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Effect of bisphenol A on the cauda epididymis of adult male albino rats and the possible protective role of quercetin: a histological and immunohistochemical study

Mazroa, Shireen A.

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The Egyptian Journal of Histology: June 2011 - Volume 34 - Issue 2 - p 377-390
doi: 10.1097/01.EHX.0000397468.63291.0c
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Abstract

Introduction

The epididymis is a key structure in the male reproductive system. It is a highly coiled, tubular structure divided into initial segment, head (caput), body (corpus), and tail (cauda) [1]. The cauda epididymis is the terminal part of the epididymis where spermatozoa complete their maturation processes and acquire the potential ability to fertilize oocytes. In addition, it is a site for storage of mature sperms till the time of ejaculation [2]. The epididymis plays a major role in protecting spermatozoa from oxidative stress by secreting a variety of antioxidant enzymes, such as glutathione reductase (GR) into the epididymal lumen [3]. The integrity of the epididymis is essential to maintain the proper microenvironment for viability of the sperms and male fertility [4].

Different chemicals released into the environment display their effects on the male reproductive system [5]. Bisphenol A (BPA) is a small organic compound (228 Da) used primarily to manufacture polycarbonate plastics and epoxy resins incorporated in the industrial processing of water pipes, toys, some dental sealants as well as lining of metal cans and containers of food and beverage [6]. BPA is one of the highest volume of chemicals produced worldwide, and over 100 tons are released into the environment yearly [7]. Many concerns have been raised about the effects of BPA. A controversy about the safety of BPA has resulted in abundant scientific studies [8]. Some studies [9,10] have failed to find any reproductive consequences associated with BPA. However, other studies confirmed the deleterious effect of BPA on different organs of the male reproductive system, such as prostate [11] and testis [12].

In recent years, dietary intervention, particularly of dietary antioxidants, has been proposed as a potential way to improve male reproduction. Flavonoid compounds of plant origin have received increasing attention due to their potential to prevent a number of chronic and degenerative diseases [13]. Flavonoids are found in significant quantities in vegetables, fruits such as grapes, and beverages such as tea. Quercetin is a potent antioxidant and an important member of dietary flavonoid family [14]. It is effective in attenuation and prevention of various disorders induced by environmental contaminants [15].

Studies concerned with the effect of BPA on the histological structure of epididymis and the expression of epididymal antioxidant enzymes such as GR are deficient. Therefore, the aim of this study was to investigate the effect of BPA on histological structure of caudal part of epididymis and the immunohistochemical (IHC) expression of GR enzyme in adult male albino rats. The possible protective role of quercetin supplementation was also evaluated.

Materials and methods

All procedures in this study were performed in accordance with the Medical Research Ethics Committee of Mansoura University (Egypt). A total of 50 adult (120 days old, 200–250 g) male albino rats were housed at 22–25°C (12 h light/dark cycle). The animals had access to normal rat chow and water supplied from glass bottles ad libitum. Rats were randomized into four groups of control and experimental animals. Group 1 (n=20) included control rats that were equally subdivided into subgroup a, including negative control rats not receiving any substance, and subgroup b, including rats subcutaneously injected with 90 mg/kg body weight/day of the solvent media of quercetin (20% glycerol in 0.9% normal saline) for 8 weeks [16]. Group 2 (n=10) included rats that received BPA powder (239658, Sigma, St Louis, Missouri, USA) dissolved in tap water as a daily oral dose of 0.1 mg/kg body weight/day by a gastric tube for 8 weeks [17]. Group 3 (n=10) included rats that were subcutaneously injected with a daily dose of quercetin (90 mg/kg body weight/day) for the same duration. Quercetin powder (Q4951 Sigma) was dissolved and diluted with 20% glycerol in 0.9% normal saline and stored in a dark bottle at 4°C. The quercetin solution was freshly prepared each week [16]. Group 4 (n=10) included rats receiving similar doses of BPA and quercetin for the same duration. After 8 weeks of the experiment, rats were anesthetized by an intraperitoneal injection of pentobarbital (50 mg/kg). Blood was collected from the aorta for hormonal assay. Rats were perfused with 2% glutaraldehyde and 2% paraformaldehyde in phosphate buffer (pH 7.4) (0.1 mol/l) by the abdominal aorta for 10 min for fixation of the tissues [18]. In each rat, right and left epididymis were dissected (Fig. 1a). The caudal end of the right epididymis was used for structural and IHC studies, whereas the caudal end of left epididymis was used for ultrastructural study and examination of sperm concentration and morphology by phase-contrast microscopy. Kidney was also obtained from control rats for preparation of positive and negative control slides for the IHC study [19].

