1 Service de Gynécologie-Obstétrique, 2 Service d'Histologie-Embryologie, Cytogénétique and 3 Unité INSERM 327, Hôpital Bichat-Claude-Bernard, Université Paris 7-Denis-Diderot, 46 rue Henri-Huchard, 75877 Paris, Cedex 18, France
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: apoptosis-related proteins/human granulosa cell/IVF/Stat-1 protein
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In all these circumstances, however, the apoptosis pathway followed by human GC was not well defined. Biochemical analyses of animal and human GC have previously shown apoptosis to be regulated by caspases and Bcl-2 gene family members (Boone and Tsang, 1998; Kugu et al., 1998
). The presence of the Fas receptor (Quirk et al., 1995
) clearly indicated that this apoptosis pathway was also involved in GC isolated from women undergoing IVF.
The capacity of cultured GC to enter apoptosis after Fas induction has been investigated previously (Sifer et al., 2000). GC from 25 women undergoing IVF were incubated with interferon (IFN)-
, a well-known Fas protein up-regulator (Choi et al., 1999
; Moers et al., 1999
), before being subjected to the action of an agonistic anti-Fas antibody. The percentage of each patient's treated GC entering apoptosis allowed distinction to be made between two groups of women. In the present study, the expression of apoptosis-related proteins implicated in the Fas pathway in relation to the rate of apoptosis was analysed.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IVF stimulation procedure
All patients received the standard stimulation protocol, as described briefly below. First, hormonal down-regulation was started on day 3 of the menstrual cycle preceding IVF with a GnRH agonist, triptorelin (Decapeptyl®; Ipsen/Biotech, Paris, France), given i.m. at a daily dose of 0.1 mg for a minimum of 2 weeks. When the plasma estradiol level was 50 pg/ml, two ampoules of rFSH (follitrophin
; Gonal-F®; 75 IU, Serono, Boulogne, France, or follitrophin ß;Puregon®; 100 IU, Organon, Saint-Denis, France) were administered i.m. every evening along with 0.05 mg of the GnRH agonist. Follicular development was monitored ultrasonographically with a 5 MHz transvaginal probe. The dose of gonadotrophin stimulation was then adjusted according to plasma estradiol levels and the size of ovarian follicles measured. When the plasma estradiol level surpassed 1800 pg/ml, and when at least two follicles with a diameter of 20 mm were observed on ultrasound examination, 10 000 IU of HCG (Gonadotrophine chorionique endo®; Organon) were given i.m. to induce follicle rupture. Transvaginal follicular aspiration was performed 3436 h later, under propofol (Diprivan®; Zeneca, Cergy, France) general anaesthesia.
Reagents
Monoclonal apoptotic agonistic anti-Fas/CD95 antibody (clone 7C11) was purchased from Immunotech (Marseilles, France), and recombinant human IFN- from R&D Systems (Abingdon, Oxford, UK). For immunological studies, the following murine monoclonal antibodies were used: anti-caspase 8/FLICE immunoglobulin (Ig)G2a (clone B9-2); anti-poly (ADP-ribose) polymerase (PARP) IgG1 (clone C2-10) and anti-heat shock protein (HSP) 70 IgG2b (clone 5G10) from Pharmingen (San Diego, CA, USA); and anti-Stat-1 IgG1 from Transduction Laboratories (Lexington, KY, USA). Polyclonal rabbit antibodies to caspase 3/CPP32 and Bax (Pharmingen) and to Bcl-2 and Bak (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were also purchased. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and conjugated goat anti-rabbit IgG was from Immunotech, and the ECL Immunoblot detection system was from Amersham (Amersham, UK).
GC isolation and culture
After oocyte identification and isolation, all FF samples were collected, pooled for each woman and centrifuged at 500 g for 30 min. The supernatant was discarded and the pellet resuspended in 10 ml of phosphate-buffered saline (PBS) (Eurobio, Les Ulis, France). An aliquot of the suspension was layered onto 10 ml of a 50% Percoll® solution (Pharmacia Biotech, Uppsala, Sweden) and centrifuged at 100 g for 30 min to sediment the red blood cells. The isolated GC were aspirated from the interface, washed and resuspended in minimal essential medium (MEM) with Earle's salts and glutamax (Gibco, Glasgow, UK) supplemented with 10% heat-inactivated fetal calf serum (Valbiotech, Paris, France) and antibiotics. Under our experimental conditions, lymphomyeloid cells were present only in the supernatant medium, this being verified as follows. GC, leukocytes and anti-CD45 antibody were mixed just before plating and on day 1 it was observed that, while GC were adherent, no lymphomyeloid cells had become attached. Purified GC were cultured for 1 day in 25 cm2 vented culture flasks (Nunc, Roskilde, Denmark) at 37°C in humidified air with 5% CO2, then washed with MEM to discard residual red and white blood cells. Fresh culture medium was added and incubation was continued according to the apoptosis-induction protocol.
