Since the beginning of humanity, people were fascinated by sex and intrigued by how the differences between sexes are determined. Ancient philosophers and middle age scholars proposed numerous fantastic explanations for the origin of sex differences in people and animals. However, only the development of the modern scientific methods allowed us to find, on the scientific ground, the right answers to these questions. In this review article, we describe the history of these discoveries, and which major discoveries allowed the understanding of the origin of sex and molecular and cellular basis of the differentiation of male and female sex characteristics during embryo development and in the adultJacek Z Kubiak, Malgorzata Kloc, Rafal P Piprek.
History of The Research on Sex Determination.
The First Concepts of Sex Determination
In the multicellular organisms, sex is a set of features of the structure, function, and behaviour of the body that allows it to be classified as a male or female individual. Sex determination is directly linked to the determination of the direction of the development of yet undifferentiated gonads into the testes or ovaries. The sexual characteristics of the individual are formed during the process of sexual differentiation. For sexually reproducing multicellular organisms, the correct structure and function of the reproductive system is a prerequisite for having a healthy and fertile offspring, which is the basis of maintaining the continuity of the species. The emergence of sex turned out to be a significant advancement in evolution, as the merger of the male and female gametes, resulting from the existence of sex, provided a high degree of genetic variability of the offspring.
Thus, in contrast to the asexually reproducing organisms, the population of sexually reproducing organisms became more diverse, and as a result, more easily adaptable to the changing environment. For centuries, humanity has been intrigued by the nature of sex, the sense of its existence and origin. The issue of sex already appeared in the book of Genesis (2: 21-24), which tells about the creation by God of the man (Adam) and the women from Adam’s rib. Plato, in his work entitled “Symposium”, written around 385-370 BC, presented his vision of the origin of human sex. In this work, Aristophanes talks about primitive people with round shapes, two pairs of arms and legs, and two faces. These people had exerted an extraordinary fear even among the gods. The god Zeus, to guard against these strong creatures, decided to cut them in half. This is how men and women were created. Both halves began to miss each other, showing the need for unity expressed in the form of love. In this way, Plato explained the essence of sex inseparable from the feeling of love.
One of the most mysterious issues remaining for centuries was what determines the sex of the individual, and thus how the sex is determined. The Greek philosopher Parmenides (540-470 BC) claimed that the sex of a child is determined by the position of the fetus in the womb. Male development would be determined by the position of the fetus on the right side of the womb and the female on the left. Around 500-428 BC, another Greek philosopher – Anaxagoras recognised that it is the paternal factor that determines the sex of the child; namely, the boys develop from the sperm from the right testicle and the girls from the left testicle. Then, Empedocles (494-434 BC) claimed that organisms consist of four elements: fire (heat), water (cold), air (moisture) and earth (dryness), and that the men have a more warm ingredient.
The most outstanding biologist of the antiquity – Aristotle (384-322 BC) did not agree with the statements of the above-mentioned thinkers. He believed that he had evidence that the female and male offspring could develop on both sides of the womb (uterus) and noted that the men with only one testicle could conceive both male and female offspring . Similar to Empedocles, Aristotle saw the mechanisms of sex determination as the predominance of hot or cold ingredients in the body.In his work, “Historia Animalium”, he pointed out that males are stronger, which is due to their higher heat, enabling the transformation of food into the concentrated seed. Females, on the other hand, in Aristotle’s opinion, were weaker and cooler, which meant that they could not convert food into sperm, and instead produced more blood that is excreted during menstruation.
According to Aristotle, the development of fetus sex was dependent on the connection of sperm with menstrual blood during conception. Sperm thickening due to the heat was to lead to the development of the male individual, while liquefaction of the sperm at a lower temperature, leading to its mixing with the mother’s blood, resulted in the development of the female individual. He also stated that both man and woman (through semen and blood) contribute to the offspring, and thought, which turned out to be true, that male and female organs develop during development from undifferentiated buds. Importantly, he stated that the testes are responsible for the development of male traits, i.e. masculinization since the testisdeprived eunuchs show feminization features. Thus, among the ancient thinkers, it was Aristotle who was closest to the truth, and his observations did indeed contribute to the broadening of knowledge about gender determination.
First Scientific Evidence of Sex Determination
The belief that the heat, positioning of the fetus in the womb and food determine sex, nestled in peoples’ minds for a long time, and survived for nearly two thousand years. In the second half of the nineteenth century, it was believed that heat and nutrition affect a child’s sex. This faith resounds even today in folk beliefs. The milestone in the biological research was the discovery of chromosomes in 1888 by the German anatomist, physiologist and pathologist Heinrich Waldeyer. Already three years later, in 1891, the German cytologist Hermann Henking, studying the meiotic division in the nuclei of a wingless blacksmith (Hemiptera), noticed that some sperm cells of this species have 12 chromosomes, and some 11. He noticed that, during meiosis, one of the 12 chromosomes behaves differently than others. By naming this strange chromosome the X chromosome, he wanted to emphasise its mysterious nature. He then searched for the X chromosome in the grasshopper cells, but to no avail. It wasn’t until 1901 that American zoologist Clarence Erwin McClung pointed out that the X chromosome, as an “additional chromosome,” could be associated with sex determination.
