1 Department of Obstetrics and Gynecology, Reproductive Biology Division and 2 Department of Medicine, McMaster University, Health Sciences Centre, Hamilton, Ontario, Canada L8N 3Z5
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, McMaster University Medical Centre, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5. Email: younglai{at}mcmaster.ca
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Abstract |
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Key words: calcium response/estradiol/granulosa cells/progesterone
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Introduction |
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Chicken and porcine granulosa cells can respond to estradiol, but not to progesterone or androgens, with a rapid increase in [Ca2+]i, and the source of Ca2+ for these cells was shown to be exclusively intracellular (Morley et al., 1992). Granulosa cells from diethylstilbestrol-treated immature rats failed to respond to estradiol with any increase in Ca2+ (Morley et al., 1992
). On the other hand, granulosa cells from equine chorionic gonadotrophin-treated, 23-day-old rats or spontaneously immortalized rat granulosa cells do show a rapid response to progesterone (Peluso et al., 2002
; Peluso 2004
). In rat granulosa cells, progesterone caused a decrease in [Ca2+]i and inhibited mitosis (Peluso et al., 2002
). This is in contrast to the studies by Morley et al. (1992)
who were not able to detect any Ca2+ changes in chicken or porcine granulosa cells when treated with progesterone. In another study, porcine granulosa cells were found to respond with increases in [Ca2+]i when stimulated with estradiol, progesterone and androstenedione (Lieberherr et al., 1999
). The current study was undertaken to determine whether estradiol and progesterone can induce changes in [Ca2+]i in human granulosa-lutein cells and whether these changes could be influenced by various antagonists.
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Materials and methods |
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Source of granulosa cells
Patients were treated with a long luteal protocol of GnRH agonist (Lupron; Abbott Laboratories, Montreal, QC, 0.5 mg per day for 1014 days) and recombinant FSH (1285 ampoules, 75 IU per ampoule Gonal F; Serono Canada Ltd, Oakville, ON) followed by HCG (Profasi; Serono). After removal of oocytecumulus complexes, the remaining follicular aspirates were transported to the research laboratory in polypropylene tubes and the granulosa cells were isolated and cultured. After 37 days in culture, areas containing small luteinized cells, characterized by the cytoplasmicnuclear ratio, were chosen for imaging.
Incubation medium conditions for imaging
Granulosa cells in culture were always exposed to 12 mmol Ca2+/l except for the experiments requiring Ca2+-free conditions, where the medium was replaced by the Ca2+-free isotonic physiological medium containing 0.1 mmol EGTA/l immediately prior to the measurement. Although distilled and de-ionized water were used for the preparation of solutions, contaminating Ca2+ from containers and other chemicals may contribute up to 10 µmol Ca2+/l. Therefore, EGTA 0.1 mmol/l was always included in the Ca2+-free medium.
Digital dynamic fluorescence ratio measurements
Changes in Ca2+ concentration were measured using a dynamic digital Ca2+ imaging system (Image-I/FL, Universal Imaging Corporation) with a Zeiss lamp (XBO 100 W/DC) coupled to a Zeiss inverted microscope (Zeiss IM 35) with a 100x oil immersion lens and a numerical aperture of 1.25, as previously described (Low et al., 1997). Images were integrated and collected by a Pulnix camera (TM-720, maximal at 3 s/frame) initially at a speed of 15 s/frame. In general, 34 probes covering an area of five pixels each were placed on different spatial areas of cells, usually near the plasma membrane and over the nuclear region. Changes in the fluorescence ratio were recorded and the data stored. Since the probes covered small areas of interest within the cell, quantitation of calcium changes was not attempted. Quantitation would vary in the different regions since our previous findings suggested that there are spatial and temporal differences within the cell upon stimulation with agonists (Kwan et al., 2003
; Younglai et al., 2004
). Images were saved and, in the event that some areas of interest showed oversaturation of colour during processing, the sequences were rerun with new areas of interest. Cells from at least three different patients had to show a response before the results were accepted as meaningful and quoted. Representative patterns of response are shown in the figures. In some instances, the positions of the probing windows were changed from the original placements to capture the spatial changes in Ca2+ concentrations. Ethical approval was obtained from the institutional research ethics board for this work, and patients signed a consent form agreeing to donate their excess cells for research.
