The role of exogenous calcium for gonadotropin-stimulated progesterone production by human granulosa-luteal cells

FERTILITY AND STERILITY

Vol. 52, No.6, December 1989

Copyright <> 1989 The American Fertility Society

Printed on acid·free paper in U.S.A.

The role of exogenous calcium for gonadotropin-stimulated progesterone production by human granulosa-luteal cells*t

Ha-Lin C. Lee, M.D.:\: Gary A. Shangold, M.D.§ Angela L. Larsen, B.S.§ James R. Schreiber, M.D.§II Department of Obstetrics and Gynecology, Michael Reese Hospital and Medical Center and The University of Chicago, Chicago, Illinois

Previous studies of cells from various species have indicated that exogenous calcium is necessary for gonadotropic stimulation of steroidogenesis. To determine whether this requirement for exogenous calcium is a universal attribute of steroidogenic cells, we studied baseline and stimulated progesterone (P) production by cultured human granulosaluteal cells obtained at the time of oocyte retrieval for in vitro fertilization (IVF). During 4 hours in culture, both cholera toxin (1.25 ~g/mL) and human chorionic gonadotropin (hCG, 1 IV /mL) stimulated a significant (P < 0.05) 2- to 4-times increase in P production. Both baseline and stimulated (cholera toxin or hCG) increases in P were unaffected when cellular uptake of exogenous calcium was inhibited by the calcium channel blocker nitrendipine (10 ~M), or by culturing the cells in calcium-free medium or in calcium-free medium with [ethylenebis(oxyethylenenitrilo)]-tetra-acetic acid (EGTA, to chelate anypossible free extracellular calcium). At later time points (24 and 48 hours), lack of available exogenous calcium began to have an inhibitory effect on P production, and the hCG effect was more sensitive to the lack of exogenous calcium than was the cholera toxin effect. We speculate that this apparent independence from exogenous calcium over a short culture period is due to the prior stimulation of these cells by exogenous gonadotropins employed in IVF cycles. Fertil Steril52:958, 1989

Calcium has been implicated as an important modulator in the gonadotropin regulation of steroid production by ovarian granulosa cells. Calcium has a multiplicity of roles in cells and the calcium messenger system is a nearly universal means by which extracellular messengers regulate cell Received May 30, 1989; revised and accepted August 8, 1989. * Presented at the Thirty-Sixth Annual Meeting ofthe Society for Gynecologic Investigation, San Diego, California. t Supported by grants HL 15062 and HD 00708 from the National Institutes of Health Bethesda, Maryland. :j: Department of Obstetrics and Gynecology, Michael Reese Hospital. § Department of Obstetrics and Gynecology, University of Chicago. II Reprint requests: James R. Schreiber, M.D., The University of Chicago, Box 446, Department of Obstetrics and Gynecology, 5841 S. Maryland Avenue, Chicago, Illinois 60637.

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function. 1,2 Calcium can bind to calmodulin, and can interact with the phospholipid-protein kinase C system, which can elicit brief responses in the cell or mediate sustained cellular activities. l -4 Studies using porcine5.6 and rat7-9 granulosa cells have demonstrated that extracellular calcium is essential for the steroidogenic action of luteinizing hormone (LH). Although the precise locus of calcium action is unknown, calcium appears to modulate the LH effect on granulosa cells in at least two sites. 5- 9 First, exogenous calcium is important for LH -stimulated cyclic adenosine monophosphate (cAMP) production, and second, calcium is involved at a step in the steroidogenic pathway distal to the cAMP production. 6 In the present work, we examined whether calcium has a similar role in human granulosa cells. We chose to study granulosa cells obtained at the time of oocyte retrieval from Fertility and Sterility

women undergoing in vitro fertilization (IVF) because of their easy accessibility and the fact that they have been previously established as a model for human steroidogenic cells. 10 MATERIALS AND METHODS Materials

