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Histological study of adult male rat seminiferous tubules following triclosan administration and the possible protective role of pomegranate juice

Mahmoud, Sahar A.; Solaiman, Amany A.

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The Egyptian Journal of Histology: June 2014 - Volume 37 - Issue 2 - p 233-247
doi: 10.1097/01.EHX.0000446590.49937.e9
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Abstract

Introduction

In recent years, there has been growing concern regarding the adverse effects of various environmental contaminants on male reproduction. With the advent of industrialization, economic development, and urbanization, drastic changes have occurred in the lifestyle and surroundings of humans that have resulted in the extensive production and use of beneficial substances. As a result, many potentially hazardous chemicals have been released into the environment at an alarming rate, and their exposure to both humans and wildlife has become inevitable. Concurrently, there has been a declining trend in the male reproductive health of humans worldwide 1.

Triclosan [5-chloro-2-(2,4-dichlorophenoxy) phenol; TCS] is a synthetic, broad-spectrum antimicrobial agent effective against a wide range of Gram-positive and Gram-negative bacteria, as well as some viruses and fungi 2. Because of its highly efficient and favorable safety profile, TCS has been widely incorporated in a variety of personal care products, including soaps, shampoos, skin creams, toothpastes, deodorants, and cosmetics. It is also included as a biocide in a large number of household items, such as plastic kitchenware, sports equipment, shoes, toys, and textiles 3,4.The popularity of antibacterial consumer products has led to increased consumer use of TCS, especially in liquid hand soaps 5.

As a consequence of its extensive use and high stability under natural conditions, TCS has been widely detected in wastewater, natural waters, and sediments, as well as in drinking water 3,6. Because of its lipophilic property, TCS has also been detected in aquatic species and food stuff samples, such as salmon and cheese, and even in human breast milk, blood, and urine samples 7. Furthermore, TCS has been observed to persist in the environment for extended periods of time, particularly under anaerobic conditions 8. The most likely routes of human exposure to TCS are ingestion and absorption through the skin 9.

The antiseptic activity of TCS is due to its ability to block the synthesis of fatty acids by inhibiting the enzyme enoyl-acyl carrier protein reductase, which catalyzes an essential step in membranes of many bacteria and fungi 10. However, the safety of TCS has come into question with respect to environmental and human health issues 11. While companies that manufacture products containing TCS claim it is safe, the US Environmental Protection Agency has registered it as a chemical with adverse effects 12. In addition, TCS is readily converted into various chlorinated dibenzo-P-dioxins and chlorophenols by heat and ultraviolet irradiation, which may also be harmful for biological systems 13,14. The literature implies that TCS-degraded products can exhibit stronger adverse effects compared with TCS 15. Recently, TCS has been included in the probable list of endocrine disruptors as the antimicrobial has been shown to disrupt thyroid hormone homeostasis and possibly the reproductive axis 7. Its phenolic structural relationship to nonsteroidal estrogens, diethylstilbestrol and bisphenol A, also raises concern 16.

Recently there has been a renewed push to identify natural remedies to fight diseases. Among the latter is pomegranate (Punica granatum L.) 17. Pomegranate is a small tree native to the Mediterranean region. It is considered one of the oldest known edible fruits and is symbolic of fertility, abundance, and prosperity. It is globally consumed fresh, in such processed forms as juice, jam, wine, and oil, and in extract supplements 18. It has been used extensively as a folk medicine since ancient times 19.

Pomegranate juice (PJ) has been shown to be the best natural antioxidant. Seeram et al. 20 have declared that PJ may contain about three times the antioxidant ability of the same quantity of green tea or red wine. The juice is a rich source of polyphenolic compounds including anthocyanins, which give the fruit its red color. It is also rich in punicalagin, quercetin, ellagic acid, and luteolin, which largely account for the antioxidant activity of the whole fruit 17. Moreover, pomegranate is an important source of vitamins A, C, and E and of folic acid 21. It has been reported that a 250 ml glass of PJ provides 50% of an adult’s recommended daily allowance (RDA) of vitamins A, C, and E, 100% RDA of folic acid, and 13% RDA of potassium and antioxidant polyphenols 22. Pomegranate is known for its antiatherogenic, anticarcinogenic, anti-inflammatory, chondroprotective, and antimicrobial effects 23,24. In addition, pomegranate has beneficial effects on spermatogenesis 23.

