Sry and Sox9 expression during canine gonadal sex determination assayed by quantitative reverse transcription-polymerase chain reaction

Abstract

Testis induction is associated with gonadal Sry and Sox9 expression in mammals, and with Sox9 expression in vertebrates where Sry is absent. In mammals, Sry might initiate testis induction by upregulating Sox9 expression; however, direct evidence supporting this hypothesis is lacking. Models of Sry-negative XX sex reversal (XXSR), in which testes develop in the absence of Sry, could provide the link between Sry and Sox9 in testis induction. To define the stages at which testis determination occurs in the canine model, Sry and Sox9 expression were measured in normal urogenital ridges (UGR) and gonads by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Testicular Sry expression rose continuously during canine developmental ages comparable to human carnegie stages (CS) 16-18, with maximal expression at CS 18. Sox9 was expressed in both male and female canine UGR up to CS 17, at which time testis expression became tenfold greater than in the ovary. Although Sox9 was detected by qRT-PCR in ovaries and mesonephroi of both sexes, expression was detected only in canine testes by whole mount in situ hybridization (WMISH). The timing of Sry and Sox9 expression is consistent with a role in testis determination: Sry expression begins at CS 16 in testes, followed by upregulation of Sox9 expression at CS 17. The quantity and temporal and spatial patterns of Sry and Sox9 expression in normal canine gonads are similar to those in humans, sheep, and pigs. These studies should provide the basis for understanding the mechanism of testis induction in the canine model of Sry-negative XXSR.