Artificial gametogenesis: scientific advances and challenges

Gametogenesis and natural fertilization are essential processes in sexual reproduction. They are the mechanism through which gametes (eggs and sperm) are generated from germ cells which, after undergoing meiosis, reduce their chromosomal load by half to allow genetic combination during fertilization. However, recent advances in biotechnology have paved the way for the production of artificial gametes from somatic cells. While this achievement promises to revolutionize reproductive medicine, it also raises profound bioethical dilemmas about the manipulation of human life and the limits of scientific intervention in natural processes.

Gametogenesis and natural fertilization

In sexually-reproducing organisms such as man, each sex has a tissue called “germinal tissue”, which is responsible for producing the gametes through a process of “gametogenesis”. To achieve this, the stem cells of the gametes (which have 2n chromosomes, one set for each parent) undergo a process called “meiosis”, which consists of two cell divisions after a single chromosomal replication. At the end of meiosis, one cell with 2n chromosomes will have become four cells with n chromosomes each. In this way, the “eggs” and “sperm” are produced in the female and male germinal tissues, respectively, each of which carry a basic set of 23 chromosomes. “Fertilization” consists of the fusion of a maternal gamete with a paternal one to give rise to the “zygote”, which already has 46 chromosomes and a unique combination of paternal and maternal genes, different from that of either parent.

It is important to know what happens during meiosis in order to understand the halving of chromosomes and the resulting genetic variation, and that it is a biological necessity for the survival and diversity of sexually reproducing species.

Before meiosis begins, the chromosomes in the cells that will give rise to the gametes consist of two sister “chromatids”, resulting from DNA replication after the last cell division. During the long prophase of meiosis I, each of the chromosomes aligns with its homologue and their chromatids pair along their lengths (maternal with paternal), forming 23 bivalents. This is followed by a reciprocal and random exchange of regions between the paternal and maternal chromatids, known as “crossing over”. These events of alignment and crossing over of non-sister chromatids are evidenced by the observation of temporary physical connections, called “chiasmata”. As a result of this exchange, new combinations of genes are produced, known as “recombination”. After prophase, during metaphase and anaphase, the homologous chromosomes in each bivalent are separated and move towards opposite poles of the cell. The daughter cells randomly receive one chromosome from each bivalent (paternal or maternal) and contain a complete set of 23 recombinant chromosomes. In the case of female gametogenesis, one of the daughter cells will become the egg and the other a polar body, remaining at the periphery of the cell. In male gametogenesis, both daughter cells are equivalent and functional. Next, meiosis II takes place, which is similar to “mitosis”, during which the sister chromatids are pulled towards opposite poles. In the case of female gametogenesis, one of the cells is still functional and will give rise to the “egg”, while the other becomes a second polar body. In male gametogenesis, the four resulting cells mature into “sperm”. At the end of meiosis, each gamete carries 23 chromosomes and a complete set of genes resulting from the exchange between the maternal and paternal chromosomes.

Artificial production of gametes

Applied biotechnology in the field of cell biology has been trying for years to generate “artificial gametes”, which would not originate from germinal tissue or from a meiotic process such as the one just described, but from other tissues or from appropriately manipulated somatic cells. For some time now, assisted human reproduction clinics have wanted to be able to offer their clients gamete-like cells, especially eggs.

In brief, their aim is to use somatic cells from a tissue other than the germinal tissue as a source of eggs or sperm. To this end, it will be necessary to induce artificial oogenesis or spermatogenesis in vitro, leading to a reduction in the number of chromosomes and potential recombination. As we shall see, the results of the research conducted thus far are only partial and, to date, only feasible for the production of artificial eggs.

Induced pluripotent stem (iPS) cells obtained from somatic cells (2n) through genetic reprogramming using the Yamanaka method^[1] have been tested as a source of non-germ cells. A first attempt was carried out by a Japanese research group from Kyoto University, the results of which were published in Science in 2012. The team used both embryonic stem cells and iPS cells from female mice to induce epiblast-like cells (epiLCs), which they were able to transform into female germ-like cells with 2n chromosomes. When aggregated with female gonadal somatic cells as reconstituted ovaries, these cells underwent reactivation of the deleted X chromosome (remember that in the early embryogenesis of mammalian females, one of the random X chromosomes—paternal or maternal—is inactivated in each cell by the phenomenon known as lyonization). The resulting cells exhibited meiotic potential and, upon transplantation under mouse ovarian bursa, grew to become functional oocytes, even giving rise to viable embryos by in vitro fertilization.^[2]

Artificial gametogenesis in humans

For many years, various research groups have been attempting to artificially create human eggs. Recently, a team led by Russian researcher Shoukhrat Mitalipov (b. 1961) from the Oregon Health and Science University (OHSU) in the USA published the results of their attempt in Nature Communications,^[3] in which they used an approach that differed from previous efforts. They fundamentally used somatic cell nuclear transfer (SCNT) technology in unfertilized enucleated oocytes, the same technique used to clone Dolly the sheep. In this case, the nuclei of the unfertilized eggs were replaced with nuclei from skin fibroblasts, a type of somatic cell. This is direct reprogramming of somatic cells into functional oocytes, albeit with a diploid genome.

