VETERINARSKI ARHIV 69 (1), 49-59, 1999

ISSN 1331-8055 Online
ISSN 0372-5480 Printed in Croatia

Quantitative study on the rat parotid gland
after orchiectomy

Davor Jezek¹*, Ljerka Banek¹, Ruzica Pezerovic-Panijan¹,
and Dzemal Pezerovic²

¹Institute of Histology and Embryology, Faculty of Medicine, University of Zagreb, Zagreb, Croatia

²Institute of Pathophysiology, Faculty of Dentistry, University of Zagreb, Zagreb, Croatia

* Contact address:
Dr. Davor Jezek,
10000 Zagreb, Majstora Radovana 24, Croatia,10000 Zagreb,
Phone: 385 1 317 097; Fax.: 385 1 424 001;
E-mail: davor@mef.hr

Jezek, D., Lj. Banek, R. Pezerovic-Panijan, Dz. Pezerovic: Quantitative study on the rat parotid gland after orchiectomy. Vet. arhiv 69, 49-59, 1999.

ABSTRACT

In this study the reaction of the rat parotid acini, duct system and the glandular connective tissue was analysed at various times after orchiectomy. Mature Fisher rats were sacrificed at 12 hours and at 3, 8, 15, 30 and 60 days after orchiectomy. Following the removal of parotid glands, histological sections were made and stereologically analysed by Weibel's multipurpose test system. When compared to controls, the volume of the parotid acini (per mm³ of the tissue) in orchiectomized rats was significantly decreased, whereas the volume of the connective tissue (per mm³ of the tissue) was significantly increased from day 8 to day 60 of the experiment. The significantly lower volume of the ductal system (per mm³ of the tissue) was noted only 60 days after orchiectomy. The statistically significant shortening of the intralobular ducts (per mm³ of the tissue) was found from day 8 to day 60 after the operation. Interlobular ducts were significantly shorter from day 8 to day 15 after orchiectomy. The shortening of the rat parotid duct system and the changes of the parotid acini and the connective tissue after orchiectomy may be provoked by a lack of testosterone or some other substance which depends on androgens, such as epidermal growth factor (EGF).

Key words: rat, parotid gland, orchiectomy, stereology

Introduction

Salivary glands in rodents display a different structure in males and females. This is especially true of the submaxillary gland in the mouse. In males, the acini of this gland are rather poorly developed in contrast to the ductal system, which has numerous intralobular and interlobular ducts. The submaxillary gland in females is rich in acini, whereas the ductal system is moderately developed. All these features of the submaxillary gland are part of the so-called "sexual dimorphism" in mammals (LACASSAGNE, 1940; POSINOVEC, 1967; SASHIMA et al., 1989). While the sexual dimorphism of the submaxillary gland and its interaction with male/female gonad is well established, the sexual dimorphism of the parotid gland remains a matter for investigation.

Some physiological studies have shown that ducts of the parotid gland are not only a passive conduit (SCHNEYER et al., 1972; GARRET, 1975), but that they may also alter the ionic composition of the saliva (SCHNEYER et al., 1972; GARRET, 1975). Moreover, it seems that their striated duct cells have an important role in the secretion of insulin, glucagon and parotin (LAWRENCE et al., 1976; IWASAKI et al., 1984; LOTTI and HAND, 1988). It has been established that striated duct cells of the mouse submaxillary gland concentrate testosterone (MORRELL et al., 1987). Some investigations have demonstrated that the parotid gland is also a target organ for testosterone (MANGONI and STEFANO, 1976). It is known that castration provokes changes in the parotid acinar cells (DZIERZYKRAY-ROGALSKA et al., 1963; ZAGREBSKA and TOCHMAN 1985). Conversely, the specific effect of castration on the morphology of the glandular acini, connective tissue, and especially on the length of the parotid duct system, has been poorly studied. The data on the effects of orchiectomy on the parotid gland may be useful for a better understanding of the function of this gland and its interactions with other endocrine glands.

For the above-mentioned reasons, in the present study we have analysed the reaction of the rat parotid gland at various periods after orchiectomy. Particular attention was paid to the acini, ducts and the glandular connective tissue.

Materials and methods

Thirty-six male Fisher rats were divided into six groups after orchiectomy. Each group consisted of six animals. Fourteen male Fisher rats served as controls.