Chemicals and reagents
Estradiol and progesterone were purchased from Steraloids Inc., Newport, RI. Fluvestrant (ICI 182,780) was obtained from Tocris Cookson Inc., Ellisville, MO. Tamoxifen and mifepristone (RU 486) were purchased from Sigma-Aldrich Chemicals, Oakville, ON. All tissue culture supplies were purchased from Life Technologies, Burlington, ON. Fura-2 acetoxymethyl ester (fura-2AM) was obtained from Molecular Probes, Portland, OR. The steroids were dissolved in dimethylsuphoxide (DMSO) and further diluted with culture medium to achieve a final concentration of 1 µg/ml which have been shown to be the optimal concentration for estradiol or progesterone to elicit effects in human sperm (Luconi et al., 1998
; Wennemuth et al., 1998
). In rat granulosa cells, progesterone at a concentration of 200 ng/ml was effective in altering [Ca2+]i uptake (Peluso et al., 2001
).
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Results |
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Discussion |
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The probes for detection of [Ca2+]cyt changes in individual cells each covered an area of five pixels. Up to nine probes have been used in the past to follow the progression of Ca2+ ion changes through the cell membrane to the nucleus (Kwan et al., 2003). Thus changes can be detected adjacent to the cell membrane, over intracellular organelles or in the nucleus, and the magnitude of responses in individual cells would depend on the location of the probes. Usually two probes were used per cellone near the membrane and the other near the nucleus. The differences in responses could also be due to patient variation as well as cell type. Small and large cells derived from the mural, cumulus or theca layers in the corpus luteum have different abilities to produce progesterone (Lemoin and Mauleon, 1982
).
Non-specific effects of supraphysiological doses of steroids have been described (Losel et al., 2003). The concentrations of estradiol and progesterone used are consistent with levels commonly found in mature human antral follicles (Kreiner et al., 1987
; Yie et al., 1995
) and used for demonstrating non-genomic effects in human spermatozoa (Wennemuth et al., 1998
; Luconi et al., 2001
). We have also used 10-fold concentrations of non-active steroids such as estriol, which has a similar structure to estradiol, to show the absence of non-specific effects.
The genomic estrogen receptors have been located in human granulosa cells (Suzuki et al., 1994; Saunders et al., 2000
; Jakimiuk et al., 2002
), but not in corpus luteum which contained progesterone receptors (Suzuki et al., 1994
). Genomic progesterone receptors have also been detected in human corpus luteum cells by immunohistochemistry (Maybin and Duncan, 2004
). Although there have been suggestions that the genomic and membrane receptors may have some similarities (Saner et al., 2003
; Sak and Everaus, 2004
) and may arise from a single transcript (Razandi et al., 1999
), there is no evidence to date on the nature of the human granulosa-lutein membrane receptor for estradiol or progesterone. In addition, there may be multiple classes of proteins which can function as non-genomic steroid receptors (Watson and Gametchu, 2003
), and recent evidence suggests that these membrane receptors may be G protein-like-coupled receptors (Edwards, 2005
).
The different patterns of [Ca2+]cyt responses observed with estradiol and progesterone suggest that there are distinct and separate mechanisms by which these agonists act on the granulosa-lutein cell. The [Ca2+]cyt response to estradiol in Ca2+-free medium suggests that Ca2+ is mobilized from intracellular stores such as the smooth endoplasmic reticulum, and Ca2+ influx could also occur. The Ca2+ response to progesterone, however, suggests that the mechanism by which progesterone acts is different from that of estradiol, in that intracellular sources are not utilized. The failure of the cells in the presence of progesterone to respond on addition of Ca2+ to the medium was unexpected. This suggests that the membrane factors through which progesterone acts are more sensitive to the destabilizing effect of the lack of calcium (Webb and Bohr, 1978) and that calcium is required for restoration of membrane stability (Ou et al., 1997
). In porcine granulosa cells, on the other hand, progesterone triggers rapid transmembrane Ca2+ influx and/or calcium mobilization from endoplasmic reticulum (Machelon et al., 1996
). The concentration of Ca2+ within the cell is controlled by two general mechanisms: (i) entry from extracellular fluid by voltage-operated channels; or (ii) release from endoplasmic reticulum by capacitative calcium entry which activates the store-operated channel permitting influx from the extracellular fluid (Brini and Carafoli, 2000
). Calcium oscillations have been shown to be required for cell division (Swann et al., 2004
) and it is possible that these oscillations trigger mitosis in the cultured granulosa cells. Calcium oscillations also represent a physiological mechanism to prevent a rapid rise of cytosolic Ca2+ concentration to toxic levels (Miyazaki, 1995
; Bootman et al., 2001
). Further studies with the use of inhibitors are required to eludicate the different intracellular mechanisms involved in estradiol- and progesterone-induced Ca2+ fluxes in human granulosa-lutein cells.
