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6J mice following chronic alcohol ingestion
Tissue & Cell, 2002 34 (3) 177–186 © 2002 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0040-8166(02)00029-0, available online at http://www.idealibrary.com
Tissue&Cell
Stereology and ultrastructure of the seminal vesicle of C57/BL/6J mice following chronic alcohol ingestion I. C. Gomes, 1 V. H. A. Cagnon, 1 C. A. F. Carvalho, 1 I. M. S. De Luca 2
Abstract. Excessive alcohol consumption causes metabolic changes and pathologic alterations in testes and accessory sex organ in different animal species. The aim of the present study was to evaluate the macroscopic, histologic and ultrastructural alterations provoked by chronic ingestion of different ethanol concentrations over increasing periods of time on the secretory epithelium of the seminal vesicle of C57/BL/6J mice in using stereological methods. Sixty male adult mice were divided into three experimental groups: Control, Alcoholic 25% and Alcoholic 35%, respectively, receiving tap water and tap water containing ethanol diluted to 25 and 35 ◦ Gay Lussac. All mice were fed with the same solid diet. After 150 and 250 days of treatment the animals were sacrificed and the seminal vesicles were collected and processed for light and transmission electron microscopy. The cellular, cytoplasmic and nuclear volumes and the area density of autophagic and secretory vacuoles were measured. The histologic alterations observed in the alcoholic mice consisted of a reduction in epithelial size and cell volume, with maintenance of the same nuclear and cytoplasmic ratio as verified in the control groups. The ultrastructural alterations were: increased density of dense body area, decreased density of secretory granule area, and dilated rough endoplasmic reticulum and Golgi cisternae. We conclude that chronic ethanol ingestion causes depleting morphologic alterations in the epithelial cells of the seminal vesicle and negatively affects the secretory process of this gland. © 2002 Elsevier Science Ltd. All rights reserved.
Keywords: seminal vesicle, alcohol, ultrastructure, morphology
Introduction Alcohol and its metabolites provoke generalized disorders in different organ systems such as the central nervous sys1 Department of Anatomy, Institute of Biology, State University of Campinas—UNICAMP, Campinas, São Paulo, Brazil, 2 Department of Histology, Institute of Biology, State University of Campinas—UNICAMP, Campinas, São Paulo, Brazil
Received 2 November 2001 Revised 12 April 2002 Accepted 22 April 2002 Correspondence to: Dr Valeria H. A. Cagnon, Departamento de Anatomia, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP) 13084-971, Campinas, S ão Paulo, Brazil. Tel.: +55 19 3788 7391; Fax: +55 19 3289 3124; E-mail: [email protected]
tem, the male reproductive system, and the circulatory and hematopoietic systems. They also act directly on the liver, pancreas, intestine, endocrine glands, and accessory sex glands (Marks & Wright, 1978; Galvão-Teles et al., 1986; Edwards & Peters, 1994). In humans, chronic alcoholism has been associated with hypogonadism, feminization, sexual impotence and changes in reproductive hormonal homeostasis (Anderson et al., 1987). However, risk factors such as duration and quantity of consumed alcohol, age of consumption and period of development have not been evaluated leading to gaps on information. Thus, controlled experimental studies have been important for evaluation of the results associated with impairment in male reproductive system due to excessive consumption of alcohol (Anderson et al., 1987). 177
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Experimental and clinical studies indicate that continued ingestion of ethanol contributes, through several mechanisms, such as testicular luteinizing hormone receptors depletion and gonadotropin responsiveness, besides pituitary and hypothalamic defects to disturbances in the hypothalamic– pituitary–gonadal axis, which provoke damage in sex organ genesis and in the normal secretory activity of the male reproductive tract (Harkin, 1963; Cicero & Badger, 1977; Van Thiel & Lester, 1979; Salonen & Huhtaniemi, 1990). In addition, alcohol acts directly on the male gonads by altering testicular testosterone synthesis (Gary et al., 1976; Van Thiel & Lester, 1979). In a study on rats chronically treated with alcohol, Klassen and Persaud (1978) reported reduced folding of the glandular mucosa and involution of secretory epithelial cells. Later, Semczuk and Rzeszowska (1981) also observed a significant reduction in seminal vesicle weight, atrophy of the secretory epithelium, and partial cell degeneration in rats treated with ethanol for 204 days compared to control. Martinez et al. (1997), after treating adult Wistar rats with sugar cane brandy at 30◦ Gay Lussac for 60, 120 or 180 days, observed marked morphologic changes in the secretory epithelium of the seminal vesicle, among them a significant reduction of epithelial cell height and of number of secretory vacuoles, the presence of nuclei of irregular contours, and a reduction of the amount of microvilli. A review of the literature showed that doubts persist about the effect of ethanol on the secretory epithelium of the seminal vesicle of mice, especially with respect to epithelial reduction and to the contributions of the nucleus and cytoplasm to this process, in addition to the behavior of these structures in terms of time of exposure and alcoholic dosage. Thus, the major objective of the present study was to analyze statistically data concerning seminal vesicle weight and cell, cytoplasmic and nuclear volumes and to study stereologically the density of areas of autophagic and secretory vacuoles in order to obtain a general view of the structural and ultrastructural alterations provoked by chronic alcohol ingestion and its consequences for the secretory process of this gland.
Material and methods Animals and tissue preparation A total of 60 adult male mice (C57/BL/6J), high alcohol preference, (Rodgers, 1966) of the same age were divided into three 20 mouse groups (Control, Alcoholic 25% and Alcoholic 35%) and assigned to two treatment times (150 and 250 days). The Control group received tap water, the experimental group Alcoholic 25% consumed only ethanol diluted at 25◦ Gay Lussac (25 ml ethanol/100 ml solution), and the experimental group Alcoholic 35% consumed only ethanol diluted at 35◦ Gay Lussac (35 ml ethanol/100 ml solution) for 150 or 250 days. All groups were fed solid Nuvilab CR chow ad libitum. Liquid and solid consumption was measured daily and the data obtained were used to calculate mean calorie ingestion by the experimental groups. At the
end of each period of treatment, mice from each group were anesthetized with Francotar/Virbaxyl (1:1) and weighed. The seminal vesicles were collected from five animals per group, weighed, fixed in Bouin’s solution, embedded in Paraplast Plus resin, cut into 5 m thick sections and stained with hematoxylin and eosin. Another five mice per group were perfused through the left ventricle with Karnovsky’s solution (Karnovsky, 1965; Sprando, 1990). Then the seminal vesicles were removed and immersed for 2 h in the same fixative followed by 1 h in cacodylate buffered 1% osmium tetroxide. The tissue was dehydrated using a graded series of ethanol and then embedded in Araldite. Ultrathin sections were obtained with an LKB ultramicrotome and stained with uranil acetate (Watson, 1958) and lead citrate (Reynolds, 1963) and examined and photographed with a LEO 906 electron microscope. Morphometric procedures The cellular, cytoplasmic and nuclear volumes were measured on the sections stained for light microscopy. Data for nuclear volume were recorded as the average obtained in 75 measurements per animal. Long and short axes were measured and the mean nuclear volume was calculated considering nuclei as ellipsoids. The sections were studied with an ocular micrometer coupled to a Zeiss microscope with a ×100 objective. For the determination of cytoplasm volume, an ocular with a 400 grid coupled to a ×100 objective was used. Points on the cytoplasm and nuclei were counted in 15 areas per group and the cytoplasmic and nuclear fractions were obtained. These data and the nuclear volumes were used to estimate the cytoplasm volume for each animal. The cellular volume was calculated by summing the nuclear and cytoplasm volumes. For the determination of density of autophagic and secretory vacuole areas we used current stereology methods and formulae as described by Weibel (Weibel, 1979). A multipurpose test system with 84 lines and 168 points was applied to 15 electronmicrographs (×10 000) per group and the numerical density of the area was estimated. Statistical analysis Mean data for total ingested calories, final body weight minus initial seminal vesicle weight and cell volumes were analyzed statistically by 2 × 3 analysis of variance (ANOVA) for factorial experiments in fully randomized designs, followed by the Tukey multiple range test for comparisons between group means at each time point (Montgomery, 1991).
