The science of genetics was advanced by two failed attempts to identify the gene on the human Y chromosome that triggers male development.

Ever since the discovery that male development in humans is a result of the presence of a Y chromosome, biologists have looked intensively for the responsible gene(s). As is so often the case, researchers have thought on many occasions that they had the answer, only to find out later that their theories were in error. One promising attempt to identify the gene for “maleness” was announced in 1987 by David Page, a molecular biologist at the Massachusetts Institute of Technology who thought that a specific gene called ZFY ZFY gene was the key. New evidence showed that Page was in error, however, and the search for the gene for male development continued. Genetics;male development
Testis-determining factor[Testis determining factor]
Sex determination
[kw]Search for the Gene That Begins Male Development (1987-1990)
[kw]Gene That Begins Male Development, Search for the (1987-1990)
[kw]Male Development, Search for the Gene That Begins (1987-1990)
[kw]Development, Search for the Gene That Begins Male (1987-1990)
Genetics;male development
Testis-determining factor[Testis determining factor]
Sex determination
[g]North America;1987-1990: Search for the Gene That Begins Male Development[06350]
[g]United States;1987-1990: Search for the Gene That Begins Male Development[06350]
[g]United Kingdom;1987-1990: Search for the Gene That Begins Male Development[06350]
[g]England;1987-1990: Search for the Gene That Begins Male Development[06350]
[c]Science and technology;1987-1990: Search for the Gene That Begins Male Development[06350]
[c]Biology;1987-1990: Search for the Gene That Begins Male Development[06350]
[c]Genetics;1987-1990: Search for the Gene That Begins Male Development[06350]
Wachtel, Stephen
Page, David
Burgoyne, Paul S.

Sex determination is controlled by a variety of different genetic and/or environmental mechanisms. In some animals, such as turtles, the temperature at which the eggs are incubated determines the sex of the hatchlings. In certain species of tropical fish, a single dominant animal becomes a male and produces a chemical that keeps all the other members of the school female. Once the dominant male dies or swims away, another adult female from the school changes sex and becomes male to take his place.

In most animals, however, sex determination is controlled by the chromosomes Chromosomes;sex determination rather than by the environment. Flies are female if they have two X chromosomes; males have only a single X chromosome. In mammals, males have one X chromosome and females have two, but it is the presence of another chromosome, the Y, rather than possession of one X chromosome, that determines whether an individual will be a male. In 1959, C. E. Ford, Ford, C. E. W. J. Welshons, Welshons, W. J. and L. B. Russell Russell, L. B. published papers that implicated the human Y chromosome as critical for male development. It was believed that no human without a Y chromosome can be male, and all males possess a Y chromosome.

Over the next thirty years, the role of the Y chromosome in development became clear. Early in human development, the embryo produces gonads, which are indistinguishable between males and females but will eventually become either testes or ovaries. In addition to the gonads, two complete sets of ducts are present: one set destined to become part of the male urogenital system and the other, the functional female system. At the sixth week of development, a major change occurs. If the embryo is XY, the gonads begin to develop into testes, and the male ducts continue to grow. If the embryo is XX, development proceeds to form ovaries with a functional female urogenital system.

Once triggered to develop, the testes of the early embryo produce two critical products that then guide the later development along the male path. One of these products is the hormone testosterone, which acts to “turn on” a number of genes and processes that initiate male development. The second product, the protein AMDF (anti-Müllerian duct factor), causes the degeneration of the superfluous female duct system. The key to male development, then, is the trigger that causes testis development and the production of these two critical products. Supporting this conclusion is the experimental observation that if the gonad is removed from a mammal prior to the time it can produce testosterone and AMDF, the animal develops as an anatomically normal, but sterile, female. Human development thus follows a female path unless an event occurs that can trigger testis formation.

The evidence for the involvement of the Y chromosome in testis formation was overwhelming. The Y chromosome contains many genes, and the question therefore arose as to whether the entire Y chromosome or simply one or more genes located on it is responsible for testis development. This gene or set of genes is called testis-determining factor (TDF).

As geneticists studied the chromosomes of humans, they found certain surprising results. About one in twenty thousand males has two X chromosomes, and a similar number of females have an X and a Y. Explaining these unusual individuals gave geneticists the tools they needed to seek out the TDF gene. All the XX males studied had a small portion of the Y chromosome attached to one of their other chromosomes. This rare genetic event is called a translocation. Geneticists have techniques for viewing chromosomes in the microscope and observing translocations. Likewise, all the XY females did not have a complete Y chromosome; in every case, a part of the Y chromosome was lacking. Obviously, the missing part was the same part that was translocated in the XX males. Researchers reasoned that this piece of the Y chromosome must contain TDF, the gene for maleness.

Conceptually, all that remained was to determine the exact boundaries of the piece of the Y chromosome that contained the gene and to search this piece using the powerful methods of molecular biology and recombinant deoxyribonucleic acid (DNA) technology to locate and study the TDF gene. Recombinant DNA technology

One of the first candidates for TDF was a gene called HY. HY gene In the 1970’s, immunologist Stephen Wachtel and his colleagues at Memorial Sloan-Kettering Cancer Research Center led a research group that studied the properties of testis cells and ovary cells. Using the tools of immunology, they discovered a protein called HY, which is found on the surface of testis cells but not on the cells of ovaries. Given that the gonad of a six-week-old human embryo requires a signal to develop into a testis, Wachtel and others concluded that the signal was HY, as this protein is produced by a gene that resides on the Y chromosome. Supporting their hypothesis was the observation that in several of the exceptional XX males and XY females, the lack or presence of the HY protein seemed to govern whether development followed a male or a female path.