Apoptosis induction
Cultured GC were divided into two aliquots. The first was treated to induce apoptosis, on day 1, with IFN- (200 IU/ml) and, on day 2, with the agonistic anti-human Fas monoclonal antibody (0.5 µg/ml). The second aliquot served as an untreated control. On day 3, treated and control GC were harvested by scraping, and the rate of apoptotic GC was measured by 4,6-diamidino-2-phenylindole (DAPI) staining.
DAPI staining
After apoptosis induction, GC were fixed with 70% ethanol for 10 min at room temperature, smeared on a glass slide and air-dried. Cell nuclei were stained with 0.1 µg/ml DAPI (Sigma, St Louis, MO, USA) in PBS containing 0.1% Tween 20 (Sigma). The slides were washed three times in PBS, air-dried and mounted in Mowiol® solution (Calbiochem, San Diego, CA, USA). For each woman, GC with morphological characteristics of apoptosis, such as chromatin condensation and/or nuclear fragmentation, were identified and counted among 1000 cells in randomly selected fields using ultraviolet microscopy. DAPI staining of treated GC allowed identification of two groups of women based on the percentage of apoptotic cells (setting threshold at the median): group 1 (n = 12) had low percentages (mean 11.6 ± 4.8%) while group 2 (n = 13) had high percentages (mean 59.5 ± 14.8%; P < 0.001) (Sifer et al., 2000) (Figure 1
). The percentages of apoptotic GC in the respective untreated control samples were comparable: 5.6 ± 2.7 versus 4.7 ± 2.7% (not significant). The remaining treated and untreated cells from each group were pooled and subjected to immunoblotting. Results were analysed according to the group assignment.
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunoblot analysis demonstrated that differences also existed in the apoptosis proteins when comparing the two groups of women after separation on the basis of their percentages of apoptotic cells. PARP cleavage, which is an established marker of apoptosis (Oliver et al., 1998) and was (to the best of our knowledge) used here for the first time to investigate apoptosis of human GC, was markedly pronounced in group 2 treated GCa finding that confirmed the DAPI results. However, for unknown reasons, the PARP 85-kDa band (which results from cleavage of the 116 kDa protein) appeared to be only slightly enhanced in groups 1 and 2.
Likewise, the expression of caspase 8 and 3 proforms was more strongly decreased for group 2 treated GC than group 1, thus indicating an enhanced activation of these enzymes in group 2 patients. These findings are not surprising because it has been well demonstrated that caspases 8 and 3 are directly associated with the Fas-mediated apoptosis pathway, as already reported in other cells (Nagata, 1997). Caspase 3 was previously detected in GC from women undergoing IVF (Izawa et al., 1998
). However, in contrast to studies in rats where caspase 3 was present in GC atretic follicles but absent from healthy follicles (Boone and Tsang, 1998
), the expression of this protein in human GC observed in the present study might suggest that caspase 3 is induced by gonadotrophins during ovarian stimulation, as outlined previously (Boone and Tsang, 1998
).
With regard to the anti-apoptosis proteins, it is well established that Bcl-2 (Adams and Cory, 1998) and HSP70 (Samali and Cotter, 1996
) can inhibit many apoptosis signals in very different cell types and in various organisms. Bcl-2 has previously been detected immunohistochemically in human ovaries (Lu et al., 1993
; Rodger et al., 1995
). It was found that this protein was weakly expressed in both control GC groups and decreased in both treated groups. Similarly, HSP70which was clearly expressed in control group GC and particularly in group 2was slightly decreased in treated groups 1 and 2. Therefore, it was concluded that Bcl-2 was probably not directly responsible for the lower percentages of apoptosis induced by IFN-
and anti-Fas antibody in group 1. In contrast, HSP70 may have played a positive role in the protection of GC in this experiment. Indeed, it has been reported that when HSP70 expression declined, cells became more sensitive to apoptosis (Robertson et al., 1999
; Sreedhar et al., 1999
).