Results Macroscopic observations Seminal vesicle weight After the treatment with ethanol the seminal vesicle weight fell from the control level of 0.23 to 0.18 mg (Alcoholic 25%, P < 0.05) and 0.11 mg (Alcoholic 35%, P < 0.05) after
stereology and ultrastructure of the seminal vesicle 179
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Fig. 1 Photomicrographs of the secretory epithelium of the seminal vesicle of control and alcoholic rats after 250 days of treatment. (A) Acini with folded mucosa. Epithelial cells (EC) of the tall columnar type. Light zones corresponding to the Golgi complex area can be seen above the nucleus (*). Nucleus (N) located in the basal region. Basal cells (bc) are intermingled with epithelial cells, presenting a clearly visible nucleolus. Lumen (L) with secretion of homogeneous aspect. Stroma (S). ×1300. (B) Secretory epithelium of a 25% alcoholic animal. Mucosa with few folds. Atrophied epithelial cells (EC). Absence of light zones above the nuclei. Centralized nuclei (N). Basal cells (bc) also atrophied. Lumen (L) with heterogeneous secretion. Stroma with different cell types (S). ×1300. (C) Secretory epithelium of an Alcoholic 35% animal. Mucosa with few folds. Markedly atrophied epithelial cells (EC). Absence of light zones above the nuclei. Centralized nuclei (N). Atrophied basal cells (bc). Lumen (L) with heterogeneous secretion. Stroma with different cell types (S). ×1300.
150 days and from 0.32 mg (Control) to 0.21 mg (Alcoholic 25 and 35%, P < 0.05) after 250 days. Caloric analysis Mean total caloric consumption at 150 days of treatment was numerically lower in control animals compared to the Alcoholic 25 and 35% groups, although the difference was not statistically significant. A similar result was observed at 250 days (Table 1). In the Alcoholic 25% group, liquid consumption was 41 and 35% of the total number of calories ingested at 150 and 250 days of treatment, respectively, while in the Alcoholic 35% group liquid consumption was 43 and 45% of the total number of calories ingested at 150 and 250 days, respectively. Body weight Body weight gain was lower in the Alcoholic 25 and 35% animals at the two treatment times compared to control. However, minimum significant differences at 250 days of treatment were observed only for the two experimental groups compared to control (Table 1). Light microscopy In the control groups, the glandular epithelium of the seminal vesicle had a tubular arrangement covered with smooth muscle and containing connective tissue resting on the basement membrane. The secretion contained in the tubular lumen had a homogeneous colloidal appearance. The epithelium consisted of basal cells sandwiched between the basis of the epithelial cells (Fig. 1A). The cytoplasm showed a light supranuclear region probably corresponding to the Table 1 Minimum significant difference (MSD) determined by the Tukey test and coefficient of variation (CV) for the mean number of total calories (MC, kcal) ingested daily and for mean body weight increase (WI, g) in the 25% (A25) and 35% (A35) alcoholic groups and controls (C) for each treatment period
MC
WI
Group variables
Period of treatment (days) 150
250
MSD (5%), CV (%)
C A25 A35
15.36a ± 1.74 16.14a,b ± 0.89 17.54b ± 1.08
13.92a ± 1.24 15.70b ± 1.41 14.99a,b ± 1.67
1.46 8.74
C A25 A35
9.22a ± 1.24 9.00a ± 1.81 7.57a ± 1.90
13.93b ± 4,39 10.44a ± 1.76 9.90a ± 4.00
3.12 29.26
a,b: Means followed by the same letter did not differ according to the Tukey multiple range text.
Table 2 Mean cell volume (m3 ) in the Control (C) and 25% (A25%) and 35% (A35%) alcoholic groups for each period of treatment
Cell volume
Group variables
Period of treatment (days) 150
250
C A25% A35%
187.00a∗
180.00b A ± 18.00 175.00a A ± 26.00 177.00a A ± 23.00
A∗∗ ± 46.00 155.00b∗ A ± 30.00 162.00a,b∗ A ± 15.00
MSD (groups) significant mean difference = 44.00. MSD (period) significant mean difference/period = 36.00. CV, coefficient of variation = 18.54%. ∗ According to the Tukey multiple range test, means followed by the same lower case letter did not differ within the group, fixed the time. ∗∗ Means followed by the same capital did not differ within time, fixed the group.
Golgi complex and the secretory vacuoles (Fig. 1A). The nuclei of the epithelial cells rested on the basal third of the cell. They were ovoid and showed one or two nucleoli (Fig. 1A). The data for the control groups of the two experimental times were pooled because cell morphology and volume were similar (Table 2). In the alcoholic groups (25 and 35%), the characteristics of the stroma were similar to those of the control groups (Fig. 1A–C). Intraluminal secretion showed a homogeneous colloidal aspect similar to that of control animals (Fig. 1B & C). The secretory epithelium was pseudostratified, with proven epithelial cell involution compared to control animals (Table 2), (Fig. 1B & C), both at 150 and 250 days of treatment (Table 2). In the alcoholic groups, cell volume was similar along the experimental period. The mean percent fractions occupied both by nuclei and by cytoplasm were similar in the alcoholic groups and in the controls (Table 3). The alcoholic groups 25 and 35% were described together at the two experimental times because they presented similar morphologic characteristics. Table 3 Mean percent value of the cell fraction represented by the nucleus and the cytoplasm in seminal vesicle epithelial cells from the control (C), Alcoholic 25% (A25%) and Alcoholic 35% (A35%) groups during each treatment period Organelles
Group variables
Period of treatment (days) 150 (%)
250 (%)
Nucleus
C A25% A35%
23.84 22.86 22.28
19.96 20.98 20.36
Cytoplasm
C A25% A35%
76.10 77.14 77.72
80.04 79.02 79.64
stereology and ultrastructure of the seminal vesicle 181
Fig. 2 Electronmicrographs of the secretory epithelium of the seminal vesicles of control animals at 150 and 250 days of treatment. (A) Tall columnar epithelial cells. Basal nucleus (N) and clearly visible nucleolus (Nu). Mitochondria (M) dispersed throughout the cytoplasm. GER localized in the perinuclear and basal regions. Golgi complex (*) and vacuoles containing an eccentric electrondense granule are visible in the supranuclear region. Clearly visible microvilli (MV) and junctional complex. Basal lamina (arrow). Lumen (L). ×5000. (B) Detail of the apical region at 150 days of treatment. Microvilli (MV) on the cell surface facing the lumen. Junctional complex (circle). Secretory vacuoles containing an eccentric electrondense granule (夹). ×15 000. (C) Detail of the basal region at 250 days of treatment. Basal lamina (arrow). Extracellular matrix with a nerve bundle (lozenge) and collagen and smooth muscle (SM) fibers. In the cytoplasm, GER and nucleus (N) of the epithelial cell. ×7500.
