Hormonal induction of functional differentiation and mammary-derived growth inhibitor expression in cultured mouse mammary gland explants

  • B. Binas
  • E. Spitzer
  • W. Zschiesche
  • B. Erdmann
  • A. Kurtz
  • T. Müller
  • C. Niemann
  • W. Blenau
  • R. Grosse
Regual Papers


A method for the cultivation of organ explants from abdominal mammary glands of virgin mice has been established. In a serum-free medium containing aldosterone, prolactin, insulin, and cortisol (APIH medium) mammary gland development was documented by lobuloalveolar morphogenesis. The hormonal requirements for in vitro expression of beta-casein and of the mammary-derived growth inhibitor (MDGI) were tested. To this end, a full length cDNA coding for mouse MDGI was prepared displaying strong homologies to a mouse heart fatty acid binding protein, which is also expressed in the mammary gland. MDGI and beta-casein transcripts were found to be absent in the mammary tissue from primed virgin mice, and were induced upon culture of mammary explants in the APIH medium. An immunohistochemical analysis with specific antibodies against MDGI and casein revealed a different pattern of expression for the two proteins. In the APIH medium, MDGI was expressed mainly in differentiating alveolar cells of the lobuloalveolar structures, whereas beta-casein was present in both ductules and alveoli. The relationship between functional differentiation and MDGI expression was further studied in explants from glands of late-pregnant mice. At this stage of development, MDGI is found both in ducts and in alveoli. If explants were cultured with epidermal growth factor (EGF) and insulin, the lobuloalveolar structure was still present, whereas MDGI disappeared. Reinduction of MDGI expression was achieved by subsequent PIH treatment. Independent on developmental stage, EGF strongly inhibits MDGI mRNA expression. It is concluded that MDGI-expression is associated with functional differentiation in the normal gland.

