Journal of Histochemistry and Cytochemistry, Vol. 49, 801-802, June 2001, Copyright © 2001, The Histochemical Society, Inc.


BRIEF REPORT

Androgen Regulation of the Cellular Distribution of the True Tissue Kallikrein mK1 in the Submandibular Gland of the Mouse

S. Kurabuchia, K. Hosoib, and E. W. Gresikc
a Department of Histology, Nipon Dental University, Tokyo, Japan
b Department of Physiology, Tokushima University School of Dentistry, Tokushima, Japan
c Department of Cell Biol & Anatatomic Science, City University of New York School of Medicine, New York, New York

Correspondence to: E. W. Gresik, Dept. of Cell Biology & Anatomical Sciences, CUNY Medical School, 138th St. and Convent Avenue, New York, NY 10031.


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The kallikrein gene family encodes for at least four different proteases in the mouse submandibular gland (SMG): mK1 (true tissue kallikrein), mK9, mK13, and mK22. These enzymes and many other biologically active proteins are synthesized by the granular convoluted tubule (GCT), a specialized segment of the SMG duct system. The GCT is under multihormonal regulation by androgens, thyroid hormones, and adrenocortical hormones. Androgens suppress synthesis of mK1 in the SMG but enhance expression of the other three kallikreins. We prepared an antibody with limited immunoreactivity for mK1 and used it to examine the effects of androgen status on the distribution of this isozyme in the SMGs of developing and mature mice by immunoperoxidase staining for the light microscope and immunogold labeling for the electron microscope. In prepubertal mice, every immature GCT cell contains mK1, confined to an accumulation of small granules in the subluminal cytoplasm. In mature mice, not every GCT cell contains mK1, and in those cells that do there is considerable intergranular variation in the intensity of staining for mK1. GCT cells containing mK1 are much more abundant in the glands of females than of males, resulting in a peculiar sexually dimorphic mosaic distribution of this isozyme in the mature SMG. Castration of adult males increases the number of GCT cells expressing mK1. Administration of androgen to intact or castrated males or to intact females reduces the number of cells staining for mK1. In all cases, immunogold labeling for mK1 is confined to secretory granules. No fine structural differences were noted between cells that were positively or negatively stained for mK1. Therefore, although GCT cells appear to be composed of a uniform population of cells on the basis of morphology alone, they are not homogeneous in their content of secretory proteins. These results indicate that androgen regulation of GCT cells is more complex than has been appreciated to date. (J Histochem Cytochem 49:801–802, 2001)

Key Words: mouse, submandibular, kallikrein, androgen


  Introduction
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Summary
Introduction
Literature Cited

The granular convoluted tubule (GCT) is a specialized segment of the duct system of the submandibular gland (SMG) of mice and other rodents (Mori et al. 1992 ; Gresik 1994 ). The GCT synthesizes and secretes many biologically active polypeptides, including growth factors (e.g. EGF, NGF), renin, and several members of the kallikrein gene family (Barka 1980 ). Androgens, thyroid hormones, and adrenocortical steroids have a trophic effect on GCT cells and upregulate expression of most of these proteins (Gresik 1994 ), except for the true tissue kallikrein mK1, which is less abundant in the glands of males than of females (Hosoi et al. 1984 ). The sensitivity of GCT cells to androgens causes them to be sexually dimorphic, and they are larger in males (Gresik 1994 ). Recently we showed that not all GCT cells contain mK1, that only a few GCT cells in males are positive for this kallikrein, and that the distribution of this enzyme creates a sexually dimorphic mosaic pattern in the two sexes (Kurabuchi et al. 1999 ). Moreover, in any given cell there is considerable intergranular variation in intensity of staining for mK1, and cells positive for mK1 occur next to cells completely lacking this kallikrein. A second type of cell that is slender and finely granulated, called the SG cell, is intensely positive for mK1; this cell is present only in the male GCT.We have now studied the effects of androgen status on the cellular distribution of mK1 in developing and mature mice, using immunoperoxidase staining for light microscopy and immunogold localization for electron microscopy. In prepubertal SMGs of both sexes, when androgen levels are low, virtually every immature GCT cell showed a strongly stained subluminal band of staining for mK1; this corresponded to an apical accumulation of small granules that were labeled by immunogold for mK1.

