Skip to main content
SpringerLink
Log in

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

  • Published:
In Vitro Cellular & Developmental Biology - Animal Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.

Similar content being viewed by others

References

  • Akerstrom V. L.; Walters M. R. Physiological effects of 1,25-dihydroxyvitamin D3 in TM4 Sertoli cell line. Am. J. Physiol. Endocrinol. Metab. 262: E884–E890; 1992.

    CAS  Google Scholar 

  • Bassuk J. A.; Birkebak T.; Rothmier J. D.; Clark J. M.; Bradshaw A.; Muchowski P. J.; Howe C. C.; Clark J. I.; Sage E. H. Disruption of the SPARC locus in mice alters the differentiation of lenticular epithelial cells and leads to cataract formation. Exp. Eye Res. 68: 321–311; 1999. doi:10.1006/exer.1998.0608.

    Article  PubMed  CAS  Google Scholar 

  • Bolduc C.; Larose M.; Yoshioka M.; Ye P.; Belleau P.; Labrie C.; Morissette J.; Raymond V.; Labrie F.; StAmand J. Effects of dihydrotestosterone on adipose tissue measured by serial analysis of gene expression. J. Mol. Endocrinol. 33: 429–444; 2004. doi:10.1677/jme.1.01503.

    Article  PubMed  CAS  Google Scholar 

  • Brekken R. A.; Sage E. H. SPARC, a matricellular protein: at the crossroads of cell–matrix communication. Matrix Biol. 19: 815–827; 2001. doi:10.1016/S0945-053X(00)00133-5.

    Article  CAS  Google Scholar 

  • Byers S. W.; Hadley M. A.; Djakiew D.; Dym M. Growth and characterization of polarized monolayers of epididymal epithelial cells and Sertoli cells in dual environment culture chambers. J. Androl. 7: 59–68; 1986.

    PubMed  CAS  Google Scholar 

  • Catalano S.; Pezzi V.; Chimento A.; Giordano C.; Carpino A.; Young M.; McPhaul M. J.; Ando S. Triiodothyronine decreases the activity of the proximal promoter (PII) of the aromatase gene in the mouse Sertoli cell line, TM4. Mol. Endocrinol. 17: 923–934; 2003. doi:10.1210/me.2002-0102.

    Article  PubMed  CAS  Google Scholar 

  • Cheng J.; Watkins S. C.; Walker W. H. Testosterone activates mitogen-activated protein kinase via Src kinase and the epidermal growth factor receptor in Sertoli cells. Endocrinology 148: 2066–2074; 2007. doi:10.1210/en.2006-1465.

    Article  PubMed  CAS  Google Scholar 

  • Coulombre J. L.; Russell E. S. Analysis of the pleiotropism at the W-locus in the mouse. The effects of W and Wv substitution upon postnatal development of germ cells. J. Exp. Zool. 126: 277–291; 1954. doi:10.1002/jez.1401260207.

    Article  Google Scholar 

  • Crouch S.; Spidel C. S.; Lindsey J. S. HGF and ligation of αvβ5 integrin induce a novel, cancer cell-specific gene expression required for cell scattering. Exp. Cell Res. 292: 274–287; 2004. doi:10.1016/j.yexcr.2003.09.016.

    Article  PubMed  CAS  Google Scholar 

  • De Gendt K.; Swinnen J. V.; Saunders P. T. K.; Schoonjans L.; Dewerchin M.; Devos A.; Tan K.; Atanassova N.; Claessens F.; Lecureuil C.; Heyns W.; Carmeliet P.; Guillou F.; Sharpe R. M.; Verhoeven G. A Sertoli cell-selective knockout of the androgen receptor causes spermatogenic arrest in meiosis. PNAS 101: 1327–1332; 2004. doi:10.1073/pnas.0308114100.

    Article  PubMed  Google Scholar 

  • de Winter J. P.; Vanderstichele H. M.; Timmerman M. A.; Blok L. J.; Themmen A. P.; de Jong F. H. Activin is produced by rat Sertoli cells in vitro and can act as an autocrine regulator of Sertoli cell function. Endocrinology 132: 975–982; 1993. doi:10.1210/en.132.3.975.

    Article  PubMed  Google Scholar 

  • Denolet E.; De Gendt K.; Allemeersch J.; Engelen K.; Marchal K.; Van Hummelen P.; Tan K. A. L.; Sharpe R. M.; Saunders P. T. K.; Swinnen J. V.; Verhoeven G. The effect of a Sertoli cell-selective knockout of the androgen receptor on testicular gene expression in prepubertal mice. Mol. Endocrinol. 20: 321–334; 2006. doi:10.1210/me.2005-0113.

