Cytoskeleton proteins F-actin and tubulin distribution and interaction with mitochondria in the granulosa cells surrounding stage III zebrafish (Danio rerio) oocytes

Abstract

The distributional arrangement of mitochondria in the granulosa cells surrounding stage III zebrafish oocyte has been reported as a contiguous aggregation of mitochondria at the margin of the each granulosa cell. The aim of the present study was to further investigate the mitochondrial distribution in the granulosa cell layer in stage III ovarian follicles and the interaction between mitochondria and cytoskeleton elements actin and tubulin. To determine mitochondrial distribution/transport, immunocytochemistry analysis of tubulin and mitochondrial COX-I was carried out along with phalloidin staining of polymerised F-actin. The follicles were also exposed to a range of conditions that are known to affect mitochondria and the cytoskeleton proteins actin and tubulin. The mitochondrial inhibitor FCCP, the anti-mitotic drug nocodazole, and actin polymerisation inhibitor cytochalasin B were used. Levels of ATP, mtDNA copy number, and viability assessed by Trypan blue were also studied after exposure to inhibitors in order to determine the relationship between mitochondrial distribution/activity and ATP production. F-actin showed a hexagonal-polygonal distribution surrounding the mitochondria in granulosa cells, with the F-actin network adjacent to the plasma membrane of each granulosa cell. Tubulin structure presented a less organised distribution than F-actin, it was sparse in the cytosol. Interaction between mitochondria and tubulin was found indicating that mitochondria and tubulin are colocalised in zebrafish ovarian follicles. The exposure of ovarian follicles to inhibitors induced the loss of mitochondrial structural integrity showing that mitochondria distribution in granulosa cells of stage III zebrafish ovarian follicles is determined by the microtubules network.