Figure 1
Figure 1:
(a) Initial segment (i), caput (ca), corpus (cor), and cauda (cau) of epididymis with attached vas deferens (v). (b) Phase-contrast micrographs of (i) normal sperm formed of hook-shaped head (h), midpiece (mp), principal piece (pp), and end piece (e) (×400). (ii) Detached head (dh) (×1000). (iii) Short tail (st) (×400). (iv) Cytoplasmic droplets (cd) (×400). (v) Two fused sperms (fs) (×400).

Histological Study

Structural study

Specimens were postfixed in 10% neutral-buffered formalin overnight at a temperature of 4°C. Tissue samples were dehydrated in alcohols, cleared in xylol, and embedded in paraplast. Tissue sections (5-μm thickness) were stained with hematoxylin and eosin stain and Masson trichrome stain [20] for light microscopic examination and for morphometric analysis of epithelial height.

Morphometric analysis of the epithelial height

Approximately 20 cross sections of each cauda epididymis were examined for epithelial height using a calibrated eye-piece micrometer. The height was measured from the basal lamina to the top of the cell. Stereocilia were not included [21].

Immunohistochemical technique for localization of glutathione reductase (GR) enzyme

Sections of 4-μm thickness were used for the IHC localization of the antioxidant GR enzyme. Sections from the kidney of control rats were used as positive control.

Kits used: Ready-to-use target retrieval solution (S1700, DakoCytomation, Denmark), primary polyclonal antibody to GR specific for rat (ab16801, Abcam Inc., Cambridge), and ready-to-use antibody diluent with background-reducing components (S3022, Dakocytomation, Denmark). Universal detection kits (K 0673, DakoCytomation) were based on a modified avidin–biotin technique in which a biotinylated secondary antibody forms a complex with peroxidase-conjugated streptavidin molecule [22].

Glutathione reductase immune-staining: Sections were dewaxed in xylol and hydrated in descending grades of alcohol down to distilled water. They were immersed into a preheated target retrieval solution at 95–99°C (without boiling) in a water bath for 40 min and then allowed to cool for 20 min at room temperature. Sections were rinsed thrice with phosphate-buffered saline (PBS). Excess liquid was tapped off the slides. Enough hydrogen peroxide was applied to cover the specimen for 5 min, then the slides were rinsed gently with PBS, and excess liquid was tapped off. Primary antibody (concentration of 2 μg/ml, according to the instruction of manufacturing company) was applied on specimens, and they were incubated for 2 h in a humidity chamber at room temperature. Slides were rinsed in PBS. Biotinylated link was applied on specimens for 10 min and sections were rinsed in PBS. Streptavidin horseradish peroxidase reagent was applied on specimens for 10 min and sections were then rinsed in PBS. Freshly prepared 3,3-diaminobenzidine tetrahydrochloride substrate chromogen solution was removed from 2–8°C storage and applied on specimens for 10 min. Slides were rinsed gently in distilled water, immersed in hematoxylin for 1/2 min, and then rinsed in tap water until blue. Slides were dehydrated in ascending grades of alcohol, cleared in xylol, mounted by Canada balsam, and covered with a cover slip. Negative control slides from both epididymis and kidney were prepared by the same steps, except that they were incubated with the antibody diluent instead of primary antibody. Positive control slides were prepared from the kidney to demonstrate the expression of the enzyme in the epithelium of the medullary renal tubules [19]. Positive reaction appeared brown in color [22].

