ARTICLE |
Correspondence to: Edward W. Gresik, Dept. of Cell Biology and Anatomical Sciences, The City University of New York Medical School, New York, NY 10031. E-mail: ewg@med.cuny.edu
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Summary |
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The action of androgens on the immunocytochemical distribution of mK1, a true tissue kallikrein, was examined in the submandibular gland (SMG) of developing and adult mice by indirect enzyme-labeled and immunogold-labeled antibody methods for light and electron microscopy, respectively. In both sexes at 3 weeks of age, essentially all of the immature granular convoluted tubule (GCT) cells were uniformly immunostained. At 4 weeks of age (the onset of puberty), morphological differences between the two sexes appeared in the GCTs, in which some cells became immunonegative. Thereafter, the immunonegative GCT cells became more abundant in the SMG of males than of females and considerable intercellular variation in staining intensity for mK1 was seen, especially in males. A few slender GCT cells with strong immunoreactivity appeared in GCT segments only in males. Castration of males resulted in an increase in the number of immunopositive GCT cells, whereas administration of dihydrotestosterone (DHT) decreased the number of immunopositive GCT cells in the SMGs of both sexes. Slender GCT cells immunoreactive for mK1 were seen in females treated with DHT for 6 days. However, there were no immunostained slender GCT cells in female SMGs after injection of DHT for 2 weeks. Immunoelectron microscopy disclosed this type of cell in male SMGs, which closely resembles immature GCT cells of prepubertal mice, with a few small secretory granules uniformly labeled with gold particles, a sparse Golgi apparatus and RER, and basal infoldings. In mature male SMGs and in SMGs of DHT-treated females and castrated males, typical GCT cells had a well-developed Golgi apparatus and a net-like RER but few to no basal infoldings, whereas in the female gland equivalent cells had moderately developed RER and some basal infoldings. These results suggest that mK1 is one of the enzymes characteristically present in immature GCT cells and that its synthesis is inhibited in part by androgens, resulting in decreased numbers of immunopositive cells. (J Histochem Cytochem 50:135145, 2002)
Key Words: true tissue kallikrein, immunocytochemistry, submandibular gland, androgens, sexual dimorphism, development, mouse
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Introduction |
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THE SEXUAL DIMORPHISM of the granular convoluted tubule (GCT) cells of the mouse submandibular gland (SMG) has been extensively documented by morphological studies at the light and electron microscopic levels and by the techniques of molecular biology, biochemistry, and immunocytochemistry (reviewed in
Tissue kallikreins are a family of enzymes that produce the bioactive peptide kinin, which provokes pain, leukocyte migration, and vasodilatation. The SMG has long been known to produce large amounts of tissue kallikreins (
Because the kallikrein isozymes share high amino acid-sequence homology and the antisera against any particular kallikrein frequently crossreact with other members of this family (
The GCT compartment develops postnatally from striated duct cells (
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Materials and Methods |
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Animals
ICR strain mice were housed under controlled conditions of temperature (22C) and lighting (light 12 hr: dark 12 hr), and allowed food and water ad libitum. The GCT compartment was studied in the following groups, each of which consisted of four mice. Postnatal developmental changes were studied in mice of both sexes at 1, 2, 3, 4, 6, 8, 11, 14, 18, and 24 weeks of age. Effects of androgen withdrawal were examined in castrated adult male mice (14 weeks old), orchiectomized at 6 weeks of age. To examine the direct effect of androgens, 14-week-old male and female mice and castrated males were injected SC at a dose of 20 µg/g body weight with 5-dihydrotestosterone (DHT) dissolved in sesame oil, seven times (during 2 weeks) before sacrifice. In addition, one group of females was sacrificed after only three injections, 6 days after the start of the experiment. Before examination, all animals were fasted overnight. Under anesthesia with sodium pentobarbital (30 mg/kg body weight), SMGs were quickly removed and processed for light and electron microscopic immunocytochemical (ICC) examination.
Antiserum
The antiserum against mK1, previously generated in rabbits, was found to be weakly crossreactive with three other major kallikreins, mK9, mK13, and mK22 (
Immunocytochemistry
Small pieces of the SMG were fixed by immersion in a mixture of 2% glutaraldehyde and 2% paraformaldehyde in 0.05 M cacodylate buffer, pH 7.4, for 2 hr at 4C, then dehydrated through a graded ethanol series, cleared in propylene oxide, and embedded in an Epon/Araldite mixture (
For light microscopy, 2-µm-thick sections were mounted on silane-coated slides (Matsunami; Tokyo, Japan), treated with methanol saturated with NaOH to remove the resin, and incubated in a solution of 0.3% H2O2 in methanol for 30 min to inactivate endogenous peroxidase. The sections were washed in distilled water and then in PBS (10 mM sodium phosphate buffer, 140 mM NaCl, pH 7.5) and then incubated for 2 hr at room temperature with the preabsorbed anti-mK1 antiserum diluted 1:40,000. After three rinses in PBS, the sections were immunostained by the avidinbiotinperoxidase complex method according to the manufacturer's protocol (Vectastain Elite ABC kit; Vector Labs, Burlingame, CA). They were then dehydrated with ethanol and mounted in Entellan (Merck; Gibbstown, NJ), and examined with a BX-50 microscope equipped with Nomarski differential interference contrast optics (Olympus; Tokyo, Japan). In the immunostained sections, the percentage of mK1-immunopositive cells in the GCT segments was determined by counting a total of at least 1000 cells for each mouse in each group. The means ± standard deviation (SD) for each group (consisting of four animals) were calculated.
