Takashi Kuwana

Identification and characterization of stem cells in prepubertal spermatogenesis in mice

The stem cell properties of gonocytes and prospermatogonia at prepubertal stages are still largely unknown: it is not clear whether gonocytes and prospermatogonia are a special cell type or similar to adult undifferentiated spermatogonia. To characterize these cells, we have established transgenic mice carrying EGFP (enhanced green fluorescence protein) cDNA under control of an Oct4 18-kb genomic fragment containing the minimal promoter and proximal and distal enhancers; Oct4 is reported to be expressed in undifferentiated spermatogonia at prepubertal stages. Generation of transgenic mice enabled us to purify gonocytes and prospermatogonia from the somatic cells of the testis. Transplantation studies of testicular cells so far have been done with a mixture of germ cells and somatic cells. This is the first report that establishes how to purify germ cells from total testicular cells, enabling evaluation of cell-autonomous repopulating activity of a subpopulation of prospermatogonia. We show that prospermatogonia differ markedly from adult spermatogonia in both the size of the KIT-negative population and cell cycle characteristics. The GFP(+) KIT(-) fraction of prospermatogonia has much higher repopulating activity than does the GFP(+)KIT(+) population in the adult environment. Interestingly, the GFP(+)KIT(+) population still exhibits repopulating activity, unlike adult KIT-positive spermatogonia. We also show that ALCAM, activated leukocyte cell adhesion molecule, is expressed transiently in gonocytes. Sertoli cells and myoid cells also express ALCAM at the same stage, suggesting that ALCAM may contribute to gonocyte-Sertoli cell adhesion and migration of gonoyctes toward the basement membrane.

Migration of Avian Primordial Germ Cells toward the Gonadal Anlage

Primordial germ cells (PGCs), like neural crest cells, lymphoid stem cells and blood stem cells, originate far from their target organs in the early embryonic stage and then migrate to their respective organs. Avian PGCs are thought to originate from epiblasts as evidenced by experiments using chick-quail chimeras (70). These chimeric embryos were made by combination of epiblasts of one species and hypoblasts of another species at the blastoderm stage, and were cultured until the 3-6 somite stage. The PGCs in the chimeric embryos were identified histologically using a nuclear marker with Feulgen staining, and results showed that most of the PGCs derived from combined epiblasts of an avian germ line were of epiblastic origin. However, Smith et al. (52) pointed out that the presumptive PGCs could have entered the epiblast from the hypoblasts before the formation of chimeras at the blastoderm stage. The PGCs gradually move to the lower layer during the early stages of primitive streak formation (53) and become located in the hypoblast layer of the so-called “germinal crescent” region in the primitive streak stage (6). In birds, the germinal crescent region is a long way from the presumptive gonadal region, and so the manner of PGCmigration is different from that in mammals and some reptiles, in which PGCs start to migrate from the posterior region of the stalk of the yolk sac toward the gonadal anlage. Thus, avian PGCs show a unique circulating pathway in addition to the mammalian type patheay of migration. As avian PGCs circulate in the blood stream for a while in the early embryonic stage, their isolation and direct artificial manipulation may be easier than those of mammals and amphibians. In fact, in mammals and amphibians, PGCs cannot be isolated without enzymatic treatment or physical dispersion, better which could damage the PGCs membrane. Moreover, surgical techniques are available for avian embryos (22, 23, 51) than for mammalian embryos in utero (33, 34), there being many difficulties in inducing embryonic development in utero after surgical operation. Three main problems regarding the developmental biology of PGCs are their origin, migratory mechanisms, and differentiation into functional germ cells. The present review focuses attention on the migratory mechanisms of avian PGCs and related problems. In chicks, PGCs separate from hypoblasts at stage 4 (21) and become located in the lacuna between hypoblasts and hyperblasts (55, 18). At stage 10 or 11, PGCs become located inside the blood vessels forming in the germinal crescent region, and begin to circulate throughout the embryonic disk in the blood until stage 11 (initial phase of PGC-circulation) (Fig. 1). Mitosis of

ANALYSIS OF CELL PROLIFERATION DURING EARLY EMBRYOGENESIS

Cell proliferation was examined during early embryogenesis of the newt (Triturus pyrrhogaster) by various methods. After the two‐cell stage, at 23°C, the blastomere (cell) number per whole embryo increased logarithmically until the mid‐blastula stage (for about 19 hr) and the rate of increase slowed down in and after the late blastula stage. On the other hand, the synchronous cleavage of the blastomeres at the animal pole continued for 18 hr until the twelfth cleavage (mid‐blastula) and the transition from synchronous to asynchronous division occurred abruptly at and after the thirteenth cell division (late blastula). The study also showed that the presumptive neuro‐ectoderm consisted mainly of cells of the fifteenth generation (G‐15) at the onset of gastrulation (pigment stage).

Interspecific melanocyte chimaeras made by introducing rat cells into postimplantation mouse embryos in utero

Dissociated cells of whole midgestation rat embryos were injected into implanted albino mouse embryos on Day 8.5 of gestation in utero. This successfully produced viable interspecific chimaeras which were found to have pigmented hairs. Two of them had many pigmented hairs covering a large area of their bodies, including a forelimb and a hindlimb. The fact that some of the introduced rat cells differentiated into functional melanocytes suggests that embryonic cells of both species were able to interact with each other normally and that the foreign cells were kept from maternal immunological assault.

Changes in the interior surface of newly mesodermalized ectoderm and its contact activity with competent ectoderm in the newtCynops (Amphibia)

SummaryThe presumptive ectoderm (pE) ofCynops gastrulae was artificially mesodermalized by contact with teleost swimbladder. The newly mesodermalized ectoderm (mE) acquired the capacity for neural induction (Suzuki et al. 1986a). SEM observations revealed that the mE cells altered their cellular profiles immediately after mesodermalization. The characteristics of the cell surface and the cell architecture became similar to those of invaginated mesoderm cells. There were distinct differences in the cellular contact between mE—pE and pE—pE combinations. The mE-pE combinations kept close contact at their interior surfaces, while the pE—pE combinations did not keep contact. Both TEM and SEM observations also indicated that there were tight contacts between mE and pE cells. These findings suggest that neural-inducing activity of the newly mesodermalized ectoderm cells is coupled with acquisition of cellular affinity toward the interior surface of competent ectoderm cells, and probably requires close cell contacts.

Pigmentation Patterns of Mouse Chimaeras following Injection of Embryonic Cells into Postimplantation Embryos In Utero

Chimaeric mice were produced by introducing dissociated embryonic cells of C57BL/6N mice into the embryos of Jcl: ICR albino mice at mid‐gestation in utero. The patterns and the existence of pigmented areas were investigated over the long term. The pigmentation of the chimaeras was observed in several locational patterns; mainly in the head and the breast, rarely in the tail, the abdomen, the anterior and posterior trunk. During long‐term observation, the pigmentation became faint in 6 of 7 chimaeras and completely disappeared in 2 of 7 chimaeras 6 months after birth, as was true in our previous observation in rat/mouse chimaeras. The reason for this discoloration, however, is unknown at present; melanocytes derived from donor cells may have failed to function or have been eliminated. To examine the entry routes of injected cells into the embryos, pollen particles, similar to embronic cells in size, were injected as a donor material. The particles were localized mainly on the mid‐dorsal line in the head, and breast near fore‐limb buds 48 hr after injection. These patterns were similar to those of areas where the pigmentation were observed in the chimaeras. The results suggested that the cells were passively incorporated into embryos on the dorsal midline and the abdomen through the neural tube and somatopleure closure, respectively.