With regard to the pro-apoptosis proteins, it was not possible to detect Bax by immunoblotting of cultured GCa result which is in agreement with the reported absence of this protein in human GC from healthy follicles, as assessed by immunolocalization (Kugu et al., 1998). In contrast, Bak was well represented in both controls and, after treatment, its expression was slightly weaker in group 1 and more markedly diminished in group 2.
In the present study, IFN- was used to stimulate Fas expression, though it is well known that IFN-
can induce apoptosis by itself (Stark et al., 1998
) by activating the janus kinases (JAK), and the signal transducers and activators of transcription (STAT). This was the reason why, in the present study, Stat-1 was examined as it is particularly enhanced by IFN-
. The present results showed that GC from both treated groups exhibited comparably enhanced Stat-1 expression, though group 2 control GC expressed this protein more strongly than group 1 control GCa finding which might explain why Stat-1 enhancement was less marked in treated group 2 than in treated group 1. As reported previously, activation of the STAT pathway might cause apoptosis by overexpression of some caspases, particularly caspase 1 (Chin et al., 1997
). However, this caspase was not investigated in the present study, and the difference observed between the two treated GC groups, according to the Stat-1 status in control samples, may reflect the synergistic expression of Stat-1 and Fas pathways. It is hypothesized that the stronger Stat-1 expression in group 2 control GC might facilitate apoptosis induction (Schindler, 1998
).
The different expression of the anti- and pro-apoptosis proteins in cultured GC from women undergoing IVF might suggest a potential role for hormones in Fas-mediated apoptosis. Indeed, FSH is known to attenuate in-vitro apoptosis in cultured human GC (Matsubara et al., 2000; Sifer et al., 2001
), whereas GnRH agonists, at different concentrations, increased the incidence of human GC apoptosis (Zhao et al., 2000
; Sifer et al., 2001
). However, those in-vitro results must be confirmed in vivo according to the women stimulation protocol. Finally, further studies are required to demonstrate a prognostic role for, and to understand the mechanism of, apoptosis of GC from women undergoing IVFespecially to explore the potential impact of hormonal regulation of interactions among the Fas-mediated pathway and cytokines.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andrews, N.C. and Faller, D.V. (1991) A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res., 19, 24992500.[ISI][Medline]
Boone, D.L. and Tsang, B.K. (1998) Caspase-3 in the rat ovary: localisation and possible role in follicular atresia and luteal regression. Biol. Reprod., 58, 15331539.[Abstract]
Chin, Y.E., Kitagawa, M., Kuida, K. et al. (1997) Activation of the STAT signaling pathway can cause expression of caspase 1 and apoptosis. Mol. Cell. Biol., 17, 53285337.[Abstract]
Choi, C., Park, J.Y., Lee, J. et al. (1999) Fas ligand and Fas are expressed constitutively in human astrocytes and the expression increases with IL-1, IL-6, TNF-alpha or IFN-gamma. J. Immunol., 162, 18891895.
Hsueh, A.W.J., Billing, H. and Tsafriri, A. (1994) Ovarian follicle atresia: a hormonally controlled apoptotic process. Endocr. Rev., 15, 707724.[ISI][Medline]
Izawa, M., Nguyen, P.H., Kim, H.H. et al. (1998) Expression of the apoptosis-related genes, caspase-1, caspase-3, DNA fragmentation factor, and apoptotic protease activating factor-1, in human granulosa cells. Fertil. Steril., 70, 549552.[ISI][Medline]
Kugu, K., Ratts, V.S., Piquette, G.N. et al. (1998) Analysis of apoptosis and expression of Bcl-2 gene family members in the human and baboon ovary. Cell Death Differ., 5, 6776.[ISI][Medline]
Lu, Q.L., Poulson, R., Wong, L. et al. (1993) Bcl-2 expression in adult and embryonic non-haematopoietic tissues. J. Pathol., 169, 431437.[ISI][Medline]
Matsubara, H., Ikuta, K., Ozaki, Y. et al. (2000) Gonadotropins and cytokines affect luteal function through control of apoptosis in human luteinized granulosa cells. J. Clin. Endocrinol. Metab., 85, 16201626.