Ultrastructural study

A part of the caudal end of the left epididymis was sliced into small pieces (1 mm3) and fixed in a mixture of 2.5% gluteraldehyde and 2.5% paraformaldehyde (pH:7.3) overnight at 4°C. Specimens were postfixed in 1% osmium tetroxide. The specimens were dehydrated in ascending grades of alcohol, passed through two changes of propylene oxide, and then embedded in epon. Semithin sections (1 μm thickness) stained with 1% aqueous toluidine blue were examined with the light microscope. Ultrathin sections (60 nm thick) were cut, mounted on copper grids, and stained with uranyl acetate and lead citrate [23]. The grids were then examined with the transmission electron microscope (TEM, SEO, Russia) at the Military Veterinary Medicine Hospital, Cairo, Egypt.

Epididymal sperm concentration and morphology

The remaining part of the left cauda epididymis was gently squeezed to discharge the epididymal sperm fluid onto a slide, and then aspirated using the leukocyte pipette of the hemocytometer to calculate its volume. The viscid epididymal sperm fluid was allowed to liquefy with 0.5 ml of washing media (FP10FL06, FertiPro NV, Beernem, Belgium) formed of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid -buffered Earle's balanced salt solution and 0.4% human serum albumin. The sperm concentration (million/milliliter) was assessed by a computer-assisted microscopic system (Auto Sperm, FertiPro NV). For sperm morphology, a drop (10 μl) of diluted sperm sample was placed on a glass slide, cover-slipped, and examined with a phase-contrast microscope (Olympus CH 30, Olympus Optical CO., LTD, Tokyo, Japan). Two hundred sperms from each animal were examined to calculate the percentage of normal and abnormal morphological forms of the sperms. Abnormal morphological forms of the sperms (Fig. 1b) included abnormalities in the head (as detached head), abnormalities in the midpiece (as cytoplasmic droplets), abnormalities in the principal and end pieces (short or coiled tail), and other abnormalities (fused or amalgamated spermatozoa). In sperm with two or more defects, only the defect affecting its more proximal region was recorded (e.g. in a sperm with cytoplasmic droplet and short tail, only the cytoplasmic droplet was recorded) [24]. The data were statistically analyzed.

Serum testosterone level analysis

The collected blood was placed in tubes, and the serum was obtained and frozen at −20°C for measuring the level of testosterone. Testosterone was assessed by an enzyme-linked immunosorbent assay kit (IB79174, IBL-America, Minneapolis, Minnesota, USA). The values were expressed as nanograms of testosterone/milliliter serum [24].

Statistical analysis

The statistical data included epithelial cell height, serum testosterone level, and sperm assay including sperm concentration and the percentage of normal and abnormal morphological forms of the sperms. Data were expressed as arithmetic mean±standard deviation. The Student's t-test was used to test the significant change in the rats of different groups in comparison with negative control rats in group 1. Statistical significance was realized at probability P less than 0.05. Statistical analysis of the data was carried out by MedCalc software (Mariakerke, Belgium) for medical statistics [25].