For electron microscopy, ultrathin sections on gold grids were etched with 3% H2O2 for 5 min, rinsed well with distilled water, and then subjected to an immunogold procedure. They were first incubated with 20% normal goat serum for 2 hr, then transferred into a drop of 1:40,000 preabsorbed anti-mK1 antiserum and kept overnight at 4C. After three rinses in PBS, sections were incubated with biotinylated goat anti-rabbit IgG (Vectastain Elite ABC kit) for 1 hr. They were finally incubated for 2 hr with 1:60 streptavidin conjugated with 10-nm gold particles (Zymed; San Francisco, CA). After staining with uranyl acetate and lead citrate, sections were viewed in a transmission electron microscope (JEM-2000EXII; JEOL, Tokyo, Japan) at 80 kV accelerating voltage.
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Results |
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Postnatal Development
At 1 week of age in both sexes, very faint immunoreactivity for mK1 was restricted to the subluminal rim of intralobular ducts of the SMG (data not shown). At 2 weeks of age the subluminal immunoreactive zone increased in extent and intensity in both sexes (Fig 1a and Fig 1b). Some segments of the intralobular ducts contained cells, presumably developing GCT cells, with small secretory granules that were strongly and uniformly stained for mK1. These granules were densely accumulated in the subluminal cytoplasm and more dispersed in the perinuclear regions. At 3 weeks of age the secretory granules increased in number and size in developing GCT cells, all of which were immunostained (Fig 2).
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At 4 weeks of age (onset of puberty) sexual dimorphism was clearly evident: GCT segments in males were more extensive and their secretory granules were larger and more abundant (Fig 1c and Fig 1d). Moreover, from this age onward the GCT cells showed intercellular variation in immunostaining for mK1, especially in males. The staining was moderate in some cells and strong in others, and immunopositive and immunonegative secretory granules were sometimes present in the same GCT cell. In addition, a few (approximately 30%) GCT cells were completely unstained; such cells appeared first in males (Fig 2). In females, almost all GCT cells were immunoreactive at this age (Fig 1d and Fig 2).
By 6 weeks of age the GCT cells had a clear sexually dimorphic mosaic ICC distribution of mK1-positive secretory granules (Fig 1e and Fig 1f). In males, about 70% of GCT cells with large secretory granules showed no immunostaining for mK1, whereas the remaining GCT cells had secretory granules with strong to moderate immunoreactivity (Fig 1e and Fig 2). Occasional slender GCT cells were observed next to immunonegative or weakly stained typical GCT cells (see Fig 3 below). In females, the GCT segments were smaller and had many typical GCT cells with moderate to strong immunostaining, but a few completely lacked immunoreactivity (Fig 2).
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At older ages the sexually dimorphic mosaic pattern of immunoreactive cells was even more pronounced. In mature males, remarkably few cells in the GCT segments were stained for mK1 (Fig 2), and many of these immunostained cells were slender GCT cells (Fig 1g). In females, about two thirds of the GCT cells were immunostained for mK1, and this proportion remained relatively constant between 11 and 24 weeks of age (Fig 1h and Fig 2). Immunopositive slender GCT cells were commonly found in male GCTs; at least one cell of this type was routinely found in every lobule of male SMGs. However, no such cells were found in the female GCTs, even after sections of at least 40 SMGs of untreated adult females of 1124 weeks of age were examined.
Fine structural immunolocalization of mK1 demonstrated that gold particles were essentially confined to secretory granules in the GCT cells. At 2 weeks of age, small irregularly shaped and uniformly dense secretory granules occupied the subluminal region of all ductal cells, and they were strongly labeled with gold particles (Fig 3). The secretory granules became rounded and increased in number at 3 weeks of age (data not shown). In these immature GCT cells, only a few segments of RER with narrow cisternae and small Golgi fields were seen (Fig 3). The basal plasmalemma was usually elaborately infolded and associated with large mitochondria.
