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Gonadotropins and Cytokines Affect Luteal Function through Control of Apoptosis in Human Luteinized Granulosa Cells

Department of Obstetrics and Gynecology, Nagoya City University Medical School, Nagoya 467-8601, Japan

Address all correspondence and requests for reprints to: Dr. Hirokazu Matsubara, Department of Obstetrics and Gynecology, Nagoya City University Medical School, Kawasumi 1, Mizuho-Cho, Mizuho-ku, Nagoya, Japan 467-8601.

	Abstract

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The luteal phase in the normal human menstrual cycle is known to be about 14 days. The physiological mechanisms that regulate the corpus luteum remain to be clarified, although apoptosis is reported to be involved. This study was undertaken to investigate the regulation of luteal function by gonadotropins, cytokines, and PGs, concentrating attention on the incidence of apoptosis and its molecular mechanisms in cultured human luteinized granulosa cells collected at oocyte pick-up from patients undergoing in vitro fertilization and embryo transfer. Clusters of granulosa cells were pipetted in 0.1% hyaluronidase in phosphate-buffered saline. After cell separation by centrifugation using Ficoll-Paque, 1 x 10⁴ viable cells/mL in RPMI 1640 medium with 10% FCS were used for experimentation. Substances added were FSH (100 ng/mL), hCG (100 ng/mL), LH (100 ng/mL), interleukin-1ß (IL-1ß; 10 ng/mL), transforming growth factor-ß1 (TGFß1; 10 ng/mL), macrophage colony-stimulating factor (M-CSF; 10 ng/mL), tumor necrosis factor- (TNF; 10 ng/mL), and PGF₂ (10 ng/mL). After 24-h culture at 37 C under 5% CO₂ and air, cells were fixed with 4% neutral buffered formalin and stained with Hoechst 33258. Apoptotic bodies were counted under a fluorescence microscope, and immunostaining was performed using anti-Fas, Fas ligand, Bcl-2, Bax, and p53 antibodies. Incidences of apoptotic bodies in the group without substance addition were 0.7 ± 0.2% (0 h), 5.9 ± 0.6% (24 h), and 7.9 ± 1.2% (48 h); spontaneous increase was significant at the latter time points. Defining the incidence at 24 h as 100%, values after treatment were: FSH, 57%; LH, 84%; hCG, 44%; IL-1ß, 76%; TGFß1, 52%; M-CSF, 50%; TNF, 177%; and PGF₂, 147%. Significant suppression was observed with FSH, hCG, TGFß1, and M-CSF (P < 0.01). On the other hand, significant induction occurred with TNF and PGF₂ (P < 0.01). On immunostaining, the incidence of stained cells with anti-Fas, Fas ligand, Bax, and p53 antibody was increased after 24-h incubation without addition. This was reduced by hCG, TGFß1, and M-CSF. No stained cells were observed with anti-Bcl-2 antibody before or after incubation. In conclusion, our results suggest that both gonadotropins (FSH and hCG) and cytokines (TGFß1 and M-CSF) may be involved in the support of luteal function via suppression of apoptosis, and that TNF and PGF₂ may contribute to ovarian dysfunction and/or luteal regression via its induction in human luteinized granulosa cells. Our results also suggest that Fas, Fas ligand, p53, and Bax may play roles in this apoptosis controlled by hCG, TGFß1, and M-CSF.

	Introduction

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THE LUTEAL phase usually lasts for about 14 days in humans, and if pregnancy does not result, menstruation starts. The detailed mechanisms of support and regression of the human corpus luteum, however, remain unknown. In recent years, single cell death with fragmentation of DNA, namely, apoptosis, has been reported to occur in the human corpus luteum from the midluteal phase on (1).

It is well known that LH and hCG have luteotropic effects, affecting morphological and functional differentiation of granulosa cells to luteal cells. Cytokines appear to have more complicated mechanisms of control of luteal function than gonadotropins. Interleukin-1ß (IL-1ß) exerts luteolytic effects on human luteinized granulosa cells in culture (2), whereas transforming growth factor-ß1 (TGFß1) and tumor necrosis factor- (TNF) have dichotomic effects, which are luteotropic or luteolytic depending on the species (3, 4, 5, 6, 7, 8, 9, 10, 11, 12). Although macrophage colony-stimulating factor (M-CSF), which is detected in high concentrations in follicular fluid, has been reported to have an important influence on follicle maturation (13), there have been no reports, to our knowledge, of effects of M-CSF on luteal function.

