PARENT SESSION
PLATFORM SESSION 5. MATURATION AND AGING OF THE REPRODUCTIVE TRACT AND SEX DETERMINATION

Sunday, August 1, 2004
3:00 PM–5:00 PM
Buchanan A202
Chair: Jock Findlay Co-Chair: Alan J. Conley

(37) USING MULTI GENE REAL TIME PCR EXPRESSION ANALYSIS TO EXAMINE TESTIS DEVELOPMENT IN Nr0b1 NULL XY B6 MICE.

Bouma, Jerry¹, Albrecht, Kenneth², Washburn, Linda¹, Recknagel, Andrew¹, Eicher, Eva¹, ¹ The Jackson Laboratory, Bar Harbor, ME² Boston University School of Medicine, Boston, MA

ABSTRACT- The X-linked transcription factor Nr0b1 (Dax1) is an orphan nuclear receptor belonging to the nuclear hormone receptor superfamily. Studies in our laboratory indicate that C57BL/6J (B6) XY mice carrying a Dax1 null (Dax1-) allele are sex reversed (i.e. develop ovaries) and infertile. In contrast, DBA/2J XY mice carrying the Dax1- allele develop testes and are fertile. To further elucidate the function of Dax1 in B6 testis development, we used a real time RT-PCR assay to examine the relative expression levels of 51 genes expressed during fetal gonad differentiation. Embryonic age (E) of the fetuses was assessed and tissue was collected from E10 through E14. Real time PCR results on cDNA from individual fetal tissue indicate that Sry was expressed at significantly higher (P<0.05) levels in E13 and E14 B6 Dax1-/Y than normal B6 XY gonads. The relative transcript levels of genes normally expressed at higher levels in XX gonads, including Adamts19 (a disintegrin-like and metalloprotease with throbospondin type 1 motif, 19), Bmp2 (bone morphogenic protein 2), Emx2 (empty spiracles homolog 2), Fgfr2 (fibroblast growth factor receptor 2), and Fst (follistatin), were significantly higher (P<0.05) in E13 and E14 B6 Dax1-/Y than B6 XY gonads. In contrast, the expression level of two testes-specific genes (Amh, anti-mullerian hormone and Dhh, dessert hedgehog) and two genes normally expressed at higher levels in XY than XX gonads (Gata4, GATA binding protein 4 and Sox9, SRY-box containing gene 9) were significantly lower in B6 Dax1-/Y than B6 XY gonads. We also examined the cellular localization pattern for several proteins known to be involved in gonadal differentiation. Wholemount in situ hybridization using antibodies to GATA4, SF1, SOX9, and WT1 proteins indicated that the cellular localization in B6 Dax1-/Y gonads was similar to that observed in normal B6 XX ovaries. We conclude that development of ovarian tissue in B6 Dax1-/Y mice is due to impaired Sry function.

KEY WORDS: Dax1, sex reversal, gonad differentiation