|Budget Amount *help
¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 1996: ¥4,100,000 (Direct Cost: ¥4,100,000)
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
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