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DOI: 10.1369/jhc.4A6333.2004
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Journal of Histochemistry and Cytochemistry
Volume 52 (11): 1437-1446, 2004
Copyright ©The Histochemical Society, Inc.

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

Shingo Kurabuchi, Edward W. Gresik and Kazuo Hosoi

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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 Materials and Methods
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We investigated the effects of 5{alpha}-dihydrotestosterone (DHT), 3,5,3'-triiodo-L-thyronine (T3), and dexamethasone (Dex) on the expression of mK1 in the granular convoluted tubule (GCT) cells of the submandibular gland (SMG) of hypophysectomized (Hypox) male mice by indirect enzyme-labeled antibody and immunogold antibody methods for light and electron microscopy. Hypox resulted in considerable atrophy of the GCT cells, which were always immunoreactive for mK1, and the cells were characterized by apical small dense secretory granules labeled with gold particles suggesting the presence of mK1, small Golgi apparatus, sparse rough endoplasmic reticulum (RER), and developed basal infoldings. Each of the hormones, DHT, T3, and Dex, enhanced the GCT phenotype to various degrees in Hypox male mice. Both DHT alone and T3 alone moderately inhibited mK1 synthesis by increasing the number of mK1-immunonegative GCT cells in Hypox males, but Dex alone had no inhibitory effect on mK1 synthesis. A significant trophic effect on GCT cells was induced by combined injection of DHT and T3 or of all three hormones, and was reflected in the appearance of abundant large secretory granules, well-developed Golgi apparatus and RER, and reduced basal infoldings. Only a few such GCT cells were immunopositive for mK1, and the pattern of immunopositive and immunonegative cells very closely resembled the mosaic pattern seen in normal male GCTs. These findings suggested that the sexual dimorphism of mK1 expression and the morphological appearance of GCT cells can be induced by treatment with two hormones, DHT and T3, but not by either of them alone. T3 appears to have a permissive effect on committed GCT cells that results in downregulation of mK1 expression in these cells. (J Histochem Cytochem 52:1437–1446, 2004)

Key Words: mK1 • immunocytochemistry • submandibular gland • hypophysectomy • 5{alpha}-dihydrotestosterone • 3,5,3'-triiodothyronine • dexamethasone • mouse


   Introduction
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 Introduction
Materials and Methods
 Results
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 Literature Cited
 
A TRUE TISSUE KALLIKREIN, mK1, identified by Hosoi et al. (1994)Go, is the product of one member of the large kallikrein gene family of the mouse (Berg et al. 1992Go), which consists of at least 26 members (Evans et al. 1987Go). Four kallikreins, mK1, mK9, mK13, and mK22, which correspond to true tissue kallikrein, the binding protein for epidermal growth factor (EGF) (Taylor et al. 1974Go), prorenin converting enzyme (Kim et al. 1991Go; Hosoi et al. 1994Go), and ß-nerve growth factor (NGF) endopeptidase (Smith et al. 1968Go; Stach et al. 1976Go, 1980Go), respectively, are detected in the mouse submandibular gland (SMG) by isoelectric focusing analysis. The amount of mK1 is much more abundant in the glands of females (Hosoi et al. 1983Go,1984Go), whereas the vast majority of biologically active substances, such as EGF, NGF, renin, and members of kallikrein gene family (except for mK1), are known to be more abundant in the glands of males (reviewed in Barka 1980Go).

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. 1999Go). 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. 2002Go). 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. 2001Go,2002Go). 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 1977Go; Gresik 1994Go). 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 1987Go; Hosoi et al. 1992Go; Maruyama et al. 1993Go; Kurihara et al. 1999Go).


