Determination of proliferation index in primary bovine cell cultures of corpus luteum stimulated with leuprolide acetate, GnRH analogue

HAMER, CS.; GERMANO, P.; GAMBAROTTA, MC.; LOMBARDO, DM.; SOÑEZ, MC.

Buenos Aires

Jornada; III Jornadas Internacionales del Instituto de Investigación y Tecnología en Reproducción Animal – INITRA; 2012

Instituto de Investigación y Tecnología en Reproducción Animal - INITRA. Facultad de Ciencias Veterinarias. UBA

GnRH, ovarian endogenous peptide, modulates follicular development and probably the formation of the corpus luteum in bovine through the regulation of apoptosis, role which was demonstrated on the granulosa cells of this species. Their participation on cell proliferation has been partially studied. The objective was to determine the effects in vitro of leuprolide acetate (LA, GnRH analogue) on the proliferation of bovine luteal cells (BLC) at doses of 10 and 100 nM during 48 hours. BLC from slaughter animals were cultured during 7 days in DMEM + F12 supplemented medium. Three experiments, in triplicates in each, were performed with the following treatments: 5% FBS (control), LA 10nM and LA 100 nM. After 48 hours the cells were fixed with Bouin solution,15 min. To identify proliferative cells, an indirect immunocytochemical LSAB method (LSAB System Kit HRP, Dako) was applied, using anti-human monoclonal antibody Ki-67 Clone MIB-1 (Dako) 1:100, with negative controls omitting the primary antibody incubation and positive controls on sections of human tonsil. The antigen-antibody complex was revealed with 3,3’ diaminobencidine (DAB) 0.003% (Dako), ending with slight contrasting with Mayer´s hematoxylin, dehydrated and mounted. 1260 images were captured using XnView and the total Ki67 positive and negative nuclei were counted. The data were statistically analyzed by InfoStat software applying parametric ANOVA (p <0.05). The amount of positive nuclei anti-Ki67, an indicator of cell proliferation, was similar for both doses of LA with significant decrease compared with the control (p = 0.002). It is concluded that the rate of proliferation of bovine luteal cells which were treated in culture for 48 hours with leuprolide acetate, GnRH analogue, was doses independent, decreasing significantly related to the controls, indicating that the analogue reduced the level of proliferation of bovine luteal cells in vitro under these conditions.