    Article  PubMed  CAS  Google Scholar 

  • Ducray A.; Bloquel M.; Hess K.; Hammond G. L.; Gerard H.; Gerard A. Establishment of a mouse Sertoli cell line producing rat androgen-binding protein (ABP). Steroids 63: 285–287; 1998. doi:10.1016/S0039-128X(98)00030-0.

    Article  PubMed  CAS  Google Scholar 

  • Gaasch J. A.; Bolwahnn A. B.; Lindsey J. S. Hepatocyte growth factor-regulated genes in differentiated RAW 264.7 osteoclast and undifferentiated cells. Gene 369: 142–152; 2006. doi:10.1016/j.gene.2005.10.036.

    Article  PubMed  CAS  Google Scholar 

  • Giesemann T.; Rathke-Hartlieb S.; Rothkegel M.; Bartsch J. W.; Buchmeier S.; Jockusch B. M.; Jockusch H. A role for polyproline motifs in the spinal muscular atrophy protein SMN. Profilins bind to and colocalize with SMN in nuclear gems. J. Biol. Chem. 274: 37908–37914; 1999. doi:10.1074/jbc.274.53.37908.

    Article  PubMed  CAS  Google Scholar 

  • Grima J.; Zhu L. J.; Cheng C. Y. Testin is tightly associated with testicular cell membrane upon its secretion by Sertoli cells whose steady-state mRNA level in the testis correlates with the turnover and integrity of inter-testicular cell junctions. J. Biol. Chem. 272: 6499–64509; 1997. doi:10.1074/jbc.272.10.6499.

    Article  PubMed  CAS  Google Scholar 

  • Guo F.; Li S. Q.; Chu Y. H.; Huang X. F.; Sun L. M.; Li Y. Q.; Li H. J.; Zhou T. H. High-level expression, polyclonal antibody preparation and sub-cellular localization analysis of mouse Rhox5 protein. Protein Expr. Purif. 54: 247–252; 2007. doi:10.1016/j.pep.2007.03.021.

    Article  PubMed  CAS  Google Scholar 

  • Hassan A. J.; Zeng J.; Ni X.; Morales C. R. The trafficking of prosaposin (SGP-1) and GM2AP to the lysosomes of TM4 Sertoli cells is mediated by sortilin and monomeric adaptor proteins. Mol. Reprod. Dev. 68: 476–483; 2004. doi:10.1002/mrd.20096.

    Article  PubMed  CAS  Google Scholar 

  • Janecki A.; Steinberger A. Polarized Sertoli cell functions in a new two-compartment culture system. J. Androl. 7: 69–71; 1986.

    PubMed  CAS  Google Scholar 

  • Kasumi H.; Komori S.; Sakata K.; Yamamoto N.; Yamasaki T.; Kanemura Y.; Koyama K. Upregulation of macrophage migration inhibitory factor and calgizzarin by androgen in TM4 mouse Sertoli cells. Asian J. Androl. 8: 549–554; 2006. doi:10.1111/j.1745-7262.2006.00196.x.

    Article  PubMed  CAS  Google Scholar 

  • Kelly C. W.; Janecki A.; Steinberger A.; Russell L. D. Structural characteristics of immature rat Sertoli cells in vivo and in vitro. Am. J. Anat. 192: 183–193; 1991. doi:10.1002/aja.1001920207.

    Article  PubMed  CAS  Google Scholar 

  • Kleinman H. K.; McGarvey M. L.; Hassell G. R.; Star V. L.; Cannon F. B.; Laurie G. W.; Martin G. R. Basement membrane complexes with biological activity. Biochemistry 25: 312–318; 1986. doi:10.1021/bi00350a005.

    Article  PubMed  CAS  Google Scholar 

  • Kleinman H. K.; McGarvey M. L.; Liotta L. A.; Robey P. G.; Tryggvason T.; Martin G. R. Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry 21: 6188–6193; 1982. doi:10.1021/bi00267a025.

    Article  PubMed  CAS  Google Scholar 

  • Lindsey J. S.; Wilkinson M. F. An androgen-regulated homeobox gene expressed in rat testis and epididymis. Biol. Reprod. 55: 975–983; 1996a. doi:10.1095/biolreprod55.5.975.

    Article  PubMed  CAS  Google Scholar 

  • Lindsey J. S.; Wilkinson M. F. Pem: a testosterone- and LH-regulated homeobox gene expressed in mouse Sertoli cells and epididymis. Dev. Biol. 179: 471–484; 1996b. doi:10.1006/dbio.1996.0276.

    Article  PubMed  CAS  Google Scholar 

  • Lui W. Y.; Mruk D. D.; Cheng C. Y. Interactions among IQGAP1, Cdc42, and the cadherin/catenin protein complex regulate Sertoli-germ cell adherens junction dynamics in the testis. J. Cell. Physiol. 202: 49–66; 2005. doi:10.1002/jcp.20098.