In 1905, American geneticist Edmund Beecher Wilson, studying spermatogenesis of several insect species, showed that there are two types of sperm, which differ in the presence or absence of one of the chromosomes. In the same year, the American geneticist Nettie Maria Stevens, studying the gametogenesis of the mealworm beetle, found that in males, but not females, one chromosome was significantly different in size. Therefore, Stevens stated that the larger chromosome is the X chromosome, while the smaller chromosome, which she called Y, must be responsible for male determination. She also suggested that there must be some unknown factor in the Y chromosome that determines male development. Wilson, on the other hand, believed that both chromosomes, X and Y, determine sex equally . Stevens died in 1912 without confirming her hypothesis, and Wilson has been described in scientific literature as a discoverer of sex-determining X and Y chromosomes.
Description of sex chromosomes contributed to the statement that sex is determined by genes. Around the same period (early 20th century), chromosomes were found to carry genetic information. Walter Sutton and Theodor Boveri discovered this, independently, in 1903. The term “gene” was introduced in 1909 by the Danish botanist Wilhelm Johannsen. However, in 1910, the American geneticist Thomas Hunt Morgan, studying the inheritance of features in the fruit fly, showed that the genes are located in the chromosomes where they are linearly arranged, and occupy strictly defined places, the so-called loci. In addition, he showed that some features (e.g. eye color) are sex-linked because their genes are located in the sex chromosomes. Interestingly, the understanding of the basics of inheritance mechanisms was possible at the beginning of the 20th century, again, thanks to the research on insects. Nevertheless, the mechanism by which sex chromosomes determine sex was to remain unexplained for a long time.
Only a few decades later, in 1956, in Great Britain, Charles Ford and John Hamerton, using cytogenetic methods, determined the number of chromosomes in humans and found that men have X and Y chromosomes (karyotype 46, XY), and women two chromosomes X (karyotype 46, XX) . Three years after this discovery, the first chromosomal aberrations in humans were described. It was noticed that people with Klinefelter syndrome are men with 47, XXY karyotype, while people with Turner syndrome are women with 45, X0 karyotype. This was the first scientific evidence confirming that the Y chromosome in humans determines the male sex.
Searching for the Factor Determining Male Sex
The discovery of the function of the Y chromosome in the determination of the male sex in humans initiated the search for a hypothetical factor determining this particular sex. This hypothetical factor was named TDF (Testis-Determining Factor) in humans, and Tdy (testis-determining factor on the Y chromosome) in mice. At that time, the most informative objects of research on sex determination were patients with sex reversal symptoms, i.e. incompatibilities between the presence of sex chromosomes and phenotypic features. The analyses of chromosomal aberrations, such as translocations and fusions, indicated that it is the short arm of the Y chromosome that is responsible for male determination. Also, the search area for the TDF factor has been gradually narrowing. Among the genes of the smaller arm of the Y chromosome, the first candidates were the H-Y antigen and BKM genes (Banded Krait Minor Gene), but their involvement in sex determination has not been demonstrated. The next gene tested was ZFY (Zinc-Finger Protein Y-Linked). It turned out that this gene also does not determine sex because it is not expressed in the mouse gonads. In addition, it was observed that the male patients with karyotype 46, XX developed male traits despite the absence of the ZFY gene.
Patients described by Palmer and colleagues (four men with karyotype 46, XX) had in their genome a small, 35,000 base pair, Y chromosome-specific region. This Y chromosome region was isolated, divided into fragments and compared with Y chromosome fragments of other mammals in Southern blotting analysis. It turned out that there is only one conserved fragment of the Y chromosome in mammals, which also does not show much variation between species. Thus, it was assumed that this fragment of the Y chromosome must contain the sex-determining gene. Sequencing of this fragment indicated only one Open Reading Frame (ORF) coding for the gene consisting of a single exon. This gene was called “sex determining region on the Y chromosome” (SRY in humans and Sry in mice). RT-PCR gene expression analysis showed that the Sry gene is expressed in developing gonads of XY mice at embryonic day 11.5, just before the first signs of testicular sexual differentiation.
The final evidence confirming the determination of male sex by the Sry gene was provided through the study of XX transgenic mice into which the Sry transgene was introduced. These individuals, despite having the female karyotype XX, developed into typical males with normally developed testes (sex reversal), but they were sterile due to the lack of Y chromosome, which, as it turned out, also contains genes responsible for the proper course of spermatogenesis. These studies proved that the Sry gene is both necessary and sufficient to determine male sex. In addition, it was shown that the Sry gene is present only in marsupials and placental mammals, i.e.in all mammals except monotremes.
Sex differences have puzzled humanity for a very long time in both scientific and social contexts. A thorough understanding of the mechanisms determining sexual development has been achieved through the use of advanced scientific techniques developed in the 20th century. However, surprisingly, the network of genes that control sex determination is still incomplete, and relatively poorly understood. Further research is likely to find novel genes involved in gonadal development and to show the complexity of the mechanisms controlling sexual development. It appears that the cascade of events leading to the sex determination is much more complicated than originally imagined. In addition, in many cases of human sex disorders, their source remains unknown, indicating that the research on the genes involved in gonadal development has a long way to go.