SPONTANEOUS OVARIAN GRANULOSA CELL LUTEINIZATION IN CATTLE: EXPERIMENTAL MODEL in vitro.

CAROU, MC.; MARURI, A.; OLEA, GB.; LOMBARDO, DM.

Ciudad Autónoma de Buenos Aires

Jornada; IV Jornadas Internacionales del Instituto de Investigación y Tecnología en Reproducción Animal; 2014

Instituto de Investigación y Tecnología en Reproducción Animal

Luteinization is a cellular diff erentiation process essential for the success of early pregnancy involving the internal theca and granulosa cells that are induced by the ovulatory stimulus to become the corpus luteum (CL). The objective was to obtain a suitable and reliable experimental model for studying the process of spontaneous luteinization of primary culture cells of bovine ovarian granulosa (CPGB) in vitro. Cultures were classifi ed according to if were obtained from ovaries with or without the presence of CL, assessing the production of P4, apoptosis and mitosis according to the seeding density (3125, 6250, 12500 and 25000 cells / ml) and time of culture (4, 6, 8 and 10 days). Th e CPGB were sown over 18X18 mm coverslips in 6-well plates. At 4, 6, 8 and 10 days of incubation, 1 ml culture medium aliquots were frozen for P4 determination by RIA. Th e cells were fi xed with methanol and stained with hematoxylin to assess apoptosis and mitosis morphologically. All treatments were performed in triplicate. The results showed an increase in the P4 production in function of time (from day 4 to 10) in cultures seeded with 6250 cells / ml, at 8 (59%) and 10 (49%; 3.13 ng P4 / ml / density at day 4) days of culture for CPGB without CL and 8 (62%) and 10 (269%; 2,15 ng P4 / ml / density at day 4) days to CPGB with CL. Th e increase in P4 production was presented in conjunction with an increase in apoptosis since day 10 (86% with CL and 92% without CL) and the decrease in mitosis since day 8. It was demonstrated that cells undergoing apoptosis produce signifi cant levels of P4.So, it is important to distinguish both, diff erentiation and apoptosis. Th erefore, under the conditions imposed to achieve spontaneous luteinization of bovine granulosa cells from ovaries with and without CL, a seeding density of 6250 cells / ml and 8 days of culture is needed. Being this a simple in vitro model for studying the ability of granulosa cells to luteinize under diff erent stimuli.