Long-Term Exposure to Supraphysiological Levels of Testosterone Impacts Rat Submandibular Gland Proteome

Abstract

The salivary glands play a central role in the secretion of saliva, whose composition and volume affect oral and overall health. A lesser-explored dimension encompasses the possible changes in salivary gland proteomes in response to fluctuations in sex hormone levels. This study aimed to examine the effects of chronic exposure to testosterone on salivary gland remodeling, particularly focusing on proteomic adaptations. Therefore, male Wistar rats were implanted with subcutaneous testosterone-releasing devices at 14 weeks of age. Their submandibular glands were histologically and molecularly analyzed 47 weeks later. The results underscored a significant increase in gland mass after testosterone exposure, further supported by histologic evidence of granular duct enlargement. Despite increased circulating sex hormones, there was no detectable shift in the tissue levels of estrogen alpha and androgen receptors. GeLC-MS/MS and subsequent bioinformatics identified 308 proteins in the submandibular glands, 12 of which were modulated by testosterone. Of note was the pronounced upregulation of Klk3 and the downregulation of Klk6 and Klk7 after testosterone exposure. Protein-protein interaction analysis with the androgen receptor suggests that Klk3 is a potential target of androgenic signaling, paralleling previous findings in the prostate. This exploratory analysis sheds light on the response of salivary glands to testosterone exposure, providing proteome-level insights into the associated weight and histological changes.

Keywords: GeLC-MS/MS; kallikrein-3; rats; salivary glands; submandibular gland’s proteome; testosterone.

Figure 1

Impact of chronic testosterone exposure on anthropometric parameters in rats at 61 weeks of age (n = 10 for CTRL and n = 14 for TEST). The parameters analyzed were (A) body weight; (B) submandibular gland (SM) mass; and (C) submandibular gland (SM) mass-to-body weight (BW) ratio (* p < 0.05; ** p < 0.01).

Figure 2

Effect of testosterone exposure on systemic parameters in rats at 61 weeks of age (n = 8 for CTRL and n = 14 for TEST). The parameters analyzed were (A) testosterone; (B) albumin; (C) total proteins; (D) glucose; (E) cholesterol; and (F) triglycerides levels (* p < 0.05; *** p < 0.001; **** p < 0.0001).

Figure 3

Histological comparison of submandibular gland sections between CTRL and TEST rats. Hematoxylin and eosin (H&E) staining was used to visualize the cellular and tissue structures (A). The glandular tissue appears organized, with well-defined secretory acini and ductal structures (intercalated, granular and striated ducts). The acini exhibit typical morphology, with centrally located nuclei and basophilic cytoplasm. Ducts (black arrows) appear lined with cuboidal or columnar epithelial cells. The submandibular glands from the TEST group exhibit larger granular ducts that are lined with a taller epithelium. The ductal-to-whole gland area ratio was also evaluated in both groups (B) (* p < 0.05).

Figure 4

Impact of testosterone administration on the content of (A) androgen receptor (n = 5 for CTRL and n = 6 for TEST) and (B) estrogen receptor alpha (n = 6 for CTRL and TEST) evaluated through Western blotting. Above each graph are representative immunoblot images. The optical densities (O.D.) obtained are expressed in arbitrary units.

Figure 5

Modeling data projection to optimize separation between CTRL and TEST groups of submandibular glands samples. (A) Principal component analysis (PCA) and (B) partial least squares discriminant analysis (PLS-DA) of submandibular gland proteome data from animal groups (n = 6 per group), performed with MetaboAnalyst.

Figure 6

Quantitative analysis of proteins from CTRL and TEST groups with gene identification and corresponding expression levels (Log2). This analysis encompasses all 87 proteins common in both studied groups that appear at least in 4 animals from each group. Proteins were considered with a cut-off of 1.3 for the TEST/CTRL ratio.

Figure 7

PPI network showing uniquely expressed proteins in TEST group, and common but upregulated ones in TEST group, and sex hormone receptors (AR and ERα). Darker network edges represent a higher level of confidence. Confidence level value range: [0.150; 0.900]. PPI network was constructed with STRING server.