Results

Histological results

Control group 1

Structure of cauda epididymis: light microscopic examination of sections in cauda epididymis stained with hematoxylin and eosin revealed no apparent differences in the structure of subgroups a and b. Rats in both subgroups revealed the presence of a connective tissue capsule surrounding a highly convoluted wide epididymal duct cut in various directions and containing spermatozoa in the lumen (Fig. 2). The epididymal duct was surrounded by a thin layer of smooth muscle fibers (Fig. 3) and connective tissue (Fig. 2). The duct was lined by pseudostratified columnar epithelium resting on a basal lamina. The epithelial height of the cells lining the duct of cauda epididymis was demonstrated in Table 1. Principal columnar cells were the predominant cell type, which had a basally located nucleus and apical stereocilia projecting into the lumen. Basal cells were found resting on the basal lamina among the base of principal cells. Clear cells with pale vacuolated cytoplasm and central nucleus were identified. Few halo cells were also seen at all levels of the epithelium from the basal lamina to the lumen. They were small rounded cells with a scanty amount of pale cytoplasm (Fig. 3). In sections stained with Masson trichrome, few blue-stained collagen fibers were found in the connective tissue surrounding sections of cauda epididymis duct (Fig. 4).

Table 1
Table 1:
Mean values±standard deviation of different parameters in rats of all groups
Figure 2
Figure 2:
A connective tissue capsule (arrow) surrounding different sections in cauda epididymis duct containing spermatozoa (stars) and surrounded by connective tissue (crossed arrows).Group 1 H&E ×100.
Figure 3
Figure 3:
A higher magnification of the previous figure showing columnar principal cells with stereocilia (arrow), clear cells with pale vacuolated cytoplasm (crossed arrows), basal cells resting on the basal lamina (arrowhead), and a halo cell with a scanty pale cytoplasm (curved arrow). Note the thin layer of smooth muscle fibers surrounding sections in the duct (thick arrow).Group 1 H&E ×400.
Figure 4
Figure 4:
Few blue-stained collagen fibers (arrows) in the connective tissue surrounding sections of cauda epididymis duct.Group 1 (Masson trichrome stain, ×400.

Immunohistochemical detection of glutathione reductase enzyme: positive control slides prepared from the kidney of control animals showed the positive immune reaction of GR enzyme in the form of fine brown granules in the epithelium lining medullary renal tubules (Fig. 5). In rats of control group 1, a positive immune reaction of GR was detected in the epithelium lining the cauda epididymis duct (Fig. 6).

Figure 5
Figure 5:
Positive immune reaction of glutathione reductase enzyme in the form of fine brown granules in the epithelium lining the medullary renal tubules.A positive control slide of immunohistochemical for glutathione Reductase ×400.
Figure 6
Figure 6:
Positive immune reaction of glutathione reductase in the epithelium lining cauda epididymis duct (arrowheads).Group 1 Immunohistochemical for glutathione reductase ×400.

Ultrastructure of cauda epididymis: principal cells showed stereocilia projecting from the apical surface into the lumen. The nucleus was basal, euchromatic, and slightly indented (Fig. 7). The cytoplasm showed well-developed Golgi complex, rough endoplasmic reticulum, mitochondria, lysosomes, and lipid droplets. In addition, numerous vesicles and multivesicular bodies were found in the upper part of the cells. The principal cells were closely connected by tight junction, near the luminal surface of the cells (Figs 8 and 9). The basal cells were small rounded or oval in shape with large oval euchromatic nucleus and cytoplasm containing few organelles (Fig. 7). Clear cells contained numerous clear vacuoles of variable sizes and shapes, many lysosomes of different electron densities, and lipid droplets (Figs 7 and 9). Halo cells were recognized among the principal cells. They showed extremely pale cytoplasm with few cytoplasmic organelles and central rounded-to-oval slightly indented euchromatic nuclei with prominent nucleoli (Fig. 10). The lumen contained sections in different regions of the sperm. The hook-shaped head contained the nucleus, whereas the flagellum was formed of midpiece, principal piece, and end piece (Fig. 11). The midpiece showed mitochondrial sheath surrounding nine outer dense fibers and a centrally located axoneme consisting of nine peripheral doublets plus two central singlet microtubules. In the principal piece, mitochondrial sheath was replaced by a fibrous sheath surrounding seven outer dense fibers. The fibrous sheath was formed of two longitudinal columns and transverse ribs. End piece showed only axoneme (Fig. 12).