   Materials and Methods
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 Materials and Methods
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 Literature Cited
 
Animals and Procedures
Intact male and female and hypophysectomized (hypox) male ICR mice were purchased from Japan SLC (Shizuoka, Japan). Hypophysectomy was performed at 4 weeks of age via the external auditory canal under pentobarbital anesthesia. The animals were housed under controlled environmental conditions (22C, light:dark 12 hr:12 hr) and were provided with food and water ad libitum. At 14 weeks of age the mice were divided into eight groups consisting of at least five animals each and were injected with hormones as described in our previous reports (Hosoi et al. 1992Go; Kurabuchi 2002Go; Kurabuchi et al. 2002Go). Briefly, the doses (per kg body weight) per injection were 5{alpha}-dihydrotestosterone (DHT; Wako, Osaka, Japan) 20 mg; dexamethasone (Dex; Wako) 10 mg; and 3,5,3' triiodo-L-thyronine (T3; ICN Biochem., Aurora, OH) 1 mg. The hormones were injected either singly or in combination every other day for 2 weeks (seven injections) before the animals were sacrificed. Three of the eight Hypox groups (males) were injected with one of the hormones, three other groups were injected with two hormones, and the remaining group was injected with all three hormones. Groups of Hypox mice not injected with any hormones and of normal male and female mice of the same age were established as controls. On the day after the final injection, the animals were fasted overnight and then sacrificed. Under Nembutal anesthesia (30 mg/kg body weight), the submandibular glands (SMGs) were quickly removed and processed for light and electron microscopic immunocytochemical examination.

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 avidin–biotin–peroxidase 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. 1999Go,2002Go) diluted 1:40,000. Peroxidase activity was detected by incubation for 3–5 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 A–gold 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.


   Results
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 Materials and Methods
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 Literature Cited
 
Light Microscopic Immunocytochemistry
Normal Male and Female Mice
The cellular distribution of mK1 exhibited sexually dimorphic mosaic expression in the GCT segments, consistent with the results of our previous studies (Kurabuchi et al. 1999Go, 2001Go,2002Go). Briefly, only a few scattered immunostained cells were observed in the GCT segments in the male gland, and some of them were slender and showed strong immunostaining. Such cells are referred to as slender GCT cells (SG cells). The other cells were typical GCT cells and stained moderately (Figure 1a) . In the female gland, on the other hand, many GCT cells showed moderate to strong immunostaining (Figure 1b). There were no morphological differences between the immunopositive and -negative GCT cells, except for the SG cells, in either sex. The GCT cells of the males were ~1.5-fold more numerous than in the females (p<0.0001; Figure 3A). Approximately 1% of cells immunostained in the male gland, as opposed to over 60% in the female gland (p<0.0001; Figure 3B).



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  		Figure 1

Nomarski differential interference microscopy of Epon/Araldite-embedded 2-µm-thick sections of SMGs of adult mice (16 weeks old) after immunostaining with preabsorbed anti-mK1 antiserum. Only a few scattered GCT cells are immunostained in the male gland (a), whereas many cells in the GCT segment are stained in the female gland (b), resulting in an unusual sexually dimorphic mosaic distribution of mK1 in the GCTs. Arrows, mK1-immunonegative cells (b,c,f,g). Bar = 50 µm.

 


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  		Figure 3

Summary of effects of Hypox and subsequent injection of DHT, T3, and Dex, alone or in combination, on the cell height (distance between apical edge and basal layer) of GCT cells (A) and the percentage of mK1-immunopositive GCT cells in the GCT segments (B). Results are expressed as means ± SEM (each, n=5). Data were analyzed by ANOVA and Bonferroni/Dunn's tests. *p<0.01; **p<0.001; ***p<0.0001, significantly different from the uninjected Hypox male (No injection) group. #, p<0.0001 vs a group of normal males. , p<0.0001 vs normal male and female groups. §, p<0.0001 vs other hormone-injected groups.

 
Hypox Male Mice
The GCT cells were drastically reduced in the SMGs of Hypox male mice (Figure 2a) . All of the cells had fewer and smaller apical secretory granules and a pale round nucleus at the center of the cell, differing from the GCTs of normal male and female mice (Figures 1a and 1b). Almost of all of these GCT cells were strongly immunostained. The cell height of the GCT cells of the Hypox males was significantly lower than in normal male or normal female glands (p<0.0001; Figure 3A) . Approximately 99% of the GCT cells immunostained positive for mK1, and the rest (less than 1%) were immunonegative. The percentage of the immunopositive cells in Hypox males was significantly greater than that of normal males and females (p<0.0001; Figure 3B).



