|Year : 2014 | Volume
| Issue : 3 | Page : 161-176
Alpha-chlorohydrin effects on the epididymis of adult albino rat: A histological and immunohistochemical study
Gehan Soliman, Sadika M.T. Al Ebs, Amal A.A. Abd-El-Hafez
Tanta University, Faculty of Medicine, Histology Department, Egypt
|Date of Web Publication||6-Feb-2018|
Amal A.A. Abd-El-Hafez
Tanta University, Faculty of Medicine, Histology Department
Source of Support: None, Conflict of Interest: None
α-Chlorohydrin (ACH) is a well-known food contaminant, has shown anti-fertility activity in males. In this research we studied the histological and immunohistochemical changes in the epididymis of adult albino rat. Twenty adult male albino rats were divided into control (group 1) (10 rats) and experimental (groups 2 and 3) (10 rats). The experimental rats received α-Chlorohydrin at a single oral dose of 100 mg/kg body weight. After 6 h (group 2) and 24 h (group 3) of ACH treatment, the specimens were submitted for the light, electron microscopic and β-tubulin immunohistochemical evaluations. Light microscopy after 6 h showed focal separation of lining epithelium, while EM examinations showed dilated rER, and Golgi complex. After 24 h LM demonstrated different stages of cell exfoliation; however, EM proved multilamellar bodies and dilated rER besides the granules. Immunohistochemi- cal examinations revealed decreased reactions in experimental groups. However, the mean total optical density of the β-tubulin immunostaining revealed statistical nonsignificant difference between the three groups. Thus ACH as an undesirable unavoidable food contaminant induced rapid histological alterations on caput epididymis with duration-dependent manner at the level of the cell and tissue, that puts another obstacle on the road of fertility.
Keywords: α-Chlorohydrin, Epididymis, Histological and immunohistochemical, study
|How to cite this article:|
Soliman G, Al Ebs SM, Abd-El-Hafez AA. Alpha-chlorohydrin effects on the epididymis of adult albino rat: A histological and immunohistochemical study. J Microsc Ultrastruct 2014;2:161-76
|How to cite this URL:|
Soliman G, Al Ebs SM, Abd-El-Hafez AA. Alpha-chlorohydrin effects on the epididymis of adult albino rat: A histological and immunohistochemical study. J Microsc Ultrastruct [serial online] 2014 [cited 2021 Mar 2];2:161-76. Available from: https://www.jmau.org/text.asp?2014/2/3/161/224880
| 1. Introduction|| |
α-Chlorohydrin [3-chloro-1,2-propanediol, ACH], is an organic chemical compound which is carcinogenic, highly suspected to be genotoxic in humans and has male anti- fertility effects . α-Chlorohydrin also has harmful effects in many organs such as testis, kidney, liver and brain. ACH or [3-monochloropropane-1,2-diol or 3-chloropropane-1,2-diol] crosses the blood testis barrier and the blood brain barrier and distributed widely in the body fluids .
It is primarily created in foods by protein hydrolysis by adding hydrochloric acid to speed up the reaction of the [soya] protein with lipids at high temperatures, in foods contact with materials containing epichlorohydrin- as used in the production of some tea bags and sausage casings. A wide range of household food products from sliced bread to crackers, beefburgers, popular brands such as Mother’s Pride and cheese are found with [ACH] above safe limits .
The epididymis is the site of accumulation and storage of spermatozoa of mammals. It is an excellent target for the development of a male contraceptive. This is because the process of sperm maturation occurs in this organ; the immature spermatozoa undergo changes in the morphology, surface properties and biochemical composition during epididymal transit then, spermatozoa become motile and are able to recognize and fertilize an egg once they traverse the epididymal duct. These changes depend on the epididymal microenvironment which is formed by the absorptive and secretory functions of the epididymal lining epithelium ,,.
The epididymal epithelial cells were versioned into four major cell types: principal, basal, clear, and halo cells. The narrow cells were added by Robaire et al. . Moreover Domeniconi et al.  furthered two other cell types to that categorization; apical cells and dark cells. The migratory cells demonstrated crossing the epithelium, as intraep- ithelial lymphocytes and macrophages are thought to be component of the lining epithelium .
