Sex Differentiation and Germ Cell Production in Chimeric Pigs Produced by Inner Cell Mass Injection into Blastocysts

Abstract

This study aimed at collecting background knowledge for chimeric pig production. We analyzed the genetic sex of the chimeric pigs in relation to phenotypic sex as well as to functional germ cell formation. Chimeric pigs were produced by injecting Day 6 or Day 7 inner cell mass (ICM) cells into Day 6 blastocysts. Approximately 20% of the piglets born from the injected blastocysts showed overt coat color chimerism regardless of the embryonic stage of donor cells. The male:female sex ratio was 7:2 and 6:1 in the chimeras derived from Day 6 and Day 7 ICM cells, respectively, showing an obvious bias toward males. When XX donor cells were injected into XY blastocysts at the same embryonic stage, the phenotypic sex of the resulting chimera was male with no germ-line cells formed from the donor cell lineage. On the other hand, when the donor was XY and the recipient blastocyst was XX, the phenotypic sex of the chimera was male, and germ-line cells were derived only from the donor cells. The combination of XY donor cells and XY blastocysts produced some chimeras in which the donor cell lineage did not contribute to germ-line formation even when it appeared in coat color. When the embryonic stage of the donor was advanced by 1 day in the XY-XY combination, 100% of the germ-line cells of the chimeras were derived from the donor cell lineage. These data showed that characteristics of sex differentiation and germ cell formation in chimeric pigs are similar to those in chimeric mice.

Blastocyst complementation generates exogenic pancreas in vivo in apancreatic cloned pigs

“…Generation of human germ cells in chimeras generated by xenogenic blastocyst complementation may not be acceptable, and measures to prevent human PSCs from contributing to reproductive cells in chimeric animals may be in order. Our data and those of others (8,10,11) suggest that formation of male germ cells from exogenic cells is suppressed when a male host embryo is complemented with female donor cells. Cell fate control (16,17) also may permit suppression of formation of "unwanted" cells in animals generated by blastocyst complementation using human PSCs.…”

“…We have confirmed that when male and female embryos are combined to produce a chimeric pig embryo, the chimera develops as a male (8). Fetuses with the host embryo's male sex that expressed donor cells' orange fluorescence were accordingly viewed as likely chimeric.…”

Putative Embryonic Stem Cell Lines from Pig Embryos

“…Up to now, chimeric offspring in the pig have been obtained from blastocysts injected with either fresh ICM cells [21,[71][72][73], cultured EG cells [74] or ES cells [20,61] or eventually from injections of ES cells into the perivitelline spaces of 4-to 8-cell stage embryos [75]. Production of chimeric piglets either from blastomere aggregation [76] or morula injection [72] has been unsuccessful.…”

“…Production of chimeric piglets either from blastomere aggregation [76] or morula injection [72] has been unsuccessful. Germ-line chimerism in the pig has only been reported by Anderson et al [21], Onishi et al [72] and Nagashima et al [73], but these authors used freshly isolated ICM cells for injection into the blastocoel.…”

Recent advances and future options for New Zealand agriculture derived from animal cloning and transgenics

“…In breeding from cloned sires, it is not the clone himself that is important but rather what is transmitted through his germline: haploid copies of the donor animal's genome, in the form of sperm. In this regard, either germline transmitting male chimaeras (Nagashima et al 2004) or males in which their testes have been re-populated with spermatogonial stem cells obtained from an elite sire (Hill & Dobrinski 2006) are both attractive, alternative strategies to achieve the same goal of effectively disseminating genetic gain in livestock industries.…”