Both Prolactin and Progesterone in Proestrus Are Necessary for the Induction of Apoptosis in the Regressing Corpus Luteum of the Rat¹

^a Departments of Cell Biology, ^b Physiology, and ^c Pathology, Faculty of Medicine, University of Córdoba,14071 Córdoba, Spain

	ABSTRACT

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This study was conducted to analyze the roles of prolactin (PRL) and progesterone in the induction of luteal cell apoptosis and accumulation of macrophages in the regressing corpus luteum. We studied the number of apoptotic cells and macrophages in regressing corpora lutea in estrus 1) in cycling rats or after blocking PRL secretion with the dopaminergic agonist CB154, and 2) after blocking progesterone actions with the progesterone receptor antagonists RU-486 or ZK98299. Cells showing the morphological features characteristic of apoptosis contained fragmented DNA as indicated by in situ 3' end labeling.

In cycling rats, a 100-fold increase in the number of apoptotic cells and a 4-fold increase in the number of macrophages was found from the evening (1600 h) of proestrus to the morning (1100 h) of estrus. Both increases were blocked by PRL suppression with CB154. Furthermore, blocking progesterone actions with progesterone receptor antagonists RU-486 or ZK98299 without affecting PRL secretion inhibited apoptosis but did not affect the accumulation of macrophages, whether treatment was started on the morning of metestrus (blocking diestrous and proestrous progesterone) or on proestrus (blocking only proestrous progesterone). Otherwise, exogenous progesterone was not effective in inducing apoptosis in the absence of PRL. These results indicate that both PRL and progesterone in proestrus are necessary for the induction of apoptosis in the regressing corpora lutea, whereas the accumulation of macrophages seemed to be dependent exclusively on the PRL surge.

	INTRODUCTION

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In the absence of pregnancy in the rat, the corpus luteum (CL) has a very limited life span. Functional demise and structural luteolysis are necessary to maintain cyclicity and to avoid accumulation of nonfunctional luteal tissue within the ovary, respectively [1]. The mechanisms controlling luteolysis are complex and show a considerable interspecies variation [2, 3]. However, a common feature in the regressing CL of many species is the abundance of macrophages [4–12]. Furthermore, treatments that induce luteolysis also induce accumulation of macrophages in the regressing CL. This has been observed in the rat after prolactin (PRL) treatment [11, 12], in the rabbit after estrogen withdrawal [13], and in the pig during prostaglandin F₂-induced luteolysis [9]. Since macrophages have the ability to damage cells and to phagocytize them, a pivotal role for these cells in luteolysis has been proposed [6].

In the rat, PRL induces structural luteolysis in the CL that has ceased producing progesterone [14]. The preovulatory PRL surge in cycling animals, or the administration of exogenous PRL, induces luteolysis, expression of monocyte chemoattractant protein-1, and accumulation of macrophages in regressing CL [10–12]. However, the existence of cause-and-effect relationships between PRL-induced cell death and invasion of macrophages is not certain, and several possibilities exist. First, PRL could induce accumulation of macrophages that could then damage luteal cells through the release of cytotoxic cytokines or reactive oxygen species. Second, PRL could induce luteal cell death, and macrophages could be consequently attracted through the release of chemotactic factors from damaged cells. Otherwise, both events (induction of cell death and accumulation of macrophages) could be coupled by sharing some common endocrine/paracrine signals, but not be causally related.

In addition to PRL, progesterone has long been suggested to have a role in CL regulation [1, 14–16]. Previous studies blocking progesterone actions with the antiprogesterone RU-486 showed the existence of enlarged CL [17, 18], which could be due to enhanced luteogenesis and/or to decreased luteolysis. Progesterone prevents apoptosis in the uterine epithelium and mammary gland [19, 20], and apoptotic changes in the cattle CL are preceded by a decline in progesterone secretion [21]. Progesterone has been found to modulate interleukin-1 [22] and superoxide anion [23] production by macrophages. Therefore, progesterone could affect luteolysis either directly or indirectly through actions on macrophages.

In order to investigate the relationships between luteolysis and accumulation of macrophages, as well as the roles of PRL and progesterone, we have studied the presence and numbers of apoptotic cells and macrophages in regressing CL of rats both cycling and after treatment with the dopaminergic agonist CB154 or antiprogestins RU-486 or ZK98299.