Transmission electron microscopy In control mice, the basal cells presented only traces of the cellular endoplasmic reticulum and no signs of the Golgi complex (Fig. 4A). The ultrastructure of the epithelial cells showed a granular endoplasmic reticulum with flat and parallel cisternae (Fig. 2A–C). The Golgi complex was prominent (Fig. 2A). There were large and small membrane-bound vacuoles, each containing a dense secretion granule (Fig. 2B). The area density of these secretory vacuoles was similar in both control groups (Fig. 5). Eventual autophagic vacuoles were seen in the cytoplasm (Fig. 2C) with a far lower area density than that of the secretory vacuoles, corresponding to 0.04 m2 /m3 at 150 days and 0.020 m2 /m3 at 250 days (Fig. 5). The mitochondria showed no structural specialization and were scattered throughout the cytoplasm. The nucleus was elongated and usually showed one nucleolus (Fig. 2A). The heterochromatin was located at the nuclear periphery and as irregular
clumps in the nucleoplasm. Microvilli of varying length and numbers were present on the apical surface (Fig. 2A & B). In alcoholic mice, the epithelial and basal cells showed signs of degeneration at the different times of treatment and at the different alcohol doses, although these changes did not intensify along time of treatment or with the doses used. Basal cells showed different stages of degeneration (Fig. 4B–D) with rounded nuclei and points of chromatin condensation scattered throughout the nucleus (Fig. 4B & C). In the secretory epithelial cells, the granular endoplasmic reticulum and the Golgi complex showed dilated cisterns (Fig. 3C–E). An increase in area density of the autophagic vacuoles was observed, as opposed to a decrease in the area density of the secretory vacuoles, with an eccentric core, in agreement with the characteristics observed in the control groups (Fig. 5). Nevertheless, secretion granules were still observed in the glandular lumen. The mitochondria were similar to those
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Fig. 3 Electronmicrographs of the secretory epithelial cells of the seminal vesicle of alcoholic mice 25 and 35% at 150 and 250 days of treatment. (A) Tall and basal columnar cells of the Alcoholic 25% group at 250 days of treatment. Vacuoles (夹) containing little or no secretion. Sparse microvilli (MV) covering the cell surface. Nucleus (N) with envelope dents. Clearly visible nucleolus (Nu). Basal cell with the nucleus presenting envelope dents (bn). Lumen (L) with granules secretion. ×5000. Inset: detail of a multivesicular body containing cell remnants and secretory vacuoles. ×15 000. (B) Low columnar epithelial cell from an animal of the Alcoholic 35% group at 250 days of treatment. The cytoplasm shows secretory vacuoles (夹) with an eccentric granule. Nucleus (N) with dents and condensed chromatin at the periphery. Nucleolus (Nu). Basal cell with a lipid droplet (Li). Degenerated nucleus of the basal cell (bn). Basal lamina (arrow). Glandular lumen (L) containing granules secretion. ×5000. (C) Detail of the apical region of the Alcoholic 25% group at 150 days of treatment. Mitochondria (M). Golgi complex (*). Accumulation of autophagic vacuoles resembling figures of the ‘myelinic’ type (hatched area). Sparse microvilli (MV) cover the cell surface. The junctional complex (circle) between adjacent cells is clearly visible. Nucleus (N). ×7500. (D) Detail of a nucleus of the Alcoholic 35% group at 150 days of treatment. Degenerative process with large electrondense inclusions. 30 000. (E) Detail of the basal region of the Alcoholic 25% group at 150 days of treatment. Dilated cisterns GER. Basal lamina clearly visible in the extracellular layer (arrow). Collagen fibers cut transversely and longitudinally and smooth muscle fibers (SM). Observe the nucleus of the basal cell (bn). ×7500.
stereology and ultrastructure of the seminal vesicle 183
Fig. 4 Electronmicrographs of the secretory epithelial cells of the seminal vesicle at 150 and 250 days of treatment. (A) Basal cell of the control group at 150 days of treatment. Small and compact mitochondria (M). Pleomorphic nucleus with envelope dents (bn). Basal lamina (arrow). ×7500. (B) Basal cell of the Alcoholic 25% group at 250 days of treatment. Mitochondria (M). Condensed chromatin throughout the nucleus (bn). Basal lamina (arrow). ×7500. (C) Basal cell from the Alcoholic 25% group at 250 days of treatment. Nucleus (bn) with spiraling chromatin intermingled with condensation points (arrowhead). Basal lamina (arrow). ×7500. (D) Basal cell from the Alcoholic 35% group at 250 days of treatment. Lipid droplets (Li). Degenerated nucleus (bn). Folded basal lamina (arrow). ×7500.
described for the control groups but seemed to be more concentrated in the supranuclear region (Fig. 3A–C). The nuclear shape was irregular compared to control with increasing alcohol concentration and time of ingestion (Fig. 3A & B). The basement membrane showed deep infoldings and the microvilli were small and scattered (Fig. 3B & C). The area density of autophagic vacuoles was numerically higher at 250 days of alcoholism with 35% ethanol (Fig. 4).
Discussion and conclusion The sequels of chronic ethanol ingestion for the accessory sex glands of rodent have been documented by both light microscopy and transmission electron microscopy, being mainly represented by regressive changes in epithelial cell height after different periods of alcohol ingestion (Semczuk & Rzeszowska, 1981; Anderson et al., 1989; Cagnon et al.,
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Fig. 5 Density (m2 /m3 ) of the area occupied by secretory and autophagic vacuoles in the cytoplasm of seminal vesicle epithelial cells in the different experimental groups and during the various treatment periods.