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  		Figure 2

Nomarski differential interference microscopy of Epon/Araldite-embedded 2-µm-thick sections of SMGs of hormone-injected Hypox male mice after immunostaining with preabsorbed anti-mK1 antiserum. In the untreated Hypox male, almost all GCT cells are atrophic but are strongly immunopositive (a). Injection with DHT (b), T3 (c), DHT + T3 (e), DHT + Dex (f), T3 + Dex (g), or all three hormones (h) somewhat reduced the number of mK1-immunopositive GCT cells. Dex alone (d) had no effect on mK1 expression. Bar = 50 µm.

 
Hormone-treated Hypox Male Mice
When Hypox male mice were injected with DHT alone, all of their GCT cells were large and contained many large secretory granules in their apical cytoplasm and a pale round nucleus at their base. The mK1-immunopositive and -negative cells were distributed in a mosaic pattern throughout the GCT segments (Figure 2b), and the immunoreactivity of the positive cells was sometimes moderate. As shown in Figure 3A, these GCT cells were much higher than the GCT cells of uninjected Hypox males (p<0.0001) and somewhat higher than the GCT cells of normal females, but not as high as the GCT cells of normal males. The percentage of immunopositive cells (~80%) in the DHT-injected Hypox male mice was lower than in the uninjected Hypox males (p<0.001; Figure 3B) but higher than in normal females and much higher than in normal males. After injection with T3 alone, the cell phenotype was enhanced and the height of the GCT cells in the Hypox mice increased (Figures 2c, p<0.0001, and 3A), and approximately 75% of the GCT cells immunostained positive (significantly decreased, p<0.0001; Figure 3B), producing a mosaic pattern closely resembling the pattern seen in normal females. Dex alone slightly increased the size of the GCT cells (Figures 2d and 3A) compared with those of uninjected Hypox males (p<0.01; Figure 3A). Immunonegative GCT cells were rarely seen in the GCT segments of the SMGs of the Dex-injected Hypox mice. The percentages of immunopositive GCT cells in the SMGs of the Dex-injected and uninjected Hypox males were similar (Figure 3B).

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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  		Figure 4

Electron micrograph of a cross-section of a GCT segment in the SMG of an uninjected Hypox male. Strong labeling of mK1 is seen on the small secretory granules in the apical cytoplasm. L, lumen. Bar = 2 µm. (Inset) An enlarged view of the boxed area, showing the immunogold labeling for mK1 restricted to the secretory granules. M, mitochondria. Bar = 0.5 µm.

 
The trophic action of DHT alone resulted in abundant large and small secretory granules packed in the apical cytoplasm and a basal euchromatic nucleus. Stacked cisternae of the RER and swollen cisternae of the Golgi apparatus were observed in the perinuclear region, and short basal infoldings were present at the base of the cell. In the mK1-immunopositive cells, large secretory granules were always labeled with gold particles, but some small granules were not, demonstrating intergranular variation (Figure 5) . No or only rare labeling was seen in the secretory granules in the immunonegative cells although they had the same fine structure as the immunopositive cells. The nuclei of the hypertrophic GCT cells in the group of Hypox males injected with DHT + T3 and the group injected with all three hormones were basal and euchromatic, and the cells contained a well-developed net-like RER, a large Golgi apparatus with swollen cisternae in the perinuclear region, and rare basal infoldings. The apical two thirds of the GCT cells of the Hypox mice injected with DHT + T3 was filled with abundant large secretory granules. The number of secretory granules in each cell in the Hypox mice treated with all three hormones was lower, but the secretory granules were sometimes irregular in shape and larger than the nucleus. In both groups, the gold particles indicative of mK1 were restricted to the secretory granules, but there were strong intergranular and intercellular variations in the number of gold particles, and strongly labeled secretory granules were scattered among completely immunonegative or weakly labeled granules (Figure 6) . No fine structural differences were observed between mK1-immunopositive and -negative GCT cells in the SMGs of these hormone-injected Hypox males (Figures 5 and 6).