	MATERIALS AND METHODS

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Animals and Chemicals

Female cycling rats of the Wistar strain (250 g average BW) were used. The animals were maintained under controlled light (14L:10D; lights-on at 0500 h) and temperature (21°C) and had free access to rat chow and tap water. The stage of the cycle was checked daily by examining vaginal smears. Only rats showing at least two consecutive 4-day cycles were used.

The dopaminergic agonist 2-Br--ergocryptine (CB154), which specifically inhibits PRL secretion, was purchased from Sandoz (Basel, Switzerland). The antiprogestins RU-486 and ZK98299 were obtained from Exelgyn (Paris, France) and Schering (Berlin, Germany), respectively. The LHRH antagonist used was ORG.30276 (Ac-D-p-Cl-Phe-D-p-Cl-Phe-D-Trp-Ser-Tyr-D-Arg-Leu-Arg-Pro-D-Ala-NH₂·CH₃·COOH; Organon, Oss, The Netherlands). Progesterone was obtained from Sigma Chemical Company (St. Louis, MO).

Experimental Designs

First experiment This experiment was conducted to analyze the effects of the suppression of the proestrous afternoon PRL surge on apoptosis and macrophage accumulation in regressing CL of the previous cycle. Cycling rats received injections of CB154 (1 mg/rat at 1000 h on proestrus) or vehicle (250 µl of 70% ethanol). Vehicle-injected rats were killed on proestrus (1600 h) and estrus (1100 h), to study apoptosis and macrophage numbers before and after PRL surge. CB154-treated rats were killed on estrus (1100 h). Five animals per group were studied.

Second experiment To analyze the effects of blocking progesterone actions, cycling rats received injections of RU-486 (4 mg/day) at 1000 h on metestrus, diestrus, and proestrus (to block both diestrous and proestrous progesterone actions) or at 1000 h on proestrus (to block only proestrous progesterone actions) or vehicle (250 µl of olive oil). Additional rats received injections of ZK299 (4 mg/rat in 250 µl of olive oil) on the morning of proestrus (1000 h). Five animals per group were killed at 1100 h on estrus. Results for vehicle-injected rats in the different groups were equivalent and were therefore pooled.

Third experiment To analyze the effects of exogenous progesterone in the absence of PRL and endogenous follicular progesterone, cycling rats received s.c. injections of LHRH antagonist (1 mg/rat in 200 µl of NaCl) on the morning (1000 h) of proestrus to suppress the preovulatory LH surge-dependent progesterone secretion, and of CB154 (1 mg/rat) to suppress the PRL surge. These animals then received injections of progesterone (5 mg/rat) or vehicle (200 µl of olive oil) at 1430 h on proestrus. Five animals per group were killed at 1100 h on estrus.

Fourth experiment In order to determine whether cells with the morphological features considered characteristic of apoptosis in experiments 1–3 contained fragmented DNA, three cycling rats were killed in the morning of estrus. Right ovaries were fixed in Bouin-Hollande's fluid and left ovaries in 4% paraformaldehyde (PFA) as in previous experiments. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method for detection of fragmented DNA was carried out in 4-µm-thick sections fixed in either Bouin-Hollande's fluid or 4% PFA. Morphological identification of apoptotic cells was performed in Bouin-Hollande-fixed tissues stained with hematoxylin and eosin.

The number of apoptotic cells/bodies recognized by their morphological features, and the number of cells or apoptotic bodies immunostained with the TUNEL method (in 4% PFA-fixed tissues) were counted in five sections per CL, from at least 3 different regressing CL (those of the previous cycle) per rat.

Tissue Processing

The right ovaries were dissected and fixed in Bouin-Hollande's fluid for 24 h and processed for paraffin embedding. The left ovaries were fixed in 4% paraformaldehyde in Sorensen buffer, pH 7.3, for 24 h and processed for paraffin embedding.

The ovaries were serially sectioned (4-µm-thick sections). Under a phase contrast microscope, nonconsecutive sections showing newly formed or regressing CL were selected for immunohistochemistry. Apoptotic cells were studied in Bouin-Hollande-fixed tissues, whereas macrophages were detected in paraformaldehyde-fixed tissues.

