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Immunocytochemical study of proteinase F in the mouse submandibular gland
Abstract:
Esteroproteinases extracted from the submandibular gland (SMG) of normal male mice were fractionated by isoelectric focusing into three major peaks with isoelectric point (pI) values of 9.9 (P-esterase), 5.8 (proteinase A), and 5.6 (proteinase D). In castrated males or normal females, an additional esteroproteinase with a pI of 4.6 (proteinase F) appeared. By single radial immunodiffusion analysis using a specific anti-proteinase F serum, the proteinase F content in females or castrated males was found to be 15 times as high as that in normal males. These facts suggest that the synthesis of proteinase F is inhibited by androgens. Immunocytochemical localization of proteinase F in the SMG was examined by indirect enzyme-labeled antibody and ferritin-labeled antibody methods for light and electron microscopy, respectively. Castration of normal males caused morphological changes in granular convoluted tubular (GCT) cells, i.e., GCT cells with both several small secretory granules in their apical region and some striations at their basal region (light cells) were observed in addition to typical GCT cells. Immunoreactive proteinase F was exclusively localized in such small secretory granules of the light cells, but was only minimally present in large secretory granules of the typical GCT cells. In females, however, uniform localization of proteinase F among secretory granules of all GCT cells was observed. It is suggested that the small secretory granules in light cells are formed after castration.
Insights
Androgens inhibit the synthesis of esteroproteinase proteinase F in mouse submandibular glands. Castration or female sex increases proteinase F, which localizes to specific secretory granules in granular convoluted tubular cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Endocrinology
Background:
- Submandibular glands (SMG) produce esteroproteinases, enzymes with esterase and protease activity.
- Hormonal regulation of SMG proteinase synthesis is not fully understood.
- Previous studies identified several esteroproteinases in male mouse SMG.
Purpose of the Study:
- To investigate the hormonal regulation of esteroproteinase synthesis in mouse SMG.
- To characterize the specific esteroproteinase (proteinase F) whose expression is altered by sex hormones.
- To determine the cellular localization of proteinase F in relation to its hormonal regulation.
Main Methods:
- Isoelectric focusing to fractionate esteroproteinases from mouse SMG.
- Single radial immunodiffusion using specific anti-proteinase F serum for quantification.
- Immunocytochemistry (enzyme-labeled antibody and ferritin-labeled antibody) for light and electron microscopy localization.
Main Results:
- Three major esteroproteinases (P-esterase, proteinase A, proteinase D) identified in normal males.
- An additional esteroproteinase, proteinase F (pI 4.6), appeared in castrated males and females.
- Proteinase F levels were 15-fold higher in females/castrated males than normal males, suggesting androgen inhibition.
- Immunoreactive proteinase F localized to small secretory granules in granular convoluted tubular (GCT) cells of castrated males ('light cells'), and uniformly in females.
- Typical GCT cells in normal males showed minimal proteinase F in large secretory granules.
Conclusions:
- Androgens significantly inhibit the synthesis of esteroproteinase proteinase F in the mouse SMG.
- The appearance of proteinase F in castrated males is associated with morphological changes in GCT cells, suggesting the formation of new secretory granules.
- Sex-dependent regulation of proteinase F expression and localization provides insights into hormone-mediated gland function.