Whether we develop as males or females is one of the most profound issues that shapes us as individuals, affecting us each day of our lives – how we think, how we behave, choices we make, opportunities we have. Amazing, then, to think that such a profound step in our development is determined by a simple genetic lottery – whether our mother’s egg was fertilized by an X- or a Y-chromosome-bearing sperm cell – and that all the complexities associated with maleness and femaleness are the result of whether embryonic tissue primordia develop as testes or ovaries.

But the genetic control networks that underpin testis or ovary development are far from simple. Little wonder that they so often fail, resulting in sex reversal or intersex development. These disorders are very common, almost always traumatic, and cause enormous healthcare expense in our society.

Our work in this area focuses on two major issues:

• Study of the key sex-determining genes Sry and Sox9
• Discovery and analysis of genes involved in sex determination and sex reversal

Study of the key sex-determining genes Sry and Sox9

Sex determination in mammals hinges on the action of a Y-chromosomal gene called Sry. We showed some years ago, using transgenic mouse technology, that Sry can trigger male development when added to an XX genome. Sry does not act alone, but rather initiates a cascade of gene regulation that leads to testis formation. We have also identified another gene, Sox9, that acts downstream from Sry in the sex determining pathway. Unlike Sry, which is found only in mammals, Sox9 is common to all vertebrates, and is likely to be the universal regulator of male sex determination in vertebrates.

We focus on the structure and regulation of Sry and Sox9 because these two genes above all others hold the key to understanding normal male sex determination and defects that can result in sex reversal. Both genes encode transcription factors (proteins that regulate activity of other genes). Major questions include what genes the SRY and SOX9 proteins regulate, what proteins interact with SRY and SOX9 to allow them to regulate their target genes, and how the Sry and Sox9 genes are themselves regulated.

Key technologies

• Molecular: transfection/reporter assays, electrophoretic mobility shift assay (EMSA), DNA footprinting, DNA microarray, chromatin immunoprecipitation (ChIP) assay, co-immunoprecipitation (co-IP), time-of-flight mass spectroscopy, quantitative real-time PCR
• Cellular: in situ hybridization, antibody production, immunofluorescence, confocal laser microscopy
• Functional/genetic: gonadal organ culture, ex-vivo electroporation, siRNA gene knockdown, transgenic and knockout mouse production

Key publications

Discovery and analysis of genes involved in sex determination and sex reversal

A small number of genes important for male sex determination have been identified, including Sry and Sox9, but it is not always clear how they fit together. Also, the majority of cases of human sex reversal are not explained by defects in the known genes. We conclude from these facts that many of the genes important for male sex determination and testis development are still at large. We aim to find these genes and to deduce how and where they fit into the regulatory network. In addition, ovarian development has remained incredibly mysterious in the face of spectacular advances in understanding the development of other tissues. We also aim to shed light on the genes that regulate the early development of the ovary.

In these studies we are using a combination of candidate gene approaches, DNA microarray and proteomic expression screening, and are carrying out molecular, cellular and whole animal studies to try to integrate these genes into the existing regulatory framework.

In this way, we have identified several genes whose expression is restricted to male or female fetal gonads.

Key technologies

• Molecular: DNA microarray, proteomic screening, time-of-flight mass spectroscopy, bioinformatic analysis, transfection/reporter assays, electrophoretic mobility shift assay (EMSA)
• Cellular: in situ hybridization, antibody production, immunofluorescence, confocal laser microscopy, fluorescence-activated cell sorting (FACS) analysis
• Functional/genetic: gonadal organ culture, ex-vivo electroporation, siRNA gene knockdown, transgenic and knockout mouse production

Key Publications