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KMID : 1023620050290020127
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Reproductive and Developmental Biology
2005 Volume.29 No. 2 p.127 ~ p.131
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Effect of Epidermal Growth Factor (EGF) on Meiotic Maturation and Pronuclear Formation of Porcine Oocytes Produced In Vitro
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Song S.H.
Kim J.G.
Song H.J.
Kumar B. Mohana
Cho S.R.
Choe C.Y.
Choi S.H.
Rho G.J.
Choe S.Y.
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Abstract
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The objective of this study was to examine the effect of EGF on meiotic maturation and pronuclear (PN) formation of porcine oocytes. Prepubertal gilt cumulus-oocyte-complexes (COCs) aspirated from follicles of abbatoir ovaries were matured in TCM199 containing 0.1mg/ml cysteine, FSH and LH, and EGF (0, 5, 10, 20, 40 ng/ml) for 22 hr at in a humidified atmosphere of in air. They were then cultured for an additional 22hr without hormones. In Experiment 1, to examine the nuclear maturation at 44hr of culture, the expanded cumulus cells were removed by vortexing for 1 min in 3 mg/ml hyaluronidase. The oocytes were fixed in acetic acid: methanol (1:3, v/v) at least for 48 hr and stained with orcein solution for 5 min. Nuclear status was classified as germinal vesicle (GV), germinal vesicle breakdown (GVBD), prophase-metaphase I (PI-MI), and PII-MII under microscope. In Experiment 2, to investigate PN formation, oocytes were fertilized with Percoll-treated freshly ejaculated sperm in mTBM with BSA and 2mM caffeine for 5hr, and cultured in NCSU-23 medium with BSA. At 6hr of culture, the embryos were fixed in formaldehyde for 48hr and stained with 10ug/ml propidium iodide for 30 min. PN status was classified as no or one PN (unfertilized), 2 PN (normal fertilized) and PN (polyspermy). Differences between groups were analyzed using one-way ANOVA after arc-sine transformation of the proportional data. The rate of oocytes that had reached to PII-MII were significantly (P<0.05) higher in all groups added EGF than that of non-treated group , but it did not differ among the all added groups , in 5, 10, 20 and 40 ng/ml EGF, respectively). No differences on the incidence of 2PN were observed in all treated groups , in 0, 5, 10, 20 and 40 ng/ml EGF, respectively), however, in non-treated group, polyspermy tended to be increased (, 0 vs. 5, 10, 20, 40 ng/ml EGF, respectively). These results suggest that EGF can be effectively used as an additive for enhancing oocyte maturation and reducing the incidence of polyspermy in pig.
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KEYWORD
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Pig oocytes, In vitro maturation, Pronuclear formation, EGF
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