Dulbecco's modified Eagle medium (DMEM, Cat #430-2100) containing 200 mg/L (1.8 mM) of anhydrous calcium chloride and 4.5 gm/L glucose (pH 7.4) was obtained from Gibco Laboratories (Grand Island, NY). The following were added per liter of medium: 0.116 gm L-arginine, 1.3 gm L-glutamine, 0.006 gm folic acid, 0.036 gm L-asparagine, 2.0 gm sodium bicarbonate, 0.11 gm sodium pyruvate, and 50 mg gentamicin. Nominally calciumfree Dulbecco's modified Eagle medium (#86-5021) was also obtained from Gibco Laboratories and had the same constituents and additives as DMEM except that it contained no calcium. Human chorionic gonadotropin (hCG), cholera toxin, and Ficoll type 400 were obtained from Sigma Chemical (St. Louis, MO). Tritiated progesterone (P) (48 Cij mmole) was obtained from Amersham (Arlington Heights, IL). Male human serum was donated by volunteers. Low-density lipoprotein (LDL, d = 1.019 to 1.063 g/mL) was isolated from donor serum by preparative ultracentrifugation as previously described. 11 Methods

Granulosa cells were aspirated from preovulatory follicles of women undergoing ovum retrieval for IVF. These women were treated with either urinary menotropins (Pergonal, Serono Laboratories, Randolph, MA) or urinary follicle-stimulating hormone (FSH Metrodin, Serono Laboratories) at a daily intramuscular (1M) dose of 150 IV to 300 IV starting day 2 of their cycle. Exogenous gonadotropin was continued until serum estradiol (E 2 ) was ;:::500 pg/mL and at least two follicles had a diameter of;:::16 mm by abdominal or vaginal ultrasonography. Ten thousand IV hCG was given 1M 34 hours before ovum retrieval. Follicular aspirates from an individual patient were combined, and the granulosa cells were isolated and cultured as previously described. 12 Briefly, granulosa cells were separated from erythrocytes by centrifugation through Ficoll-type 400 density gradient, plated in DMEM (with additives Vol. 52, No.6, December 1989

as described in Materials) plus 20% vol/vol human male serum, and cultured at 37°C in humidified atmosphere of95% air and 5% CO 2 • Cell viability was determined by trypan blue dye. A total of 200,000 viable cells was plated in each well. Each data point was performed in triplicate wells. Experiments were repeated three times. At the end of 24 hours, the serum-supplemented medium was replaced with serum-free DMEM (with additives) containing 200 Ilg/2 mL LDL and the cells were cultured for an additional 24 hours. Low-density lipoproteins was added to provide the cells with adequate cholesterol substrate for steroidogenesis. The dose of LDL was selected from preliminary studies which demonstrated that this dose of LDL provided more than 10 times the needed amount of cholesterol for maximal stimulated P production. At 48 hours of culture, the culture media were again replaced with media containing the treatments as described in the figure legends. Aliquots of medium were then taken at 4 hours, 24 hours, and 48 hours after this last medium change and frozen at -20°C. All aliquots of the same experiment were assayed for P content in the same radioimmunoassay (RIA). Bound and free steroid were separated by dextran charcoal following an overnight incubation at 4°C. Cross-reactivity of the antibody was: P = 100%, 17-hydroxypregn-4-ene-3,20-dione = 0.09%, 20-a-hydroxypregn-4-ene-3-one = 0.1 %, cortisol = <0.02%, deoxycorticosterone = 1.5%, testosterone = 0.2%, and estrone, 17f1-E2 , estriol = <0.02%. Data are presented as the mean ± standard error of the mean (SEM). Analysis of variance followed by Tukey test13 for multiple pair wise comparisons was used to test for significant differences between groups. These analyses were carried out on a Macintosh SE (Apple Computer, Cupertino, CAl personal computer using the Statview 512+ (BrainPower, Inc., Calabasas, CAl software package. Probability < 0.05 was considered a significant difference between groups. RESULTS

Preliminary experiments were conducted to determine the optimal culture protocol which would allow testing of the necessity of exogenous calcium on both baseline and cholera toxin-stimulated P production. Human ovarian granulosa cells were initially cultured for 24 hours in medium consisting of DMEM plus 20% male serum. The serum was added to assure the plating of cells to the plastic Lee et al.

