Normal mammalian development requires participation of both maternal and paternal genomes because of the existence of genomic imprinting, whereas the gynogenetic and androgenetic embryos die shortly after implantation1,2,3. Generation of gynogenetic bimaternal mice containing two sets of maternal genomes was achieved using non-growing oocytes with imprinting modifications4; however, the approach was technically challenging and impractical for further applications. Recently, we and others have derived mammalian androgenetic and parthenogenetic haploid embryonic stem cells (ahESCs and phESCs), and showed that ahESCs can replace gametes to produce offspring5,6,7, which provided alternative resources for reproduction6,7. Here we report that after proper imprinting modifications, the mouse phESCs can efficiently produce viable fertile offspring upon intracytoplasmic injection into MII oocytes. We thus establish a novel strategy of generating bimaternal mammals, which is valuable to uncover the function of genomic imprinting, and to improve assisted reproduction in diverse mammalian species.

To improve the development of phESC-produced gynogenetic embryos, we modified the imprinting status of the H19-Igf2 and Dlk1-Dio3 clusters that controlled fetal growth by knocking out the DMRs, which may cause bidirectional loss of imprinting of all genes within the clusters. According to previous studies9,10,11,12,13, three types of genetic deletions were designed, including a ∼5 kb deletion of the H19-Igf2 locus (from −4 kb to +1 kb of H19 transcription start site (TSS)), a ∼15 kb deletion (from −10 kb to +4 kb of H19 TSS) spanning the H19 gene, H19-ICR (from −4 kb to −2 kb of H19 TSS) and the upstream region of H19-ICR, and a ∼4 kb deletion spanning the IG-DMR of the Dlk1-Dio3 cluster (Supplementary information, Figure S2A and S2E). To boost the efficiency of homologous recombination-mediated genetic knockout, we used the CRISPR/Cas9 system to introduce a targeted DNA double strand break in the knockout region. The correctly targeted clones were identified by PCR with specific primer pairs that could distinguish the WT and knockout alleles (Supplementary information, Figure S2B, S2F and Table S1C). Consistent with the genetic deletions, H19 expression was abolished in phESCs carrying the 5 kb deletion (phESCΔ5H19) or the 15 kb deletion (phESCΔ15H19) of the H19-Igf2 locus, and Gtl2 expression was silenced in phESCs carrying the IG-DMR deletion (phESCΔIG) (Supplementary information, Figure S2C and S2F). Then we purified the haploid cells of these genetically modified phESC lines by FACS and generated bimaternal embryos by MII oocyte injection to investigate the gynogenetic development (Supplementary information, Figure S2D and S2G). A total of 624 phESCΔ5H19 embryos from 2 phESCΔ5H19 sublines, and 611 phESCΔ15H19 embryos from 2 phESCΔ15H19 sublines were transferred into pseudopreganant mothers. None developed to term (E19.5), though some embryos developed beyond E13.5 (Supplementary information, Table S1A). However, of the 568 phESCΔIG embryos transplanted, 13 (2.3%) full-term pups were recovered by cesarean section (Supplementary information, Table S1A). These phESCΔIG pups were severely growth-retarded (0.57 ± 0.02 g, n = 10), with about 46% of normal body weight of WT pups (1.25 ± 0.04 g, n = 12; Figure 1E and 1F), and all died within several hours after birth. Comparison of global imprinting status between phESCΔIG and WT pups showed that of 16 DMRs recovered by RRBS, 15 maternally methylated DMRs had similar methylation levels, but the H19-DMR was hypomethylated in the phESCΔIG pups (Figure 1H and Supplementary information, Figure S2L). Bisulfite sequencing analysis confirmed the normal methylation level of maternally methylated Igf2r- and Snrpn-DMRs, and hypomethylation of H19-DMR (Figure 1G).

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