Key words

organ culture mammary explants mouse MDGI differentiation 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Ball, R. K.; Friis, R. R.; Schoenenberger, C. A., et al. Prolactin regulation ofβ-casein gene expression and of a cytosolic 120-kd protein in a cloned mouse mammary epithelial cell line. EMBO J. 7:2089–2095; 1988.PubMedGoogle Scholar
  2. 2.
    Banerjee, M. R.; Antoniou, M. Steroid and polypeptide hormone interaction in milk-protein gene expression. In: Litwack, G., ed. Biochemical actions of hormones, vol. 12. New York: Academic Press; 1985:237–289.Google Scholar
  3. 3.
    Billich, S.; Wissel, T.; Kratzin, H., et al. Cloning of a full-length complementary DNA for fatty-acid-binding protein from bovine heart. Eur. J. Biochem. 175:549–556; 1988.PubMedCrossRefGoogle Scholar
  4. 4.
    Boehmer, F.-D.; Lehmann, W.; Noll, F., et al. Specific neutralizing antiserum against a polypeptide growth inhibitor for mammary cells purified from bovine mammary gland. Biochem. Biophys. Acta 846:145–154; 1985.CrossRefGoogle Scholar
  5. 5.
    Boehmer, F.-D.; Kraft, R.; Otto, A., et al. Identification of a polypeptide growth inhibitor from bovine mammary gland. J. Biol. Chem. 262:15137–15143; 1987.Google Scholar
  6. 6.
    Chen, L.-H.; Bissell, M. J. A novel mechanism for whey acidic protein gene expression. Cell Regul. 1:45–54; 1989.PubMedGoogle Scholar
  7. 7.
    Chomczynsky, P.; Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform-extraction. Anal. Biochem. 162:156–159; 1987.Google Scholar
  8. 8.
    Church, G. M.; Gilbert, W. Genomic sequencing. Proc. Natl. Acad. Sci. USA 81:1991–1995; 1984.PubMedCrossRefGoogle Scholar
  9. 9.
    Coleman, S.; Daniel, C. W. Inhibition of mouse mammary ductal morphogenesis and down-regulation of the EGF-receptor by epidermal growth factor. Dev. Biol. 137:425–433; 1990.PubMedCrossRefGoogle Scholar
  10. 10.
    Du Bois, M.; Elias, J. J. Subpopulations of cells in immature mouse mammary gland as detected by proliferative responses to hormones in organ culture. Dev. Biol. 106:70–75; 1984.PubMedCrossRefGoogle Scholar
  11. 11.
    Emerman, J. T.; Pitelka, D. R. Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes. In Vitro 13:316–328; 1977.PubMedCrossRefGoogle Scholar
  12. 12.
    Ervin, P. R., Jr.; Kaminski, M. S.; Cody, R. C., et al. Production of mammastatin, a tissue specific growth inhibitor, by normal human mammary cells. Science 244:1585–1587; 1989.PubMedCrossRefGoogle Scholar
  13. 13.
    Feinberg, A. P.; Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137:266–267; 1984.PubMedCrossRefGoogle Scholar
  14. 14.
    Ginzberg, J.; de Baetselier, A.; Walker, M. D., et al. Brain tubulin and actin cDNA sequences: isolation of recombinant plasmids. Nucleic Acids Res. 8:3553–3564; 1980.CrossRefGoogle Scholar
  15. 15.
    Grosse, R.; Langen, P. Mammary-derived growth inhibitor. In: Born, G. V. R.; Cuatrecasas, P.; Herken, H., et al. Handbook of experimental pharmacology, vol. 95/II. Heidelberg: Springer-Verlag; 1990:249–265.Google Scholar
  16. 16.
    Hennighausen, L.; Sippel, A. E. Characterization and cloning of the mRNAs specific for the lactating mouse mammary gland. Eur. J. Biochem. 125:131–141; 1982.PubMedCrossRefGoogle Scholar
  17. 17.
    Ichinose, R. R.; Nandi, S. Influence of hormones on lobuloalveolar differentiation of mouse mammary glands in vitro. J. Endocrinol. 35:331–340; 1966.PubMedCrossRefGoogle Scholar
  18. 18.
    Imagawa, W.; Bandyopadhyay, G. K.; Nandi, S. Regulation of mammary epithelial cell growth in mice and rats. Endocr. Rev. 11:494–523; 1990.PubMedGoogle Scholar
  19. 19.
    Juergens, W. G.; Stockdale, F. E.; Topper, Y. J., et al. Hormone-dependent differentiation of mammary gland in vitro. Proc. Natl. Acad. Sci. USA 54:629–634; 1965.PubMedCrossRefGoogle Scholar
  20. 20.
    Kurtz, A.; Vogel, F.; Funa, K., et al. Developmental regulation of mammary-derived growth inhibitor expression in bovine mammary tissue. J. Cell Biol. 11:1779–1789; 1990.CrossRefGoogle Scholar
  21. 21.
    Levay-Young, B. K.; Hamamoto, S.; Imagawa, W., et al. Casein accumulation in mouse mammary epithelial cells after growth stimulated by different hormonal and nonhormonal agents. Endocrinology 126:1173–1182; 1990.PubMedGoogle Scholar
  22. 22.
