Localization Of Murine Primordial Germ Cells In Genital Ridge And The Isolation And Culture Of Primordial Germ Cells In Mouse And Pig

Development and migration of primordial germ cells (PGCs) is one of basic issues in mammalian gametogenesis and developmental biology. The isolation and culture of PGCs is so important for the establishment of embryonic germ (EG) cell lines. EG cells are one type of pluripotent stem cells that has morphological, biochemical, immunological and developmental properties in common with ES cells, including pluripotency, molecular markers and the capacity to contribute to the germ line of chimeras. Therefore, isolation and culture of PGCs become an important approach to obtain pluripotent stem cells. In the present study, mouse and porcine embryos were adopted to investigate the morphological development of murine genital ridges on different days post-coitum (dpc) and isolation of PGCs in both species. The proliferation regularity of murine PGCs was assessed via observation of their number located in the genital ridge after AP staining. Isolation and culture procedures were examined and optimized via using murine embryonic PGCs on different dpc. Porcine PGCs were isolated as well and used for the primary culture and subculture in order to investigate factors that influenced the isolation and culture of PGCs. The following experiments were included in this study:Experiment 1. Morphological investigation of embryonic genital ridges at different developmental stage in mouseThe HE stained paraffin sections and AP stained cryostat sections of mouse embryonic genital ridges at different developmental stages were observed to conduct morphological study. The morphological changes of embryonic genital ridges and localization of PGCs in them were compared to find the proliferation regularity of PGCs and furnish a reference for the isolation and culture of PGCs. The results showed that the morphology of genital ridge derived from 11.5-13.5 dpc embryos changed with embryonic age: changed into oval materiality organ, the gonad, from the primordial gonad. The AP staining showed that during the process, numbers of murine PGCs located in the genital ridge varied greatly with embryonic age and were increased quantitatively with the increase of age. Mouse embryos...