Calcium and progesterone production

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Figure 1 Effect of nitrendipine and cholera toxin on granulosa cell progesterone production. Human ovarian granulosaluteal cells were isolated and cultured for 24 hours in DMEM + 20% male serum. The media were changed and the cells were cultured for another 24 hours in DMEM + 200 ~g/2mL LDL. The media were changed again and the cells were cultured in DMEM (control = C), medium + 10 ~M nitrendipine (Nt), medium + 1.25 ~g/mL cholera toxin (CT), or medium + CT + Nt. Aliquots of medium were removed at 4, 24, and 48 hours and the progesterone content was measured by specific RIA. All media after the first 24 hours of culture contained 200 ~g/2mL LDL. Values are mean ± SEM, n = 3 tissue culture wells.

dishes. This medium was removed and the cells were then cultured for 24 hours in serum-free DMEM. The cells responded to the addition of cholera toxin at 48 hours of culture with increased P production only after being cultured in serumfree medium. Cells that were cultured for 48 hours in the continued presence of serum produced P with no further increase in response to added cholera toxin. Low-density protein was added to the serum-free medium to assure the availability of exogenous cholesterol substrate. This protocol for the first 48 hours of culture was used in all the experiments described in this report. The amount of P produced by the cultured cells varied from experiment to experiment (Figs. 1 to 4). For this reason, all treatment groups in an individual experiment used the same pooled isolated granulosa cells. Repeat experiments yielded similar results and the data presented in the figures are representative individual experiments. In the first series of experiments, we asked whether the calcium channel blocker, nitrendipine,14 would have an effect on baseline or cholera toxin-stimulated P production. The results are shown in Figure 1. Fresh medium was added at 48 960

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Calcium and progesterone production

hours which consisted of DMEM and LDL alone (control), or with the addition of nitrendipine and/ or cholera toxin. Aliquots of medium were removed for measurement of P at 4, 24, and 48 hours. After 4 hours of culture (Fig. 1, left panel), the control cells produced about 50 ng of P and the addition of 10 p.M nitrendipine had no effect. The addition of 1.25 p.g/mL cholera toxin doubled the production of P and again the addition of nitrendipine had no effect. After 24 hours of culture (Fig. 1, middle panel) and 48 hours of culture (Fig. 1, right panel), the control granulosa cells continued to produce P and cholera toxin stimulated a 2.5- to 3.8-times increase in P production. The addition of nitrendipine had no consistent inhibitory effect on either control or cholera toxin-stimulated production. The slight increase in P in the cholera toxin plus nitrendipine group at 24 hours was not seen in two repeat experiments where the nitrendipine had no effect. These data, surprisingly, suggest that exogenous calcium uptake via the L-calcium channels, (the calcium channels blocked by nitrendipine 14 ), is not necessary for either baseline or cholera toxin-stimulated P production during a 48-hour culture period. As nitrendipine did not have any inhibitory effect on P production in our cell system, we tested the same batch of nitrendipine on a different cell system where it is known to have an inhibitory effect. When we added this same nitrendipine to single identified rat pituitary gonadotropes concomitantly with gonadotropin-releasing hormone (GnRH), 10 p.M nitrendipine effectively suppressed the second phase of cytosolic calcium elevation elicited by GnRH. This second phase is the portion of the GnRH-induced response which has been shown to be the result of the influx of extracellular Ca++ through dihydropyridine-sensitive membrane calcium channels. 15 To further test the notion of the lack of importance of exogenous calcium, we elected to culture the human granulosa cells in calcium-free DMEM or in calcium-free DMEM plus 100 p.M [ethylenebis(oxyethylene nitrilo)]-tetra-acetic acid (EGTA, added to chelate any possible Ca++ in the medium) and the results are shown in Figure 2. After 4 hours of culture (Fig. 2, left panel), cholera toxin again doubled P production. Both control and cholera toxin-stimulated cells cultured in either calciumfree DMEM, or in calcium-free DMEM plus EGT A, produced as much P as did cells cultured in DMEM. After 24 hours in culture, the control cells continued to produce P and neither the calciumfree medium nor the calcium-free medium plus Fertility and Sterility

