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Comparative Morphological Study on Parotid and Submandibular Salivary Glands in Ovariectomized Rats
J Dent Hyg Sci 2022;22:83-9
Published online June 30, 2022;  https://doi.org/10.17135/jdhs.2022.22.2.83
© 2022 Korean Society of Dental Hygiene Science.

Moon-Jin Jeong1 , Myoung-Hwa Lee1 , Do-Seon Lim2 , Myeongju Jeong3 , Soon-Jeong Jeong4 ,5,†

1Department of Oral Histology and Developmental Biology, School of Dentistry, Chosun University, Gwangju 61452, 2Department of Dental Hygiene, Graduate School of Public Health Science, Eulji University, Seongnam 13135, 3Department of Biosafety, College of Life and Health, Kyungsung University, Busan 48434, 4Department of Dental Hygiene, College of Health Science, Youngsan University, Yangsan 50510, 5Institute of Basic Science for Well-Aging, Youngsan University, Yangsan 50510, Korea
Correspondence to: Soon-Jeong Jeong, https://orcid.org/0000-0002-8959-4663
Department of Dental Hygiene & Institute of Basic Science for Well-Aging, Youngsan University, 288 Junam-ro, Yangsan 50510, Korea
Tel: +82-55-380-9453, Fax: +82-55-380-9305, E-mail: jeongsj@ysu.ac.kr
Received May 25, 2022; Revised May 30, 2022; Accepted June 1, 2022.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: Estrogen deficiency affects the structure and function of the salivary glands in women, leading to a decrease in salivary secretion and a change in the composition of saliva. Previous studies on changes in the salivary glands that cause estrogen deficiency have reported only partial results for the parotid and submandibular glands, and there are few comparative morphological studies of histological changes between the parotid and submandibular glands in ovariectomized rats (OVX) leading to estrogen deficiency. This study aimed to analyze the histopathological and histochemical changes in the parotid and submandibular salivary glands causing estrogen deficiency by using OVX, and to discuss the mechanism on these changes.
Methods: The parotid and submandibular glands from sacrificed control and OVX groups were fixed with cold 4% paraformaldehyde in phosphate buffer (pH 7.2). The tissues were dehydrated using a series of graded ethyl alcohol and embedded in paraffin. For histopathological analysis, sections cut to a thickness of 6 to 7 μm were stained with hematoxylin and eosin (H&E). For histochemical analysis, Periodic acid-Schiff (PAS), Alcian blue (AB, pH 2.5), and PAS+AB (pH 2.5 and pH 1) staining was performed.
Results: Histopathological analysis of OVX tissue showed that the parotid and submandibular salivary glands were broadly and clearly separated and divided into lobes. In OVX, acinar and ductal cells with condensed polymorphic or pyknotic nucleus, which are presumed to be characteristic of apoptotic cells, and degenerated cells with lipid deposition in cytoplasmic granules and ruptured membranes were increased. Histochemical analysis of OVX, confirmed an increase in the number and acidification of acinar secretory granules.
Conclusion: Histopathological and histochemical changes and the effects of estrogen deficiency are more evident in the submandibular salivary gland than in the parotid gland.
Keywords : Estrogen deficiency, Ovariectomized rat, Parotid gland, Submandibular gland
Introduction

Estrogen is a steroid hormone that directly and indirectly affects many organs and tissues, including the cardiovascular system, bone tissues, and salivary glands1,2). Estrogen regulates the growth, differentiation and function of cells that form the reproductive and non-reproductive organs and tissues3). Changes and levels of estrogen in women, such as menstrual cycle4,5), pregnancy6,7), menopause8,9), hormone therapy10), and the use of oral contraceptives11) affect the function of the salivary gland. Estrogen deficiency is also related to the development of oral diseases, dysfunction of the salivary gland, decrease in secretion, and changes in the composition of saliva12,13).

