234. Hedgehog signalling components in adult rat testis spermatogonial cells

Abstract

In normal tissues, Hedgehog-induced progenitor cell proliferation is transient and tightly regulated, preventing continuous regeneration. However, activation of constitutive Hedgehog signalling results in unregulated self-renewal of progenitor cells in association with several human cancers. Although the contribution of Hedgehog signalling to cancers is widely accepted, its impact on spermatogonial stem cells and impact on male fertility are unknown. In this study, we aimed to clarify the possible role of Hh signalling on normal spermatogenesis in the adult rat and in adult testicular stem cells in the irradiated model {1}. Adult male rats were obtained from Monash University Central Animal Service and killed by cervical dislocation before tissue removal and fixation in Bouins for routine histochemical procedures. For studies on irradiated testes, adult LBNF1 male rats (hybrids between Lewis and Brown–Norway) were purchased from Harlan Sprague–Dawley, Inc. (Indianapolis, IN, USA). Testes were irradiated with 6 Gy to deplete all maturing germ cell types. At 15 weeks after irradiation the animals were injected simultaneously with 1.5 mg each of Cetrorelix pamoate and Cetrorelix acetate. Testes were collected 1, 2 or 4 weeks after injection. In situ hybridisation combined with immunohistochemistry was performed using DIG-labelled cRNA probes to identify the cells in which Hedgehog signalling components are made {2}. Signals for mRNAs encoding t he transmembrane receptors Ptc2 and Smo are most intensely detected in spermatogonia and spermatocytes and are much less intense in the round spermatids. The mRNA for the cytoplamic regulator, Fused, is restricted to the earliest germ cell types, whereas expression of the negative cytoplasmic regulator, SuFu, only begins in the round spermatids and persists in elongating spermatids. Gli1 and Gli3 are expressed from spermatogonia through to round spermatids, whereas Gli2 is restricted to spermatogonia and spermatocytes. This pattern mimics what was reported for mouse {2}. Examination of the irradiated rat testis model revealed that Hedgehog signalling machinery is produced by resting spermatogonial stem cells but is turned off when they differentiate in response to hormones. This matches the emerging understanding of Hedgehog signals in cancer stem cells and provides the first demonstration that Hedgehog signalling may influence stem cells in the adult testis.

(1) Shuttlesworth G.A. et al. 2000. Endocrinology. 141: 37–49

(2) Szczepny A. et al. 2006. Dev Dyn. 235:3063–3070.