All animals were housed under standard conditions (fed with commercial rat pellets and with water available ad libitum ). They were sacrificed after the following periods of time: 12 hours, 3 days, 8 days, 15 days, 30 days and 60 days. Rat parotid glands were removed and fixed in Bouin's fluid. The tissue was then processed using a standard histological procedure (embedded in paraffin; sections of 5-7 µm were made and stained with hematoxylin and eosin).

A histological analysis of the gland was performed by light microscopy. For the stereological analysis, Weibel's multipurpose test system with 42 points was used. The following stereological parameters were determined: a) volume of the acini (Vva), ducts (Vvd) and connective tissue (Vvct) (volume density - Vv); b) length of intralobular (intercalated and striated, Lvi) and excretory interlobular (Lve) ducts per mm³ of the tissue (length density - Lv). Volume density (Vv) was determined by the point counting method (WEIBEL, 1979). Length density of the mentioned ducts (Lv) was calculated as follows (according to ELIAS and HYDE, 1980):

Lv=2 P_A

where Lv is the length of parotid duct system per unit volume, the number of profiles of duct system equals P, and the total test area used equals A. Every stereological variable was assessed at a magnification of ×400, where the length of the test surface area (A) was 0.02094 mm² for each analysed microscopic field.

A statistical evaluation was made by a three-way ANOVA (with factors: group, time, tissue). Data were processed by a personal computer.

Results

Qualitative histological analysis

Parotid gland of control animals was composed of several lobes of different size. Each lobe comprised several lobules, which were divided by an interlobular connective tissue. Within the interlobular connective tissue, excretory ducts could be observed. Depending on their dimensions, these ducts were layered by one or two layers of columnar epithelial cells. Lobules consisted of numerous acini. The neighbouring acini were divided by a gentle network of an interacinar connective tissue. Acinar cells were pyramidal with a regular, round nucleus situated at the base of the cell. Intralobular ducts (intercalated and striated) were located between the glandular acini. Intercalated ducts bore flat or cubic cells with an elongated or oval nucleus, whereas striated ducts were layered by cubic or columnar cells with a mostly round nucleus (Fig. 1).

Fig. 1. Parotid gland of the control animal. Among the abundant acini of the gland (A), a number of intralobular (secretory) ducts (arrowheads) are visible. ×200, Bar=100 µm.

Orchiectomized rats displayed no difference in morphology of the parotid tissue until day 8 of the study. In groups of rats sacrificed after 8-60 days of the experiment, a gradual increase in the interacinar and interlobular connective tissue could be noted (Figs. 2, 3). Because of the increased presence of the connective tissue, the acini of orchiectomized animals seemed to be less represented in the section. However, no significant changes with regard to the morphology of the acinar tissue itself, as well as to the structure of the duct system between the orchiectomized and control rats, could be observed (Figs 2, 3).

Fig. 2. Parotid gland in the orchiectomized rat 30 days after the operation. Several lobules composed of the acini (A) are divided by the enlarged connective tissue (c). Some intralobular ducts are indicated by arrowheads. Within the interlobular tissue, one excretory duct can be noted (arrow). ×200, Bar=100 µm.

Fig. 3. Orchiectomized rat after 60 days of the experiment. Within the augmented connective tissue (c), interlobular ducts (arrows) are situated. The acini (A) of the neighbouring lobules seem to be less abundant. ×200, Bar=100 µm.

Quantitative (stereological) analysis

There was no difference in the volume of the acini per mm³ of the tissue between the control and orchiectomized animals up to day 8 of the experiment (Table 1). In the period of 8-60 days of  the experiment a statistically significant decrease in the volume of the acini in orchiectomized rats could be recorded (day 8 - P<0.001, day 60 - P<0.0001). The interacinar and the interlobular connective tissue (per mm³ of the tissue) was significantly increased in orchiectomized animals sacrificed after 8-60 days of the experiment (P<0.0001). In contrast to the acini and the connective tissue of the gland, a significant decrease in the volume of the ductal system was observed no earlier than 60 days after orchiectomy (P<0.01) (Table 1).

Table 1. Volume of acini (Vva), ducts (Vvd) and connective tissue (Vvct) per mm³ of the parotid gland tissue in controls and orchiectomized rats.

Lv (mm/mm³); ^a-P<0.01; ^b-P<0.001; ^c-P<0.0001

The length of intralobular ducts (per mm³ of the tissue) of the parotid gland after a shorter period of orchiectomy (i.e., up to day 8) showed no significant difference between the orchiectomized and control rats (Table 2). However, in the following groups (day 8 - day 60) there was a significant decrease in the length of intralobular ducts of orchiectomized animals when compared to controls (P<0.001) (Table 2).

Table 2. Length of the rat parotid duct system per mm³ of the tissue in controls and orchiectomized rats (Lvi - length of intralobular ducts; Lve - length of excretory or interlobular ducts).

Lv (mm/mm³); ^a-P<0.01; ^b-P<0.001

Interlobular ducts in the majority of orchiectomized groups manifested no significant difference in length when compared to controls. Only in two groups (sacrificed on days 8 and 15) did interlobular ducts become significantly shorter per mm³ of the parotid gland tissue (P<0.01)(Table 2).

Discussion

In rodents, the sexual dimorphism of the submaxillary gland is well established (MUDD and WHITE, 1975). Conversely, the relation between the testis and the parotid gland is somewhat unclear. The reason for this may lie in the fact that, in the rat, two different parts of the parotid gland can be distinguished: an inferior lobe with a typical serous structure, and a superior lobe (also called Loewenthal's gland or glandula exorbitalis lacrimalis, which has ducts emptying into the conjuctival sac) (PARHON et al., 1955). According to some authors, only Loewenthal's gland exhibits sexual dimorphism and variations in histological structure under the influence of sex hormones (PARHON et al., 1955; BAQUICHE, 1959). Conversely, some stereological measurements on the rat parotid inferior lobe showed that the size of the glandular acini and their cells was significantly different in the male and female rat (ZAGREBSKA, 1981). These differences were even more pronounced following testosterone administration (ZAGREBSKA, 1981). Also, an atrophy of the parotid acinar cells and a decrease in the activity of some enzymes of these cells after castration has been described (BAQUICHE, 1959; DZIERZYKRAY-ROGALSKA et al., 1963; ZAGREBSKA and TOCHMAN, 1985).

The influence of orchiectomy on the morphology of the parotid gland ducts is not well known. After gonadectomy in the male mouse, some minor changes in the staining characteristics of parotid duct cells were observed (DZIERZYKRAY-ROGALSKA et al., 1963). Quantitative studies on the structure of the acini and connective tissue, as well as on the length of the parotid duct system in intact glands and after orchiectomy, are less numerous. The results of our investigation provide qualitative and quantitative (stereological) data on the morphology of the parotid gland prior to and after orchiectomy in male adult rats.

In control rats, the ratio between the length of interlobular and intralobular ducts is approximately 1:5. Our investigation shows that the shortening of the intralobular ducts appeared as early as the eighth day after orchiectomy. Changes in the staining characteristics of the mouse parotid duct cells cannot be seen earlier than two weeks after operation (DZIERZYKRAY-ROGALSKA et al., 1963). It would appear that the length of the intralobular ducts is a more sensitive indicator of morphological changes in the parotid gland provoked by orchiectomy. It is very likely that the described changes in the structure of the rat parotid gland presented in our study are due to a lack of testosterone.

Studies in the mouse have shown that the effects of castration on the parotid gland after a longer period of time become less pronounced (DZIERZYKRAY-ROGALSKA et al., 1963). This is due to the compensatory activity of the adrenal cortex. However, our results demonstrated that in the rat the length of intralobular ducts was shorter after 60 days of treatment. There was no return to its normal value during this period of orchiectomy. It could be presumed that during the investigated period after the operation, the adrenal cortex in the rat did not develop compensatory activity or produced testosterone, as was described in the mouse (DZIERZYKRAY-ROGALSKA et al., 1963).

One electron microscopic study has demonstrated that the cells of intralobular ducts of the human parotid gland do not differ significantly from those of the submaxillary gland (RIVA et al., 1976). Similarly, the occurrence of the shortening of intralobular parotid ducts in our experiment could be explained by the analogy with the mouse submaxillary gland. It is known that the female gland comprises less numerous intralobular ducts, while the male gland contains hypertrophic intralobular ducts (MUDD and WHITE, 1975). The larger number of intralobular ducts on sections of the male submaxillary gland suggests their greater length, while their smaller number in the female mouse confirms that these ducts are shorter. If testosterone is given to the female mouse, or estrogen to the male mouse, the described morphologic characteristics of their submaxillary duct system will be changed (MUDD and WHITE, 1975).