Moers, C., Warskulat, U., Muschen, M. et al. (1999) Regulation of CD95 (Apo-1/Fas) ligand and receptor expression in squamous-cell carcinoma by interferon-gamma and cisplatin. Int. J. Cancer, 80, 564572.[ISI][Medline]
Nagata, S. (1997) Apoptosis by death factor. Cell, 88, 355365.[ISI][Medline]
Nakahara, K., Saito, H., Saito, T. et al. (1997a) Incidence of apoptotic bodies in membrana granulosa of the patients participating in an in vitro fertilization program. Fertil. Steril., 67, 302308.[ISI][Medline]
Nakahara, K., Saito, H., Saito, T. et al. (1997b) The incidence of apoptotic bodies in membrana granulosa can predict prognosis of ova from patients participating in in vitro fertilization program. Fertil. Steril., 68, 312317.[ISI][Medline]
Oliver, F.J., de la Rubia, G., Rolli, V. et al. (1998) Importance of Poly(ADP-ribose) Polymerase and its cleavage in apoptosis. J. Biol. Chem., 273, 3353333539.
Oosterhuis, G.J.E., Michgelsen, H.W., Lambalk, C.B. et al. (1998) Apoptotic cell death in human granulosa-lutein cells: a possible indicator of in vitro fertilization outcome. Fertil. Steril., 70, 747749.[ISI][Medline]
Palumbo, A. and Yeh, J. (1994) In situ localization of apoptosis in the rat ovary during follicular atresia. Biol. Reprod., 51, 888895.[Abstract]
Piquette, G.N., Tilly, J.L., Prichard, L.E. et al. (1994) Detection of apoptosis in human and rat ovarian follicles. J. Soc. Gynecol. Invest., 1, 297301.[ISI][Medline]
Quirk, S.M., Cowan, R.G., Joshi, S.G. et al. (1995) Fas antigen-mediated apoptosis in human granulosa/luteal cells. Biol. Reprod., 52, 279287.[Abstract]
Robertson, J.D., Datta, K., Biswal, S.S. et al. (1999) Heat-shock protein 70 antisense oligomers enhance proteasome inhibitor-induced apoptosis, Biochem. J., 344, 477485.[ISI][Medline]
Rodger, F.E., Fraser, H.M., Duncan, W.C. et al. (1995) Immunolocalization of Bcl-2 in the human corpus luteum. Hum. Reprod., 10, 15661570.[Abstract]
Samali, A. and Cotter, T.G. (1996) Heat shock proteins increase resistance to apoptosis. Exp. Cell Res., 223, 163170.[ISI][Medline]
Schindler, C. (1998) STATs as activators of apoptosis. Trends Cell Biol., 8, 9798.[ISI][Medline]
Seifer, D.B., Gardiner, A.C., Ferreira, K.A. et al. (1996) Apoptosis as a function of ovarian reserve in women undergoing in vitro fertilization. Fertil. Steril., 66, 593598.[ISI][Medline]
Sifer, C., Benifla, J.L., Bringuier, A.F. et al. (2000) Incidence of induced apoptosis of human granulosa cells could predict in vitro fertilization-embryo transfer outcome. Hum. Reprod., 15 (Abstract Book 1), 207.[Medline]
Sifer, C., Blanc-Layrac, G., Bringuier, A.F. et al. (2001) Effects of a gonadotropin releasing hormone agonist and follicle stimulating hormone on the incidence of apoptosis in human granulosa cells. Hum. Reprod., 16 (Abstract Book 1), 65.
Sreedhar, A.S., Pardhasaradhi, B.V., Begum, Z. et al. (1999) Lack of heat shock response triggers programmed cell death in a rat histiocytic cell line. FEBS Lett., 456, 339342.[ISI][Medline]
Stark, G.R., Kerr, I.M., Williams, B.R.G. et al. (1998) How cells respond to interferons. Ann. Rev. Biochem., 67, 227264.[ISI][Medline]
Sugino, N., Takiguchi, S., Ono, M. et al. (1996) Nitric oxide concentrations in the follicular fluid and apoptosis of granulosa cells in human follicles. Hum. Reprod., 11, 24842487.[Abstract]
Tilly, J.L., Kowalsky, K.I., Johnson, A.L. et al. (1991) Involvement of apoptosis in ovarian follicular atresia and postovulatory regression. Endocrinology, 129, 27992801.[Abstract]
Yuan, W. and Judice, L. (1997) Programmed cell death in human ovary is a function of follicle and corpus luteum status. J. Clin. Endocrinol. Metab., 82, 31483155.
Zhao, S., Saito, H., Wang, X. et al. (2000) Effects of gonadotropin-releasing hormone agonist on the incidence of apoptosis in porcine and human granulosa cells. Gynecol. Obstet. Invest., 49, 5256.[ISI][Medline]
Submitted on December 14, 2000; resubmitted on July 11, 2001; accepted on November 13, 2001.