After various luteolytic factors, such as PGF₂ and PRL, were found (14, 15), researchers began to pay attention to their possible involvement in the induction of apoptosis. However, whether these gonadotropins and cytokines impact on apoptosis in the human corpus luteum remains to be clarified. Corpus luteum dysfunction can be classified into a functional abnormality due to poor hormone secretion and a structural abnormality with a decrease in the luteal cell number. The effects of gonadotropins and cytokines on progesterone production in human granulosa cells in vitro may be important for the former (6, 16, 17), whereas induction of apoptosis is related to the latter.

Recently, genes that encode proteins relevant to activation of apoptosis in the follicle and the corpus luteum have been reported. In the human corpus luteum, the expression of Fas antigen becomes positive in the early luteal phase, and this may be strengthened with subsequent regression (18). In the rat ovary, Bax seems to antagonize the apoptosis suppressive effects of Bcl-2, and cell fate seems to be decided by this balance (19). In the bovine corpus luteum, although the level of Bax mRNA increases during structural regression, those of Bcl-2 and p53 mRNAs do not change (20). However, there has been no report of simultaneous assessment of expression of all of these genes in the human corpus luteum.

By examining the effects of gonadotropins and cytokines on the generation of apoptosis of human luteinized granulosa cells in the early luteal phase, we tried to clarify whether corpus luteum function may be in part regulated by these factors acting through apoptosis. In addition, possible molecular mechanisms of apoptosis and its regulation were targeted.

	Materials and Methods

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Hormones and reagents

RPMI 1640 medium was purchased from Life Technologies, Inc.(Grand Island, NY), and supplemented with sodium bicarbonate (2.0 g/1000 mL), penicillin (700 mg/1000 mL), streptomycin (100 mg/1000 mL), and 10% FCS. Human FSH (F-4021; 7,000 IU/mg), human LH (L-5259; 11,000 IU/mg), hCG (C-0434; 14,000 IU/mg), recombinant human IL-1ß (I-4019), recombinant human TGFß1 (T-7039), recombinant human M-CSF (M-6518), recombinant human TNF (T-6674), and PGF₂ (P-0424) were purchased from Sigma (St. Louis, MO). Antihuman Fas (Sc-715; rabbit polyclonal antibody), antihuman Fas ligand (Sc-957; rabbit polyclonal antibody), antihuman Bax (Sc-526; rabbit polyclonal antibody), antihuman Bcl-2 (Sc-509; mouse monoclonal IgG1 antibody), and antihuman p53 (Sc-98; mouse monoclonal IgG1 antibody) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Patients and granulosa cell isolation

Granulosa cells were obtained by ultrasound-guided follicular aspiration from patients undergoing in vitro fertilization (IVF) and embryo transfer at Nagoya City University Medical School after informed consent had been given by the patients. The mean age was 33.2 yr (range, 28–37 yr). The indications for infertility treatment were bilateral tubal obstruction and male factor presence. Patients received the GnRH analog buserelin acetate (Suprecur, Hoechst, Tokyo, Japan; 900 µg/day, nasally) starting in the midluteal phase of the cycle before the induction of ovulation. Follicle development was stimulated with human menopausal gonadotropin (150–300 IU/day; Humegon, Organon, Tokyo, Japan) or FSH (Fertinom P, Serono, Tokyo, Japan), and follicles were aspirated 36 h after the administration of hCG (10,000 IU; Mochida, Tokyo, Japan). Follicular fluid, including granulosa cells, was centrifuged (250 x g, 10 min) after oocytes were removed. Hyaluronidase (Wako Chemical Co., Osaka, Japan; 0.1%, wt/vol) in phosphate-buffered saline (PBS) was added to the pellet, and after incubation (5% CO₂ and air, 37 C for 15 min), cell masses were repipetted and filtered through a 70-µm pore size nylon filter (Becton Dickinson and Co., Franklin Lakes, NJ). Then, they were underlayered with Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) and centrifuged (450 x g, 30 min) for cell separation. The granulosa cells, obtained from the interface, were washed and centrifuged (250 x g, 10 min) with culture medium (10% FCS in RPMI 1640). Pellets were resuspended in 2 mL culture medium, and cell numbers and viability were accessed by the trypan blue exclusion test with a hemocytometer.

