Effects of leuprolide acetate (GnRH analogue) as inductor of apoptosis in primary cultures of bovine luteal cells

GERMANO P; MARURI A; SOÑEZ MC; LOMBARDO DM

CABA

Jornada; Segundas Jornadas Internacionales del Instituto de Investigación y Tecnología en Reproducción Animal; 2010

Instituto de Investigación y Tecnología en Reproducción Animal - INITRA. FCV. UBA

Gonadotropin-releasing hormone (GnRH) has been involved in mechanisms associated with apoptosis in mammalian ovary in relation with the processes of folliculogenesis, follicular atresia and luteolysis. The effects of GnRH agonists / antagonists on bovine luteal cells have not been explored. This study aims to evaluate the effects of Leuprolide Acetate (LA - GnRH agonist) and Antide (ANT - GnRH antagonist) on apoptosis in primary cultures of bovine luteal cells. Bovine ovaries were collected from slaughter animals and corpora lutea were isolated. Luteal cells were dispersed by mechanical and enzymatic treatment (trypsin 0.25%), 1x 105/well were seeded and cultured for 7 days with DMEM + F12 supplemented and 10% FBS. Oil red stain was applied to detect lipid vacuoles typical of luteal steroidogenic cells. Treatments for 24 and 48 hours whith 5% FBS (Control), ANT 100 nM, LA 1,10 and 100 nM and ANT plus LA 1,10 and 100 nM were run by triplicate. Apoptotic bodies were assessed following morphological criteria by staining with Mayer Hematoxylin. Images were captured using IM50 software (Leica). Data were statistically analyzed by two-way ANOVA and Bonferroni test (p ≤ 0.05). Results indicate an increased number of apoptotic cells in relation to the dose of LA. In treatments of 24 hours there was no signifi cant statistical differences, where as in cultures treated for 48 hours with increasing doses of LA (1,10,and 100 nM), there was an increase in the percentage of apoptotic bodies compared to control indicating signifi cant differences with the dose of 100 nM (p=0,0048). ANT seems to behave as an antagonist partial since increased the percentage of apoptotic bodies in respect of control, but not completely abolished the inducing effect of LA in all the doses used. We conclude that LA is an inducer of apoptosis in a dose-dependent in bovine luteal cells in primary cultures stimulated for 48 hours, resulting partially blocked this effect by the antagonist ANT 100 nM dose.