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Endocrinology Vol. 140, No. 8 3573-3580
Copyright © 1999 by The Endocrine Society

ARTICLES

Alterations in Intracellular Messengers Mobilized by Gonadotropin-Releasing Hormone in an Experimental Ovarian Tumor1

Astrid Chamson-Reig, Omar P. Pignataro, Carlos Libertun and Victoria A. R. Lux-Lantos

Instituto de Biología y Medicina Experimental-CONICET, 1428 Buenos Aires, Argentina

Address all correspondence and requests for reprints to: Dr. Carlos Libertun, Instituto de Biología y Medicina Experimental, Vuelta de Obligado 2490, 1428 Buenos Aires, Argentina. E-mail: libertun{at}dna.uba.ar


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cells derived from an experimental luteinized ovarian tumor are more sensitive to GnRH endocrine action than control luteal cells. In an attempt to understand the possible causes of the differential sensibility to GnRH action, we examined the number and affinity of GnRH receptors and the second messenger response to GnRH stimulation in both tissues. For GnRH receptor studies membranes were obtained from 4- to 6-week-old ovarian tumors (luteoma) and ovaries from prepubertal rats treated with 25 IU PMSG and 25 IU hCG (SPO) and were incubated with [125I]Buserelin. The number of GnRH receptors were increased in luteoma compared with that in SPO ovaries; dissociation constants were similar in both tissues. GnRH stimulation of second messenger release was assessed in cells obtained from luteoma and SPO ovaries by collagenase treatment. Buserelin (100 ng/ml) induced a significant 35% calcium increase in SPO cells, as determined by the fura-2 method; in luteoma cells no response was observed after buserelin stimulation, although a calcium transient was induced by thapsigargin (0.5 µM), an inhibitor of Ca2+-adenosine triphosphatase associated with the endoplasmic reticulum. The effect of buserelin on inositol phosphates was evaluated after incubation of luteoma and SPO cells with [3H]myo-inositol for 48 h. Buserelin induced a 400% increase in inositol trisphosphate in SPO cells. Again, luteoma cells did not respond to buserelin stimulation, although NaF (10 mM), an activator of G proteins coupled to phospholipase C, induced an 800% increase in inositol trisphosphate. Although the number of GnRH receptors is augmented in luteoma cells, justifying an increased endocrine response, neither inositol phosphates nor intracellular calcium were released by a GnRH analog, indicating the uncoupling of GnRH receptors from phospholipase C. These data provide evidence that the transformation of the ovary into a luteoma implies the acquisition of novel characteristics in the GnRH receptor second messenger-generating system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN PREVIOUS studies we have reported that intrasplenic ovarian tumors, which develop in response to the high gonadotropin levels characteristic of this model (1, 2, 3), regress significantly under a GnRH analog treatment, principally due to desensitization of the pituitary (4). Nevertheless, a direct effect of GnRH agonists on this ovarian tumor was also described, as it possesses GnRH receptors, and GnRH analogs inhibit the LH-induced progesterone secretion in vitro (5). In fact, the inhibitory effect elicited by GnRH on steroidogenesis was more intense in tumor cells than in control luteal cells from superovulated prepubertal rats (SPO) under the experimental conditions studied. Differences in GnRH action between both kinds of cells could be due to a variety of factors, including receptor number or affinity and second messenger mobilization.

GnRH is primarily recognized for its regulation of LH and FSH release from the pituitary. However, it is also thought to be an important paracrine/autocrine regulator in the gonads. A GnRH-like peptide and GnRH receptors have been isolated from ovarian extracts, and transcription from the genes has also been confirmed in this tissue (6, 7, 8, 9, 10). The identification of ovarian GnRH receptors and evidence of direct effects of the decapeptide on steroidogenesis (11, 12, 13) lend credence to its putative role as an intraovarian hormone. With regard to the mechanism of action of this peptide, it has been shown that activation of GnRH receptors in ovarian cells, like that in pituitary cells, is associated with G protein-mediated activation of phospholipase C (PLC) (11, 14, 15). Rapid incorporation of [32P]orthophosphate into phosphatidic acid and phosphatidylinositols and hydrolysis of phosphatidylinositol (PI) mono- and bis-phosphates with rapid formation of inositol mono-, bis-, and tris-phosphates (InsP, InsP2, and InsP3) and diacylglycerol (DAG) have been described in ovarian tissue. Inositol-1,4,5-trisphosphate (InsP3), acting on InsP3-specific receptors at the endoplasmic reticulum, induces a rapid increase in intracellular Ca2+ ([Ca2+]i), although actions at the plasma membrane have also been described. In addition to its now classic effects through activation of phospholipase C, GnRH may exert its action through phospholipase A2 and phospholipase D stimulation (15).