    Mieth, M.; Boehmer, F.-D.; Ball, R., et al. Transforming growth factor-β inhibits lactogenic hormone induction ofβ-casein-expression in HC11 mouse mammary epithelial cells. Growth Factors 4:9–15; 1990.PubMedGoogle Scholar
  23. 23.
    Mueller, T.; Kurtz, A.; Vogel, F., et al. A mammary-derived growth inhibitor (MDGI) related 70 kDa antigen identified in nuclei of mammary epithelial cells. J. Cell. Physiol. 138:415–423; 1989.CrossRefGoogle Scholar
  24. 24.
    Nandi, S. Endocrine control of mammary gland development and function in the C3H/He Crgl mouse. JNCI 21:1039–1063; 1958.PubMedGoogle Scholar
  25. 25.
    Nandi, S. Hormonal control of mammogenesis and lactogenesis in the C3H/He Crgl mouse. Univ. Calif. Publ. Zool. 65:1–128; 1959.Google Scholar
  26. 26.
    Saiki, R. K.; Gelfaud, D. H.; Stoffel, S., et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA-polymerase. Science 239:487–491; 1988.PubMedCrossRefGoogle Scholar
  27. 27.
    Sanger, F.; Nicklen, S.; Coulson, A. R. DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463–5467; 1977.PubMedCrossRefGoogle Scholar
  28. 28.
    Savage, C. R., Jr.; Cohen, S. Epidermal growth factor and a new derivative. Rapid isolation procedures and biological and chemical characterization. J. Biol. Chem. 247:7609–7611; 1972.PubMedGoogle Scholar
  29. 29.
    Slot, J. W.; Geuze, H. J. A new method of preparing gold probes for multiple-labelling studies. Eur. J. Cell Biol. 38:87–93; 1985.PubMedGoogle Scholar
  30. 30.
    Smith, G. H. Functional differentiation of virgin mouse mammary epithelium in explant culture is dependent upon extracellular proline. J. Cell. Physiol. 131:190–199; 1987.PubMedCrossRefGoogle Scholar
  31. 31.
    Thomas, P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77:5201–5205; 1980.PubMedCrossRefGoogle Scholar
  32. 32.
    Tonelli, Q. J.; Sorof, S. Epidermal growth factor requirement for development of cultured mammary gland. Nature 285:250–252; 1980.PubMedCrossRefGoogle Scholar
  33. 33.
    Tonelli, Q. J.; Sorof, S. Induction of biochemical differentiation in three-dimensional collagen cultures of mammary epithelial cells from virgin mice. Differentiation 22:195–200; 1982.PubMedCrossRefGoogle Scholar
  34. 34.
    Tonelli, Q. J.; Sorof, S. Expression of a phenotype of normal differentiation in cultured mammary glands is promoted by epidermal growth factor and blocked by cyclic adenine nucleotide and prostaglandins. Differentiation 20:253–259; 1981.PubMedCrossRefGoogle Scholar
  35. 35.
    Tweedie, S.; Edwards, Y. cDNA sequence for mouse-heart fatty acid binding protein, H-FABP. Nucleic Acids Res. 17:4374; 1989.PubMedCrossRefGoogle Scholar
  36. 36.
    Vonderhaar, B. K. Local effects of EGF, alpha TGF and EGF-like growth factors on lobulo-alveolar development of the mouse mamary gland in vivo. J. Cell. Physiol. 132:581–584; 1987.PubMedCrossRefGoogle Scholar
  37. 37.
    Vonderhaar, B. K.; Nakhasi, H. C. Bifunctional activity of epidermal growth factor on alpha- and kappa-casein gene expression in rodent mammary glands in vitro. Endocrinology 119:1178–1184; 1986.PubMedGoogle Scholar
  38. 38.
    Wood, B. G.; Washburn, L. L.; Mukherjee, A. S., et al. Hormonal regulation of lobulo-alveolar growth, functional differentiation and regression of whole mouse mammary gland in organ culture. J. Endocrinol. 65:1–6; 1975.PubMedGoogle Scholar
  39. 39.
    Yoshimura, M.; Oka, T. Hormonal induction ofβ-casein gene expression: requirement of ongoing protein synthesis for transcription. Endocrinology 126:427–433; 1990.PubMedCrossRefGoogle Scholar
  40. 40.
    Yoshimura, M.; Oka, T. Transfection ofβ-casein chimeric gene and hormonal induction of its expression in primary murine mammary epithelial cells. Proc. Natl. Acad. Sci. USA 87:3670–3674; 1990.PubMedCrossRefGoogle Scholar

Copyright information

© Tissue Culture Association 1992

Authors and Affiliations

  • B. Binas
    • 1
  • E. Spitzer
    • 1
  • W. Zschiesche
    • 1
  • B. Erdmann
    • 2
  • A. Kurtz
    • 1
  • T. Müller
    • 1
  • C. Niemann
    • 1
  • W. Blenau
    • 1
  • R. Grosse
    • 1
  1. 1.Department of Cellular Biochemistrythe Max-Delbrück Center for Molecular MedicineBerlinGermany
  2. 2.Department of Electron Microscopythe Max-Delbrück Center for Molecular MedicineBerlinGermany

Personalised recommendations