Culture procedure

Cells (10,000 viable cells/mL) were cultured for 24 or 48 h at 37 C under 5% CO₂ and air in culture medium in plastic plates (Falcon 3002, Becton Dickinson and Co., Lincoln Park, NJ) with or without gonadotropins, cytokines, and PGs. Using the known concentrations of gonadotropins, cytokines, and PGs in the follicular fluid on ovum pick-up with IVF treatment as references (21, 22, 23, 24, 25, 26, 27, 28), various concentrations of FSH (30, 100, and 300 ng/mL), LH (1, 10, 30, 100, and 300 ng/mL), and hCG (1, 10, 30, 100, and 300 ng/mL), IL-1ß (0.1, 1, 10, 30, and 100 ng/mL), TGFß1 (1, 10, 30, and 100 ng/mL), M-CSF (0.1, 1, 10, 30, and 100 ng/mL), and PGF₂ (1, 10, and 100 ng/mL) were prepared for testing. TNF was not detected in the follicular fluid, but TNF was added to this experiment as an unfavorable exogenous factor because it is reported to have a luteolytic action on granulosa cells (10). Therefore, comparable concentrations (1, 10, and 100 ng/mL) to those used for other cytokines were applied.

Cell fixation and counting of apoptotic bodies

In the present study fluorescence microscopy for confirming apoptotic bodies as a result of morphological change in the nucleus was employed. Cultured cells were stripped off from plastic plates by cell remover (Costar, Cambridge, MA) and fixed with 4% (wt/vol) of neutral buffered formalin for 30 min. After fixation, they were washed by PBS twice, stained with 0.6 µg/mL Hoechst 33258 (Wako Chemical Co.), and placed on glass slides. After several photographs were taken at x200 magnification under a fluorescence microscope, apoptotic bodies were counted in 1000 separated granulosa cells at random on microphotographs.

Progesterone assay

Progesterone RIAs were performed with commercial kits (Diagnostic Products, Los Angeles, CA). The progesterone assay had a sensitivity of 20 pg/mL, and, using medium standards, the intra- and interassay coefficients of variation were 3.9% and 5.6%, respectively.

Immunostaining

After fixation with 4% (wt/vol) neutral buffered formalin for 30 min, human luteinized granulosa cells were washed with PBS, incubated with 0.3% H₂O₂-methanol for 30 min to exhaust endogenous peroxidase activity, and further washed three times. Immunostaining was performed by the streptavidin-biotin complex method with a Histofine streptavidin-biotin-peroxidase complex kit (Nichirei Co., Tokyo, Japan). Cells were incubated with dilutions of anti-Fas, Fas ligand, Bax, Bcl-2, and p53 antibodies at 4 C overnight and washed with PBS. Immunodetection was performed by incubating the cells for 4 min with 3,3'-diaminobenzidine. After washing, cells were nuclear stained with methyl green. Permanent specimens were made by dehydration through an ethanol and xylene series and embedding in Harleco Synthetic Resin (Matsunani Glass Industries Ltd., Osaka, Japan). The presence of staining was evaluated in 1000 separated cells at random at x200 magnification under a microscope. Fas, Fas ligand, Bax, Bcl-2, and p53 immunoreactivity was expressed as the percentage of cells exhibiting specific staining. The same concentration of affinity-purified normal rabbit IgG or mouse IgG1 was employed instead of primary antibody as a negative control.

Statistical analysis

Data are presented as the mean ± SEM. The statistical significance of differences was assessed by ANOVA followed by t test, with P < 0.01 as the cut-off.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Progesterone concentration and the incidence of apoptotic bodies in cultured human luteinized granulosa cells without substance addition

Progesterone production in the group without substance addition was 258.0 ± 28.7 ng/mL (24 h) and 682.0 ± 82.4 ng/mL (48 h), with a time-dependent increase (Fig. 1). Apoptotic bodies with nuclear fragmentation were assessed under a fluorescence microscope (Fig. 2). Incidences of apoptotic bodies were 0.7 ± 0.2% (0 h, preculture), 5.9 ± 0.6% (24 h), and 7.9 ± 1.2% (48 h; Fig. 3). A significant spontaneous increase was observed during culture, with no difference between 24 and 48 h. Therefore, the 24-h culture time was adopted for the present study. Moreover, the incidences of apoptotic bodies and progesterone production were not significantly affected by the age of the patients and indications for IVF-embryo transfer treatment.

