Polyploidy of semi-cloned embryos generated from parthenogenetic haploid embryonic stem cells

Abstract

In mammals, the fusion of two gametes, an oocyte and a spermatozoon, during fertilization forms a totipotent zygote. There has been no reported case of adult mammal development by natural parthenogenesis, in which embryos develop from unfertilized oocytes. The genome and epigenetic information of haploid gametes are crucial for mammalian development. Haploid embryonic stem cells (haESCs) can be established from uniparental blastocysts and possess only one set of chromosomes. Previous studies have shown that sperm or oocyte genome can be replaced by haESCs with or without manipulation of genomic imprinting for generation of mice. Recently, these remarkable semi-cloning methods have been applied for screening of key factors of mouse embryonic development. While haESCs have been applied as substitutes of gametic genomes, the fundamental mechanism how haESCs contribute to the genome of totipotent embryos is unclear. Here, we show the generation of fertile semi-cloned mice by injection of parthenogenetic haESCs (phaESCs) into oocytes after deletion of two differentially methylated regions (DMRs), the IG-DMR and H19-DMR. For characterizing the genome of semi-cloned embryos further, we establish ESC lines from semi-cloned blastocysts. We report that polyploid karyotypes are observed in semi-cloned ESCs (scESCs). Our results confirm that mitotically arrested phaESCs yield semi-cloned embryos and mice when the IG-DMR and H19-DMR are deleted. In addition, we highlight the occurrence of polyploidy that needs to be considered for further improving the development of semi-cloned embryos derived by haESC injection.

Fig 1. Generation of the IG-DMR and H19-DMR deletions in haESCs.

(A) IG-DMR and H19-DMR deletions were engineered in phaESCs, which were established from haploid blastocysts obtained from activated mouse oocytes, by simultaneous transfection with four vectors encoding CRISPR-Cas9 nucleases, a CAG-EGFP-IRES-hygro piggyBac transposon vector, and a transposase vector. (B) Sequences of PCR fragments amplified over the deleted regions confirmed the loss of both DMRs in DKO-phaESC-1 and DKO-phaESC-2. (C) Morphology of DKO-phaESC lines. Scale bar, 200 μm. (D) Haploid karyotypes were observed in both DKO-phaESC-1 and DKO-phaESC-2. (E) Transcription of imprinted genes Gtl2 and H19, both of which are maternally expressed and regulated by the IG- and H19-DMRs, was reduced in DKO-phaESC-1 and DKO-phaESC-2. Gene expression was normalized to Gapdh relative to the parental cell line. Data represents relative expression of each sample with the mean values and standard deviation (n = 4). **** P < 0.0001; ** P < 0.01; ns, non-significant.

Fig 2. Characterization of scESC lines derived by injection of DKO-phaESCs into oocytes.

(A) A scheme of the generation of scESC lines by injection of DKO-phaESCs into MII oocytes. (B) DKO-phaESCs were arrested in metaphase with demecolcine for 8 hours and sorted for a 2n DNA content. The peak of the Hoechst intensity corresponding to 2n DKO-phaESCs is indicated (asterisk). (C) Semi-cloned embryo development after injection of DKO-phaESCs into oocytes. EGFP fluorescence merged with bright field images are shown. At day 4, morulae developed to blastocysts. Scale bar, 200 μm. (D, E) DNA content analysis of 4 scESC lines, which were either untreated (D) or treated with demecolcine (E), by flow cytometry after Hoechst staining. (D) DNA content at the G1 phase of scESC-1 and scESC-3 appeared in the middle between the DNA content of G1 and G2 phase control diploid ESCs, indicating scESC-1 and scESC-3 are triploid. scESC-4 contained both diploid and tetraploid cells. (E) Only one population of DNA content was observed in each scESC-1, scESC-2 and scESC-3, while scESC-4 showed two populations of different DNA contents. A haploid-diploid mixed ESC line and a diploid ESC line were included as a reference (top). The percentage of cells within the peaks is indicated in the histograms (top and bottom). (F) Metaphase spreads show triploid karyotypes of scESC-1 and scESC-3, and a tetraploid karyotype of scESC-4.

Fig 3. Generation of semi-cloned mice by transfer of semi-cloned embryos into recipient mothers.

(A) 7 offspring (F0 no. 1–7) were obtained from 2 albino recipient mothers after transfer of semi-cloned and albino control 2-cell embryos. F0 no. 6 (indicated by asterisk) and no. 7 displayed black eyes and agouti coat color indicating DKO-phaESC derived pigmentation. (B) Toe biopsies of F0 no. 1–7. Biopsies of F0 no. 6 and 7 expressed EGFP under UV illumination. (C) Genotyping of F0 no. 1–7. F0 no. 6 and 7 possessed wild type and deletion alleles of the IG-DMR and H19-DMR. (D) Mating of semi-cloned F0 no. 6 and 7 with wild type males yielded healthy F1 pups (indicated by asterisk). (E) Bisulfite DNA methylation analysis of Kcnq1, Igf2r and Peg13 in biopsies of F0 and a control mouse. White circles represent unmethylated CpGs; black circles represent methylated CpGs. The ratio of methylated CpGs is shown in brackets.