From early pubertal (6 weeks of age) to fully adult (24 weeks) stages, the fine structural features of the cells of the GCT compartment remained relatively constant (Fig 4). Most GCT cells had the characteristic sexually dimorphic morphology previously described by several authors (see reviews by
In addition, from 6 weeks onward the GCT compartment of males became hypertrophic and contained cells that were slender in shape with morphological features similar to immature GCT cells (
Effect of Castration and Administration of DHT on the SMG
GCTs of castrated adult males resembled those of normal mature females (Fig 5a, Fig 5c, and Fig 5e). However, the GCT cells in the castrated males (Fig 5c) were smaller, and the secretory granules were somewhat less abundant, compared to those of the normal females (Fig 5e). In castrated males, about two thirds of the GCT cells showed strong to moderate immunostaining, while the others were immunonegative (Fig 6). Both positive and negative cells showed the same morphological features. Slender GCT cells were not detected in the GCT segments of castrated males.
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Electron microscopy demonstrated that the secretory granules varied in size and density (Fig 7). In some cells, uniformly small, dense apical secretory granules were common, but in other cells lucent granules (usually large) and variably sized dense granules were present along with several vacuoles (Fig 7). Perinuclear RER and Golgi apparatus were sparse, but basal infoldings associated with large mitochondria were prominent. In immunopositive GCT cells there was significant intergranular variation in the number of gold particles: both large and small dense secretory granules usually were labeled well, but large lucent granules were weakly labeled or unlabeled. The fine structural feature of the positively and negatively stained cells were essentially the same.
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DHT had a strong trophic effect on GCT cells of intact males (Fig 5b), castrated males (Fig 5d), and intact females (Fig 5f). Although DHT reduced immunostaining for mK1, the pattern of immunostaining was not identical in the three groups. In intact males treated with DHT the percentage of positively stained cells was even lower than in untreated normal males (Fig 6). After administration of DHT for 2 weeks to castrated males (Fig 5d) or intact females (Fig 5f), the percentage of GCT cells positive for mK1 declined (Fig 6), and considerable intergranular variation in staining intensity persisted in those cells that were positive. However, more positively stained GCT cells were present than in intact or DHT-treated normal males (Fig 5a5f). Strongly stained slender GCT cells reappeared in castrated males after treatment with supraphysiological doses of DHT. These slender GCT cells were also seen in females treated with DHT for 6 days (three injections) (Fig 5f) but were never seen in females treated for 2 weeks (seven injections) (Fig 5g) or in intact females. Finally, in all three groups many large typical GCT cells were completely unstained for mK1.
The persistence of slender GCT cells was confirmed by electron microscopy (Fig 8). The cytoplasm of these cells was lighter than in typical GCT cells; the RER was sparse, and basal infoldings were well developed. The apical secretory granules were strongly labeled with gold particles for mK1. The electron microscopic appearance of the hypertrophied GCT cells of DHT-treated females and of DHT-treated castrated males was essentially the same (data not shown).
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Discussion |
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The ICC data in this study indicate that all immature GCT cells in the mouse SMG at prepubertal ages synthesize mK1. It has been documented previously by isoelectric focusing that mK1 appears earliest, by Day 15 of age, and other members of the kallikrein familymK9, mK13, and mK22follow thereafter (
In castrated males, GCTs regressed and resembled those of normal females, as noted previously (
The present study found that some structural features typical of female GCT cells did not completely disappear after androgen treatment, i.e., a few basal infoldings remained even after hormone treatment. The present ICC data indicate that the immunopositive GCT cells decrease in number in both female mice and castrated male mice exposed to DHT, and show that androgens inhibit the synthesis of mK1 in many GCT cells. This is in good agreement with the previous report that DHT treatment decrease mK1 content in homogenates of the SMG (
Intergranular variation in immunostaining for mK1 observed by light and electron microscopy was most obvious in the GCT cells of young males (about 6 weeks of age) and of females and castrated males treated with excess DHT. The present electron microscopic analysis confirms our previous report (
Previously we suggested that the slender GCT cells may represent a cell type that is distinct from the typical large, mature GCT cells (
In summary, in this study we have followed the postnatal development of the sexually dimorphic mosaic distribution of mK1 in mature GCT cells from the uniformly immunopositive immature GCT cells and have characterized the role of androgen status on this peculiar distribution pattern. Lastly, our data suggest that the slender GCT cells seen in adults may represent newly forming GCT cells that have an immature phenotype as they develop into typical large GCT cells.
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Footnotes |
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1 Present address: Dept. of Histology, The Nippon Dental University School of Dentistry, Tokyo, Japan.
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Acknowledgments |
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This work was supported in part by a grant-in-aid for scientific research (11671820) from the Ministry of Education, Science and Culture, Japan to S. Kurabuchi, and NIH Grant DE10858 to E. W. Gresik.
Received for publication March 27, 2001; accepted September 26, 2001.
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