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.
In our previous studies, we demonstrated that female primordial germ cells (PGCs) have the ability to differentiate into W chromosome-bearing (W-bearing) spermatozoa in male gonads of germline chimeric chickens. In this study, to investigate the differentiation pattern of female PGCs in male gonads in chickens, three germline chimeric chickens were generated by injecting female PGCs into the male recipient embryos. After these male chimeras reached sexual maturity, the semen samples were analyzed for detecting W-bearing cells by PCR and in situ hybridization analyses. The results indicated that the female PGCs had settled and differentiated in their testes. A histological analysis of the seminiferous tubule in those chimeras demonstrated that the W-bearing spermatogonia, spermatocytes, and round spermatids accounted for 30.8%, 32.7%, and 28.4%, respectively. However, the W-bearing elongating spermatid was markedly lower (7.7%) as compared to the W-bearing round spermatid. The W-bearing spermatozoa were hardly ever observed (0.2%). We concluded that although female PGCs in male gonads are capable of passing through the first and second meiotic division in adapting themselves to a male environment, they are hardly complete spermiogenesis.
The D-loop region in mitochondrial DNA (mtDNA) was sequenced and compared among six chickens. Eleven single nucleotide polymorphisms (SNP) were observed. For six of the SNP sites, polymerase chain reaction (PCR) primers that had each base (A, C, G and T) as the penultimate base at the 3 ¢ end (N2 base) and bases specific to both alleles at the 3 ¢ end were produced to type the polymorphisms. Twenty-one out of 96 primers succeeded in distinguishing the SNP by the presence or absence of PCR product. This method provides an easy way to discriminate SNP in chicken mtDNA.
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