Immunohistochemistry

Macrophages were detected by immunohistochemistry with the monoclonal antibody ED1, following previously described methods [12, 24]. This antibody recognizes a lysosome-associated antigen in macrophages [25]. Briefly, paraformaldehyde-fixed sections (4-µm thick) were placed on poly-L-lysine-coated slides, and after dewaxing and inhibition of peroxidase with 2% hydrogen peroxide in methanol for 30 min, sections were rinsed in PBS, blocked with 10% normal rabbit serum for 2 h, and incubated overnight with mouse monoclonal ED1 antibody (diluted 1:400). The sections were processed according to the avidin-biotin complex (ABC) method. Briefly, sections were treated sequentially with rabbit anti-mouse IgG-biotin conjugate (Sigma, London, UK; 1:1000 1 h at room temperature), and avidin-biotin peroxidase complex (Vector Labs, Burlingame, CA; 1 h at room temperature). Tissue-bound peroxidase was visualized by incubation in 0.03% diaminobenzidine-tetrahydrochloride (Type IV; Sigma, St. Louis, MO), 0.01% hydrogen peroxide in 0.1 M Tris-buffer (pH 7.6) for 1 min. Afterwards, sections were darkened in 1% copper sulphate for 5 min and counterstained with hematoxylin.

Negative controls for immunohistochemistry were run by incubating the sections with nonimmune serum instead of the primary antibody.

Cell Counting

The regressing CL of the previous cycle were studied on the morning (1100 h) of estrus. In vehicle-injected rats, CL were also studied on the evening (1600 h) of proestrus (experiment 1). Regressing CL were easily distinguishable from late-regressing CL by their general appearance, size, and ratio of parenchymal to stromal cells. They were also easily distinguishable from newly formed CL, when present, due to the incomplete morphological luteinization of the new CL. At least three CL per rat were studied. Five nonconsecutive sections were scored. For this, the apoptotic cells and macrophages (showing ED1 immunoreactivity, and counted only if the nucleus was present in the section) were counted systematically throughout the section, following previously described procedures [12]. Numbers were expressed per square millimeter of tissue section. Apoptotic cells were recognizable by their characteristic morphological features [26] such as shrunken eosinophilic cytoplasm and condensed chromatin, which was either delineated into discrete masses aligned with the nuclear membrane or fragmented into multiple densely stained masses clustered together.

In Situ 3' End-Labeling (TUNEL)

Apoptotic cell detection was based in the method described by Gavrieli et al. [27], with the In Situ Cell Death Detection Kit, POD (Boehringer, Mannheim, Germany), according to the instructions of the supplier, with slight modifications. After dewaxing and hydration, endogenous peroxidase was blocked by incubation for 30 min in 2% H₂O₂ in methanol. After being washed with PBS, sections were submitted to protease treatment. Three different proteases were assayed, since results were highly dependent on this step. Proteinase K (5 mg/ml in 20 mM Tris, 2 mM CaCl₂, pH 7.2, from 5 to 25 min at 37°C; Dako Diagnostica, Glastrup, Denmark), Protease XIV Pronase E (0.1% w:v in 50 mM phosphate buffer, pH 7.2, from 5 to 15 min at room temperature; Sigma Chemical Co.), and trypsin (0.2% w:v in 50 mM phosphate buffer, pH 7.2, from 5 to 20 min at 37°C; Difco, Detroit, MI). Sections were washed again and treated with TUNEL reaction mixture (containing terminal deoxynucleotidyl transferase and label solution) for 60 min at 37°C. After being washed in PBS, sections were incubated with the anti-label antibody conjugated with horse-radish peroxidase (POD). Negative controls were incubated with label solution lacking terminal deoxynucleotidyl transferase. Afterwards, 3' end-bound POD was visualized by incubation in 0.03% diaminobenzidine-tetrahydrochloride (Type IV; Sigma), 0.01% hydrogen peroxide in 0.1 M Tris-buffer (pH 7.6) for 1 min. Afterwards, sections were darkened in 1% copper sulphate for 5 min and counterstained with hematoxylin.

Statistical analysis was carried out by ANOVA and Tukey's test comparison method.

