Primordial germ cells (PGCs), collected from the blood of 2-day-old chick embryos, were concentrated by Ficoll density centrifugation. The blood contained 0.048% PGCs and the concentrated fraction contained 3.9% PGCs in blood cells. The PGCs were picked up with a fine glass pipette, and one hundred were then injected into the terminal sinuses of 2-day-old Japanese quail embryos (24 somites); bubbles were then inserted to prevent haemorrhage. The embryos were further incubated at 38 degrees C for 24 h, and then fixed. Serial sections were stained with the periodic acid-Schiff reagent (PAS) to demonstrate chicken PGCs and with Feulgen stain to identify quail cells. On the basis of the differences in staining properties, 63.6 +/- 5.3 chick PGCs were detected in the quail embryo in the area where the gonads develop. Furthermore, 39.3 +/- 4.5 chick PGCs were incorporated into the quail germinal epithelium within 24 h of the injection. A similar percentage of the host (quail) PGCs had also migrated to the germinal epithelium at the same stage of development. This technique for obtaining germ-line chimaeras will facilitate research on avian germ-line differentiation.

Germline chimeric chickens were produced by transfer of primordial germ cells from White Leghorn to Barred Plymouth Rock, and vice versa. Blood was collected from stage 13-15 embryos and primordial germ cells were concentrated by Ficoll density gradient centrifugation. Approximately 200 primordial germ cells were injected into the bloodstream through the dorsal aorta of stage 14-15 recipient embryos from which blood had been drawn via the dorsal aorta prior to the injection. Intact embryos were also prepared as recipients for White Leghorns only. The manipulated embryos were cultured in recipient eggshells until hatching. Germline chimerism of the chickens reaching maturity was examined by mating them with Barred Plymouth Rocks and donor-derived offspring were identified based on their feather color. The efficiency of production of germline chimeras was 95% (19/20). When primordial germ cells were transferred from White Leghorn to Barred Plymouth Rock, the average frequency of donor-derived offspring was 81% for three male chimeras (96% for one female chimera), and it was approximately 3.5 times higher for transfer in the opposite direction (23% for 6 male chimeras). Removing blood from recipient embryos prior to primordial germ cell injection enhanced the frequency of donor-derived offspring by 10% in resulting male chimeras. Male chimeras produced donor-derived offspring more frequently (approximately 3.8 times) than female chimeras. Increases, decreases, or no changes were observed in the frequency of donor-derived offspring from the germline chimeras with increasing age.(ABSTRACT TRUNCATED AT 250 WORDS)

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This study was carried out to elucidate whether primordial germ cells, obtained from embryonic blood and transferred into partially sterilized male and female recipient embryos, could differentiate into functional gametes and give rise to viable offspring. Manipulated embryos were cultured until hatching and the chicks were raised until maturity, when they were mated. When the sex of the donor primordial germ cells and the recipient embryo was the same, 15 out of 22 male chimaeric chickens (68.2%) and 10 out of 16 female chimaeric chickens (62.5%) produced donor-derived offspring. When the sex of the donor primordial germ cells and the recipient embryo was different, 4 out of 18 male chimaeric chickens (22.2%) and 2 out of 18 female chimaeric chickens (11.1%) produced donor-derived offspring. The rates of donor-derived offspring from the chimaeric chickens were 0.6-40.0% in male donor and male recipient and 0.4-34.9% in female donor and female recipient. However, the rates of donor-derived offspring from the chimaeric chickens were 0.4-0.9% in male donor and female recipient and 0.1-0.3% in female donor and male recipient. The presence of W chromosome-specific repeating sequences was detected in the sperm samples of male chimaeric chickens produced by transfer of female primordial germ cells. These results indicate that primordial germ cells isolated from embryonic blood can differentiate into functional gametes giving rise to viable offspring in the gonads of opposite-sex recipient embryos and chickens, although the efficiency was very low.

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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.

1. Primordial germ cells (PGCs) are the progenitor cells for gametes. In aves, PGCs show a unique migration pathway, that is, they circulate temporarily through the bloodstream during early development. 2. In this study we developed a method to purify circulating primordial germ cells (cPGCs) in quail and chicks by Nycodenz density gradient centrifugation. 3. The process consisted of primary and secondary purification. In primary purification, cPGCs were enriched at the interface of 8 and 12% Nycodenz fractions. In secondary purification, cPGCs were harvested from 8% Nycodenz fraction at a purity of 90% and from 10% Nycodenz fraction at a purity of 70%. The recovery rate of cPGCs was over 70%. 4. This method would facilitate research on cPGCs' culture and the production of transgenic birds using cPGCs.

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