321. HEDGEHOG SIGNALLING COMPONENTS IN DEVELOPING RAT TESTIS

Hedgehog (Hh) signalling regulates normal development of many tissues and is upregulated in some cancers. Mice which lack the testicular desert hedgehog (Dhh) ligand exhibit disrupted embryonic gonad formation and male infertility in adulthood. However, the roles and sites of Hedgehog (Hh) signalling activity in the developing rat testis are unknown. Transcripts encoding Hh pathway components in embryonic and juvenile rat testes were localised by in situ hybridization with DIG-labelled cRNA probes. On embryonic day (E) 17.5, Sertoli cells contained transcripts encoding Dhh and both gonocytes and Sertoli cells contained the Ptc2 and Smo receptor transcripts. The cytoplasmic regulators (fused [Fu], suppressor of fused [SuFu]) and the three Gli transcriptional mediators were also detected in gonocytes. On E21.5 and postnatal day 4, all transcripts were present in Sertoli cells, but not gonocytes. To test the function of Hh signalling at the onset of rodent spermatogenesis, the newborn (day 1) mouse testis was cultured in hanging drop cultures. Addition of the selective Hh signalling inhibitor, cyclopamine, yielded a significantly lower level of the Hh target, Gli1 mRNA, as measured by real time PCR compared to vehicle controls. This confirms Hh pathway activity and inhibition in this system. Meiotic markers, SYCP3 and Stra8, were consistently upregulated (n = 6) following cyclopamine addition, however the fold-change varied between experiments. Overall, the cellular expression data indicate that Hh signals are active in both embryonic and juvenile rodent testes. The finding that Hh genes analysed in this study are expressed in both Sertoli and germ cells shows that both cell types are potential Hh targets, depending on the developmental stage of testis. The upregulation of candidates for Dhh target genes in the juvenile mouse testis in vitro suggests that Hedgehog signaling downregulates the expression of meiotic genes in gonocytes via paracrine mechanisms.