Although a previous communication revealed that human luteinizing granulosa cells did not show a [Ca2+]cyt response to testosterone as they did to androstenedione (Machelon et al., 1998), in four of six patients we found that testosterone could induce [Ca2+]cyt uptake in granulosa-lutein cells. In addition, cells from only four of eight patients responded to androstenedione. This may reflect patient variability or possible high levels of androgens or estrogens produced during stimulation protocols, attenuating the effects of these steroids. A similar attenuation has been found in estrogen-treated microvessels (Kakucs et al., 2001
). Moreover, testosterone at a concentration of 105 mol/l can induce [Ca2+]cyt influx in chicken granulosa cells (Morley et al., 1992
) as well as activated T cells (Benten et al., 1997
). This observation with testosterone is validated further by the absence of [Ca2+]cyt responses to the structurally similar steroids, DHEA and dihydrotestosterone.
Neither DMSO, estrone, estriol, pregnenolone, DHEA or dihydrotestosterone stimulated Ca2+ uptake in the human granulosa-lutein cells. The use of these control steroids, while not eliciting increases in [Ca2+]cyt concentrations, raised the possibility that these treatments could enhance or inhibit the responses to estradiol and progesterone. Such a possibility is reflected in Figure 3A and B where the rapid response to progesterone is apparently delayed. This inhibition of a steroid effect by another steroid is being examined in more detail. The media used in our experiments do not exceed 0.5% DMSO. A concentration of 0.21% DMSO in the culture medium can induce a 2- to 6-fold increase in Ca2+ uptake in chicken granulosa cells (Morley and Whitfield, 1993), pointing to species variation in sensitivity of membrane receptors.
It is interesting that tamoxifen by itself had a membrane effect in stimulating an increase in [Ca2+]cyt. While this was unexpected, it has been reported that tamoxifen has a non-genomic effect in stimulating membrane-bound guanylate cyclase in porcine proximal tubular LLC-PK1 cells (Chen et al., 2003) and can stimulate an increase in [Ca2+] uptake in MCF-7 breast cancer cells (Chang et al., 2002
). Tamoxifen has also been found to stimulate Ca2+ influx in human sperm and to inhibit the progesterone-induced Ca2+ influx (Luconi et al., 2001
). In chicken granulosa cells, tamoxifen had no effect (Morley et al., 1992
). Tamoxifen, which is a selective estrogen receptor modulator, can have antagonist activity in breast cancer cells but is an agonist for endometrial growth (Hermenegildo and Cano, 2000
). In our study, tamoxifen completely inhibited the effects of both estradiol and progesterone. With chicken granulosa cells, tamoxifen prolonged the carbachol-triggered [Ca2+]cyt surges (Morley and Whitfield, 1994
).
ICI-182780 prevented the [Ca2+]cyt response to estradiol, but further addition of estradiol could reverse this inhibition. The relative binding of ICI-182780 is 0.89, compared with that of estradiol 1.0 (Wakeling et al., 1991), and therefore excess estradiol would be expected to reverse the non-genomic effect of ICI-182780. The inhibitory effect of ICI-182780 on the non-genomic action of estradiol has also been observed in rat astrocytes (Chaban et al., 2004
). This estrogen receptor antagonist can activate large conductance, calcium-activated potassium channel (BKCa) activity in smooth muscle (Dick, 2002
) just like estradiol and tamoxifen (Valverde et al., 1999
; Dick et al., 2001
), but in cultured endothelial cells of human coronary artery it is inhibitory for BKCa channel activity (Liu et al., 2003
). Thus these two estrogen antagonists, tamoxifen and ICI-182780, act by different mechanisms at the cell membrane. However, more recent evidence suggests that tamoxifen and ICI-182780 have high affinities for the membrane receptor for estradiol in the SKBR3 breast cancer cell line (Thomas et al., 2005
), confirming our observations on the non-genomic effects of these compounds. Competition between estradiol and the antagonists can explain the reversal of effects when excess steroid is added.
