• Clinical and Experimental Reproductive Medicine
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• v.34 no.2
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• pp.95-106
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• 2007

Objective: We previously identified differentially expressed genes (DEGs) between germinal vesicle (GV) and metaphase II (MII) mouse oocyte. The present study was accomplished as a preliminary study to elucidate the role of ribose 5-phosphate isomerase A (Rpia), the essential enzyme of the pentose phosphate pathway (PPP), in oocyte maturation. We observed expression of Rpia in the mouse and porcine oocytes. Methods: Expression pattern of the 11 MII-selective DEGs in various tissues was evaluated using RT-PCR and selected 4 genes highly expressed in the ovary. According to the oocyte-selective expression profile, we selected Rpia as a target for this study. We identified the porcine Rpia sequence using EST clustering technique, since it is not yet registered in public databases. Results: The extended porcine Rpia nucleotide sequence was submitted and registered to GenBank (accession number EF213106). We prepared primers for porcine Rpia according to this sequence. In contrast to the oocyte-specific expression in the mouse, Rpia was expressed in porcine cumulus and granulosa cells as well as in oocytes. Conclusion: This is the first report on the characterization of the Rpia gene in the mouse and porcine ovarian cells. Results of the present study suggest that the mouse and porcine COCs employ different mechanism of glucose metabolism. Therefore, the different metabolic pathways during in vitro oocyte maturation (IVM) in different species may lead different maturation rates. It is required to study further regarding the role of Rpia in glucose metabolism of oocytes and follicular cell fore exploring the regulatory mechanism of oocyte maturation as well as for finding the finest culture conditions for in vitro maturation.

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.17-23
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• 2002

The present study was carried out to investigate the effects of the maturation media such as a modified TCM-199 (mTCM-199) medium, modified Waymouth MB 752/1 (mWaymouth MB 752/1) medium or NCSU-23 medium on penetrability of pig oocytes by liquid boar sperm. Oocytes (30~40) were transferred into each well of a Nunc 4-well multidish containing 0.5 $m\ell$ maturation medium. When immature pig oocytes were cultured in mTCM-199, mWaymouth MB 752/1 and NCSU-23 maturation media for 44 h in 5% $CO_2$, in air at 38.5$^{\circ}C$, the germinal vesicle breakdown (CVBD) rates of the oocytes were 95.6, 94.1 and 94.9%, respectively, and the maturation rates (metaphase II) of oocytes were 92.5, 90.1 and 91.1%, respectively. No differences were observed among the maturation media. The spermrich portion of ejaculates with greater than 90% motile sperm were used in the experiment. The semen was cooled 22 to 24$^{\circ}C$ over 2 h period. The semen was diluted with Beltsville Thawing Solution (BTS) extender at room temperature to give 2$\times$10$^{8}$ sperm/$m\ell$ in 100 $m\ell$ plastic bottle. Liquid boar semen of 30 $m\ell$ in 100 $m\ell$ plastic bottle was kept at 17$^{\circ}C$ for 5 days. The sperm with greater than 70% motility after day 5 of storage were used for in-vitro fertilization (IVF). After 44 h maturation of immature oocytes, cumulus cells were removed and oocytes (30~40) coincubated far 6 h in 0.5 $m\ell$ mTCM-199 and mTBM fertilization media with 2$\times$1061$m\ell$ sperm concentration. At 6 h after IVF, oocytes were transferred into 0.5 $m\ell$ mTCM-199 and NCSU-23 culture media for further culture 6 or 42 h. Sperm penetration, polyspermy and male pronuclear formation of oocytes at 12 h after IVF, and developmental ability of oocytes at 48 h after IVF were evaluated. The oocytes in combination with NCSU-23 medium for maturation and mTBM medium for IVF increased male pronuclear formation (48.0%) compared to those in combination with mTCM-199 media for maturation and IVF, and mWaymouth MB 752il medium for maturation and mTCM-199 medium far IVF. The rates of cleaved embryos (2~4 cell stage) at 48 h after IVF were 24.1% in combination with mTCM-199 media for maturation, IVF and culture, 43.6% in combination with mWaymouth MB 75211 medium fur maturation and mTCM-199 media for IVF and culture, and 71.2% in combination with NCSU-23 medium for maturation, mTBM medium for IVF and NCSU-23 medium for culture. In conclusion, we found out the oocytes matured in vitro were fertilized by liquid boar sperm stored in BTS extender at 17$^{\circ}C$ for 5 days. We recommend the simple defined NCSU-23 medium for nuclear maturation, mTBM medium and liquid boar sperm for IVF, and NCSU-23 medium for embryo culture.