87 EFFECTS OF HUMAN RECOMBINATION GRANULOCYTE–COLONY STIMULATING FACTOR (hrG-CSF) ON IN VITRO CULTURE OF PORCINE CLONED EMBRYOS DERIVED FROM THIN CUMULUS CELL LAYER OF OOCYTES MATURED IN VITRO

Abstract

Although several cloned pigs have been successfully produced, the developmental competence of cloned embryos in vitro is still very low. Granulocyte colony-stimulating factor receptor (G-CSFR) was founded in the human trophoblastic cell line that is implicated in regulation and proliferation of trophoblast. In the present study, the somatic cell NT embryos derived from oocytes that have more than 3 cumulus cells layer were cultured and supplemented with various concentrations of hrG-CSF (0, 10, 50, and 100 ng mL⁻¹, respectively). Although there were no significant effects on the various concentration of hrG-CSF treatment groups compared with control, the somatic cell NT blastocysts formation tended to increase after 10 ng mL⁻¹ hrG-CSF treatment (24.19 ± 2.90%) compared with control (21.37 ± 2.98%). Moreover, we investigated the effects of 10 ng mL⁻¹ hrG-CSF on in vitro culture of porcine cloned embryos derived from oocytes that were categorized into grade A (cumulus cell layer >10), grade B (10 > cumulus cell layer ≥ 3), and grade C (cumulus cell layer <3). After supplementation of 10 ng mL⁻¹ hrG-CSF on in vitro-culture of different groups, the developmental competence, blastocyst quality, and gene transcript levels were observed. The results showed that 10 ng mL⁻¹ hrG-CSF has no beneficial effects on cloned embryos derived from grade A oocytes (10 ng mL⁻¹ hrG-CSF 25.35 ± 2.53% v. control 25.00 ± 2.66%), but it significantly increased blastocyst formation of embryos derived from grade B oocytes (22.09 ± 2.10%) compared with grade B control (12.09 ± 2.31%, P < 0.05). There were obvious increases in blastocyst formation derived from grade C oocytes after 10 ng mL⁻¹ hrG-SCF treatment (25.74 ± 1.65%) compared with grade C control (16.82 ± 2.30%, P < 0.05). However, there were no significantly differences in cleavage rate and total cell number of blastocysts among each group. Otherwise, the PCNA, POU5F1, Dnmt1, Bcl2, and Bax transcript levels were significantly increased in blastocysts that were derived from grade C oocytes after 10 ng mL⁻¹ hrG-SCF treatment compared with grade C control. In conclusion, supplementation of 10 ng mL⁻¹ hrG-CSF in in vitro-cultured porcine embryos increased blastocyst formation of embryos derived from thin cumulus layer of oocytes by reducing apoptosis while increasing cell proliferation and nuclear reprogramming. These results provide an experimental basis for the use of poor quality oocytes for agricultural production.

This work was supported by a grant from Research Program (No. 307–02) Gyeonggi-province project and the Next-Generation BioGreen21 Program [no. PJ01107702], Rural Developmental Administration (RDA), Republic of Korea.