RU 486 has a Kd of 109 mol/l (Cadepond et al., 1997
) and has both anti-progestational and anti-glucocorticoid activities. At a concentration of 1 µg/ml used in these experiments, RU 486 showed a slight increase in [Ca2+]cyt uptake but inhibited the [Ca2+]cyt response to progesterone. Similar to the effects of ICI-182780, excess progesterone can over-ride the inhibitory effect of RU 486. Concentrations of RU 486 used in the past have ranged from 640 nmol/l to inhibit the effects of a similar concentration of progesterone (Peluso et al., 2001
) to 100 µmol/l to show an increase in apoptosis in luteinizing granulosa cells (Svensson et al., 2001
). The inability of RU 486 and ICI-182780 to inhibit the [Ca2+]cyt responses to tamoxifen suggests that tamoxifen is acting at a different site compared with the other two antagonists. Taken together, these data suggest that the sex steroids, estradiol and progesterone, as well as the antagonists, tamoxifen, ICI-182780 and RU 486, can act as genomic and non-genomic agents.
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Acknowledgements |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bootman MD, Lipp P and Berridge MJ (2001) The organization and functions of local Ca2+ signals. J Cell Sci 114, 22132222.[ISI][Medline]
Bramley T (2003) Non-genomic progesterone receptors in the mammalian ovary: some unresolved issues. Reproduction 125, 315.
Brini M and Carafoli E (2000) Calcium signaling: a historical account, recent developments and future perspectives. Cell Mol Life Sci 57, 752764.
Cadepond F, Ulman A and Baulieu E-E (1997) RU486 (Mifepristone): mechanisms of action and clinical uses. Annu Rev Med 48, 129156.[CrossRef][ISI][Medline]
Chaban VV, Lakhter AJ and Micevych P (2004) A membrane estrogen receptor mediates intracellular calcium release in astrocytes. Endocrinology 145, 37883795.
Chang H-T, Huang J-K, Wang J-L, Cheng J-S, Lee K-C, Lo Y-K, Liu C-P, Chou K-J, Chen W-C et al. (2002) Tamoxifen-induced increases in cytoplasmic free Ca2+ levels in human breast cancer cells. Breast Cancer Res Treat 71, 125131.[CrossRef][ISI][Medline]
Chen ZJ, Vetter M, Chang GD, Liu S, Ding Y and Chang CH (2003) Non-genomic effects of tamoxifen on the activation of membrane bound guanylate cyclase GC-A. J Pharm Pharmacol 55, 15391545.[CrossRef][ISI][Medline]
Dick GM (2002) The pure anti-oestrogen ICI 182,780 (FaslodexTM) activates large conductance Ca2+-activated K+ channels in smooth muscle. Br J Pharmacol 136, 961964.[CrossRef][ISI][Medline]
Dick GM, Rossow CF, Smirnov S, Horowitz B and Sanders KM (2001) Tamoxifen activates smooth muscle BK channels through the regulatory 1 subunit. J Biol Chem 267, 3459434599.[CrossRef]
Edwards DP (2005) Regulation of signal transduction pathways by estrogen and progesterone. Annu Rev Physiol 67, 335376.[CrossRef][ISI][Medline]
Falkenstein E, Norman AW and Wehling M (2000) Mannheim classification of nongenomically initiated (rapid) steroid action(s). J Clin Endocrinol Metab 85, 20722075.
Gerdes D, Christ M, Haseroth K, Notzon A, Falkenstein E and Wehling M (2000) Nongenomic actions of steroidsfrom laboratory to clinical implications. J Paediatr Endocrinol Metab 13, 853878.
Hermenegildo C and Cano A (2000) Pure anti-oestrogens. Hum Reprod Update 6, 237243.
Jakimiuk AJ, Weitsman SR, Yen H-W, Bogusiewicz M and Magoffin DA (2002) Estrogen receptor alpha and beta expression in theca and granulosa cells from women with polycystic ovary syndrome. J Clin Endocrinol Metab 87, 55325538.
Kakucs R, Varbiro S, Nadasy GL, Monos E and Szekacs B (2001) Acute, nongenomic vasodilatory action of estradiol is attenuauted by chronic estradiol treatment. Exp Biol Med 226, 538542.
