Toyoaki Fujimoto

Observations of the primordial germ cells in blood samples from the chick embryo

Abstract Primoridal germ cells (PGCs) from the circulating blood in 2-day chick embryos were observed by histochemical techniques in smear preparation and by phase contrast microscopy in fresh samples. In the blood smear, PGCs were readily distinguished from blood cells by their large size (15–20 μm in diameter) and by the round and large nuclei (8–10 μm in diameter) occupying eccentrically the greater parts of the cells. Histochemically, they were demonstrated to contain abundant glycogen, a lesser amount of yolk granules (by the PAS reaction), and a large amount of lipid droplets (by the Oil red O and Sudan black B stains) in their cytoplasm. Alkaline phosphatase activity was proved slightly in their cytoplasm by the simultaneous diazo coupling method. In phase contrast microscopy, living PGCs in the blood and from the primitive gonad of 3-day embryos in parts of the examinations were observed to change their shape in somewhat amoeboid fashion, suggesting their capability for locomotion as a possible mechanism of the migration of PGCs in vivo .

Ultrastructural Evidence that Chick Primordial Germ Cells Leave the Blood‐Vascular System Prior to Migrating to the Gonadal Anlagen

It is known that chick primordial germ cells (PGCs), after separation from the endoderm in early embryonic development, temporarily circulate via the blood‐vascular system and eventually migrate to the gonadal anlagen. However, direct evidence that circulating PGCs leave the blood vessels is lacking. The purpose of present study is to describe the ultrastructural features of PGCs as they emerge from the blood vessels. PGCs leaving the blood vessels were first examined with semi‐thin sections stained with toluidine blue. Then, some of the sections were re‐embedded in Epon 812, and sectioned for electron microscopy. PGCs were observed emerging from the capillaries in the region posterior to the omphalomesenteric arteries of the embryo, between the splanchnic mesoderm and open‐gut endoderm, at stages 15–18 (about 2.5 days of incubation). Ultrastructurally, PGCs exhibited the protruding, bulge‐like cytoplasmic processes through the endothelial gaps in the capillary walls. Prior to emerging, intravascular PGCs seemed to stick to the endothelium of the blood vessels. Thus, our results offer ultrastructural evidence that the circulating PGCs exit the blood vessels prior to migrating to the gonadal anlagen.

Teratogenic effects of a single oral administration of methylmercuric chloride in mice

The teratogenic effects of methylmercuric chloride (MMC) given orally as a single dose to pregnant ICR mice on day 10 of gestation were examined. The doses tested were 25, 20, 15 and 10 mg/kg. Controls received distilled water orally. Each group consisted of 20 females. Fetuses were taken on day 18 of gestation for teratological study. The number of resorbed or dead embryos was moderately increased in the 25 mg/kg group. Fetuses from dams given 25, 20 and 15 mg/kg MMC weighed significantly less than those in the control group. Many fetuses with malformations were observed in the treated groups; cleft palate occurred in 100, 58.6 and 28.0% of fetuses from dams given 25, 20 and 15 mg/kg MMC, respectively (statistically significant). Hydronephrosis appeared in 23.8 and 18.5% of fetuses from dams given 25 and 20 mg/kg MMC, respectively (statistically significant). Skeletal variations, incomplete ossification of sternebrae, for example, were also observed in the treated groups. These results indicate that MMC is teratogenic so far as cleft palate is concerned and embryotoxic in ICR mice.

Prealbumin gene expression during mouse development studied by in situ hybridization

Localization of prealbumin mRNA in tissues from mice at various stages of gestation was investigated using in situ hybridization procedures. Prealbumin mRNA was detected as early as the 10th day of gestation. It was specifically localized in endodermal cells of the visceral yolk sac, tela choroidea, and hepatocytes. In the adult mice, prealbumin mRNA was localized in the hepatocytes and choroid plexus epithelial cells. These observations indicate that synthesis of prealbumin mRNA is initiated in several different types of cells at early stages of fetal development.

