Van chromosoom naar DNA in zes stappen.

Image: iStockPhoto toricheks

From chromosomes to genes

It is known from life descriptions prior to 1900 that sex diversity had long been known even then. At that time, viewing the genitals was the only way to determine a person’s gender (Rousseau 1980). Since then, technical knowledge about the body has increased. However, it remained that the knowledge of the moment determined who was male and who was female. Despite the fact that knowledge increasingly confirmed that sex was not a dichotomy, sex diversity became less accepted: people who did not fit into that imaginary dichotomy were forced to confirm with operations that were not possible before. In doing so, the principle was again and again that with better medical technology, doctors could determine who was really a man or really a woman. And time and again, new knowledge showed that the old views were wrong.

The history of the chromosome makes this painfully clear:

Buste van Aristoteles. Marmer, Romeinse kopie naar een Grieks origineel van brons door Lysippos uit 330 voor onze jaartelling. De mantel van albast is een moderne toevoeging. Foto: Jastrow 2006.

Bust of Aristotle. Marble, Roman copy after a Greek original of bronze by Lysippos from 330 BC. The mantle of alabaster is a modern addition. Photo: Jastrow 2006.

That traits can be passed on to offspring was also not lost on the philosopher Aristotle (384-322 before the Western era). He assumed that the man contributed through the sperm and that the woman’s characteristics were transmitted through blood left in the womb after menstruation. Sperm, according to Aristotle, arose from blood that boiled in the veins of the pelvis during sexual arousal. This idea of procreation and heredity lives on in words and expressions like blood relatives, my blood child, my own flesh and blood and blood is thicker than water (Kremer 2003)1Aristotle was wrong, that much clear.

Although the existence of sperm had of course long been known, Anthoni van Leeuwenhoek was the first to see male sperm cells with his microscope. He called them “sperm animals” – the energetically swimming, nematode-like animals played an important role in the creation of the embryo, according to him. That idea did not fit well with the prevailing idea that all creatures, including viviparous creatures, originated from an egg. (Mittwoch 2005)

Back in the days of Antoni van Leeuwenhoek, Renier de Graaf was doing research on female oocytes in rabbits. He never got to see the egg, but concluded on the basis of the follicles that now bear his name that the embryo is formed from the female egg and not from a ‘sperm’. It was not until 1827, more than a century and a half after Van Leeuwenhoek’s publication, that Carl von Baer published on the oocyte located in the Graaf’s follicle. Baer considered the ‘spermids’ to be parasites and gave them the name ‘spermatozoa’. The view we now have of the roles of sperm and egg came about when Alfred Kölliker showed in 1841 that the ‘sperm animals’ are cells produced in the testes (i.e. not animals) and Oscar Hertwig described in 1876 how the nuclei of egg and sperm cells come together. (Mittwoch 2005)

But with all this knowledge, science still had no idea how the difference between men and women arose.

Based on a book that was published about 40 years earlier (but largely ignored at the time) by Moravian-Austrian Augustinian Gregor Mendel (1822-1884), popular belief started to be that characteristics were passed from one generation to the next. In the process, it was soon discovered that heredity is related to chromosomes located in the gametes (Morgan 1966). Mendel’s ideas are still valid today. For that reason, heredity in organisms in which the offspring receive half of the genes from each parent is still called Mendelian inheritance: characteristics are passed on according to Mendel’s Laws.

Although the existence of chromosomes was already known around 1890, for a long time there was uncertainty about the number of chromosomes and the possible presence of a Y chromosome (Gartler 2006). In 1921 the American zoologist Theophilus Painter published research showing that humans must have 46 or 48 chromosomes and that there was a Y chromosome (Painter 1921). It would be another thirty years before two publications described how chromosomes could be used to determine the ‘chromosomal sex’ and variations thereof.

In 1928, British bacteriologist Fred Griffith discovered that a mixture of live harmless bacteria and dead harmful bacteria was capable of taking the life of a mouse. He concluded that something in the dead bacteria had changed the living bacteria. He called that process “transformation. (Griffith 1928) The peculiarity of this experiment is the lack of sperm or an egg; two things that even today many people consider essential for passing on characteristics to offspring.

After Griffith demonstrated that living bacteria can inherit characteristics from dead bacteria, disagreement arose over the cause of this. One camp said that proteins were the carrier of the hereditary information, the other camp said that DNA was responsible for passing on characteristics. Canadian physician and researcher Oswald Avery, along with his colleagues Colin MacLeod and Maclyn McCarty, foolishly started trying it out: each time, a new part of the dead bacteria was introduced to living bacteria. This research showed that only DNA and nothing else was responsible for passing on hereditary characteristics (Avery 1944).

It may sound strange, but linking genes to hormones does not necessarily require knowledge of the DNA structure of a hormone. As a result, a gene map for the fruit fly already existed in 1913 (Sturtevant 1913). Such a map was possible by studying many generations of fruit flies and analyzing the data statistically. For the human genome, however, this proved more complicated. The necessary mathematical basis was not described until the early 1930s (Bernstein 1931, Hogben 1934) and practical problems (e.g., the speed of reproduction and the lack of fast computers) made it virtually impossible to localize autosomal genes. The first hereditary disorders linked to a chromosome in humans, hemophilia and color blindness, were located on the X chromosome for this reason (Bell 1937).