Principal cells [PCs] are the most abundant cells and play a major role in secretion and absorption . Tight junctions between principal cells form the blood-epididymis barrier ,. This barrier, in prolongation with the blood-testis barrier, is critical for the avoidance of autoimmune responses against antigenic germ cells . Basal cells are flat elongated cells that may have a defensive role, both by detoxifying electrophiles , or by acting like macrophages . It is believed that the basal cells function as stem cells, regenerating themselves as well as the other cells as the need arises . Clear cells participate in the uptake of luminal components and the discarding of cytoplasmic droplets detached from spermatozoa .
The halo cells are rounded or irregular in shape with dark, small nuclei surrounded by a light halo of pale cytoplasm. Halo cells have been postulated to be lymphocytes or monocytes and are supposed to play a role in the immunological barrier of the male reproductive duct . The apical cells were found to be fewer in number than any other cell type. They have a wide apical portion with apically located spherical nucleus and narrow stem not extending to the basal lamina . Dark cells [DCs] appear among the PCs as narrow, tall, and darkly stained cells, extending from the basement membrane to the lumen . The narrow cells are narrower than the principal cells, attenuated, and send a thin process of cytoplasm to reach the basement membrane. They are characterized by numerous apically located cup-shaped vesicles that are involved in endocytosis and function in secreting H+ ions into the lumen by recycling to and from the apical plasma membrane .
As ACH is recognized to interfere with reabsorption in the epididymis, microtubule dysfunction may compromise these essential processes of endocytotic vesicle transport . To address this, β-tubulin was immunolo- calized within epididymis.
ACH is considered to be carcinogenic, also has male antifertility effects, at the same time it is found in our food at levels considerably higher than those deemed safe by the European Union . The legal limit for ACH is 20 μg per kg, but the safety guideline on daily intake is 120 μg for a 60 kg person per day. Consequently, it was mandatory to explore its role in nowadays widespread male infertility.
Many studies were done to determine whether [ACH] affected the metabolism of spermatozoa directly or through disruption of the epididymal metabolism, all of these researches studied the physiological or the biochemical effects, so our study was carried out to determine the epididymal histological and ultrastructure alterations. Moreover the second goal was to evaluate the ACH effect on B-tubulin immunolocalizations.
| 2. Materials and methods|| |
2.1. The used animals and study design
Twenty adult male albino rats weighing 150–200 gm each, were maintained on standard balanced diet and allowed free access to water and food. All rats were housed five per cage in metal cages. The rats were assigned randomly into three groups: 10 animals were kept as control (group 1), while the rest 10 animals were used as experimental, received α-Chlorohydrin. These rats were dissected at two points from the start of the experiment; at 6 h five experimental (group 2) and five control rats while the other five experimental rats at 24 h (group 3) with five controls.
2.2. α-Chlorohydrin and dosage
The experimental animals received α-Chlorohydrin (Sigma) at a single oral (sublethal) dose 100 mg/kg bw. It was chosen on the basis that it caused severe toxicity at an oral dose of 150 mg/kg bw . The oral LD50 (median lethal dose, 50%) of ACH was reported to be 152 mg/kg bodyweight in rats .
2.3. Histological and ultrastructure preparations
At the planned time the rats were anaesthetized with ether inhalation and the epididymis was carefully dissected. The right caput epididymidis was taken and fixed with 10% neutral buffered formalin solution. The tissues were routinely processed, embedded in paraffin and sectioned at 3–5 μm. These sections were stained with hematoxylin-eosin for histopathologic examination.
The left caput epididymis was fixed in 5% phosphate buffered glutaraldehyde (pH 7.3) for 2 h, and then post fixed in 1% phosphate buffered osmium tetraoxide for 1–2 h. After dehydration, the specimens were embedded in Epoxy resin mixture and semithin sections were cut and stained with toludine blue and examined by light microscope to choose the selected areas for proper orientation. Ultrathin sections were cut with LKB ultramicrotome and stained with uranyl acetate and lead citrate for detailed examination and photographed at JEOL-TEM 100 SX E/M (UK) Ltd) in the EM unit, Faculty of Medicine, Tanta University .
