Human Luteinized Granulosa Cells Research Articles

Human granulosa-lutein cell in vitro production of progesterone, inhibin A, inhibin B, and activin A are dependent on follicular size and not the presence of the oocyte

To investigate inhibin A, inhibin B, activin A, and P production by cultured granulosa cells (GCs) and what relationship this hormone production has to fertility. Luteinized GCs from individual follicles were cultured, and inhibin A, inhibin B, activin A, and P production were measured by ELISA at 24 and 72 hours. Research laboratory and university hospital. Fifteen women who undertook an IVF-ICSI program, yielding 58 follicles. Individual follicular aspiration and preparation of GCs for culture. Inhibin A, inhibin B, activin A, and P production; oocyte retrieval; and fertility outcome. Inhibin A, inhibin B, and P continued to be secreted by GCs in vitro, and activin A levels were detected only marginally in 56% of cultures. The rate of production also was dependent on the size of follicle from which the GCs originated but not on oocyte presence or ability to fertilize. Granulosa cell stimulation with hCG had no effect on inhibin A but increased P and decreased inhibin B production. A marked effect of luteal differentiation appears to be the inhibition of inhibin B production in response to hCG stimulation. Luteinized GC function, with respect to inhibins, activin A, and P production, was not influenced by the presence or absence of an oocyte and did not correlate with fertility outcome. However, follicle size did influence rates of local hormone production.

Müllerian-inhibiting substance inhibits cytochrome P450 aromatase activity in human granulosa lutein cell culture

To investigate the effects of Müllerian-inhibiting substance (MIS) on cytochrome P450 aromatase (CYP19) gene expression in cultured human granulosa lutein cells (GLC). In vitro primary cell culture study. Academic research laboratory and hospital-based fertility center. Eight normo-ovulatory patients undergoing IVF procedures due to male factor or tubal infertility. Serum and follicular fluid (FF) collected and stored at -80 degrees C until assayed. Granulosa lutein cells were harvested from follicular aspirates obtained during oocyte retrieval and cultured for 7 days with media in the presence or absence of MIS (10 ng/mL) or FSH 0.2 IU/mL. Serum and FF levels of E2 and MIS, and E2 production by GLC in culture. Levels of CYP19 mRNA in cultured GLC were determined by quantitative polymerase chain reaction (PCR) and CYP19 protein by Western blot. Statistical comparison used ANOVA and post hoc Tukey tests. Follicle-stimulating hormone significantly increased E2 production in cultured GLC compared with control. The increase in E2 production is associated with higher levels of CYP19 mRNA and protein in GLC. The presence of MIS significantly inhibited FSH-induced E2 production, with concomitant reduction in CYP19mRNA and protein levels. Müllerian-inhibiting substance inhibits FSH augmentation of CYP19 enzyme activity and CYP19 gene expression in GLC. These findings may help to explain the association of high MIS levels and low FF E2 levels reported in women with polycystic ovary syndrome (PCOS).

Luteogenic Hormones Act through a Vascular Endothelial Growth Factor-Dependent Mechanism to Up-Regulate α 5β 1 and α vβ 3 Integrins, Promoting the Migration and Survival of Human Luteinized Granulosa Cells

The formation of the corpus luteum (CL) is critical for the establishment of a successful pregnancy. After ovulation, the CL develops from the remnants of the ovulated ovarian follicle. This process, which involves varying cell-matrix interactions, is poorly characterized. To understand the role and potential regulation of cell-matrix interactions in the formation of the CL, we investigated the expression and activity of the matrix protein fibronectin (FN) and several of its integrin receptors on luteinized granulosa cells (GCs). In situ, FN and several FN-binding integrins were detected around luteinizing GCs during the early luteal phase, although expression declined in the late luteal phase. In vitro, GCs released FN, and stimulation of these cells with human chorionic gonadotropin increased the surface expression of FN, alpha(5)beta(1), and alpha(v)beta(3). Up-regulation of these proteins on GCs was reproduced by stimulation with vascular endothelial growth factor (VEGF) and was inhibited by anti-VEGF antibody. Lastly, expression of alpha(5)beta(1) and alpha(v)beta(3) mediated adhesion to FN, facilitated migration, and prevented apoptosis. These data suggest that in vivo luteogenic hormones, in part through a VEGF-dependent mechanism, stimulate selected integrin-matrix adhesive interactions that promote the motility and survival of GCs and thus contribute to the formation and preservation of the CL.