4 Hours

Figure 2 Effect of Calcium-free medium (±EGTA) and cholera toxin on granulosa cell progesterone production. Granulosa cells were isolated and cultured for 48 hours as described in Fig. 1. After 48 hours of culture, the media were changed and the cells were cultured in DMEM (control = C), calcium-free DMEM (-Ca), calcium-free DMEM + 100 ~M EGTA (-Ca + EGTA), DMEM + 1.25 ~g/mL cholera toxin (CT), calcium-free DMEM + CT (CT + -Ca), or calcium free DMEM + EGTA + CT (CT + -Ca + EGTA). Aliquots of medium were removed at 4, 24, and 48 hours for progesterone assay. All media after the first 24 hours contained 200 ~g/2mL LDL. Values are mean ± SEM, n = 3.

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EGTA had a significant effect on this baseline production (Fig. 2, middle panel). Cholera toxin stimulated a 5-times increase in P production by cells in DMEM. Culturing the cells in calcium-free DMEM resulted in a nonsignificant 38% reduction in the cholera toxin-stimulated increase, whereas culturing the cells in calcium-free DMEM plus EGTA caused a significant (P < 0.05) 75% inhibition ofthe cholera toxin-stimulated increase. After 48 hours of culture, the calcium-free DMEM, and to a much greater extent, the calcium-free DMEM plus EGTA, significantly (P < 0.05) inhibited both baseline and cholera toxin-stimulated P production (Fig. 2, right panel). Calcium-free medium plus EGTA inhibited the cholera toxin-stimulated 13times increase in P by about two-thirds. However, even though the calcium-free DMEM and calciumfree DMEM plus EGTA had a significant effect on human granulosa cell steroidogenesis during the 48 hours of culture, the cells still produced large amounts of P as a result of cholera toxin-stimulation (3.5 and 2.0 ,."g P per 200,000 cells in these media, respectively). We next examined for possible effects of calcium channel blockade and calcium-free media on hCGstimulated steroidogenesis by these human ovarian granulosa cells. Human chorionic gonadotropin is a physiological regulator ofluteal cell steroidogenesis, and manipulations of exogenous calcium availability could affect hCG stimulation of P producVol. 52, No.6, December 1989

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tion differently from cholera toxin stimulation. The effect of the calcium channel blocker nitrendipine on baseline and hCG-stimulated P production is shown on Figure 3. As was seen in Figure 1, 10 ,."M nitrendipine had no effect on baseline P production at any of the time points tested (4, 24, and 48 hours). The addition of 1 IU/mL hCG caused a 3-, 3.9-, and 4.8-times increase in P production at 4, 24, and 48 hours, respectively. This dose ofhCG was selected to achieve maximal stimulation of steroidogenesis and LDL receptor synthesis. 12 At 4 and 24 hours, the addition of nitrendipine had no significant effect on hCG-stimulated steroidogenesis. However, as opposed to the results with cholera toxin at 48 hours (shown in Fig. 1, right panel), nitrendipine caused a statistically significant (P < 0.05) 50% inhibition of the hCG effect (Fig. 3, right panel). We next examined the necessity of exogenous calcium for hCG-stimulated P production by incubating the cells in calcium-free DMEM or in calcium-free DMEM plus EGTA, and the results are shown on Figure 4. After 4 hours of culture (Fig. 4, left panel), hCG stimulated a 1.7-times increase in P production and neither calcium-free nor calcium-free DMEM plus EGTA had any effect on this increase. At 24 and 48 hours, hCG stimulated a 2.7 and 5.6-times increase, respectively (Fig. 4, middle and right panels). Calcium-free DMEM resulted in a small (-20%) inhibition of the hCG effect (P < 0.05) at 48 hours, Lee et al. Calcium and progesterone production