    Article  PubMed  CAS  Google Scholar 

  • Lui W.-Y.; Lee W. M.; Cheng C. Y. Transforming growth factor-β3 perturbs the inter-Sertoli tight junction permeability barrier in vitro possibly mediated via its effects on occludin, zonula occludens-1, and claudin-11. Endocrinology 142: 1865–1877; 2002. doi:10.1210/en.142.5.1865.

    Article  Google Scholar 

  • Maiti S.; Doskow J.; Sutton K.; Nhim R. P.; Lawlor D. A.; Levan K.; Lindsey J. S.; Wilkinson M. F. The Pem homeobox gene: rapid evolution of the homeodomain, X chromosomal localization, and expression in reproductive tissue. Genomics 34: 304–316; 1996. doi:10.1006/geno.1996.0291.

    Article  PubMed  CAS  Google Scholar 

  • Mather J. P. Establishment and characterization of two distinct mouse testicular epithelial cell lines. Biol. Reprod. 23: 243–252; 1980. doi:10.1095/biolreprod23.1.243.

    Article  PubMed  CAS  Google Scholar 

  • McLachlan R. I.; O’Donnell L.; Meachem S. J.; Stanton P. G.; de Kretser D. M.; Pratis K.; Robertson D. M. Identification of specific sites of hormonal regulation in spermatogenesis in rats, monkeys, and man. Recent Prog. Horm. Res. 57: 149–179; 2002. doi:10.1210/rp.57.1.149.

    Article  PubMed  CAS  Google Scholar 

  • Meinhardt A.; Bacher M.; McFarlane J. R.; Metz C. N.; Seitz J.; Hedger M. P.; de Kretser D. M.; Bucala R. Macrophage migration inhibitory factor production by Leydig cells: evidence for a role in the regulation of testicular function. Endocrinology 137: 5090–5095; 1996. doi:10.1210/en.137.11.5090.

    Article  PubMed  CAS  Google Scholar 

  • Metsis M.; Timmusk T.; Allikmets R.; Saarma M.; Perrson H. Regulatory elements and transcriptional regulation by testosterone and retinoic acid of the rat nerve growth factor receptor promoter. Gene 121: 247–254; 1992. doi:10.1016/0378-1119(92)90128-C.

    Article  PubMed  CAS  Google Scholar 

  • Ming M.; Sikstrom R.; Lachance S.; Delalandre A.; Carrière S.; Chan J. S. Hormonal regulation of expression of the angiotensinogen gene in cultured mouse hepatoma cells. Am. J. Hypertens. 6: 141–148; 1993.

    PubMed  CAS  Google Scholar 

  • Nakhla A. M.; Mather J. P.; Jane O. A.; Bardin C. W. The action of calcitonin on the TM4 Sertoli cell line and on rat Sertoli cell-enriched cultures. J. Androl. 10: 321–331; 1989.

    PubMed  CAS  Google Scholar 

  • Obermann H.; Raabe I.; Balvers M.; Brunswig B.; Schulze W.; Kirchhoff C. Novel testis-expressed profilin IV associated with acrosome biogenesis and spermatid elongation. Mol. Hum. Reprod. 11: 53–64; 2005. doi:10.1093/molehr/gah132.

    Article  PubMed  CAS  Google Scholar 

  • Petty A. P.; Garman K. L.; Winn V. D.; Spidel C. M.; Lindsey J. S. Overexpression of carcinoma and embryonic cytotrophoblast cell-specific Mig-7 induces invasion and vessel-like structure formation. Am. J. Pathol. 170: 1763–1780; 2007. doi:10.2353/ajpath.2007.060969.

    Article  PubMed  CAS  Google Scholar 

  • Rawe V. Y.; Payne C.; Schatten G. Profilin and actin-related proteins regulate microfilament dynamics during early mammalian embryogenesis. Hum. Reprod. 21: 1143–1153; 2006. doi:10.1093/humrep/dei480.

    Article  PubMed  CAS  Google Scholar 

  • Russell L. D.; Ettlin R. A.; Hikim A. P.; Clegg E. D. Histological and histopathological evaluation of the testis. Cache River, Clearwater; 1990.

    Google Scholar 

  • Russell L. D.; Griswold M. D. The Sertoli cell. Cache River, Clearwater; 1993.

    Google Scholar 

  • Seftor E. A.; Meltzer P. S.; Kirschmann D. A.; Margaryan N. V.; Seftor R. E. B.; Hendrix M. J. C. The epigenetic reprogramming of poorly aggressive melanoma cells by a metastatic microenvironment. J. Cell. Mol. Med. 10: 174–196; 2006. doi:10.1111/j.1582-4934.2006.tb00299.x.

