Spermatogonial transplantation provides access to the mammalian germline and has been used in experimental animal models to study stem cell/niche biology and germline development, to restore fertility, and to produce transgenic models. The potential to manipulate and/or transplant the germline has numerous practical applications that transcend species boundaries. To make the transplantation technology more broadly accessible, it is necessary to develop practical recipient preparation protocols. In the current study, mouse recipients for spermatogonial transplantation were prepared by treating pregnant females with the chemotherapeutic agent busulfan at different times during gestation. Donor germ cells were introduced into the testes of male progeny between 5 and 12 days postpartum. Analysis of recipient animals revealed that busulfan treatment of pregnant females on 12.5 days postcoitum was the most effective; male progeny transplanted with donor germ cells became fertile and passed the donor genotype to 25% of progeny. This approach was effective because 1) the cytoablative treatment reduced (but did not abolish) endogenous spermatogenesis, creating space for colonization by donor stem cells, 2) residual endogenous germ cells contributed to a healthy testicular environment that supported robust donor and recipient spermatogenesis, and 3) fetal busulfan-treated males could be transplanted as pups, which have been established as better recipients than adults. Laboratory mice provide a valuable experimental model for developing the technology that now can be applied and evaluated in other species.

The aim of the present study was to examine the effect of culture under 5 and 20% oxygen on the development, differentiation and viability of zygotes and in-vivo-produced embryos at the 2-cell and 8-cell stages of development. First, zygotes collected in a common pool were cultured in 20% O2 for 0, 23, 46 and 95 h. Zygotes and in-vivo-produced embryos at the 2-cell and 8-cell stages of development were then cultured in 5 or 20% O2. The proportion of embryos reaching the compaction and blastocyst stages of development did not differ between groups regardless of the period of time embryos were cultured in 20% O2 or the stage at beginning of culture. Duration of culture under 20% O2 had a significant effect on total number of blastocyst cells. A stage-specific effect was observed on total and trophectoderm cell numbers in blastocysts resulting from the culture of zygotes and in-vivo-produced embryos under 20% O2. ICM and percent ICM development was significantly decreased by culture in 20% O2 at all stages examined. Oxygen concentration had no effect on implantation rate and fetal weights upon embryo transfer. However, transfer of zygotes grown to the blastocyst stage in 20% O2 resulted in a dramatic decrease in fetal development per blastocyst and fetal development per implantation. These results demonstrate that culture of F1 mouse zygotes in 20% O2 compromises the developmental potential of resultant blastocysts, which appear to be normal on morphological assessment.

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The aim of the current study was to examine the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the development and differentiation of preimplantation mouse embryos from different strains and under different culture conditions. Embryos from F1 hybrid mice were cultured in a modified G1 medium lacking amino acids and EDTA (simple G1), human tubal fluid medium (HTF) or in G1/G2 sequential media, supplemented with GM-CSF (0, 2, 4, 8, and 16 ng/ml). Embryos from CF1 mice were subsequently cultured in G1/G2 with (5 mg/ml) or without HSA, in the absence or presence of GM-CSF (2 ng/ml). GM-CSF had no effect at any concentration on F1 embryo development and blastocyst cell numbers, irrespective of the culture media used. Similarly, GM-CSF had no effect on CF1 blastocyst development. However, a stimulatory effect of GM-CSF was evident on total blastocyst cell number and ICM development when CF1 embryos were cultured in the absence of HSA. When HSA was present in the media the beneficial effect of GM-CSF was negated. There was no difference in the number of apoptotic cells in CF1 blastocysts when G1/G2 were supplemented with GM-CSF with or without HSA. These data indicate that there is no beneficial effect of supplementing either simple (simple G1 or HTF) or more complete (G1/G2) media with GM-CSF when protein is present in the medium. However, when culture conditions are suboptimal and non-physiological, i.e. the absence of protein, GM-CSF stimulates development of both total cell numbers and ICM development of CF1 blastocysts.

The origin of the germ cell lineage in vertebrates is a fundamental question that has preoccupied developmental biologists. Recent work on the origin of the avian germ line has extended and clarified our understanding of the temporal and spatial segregation of primordial germ cells (PGC) during prestreak stages of development. The germ cells first appear at Stage X (Eyal-Giladi and Kochav, 1976) in the ventral surface of the area pellucida in a scattered pattern among polyingressing cells. Subsequently, the PGC gradually translocate from the epiblast to the hypoblast. The entire process appears to be dependent upon the maintenance of an organized area pellucida. Little is known about the regulatory events governing germ cell emergence during this period; however, the culture of dispersed blastodermal cells on a mouse fibroblast feeder layer can compensate for a disorganized area pellucida and offers an in vitro system to examine the molecular basis of germ cell development. Such basic information is valuable for current approaches towards the production of transgenic poultry with targeted changes to the genome through the use of avian embryonic stem cells or primordial germ cells. Refinement of the culture of primordial germ cells or their precursors should allow academic and industrial research laboratories to answer significant biological questions and to improve the genetic potential of commercial poultry stocks. A better understanding of the biology of avian primordial germ cells during early embryo development can only enhance this process.