The present set of experiments was designed to evaluate both GnRH receptor number and affinity and second messenger response to GnRH stimulation comparatively in luteoma and control luteal cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Adult female virgin Sprague Dawley rats (200–250 g) from the Instituto de Biología y Medicina Experimental colony were housed in groups in an air-conditioned room, with lights on from 0700–1900 h. They were given free access to laboratory chow and tap water. At the end of the experimental procedures, animals were killed by decapitation according to protocols for animal use approved by the institutional animal care and use committee (IBYME-CONICET) that follows NIH guidelines.

Tumor-bearing animals were bilaterally ovariectomized, and one ovary was implanted into the spleen 4–6 weeks before the experiments (luteoma), as previously described (3, 4, 5).

Control animals were 23- to 25-day-old female rats injected with 25 IU PMSG (Novormon, Syntex, Buenos Aires) and 25 IU hCG (Endocorion, Elea, Buenos Aires) 48 h later. These animals were used 5 days after hCG injection (SPO).

For GnRH receptor studies only, a second control group was used: 23- to 25-day-old female prepubertal rats without any treatment (PP). This group was included in receptor studies because it has been shown that prepubertal ovaries possess the maximal amount of ovarian GnRH receptors (16, 17); therefore, it served as a control of receptor levels for the experimental groups (luteoma and SPO).

GnRH receptors
Iodination of tracer ([125I]Buserelin) and receptor assays were performed as described previously (5). Briefly, for saturation analysis, membranes from ovaries from SPO and PP rats or luteoma were obtained and incubated with 5–8 x 104 cpm [125I]GnRH agonist. Ligand concentrations were near saturating, representing about 85% receptor occupancy. Nonspecific binding was determined by addition of 1 x 10-6 M unlabeled GnRH agonist and represented 5–8% of the total iodinated tracer. For Scatchard analysis, membranes were incubated with increasing concentrations of the labeled analog (5,000–120,000 cpm). In all cases tubes were incubated for 120 min on ice, and the reaction was terminated by centrifugation at 13,000 rpm for 20 min at 4 C. The supernatants were aspirated and discarded, and the pellets were counted in a {gamma}-spectrometer.

Luteal cells
Animals were operated on as described above to induce the development of the luteoma and were left undisturbed for 4–6 weeks. Cells from ovarian tumors as well as from 23- to 25-day-old SPO were isolated with collagenase, as described previously (4, 5). Cells were then used either the same day for calcium measurements or plated in plastic 24-multiwell plates coated with rat tail collagen (~750,000 cells/ml in DMEM-Ham’s F-12 with 2.2 g/liter sodium bicarbonate, 10% FCS, Nystatin, and gentamicin) for inositol phosphate studies. Note the similitude between both cell types, as observed by light microscopy in fresh cell cultures (10-fold; Fig. 1Go).



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  		Figure 1. Light microscopy photomicrographs of luteoma (upper panel) and superovulated prepubertal ovarian cells (lower panel) after 72 h in culture (magnification, x10).