	RESULTS

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Effects of PRL Suppression

In the evening (1600 h) of proestrus, vehicle-injected rats showed CL in which apoptotic cells were very scarce. Macrophages with elongated shape and scant cytoplasm were present in low numbers. In the morning (1100 h) of estrus, apoptotic cells were extremely abundant and uniformly distributed throughout the CL section (Fig. 1A). Most of these cells corresponded to advanced apoptotic cells that showed eosinophilic cytoplasm and fragmented nuclei. Macrophages were also abundant, and most of them contained phagocytized material (Fig. 1B). In rats treated with CB154, apoptotic cells were nearly absent (Fig. 1C), and macrophages (Fig. 1D) were similar, in both number and size, to those present in vehicle-injected rats in the evening of proestrus. Quantitative data are shown in Figure 2. There was a 100-fold increase in the number of apoptotic cells, and a 4-fold increase in the number of macrophages in vehicle-injected rats from the evening (1600 h) of proestrus to the morning (1100 h) of estrus. The increase in the number of both apoptotic cells and macrophages was blocked by treatment with CB154.

View larger version (159K): [in this window] [in a new window]   FIG. 1. Micrographs of the CL on the morning of estrus from rats treated with vehicle (A, B) or CB154 (C, D) on proestrus; RU-486 on metestrus, diestrus, and proestrus (E, F); or RU-486 on proestrus (G, H). A,C,E,G) Hematoxylin and eosin-stained sections in which apoptotic cells (arrows) can be observed. B,D,F,H) Immunostained sections for ED1 antigen. Macrophages (arrows) can be observed. x625.

View larger version (24K): [in this window] [in a new window]   FIG. 2. Numbers of macrophages and apoptotic cells in the CL of rats in proestrus and estrus, and treated with vehicle or CB154 on proestrus. a, p < 0.01 vs. proestrus; b, p < 0.01 vs. vehicle.

Effects of Antisteroids RU-486 and ZK98299

Rats treated with RU-486 from the morning of metestrus showed unruptured luteinized follicles in the morning of estrus. The regressing CL of the previous cycle were large and showed only occasional apoptotic cells (Fig. 1E). Macrophages were abundant (Fig. 1F), although they did not contain phagocytized apoptotic cells. In rats treated with RU-486 or ZK299 on the morning of proestrus, newly formed CL were present, indicating the occurrence of ovulation, although unruptured unluteinized follicles were also found in some animals. Regressing CL showed occasional apoptotic cells (Fig. 1G) and abundant macrophages (Fig. 1H), similar to those found in rats treated with RU-486 from the morning of metestrus. Quantitative data are presented in Figure 3. The number of apoptotic cells were highly decreased (about 86%) in RU-486-treated rats, whether the treatment was started on metestrus or on proestrus, as well as in ZK299-treated rats. However, the number of macrophages was not affected by any antiprogesterone treatment.

View larger version (34K): [in this window] [in a new window]   FIG. 3. Numbers of macrophages and apoptotic cells in the CL of rats in estrus and treated with oil or RU-486 from metestrus (M) or on proestrus (P), or with ZK89299 (ZK299) on proestrus (P). a, p < 0.01 vs. oil.

Effects of Exogenous Progesterone in the Absence of PRL

Rats treated with LHRH antagonist and CB154 showed unruptured preovulatory follicles, and newly formed CL were absent. Regressing CL showed absence of apoptotic cells and low numbers of macrophages, equivalent to those present in CB154-treated rats (experiment 1). The administration of progesterone in the early evening of proestrus did not increase the number of apoptotic cells or macrophages. Quantitative data are shown in Figure 4. No significant changes were found after progesterone treatment.

View larger version (23K): [in this window] [in a new window]   FIG. 4. Numbers of macrophages and apoptotic cells in the CL of rats in estrus and treated with vehicle or LHRH antagonist (LHRH-ANT) plus CB154 on proestrous morning and with oil or progesterone (P4) on proestrous afternoon. a, p < 0.01 vs. vehicle.

