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

本研究旨在确定猪原始生殖细胞（PGC）特有的糖缀合物，并将其作为标记物，观察标记细胞在形态学和超微结构上是否具有典型的PGC特征。为了选择合适的标记物，建立一种有效的猪PGC收集方法，我们还对不同发育阶段的猪PGC细胞内糖缀合物的分布和定位进行了研究。将三种抗Lewis^x、抗Lewis^y和抗sialyl Lewis^x抗体与石蜡和树脂包埋切片（K4M）反应。Anti-Lewis^x与PGC阳性反应最强，后肠、间质、生殖脊等周围细胞阴性。用抗lewis ^x抗体对16 ~ 24胎石蜡包埋标本进行处理，发现16胎卵黄袋和后肠上皮区存在PGC， 19 ~ 22胎PGC主要通过卵黄囊的内切酶迁移到生殖脊。大多数PGC在分娩24天前在生殖器脊处沉淀。直到20dpc，抗lewis ^x阳性细胞数量增加，未见具有PGC形态特征的阴性细胞。22例dpc胎儿出现抗lewis ^x阴性PGC，抗lewis ^x阳性PGC数量减少。阴性细胞最多的是生殖器脊，提示Lewis^x可能在PGC的迁移中起作用。免疫金法显示，除细胞核外，Lewis^x在细胞表面呈规则分布，在细胞质中呈非均匀分布。电镜观察到大量球形线粒体和粗糙的内质网。迁移的PGC被小空间包围，不与体细胞接触，常观察到带假足的PGC。从这些结果来看，18 - 22胎分离的腹侧中肾包括生殖脊和背侧肠系膜是收集猪PGC的最佳选择。用该方法收集的胚胎源细胞经胰蛋白酶处理分离后，在小鼠典型ES培养基中培养。增殖细胞形成抗lewis ^x阳性菌落，但没有证据表明这些细胞来源于PGC。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