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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2008-0127
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The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 8 3179-3185
Copyright © 2008 by The Endocrine Society

Gonadotropin-Releasing Hormones I and II Induce Apoptosis in Human Granulosa Cells

In-Sun Hong, Anthony P. Cheung and Peter C. K. Leung

Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada, V6H 3V5

Address all correspondence and requests for reprints to: Peter C. K. Leung, Ph.D., Department of Obstetrics and Gynecology, University of British Columbia, 2H-30, 4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5. E-mail: peleung{at}interchange.ubc.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: The direct effects of GnRH-I or GnRH-II on apoptosis in human granulosa cells are unknown and, if present, can be influenced by FSH. Apoptosis involves activation of the intracellular proteolytic cascade of caspases. We therefore evaluated the roles of GnRH-I and -II, and the effects of FSH, on apoptosis in human granulosa cells and on caspases.

Methods: Human immortalized granulosa cells treated with GnRH-I or GnRH-II or nothing were cultured with and without antide (a GnRH-I antagonist), a broad-spectrum caspase inhibitor or selective caspase-8, -3, or -7 inhibitor, or FSH in replicates for 72 h. Apoptotic changes were evaluated by terminal deoxynucleotidyl-transferase-mediated biotin-dUTP nick-end labeling (TUNEL) assays, immunoblotting, and expression levels of caspases and compared by ANOVA.

Results: GnRH-I and -II induced TUNEL-positive apoptotic cells and increased cleavage activities of caspase-8, -3, and -7 by 48 h and peaked at 72 h, changes that were blocked by FSH cotreatment. Antide also effectively blocked these TUNEL-positive changes and expression levels of caspase-3 induced by GnRH-I or -II. Activation of caspase-8, -3, and -7 was inhibited by the corresponding caspase inhibitor. Caspase-8 inhibitor also abolished cleavages of caspase-3 and -7 induced by GnRH-I and -II.

Conclusion: GnRH-I and -II induce apoptosis in human granulosa cells through GnRH-I receptors, which mediate the proteolytic caspase cascade involving caspase-8 (the initiator) and caspase-3 and -7 (the effectors). FSH protects human granulosa cells from apoptosis induced by GnRH-I or -II. This raises potentially important roles of GnRH-I and GnRH-II in regulating follicle development and atresia together with FSH.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Apoptosis is the biological process by which unwanted cells are eliminated in response to developmental signals or toxic stimuli. The major features of apoptosis are DNA fragmentation, cell shrinkage, plasma membrane blebbing, and the formation of apoptotic bodies (1, 2). Apoptosis also plays an important role in the process of maintaining the reproductive apparatus (3, 4). During growth and development of human ovarian follicles, only a limited number of follicles proceed to the ovulatory stage, whereas more than 99% of follicles undergo the apoptotic process of atresia (5, 6). Recent studies have revealed that apoptosis of ovarian granulosa cells play an important role in follicular atresia (7, 8). Granulosa cells maintain the developing oocyte until its release from the follicle during ovulation and also synthesize hormones that are related to oocyte maturation and ovulation. Many investigators have studied factors that directly or indirectly regulate apoptosis of granulosa cells. Recent studies have suggested a physiological role of GnRH on granulosa cell apoptosis (9).

