Distinct hormonal regulation of two types of sexual dimorphism in submandibular gland of mice.

The submandibular gland (SMG) of mice exhibits prominent sexual dimorphism in two aspects: the preferential development of granular convoluted tubule (GCT) cells and the earlier disappearance of granular intercalated duct (GID) cells in males after puberty. The former is dependent on androgens and thyroid hormones, whereas the hormonal dependence of the latter remains obscure. In the present study, we examined the effects of the postnatal administration of androgens and thyroid hormones to wild-type (WT) and androgen-receptor-knockout (ARKO) mice on these two types of sexual dimorphism by counting the numbers of GCT and GID cells labeled with nerve growth factor and submandibular gland protein C, respectively, as immunohistochemical markers. WT females and ARKO males and females exhibited a lower number of GCT cells and higher number of GID cells at 5 and 11 weeks postpartum than WT males. The administration of dihydrotestosterone for 1-2 weeks prior to these ages caused an increase in GCT cells and decrease in GID cells in WT females to similar levels as those in WT males, whereas it had no effects in ARKO, indicating that both types of sexual dimorphism are androgen-dependent. In contrast, the administration of thyroxine caused an increase in GCT cells but did not cause a decrease in GID cells in WT females or ARKO, indicating that the former is dependent on thyroid hormones, whereas the latter is not. The present results suggest that the two types of sexual dimorphism in the mouse SMG undergo distinct forms of hormonal regulation and, therefore, have different mechanisms.

Synthesis, localization and possible function of serine (or cysteine) peptidase inhibitor, clade B, member 6a (Serpinb6a) in mouse submandibular gland.

The granular convoluted tubule (GCT) in the duct system of the submandibular gland (SMG) develops preferentially in male mice and produces a number of bioactive peptides including proteases such as renin and kallikrein. We examine the synthesis and localization of the serine (or cysteine) peptidase inhibitor, clade B, member 6a (Serpinb6a), the mouse ortholog of the human intracellular serine protease inhibitor SERPINB6, in the mouse SMG by using reverse transcription plus the polymerase chain reaction, in situ hybridization, immunoblotting and immunohistochemistry. Serpinb6a mRNA expression was more abundant in the male than in the female SMG and in the GCT than in other duct portions or acini. Within GCT cells, immunoreactivity for Serpinb6a was localized in the nucleus and cytosol but was absent in the secretory granules. The binding target of Serpinb6a in the SMG was investigated by using a mass spectrometric analysis of immunoprecipitation products and kallikrein-1-related peptidase b26 (Klk1b26), a serine protease, was identified. These results raise the possibility that Serpinb6a functions in the protection of GCT cells from intracellular kallikreins that may leak from secretory granules.

Expression and localization of calpain 3 in the submandibular gland of mice.

OBJECTIVES: Calpains comprise a family of intracellular Ca2+-dependent cysteine proteases and are considered to play roles in various physiological phenomena with limited proteolytic activities against specific substrates. We herein revealed the expression and localization of calpain 3, the muscle-type calpain, in the submandibular gland (SMG) of mice. DESIGN: The expression of the mRNA for conventional, ubiquitous calpains 1 and 2 and skeletal muscle-specific calpain 3 was examined in the major salivary glands of mice using RT-PCR, and the expression and localization of calpain 3 protein was examined in the SMG of mice using immunohistochemistry and Western blotting. RESULTS: The large catalytic subunits of calpains 1 and 2 and the small regulatory subunit common to calpains 1 and 2 were weakly expressed in the parotid gland, sublingual gland, and SMG at similar levels in males and females. In contrast, the single large catalytic subunit of calpain 3 was expressed predominantly in the SMG at markedly higher levels in males than in females and in a manner dependent on androgens. Immunoreactivity for calpain 3 was mainly localized in cells of the granular convoluted tubules (GCT) that developed preferentially in the male SMG. In GCT cells, calpain 3 immunoreactivity was localized predominantly in the cytosolic region and was absent in the secretory granules. CONCLUSIONS: These results revealed that the GCT is the primary site of production of calpain 3 in the mouse SMG.

Immunohistochemical localization of keratin 5 in the submandibular gland in adult and postnatal developing mice.

Keratin 5 (K5) is a marker of basal progenitor cells in the epithelia of a number of organs. During prenatal development of the submandibular gland (SMG) in mice, K5(+) progenitor cells in the developing epithelia play important roles in its organogenesis. Although K5(+) cells are also present in the adult mouse SMG and may function in tissue regeneration, their histological localization has not yet investigated in detail. In the present study, we examined the immunohistochemical localization of K5 in the SMG in adult and postnatal developing mice. At birth, K5 immunoreactivity was detected in the entire duct system, in which it was localized in the basal cells of a double-layered epithelium, but was not detected in the terminal tubule or myoepithelial cells. At postnatal weeks 1-3, with the development of intercalated ducts (ID), striated ducts (SD), and excretory ducts (ED), K5-immunoreactive basal cells were gradually restricted to the ED and the proximal double-layered portions of the ID connecting to the SD. At the same time, K5 immunoreactivity appeared in myoepithelial cells, in which its positive ratio gradually increased. In adults, K5 immunoreactivity was localized to most myoepithelial cells, most basal cells in the ED, and a small number of ID cells at the boundary between the ID and SD in the female SMG or between the ID and granular convoluted tubules in the male SMG. These results suggest that K5 is a marker of differentiated myoepithelial cells and duct progenitor cells in the mouse SMG.