The fact that the reprogrammed oocytes contain a diploid (2n) set of chromosomes would invalidate their use as artificial gametes, since eventual in vitro fertilization with haploid sperm would give rise to zygotes with three sets of chromosomes (triploids). Therefore, in the aforementioned research, the reduction of chromosomes from 2n to n was addressed through an experimental process of reductive cell division, termed “mitomeiosis”, wherein the somatic genomes are prematurely forced to divide following transplantation into the cytoplasm of previously enucleated oocytes at the metaphase stage of meiosis I. It should be noted that female gametogenesis to form the eggs begins during the first few months of fetal development, but the process arrests at the end of prophase of meiosis I. The oocyte-generating cells will resume meiosis upon reaching sexual maturity. Maternal imprinting occurs asynchronously as oogenesis is completed, when meiosis resumes to produce a single egg in each menstrual cycle.^[4]

Because of this, oocytes that receive the somatic cell nucleus by SCNT remain arrested at metaphase due to a failure in the activation required to continue to metaphse. To overcome this difficulty, Mitalipov’s team tested artificial activation using an enzymatic treatment, successfully bypassing the arrest and inducing the segregation of somatic chromosomes and cell division to give rise to two cells, which would generate the artificial egg and a polar body. Although the difficulty of reducing 2n to n chromosomes was overcome, the distribution of paternal and maternal chromosomes to the two daughter cells was irregular and without recombination due to the absence of pairing and crossover of homologous chromosomes. However, some of the artificial SCNT oocytes produced generated viable zygotes when subjected to fertilization, demonstrating the possibility of experimentally reducing the number of diploid chromosomes.

Viable zygotes with 46 chromosomes underwent normal embryonic cell divisions and developed into embryos with integrated somatic chromosomes. While this study demonstrates the potential of “mitomeiosis” for the artificial gametogenesis of oocytes with 23 chromosomes, it is a first step—or as its authors prefer to say, a proof of concept—that requires further research to ensure its efficacy and safety before its application for the intended purposes.

Bioethical aspects

Biotechnology in relation to human reproduction has drawn criticism for years, largely due to its applications for various purposes that disregard some basic principles of bioethics and human dignity, especially at the beginning of life. Science has made it possible to create embryos in the laboratory, freeze them, select them, and use them in research; it has also allowed scientists to generate embryo models, three-parent embryos, embryo chimeras, etc. Why not then produce gamete substitutes from non-germ cells? To what end?

The idea of artificial gametes is more about filling a desire than a need. It is about producing gamete-like cells in vitro from cells of those who wish to have a genetically related child when this is not possible through normal fertilization. In theory, it is presented as a way of tackling infertility, when a woman cannot have children for natural reasons, or is postmenopausal, or has a high risk of transmitting a serious disease. However, it goes further and seeks to provide a way for same-sex couples, single people, and deceased donors to have children. Are all these reproductive scenarios ethically acceptable?

Another potential use of artificial gametes and the embryos derived from them is of course for research in relation to infertility, developmental problems, etc.

In principle, obtaining human eggs and sperm from non-germ cells in the laboratory should not in itself raise any ethical concerns—as long as they are not fertilized—even if this research involves genetic modifications of the gametes, since there are no lives at stake. Lives are endangered, however, from the moment of fertilization, specifically of the zygotes and embryos that may result from these experiments, with the added risk of possible harmful effects that could arise during their development, which may be impaired due to manipulation of the cells from which they are derived. There are many issues related to epigenetic modifications that determine the necessary differential imprinting of future male or female gametes. There is also the ever-present risk of possible epigenetic alterations due to the conditions of in vitro cell manipulation, with more serious consequences in this case as they are germ cells, which could even transmit errors to subsequent generations.^[3]

Another very important aspect is the destruction of embryos during their production due to the very artificiality of the process by which they are obtained, or because they do not satisfy the expectations or certainty of their development, which is not without risks and uncertainties.

In the case of the research by Mitalipov and his team for the production of artificial eggs using the SCNT method, a very important phenomenon that takes place in natural meiosis but not in artificial “mitomeiosis” is overlooked. It concerns genetic recombination. The exchange of homologous regions between maternal and paternal homologous chromosomes is the main source of genetic diversity, apart from mutations. This fact alone goes against natural logic, as it assumes a reduction in the genetic variation characteristic of sexually-reproducing beings, with unpredictable consequences in the case of humans.

Furthermore, the gametes derived from these methodologies could be used in genome editing experiments, such as CRISPR/Cas9, as a means of investigating hereditary diseases, or worse, for genetic enhancement. This is a topic that connects with genome editing applications and that raises huge questions and ethical concerns.

Moreover, for cases of infertility, to satisfy the desire to have a child when this is not possible due to circumstances, is it not simpler and more humane to adopt, knowing also the human tragedies faced by so many children who have been abandoned or at risk of social exclusion?

The advent of artificial gametes and, consequently, the potential individuals originating from them, raises important bioethical questions within the scope of unnatural practices in relation to human reproduction, such as the distinction between embryos of artificial and natural origin, the human rights of children that may result from these practices, etc. Regardless, a regulatory framework must be established that outlines ethically acceptable pathways for human reproduction in general and for the manipulation of gametes and embryos in particular.