2.4. Immunohistochemical preparations
- Epididymal tissues: β-Tubulin immunohistochemical detection was performed on formalin-fixed, paraffin- embedded epididymal tissues. 5 μm cross sections were deparaffinized with xylene, and hydrated in graded ethanol solutions (5 min each). Tissue sections were immersed for 10 min in phosphate buffered saline (PBS) containing 3% hydrogen peroxide to inactivate endogenous peroxidase activity.
- Primary antibody handling: Prior to incubation with the primary antibody, the tissue sections were blocked for 30 min in PBS containing 10% blocking serum (horse serum). The sections were then incubated overnight in a humid chamber with primary rabbit anti-rat β-tubulin antibody at a dilution of 1:250 (Boehringer Mannheim; West Germany).
- Secondary antibody handling: The sections were then rinsed three times in PBS and incubated with goat anti-rabbit peroxidase-conjugated secondary antibody (peroxidase-labeled streptavidin) for 1 h and rinsed again three times in PBS. The immunoreactivity was visualized using 3,3’-diaminobenzidine hydrogen peroxide as a chromogen and sections were counterstained with hematoxylin (14).
Optical density and statistical analysis
The image analysis system (Leica Q 500 MC program) in the Tanta Faculty of Medicine Central Research Lab., Tanta, was used to measure the mean total optical density of the β-tubulin. The values were represented as mean ± standard deviation (SD). The data were analyzed by unpaired Student’s t-test using SPSS software. Differences were regarded as significant if probability value P <0.05 and highly significant if P <0.01.
| 3. Results|| |
3.1. Light microscope
By light microscopy, sections of the control (group 1) caput epididymal tissues revealed that the epididymis was a tubule of smooth muscle cells lined by a tall pseudostrat- ified columnar epithelium with stereocilia and filled with spermatozoa. The sections of the tubules were separated by intertubular connective tissue having blood vessels [Figure 1]. The intraepithelial glands were demonstrated in different stages of their formation; small spaces within the epithelium or larger ones surrounded by the neighboring cells [Figure 1]. The lining pseudostratified epithelium composed of a lot of cell types which were regularly arranged. Two cell types were prominent and easily noticeable: the short basal cells appeared as pyramidal to polyhedral. They had round nuclei with dense appearance. The tall principal cells (PCs) had irregular, oval nuclei with one or two large nucleoli. These nuclei were paler than those of the basal cells and are located basally within the cell. The principal cells exhibited apical stereocilia and noticeable vacuoles [Figure 1] and [Figure 2].
|Figure 1: Photomicrographs of the control epididymis caput tubules appear as tubules of smooth muscle (SM) separated by intertubular connective tissue (ct) with blood vessels (bv), lined by a tall pseudostratified columnar epithelium and filled with spermatozoa. There are short basal pyramidal cells (B) and tall principal cells (P) exhibited apical stereocilia. Intraepithelial glands (Gl) and hallo cells (H) are noticed. H&E, Mic. Mag. ×400 (a), ×1000 (b and c).|
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|Figure 2: Photomicrographs of semithin sections for control group show tubules of smooth muscle (SM) cells, regularly arranged cells of the lining pseudostratified epithelium. Basal cells (B) appear as pyramidal cells with dense nuclei. The principal (P) cells are tall cells, have irregular, oval basal nuclei with one or two nucleoli, paler than those of the basal cells, apical stereocilia (S) and noticeable vacuoles. Hallo (H) cell with its narrow rim of clear cytoplasm, apical (A) cell with its characteristic apically located spherical nucleus and dark (D) cell with its dark, elongated fusiform nuclei are forcibly defined. Connective tissue (ct). Blood vessels (bv). Toludine blue, Mic. Mag. ×1000.|
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However, the other cells were forcibly defined by the light microscope; halo cells with its narrow rim of clear cytoplasm were usually located at the base of the epithelium. Apical cells had a characteristic apically located spherical nucleus and did not contact the basement membrane. Dark cells were narrow, tall and darkly stained cells extending from the basement membrane to the lumen between the principal cells. They had dark, elongated fusiform nuclei [Figure 2].
