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Mice with two fathers were born alive for the first time.

Adult mouse with the genomes of two mothers, which gave its own living offspring
Li et al, Cell Stem Cell

Chinese scientists have significantly increased the efficiency of growing adult mice with genomes from two mothers, but without their father. Also for the first time they managed to get the living mice from two fathers. This was achieved through the use of embryonic stem cells and the shutdown of certain sections in their genome, which work differently depending on which parent they are derived from. A study published in the journal Cell Stem Cell .

Parthenogenesis is a form of reproduction in which germ cells develop into an adult organism without any fertilization at all, or without merging the sperm nucleus with the egg cell after fertilization (this variant is called genogenesis). This process occurs in several dozen species of animals, including vertebrates (lizards, amphibians, fish, birds). Most often, females breed in this way, however, similar cases are shown in males of some fish. For mammalian cases of parthenogenesis in nature is not yet known. Cases where viable offspring have same-sex parents, also has not yet been observed.

In this project, scientists worked with the phenomenon of genomic imprinting. This is an epigenetic process, in which the gene works differently depending on whether it is received from the father or from the mother. The inheritance of the relevant features is therefore not based on the classical Mendeleev principle. For example, the allele of the IGF2 gene (insulin-like growth factor) works if it is inherited from the father. Inherited from the mother the same allele will not work. Imprinting is carried out by DNA methylation in the promoter region of the gene to be turned off, as a result of which transcription is blocked. In the genome, such genes are most often located in groups (imprinting chromosome regions). In mammals, they know about a hundred.

One of the main theories explaining the phenomenon of genomic imprinting is the theory of parental opposition. It implies that parents have different “goals” in the context of evolutionary fitness and procreation. Father’s genes “strive” to increase the success of offspring, including at the expense of the mother’s health during pregnancy and after. Mother’s genes, on the contrary, “strive” to preserve maternal resources in order for it to survive and ensure the viability of not only this, but also the next generations.

The difference between the genomes of father and mother, including those related to genomic imprinting, underlies the barriers that prevent same-sex reproduction, and stimulates the mutually beneficial exchange of genetic information between different sexes, and also promotes the spread of useful mutations in the population and maintain competitiveness among offspring.

Back in 2004, another group of researchers had already  succeeded inobtaining mice from two motherless mothers, using immature eggs and turning off the portion of the H19 gene undergoing imprinting in the genomes added to them. One such mouse even grew up as an adult and was able to produce its own offspring.

At the moment, scientists note, it is clear that many aspects that determine the barriers associated with same-sex reproduction are not yet known. So, there are all new data on genes susceptible to imprinting, which so far do not provide a complete picture of what is happening.

In this project, scientists took DNA from embryonic stem cells (ESCs) – these are cells obtained from the blastocyst at an early stage of embryo development (5-6 days after fertilization, such an embryo consists of 50-150 cells). ESCs are pluripotent – they can develop into all types of cells of an adult organism. They can be subjected to cultivation, including in the haploid form (HAASK), when DNA from only one of the parents is stored in the cell.

It turned out that HESCs are suitable by their properties as DNA donors. They are hypomethylated, that is, their DNA is much less methylated than the DNA of eggs, spermatozoa, and fibroblasts. Most of all, their type of methylation resembled primary germ cells, gonocytes, which appear on the 10.5th day of embryo development. A more detailed analysis of specific sections of the ESC genome revealed that in their case, indeed, one can speak about the absence of imprinting effects.

Due to the injection into the immature oocytes of the HESCR with the H19 and IG imprinting sections turned off using CRISPR-Cas9 technology (as it turned out, the mice survived much better if the sections were both turned off) they managed to obtain viable mice from two mothers. However, such mice had significant developmental defects, in addition, they moved slower and could overcome smaller distances compared to the wild type. A comparative analysis of the work of the genes of their brain and the brain of wild-type mice revealed a difference in the work of the three genes susceptible to imprinting – Th , Xlr3b and  Rasgrf1 . They were responsible for low activity, cognitive activity and memory.

Surprising was the fact that experimental mice lived longer than wild-type mice. A comparative analysis of factors affecting metabolism revealed a decrease in the work of the lgf1 gene , which in humans is a factor adversely affecting longevity. In addition, cholesterol was reduced in parthenogenetic mice.

In the new experiment, scientists in addition to H19 and IG also turned off the region of the Rasgrf1 gene . As a result, as many as 29 out of 210 embryos survived (14 percent – compared to the work in 2004, this is more than a tenfold increase in the efficiency of the method). The total levels of genome methylation in these mice were similar to the control ones, and all their imprinting genes were expressed normally. The work of the lgf1 gene also corresponded to the norm.

Mice from two mothers were crossed with normal mice, resulting in a total of 22 mice (out of 6 broods). Nine mice (with the H19 or IG sections turned off separately) died, and the remaining 13 grew up in adult individuals.

For the experiment with two fathers, it was necessary to turn off more sites. In previous studies, it was already shown that the embryos with the DNA of two fathers died at much earlier stages compared with experiments with the DNA of females. Scientists chose six imprinting sites (Nespas, Grb10, Igf2r, Snrpn, Kcnq1, Peg3), turned them off from male GAECs, and co-injected these genomes with sperm into the non-nuclear eggs. Of the 1144 embryos, however, none were able to grow the placenta and did not survive.

As an alternative strategy, scientists made diploid ESCs to create tetraploid embryos, in the hope that they could develop a normal placenta. A total of 1023 embryos were made, of which 12 (1.2 percent) were born alive. Shortly after birth, however, they died. Their weight was twice as high as usual and they had other developmental defects, including suffered from edema and could not breathe properly and suck milk.

It turned out that in such mice hypomethylation of the Gnas region was observed and the corresponding gene was poorly expressed. The researchers turned off another site related to this gene (it was included in the Nespas area) and created 477 embryos with 7 sites already turned off. Twelve mice were born alive (2.5 percent), their weight and other signs were significantly closer to the wild type. Two mice with signs of dropsy were completely absent, managed to live two days.

Analysis of the methylation of the genomes of such mice revealed that, although in general it also turned out to be similar to the wild type, a number of imprinting sites in experimental mice were not methylated, and the corresponding genes (for example, Cdkn1c , Sgce , Plagl1 , Zim1 , Calcr and  Asb4 ) were not expressed like wild type. Obviously, in solving this problem lies the key to further experiments.

Separately, scientists note that the fact that two maternal genomes reduced mouse weight and two paternal ones increased it, in their opinion, supports the theory of “parental confrontation”, since a mother will spend much more than her own resources to grow a large embryo and small offspring will provide more chances for her to survive.

Recall that if such experiments with mammals are complex and require a lot of trial and error, then  some termites living in the wild, in all likelihood, do not develop from fertilized eggs or from unfertilized ones.

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