Direct effect of gonadotropin-releasing hormone agonist and antagonist on the growth, apoptosis and steroidogenesis in human granulosa cells from women undergoing in vitro fertilization

VITALE, AM; ABRAMOVICH, D; PELUFFO, MC; MERESMAN, G; TESONE, M

Philadelphia, Pennsylvania

Congreso; 60th ASRM Annual Meeting; 2004

ASRM (American Society of Reproductive Medicine)

Objective To evaluate the direct effect of a GnRH-a (leuprolide acetate, LA) and a GnRH-ant (antide, ANT) on the steroidogenesis, growth and programmed cell death (apoptosis) of human granulosa-luteal cells (GLC) obtained from patients undergoing ART. Design Human GLC were cultured after isolation from follicular fluid aspirated of patients undergoing ART. Materials and methods Proliferation assays: 100,000 GLC were plated in SFM and incubated 24 h with different agents; EGF (10 ng/ml); LA as GnRH-a (1, 10 and 100 ng/ml) and ANT (10-7 M), as GnRH antagonist; or a combination of ANT with LA, adding LA 100 ng/mL 3 hours after the addition of ANT 10-7M. 24h before harvesting, 1 ìCi 3H-thymidine was added to each well, and DNA synthesis was assessed by 3H-thymidine incorporation. Apoptosis: % of apoptotic cells was assessed by the acridine orange-ethidium bromide technique in GLC cultures at basal conditions and after exposure to LA 100 ng/mL or ANT 10-7 M, or a combination of ANT with LA, adding LA 3 hours after the addition of ANT. After addition of the acridine orange-ethidium bromide mix, the cells were viewed by a fluorescence microscope and the apoptotic cells were counted as a percentage of the total. One-way ANOVA was used to compare the mean values among the treatments. When the values were significant, ANOVA analysis was followed by Tukey test. Values of p< 0.05 were considered significant. Results The GnRH-a LA alone had no effect on basal DNA synthesis in GLC cultures. Unexpectedly, ANT alone produced a significant stimulatory effect (350%) of 3H-thymidine incorporation on basal conditions. To further determine the role of LA in cell proliferation, different concentrations of LA were added to EGF (10 ng/ml) stimulated cell cultures (SCCs). In these conditions 3H-thymidine uptake was down-regulated by LA at 10 and 100 ng/ml (LA 1 ng/ml: 7385.3±1164.4 cpm/well, P>0.05; LA 10 ng/ml: 1798±268 cpm/well and LA 100 ng/ml: 1698.6±259.3 cpm/well, p<0.05 vs. SCC: 7556±758 cpm/well), while ANT did not produce any change in DNA synthesis in the presence of EGF. The inhibitory LA effect was completely reversed by the addition of ANT to the culture medium. To assess whether this DNA synthesis inhibition was produced by an increase of the apoptosis of GLC cells, these cells were cultured in the presence of LA (100 ng/ml). This treatment showed a significant increase in the apoptotic levels from 39.6±2.5 % at basal conditions to 58.6±4% (expressed as a % of apoptotic cells, p<0.05). This effect was reversed by ANT: 40.4±4 (vs. basal, P>0.05), while ANT alone had no effect on basal apoptosis. Conclusion These findings suggest that the direct inhibitory effect of GnRH agonists on ovarian follicular growth described in conventional ART procedures, could be avoided with the use of GnRH antagonists, and besides these molecules could improve follicular growth. Supported by: ANPCYT (BID 1201 OC-AR PICT 99:05–06384) and Universidad de Buenos Aires (X909).