While, 6 h after α-Chlorohydrin treatment (group 2) changes were present in all tubules but they were not uniformly distributed. Some tubules appeared with low columnar cells with scarce of marginal stereocilia whereas the intertubular connective tissue became more prominent and their blood vessels were congested and dilated [Figure 3]. In other specimens there were less affected tubules with focal separation of their lining epithelium from the basement membrane [Figure 3] and [Figure 4]. There were slight disturbance in the cellular array. The wandering cells were more noticed than control, the clear cells showed some degeneration, while the dark cells appeared in clusters and sloughed cells emerged in the lumen beside the sperms [Figure 4].
|Figure 3: Photomicrographs of caput epididymis tubules (group 2) 6 h after α-chlorohydrin treatment show some tubules lined by low columnar cells with dislodge stereocilia (arrows), others with focal separation of their lining epithelium from the basement membrane (arrow heads). The intertubular connective tissue is prominent with dilated blood vessels (BV).|
H&E, Mic. Mag. ×400.
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|Figure 4: Photomicrographs of semithin sections for caput epididymis tubules (group 2) 6 h after α-chlorohydrin treatment showing slight disturbance in the cellular array. The wandering cells (arrows) are noticed; the clear (C) cells show some degeneration, while the dark (D) cells appear sporadic or in clusters. Sloughed (S) cells emerge in the lumen beside the sperms. There is focal separation of the lining epithelium from the basement membrane (arrow heads). Toludine blue, Mic. Mag. ×1000.|
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Twenty-four hours after α-Chlorohydrin treatment (group 3), the tubules showed different stages of cell exfoliation. Most of the tubules showed luminal migration of some wandering and principle cells [Figure 5]. Others showed fully expelled cells which were alienated from each others [Figure 5]. Further tubules contained separated sheets of epithelial cells with loss of their stereocilia [Figure 5]. Some tubules appeared occluded with hyperenfolding of the wall giving arberisation look [Figure 6]. In these hyperfolded tubules the nuclei of the (PCs) were also hyperfolded, the clear cells were disintegrated while the halo cells were multiple [Figure 6]. Most of the tubules of this group by the first look showed the characteristic distorted cellular array [Figure 7]. The abundant cell granules were the feature of this group. Nearly, all distinct cell types contained granules but with different quantities and positions [Figure 7]. They were supra nuclear in most of the PCs, or may be sporadic in other cells while rarely observed filling the narrow cells [Figure 7].
|Figure 5: Photomicrographs of caput epididymis tubules (group 3) 24 h after α-chlorohydrin treatment showing luminal migration of some wandering or principle cells (arrows), fully expelled cells which are alienated from each others (arrow heads). Other tubules contain separated sheets of epithelial cells with loss of their stereocilia (thick arrows). H&E, Mic. Mag. ×400.|
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|Figure 6: Photomicrographs of caput epididymis tubules (group 3) 24 h after α-chlorohydrin treatment show tubules nearly occluded with hyperenfolding of the wall giving arberisation look (a–c). The nuclei of the PCs are hyperfolded (arrows), the clear (C) cells are disintegrated while the hallo (H) cells are multiple. H&E, Mic. Mag. ×400 (a and c), Toludine blue, Mic. Mag. (b) ×(400). (d) ×1000.|
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|Figure 7: Photomicrographs of semithin sections caput epididymis tubules (group 3) 24 h after α-chlorohydrin treatment showing distorted cellular arrangement. All cell types contain granules, it is supra nuclear in most of the PCs (arrows), may be sporadic in others (arrow heads) while rarely filling the narrow (N) cells. Toludine blue, Mic. Mag. ×1000.|
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3.2. Electron microscope
The control group (group 1) showed many tubules lined by pseudostratified columnar epithelium with stereocilia; it was consisted of many cell types. Basal cells were flat elongated cells situated between the myoid and the other cells. They had flat nuclei in which accumulations of hetero- chromatin take place. The thin cytoplasm of these cells was relatively clear, with a scarcity of organelles [Figure 8]. The principal columnar cells were the most numerous; their cytoplasm also had supranuclear lipid droplets, numerous profiles of apically located endolysosomes, and multivesicular bodies. The apical cell membranes of those cells displayed a profusion of pinocytotic and coated vesicles at the bases of the many stereocilia that projected into the lumen of the epididymis [Figure 8]. The clear cells were characterized by an apical region containing few microvilli, but numerous coated pits, small and large apical vesicles, endosomes, and lysosomes, all were involved in endocytosis [Figure 8].
