INHIBITION OF APOPTOSIS INDUCED BY ALLOPREGNANOLONE IN THE RAT CORPUS LUTEUM.

LACONI MYRIAM; CABRERA RICARDO

Kailua Kona, Hawaii

Congreso; First World Congress on Reproductive Biology; 2008

We report the effect of allopregnanolone (3á-hydroxy-5á-pregnan-20-one),  one of the best characterized neurosteroids, on the ovulation rate and the corpus luteum histology associated to morphological feature of apoptotic cell death in cycling rats. Allopregnanolone (ALLO) was injected intracerebroventriculary (i.c.v.) during proestrus.  The luteinizing hormone (LH) and progesterone (Pg) serum levels, the ovulation test and the histological analysis were performed in the estrum morning. The ALLO injection significantly decreases LH serum level (p<0,001) and the number of oocytes collected on estrus (p<0002). Simultaneously, an inhibition of apoptotic figures was detected in the already formed corpora  lutea belonging to the previous ovary cycle. When the GABAA  antagonist, (Bi) was i.c.v. injected alone or together with ALLO any effect on the ovulation rate occurred, but the LH level or the apoptotic cell number  were  recovered. Paraffin sections of the corpora lutea  and hematoxilin - eosin stain were used to detect apoptotic cells. To confirm that the cell dead was due to apoptosis  some paraffin sections were processed  for the TUNEL assay, terminal deoxynucleotidyl transferase – mediated dUTP nick end labeling (Dead End colorimetric TUNEL system, Promega). Corpora lutea were also processed  for electron microscopy  to study the ultrastructural features of the apoptotic cells. Under light microscopy apoptotic cells were characterized by their nuclear pyknotic appearance, marginated chromatin and by the presence of multiple densely stained apoptotic bodies. Ultrastructurally, the luteal cells undergoing apoptosis exhibits their cytoplasm organization heavily deteriorated due to organelle swelling and fragmentation, mainly of the smooth endoplasmic reticulum. The nuclei, in an initial step of regression, displayed chromatin  condensation and a prominent perinuclear space. In a more advanced step of degeneration, it evidences large irregular clusters of condensed chromatin and finally, fragmentation and  formation of apoptotic bodies. In conclusion, the present results indicate that i.c.v. injected ALLO inhibits apoptosis, in the corpus luteum and this action was blocked by Bi.