 
Intracellular Ca2+ measurements
Intracellular calcium was measured as described previously (18). Briefly, fura-2/AM (tetracetoxymethylesther fura-2) was used as a fluorescent indicator. The pellet of luteal cells of each experimental group (luteoma and SPO) was redispersed and incubated in the presence of 2 µM fura-2/AM for 45 min at 37 C in an atmosphere of 5% CO2. Cells were then washed twice and prepared at a density of 2 x 106 cells/ml. Fluorescence was measured in a spectrofluorometer (Jasco Corp., Tokyo, Japan) provided with the accessory CA-261 to measure Ca2+ with continuous stirring, thermostat adjusted to 37 C, and injection chamber. Intracellular Ca2+ levels were registered every second by exposure to alternating 340- and 380-nm light beams, and the intensity of light emission at 505 nm was measured. Drugs (5 µl) were injected into the chamber at 2 min (concentration in chamber, 100 ng/ml buserelin, 1 x 10-12 M hCG, or 0.5 µM thapsigargin). The preparation was calibrated determining maximal fluorescence induced by 0.1% Triton X-100 (Fmax) and minimal fluorescence (Fmin) in the presence of 5 mM EGTA. [Ca2+]i was calculated according to the method of Grynkiewicz (19). Basal values were considered to be those measured during the first minute of the experiment. Resulting graphs were scanned, processed, and quantified using Ungraph 2.0 and Excel 5.0 software. Results were normalized with respect to average basal levels. Experiments were repeated three to six times.

Measurement of inositol phosphates
Inositol phosphates were measured as described previously (20) with minor modifications. Briefly, 1 day after plating, the medium in the wells was changed to fresh medium containing 4 µCi/ml [3H]myo-inositol and incubated for 48 h before the experiment. At the end of the labeling period, the cells were washed twice with DMEM-Ham’s F-12 with 2.2 g/liter sodium bicarbonate containing 0.1% BSA (buffer 1). Cells were then incubated in buffer 1 with 20 mM LiCl for 15 min. Thereafter, stimuli (10 µl) were added (final concentrations in the well, 1 and 100 ng/ml Buserelin and 10 mM NaF), and cells were further incubated for 30 min. After the incubation, the cells were placed on ice, treated with 0.5 M HClO4, and scraped. Well contents were transferred to tubes and centrifuged. The pellets were kept for DNA measurement. The neutralized supernatants (0.72 M KOH and 0.6 M HKCO3) were chromatographed on Dowex (Bio-Rad Laboratories, Inc., Hercules, CA) columns (formate form) to elute InsP, InsP2, and InsP3. Two-milliliter aliquots of each wash were mixed with 6 ml Optiphase Hisafe 3 (Wallac, Turku, Finland) and counted in a liquid scintillation counter. Experiments were repeated three to five times.

Statistical analysis
Scatchard analysis of binding data was performed using a computer curve-fitting program (Ligand) for a single class of binding sites. Changes in receptor number among groups were analyzed using one-way ANOVA followed by Tukey’s test. In intracellular calcium studies, the amount of calcium released was assessed by the area under the curve between 2 and 3 min or 2 and 4.30 min, depending on the stimulus; differences in areas were analyzed by multiple variance analysis for paired samples, followed by Tukey’s test. For inositol phosphate studies, differences among groups were analyzed by multiple variance analysis for paired samples, followed by Tukey’s test. In all cases P < 0.05 was considered significant.

Drugs
[D-Ser(tBu)6-des-Gly10]GnRH-N-ethylamide (Buserelin), a GnRH agonist, was a gift from Hoechst (Buenos Aires, Argentina). PMSG (Novormon) was a gift from Syntex (Buenos Aires, Argentina), and hCG (Endocorion) was purchased from Elea (Buenos Aires, Argentina). NaF, myo-inositol, LiCl, fura-2/AM, and thapsigargin were purchased from Sigma Chemical Co. (St. Louis, MO). 125Iodine and [2-N-3H]myo-inositol (20 Ci/mmol) were obtained from NEN Life Sciences Products (Boston, MA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GnRH receptors in ovarian tissues from luteoma-bearing rats, prepubertal female rats, and SPO rats
According to Scatchard analysis ovarian tissues showed a single class of high affinity binding sites (Fig. 2Go, upper panel). Kd values were similar among the groups (PP, 0.054 ± 0.020 nM; SPO, 0.048 ± 0.039 nM; luteoma, 0.047 ± 0.032 nM).



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  		Figure 2. GnRH receptors in ovarian tissues. Upper panel, Scatch-ard analysis of GnRH binding to membranes of ovarian tumors (LUTEOMA), prepubertal ovaries (PP), and superovulated prepubertal ovaries (SPO). One experiment representative of three is shown. Lower panel, Number of GnRH receptors in the different tissues determined by saturation analysis (n = 8 for each tissue). a, Significantly different from SPO; b, significantly different from PP; c, significantly different from LUTEOMA.

