Difficult, but possible
Spermatozoa from stem cells helped to conceive baby rats
Polina Loseva, N+1
Japanese scientists have grown spermatozoa from rat embryonic cells. Until now, such experiments have been carried out only on mice, it has not been possible to do this for other species. The resulting spermatozoa turned out to be functional — they can be used to conceive viable offspring. However, they cannot fertilize an egg on their own yet, and they do not develop without donor supporting cells. The work was published in the journal Science (Oikawa et al., Functional primordial germ cell–like cells from pluripotent stem cells in rats).
Artificial insemination technologies help to conceive a child for those for whom, for some reason, natural insemination is impossible. But this only works when there are germ cells, even if they are not completely healthy. But for those people in whose body germ cells are not produced for some reason, there is nothing to help yet.
Germ cells could be grown from stem cells, but there are several difficulties with this. Firstly, to do this, you will have to take the primary germ cells — a group of cells that separate from the rest quite early, in humans it is around two weeks of development. Secondly, there are very few primary germ cells in a real embryo, literally several dozen, so it is quite difficult to study their formation and development.
For the first time, a group of Japanese scientists managed to grow primary germ cells and get sperm from them in 2011. They worked on mice, and no one has repeated their results for any other species yet. Now another group of Japanese scientists, Mami Oikawa from The University of Tokyo and her colleagues tried to do the same for a rat.
Despite the fact that both types of rodents are used in many works on physiology and behavior, a significant part of embryological studies has been done on mice. Therefore, the authors of the work had to adapt all the stages of the protocol for rats, and at the same time understand the features of rat embryogenesis.
They used rat embryonic stem cells as raw materials for sperm production. They had to be turned into an epiblast — in an embryo, this is a layer of cells that are ready to specialize and divide into several layers of the embryo, including budding off primary germ cells. It turned out that, although mouse epiblast is easy to cultivate on a plane, rat cells do not keep in a single layer, and I had to learn how to grow them in the form of three-dimensional spheres.
From left to right: rat blastocyst, embryo after implantation (epiblast lining it "from the inside"), culture of embryonic stem cells from blastocyst, culture of epiblast-like cells. Drawings from the article by Oikawa et al.
Then scientists had to select a set of signaling substances with which epiblast cells can be turned into primary germ cells. And in order for them to mature to the real precursors of spermatozoa, they had to be settled together with the supporting cells of the developing sex glands. Researchers took such glands from older rat embryos and cleaned them of their own sperm precursors (for this, again, they had to develop a new technique, because they do not produce the same molecular markers as mouse cells). However, the design worked only with the female sex glands — for some reason, the precursors of spermatozoa refused to develop in the male ones.
The protocol for obtaining spermatozoa: embryonic stem cells, then epiblast-like cells, then primary germ cells, then co-cultivation with supporting cells, then implantation into empty sex glands and sperm at the exit.
As a result, a multi-stage method was obtained: first, embryonic rat stem cells, then transformation into an epiblast, then separation of primary germ cells, then cultivation in immature glands with transformation into mature sperm precursors. After all these manipulations, the scientists checked what epigenetic changes occur in the resulting precursors — that is, on which parts of DNA and histones characteristic markers appear. And they confirmed that these changes are similar to those that occur in real cells in the rat body.
Finally, it remains to make sure that these precursors give full-fledged spermatozoa. To do this, the researchers raised rats with a defect in one gene that is responsible for the development of germ cells. They develop sex glands, but "empty", without spermatozoa. Researchers planted progenitor cells in such rats - and they began to produce spermatozoa. There were fewer of them than in mice in similar experiments. And the mice could not fertilize the female with them on their own. But with the help of artificial insemination, scientists introduced these spermatozoa into eggs — and the rats turned out to be healthy and fertile.
On the left: baby rats obtained from artificially grown spermatozoa, on the right: a rat conceived with the help of such a sperm grows healthy and fertile and gives its own offspring.
This work is a clear example of how difficult it can be to transfer a working technique from one type to another and what tricks you have to go to for this. Therefore, although the authors mention that the rat is physiologically closer to a human than a mouse, we can expect that the next step will also be difficult. In addition, in order to fully achieve their goal, researchers need to learn how to grow full-fledged spermatozoa capable of independent conception. And ideally, do it without supporting testicular cells.
With eggs, the situation is no easier than with spermatozoa. In 2021, scientists have finally assembled an artificial follicle with which it is possible to grow mouse eggs in vitro "from scratch". We talked about what technologies will become possible if we learn how to do this with human and other species' eggs in the text "Mother from a test tube".
Portal "Eternal youth" http://vechnayamolodost.ru