Canadian physician and researcher Murray Barr and his student Ewart Bertram discovered a globule of matter in cells of women that is not present in cells of men (Barr 1949). That globule is today named after its discoverer: the so-called “Barr body” (also known as sex chromatin). The Barr body occurs upon X-inactivation and is a sign that more than one X chromosome is present. This invention led to the sex chromatin test in 1955 which allowed one’s chromosomal sex to be determined with only a microscope and staining of the cells (Marberger 1955, Moore 1955).

Between 1968 and 1991, this test was used at the Olympics to determine whether someone should compete as a woman (did have a Barr body in their cells) or a man (no Barr body). Remarkable detail: only people who wanted to compete as women were tested; if men had also been tested, men with Klinefelter Syndrome (47,XXY) would theoretically have been allowed to compete among the women. Polish sprinter Ewa Klobukowska was the first athlete to be “unmasked” by this test ; her medals were taken away from her and she was publicly shamed. It is now assumed that her chromosome pattern was XX/XXY mosaic. (Ritchie 2008)

Many people think that James Watson and Francis Crick discovered DNA. That is incorrect – they described the structure of DNA (Watson 1953). The schematic graphic representation of that structure, the double helix (drawn by Odile Crick – Francis Crick’s wife (Olby 2003)), is so simple that it seems anyone can understand what DNA is.

Yet 84 years and dozens of publications lie between the discovery of DNA and the description of its structure. DNA had already been discovered by Fritz Miescher in 1869, although he called it “Nuclein” (Olby 1974). Until the publication of Watson and Crick, numerous scientists had tried to achieve the same thing and sometimes they were close.

The work of Watson and Crick was built on the work of many other scientists. Of these, Maurice Wilkins and Rosalind Franklin may have been the most important. Franklin’s X-ray diffraction photographs of DNA arguably underpinned Watson’s ideas about the double helix structure of DNA (Maddox 2002). In 1962, Maurice Wilkins, Francis Crick, and James Watson received a Nobel Prize for their work. Rosalind Franklin had already died by then. Whether she would have shared the prize is unknown; the Nobel Prize is not awarded posthumously nor to more than three people.

For thirty years, scientists thought that humans had 48 chromosomes (Harper 2008). It was not until 1957 that Indonesian-American cytogeneticist Joe-Hin Tjio and Swedish botanist and geneticist Albert Levan proved that most people have 46 chromosomes(Tjio 1956).

From then on, the technique was available to investigate chromosomal variations in miscarriages (Boué 1975, Kim 1975) and perinatal mortality (Machin 1974). This is not to say, incidentally, that chromosomal variations now seen as forms of sex diversity were not known before. Based on physical characteristics, Turner Syndrome (45,X) had been described as early as 1938 (Turner 1938) Klinefelter Syndrome (47,XXY) as early as 1942 (Klinefelter 1942).

After the structure of DNA became known, it was obvious that research would be done to determine its exact composition.

By 1977, a method had been published to determine the sequence of nucleotides in DNA. Ten years later, in 1987, the first machine to automate this work hit the market (Venter 2001).

The fact that chromosomes had not proven to be a perfect indicator of people’s sex and gender identity was all the more reason to look for genetic evidence of the difference between men and women. A 1990 publication introduced a new gene that was confidently called the SRY gene: Sex Determining Region Y (Sex Determining Region [on the] Y-[chromosome]) (Sinclair 1990).

In 1991 they had already decided to use the SRY gene in the sex test at the Olympic Games from then on. Because the SRY test also proved not to be useful, it was decided in 1991 to stop such sex tests (Ritchie 2008).

Also, in the search for genetic evidence for the cause of different forms of sex diversity, the results were not universally applicable. For example, several research groups, including one in the Netherlands, worked on information about the Androgen Receptor gene in the late 1980s (Brinkmann 1989). By mapping the Androgen Receptor gene, for example, it became possible to determine in girls with XY chromosomes whether they had androgen insensitivity syndrome or yet another form of sex diversity.  But again, there appeared to be more exceptions than expected.

Even in 2018, no genetic variation can be found in about half of the people affected by a form of 46,XY sex diversity (Cools 2018). A 2012 review article providing insight into the large number of genes involved in different forms of sex diversity even states that genetic variation was found in 20 percent of cases (Ono 2012). In other words, genetic testing can make a diagnosis with certainty, but cannot exclude a diagnosis with certainty.

In 1990, work began to map all of the approximately 3 billion base pairs of the human genome. Despite the fact that the state of the art made the Human Genome Project seem unfeasible at first, a rough and incomplete map was presented on July 7, 2000. On February 15, 2001, the accompanying scientific article was published in Nature. (IHGSC 2001, Collins 2003)

That the data was placed in the public domain while the research was ongoing had a reason. Celera Genomics, a commercial laboratory, was also mapping the human genome and was using a faster technique than the Human Genome Project. Celera’s goal was to patent genes (Collins 2003). On February 16, 2001, Celera published an article in Science about their gene map that had been worked on for only nine months and yet was based on a number of base pairs equivalent to 5.11 times the complete human genome (Venter 2001). With the move by the Human Genome Project, Celera was immediately unable to patent a significant portion of the genes they found (Collins 2003).

