EXPRESSION OF A UNIQUE GLYCOCONJUGATE IN PRIMORDIAL GERM CELLS AND CULTURE OF EMBRYO DERIVED CELLS COLLECTED FROM THE GENITAL RIDGE AND ITS PERIPHERAL ORGANS

本研究旨在确定一种猪原始生殖细胞(PGC)特有的糖偶联物作为其标志物，观察标记细胞在形态和超微结构上是否具有典型的PGC特征，并观察其在不同发育阶段细胞内的分布和PGC在胚胎中的定位，以选择合适的标志物，建立一种有效的收集猪原始生殖细胞的方法。抗Lewis^x与PGC呈强阳性反应，周围细胞，即后肠、间充质和生殖器脊呈阴性反应。用抗lewis^x抗体处理16~24dpc的胎儿石蜡包埋标本，发现16dpc的卵黄囊和后肠上皮区存在pGC，并通过MES…迁移。更迷人的是从19到22DPC到生殖器脊。大多数生殖器癌在24天前定居在生殖器沟内。至20DPC，抗Lewis^x阳性细胞增多，未见具有PGC形态特征的阴性细胞。22例DPC胎儿PGC呈抗Lewis^x阴性，抗Lewis^x阳性的PGC数量减少，最阴性的细胞位于生殖器脊，提示Lewis^x可能参与了PGC的迁移。免疫金标法显示Lewis^x呈规律性分布于细胞表面，除胞核外，呈异质性分布于胞浆。电子显微镜下可见大量球形线粒体和粗面内质网。移行中肾细胞被小间隙包围(S)，而不是与体细胞接触，经常观察到伪足的原生殖细胞。从这些结果来看，腹侧中肾包括带有背侧肠系膜的生殖器脊是收集猪PGC的最佳方法。用这种方法收集并经胰酶处理分离的胚胎来源细胞在典型的ES培养基中培养。增殖细胞形成抗Lewis^x阳性克隆，但没有证据表明这些细胞来自PGC。较少

This study was designed to determine a glycoconjugate which was unique to porcine primordial germ cells (PGC) and was employed as their marker and to examine whether marked cells morphologically and ultrastructurally showed typical features of PGC.Intracellular distribution of the glycoconjugates and localization of PGC in fetuses at various developmental stages were also examined in order to select a suitable marker and to establish an effective method for collection of porcine PGC.Three kinds of anti-Lewis^x, anti-Lewis^y and anti-sialyl Lewis^x antibodies were reacted with sections embedded in paraffin and resin (K4M). Anti-Lewis^x showed the strongest positive reaction with PGC,and surrounding cells, i.e.the hindgut, mesenchyme and genital ridge, were negative. Treatments of Paraffin embedded specimens from fetuses at 16 to 24 dpc with anti-Lewis^x antibody revealed that existence of PGC in yolk sack and epithelial regions of the hind gut at 16 dpc, and PGC migrated through the mes … More enchyme from 19 to 22 dpc to the genital ridge. Majority of PGC settled in the genital ridge until 24 dpc. Until 20 dpc, number of anti-Lewis^x positive cells increased and no negative cell with the morphological features of PGC was observed. Anti-Lewis^x negative PGC,however, were observed on 22 dpc fetuses with decrease of number of anti-Lewis^x positive PGC.The most negative cells were in the genital ridge, suggesting that Lewis^x may have a role in migration of PGC.Immunogold method showed that Lewis^x were regularly distributed on the cell surface and heterogeneously distributed in cytoplasm except for nucleus. A lot of spherical mitochondria and rough endoplasmic reticulum were also observed by electronmicrography. Migrating PGC were surrounded by small space (s) rather than contacted with somatic cells, PGC with pseudopod were observed often.From these results, the ventral mesonephros including genital ridge with the dorsal mesentery dissected from 18 to 22 dpc fetuses are optimum for collection of porcine PGC.Embryo derived cells collected by this method and isolated by trypsin treatment were cultured in typical ES medium for the mouse. Proliferating cells formed anti-Lewis^x positive colonies, but there is no evidence that these cells are derived from PGC. Less