@misc{oai:ir.soken.ac.jp:00000939,
 author = {中馬, 新一郎 and チュウマ, シンイチロウ and CHUMA, Shinichiro},
 month = {2016-02-17},
 note = {Mechanisms of germ cell formation have been one of intriguing questions in biology. Segregation of the germ line responsible for continuity of the genome through generations from the somatic cells specialized for development of each individual occurs at early stages of development in many multicellular organisms. In mice, presumptive primordial germ cells originate in the proximal region of the pluripotent epiblast abutting on the extraembryonic ectoderm before and at around the onset of gastrulation. Germline specification in mice is thus considered to take place at relatively later stages of embryogenesis among the pluripotent stem cells under the influence from still unidentified environmental factors.<br />　　　　Primordial germ cells (PGCs), which are considered to be restricted to the germ line, are first recognized as about 50 cells showing strong alkaline phosphatase (ALPase) activity in the posterior extra-embryonic region of the primitive streak-stage embryos at around 7.25 days post coitum (dpc). They migrate to the gonad anlagen, genital ridge, and proliferate up to about 25000 cells by around 13.5dpc. These PGCs express several surface markers including ALPase and a transcription factor (Oct3/4) which are also expressed in earlier totipotent embryonic stem cells. But they are distinct from totipotent stem cells by their migratory behavior. PGCs are considered to be repressed from totipotency because of their inability to contribute to chimeras when transplanted into early embryos.<br />　　　　During investigations of growth factors effective to PGCs in vitro, colonies of cells resembling undiffrentiated pluripotent embryonic stem (ES) cells appeared in the presence of LIF (leukemia inhibitory factor) and bFGF (basic fibroblast growth factor). These cells, named as EG (embryonic germ) cells, showed similarity to ES cells in their morphology, surface markers, and totipotency demonstrated by formation of germline chimeras. Derivation of the totipotent cells from PGCs that are thought to be determined to the germ line, implied dedifferentiation of PGCs and recovery of totipotency. A recent study reported that other combinations of growth factors, LIF and RA (retinoic acid) or FK (forskolin) also supported long term proliferation of PGCs in vitro, but the differentiation potency of these cells was not examined. In this study, I established EG cell lines in the presence of LIF, FK and RA, and performed transplantation of these cells in reaggregates with fetal gonadal somatic cells into the adult mouse testis to test their potency of differentiation. Transplants gave rise to varieties of differentiated somatic tissues, indicating either dedifferentiation of PGCs to pluripotent stem cells, or proliferation of the remaining stem cells within PGCs occured in this combination of growth factors.<br />　　　　PGCs after arrival at genital ridges at around 10.5dpc continue to divide mitotically until about 13.5dpc. Then, they take different fates of development according to their surrounding gonadal sex. Germ cells in male gonads are considered to arrest at the G0 stage of cell cycle as prospermatogonia. In female, germ cells enter into meiosis and arrest at prophase of the first meiotic division. Surface markers of PGCs which are common to early totipotent stem cells start to diminish gradually after transition into mitotic arrest or meiosis. These fetal germ cells resume mitosis as spermatogenic stem cells or meiotic progression and oocyte maturation after birth. It has been suggested that sex-differentiation of fetal germ cells is determined by the surrounding gonadal somatic environments, rather than their own chromosomal constitution. However, studies on differentiation of PGCs have been limited to experiments using organ or reaggregate cultures. Thus, examinations of meiotic transition by PGCs and identification of its regulative factors have been impeded, due to the lack of dissociation culture system of PGCs in which meiotic transition could be detected and analyzed in detail.<br />　　　　I developed a dissociation culture system in which female PGCs entered into the prophase of first meiotic division. I demonstrated, using this system, that expression of meiotic protein by PGCs occured in the absence of embryonic gonadal somatic cells. This indicates that meiotic transition by PGCs could be determined independently on gonadal environment. I observed a tendency of synchrony of meiotic transition by PGCs in each cell colony formed in vitro. I also demonstrated that LIF-gp130 signal, previously known as an inhibitor of differentiation of ES cells and as a PGCs' survival or growth factor, repressed meiotic transition by PGCs and observed consistent difference of LIF expression between male and female embryonic gonads. In vitro system developed in this study shall provide a novel basis for analysis on mechanisms and factors involved in the development and differentiation of fetal germ cells in mammals., 総研大甲第404号},
 title = {マウス胎仔生殖細胞の培養系における増殖・分化に関する研究},
 year = {}
}