While some species of lizards and snakes can create new life via asexual reproduction, mammals rely on the union of male and female gametes for reproduction.1 That was until a few years ago, when researchers generated mouse pups using genetic material from two same-sex parents.2 While the pups born to two female mice survived to adulthood, those produced from two male mice died shortly after birth.
Now, after spending almost six years improving their method, the same research team, led by developmental biologist Wei Li at the Chinese Academy of Sciences (CAS), has successfully generated mouse pups from two male parents, with some surviving into adulthood.3 The results, published in Cell Stem Cell, uncover mechanisms that can advance the generation of genetically modified animal models and improve understanding of some congenital diseases.
“This work will help to address a number of limitations in stem cell and regenerative medicine research,” said Li in a press release.
Mammalian reproduction involves genomic imprinting, a process in which epigenetic modifications restrict the expression of certain genes to a single parental allele. The genes that are silenced or expressed depend on their parental origin. As a result, offspring that do not receive genetic material from both the male and female parents may exhibit developmental defects, some of which could be lethal.
In their 2018 study, Li and his team derived haploid embryonic stem cells (ESCs) from mouse sperm that contained half the number of chromosomes.2 Using CRISPR/Cas9 editing tools, the researchers manipulated seven imprinted genomic regions in these cells. The researchers then injected the ESCs along with unedited sperm into an oocyte that lacked a nucleus, and therefore any female genetic material. Although this strategy successfully generated embryos, they failed to develop a placenta. When the researchers supplemented the embryos with pluripotent stem cells that differentiated to form placental tissue some of the embryos survived to term, but the pups died soon after birth.
In the present study, the researchers took a closer look at the nonviable pups to determine what was going wrong. They observed physical abnormalities such as excessive weight, open tongue, umbilical hernias, and genetic abnormalities like overexpression of paternally expressed imprinted genes. Researchers have previously hypothesized that paternal genes promote growth, while maternal genes restrict it.4 Consistent with this, Li and his team observed that excessive organ growth in bi-paternal pups compressed their chest cavity, resulting in death.
To coax the bi-paternal embryos to exhibit more typical developmental characteristics, the researchers modified up to 20 imprinted regions linked to overgrowth and mis-regulated paternal imprinting. These modifications not only increased the embryos’ ability to develop to full term, but compared to pups generated from unedited ESCs, the resulting pups showed improved developmental outcomes, including the formation of a functional placenta. Nearly 12 percent of the embryos survived until birth, an improvement over 1.2 percent observed in their previous study. However, in a monumental first, some of the pups lived to adulthood. Researchers also showed that the 20 modifications improved their ability to clone these mice from ESCs.
“These findings provide strong evidence that imprinting abnormalities are the main barrier to mammalian unisexual reproduction,” said study coauthor Guan-Zheng Luo, a biochemist at Sun Yat-sen University.
However, the bi-paternal animals that survived to adulthood showed overgrowth and had reduced lifespans. Although they developed normal sexual characteristics, they were sterile.
“Further modifications to the imprinting genes could potentially facilitate the generation of healthy bi-paternal mice capable of producing viable gametes and lead to new therapeutic strategies for imprinting-related diseases,” said co-author Zhi-Kun Li, a developmental biologist at CAS.
Imprinting disorders are congenital diseases that can manifest as neurodevelopmental impairment, endocrine and metabolic disturbances, and organ or skeletal abnormalities.5 In addition to causing diseases in people, imprinting abnormalities present a challenge in the maintenance of stem cell pluripotency.6 This technology, along with animal cloning, forms the basis of regenerative medicine by offering experimental platforms such as genetically modified animal models and stem cell-derived organoids.
“This approach can significantly improve the developmental outcomes of embryonic stem cells and cloned animals, paving a promising path for the advancement of regenerative medicine,” said Luo.
- Lampert KP. Facultative parthenogenesis in vertebrates: Reproductive error or chance? Sex Dev. 2008;2(6):290-301.
- Li ZK, et al. Generation of bimaternal and bipaternal mice from hypomethylated haploid ESCs with imprinting region deletions. Cell Stem Cell. 2018;23(5):665-676.e4.
- Li et al. Adult bi-paternal offspring generated through direct modification of imprinted genes in mammals. Cell Stem Cell. 2025
- Haig D. Genomic imprinting and kinship: How good is the evidence? Annu Rev Genet. 2004;38(1):553-585.
- Eggermann T, et al. Imprinting disorders. Nat Rev Dis Primers. 2023;9(1):33.
- Cui T, et al. Current advances in haploid stem cells. Protein Cell. 2020;11(1):23-33.
