303 HUMAN RECOMBINATION GRANULOCYTE-COLONY STIMULATING FACTOR (HRG-CSF) HAVE BENEFICIAL EFFECTS ON PORCINE OOCYTES QUALITY DURING IN VITRO MATURATION AND SUBSEQUENT VIABILITY OF EMBRYONIC DEVELOPMENT

Abstract

Granulocyte colony-stimulating factor (G-CSF), also known as colony-stimulating factor 3 (CSF3), is required for the proliferation, differentiation, and survival of cells. In humans, G-CSF is a biomarker of human oocyte developmental competence for embryo implantation. Furthermore, G-CSF concentration increases during the menstrual cycle and levels were significantly higher during ovulatory phase than the other phases. In this study, we examined G-CSF and its receptor gene expression in the porcine granulosa cells, corpus luteum, cumulus cells, and oocytes. The cumulus-oocyte complexes (COC) were aspirated from antral follicles 1 to 3 mm (small follicle) and 3 to 6 mm (medium follicle). The COC from 2 kinds of follicles were matured in protein-free maturation medium supplemented with various concentrations of hrG-CSF (0, 10, and 100 ng mL^–1, respectively). Statistical analyses were done by one-way analysis of variance (ANOVA) followed by Duncan's multiple range tests. After real time-PCR was performed, the CSF3 and its receptor (CSF3R) were observed all of granulosa cells, corpus luteum, cumulus cells, and oocytes. Interestingly, the CSF3 transcript levels were significantly lower in oocytes compared with other cell types, but the CSF3R transcript levels in oocytes were almost similar with granulosa cells. After 44 h of IVM, the rates of nuclear maturation had no difference, and the intracellular ROS levels of oocytes from both kind of follicle groups matured with 10 ng mL^–1 were significantly decreased compared to other groups (P < 0.05). After PA, the cleavage and blastocyst formation rates were significantly (P < 0.05) increased for the 100 ng mL^–1 of small follicle (SF; 63.29 and 31.18%) group compared to control and 10 ng mL^–1 of SF (38.64, 10.4, and 49.0, 15.6%, respectively) group, and significantly (P < 0.05) increased in the 10 ng mL^–1 of medium follicle (MF; 76.32 and 45.61%) group compared with control and 100 ng mL^–1 of MF (52.1, 32.8 and 61.3, 33.9%, respectively). The total cell numbers of blastocyst from SF and MF groups were significantly increased in the 10 ng mL^–1 (73.67 and 106.52) groups. At IVF, the blastocysts formation rates were significantly increased in the 10 ng mL^–1 of MF group compared to control, 100 ng mL^–1 of SF, and control of MF (21.1, 22.8, and 27.8%, respectively). We also examined the Bcl2 and ERK2 transcript levels, which were significantly increased at 100 ng mL^–1 of SF and 10 ng mL^–1 of MF. These results suggest that hrG-CSF improved the quality of porcine oocyte and embryonic viability.

This was supported, in part, by a grant from the National Research Foundation of Korea Grant Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.