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ブタ活性化卵子の初期発生能ならびに単為発生胚の初期胚特異タンパク質の発現性

活化猪卵母细胞的早期发育潜力和孤雌胚胎中早期胚胎特异性蛋白的表达

基本信息

批准号：
09460129
负责人：
MIYAKE Masashi
金额：
$ 3.65万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09460129/
关键词：
parthenogenesis in vitro early development postimplantation development gene expression ブタ

项目摘要

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 … More +/K+ ratio.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

本研究旨在建立猪早期胚胎的无血清培养体系，并对猪二倍体孤雌生殖的发育限制进行研究。体外成熟的猪卵母细胞经电刺激激活后，用细胞松弛素B处理。这些卵母细胞在各种简单培养基中培养168小时，这些培养基基于含有0.5mg/ml透明质酸（mWM）的改良Whitten'培养基。考察了培养液的渗透压、聚乙烯醇（PVA）替代牛血清白蛋白（BSA）的浓度以及氨基酸的影响。这些研究阐明了以下几个问题：1.二倍体在等渗mWM（309 mOsmol）中培养，由于致密化和囊胚腔形成的失败，导致发育到囊胚期的频率很低（几个%）。在低渗mWM（254 mOsmol）中培养的二倍体中有14 - 20%发育到囊胚期。渗透效应的原因只是介质的渗透压，而钠离子的渗透压是介质渗透压的主要原因。 ...更多信息 2.等渗条件（280 - 320 mOsmol）更有利于活化后48小时的二倍体培养，（220 ~ 270 mOsmol）对二倍体的发育有较好的促进作用。（3）0.5 mg/ml以上的PVA替代BSA对二倍体发育到囊胚期有较好的促进作用，从二次培养开始向PVA-mWM中添加混合必需氨基酸（EA）和非必需氨基酸（NEA）的氨基酸溶液（AA），引起二倍体的强烈4-细胞阻断。激活后0 ~ 48 h再加入EA，二倍体细胞的4-细胞阻滞程度较重。NEA的加入减弱了这种阻断作用，并且NEA可以像BSA一样支持二倍体细胞发育到囊胚期。6.激活后加入EA 48可以像BSA一样支持二倍体细胞发育到囊胚期。将激活后48 h的3 ~ 4细胞期卵母细胞移植给受体，观察孤雌二倍体的体内发育情况。在激活后18 ~ 30天处死动物，检查胎仔数和黄体的存在情况。1.在4个试验中，有1个试验获得了30天的胎仔（35%），而在另外3个试验中没有获得胎仔。在激活后28- 29天，在一只动物中观察到流产胎仔。这些结果表明，孤雌激活二倍体的发育极限在激活后30天左右; 2.在许多试验中获得了25天左右的孤雌激活胚胎，这一阶段的孤雌激活二倍体的发育能力相对较高（41%的转移卵子），然而，原始心脏和肝脏的囊性结构，在20- 30岁左右收集的一些胎儿中观察到了无头畸胎。核磁共振图像显示，一些外观正常的胎儿大脑中有异常的空腔形成。1.根据猪体内大部分印迹基因的序列，设计了猪基因探针，并对猪基因探针的结构进行了分析。用p57 KIP 2、PEG 1/MEST、SNRPN和H19.2探针从正常猪胎儿中扩增出印迹基因的PCR产物，并结合IGF 2、IGF 2 R和WT 1的PCR产物和亚定量PCR，采用Northern杂交法比较了印迹基因候选基因在正常和孤雌生殖胎儿中的表达。IGF 2在正常胚胎中的表达强于孤雌生殖胚胎，而p57 KIP 2在正常胚胎中的表达与孤雌生殖胚胎无明显差异。少