Michiharu Sakurai

The developmental origin of primordial germ cells and the transmission of the donor-derived gametes in mixed-sex germline chimeras to the offspring in the chicken

A novel system has been developed to determine the origin and development of primordial germ cells (PGCs) in avian embryos directly. Approximately 700 cells were removed from the center of the area pellucida, the outer of the area pellucida, and the area opaca of the stage X blastoderm (Eyal‐Giladi and Kochav, 1976; Dev Biol 49:321–337). When the cells were removed from the center of the area pellucida, the mean number of circulating PGCs per 1 μl of blood was significantly decreased to 13 (P < 0.05) in the embryo at stage 15 (Hamburger and Hamilton, 1951: J Morphol 88:49–92) as compared to intact embryos of 51. When the removed recipient cells from the center of the area pellucida were replenished with 500 donor cells, no reduction in the PGC number was observed. The removal of cells from the outer of area pellucida or from the area opaca had no effect on the number of PGCs. When another set of the manipulated embryos were cultured ex vivo to hatching and reared to sexual maturity, the absence of germ cells and the degeneration of seminiferous tubules were observed in resulting chickens derived from the blastoderm from which the cells were removed from the center of the area pellucida.

Production and characterization of monoclonal antibodies that recognize bovine Kit receptor

Kit receptor is a transmembrane tyrosine kinase that is the receptor for stem cell factor (SCF). The extracellular domain of bovine Kit receptor (boKit) was produced by a baculovirus expression system. Six monoclonal antibody (MAb) clones designated as bK-1 to bK-6 were obtained upon immunization of mice with the recombinant protein. Immunoprecipitation and flow cytometric analysis indicated that all of the MAbs specifically bound to boKit expressed in COS-7 cells transfected with boKit cDNA. Four of the six MAbs neutralized the biological activity of recombinant bovine SCF, whereas the other two did not. The boKit-positive and boKit-negative cell fractions were sorted from cryopreserved bovine bone marrow cells by the use of MAb bK-1. Colony formation assays indicated that the cells which were able to grow in response to bovine SCF were enriched in the boKit-positive fraction. These MAbs would be valuable in studying possible boKit-positive cell species such as bovine hematopoietic cells, and in defining the biological role of Kit receptor in cattle.

ephA9, a novel avian receptor tyrosine kinase gene.

Protein tyrosine kinase (TK) receptors are known to play crucial roles in various aspects of normal development and in tumorigenesis. Germ cells before their colonizing to the gonads during embryogenesis are called primordial germ cells (PGCs). To identify TK genes expressed in chicken PGCs, we purified these cells from the blood of 2.5-day-old embryos, extracted the polyA(+) RNA, and subjected it to reverse transcription-polymerase chain reaction (RT-PCR) amplification with TK gene-specific primers. As a result, we identified 13 receptor TK genes and 6 non-receptor TK genes. One of the receptor TK genes appeared to be novel, and thus the full-length DNA complementary to RNA (cDNA) sequence was retrieved by the rapid cloning of cDNA ends method. Sequence analysis of this cDNA indicated that it encoded a novel TK receptor of the Eph family. The putative amino-acid sequence of this TK was 63.0% identical to that of human EphA1; therefore, we designated our novel TK as EphA9. Northern blot hybridizations indicated that ephA9 RNA transcripts were present in the kidney, lung, testis, and thymus but not in the spleen, brain, or liver. Expression of a fusion protein containing the intracellular domain of EphA9 in bacterial cells showed that this domain had TK enzymatic activity. The EphA9 species produced in Cos-1 cells transfected with an expression vector were tyrosine-phosphorylated. These data suggest that EphA9 plays its biological role(s) in various organs of chickens as an active TK.

Production of a monoclonal antibody that recognizes bovine stem cell factor (SCF) and its use in the detection and quantitation of native soluble bovine SCF in fetal bovine serum

Stem cell factor (SCF) is a pluritropic hematopoietic cytokine that acts predominantly on the proliferation and differentiation of hematopoietic progenitor cells. SCF has long been thought to be present in fetal bovine serum (FBS) as an endogenous factor that stimulates the growth of hematopoietic progenitor cells in FBS-supplemented cultures. To detect and quantitate bovine SCF in FBS, we produced a monoclonal antibody (mAb) by immunizing mice with recombinant soluble bovine SCF protein, which was expressed in insect cells by using a baculovirus system. Using the mAb, we purified native soluble bovine SCF from FBS by immunoaffinity chromatography. Western blot analysis revealed that the purified SCF protein had a molecular weight of 33 kDa. In addition, an enzyme-linked immunosorbent assay (ELISA) incorporating the mAb revealed that the levels of native soluble SCF in commercially available FBS were likely to be <100 pg/ml. These results suggest that the concentration of native soluble bovine SCF present in FBS may be insufficient to promote additive biologic effects on the growth of hematopoietic progenitor cells in FBS-supplemented cultures.