1996; Garcia et al., 1999). In the present study, we demonstrated macroscopic atrophy of the seminal vesicle of 21.74% (Alcoholic 25%) and 52.18% (Alcoholic 35%) at 150 days of treatment and of 34.37% for both alcoholic groups at 250 days of treatment. These observations agree with data reported by Klassen and Persaud (1978), who observed a significant decrease in seminal vesicle weight in Sprague–Dawley rats receiving 10% ethanol for 5 weeks. Similarly, Semczuk and Rzeszowska (1981) reported that the weights of the seminal vesicles of Wistar rats receiving 40% ethanol at the dose of 3 g/kg body weight were significantly decreased compared to control after 204 days of treatment. Anderson et al. (1987), after treating C57/BL/6J mice with 5% ethanol for 43 days, observed a significant decrease in seminal vesicle weight compared to control. On this basis, we may conclude that alcohol negatively affects the macroscopic appearance of the seminal vesicle, with moderate intensification in the Alcoholic 35% group. The present results demonstrate that all animals gained weight along the experimental period, with weight being significantly lower in alcoholic animals only at 250 days compared to control. Furthermore, total caloric ingestion was numerically higher in alcoholic animals compared to control, with a mean ingestion of 16.0 kcal. Of this total, the rates corresponding to ethanol ingestion were lower than 50% in all experimental groups. According to the National Research Council (1978), mice show adequate growth rates by ingesting about 14.5 kcal of energy per day. Thus, the animals used in the present study ingested adequate amounts of energy for growth. Different investigators have reported that alcoholic animals show deficient gain weight, which is numerically lower compared to control (Ratcliffe, 1972; Cicero & Badger, 1977; Klassen & Persaud, 1978; Oliveira & Ferreira, 1987). In contrast, Anderson et al. (1989), in a study in which they administered increasing ethanol doses (3, 4 and 5%) to mice starting at 18 days
of age, did not detect a significant difference in final body weight between alcohol-treated and control animals. Willis et al. (1983) also did not detect differences in weight gain between mice chronically treated with 5% ethanol for 10 and 20 weeks and control animals. According to Lieber (1984), alcohol is an energy-rich drug and in many societies alcoholic beverages are considered to be part of the basic feeding supply. Large doses of alcoholic beverages have a profound effect on nutritional status both in humans and in laboratory animals, eventually causing primary and secondary malnutrition. Primary malnutrition is characterized by the ability of alcohol to shift high-energy nutrients away from the diet. Secondary malnutrition is characterized by difficulties in nutrient digestion and absorption due to the gastrointestinal problems associated with alcoholism. According to Campana et al. (1975), in rodents the state of protein malnutrition is characterized by the occurrence of behavioral disorders, hair loss or changes in hair distribution, diarrhea and edema, in addition to a marked weight loss. In the present study, these symptoms were not observed in alcohol-treated mice and therefore it may be stated that the animals were not in a condition of protein malnutrition. In addition, since all mice in the alcoholic groups presented weight gain and adequate energy ingestion for growth during the course of the experiment, we may conclude that alcohol acted on the organs of the male reproductive system independently of nutritional factors. We believe that the difference is literature data concerning animal weight gain are due to methodological differences. Light microscopy examination demonstrated a reduced cell volume in the animals treated with 25 and 35% ethanol along the experimental periods. However, there was no predominant reduction of any one cell compartment, a fact leading us to conclude that epithelial reduction occurred in the cell as a whole. We found no reports in the literature about the volumes of epithelial cells of the seminal vesicle in the
stereology and ultrastructure of the seminal vesicle 185
presence of chronic alcohol ingestion. In general, the present results are compatible with those reported by Martinez et al. (1997), who demonstrated a reduction in the height of secretory epithelial cells of the seminal vesicle of Wistar rats treated with sugar cane brandy at 30◦ Gay Lussac for 60, 120 and 180 days. Willis et al. (1983), in agreement with the present data, also reported a significant decrease in the height of epithelial cells of the seminal vesicle of mice treated with 5 and 6% ethanol for 21 days, with the severity of the effects on the tissues being proportional to the duration of treatment and to alcohol concentration. However, in the present study we did not observe a significant intensification of cytoplasmic volume atrophy along the experimental period, even with the addition of 10◦ Gay Lussac to the alcohol dose. Transmission electron microscopy observations in the different alcoholic groups showed cellular alterations indicating degenerative features. The basal cells showed marked ultrastructural changes with the occurrence of degenerated cells. The functions of the basal cells of the seminal vesicle epithelium are still unknown. However, some investigators have proposed that, under normal hormonal conditions, the basal cells play a role in the distribution and transport of substances to the secretory epithelial cells (Deane & Wurzelmann, 1965; Aumüller et al., 1981). In the present study, the morphologic lesions of the basal cells from alcoholic mice led us to infer that the distribution and transport of substances to the epithelial secretory cells were impaired. Even though the various organelles of secretory epithelial cells involved in the secretory process of the seminal vesicle showed signs of degeneration, vacuoles containing secretory granules as well as secretion into the glandular lumen were still detectable. According to Aumüller et al. (1981), the formation of autophagic vacuoles, the dilatation of cisterns of the Golgi complex and of the granular endoplasmic reticulum can be interpreted as signs of degradation of biological membranes. Furthermore, the present results are similar to those reported by Martinez et al. (1997) for rats with chronic alcoholism induced by the ingestion of sugar cane brandy at 30◦ Gay Lussac for 60, 120 and 180 days, which also showed reduction of vacuoles with secretion granules, increased dense bodies, and decreased number of microvilli. On the basis of the present results, we may conclude that, even though the cell organelles of the secretory epithelium of the seminal vesicle showed degenerative characteristics as a consequence of abusive alcohol ingestion, the secretory process was not totally interrupted. Chronic alcoholism induced important histologic and ultrastructural changes in the secretory epithelial cells of the seminal vesicle of mice along the experimental period and with the alcohol doses used. However, we cannot state that the severity of the changes is directly proportional to the time of exposure to alcohol or to alcohol dose.
ACKNOWLEDGEMENTS Supported by CAPES.
REFERENCES Anderson Jr., R.A., Willis, B.R., Phillips, J.F., Oswald, C. and Zaneveld, L.J.D. 1987. Delayed pubertal development of the male reproductive tract associated with chronic ethanol ingestion. Biochem. Pharmacol., 36 (13), 2157–2167. Anderson Jr., R.A., Phillips, J.F., Berryman, S.H. and Zaneveld, L.J.D. 1989. Ethanol-induced delayed male puberty in mice is not due to impaired Leydig cell function. Reprod. Toxicol., 3, 101–113. Aumüller, G., Giers, K., Giers, U., Völk, A. and Seitz, J. 1981. p-Chlorophenylalanine-induced proliferation of the seminal vesicle epithelium. Cell Tissue Res., 219, 159–172. Cagnon, V.H.A., Garcia, P.J., Martinez, M. and Padovani, C.R. 1996. Ultrastructural study of the coagulating gland of Wistar rats submitted to experimental chronic alcohol ingestion. Prostate, 28, 341–346. Campana, A.O., Burini, R.C., Outa, A.Y. and Camargo, J.L.V. 1975. Experimental protein deficiency in adult rats. Rev. Bras. de Pesquisas Méd. e Biol., 8 (3/4), 221–226. Cicero, T.J. and Badger, T.M. 1977. Effects of alcohol on the hypothalamic–pituitary–gonadal axis in the male rat. J. Pharmacol. Exp. Ther., 201 (2), 427–433. Deane, H.W. and Wurzelmann, S. 1965. Electron microscopic observations on the postnatal differentiation of the seminal vesicle epithelium of the laboratory mouse. Am. J. Anat., 117, 91–134. Edwards, G. and Peters, T.J. 1994. Alcohol and