Induced apoptosis and expression of related proteins in granulosa cells from women undergoing IVF: a preliminary study.
BACKGROUND
Because apoptosis may be involved in the outcome of IVF, the expression of pro- and anti-apoptosis proteins in a model of induced granulosa cell (GC) apoptosis was evaluated in 25 women with normal FSH levels undergoing IVF.


METHODS
After 1 day of culture, apoptosis was induced with interferon (IFN)-gamma (200 IU/ml), followed 24 h later by an agonistic anti-Fas antibody (0.5 microg/ml). On day 3, apoptotic GC, identified by chromatin condensation and/or nuclear fragmentation after DAPI staining, were counted among 1000 cells in randomly chosen fields under UV microscopy, and enabled allocation of women into two groups with either low (group 1) or high (group 2) percentages of apoptosis (11.6 +/- 4.8 and 59.5 +/- 14.8% respectively; P < 0.001). Immunoblotting was used to evaluate the following in proteins: poly (ADP-ribose) polymerase (PARP), caspases 8 and 3, Bcl-2, heat shock protein (HSP) 70, Bax, Bak and Stat-1 (a protein known to be inducible by IFN-gamma).


RESULTS
Based on densitometric analysis of immunoblots, the PARP 116 kDa bands were respectively 4.3- and 33.3-fold lower for treated groups 1 and 2. Caspase 8, caspase 3 and HSP70 were expressed slightly less in treated group 2 than treated group 1. Densitometric analysis of bands corresponding to Bcl-2 showed respectively for treated groups 1 and 2, 3.2- and 2.5-fold decreases. Bak expression was similar in both control groups, and comparably lower in the two treated groups. With regard to Stat-1, densitometry showed 3.3- and 1.3-fold increases respectively in treated groups 1 and 2.


CONCLUSIONS
These results suggested that Fas-mediated apoptosis of GC is accompanied by significant changes in proteins acting in apoptosis, and that this type of programmed cell death might play a potential prognostic role for women undergoing IVF.
Benifla
J L
JL
Service de Gynécologie-Obstétrique, and Unité INSERM 327, Hôpital Bichat-Claude-Bernard, Université Paris 7-Denis-Diderot, 46 rue Henri-Huchard, 75877 Paris, Cedex 18, France. Jl.benifla@rth.ap-hop-paris.fr
Sifer
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C
Bringuier
A F
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Blanc-Layrac
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Camus
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Madelenat
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Feldmann
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D000328:Adult; D017209:Apoptosis; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D005260:Female; D005307:Fertilization in Vitro; D006107:Granulosa Cells / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000378:metabolism
D008565:Membrane Proteins; D020033:Protein Isoforms; D011506:Proteins; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases