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: [email-masked]

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 PA

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.

Group

Vva
Mean±SE
(×10-2)

Vvd
Mean±SE
(×10-3)

Vvct
Mean±SE
(×10-3)

Controls

73.2±1.5

85.2±7.8

186.9±9.4

I (12 hours)

71.3±3.9

74.4±21.8

199.5±6.9

II (3 days)

72.8±5.3

74.6±11.9

255.4±11.6

III (8 days)

66.4±9.0b

58.6±7.3

305.1±7.1c

IV (15 days)

64.1±0.6b

50.2±6.2

314.1±1.1c

V (30 days)

63.1±8.0b

44.3±5.3

354.4±9.3c

VI (60 days)

57.2±7.3c

22.4±1.4a

372.7±6.9c

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).

Group

Lvi
Mean±SE

Lve
Mean±SE

Controls

34.4±1.4

6.9±0.3

I (12 hours)

34.6±1.3

7.3±0.4

II (3 days)

34.4±1.0

6.4±0.4

III (8 days)

25.7±0.7b

5.5±0.4a

IV (15 days)

26.1±0.9b

5.0±0.5a

V (30 days)

25.9±1.2b

7.2±0.5

VI (60 days)

25.8±1.2b

7.6±0.6

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

BAQUICHE, M. (1959): Le dimorphisme
sexuel de la glande de Loewenthal chez le rat albinos. Acta Anat. 36, 247-280.

COPE, G.H. (1978): Stereological
analysis of the duct system of the rabbit parotid gland. J. Anat. 126, 591-604.

DZIERZYKRAY-ROGALSKA, I., S.
CHODYNICKI, L. WISNIEWSKI (1963): The effect of gonadectomy on the parotid
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GARRETT, J. R. (1975): Recent advances
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IWASAKI, I., H. HORIE, J. TOMARY, G.
IDE, G. AONUME (1984): Osteogenesis bioassay and immunohistochemical and
radioisotopic studies of a subunit of parotin, a parotid gland extract and
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LACASSAGNE, A. (1940): Réaction de la
glande sousmaxillaire á l'hormone mâle chez la souris et le rat. C. R. Séances
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LAWRENCE, A. M., L. KIRSTEINS, J.
MITTON, I. L. HINES (1976): Parotid gland insulin: an extrapancreatic source of
insulin in rats. Diabetes 25, 328.

LOTTI, L. V., A. R. HAND (1988):
Endocytosis of parotid salivary proteins by striated duct cells in
streptozatocin-diabetic rat. Anat. Rec. 221, 802-811.

MANGONI, di S., C. STEFANO (1976): Le
parotidi di ratto organo bersaglio per il testosterone. Boll. Soc. Ital. Bio.
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MORRELL, J. I., E. W. GRESIK, T. BARKA
(1987): Autoradiographic localization of dihydrotestosterone binding in the
major salivary glands and other androgen-responsive organ of the mouse. J.
Histochem. Cytochem. 35, 1053-1058.

MUDD, B. D., S. C. WHITE (1975): Sexual
dimorphism in the rat submandibular gland. J. Dent. Res. 54, 193-198.

PARKS, H. F. (1961): On the fine
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PARHON, C., A. BABES, I. PETREA, E.
BURGHER (1955): Testicul la sobolanii albi parotidectomizati uni-si bilateral.
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POSINOVEC, J. (1967): Sexual dimorphism
of human parotid gland. Rad. Med. Fak. Zagreb 15, 171-184.

RIVA, H., F. TESTA-RIVA, M. DEL FIACCO,
M. S. LANTINI (1976): Fine structure and cytochemistry of the intralobular ducts
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SASHIMA, M., S. HATAKEYAMA, M. SATOH,
A. SUZUKI (1989): Harderianization is another sexual dimorphism of rat exorbital
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SCHNEYER, L. H., J. A. YOUNG, C. A.
SCHNEYER (1972): Salivary secretion of electrolytes. Physiol. Rev. 52, 720-777.

TANDLER, B., ERLANDSON R. A. (1976):
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THESLEFF, I., L. VIINIKA, L. SAXEN, E.
LEHTONEN, J. PERHEETUPA (1988): The parotid gland is the main source of human
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ZAGREBSKA, H. (1981): The effect of
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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

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