Inhibition of progesterone production in human luteinized granulosa cells treated with LXR agonists

Progesterone production by luteal cells is dependent on the supply of cholesterol by lipoproteins. The aim of this study was to determine whether the liver X receptors (LXRs) contribute to cholesterol homeostasis and progesterone secretion in human luteinized granulosa cells. Cells were isolated from follicular aspirates of patients undergoing in vitro fertilization. Luteinization was induced by a 7-day treatment with human chorionic gonadotrophin. LXR beta was expressed at higher levels than LXR alpha in granulosa cells and its expression was increased during luteinization. Treatment of luteinized granulosa cells by LXR agonists induced a significant time- and dose-dependent reduction in progesterone secretion (50% reductions after a 7-day treatment with 1-microM of either GW3965 or T0901317). mRNA levels of steroidogenic genes including steroidogenic acute regulatory protein and P450 side-chain cleavage were only moderately affected by LXR activation, with a significant reduction that was observed at 10 microM agonist concentration. Cellular cholesterol was markedly reduced after treatment with LXR agonists as a result of an increased cholesterol efflux that was related to the induction of LXR target genes (ABCA1, ABCG1, apo E, PLTP). Our study identifies LXRs as new, key actors contributing to regulation of cholesterol metabolism and steroidogenesis in luteinized granulosa cells.

Distinct responses of human granulosa lutein cells after hCG or LH stimulation in a spheroidal cell culture system

The growth and development of the corpus luteum (CL) is regulated by gonadotropic hormones. It is formed by granulosa cells (GCs), theca cells, and endothelial cells, and is the primary source of circulating progesterone. During early pregnancy only human chorionic gonadotropin (hCG) but not luteinizing hormone (LH) extends the life span of the CL, although hCG and LH interact with the same receptor and have similar actions on the CL. In this study a recently by our group established spheroidal GC culture assay served as a model of CL development on which we compared the actions of the gonadotropic hormones LH and hCG. To find out which signal pathways take part in the hormonal regulation of GC we stimulated GC-spheroids with modulators of protein kinases A and C dependent signaling cascades and determined their impact on sprout forming activity in GC. Our results indicate that PKA-dependent signaling pathways play a major role in mediating the hormonal-induced signaling cascades leading to sprouting in GC. Furthermore, this study strongly indicates that the different effects of hCG and LH in the maintenance of the CL may be reasoned in different signal transduction pathways triggered by hCG or LH.

Effects of trichlorfon on progesterone production in cultured human granulosa–lutein cells

Trichlorfon, a widely used organophophorus pesticide, has been reported to disrupt reproductive function in human and animal. However, the mechanisms have not been clearly elucidated. In the present study, the effects of trichlorfon on the biosynthesis of progesterone in the primary human granulosa–lutein cells (hGLCs) and the related pathway were investigated. Results showed that progesterone production in hGLCs treated with trichlorfon decreased significantly while cell viability was not affected. Trichlorfon also inhibited FSH-stimulated progesterone production, and this inhibition could not be reversed by 8-bromo-cAMP. However, trichlorfon did not affect the intracellular cAMP contents in the basal and FSH-stimulated conditions. These results suggested that the site in the steroid biosynthesis pathway affected by trichlorfon occurred downstream of PKA activation in hGLCs. Furthermore, our results found that 22(R)-hydroxycholesterol (22R-HC) could remove the inhibitory action of trichlorfon on progesterone biosynthesis, indicating that trichlorfon caused a disruption of cholesterol transport across mitochondrial membranes, which was further confirmed by the observation that trichlorfon dose-dependently inhibited the mRNA level of the steroidogenic acute regulatory protein (StAR). These results suggested that trichlorfon inhibited steroidogenesis in hGLCs by reducing StAR gene expression, which may further contribute to the pathogenesis of trichlorfon-induced reproductive dysfunction.

