| Project/Area Number |
07456123
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied animal science
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| Research Institution | Okayama University |
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Principal Investigator |
NIWA Koji Okayama University Faculty of Agriculture, Professor, 農学部, 教授 (40089115)
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| Project Period (FY) |
1995 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥6,800,000 (Direct Cost: ¥6,800,000)
Fiscal Year 1997: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1996: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1995: ¥3,100,000 (Direct Cost: ¥3,100,000)
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| Keywords | Pig / Oocyte maturation / In-vitro fertilization / Pronucleus / Follicular cells / Maturation medium / Epidermal growth factor / Cysteine / グルタチオン / シスチン |
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| Research Abstract |
The present study was conducted to develop an effective system for in vitro fertilization of pig oocytes. A series of experiments had been done to examine the effects of various factors during culture of oocytes on normal cytoplasmic maturation that leads normal male pronuclear formation or monospermy after fertilization in vitro. It was found that epidermal growth factor and/or gonadotropins (eCG+hCG) can stimulate oocyte maturation in serum-free medium and this stimulative effect is mediated by the presence of cumulus cells. When cumulus-enclosed oocytes were cultured in BSA-free Whitten's medium (mWM) and modified TCM-199 (TCM-199B), cytoplasmic maturation of oocytes was affected by gonadotropins and/or fetal calf serum and/or pig follicular fluid (PFF) in different ways in different media. Further experiments were conducted to examine the ability of oocytes to achieve male pronuclear formation when they are matured and penetrated in vitro under various culture conditions. The resul
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ts indicated that male pronuclear formation in oocytes is promoted by the addition of 20 amino acids and/or cysteine in simple maturation medium (mWM supplemented with gonadotropins and PFF) and by the presence of cumulus cells at fertilization in vitro. It was also clarified that the presence of cysteine in serum-free maturation medium is required only between 42 and 48 h after initiation of maturation for promotion of male pronuclear formation after sperm penetration in vitro. Male pronuclei can be formed in cumulus-free oocytes matured in the presence of cysteine, but the presence of cumulus cells is essential for maintainingmale pronuclear formation in oocytes matured in the presence of cystine. In the study examined penetration of immature oocytes, it was demonstrated that the cytoplasm of maturing oocytes possesses an activity for transforming sperm nuclei into metaphase chromosomes. Although further improvements in the conditions used for in vitro maturation and fertilization are needed, the results obtained in the present study would contribute to ensure the production of larger numbers of normal pig embryos. Less
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