By Lucy Hamer
Parthenogenesis describes a natural form of reproduction which occurs without any male contribution. An embryo develops from spontaneous activation of an unfertilised egg cell which contains a complete set of somatic chromosomes from the mother. Another related mode of reproduction, gynogenesis, requires the presence of sperm to activate the egg but the paternal genes similarly do not contribute to the chromosome complement of the offspring (Neaves & Baumann, 2011). Conversely, natural androgenesis occurs if the maternal genome is excluded shortly after fertilisation and as a result the full genetic content of the offspring was derived from the father (Morgado-Santos et al., 2017). In mammals, an epigenetic phenomenon known as genomic imprinting prevents embryogenesis from taking place without maternal and paternal genomic contribution (Galis & van Alphen, 2019).
Genomic imprinting refers to a non-Mendelian form of gene expression in which particular genes are monoallelically expressed in a parent-of-origin specific manner (Galis & van Alphen, 2019, Wilkins & Haig, 2003). During gametogenesis, sex-specific epigenetic marks are laid down which dictate whether the paternal or maternal copy of that gene is responsible for gene expression in any resulting offspring (Ishia & Moore, 2013). For example, in male gametes a methylation mark is established on chromosome 15 which promotes paternal expression of the Igf2 gene but blocks expression of H19. Conversely, the maternal allele remains unmethylated so Igf2 is not maternally expressed whilst H19 is (Huang et al., 2012). Recent genome-wide studies estimate that there are around 100 imprinted genes in humans (Wilkins et al., 2016), with imprinting mostly affecting genes involved in foetal growth (Wilkins & Haig, 2003, Ishia & Moore, 2013).
Experiments in the 1980s demonstrated the importance of both male and female genetic information for normal embryonic development in mice (Li et al., 2018). Embryos of various genetic constitution were constructed by transferring pronuclei between fertilised eggs, before being transferred to pseudopregnant females and allowed to develop. Researchers were unable to obtain any viable embryos from androgenetic eggs reconstituted with two male pronuclei (McGrath & Solter, 1984, Surani & Barton, 1983, Surani et al., 1984), and whilst Surani et al. managed to obtain a handful of embryos from parthenogenetic eggs with two female pronuclei, they were all highly abnormal (Surani & Barton, 1983, Surani et al., 1984). These findings are invaluable evidence that the parental sexes do not contribute functionally equivalent information to the zygote, leading to the hypothesised existence of imprinted genes. Following this discovery, it didn’t take long for the first imprinted gene to be identified (Kelsey & Bartolomei, 2012), by Barlow et al. in 1991 (Barlow et al., 1991).
Numerous attempts have been made to cross the reproduction barriers presented by genomic imprinting and generate offspring without contribution from both parental sexes. In 2004, Kono et al. produced the first surviving bimaternal mice by artificially modulating the expression of particular imprinted genes (Kono et al., 2004). Parthenogenetic embryos are not viable in mammals because offspring receive incorrect dosages of imprinted genes that are vital for foetal growth, leading to abnormal development (Ishia & Moore, 2013). Theoretically, if these gene dosages are corrected by genetic modifications, viable bimaternal and bipaternal embryos can be generated.
Noting that the approach used by Kono et al. was impractical for further applications, Li and a team at the Chinese Academy of Sciences recently set out to explore whether newly characterised mammalian haploid cell lines could be used to produce bimaternal and bipaternal mouse pups (Li et al., 2016). The team established androgenetic haploid embryonic stem cells (ahESCs) in 2012 by transferring sperm into enucleated oocytes (Li et al., 2012), whilst parthenogenetic hESCs (phESCs) were derived from early-stage parthenogenetic embryos, by a different research group (Wan et al., 2013). These cell lines exhibit unimpaired pluripotency similar to germ cells, suggesting that ahESCs and phESCs could functionally replace the male and female gametes in supporting embryonic development (Li et al., 2012, Wan et al., 2013). The characteristic that makes these cells promising tools for the construction of bimaternal or bipaternal embryos is their methylation status. After prolonged culture, hESCs are hypomethylated compared to mature gametes and instead resemble early gamete progenitors called primordial germ cells (PGCs) which have not yet received their sex-specific imprints (Li et al., 2018). This provides researchers with an easier starting point to manipulate gene expression from, increasing their chances of creating offspring with two sets of maternal or paternal chromosomes but normal gene dosages.
The researchers first set out to determine which imprinted genes needed to be modified to permit full-term development. Parthenogenetic embryos were generated by injecting unmodified phESCs into oocytes and whilst no viable offspring were recovered, genome-wide methylation analysis of the most developmentally advanced embryo shed valuable insight on which imprinted genes were sabotaging development. There were three imprinted loci at which the methylation status differed significantly between the phESC-derived bimaternal embryos and the wild-types: H19, Rasgrf1 and IG. It was assumed that development of the constructed bimaternal embryos was mainly impaired as a result of aberrant expression of genes under the control of these imprints (Li et al., 2016).
Subsequently, the H19, IG and Rasgrf1 imprinted regions were deleted, generating 3KO-phESCs which were then used to generate bimaternal embryos. 210 embryos were transferred to pseudopregnant mice, resulting in 29 live mice which all exhibited normal development when compared to controls. Upon mating with wild-type males, female 3KO-bimaternal mice showed only slightly impaired fertility, with 13 out of 22 delivered pups developing normally into adults. These results demonstrate the successful traversal of the bimaternal reproduction barriers, allowing the researchers to focus their efforts on generating viable androgenetic embryos. The process is substantially more complicated in males as the edited ahESCs have to be injected into an enucleated egg cell alongside sperm from another male mouse. Seven imprinted regions had to be deleted from the ahESCs in order to successfully recover any bipaternal mice from the pregnant surrogates, but unfortunately none of these pups survived to adulthood (Li et al., 2018).
Whilst the work of Li et al. is hugely promising and indicates that the generation of viable offspring containing genetic material from two parents of the same sex is possible, there is still a long way to go. The limited success of the research team in generating offspring with two sets of paternal chromosomes demonstrates that our knowledge of genomic imprinting is not complete and that more research needs to be conducted to fully understand the reproductive obstacles presented by genomic imprinting. Additionally, in order for these techniques to be translated to other mammals, the full set of imprinted genes that pose problems for bimaternal or bipaternal embryogenesis need to be verified for each species which will require comprehensive genome-wide studies. Ethical concerns surrounding the generation of unviable or abnormal offspring are also major barriers to human translation (Cell Press, 2020).
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