Despite the widespread use of TCS, few independently published studies have investigated the emerging health concerns related to the use of this antimicrobial 7. Thus, the goal of this work was to clarify the impact of TCS on the histological structure of testicular seminiferous tubules (STs) in adult male albino rats. It also aimed to evaluate the efficacy of PJ coadministration.

Materials and methods

A total of 32 adult male albino rats (140–160 g) were included in the work. They were housed under the same laboratory conditions and provided standard rat chow and water ad libitum. All experimental procedures were approved and performed in compliance with the guidelines of the local ethical committee of the Faculty of Medicine, University of Alexandria. TCS of 99.6% purity was obtained from Aldrich Chemical Company (St Louis, Missouri, USA) in the form of white powder. To prevent its photolysis, stock solutions of TCS were stored in brown flasks and kept refrigerated at 4°C before use. TCS was administered daily by gavage. A uniform suspension of TCS in PBS was made fresh every day just before intubation.

Preparation of PJ: fresh pomegranate fruit was washed and manually peeled, without separation of the seeds. PJ was obtained by using a commercial blender (Braun, Kronberg, Germany) and filtered to remove the residue. The juice was used within 1 h and given orally by gavage 18.

Rats were acclimatized to the animal house condition for 7 days before the experiment. Thereafter, animals were randomly assigned to four equal groups of eight rats each.

  • Group I (the control group): rats in this group received PBS orally (1 ml/kg/day) and served as controls.
  • Group II (the PJ group): rats in this group received PJ at a dose of 10 ml/kg/day 25.
  • Group III (the TCS group): rats in this group received TCS (20 mg/kg/day) in PBS 13.
  • Group IV (the TCS+PJ group): rats in this group received TCS at the same dose as in group III together with PJ (10 ml/kg/day). The experiment continued for 60 days 13. At the end, blood samples were collected from the retro-orbital venous plexus of all rats. Centrifugation was performed and the sera were kept at −20°C for further analysis of testosterone levels by means of the ELISA technique (Abbot, Vienna, Austria) 26. Rats were then euthanized and the testes of all rats were harvested for histological examination. The right testis of each rat was immediately dissected out and cut into small pieces of 1×1 mm3 and fixed in 3% phosphate buffered gluteraldehyde. The testes were further processed and prepared for transmission electron microscopic examination by Jeol 100 CX electron microscope (Jeol, Tokyo, Japan) at the Faculty of Science, University of Alexandria 27. The left testis of each rat was fixed in Bouin’s fixative, processed to get 5–6 μm-thick paraffin sections, and stained with H&E 28.

Morphometric study

The morphometric measurements were recorded using an Olympus BX41TF (Olympus, Tokyo, Japan) image analyzer computer system (Medical Research Institute, Alexandria University). The germinal epithelial height of STs was measured from the spermatogenic cells on the inner surface of the basement membrane through the most advanced cell types lining the lumen of the tubules. Data were obtained from 10 random microscopic fields per animal at ×200 magnification in H&E-stained sections. The number of germ cells was also counted in each high-power field (HPF). Ten HPFs from each slide of each animal of each group were used.

Statistical analysis

Data obtained from hormonal assay and morphometry were expressed as mean±SD. They were fed into the computer using statistical package for the social sciences (SPSS, version 20; IBM, Armonk, New York, USA) software package. Statistical analysis was carried out using one-way analysis of variance and the post-hoc test (Scheffe) for pairwise comparison. The level of significance was set at P value less than 0.05.

Results

Hormonal assay (serum testosterone level)

The mean value of serum testosterone levels in PJ-alone-treated rats (group II) did not show any significant difference when compared with the control group (P1=0.990). In contrast, the mean value of testosterone levels in TCS-treated rats (group III) was significantly lower as compared with the control group (P1<0.001), whereas coadministration of PJ with TCS (in group IV) was associated with significantly higher serum testosterone levels as compared with the TCS group (P2=0.001), although still significantly lower compared with the control group (P1=0.013) (Table 1).