Comparison of Morphological with In Situ 3' End Labeling Identification of Apoptotic Cells

Best results (intensity of the label, nuclear morphology, and tissue architecture preservation) with the TUNEL method were obtained after Proteinase K pretreatment for 15 min in 4% PFA-fixed tissues. In Bouin-Hollande-fixed pieces, tissue architecture was disrupted even with the minimal protease incubation times necessary to label fragmented chromatin. Otherwise, nuclear morphology in hematoxylin and eosin-stained tissues was better preserved in Bouin-Hollande-fixed tissues. Comparison between the morphological features and immunostaining (TUNEL) of apoptotic cells is shown in Figure 5. All cells showing morphological features considered characteristic of apoptosis contained fragmented DNA as indicated by immunostaining with the TUNEL method. This was evident not only for apoptotic bodies or advanced apoptotic cells but also for cells with slight signs of apoptosis. Otherwise, cells with normal morphological features were not immunostained. Dying granulosa cells in atretic follicles, previously found to be apoptotic [28] were intensely stained with the TUNEL method and serve as positive controls. No significant differences for counts of apoptotic cells in hematoxylin and eosin-stained vs. immunostained apoptotic cells with the TUNEL method (812 ± 63 vs. 779 ± 81 cells/mm², respectively; mean ± SEM for n = 3, Student's t-test) were found.

View larger version (71K): [in this window] [in a new window]   FIG. 5. Micrographs of regressing CL (A–D) and an atretic follicle (E) on the morning of estrus, stained with hematoxylin and eosin (A, C) or in situ 3' end labeling (TUNEL) (B, D, E). Apoptotic bodies (arrowheads) and early apoptotic cells (arrows) can be observed. A–D) x500; E) x225.

	DISCUSSION

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On the morning of estrus, abundant cells with the characteristic morphological features of apoptosis were present in the CL of the previous cycle. These included cells with slight signs of apoptosis (margination of the chromatin with preservation of the general nuclear morphology) and cells with advanced signs of apoptosis (containing a single chromatin mass or multiple nuclear fragments, sometimes observed inside the cytoplasm of macrophages). All these cells contained fragmented DNA, as revealed by in situ 3' end-labeling. No significant differences in cell counts were found between morphological evaluation and the TUNEL method for the identification of apoptotic cells. This agrees with previous studies that have shown that all cells classified as apoptotic on the basis of morphological criteria contained fragmented DNA [28] and that no differences were found between counts in hematoxylin and eosin-stained sections and in situ 3' end-labeled tissues [29]. These results confirmed that apoptosis is involved in CL regression in the rat and validated the evaluation of apoptosis by morphological criteria during structural luteolysis.

This study demonstrated that the induction of luteal cell death and accumulation of macrophages during luteolysis involve different mechanisms. In control rats, macrophage accumulation occurred from the evening of proestrus to the morning of estrus and seemed to be dependent exclusively on the preovulatory PRL surge. This agrees with previous studies indicating that the PRL surge in cycling rats [12] or the administration of PRL [11] induce macrophage accumulation in regressing CL. The entrance of macrophages is probably dependent on the release of chemotactic factors from the CL. Previous authors have shown that regressing CL in the rat [10] and sheep [30] express monocyte chemoattractant protein 1 (MCP-1) and that PRL induces the expression of this protein in the CL of the rat [11]. In the present study, macrophage accumulation was not affected by any treatment except CB154, which specifically inhibits PRL secretion. This suggests that PRL is the main factor determining the accumulation of macrophages in the regressing CL of the rat. However, the relationships between the accumulation of macrophages and the induction of cell death are not clear. It has been suggested that luteolysis could be the result of the interaction between inflammatory cells and luteal cells [6]. Conversely, the entrance of macrophages could be due to the presence of damaged luteal cells in the regressing CL. The data of this study indicated that the accumulation of macrophages was not dependent on the presence of dying luteal cells, since they also accumulated in antiprogestin-treated rats, in which apoptotic cells were nearly absent. These data suggest that the mere presence of macrophages is not enough to induce luteal cell apoptosis. Previous studies [13] in the rabbit CL have shown that the presence of macrophages induced by estrogen withdrawal does not preclude the continuation of progesterone production after estradiol replacement. Similarly, treatment of ewes with phorbol 12-myristate 13-acetate (PMA) increases the expression of MCP-1 mRNA in the CL [31] but does not induced structural luteolysis [32]. Taken together, these results suggest that the expression of MCP-1 and the subsequent accumulation of macrophages is not sufficient to cause luteal cell death. However, it cannot be disregarded that an additional signal could be necessary to activate PRL-recruited macrophages to a cytocidal state.