|Figure 8: Electromicrographs of the control epididymis caput tubules lined by a tall pseudostratified columnar epithelium, with stereocilia filled with spermatozoa. The lining epithelium consists of flat elongated basal (B) cells, situated between the myoid (M) cells and the other cells (a–c). The most numerous is the principal (P) columnar cells; their cytoplasm has a large supranuclear lipid (l) droplets, endolysosomes, and multivesicular bodies (arrows). The apical cell membranes display pinocytotic vesicles at the bases of the many stereocilia (s) that projected into the lumen of the epididymis. (d) is higher magnification of the clear (C) cell in (a), it is characterized by an apical region (A) containing few microvilli, but numerous coated pits, small and large apical vesicles, endosomes, and lysosomes. The basal region containing the nucleus is out of the section.|
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Halo cells were small cells with a narrow rim of cytoplasm that’s more clear than that of (PCs). These cells were usually located at the base of the epithelium. Apical cells had a characteristic apically located spherical nucleus and do not contact the basement membrane [Figure 9]. The narrow cells were usually narrower than the principal cells, and send a thin process of cytoplasm to reach the basement membrane. They were characterized by numerous apically located cup-shaped expansions that are involved in endocytosis with absent microvilli (dislike clear cells) [Figure 9].
|Figure 9: Electromicrographs of the control epididymis caput tubules showing halo (H) cell as small cell with a narrow rim of cytoplasm that is more clear than that of (PCs). Apical cell (A) has a characteristic apically located spherical nucleus and do not contact the basement membrane. The narrow (Nc) cell is narrower than the principal cells, and send a thin process of cytoplasm to reach the basement membrane. It is characterized by apically cup-shaped expansion (e) that is involved in endocytosis with absent microvilli. Notice, the dark granules present in basal cells (right) and the less dark in the PCs (left).|
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Electron microscopic examinations of samples achieved 6 h after α-Chlorohydrin treatment (group 2) revealed nearly uniform affection of the tubular cells. Principal cells (PCs) were the most abundant cells, their cytoplasm showed condensations of its contents until it was difficult to discriminate between the organelles except for the supranuclear Golgi [Figure 10]. They contained some dense particles, and phagosomes. Often, the cytoplasm showed subnuclear vacuolization [Figure 10] and [Figure 11].
|Figure 10: Electromicrographs of caput epididymis tubules (group 2) 6 h after α-chlorohydrin treatment showing predominant principle (P) cells, their cytoplasm shows supranuclear Golgi (G) some dense particles, and phagosomes (arrows), subnuclear vacuolization (v) and irregular or hyperfolded nuclei (N). Clear (C) cell has phagosomes (arrows) and lysosomes (l). Notice two halo cells (H) (where the section going away from the nucleus of one of them) resting on both sides of basal cell (B). Myoid cell (M).|
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|Figure 11: An electromicrograph of caput epididymis tubules (group 2) 6 h after α-chlorohydrin treatment show principle (P) cells with subnuclear vacuolization (v). Basal cells (B) resting on basement membrane supported by the myoid cell (M). Membrane-bounded lipofuscin-like structures, large lipid droplets, and electron-dense granules (arrows) with dissimilar size or electron density are observed in basal and dark (D) cells.|
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Their nuclei were mostly irregular or hyperfolded [Figure 10] and [Figure 11].
The most conspicuous feature in the majority of basal cells was the electron-dense granules. Such inclusions appeared either above or by the side of the nucleus, which was frequently indented when these structures were large [Figure 11] and [Figure 12]. Some of its apical processes are continuous with the above principle cell [Figure 12]. Granules with dissimilar size or electron density were also observed scattered in dark, principle and clear cells [Figure 12].
|Figure 12: Electromicrographs of caput epididymis tubules (group 2) 6 h after α-chlorohydrin treatment showing (a and b) higher magnifications of (a) basal cell resting on basement membrane with collagen (cl) fibers under it. Some of its apical processes are continuous with the above principle cell (arrow) and contains electron dense granules scattered in the cytoplasm. (b) Dark cell contains lipofuscin-like structures, granules with dissimilar size or electron density (g) and some apical vesicles (v) giving the cell sieve like appearance. (c) Part of principle cell studied with granules nearly similar to that of the basal cell. (d) Clear cell has homogenous moderately dense granules (arrow heads).|
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The clear cells multiplied a lot; where it appeared singly in between PCs, alternating with them or in clusters [Figure 13]. Occasionally they contained lysosomes [Figure 13]. Their nuclei became irregular, rER were noticeably dilated between the proliferated mitochondria after the 6 h of treatment [Figure 13].
|Figure 13: Electromicrographs of caput epididymis tubules (group 2) 6 h after α-chlorohydrin treatment showing the clear cells singly (a), alternating with the principle cells (b and c) and in clusters (d). (b and d) Clear cell with irregular nucleus hosts mitochondria and dilated rRE (r) while one principle cell holds variable sized dark granules (g). (c and d) The cells contain lysosomes (l) and their nuclei are mostly irregular.|
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Halo cells with its narrow rim of clear cytoplasm were usually located at the base of the epithelium and contained variable numbers of granules [Figure 10].
The dark cells with their irregular dim nucleus and narrow cells with the characteristic apical cup-shaped expansion holding many vesicles and multivesicular bodies [Figure 14] were uncommon findings. Migrating monocyte housing many lysosomes were only characterized once by its nucleus, its cytoplasm that is more clear than that of PCs when it migrated up between them [Figure 15], also by the absence of any junctions between it and the surrounding cells.
|Figure 14: An electromicrograph of caput epididymis tubules (group 2) 6 h after α-chlorohydrin treatment showing three cell types; the clear (C) cells contains a lot of dilated rER and Golgi, a dark (D) cell with irregular darker nucleus, and a narrow (Nc) cell with apical cup-shaped expansion (e), many vesicles and multivesicular bodies (v).|
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|Figure 15: An electromicrograph of caput epididymis tubules (group 2) 6 h after α-chlorohydrin treatment showing migrating monocyte housing many lysosomes. Notice the absence of any junctions between it and the surrounding cells.|
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Twenty-four hours after α-Chlorohydrin treatment (group 3), in addition to the changes found in group 2, principal cells revealed granules while clear cells housed multilamellar bodies and dilated rER besides the granules [Figure 16]. The engulfing of the sperms and large vacuoles were the character of the cells of that group [Figure 17]. Mic. Mag at the pare in all EM photos.
|Figure 16: An electromicrograph of caput epididymis tubules (group 3) 24 h after α-chlorohydrin treatment showing principal (P) cells with granules while clear (C) cells housing multilamellar bodies (arrows) and dilated rER besides the granules. The nucleoli of some cells are enlarged with obvious details (Nl).|
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|Figure 17: An electromicrograph of caput epididymis tubules (group 3) 24 h after α-chlorohydrin treatment showing the engulfed sperms (es) and huge vacuoles (v).|
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3.3. Immunohistochemical staining for β-tubulin
In control rats, β-tubulin appeared concentrated in the apical region of epithelial cells of caput region of the epididymis. It appeared to be uniformly distributed throughout all cell types [Figure 18]. The dense immunostain- ing in the apical region of cells gradually diminished toward the base [Figure 18].
|Figure 18: Photomicrographs caput epididymis for control and ACH-treated rats, immunostained with the anti-β tubulin antibody. β-Tubulin immunostaining is uniformly present and distributed throughout the apical region of all cell types in the epithelial cells of control rats, gradually diminishes toward the base (a–c). At 6 h, β-tubulin appears clumped within the epithelia. The overall immunostaining has diminished, gradually fads toward the base and loses the uniform distribution (areas with low reactions (arrows) and areas with high reactions (arrow heads)) (d). At 24 h following ACH treatment, moreover the reactions at 6 h, the desquamated epithelial cells show heavy reaction (e). Mic. Mag. (a) ×400, (b and c) ×1000, (d and e) ×400.|
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At 6 h following ACH treatment, β-tubulin appears clumped within the epithelia. The overall immunostaining has diminished, gradually faded toward the base and lost the uniform distribution [Figure 18]. At 24 h following ACH treatment, β-tubulin immunostaining was more clumped and the desquamated epithelial cells showed heavy reaction [Figure 18].
3.4. Optical density and statistical analysis
The mean total optical density of the β-tubulin immunostained control sections was (83.19 ±4.17), while it was (83.49 ±4.48) and (81.03 ±6.04) for 6 and 24 h, respectively. The statistical analysis revealed nonsignificant differences between the three groups where the P values were >0.05.
| 4. Discussion|| |
Many of the reproductive toxicants have primary effects on the testis, which overshadow effects downstream on the efferent ducts and epididymis. The specific target of these effects depends upon the dosage and the exposure time .
Toxicology of the epididymis has received less attention than other regions of the male reproductive system. It is often necessary to design experiment that separate testosterone-dependent responses (arising in the testis) from direct effects on epididymal tissues, to discover the mechanisms of toxicity. So we depended while selecting the dose and duration, on the fact that short-term toxicity targets directly the epididymis away from the remote effects of hormones.
It has been documented in other studies also, that, [ACH] appears to have direct effects on epididymal spermatozoa without testicular effects; if the dosage is low and exposure time is short .
In agreement with AbouHäila and Fainmaurel , the epididymal epithelium of the controls in the present study demonstrated numerous intraepithelial glands. It started as small spaces within the epithelium; these spaces coalesce to form larger ones which become surrounded by the neighboring cells. However in the experimental groups these glands were not observed. This observation may be attributed to the decreased epithelial height and activity noticed after ACH administration.
Dilatation and congestion of the interstitial blood vessels were one of the earliest changes that could be detected in the present work, similar results was explained by Huether and McCance , as due to the decrease in the ratio of oxygen supply to the oxygen need that causes an increase in the rate of adenosine production. This lead to dilatation of the vessels and increased blood flow in order to restore the ratio to the equilibrium value. The intertubular connective tissue was prominent with dilated blood vessels; this could be a result of the exudations from the blood vessels and the secretions from the connective tissue cells.
Light microscopic examination of the epididymal sections of this work also revealed many scattered vacuoles that varied in the size and shape. There was also separation of the epithelial cells lining the epididymal tubules. These findings were previously detected by Ahmed et al. , who considered these as degenerative changes. However, Domeniconi et al.  demonstrated that many vacuoles were detected normally in the cytoplasm of the principal cells. Aire et al.  described those vacuoles as a single large heteorogeneous lipid droplet of unknown function that was characteristically situated immediately proximal to the nucleus.
The focal separation of the lining epithelium from the basement membrane and slight disturbance in the cellular array observed at 6 h may be attributed for the hypermotility of the smooth muscle cells as a result of the inflammatory process. This could be also considered the cause for the 24 h observations of hyperenfolding of the tubular wall giving arberisation look. We can explain those findings as well by, epithelial hyperplasia causing recanalization. This is in agreement with Nakai et al. , who found that epithelia with medium inflammatory responses often exhibit irregular epithelial growth along the edge of luminal contents and form multiple abnormal ductules.
The wandering cells and different stages of cell exfoliation may be attributed to the inflammatory processes and cells. In coincidence with this is Nakai et al. , who stated that, the response of epithelium is dependent upon the degree of inflammation. An acute inflammatory reaction may cause ductal epithelium to stretch excessively by the released chemotactic substance, possibly a cytokine of the interleukin superfamily, which then recruits large numbers of neutrophils and other inflammatory cells.
