-6 endogenous levels in cultures, adding recombinant human IL-6 from 10-200 IU/mL had no effect on progesterone production or aromatase activity in GC cultures free of macrophages, whereas in GC cultures including macrophage contaminants, stimulatory effects on basal and hCG-stimulated progesterone production and on basal and FSH-stimulated aromatase activities were observed. The present study provides strong support for the view that IL-6 is produced by GCs in the preovulatory follicle at the time of ovulation. In addition, we showed that IL-6 might be an intraovarian regulatory factor concerned with steroidogenesis.

Comparative IL-6 effects on FSH and hCG-induced functions in porcine granulosa cell cultures.

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.

Relationships between in vitro progesterone production by granulosa-luteal cells and certain characteristics of human stimulated cycles.

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.