Salivary glands are important organs of the oral cavity that play a role in salivation1,14). There are three major salivary glands in the oral cavity, the parotid, submandibular and sublingual glands, which secrete approximately 95% of saliva, while the minor salivary glands secrete the remaining 5% of saliva1). The acini of the parotid gland consist only of serous cells, those of the submandibular gland contain serous and mucous cells, and those of the sublingual gland consist of only mucous cells14). Salivation is regulated by the parasympathetic and sympathetic nervous systems1); the parasympathetic nerve secretes water and ions, whereas the sympathetic nerve stimulates the secretion of proteins15). In addition, the composition of proteins and inorganic substances of saliva is related to changes in the estrogen hormone7,16), which plays an important role in the regulation of salivary glands3).

Studies on changes in the salivary glands causing estrogen deficiency have reported only partial results for the parotid and submandibular glands, and there are few comparative morphological studies of histological changes between the parotid and submandibular glands in ovariectomized rats (OVX) leading to estrogen deficiency. The mechanism by which changes in the salivary glands cause hormone deficiency remains unclear. This study aimed to analyze the histopathological and histochemical changes in the parotid and submandibular salivary glands causing estrogen deficiency by using OVX, and to discuss the mechanism of changes.

Materials and Methods

1.Animals

Ten healthy female Sprague Dawley rats (Samtako Bio, Osan, Korea) weighing 210 to 230 g and aged 8 to 10 weeks were used. Rats were bred in a laboratory maintained at a temperature of 23 ± 2°C, humidity of 60 ± 10%, and a light/dark cycle of 12 hours, and allowed free access to feed and water. Rats were randomly divided into control and OVX groups, with each group consisting of five rats. All the animal studies were approved by the Institutional Animal Care and Use Committee of Chonnan National University (No. CNU IACUC-YB-R-2018-59) and animal care was carried out in specific pathogen free (SPF) level systems by following the ‘Guide for the Care and Use of Laboratory Animals’.

2.Establishment of ovariectomized rat

Rats in the OVX group were anesthetized with intramuscular injections of 2% Rumpun (Bayerkorea, Seoul, Korea) and 10% ketamine (Yuhan, Seoul, Korea), and their bilateral ovaries were ligated and cut off. The rats were then given two weeks to recover from the procedure.

3.Histopathological and histochemical analysis

Parotid and submandibular glands from sacrificed rats were fixed with cold 4% paraformaldehyde in phosphate buffer (pH 7.2) for 12 hours. The tissues were then dehydrated with a series of graded ethyl alcohol and embedded in paraffin. The blocks were cut into 6 to 7 µm sections using a motorized rotary microtome (RMC Products, Tucson, AZ, USA). For histopathological analysis, sections of each sample were stained with hematoxylin and eosin (H&E) stained, and for histochemical analysis, Periodic acid-Schiff (PAS), Alcian blue (AB, pH 2.5) and PAS+AB (pH 2.5 and pH 1) staining was performed. PAS staining reacts with various carbohydrates, vicinal hydroxyl groups near the glycoprotein, and neutral muco-polysaccharides to produce a red color. AB staining was used for acidic glycoproteins and acidic muco- polysaccharides. In PAS+AB staining, AB shows acidic muco-substances in blue, PAS-positive substances are stained red, epithelial mucin is stained purple, and neutral muco-substances are reddish purple. Histopathological and histochemical changes observed through the microscopy were scored according to the results: negative or none was represented as −, mild as +, moderate as ++, and severe as +++.

Results

1.Parotid salivary gland

The parotid gland of the control group was composed of parenchyma and stroma of a general type consisting of acini made up of serous cells, and ducts composed of intercalated ducts, granular ducts, striated ducts, and connective tissue (Fig. 1A, Table 1). The acini and ducts were separated into lobes, and narrowly separated interlobular septa were observed in the parotid gland. Acinar cells with a large round nucleus and darkly stained serous granules, intercalated ducts forming a monolayer cubic cell with round nucleus, and granular ducts forming a monolayer long columnar cell with a round nucleus in the middle of the cytoplasm were observed. The striated duct forming columnar cells with a central round nucleus exhibited basal infolding along the long axis. In the parotid gland of the OVX group, widely separated interlobular septa were observed, and cells with condensed polymorphic or pyknotic nucleus and degenerated cells with ruptured membrane increased in the acini, granular and striated ducts (Fig. 1B, Table 1).