Figure 7
Figure 7:
Principal cells (P) with stereocilia projecting into the lumen (stars) and euchromatic indented nucleus (arrows). Note the clear cells (C) with numerous vacuoles (arrowheads) and the small basal cell (crossed arrow) on the basal lamina (curved arrow).Group 1 TEM ×1500.
Figure 8
Figure 8:
Well-developed Golgi complex (arrows) and apical numerous vesicles (arrowheads) in principal cells (P). Note the tight junction (thick arrow) between the cells near the luminal surface.Group 1 TEM ×10 000.
Figure 9
Figure 9:
A clear cell (C) containing numerous vacuoles (v) variable in size and shape. Note the lysosomes (curved arrows) and lipid droplets (thick arrow) in clear and principal cells, the small vesicles (arrowheads) in the upper part of the principal cells (P), and the tight junction (arrow) between the cells.Group 1 TEM ×3000.
Figure 10
Figure 10:
Halo cells (H) among principal cells (P). Note the central euchromatic indented nuclei (arrowheads) containing prominent nucleoli and the pale cytoplasm with few cytoplasmic organelles (arrows).Group 1 TEM ×3000.
Figure 11
Figure 11:
Sections in head containing the nucleus (N), midpiece (MP) with mitochondrial sheath (arrowheads), principal piece (arrows), and end piece (curved arrows) of spermatozoa.Group 1 TEM ×8000.
Figure 12
Figure 12:
Mid-pieces (MP) with mitochondrial sheath (arrowheads) surrounding nine outer dense fibers (ODF) and central axoneme formed of nine peripheral doublets (curved arrow) and two central singlet (crossed arrow) microtubules. Note the fibrous sheath with two longitudinal columns (white stars) connected with transverse ribs (thick arrow), surrounding seven outer dense fibers (arrow) in a principal piece (PP).Group 1 TEM ×60 000.

Group 2

Structure of cauda epididymis: the epithelial height of cells lining the duct of cauda epididymis was significantly (P<0.05) decreased compared with negative control rats of group 1 (Table 1). Most of cells lining the duct appeared low cubical-to-flat with pyknotic nuclei. The smooth muscle layer and connective tissue surrounding the epididymal duct appeared thick (Fig. 13). Masson trichrome stain revealed excess blue-stained collagen fibers in the connective tissue surrounding the epididymal duct (Fig. 14).

Figure 13
Figure 13:
Low cubical-to-flat epithelial cells with pyknotic nuclei (arrows) lining the epididymal duct. Note the thickening in the surrounding smooth muscle layer (crossed arrows) and the connective tissue (stars) surrounding the duct.Group 2 H&E ×400.
Figure 14
Figure 14:
Excess blue-stained collagen fibers (arrow) in connective tissue surrounding epididymal duct.Group 2 Masson trichrome stain ×400.

Immunohistochemical detection of glutathione reductase enzyme: the immune reaction of GR was negative in the epithelial cells lining cauda epididymis (Fig. 15).

Figure 15
Figure 15:
Negative immune reaction of glutathione reductase in the epithelial cells (arrow).Group 2 Immunohistochemical for glutathione reductase ×400.

Ultrastructure of cauda epididymis: principal cells showed pyknotic nuclei. The cytoplasm showed many lysosomes, distorted mitochondria, and extremely dilated Golgi complex and rough endoplasmic reticulum. The lumen showed shedding of epithelial remnants. Stereocilia and tight junction were still seen (Figs 16 and 17). Clear cells were less frequently detected and showed a disintegration of the cell membrane. The cytoplasm contained large apical vacuoles, many lysosomes, and few lipid droplets. In addition, the nucleus appeared small and eccentric (Fig. 18). Many spermatozoa retained cytoplasmic droplets confluent with the flagella. Several heterogeneous vesicles and lamellae were recognized in the cytoplasmic droplets (Fig. 19).