Kelce WR, Stone CR, Laws SC, Gray LEJ, Kemppainen JA and Wilson EM (1995) Persistent DDT metabolite, p,p'DDE is a potent androgen receptor antagonist. Nature 375, 581585.[CrossRef][ISI][Medline]
Kreiner D, Veeck L, Liu H-C, Rosenwaks Z and Itskovitz J (1987) Follicular fluid estradiol and progesterone are markers of preovulatory oocyte quality. Fertil Steril 48, 991994.[ISI][Medline]
Kwan CY, Harrison PH and Kwan TK (2003) Pramanicin, an antifungal agent, raises cytosolic Ca2+ and causes cell death in vascular endothelial cells. Vascul Pharmacol 40, 3542.[CrossRef][ISI][Medline]
Lemon M and Mauleon P (1982) Interaction between two cell types from the corpus luteum of the sow in progesterone synthesis in vitro. J Reprod Fertil 64, 315323.[ISI][Medline]
Levin ER (2001) Cell localization, physiology, and nongenomic actions of estrogen receptors. J Appl Physiol 91, 18601867.
Levin ER (2002) Cellular functions of plasma estrogen receptors. Steroids 67, 471475.[CrossRef][ISI][Medline]
Lieberherr M, Grosse B and Machelon V (1999) Phospholipase C- and ovarian sex steroids in pig granulosa cells. J Cell Biochem 74, 5060.[CrossRef][ISI][Medline]
Liu Y-C, Lo Y-C, Huang C-W and Wu S-N (2003) Inhibitory action of ICI-182780, an estrogen receptor antagonist, on BKCa channel activity in cultured endothelial cells of human coronary artery. Biochem Pharmacol 66, 20532063.[CrossRef][ISI][Medline]
Losel RM, Falkenstein E, Feuring M, Schultz A, Tillmann H-C, Rossol-Haseroth K and Wehling M (2003) Nongenomic steroid action: controversies, questions and answers. Physiol Rev 83, 9651016.
Low AM, Sormaz L, Kwan CY and Daniel EE (1997) Mobilization of internal Ca2+ by intestinal polypeptide in endothelial cells. Eur J Pharmacol 339, 227235.[CrossRef][ISI][Medline]
Luconi M, Bonaccorsi L, Maggi M, Pecchioli P, Krausz C, Forti G and Baldi E (1998) Identification and characterization of functional nongenomic progesterone receptors on human sperm membrane. J Clin Endocrinol Metab 83, 877885.
Luconi M, Muratori M, Forti G and Baldi E (1999) Identification and characterization of a novel functional estrogen receptor on human sperm membrane that interferes with progesterone effects. J Clin Endocrinol Metab 84, 16701678.
Luconi M, Bonaccorsi L, Forti G and Baldi E (2001) Effects of estrogenic compounds on human spermatozoa: evidence for interaction with a nongenomic receptor for estrogen on human sperm membrane. Mol Cell Endocrinol 178, 3945.[CrossRef][ISI][Medline]
Machelon V, Nome F, Grosse B and Lieberherr M (1996) 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. J Cell Biochem 61, 619628.[CrossRef][ISI][Medline]
Machelon V, Nome F and Tesarik J (1998) Nongenomic effects of androstenedione on human granulosa luteinizing cells. J Clin Endocrinol Metab 83, 263269.
Maybin JA and Duncan WC (2004) The human corpus luteum: which cells have progesterone receptors? Reproduction 128, 423431.
Miyazaki S (1995) Calcium signaling during mammalian fertilization. In Bock GR and Ackrill K (eds), Calcium Waves, Gradients and Oscillations. Ciba Foundation Symposium 188. John Wiley and Sons, NY, pp. 235251.
Morley P and Whitfield JF (1993) The differentiation inducer, dimethyl sulfoxide, transiently increases the intracellular calcium ion concentration in various cell types. J Cell Physiol 156, 219225.[CrossRef][ISI][Medline]
Morley P and Whitfield JF (1994) Effect of tamoxifen on carbachol-triggered intracellular calcium responses in chicken granulosa cells. Cancer Res 54, 6974.[Abstract]
Morley P, Whitfield JF, Vanderhyden BC, Tsang BK and Schwartz JL (1992) A new nongenomic estrogen action: the rapid release of intracellular calcium. Endocrinology 131, 13051312.[Abstract]
Ou Y-J, Leung Y-M, Huang S-J and Kwan C-Y (1997) Dual effects of extracellular Ca2+ on cardiotoxin-induced cytotoxicity and cytosolic Ca2+ changes in cultured single cells of rabbit aortic endothelium. Biochim Biophys Acta 1330, 2938.[ISI][Medline]
Peluso JJ (2004) Rapid actions of progesterone on granulosa cells. Steroids 69, 579583.[CrossRef][ISI][Medline]
Peluso JJ, Fernandez G, Pappalardo A and White BA (2001) Characterization of a putative membrane receptor for progesterone in rat granulosa cells. Biol Reprod 65, 94101.
