Additive and/or Synergistic Action (Downregulation) of Androgens and Thyroid Hormones on the Cellular Distribution and Localization of a True Tissue Kallikrein, mK1, in the Mouse Submandibular Gland
Department of Histology, Nippon Dental University School of Dentistry at Tokyo, Tokyo, Japan (SK); Department of Cell Biology and Anatomical Sciences, City University of New York Medical School, New York, New York (EWG); and Department of Molecular Oral Physiology, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan (KH)
Correspondence to: Shingo Kurabuchi, PhD, Dept. of Histology, Nippon Dental University School of Dentistry at Tokyo, Fujimi 1-9-20, Chiyoda-ku, Tokyo 102-8159, Japan. E-mail: kurabuchi{at}tokyo.ndu.ac.jp
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Summary |
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Key Words: mK1 immunocytochemistry submandibular gland hypophysectomy 5-dihydrotestosterone 3,5,3'-triiodothyronine dexamethasone mouse
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Introduction |
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We previously prepared an antiserum with specific immunoreactivity for mouse mK1, and an immunocytochemical study with the antiserum showed that the lower content of mK1 in the SMGs of males was due not to uniformly lower synthesis of this enzyme in each granular convoluted tubule (GCT) cell but to a smaller number of GCT cells that express it. Only a few scattered GCT cells were immunopositive for mK1 in the male gland, and many more cells were mK1-immunopositive in the female gland, revealing an unusual sexually dimorphic cellular mosaic distribution of mK1 in the GCT segments (Kurabuchi et al. 1999). We also found that the sexually dimorphic mosaic expression of mK1 was established after the onset of puberty, whereas virtually all immature GCT cells were uniformly mK1-immunoreactive at prepubertal ages (Kurabuchi et al. 2002
). Experiments have also demonstrated that the number of mK1-immunopositive cells in the male gland is increased by castration and that the proportion of these immunopositive cells in the glands of female and castrated male mice is decreased by DHT administration (Kurabuchi et al. 2001
,2002
). Taken together, these immunocytochemical findings suggest that androgens are the key hormonal stimulus driving the development of the GCT phenotype and the sexually dimorphic mosaic distribution of mK1 in this segment.
Submandibular GCT cells are known to be regulated by a variety of hormones, and thyroid hormones and adrenocortical hormones, as well as androgens, are involved (reviewed in Chrétien 1977; Gresik 1994
). Hypophysectomized (Hypox) animals are a useful model in which to investigate the actions of these pituitary-dependent hormones because they allow assessment of the direct effect of one or more hormones on target tissues. The aim of the present study using Hypox mice was to identify the roles of three hormones, DHT, T3, and Dex, alone and in combination, in regulating the sexually dimorphic GCT phenotype and mosaic cellular expression of mK1 by light and electron microscopic analyses, and to use the morphological findings to interpret the results of previously published biochemical analyses of mK1 in the mouse SMG (Murai 1987
; Hosoi et al. 1992
; Maruyama et al. 1993
; Kurihara et al. 1999
).
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Materials and Methods |
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Immunocytochemistry
The SMGs were minced into small pieces and immersed for 4 hr in a cold mixture of 2% glutaraldehyde and 2% paraformaldehyde in 0.05 M cacodylate buffer, pH 7.4. After dehydration through a graded series of ethanol solutions, they were embedded in Epon/Araldite.