Figure 16
Figure 16:
Principal cells (P) with pyknotic nucleus (thick arrow). Note stereocilia (arrow) and epithelial remnants (arrowheads) in the lumen, and the tight junction between the cells (curved arrow).Group 2 TEM ×3000.
Figure 17
Figure 17:
Dilated Golgi complex (arrows) and lysosomes (arrowheads).Group 2 TEM ×10 000.
Figure 18
Figure 18:
A clear cell (C) with disintegration of the cell membrane (arrowhead), large apical vacuoles (v), lysosomes (arrows), and small nucleus (N) just above the basal lamina (b).Group 2 TEM ×3000.
Figure 19
Figure 19:
Heterogeneous vesicles and lamellae (arrowheads) within cytoplasmic droplets confluent with sperm flagella.Group 2 TEM ×10000.

Group 3

The structure, ultrastructure, and IHC reaction detected in the rats of this group showed no apparent differences from those detected in control group 1.

Group 4

Structure of cauda epididymis: the epithelial height of cells lining the duct of cauda epididymis was not significantly (P>0.05) different from negative control rats of group 1 (Table 1). The duct of cauda epididymis was lined by pseudostratified columnar cells consisting of principal cells, basal cells, clear cells, and halo cells. Few cells showed pyknotic nuclei. Smooth muscle layer and connective tissue surrounding the duct appeared thin (Fig. 20). Few blue-stained collagen fibers were found in the connective tissue surrounding the duct (Fig. 21).

Figure 20
Figure 20:
Principal cells (arrow), basal cells (arrowhead), clear cells (crossed arrows), and halo cells (curved arrow) lining the cauda epididymis duct. Note the pyknotic nuclei in some cells (empty arrow), the thin layer of smooth muscle (thick arrow), and connective tissue (stars) surrounding the duct.Group 4 H&E ×400.
Figure 21
Figure 21:
Few blue-stained collagen fibers (arrow) in the connective tissue surrounding the duct.Group 4 Masson trichrome stain ×400.

Immunohistochemical detection of glutathione reductase enzyme: a positive immune reaction of GR was detected in the epithelium lining the cauda epididymis duct (Fig. 22).

Figure 22
Figure 22:
Positive immune reaction of glutathione reductase in the epithelial cells (arrows).Group 4 Immunohistochemical for glutathione reductase ×400.

Ultrastructure of cauda epididymis: cauda epididymis revealed the presence of principal cells with stereocilia and clear cells with intact cell membrane and numerous apical vacuoles of variable sizes and shapes (Fig. 23). The lumen contained sections in different parts of spermatozoa with no cytoplasmic droplets (Fig. 24).

Figure 23
Figure 23:
Principal cells (P) with stereocilia (arrow) and clear cells (C) with numerous apical vacuoles (v) variable in size and shape.Group 4 TEM ×3000.
Figure 24
Figure 24:
Sections in head containing the nucleus (N), midpiece (MP), principal piece (crossed arrow), and end piece (arrow) of spermatozoa.Group 4 TEM ×6000.

Epididymal sperm morphology and concentration

Compared with negative control rats in group 1, a significant (P<0.05) decrease in sperm concentration and the percentage of normal sperms, associated with a significant (P<0.05) increase in the abnormal morphological forms of sperms, particularly the retention of cytoplasmic droplets in the midpiece, was detected in rats receiving BPA in group 2. However, no significant (P>0.05) difference was reported in all the parameters in group 3 receiving quercetin and in group 4 receiving quercetin with BPA (Table 1).

Serum testosterone level

Compared with negative control rats in group 1, a significant (P<0.05) decrease in the mean value of serum testosterone was detected in group 2. There was no significant difference (P>0.05) detected in the mean values of serum testosterone level in groups 3 and 4 compared with group 1 (Table 1).

