Effect of conditioned medium of mouse embryonic fibroblasts produced from EC-SOD transgenic mice in nuclear maturation of canine oocytes in vitro.
The rate of in vitro maturation (IVM) of canine oocytes has not improved in comparison to that of other mammalian species. This study aims to improve the efficiency of canine oocytes IVM using the antioxidant, extracellular superoxide dismutase (EC-SOD). Thus, the effect of conditioned medium of EC-SOD transgenic mouse embryonic fibroblasts cultured with MEF culture medium (DMEM + 5% FBS) for in vitro nuclear maturation in canine oocytes was investigated. In experiment I, oocytes were collected from the ovaries of domestic bitches, which were allotted to one of two groups: (1) TCM199 + 1% FBS (n = 108) or (2) DMEM + 5% FBS (n = 112), cultured for 48 h and investigated for in vitro nuclear maturation of canine oocytes using Hoechst staining. Meiotic progression to metaphase II in group 1 was 1.8% compared to 1.8% in group 2. In experiment II, EC-SOD levels were examined in NTg-CMEF and Tg-CMEF at 0, 2 and 4 days obtained from EC-SOD transgenic mice generated in our laboratory. The concentration of EC-SOD in Tg-CMEF at day 2 (371.7 +/- 3.1 ng/ml) was the highest for all groups (P < 0.05). EC-SOD levels in Tg-CMEF were higher than in NTg-CMEF; therefore, the efficiency of Tg-CMEF for IVM was investigated. In experiment III, oocytes were allotted to one of three groups: (1) Tg-CMEF at day 0 (n = 84), (2) Tg-CMEF at day 2 (n = 92) or (3) Tg-CMEF at day 4 (n = 98), cultured for 48 h and the IVM of canine oocytes investigated. The mean percentage of MII oocytes in IVM was 2.4, 4.4 and 2.0% for groups 1, 2 and 3, respectively. In experiment IV, the effects of conditioned medium of EC-SOD transgenic mouse embryonic fibroblasts (Tg-CMEF) cultured in MEF culture medium were compared with conditioned medium acquired from non-transgenic mouse embryonic fibroblasts (NTg-CMEF) on IVM of canine oocytes. In this experiment, meiotic progression to metaphase II was 7.1% in Tg-CMEF versus 0% in NTg-CMEF (P < 0.05). Tg-CMEF was more effective than NTg-CMEF. In conclusion, it was verified that canine oocytes were able to effectively progress to metaphase II in IVM when cultured in Tg-CMEF.
Lee
Sang Ryeul
SR
School of Life Science and Biotechnology, Kyungpook National University, 1370 Sankyuk-dong, Buk-ku, Daegu 702-701, South Korea.
Kim
Myoung Ok
MO
Kim
Sung Hyun
SH
Kim
Bong Soo
BS
Yoo
Dong Hun
DH
Park
Young Sik
YS
Park
Yong Bok
YB
Ha
Ji Hong
JH
Ryoo
Zae Young
ZY
D000818:Animals; D002467:Cell Nucleus / Q000187:drug effects*; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004285:Dogs / Q000502:physiology*; D005260:Female; D005347:Fibroblasts / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic / Q000378:metabolism; D009865:Oocytes / Q000187:drug effects* / Q000254:growth & development; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*
D017077:Culture Media, Conditioned; C499090:Sod3 protein, mouse; D013482:Superoxide Dismutase