Objective: To produce canine gene probes and define the onset of normal testis induction and differentiation by gene expression. These studies will provide molecular diagnostic tools for inherited reproductive abnormalities, such as hermaphroditism.

Design: Embryos were sexed by polymerase chain reaction (PCR) for Sry in genomic DNA. One urogenital ridge (UGR) per embryo (d27-37 gestation) was assayed by quantitative reverse transcription polymerase chain reaction (qRTPCR), and the other by whole mount in situ hybridization (WMISH).

Materials and Methods: Products from triplicate sample and duplicate control cDNA (RT+, RT-) templates (TaqMan Gold RT-PCR, Perkin Elmer) were quantified by automated fluorescence detection (ABI Prism 7700). Starting template was calculated from standard curves and means were compared by Student’s t test (P<.05). For WMISH, tissues were hybridized with digoxigenin-labeled riboprobes, then stained and photographed.

Results: Sry expression peaked in male UGR at d32-33. Sox9 was expressed in male but not female gonads (d32-37).MIS was expressed only in male gonads (> d32).

Conclusions: Testis induction begins with high level Sry and Sox9 expression (d31-32) and differentiation begins with MIS expression (d32).

Supported by NIH, Marilyn Simpson Trust and Dorothy R. Donnelley Faculty Development Award to the J.A. Baker Institute for Animal Health.