04 June 2012

Rejuvenate the egg

American scientists have found a way to rejuvenate human eggs with the help of donor mitochondria

Everyone in the cage!
Age is not a hindrance to childbirth

Alla Astakhova, the magazine "Results" No. 23-2012

The head of the laboratory of the Stem Cell Center of the Oregon Health and Science University in the USA, Shukhrat Mitalipov, admits that in the 90s he spent a lot of money from his postgraduate scholarship to feed experimental mice at the Moscow Institute of Medical Genetics. Today he is one of the most successful scientists working in the field of stem cells and reproductive technologies. Several primates have already been born in his laboratory, emerging from combined eggs. As the scientist says, the technology of cytoplasm transplantation will soon benefit people: with its help, it will be possible not only to treat genetic diseases, but also to remove traces of age from eggs.

Photo: Dmitry Plenkin

– Shukhrat, in scientific circles you are known as the person who cloned a monkey for the first time...

– We really managed to get a cloned embryo, but it has not yet come to the birth of clone monkeys. It is necessary for the female to be able to carry such an embryo. But monkeys have miscarriages at an early stage all the time. There are still a lot of problems.

– Does this mean that it is also impossible to clone a person yet? Not for ethical reasons, but because the technology is imperfect?

– As far as I know, yes. Several serious laboratories in the world are working on so–called therapeutic cloning - they are trying to obtain embryonic stem cells by nuclear transplantation, but this has not yet been done. Embryos cannot be grown even to the blastocyst stage – this is about a week. They stop developing earlier. Actually, this technology was not called cloning quite correctly. This is a technique of cell nucleus transplantation. The nucleus of a somatic cell of one organism is transplanted into the cytoplasm of the germ cell of another organism. But that's not all. For example, we are now learning how to transplant cytoplasm from one cell to another and thus treat eggs in women. This will help to fight some diseases inherited through the female line. Other applications of this technology are also possible. For example, for the treatment of age-related infertility. It is known that by the age of 35-40, the quality of eggs decreases. Now in the United States, up to 25 percent of women are starting a family at this age. But their chances of having a healthy baby are decreasing. The percentage of pregnancies after IVF (in vitro fertilization) in the United States in women under 35 is about 30 percent, and after 40 – less than 5 percent. But if the same women use young donor eggs, the percentage of successful implantations increases and they can have healthy children up to the age of 60. IVF specialists have already made it a practice to immediately offer donor cells to those over 40. It turns out that the state of the reproductive system in women remains normal for a long time and the main problem is in the eggs.

– Have you decided to cure eggs of old age?

– We tried to use our technologies to rejuvenate their cytoplasm.

– Do you use the same method to treat genetic diseases?

– In both cases, we replace the cytoplasm of the eggs with a donor one. It is known that not all genes are located in the nucleus. Mitochondria, the energy factories of cells, are located in the cytoplasm.

Mitochondria are the energy factories of the cell.
Now scientists are able to replace them with donor material,
if it becomes necessary (figure from wikipedia.org ).

There are only 37 mitochondrial genes, but they are very important because they are responsible for the use of energy. Mitochondria oxidize organic compounds and use the released energy to synthesize molecules of adenosine triphosphate, a nucleotide that is a universal source of energy for all biochemical processes. Mitochondrial genes do not undergo recombination and are transmitted only through the maternal line. It is believed that in relation to the mitochondrial genome, we are all clones on the maternal side. But it turned out that this was not the case. The problem is that the mitochondrial genome, unlike the nuclear one, is capable of undergoing strong mutations. Now it is thought that there are about 100 times more mutations in it than in the cell nucleus. By the way, one of the theories of aging is based on this: with age, mitochondrial genes in human somatic cells mutate. The older we get, the more such mutations accumulate in tissues and organs. Because of them, mitochondria cannot produce energy, and cells die. Actually, more than 1,000 genes are involved in the process of energy processing, not only 37 mitochondrial genes, but the rest turned out to be in the cell nucleus during evolution. Once mitochondria were separate living bacteria, then there was a symbiosis of bacteria with a cell. By the way, scientists suggest that mitochondria have become an apple from the tree of knowledge of good and evil for living organisms. Early unicellular eukaryotes were immortal before bacteria appeared in them, which later became mitochondria. After that, cells acquired the ability to efficiently receive energy, organisms became multicellular and evolution went up. But now mitochondrial genes determine the life span. At first, all thousands of genes were probably in mitochondria. Then most of them migrated to the nuclear genome. These 37 remained in the cytoplasm, inside the mitochondrion, which produces energy. But it's a dangerous place to store genetic information. It's like storing genes in a nuclear reactor. Mutations occur because reactive oxygen molecules are produced during electron transport, capable of attacking DNA. As a result, we are getting old. Why do we eat antioxidants, vitamins? To protect the mitochondria in cells, whether we know it or not.