alcohol problems. Br. Med. Bull., 50 (1), 186–199. Galvão-Teles, A., Monteiro, E., Gavaler, J.S. and Van Thiel, D.H. 1986. Gonadal consequences of alcohol abuse: lessons from the liver. Special article by the American Association for the Study of Liver Diseases. Hepatology, 6 (1), 135–140. Garcia, P.J., Cagnon, V.H.A., Mello, W., Martinez, M. and Martinez, F.E. 1999. Ultrastructural observations of the dorsal lobe of the prostate of rats (Rattus novergicus) submitted to experimental chronic alcohol ingestion. Rev. Chil. Anat., 17 (2), 153–159. Gary, G.G., Actman, K. and Southrgn, L. 1976. Effect of alcohol (ethanol) administration on sex hormone metabolism in normal men. N. Engl. J. Med., 295, 793–797. Harkin, J.C. 1963. Prostatic ultrastructure. Natl. Cancer Inst. Monogr., 12, 85–97. Karnovsky, J.M. 1965. A formaldehyde–glutaraldehyde fixative in high osmolality for use in electron microscopy. J. Cell Biol., 27, 137–138. Klassen, R.W. and Persaud, T.V.N. 1978. Influence of alcohol on the reproductive system of the male rat. Int. J. Fertil., 23 (3), 176–184. Lieber, C.S. 1984. Alcohol and the liver. Hepatology, 4, 1243–1260. Marks, V. and Wright, J. 1978. Metabolic effects of alcohol. Clin. Endocrinol. Metab., 7, 245–466. Martinez, F.E., Garcia, P.J., Padovani, C.R., Cagnon, V.H.A. and Martinez, M. 1997. A morphometric ultrastructural study of the seminal vesicle of rats submitted to experimental chronic alcoholism. J. Submicrosc. Cytol. Pathol., 29 (4), 537–542. Montgomery, D.C. 1991. Design and Analysis of Experiments, 3rd edn, Wiley, New York, p 649. National Research Council, 1978. Subcommittee on Laboratory Animal Nutrition. Nutrient Requirements of Laboratory Animals. 10, 3rd edn, National Academy of Sciences, Washington, DC, p 96. Oliveira, C. and Ferreira, A.L. 1987. Effect of alcohol on the rat justa prostatic pelvic ganglia and gonads. Genenbaurus Morphol. Jahrb., 133, 771–780. Ratcliffe, F. 1972. The effect of chronic ethanol administration on the growth of rats. Arch. Int. Pharmacodyn., 197, 19. Rodgers, D.A. 1966. Factors underlying differences in alcohol preference among inbred strains of mice. Psychosomat. Med., 28, 498–513. Reynolds, E.S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol., 17, 208. Salonen, I. and Huhtaniemi, I. 1990. Effects of chronic ethanol diet on pituitary–testicular function of the rat. Biol. Reprod., 42, 55–62. Semczuk, M. and Rzeszowska, G. 1981. Chronic alcohol intoxication and the morphological state of seminal vesicles and prostate gland in a white rat. Materia Medica Polona, 2, 130–135. Sprando, R.L. 1990. Perfusion of rat testis through the hearth using heparin. In: Russel, L.D., Ettilin, A.P.S. and Cleeg, E.D. (eds) Histological and Histopathological Evaluation of the Testis. Cache River Press, Clearwater, pp 277–280. Van Thiel, D.H. and Lester, R. 1979. The effect of chronic alcohol abuse on sexual function. Clin. Endocrinol. Metab., 8 (3), 499–510.
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Tissue&Cell
Stereology and ultrastructure of the seminal vesicle of C57/BL/6J mice following chronic alcohol ingestion I. C. Gomes, 1 V. H. A. Cagnon, 1 C. A. F. Carvalho, 1 I. M. S. De Luca 2
Abstract. Excessive alcohol consumption causes metabolic changes and pathologic alterations in testes and accessory sex organ in different animal species. The aim of the present study was to evaluate the macroscopic, histologic and ultrastructural alterations provoked by chronic ingestion of different ethanol concentrations over increasing periods of time on the secretory epithelium of the seminal vesicle of C57/BL/6J mice in using stereological methods. Sixty male adult mice were divided into three experimental groups: Control, Alcoholic 25% and Alcoholic 35%, respectively, receiving tap water and tap water containing ethanol diluted to 25 and 35 ◦ Gay Lussac. All mice were fed with the same solid diet. After 150 and 250 days of treatment the animals were sacrificed and the seminal vesicles were collected and processed for light and transmission electron microscopy. The cellular, cytoplasmic and nuclear volumes and the area density of autophagic and secretory vacuoles were measured. The histologic alterations observed in the alcoholic mice consisted of a reduction in epithelial size and cell volume, with maintenance of the same nuclear and cytoplasmic ratio as verified in the control groups. The ultrastructural alterations were: increased density of dense body area, decreased density of secretory granule area, and dilated rough endoplasmic reticulum and Golgi cisternae. We conclude that chronic ethanol ingestion causes depleting morphologic alterations in the epithelial cells of the seminal vesicle and negatively affects the secretory process of this gland. © 2002 Elsevier Science Ltd. All rights reserved.
Keywords: seminal vesicle, alcohol, ultrastructure, morphology
Introduction Alcohol and its metabolites provoke generalized disorders in different organ systems such as the central nervous sys1 Department of Anatomy, Institute of Biology, State University of Campinas—UNICAMP, Campinas, São Paulo, Brazil, 2 Department of Histology, Institute of Biology, State University of Campinas—UNICAMP, Campinas, São Paulo, Brazil
Received 2 November 2001 Revised 12 April 2002 Accepted 22 April 2002 Correspondence to: Dr Valeria H. A. Cagnon, Departamento de Anatomia, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP) 13084-971, Campinas, S ão Paulo, Brazil. Tel.: +55 19 3788 7391; Fax: +55 19 3289 3124; E-mail: [email protected]
tem, the male reproductive system, and the circulatory and hematopoietic systems. They also act directly on the liver, pancreas, intestine, endocrine glands, and accessory sex glands (Marks & Wright, 1978; Galvão-Teles et al., 1986; Edwards & Peters, 1994). In humans, chronic alcoholism has been associated with hypogonadism, feminization, sexual impotence and changes in reproductive hormonal homeostasis (Anderson et al., 1987). However, risk factors such as duration and quantity of consumed alcohol, age of consumption and period of development have not been evaluated leading to gaps on information. Thus, controlled experimental studies have been important for evaluation of the results associated with impairment in male reproductive system due to excessive consumption of alcohol (Anderson et al., 1987). 177
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Experimental and clinical studies indicate that continued ingestion of ethanol contributes, through several mechanisms, such as testicular luteinizing hormone receptors depletion and gonadotropin responsiveness, besides pituitary and hypothalamic defects to disturbances in the hypothalamic– pituitary–gonadal axis, which provoke damage in sex organ genesis and in the normal secretory activity of the male reproductive tract (Harkin, 1963; Cicero & Badger, 1977; Van Thiel & Lester, 1979; Salonen & Huhtaniemi, 1990). In addition, alcohol acts directly on the male gonads by altering testicular testosterone synthesis (Gary et al., 1976; Van Thiel & Lester, 1979). In a study on rats chronically treated with alcohol, Klassen and Persaud (1978) reported reduced folding of the glandular mucosa and involution of secretory epithelial cells. Later, Semczuk and Rzeszowska (1981) also observed a significant reduction in seminal vesicle weight, atrophy of the secretory epithelium, and partial cell degeneration in rats treated with ethanol for 204 days compared to control. Martinez et al. (1997), after treating adult Wistar rats with sugar cane brandy at 30◦ Gay Lussac for 60, 120 or 180 days, observed marked morphologic changes in the secretory epithelium of the seminal vesicle, among them a significant reduction of epithelial cell height and of number of secretory vacuoles, the presence of nuclei of irregular contours, and a reduction of the amount of microvilli. A review of the literature showed that doubts persist about the effect of ethanol on the secretory epithelium of the seminal vesicle of mice, especially with respect to epithelial reduction and to the contributions of the nucleus and cytoplasm to this process, in addition to the behavior of these structures in terms of time of exposure and alcoholic dosage. Thus, the major objective of the present study was to analyze statistically data concerning seminal vesicle weight and cell, cytoplasmic and nuclear volumes and to study stereologically the density of areas of autophagic and secretory vacuoles in order to obtain a general view of the structural and ultrastructural alterations provoked by chronic alcohol ingestion and its consequences for the secretory process of this gland.