For light microscopy, 2-µm-thick sections were mounted on silane-coated slides (Matsunami; Tokyo, Japan). The sections were treated for 10 min with methanol saturated with NaOH to remove the resin and incubated for 30 min in 0.3% H2O2 in methanol to inhibit endogenous peroxidase activity. The sections were washed in distilled water (DW) and phosphate-buffered saline (PBS; 0.14M NaCl in 0.01 M sodium phosphate buffer and 0.14 M NaCl, pH 7.5) and were immunostained by the avidinbiotinperoxidase complex method according to the manufacturer's protocol (Vecstain Elite ABC kit; Vector Labs, Burlingame, CA). The sections were then incubated for 4 hr at room temperature with the primary antibody, rabbit anti-mK1 antiserum (Kurabuchi et al. 1999,2002
) diluted 1:40,000. Peroxidase activity was detected by incubation for 35 min in 1% 3,3'-diaminobenzidine tetrahydrochloride (Dojin Labs; Kumamoto, Japan), 0.01% H2O2, 0.05 M Tris-HCl, pH 7.6. The sections were then dehydrated with ethanol, mounted in Entellan (Merck; Gibbstown, NJ), and examined with a BX-50 microscope and Nomarski differential interference contrast optics (Olympus; Tokyo, Japan). Cross-sections of GCT segments were identified in the immunostained sections, and the distance between the apical edge and basal layer (cell height) of more than 100 GCT cells per animal was measured. In addition, the total number of GCT cells was counted, the number of mK1-immunopositive GCT cells was counted, and their percentages per section were calculated. The means ± SEM of these values were calculated for five animals. One-way ANOVA analyses were performed using StatView software (SAS; Cary, NC). Significant differences between treatment groups were determined by the Bonferroni/Dunn's multiple comparison tests. Statistical significance was inferred when the p value was less than 0.05.
For electron microscopy, ultrathin sections on gold grids were etched with 3% H2O2 for 5 min, thoroughly rinsed with DW, and subjected to immunostaining for mK1 by the protein Agold technique. The sections were first incubated with 20% normal goat serum for 2 hr, and after direct transfer into a drop of antiserum specific for mK1 (1:40,000 diluted) they were incubated overnight at 4C. The sections were then rinsed three times in PBS and incubated with biotinylated goat anti-rabbit IgG (Vecstain Elite ABC kit) for 1 hr, and then washed. Finally, they were incubated for 2 hr with a 1:60 dilution of streptavidin conjugated with 15-nm gold particles (Zymed; San Francisco, CA). The sections were contrasted with uranyl acetate and lead citrate and viewed in a transmission electron microscope (JEM-2000; EXII, JEOL, Tokyo, Japan) at 80 kV accelerating voltage.
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Results |
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Both combined injection of DHT and T3 and injection of all three hormones had a significant trophic effect on the GCT cells (Figures 2e and 2h). After injection with DHT and T3, the GCT cells in Hypox SMGs were larger and contained abundant large secretory granules in their apical cytoplasm (Figure 2e), similar to the GCT cells of normal males (Figure 1a). After injection of all three hormones, the secretory granules were much larger but fewer in number, and in some cells one or a few secretory granules were larger than the basal nucleus (Figure 2h). Only a few GCT cells in the GCT segments of these two groups were immunopositive, and all of the others were immunonegative (Figures 2e and 2h). Most of the immunopositive cells were typical GCT cells, but smaller SG cells were also present (data not shown). The GCT cells in these two groups were significantly higher than in the Hypox males and the other hormone-injected groups (p<0.0001; Figure 3A). The percentages of immunopositive cells in these two groups were also significantly lower than in the Hypox males and in the other hormone-injected groups (p<0.0001; Figure 3B), in which the percentages were close to that in the normal male gland. Combined treatment with DHT and Dex (Figure 2f) or T3 and Dex (Figure 2g) also had a trophic effect on the GCT cells (p<0.0001; Figure 3A) and decreased the number of mK1-immunopositive GCT cells (p<0.0001; Figure 3B). However, there were no differences in the sizes of GCT cells and percentages of immunopositive cells in these groups injected with DHT + Dex or T3 + Dex, compared with those in the groups injected with DHT alone or T3 alone, respectively. The secretory granules in some of the GCT cells of the Hypox group injected with both T3 and Dex (Figure 2g) were larger than in the other groups, i.e., those injected with hormones alone or in combination and normal males or females. However, the granules were not as large as those in the group injected with all three hormones (Figure 2h).
Electron Microscopic Immunocytochemistry
The ultrastructural analysis by immunogold staining extended the findings of light microscopy. Almost all of the atrophic GCT cells in the gland of Hypox male mice had small secretory granules near the apical lumen, and there was variation in the size and density of the secretory granules among the cells (Figure 4) . The round euchromatic nucleus was at the center of the cell, and the basal area was occupied by well-developed infoldings of the basal plasma membrane, associated with elongated mitochondria. A small Golgi apparatus with several narrow cisternae and sparse segments of flattened rough endoplasmic reticulum (RER) were present in the perinuclear cytoplasm. Gold particles, indicating the presence of mK1, were restricted to the secretory granules (Figure 4).