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Figure 3 Effect of nitrendipine and human chorionic gonad0tropin (hCG) on granulosa cell progesterone production. Granulosa cells were isolated and cultured for 48 hours as described in Fig. 1. After 48 hours of culture, the media were changed and the cells were cultured in DMEM (control = C), 10 /LM nitrendipine (Nt), 1 IU/mL hCG, or hCG + Nt. Aliquots of media were removed at 4, 24, and 48 hours for progesterone assay. All media after the first 24 hours contained 200 /Lg/2mL LDL. Values are mean ± SEM, n = 3.

whereas the calcium-free DMEM plus EGTA blocked the hCG stimulation completely at 24 and 48 hours (Fig. 4, middle and right panels). The hCG-stimulated cells cultured in calciumfree medium plus EGTA (Fig. 4) produced P during each of the time periods tested. However, the rate of production after the initial 4 hours of culture was very modest (cumulative P: 4 hours-200 ng, 24 hours-280 ng, 48 hours-360 ngper 200,000 cells). In addition, as was stated previously, the hCG-stimulated increase in P production was blocked completely after the first 4 hours in calcium-free DMEM + EGTA. To determine whether the addition of EGTA to calcium-free medium was toxic, we examined the viability of cells after 24 hours in this medium. Viability was tested by adding trypan blue stain to the culture medium and counting the number of cells and the percent of cells that excluded the dye. Calcium-free medium plus EGTA had no effect on cell viability. Greater than 95% of cells in DMEM medium + hCG or cells in calciumfree DMEM + EGTA + hCG excluded the trypan blue and there was no change in the number of plated cells. DISCUSSION

Our present studies indicate that extracellular calcium is not essential, over a short culture period 962

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Calcium and progesterone production

(4 hours), for either baseline or hCG/cholera toxinstimulated steroid production by human ovarian granulosa cells obtained after ovarian hyperstimulation for IVF. We demonstrated this by incubating human granulosa cells for 4 hours in the presence of the calcium channel blocker nitrendipine, or by denying the cells extracellular calcium by incubating them in calcium free medium ± EGTA. None ~f these manipulations had an effect on the baseline or hCG/cholera toxin-stimulated P production. These results differ from previous reports. Studies with porcine ovarian granulosa cells 5 •6 indicate that there is a significant reduction in LHstimulated P production at 4 hours of culture when the cells are incubated in the presence of calcium channel blockers, including verapamil (50 ~M, which blocks voltage sensitive calcium channels) or diltiflzem (50 ~M, which blocks stimulated sodium efflux and limits cellular calcium influx). In addition, culturing porcine cells in calcium-free DMEM, with or without 100 ~M EGTA, significantly inhibits LH -stimulated P production during a short-term (4 hours) incubation. Similarly, studies with rat ovarian granulosa cells 7-9 demonstrate that baseline and cholera toxin (10 ~M) or FSH (100 to 500 ng/mL)-stimulated P production at 4 4 Hours

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Figure 4 Effect of Calcium-free medium (±EGTA) and human chorionic gonadotropin (hCG) on granulosa cell progesterone production. Granulosa cells were isolated and cultured for 48 hours as described in Fig. 1. The media were changed and the cells were cultured in DMEM (Control = C), DMEM + 1 IU/mL hCG, calcium-free DMEM + hCG (-Ca + hCG), or calcium-free DMEM + 100 /LM EGTA + hCG (-Ca + EGTA + hCG). Aliquots of medium were removed at 4, 24, and 48 hours for progesterone assay. All media after the first 24 hours contained 200 /Lg/2mL LDL. Values are mean ± SEM, n = 3.

