This study has demonstrated, for the first time, that bi-paternal reproduction in mammals is possible through the correction of genomic imprinting defects. Using 20 targeted genetic modifications, the researchers were able to generate healthy mice from two biological fathers, overcoming barriers that previously made these embryos unviable. The research proves that imprinting is the main obstacle to unisexual reproduction in mammals and opens up new possibilities for genetic engineering and regenerative medicine, although challenges such as abnormal growth and infertility still need to be resolved.
Pluripotent stem cell technology and animal cloning are important advances in science, enabling the development of new approaches in regenerative medicine.
This means that these techniques help in the creation of innovative treatments for diseases and in the study of living organisms in a more controlled manner.
They have also made it possible to generate genetically modified animal models to study human diseases, as well as to create artificial embryos in the laboratory and organoids, small structures that mimic human organs and are made from stem cells.
In addition, these technologies are already being applied in medicine, such as the use of stem cells to treat diseases and even the idea of transplanting organs from animals to humans (xenogeneic transplantation).
However, their true potential is limited by a biological problem called genomic imprinting. This term refers to a mechanism by which certain genes are activated or silenced depending on whether they were inherited from the mother or the father.
When this process fails, major difficulties arise, such as premature death of cloned embryos, problems in generating offspring from embryonic stem cells, and reduced ability of induced stem cells to transform into other types of cells.
Resolving this instability in imprinting could greatly improve the efficiency of these technologies and expand their applications. A few years ago, a major breakthrough was achieved in bi-maternal reproduction (i.e. offspring generated only from two mothers, without the participation of a father).
This was possible by modifying specific regions of DNA called gametic differentially methylated regions (gDMRs), which control the activation of genes inherited from the parents.
By making these changes to haploid embryonic stem cells derived from eggs, the researchers were able to correct genetic flaws and produce healthy offspring. This experiment also paved the way for more advanced gene-editing and chromosome-modification techniques in mammals.
However, while some species of lower vertebrates (such as certain reptiles and amphibians) can reproduce spontaneously without the need for two sexes, this does not occur naturally in mammals.
Bi-paternal reproduction (that is, the creation of embryos from two parents) has never been observed in nature. In fact, even when scientists try to create bi-maternal or bi-paternal embryos in the lab, they do not survive without genetic modifications.
And bi-paternal embryos die even earlier than bi-maternal ones, suggesting that the problem of imprinting has an even more severe impact in these cases.
The aim of this study, conducted by researchers from the Chinese Academy of Sciences, China, is to develop a strategy to enable bi-paternal reproduction, increasing the viability of these embryos and allowing more complex genetic modifications in regenerative medicine.
Previous research has already attempted to modify seven imprinted regions in DNA that are associated with the early death of bi-paternal embryos.
Scientists have shown that haploid embryonic stem cells derived from gametes, when cultured under certain conditions, lose the epigenetic imprinting marker and enter a “neutral” state, with no specific maternal or paternal inheritance marks.
When seven imprinted regions were deleted in this state, bi-paternal mice were able to be born but did not survive, indicating that simply erasing imprinting was not enough to ensure viability.
However, even after these modifications, the embryos still had severe defects and were unable to survive. This raised a fundamental question: are imprinting genes the only barrier that prevents bi-paternal reproduction in mammals?
To answer this question, this study used different gene editing techniques, including gene deletion, specific mutations and changes in regulatory regions of DNA, to try to correct the problems caused by imprinting.
In total, 20 genetic modifications were made, affecting hundreds of genes. Using this approach, the researchers were able to develop a strategy that corrected major developmental defects in bi-paternal embryos.
This allowed, for the first time, the generation of healthy adult mice from two biological parents of the same sex. The study used three different methods to achieve this feat: complementation with embryonic stem cells (ESCs), injection of haploid cells, and nuclear cloning (SCNT).