The granules were a feature of most of the cells after ACH management. Nearly, all distinct cell types contained granules but with different quantities, positions and densities. Electron microscopy declared the nature of most of them as being, smooth surfaced vesicles containing a material, membrane-bound electron-dense granules, endosomes, phagosomes, lipofusion or fat inclusions. It is suggested that such dark granules are secretory granules, owing to the fact that the epididymal epithelium carrying out a dual function of absorption and secretion.
In favor with us are the findings of Awobajo et al. .
The membrane-bound lipofuscin substances and granules in the basal cells perhaps originated from the principal cells; these structures often appeared in the supra-nuclear region of the principal cells. Occasionally these materials were isolated within plasma membranes in cross-sections between principal and basal cells; it was not clear in which cell type they were contained. Although these observations are suggestive of a process of transfer of such structures from the principal cell to the basal cell , direct evidence is lacking; however, this phenomena were observed once in this work after 6 h of ACH administration, where apical processes of a basal cell are continuous with the above principle cell.
The absorptive processes are carried out by the formation of coated and smooth-surfaced pinocytotic invaginations. The secretory products of the cells are formed in the Golgi apparatus and they are of two types. The smooth-surfaced vesicles are found mostly in the apical part of the cell while the electron-dense, membrane- bound granules are found more toward the basal part of the cell. Polarization of secretory products within the cell could perhaps indicate the sites at which they are secreted . The smooth-surfaced vesicles are extruded into the lumen while the electron-dense granules discharged into the intercellular spaces. They may thus have an endocrine function, as the products secreted by the electron-dense granules into the intercellular spaces may find their way into the sub-epithelial blood capillaries .
The engulfing of the sperms and large vacuoles in this investigate may be the response of epithelium to acute injury induced by the chemical. It depends upon the degree of inflammation caused. It is likely that the epithelium releases a chemotactic substance, which then recruits large numbers of neutrophils and stimulate cell phagocytic activity .
Halo cells with its narrow rim of clear cytoplasm were usually located at the base of the epithelium were considered to be the lymphocytes or monocytes before migration as suggested by, Holschbach and Cooper . Its multiplication in the current study may be the result of inflammatory process occurred by ACH.
A lot of studies were conducted to evaluate the effect of ACH on β-tubulin. It was reported that, microtubule disruption leads to inhibition of endocytosis in kidney proximal tubule cells . Like the kidney, the efferent ducts and initial segment epididymis are responsible for fluid and protein reabsorption through receptor-mediated endocy- tosis as well as passive diffusion . Since ACH is known to interfere with reabsorption in the epididymis, microtubule dysfunction may compromise these essential processes of endocytotic vesicle transport . It is interesting that ACH a chemical responsible for the first well known case of infertility in humans caused by chemical exposure .
Ducts respond to toxic insult by at least two different means: an increased rate of fluid reabsorption or decreased secretions; or a decreased rate of reabsorption or increased secretions. The first response leads to increased viscosity of luminal fluids, sperm stasis, ductal occlusions, granulomas and possibly fibrosis. The second response dilutes the luminal fluid, decreases sperm concentration, and leads to a decrease in sperm transit time through the epididymis .
Thus, it appears that the general mechanism for epididy- mal toxicity by ACH in this research is an increase of fluid reabsorption, denoted by the multiplied clear cells whose main function is absorption, together with alterations in granular secretions of proteins, besides the micro ductules formed, all are followed by sperm stasis and ductal occlusion.
| 5. Conclusion|| |
As histological evaluations of epididymis is said to have a prominent role in male reproductive risk assessment and provides information on the severity of the toxicity and cellular site of the damage, it is accomplished that ACH as a food contaminant, has harmful effects on the cell structure and tissue architecture of the epididymis; that adds another rock on the build of infertility. Its level must be adjusted to the safe levels which need to be further elucidated.
Conflicts of interest
The authors certify that there is no conflicts of interest with any financial organization regarding the materials used and/or discussed in the paper.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16], [Figure 17], [Figure 18]