Table 1
Table 1:
Comparison between the different studied groups according to serum testosterone level

Histological results (H&E stain)

Group I (the control group)

The control rat testis was covered with a connective tissue capsule, tunica albuginea. The parenchyma was made up of many STs, nearly of the same shape and size. Each tubule was bound by a basement membrane and flat peritubular myoid cells, and was lined by Sertoli cells as well as several layers of spermatogenic cells. Sertoli cells showed large vesicular nuclei with prominent nucleoli. The basal layer of spermatogenic cells was formed of spermatogonia that exhibited rounded to oval nuclei. Primary spermatocytes were seen above the spermatogonia and were the largest cells in the spermatogenic series. They exhibited large rounded nuclei with variable patterns of chromatin distribution according to the prophase stage of the first meiotic division. The spermatids were seen as several rows of cells at the adluminal compartment of the tubules. They were smaller than primary spermatocytes with pale nuclei. Spermatozoa were seen in the lumen of the tubules. The narrow interstitial spaces in between the tubules were occupied by stromal elements and clusters of Leydig cells with acidophilic cytoplasm and vesicular nuclei (Fig. 1a and b).

Figure 1
Figure 1:
Light photomicrographs of control rat testis (group I) showing (a) sections of closely packed well-organized seminiferous tubules (STs) with normal epithelial stratification. Clusters of interstitial Leydig cells (L) are seen in between the tubules. (b) High-power view of a cross-section of an ST illustrating several layers of spermatogenic cells: spermatogonia (1), primary spermatocytes (2), spermatids (3), and spermatozoa (4). Sertoli cells with large vesicular nuclei (↑) are seen resting on the basement membrane. The interstitial spaces contain clusters of Leydig cells (L). Note flat myoid cells (∧) surrounding the tubules. H&E, (a) ×200, (b) ×400.

Group II (the pomegranate juice group)

The histological characteristics of the STs in this group were quite similar to those of the control group. Well-organized tubules lined with Sertoli cells and full complement of spermatogenic cells were evident.

Group III (the triclosan group)

Examination of H&E-stained sections of the testis in this group revealed pronounced morphological alterations of most of the STs. Considerable reduction in the spermatogenic cell mass was evident with a consequent thinning out of the layers of lining cells that exhibited deeply stained nuclei. Individual cellular loss was evidenced by empty spaces between the spermatogenic cells (Figs 2 and 3). In addition, many STs revealed cytoplasmic vacuolation of the lining cells (Fig. 2a). The STs further attained relatively wide lumina with paucity of sperm in the lumen. Exfoliated germ cells in the tubular lumen were frequently encountered as well. The interstitial Leydig cells were seen in between the STs, many of which exhibited dark nuclei (Fig. 3a).

Figure 2
Figure 2:
(a, b) Light photomicrographs of rat testis of group III (triclosan group) showing a noticeable diminution in the number of lining spermatogenic cells and mature sperms. Many lining cells exhibit vacuolated cytoplasm and deeply stained nuclei (↑). Note focal loss of spermatogenic cells leaving empty spaces (∧). L, clusters of Leydig cells. H&E, (a) ×200, (b) ×400.
Figure 3
Figure 3:
(a, b) Light photomicrographs of rat testis of group III (triclosan group) showing the lining cells of seminiferous tubules (STs) with deeply stained nuclei, some of which have vacuolated cytoplasm (↑). One ST reveals exfoliated cells in the lumen (*). Note focal loss of germ cells (∧). L, clusters of Leydig cells with dark nuclei. H&E, (a) ×200, (b) ×400.

Group IV (the triclosan+pomegranate juice group)

Coadministration of PJ with TCS resulted in evident improvement in the histological characteristics of the STs that revealed preserved signs of active spermatogenesis and retained normal appearance and thickness of their lining (Fig. 4). However, some STs still depicted vacuolated cells with deeply stained nuclei in the basal compartment (Fig. 5a and b). Apparently normal clusters of Leydig cells occupied the interstitial spaces between the tubules.