Histopathological Analysis of the Parotid and Submandibular Salivary Glands in the Control and OVX Groups

Analysis item Parotid gland Submandibular gland


Control OVX Control OVX
Polymorphic nucleus ++ ++
Pyknotic nucleus + +++
Lipid deposition +++
Degenerative cell ++ ++

OXV: ovariectomized rats.



Fig. 1. Micrographs of the hematoxylin and eosin stained parotid gland in the control (A) and OVX (B) groups. (1) Lobes composed of acini and salivary ducts. (2) Serous cells and secretory granules of acini. (3) Intercalated ducts. (4) Granular duct. (5) Striated ducts. ↖: polymorphic or pyknotic nucleus, OVX: ovariectomized rats.

Histochemical staining of acinar secretory granules in the parotid gland revealed that the control group had a moderate reaction to PAS and PAS+AB staining, but no reaction to AB staining (Fig. 2A, Table 2). On the other hand, the reactions of PAS and PAS+AB staining in the OVX group were similar to those of the control, but there was a mild reaction to AB staining (Fig. 2B, Table 2).

Histochemical Analysis of the Parotid and Submandibular Salivary Glands in the Control and OVX Groups

Salivary glands PAS staining AB staining PAS+AB staining



Control OVX Control OVX Control OVX
Parotid gland ++ ++ + ++ ++
Submandibular gland ++ ++ ++ +++ ++ +++

OXV: ovariectomized rats, PAS: Periodic acid-Schiff, AB: Alcian blue.



Fig. 2. Micrographs of the parotid gland in the control (A) and OVX (B) groups according to histochemical staining. (1) PAS staining. (2) AB staining. (3) PAS+AB staining. OXV: ovariectomized rats, PAS: Periodic acid-Schiff, AB: Alcian blue.

2.Submandibular salivary gland

The submandibular salivary gland was composed of parenchyma and stroma, consisting of acini made up of serous and mucous cells, ducts, and connective tissues (Fig. 3A). Narrowly separated interlobular septa, acini closely filled with serous and mucous cells, and ducts were observed. The granules of serous cells with nucleus located at the bottom were stained relatively dark, and the granules of mucous cells with large, round nucleus were stained brightly. The submandibular gland was characterized by well-developed granular ductal cells filled with many granules, and the intercalated ducts and striated ducts were similar to those of the parotid gland. The acinar and ducts of the submandibular gland in the OVX group showed widely separated interlobular septa (Fig. 3B). There was an increase in the number of acinar cells with polymorphic or pyknotic nucleus and degenerated cells with lipid deposition in the cytoplasmic granules and ruptured membranes (Fig. 3B, Table 1).

Fig. 3. Micrographs of the hematoxylin and eosin stained submandibular gland in the control (A) and OVX (B) groups. (1) Lobes composed of acini and salivary ducts. (2) Serous (#) and mucous cells and secretory granules of acini. (3) Intercalated duct. (4) Granular duct. (5) Striated ducts. *: lipid deposition, ↖: polymorphic or pyknotic nucleus, OXV: ovariectomized rats.

Histochemical staining of acinar secretory granules in the submandibular gland indicated that the control group had a moderate response to all staining (Fig. 4A, Table 2), but the reactions of AB and PAS+AB staining in the OVX group were severe, indicating a change in the acidification of the acinar secretory granules (Fig. 4B, Table 2).