Regulation of Proliferation and Steroidogenesis in Human Granulosa Cells

Background and Aims : Human follicles selected for further development are thought to receive precise signals from gonadotropins and locally produced growth factors for survival. In this experiment, the mechanisms of the regulation of proliferation and steroidogenesis by GDF-9 and Br-cAMP in human ovarian cells were investigated. Methods : SVOG cells were cultured and treated with GDF-9 and Br-cAMP. The expression of HOXA7 and cofactors were examined by Western blot analysis and/or RT-PCR. The cell proliferation was measured by thymidine incorporation assay. Results : Treatment with GDF-9 resulted in a significant increase of cell proliferation. Although the treatment with Br-cAMP increased the expression of StAR protein, this treatment resulted in a significant decrease of cell proliferation compared with the control and no change in HOXA7 or its cofactors. Conclusion : The present study demonstrated that GDF-9 had a stimulatory effect and cAMP had an inhibitory effect on the proliferation of SVOG cells. These results may reflect the characteristics of this human cell line as luteinized granulosa cell. Therefore, this culture system will be useful for studying the effects of local factors and gonadotropin on the regulation of human luteinized granulosa cells in the future.

Expression of Eph Receptor Tyrosine Kinases and their Ligands in Human Granulosa Lutein Cells and Human Umbilical Vein Endothelial Cells

Corpus luteum development is regulated by gonadotropins and accompanied by extremely rapid vascularization of the avascular granulosa cell compartiment by endothelial cells (EC). The proliferation of Granulosa cells (GC) and EC is a complex interplay and takes place in a spatially and temporarily coordinated manner. The erythropoietin-producing hepatoma amplified sequence (Eph) receptors and their ligands-the ephrins- are a recently detected family of membrane located protein tyrosine kinases which play a crucial role in the growth and development of nerve and blood vessel network. We report about the mRNA expression pattern of Ephs and their ligands in human GC, in human EC, and in carcinoma cell lines OvCar-3 and Hela. The mRNA of EphA4, EphA7, ephrinA4, ephrinB1 and ephrinB2 was detected in GC and EC, while EphA2 was expressed only in GC. The expression of various Ephs and ephrins did not change in GC after stimulation with human chorion gonadotropin. Our study analyzes for the first time the expression of the complete human Eph/ephriny-system in GC and in EC. The remarkable similarity between these two cell types supports the theory of a functional relationship of EC and GC. In addition, it was shown that hCG is not a major determinant of Eph/ephrin regulation in GC.

Ethanolic extracts of black cohosh ( Actaea racemosa) inhibit growth and oestradiol synthesis from oestrone sulphate in breast cancer cells

Extracts of black cohosh ( Actaea racemosa) and soy are used as ‘natural’ alternatives to conventional hormone replacement therapy (HRT) and there is some evidence that soy may protect against breast cancer by inhibiting the production of active oestrogens. This study compares the action of ethanolic extracts of black cohosh (BCE) and genistein on growth and enzyme activity in MCF-7 and MDA-MB-123 breast cancer cells. BCE inhibited growth at the two highest doses tested, i.e. 50 and 100 μg/ml, whilst genistein stimulated growth in the oestrogen receptor positive (ER +) MCF-7 cells, but at high doses it inhibited growth in both cell lines. BCE did not affect the conversion of androstenedione to oestradiol and only the highest doses (50 and 100 μg/ml) significantly inhibited the conversion of oestrone to oestradiol in MDA cells. In contrast, BCE induced a dose-dependent inhibition of the conversion of oestrone sulphate to oestradiol in both cell lines, whilst in human granulosa lutein (GL) cells enzyme activity was only inhibited at the highest dose of BCE. Genistein had no significant effect on enzyme activity in breast cancer cells and like BCE only the highest doses (10 and 50 μM) inhibited enzyme activity in human GL cells. In vivo genistein may have growth stimulatory effects on breast tissue but BCE not only inhibits growth but inhibits the conversion of oestrone sulphate to active oestradiol, considered by some, to be the preferred pathway of oestradiol synthesis in breast tissue.

Regulation of angiogenic factors TIMP-1, CTGF and Mac25 by chorionic gonadotropin in human granulosa lutein cells

As prerequisite for development and maintenance of many diseases angiogenesis is of particular interest in medicine. Pathologic angiogenesis takes place in chronic arthritis, collagen diseases, arteriosclerosis, retinopathy associated with diabetes, and in particularly in cancers.

