Semen or Semenya: problems of choosing a sex
The gender of the South African runner Caster Semenya is one of the most popular topics for speculation today. Gender seems to be an unambiguous obvious fact, but in reality, the development of the sex glands – the main stage of acquiring one or another sex – is an unusually complex and changeable process.
In the article presented to your attention, Professor of the Department of Cell Biology at Duke University Medical Center Blanche Capel, a well-known researcher (more precisely, a well-known researcher of the NICA) in the field of gender issues, discusses atagonistic molecular and cellular interactions occurring at the early stages of embryo development. Her work (the article is included in the October issue of The Scientist magazine) allows the reader to imagine the complexity and intensity of the struggle taking place between the signals that determine the formation of gender. In some cases, none of the programs can get absolute primacy.
The recent controversy over the gender of the South African runner Caster Semenya is a demonstration of the possible difficulties that arise when determining the sex of a person. Experts are forced to choose which criteria are decisive: DNA, genitals or hormones. In most people, these three characteristics correspond to each other, but there are cases when genetic women with two X chromosomes (XX genotype) form male genitals, and vice versa. This happens because in humans, as in most mammals, the genetic sex (genotype XX or XY) controls the development of the genital glands (testes or ovaries) in the embryonic period, while the formation of the genital ducts, external genitalia and secondary sexual characteristics is already regulated by hormones of the genital glands.
In many animals, the signs of sex are quite plastic even in adulthood. For example, in some species of fish, adult females are able to quickly change sex and turn into males. If for some reason the dominant male of the shoal disappears, one of the females changes sex, while acquiring the habits of an alpha male. Such females lose the ability to spawn and begin to produce sperm. A more sophisticated example is some species of moles that have hermaphrodite sex glands with testicular and ovarian components. The sexual characteristics of such animals can repeatedly change in adulthood, depending on the time of year and the situation: sometimes it is more profitable to be submissive, and sometimes, on the contrary, to produce high levels of testosterone and behave aggressively.
What provides such sexual plasticity observed in many animals? Perhaps there is an innate variability of sexual characteristics. For most development processes, there is only one possible outcome. For example, the rudiment of a kidney can only turn into a kidney, and the rudiment of a lung can only turn into a lung. The germ of the sex gland, on the contrary, can develop into both the seminal gland and the ovary. The "choice of sex" occurs in the embryonic period and usually does not change throughout life, however, as we have already said, some species may later reconsider this choice.
Another striking difference between the sex determination process and other developmental processes is that the genes regulating most of the mechanisms that ensure this process are practically unchanged in all representatives of the animal kingdom. The mechanisms themselves that ensure the determination of sex vary very much. In some animals, the determination of the sex of the offspring depends on the density of the population, while in others it depends on the temperature. The human embryo develops inside the uterus, where in most cases it is protected from what is happening in the environment. The determination of his sex occurs with the help of a genetic mechanism, which is based on the X and Y sex chromosomes. There is no single mechanism that would control this process in all vertebrates, but it seems impossible that none of the stages of such an important process is universal for all living things.
When I started working in my own laboratory, it seemed to me that the secret to solving this problem, perhaps, lies in a better understanding of how sex determination occurs at the level of cell biology of organ development. What makes gonadal cells decide in favor of the formation of testes or ovaries, and how do the various mechanisms of sex determination characteristic of different representatives of the animal kingdom regulate this process? The results of a recent study conducted by the staff of my laboratory together with other specialists indicate that, most likely, a common underlying mechanism still exists.
For me, the story began in 1991 with the discovery of a gene that controls the determination of sex in mammals. This discovery, which received a great response in the media, was made in the laboratory of Robin Lovell-Badge at the National Institute of Medical Research in London, where I worked after defending my doctoral dissertation. We isolated the mouse Sry gene, localized on the Y chromosome, and embedded it in the genome of mouse embryos with the XX genotype (female). As a result, the sex of the embryos changed to male.
The removal of the Sry gene from the Y chromosome of embryos with the X-genotype, on the contrary, forced these initially male embryos to develop into female animals. Further work done in collaboration with the Peter Goodfellow laboratory of the Imperial Cancer Research Fund, who studied the human version of the Sry gene, showed that species variations of this gene are part of the Y chromosomes of horses, chimpanzees, rabbits, pigs, cattle and tigers. This fact indicates the preservation of the studied mechanism of sex determination in mammals.