Figure 4
Figure 4:
Light photomicrograph of rat testis of group IV (triclosan+pomegranate juice group) showing seminiferous tubules with normal looking spermatogenic lining cells and Sertoli cells (↑). L, Leydig cells with vesicular nuclei. H&E, ×200.
Figure 5
Figure 5:
Light photomicrographs of rat testis of group IV (triclosan+pomegranate juice group) showing (a) some closely packed seminiferous tubules (STs) with several layers of spermatogenic lining cells. Some germ cells in the basal compartment exhibit vacuolated cytoplasm and deeply stained nuclei (↑). The lumina are full of sperms. L, Leydig cells. (b) A high-power view of a cross-section of an ST showing multiple germ cells in the basal compartment with vacuolated cytoplasm and dark nuclei (↑). H&E, (a) ×200, (b) ×400.

Ultrastructural results

Group I (the control group)

Electron microscopic examination of control rat testis revealed several STs bound by elongated myoid cells encircling the basal lamina. The tubules were lined by Sertoli cells and spermatogenic cells. Sertoli cells were identified by their large indented euchromatic nuclei and prominent nucleoli. Their lateral processes were interconnected by intact junctional complexes. Their cytoplasm revealed multiple mitochondria, profiles of endoplasmic reticulum (ER), and lysosomes (Fig. 6). Two types of spermatogonia were further encountered. Type A spermatogonia appeared as dome-shaped cells lying immediately over the basal lamina. Both pale (Fig. 7a) and dark (Fig. 7b) varieties of type A cells were identified. Type B spermatogonia could be distinguished by their rounded nuclei with coarser clumps of peripheral heterochromatin (Fig. 7c). Primary spermatocytes were identified by their relatively large rounded nuclei that showed many patterns of chromatin condensation. Their cytoplasm was relatively electron lucent and exhibited many dispersed mitochondria (Fig. 8a). Spermatids were smaller than primary spermatocytes and seen in various stages of differentiation. They showed euchromatic nuclei, related to an acrosomal cap. Their cytoplasm revealed several peripherally located mitochondria, profiles of ER, and lysosomes (Fig. 8b). A large number of spermatozoa were seen in the tubular lumina with their characteristic head and tail (Fig. 8c).

Figure 6
Figure 6:
Electron photomicrograph of control (group I) rat seminiferous tubule showing a Sertoli cell resting on the basal lamina (b). The cell depicts a large indented nucleus (N) with prominent nucleolus (n). The cytoplasm shows some mitochondria (M) and profiles of smooth endoplasmic reticulum (R). A part of primary spermatocyte (P) is also seen. L, lysosome; m, myoid cell. ×3000.
Figure 7
Figure 7:
Electron photomicrographs of control (group I) rat seminiferous tubule showing (a) pale type A spermatogonium broadly applied to the basal lamina (b). It exhibits an oval nucleus (N) with scanty heterochromatin. A part of Sertoli cell (S) is also seen. L, lysosome; m, myoid cell; M, mitochondria; R, smooth endoplasmic reticulum; ↑, intercellular junctional complex. (b) A dark type A spermatogonium with a rounded nucleus (N) and prominent nucleolus (n) is seen. Parts of Sertoli cell (S) and primary spermatocyte (P) are also evident. L, lysosome; m, myoid cell; M, mitochondria; R, smooth endoplasmic reticulum; ↑, junctional complex. (c) A type B spermatogonium with a rounded nucleus (N) and coarser clumps of peripheral heterochromatin is seen. The adjacent and overlying Sertoli cells depict some mitochondria (M) and lysosomes (L). b, basal lamina; m, myoid cell; R, endoplasmic reticulum; ↑, junctional complex. (a, c) ×4000, (b) ×2500.
Figure 8
Figure 8:
Electron photomicrographs of control (group I) rat seminiferous tubule showing (a) a primary spermatocyte (P) exhibits a large rounded nucleus (N) with dispersed chromatin. The cytoplasm reveals several mitochondria (M). (b) A spermatid with an acrosomal cap (↑) covers the anterior hemisphere of the nucleus (N). Its cytoplasm depicts peripherally located mitochondria (M). Parts of two adjacent spermatids are also seen. (c) Cross-sections through sperm tails. The middle piece (MP) shows an axoneme and nine outer dense fibers surrounded by circumferentially oriented mitochondria (inset 1). The principal piece (PP) appears with its fibrous sheath and the end piece (EP) is formed of an axoneme surrounded only by a flagellar membrane. A longitudinal section of sperm head with deeply condensed nucleus and fully formed acrosome (↑) is seen in inset 2. (a) ×2500, (b) ×4000, (c) ×5000, inset 1 ×13 000, inset 2 ×15 000.