GnRH is the central hypothalamic regulator of reproductive functions (10, 11). This decapeptide is synthesized and released by hypothalamic secretory neurons and delivered to the pituitary gland via the hypophyseal portal blood system. In the anterior pituitary, GnRH stimulates the synthesis and the release of the gonadotropins, LH and FSH. The effects of GnRH are mediated by a cell surface receptor (GnRH-R) belonging to the G protein-coupled receptor superfamily (12, 13). In addition to its well-known roles in regulating the hypothalamus-pituitary-gonadal axis, GnRH has been found in extrahypothalamic regions of the central nervous system (14). Two forms of GnRH, termed GnRH-I and GnRH-II, encoded by separate genes, have been identified in humans (15). GnRH-I and GnRH-I receptors have been localized in human reproductive tissues, such as the placenta (16), the ovary (17), the endometrium (18), and testis (19). Functional studies have established that GnRH-I regulates steroidogenesis and inhibits cell growth in human ovarian cells (10). The presence of GnRH-I mRNA in granulosa cells of primary, secondary, and tertiary follicles have been identified from in situ hybridization studies (20). Recent studies from our laboratory have also confirmed GnRH-II expression in human granulosa-luteal cells, immortalized ovarian surface epithelial cells, and ovarian cancer (20). GnRH-II is also broadly distributed and highly expressed throughout the extrahypothalamic regions, including ovarian (21), endometrial (22), and breast tissue (23). Only the truncated but not the functional forms of GnRH-II receptors are known to exist in humans, even though GnRH-II receptor mRNA expression has been reported in many reproduction-related tissues, such as mammary gland, prostate, endometrium, placenta, and gonads (22, 23). Hence, the functions of GnRH-II in reproductive tissues and the mechanisms involved remain unclear.

The direct effects of GnRH-I on apoptosis in granulosa cells have been investigated by several researchers and shown to increase the incidence of apoptosis in porcine granulosa cells (24, 25) and down-regulate the proliferation of human granulosa cells (26, 27). Because of their close association with the oocyte, increased apoptosis in granulosa cells may influence the development and quality of the oocyte (28, 29), increase the occurrence of empty follicles and poor oocyte fertilization, and result in lower pregnancy rates (30). However, the direct effects of GnRH-I and GnRH-II on apoptosis in human granulosa cells and the underlying mechanisms involved remain unknown.

The purpose of this study was to characterize the effects of GnRH-I and -II on granulosa cells, using human immortalized granulosa cells as the experimental model. We used terminal deoxynucleotidyl-transferase-mediated biotin-dUTP nick-end labeling (TUNEL) assay to investigate apoptosis induced by GnRH-I and GnRH-II. In addition, the expression levels of the initiator caspase-8 and the effectors caspase-3 and -7 involved in apoptosis were analyzed by Western blotting. We also used inhibitors specific for these caspase activities to detail whether GnRH-I and GnRH-II induced apoptosis in human granulosa cells through this intracellular proteolytic cascade of caspases. Furthermore, because FSH is a major survival factor of ovarian follicles, we examined its antiapoptotic effects on human granulosa cells after cotreatment with GnRH-I or -II.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental design

The use of immortalized human granulosa cells for this study was approved by the University of British Columbia Research Ethics Board. In each set of experiments, human immortalized granulosa cells were cultured without any agonists or inhibitors (controls) or with GnRH-I or GnRH-II, each at a dose of 10–7 mol/liter. In separate experiments involving the same three groups, each of following treatments was added to the culture medium of the GnRH-I and GnRH-II groups (but not to that of controls) at the specified time: 1) 10–7 mol/liter of the GnRH antagonist antide added 2 h before GnRH-I or GnRH-II; 2) 50 µmol/liter of a broad-spectrum caspase inhibitor (Boc-D-FMK), a selective caspase-8 inhibitor (Ac-IEPD-CHO), a selective caspase-3 inhibitor (Ac-DMQD-CHO), or a selective caspase-7 inhibitor (Ac-DNLD-CHO) added 2 h before GnRH-I or GnRH-II treatment; and 3) 50 ng/ml FSH added to the culture medium at the same time as GnRH-I or GnRH-II treatment. All experiments were cultured in duplicate or triplicate and evaluated in a time-dependent manner. TUNEL assays, immunoblotting, and expression levels of caspases were used to compare the effects of these various treatments alone or in combinations on apoptosis.