Discussion

Spermatozoa leaving the testis and entering the epididymis are nonfunctional gametes. The activities performed by the epithelial cells of the epididymis are responsible for the creation of regional microenvironments in which spermatozoa undergo maturation [1]. In this study, the histological structures of cauda epididymis and epididymal spermatozoa in control rats were similar to that described in other studies [26,27]. Cauda epididymis duct was lined by four types of cells: principal cells, basal cells, clear cells, and halo cells. Principal cells were characterized by the presence of tall stereocilia. It has been reported that stereocilia provide a large surface area to interact with molecules present in the lumen. Furthermore, they contain a variety of membrane pumps, channels, and transporter systems to maintain the proper volume and pH of epididymal lumen [28]. The presence of well-defined rough endoplasmic reticulum, mitochondria, and Golgi complex in the principal cells suggested an active secretory function, whereas the presence of numerous vesicles and multivesicular bodies in the upper part of the cells may reflect an absorptive function. It has been reported that principal cells are active in the transport and secretion of ions and small organic molecules. They are also involved in secretion and absorption of fluid and in protein synthesis. The secretory products of principal cells allow for the formation of a specific luminal microenvironment [29]. This microenvironment is maintained by the presence of a blood–epididymal barrier in the form of tight junctions between principal cells, which is necessary for sperm maturation [1].

In this study, clear cells were characterized by numerous apical vacuoles and lysosomes. It has been reported that the endocytic activity of clear cells is greater than any other cell type in the epididymis. They are responsible for the uptake of a number of different proteins and luminal components, as well as the disposal of cytoplasmic droplets detached from spermatozoa [30]. Basal cells were characterized by their location against the basement membrane. It is believed that they regulate electrolyte and water transport by the principal cells [30] and may help to regulate principal and clear cell functions [31]. Halo cells were found throughout the epididymis. They are the main type of immune cells in the epididymis as they have been postulated to be lymphocytes or monocytes [30].

Epididymis can be a target for different toxicants. Environmental chemicals may affect epididymal sperm maturation and hence the fertility of individuals exposed to such toxicants [32]. BPA is a small organic compound widely used in food packaging and dental appliances that can leach from polycarbonate plastics. Therefore, there is a general concern for its potential to cause adverse effects on human health [6,8]. The potential for BPA exposure was demonstrated when BPA was detected in 95% of the urine samples of people in the United States [33]. It was also detected in amniotic fluid, neonatal blood, placenta, and breast milk [34].

The organizations such as the United States Food and Drug Administration and the European Food Safety Authority confirmed that the current human exposure levels of BPA are safe. However, extensive studies reported deleterious effects of BPA [6,8,35]. Moreover, it can interfere with the endocrine signaling pathways, even at doses below the calculated safe dose [8].

In this study, the height of the epithelium lining cauda epididymis duct in rats receiving BPA (group 2) showed a significant decrease compared with the control group. The epithelium appeared low cubical-to-flat and showed pyknotic nuclei. A previous study reported the degeneration of the cells of epididymis after exposure to BPA [36]. In addition, this study showed a thickening in the smooth muscle layer and in the connective tissue surrounding the duct. It was reported that BPA can induce fibroblast and smooth muscle stromal hyperplasia in other organs [11,37].

In rats of group 2 subjected to BPA, the principal cells showed distorted mitochondria, many lysosomes, and extremely dilated Golgi complex and rough endoplasmic reticulum. Moreover, some epithelial remnants were seen in the lumen. However, stereocilia and tight junctions were not affected. Clear cells were less frequently detected and showed disintegration of the cell membrane. Many spermatozoa in the lumen showed retention of cytoplasmic droplets confluent with their flagella. No studies were available to discuss the effect of BPA on the ultrastructure of the cauda epididymis. However, the dilatations of Golgi complex in this study may explain the toxic effect of BPA through alteration of the secretory function of epididymal cells and by increasing the lysosome activities, resulting in degeneration of the cells. Similar degenerative findings were reported in the epididymis by other toxicants [38,39]. The presence of cytoplasmic droplets was reported in rat cauda epididymal spermatozoa after treatment with cytotoxic and xenobiotic agents with estrogenic activity [40]. It has been known for decades that BPA has the efficacy of the hormone estradiol in some tissues [41]. The retention of cytoplasmic droplets reported in this study might also be due to the degenerative changes in clear cells, which are responsible for removal of cytoplasmic droplets of spermatozoa as reported before [29]. It has been reported that the retention of cytoplasmic droplet may result in impairment of sperm motility and can consequently influence male infertility [42].