– Are the mitochondria of eggs also not protected from mutations?

– They are much better protected due to the fact that they are not used as energy factories. Apparently, this happened in the course of evolution – if they turn on their "nuclear reactors", they will begin to mutate quickly. And the view will quickly disappear. Therefore, in the lines of female germ cells, energy comes from glycolysis – the cleavage of small pieces from glucose molecules. This is very inefficient, but it is safe for the genome. Eggs have another source of energy. Thousands of surrounding cells in the ovaries share it with them. In general, it's like bees – their uterus does not work, but only produces germ cell lines. Something similar is happening here. But until a certain date. It is believed that after 40 years, nature ceases to protect a woman's eggs from mitochondrial DNA mutations. For this reason, the survival rate of embryos drops several times. Apparently, evolution has decided that it is better for such cells not to survive.

– Don't animals have this?

– There are, but such reproductive problems are more or less pronounced only in humans. Therefore, researchers have some hopes that mitochondria can be replaced with younger ones. We have tested this technology on mice, on monkeys. The females successfully produced offspring. Yes, we can transplant mitochondria from young eggs. But the fact is that these animals do not have pronounced reproductive aging. Neither mice nor monkeys simply survive to reproductive old age. Since we don't have a good model, it looks like we will have to show the effectiveness of this method on patients' eggs. Our task is to create a combined cell. First we will get a few eggs from the woman. Then we will transplant cytoplasm from the egg of a young female donor into each of them. We have proved that in this way it is possible to replace mitochondrial genes almost completely or 99 percent. At the same time, both women should not be relatives at all or have any special compatibility. We have done similar things with mice, with monkeys, now we are working with human cells. We do not yet have permission from the FDA (Food and Drug Administration) to conduct clinical trials. Therefore, we fertilize combined human eggs in a test tube and grow them to the stage of weekly development. This is just the beginning. We hope that in a year or two we will get permission to transplant such embryos to patients.

The process of "constructing" an egg
for the subsequent birth of a child without hereditary diseases
(here and further photos – oregonlive.com )

– Do you think it will be difficult to get permission?

– These works are generally not easy to conduct. In the USA, a law has been passed prohibiting the allocation of state funds for the research of human embryos. Therefore, the topic turned out to be out of state funding. No one forbids it, but you can't work without money either. Therefore, we have created a special parallel laboratory so that other funds can be invested in it. After all, we have no right to use the money of private investors in a state laboratory. Now we receive money from private foundations in Oregon and from France. In addition, there is a lot of paperwork to be done – we have to go through a special council authorizing studies in which patients are involved. There are other prohibitions – in many states, for example, it is impossible to pay for egg donation. But if you don't pay, who will give them for free? I fought for a long time at our university – almost three years. Now we have been given permission to pay compensation to women donors. It's not easy.

– Have you already started working with genetic diseases?

– In this case, it is necessary to take an egg from a patient who, as we know, has mutations of mitochondrial genes. How will we find out? Unfortunately, even after at least one sick child was born, because mitochondrial mutations are not reflected in nuclear genes. These mutations mainly cause neurological abnormalities, muscular dystrophy, heart disorders, affect vision and hearing. We ask such women to come and donate eggs. And healthy young women donate their own – we will use donor mitochondria. To date, we do not have permission to transplant embryos to patients. And it also needs FDA approval. By the way, such problems exist not only in the USA. This technology is considered the technology of the future, so in many countries it is also prohibited by law. All we can do is grow embryos in a test tube up to a week old. And wait for FDA approval.

– Will you wait?

– I hope it will happen sooner or later. Treatment methods are developing. For example, we are now thinking about how to learn how to treat not only mitochondrial, but also nuclear DNA. The mutation can be replaced with a healthy copy of the gene. Within 5-10 years, this technology will be fully developed on animals. We are currently conducting some research in this area on monkeys.

Genetically modified monkeys from the laboratory of Shukhrat Mitalipov.
Each cell contains the DNA of two mothers and one father

But you'll have to work hard here. Firstly, it should be a very effective technology. We need to be sure that the gene in the egg is corrected. Secondly, the technique should be safe. But even in this case, ethical discussions are still waiting for us. Now the issue is being debated even in relation to mitochondrial genes. In England, for example, they have been arguing for almost two years whether it is necessary or not to replace mitochondrial DNA. However, permission to transplant embryos is likely to be granted to British scientists. And the first clinical trials will be there.

Portal "Eternal youth" http://vechnayamolodost.ru04.06.2012

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