On April 14, 2003, the Human Genome Project was completed: 98% of the human genome was then said to have been determined with an accuracy of 99.99 percent.(Collins 2003)

What caused particular surprise was the number of genes found. That was at about 22,300 genes (according to Celera) or 30,000 to 40,000 genes (according to the International Human Genome Sequencing Consortium) – about the same number as in other mammals. It was originally thought that a complicated animal like humans would have more genes. Some areas (January 2019) are still uncharted.

Nothing came of Celera’s patenting of genes. After U.S. President Bill Clinton and British Prime Minister Tony Blair indicated in a joint statement their opposition to patenting newly found genes, companies like Celera lost much of their stock market value. The Nasdaq lost 200 points, the second-largest loss in history, and ten biotechnology companies were worth a combined $30 billion less that day. (Sulston 2002)

Anyone who has read through the history above will notice that the definition of man and woman through time is highly dependent on state of the art biological and technological research from that time.

Before there was any knowledge of chromosomes and genes, external genitalia was considered: penis = male, no penis = female. Before 1900, assigning a gender to an intersex child was a legal and social problem – in fact, doctors knew as little about intersex as, say, judges or pastors. But with the new knowledge about heredity, chromosomes, and gene, doctors became the experts who could determine a person’s true sex. And as knowledge increased, so did confusion, for each time determining one’s gender was more complicated than expected:

Sex determination throughout the years

  • Before
  • Looking at appearance
  • Although this seems like the simplest way, there is a lot of overlap between the sexes. For example, just because men are taller on average than women does not mean that someone who is 6 feet tall is a man. Even from the shape of the skull or pelvis, sex cannot always be determined with certainty.
    Penis = man, no penis = woman.2 Speaking in medical terms, men with Klinefelter syndrome have wide hips that can easily be seen as feminine. Due to the influence of testosterone, the body of women with congenital adrenal hyperplasia can have masculine features. For these reasons, the body is not a good indicator of sex.
  • 1950
  • Looking at the gonads
  • Around this time, anesthesia for children became available. This made it possible to perform surgery and remove the gonads in children. A microscope could then be used to determine whether testicular or ovarian tissue was present.
    Testicular tissue = boy, ovarian tissue = girl.3The test is not useful for determining gender because women who have a medical diagnosis of androgen insensitivity syndrome have testes AND in the vast majority of cases have a female body and a female gender identity.
  • 1950
  • Looking at the Barr body
  • If a cell contains more than one X chromosome, the extra X chromosomes are deactivated. The deactivated X chromosome in cells is visible under the microscope as a dark spot named after its discoverer: the Barr body.
    Barr-body = boy, no Barr-body = girl.4The test is not accurate enough to determine sex because women with Turner Syndrome and androgen insensitivity syndrome do not have Barr bodies, whereas men with Klinefelter Syndrome, some men with congenital adrenal hyperplasia and XX chromosomes do have Barr bodies.
  • 1957
  • Searching for a Y chromosome
  • Although it has been known since 1890 that humans have chromosomes, it has only been known with certainty since 1957 that humans have 46 chromosomes.
    Y chromosome = boy, no Y chromosome = girl.5The test is not suitable for determining sex because in different forms of sex diversity women are born with a Y chromosome.
  • 1990
  • Searching for an SRY gene
  • A 1990 publication introduced a new gene that was confidently called the SRY gene: Sex Determining Region Y (Sex Determining Region [on the] Y-[chromosome]) (Sinclair 1990). It was later found that many more genes than just the SRY gene determine whether a person’s body appears more male or female.
     SRY gene = boy, no SRY gene = girl.6The test is not suitable for determining sex because intersex women for instance with a medical diagnosis of androgen insensitivity syndrom also have an SRY gene, without being able to masculinize.
  • 2011
  • Measuring hormone levels
  • Since all previous methods of sex determination failed, a new approach was adopted in 2011 (Football Association FIFA) and 2012 (International Olympics Committee IOC): the testosterone level of female athletes was not allowed to exceed 10 nmol/l. Women with hyperandrogenism were allowed to compete in women’s competitions only if they underwent medical treatment to lower testosterone levels.7In 2015, the international athletics federation IAAF lost a lawsuit filed by Indian athlete Dutee Chand at the International Court of Arbitration for Sport (CAS) because it could not be scientifically proven that testosterone would actually contribute to improved athletic performance. Despite this, an even lower limit of 5 nmol/l is now used by the IAAF. More than 5 nmol testosterone per liter of blood = boy, less than 5 nmol testosterone per liter of blood = girl.8 The test is not suitable for determining a person’s sex because sensitivity to androgens also plays a role and, furthermore, there is a lot of overlap between men and women – in a study of athletes, 16.5% of men were found to have very low testosterone levels, while 13.7% of women actually had high testosterone levels(Healy 2014).
  • now


Tekening van de dubbele helix van DNA.