Reagents and antibodies

GnRH-I (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), GnRH-II (pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2), and the GnRH antagonist antide (N-Ac-D-Nal1-D-Cpa2-D-Pal3-Ser4-Lys(Nic)5-D-Lys(Nic)6-Leu7-Ilys8-Pro9-D-Ala10-NH2), were purchased from Bachem (King of Prussia, PA). All stock solutions were aliquoted, stored at –20 C, and diluted in cell culture medium immediately before use. Broad-spectrum caspase inhibitors (Boc-D-FMK) and selective inhibitors of caspase-8 (Ac-IEPD-CHO), caspase-3 (Ac-DMQD-CHO), and caspase-7 (Ac-DNLD-CHO) were obtained from Calbiochem (San Diego, CA). Antibodies to caspase-8, caspase-3, and caspase-7 were purchased from Cell Signaling Technology, Inc. (Danvers, MA).

Cell culture

Human granulosa cells were obtained from in vitro fertilization procedures and immortalized with Simian Virus 40 large T antigen as described previously (31). The human immortalized granulosa cells were cultured in M199/MCDB105 (1:1) supplemented with 10% fetal bovine serum (Hyclone Laboratories Inc., South Logan, UT) at 37 C in a humidified atmosphere of 5% CO2 in air. Cells were then dissociated with 0.06% trypsin (1:250)/0.01% EDTA (Life Technologies, Inc., Rockville, MD) in Mg2+/Ca2+-free Hank’s Buffered Salt Solution and seeded at a density of 2 x 105 cells in 35-mm dishes (Falcon; Becton Dickinson, Franklin Lakes, NJ). After culturing for 2 d, the cells were washed once with medium and serum starved for 6 h before GnRH-I or GnRH-II treatment.

Immunoblotting analysis

After the specific treatment for each experiment, cells were washed twice with ice-cold PBS and lysed in ice-cold RIPA buffer [150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris (pH 7.5), 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 100 µg/ml aprotinin] containing phosphatase inhibitor cocktail (Sigma Chemical Co., St. Louis, MO). The extract was placed on ice for 15 min and centrifuged to remove cellular debris, and the protein content of the supernatant was determined by the Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA). The protein sample was further separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blotted in 1x PBS containing 5% (wt/vol) skim milk at room temperature for 1 h, washed in a mixture of PBS and 0.05% Tween 20 (Tween-PBS; Sigma), and then incubated overnight at 4 C with the relevant antibody to detect the specific protein under investigation. The peak apoptotic signal of each band on Western blotting was quantitated with the Scion Image Software (Scion, Frederick, MD) using β-actin as the internal control and then expressed as fold increase from that of the control at 24 h in each set of replicate experiments.

TUNEL assay

DNA strand breaks in apoptotic cells were measured by TUNEL assay using the in situ detection kit, POD (Roche Molecular Biochemicals, Mannheim, Germany). Treated cells were fixed with 4% paraformaldehyde solution and incubated in a 0.1% Triton X-100 permeabilization solution on ice according to the manufacturer’s instructions. Cell were then rinsed twice in PBS and reacted with 50 µl of the TUNEL reaction mixture at 37 C for 60 min in a dark, humidified chamber. Cells were again rinsed three times in PBS and incubated for 30 min with 50 µl of the Converter-POD (Roche Diagnostics, Castle Hill, Australia) followed by another 15 min with diaminobenzidine to ensure the detection of TUNEL-labeled cells. Under light microscopy, the number of TUNEL-positive cells per high-power field (x100) was counted and expressed as a percentage of the total number of cells present in that field. The mean over at least three random fields was then calculated and expressed as fold increase from that of the control at 24 h in each set of replicate experiments.

Statistical analysis

Results from each experiment replicate were presented as the mean ± SEM. Data were first assessed by two-way ANOVA with time as the repeated measures. Significant results were followed by one-way factorial and repeated-measures ANOVA for between- and within-group differences, respectively, and corresponding significant results were further assessed by Tukey’s multiple comparison tests. Statistical significance was defined at a P level of <0.05. Because normality of data was not assumed, results were furthered analyzed by the nonparametric equivalents of Kruskal-Wallis test and Friedman test for between- and within-group comparison, respectively. Because the pattern of statistical significance remained the same, results were reported according to ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Apoptotic effects of GnRH-I and -II in human granulosa cells

To investigate that GnRH-I and -II induced apoptosis, we performed TUNEL assays on cells obtained from cultures at 24, 48, and 72 h to identify apoptotic cells, characterized by the inclusion of densely stained circular bodies representing fragmented DNAs of apoptosis. GnRH-I or -II treatment significantly increased the number of these apoptotic or TUNEL-positive cells in a time-dependent manner compared with controls. These apoptotic changes induced by GnRH-I and -II were clearly observed by 48 h and peaked at 72 h. The number and distribution of TUNEL-positive cells after GnRH-I or -II treatment were similar (Fig. 1Go).


