1991 Fiscal Year Final Research Report Summary
Fundamental study on analysis of early embryonic differentiation and identical farm animal production by embryonic blstomere cloning
| Project/Area Number |
01480095
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
畜産学(含草地学)
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| Research Institution | Kyoto University |
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Principal Investigator |
IRITANI Akira Kyoto Univ., Col. of Agri. Professor, 農学部, 教授 (80026385)
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| Co-Investigator(Kenkyū-buntansha) |
HOSOI Yoshihiko Kyoto Univ., Col. of Agri. Res. Assist., 農学部, 助手 (70192739)
UTSUMI Kyozo Kyoto Univ., Col. of Agri. Assoc. Prof., 農学部, 助教授 (90033266)
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| Project Period (FY) |
1989 – 1991
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| Keywords | Embryonic stem cell / Bovine / Nuclear transpalantation / Electrofusion / Cloning / In vitro fertilized embryos / Oocytes maturation / Nuclear-cytoplasmic ratio |
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| Research Abstract |
1. Production of in vitro matured oocytes and in vitro fertilized (IVF) embryos aiming to utilized IVF embryos for cloning was tried. Development of embryos derived from oocytes with intact cumulus cell were not improved even when they were cultured with additional granulosa cells (13%), suggesting that an amount of factors (s) secreted from granulosa cells surrounding oocyte might be enough for oocytes maturation and subsequent embryonic development. Developmental ability of embryos to the blastocyst stage was improved from 13% to 30% by addition of 30% follicular fluid into maturation medium.
2. Developmental ability of reconstituted embryos having 5 sorts of different nuclear-cytoplasmic ratios derived from rat 2-cell eggs were compared. All of the eggs having 1 : 1 or 1 : 2 of the nuclear-cytoplasmic ratio developed into late morula stage, Developmental rate of eggs having 1 : 3 ratio was decreased and all of the eggs failed to develop furthermore and degenerated.
3. Embryonic stem cell expected to be used as donor cell for nuclear transplantation. Mouse blastocyst and bovine blastocyst were cultured on the proper feeder layer in order to establish the stem cell line. However, mouse inner-cell mass was cultured for 3 generation, but degenerated after that. No generation of the bovine cell line was gained.
4. The relationship between activation of oocytes by electric stimulation and developmental ability of reconstituted embryos were examined. Oocytes matured in vitro for 28 h were stimulated by direct current pulse under several conditions. High proportion of the reconstituted embryos were cleaved 48 to 50 h after electrofusion under the condition of either 10 V/eggs for 50 to 150 usec. Twenty tree of the 69 reconstituted embryos developed to more than 9-cell stage, and 4 embryos developed to morula to blastocyst stage. Then each of three blastocysts was nonsurgically transferred to the uterus of recipient cow, but no pregnancy was obtained.
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