 
Significant differences in GnRH receptor numbers were observed among all ovarian tissues (P < 0.001; Fig. 2Go, lower panel). Ovaries of PP animals had the largest number of GnRH receptors, followed by the luteoma group and finally by the ovaries of SPO rats.

[Ca2+]i mobilization in ovarian cells from luteoma and ovaries from SPO rats
Changes in intracellular calcium induced by different stimuli were monitored in luteoma and SPO cells. No differences were observed in basal calcium levels between the groups (basal [Ca2+]i, 171.6 ± 23.5 and 194.5 ± 18.1 nM in luteoma and SPO cells, respectively; n = 5). As expected, Buserelin induced a significant and classical release of [Ca2+]i in SPO cells. In contrast, no mobilization of calcium was observed in luteoma cells (Fig. 3Go and Table 1Go). hCG, an agent proposed to activate PLC in the ovary, was also able to induce a significant increase in [Ca2+]i levels in SPO cells, although of less magnitude than that induced by Buserelin, both at concentrations that induce maximal endocrine responses (Table 1Go). Again, luteoma cells were unresponsive (Fig. 4Go). Thapsigargin, an inhibitor of Ca2+-adenosine triphosphatase associated with the endoplasmic reticulum, induced calcium release in both cell types (Fig. 5Go), although the levels achieved were significantly higher in luteoma than in SPO cells [area under the plateau from 2.05–4.55 min: luteoma, 2195 ± 371.3 (7%); SPO, 1187 ± 195.3 (6%); P < 0.05].



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  		Figure 3. [Ca2+]i mobilization induced by Buserelin (100 ng/ml in chamber) in luteoma and SPO cells. Curves represent the average of five experiments (percent increase with respect to basal levels), and lines on top represent the SE for each point. Basal levels are cited in the text. Buserelin was administered at 2 min.

 

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  		Table 1. Areas (percentages) under the peak of intracellular calcium curves induced by different stimuli in dispersed luteoma and SPO cells from 2.05–3.05 min

 


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  		Figure 4. [Ca2+]i mobilization induced by hCG (1 x 10-12 M) in luteoma and SPO cells. Curves represent the average of four experiments. hCG was administered at 2 min.

 


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  		Figure 5. [Ca2+]i mobilization induced by thapsigargin (0.5 µM) in luteoma and SPO cells. Curves represent the average of six or seven experiments. Thapsigargin was administered at 2 min.

 
Inositol phosphates in cell cultures from luteoma and ovaries from SPO rats
No significant differences in basal total inositol phosphates, including InsP, InsP2, and InsP3, were observed between luteoma- and SPO-derived cells. In SPO cells, Buserelin (100 ng/ml) and NaF (10 mM) induced significant increases in total inositol phosphates (P < 0.01; Fig. 6Go). Buserelin in concentrations as low as 1 ng/ml was still able to significantly increase inositol phosphate levels in SPO cells, although to a lesser degree than 100 ng/ml Buserelin. This indicated a concentration-dependent effect [1 ng/ml Buserelin, 4210 ± 380 cpm; 100 ng/ml Buserelin, 5970 ± 590 cpm; n = 3; by one-way ANOVA of control, 1 ng/ml Buserelin, and 100 ng/ml Buserelin followed by Student-Newman-Keuls test: 1 ng/ml Buserelin vs. 100 ng/ml Buserelin, P < 0.05]. In contrast, in luteoma cells only NaF was able to significantly augment total inositol phosphate levels (P < 0.05). The percent increase induced by NaF was significantly higher in SPO than in luteoma cells (SPO, 950 ± 132%; luteoma, 700 ± 106%; P < 0.05). When the effects of the different stimuli were analyzed on separate inositol phosphates (InsP, InsP2, and InsP3) formed from the hydrolysis of phosphatidylinositols in both kinds of cells, it was observed that in SPO cells both Buserelin and NaF significantly stimulated the increase of all three inositol phosphates (Fig. 7Go). Again, a concentration-dependent response to Buserelin (1 and 100 ng/ml) was observed in InsP, InsP2, and InsP3 in control cells (not shown), like that observed in total inositol phosphates. In luteoma cells, NaF significantly affected the three inositol phosphates analyzed, whereas Buserelin had no significant effect on any of them. Interestingly, for Buserelin the highest percent increase over basal levels in the different inositol phosphates was noted in InsP2, whereas for NaF, which stimulated the G protein directly, the maximal increase was observed in InsP3 (Table 2Go). Moreover, the percent increase in InsP3 induced by NaF in both cell kinds was very similar (Table 2Go). Although no significant differences in basal levels of total inositol phosphates, InsP, InsP2, or InsP3 were observed between luteoma and SPO cells, intracellular free [3H]inositol levels were significantly higher in SPO cells (Fig. 8Go). This difference could not be accounted for by differences in [3H]inositol incorporation (luteoma, 300,346 ± 27,934 cpm; SPO, 349,742 ± 17,802 cpm; n = 3; P = NS), which was not significantly different between the groups, or by differences in cell number at the end of the experiments, as these varied maximally by 25% in favor of SPO, as assessed by DNA measurement in the pellets after cell lysis (not shown). After 30-min incubation with the different stimuli, a significant decrease in free [3H]inositol was only observed after Buserelin treatment in SPO cells (Fig. 8Go). Decreases in free inositol in response to PLC-activating agents have been reported previously (21). It is interesting to note that NaF, which elicited the largest amount of inositol phosphates, did not alter free [3H]inositol in either cell type.