Figure 1
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  		FIG. 1. Time-dependent apoptotic effects of GnRH-I and -II on human immortalized granulosa cells. Apoptotic nuclei were identified with the TUNEL assay after treatment with GnRH-I or -II (each at a dose of 10–7 mol/liter). GnRH-I and -II significantly increased the number of TUNEL-positive cells after 48 h in a time-dependent manner. The number and distribution of apoptotic or TUNEL-positive cells after GnRH-I or -II treatment were similar. Significant within-group differences on ANOVA were followed by pairwise comparison (Tukey’s test): * and {dagger}, significant differences from values at 24 and 48 h, respectively. Significant between-group differences on ANOVA at each time were similarly compared. The P value represents between-group differences at each time point: {ddagger}, significant differences from control values.

 
To further confirm that GnRH-I and -II induced apoptosis, Western blot analysis was used to evaluate the expression levels of some of the critical intracellular mediators of apoptosis, the initiator caspase-8 and the effectors caspases-3 and -7, in human immortalized granulosa cells treated with GnRH-I or -II (10–7 mol/liter) in a time-dependent manner (24, 48, and 72 h). As shown in Fig. 2Go, both GnRH-I and -II significantly activated caspase-8, -3, and -7. Furthermore, these data from Western blot analysis were consistent with our TUNEL assay results. In the intracellular proteolytic cascade of apoptosis, caspase-8 directly cleaves the relevant procaspases and activates effectors such as caspases-3 and -7 downstream (32, 33). Hence, our results revealed that GnRH-I and -II indeed induced apoptosis and triggered the corresponding cascade mediated by caspases.


Figure 2
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  		FIG. 2. GnRH-I and -II induced cleavage activities of caspase-3, -7, and -8 on Western blot analysis. Human immortalized granulosa cells were treated with GnRH-I or -II (each at a dose of 10–7 mol/liter) in a time-dependent manner (24, 48, and 72 h). Both GnRH-I and -II significantly activated the initiator caspase-8 and the effectors caspase-3 and -7. See Fig. 1 for annotations.

 
GnRH antagonist antide inhibited GnRH-I- and -II-mediated apoptosis

In contrast, antide, when added at a dose of 10–7 mol/liter to the media before GnRH-I and -II treatment, effectively blocked the TUNEL-positive changes observed with GnRH-I or -II alone (Fig. 3AGo) in human granulosa cells. Furthermore, the corresponding expression levels of caspase-3 (a key effector caspase) as assayed by Western blotting were significantly attenuated (Fig. 3BGo). Because the functional receptor for GnRH-II has not yet been identified in human, and antide, a GnRH-I antagonist, successfully blocked GnRH-II-induced apoptotic changes in our granulosa cell cultures, our findings strongly suggest that both GnRH-I and -II mediate apoptosis by binding to GnRH-I receptors.


Figure 3
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  		FIG. 3. GnRH antagonist antide attenuated GnRH-I- and -II-mediated apoptosis. A, In the presence of antide (10–7 mol/liter), the apoptotic effects of GnRH-I or -II on human immortalized granulosa cells were significantly attenuated, reducing the number of apoptotic or TUNEL-positive cells seen. B, Western blot analysis showed that the corresponding expression levels of caspase-3 (a key effector of apoptosis) induced by GnRH-I or -II were effectively blocked by antide. See Fig. 1 for annotations (* and {dagger}); {ddagger}, significant differences from values in treatment groups with antide added.