Role for Prostaglandins in the Regulation of Type 1 11β-Hydroxysteroid Dehydrogenase in Human Granulosa-Lutein Cells

11beta-hydroxysteroid dehydrogenase (11betaHSD) enzymes regulate glucocorticoid availability in target tissues. 11betaHSD1 is the predominant isoenzyme expressed and active in human granulosa-lutein (hGL) cells. This study investigated the effects of pharmacological inhibitors of prostaglandin (PG) synthesis on 11betaHSD1 activities and expression in hGL cells. The consequences for 11betaHSD1 of increasing exposure of hGL cells to PGs, either by treatment with exogenous PGs or by challenging cells with IL-1beta, were also assessed. Suppression of basal PG synthesis using four different inhibitors of PG H synthase enzymes [indomethacin, niflumic acid, meclofenamic acid (MA) and N-(2-cyclohexyloxy-4-nitorophenyl) methane sulfonamide (NS-398)] each resulted in significant decreases in both cortisol oxidation and cortisone reduction. Both activities of 11betaHSD1 were suppressed by up to 64+/-6% (P<0.05). Over 4 and 24 h, neither MA nor NS-398 affected the expression of 11betaHSD1 protein, suggesting enzyme regulation by PGs at the posttranslational level. When cells were cotreated for 4 h with PGHS inhibitors plus 30 nm PGD2, PGF2alpha, or PGE2, each PG overcame the suppression of cortisol oxidation by indomethacin or MA. Treatment of hGL cells with IL-1beta increased the concentrations of both PGE2 and PGF2alpha, accompanied by a 70+/-25% increase in net cortisol oxidation. All three responses to IL-1beta were abolished when cells were cotreated with MA. These findings suggest a role for PGs in the posttranslational regulation of 11betaHSD1 activities in hGL cells.

O-204: Angiotensin II (Ang II) regulates apoptosis in human granulosa-lutein cells (GL)

The human ovary has a complete and functional gonadotrophin-sensitive renin-angiotensin system and both types of AngII receptors, AT1 and AT2, are present in ovarian cells. We have shown that AngII is present in high concentrations in granulosa cells and follicular fluid of human preovulatory follicles and in luteal cells. AngII regulates ovulation and modulates steroidogenesis in rat luteal cells and human GL cells. Other investigators have shown that AngII regulates luteal cell apoptosis. Recently, we have shown that apoptosis of human GL cells correlates with several indicators of IVF outcome, such as embryo fragmentation and pregnancy rate, and is regulated by intraovarian growth factors. The aim of the present study was to test whether AngII could induce in vitro apoptosis of gonadotrophin-exposed human IVF GL cells. Following AngII exposure, apoptosis was assessed by caspace expression. Controls included the non-specific angiotensin receptor blocker Saralasin and the specific AT2 receptor (AT2-R) blocker CGP-42112A. GL cells from 87 IVF-intra-cytoplasmic sperm injection (ICSI) cycles were analyzed. Ovarian stimulation was performed using recombinant FSH and LH or HMG. The pregnancy rate for these cycles was 56.32%. Following oocyte recovery GL cells were collected from pooled follicular fluid (FF), washed and purified using a 50% percoll gradient and anti-CD45-coated magnetic beads. Cells were cultured without serum for 48 hours in the presence or absence of 10-11, 10-9, 10-7M AngII, with and without Saralasin10-5 or CGP-42112A 10-5M. The percentage of apoptotic cells was determined under a fluorescence microscope by staining with 10μm caspace FITV-VAD-FMK (a fluorescent marker for activated caspaces) and 0.5μg/ml propidium iodide. Statistical analysis was carried out using the student-t test. AngII in concentrations present in human follicle fluid caused increased apoptosis of GL cells compared to untreated control cells (10-11M p=0.312; 10-9M p<0,05; 10-7M p<0,05, respectively). This effect was partially inhibited by both Saralasin and CGP-42112A (18.15% and 33.01%, respectively, p<0,05). In these preliminary studies on human GL cells Ang II at concentrations that are present in FF from human IVF cycles (J Soc Gynecol Investig. 1994 1:118-27) is proapoptotic. The partial blockade by angiotensin receptor antagonists is consistent with a receptor-mediated action. Ovarian AII has been linked to fertility, steroidogenesis and polycystic ovarian syndrome. Further studies are underway to determine the relevance of these results to the outcome of IVF cycles and other clinical scenarios.

P-731: Intra-ovarian mechanisms in diminished ovarian reserve (DOR): Exploring apoptotic pathways using gene expression analysis in human luteinized granulosa cells

DOR and poor pregnancy outcomes are linked to increased apoptosis in human preovulatory granulose cells (GC), though molecular pathways involved are still poorly understood. We explored the expression of genes involved in the apoptotic cascade, specifically the anti-apoptotic Bcl-2, and the pro-apoptotic FAS and BAX, in GCs obtained from women with DOR versus normal ovarian reserve undergoing IVF-ET in an academic infertility center.