Group II (the pomegranate juice group)

The ultrastructural features of the STs in rats of this group were apparently similar to those of the control group.

Group III (the triclosan group)

Ultrastructural examination of rat testis clarified evident degenerative and microvacuolar changes in the STs. The Sertoli cells exhibited considerable cytoplasmic vacuolation with accompanying dilatation of ER. The cells further revealed degenerated mitochondria with disrupted cristae and a relative increase in the lysosomal content (Fig. 9a and b). Some cells depicted areas of rarified cytoplasm (Figs 10 and 11a). Nevertheless, their nuclei were apparently normal (Fig. 11a). All types of spermatogenic cells were affected as well. Many spermatogonia revealed wide perinuclear cisternae and cytoplasmic vacuolation (Figs 9a and 10), whereas others exhibited small dense nuclei with clumped chromatin and dense cytoplasmic contents (Fig. 11b). Primary spermatocytes showed irregular nuclei with wide perinuclear cisternae along with cytoplasmic vacuolation (Figs 9a and b and 12a). As for the spermatids, they revealed nuclear deformities and multiple cytoplasmic vacuoles of variable sizes (Fig. 12b) as well as localized areas of cytoplasmic depletion (Fig. 12c). In addition, many deformed sperms with abnormal defective heads (Fig. 13a) and swollen degenerated mitochondria (Fig. 13b) were frequently encountered. Sperms with interrupted circumferential ribs of the principal piece were further revealed in the tubular lumina (Fig. 13c).

Figure 9
Figure 9:
Electron photomicrographs of group III rat testis (triclosan group) showing (a) Sertoli cell with its characteristic nucleus (N) resting on the basal lamina (b). Its cytoplasm exhibits multiple vacuoles (V) and numerous lysosomes (L). An adjacent type A spermatogonium (A) reveals an oval nucleus with irregularly dilated perinuclear cisterna (↑) and vacuolated cytoplasm (∧). A primary spermatocyte (P) with a dilated perinuclear cisterna (↑↑) is also seen. (b) Sertoli cell (S) depicts multiple swollen mitochondria (M) with disrupted cristae, dilated endoplasmic reticulum (R), and some cytoplasmic vacuoles (V). A primary spermatocyte (P) shows irregularly dilated perinuclear cisterna(↑). b, basal lamina; m, myoid cell; N, Sertoli cell nucleus. (a) ×2000, (b) ×4000.
Figure 10
Figure 10:
Electron photomicrograph of group III rat testis (triclosan group) showing two adjacent spermatogonia (SP) that exhibit irregular nuclei (N) with wide perinuclear cisternae (↑) and multiple cytoplasmic vacuoles (V1). The intervening Sertoli cell (S) shows some cytoplasmic vacuoles (V2) and localized areas of rarified cytoplasm (*). b, basal lamina; M, mitochondria. ×2000.
Figure 11
Figure 11:
Electron photomicrographs of group III rat testis (triclosan group) showing (a) Sertoli cell with normal nucleus (N), large areas of rarified cytoplasm (*), and some cytoplasmic vacuoles (V). One spermatogenic cell reveals many cytoplasmic vacuoles (↑). b, basal lamina; L, lysosome; M, mitochondria; P, primary spermatocyte; ∧, intercellular junction. (b) Two adjacent spermatogonia (SP): the left one reveals a small dense nucleus (N1) with clumped chromatin and large cytoplasmic vacuoles (V1); the right one shows localized widening of the perinuclear cisterna (↑) and rarified cytoplasm (*). The intervening Sertoli cell (S) reveals multiple cytoplasmic vacuoles (V2). b, basal lamina; L, lysosome; m, myoid cell; M, mitochondria; ∧, intercellular junctional complex. (a) ×2500, (b) ×3000.
Figure 12
Figure 12:
Electron photomicrographs of group III rat testis (triclosan group) showing (a) primary spermatocytes with irregular nuclei (N) with wide perinuclear cisternae (↑) and some cytoplasmic vacuoles (V). (b) Multiple spermatids with many cytoplasmic vacuoles (V) of variable sizes. One spermatid shows defective nucleus (N) with blebbing of the nuclear envelope (↑). M, peripherally located mitochondria. (c) Adjacent spermatids reveal some cytoplasmic vacuoles (V) and areas of rarified cytoplasm (*). One spermatid reveals uneven chromatin distribution of the nucleus (N1). The other depicts a defective nucleus (N2). M, mitochondria. (a, c) ×2500, (b) ×3000.
Figure 13
Figure 13:
Electron photomicrographs of group III rat testis (triclosan group) showing (a) defective heads (↑) of spermatozoa. M, mitochondria. (b) The middle piece of many spermatozoa shows swollen mitochondria with disrupted cristae (↑). (c) Several transversely sectioned sperm tails at the principal piece level show interrupted circumferential ribs (↑). (a) ×4000, (b) ×5000, (c) ×7500.