In 1993, I started working in my own laboratory at Duke University. While research groups led by my former colleagues continued to study the Sry gene and other genes directly, I devoted my work to studying the early cellular mechanisms responsible for deciding on the formation of testes or ovaries at the stage of the beginning of the expression of the Sry gene transcription factor in the germ of the reproductive gland.
The first problem for us was the creation of a system that allows us to study mouse genitalia in the process of their development under regulated conditions. The task of creating conditions that would allow the embryonic sex glands to remain viable in the laboratory for several days until they "make" a final decision turned out to be far from easy.
In 1995, together with former colleagues, we decided to test an old hypothesis according to which a population of cells from mesonephros tissue (primary kidney), closely associated with gonads in the early stages of development, migrates to the tissues of the future sex gland. We created a recombinant organ by combining an unpainted germ of the sexual gland and mesonephros, whose cells under certain conditions acquired a blue color due to the beta-galactosidase enzyme gene embedded in their genome. To everyone's delight, after several days of joint cultivation of these fragments, the blue mesonephros cells migrated into the unpainted gonadal tissue, and exclusively possessed the male X-genotype. Penetrating into the male gonad, mesonephros cells surrounded Sertoli cells expressing Sry and formed spermatic cords – the first morphological change indicating the beginning of testicular formation. Placing a membrane between the tissue fragments blocked cell migration and prevented the initiation of testicular formation.
After the failure of many attempts to force mesonephros cells to migrate inside the germ of the female gonad with the XX genotype, we created a so-called "sandwich" culture. To do this, the developing female gonad was placed between the male gonad and the mesonephros. As a result, on their way to the male gonad, mesonephros cells penetrated into the female XX gonad, which, in the absence of the key Sry gene, triggered activation of several genes associated with the development of the male sex gland in its cells and led to the formation of spermatic cords.
These and other experiments gradually changed our ideas about the mechanisms of sex determination. Despite the fact that the Sry gene is at the origin of the sequence of events leading to the determination of sex in mammals, it became clear that the formation of testes depends on other mechanisms, and in their absence, the embryo develops secondary sexual characteristics of the female body.
By the end of the 90s, we had identified several processes that play an important role in the development of gonads, but still did not have a clear idea of the complex of genes controlling them. Luck smiled on us in the face of colleagues from the University of Washington, who created mutant mice in whose body fibroblast growth factor-9 (Fgf9) was not synthesized. Such animals died immediately after birth due to underdevelopment of the lungs. Since all the embryos of these mice developed as female, we assumed that the Fgf9 gene plays one of the key roles in testicular development. Whereas SRY is a transcription factor and can only affect cells expressing a specific protein, and Fgf9 is a secreted protein and acts as a signaling molecule for neighboring cells. Its possible role in the regulation of proliferation and involvement of mesonephros cells attracted our attention.
The results of further work showed that at the bi–potential stage of gonad development – before making a fateful decision about choosing a sex - Fgf9 is expressed by gonad cells of both sexes. However, after starting the expression of Sry, its expression greatly increases in male (XY) gonads and decreases in female (XX). In the gonads that did not have the Fgf9 gene, the development of the testes was completely blocked and a number of signs of ovarian formation were observed.
The addition of the soluble Fgf9 protein to the culture medium induced the migration of mesonephros cells into female sex glands with the XX genotype, shifting their development towards the formation of testes.
All our results pointed to the important role of the Fgf9 gene in the development of male sex glands. The development of the ovaries looked like a passive process taking place in the absence of any specialized factors.
The first refutation of this was obtained in 1999, when Harvard University scientists created mice unable to synthesize the Wnt4 protein. Just like Fgf9, Wnt4 is a secreted signaling molecule and can have an effect on cells removed from its source. In mice without the Wnt4 gene, even in the case of the XX genotype, the developing gonads had signs of seminal glands. This fact seemed particularly interesting because of its comparability with clinically registered cases of women with XX genotype who had testicles in the absence of the Sry gene. According to one possible explanation, these patients experienced a failure of the process that determines the formation of the ovaries and blocks the development of the testicles.
