(A) Population of gonadal germ cells between embryonic day (ED) 9–12. The number of germ cells was determined by their expression of GFP protein in iCaspase9 transgenic embryos (n = 3–7 gonad pairs for each sex at each day). (B) Yield of viable dissociated cells directly from freshly isolated embryonic day 9 gonads (control), cryopreserved dissociated gonadal cells subsequently thawed (frozen cells), and cryopreserved whole gonads subsequently thawed then dissociated (frozen gonads). Cell viability was determined using a trypan blue exclusion assay. Data from 13 to 20 independent experiments using mixed male and female gonads. (C) Flow cytometric analysis of Magnetic Activated Cell (MAC)-sorted female gonadal cells. Frozen female ED 9 (HH35) gonads from iCaspase9 GFP+ embryos were MAC-sorted using an anti-SSEA-1 antibody. The purified cells were then immunostained by secondary antibody against SSEA-1 to detecting the percentage of SSEA-1 cells expressing GFP. The GFP+ population was analysed for viability using Syto blue; n = 5 independent experiments. (D) Yield of MAC-sorted cells from cryopreserved ED 9 (HH35) female gonads. The average number of GFP+ cells purified by MACS from a single iCaspase9 embryo using an anti-SSEA-1 antibody. Data from five independent experiments using 12–26 gonad pairs per experiment. (E) Colonisation of sterile iCaspase9 embryos by cryopreserved male and female gonadal cells. The host ED 14 gonads are shown on the left and transverse sections from those gonads are on the right. Day 2.5 iCaspase9 host embryos were injected with gonadal donor cells at a 5GFP+:1RFP+ ratio mixed with B/B compound; 45,000 male cells/embryo. Female cells were MAC-sorted before injecting 1400 female cells/embryo. Representative embryo shown (n > 5, for each sex). Scale bar = 100 μm. (F) Seminiferous tubule of an adult testis (>6 months) from a sterile surrogate host injected with donor male cells prepared as in (C). Representative testis section from n = 7 males. Scale bar = 100 μm.