LETTER TO THE EDITOR

Re: Immunocytochemical Demonstration of Caseins in Mouse Mammary Glands

Correspondence to: Gilbert H. Smith, Bldg. 10, Room 5B56, 9000 Rockville Pike, Bethesda, MD. E-mail: gs4d@nih.gov

The purpose of this communication is to clarify and correct some of the statements published in the article, "Immunocytochemical demonstration of s1- and ß-casein in mouse mammary glands at early stages of pregnancy" by T. Kanazawa and K. Kohmoto in the Journal of Histochemistry and Cytochemistry 52: 257–264, 2002. These authors state in the Summary that they were able to localize the caseins to the rough endoplasmic reticulum and the Golgi apparatus in immunocytochemically stained paraffin sections from lactating mammary glands. These two cell organelles are not resolved in Bouin's-fixed paraffin sections viewed under the light microscope. This requires preparation of the tissue for electron microscopy, as shown in the review article they cite in support of their interpretation (Burgoyne and Duncan 1998 ). Furthermore, in the Introduction the authors state, "mammary epithelial cells in vivo need to experience pregnancy and parturition to differentiate functionally." This statement is completely without support in the scientific literature. In fact, functional differentiation, as defined by the synthesis of milk proteins in mammary epithelial cells, was demonstrated in vivo over 40 years ago in ovariectomized, hypophysectomized, and adrenalectomized female mice by the injection of insulin, hydrocortisone, and prolactin (Nandi 1958 ). This observation was later confirmed in explant cultures from unprimed nulliparous female mice placed in serum-free medium, but only in the presence of insulin, hydrocortisone, and prolactin (Juergens et al. 1965 ; Turkington et al. 1965 ; Stockdale and Topper 1966 ). Subsequent experimentation too vast to enumerate here has supported these observations, and it is a widely accepted tenet that all three of these hormones are required and sufficient for a lactogenic response in mammary epithelial cells, whether in vivo or in vitro. Topper's group demonstrated that insulin was essential over 20 years ago (Bolander et al. 1981 ). The authors dismiss these wide-ranging and multidisciplinary reports by stating "it was not clear whether these three hormones are truly sufficient for inducing differentiation in mammary epithelial cells." Subsequently, they refer to their own unpublished work, which demonstrates induction of casein in cultures (serum-free) from cycling nulliparous mice with these three hormones to provide a straw-man hypothesis to justify repeating the work of others with monoclonal antibodies to mouse - and ß-casein.

In the Discussion, the authors once again chose to ignore the published literature that convincingly demonstrates that mammary epithelial cells from unprimed cycling nulliparous mice can be induced to synthesize caseins as well as other milk proteins (Vonderhaar et al. 1973 ) both in vivo and in vitro. They misquote the in vitro results of Tonelli and Sorof 1982 and of Durban et al. 1985 to support the contention that their unpublished demonstration of casein synthesis in primary mammary epithelial cells in serum-free medium is a seminal discovery and justifies the technical improvements for casein detection which they allege that their monoclonal antibodies provide. Furthermore, their claim that three phases of casein synthesis occur in the mammary epithelium during pregnancy is based on the presence of immunologically detectable intracellular casein and discounts the movement of the casein into the luminal space. In early pregnancy at 0–4 days, the acinar lumina are small and contain no secretory product. Subsequently, at days 6–10, the acinar lumina become distended with secretion and the epithelial cells themselves less secretory in appearance. At 14 days of pregnancy both lumina and epithelial cells are distended with secretory product. It has been shown that progesterone levels rise during pregnancy and prevent the translation of casein mRNAs (Guyette et al. 1979 ; Rosen et al. 1980 ). Therefore, this observation is also not novel.

Finally, in our 1981 paper (Smith and Vonderhaar 1981 ), we demonstrated by immunoelectron microscopy that casein was present within the cisternae of the rough endoplasmic reticulum in the epithelial cells within explants incubated with insulin, hydrocortisone, and prolactin but not in control cultures, in excellent agreement with our immunohistochemical results carried out on the same samples. Our immunohistochemical analysis of casein synthesis induction was carried out on deplasticized 1-µm sections by immunostaining methodology (Hogan and Smith 1982 ) essentially identical to that reported in the Kanazawa paper under discussion. The anti-casein antibody used was characterized in Smith et al. 1984 and was shown to immunoprecipitate only casein(s) from mouse skim milk.

Literature Cited

Bolander FF, Jr, Nicholas KR, Van Wyk JJ, Topper YJ (1981) Insulin is essential for accumulation of casein mRNA in mouse mammary epithelial cells. Proc Natl Acad Sci USA 78:5682-5684[Medline]

Burgoyne RD, Duncan JS (1998) Secretion of milk proteins. J Mamm Gland Biol Neoplasia 3:275-286

Durban EM, Medina D, Butel JS (1985) Comparative analysis of casein synthesis during mammary cell differentiation in collagen and mammary development in vivo. Dev Biol 109:288-298[Medline]

Guyette WA, Matusik RJ, Rosen JM (1979) Prolactin-mediated transcriptional and post-transcriptional control of casein gene expression. Cell 17:1013-1023[Medline]

Hogan DL, Smith GH (1982) Unconventional application of standard light and electron immunocytochemical analysis to aldehyde-fixed, araldite-embedded tissues. J Histochem Cytochem 30:1301-1306[Abstract]

Juergens WG, Stockdale FE, Topper YJ, Elias JJ (1965) Hormone-dependent differentiation of mammary gland in vitro. Proc Natl Acad Sci USA 54:629-634[Medline]

Nandi S (1958) Endocrine control of mammary-gland development and function in the C3H/He Crgl mouse. J Natl Cancer Inst 21:1039-1063

Rosen JM, Matusik RJ, Richards DA, Gupta P, Rodgers JR (1980) Multihormonal regulation of casein gene expression at the transcriptional and posttransciptional levels in the mammary gland. Rec Prog Horm Res 36:157-193[Medline]

Smith GH, Vonderhaar BK (1981) Functional differentiation in mouse mammary gland epithelium is attained through DNA synthesis, inconsequent of mitosis. Dev Biol 88:167-179[Medline]

Smith GH, Vonderhaar BK, Graham DE, Medina D (1984) Expression of pregnancy-specific genes in preneoplastic mouse mammary tissues from virgin mice. Cancer Res 44:3426-3437[Abstract]

Stockdale FE, Topper YJ (1966) The role of DNA synthesis and mitosis in hormone-dependent differentiation. Proc Natl Acad Sci USA 56:1283-1289[Medline]

Tonelli QJ, Sorof (1982) Induction of biochemical differentiation in three–dimensional collagen of mammary epithelial cells from virgin mice. Differentiation 22:195-200[Medline]

Turkington RW, Juergens WG, Topper YJ (1965) Hormone-dependent synthesis of casein in vitro. Biochim Biophys Acta 111:573-576[Medline]

Vonderhaar BK, Owens IS, Topper YJ (1973) An early effect of prolactin on the formation of lactalbumin by mouse mammary epithelial cells. J Biol Chem 248:467-471[Medline]

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