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  		Figure 6. Effect of 30-min stimulation with Buserelin (Bus; 100 ng/ml) and NaF (10 mM) on total inositol phosphates (counts per min/1 x 10-3) in luteoma and SPO cells in primary culture. Cells were labeled with 4 µCi [3H]inositol and preincubated with LiCl (20 mM). Results represent the average of five experiments. Multiple ANOVA of repeated measures indicates a significant interaction (P < 0.01). For this and the following figures, the asterisk indicates a significant difference between cell types for a given stimulus. a, Significantly different from control levels in SPO cells. b, significantly different from control levels in luteoma cells. In all cases, P < 0.05 or less.

 


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  		Figure 7. Effects of Buserelin (Bus; 100 ng/ml) and NaF (10 mM) on InsP (upper panel), InsP2 (middle panel), and InsP3 (lower panel) in luteoma and SPO cells in primary culture. Conditions were the same as those described in Fig. 6Go. Results represent the average of three experiments. Multiple ANOVA of repeated measures indicates a significant interaction (P < 0.01 or less) for each inositol phosphate.

 

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  		Table 2. Percent increase in InsP, InsP2, and InsP3 with respect to control levels (100%) in cultured luteoma and SPO cells after a 30-min incubation with different stimuli

 


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  		Figure 8. Free [3H]inositol levels in luteoma and SPO cells. Experimental procedures are detailed in Figs. 6Go and 7Go. Results represent the average of five experiments. Multiple ANOVA of repeated measures indicates a significant interaction (P < 0.01).

 
To determine whether the difference in basal free [3H]inositol between the two cell types could be due to differential incorporation of this compound into the various phosphatidylinositols in the plasma membranes, these were measured, as in previous works (22, 23), after extraction with methanol/chloroform of the membrane pellet by TLC. A representative experiment shows that the incorporation of [3H]inositol into PI was specifically augmented in luteoma cells with respect to that in SPO cells (luteoma, 35.1 ± 1.1%; SPO, 21.9 ± 1.0% of radioactivity incorporated into membrane lipids; P < 0.05). This 60% increase in [3H]inositol incorporated into PI could be the explanation for the low levels of free inositol observed in luteoma cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In previous works we had established the participation of GnRH in the control of the endocrine function of these experimental luteoma, indicating a direct effect on the tumor in addition to its effect through gonadotropin modulation (4, 5). Luteoma cells were more sensitive to the GnRH-induced inhibition of progesterone elicited by a LH stimulus than control luteal cells under those experimental conditions. This difference prompted us to study the receptors and mechanisms of action of the decapeptide in more detail in luteoma and control luteal tissue.