Material and methods Animals and tissue preparation A total of 60 adult male mice (C57/BL/6J), high alcohol preference, (Rodgers, 1966) of the same age were divided into three 20 mouse groups (Control, Alcoholic 25% and Alcoholic 35%) and assigned to two treatment times (150 and 250 days). The Control group received tap water, the experimental group Alcoholic 25% consumed only ethanol diluted at 25◦ Gay Lussac (25 ml ethanol/100 ml solution), and the experimental group Alcoholic 35% consumed only ethanol diluted at 35◦ Gay Lussac (35 ml ethanol/100 ml solution) for 150 or 250 days. All groups were fed solid Nuvilab CR chow ad libitum. Liquid and solid consumption was measured daily and the data obtained were used to calculate mean calorie ingestion by the experimental groups. At the
end of each period of treatment, mice from each group were anesthetized with Francotar/Virbaxyl (1:1) and weighed. The seminal vesicles were collected from five animals per group, weighed, fixed in Bouin’s solution, embedded in Paraplast Plus resin, cut into 5 m thick sections and stained with hematoxylin and eosin. Another five mice per group were perfused through the left ventricle with Karnovsky’s solution (Karnovsky, 1965; Sprando, 1990). Then the seminal vesicles were removed and immersed for 2 h in the same fixative followed by 1 h in cacodylate buffered 1% osmium tetroxide. The tissue was dehydrated using a graded series of ethanol and then embedded in Araldite. Ultrathin sections were obtained with an LKB ultramicrotome and stained with uranil acetate (Watson, 1958) and lead citrate (Reynolds, 1963) and examined and photographed with a LEO 906 electron microscope. Morphometric procedures The cellular, cytoplasmic and nuclear volumes were measured on the sections stained for light microscopy. Data for nuclear volume were recorded as the average obtained in 75 measurements per animal. Long and short axes were measured and the mean nuclear volume was calculated considering nuclei as ellipsoids. The sections were studied with an ocular micrometer coupled to a Zeiss microscope with a ×100 objective. For the determination of cytoplasm volume, an ocular with a 400 grid coupled to a ×100 objective was used. Points on the cytoplasm and nuclei were counted in 15 areas per group and the cytoplasmic and nuclear fractions were obtained. These data and the nuclear volumes were used to estimate the cytoplasm volume for each animal. The cellular volume was calculated by summing the nuclear and cytoplasm volumes. For the determination of density of autophagic and secretory vacuole areas we used current stereology methods and formulae as described by Weibel (Weibel, 1979). A multipurpose test system with 84 lines and 168 points was applied to 15 electronmicrographs (×10 000) per group and the numerical density of the area was estimated. Statistical analysis Mean data for total ingested calories, final body weight minus initial seminal vesicle weight and cell volumes were analyzed statistically by 2 × 3 analysis of variance (ANOVA) for factorial experiments in fully randomized designs, followed by the Tukey multiple range test for comparisons between group means at each time point (Montgomery, 1991).
Results Macroscopic observations Seminal vesicle weight After the treatment with ethanol the seminal vesicle weight fell from the control level of 0.23 to 0.18 mg (Alcoholic 25%, P < 0.05) and 0.11 mg (Alcoholic 35%, P < 0.05) after
stereology and ultrastructure of the seminal vesicle 179
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Fig. 1 Photomicrographs of the secretory epithelium of the seminal vesicle of control and alcoholic rats after 250 days of treatment. (A) Acini with folded mucosa. Epithelial cells (EC) of the tall columnar type. Light zones corresponding to the Golgi complex area can be seen above the nucleus (*). Nucleus (N) located in the basal region. Basal cells (bc) are intermingled with epithelial cells, presenting a clearly visible nucleolus. Lumen (L) with secretion of homogeneous aspect. Stroma (S). ×1300. (B) Secretory epithelium of a 25% alcoholic animal. Mucosa with few folds. Atrophied epithelial cells (EC). Absence of light zones above the nuclei. Centralized nuclei (N). Basal cells (bc) also atrophied. Lumen (L) with heterogeneous secretion. Stroma with different cell types (S). ×1300. (C) Secretory epithelium of an Alcoholic 35% animal. Mucosa with few folds. Markedly atrophied epithelial cells (EC). Absence of light zones above the nuclei. Centralized nuclei (N). Atrophied basal cells (bc). Lumen (L) with heterogeneous secretion. Stroma with different cell types (S). ×1300.
150 days and from 0.32 mg (Control) to 0.21 mg (Alcoholic 25 and 35%, P < 0.05) after 250 days. Caloric analysis Mean total caloric consumption at 150 days of treatment was numerically lower in control animals compared to the Alcoholic 25 and 35% groups, although the difference was not statistically significant. A similar result was observed at 250 days (Table 1). In the Alcoholic 25% group, liquid consumption was 41 and 35% of the total number of calories ingested at 150 and 250 days of treatment, respectively, while in the Alcoholic 35% group liquid consumption was 43 and 45% of the total number of calories ingested at 150 and 250 days, respectively. Body weight Body weight gain was lower in the Alcoholic 25 and 35% animals at the two treatment times compared to control. However, minimum significant differences at 250 days of treatment were observed only for the two experimental groups compared to control (Table 1). Light microscopy In the control groups, the glandular epithelium of the seminal vesicle had a tubular arrangement covered with smooth muscle and containing connective tissue resting on the basement membrane. The secretion contained in the tubular lumen had a homogeneous colloidal appearance. The epithelium consisted of basal cells sandwiched between the basis of the epithelial cells (Fig. 1A). The cytoplasm showed a light supranuclear region probably corresponding to the Table 1 Minimum significant difference (MSD) determined by the Tukey test and coefficient of variation (CV) for the mean number of total calories (MC, kcal) ingested daily and for mean body weight increase (WI, g) in the 25% (A25) and 35% (A35) alcoholic groups and controls (C) for each treatment period
MC
WI
Group variables
Period of treatment (days) 150
250
MSD (5%), CV (%)
C A25 A35
15.36a ± 1.74 16.14a,b ± 0.89 17.54b ± 1.08
13.92a ± 1.24 15.70b ± 1.41 14.99a,b ± 1.67
1.46 8.74
C A25 A35
9.22a ± 1.24 9.00a ± 1.81 7.57a ± 1.90
13.93b ± 4,39 10.44a ± 1.76 9.90a ± 4.00
3.12 29.26
a,b: Means followed by the same letter did not differ according to the Tukey multiple range text.