Fertility and Sterility

hours is decreased significantly in the presence of verapamil (250 ~M), EGTA (100 ~M), or lanthanum (100 ~M, an inorganic calcium channel blocker). Clearly, the human ovarian granulosa cells examined in this study, which have been exposed to exogenous gonadotropins in vivo, differ from the porcine and rat cells in that the human cells do not need exogenous calcium over a short 4hour culture period to secrete P under control or stimulated conditions. Because we did not observe any inhibition of P production due to the addition of a calcium channel blocker or due to lack of extracellular calcium in human granulosa cells during 4 hours of culture, we decided to examine the dependence of these highlystimulated human cells on extracellular calcium over a longer culture period. The data in this report indicate that the calcium channel blocker nitrendipine (10 ~M) had no effect on either baseline or cholera toxin-stimulated P production up to 48 hours of culture. Human chorionic gonadotropinstimulated P production, however, was decreased by 50% at 48 hours. This differential effect of nitrendipine on cholera toxin versus hCG-stimulated cells is in accordance with previous studies with porcine and rat granulosa cells which indicate a dual role of calcium on steroidogenesis.5-8 Calcium was shown in these reports to be important for gonadotropin-stimulated cAMP production and also has a role at a site(s) distal to cAMP production. Human chorionic gonadotropin-stimulated cells would be vulnerable to both effects of calcium. Cholera toxin, however, bypasses the gonadotropin receptor in its stimulation of increased cellular cAMP by covalently modifying the guanyl nucleotide-binding protein of the adenylate cyclase system. I6 When the cells were cultured with calcium-free medium (with or without added EGTA), inhibition of cholera toxin-stimulated P production was apparent at 24 hours, and both baseline and cholera toxin-stimulated P production was significantly decreased at 48 hours. Contrary to the short term (4 hours) independence of the human granulosa cells to extracellular calcium, the data in this report indicate that calcium has a modulating role on steroidogenesis in human granulosa cells over longer time periods (24 to 48 hours). This calcium effect is similar to that observed in studies with porcine and rat granulosa cells, but with a shifting to a later time frame. Compared with cholera toxinstimulated cells, hCG-stimulated cells are more sensitive to depletion of extracellular calcium. At Vol. 52, No.6, December 1989

24 and 48 hours of culture, the calcium-free medium + EGT A completely blocked hCG-stimulated P production, whereas cholera toxin was still able to stimulate a 2-times and 5-times increase in P at 24 and 48 hours, respectively. However, it should be noted that despite the decrease in cholera toxin/ hCG-stimulated P production at 24 and 48 hours by cells cultured in medium lacking Ca++, large quantities of steroid product were made by human granulosa cells with no access to exogenous calcium. A potential site of action for the inhibitory effect of calcium-free medium + EGTA, noted at 24 and 48 hours of culture, could be on the ability of LDL cholesterol to be taken up by the cell via the LDL receptor. Studies of the membrane LDL receptor demonstrate that divalent cations are needed for maximum binding ofLDL to its receptor, and binding is diminished by the removal of divalent cations or the addition of a chelator such as EDTA.I7 Our studies suggest that ovarian granulosa cells vary in their dependence on extracellular calcium. The human granulosa cells obtained at the time of oocyte retrieval from women undergoing IVF have been stimulated with exogenous gonadotropin before they were cultured. This in vivo hyperstimulation could be a contributing factor to their relative independence of exogenous calcium for baseline and stimulated steroidogenesis. The mechanism is unknown, but it is clear from these studies that human IVF granulosa cells can make substantial amount of steroid, over an extended period of time, with little or no need for exogenous calcium. The data in this report thus demonstrate that there is no absolute need for exogenous calcium for steroidogenesis in all cells. In addition, the transport of exogenous calcium through specific channels, the L channels, is not a prerequisite for either baseline or gonadotropin-stimulated steroidogenesis.