Postpubertally, the previously described sexually dimorphic mosaic pattern of mK1 staining was seen. At 7 weeks after castration of young adult males the GCT compartment resembled that of intact adult females; more GCT cells were positive for mK1 than in intact males. Dihydrotestosterone (DHT) had a strong trophic effect on the GCT cells of intact and castrated males and of intact females. In all groups the number of GCT cells stained for mK1 declined. However, in intact and castrated males intensely stained SG cells persisted, even in the presence of supraphysiological levels of the androgen. SG cells were not seen in DHT-treated females. There was considerable intergranular variation in intensity of staining for mK1, and many cells were completely free of immunostaining. Immunogold labeling for mK1 was confined to secretory granules in all cases, but there was variation in the number of gold particles associated with different granules. Many cells contained abundant secretory granules that totally lacked immunogold labeling. The small apical granules of the SG cells were uniformly and intensely labeled. There were no differences in fine structure of cells that were positive or negative for mK1.

These immunocytochemical findings on the effects of androgen status on the distribution of mK1 in the GCT compartment of the mouse SMG are consistent with previous biochemical reports (Hosoi et al. 1983 , Hosoi et al. 1984 , Hosoi et al. 1990 , Hosoi et al. 1992 ). These results also indicate that, despite their uniform appearance, individual GCT cells may differ in their content of secretory proteins. It has previously been reported that some GCT cells lacked EGF (Gresik and Barka 1977 ), NGF (Kusakabe et al. 1986 ), or renin (Gresik et al. 1978 ), but they were infrequent and showed no sexual differences. Moreover, mK1 is present in immature GCT cells at prepubertal stages, but EGF and renin appear much later (Gresik 1994 ). Androgens regulate not only the structure of the GCT cells but also the contents of these cells. Androgens increase expression of most of the polypeptides synthesized by the GCT cell. When it was first reported that androgen reduces the concentration of mK1 in the SMG (Hosoi et al. 1984 ), it was not known whether the content of this enzyme declined uniformly in all GCT cells or whether the number of cells containing mK1 declined. Our findings show that the latter condition prevails, and indicate that androgen regulation of the GCT phenotype may be more complex than has been appreciated heretofore.


  Footnotes

Presented in part at the Joint Meeting of the Histochemical Society and the International Society for Analytical and Molecular Morphology, Santa Fe, NM, February 2–7, 2001.

Received for publication December 28, 2000; accepted February 16, 2001.
  Literature Cited
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Summary
Introduction
Literature Cited

Barka T (1980) Biologically active polypeptides in submandibular glands. J Histochem Cytochem 28:836-859[Abstract]

Gresik E (1994) The granular convoluted tubule (GCT) cell of rodent submandibular glands. Microsc Res Tech 27:1-24[Medline]

Gresik E, Barka T (1977) Immunocytochemical localization of epidermal growth factor in mouse submandibular gland. J Histochem Cytochem 25:1027-1035[Abstract]

Gresik E, Michelakis A, Barka T, Ross T (1978) Immunocytochemical localization of renin in the submandibular gland of the mouse. J Histochem Cytochem 26:855-861[Abstract]

Hosoi K, Maruyama S, Ueha T, Sato S, Gresik EW (1992) Additive and/or synergistic effects of 5-{alpha}-dihydrotestosterone, dexamethasone, and triiodo-L-thyronine on induction of proteinases and epidermal growth factor in the submandibular gland of hypophysectomized mice. Endocrinology 130:1044-1055[Abstract]

Hosoi K, Tanaka T, Ishii Y, Ueha T (1983) A new esteroproteinase (proteinase F) from the submandibular glands of female mice. Biochim Biophys Acta 756:163-170[Medline]

Hosoi K, Tanaka T, Murai T, Ueha T (1984) Inhibitory effect of androgen on the synthesis of proteinase F in the male mouse submandibular gland. J Endocrinol 100:253-262[Abstract]

Hosoi K, Ueha T, Fukuuchi H, Kohno M, Takahashi T (1990) Reversal of relative proteinase F activity and onset of androgen-dependent proteinases in the submandibular gland of postnatal mice. Biochem Int 22:179-187[Medline]

Kurabuchi S, Tada J, Gresik EW, Hosoi K (1999) An unusual sexually dimorphic mosaic distribution of a subset of kallikreins in the granular convoluted tubule of the mouse submandibular gland detected by an antibody with restricted immunoreactivity. Histochem J 31:19-28[Medline]

Kusakabe T, Ichikawa M, Ichikawa A (1986) Immunohistochemical localization of nerve growth factor and epidermal growth factor in mouse submandibular glands during postnatal development and aging. Proc XIth Int Congr Electron Microsc 2865

Mori M, Takai Y, Kunikata M (1992) Biologically active peptides in submandibular gland—role of the granular convoluted tubule. Acta Histochem Cytochem 25:325-341