GnRH receptors have been described in ovarian tissue (7, 9, 16), and we had already determined their presence in experimental luteoma (5). Significant differences in receptor number, although not in Kd values, were observed among the tested tissues. Ovarian GnRH receptor number was significantly greater in prepubertal animals than in prepubertal superovulated animals subjected to gonadotropin stimulation. This is in agreement with previous results (16, 17) and parallels the observations in the pituitary, where receptors have been described to be maximal previous to puberty onset (18, 24). Furthermore, receptor number was significantly higher in luteoma tissue than in ovaries from PMSG-hCG-treated prepubertal rats, although both were under the influence of very high gonadotropin levels. This difference in receptor number could justify a higher sensitivity of luteoma cells to GnRH action. It is important to take into account that GnRH receptor number per mg tissue does not increase when an estrous ovary turns into a luteoma (5). In these ovarian tumors, receptor levels are maintained even in the presence of very high gonadotropin levels, marking a difference from control ovaries from prepubertal rats, in which receptors abruptly fall after PMSG-hCG treatment.

Subsequently, the effect of GnRH on the increase in classical second messengers elicited by this peptide, inositol phosphates, and calcium (14, 15, 25) was analyzed to establish whether the increase in receptor number was coupled to an increase in second messenger response. Buserelin, in a dose that was maximal to exert its endocrine action, induced a typical calcium response in control SPO cells, in agreement with results from other laboratories (26, 27, 28). Surprisingly, no response was observed in tumor cells. Furthermore, a maximal hCG stimulus was also able to induce a calcium transient in SPO cells, although of lower intensity than that induced by Buserelin. The effect of LH (hCG) on calcium mobilization is controversial. Although the classical mechanism of action of LH implies stimulation of adenylate cyclase and cAMP production (15), the regulation of steroidogenesis by LH may be exerted through the stimulation of multiple pathways. The activation of PLC and intracellular calcium increases have been involved in the action of gonadotropins in the ovary in several species, including the mouse, swine, hen, and cow (29, 30, 31, 32). In other species, such as sheep and rats, LH did not induce calcium transients in the ovary (21, 33, 34), although increases in InsP3 formation after LH treatment were observed in these species (35, 36). In our experimental conditions, hCG induced significant calcium increases in rat SPO cells in a variety of concentrations (1 x 10-12 to 1 x 10-9 M; not shown). Again, no effect on intracellular calcium was observed in tumor cells under hCG stimulation. As shown above, we were unable to induce any calcium transients in luteoma cells with the stimuli tested. To evaluate the possibility that luteoma cells were unresponsive to GnRH or hCG but were still able to respond to other calcium-inducing agents, cells were tested in the presence of thapsigargin. Thapsigargin is a specific inhibitor of the endoplasmic reticulum calcium-adenosine triphosphatase (37) and therefore induces increases in cytosolic calcium due to calcium leakage, a mechanism independent of PLC activation. This drug has been shown to induce calcium release in rat ovarian cells (27, 38). In our case, both kinds of cells responded to 0.5 µM thapsigargin with calcium increases, although calcium levels attained in luteoma cells were significantly higher than those in SPO cells. It is interesting to note that although the thapsigargin-sensitive calcium stores in luteoma cells were augmented with respect to those in control cells, they were insensitive to GnRH stimulation. This resistance to increase intracellular calcium levels might be an adaptation of the luteoma cells to maintain low calcium levels, as increases in intracellular calcium, elicited by GnRH or PGF2{alpha}, have been proposed to induce cell death in ovarian tissue (39, 40, 41), which would hinder tumor growth. The use of other pharmacological agents acting on calcium metabolism will allow us to determine whether the impairment of calcium release after specific stimulation is a generalized phenomenon in these cells.

The failure of luteoma cells to respond to GnRH with a calcium transient is not an isolated observation, as it is in agreement with our results in phospholipid hydrolysis. Although both cell types had similar basal levels of inositol phosphates, the GnRH agonist and NaF induced significant increases in inositol phosphates in SPO cells, but only NaF did so in tumor cells. NaF is an activator of G proteins coupled to PLC by substituting for endogenous guanosine triphosphate (42). Therefore, the activation of phospholipid hydrolysis induced by this agent in luteoma cells implies that PLC is active. Moreover, when expressed as a percentage of control levels, the amount of InsP3 formed was identical in luteoma and SPO cells under NaF stimulation. The lack of a significant amount of InsP3 formation in response to Buserelin in luteoma cells correlates with the lack of calcium mobilization observed in these cells under this stimulus. In control SPO cells, Buserelin induced a concentration-dependent increase in all three inositol phosphates, as expected because calcium was also released by this treatment.