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Discussion |
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In agreement with the findings in a previous morphological study (Kurabuchi 2002), the results of the present study showed that administration of DHT, T3, or Dex to Hypox males enhanced the GCT phenotype, i.e., enlargement of cell size and increase in number of secretory granules. This result means that these hormones have induced the gene expression of secretory products, such as growth factors and kallikrein gene family members. However, the results of the specific immunocytochemical stainings used in this experimental system strongly suggested that each hormone, given alone or in combination, downregulated the number of mK1-immunoreactive cells, although the amount of mK1 in each positive cell was extremely increased. With respect to the hormones injected singly, DHT alone had the strongest trophic effect on the structure of GCT cells but only moderately reduced the number of mK1-positive GCT cells. Therefore, DHT alone did not fully restore the GCT phenotype in Hypox males to the extent that mK1 expression remained higher than in the SMGs of intact males. T3 alone had a moderately trophic action on GCT structure and also reduced the number of immunopositive GCT cells in Hypox males. The appearance of the GCT cells in the T3-injected Hypox males was almost identical to that of the GCT cells of normal females, strongly suggesting that thyroid hormone is one of the key hormones in maintaining the GCT phenotype in intact females. This is consistent with the results of a previous study (Kurabuchi 2002
). Although Dex alone slightly increased the size of GCT cells, it had no inhibitory effect on the immunocytochemical distribution of mK1 because essentially all of the GCT cells in these mice were positive for mK1. This observation explains why Dex itself increased the levels of SMG mK1 when given alone to Hypox mice (Hosoi et al. 1992
; Maruyama et al. 1993
; Kurihara et al. 1999
). Because all of the GCT cells expressed mK1 and were slightly larger, the final effect of this hormone is an increase in the total amount of mK1 in gland homogenates.
Stronger effects were observed after combined injection of two or three hormones. Concomitant injection of DHT + T3 or of DHT + T3 + Dex significantly enlarged the size of GCT cells in Hypox mice and greatly reduced the number of mK1-producing GCT cells, so that the GCTs very closely resembled those of intact male mice. These findings imply that the combination of DHT + T3, with or without Dex, is necessary for induction of the GCT phenotype characteristic of intact males. On the other hand, the combinations of DHT + Dex and T3 + Dex were not as effective as DHT alone or T3 alone. However, this and previous studies (Kurabuchi 2002) have shown that combinations of T3 + Dex or all three hormones make the secretory granules of GCT cells larger than in other hormone-treated groups or normal males and females. Because large secretory granules are regarded as an index of hypertrophic GCT cells (Chrétien 1977
), Dex apparently enhances the trophic action of T3 or DHT + T3 on the structure of GCT cells. Enlargement of the GCT lumen and decreased numbers of secretory granules were remarkable only with the combined injection of three hormones, presumably implying increased secretory activity. It is well known that the discharge of secretory granules is induced by secretagogue-stimulated salivation (Murphy et al. 1980
). At present, however, no report has shown that the combination of these hormones affects the secretory cycle of saliva. Such a possibility remains to be verified. The present immunocytochemical and ultrastructural observations in Hypox male mice injected with hormones in this study strongly suggest that the sexually dimorphic mosaic expression of mK1, as well as the overall cellular phenotype of the GCTs, are due to additive and/or synergistic actions of androgens and thyroid hormones. The effects of these hormones may be modulated by adrenocortical hormones.
The results of biochemical analyses (Hosoi et al. 1992; Maruyama et al. 1993
; Kurihara et al. 1999
) have indicated that injection of T3 alone has no effect on the mK1 content of the SMGs of Hypox mice and that DHT injected alone increases it. By contrast, the results of our immunocytochemical study revealed distinct differences in the expression of mK1 and the phenotype of GCT cells between the untreated Hypox mice and the T3- or DHT-injected Hypox mice, especially with regard to decreases in the proportion of mK1-immunonpositive cells. However, in these hormone-treated Hypox mice, there were increases in the number and volume of secretory granules containing mK1 in each immunopositive cell, resulting in an increase in the quantity of mK1 in these cells. Therefore, biochemical analyses alone cannot detect an inhibitory effect of T3 or DHT on mK1 expression.