Contradictory evidence began to accumulate, however, as studies of both humans and experimental animals were done in the late 1970’s. Rare XX males were found whose testes were completely devoid of HY protein, and rare XY females had functional ovaries that made HY. Clearly, HY was not TDF; later, researchers came to believe that HY plays a role in sperm development, an event that occurs much later in male development than the TDF trigger.

The next theory came in 1987, when David Page of the Massachusetts Institute of Technology’s Whitehead Institute published a paper in the journal Cell that excited the entire research community because it offered a clear and logical explanation for human sex determination. The candidate gene, ZFY (zinc finger Y chromosome), Zinc finger Y chromosome had molecular attributes that made it a plausible testis-determining factor. Page reported an analysis of DNA from the same groups of exceptional XX males and XY females. He looked at the DNA itself rather than at gonad proteins like HY. Page found 99.8 percent of a normal Y chromosome in the chromosomes of a twelve-year-old XY girl, yet she had functional ovaries. Thus the TDF must reside in this small 0.2 percent region of the Y chromosome, a region near the location of HY but distinct from it. Using the methods of recombinant DNA technology and DNA sequencing, Page isolated a candidate gene and claimed that he had found the trigger for male development, TDF.

ZFY is a gene that controls the production of a protein that can bind to DNA and alter the function of other genes. This is exactly what one would expect a triggering gene to do: throw a switch, turning on ZFY, thus initiating a cascade of other events that would lead the gonad to become a testis. The ZFY protein is a member of a well-studied group of DNA-binding proteins Proteins called the zinc fingers. These proteins all possess one or more projections, or fingers, that contain an atom of zinc onto which DNA binds. Other zinc finger proteins are known to have the ability to turn groups of genes on and off. The ZFY gene was thus an appealing candidate for the TDF.

Puzzling to Page and others in the field, however, was the fact that there was an identical gene called ZFX on the X chromosome. Page’s theory was thus confronted with the dilemma that the X chromosome would also have to play a crucial role in testis determination, unless it could be shown that the ZFX gene was nonfunctional.

Two years later, the weight of contradictory evidence to the idea that these zinc finger genes were testis-determining factors became overwhelming. Developmental biologist Paul S. Burgoyne published an article in the journal Nature that summarized work conducted in London, in his laboratory and in several others. Burgoyne wrote that further examination of rare exceptional individuals had revealed that males lacking the ZFY gene could be found and that females possessing functional ZFY genes could, nevertheless, make ovaries. Obviously, like HY a decade or so before, ZFY plays some role in male development but clearly is not the triggering gene. The gene that triggers testis determination must still reside in the small portion of the Y chromosome where both ZFY and HY are located.

In 1990, Australian researchers identified the gene known as SRY SRY gene (sex-determining region Y) as the TDF gene. SRY is known to trigger the pathway to male development, but it does not determine sexual development by itself. Scientists have continued to probe the genetic nature of sexual development.

The testis-determining factor is one of a number of critical genes that has to function in the correct order and in the correct tissues of an embryo in order for development to proceed normally. This intricately regulated process of gene function is not well understood. One way to learn more about this process is to study genes like the TDF and learn how they make the cells in an embryo different—for example, how genes cause some cells in an embryo to become liver cells while others become skin or kidney or testis. A more complete understanding of how these genes function might enable biologists to intervene in this development should the normal process fail.

The ability to manipulate the TDF and control its function could permit geneticists to alter the sex of a mammalian embryo. Given that genes like HY and ZFY have been found in all mammals studied so far, geneticists expect that the TDF also will be found in all mammals that use a Y chromosome to determine maleness. The ability to manipulate the function of the TDF would have obvious agricultural economic benefits. Animal breeders might, in some circumstances, prefer female offspring, such as in a dairy herd, whereas in other cases they may prefer males for breeding. Control of the sex of an animal’s offspring would then become yet another tool to make raising animals more productive.

A second and very different significance of the search for the TDF is that it shows how science works. Experimental evidence is collected using the best technology available at the time, and hypotheses are constructed. Later, as newer procedures or new experimental designs become available, old hypotheses, such as that the HY or the ZFY is the trigger for testis development, are discarded, and the search goes on for new data. The collaborative and competitive efforts of different research groups provide a constant set of checks and balances that ensure that hypotheses receive rigorous reevaluation and continual modification. Scientists can grow quite fond of their own hypotheses, especially those that stem from their own research. They must be willing to abandon those hypotheses, however, in the face of new and conflicting data. Progress in science depends on the continual refinement of the current view of the world, and a major strength of the scientific method is the scrutiny it brings to this view. Genetics;male development
Testis-determining factor[Testis determining factor]
Sex determination

• Burgoyne, Paul S. “Thumbs Down for Zinc Finger?” Nature 342 (December, 1989): 860-862. Summarizes the arguments against Page’s ZFY gene hypothesis. Contains all the references to the relevant research in the original literature but is written as a news summary.
• Craig, Ian. “Sex Determination: Zinc Fingers Point in the Wrong Direction.” Trends in Genetics 6 (May, 1990): 135-137. Presents the evidence against the ZFY gene being the TDF. Written for a general college-level audience. Includes references to the primary research literature.
• Gilbert, Scott F. “Sex Determination.” In Developmental Biology. 8th ed. Sunderland, Mass.: Sinauer Associates, 2006. College-level text provides an excellent description of the various mechanisms by which the sex of an animal is determined. Covers the environmental, genetic, and hormonal controls of this process.
• Raven, Peter H., et al. Biology. 7th ed. New York: McGraw-Hill, 2004. General college-level text contains well-illustrated discussions about sex chromosomes and early embryonic development. Includes detailed glossary.
• Roberts, Leslie. “Zeroing in on the Sex Switch.” Science 239 (January 1, 1988): 21-23. Details, in clear and readable fashion, the major events that led to Page’s discovery of ZFY.

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