View larger version (13K): [in this window] [in a new window]   Figure 1. Progesterone concentrations in cultured human luteinized granulosa cells. Human luteinized granulosa cells were isolated from follicular fluid of patients undergoing IVF and incubated for 24 or 48 h without any substance addition. Data are the mean ± SEM values for 11 cultures.

View larger version (47K): [in this window] [in a new window]   Figure 2. Microphotograph of apoptotic bodies of cultured human luteinized granulosa cells after treatment with TNF (10 ng/mL). Nuclear morphology was examined under a fluorescence microscope, using Hoechst 33258 stain. Note the fragmented nuclei, indicated by arrows (apoptotic bodies). Original magnification, x200.

View larger version (14K): [in this window] [in a new window]   Figure 3. Incidences of apoptotic bodies in cultured human luteinized granulosa cells isolated from follicular fluid of patients undergoing IVF and incubated for 24 or 48 h. Cells were stained with 0.6 µg/mL Hoechst 33258, and apoptotic bodies were counted in 1000 separated granulosa cells at random under a fluorescence microscope. Data are the mean ± SEM values for 11 cultures. Time 0 h represents the incidence of apoptotic bodies before incubation. *, P < 0.01 vs. 0 h.

At first, dose dependence was examined. Dose-dependent suppression of incidence of apoptotic bodies was observed with hCG, but not FSH or LH (Fig. 4). As the follicular concentration of hCG was between 10–100 ng/mL 36 h after the administration of 10,000 IU hCG, a concentration of 100 ng/mL was used for the comparison with control (without substance addition) in consideration of physiological condition. As dose-dependent suppression was not observed with either FSH or LH, the same concentration as that of hCG (100 ng/mL) was used for these hormones.

View larger version (31K): [in this window] [in a new window]   Figure 4. Effects of treatment with gonadotropins on cultured human luteinized granulosa cells. Dose dependence was examined. Cells were incubated for 24 h with various doses of FSH (30, 100, or 300 ng/mL), LH (1, 10, 30, 100, or 300 ng/mL), or hCG (1, 10, 30, 100, or 300 ng/mL). Apoptotic bodies were analyzed as described for Fig. 3. Data are the mean ± SEM values for 11 cultures. *, P < 0.01.

View larger version (30K): [in this window] [in a new window]   Figure 5. Effects of treatment with gonadotropins on cultured human luteinized granulosa cells. Cells were incubated for 24 h in the absence (Control) or presence of FSH (100 ng/mL), LH (100 ng/mL), or hCG (100 ng/mL). Apoptotic bodies were analyzed as described for Fig. 3. Data are the mean ± SEM values for 11 cultures. *, P < 0.01 vs. control.

At first, dose dependence was examined. For TGFß1 and M-CSF, at concentrations near 10 ng/mL, suppression of the incidence of apoptotic bodies almost reached a plateau (Fig. 6A). Therefore, a concentration of 10 ng/mL was used for subsequent experiments. As dose-dependent suppression was not observed with IL-1ß, TNF, or PGF₂ (Fig. 6, A and B), a concentration of 10 ng/mL was also used for the comparison with controls (without substance addition).

View larger version (24K): [in this window] [in a new window]   Figure 6. Effects of treatment with cytokines and a PG on cultured human luteinized granulosa cells. Dose dependence was examined. Cells were incubated for 24 h with various doses of A) IL-1ß (0.1, 1, 10, 30, or 100 ng/mL), TGFß1 (1, 10, 30, or 100 ng/mL), or M-CSF (0.1, 1, 10, 30, or 100 ng/mL), or B) TNF (1, 10, or 100 ng/mL) or PGF2 (1, 10, or 100 ng/mL). Apoptotic bodies were analyzed as described in Fig. 3. Data are the mean ± SEM values for 11 cultures. *, P < 0.01.

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View larger version (24K): [in this window] [in a new window]   Figure 7. Effects of treatment with cytokines and a PG on cultured human luteinized granulosa cells. Cells were incubated for 24 h in the absence (Control) or presence of IL-1ß (10 ng/mL), TGFß1 (10 ng/mL), M-CSF (10 ng/mL), TNF (10 ng/mL), or PGF2 (10 ng/mL). Apoptotic bodies were analyzed as described for Fig. 3. Data are the mean ± SEM values for 11 cultures. * and **, P < 0.01 vs. control.