We found that, like Fgf9, at the bi-potential stage of development of the sex glands, Wnt4 is expressed by gonads of both sexes. However, at the moment of making a critical decision, its expression changes in the opposite way to the change in Fgf9 expression: it increases in XX-gonads and decreases in X-gonads.
Among the results of our earlier experiments was the observation that the Fgf9 protein can block the expression of the Wnt4 gene. We assumed that these signaling mechanisms could act antagonistically, thus deciding the fate of developing gonads, and decided to test this hypothesis.
Other researchers have established that the primary role of the Sry protein is up-regulation of the Sox9 transcription factor. Various experiments have shown that the Sox9 protein is able to act as the Sry protein when activating testicular development. We wanted to establish which roles in this play belong to the proteins Wnt4 and Fgf9. The results of carefully planned experiments have shown that Fgf9 and Sox9 enhance each other's signaling effects when embryonic gonads turn into testes. It also turned out that when the Fgf9 gene is removed, male gonads with the X-genotype change sex and activate genes responsible for ovarian formation. However, the most striking discovery was the fact that the activation of both proteins, Sox9 and Fgf9, sharply increases in female XX gonads in the absence of the Wnt4 gene. This clearly demonstrates how the mechanism of formation of male genitalia can be activated in a genetically female XX organism in the complete absence of the Sry gene, as it apparently happened in the men mentioned above with the female XX genotype.
Based on the results obtained, we proposed a new model for determining sex in mammals. Fgf9, Sox9, and Wnt4 genes were simultaneously expressed in both XX and XY embryonic gonads at the early stages of development, when the sex of the sex gland has not yet been determined. The XX-gonads are dominated by the protein Wnt4, which blocks the mechanism of testicular development. At the same time, due to the action of the SRY protein, the Sox9 and Fgf9 pair dominates in the gonads, which leads to the suppression of Wnt4 activity.
Representatives of the animal kingdom use various means that determine the choice of the sex of offspring and, in some cases, adults, from population density and behavioral signals in fish to ambient temperature in turtles, alligators and other reptiles and hormonal influences in many oviparous species. However, there is no doubt that at least one fundamental stage of such an important process as sex determination, at some stage, should be universal for all animals.
I and other experts began to suggest that, despite the diversity of genes governing sex determination in different species, the basic model of antagonistic signals similar to those we observed in Fgf9 and Wnt4 mice is universal. Such a fundamental mechanism may well respond to both a genetic "switch" (Sry in mammals) and environmental conditions (temperature in turtles) as long as the initial solution is reinforced and strengthened by various processes that direct the development of all cells of the developing gland in the right direction.
As the next object of research, we chose a red-eared water turtle, the sex of the offspring of which is determined depending on the ambient temperature. Incubation of the eggs of this turtle at a temperature of 26 degrees Celsius leads to the appearance of exclusively male cubs, whereas at a temperature of 31 degrees, only female turtles are born. (At intermediate values of incubation temperature, the sex ratio of the cubs changes.) To study the cellular mechanisms of the development of the seminal glands and ovaries in turtle embryos, we returned to our old methods of organ cultures.
This work led us to the idea that the antagonistic signal system we discovered is just the tip of the iceberg and that we should devote our work not to individual genes, but to the functioning of the entire complex system of signals that ensure the determination of sex and the development of gonads. We have high hopes for our new project and plan to use many new methods and computational methods of systems biology.
Our understanding of sexual development is changing at the same time as our ability to test and evaluate the process is improving. To date, we have only begun to understand the genetic and cellular processes that influence the initial stages of gonad differentiation. The subsequent effects of hormones, the environment and the nervous system also play critical roles in the final identification of an individual as "male" or "female".
Due to the exceptional complexity of the issue, the Sry protein detection tests used by many sports organizations to obtain a final decision on determining the gender of participants in the competition look very simplistic. Among other things, such an assessment excludes the existence of a category for qualified individuals with one or another combination of male and female characteristics. At the same time, such individuals also possess a certain range of abilities. As for Caster Semenya, it is a pity that such an outstanding achievement is overshadowed by accusations caused more by her non-compliance with Western standards of female attractiveness than by deliberate misinformation about her gender.
Translated by Evgenia Ryabtseva
Portal "Eternal youth" http://vechnayamolodost.ru based on the materials of The Scientist: Choosing Sex.
29.09.2009