Table 2 Mean cell volume (m3 ) in the Control (C) and 25% (A25%) and 35% (A35%) alcoholic groups for each period of treatment
Cell volume
Group variables
Period of treatment (days) 150
250
C A25% A35%
187.00a∗
180.00b A ± 18.00 175.00a A ± 26.00 177.00a A ± 23.00
A∗∗ ± 46.00 155.00b∗ A ± 30.00 162.00a,b∗ A ± 15.00
MSD (groups) significant mean difference = 44.00. MSD (period) significant mean difference/period = 36.00. CV, coefficient of variation = 18.54%. ∗ According to the Tukey multiple range test, means followed by the same lower case letter did not differ within the group, fixed the time. ∗∗ Means followed by the same capital did not differ within time, fixed the group.
Golgi complex and the secretory vacuoles (Fig. 1A). The nuclei of the epithelial cells rested on the basal third of the cell. They were ovoid and showed one or two nucleoli (Fig. 1A). The data for the control groups of the two experimental times were pooled because cell morphology and volume were similar (Table 2). In the alcoholic groups (25 and 35%), the characteristics of the stroma were similar to those of the control groups (Fig. 1A–C). Intraluminal secretion showed a homogeneous colloidal aspect similar to that of control animals (Fig. 1B & C). The secretory epithelium was pseudostratified, with proven epithelial cell involution compared to control animals (Table 2), (Fig. 1B & C), both at 150 and 250 days of treatment (Table 2). In the alcoholic groups, cell volume was similar along the experimental period. The mean percent fractions occupied both by nuclei and by cytoplasm were similar in the alcoholic groups and in the controls (Table 3). The alcoholic groups 25 and 35% were described together at the two experimental times because they presented similar morphologic characteristics. Table 3 Mean percent value of the cell fraction represented by the nucleus and the cytoplasm in seminal vesicle epithelial cells from the control (C), Alcoholic 25% (A25%) and Alcoholic 35% (A35%) groups during each treatment period Organelles
Group variables
Period of treatment (days) 150 (%)
250 (%)
Nucleus
C A25% A35%
23.84 22.86 22.28
19.96 20.98 20.36
Cytoplasm
C A25% A35%
76.10 77.14 77.72
80.04 79.02 79.64
stereology and ultrastructure of the seminal vesicle 181
Fig. 2 Electronmicrographs of the secretory epithelium of the seminal vesicles of control animals at 150 and 250 days of treatment. (A) Tall columnar epithelial cells. Basal nucleus (N) and clearly visible nucleolus (Nu). Mitochondria (M) dispersed throughout the cytoplasm. GER localized in the perinuclear and basal regions. Golgi complex (*) and vacuoles containing an eccentric electrondense granule are visible in the supranuclear region. Clearly visible microvilli (MV) and junctional complex. Basal lamina (arrow). Lumen (L). ×5000. (B) Detail of the apical region at 150 days of treatment. Microvilli (MV) on the cell surface facing the lumen. Junctional complex (circle). Secretory vacuoles containing an eccentric electrondense granule (夹). ×15 000. (C) Detail of the basal region at 250 days of treatment. Basal lamina (arrow). Extracellular matrix with a nerve bundle (lozenge) and collagen and smooth muscle (SM) fibers. In the cytoplasm, GER and nucleus (N) of the epithelial cell. ×7500.
Transmission electron microscopy In control mice, the basal cells presented only traces of the cellular endoplasmic reticulum and no signs of the Golgi complex (Fig. 4A). The ultrastructure of the epithelial cells showed a granular endoplasmic reticulum with flat and parallel cisternae (Fig. 2A–C). The Golgi complex was prominent (Fig. 2A). There were large and small membrane-bound vacuoles, each containing a dense secretion granule (Fig. 2B). The area density of these secretory vacuoles was similar in both control groups (Fig. 5). Eventual autophagic vacuoles were seen in the cytoplasm (Fig. 2C) with a far lower area density than that of the secretory vacuoles, corresponding to 0.04 m2 /m3 at 150 days and 0.020 m2 /m3 at 250 days (Fig. 5). The mitochondria showed no structural specialization and were scattered throughout the cytoplasm. The nucleus was elongated and usually showed one nucleolus (Fig. 2A). The heterochromatin was located at the nuclear periphery and as irregular
clumps in the nucleoplasm. Microvilli of varying length and numbers were present on the apical surface (Fig. 2A & B). In alcoholic mice, the epithelial and basal cells showed signs of degeneration at the different times of treatment and at the different alcohol doses, although these changes did not intensify along time of treatment or with the doses used. Basal cells showed different stages of degeneration (Fig. 4B–D) with rounded nuclei and points of chromatin condensation scattered throughout the nucleus (Fig. 4B & C). In the secretory epithelial cells, the granular endoplasmic reticulum and the Golgi complex showed dilated cisterns (Fig. 3C–E). An increase in area density of the autophagic vacuoles was observed, as opposed to a decrease in the area density of the secretory vacuoles, with an eccentric core, in agreement with the characteristics observed in the control groups (Fig. 5). Nevertheless, secretion granules were still observed in the glandular lumen. The mitochondria were similar to those
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Fig. 3 Electronmicrographs of the secretory epithelial cells of the seminal vesicle of alcoholic mice 25 and 35% at 150 and 250 days of treatment. (A) Tall and basal columnar cells of the Alcoholic 25% group at 250 days of treatment. Vacuoles (夹) containing little or no secretion. Sparse microvilli (MV) covering the cell surface. Nucleus (N) with envelope dents. Clearly visible nucleolus (Nu). Basal cell with the nucleus presenting envelope dents (bn). Lumen (L) with granules secretion. ×5000. Inset: detail of a multivesicular body containing cell remnants and secretory vacuoles. ×15 000. (B) Low columnar epithelial cell from an animal of the Alcoholic 35% group at 250 days of treatment. The cytoplasm shows secretory vacuoles (夹) with an eccentric granule. Nucleus (N) with dents and condensed chromatin at the periphery. Nucleolus (Nu). Basal cell with a lipid droplet (Li). Degenerated nucleus of the basal cell (bn). Basal lamina (arrow). Glandular lumen (L) containing granules secretion. ×5000. (C) Detail of the apical region of the Alcoholic 25% group at 150 days of treatment. Mitochondria (M). Golgi complex (*). Accumulation of autophagic vacuoles resembling figures of the ‘myelinic’ type (hatched area). Sparse microvilli (MV) cover the cell surface. The junctional complex (circle) between adjacent cells is clearly visible. Nucleus (N). ×7500. (D) Detail of a nucleus of the Alcoholic 35% group at 150 days of treatment. Degenerative process with large electrondense inclusions. 30 000. (E) Detail of the basal region of the Alcoholic 25% group at 150 days of treatment. Dilated cisterns GER. Basal lamina clearly visible in the extracellular layer (arrow). Collagen fibers cut transversely and longitudinally and smooth muscle fibers (SM). Observe the nucleus of the basal cell (bn). ×7500.
stereology and ultrastructure of the seminal vesicle 183
Fig. 4 Electronmicrographs of the secretory epithelial cells of the seminal vesicle at 150 and 250 days of treatment. (A) Basal cell of the control group at 150 days of treatment. Small and compact mitochondria (M). Pleomorphic nucleus with envelope dents (bn). Basal lamina (arrow). ×7500. (B) Basal cell of the Alcoholic 25% group at 250 days of treatment. Mitochondria (M). Condensed chromatin throughout the nucleus (bn). Basal lamina (arrow). ×7500. (C) Basal cell from the Alcoholic 25% group at 250 days of treatment. Nucleus (bn) with spiraling chromatin intermingled with condensation points (arrowhead). Basal lamina (arrow). ×7500. (D) Basal cell from the Alcoholic 35% group at 250 days of treatment. Lipid droplets (Li). Degenerated nucleus (bn). Folded basal lamina (arrow). ×7500.
described for the control groups but seemed to be more concentrated in the supranuclear region (Fig. 3A–C). The nuclear shape was irregular compared to control with increasing alcohol concentration and time of ingestion (Fig. 3A & B). The basement membrane showed deep infoldings and the microvilli were small and scattered (Fig. 3B & C). The area density of autophagic vacuoles was numerically higher at 250 days of alcoholism with 35% ethanol (Fig. 4).
