ۥ- ][~d~dddddd||||   .......4bQ/7 QUANTITATIVE STUDY ON THE RAT PAROTID GLAND AFTER ORCHIECTOMY Davor Je`ek, Ljerka Banek, Ru`ica Pezerovi}-Panijan and D`emal Pezerovi}1 Institute of Histology and Embryology, Faculty of Medicine, University of Zagreb, HR-10000 Zagreb, [alata 3, Croatia and 1Institute of Pathophysiology, Faculty of Dentistry, University of Zagreb, HR-10000 Zagreb, Ki{pati}eva 11, Croatia Running Title: Je`ek et al.: Parotid gland after orchiectomy Corresponding author: Davor Je`ek, MD, PhD Majstora Radovana 24 HR-10000 Zagreb Croatia Phone: +385 1 317 097 Fax.: + 385 1 424 001 e-mail: davor@mef.hr ABSTRACT In this study, the reaction of the rat parotid acini, duct system and the glandular connective tissue at various times after orchiectomy was analysed. Mature Fisher rats were sacrificed 12 hours and 3, 8, 15, 30 and 60 days after orchiectomy. Following the removal of parotid glands, histological sections were made and stereologically analysed by Weibelsymbol 162 \f "Symbol" \s 12s 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. The 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 the lack of testosterone or some other substance which depends on androgens, like epidermal growth factor (EGF). KEY WORDS: rat, parotid gland, orchiectomy, stereology Je`ek D., Banek Lj., Pezerovi}-Panijan R., Pezerovi} D`.: Morfometrijsko istra`ivanje dou{ne `lijezde {takora nakon orhidektomije SA@ETAK Tijekom ovog istra`ivanja prou~avana je struktura acinusa, kanalnog sustava i veziva dou{ne `lijezde {takora nakon razli~itih razdoblja po odstranjenju mu{ke spolne `lijezde (orhidektomije). Zreli {takori soja Fisher `rtovani su 12 sati te 3, 8, 15, 30 i 60 dana poslije orhidektomije. Nakon odstranjenja dou{nih `lijezdi, histolo{ki rezovi su stereolo{ki analizirani pomo}u Weibel-ove mnogonamjeneske testne mre`ice. U usporedbi s kontrolom, volumen acinusa dou{ne `lijezde (u mm3 tkiva) orhidektomiranih {takora bio je zna~ajno sni`en, dok je volumen vezivnog tkiva (u mm3 tkiva) bio zna~ajno pove}an od 8-og pa sve do 60-og dana pokusa. Zna~ajno smanjenje volumena kanalnog sustava (u mm3 tkiva) moglo se otkriti tek 60 dana nakon orhidektomije. Morfometrijska analiza tako|er je pokazala da dolazi do zna~ajnog skra}enja intralobularnog kanalnog sustava (u mm3 tkiva) u razdoblju od 8-og do 60-og dana nakon operacije. Skra}enje interlobularnih kanala moglo se zabilje`iti u orhidektomiranih {takora `rtvovanih samo 8-og i 15-og dana pokusa. Ovo skra}enje kanalnog sustava kao i promjene na acinusima i vezivnom tkivu dou{ne `lijezde {takora mogli bi biti uzrokovani nedostatkom testosterona ili neke druge tvari koja je ovisna o androgenima, kao npr. epidermalnog ~imbenika rasta (EGF). KLJU^NE RIJE^I: {takor, dou{na `lijezda, orhidektomija, stereologija INTRODUCTION Salivary glands in rodents display different structure in males and females. This is especially true for the submaxillary gland in the mouse. In males, the acini of this gland are rather poorly developed in contrast to the ductal system that has numerous intralobular and interlobular ducts. The submaxillary gland in females is rich in the acini, whereas the ductal system is moderately developed. All this features of 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 is still a matter of an investigation. Some physiological studies have shown that ducts of the parotid gland are not only a passive conduit (SCHNEYER et al., 1972; GARRET, 1975). They may 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 of the parotid acinar cells (DZIERZYKRAY-ROGALSKA et al., 1963; ZAGREBSKA and TOCHMAN 1985). On the contrary, 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 the better understanding the function of this gland and its interactions with other endocrine glands. For the above-mentioned reasons, we have in the present study analysed the reaction of the rat parotid gland at various periods after orchiectomy. Particular attention was directed to the acini, ducts and the glandular connective tissue. MATERIALS AND METHODS Thirty six male Fisher rats were divided after orchiectomy in 6 groups. Each group consisted