Surrogate fathers
CRISPR editing turned males into sterile recipients of someone else's sperm
Daria Spasskaya, N+1
American geneticists and veterinarians have tested the creation of mutant sterile farm animals in order to further transplant spermatogenic cells from males with valuable qualities to such pigs, goats and bulls and make them "surrogate fathers". To do this, scientists had to clone animals, as well as edit embryos using CRISPR to turn off the NANOS2 gene in them. The results of the experiments are described in an article in the Proceedings of the National Academy of Sciences (Ciccarelli et al., Donor-derived spermatogenesis following stem cell transplantation in sterile NANOS2 knockout males).
Transplantation of spermatogenic stem cells into the testes may be in demand in medicine, for example, in this way it is theoretically possible to restore fertility to a man after radiation therapy for cancer (this possibility has already been shown on monkeys). Animal husbandry is even more interested in this technology – by transplanting material from a valuable male producer into healthy testes of sterile males, it is possible to significantly increase the number of offspring with the specified qualities.
The breeding of "surrogate fathers" faces two main problems. The first is actually cell transplantation. Studies on mice have shown that there is a small population of sperm precursors (spermatogonia) in the testes, which, after isolation, can be turned into mature functional sperm. In experiments on mouse models, it was confirmed that such cells can indeed be transplanted, but whether this works on large farm animals was unclear.
The second problem concerns the sterilization of potential stem cell recipients. Their own seed should be completely destroyed to avoid competition with the donor's material, while the reproductive system should function normally. In experimental models, this was solved by local irradiation of the testes or chemical sterilization with busulfan. However, these methods lead to the accumulation of mutations in the cells of the reproductive system, besides, they do not guarantee absolute sterility.
Researchers from the Center for Reproductive Biology at the University of Washington, led by Jon Oatley, tried to solve the problem of sterility of recipients by creating transgenic animals with a mutation in the NANOS2 gene. This gene plays a key role in the differentiation of germ cells by male type, and when it is turned off, spermatozoa do not form.
The scientists conducted the first round of experiments on mice, with a ready-made line of animals with NANOS2 turned off. It is possible to maintain such a line in the laboratory due to the fact that only males with both copies of the gene turned off are sterile, and heterozygous males and females retain fertility.
The testicles of mice with the deletion of the NANOS2 gene are smaller in size than normal ones. Drawings from an article in PNAS.
One-month-old white sterile mice were successfully transplanted with stem spermatogonia from donors with black hair, and confirmed that this operation leads to the restoration of fertility, and that the offspring one hundred percent carries the genetic material of donors. At the same time, the recipients did not have immune rejection of the donor material, which scientists had previously worried about.
After that, the researchers moved on to experiments on large animals – pigs and goats, genetic operations with which have already been pretty well worked out. To obtain mutant pigs with a mutation in NANOS2, genetics first obtained pig connective tissue cells with two broken copies of the gene using CRISPR-Cas9. Then, by transferring somatic nuclei into an egg (Dolly the sheep was cloned in this way), they cloned embryos, from which, after being planted to surrogate mothers, several sterile piglets were born.
Experiment on transplantation of spermatogenic stem cells to a mutant hog using ultrasound.
Unfortunately, some of the cloned pigs had problems with their bones, so only two pigs managed to transplant stem cells into the testes. As they grew, their ejaculate was regularly examined and the presence of spermatozoa in it was checked. In one animal, stem cells actually developed into spermatozoa, but they turned out to be too few for normal fertilization. Nevertheless, scientists considered the experiment successful and repeated it on goats. The mutant goats turned out to be healthy, so geneticists concluded that the bone problems in pigs were not related to the shutdown of the NANOS2 gene. Spermatogenic stem cells were injected into three goats, and 136 days after transplantation, one of them developed normal motile spermatozoa.
In the third series of experiments, geneticists tried to breed sterile mutant bulls. To do this, scientists had to carry out CRISPR editing of embryos in order to inactivate the NANOS2 gene in them (apparently, it is more difficult to clone cows from fibroblasts than pigs). Out of 32 transplanted embryos, only two calves were born, which turned out to be mosaics according to the Nanos2 gene, that is, in some tissues the gene was broken, and in some it turned out to be intact (mosaicism is the main problem of CRISPR editing of embryos, along with inappropriate editing). Nevertheless, a biopsy of the testicular tissues showed that the bulls should be sterile. Thus, the authors concluded that switching off the NANOS2 gene works effectively on different types of farm animals to obtain sterile recipients and future cultivation of "surrogate fathers".
Another potential application of this technology is the conservation of endangered species. For example, scientists are currently trying to save the northern white rhinos from the final extinction. All males of this subspecies have already died, but their frozen sperm was used to obtain viable embryos by fertilizing the eggs of the remaining females in a test tube.
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