In this study, administration of BPA resulted in a significant decrease in the serum testosterone level, epididymal sperm concentration, and the percentage of the normal forms of spermatozoa associated with a significant increase in the percentage of abnormal forms, particularly the retention of cytoplasmic droplets. Different studies reported similar results with BPA [36,42]. However, other studies reported that BPA has no effect on the sperm count [43].

Oxidative stress is associated with male infertility. The epididymis plays a major role in protecting spermatozoa from oxidative stress by removing reactive oxygen species (ROS) and by secreting antioxidant enzymes into the epididymal lumen. Therefore, a deficiency in the enzyme systems will affect the integrity of spermatozoa [2]. GR is one of the antioxidant enzymes secreted in the epididymal lumen [3]. In this study, GR revealed a positive immune reaction in the epithelial cells lining cauda epididymis in control group 1. The activity and the immune reactivity of GR were detected in the epithelium of epididymal tail in other studies [2,3]. After the administration of BPA in group 2, the immune reaction of GR became negative. It was reported that BPA may cause depletion in GR, leading to oxidative stress in epididymal sperm of rats [36]. Furthermore, BPA can generate ROS resulting in oxidative stress and damage in testes [44] and sperms [45] of rats. In group 4, receiving quercetin together with BPA, the immune reaction of GR was positive. It was reported that coadministration of antioxidant substances such as vitamin C with BPA can protect against BPA toxicity in epididymis and sperms [36].

Quercetin is an antioxidant molecule found in fruits and vegetables, mainly in grapes [14]. It is a potent antioxidant with ROS scavenger activity [46]. It can also ameliorate the reproductive toxicity induced by environmental contaminants [15] and environmental estrogens [47]. In this study, quercetin alone was given to group 3 rats at a dose that did not alter the structure, ultrastructure, or the IHC reaction. When a similar dose of quercetin was given with BPA to group 4 rats, no significant difference in the height of the epithelium lining the cauda epididymis was found compared with the control group. In addition, no structural or ultrastructural toxic changes were found, except the presence of few pyknotic nuclei in the epithelium lining the cauda epididymis. The immune reaction of the GR was also positive in this group. Serum testosterone level, sperm concentration, and the percentage of sperm morphological forms were also similar to control group 1. In agreement with these results, different studies reported that quercetin can ameliorate the BPA-induced cytotoxicity [48] and improve the sperm quality [16]. It can also ameliorate the reproductive toxicity induced in spermatogenic cells by other environmental estrogenic contaminants [47]. Quercetin supplementation was also found to enhance the expression of higher levels of GR enzyme to protect against oxidative stress in other different tissues [14]. In addition, it was reported that quercetin has an antiestrogen effect and enhances testosterone hormone in male rats [49].

Conclusion

Exposure to BPA resulted in degenerative structural and ultrastructural changes in cauda epididymis, and an inhibition in the IHC expression of the antioxidant GR enzyme in adult male albino rats. In addition, a decrease in serum testosterone and sperm concentration associated with an increase in the percentage of abnormal morphological sperm forms was detected. The administration of quercetin prevented most of the changes, suggesting its possible protective effect.

Recommendations

On the basis of the results of this study, the use of quercetin as a supplement for treatment of male infertility might be considered. The mechanisms of action and the effective dose and duration of administration are in need of further investigation.

Table
Table:
No title available.

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Keywords:

bisphenol A; cauda epididymis; epididymal sperms; glutathione reductase; quercetin; structure; ultrastructure

© 2011 The Egyptian Journal of Histology