Immunostaining was performed of cultured human luteinized granulosa cells treated with selected gonadotropins and cytokines, causing either significant suppression (FSH, hCG, TGFß1, and M-CSF) or induction (TNF and PGF₂) of apoptosis. Five antibodies were used: anti-Fas antibody (10 ng/mL), anti-Fas ligand antibody (20 ng/mL), anti-Bax antibody (20 ng/mL), anti-Bcl-2 antibody (100 ng/mL), and anti-p53 antibody (100 ng/mL). No immunoreaction was observed using a negative control.

With precultured human luteinized granulosa cells, some were positive for anti-Fas antibody, anti-Fas ligand antibody, anti-Bax antibody, or anti-p53 antibody binding. After incubation for 24 h without any substance addition, incidences of stained cells were increased with all four antibodies: anti-Fas antibody, 3.3 times; anti-Fas ligand antibody, 3.0 times; anti-Bax antibody, 3.3 times; and anti-p53 antibody, 3.9 times. With one exception, the anti-Bcl-2 antibody, no stained cells were noted before or after incubation.

Next, the effects of treatment with FSH, hCG, TGFß1, M-CSF, TNF, and PGF₂ on the incidence of stained cells were examined. Defining the incidences of stained cells incubated for 24 h without any substance addition as 100%, the following values were obtained: anti-Fas antibody (Fig. 8A): FSH, 77.7%; hCG, 40.2%; TGFß1, 56.1%; M-CSF, 58.0%; TNF, 101.5%; and PGF₂, 90.9%; anti-Fas ligand antibody (Fig. 8B): FSH, 106.7%; hCG, 46.3%; TGFß1, 36.9%; M-CSF, 46.3%; TNF, 111.8%; and PGF₂, 116.9%; anti-Bax antibody (Fig. 8C): FSH, 105.6%; hCG, 38.9%; TGFß1, 41.7%; M-CSF, 47.2%; TNF, 97.6%; and PGF_2a, 93.0%; anti-p53 antibody (Fig. 8D): FSH, 90.1%; hCG, 25.3%; TGFß1, 49.5%; M-CSF, 53.9%; TNF, 103.7%; and PGF₂, 105.1%. Significant suppression was observed in cultured luteinized granulosa cells treated with hCG, TGFß1, and M-CSF (P < 0.01).

View larger version (44K): [in this window] [in a new window]   Figure 8. Immunochemical analysis of Fas, Fas ligand, Bax, Bcl-2, and p53 proteins in cultured human luteinized granulosa cells treated with selected gonadotropins and cytokines. Cells were incubated for 24 h in the absence (24 h) or presence of FSH (100 ng/mL), hCG (100 ng/mL), TGFß1 (10 ng/mL), M-CSF (10 ng/mL), TNF (10 ng/mL), or PGF2 (10 ng/mL) and analyzed by immunostaining. Results for Fas (A), Fas ligand (B), Bax (C), Bcl-2 (no stained cells noted before or after incubation, data not shown), and p53 (D) immunoreactivity are percentages of cells exhibiting specific staining. Data are the mean ± SEM values for 5 cultures. *, P < 0.01 vs. 24 h.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

When gonadotropins, such as FSH, LH, or hCG, were added to the culture medium, FSH significantly suppressed apoptosis. FSH is the most important factor for follicle growth in the preovulatory phase, and a lack of FSH could lead to follicular atresia through apoptosis. However, human serum FSH levels also demonstrate a decrease from the midluteal phase on. Although the physiological significance of FSH for the corpus luteum seems to be limited, our results suggest that FSH may exert a promoting effect on luteinized granulosa cells in the early luteal phase when granulosa cells are luteinized and differentiated, suppressing apoptosis.

It is known that during pregnancy the corpus luteum will become corpus luteum graviditatis, when suppression of apoptosis occurs (30). The observed suppressive effect of hCG appears to justify its clinical use for treating ovarian dysfunction. An increase in LH/hCG receptors is reported to occur from the early to midluteal phase (31). Estrogen and progesterone are known to be produced in the luteal phase under the influence of both FSH and LH. The latter would be expected to be an indispensable hormone even in the nonpregnant luteal phase, supporting the corpus luteum via binding to its receptor, but addition of LH showed no significant suppression of apoptosis in this study. As LH shares the same receptor as hCG, the results appear inconsistent. In this study the collected granulosa cells were grown under the influence of exogenous FSH and hCG administered in vivo. The possibility that the response of these granulosa cells is not a reflection of the response in the natural cycle must therefore be taken into consideration. However, a possible explanation of the discrepancy is that an apoptosis-suppressive effect of LH could be attenuated by a decrease in biological activity, detailed data for which were not given by the company from which it was purchased. Also, LH is released in a pulsatile fashion in vivo, and it might not be expected to fully exert its effects in vitro.