Peluso JJ, Fernandez G, Pappalardo A and White BA (2002) Membrane-initiated events account for progesterone's ability to regulate intracellular free calcium levels and inhibit rat granulosa cell mitosis. Biol Reprod 67, 379385.
Razandi M, Pedram A, Greene GL and Levin ER (1999) Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: studies of ER and ER
expressed in Chinese hamster ovary cells. Mol Endocrinol 13, 307319.
Revelli A, Massobrio M and Tesarik J (1998) Nongenomic actions of steroid hormones in reproductive tissues. Endocr Rev 19, 317.
Sak K and Everaus H (2004) Nongenomic effects of 17beta-estradioldiversity of membrane binding sites. J Steroid Biochem Mol Biol 88, 323335.[CrossRef][ISI][Medline]
Saner KJ, Welter BH, Zhang F, Hansen E, Dupont B, Wei Y and Price TM (2003) Cloning and expression of a novel, truncated, progesterone receptor. Mol Cell Endocrinol 200, 155163.[CrossRef][ISI][Medline]
Saunders PTK, Millar MR, Williams K, Macpherson S, Harkiss D, Anderson RA, Orr B, Groome NP, Scobie G and Fraser HM (2000) Differential expression of estrogen receptor- and-
and androgen receptor in the ovaries of marmosets and humans. Biol Reprod 63, 10981105.
Suzuki T, Sasano H, Kimura N, Tamura M, Fukaya T, Yajima A and Nagura H (1994) Immunohistochemical distribution of progesterone, androgen and oestrogen receptors in the human ovary during the menstrual cycle: relationship to expression of steroidogenic enzymes. Hum Reprod 9, 15891595.[Abstract]
Svensson EC, Markström E, Shao R, Andersson M and Billig H (2001) Progesterone receptor antagonists Org 31710 and RU 486 increase apoptosis in human periovulatory granulosa cells. Fertil Steril 76, 12251231.[CrossRef][ISI][Medline]
Swann K, Larman MG, Saunders CM and Lai FA (2004) The cytosolic sperm factor that triggers oscillations and egg activation in mammals is a novel phospholipase C: PLC. Reproduction 127, 431439.
Tesarik J and Mendoza C (1997) Direct non-genomic effects of follicular steroids on maturing human oocytes: oestrogen versus androgen antagonism. Hum Reprod Update 3, 95100.
Thomas P, Pang Y, Filardo EJ and Dong J (2005) Identity of an estrogen membrane receptor coupled to a G protein in human breast cancer cells. Endocrinology 146, 624632.
Valverde MA, Rojas P, Amigo J, Cosmelli D, Orio P, Bahamonde MI, Mann GE, Vergara C and Latorre R (1999) Acute activation of maxi-K channels (hSlo) by estradiol binding to the subunit. Science 285, 19291931.
Wakeling AE, Dukes M and Bowler J (1991) A potent specific pure antiestrogen with clinical potential. Cancer Res 51, 38673873.[Abstract]
Watson CS and Gametchu B (2003) Proteins of multiple classes may participate in nongenomic steroid actions. Exp Biol Med 228, 12721281.
Webb RC and Bohr DF (1978) Mechanism of membrane stabilization by calcium in vascular smooth muscle. Am J Physiol 235, C227C232.[ISI][Medline]
Wennemuth G, Eisoldt S, Bode HP, Renneberg H, Schiemann PJ and Aumuller G (1998) Measurement of calcium influx in surface-fixed single sperm cells: efficiency of different immobilization methods. Andrologia 30, 141146.[ISI][Medline]
Yie S-M, Brown GM, Liu G-Y, Collins JA, Daya S, Hughes EG, Foster WG and Younglai EV (1995) Melatonin and steroids in human pre-ovulatory follicular fluid: seasonal variations and granulosa cell steroid production. Hum Reprod 10, 5055.[Abstract]
Younglai EV, Kwan TK, Kwan C-Y, Lobb DK and Foster WG (2004) Dichlorodiphenylchloroethylene elevates cytosolic calcium concentrations and oscillations in primary cultures of human granulosa-lutein cells. Biol Reprod 70, 16931700.
Submitted on October 27, 2004; resubmitted on March 18, 2005; accepted on April 14, 2005.