Production of Mixed-sex Germline Chimeras in the Chicken

Abstract

The overall objectives of this research were to investigate the production of mixed-sex germline by chicken gonadal primordial germ cells (PGCs) and the expression of DAZL (Deleted in Azoospermia-Like) and VASA in chicken germline.

The ideal host for the production of germline chimeras would be a sterile recipient embryo where endogenous germ cells have been completely depleted. Busulfan (1,4-butanediol dimethanesulfonate) selectively depletes stem cells in both mammals and birds. In this study, two delivery formulations: busulfan+sesame oil suspension (Bu/O) and busulfan + dimethyl formamide (DMF) +sesame oil emulsion (BuDMF/O), were compared relative to their ability to deplete endogenous germ cells. Both busulfan treatments resulted in a significant decrease in PGCs (P<0.05) when compared to controls. However, the BuDMF/O emulsion resulted in significantly fewer germ cells (P<0.05) than that observed with the Bu/O suspension above. The coefficient of variation of the Bu/O and BuDMF/O was 47% vs. 11%, indicating that BuDMF/O formulation is a more repeatable delivery method. Repopulation with exogenous PGCs after BuDMF/O treatment resulted in an average of 94 PGCs/embryo while non-repopulated embryos exhibited an average of 23 PGCs (P<0.05). Subsequently, same-sex and mixed-sex germline chimeras were produced by the transfer of male gonadal PGCs into untreated and BuDMF/O-treated recipient embryos. The frequency of germline chimerism was 2.8 % (1/36) in untreated recipients, while the frequency of germline chimerism among BuDMF/O-treated recipients was 22.6% (7/31, p<0.05). When female embryonic recipients were treated with BuDMF/O and injected with male PGCs, 1 out of 14 (7.1%) were determined to be germline chimeric; Conversely, 6 out of 17 (35.3%) male recipients were identified as germline chimeras. The frequency of germline chimerism was significantly (X2c=3.38, p<0.05) higher when the donor and recipient were the same-sex compared with the production of mixed-sex chimeras. These results demonstrated that the administration of a solublized busulfan formulation improved the degree of endogenous PGC depletion and thus enhanced the efficiency of germline chimera production. However this improved depletion did not result in a significant deviation of the sex ratio in the offspring of mixed-sex female germline chimeras. It appears that biological barriers exist which impede the subsequent proliferation of exogenous male PGCs in a female host.

DAZL and VASA proteins are components of germ plasm required for germ cell determination and differentiation in Drosophila and Xenopus. Polyclonal antibodies against synthetic peptides of DAZL and VASA were generated to characterize the expression of DAZL protein in chicken germline. Immunblotting of protein extracts from adult chicken tissues with anti-C-terminal DAZL and anti-N-terminal DAZL antibodies visualized a same size protein of ~ 40KD in testis, which suggest this protein is chicken DAZL protein. Immunohistochemical studies using anti-C-terminal DAZL, anti-N-terminal DAZL antibodies and anti-N-terminal VASA antibody demonstrated the germline-specific expression of chicken DAZL and VASA proteins throughout all stages of germline development. VASA and DAZL were detected in the cytoplasm of PGCs in the germinal crescent region, the circulating blood, the dorsal mesentery and gonads. After sexual differentiation, chicken DAZL and VASA were exclusively expressed in the cytoplasm of spermatogonia and primary spermatocytes in the testes or in the cytoplasm of oogonia and primary oocytes in the ovary. Chicken VASA protein was also espressed in the cytoplasm of early spermatids. VASA and DAZL protein were not detected in the pre-streak stage of chicken embryos, which suggest the induced model of germ cell formation in birds. These observations suggest a critical role of DAZL and VASA in avian germ cell development and provide complete germ cell markers in avian germline cell research.