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  		Figure 5

Electron micrograph of the subluminal rim on a cross-section of a GCT segment in the SMG of a DHT-treated Hypox male, showing both immunopositive (arrowheads) and -negative cells. Large secretory granules are strongly labeled for mK1, while small secretory granules are unlabeled or weakly labeled. L, lumen. Bar = 2 µm.

 


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  		Figure 6

Electron micrograph of well-developed GCT cells in the SMG of a Hypox male injected with DHT + T3 + Dex. Huge secretory granules (asterisks) are moderately labeled for mK1, while others are unlabeled. No basal infoldings are seen at the base (arrows) of the cell. G, Golgi apparatus; L, lumen; M, mitochondria. Bar = 1 µm. (Inset) An enlarged view of the boxed area, showing that the immunogold labeling for mK1 is restricted to the upper two secretory granules. Arrows, gold particles suggesting the presence of mK1. Bar = 1 µm.

 

   Discussion
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 Summary
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 Materials and Methods
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 Discussion
Literature Cited
 
The results of the immunocytochemical study with a specific anti-kallikrein antibody indicate that Hypox resulted in atrophy of GCT cells in the SMGs but that the atrophic GCT cells retained evidence of being secretory cells and keeping mK1 synthesis. Isoelectrical focusing has shown that mK1 persists in the SMG of the Hypox mouse, whereas other members of the kallikrein gene family and EGF almost completely disappear (Hosoi et al. 1992Go; Maruyama et al. 1993Go). These findings imply that the GCTs of the SMGs in Hypox males consist of only a single cell type that continues to produce mK1. The fine structure and mK1 immunostaining of the atrophic GCT cells of the Hypox males closely resemble those of the immature GCT cells seen in prepubertal mice: a small Golgi apparatus, sparse RER, well-developed basal infoldings, and small dense apical secretory granules (Kurabuchi et al. 2001Go,2002Go; Kurabuchi 2002Go). Furthermore, previous studies have shown that Hypox increases the abundance of mK1 in male SMG (Hosoi et al. 1992Go; Maruyama et al. 1993Go), and the increase can be explained by the immunocytochemical findings in the present study showing the presence of mK1 in only a very few GCT cells in normal males but in virtually all of the atrophic GCT cells in the Hypox males.

In agreement with the findings in a previous morphological study (Kurabuchi 2002Go), 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 2002Go). 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. 1992Go; Maruyama et al. 1993Go; Kurihara et al. 1999Go). 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 2002Go) 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 1977Go), 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. 1980Go). 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. 1992Go; Maruyama et al. 1993Go; Kurihara et al. 1999Go) 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. 1979Go; Aloe and Levi-Montalcini 1980Go). T3 given alone to prepubertal mice induces GCT cells precociously (Chabot et al. 1987Go) and potentiates the ability of androgens to induce the GCT phenotype and expression of kallikreins and EGF in immature mice (Gresik and Barka 1980Go). In addition, thyroid hormones upregulate the expression of androgen receptor in the SMG of developing and adult mice (Minetti et al. 1986Go,1987Go). 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 1994Go). 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. 1987Go; Sawada and Noumura 1995Go). It is also reported that expression of the SMG androgen receptor in mice is induced by thyroid hormone (Minetti et al. 1986Go, 1987Go). 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. 2002Go). 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)Go, 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. 1992Go; Maruyama et al. 1993Go; Kurihara et al. 1999Go). 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)Go 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 1987Go; Kurihara et al. 1999Go). 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. 1986Go,1987Go) 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.


   Acknowledgments
 
Supported in part by a Grant-in-Aid for Scientific Research (11671820) from the Ministry of Education, Science, Sports and Culture, Japan to S. Kurabuchi and by NIH Grant DE10858 to E.W. Gresik.


   Footnotes
 
Received for publication April 2, 2004; accepted July 10, 2004


   Literature Cited
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

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