X-irradiation Removes Endogenous Primordial Germ Cells (PGCs) and Increases Germline Transmission of Donor PGCs in Chimeric Chickens

Abstract

Abstract. Primordial germ cells (PGCs) are embryonic precursors of germline cells with potential applications in genetic conservation, transgenic animal production and germline stem cell research. These lines of research would benefit from improved germline transmission of transplanted PGCs in chimeric chickens. We therefore evaluated the effects of pretransplant X-irradiation of recipient embryos on the efficacy of germline transmission of donor PGCs in chimeric chickens. Intact chicken eggs were exposed to X-ray doses of 3, 6 and 9 Gy (dose rate = 0.12 Gy/min) after 52 h of incubation. There was no significant difference in hatching rate between the 3-Gy-irradiated group and the nonirradiated control group (40.0 vs. 69.6%), but the hatching rate in the 6-Gy-irradiated group (28.6%) was significantly lower than in the control group (P<0.05). No embryos irradiated with 9 Gy of X-rays survived to hatching. X-irradiation significantly reduced the number of endogenous PGCs in the embryonic gonads at stage 27 in a dose-dependent manner compared with nonirradiated controls. The numbers of endogenous PGCs in the 3-, 6-and 9-Gy-irradiated groups were 21.0, 9.6 and 4.6% of the nonirradiated control numbers, respectively. Sets of 100 donor PGCs were subsequently transferred intravascularly into embryos irradiated with 3 Gy X-rays and nonirradiated control embryos. Genetic cross-test analysis revealed that the germline transmission rate in the 3-Gyirradiated group was significantly higher than in the control group (27.5 vs. 5.6%; P<0.05). In conclusion, X-irradiation reduced the number of endogenous PGCs and increased the germline transmission of transferred PGCs in chimeric chickens. Key words: Chicken, Germline chimera, Primordial germ cell, X-ray (J. Reprod. Dev. 58: [432][433][434][435][436][437] 2012) P rimordial germ cells (PGCs) are the founder germline cells. In chickens, PGCs are scattered in the center of the blastodisc of freshly oviposited eggs (stage X: Roman numerals refer to the staging system of Eyal-Giladi and Kochav [1]). Following the formation of the primitive streak, PGCs move passively to the anterior border of the extraembryonic region, the so-called germinal crescent region [2]. They then enter the developing vascular network in the germinal crescent region and are transported by the embryonic circulation to the intermediate mesoderm, where they leave the blood vessels and migrate to the genital ridge [3,4]. After settling in the gonads, the PGCs proliferate and then differentiate into functional gametes.A technique for producing live offspring from isolated PGCs following their intravascular transplantation into developing embryos was initially established in chickens in 1993 [5]. Chicken PGCs can be stored at -196 C using a simple protocol, without losing their ability to transmit to the germline [6][7][8]. In addition, a novel method for long-term culture of PGCs that maintains their commitment to the germ cell lineage has recently been developed in chickens [9]. PGCs have therefore received ...

Poultry genetic resource conservation using primordial germ cells

“…Surgical removal of tissues containing PGCs such as blastodermal cells or blood from recipient chicken embryos resulted in increased germline chimerism [65, 66]. Irradiation of chicken embryos with X-rays or gamma-rays resulted in decreased number of endogenous PGCs and thus increased the proportion of donor-derived gametes [67, 68]. However these methods failed to produce stable high-grade germline of chimeric chickens, suggesting recovery of the remaining endogenous PGCs after treatment.…”

Culture Conditions for Maintain Propagation, Long-term Survival and Germline Transmission of Chicken Primordial Germ Cell-Like Cells

“…However, in this study, germline transmission rate of the long-term cultured PGC-LCs were low (6%; 3/50). Thus, sterilization of the recipient should be essential for improvement of donorgermline transmission by use of irradiation (Carsience et al, 1993, Atsumi et al, 2009, Macdonald et al, 2010, Nakamura et al, 2012, busulfan treatment (Song et al, 2005, Nakamura et al, 2008. On the other hand, gametic differentiation ability of PGC-LCs might be decreased during long-term culture, this system should be progress so as to improve the ability of gametic differentiation in PGC-LCs.…”

Production of Pure Hinai-dori with Normal Reproductive Capability from Transferred Primordial Germ Cells

“…One method to improve the efficiency of germline transmission is the removal of endogenous PGCs in recipient embryos, prior to transplantation of donor PGCs. It has been demonstrated that exposure of recipient chicken embryos to gamma rays or X-rays reduces the number of endogenous PGCs (Carsience et al, 1993;Park et al, 2010) and increases germline transmission of donor PGCs (Nakamura et al, 2012). A method to produce high-grade germline chimeras by recipient embryo sterilization followed by the administration of the alkylating agent busulfan (1, 4-butanediol dimethanesulfonate) has been developed (Nakamura et al, 2010a).…”