Fig. 4. Micrographs of the submandi-bular gland in the control (A) and OVX (B) group according to histochemical staining. (1) PAS staining. (2) AB staining. (3) PAS+AB staining. *: lipid deposition, ↖: polymorphic or pyknotic nucleus, OXV: ovariectomized rats, PAS: Periodic acid-Schiff, AB: Alcian blue.
Discussion

Ovariectomy leads to estrogen deficiency due to the loss of ovarian function, resulting in morphological and physiological changes in various regions of the salivary glands17,18). After ovariectomy, alterations in lipid deposition and lipogenesis, fibrosis, and mitochondrial morphological changes are observed in the salivary gland tissue18). Estrogen deficiency also directly or indirectly affects glucose and lipid metabolism2). In particular, the increase in lipid accumulation in acinar and ductal cells stimulates the migration of inflammatory cells and induces damage to the acinar cells involved in glucose and lipid metabolism2). In this study, the parotid and submandibular salivary glands in the OVX group were composed of clearly separated and divided lobes densely filled with acinar and ductal cells. In addition, acinar and ductal cells with condensed polymorphic or pyknotic nucleus, which are presumed to be characteristic of apoptotic cells, and degenerated cells with lipid deposition in cytoplasmic granules and ruptured membranes were increased. The wide and clearly distinguished interlobular septa of the parotid and submandibular salivary glands in OVX are the result of atrophy of acinar and ductal cells due to estrogen deficiency19,20). Atrophy occurred due to vascular damage and permeability of blood vessels between lobes and similar results have been reported in the submandibular gland of elderly postmenopausal rats21), oral mucosal epithelium of OVX22), and the sublingual region of OVX23). Estrogen exhibits inhibitory effects on apoptosis in breast cancer cells, cardiac myocytes, endothelial cells, neuronal cells, and prostate cells24). The increase in the number of cells with polymorphic or pyknotic nucleus and degenerated cells in the parotid and submandibular salivary glands of OVX is also associated with estrogen deficiency. The mechanism of salivary gland dysfunction due to hormone deficiency, including menopause, remains unclear. Lipid deposition and excessive production of reactive oxygen species are known to induce dysfunction in the salivary glands to female rats18). Recently, it was reported that xerostomia caused by salivary gland dysfunction after menopause is the result of ferroptosis caused by increased lipid peroxidation and iron accumulation in the submandibular gland18). Lipid deposition and changes in the acinar and ductal cells, causing estrogen deficiency observed in this study, are also associated with ferroptosis.

In this study, the histopathological and histochemical changes were greater in the submandibular gland than in the parotid gland. Estrogen binds to estrogen receptors (ERs) found in the oral cavity and salivary glands, and ER acts as a transcription factor at the DNA-binding sites2). There are two subtypes of ER: ER-a25) and ER-b26). It has been reported that oral dryness caused by estrogen deficiency is particularly related to ER-b20). ER-b in the oral epithelium and salivary glands (which are directly affected by estrogen), affects acinar and ductal cells3) and plays an important role in the function and maintenance of oral cavity and salivary glands1). The submandibular salivary gland contains more ER-b than the parotid gland3). Therefore, the effect of estrogen deficiency was more evident in the submandibular salivary gland than in the parotid gland, which is consistent with our results.

Acknowledgments

This study was supported by research fund from Chosun University (2021).

Conflict of Interest

No potential conflict of interest relevant to this article was reported.

Ethical Approval

This study was approved by the Institutional Animal Care and Use Committee of Chonnan National University (No. CNU IACUC-YB-R-2018-59).

Author contributions

Conceptualization: Moon-Jin Jeong and Soon-Jeong Jeong. Data acquisition: Do-Seon Lim, Myoung-Hwa Lee, and Myeongju Jeong. Formal analysis: Do-Seon Lim, Myoung-Hwa Lee, and Myeongju Jeong. Funding: Moon-Jin Jeong. Supervision: Soon-Jeong Jeong. Writing–original draft: Soon-Jeong Jeong. Writing–review & editing: Moon-Jin Jeong and Soon-Jeong Jeong.

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