Viable mice generated via imprinted region deletions in bi-paternal ESCs and tetraploid complementation. (A) Schematic of bi-paternal mouse embryo creation via co-injection of sperm and haploid ESCs with imprinted region deletions into enucleated oocytes, followed by tetraploid complementation. (B) Image of newborn bi-paternal 18KO pups. All are GFP-positive with GFP-negative placentas from tetraploid complementation. Two pups with open eyelids (asterisks) and a GFP-negative wild-type (WT) control are shown. (C–D) Comparison of edema, body weights, abdominal hernias, and macroglossia between 7KO (7 days), 10KO (10 days), and 18KO (18 days) pups. Each dot represents an individual litter or pup.
These results confirm that imprinting defects are the main barrier that prevents bi-paternal reproduction in mammals. Furthermore, the study demonstrates an unprecedented level of control over genetic modification in mammals, opening up new possibilities for genetic engineering and regenerative medicine.
Another interesting finding was that, by correcting imprinting defects, male and female embryonic stem cells had similar stability, suggesting that the differences normally observed may be linked to imprinting and not just to the sex chromosomes.
By analyzing the DNA of the bi-paternal mice, the researchers found that their methylation patterns (chemical marks that control gene activation) were similar to those of normal paternal genomes.
A particular focus was placed on the region of the Peg13 gene, which contains several critical regulatory marks. By modifying this region, it was possible to restore the correct activation of neighboring genes and correct the defects that prevented embryo development.
Despite significant progress, there are still challenges to be overcome. For example, although bi-paternal mice survived to adulthood, many did not reach full maturity and exhibited excessive growth and shorter life expectancy.
(D) Image of adult 19KO mice. Green fluorescence from GFP-tagged bi-paternal ESCs is visible in the feet, mouth, and tail (hairless areas). (E) Growth curves for WT (n = 6, male) and 19KO (n = 7, male) mice. Dashed lines with shading represent standard deviations, and aligned black dots indicate the average weekly body weight ratio of 19KO versus WT mice. Arrows denote the weaning period. (H) Computed tomography (CT) images of 6-week-old 19KO and WT mice. Red and blue arrowheads indicate interfrontal and coronal sutures in 19KO and WT mice.
This suggests that imprinting may have additional roles throughout life beyond embryonic development.
Furthermore, the bi-paternal mice were not able to reproduce naturally, and cloning methods were needed to propagate the lineage. In the future, epigenetic editing techniques, such as the use of specific proteins to modify gene regulation without permanently altering DNA, may be alternatives to avoid the challenges of permanent mutations.
Despite these limitations, the study represents a historic advance in biotechnology, proving that it is possible to overcome genetic barriers to create embryos from two same-sex parents and paving the way for new applications in genetics and regenerative medicine.
Comparison of hippocampal-to-brain volume ratio between WT (n = 3) and 19KO (n = 4) mice at 6 weeks. MRI data showing 3D reconstruction of WT and 19KO mouse brains (gray) and hippocampi (green) at 6 weeks.
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Adult bi-paternal offspring generated through direct modification of imprinted genes in mammals
Zhi-kun Li, Li-bin Wang, Le-yun Wang, Xue-han Sun, Ze-hui Ren, Si-nan Ma, Yu-long Zhao, Chao Liu, Gui-hai Feng, Tao Liu, Tian-shi Pan, Qing-tong Shan, Kai Xu, Guan-zheng Luo, Qi Zhou, and Wei Li
Cell Stem Cell. Available online 28 January 2025
Abstract:
Imprinting abnormalities pose a significant challenge in applications involving embryonic stem cells, induced pluripotent stem cells, and animal cloning, with no universal correction method owing to their complexity and stochastic nature. In this study, we targeted these defects at their source—embryos from same-sex parents—aiming to establish a stable, maintainable imprinting pattern de novo in mammalian cells. Using bi-paternal mouse embryos, which exhibit severe imprinting defects and are typically non-viable, we introduced frameshift mutations, gene deletions, and regulatory edits at 20 key imprinted loci, ultimately achieving the development of fully adult animals, albeit with a relatively low survival rate. The findings provide strong evidence that imprinting abnormalities are a primary barrier to unisexual reproduction in mammals. Moreover, this approach can significantly improve developmental outcomes for embryonic stem cells and cloned animals, opening promising avenues for advancements in regenerative medicine.
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