Taken together, these results suggest an uncoupling of GnRH and LH membrane receptors from PLC in luteoma cells, as evidenced by the lack of either calcium or inositol phosphate (or both) responses. This implies that the inhibition exerted by GnRH on LH-induced progesterone secretion in these cells, observed in former studies (4, 5), is probably not mediated by the classical GnRH activation of PLC as has been suggested for control luteal cells (14, 15, 21, 25, 26, 41). Alternative mechanisms for GnRH action in the ovary have been proposed. In addition to the generation of the calcium-mobilizing inositol phosphate(s) and protein kinase C (PKC) activator DAG, GnRH has also been reported to cause accumulation of arachidonic acid in ovarian cells (43, 44, 45) through PLC or PLA2 stimulation (43, 46). It has also been suggested that PLA2-induced increases in arachidonic acid may increase progesterone levels (47); on the other hand, an increase in PLA2 activity caused a loss in progesterone secretion in late pregnancy (45). GnRH activation of phospholipase D, with a resultant increase in phosphatidic acid, has also been described (48). Among other effects, phosphatidic acid can be converted into DAG without a concomitant increase in InsP3, and DAG has been implicated in PKC activation (49), which, in turn, is postulated to be responsible for the inhibition of LH-induced progesterone secretion (13). Several examples from the literature show that a receptor can be uncoupled from one second messenger-generating system while still being active on another. Davis (42) showed that although the phorbol ester 12-O-tetraphorbol 12-myristate 13-acetate, an activator of PKC, inhibited the actions of LH receptor stimulation on phospholipid turnover, it was without effect on receptor-induced activation of cAMP and progesterone accumulation in bovine luteal cells. Another example was presented by McCann and Flint (50), who showed that treatment of sheep luteal tissue with pertussis toxin inhibited PGF2{alpha} action on PLC, whereas the inhibitory effect of PGF2{alpha} on LH-stimulated adenylate cyclase was conserved. Therefore, it is possible that although GnRH receptors might be uncoupled from PLC in luteoma cells, they might still be coupled to other second messenger-generating systems, such as PLA2 or phospholipase D, to produce their antigonadotropic effects. Further studies will be needed to determine which pathway(s) is involved.

Although [3H]inositol incorporation was similar in both cell types, a highly significant difference in free [3H]inositol levels was observed. These levels were approximately 10 times higher in SPO cells than in luteoma cells, marking an interesting alteration in inositol metabolism in tumor cells. A representative experiment shows an important increase in membrane phosphatidylinositol in luteoma cells compared with that in control luteal cells, which could explain this difference. Unusual metabolism of phosphoinositides in tumor cells has been reported previously, as in MA-10 Leydig tumor cells (22, 23). The particular metabolism of phosphoinositides in luteoma cells will be the subject of future research.

In summary, our data show that luteoma cells, which develop under high constant gonadotropin stimulation, possess GnRH receptors that are not down-regulated in this particular endocrine milieu. Furthermore, they are uncoupled from their classic second messenger-generating system, PLC. Metabolism of inositol into phospholipids is also notably altered in luteoma cells. These data provide evidence that the transformation of the ovary into a luteoma implies the acquisition of novel characteristics in the GnRH receptor second messenger-generating system.


   Footnotes
 
1 The data in this paper are from a thesis to be submitted for the degree of Doctor of Philosophy at the University of Buenos Aires (Buenos Aires, Argentina). Portions of this work have been presented in abstract form at the meeting of the Argentine Society of Clinical Investigation, Mar del Plata, Argentina, November 1997. This work was supported by the University of Buenos Aires, Agencia Nacional de Promocion Cientifica y Tecnologica and Consejo Nacional De Investigaciones Cientificas y Tecnicas (CONICET) (Buenos Aires, Argentina). Back

Received November 5, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Biskind M, Biskind G 1944 Development of tumors in the rat ovary after transplantation into the spleen. Proc Soc Exp Biol Med 55:176–179
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