OBSERVATIONS OF PRIMORDIAL GERM CELLS IN THE TURTLE EMBRYO (CARETTA CARETTA): LIGHT AND ELECTRON MICROSCOPIC STUDIES

Primordial germ cells (PGCs) in the turtle embryo (Caretta caretta) were observed with light and transmission electron microscopes. Identification of the PGCs for light microscopy was made by the periodic acid‐Schiff (PAS) technique. PGCs were first found in the yolk‐sac endoderm through the 5th to 6th day of development. PGCs freed from the endoderm then migrated to the root area of the dorsal mesentery and the coelomic angle between the 7th and the 11th day of development, and finally settled down in the gonadal anlage by the 14th day. Turtle PGCs were characterized by a large size (16 μm in diameter) and large nuclei with distinct nucleoli, and by the presence of large numbers of lipid droplets, yolk platelets and glycogen particles in the cytoplasm. Cell organelles were well‐developed in PGCs at later stages. Amoeboid features of the PGCs were observed in the mesenchyme, indicating active locomotion. PGCs were usually surrounded or encircled by neighboring somatic cells. No intravascular PGCs were detected at any stage of development examined.

Ultrastructure of primordial germ cells in the early chick embryo

Primordial germ cells (PGCs) of vertebrates appear in the extragonadal (in amniotes, extraembryonic) site at very early stages of development and migrate to the gonadal anlagen with the advance of embryonal development (e.g., 1–4). PGCs are identifiable by morphological or histochemical properties. These properties provide effective means by which their migratory pathway to the gonads may be traced. As to the morphological properties, PGCs are large in size and have a prominent nucleolus, and large amount of glycogen and lipid. Of the histochemical properties, periodic acid-Schiff (PAS) and alkaline phosphatase reactions have been utilized. For example, the PAS staining for glycogen has been effective for the identification of PGCs in chicks (5–11), turtles (4), lizards (12) and humans (13). Alkaline phosphatase activity has been used for identification of these cells in mice (14–18) and humans (19, 20)

The Role of Fibronectin in the Interstitial Migration of Primordial Germ Cells in Amniotes

Abstract Primordial germ cells (PGCs) in amniotes separate from the endoderm of the early embryo and finally migrate through the dorsal mesentery (DM), which is the last portion of the migratory pathway, into the gonadal anlage, while in birds and some reptiles, the cells temporarily circulate via the blood vascular system before undergoing this migration. To understand the role of extracellular matrix in the migration of PGCs, the distribution and localization of fibronectin (FN) were examined using direct immunoperoxidase staining in embryos of mice, chicks and man. In mouse embryos of 10.0 to 11.0 days of gestation, the DM was rich in FN, at which time the PGCs were at the peak of their migration. FN was localized at the sites of interaction between cytoplasmic processes of PGCs and the neighboring somatic cells. In man, a positive reaction to FN was found in the DM of 5‐week old embryos, in which most of PGCs were migrating. In chick embryos at 2.5 days of incubation, the splanchnic mesoderm (SPM), into which the PGCs were passing from the blood vessels, was rich in FN. In 3.0‐day‐embryos, the DM, which was derived from the SPM, was rich in FN. FN was localized at the sites of interaction between pseudopodia of PGCs and the adjacent somatic cells. After the completion of PGC migration into the gonad, FN reaction was weakened or disappeared in every species examined.

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.

The Anterior Half of Mouse Palatal Shelf Is Elevated by a Remodeling Movement

During the development of the mammalian secondary palate, the lateral palatine process (the palatal shelf) rises from the vertical plane beside the tongue to the horizontal plane above it. To determine the mode of elevation of the palatal shelf, we have cultured the whole ICR mouse fetus on day 14 (0-2 h) of gestation with a scratch as a marker at the distal edge of the anterior fourth of the vertical palatal shelf. After 6-18 h of culture, the survival ratio was 78.9%, and the palatal shelf rose horizontally above the tongue in 22.2% of the surviving fetuses. The scratch was found as a scar on the oral epithelium laterally to the medial edge of the elevated palatal shelf. These results indicate that the medial edge of the horizontal shelf was newly formed from the medial wall of the preceding vertical-stage shelf during elevation, and that the palatal shelf was elevated by a remodeling process in the anterior half of the shelf.

Localization of Prealbumin mRNA in Fetal and Adult Mice

We searched for the cellular sites of prealbumin mRNA synthesis in fetal and adult mice by in situ hybridization technique. As early as at the 10th day of gestation (the earliest time point examined), prealbumin mRNA was detected in several different types of fetal cells, such as endo-dermal cells of the visceral yolk sac, tela choroidea, the predecessor of the choroid plexus, and hepatocytes. In the adult mice, prealbumin mRNA was localized in hepatocytes, choroid plexus epithelial cells, and inner and outer nuclear layers and ganglion cell layer of the retina. These observations suggest the possibility that prealbumin may have a function in neuronal systems different from its known functions concerning the plasma transport of both retinol and thyroxine.