Publisher Summary

The critical factor in mammalian sex determination and differentiation is the development of the gonad. It has been shown that the testes produce testosterone and anti-Müllerian hormone (AMH), which are responsible for inducing the male secondary sex characteristics. Sex determination can be reduced to its essence: the choice between forming testis or ovary. Once gonadal sex is determined, the subsequent processes—sex differentiation—commence. In mammals, sex determination is chromosomally based: females have two X chromosomes and males have an X and a Y. These results implied that the Y chromosome carried a gene required for testis development. In humans, this gene was called the “testis determining factor” (TDF) because of the uncertain nature of the genetic material involved. The role of TDF as a “switch” in sex determination is misunderstood. Many genes are required in the pathway leading to testis formation. Some of these genes are autosomal and some, X-linked, but in the absence of TDF, the pathway would not produce testes. In the presence of TDF, the pathway is completed and testes are produced, leading to male sex differentiation.

I Mammalian Sex Determination

II The Human Y Chromosome

III Early Candidates for TDF

IV Searching for TDF on the Human Y Chromosome

V Walking on the Y Chromosome: Identification of ZFY

VI Difficulties in Equating ZFY and TDF: The Role of Marsupials

VII Reevaluation of the Y-Chromosome Deletion Map

VIII Mapping the Sex-Determining Region

IX SRY, A New Candidate for TDF

X Sequence Analysis of SRY

XI Tissue Distribution and Expression of SRY

XII Genetic Evidence Equating SRY and TDF: Analysis of XY Females

XIII SRY is the Testis-Determining Gene

References

I Mammalian Sex Determination

The critical factor in mammalian sex determination and differentiation is the development of the gonad. In a series of classic experiments, Alfred Jost demonstrated the central role of the testis in male development. When male rabbit embryos were castrated in utero, subsequent development was female; when female rabbit embryos were castrated at the same stage, subsequent development was also female. These results (reviewed by Jost et al., 1973) suggested that the basic mammalian body plan is female but presence of testes determines whether the embryo will develop as a male. Further work showed that the testes produce testosterone and anti-Müllerian hormone (AMH), which are responsible for inducing the male secondary sex characteristics. Consequently, sex determination can be reduced to its essence: the choice between forming testis or ovary. Once gonadal sex is determined, the subsequent processes known as sex differentiation commence.

In mammals, sex determination is chromosomally based: females have two X chromosomes and males have an X and a Y. Ford et al. (1959) showed that individuals with Turner syndrome had a single X chromosome and were female. In the same year, Jacobs and Strong (1959) showed that patients with Klinefelter syndrome had two X chromosomes, and a Y, and were male. These results implied that the Y chromosome carried a gene required for testis development. In humans, this gene was later called the testis determining factor (TDF) because of the uncertain nature of the genetic material involved. Mouse geneticists were less coy, however, and called it the testis determining Y gene (Tdy).

The role of TDF as a “switch” in sex determination is often misunderstood. Evidently, many genes are required in the pathway leading to testis formation. Some of these genes are autosomal and some, X-linked, but in the absence of TDF, the pathway will not produce testes. In the presence of TDF the pathway is completed and testes are produced, ultimately leading to male sex differentiation.

In summary, the main event in mammalian sex differentiation is the formation of testes, which is controlled by the Y-linked TDF gene. In this chapter we will briefly review the search for TDF, the identification of SRY, and show that SRY can be equated to TDF.

II The Human Y Chromosome

The human Y chromosome is a bipartite structure with two different functions. At male meiosis the Y chromosome must pair with the X chromosome and it must encode TDF (Ellis, 1991). These functions are mediated by distinct regions on the Y chromosome. Pairing with the X chromosome occurs within a region shared between the short arms of the X and Y chromosomes known as the pseudoautosomal region (PAR) (Burgoyne, 1982). An obligate recombination event within the PAR is necessary to ensure correct meiotic segregation (Ellis, 1991). There is a distinct delineation between the two regions marked by the pseudoautosomal boundary (PAB) (Ellis et al., 1989). The boundary separates the pseudoautosomal region from the Y-specific region which, by definition, does not participate in homologous recombination with the X chromosome and must carry TDF.

III Early Candidates for TDF

For more than 10 years, many believed that the male-specific H-Y antigen was the product of TDF (Wachtel et al., 1975), but the discovery of male mice failing to express H-Y excluded the Hya gene as a possible candidate (McLaren et al., 1984). Another candidate was the Bkm repeat sequence first isolated from a snake called the banded krait (Jones and Singh, 1981). Although Bkm sequences were found on the sex chromosomes of many mammals, their absence from the human Y excluded them as a candidate for TDF (Kiel-Metzger et al., 1985; reviewed in Chapter 1, this volume).

IV Searching for TDF on the Human Y Chromosome

In order to find TDF on the Y chromosome, three types of detailed maps were constructed: a meiotic map of the pseudoautosomal region (Goodfellow et al., 1986; Weissenbach et al., 1987); a deletion map based on the analysis of sex-reversed XX males and XY females (Vergnaud et al., 1986; Guellaen et al., 1984), and a long-range restriction map linking the first two maps (Pritchard et al., 1987). By studying individuals with Y-chromosome deletions it was possible to localize TDF to the short arm (Yp) (Goodfellow et al., 1985). However, a more precise localization was made possible by examining the chromosomes of XX males. These sex-reversed individuals appear to lack the Y chromosome but have a male phenotype with infertile testes. XX males occur mainly because of an abnormal recombination event between the X and Y chromosomes at male meiosis. This aberrant exchange extends beyond the pseudoautosomal region into the Y-specific region and results in transfer of TDF to the X chromosome (Ferguson-Smith, 1966; Guellaen et al., 1984; Petit et al., 1987). Transfer of varying portions of the Y chromosome to the X in different XX males allowed construction of a deletion map. This allowed TDF to be mapped to the most distal part of the Y-specific region, adjacent to the pseudoautosomal boundary. The meiotic map of the pseudoautosomal region indicated that MIC2 was the closest pseudoautosomal gene to TDF (Goodfellow et al., 1986). In order to link the deletion map and the meiotic map (which are based on different principles) a long-range restriction map was constructed (Pritchard et al., 1987). This map began at the 5′ CpG-rich island of the MIC2 gene in the pseudoautosomal region, spanned the boundary, and identified a CpG island in the Y-specific region. As CpG-rich islands are often associated with genes, it was predicted that this Y-specific island might define a new candidate for TDF (Pritchard et al., 1987).

V Walking on the Y Chromosome: Identification of ZFY

On the basis of this information, Goodfellow and colleagues initiated a Y-chromosome walk from MIC2 across the pseudoautosomal boundary into the Y-specific region toward the CpG island identified by Pritchard et al. (1987). This walk finished 5 kb distal (telomeric) to the CpG island that marked a possible candidate for TDF. At the same time Page and...