紅林 賢臣 神戸大学, 自然科学研究科, 日本学術振興会特別研究員
HARAYAMA Hiroshi KOBE UNIVERSITY, GRADUATESCHOOL OF SCIENCE AND TECHNOLOGY, ASSOCIATE PROFESSOR, 大学院・自然科学研究科, 助教授 (30281140)
KUREBAYASHI Satoshi KOBE UNIVERSITY, GRADUATESCHOOL OF SCIENCE AND TECHNOLOGY
|Budget Amount *help
¥5,700,000 (Direct Cost : ¥5,700,000)
Fiscal Year 1999 : ¥1,800,000 (Direct Cost : ¥1,800,000)
Fiscal Year 1998 : ¥3,900,000 (Direct Cost : ¥3,900,000)
This study was designed to establish serum-free culture system for porcine early embryos and to examine the developmental limitation of parthenogenetic porcine diploids. In vitro matured porcine oocytes were electro-stimulated for activation and were treated with cytochalasin B. These oocytes were cultured for 168 hours in various simple media that were based on modified Whitten' medium with 0.5mg/ml hyaluronic acid (mWM). Osmolarity of media, concentration of polyvinyl alcohol (PVA) as substitutes for ovine serum albumin (BSA) and effects of amino acids were examined. These studies clarified following items ;
1.Diploids cultured in isotonic mWM (309mOsmol) throughout culture period resulted in very low frequency (a few %) of development to the blastocyst stage by the failure of compaction and formation of blastocoel. Fourteen to 20% of diploids cultured in hypotonic mWM (254 mOsmol) developed to the blastocyst stage. The cause of the osmotic effect is just osmolarity of a media, but Na
2.Isotonic condition (280 to 320 mOsmol) is more beneficial for the culture of diploids up to 48 hours after activation, and hypotonic condition (220 to 270 mOsmol) is better for the development of diploids from 72 hours after activation.
3.More than 0.5 mg/ml of PVA as substitutes for BSA supports well the development of diploids to the blastocyst stage, but their expansion.
The addition of amino acids solution (AA) mixed essential (EA) and nonessential amino acids (NEA) to PVA-mWM from the biginning of culture causes strong 4-cell blocking of diploids. The frequency to the blastocyst stage is significantly lower than AA-free condition.
5.The addition of EA for 0 to 48 hours after activation brings sever 4-cell blocking to the diploids. The blocking effect is weakened by the addition of NEA, and NEA can support the development of diploids to the blastocyst stage as like as BSA.
6.The addition of EA 48 after activation well supports their development to the blastocyst stage as like as BSA. Moreover, most of blastocysts cultured under the condition show the hatching process.
To determine the in vivo developmental of parthenogenetic diploids, activated oocytes at the 3- to 4-cell stage 48 hours after activation were transferred to recipients. The animals were sacrificed on 18 to 30 days after activation, and the number of fetuses and existence of corpus lutea was examined.
1.In one of four trails, 30-days fetuses were obtained (35%), but in the other 3 cases no fetuses was obtained. Aborted fetuses were observed on 28- to 29-day after activation in one animal. These results suggest that the developmental limitation of parthenogenetic diploids is around 30 day after activation.
2.Parthenogenetic fetuses on 25-day, at the early stage of post implantation, were obtained in many trials, indicating that the developmental ability of diploids to the stage was relatively high (41% of transferred eggs), however, cyst-like structure of the primitive heart and liver, and acephalus were observed in some of fetuses collected around 20-day.
3.NMR images shows that some of fetuses with normal appearance have abnormal cavity formation in the brain.
Expression of some of imprinted genes that have been clarified their imprinting in the mouse and/or human in parthenogenetic porcine diploids was also examined in the present study.
1.As the sequence of most of the imprinted genes in the pig, probes for porcine genes were designed from the sequence of mouse and human genes. PCR products were obtained from normal porcine fetuses using probes for p57KIP2, PEG1/MEST, SNRPN and H19.
2.Expression of candidates of imprinted genes were compared between normal and parthenogenetic fetuses by northern-hybridization using PCR products of IGF2, IGF2R and WT1 in addition to the above PCR products and sub quantitative PCR. The expression of IGF2 is stronger in normal fetuses than parthenogenotes, and that of p57KIP2 was almost same between them. Less