A Low-tech, Cost-effective And Efficient Method For Safeguarding ...

We assayed for gonadal cell survival after cryopreservation and thawing. Gonadal cell viability was severely reduced after the cryopreservation of dissociated gonadal cells (Figure 2B). In comparison, cryopreservation of whole gonads followed by thawing and subsequent cell dissociation did not significantly reduce gonadal cell viability when compared to the viability of directly dissociated cells from freshly isolated gonads (Figure 2B). We next assayed the capacity of cryopreserved gonadal cells to recolonise the gonad of surrogate host embryos using GFP+ and RFP+ labelled gonads from ED 8 to ED 11 transgenic embryos as donors. The migration of donor gonadal germ cells to a host gonad can be easily quantified by counting the fluorescent cells in the host gonads. Whole donor gonads were cryopreserved and subsequently dissociated cells prepared from these frozen gonads were injected into wildtype host embryos. We injected 10,000–15,000 female gonadal cells (~150 germ cells) into the host embryos and observed germ cell colonisation 5–6 days (ED 8–9) post injection. Gonadal cells from ED 8–10 donor female embryos achieved repeatable colonisation of host gonads, while injection of ED 11 donor resulted in much fewer fluorescent cells present in the host embryo (Figure 2—figure supplement 1).

Male gonadal germ cells form the SSC population of the testes that proliferates and differentiates into spermatozoa for the entire life of the cockerel. In contrast, female gonadal germ cells enter into meiosis starting at ED 15 and reach a final population of 480,000 post-replicative germ cells in the hatched chick (Hughes, 1963). We postulated that the number of fluorescent cells observed colonising the host female gonad (Figure 2—figure supplement 1) would not be sufficient to form an appropriate oocyte hierarchy in the mature ovary and a continuous egg-laying cycle lasting the 10–12-month period of a typical layer hen. The cell surface stem cell marker, SSEA-1, is highly expressed on the surface of migratory and post-migratory gonadal germ cells until ED 9–10 of incubation after which expression of SSEA-1 is rapidly lost from the germ cell (Urven et al., 1988). Selective enrichment of gonadal germ cells using SSEA-1 antibody was shown to increase the colonisation of the host gonad (Kim et al., 2004; Mozdziak et al., 2005; Mozdziak et al., 2006). We used MACS to purify SSEA-1-expressing cells from the ED 9–10 (stage 35 HH) female gonads. We first determined that SSEA-1 expression on female gonadal germ cells decreased after ED 10 (Figure 2—figure supplement 2). Cryopreserved iCaspase9 gonadal tissues were thawed and dissociated, and MACS using SSEA-1 antibody was used to enrich for female PGCs. We observed that the SSEA-1 antibody captured and enriched >40-fold GFP+ gonadal germ cells (1.17% increased to 46%). The MACS-purified cells were 94% viable and as expected coexpressed SSEA-1 antigen on their surface (Figure 2C). The yield of germ cells from the female ED 9 gonad by MACS was approximately 5000 putative germ cells per pair of gonads (Figure 2D).

We next tested the colonisation of male dissociated gonadal cells and MACS-enriched female gonadal cells using iCaspase9 host embryos treated with B/B compound. We used GFP+ and RFP+ gonad pairs mixed in a ratio of 5:1 to identify multiple colonisation events. A cell suspension of male gonadal cells or MACS-purified female gonadal cells was mixed with B/B dimerisation chemical and injected into the vascular system of ED 2.5 (stage 16 HH) iCaspase9 embryos (Figure 2E). Whole tissue imaging and cryosections of surrogate host ED 14 gonads indicated that the donor PGCs from frozen gonadal tissues colonised the iCaspase9 host embryos of the same sex. The majority of cells were GFP+ and fewer RFP+ PGCs were also present in the host gonads (Figure 2E). An analysis of cryosections of adult male testes indicated that the majority of putative germ cells in the seminiferous tubules were GFP+ and a minority of cells were RFP+ (Figure 2F).