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Matrix-coated transwell-cultured TM4 sertoli cell testosterone-regulated gene expression mimics in vivo expression

In Vitro Cellular & Developmental Biology - Animal volume 44, pages 434–443 (2008)Cite this article

Abstract

In vitro culture systems are needed to mimic in vivo epithelial cell environments for identifying cell signaling, gene expressions, and molecular mechanisms. One such system is matrix-coated transwell cultures. However, no data exist on culturing Sertoli cells in this manner with respect to testosterone-regulated gene expression. Because the TM4 mouse Sertoli-like cell line expresses androgen receptor, our objective was to determine if testosterone treatment added to the bottom chamber of a matrix-coated transwell system induces some gene expressions found in Sertoli cells in vivo. After serum starvation, transwell-cultured TM4 cells were treated with testosterone or left untreated for 24 h. Microarray analyses initially identified differentially expressed genes either induced or repressed by testosterone treatment. By Northern blot analyses, Pem mRNA, a frequently used marker of Sertoli cell testosterone responsiveness, was induced. Proteins of the transcripts induced by testosterone in the in vitro system were immunolocalized to Sertoli cells in testosterone-dependent stages of spermatogenesis in mouse testes. By immunohistochemistry analyses of sectioned mouse testes, gene expression induced by testosterone in transwell-cultured TM4 cells, profilin as well as secreted protein acidic and rich in cysteines (SPARC) are localized to Sertoli cells in testosterone-dependent stages of spermatogenesis. Findings include localizations of SPARC and profilin, as well as an apparent germ cell communication required for translation of Pem mRNA in Sertoli cells. Taken together, results of these studies suggest that this TM4 transwell-culture system could be used to study these testosterone-regulated Sertoli gene expressions in vitro.

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Acknowledgments

We thank Dr. Michael D. Griswold, Washington State University, for helpful discussions and dominant white spotting mice. We also appreciate the Pem antibodies provided by Dr. Miles F. Wilkinson, MD Anderson Cancer Research Center, Houston, TX. In addition, we thank Dr. E. Helene Sage, Hope Heart Program Benaroya Research Institute at Virginia Mason, Seattle, WA, for helpful advice for detection of SPARC by immunohistochemistry.

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  1. Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Wegner Hall, Pullman, WA, 99164-6534, USA

    Brianna C. Prante, Kiera L. Garman, Brandon N. Sims & J. Suzanne Lindsey

  2. School of Molecular Biosciences, Washington State University, Pullman, WA, USA

    J. Suzanne Lindsey

  3. Center for Reproductive Biology, Washington State University, Pullman, WA, USA

    J. Suzanne Lindsey

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  1. Brianna C. Prante
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  2. Kiera L. Garman
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  3. Brandon N. Sims
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  4. J. Suzanne Lindsey
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Correspondence to J. Suzanne Lindsey.

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This work was funded by the National Institutes of Health (DK069962 and CA93925) as well as the Washington State University Center for Reproductive Biology.

Editor: J. Denry Sato

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Prante, B.C., Garman, K.L., Sims, B.N. et al. Matrix-coated transwell-cultured TM4 sertoli cell testosterone-regulated gene expression mimics in vivo expression. In Vitro Cell.Dev.Biol.-Animal 44, 434–443 (2008). https://doi.org/10.1007/s11626-008-9135-8

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Keywords

  • Extracellular matrix
  • Transwell
  • Pem
  • Rhox5
  • SPARC
  • Profilin