Two Fathers Producing Offspring: Uncovering the Truth Behind Imprinted Genes

In mammals, the idea that two paternal genomes could combine to produce viable offspring has long seemed like science fiction. Mammalian embryonic development relies heavily on genomic imprinting, a specialized epigenetic system that governs how certain genes are expressed depending on whether they originate from the mother or the father. This regulatory system is established during the formation of sperm and eggs. Through mechanisms such as DNA methylation, histone modifications, and regulation by non-coding RNAs, specific genes are selectively silenced so that only one parental copy is active. As a result, some genes are expressed exclusively from the paternal chromosome, while others function only when inherited from the maternal chromosome.

A well-known example involves the insulin-like growth factor gene IGF-2, which in most mammals is expressed only from the paternal genome. Conversely, the long non-coding RNA gene H19 is expressed from the maternal genome while the paternal copy remains silent. These complementary expression patterns reflect an evolutionary balance—sometimes described as a conflict—between maternal and paternal genetic interests. Once established, these imprinting states remain stable throughout the organism's life.

Genomic imprinting plays essential roles throughout mammalian biology. It affects placental formation, early embryonic development, brain structure, and many physiological processes later in life. Interestingly, genes expressed from the paternal genome often promote fetal growth and enhance nutrient transfer from the mother. In contrast, maternally expressed genes tend to restrain excessive growth, thereby protecting maternal resources.

Earlier studies in mice demonstrated how crucial this balance is. Embryos containing two paternal genomes typically show severe developmental abnormalities. These include excessive fetal growth, enlarged organs, respiratory problems, and major malformations of the brain and face. Such embryos usually die before or shortly after birth. For many years, these outcomes were considered an unavoidable biological barrier.

Recent research has begun to challenge this assumption. Scientists discovered that by directly deleting multiple imprinted regions from the paternal genome, it was possible to produce bi-paternal mice that survived to adulthood. This finding revealed that the main obstacle preventing unisexual reproduction in mammals is not the absence of maternal DNA itself, but the imbalance created by imprinted gene expression.

Why does deleting these imprinted regions help? In cells that contain two paternal genomes, genes normally expressed only from the paternal side may become overactive because both copies are active. This double dosage disrupts embryonic development. Removing one copy of the relevant imprinted regions reduces gene expression levels and restores a more balanced regulatory state. Importantly, in these experiments researchers did not introduce maternal genes. Instead, they modified only paternal chromosomes.

The process began by injecting sperm into enucleated oocytes—egg cells whose nuclei had been removed—to generate haploid androgenetic embryonic stem cells. Using CRISPR/Cas9 gene editing, researchers sequentially deleted several key imprinting regions associated with developmental defects. The modified cell nucleus was then introduced into another enucleated egg together with sperm from a second male mouse, producing embryos carrying two paternal genomes.

Early experiments targeted only seven imprinting regions. Although these embryos could develop far enough to be born, they suffered from severe conditions such as edema, enlarged tongues, umbilical hernias, and severe breathing difficulties, and none survived. As researchers expanded the number of deleted imprinting regions to ten and then eighteen, these abnormalities gradually diminished. Mice with eighteen deletions were able to breathe normally at birth and showed much more typical organ sizes. Nevertheless, they still failed to suckle effectively and most died during early life unless they received artificial feeding.

Flowchart of the procedure for generating imprinting-region–edited mice（Image source：Li ZK et al. (2025)， CC BY 4.0 ）

Further investigation revealed that the suckling failure was linked to abnormalities in the Nnat/Blcap imprinting region. The Nnat gene encodes neuronatin, a protein involved in neural development and cellular differentiation during embryogenesis. In many species it is expressed primarily from the paternal chromosome. The nearby gene Blcap participates in apoptosis and inhibits cell proliferation. Although Blcap itself is not strictly imprinted, its expression is indirectly influenced by the activation of Nnat.

