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

本研究旨在建立猪早期胚胎无血清培养体系，探讨孤雌繁殖猪二倍体的发育限制。体外成熟的猪卵母细胞经电刺激活化，并经细胞松弛素b处理，卵母细胞在含0.5mg/ml透明质酸（mWM）的改良Whitten培养基中培养168小时。考察了培养基的渗透压、聚乙烯醇（PVA）替代羊血清白蛋白（BSA）的浓度及氨基酸的影响。这些研究澄清了以下事项；在等渗mWM （309mOsmol）中培养的二倍体在整个培养过程中由于压实和囊胚腔的形成失败而发育到囊胚期的频率很低（只有几%）。在低渗mWM （254 mOsmol）中培养的二倍体有14% ~ 20%发育到囊胚期。渗透效应的原因仅仅是介质的渗透压，而不是Na…More +/K+比。等渗条件（280 ~ 320 mOsmol）在激活后48小时内有利于二倍体的培养，低渗条件（220 ~ 270 mOsmol）在激活后72小时内有利于二倍体的发育。0.5 mg/ml以上的PVA替代BSA对二倍体发育至囊胚期有良好的支持作用，但对其增殖有不利影响。从培养开始，在PVA-mWM中加入必需氨基酸（EA）和非必需氨基酸（NEA）混合的氨基酸溶液（AA），对二倍体有较强的4细胞阻滞作用。进入囊胚期的频率明显低于无aa条件。激活后添加EA 0 ~ 48小时，对二倍体产生严重的4细胞阻断。加入NEA后，阻断作用减弱，NEA能像bsa一样支持二倍体发育至囊胚期。激活后添加EA 48能很好地支持它们像BSA一样发育到囊胚阶段。而且，在此条件下培养的胚泡大部分呈现出孵化过程。为了确定孤雌生殖二倍体的体内发育情况，在激活后48小时将激活的3- 4细胞期的卵母细胞转移给受体。激活后18 ~ 30 d处死，观察胎数和黄体是否存在。其中1例获得30天胎儿（35%），其余3例未获得胎儿。其中一只动物在激活后28至29天观察到流产胎儿。这些结果表明，孤雌生殖二倍体的发育限制在激活后30天左右。在植入后的早期阶段，多次试验获得了25天的孤雌胚胎，表明二倍体发育到该阶段的能力相对较高（移植卵子的41%），但在20天左右收集的一些胎儿中，观察到原始心脏和肝脏的囊肿样结构，以及头部3。核磁共振图像显示，一些外观正常的胎儿在大脑中有异常的空洞形成。本研究还检测了一些在小鼠和/或人身上已经明确的印迹基因在孤雌生殖猪二倍体中的表达。由于猪的大部分印迹基因序列都是由小鼠和人类的基因序列设计而成的，因此猪基因探针的设计主要基于小鼠和人类的基因序列。用p57KIP2、PEG1/MEST、SNRPN和H19.2探针从正常猪胎儿中获得PCR产物。利用IGF2、IGF2R和WT1的PCR产物及亚定量PCR进行北杂交，比较正常和孤雌胎中候选印迹基因的表达情况。IGF2在正常胎中的表达强于孤雌胎，而p57KIP2在正常胎中的表达几乎相同。少

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