Binding of progesterone to cell surfaces of human granulosa-lutein cells

Progesterone is produced by granulosa cells under the influence of luteinizing hormone. Nuclear progesterone receptors have been found in rat granulosa cells. Human granulosa-lutein cells rapidly respond to progesterone with an increase in intracellular calcium suggesting the existence of a nongenomic mechanism. This study was conducted to determine whether binding of progesterone to granulosa cells could occur at the membrane. Granulosa cells were obtained from an in vitro fertilization program and examined immunohistochemically with an antiserum to membrane progesterone receptors. Approximately 14–70% of freshly harvested or cultured granulosa cells of six patients showed a positive reaction to the antiserum, limited to the cell membrane. Western blot analysis of homogenates of granulosa cells and a granulosa cell tumour confirmed the presence of progesterone receptors A, B and C and low amounts of a putative membrane receptor. These results demonstrate that the plasma membranes of human granulosa cells possess binding components for progesterone which may be involved in its nongenomic mechanism of action.

Influence of tumor necrosis factor-α on estradiol, progesterone, insulin-like growth factor-II, and insulin-like growth factor binding protein-1, 2, and 3 in cultured human luteinized granulosa cells

Objective The objective was to investigate the influence of tumor necrosis factor (TNF)-α on estradiol, progesterone, insulin-like growth factor (IGF)-II, and insulin-like growth factor binding protein (IGFBP)-1, 2, and 3 in cultured human luteinized granulosa cells. Study design Human luteinized granulosa cells were obtained from follicular fluid by transvaginal oocyte aspiration from infertile patients undergoing controlled ovarian hyperstimulation (COH) for in vitro fertilization (IVF). The cells were cultured for 72 h with TNF-α at concentrations of 1.0, 10.0, and 100.0 ng/ml. The cells not treated with TNF-α served as controls. Radioimmunoassay (RIA) and reverse transcription-polymerase chain reaction (RT-PCR) were used to examine the influence of TNF-α on estradiol, progesterone, IGF-II, and IGFBP-1, 2, and 3. Results were analyzed using the Kolmogorov–Smirnov test and analysis of variance (ANOVA). Statistical significance was defined as p < 0.05. Results The concentrations of progesterone seemed to decrease as the concentrations of TNF-α increased and the concentration of progesterone in the 100.0 ng/ml TNF-α group was significantly lower than that in the control and other TNF-α groups. The expressions of IGF-II mRNA in the 10.0 and 100.0 ng/ml TNF-α groups were significantly lower than that in the control group. The expressions of IGFBP-2 mRNA seemed to be decreased in the 10.0 and 100.0 ng/ml TNF-α groups compared with that in the control group, but there were no statistical significances. Conclusion TNF-α may play a role as a regulator of human ovarian physiology by modulating the IGF systems in luteinized granulosa cells.

Ion channels of primate ovarian endocrine cells: identification and functional significance

Ion channels are crucially involved in cellular functions, but little is known about molecular identity, subunit composition and the specific role of ion channels in ovarian endocrine cells in human and nonhuman primates. Using human luteinizing granulosa cells, a few groups have started to address these questions and have begun to show molecular identity of ion channels, electrophysiological functions and the relationship to hormone production, as well as regulation by hormones and intraovarian factors. Functional ion channels that have been identified so far include T- and L-type Ca2+ channels (Cav3.2, Cav1.2), a voltage-dependent Na+ channel (Nav1.7), as well as voltage- (Kv4.2) and Ca2+-dependent K+ channels (BKCa). Since all these ion channels were found to be involved in steroid hormone synthesis and are expressed by endocrine ovarian cells in human and nonhuman ovary, it has been proposed that they are physiological key molecules for ovarian function. Furthermore, they may be novel targets for modulating ovarian functions.