Acknowledgments. We thank Ms. Brenda Brown for the preparation of this manuscript. Nitrendipine was the kind gift of Alexander Scriabine, M.D. (Miles Laboratories, West Haven, Connecticut). The P RIA was performed using an antibody generously provided by Robert Beneveniste, Ph.D. (Michael Reese Hospital, Chicago, Illinois).

REFERENCES 1. Rasmussen H: The calcium messenger system. N Engl J Med 314:1094, 1986 2. Rasmussen H: The calcium messenger system. N Engl J Med 314:1164,1986

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3. Takai Y, Kishimoto A, Iwasa Y, Kawabara Y, Mori T, Nishizuka Y: Calcium-dependent activation of a multifunctional protein kinase by membrane phospholipids. J BioI Chem 254:3692, 1979 4. Nishizuka Y, Takai Y, Kishimoto A, Kikkawa U, Kaibuchi K: Phospholipid turnover in hormone action. Rec Prog Horm Res 40:301, 1984 5. Veldhuis JD, Klase PA, Demers LM, Chafouleas JG: Mechanisms subserving calcium's modulation ofluteinizing hormone action in isolated swine granulosa cells. Endocrinology 114:441, 1984 6. Veldhuis JD, Klase P A: Mechanisms by which calcium ions regulate the steroidogenic actions of luteinizing hormone in isolated ovarian cells in vitro. Endocrinology 111:1, 1982 7. Carnegie JA, Tsang BK: Follicle-stimulating hormone-regulated granulosa cell steroidogenesis: Involvement of the calcium-calmodulin system. Am J Obstet GynecoI145:223, 1983 8. Tsang BK, Carnegie JA: Calcium requirement in the gonadotropic regulation of rat granulosa cell progesterone production. Endocrinology 113:763, 1983 9. Tsang BK, Carnegie JA: Calcium dependent regulation of progesterone production by isolated rat granulosa cells: effects of the calcium ionophore A 23187, prostaglandin E 2 , dl-isoproterenol and cholera toxin. BioI Reprod 30:787, 1984 10. Golos TG, Strauss JF III: Regulation of low density lipoprotein receptor synthesis in cultured luteinized human

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granulosa cells by human chorionic gonadotropin and 8bromo-cyclic AMP. J BioI Chem 260:14399,1985 11. Schreiber J, Nakamura K, Schmit V, Weinstein D: Androgen and FSH synergistically stimulate lipoprotein degradation and utilization by ovary granulosa cells. Steroids 43: 25,1984 12. Golos TG, August AM, Strauss JF III: Expression of low density lipoprotein receptor in cultured human granulosa cells: regulation by human chorionic gonadotropin, cyclic AMP, and sterol. J Lipid Res 27:1089, 1986 13. Kleinbaum DG, Kupper LL, Muller KE: One-way analysis of variance. In Applied Regression Analysis and Other Multivmable Methods, Second Edition, Boston, PWSKENT Publishing Company, 1988, p 365 14. Nowycky MC, Fox AP, Tsien RW: Three types of neuronal calcium channel with different calcium agonist sensitivity. Nature 316:440,1985 15. Shangold GA, Murphy SN, Miller RJ: Gonadotropin-releasing hormone-induced Ca2+ transients in single identified gonadotropes require both intracellular Ca2+ mobilization and Ca2+ influx. Proc Nat! Acad Sci 85:6566, 1988 16. Cassel D, Pfeuffer T: Mechanism of cholera toxin action: covalent modification of the guanyl nucleotide-binding protein of the adenylate cyclase system. Proc Natl Acad Sci USA 75:2669, 1978 17. Kovanen P, Basu S, Goldstein J, Brown M: Low density lipoprotein receptors in bovine adrenal cortex. II. Low density lipoprotein binding to membranes prepared from fresh tissue. Endocrinology 104:610, 1979

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