FIGURE 4. EBs support differentiation of haploid male germ cells that support fertilization of oocytes.

a, Expression of Sry in developing EBs analysed by RT–PCR (top panel). b, Expression of genes regulating male germ cell specification during EB development. Gcnf helps restrict Oct4 expression to the germ line¹⁹. Acrosin, acrosomal protein with a role in oocyte interaction²⁰; haprin, haploid germ-cell-specific RING finger protein; LH-R and MIS, markers for Leydig and Sertoli cells, respectively; Zp1–3, proteins of the oocyte zona pellucida. Hprt served as a control. c, Expression of male germ-cell-specific genes in male or female EBs assayed by RT–PCR. Control RNA from ovary or testis is shown. d, Identification of haploid male germ cells in adult testis (left) or day 13 EBs (right) by immunostaining with the FE-J1 antibody²⁴. Cells were stained with the DNA-binding dye Hoechst 33342 to reveal DNA content. 1C, haploid; 2C, G0/G1 phase; 4C, G2/M phase. e, Immunohistochemistry of the FE-J1⁺ cells sorted in d. FE-J1–PE, red; nuclear Hoechst 33342, blue. f, Fluorescence image of blastocysts derived from intracytoplasmic injection of oocytes with EB-derived FE-J1⁺ haploid cells. g, FISH analysis of control ES cells and morula stage embryos, with fluorescence signals indicated by arrows. Panels 1 (X and Y chromosome markers) and 2 (autosomal ( light chain) and Y chromosome markers) show embryos derived from intracytoplasmic oocyte injection of FE-J1⁺ haploid cells. Panels 3 (X and Y chromosome markers) and 4 (autosomal ( light chain) and Y chromosome markers) show control male ES cells. Panels 5 (X and Y chromosome markers; note two X and absence of Y signals) and 6 (autosomal ( light chain) and Y chromosome markers; note absence of Y signal) show control female ES cells.