of 6 animals. Fourteen male Fisher rats served as controls. All animals were housed under standard conditions (fed with commercial rat pellets and 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 Bouinsymbol 162 \f "Symbol" \s 12s fluid. The tissue was then processed by a standard histological procedure (embedded in paraffin; sections of 5-7symbol 109 \f "Symbol" \s 12m were made and stained with hematoxylin and eosin). A histological analysis of the gland was performed by a light microscopy. For the stereological analysis, Weibelsymbol 162 \f "Symbol" \s 12s multipurpose test system with 42 points was used. The following stereological parameters were determined: a) the volume of the acini (Vva), ducts (Vvd) and connective tissue (Vvct) (volume density - Vv) b) the 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 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 the magnification of x400, 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). The data were processed by a personal computer. RESULTS Qualitative histological analysis Parotid gland of control animals was composed from 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 the regular, round nucleus situated at the base of the cell. Between the glandular acini, intralobular ducts (intercalated and striated) were located. Intercalated ducts bore flat or cubic cells with elongated or oval nucleus, whereas striated ducts were layered by cubic or columnar cells with mostly round nucleus (Fig. 1). Orchiectomized rats did not display any difference in morphology of the parotid tissue until day 8 of the study. In groups of rats sacrificed after 8-60 days of 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 concerning the morphology of the acinar tissue itself as well as the structure of the duct system between the orchiectomized and control rats could be observed (Figs 2,3). Quantitative (stereological) analysis The volume of the acini per mm3 of the tissue was not different between the control and orchiectomized animals up to day 8 of the experiment (Table 1.). In the period of 8-60 days of 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 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 not earlier than 60 days after orchiectomy (P < 0.01) (Table 1). 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) did not show any significant difference between the orchiectomized and control rats (Table 2). However, in the following groups (day 8 - day 60) there was a significant decrease of the length of intralobular ducts of orchiectomized animals when compared to controls (P < 0.001) (Table 2). Interlobular ducts in the majority of orchiectomized groups did not manifest any significant difference in the length when compared to controls. Only in two groups (sacrificed on days 8 and 15) interlobular ducts became 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). On the contrary, the relation between the testis and the parotid gland is not quite clear. The reason may be 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 Loewenthalsymbol 162 \f "Symbol" \s 12s gland or glandula exorbitalis lacrimalis which has ducts emptying into the conjuctival sac) (PARHON et al., 1955). According to some authors, only Loewenthalsymbol 162 \f "Symbol" \s 12s gland exhibits sexual dimorphism and variations in histological structure under the influence of sex hormones (PARHON et al., 1955; BAQUICHE, 1959). On the contrary, some stereological measurements on the rat parotid inferior lobe showed that the size of the glandular acini and their cells was significantly different between 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 small 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 and after orchiectomy in the 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 appears already on the eighth day after orchiectomy. Changes in the staining characteristics of the mouse parotid duct cells can not be seen earlier than two weeks after operation (DZIERZYKRAY-ROGALSKA et al., 1963). It seems that the length of the intralobular ducts is a more sensitive indicator of morphological changes of the