Morphological Changes of Porcine Granulosa Cells during in vitro Expansion

This study aimed to assess morphological changes of porcine granulosa cells (pGCs) following in vitro long-term culture. The pGCs from passage 5 showed a dramatic enlargement of cytoplasm (14422.600 +/- 1300.704 mu m(2)) which was 8.23-fold higher than pGCs from primary culture (1752.100 +/- 102.244 mu m(2)). Nuclear areas were increased from passage 0 (164.990 +/- 3.461 mu m(2)) to passage 4 (399.514 +/- 15.110 mu m(2)) and passage 5 (416.326 +/- 32.683 mu m(2)), respectively. The nucleocytoplasmic ratio of pGCs from passage 0 was 0.089 +/- 0.002 which was 2-fold higher than passage 4 (0.045 +/- 0.002) and 3-fold higher than passage 5 (0.029 +/- 0.002). The diameter of microfilament bundles of pGCs was increased from passage 0 (1.171 +/- 0.031 mu m) to passage 4 (1.550 +/- 0.056 mu m) and passage 5 (1.579 +/- 0.053 mu m), respectively. The weak expression of beta-galactosidase was observed in the several pGCs from passage 2, while pGCs from passage 4 showed the strong activity of beta-galactosidase. The flow cytometly analysis demonstrated that the ratio of apoptotic pGCs was increased through in vitro expansion. These results revealed that pGCs exposed replicative senescent characteristics including the increase of nuclear and cell size, as well as the increase of diameter of actin filament bundles in cytoplasm, and the reduction of nucleocytoplasmic ratio. (C) 2019 PVJ. All rights reserved