The role of the SRY gene in determing sex. Ahmed Mahmoud 10-29-2009

What makes a male or female? In mammals, gonadal development is determined by the sex chromosomes. XX usually constitutes female XY constitutes male How ever, some males have XX Some females have XY Why is this?

Key players in the sex determining game. SRY gene Stands for Sex determining region of Y chromosome. WNT signaling pathway a network of proteins used to control the production of wnt signaling molecules. WNT4- the gene that codes for a signaling protein that is involved in female gonadal development. β-catenin- protein complex that is used in the WNT signaling pathway. RSPO1 gene- encodes for a small secreted protein that is able to encourage the Wnt/β-Catenin signaling pathway.

History of the SRY Cladogram showing evolution of SRY Gene. Ucl.ac.uk

The Exact region was found out in 1990. The Y chromosome’s role in sex determination was known since the early twentieth century. The Exact region was found out in 1990. SRY region extends 897 base pairs long Exact location of the SRY gene on the Y chromosome

The SRY gene: The Male determining factor. SRY gene is located on the short arm of the Y chromosome This Gene causes male gonadal development.

SRY and The SOX family SRY is part of the SOX family of proteins that is characterized by a DNA binding domain. There are 20 SOX genes present in humans. SRY however does not have a transcription domain and closely works with another member of the SOX family; SOX9(SRY box-9). In XY individuals, SOX9 was found to increase in expression shortly after the beginning of SRY being expressed.

Sox9 Gene location & Info SOX9 is located on Chromosome 17 It is 5,401 pairs long genecards.org Location of SOX9 Gene

The female determining factors. R-Spondin 1 (Rspo 1) and The Wnt/β-Catenin pathway. Rspo 1 was found to increase in expression in XX females at the time of ovarian differentiation(1). Rspo 1 is needed to express the Wnt4 gene and it works through the stabilization of β-Catenin to develop ovaries and block testis. Rspo 1, Wnt4 and β-Catenin are all parts of one pathway to a ovarian result and block the development of testis.

RSPO1 Loci. 23,645 bases long WNT4 Loci. 25,722 bases long Genecards.org RSPO1 Loci. 23,645 bases long Genecards.org WNT4 Loci. 25,722 bases long

Pathways of determination. Int J Biochem Cell Biol. 2008; 40(12): 2889–2900

SRY blocks Wnt Pathway Lab experiment performed by Pascal Bernard HEK293T cells –Human embryonic kidney cells NT2/D1 cells-Human embryonic carcinoma cells TCF- T-Cell specific HMG box factor. Protein found in the above mentioned cells that plays a key role in Wnt signaling TOP-Short for TOPFLASH which is an assay which has a binding site for β-catenin to bind to the cell’s TCF protein FOP- short for FOPFLASH which is a a control for TOP. It contains a mutant binding site that is unable to bind to TCF protein. BIO- 6-bromoindirubin-3’-oxime. Used to Activate TOPFLASH Int J Biochem Cell Biol. 2008; 40(12): 2889–2900 When SRY was added, it reduced the activation of TCF/β-Catenin by two fold When BIO is added and no SRY is present, TCF/β-Catenin binding is strongly activated. As SRY is introduced, the cells with the binding site for TCF begin to slowly decrease.

Rspo1 activates β-Catenin signaling Experiment performed by Anne-Amandine Chassot Experiment sought out to show that Rspo1 controls the activation of the β-catenin signaling pathway. Urogenital ridges were stained for finding the Lef1 which is a gene involved in β-catenin signaling. An XX gonad positive for Rspo1 shows staining that represents the Lef1 gene for β-catenin signaling. Human Molecular Genetics 2008 17(9):1264-1277; doi:10.1093/hmg/ddn016 An XY gonad also positive for Rspo1 does not show staining for the Lef1 gene which may be due to the SRY genes inhibition of the signaling pathway. An XX gonad negative for Rspo1 showing no staining for the Lef1 gene. Proving that Rsp01 mediates the β-Catenin Signaling pathway.

SRY and Sex Reversal Sex Reversal syndrome (SRS) is a kind of genetic disorder which creates a conflict between gonad development and chromosomal phenotype. The incidence rate of Sex reversal syndrome occurs in about one in every 100,000 individuals SRS proves once more that the SRY gene is the most important male determining factor

A study male SRS 46 XX male sex reversal Study conducted on a 20 year male. Patient had undescended testicles. Physically, the patient had the sexual characteristics of a normal male. Slender skeleton and light beard.

Translocation of SRY. M NM F P The patients DNA and control samples were tested by PCR(Polymerase chain reaction)amplification in figure A. Figure B shows fluorescent microscopy on the patients DNA. Fluorescence in situ hybridsation of patients DNA showed a green fluorescence on the short arm of the X chromosome. This gen was translocated to Chromosome X from Chromosome Y. M NM F P PCR amplification showed that both the Patient (P) and the Normal male (NM) were consistent in showing a SRY fragment while the Normal female (NM) had no such fragments of SRY.

SRS in the Olympic games In the 1992 Barcelona Olympic games, female athletes were tested through PCR Over 2000 test were performed and of those, 15 were reported positive. In the 1996 Atlanta games, 8 reported positive. Gender Verification tests were abandoned in 1999 and still are to this day.

Conclusion SRY gene is the most important male determining factor. The pathway leading to ovarian development goes through the β-catenin/Wnt signaling pathway. The SRY gene can inhibit the usual pathway leading to ovarian differentiation. SRS is due to the translocation to an X chromosome.

References Bernard, Pascal.(2008). Human SRY Inhibits β-Catenin-mediated transcription. Int J Biochem Cell Biol,40(12),2889-2900 Nef, S., Vassalli,J.(2009). Complementary Pathways in mammalian female sex determination. Journal of Biology,8(74). Chassot, A.,Ranc, Fariba., Gregoire, E., Roepers-Gajadien, H., Teketo,M., Camerino, G., Rooij,D., Schedl,A. And Chaboissier, M.(2008). Activation of β-catenin signaling by Rspo1 controls differentiation of the mammalian ovary. Human Molecular Genetics,17(9), 1264-1277. Smith, C., Shoemaker,C.,Roeszler, K., Queen, J., Crews, D. and Sinclair, A.(2008). Cloning and expression of R-Spondin1 in different vertebrates suggests a conserved role in ovarian development. BMC Developmental Biology,8(72). Wang, T., Liu, J., Yang,J.,Chen,J. And Ye, Z.(2008). 46, XX male sex reversal: a case report and review of the genetic basis. First Int. Journal of Andrology, 41, 59-62. Ritchie, R., Reynard,J. And Lewis, T.(2008). Intersex and the Olympic games. J R Soc Med,101, 395-399. Marchal, J., Acosta, M., Bullejos, M., Diaz de la Guardia, R. And Sanchez, A. (2007).Genomics,91,142-151. Sinclair, A. (2001). Eleven years of sexual discovery. Genome Biology, 2(7), 4017.1-4017.3 Newton, G. (2003). SRY and sex reversal. Retrieved October 1, 2009, from http://genome.wellcome.ac.uk/doc_WTD020752.html.