Morphology and gene expression profile of the submandibular gland of androgen-receptor-deficient mice.

OBJECTIVES: In the submandibular gland (SMG) of mice, the granular convoluted tubule (GCT) develops preferentially in males dependent on androgens. To clarify the molecular mechanism of androgen action in SMG, we examined the SMG of mice deficient for the androgen receptor (ARKO). DESIGN: The morphological features and gene expression in the SMG of control and ARKO mice with or without hormone treatments were analysed by immunohistochemistry, DNA microarray, and RT-PCR. RESULTS: The development of GCT and expression of GCT-specific products such as NGF were even lower in ARKO male SMG than in control female SMG. The administration of androgens to ARKO males had no effect on SMG, whereas the administration of thyroid hormone (T4) caused the extensive conversion of striated duct cells to GCT cells with the increase of NGF mRNA. Gene expression profiles in control and ARKO male SMG were analysed by DNA microarrays, and genes with higher or lower expression in ARKO male SMG were determined. They were then classified into groups according to their responsiveness to the administration of dihydotestosterone (DHT) or T4 to ARKO males. RT-PCR revealed that, while no gene was responsive to DHT, expression of many genes was up- or down-regulated by T4. CONCLUSIONS: These results confirmed that GCT cell differentiation induced by androgens is dependent on the classical androgen receptor (AR), whereas that by T4 is independent of AR. Differential reactivity of genes to androgens and thyroid hormone in ARKO mice may shed light on the mechanism of androgen action in the SMG.

Identification of 5-bromo-2'-deoxyuridine-labeled cells during mouse spermatogenesis by heat-induced antigen retrieval in lectin staining and immunohistochemistry.

DNA replication occurs during S-phase in spermatogonia and preleptotene spermatocytes during spermatogenesis. 5-Bromo-2'-deoxyuridine (BrdU) is incorporated into synthesized DNA and is detectable in the nucleus by immunohistochemistry (IHC). To identify BrdU-labeled spermatogenic cells, the spermatogenic stages must be determined by visualizing acrosomes and detecting cell type-specific marker molecules in the seminiferous tubules. However, the antibody reaction with BrdU routinely requires denaturation of the DNA, which is achieved by pretreating tissue sections with hydrochloric acid; however, this commonly interferes with further histochemical approaches. Therefore, we examined optimal methods for pretreating paraffin sections of the mouse testis to detect incorporated BrdU by an antibody and, at the same time, visualize acrosomes with peanut agglutinin (PNA) or detect several marker molecules with antibodies. We found that the use of heat-induced antigen retrieval (HIAR), which consisted of heating at 95C in 20 mM Tris-HCl buffer (pH 9.0) for 15 min, was superior to the use of 2 N hydrochloric acid for 90 min at room temperature in terms of the quality of subsequent PNA-lectin histochemistry with double IHC for BrdU and an appropriate stage marker protein. With this method, we identified BrdU-labeled spermatogenic cells during mouse spermatogenesis as A1 spermatogonia through to preleptotene spermatocytes.

Expression and Localization of α-amylase in the Submandibular and Sublingual Glands of Mice.

In the major salivary glands of mice, acinar cells in the parotid gland (PG) are known to be the main site for the production of the digestive enzyme α-amylase, whereas α-amylase production in the submandibular gland (SMG) and sublingual gland (SLG), as well as the cell types responsible for α-amylase production, has been less firmly established. To clarify this issue, we examined the expression and localization of both the mRNA and protein of α-amylase in the major salivary glands of male and female mice by quantitative and histochemical methods. α-amylase mRNA levels were higher in the order of PG, SMG, and SLG. No sexual difference was observed in α-amylase mRNA levels in the PG and SLG, whereas α-amylase mRNA levels in the female SMG were approximately 30% those in the male SMG. Using in situ hybridization and immunohistochemistry, signals for α-amylase mRNA and protein were found to be strongly positive in acinar cells of the PG, serous demilune cells of the SLG, and granular convoluted tubule (GCT) cells of the male SMG, weakly positive in seromucous acinar cells of the male and female SMG, and negative in mucous acinar cells of the SLG. These results clarified that α-amylase is produced mainly by GCT cells and partly by acinar cells in the SMG, whereas it is produced exclusively by serous demilune cells in the SLG of mice.