Discussion and conclusion The sequels of chronic ethanol ingestion for the accessory sex glands of rodent have been documented by both light microscopy and transmission electron microscopy, being mainly represented by regressive changes in epithelial cell height after different periods of alcohol ingestion (Semczuk & Rzeszowska, 1981; Anderson et al., 1989; Cagnon et al.,
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Fig. 5 Density (m2 /m3 ) of the area occupied by secretory and autophagic vacuoles in the cytoplasm of seminal vesicle epithelial cells in the different experimental groups and during the various treatment periods.
1996; Garcia et al., 1999). In the present study, we demonstrated macroscopic atrophy of the seminal vesicle of 21.74% (Alcoholic 25%) and 52.18% (Alcoholic 35%) at 150 days of treatment and of 34.37% for both alcoholic groups at 250 days of treatment. These observations agree with data reported by Klassen and Persaud (1978), who observed a significant decrease in seminal vesicle weight in Sprague–Dawley rats receiving 10% ethanol for 5 weeks. Similarly, Semczuk and Rzeszowska (1981) reported that the weights of the seminal vesicles of Wistar rats receiving 40% ethanol at the dose of 3 g/kg body weight were significantly decreased compared to control after 204 days of treatment. Anderson et al. (1987), after treating C57/BL/6J mice with 5% ethanol for 43 days, observed a significant decrease in seminal vesicle weight compared to control. On this basis, we may conclude that alcohol negatively affects the macroscopic appearance of the seminal vesicle, with moderate intensification in the Alcoholic 35% group. The present results demonstrate that all animals gained weight along the experimental period, with weight being significantly lower in alcoholic animals only at 250 days compared to control. Furthermore, total caloric ingestion was numerically higher in alcoholic animals compared to control, with a mean ingestion of 16.0 kcal. Of this total, the rates corresponding to ethanol ingestion were lower than 50% in all experimental groups. According to the National Research Council (1978), mice show adequate growth rates by ingesting about 14.5 kcal of energy per day. Thus, the animals used in the present study ingested adequate amounts of energy for growth. Different investigators have reported that alcoholic animals show deficient gain weight, which is numerically lower compared to control (Ratcliffe, 1972; Cicero & Badger, 1977; Klassen & Persaud, 1978; Oliveira & Ferreira, 1987). In contrast, Anderson et al. (1989), in a study in which they administered increasing ethanol doses (3, 4 and 5%) to mice starting at 18 days
of age, did not detect a significant difference in final body weight between alcohol-treated and control animals. Willis et al. (1983) also did not detect differences in weight gain between mice chronically treated with 5% ethanol for 10 and 20 weeks and control animals. According to Lieber (1984), alcohol is an energy-rich drug and in many societies alcoholic beverages are considered to be part of the basic feeding supply. Large doses of alcoholic beverages have a profound effect on nutritional status both in humans and in laboratory animals, eventually causing primary and secondary malnutrition. Primary malnutrition is characterized by the ability of alcohol to shift high-energy nutrients away from the diet. Secondary malnutrition is characterized by difficulties in nutrient digestion and absorption due to the gastrointestinal problems associated with alcoholism. According to Campana et al. (1975), in rodents the state of protein malnutrition is characterized by the occurrence of behavioral disorders, hair loss or changes in hair distribution, diarrhea and edema, in addition to a marked weight loss. In the present study, these symptoms were not observed in alcohol-treated mice and therefore it may be stated that the animals were not in a condition of protein malnutrition. In addition, since all mice in the alcoholic groups presented weight gain and adequate energy ingestion for growth during the course of the experiment, we may conclude that alcohol acted on the organs of the male reproductive system independently of nutritional factors. We believe that the difference is literature data concerning animal weight gain are due to methodological differences. Light microscopy examination demonstrated a reduced cell volume in the animals treated with 25 and 35% ethanol along the experimental periods. However, there was no predominant reduction of any one cell compartment, a fact leading us to conclude that epithelial reduction occurred in the cell as a whole. We found no reports in the literature about the volumes of epithelial cells of the seminal vesicle in the
stereology and ultrastructure of the seminal vesicle 185
presence of chronic alcohol ingestion. In general, the present results are compatible with those reported by Martinez et al. (1997), who demonstrated a reduction in the height of secretory epithelial cells of the seminal vesicle of Wistar rats treated with sugar cane brandy at 30◦ Gay Lussac for 60, 120 and 180 days. Willis et al. (1983), in agreement with the present data, also reported a significant decrease in the height of epithelial cells of the seminal vesicle of mice treated with 5 and 6% ethanol for 21 days, with the severity of the effects on the tissues being proportional to the duration of treatment and to alcohol concentration. However, in the present study we did not observe a significant intensification of cytoplasmic volume atrophy along the experimental period, even with the addition of 10◦ Gay Lussac to the alcohol dose. Transmission electron microscopy observations in the different alcoholic groups showed cellular alterations indicating degenerative features. The basal cells showed marked ultrastructural changes with the occurrence of degenerated cells. The functions of the basal cells of the seminal vesicle epithelium are still unknown. However, some investigators have proposed that, under normal hormonal conditions, the basal cells play a role in the distribution and transport of substances to the secretory epithelial cells (Deane & Wurzelmann, 1965; Aumüller et al., 1981). In the present study, the morphologic lesions of the basal cells from alcoholic mice led us to infer that the distribution and transport of substances to the epithelial secretory cells were impaired. Even though the various organelles of secretory epithelial cells involved in the secretory process of the seminal vesicle showed signs of degeneration, vacuoles containing secretory granules as well as secretion into the glandular lumen were still detectable. According to Aumüller et al. (1981), the formation of autophagic vacuoles, the dilatation of cisterns of the Golgi complex and of the granular endoplasmic reticulum can be interpreted as signs of degradation of biological membranes. Furthermore, the present results are similar to those reported by Martinez et al. (1997) for rats with chronic alcoholism induced by the ingestion of sugar cane brandy at 30◦ Gay Lussac for 60, 120 and 180 days, which also showed reduction of vacuoles with secretion granules, increased dense bodies, and decreased number of microvilli. On the basis of the present results, we may conclude that, even though the cell organelles of the secretory epithelium of the seminal vesicle showed degenerative characteristics as a consequence of abusive alcohol ingestion, the secretory process was not totally interrupted. Chronic alcoholism induced important histologic and ultrastructural changes in the secretory epithelial cells of the seminal vesicle of mice along the experimental period and with the alcohol doses used. However, we cannot state that the severity of the changes is directly proportional to the time of exposure to alcohol or to alcohol dose.
ACKNOWLEDGEMENTS Supported by CAPES.
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