Using the Nnat gene as an example: this gene is expressed only from the paternal chromosome, while the maternal copy remains silent. In a bi-paternal condition this leads to excessive expression. Removing one allele through targeted deletion can reduce the dosage and alleviate the imbalance（By Shui-Ye You）

When researchers removed the imprinting region controlling Nnat and Blcap—representing the nineteenth targeted deletion—the results improved dramatically. Facial proportions, eyelid development, brain structure, and suckling behavior all normalized. These mice could nurse naturally, grow normally, and show active behavior with stable body weight.

Despite these improvements, a major obstacle remained: placental development. Mammalian placentas rely heavily on maternally regulated imprinting genes, and embryos containing only paternal genomes cannot form a functional placenta on their own. In the experiments described above, the mice survived because researchers used a technique called tetraploid complementation. This method provides a functional placenta derived from auxiliary cells, allowing the embryo itself to develop normally.

To overcome this final barrier, scientists examined additional imprinted regions affecting placental formation. They identified the Sfmbt2 gene and a cluster of seventy-two microRNAs located in its first intron as key factors. These microRNAs were abnormally overexpressed in embryos lacking maternal genomes. By deleting the entire Sfmbt2 region—including all seventy-two microRNA genes—researchers produced embryos capable of forming functional placentas. These embryos developed normally to term and healthy bi-paternal mice were delivered by Caesarean section.

To confirm that the extensive genetic modifications had restored normal regulatory balance, scientists analyzed RNA expression across multiple organs of adult mice carrying twenty targeted deletions. Gene expression patterns were highly similar to those of normal mice. Previously misregulated imprinted genes such as Nnat, Peg10, and Kcnk9 returned to stable expression levels. These results indicate that deleting twenty specific imprinting regions can restore a near-normal epigenetic balance.

An unexpected observation emerged when researchers attempted animal cloning using cells from these genetically modified mice. Somatic cells taken from bi-paternal mice produced cloned embryos with a significantly higher success rate than those derived from ordinary mice. This suggests that imprinting abnormalities may be a major reason cloned animals often fail to survive. Because many imprinting defects had already been corrected in these animals, their cells were better suited for nuclear transfer cloning.

Researchers also attempted to breed male and female bi-paternal mice together. These efforts were unsuccessful. Even when imprinting barriers are removed, mammalian reproduction still relies heavily on female germ cells and the specialized cytoplasmic environment of the egg. Genetic modifications alone cannot fully replace the biological roles of the female reproductive system.

Research overview. The upper panel shows the workflow for producing imprinting-region–edited mice, while the lower panel summarizes the outcomes associated with different numbers of imprinting-region deletions（Image source：Li ZK et al. (2025)， CC BY 4.0 ）

The significance of this work extends far beyond the creation of animals with two fathers. The study highlights the fundamental role of imprinting genes in shaping mammalian life history. From embryo size and placental development to organ proportions, behavior, metabolism, and even lifespan, imprinting acts as a regulatory system balancing parental genetic influences.

According to the parental conflict theory, paternal genes tend to promote growth while maternal genes restrain it. The early bi-paternal mice strongly supported this idea: embryos initially showed excessive growth and lethal organ enlargement. As additional imprinting regions were corrected, these developmental imbalances gradually disappeared.

The research also suggests that imprinting may influence longevity. Bi-paternal mice tend to have shorter lifespans, whereas previous studies of bi-maternal mice showed extended longevity. These observations imply that parent-of-origin gene expression may affect physiological processes long after development is complete.

From a broader perspective, this work represents a major advance in developmental biology. It offers new insights into how life emerges from the interaction between maternal and paternal genomes. The approach may eventually provide valuable tools for biomedical research. For example, correcting the Nnat-Blcap region eliminated facial malformations and feeding defects in mice, potentially offering clues for understanding related human developmental disorders. Similarly, the ability to construct a fully functional placenta by modifying imprinting genes could help improve cloning technologies and assisted reproductive techniques in the future.

Author: Shui-Ye You

Reference:

Li ZK et al. (2025). Adult bi-paternal offspring generated through direct modification of imprinted genes in mammals. Cell Stem Cell.

（Paid content. Unauthorized reproduction or use is prohibited.）