Group IV (the triclosan+pomegranate juice group)

The majority of different spermatogenic cell types and Sertoli cells retained their normal ultrastructural appearance (Fig. 14a, b, and c). Yet, some Sertoli cells still depicted an apparent increase in the lysosomal content (Fig. 15a) and some degenerated mitochondria with disrupted cristae (Fig. 15b). Some spermatogonia (Figs 15b and 16a) and primary spermatocytes (Fig. 16b) still exhibited wide perinuclear cisternae. Individual spermatids revealed nuclear disruption and mild cytoplasmic vacuolation (Fig. 17a and b).

Figure 14
Figure 14:
Electron photomicrographs of a rat seminiferous tubule of group IV (the triclosan+pomegranate juice group) showing (a) an intact Sertoli cell (S) and dark type A spermatogonium (A) resting on the basal lamina (b). M, mitochondria; R, endoplasmic reticulum; ↑, intercellular junctional complex. (b) An early spermatid with an oval nucleus (N) and intact overlying acrosomal cap (↑). The cytoplasm depicts several peripherally located mitochondria (M). L, lysosome. (c) Cross-section through the tails of spermatozoa reveals intact middle piece (↑), principal piece (∧), and end piece (↑↑). (a, b) ×3000, (c) ×5000.
Figure 15
Figure 15:
Electron photomicrographs of a rat seminiferous tubule of group IV (the triclosan+pomegranate juice group) showing (a) Sertoli cell (S) with numerous lysosomes (L) and normal mitochondria (M). Note wide intercellular space (*). b, basal lamina; m, myoid cell; P, primary spermatocyte. (b) Sertoli cell (S) exhibits numerous lysosomes (L) and swollen mitochondria (M) with disrupted cristae. An adjacent dark type A spermatogonium (SP) reveals an irregular nucleus (N) with a wide perinuclear cisterna (↑). b, basal lamina; ld, lipid droplet; ∧, intercellular junctional complex. (a) ×2000, (b) ×4000.
Figure 16
Figure 16:
Electron photomicrographs of a rat seminiferous tubule of group IV (the triclosan+pomegranate juice group) showing (a) pale type A spermatogonium (SP) resting on an irregular basal lamina (b) and exhibiting a large oval nucleus (N1) with localized dilatation of perinuclear cisterna (↑). N2, Sertoli cell nucleus; ∧, intercellular junctional complex. (b) Primary spermatocyte with a large nucleus (N) depicts irregularly dilated perinuclear cisterna (↑). Part of an adjoining primary spermatocyte (P) is also seen. (a) ×3000, (b) ×2500.
Figure 17
Figure 17:
Electron photomicrographs of a rat seminiferous tubule of group IV (the triclosan+pomegranate juice group) showing (a) a spermatid with an oval nucleus (N) and intact acrosomal cap (↑) exhibiting some cytoplasmic vacuoles (V). L, lysosome; M, mitochondria. (b) Adjacent spermatids reveal localized nuclear disruption (↑) and some cytoplasmic vacuoles (V). G, prominent Golgi apparatus; M, peripherally located mitochondria; ∧, intact acrosomal cap. ×3000.

