In Vivo Effect Of Gonadotropin Releasing Hormone Analogs On Caspase-3 Expression In Rat Ovarian Follicles

PARBORELL FERNANDA; ABRAMOVICH DALHIA; TESONE MARTA

Quebec, Canadá.

Congreso; 38th Annual Meeting of the Society for the Study of Reproduction; 2005

Gonadotropin-releasing hormone (GnRH) analogs: agonists (GnRH-a) or antagonists (GnRH-ant) have been widely used to inhibit gonadotropin pituitary release. However,  studies have also shown that GnRH has extrapituitary effects, particularly on the rat and human ovary. In previous studies from our laboratory, we showed an apoptotic follicular effect of GnRH-a correlated with an imbalance in the ratio of antiapoptotic:proapoptotic proteins (Bcl-xL/Bcl-xS), as well as, a decrease of Bax traslocation to mitochondria in  preovulatory follicles (POF). To elucidate whether these changes have an effect on the caspase cascade, we studied the main effector caspase-3 analysing its levels and localization. Prepubertal rats superovulated with eCG (Control group) were injected during 48 h, each 12 h with GnRH-a (Leuprolide Acetate, 1µg/rat/day, LA group) and/or GnRH-ant (Antide, 10 ug/rat/day; LA+Ant group or Ant group). Healthy POF (>400 mm in diameter) were dissected from ovaries under a stereoscopic microscope. Proteins from POF were isolated for Western blot analyses to measure the inactive and active form of caspase-3. In addition, ovaries were fixed in formalin and used for immunohistoquemistry (IHC) of caspase-3 (inactive and active form). LA group showed a significant increase (arbitrary units), in the level of the active form (17 kDa) as compared to the control group (C: 9,200±300, LA: 11,636±321, p<0.05).  The co-injection of Ant interfered with this LA effect (LA: 11,636±321, LA+Ant: 9,467±267, p<0.05) and Ant alone significantly decreased caspase-3 cleavage (C: 9,200±300, Ant: 7,450±250, p<0.05). None of treatments changed the procaspase-3 levels (33 kDa). IHC analyses showed that theca cells (TC) from preantral follicles (PF) exhibited a moderate signal for caspase-3 only in control and LA group, whereas TC from antral follicles (AF) showed immunoreactivity in all groups, appearing more intense in LA group and in addition in this group the staining was more evident in the nucleus of TC. On the other hand, granulosa cells of PF and AF exhibited a weak or absent immunostaining for caspase-3 for all treatments. We conclude that LA enhances the caspase-3 expression in POF, whereas Ant decreased it; in addition, IHC results would suggest that these changes mainly occur in TC.