Furthermore, it has been established that parotid intralobular duct cells contain a number of granules in their cytoplasm (PARKS, 1961; RIVA et al., 1976). The granules of intercalated duct cells correspond to the secretory granules in acinar cells (PARKS, 1961; RIVA et al., 1976; TANDLER and ERLANDSON, 1976). Hence, it is presumed that intercalated duct cells assist in the formation of saliva (RIVA et al., 1976). They also represent a reservoir of pluripotent cells that may differentiate into acinar cells (RIVA et al., 1976). As in the human parotid gland, all transition stages of duct cells are discernible. It is concluded, therefore, that cells of intercalated ducts could differentiate into those of striated duct cells (RIVA et al., 1976). The results of the experiments with steroids on the mouse submaxillary gland (MUDD and WHITE, 1975) may suggest that the direction of duct cells differentiation could be opposite. It would appear that the lack of testosterone (or some factors that depend on it) direct the transition of striated duct cells into intercalated duct cells, or even their later differentiation into acinar cells. The results of our study could not confirm these data for the rat parotid duct cells. We presume that the lack of testosterone in our study induced a reduction in the volume of the acini and the ductal system, as well as the augmentation of the connective tissue. It would appear that the cells of the rat parotid interlobular ducts also react on orchiectomy by shortening 8 to 15 days after the operation.

It has been established that production of EGF in the submaxillary gland depends on androgens (DZIERZYKRAY-ROGALSKA et al., 1963). Moreover, it has been demonstrated that the parotid gland is the main source of EGF in the human saliva, although the saliva from the submaxillary gland also contains this factor (THESLEFF et al., 1988). Hence, the lack of testosterone in our experiment (provoked by orchiectomy) could also lead to a decrease of EFG in the parotid gland and may influence the shortening of the intralobular and interlobular ducts, as well as the atrophy of the acinar cells. This possibility becomes particularly interesting if one bears in mind the fact that the parotid gland binds more testosterone than the prostate or seminal vesicles (MANGONI and STEFANO, 1976). Therefore, it can be supposed that this hormone is essential for the function of the parotid gland.

Acknowledgements
This study was supported by Grants for Scientific Research Nos. 108012 and 108900 from the Ministry of Science and Research, Republic of Croatia.

References

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Received: 4 November 1998
Accepted: 15 January 1999

Jezek, D., Lj. Banek, R. Pezerovic-Panijan, Dz. Pezerovic: Morfometrijsko istrazivanje zausne zlijezde stakora nakon orhidektomije. Vet. arhiv 69, 49-59, 1999.

SAZETAK

Tijekom ovog istrazivanja proucavana je struktura acinusa, kanalnog sustava i veziva zausne zlijezde stakora nakon razlicitih razdoblja po odstranjenju muske spolne zlijezde (orhidektomije). Zreli stakori soja Fisher usmrceni su 12 sati te 3, 8, 15, 30 i 60 dana poslije orhidektomije. Nakon odstranjenja zausnih zlijezdi, histoloski rezovi su stereoloski analizirani pomocu Weibel-ove mnogonamjeneske testne mrezice. U usporedbi s kontrolom, volumen acinusa zausne zlijezde (u mm³ tkiva) orhidektomiranih stakora bio je znacajno snizen, dok je volumen vezivnog tkiva (u mm³ tkiva) bio znacajno povecan od 8-og pa sve do 60-og dana pokusa. Znacajno smanjenje volumena kanalnog sustava (u mm³ tkiva) moglo se otkriti tek 60 dana nakon orhidektomije. Morfometrijska analiza takoder je pokazala da dolazi do znacajnog skracenja intralobularnog kanalnog sustava (u mm³ tkiva) u razdoblju od 8-og do 60-og dana nakon operacije. Skracenje interlobularnih kanala moglo se zabiljeziti u orhidektomiranih stakora usmrcenih samo 8-og i 15-og dana pokusa. Ovo skracenje kanalnog sustava kao i promjene na acinusima i vezivnom tkivu zausne zlijezde stakora mogli bi biti uzrokovani nedostatkom testosterona ili neke druge tvari koja je ovisna o androgenima, kao npr. epidermalnog cimbenika rasta (EGF).

Kljucne rijeci: stakor, zausna zlijezda, orhidektomija, stereologija