TGFß1 has different effects on the corpus luteum, which may be luteotropic or luteolytic depending on the subjects (3, 4, 5, 32). The present study showed that it could suppress apoptosis in human luteinized granulosa cells.

The role of M-CSF in granulosa cells is unclear, although some researchers claim that it acts to increase their numbers in mice (33). Because M-CSF is detected in high concentrations (0.5–5 ng/mL) in the follicular fluid just before ovulation, it was chosen for investigation here. The results clearly showed that it had an inhibitory effect on apoptosis of human luteinized granulosa cells. This could explain the promoting effects of macrophages on progesterone production of granulosa cells described previously (34).

Regarding TNF, promotion of apoptosis in mouse lutein cells has been reported (7), along with luteal regression in bovine and porcine luteal cells (8, 9). Expression of TNF is strongest in the human corpus luteum during the midluteal phase and abates in the regressive corpus luteum (35). To our knowledge there has been no previous report of the effects of TNF on a apoptosis in the human corpus luteum. Our results clearly showed a stimulation, in agreement with that found in the mouse, where interferon- also plays a role (7). TNF thus seems to influence apoptosis in the corpus luteum in balance with various other factors in vivo.

In sheep, regression of the corpus luteum can be blocked by removing the uterus (36). PGF₂ from endometrium is known to be the agent responsible for luteolysis in this case (37). However, the same regression does not occur in human. Cultured human luteal cells produce PGF₂, and its addition to the culture system suppresses progesterone production (14). Moreover, it is documented that injection of PGF₂ into the corpus luteum in vivo similarly causes luteal regression and reduces progesterone production (38). Reported actions of PGF₂ on luteal cells are the suppression of progesterone production via a rise in intracellular calcium on activation of cyclic kinase (39) and inhibition of binding of LH to luteal cell, possibly through induction of reactive oxygen species (40, 41). Therefore, the local action of PGF₂ in the suppression of human corpus luteum is noteworthy. From the present experiment, the possibility that the effects of PGF₂ are mediated by a decrease in cell number through induction of apoptosis must be considered. Local PGF₂ could thus take part in luteal regression in the normal menstrual cycle by autocrine and/or paracrine actions.

Our present examination of Fas, Fas ligand, Bax, and p53 demonstrated that their expression was remarkably increased after 24 h in culture. These genes are all known to be related to apoptosis, so that the results are in line with the literature. Furthermore, protein levels were reduced by the addition of hCG, TGFß1, and M-CSF, which suppressed apoptosis in human luteinized granulosa cells. In animal studies using the rat and porcine, it has been found that both Fas and Fas ligand appear in luteinized granulosa cells (42, 43). Thus, they could contribute to the mechanisms of induction of apoptosis.

The lack of Bcl-2 protein in human luteinized granulosa cells in the present study might have been related to its suppression by p53 (44). The role of Bcl-2 in apoptotic control of human luteinized granulosa cells appears to be minimal if any, as the protein was undetectable even without addition of suppressive factors. The fact that addition of FSH failed to reduce the expression of these apoptosis proteins is not unexpected, as its suppressive effect on human granulosa cells may be mediated through other signal transmission pathways after induction of apoptosis signaling by Fas and Fas ligand, possibly involving caspase. It has been reported that signal transmission by Fas and Fas ligand is interrupted by insulin-like growth factor-I, which inhibits the activity of caspase-3 (45). Induction of apoptosis by PGF₂ and TNF without any clear effect on the expression of Fas, Fas ligand, Bax, and p53 also suggests that other factors play roles.

In conclusion, both gonadotropins (FSH and hCG) and cytokines (TGFß1 and M-CSF) can serve as promoting (luteotropic) factors, affecting luteinization in the early luteal phase via suppression of apoptosis of human luteinized granulosa cells. The finding that PGF₂ and TNF, in contrast, induce apoptosis suggests that both may be involved in midluteal phase ovarian dysfunction and/or luteal regression in the late luteal phase under environmental conditions where these two factors increase. Although it was confirmed that factors such as Fas, Fas ligand, p53, and Bax are involved in the control of apoptosis in human luteinized granulosa cells, our results also suggest that other agents play a role.

Received June 16, 1999.

Revised October 15, 1999.

Accepted December 16, 1999.

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