Morphometric results

Data in Table 2 revealed that the mean number of germ cells/HPF was significantly reduced in TCS-treated rats (group III) as compared with the control group. There was likewise a significant decrease in the germinal epithelial height of the STs as compared with control (P1<0.001). In contrast, in comparison with the TCS-treated group III, animals cotreated with TCS and PJ (group IV) showed a significant increase in both parameters (P2<0.001).

Table 2
Table 2:
Comparison between the different studied groups according to number of germ cells/high-power field and seminiferous tubules germinal epithelial height

Discussion

Public concern regarding environmental hazards is perhaps greatest when potential exposures are related to reproductive health 29. TCS is an environmental contaminant of emerging concern because of its presence in a wide variety of personal care and household products, as well as in the ecosystem and in human body fluids. TCS has been subjected to several toxicological studies; yet, little information is currently available concerning its adverse effects on the reproductive system 30,31. Moreover, the highly sensitive cellular composition of the spermatogenic epithelium makes the testis more vulnerable to environmental hazards compared with other tissues. A research work concerned with treatment of male infertility problems has issued the existence of a link between the antioxidant-rich PJ and male fertility 23. Therefore, the study aimed to histologically clarify the possible testicular alterations that might occur in the STs of adult rats following TCS administration, as well as the possible protective role of PJ coadministration.

In the current work, at the chosen dose of TCS (20 mg/kg/day), the mean serum testosterone level in group III rats was significantly reduced as compared with controls, suggesting that TCS has an inhibitory effect on testosterone production. A similar reduction in serum testosterone levels following TCS administration has been reported by previous researchers 13,32. Such hormonal alteration was parallel with the degenerative changes demonstrated histologically in the STs of treated animals. For instance, the tubules revealed considerable reduction in the number of spermatogenic lining cells with the frequent appearance of deeply stained nuclei. Many tubules exhibited cytoplasmic vacuolation of the lining cells along with exfoliated germ cells in the tubular lumen. Ultrastructurally, Sertoli cells evidenced considerable vacuolation of the cytoplasm with degenerated mitochondria and prominent lysosomal activity. Although spermatogonia are considered the most resistant germ cells, 60-day exposure to TCS appeared to overcome their resistance to injury. Many cells exhibited small dense nuclei and dense vacuolated cytoplasm, whereas others depicted less degenerative changes with wide perinuclear cisternae. Primary spermatocytes and spermatids were also among the affected germ cells. They showed nuclear irregularities, wide perinuclear cisternae, and cytoplasmic vacuolation. Moreover, spermatozoa exhibited deformed heads and tails with the appearance of swollen mitochondria in the middle piece, and interrupted circumferential ribs. Morphometric and statistical analyses of the germinal epithelial height of STs and the number of germ cells/HPF confirmed these results as they showed significantly reduced mean values compared with the control group. These findings suggest that long-term exposure to low doses of TCS results in considerable damaging effect on all spermatogenic stages starting from spermatogonia.

The results of this work are consistent with those of Kumar et al. 13, who reported that TCS administration was associated with degenerative changes in the STs as well as in sex accessory organs of adult rats – namely, the cauda epididymis, vas deferens, and prostate. They attributed such histological alterations to the decrease in the levels of testosterone and androgen receptors in treated rats. They further recorded low sperm count in the testis of treated animals as compared with controls, probably because of reduced testicular spermatogenesis induced by TCS.