 
Effect of caspase inhibitors on GnRH-I- and -II-induced apoptosis of human granulosa cells

Caspase inhibitors have been widely used to confirm the dependency of apoptotic processes on caspase activation. To determine whether the initiator caspase-8 as well as the effectors caspase-3 and -7 mediated apoptosis induced by GnRH- I and GnRH-II, human granulosa cells were preincubated with a broad-spectrum caspase inhibitor (Boc-D-FMK), a selective inhibitor of caspase 3 (Ac-DMQD-CHO), caspase 7 (Ac-DNLD-CHO), or caspase 8 (Ac-IEPD-CHO) before treatment with GnRH-I or -II. In the absence of these caspase inhibitors, increased expression of cleaved caspase-8, -3, and -7 after GnRH-I or -II treatment was detected by Western blot analysis from 48–72 h in a time-dependent manner. In contrast, activation of caspase-8, -3, and -7 in human immortalized granulosa cells was inhibited by the respective caspase inhibitors (Fig. 4AGo).


Figure 4
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  		FIG. 4. Effects of caspase inhibitors on GnRH-I- or -II-induced apoptosis of human immortalized granulosa cells. A, Cells were preincubated for 2 h with 50 µmol/liter of a broad-spectrum caspase inhibitor (Boc-D-FMK), a selective inhibitor of caspase-8 (Ac-IEPD-CHO), caspase-3 (Ac-DMQD-CHO), or caspase-7 (Ac-DNLD-CHO) followed by a 48- and 72-h exposure of GnRH-I or -II (both at a dose of 10–7 mol/liter). GnRH-I- or -II-induced cleavage activities of caspase-8, -3, and -7 on Western blot analysis were successfully attenuated by the broad-spectrum caspase inhibitor; furthermore, GnRH-I- or -II-induced cleavage of each specific caspase was attenuated by its corresponding caspase inhibitor. B, The specific caspase-8 inhibitor (Ac-IEPD-CHO) successfully attenuated GnRH-I- and -II-induced caspase-3 and -7 activation on Western blot analysis. These results indicate that both GnRH-I and -II induce cleavage of initiator caspase-8, which in turn activates effectors caspase-3 and -7.

 
It is also well known that activated initiator caspase-8 directly cleaves and activates effector caspases-3 and -7. To confirm that GnRH-I and -II induced apoptosis in human granulosa cells was caspase-8 dependent, the specific caspase-8 inhibitor, Ac-IEPD-CHO, was added 2 h before GnRH-I or GnRH-II treatment and, indeed, was found to abolish GnRH-I- and -II-induced cleavage of caspase-3 and -7 (Fig. 4BGo). These results indicate that both GnRH-I and -II induce cleavage of initiator caspase-8, which in turn activates effectors caspase-3 and -7.

FSH protected human granulosa cells from undergoing GnRH-I- and -II-mediated apoptosis

FSH is a major survival factor for early antral follicles (34). In vitro, FSH prevents atresia of cultured preovulatory follicles (35). To investigate these antiapoptotic effects of FSH, human immortalized granulosa cells were cultured with both FSH (50 ng/ml) and GnRH-I or -II (10–7 mol/liter). The time-dependent increase in TUNEL-positive cells induced by GnRH-I or -II was effectively attenuated by FSH (50 ng/ml) cotreatment as shown in Fig. 5AGo. Correspondingly, increased expression of cleaved caspase-8, -3, and -7 on Western blot analysis induced by GnRH-I or -II from 48–72 h was attenuated by FSH cotreatment. These results demonstrate that FSH indeed acts as a survival factor, protects human granulosa cells from undergoing apoptosis, and attenuates the apoptotic effects of GnRH-I and -II (Fig. 5BGo).