    Article  PubMed  CAS  Google Scholar 

  • Sharpe R. M. Regulation of spermatogenesis. In: Knobil E.; Neill J. D. (eds) The physiology of reproduction. Raven, New York, pp 1363–1434; 1994.

    Google Scholar 

  • Sharpe R. M. Sertoli cell endocrinology and signal transduction: androgen regulation. In: Skinner M. K.; Griswold M. D. (eds) Sertoli cell biology. Elsevier, San Diego, pp 199–216; 2005.

    Chapter  Google Scholar 

  • Show M. D.; Anway M. D.; Zirkin B. R. An ex vivo analysis of Sertoli cell actin dynamics following gonadotropic hormone withdrawal. J. Androl. 25: 1013–1021; 2004.

    PubMed  CAS  Google Scholar 

  • Skare P.; Kreivi J. P.; Bergstrom A.; Karlsson R. Profilin I colocalizes with speckles and Cajal bodies: a possible role in pre-mRNA splicing. Exp. Cell Res. 286: 12–21; 2003. doi:10.1016/S0014-4827(03)00102-2.

    Article  PubMed  CAS  Google Scholar 

  • Streuli C. H.; Schmidhauser C.; Bailey N.; Yurchenco P.; Skubitz A. P.; Roskelley C.; Bissell M. J. Laminin mediates tissue-specific gene expression in mammary epithelia. J. Cell. Biol. 129: 591–603; 1995. doi:10.1083/jcb.129.3.591.

    Article  PubMed  CAS  Google Scholar 

  • Sutton K. A.; Maiti S.; Tribley W. A.; Lindsey J. S.; Meistrich M. L.; Bucanna C. D.; Sanborn B. M.; Joseph D. R.; Griswold M. D.; Cornwall G. A.; Wilkinson M. F. Androgen regulation of the Pem homeodomain gene in mice and rat Sertoli and epididymal cells. J. Androl. 19: 21–30; 1998.

    PubMed  CAS  Google Scholar 

  • Tan K. A. L.; De Gendt K.; Atanassova N.; Walker M.; Sharpe R. M.; Saunders P. T. K.; Denolet E.; Verhoeven G. The role of androgens in Sertoli cell proliferation and functional maturation: Studies in mice with total or Sertoli cell-selective ablation of the androgen receptor. Endocrinology 146: 2674–2683; 2005. doi:10.1210/en.2004-1630.

    Article  PubMed  CAS  Google Scholar 

  • Tokuda N.; Mano T.; Levy R. B. Phagocytosis by the murine testicular TM4 Sertoli cell line in culture. J. Urol. 147: 278–282; 1992.

    PubMed  CAS  Google Scholar 

  • Vernon R. B.; Sage H. The calcium-binding protein SPARC is secreted by Leydig and Sertoli cells of the adult mouse testis. Biol. Reprod. 40: 1329–1340; 1989. doi:10.1095/biolreprod40.6.1329.

    Article  PubMed  CAS  Google Scholar 

  • Wayne C. M.; Sutton K.; Wilkinson M. F. Expression of the Pem homeobox gene in Sertoli cells increases the frequency of adjacent germ cells with deoxyribonucleic acid strand breaks. Endocrinology 143: 4875–4885; 2002. doi:10.1210/en.2002-220558.

    Article  PubMed  CAS  Google Scholar 

  • Wolgemuth D. J.; Viviano C. M.; Watrin F. Expression of homeobox genes during spermatogenesis. Ann. NY Acad. Sci. 637: 300–312; 1991.

    Article  PubMed  CAS  Google Scholar 

  • Yomogida K.; Ohtani H.; Harigae H.; Ito E.; Nishimune Y.; Engel J. D.; Yamamoto M. Developmental stage- and spermatogenic cycle-specific expression of transcription factor GATA-1 in mouse Sertoli cells. Development 120: 1759–1766; 1994.

    PubMed  CAS  Google Scholar 

  • Zhou Q.; Nie R.; Prins G. S.; Saunders P. T. K.; Katzenellenbogen B. S.; Hess R. A. Localization of androgen and estrogen receptors in adult male mouse reproductive tract. J. Androl. 23: 870–881; 2002.

    PubMed  CAS  Google Scholar 

  • Zhuang L. Z.; Phillips D. M.; Gunsalus G. L.; Bardin C. W.; Mather J. P. Effects of gossypol on rat Sertoli and Leydig cells in primary culture and established cell lines. J. Androl. 4: 336–344; 1983.

    PubMed  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  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

Authors
  1. Brianna C. Prante
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kiera L. Garman
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Brandon N. Sims
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J. Suzanne Lindsey
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J. Suzanne Lindsey.

Additional information

Editor: J. Denry Sato

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

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11626-008-9135-8

Keywords

Search

Navigation