Thyroid hormone itself has a trophic effect on the GCT cells, as shown by the ability of T3 to promote development of GCT cells in the androgen-insensitive Tfm/Y mouse (Hosoi et al. 1979; Aloe and Levi-Montalcini 1980
). T3 given alone to prepubertal mice induces GCT cells precociously (Chabot et al. 1987
) and potentiates the ability of androgens to induce the GCT phenotype and expression of kallikreins and EGF in immature mice (Gresik and Barka 1980
). In addition, thyroid hormones upregulate the expression of androgen receptor in the SMG of developing and adult mice (Minetti et al. 1986
,1987
). The trophic effect of androgens on the GCT compartment is biphasic. It recruits striated duct or intercalated duct cells to become newly formed GCT cells and then promotes the conversion of these immature GCT cells to fully differentiated GCT cells typical of mature male mice (Gresik 1994
). Our observation that DHT given alone did not fully restore the adult male mosaic pattern of mK1 distribution to Hypox mice, in that many more cells stained for mK1 than in intact male mice, might also be explained by relatively low androgen sensitivity because of inadequate levels of thyroid hormone to maintain proper levels of androgen receptors in the glands. This idea is supported by the ability of simultaneous treatment with DHT and T3 to fully restore the mK1 staining pattern typical of intact adult males.
It is known that some GCT cells lack androgen receptors, although most of them possess (Morrel et al. 1987; Sawada and Noumura 1995
). It is also reported that expression of the SMG androgen receptor in mice is induced by thyroid hormone (Minetti et al. 1986
, 1987
). Therefore, depending on whether or not the GCT cells possess the receptors for the hormone, they would become either mK1-immunopositive or mK1-immunonegative (Kurabuchi at al. 2002
). The expression level of androgen receptor (and probably other hormone receptors, such as thyroid hormone receptors) would therefore strongly affect both the number of mK1-negative cells and the mK1 expression level in each GCT cell.
Finally, according to Kurihara et al. (1999), DHT injection paradoxically increased mK1 levels in intact females and castrated males of the C3H/HeN strain mice, but if the C3H/HeN mice were also given T3 the DHT caused a reduction in mK1 content. The interpretation by these authors is that androgens exert an inductive effect on mK1, as they do on the other kallikreins, but that this action of androgen is suppressed by thyroid hormones. As described above, this idea is inconsistent with the findings of our immunocytochemical study, because T3 alone and DHT alone each decreased mK1-immunopositive GCT cells in the Hypox ICR males, even though the total content of mK1 in the gland homogenates increased (Hosoi et al. 1992
; Maruyama et al. 1993
; Kurihara et al. 1999
). They also found that a fixed dose of androgens caused progressive reduction in the content of SMG mK1 in the presence of increasing doses of T3, and suggested that C3H/HeN mice are relatively insensitive to thyroid hormones because increasing doses of T3 were necessary to elicit a greater inhibitory action on the effect of androgens on mK1 content. The insensitivity to thyroid hormones postulated by Kurihara et al. (1999)
predicts that the GCT phenotype and mK1 expression in the SMGs of C3H/HeN males and females are very similar to those of Hypox mice (ICR strain) given DHT + Dex and Dex alone, respectively. Although no immunocytochemical studies of mK1 have been performed in C3H/HeN mouse SMGs, it is possible that the GCT phenotype is more developed in males than in females of this strain but that the number of mK1 immunonegative cells is low even in the males. This would be consistent with previous studies demonstrating that the mK1 content of the SMGs of C3H/HeN mice is more abundant in males than in females (Murai 1987
; Kurihara et al. 1999
). However, it appears more plausible that T3 potentiates the action of androgens on the GCT cells by upregulating expression of the androgen receptor in both strains of mice (Minetti et al. 1986
,1987
) and that the direct actions of androgens are enhancement of the structure of GCT cells, upregulation of most secretory products, and inhibition of expression of mK1 in these cells. Coupling the biochemical analyses described above and the immunocytochemical analysis of the distribution of mK1 in this study provides evidence that should clarify these issues.
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Acknowledgments |
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Footnotes |
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