A new study has shown that targeted editing of epigenetic marks in the oocyte can be used to produce viable offspring without the need for fertilisation. The epigenetically-modified female mouse, born from the unfertilised oocyte, not only survived to adulthood, but also gave birth to viable pups. Mina Popovich and Susana Chuva de Sousa Lopes from the SIG Stem Cells report.
Successful mammalian development relies on the complementary contribution of both sexes, as differences between the maternal and paternal genome ensure normal embryogenesis following fertilisation. These differences include sex-specific epigenetic marks, or genomic imprints that lead to the monoallelic, parent-specific expression of a small number of genes (about 150). This phenomenon of genomic imprinting has a critical influence on the regulation of mammalian development, providing a balance in gene expression between paternal and maternal genomes.
Imprinted genes are associated with regions of differential DNA methylation, referred to as ‘imprinting control regions’. The wave of DNA demethylation that occurs just after the formation of primordial germ cells erases a large majority of the epigenetic imprints of the previous generation, which then must be reset during male or female gametogenesis, depending on the sex of the embryo.
The importance of genomic imprinting came to light following attempts to create mice with either two maternal genomes (gynogenetic embryos) or two paternal genomes (androgenetic embryos).(1,2) The failure of such embryos to develop beyond implantation demonstrated that the maternal and paternal genomes were not functionally equivalent. Accordingly, a diploid genome derived from only one sex by parthenogenesis (replication of DNA transforming a haploid gamete into a diploid cell) is incapable of supporting complete embryo development. In the case of males, parthenogenesis has been attempted by placing one sperm cell into an enucleated mature oocyte. Yet in both male and female mammals, parthenogenesis has been exceedingly limited. In parthenogenetic embryos, dysregulation stems from the two-fold establishment of maternal-specific or paternal-specific imprints.
In a remarkable effort to overcome this reproductive barrier in females, researchers led by Yanchang Wei from the Ren Ji Hospital in Shanghai used a novel methylation-rewriting approach to edit several imprinting control regions in mouse oocytes.(3) Based on CRISPR-Cas technology, this method allows allele-specific targeting of imprinting control regions. The researchers chose to edit two paternally and five maternally DNA methylated regions, associated with genes known to be critical for embryonic development. The dysregulation of these genes leads to embryonic or postnatal lethality, or severe developmental disorders.
The researchers edited a total of 389 oocytes. Following their parthenogenetic activation, 227 (58.4%) of the oocytes formed diploid ‘zygotes’. Around 85% of the parthenotes developed into blastocysts, which were then transferred into recipient females. A total of three pups were recovered, two of which died within the first 24 hours. The remaining female pup grew to adulthood. Further testing of the obtained mice revealed that correct DNA methylation patterns of all seven imprinting control regions are necessary for full-term development, as the surviving mouse was the only one to exhibit correct imprinting patterns across all seven regions.
However, despite reaching adulthood, the parthenogenetic mouse displayed postnatal growth delays. The authors attributed these defects to another critical paternally methylated imprinting control region that was not edited. Nonetheless, the mouse was able to mate and gave birth to normal, viable pups. This outcome was expected, as the germ cells of the parthenote undergo epigenetic reprogramming, thus resetting practically all epigenetic marks.
Notably, gene editing has been previously used to generate bimaternal mice and more recently Li et al generated bimaternal and bipaternal mice by deleting imprinting regions in haploid embryonic stem cells, which were then injected into mature oocytes.(4,5) However, this latest PNAS study is the first to create offspring from a single unfertilised mammalian oocyte.
The success of parthenogenesis in mammals is of exceptional value for understanding the role of imprinted genes in development and disease. This work also has potential to improve animal cloning efficiency, with important applications in agriculture, as well as the preservation of endangered species. Crucially, however, the low success rate and abnormal development render any applications in humans unfeasible. Further research into genomic imprinting is necessary to ensure the safety of genetic manipulations involving imprinted genes. Genomic imprinting impacts an extremely wide range of biological processes, the effects of which extend into adulthood, and may have profound roles in a variety of diseases. Nevertheless, future studies in animal models will certainly provide precious insights into key aspects of reproduction, including the epigenetic regulation of genes during development.
1. McGrath J, Solter D. Completion of mouse embryogenesis requires both the maternal and paternal genomes.
Cell 1984; 37: 179–1832. Yao Q-Y, Yuan X-Q, Liu C, et al.
2. Surani MH, Barton SC, Norris ML. Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis
. Nature 1984; 308: 548–550.
3. Wei Y, Yang CR, Zhao ZA. Viable offspring derived from single unfertilized mammalian oocytes. PNAS 2022; doi.org/10.1073/pnas.2115248119
4. Kono T, Obata Y, Wu Q, et al. Birth of parthenogenetic mice that can develop to adulthood
. Nature 2004; 428: 860–864.
5. Li Z-K, Wang L-Y, Wang L-B, et al. Generation of bimaternal and bipaternal mice from hypomethylated haploid ESCs with imprinting region deletions
. Cell Stem Cell 2018; 23: 665-676.e4.