Figure 5
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  		FIG. 5. FSH as a survival factor, preventing apoptosis of human immortalized granulosa cells induced by GnRH-I or -II. A, Human immortalized granulosa cells were treated with FSH (50 ng/ml) and GnRH-I or -II (10–7 mol/liter). GnRH-I and -II significantly increased the number of TUNEL-positive cells after 48 h in culture. The number of these apoptotic cells was markedly reduced with FSH cotreatment. B, GnRH-I- or -II-induced cleavages of the initiator, caspase-8, and the effectors, caspase-3 and -7, were blocked by FSH (50 ng/ml) cotreatment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Granulosa cells play an important role in regulating ovarian physiology, maintaining and controlling ovarian function, growth, and differentiation (36, 37). The growing oocyte is surrounded by layers of granulosa cells. Granulosa cells synthesize and secrete a variety of growth factors and cytokines that may regulate the differentiation and growth of the oocytes. Hence, communication between the granulosa cells of the growing follicle and the oocyte is essential for its development and maturation (38). Furthermore, apoptosis of granulosa cells not only is related to follicle atresia but also may be linked to poor oocyte quality and lower pregnancy rates after suboptimal ovarian stimulation with FSH and GnRH agonist exposure during in vitro fertilization treatment for infertility.

Our study has focused on the roles of GnRH-I and -II on apoptosis in human granulosa cells, the underlying molecular and cellular processes involved, and the antiapoptotic effects of FSH. Our results show that GnRH-I and -II can indeed increase apoptosis in human granulosa cells in a time-dependent manner, findings that are consistent with the apoptotic effects of GnRH agonists documented in previous studies (9, 39). We have also demonstrated that GnRH-I and -II induce apoptosis through the GnRH-I receptor because antide, a GnRH-I antagonist, can successfully block apoptosis induced by GnRH-II in human immortalized granulosa cells.

Apoptosis is a physiological process by which unwanted cells are eliminated in response to the developmental signals or toxic stimuli without inducing an immune response or inflammatory reaction (1, 2, 40). Apoptosis is mediated by the activation of a family of cysteine proteases, known as caspases. Among the caspases, caspase-3 and -7 are effectors responsible for cleaving a variety of key proteins in the cell, including poly (ADP-ribose) polymerase and lamins. Caspase-8 is an initiator that activates the effector caspases, producing an amplifying chain reaction (41). Our results show that both GnRH-I and -II can significantly activate initiator caspase-8 and effectors caspase-3 and -7 (Fig. 2Go). Our experiments using inhibitors specific for these caspases further confirm that activation of caspase-8, -3, and -7 are required for the apoptotic process induced by GnRH-I and -II. As shown in Fig. 4Go, GnRH-I- and -II-induced cleavages of caspases-8, -3, and -7 in human immortalized granulosa cells were inhibited by the broad-spectrum caspase inhibitor (Boc-D-FMK), the specific inhibitor of caspase-8 (Ac-IEPD-CHO), caspase-3 (Ac-DMQD-CHO), and caspase-7 (Ac-DNLD-CHO), respectively; furthermore, the caspase-8 inhibitor, Ac-IEPD-CHO, was able to abolish caspase-3 and -7 activation. Thus, our results indicate that both GnRH-I and -II activate the initiator caspase-8, which in turn activates effectors caspase-3 and -7.

FSH is the most important survival factor for follicle growth in the preovulatory phase, and a lack of FSH at the critical time will lead to follicular atresia through apoptosis (3, 26, 42). It has been well documented that FSH increases estradiol and progesterone production by granulosa cells in vitro. Estrogens, in association with gonadotropins, are known to effect granulosa cell growth (43). Furthermore, recent evidence has shown that progesterone acts as a survival factor that prevents apoptosis in rat granulosa cells by inhibiting the cell oxidation pathway (44). FSH is routinely administered during in vitro fertilization treatment to stimulate the development of multiple follicles after pituitary down-regulation with GnRH agonist. Hence, these two hormones can regulate apoptosis of granulosa cells but in opposite directions; GnRH agonist acts as an inducer, whereas FSH acts as an inhibitor of apoptosis (25, 26, 45). We therefore hypothesized that FSH could similarly counteract apoptosis induced by GnRH-I and -II in human granulosa cells. Indeed, GnRH-I- or GnRH-II-induced TUNEL-positive reactions and cleavage of caspases 8, 3, and 7 were attenuated by FSH cotreatment. These data confirm that FSH protects granulosa cells from undergoing apoptosis induced by GnRH-I or GnRH-II.