parotid gland provoked by orchiectomy. It is very likely that the described changes in the structure of the rat parotid gland given in our study are due to a lack of testosterone. Studies in mouse showed that the effects of castration on the parotid gland after a longer period of time became 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 the 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 bigger 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 supposed that intercalated duct cells help in the formation of the 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. Therefore, it has been concluded 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 appears 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 seems 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 salivary gland and Loewenthalsymbol 162 \f "Symbol" \s 12s gland in white mice. Acta Med. Pol. 4, 221-228. ELIAS, H., D.M. HYDE (1980): An elementary introduction to stereology (quantitative microscopy). Am. J. Anat. 159, 411-416. GARRETT, J.R. (1975): Recent advances in physiology of salivary glands. Br. Med. Bull. 31, 152-155. 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 subunit. Exp. Mol. Pathol. 40, 51-60. LACASSAGNE, A. (1940): Raction de la glande sousmaxillaire l'hormone mle chez la souris et le rat. C. R. Sances Soc. Bio. Fil. 133, 539-540. 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. Sper. 52, 397-403. 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 structure of the parotid gland of mouse and rat. Am. J. Anat. 108, 303-329. PARHON, C., A. BABES, I. PETREA, E. BURGHER (1955): Testicul la sobolanii albi parotidectomizati uni-si bilateral. Stud. Cercet. Endocr. 6, 361-365. 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 of the human parotid gland. J. Anat. 122, 627-640. SASHIMA, M., S. HATAKEYAMA, M. SATOH, A. SUZUKI (1989): Harderianization is another sexual dimorphism of rat exorbital lacrimal gland. Acta Anat. 135, 303-306. 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): Ultrastructure of baboon parotid gland. Anat. Rec. 184, 114-132. THESLEFF, I., L. VIINIKA, L. SAXEN, E. LEHTONEN, J. PERHEETUPA (1988): The parotid gland is the main source of human salivary epidermal growth factor. Life Sci. 43, 13-18. WALKER, P., M.E. WEICHSEL, S.B. HOATH, R.E. POLAND, D.A. FISHER (1981): Effect of thyroxine, testosterone and corticosterone on nerve growth factor (NGF) and epidermal growth factor (EGF) concentrations in adult female mouse submaxillary gland. Dissociation of NGF and EGF responses. Endocrinology 109, 582-587. WEIBEL, E., R. (1979): Stereological methods. Practical methods for biological morphometry. Vol. 1. Academic Press. London- New York - Toronto - Sydney - San Francisco. ZAGREBSKA, H. (1981): The effect of testosterone on secretory segments of rat parotid gland. Folia Morphol. (Warsz.) 40, 97-104. ZAGREBSKA, A., A. TOCHMAN (1985):Ultrastructural investigations of the influence of castration on parotid cells of the rat. Z. Mikrosk. Anat. Forsch. (Leipzig) 99, 717-724. LEGENDS FOR FIGURES 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. x200, Bar = 100m. 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). x200, Bar = 100m. 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. x200, Bar = 100m. TABLES Table 1. Volume of acini (Vva), ducts (Vvd) and connective tissue (Vvct) per mm3 of the parotid gland tissue in controls and orchiectomized rats. 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). Table 1. VARIABLE/ GROUP Vva Mean SE (x10-2) Vvd Mean SE (x10-3) 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 9.0*** 58.6 7.3 305.1 7.1****  IV (15 days) 64.1 0.6*** 50.2 6.2 314.1 1.1****  V (30 days) 63.1 8.0*** 44.3 5.3 354.4 9.3****  VI (60 days) 57.2 7.3**** 22.4 1.4** 372.7 6.9****  Vv[mm0] **-P<0.01; ***-P<0.001; ****-P<0.0001 Table 2. VARIABLE/ 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 0.7*** 5.5 0.4**  IV (15 days) 26.1 0.9*** 5.0 0.5**  V (30 days) 25.9 1.2*** 7.2 0.5  VI (60 days) 25.8 1.2*** 7.6 0.6   Lv[mm/mm3] **-P<0.01; ***-P<0.001 page 17 v.AA. 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