APOPTOSIS REGULATION ON A BOVINE GRANULOSA CELL STABLISHED LINE (BGC-1) WITH A GNRH ANALOG (Leuprolide Acetate)

CAROU C; REVILLA, M; FIORITO, C; LOMBARDO, DM

Universidad Nacional de Cordoba

Congreso; XI Congreso Argentino de Ciencias Morfológicas; 2008

Gonadotropine releasing hormone receptors wereidentified on ovaric granulose (GC) and luteal cells. GnRHractivation on GC leads to gonadal function activation orinhibition. In vivo and in vitro assays over hormonal regulationwith GnRH agonists (GnRHa), show an increase on ratovarian follicular atresia. Furthermore, it was determined thatin vivo administration of GnRHa, increase cellular death byapoptosis on ovary tissue and GC. We have previouslyidentified, in our laboratory, a significant increase on BGC-1apoptotic rate when induced for 24 Hs with 100 nM Leuprolideacetate (LA) using DAPI and TUNEL apoptotic detectiontechniques. To corroborate and more specifically analyzedthese results, the porpoise of this work was to realizedcitometric assay (FACS) through Anexina V and PI BGC-1label. We tested three LA doses (1, 10 and 100 nM), withand without 5% bovine fetal calf for 24 and 48 incubationtime. Three experiments were done for 24 and two for 48 Hs,both by triplicate, analyzing results by two way ANOVA andBonferroni comparison of means test. 100 nM LA for 48 Hsshows a significant increase on total apoptosis of 74% overcontrol (p = 0.04). The increases on early apoptosis werealso significant for 10 and 100 nM LA, with an increase of69.84 for 10 and 70% for 100 nM LA over control (p = 0.015).For 24 Hs incubation, we observed a significant increase ofearly apoptosis at 100 nM la (68 % over control, p = 0.018),being the increase for total apoptosis at the same LA doses35.87 % over control (p = 0.0244). These results confirm ourprevious observations with DAPI and TUNEL. By the otherhand, FACS with Anexina V and PI, make possible to seethe temporal dynamics for the apoptotic process withprolonged incubation times.