In vitro exposure to environmental tobacco smoke induces CYP1B1 expression in human luteinized granulosa cells

Women smokers and women exposed to environmental tobacco smoke (ETS) have reduced ovarian function as evidenced by an earlier menopause, reduced follicular numbers, decreased levels of circulating estradiol, and decreased conception rates; however, the mechanism of action of altered ovarian function by ETS is poorly understood. The direct effects of ETS were evaluated using human luteinized granulosa cells (HLGCs) exposed to ETS in primary cell culture. Exposure to ETS caused a decrease in both estradiol and progesterone production. There was a concentration dependent increase in CYP1B1 gene and protein expression without a change in catechol- O-methyltransferase (COMT) expression. This is the first report of CYP1B1 induction secondary to ETS exposure in cells from the human ovary. CYP1B1 metabolizes both endogenous estrogens and polyaromatic hydrocarbons in ETS to a variety of reactive species and may contribute to the complex effects of ETS on ovarian function.

HCG‐dependent regulation of angiogenic factors in human granulosa lutein cells

As prerequisite for development and maintenance of many diseases angiogenesis is of particular interest in medicine. Pathologic angiogenesis takes place in chronic arthritis, collagen diseases, arteriosclerosis, retinopathy associated with diabetes, and particularly in cancers. However, angiogenesis as a physiological process regularly occurs in the ovary. After ovulation the corpus luteum is formed by rapid vascularization of initially avascular granulosa lutein cell tissue. This process is regulated by gonadotropic hormones. In order to gain further insights in the regulatory mechanisms of angiogenesis in the ovary, we investigated these mechanisms in cell culture of human granulosa lutein cells. In particular, we determined the expression and production of several angiogenic factors including tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), Leptin, connective tissue growth factor (CTGF), meningioma-associated complimentary DNA (Mac25), basic fibroblast growth factor (bFGF), and Midkine. In addition, we showed that human chorionic gonadotropin (hCG) has distinct effects on their expression and production. hCG enhances the expression and production of TIMP-1, whereas it downregulates the expression of CTGF and Mac25. Furthermore it decreases the expression of Leptin. Our results provide evidence that hCG determines growth and development of the corpus luteum by mediating angiogenic pathways in human granulosa lutein cells. Hence we describe a further approach to understand the regulation of angiogenesis in the ovary.

Effect of gonadotropin-releasing hormone agonist and antagonist on proliferation and apoptosis of human luteinized granulosa cells

The GnRH agonist leuprolide acetate (LA) inhibited DNA synthesis in epidermal growth factor-stimulated human granulosa luteinized cell cultures. This effect was blocked by the prior addition of a GnRH antagonist antide (ANT), and this compound per se was able to produce a stimulatory effect of DNA synthesis on basal conditions. Leuprolide acetate produced an increase in the percentage of apoptotic cells, and when these two factors were co-incubated, ANT blocked the apoptotic effect produced by LA.

Rapid effects of pesticides on human granulosa-lutein cells

Following our previous demonstration that p,p'-DDE (dichlorodiphenylchloroethylene), at environmentally relevant concentrations, can rapidly increase intracellular calcium [Ca2+]i concentrations in human granulosa-lutein cells, we examined whether other pesticides, such as Kepone, o,p-DDE and methoxychlor, have similar effects. Cultured human granulosa-lutein cells were loaded with Fura-2 AM, and changes in [Ca2+]i concentrations within small areas of single cells were studied with a dynamic digital Ca2+ imaging system. Kepone, at concentrations of 0.2-2 nmol/ml, consistently increased [Ca2+]i concentrations 2-6 times higher than baseline values within minutes of exposure. Methoxychlor at concentrations of 2.8-280 nmol/ml failed to alter [Ca2+]i levels consistently in cells from 10 patients. However, at 0.28 and 1.4 nmol/ml, increases in [Ca2+]i concentrations could be elicited by methoxychlor. The isomer o,p-DDE at 3 nmol/ml increased [Ca2+]i in granulosa cells of 11/20 patients. Pertussis toxin treatment inhibited the [Ca2+]i increases induced by estradiol, p,p'-DDE, o,p-DDE and methoxychlor, but not by Kepone or progesterone, indicating that Kepone and progesterone may act through an insensitive G protein-coupled receptor. The [Ca2+]i increases induced by Kepone also occurred in Ca2+-free medium, suggesting that [Ca2+]i mobilization occurred from the smooth endoplasmic reticulum. Thapsigargin and cyclopiazonic acid, two inhibitors of the endoplasmic reticulum Ca2+ pump, also stimulated [Ca2+]i increases but did not inhibit the Ca2+ response to all the pesticides. These results demonstrate that pesticides can have a rapid effect on human granulosa-lutein cells, and a nongenomic mechanism of action is suggested.