EGF, epithelium and Monoclonal antibody against the human SRY protein 135 The human SRY protein is present in fetal and adult Sertoli cells and germ cells LAURA SALAS-CORTÉS 1,2* FRANCIS JAUBERT 3 , SANDRINE BARBAUX 2 , CATHERINE NESSMANN 4 , MARÍA R. BONO 1 , MARC FELLOUS 2 , KEN McELREAVEY 2 and MARIO ROSEMBLATT 1,5 1 Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile, 2 Unité d’Immunogénétique Humaine, INSERM U. 276, Institut Pasteur, Paris, France, 3 Laboratoire d’Anatomie Pathologique, Hôpital Necker-Enfants Malades, Paris, France, 4 Laboratoire d’Embryologie et Cytogénétique, Hôpital Robert Debrè, Paris, France and 5 Fundación Ciencia para la Vida, Santiago, Chile ABSTRACT Sex determination in mammals is controlled by the Y chromosome located SRY gene. Despite recent advances tow ards understanding the mechanisms that regulate sex determination in mammals, the expression profile of the SRY protein in human tissues is unknow n. To localize the SRY protein and determine its cellular distribution, w e prepared monoclonal antibodies (mAb) against the recombinant SRY protein. One antibody, LSRY1.1, recognizes a SRY-specific epitope and was used to localize the protein in different cells and tissues. The mAb recognizes a protein of 27 kDa in total lysates of HeLa SRYB3 cells. Immunocytochemical staining showed a nuclear localization of the protein. Immunohistochemical studies performed on gonadal tissue of a fetus, a one month-old boy and an adult man, demonstrated the presence of SRY protein in the nucleus of Sertoli and germ cells. In addition tw o 46,XX SRY(+) males had the SRY protein in their gonadal tissues. All other samples w ere negative, including all female tissue studied and the testis of a 46,XX SRY(-) male. The presence of SRY protein in fetal and adult gonadal tissues including germ cells suggests that SRY may have other male-specific functions in addition to sex determinism. KEY WORDS: SRY protein, monoclonal antibody, testis, germ cells, Sertoli cells Int. J. Dev. Biol. 43: 135-140 (1999) 0214-6282/99/$15.00 © UBC Press Printed in Spain www.lg.ehu.es/ijdb *Address for reprints: Unité d’Immunogénétique Humaine, INSERM U. 276, Institut Pasteur, 25 Rue du Dr. Roux, 75724 Paris cedex 15, France. FAX: (562) 40 61 31 53. e-mail: lsalasc@pasteur.fr Introduction The mechanism regulating mammalian sex determination depends on a molecular switch that is genetically controlled by a gene on the Y chromosome. The factor controlling this molecular switch has been termed Testis Determining Factor (TDF) in humans and Tdy in mice. Using 46,XX males that carried a portion of the Y chromosome translocated to an X chromosome, the SRY (Sex determining region Y chromo- some) gene was cloned (Gubbay et al., 1990; Sinclair et al. , 1990). Several lines of evidence indicate that Sry corresponds to TDF: 1) individuals with a 46,XY karyotype but presenting a female phenotype with complete gonadal dysgenesis, carry mutations in the SRY open reading frame (Berta et al., 1990; Jäger et al., 1990). 2) In normal developing mouse embryos, expression of Sry occurs in the genital ridge around 10.5-12.5 days post coitum (dpc), just before the differentiation of the genital ridge into testis (Koopman et al., 1990). 3) Mice with a female karyotype developed testis when a 14 kb fragment of DNA containing the Sry gene was introduced as a transgene, Original Article Abbreviations used in this paper: SRY, Sex-determining region Ychromosome; TDF, Testis Determining Factor; HMG, High Mobility Group; mAb, mono- clonal antibody; GST, glutathione S-transferase; Sox protein, SRY-box containing protein; AMH, anti-Müllerian hormone. demonstrating that Sry is both necessary and sufficient to determine testis formation (Koopman et al. , 1991). The SRY protein has a 79-amino acid HMG (High Mobility Group) domain, a conserved motif present in many DNA-binding proteins including several transcription factors (Ner, 1992). SRY binds to DNA and induces substantial bending in target DNA sequences, suggesting that SRY may act as a transcriptional regulator of other downstream sex-determining genes (Ferrari et al., 1992; Giese et al., 1992; Van der Wetering and Clevers, 1992). In mice, fetal expression of Sry is limited to male gonadal somatic tissues and does not require the presence of germ cells (Koopman et al., 1990). High levels of Sry mRNA are also found in adult mouse testis, but this transcript has a circular structure, is not