Abundances of autophagy-related protein LC3B in granulosa cells, cumulus cells, and oocytes during atresia of pig antral follicles

Abstract

In mammals, apoptosis has been accepted as the type of programmed cell death (PCD) that occurs in ovarian follicles undergoing atresia. Results of recent studies, however, indicate autophagy may be an alternative mechanism involved in follicle depletion through independent or tandem actions with apoptosis. Western blotting and immunofluorescence procedures were used in the present study to investigate the abundances of LC3B protein in freshly collected granulosa cells (GCs), cumulus cells (CCs), and oocytes to evaluate whether autophagy is an important process of antral follicle atresia in sexually mature sows. Furthermore, apoptosis was analyzed using annexin V and TUNEL assays in the same cellular cohorts to evaluate the correlation between the two processes. Immunostaining results indicate autophagy was induced in the majority of GCs, CCs, and oocytes from early and advanced stage atretic follicles. The quantitative results of western blot analysis indicate there is a progressive increase (P < 0.05) in abundance of autophagy-related protein (LC3B-II) in these cells compared with cells in non-atretic follicles. Furthermore, there is confirmation that apoptosis occurs in the GCs of atretic follicles, thus indicating that in pigs apoptosis and autophagy are processes in GCs that regulate PCD and as a consequence antral follicle depletion. There was a greater abundance of LC3B-II in CCs and oocytes of atretic follicles, while apoptosis was not detected. It, therefore, is suggested that in these cells the two processes function independently, with autophagy having a cytoprotective rather than PCD mechanism of action.

Keywords: Atresia; Autophagy; Cumulus cells; Granulosa cells; LC3B; Pig.