232. Developmentally regulated activin A signal transduction by Sertoli cells is required for normal mouse testis development

Abstract

Activin A, a TGF-β superfamily ligand, is critical for normal mouse testis development and quantitatively normal sperm production. Testicular activin production changes during development, being substantially higher in the immature testis relative to the adult [1, 2]. Activin influences the Sertoli cell, the nurse cell to developing sperm, enhancing proliferation during its immature phase, but not following terminal differentiation [3]. In the Inha−/− mouse, chronic excessive activin production results in Sertoli cell-derived tumours [4] whereas reduced activin bioactivity, in the InhbaBK/BK mouse, delays fertility [5]. Activin signals are transduced by the phosphorylation and nuclear accumulation of the transcription factors SMAD2 and SMAD3. By comparing activin signal transduction in immature v. terminally differentiated Sertoli cells, using quantitative confocal microscopy and western blot analysis of total and phosphorylated SMAD2 and SMAD3, we discovered that mouse Sertoli cells exhibit developmentally regulated activin responses. Activin induces nuclear accumulation of SMAD3, but not SMAD2, in immature cells, although both proteins are phosphorylated. In contrast, terminally differentiated cells exhibit nuclear accumulation of both SMAD2 and SMAD3. We observed that this shift coincides with decreased SMAD3 production at puberty and changes in FSH-induced Smad transcription, which favours Smad3 in immature cells but promotes Smad2 synthesis in terminally differentiated cells. Furthermore, whereas removal of SMAD3 from the Inha−/− mouse rescues the tumour phenotype [6], we demonstrated that insufficient SMAD3 production impairs testis growth. We hypothesised that this developmentally regulated SMAD utilisation drives specific transcriptional outcomes. Using microarray and quantitative PCR, we identified novel activin target genes displaying developmental stage-specific expression patterns coinciding with differential SMAD usage, including Gja1 and Serpina5 which are required for male fertility. These mRNAs are also modulated in vivo, increased 1.5–2 fold in Inha−/− testes and decreased by half in InhbaBK/BK testes, confirming that normal testis development requires carefully regulated activin production and responsiveness. (1) Buzzard J et al. 2004. Endocrinology 145(7): 3532–3541 (2) Barakat et al. 2008. Reproduction 2008 Epub ahead of print (3) Boitani C et al. 1995. Endocrinology 136(12): 4538–4544 (4) Matzuk M et al. 1992. Nature 360: 313–319 (5) Brown C et al. 2000. Nature Genetics 25(4): 453–457 (6) Li Q et al. 2007. Molecular Endocrinology 21(10: 2472–2486