Genetic Control of Testis Determination

V.R. Harley

I Introduction

Jost (1947; Jost et al., 1973) showed that castrated rabbit embryos of either chromosomal sex develop as females, indicating that the presence of testes is necessary for the development of male characteristics. During embryogenesis, the gonad arises as an indifferent tissue, the fate of which follows either the testicular or the ovarian pathway. In mammals, this developmental switch is controlled chromosomally; females have two X chromosomes and males have an X and a Y chromosome. Individuals with a single X chromosome and no Y chromosome (that is, XO) are female (Ford et al., 1959). The presence of a Y chromosome results in male development, regardless of the number of X chromosomes the individual possesses (Jacobs and Strong, 1959; Table 1). Thus, the Y chromosome must encode a dominant inducer of testis formation. The Y-linked gene(s) controlling this process has been named the testis determining factor (TDF; Tdy in mouse). TDF is activated at some time during embryogenesis to commit the undifferentiated genital ridge to the testicular pathway. As a consequence of the action of TDF, subsequent hormonal production induces male sexual differentiation.

The gonad is composed of cells derived from four lineages: supporting cells, steroid-producing cells, connective tissue cells, and germ cells. The first signs of male development can be recognized in the genital ridges of mice at 12.5 days post coitum (dpc) when Sertoli cells organize into “testis cords.” Female gonads show no apparent change until 13.5 dpc when the germ cells first enter meiosis (McLaren, 1984). Tdy is thought to act solely in the supporting cell lineage to divert the indifferent gonad away from the ovarian pathway and into the testicular pathway, by triggering supporting cells to differentiate into Sertoli cells rather than into the follicle cells of the ovary (Palmer and Burgoyne, 1991a). Sertoli cells, without further Tdy involvement, then commit the fate of the steroid-producing cells to produce Leydig cells and induce mitotic arrest in the germ cells (Burgoyne et al., 1988). As in other developmental processes, Tdy is presumed to act with other regulatory molecules in the genital ridge to give rise to testes and subsequent male development.

Among humans, rare individuals arise who carry two X chromosomes but are phenotypically male (XX males) or carry a Y chromosome but are phenotypically female (XY females). The molecular analysis of the genomes of these so-called sex-reversed patients led to the isolation of the male sex-determining region Y gene, SRY. Prior to this discovery, candidate Y-located genes had been isolated that subsequently failed to meet established criteria.

This chapter chronicles the identification of a new candidate for the testis determining gene: SRY. Evidence, both circumstantial and direct, that SRY is TDF is outlined. Emphasis is placed on biochemical properties of SRY. Detailed information on the biology of mouse Sry can be found elsewhere (Capel and Lovell-Badge, 1992). The cell and tissue biological processes during sexual differentiation of the gonad are discussed in subsequent chapters.

II SRY, a New Candidate for the Testis Determining Factor

A Mapping the Human Y Chromosome

The approach that proved successful for the isolation of TDF was deletion mapping of the Y chromosome. Almost 60 years ago, Koller and Darlington (1934) observed that X and Y rat chromosomes were dissimilar but could pair along part of their lengths. These researchers proposed that the Y chromosome was composed of a shared region and a Y specific region. The shared or “pseudoautosomal” region (PAR) is terminal on the short arms of the X and Y chromosomes (Pearson and Bobrow, 1970) and would be required for correct pairing during male meiosis. The Y-specific region would encode TDF. Recombination in the PAR region would maintain homology between the X and Y shared regions but recombination should not occur in the Y-specific region (Ferguson-Smith, 1966; Fig. 1). Although cytogenetic analysis of individuals with Y chromosome deletions suggested that TDF was located on the short arm (Goodfellow et al., 1985), precise localization was achieved by studying XX males.

Analysis of the genomes of some XX males showed the presence of Y-derived sequences (Guellen et al., 1984) that presumably arose by aberrant recombination between the X and Y chromosomes (Ferguson-Smith, 1966). Different XX males inherited different terminal fragments, which allowed the construction of a deletion map of the Y chromosome (Affara et al., 1986; Muller et al., 1986; Vergnaud et al., 1986). These maps began to define the minimum region required for male sex determination (Page et al., 1987; Palmer et al., 1989).

B ZFY

Page et al. (1987) further narrowed the region in which TDF must lie as between 140 and 280 kb from the PAR boundary, because an XX male had inherited only 280 kb of Y-derived sequence and part of this region was deleted in an XY female patient with a Y;22 translocation (Fig. 2). On cloning this region and screening by Southern analysis for Y-specific sequences among eutherian mammals, a gene dubbed ZFY was identified. Since ZFY could encode a zinc finger protein and therefore might be a transcription factor, and since mouse Y homologs (Zfy-1 and -2) mapped to a small region known to contain Tdy (McLaren et al., 1988; Roberts et al., 1988; Mardon et al., 1989; Nagamine et al., 1989), ZFY seemed a good candidate for TDF.

However, inconsistencies arose. First it was shown by Sinclair et al. (1988) that the genes homologous to ZFY were not on the marsupial Y chromosome. Further, Koopman et al. (1989) showed that, although Zfy-1 was expressed in the fetal testis of normal mice, expression was absent in homozygous WW mice, which have normal testes but lack germ cells. Finally, Palmer et al. (1989) found four XX males/intersexes who carried less than 60 kb of Y-specific DNA derived from the region adjacent to the PAR boundary but lacked ZFY in their genomes. Collectively, these data firmly rule out ZFY as TDF. The variation in sexual phenotype was suggested to be caused by the proximity of the Y breakpoints to SRY (Palmer et al., 1989).

C Discovery of SRY

Palmer et al. (1989) found that different sized DNA fragments were detected from the four ZFY-negative XX patients when hybridized to a probe 35 kb from the PAR...