Induction of apoptosis has been found to be involved in luteolysis in several species [33–35]. Although most dying cells seemed to correspond to vascular cells, apoptotic steroidogenic cells were also found. This study confirms previous reports indicating that PRL induces programmed cell death in regressing CL in rats [34]. Blocking the PRL surge with the dopaminergic agonist CB154 completely inhibited luteal cell apoptosis. This agrees with the persistence of the CL in CB154-treated rats [36]. Interestingly, the induction of programmed luteal cell death was also dependent on progesterone. This was evidenced by the extreme scarcity of apoptotic cells in rats lacking progesterone actions. The inhibition of luteal cell apoptosis in antiprogesterone-treated animals was not due to the disturbance of PRL secretion since it has been previously reported that the preovulatory PRL surge was unaffected by the antiprogesterone treatment [37, 38]. This was also supported by the PRL-dependent accumulation of macrophages, which was unaffected in antiprogestin-treated rats. The inhibition of apoptosis was equivalent in rats treated with RU-486 both from the morning of metestrus (blocking the actions of both diestrous and proestrous progesterone) and on proestrus (blocking the actions of proestrous progesterone). This indicated that progesterone on the afternoon of proestrous is responsible for this luteolytic effect. Proestrous progesterone is produced by preovulatory follicles and is dependent on the LH surge [39], and at this time, the CL has already ceased autonomous production of progesterone [39, 40]. This indicated that, in agreement with previous concepts [1], the loss of the ability of the CL to secrete progesterone is associated with the luteolytic effect of progesterone, whereas this effect is absent in the diestrous CL, which is actively secreting progesterone.

The equivalence of the results obtained after treatment with two different progesterone antagonists, RU-486 and ZK98299, reinforced the evidence for a role of progesterone in the luteolytic process in the rat. RU-486 and ZK98299 have a different mode of action at the molecular level [41, 42] and affect the secretion of gonadotropins differentially [43]. This has been interpreted to mean that RU-486 also blocks ligand-independent activation of progesterone receptor [44, 45]. The present results showed that, at the ovarian level, these antagonists affected apoptosis in regressing CL similarly. Both progesterone and glucocorticoid receptors are present in the rat ovary [46, 47]. However, the finding that ZK98299, which has about 20-fold lower binding affinity to glucocorticoid receptor than does RU-486 [48–50], inhibited apoptosis similarly, suggests that the blockade of progesterone action at the receptor is the cause of this effect. Nevertheless, during the rat estrous cycle, progesterone receptors are expressed in granulosa cells during the period of the LH surge [51], but receptor expression has been reported to be absent in the steroidogenic luteal cells [52]. Other cells of the CL, such as vascular cells or macrophages, could be involved in these effects of progesterone and their antagonists. Since progesterone has been found to modulate the production of cytokines and reactive oxygen species by macrophages [22, 23], it could act as a second signal to activate PRL-recruited macrophages in the regressing CL. The existence of a possible role for progesterone on luteal function has long been suggested [1, 14, 15]. In this study, exogenous progesterone was not effective in inducing luteal cell apoptosis in CB154-treated rats. This strongly suggests that both PRL and progesterone are necessary for the induction of apoptosis in the regressing CL. Experiments are in progress to further investigate these possibilities.

In summary, luteolysis in the rat seems to be under the control of two coupling endocrine mechanisms. First, the preovulatory LH surge couples ovulation (i.e., formation of a new CL) to the induction of cell death in the CL of the previous cycle, through the production of progesterone on proestrous afternoon. Second, the preovulatory PRL surge couples the induction of cell death and the accumulation of macrophages to phagocytize apoptotic cells in regressing CL.

	ACKNOWLEDGMENTS

 
The authors are very grateful to J. Molina, P. Cano, and E. Tarradas for their technical assistance. The authors wish to thank Drs. Sitruk-Ware (Exelgyn, Paris, France) and W. Elger (Schering, Berlin, Germany) for the supply of the antiprogestagens RU-486 and ZK98299. Dr. H. Kloosterboer (Organon, Oss, The Netherlands) generously supplied the LHRH antagonist Org30276.

	FOOTNOTES

 
¹ This work has been subsidized by DGES, Spain.

² Correspondence. FAX: 34 57 218288; fi1begac{at}lucano.uco.es

Accepted: July 6, 1998.

Received: March 3, 1998.

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