We have previously characterized in detail mRNA expression of GnRH-I, GnRH-II, and GnRH-I receptors not only in our immortalized cell lines but also in human luteinized granulosa cells obtained from in vitro fertilization procedures (46). In the same study, we have further localized by immunocytochemistry the presence of GnRH-I, GnRH-II, and GnRH-I receptors in the granulosa cell layer of preovulatory follicles as well as granulosa luteal cells of the corpus luteum from human ovaries. Hence, findings from our current study further attest to our original suggestion that immortalized granulosa cell lines, in addition to primary granulosa cells, provide a suitable experimental model for functional studies on the autocrine effects of GnRH-I and GnRH-II and their receptors (46) in the human ovary.

Our in vitro findings appear to be incongruent with clinical practice when the commonest in vitro fertilization regimen involves GnRH agonist treatment before and during FSH administration as a strategy to increase multiple follicle development and growth and, hence, oocyte yield. However, this apparent discrepancy can be explained. First, our findings are not referring to the known endocrine effects of continuous GnRH or GnRH agonist treatment on the pituitary-ovarian axis. Rather, we are modeling the effects of GnRH-I and GnRH-II produced locally in the ovary on granulosa cell function. Second, if our in vitro findings were to apply to the clinical setting, the availability of FSH from exogenous administration would counteract the apoptotic effects of the GnRH system, just as in our in vitro model. Our in vitro findings may also have clinical relevance in women at risk of developing ovarian hyperstimulation syndrome, when FSH administration is withdrawn (coasting) to allow high estradiol levels to fall to a lower range before human chorionic gonadotropin administration while GnRH agonist treatment is continued. Indeed, low pregnancy rates have been reported if coasting is greater than 4 d (47). It is tempting to speculate that, among other causes, the GnRH system may induce apoptosis of the granulosa cells after ambient FSH levels have declined to a critical threshold during prolonged coasting, which in turn affects oocyte quality and, consequently, pregnancy rates. Finally, with the newer ovarian stimulation regimen using GnRH antagonists instead of GnRH agonists to prevent premature LH surge, future studies evaluating the role of GnRH antagonists alone on granulosa cell apoptosis and the related caspase signaling pathways will be of interest.

In conclusion, our study shows that in addition to its well-established actions on the pituitary-gonadal axis, GnRH-I (as well as GnRH-II) can directly induce apoptosis in human granulosa cells. This raises a potentially important role of GnRH-I or GnRH-II in regulating follicle development and atresia. The direct apoptotic effects of GnRH-I and -II are mediated by GnRH-I receptors. The intracellular signals of apoptosis induced by GnRH-I or -II, in turn, are mediated by activation of the proteolytic caspase cascade, involving caspase-8 (the initiator) and caspase-3 and caspase-7 (the effectors). In addition, FSH protects human granulosa cells from undergoing apoptosis induced by GnRH-I and -II. Our study provides a novel insight into the caspase signaling pathways involved in GnRH-I- and -II-induced apoptosis in human granulosa cells. Moreover, our in vitro model systems suggest potential mechanisms responsible for follicular atresia through regulation of apoptosis of granulosa cells by GnRH-I or GnRH-II, which warrants further investigation.


   Footnotes
 
This research was supported by the Canadian Institutes of Health Research. P.C.K.L. is a recipient of a Distinguished Scientist Award of the Child and Family Research Institute. I.S.H. was awarded a graduate studentship from the Child and Family Research Institute.

Disclosure Summary: I.S.H., A.P.C., and P.C.K.L. have nothing to declare.

First Published Online May 13, 2008

Abbreviation: TUNEL, Terminal deoxynucleotidyl-transferase-mediated biotin-dUTP nick-end labeling.

Received January 18, 2008.

Accepted May 2, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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H.-M. Wu, J.-C. Cheng, H.-S. Wang, H.-Y. Huang, C. D. MacCalman, and P. C.K. Leung
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