The testosterone hormone plays a crucial role in the initiation, maintenance, and quantitative and qualitative regulation of spermatogenesis and spermiogenesis processes 33 mainly through its stabilizing action on Sertoli cells. It has been postulated by several investigators that deprivation of testosterone affects these processes and causes a decrease in the number of spermatogenic cells. The underlying mechanisms have been attributed to the altered function of Sertoli cells that deprives the developing spermatogenic cells from the necessary survival factors rather than to a direct effect on the germ cells 34. In this context, the decrease in plasma testosterone levels observed in the present study might explain the decreased height of the spermatogenic epithelium and the number of lining germ cells in TCS-treated rats. Moreover, it has been previously shown that decreased testosterone levels are associated with histological alterations in Sertoli and Leydig (androgen target) cells 35. In this context, the possibility exists that the ultrastructural alterations of Sertoli cells observed here might be related to the decreased testosterone levels.

The mechanism responsible for low serum testosterone concentrations following TCS administration is not exactly known 5. Nevertheless, it has been speculated that TCS suppresses steroidogenesis as a result of an effect on luteinizing hormone (LH) secretion, thereby implicating the pituitary–gonadal axis as a target for endocrine disruption 13. It is well known that when LH binds to its receptor in a Leydig cell it results in activation of various agents of steroidogenic cascade, causing an increased production of testosterone. Kumar et al. 36 confirmed the antiandrogenic activity of TCS in Leydig cells in vitro through disruption of the entire cAMP-dependent steroidogenic pathway. Moreover, TCS has been speculated to decrease serum cholesterol level, which, in turn, results in downregulation of steroidogenesis 37.

The steroidogenic acute regulatory (StAR) protein is a factor that plays a crucial role in regulating steroidogenesis by transporting cholesterol to the inner mitochondrial membrane for its utilization by steroidogenic enzymes. Kumar et al. 13 further reported a decreased transcription and translation of StAR in the testis following TCS administration. They attributed such alteration to the decreased level of serum LH as it simulates steroidogenesis mostly by regulating the level and activity of StAR protein in steroidogenic cells. In addition, TCS may have direct effects on mitochondria, impairing its function through an uncoupler effect and disrupting mitochondrial membrane fluidity. Besides, it directly affects ATP, causing a significant inhibition of the enzyme activity, considering the results obtained with disrupted mitochondria 38.

Although the decrease in testosterone production is an important factor implicated in TCS-induced testicular injury, oxidative stress (OS) has been recently suggested as another mechanism responsible for the cytotoxic effect of TCS. Tamura et al. 39 postulated that TCS at sublethal concentrations induces OS, which decreases the cellular thiol content with a consequent increase in intracellular zinc concentration by its release from intracellular stores in rat cells. It has been postulated that zinc impairs the antioxidative system through NADPH-dependent mechanisms and promotes the intracellular production of reactive oxygen species (ROS) 40. Thus, TCS-induced disturbance of cellular zinc homeostasis may induce adverse actions on the cells.

Kawanai 41 has reported that TCS increases intracellular Ca2+ concentration by promotion of Ca2+ influx, which consequently activates Ca2+-dependent K+ channels and induces membrane hyperpolarization in rat cells. The persistent increase in membrane K+ permeability by intracellular Ca2+ may further induce adverse action on the cells that is independent from the change in membrane potential. It is documented that mitochondrial uptake of intracellular Ca2+ is associated with ROS generation and subsequently OS. The increase in ROS triggers the opening of mitochondrial permeability transition pore and inner membrane anion channel. This, in turn, leads to simultaneous collapse of the mitochondrial membrane potential and increased ROS generation. The generated ROS can be released into the cytoplasm and trigger ROS-induced ROS release in neighboring mitochondria. This mitochondrion-to-mitochondrion ROS signaling constitutes a positive feedback mechanism for enhanced ROS production, leading to significant mitochondrial and cellular injury 42. In agreement with such a concept, Lin et al. 43 have reported a significant increase in the concentration of malondialdehyde, a lipid peroxidation marker, in earthworms following TCS exposure, indicating the production of ROS.

There is evidence that TCS can influence the endocrine function indirectly through effects on the metabolism of key hormones, including thyroid hormones, because it chemically mimics the thyroid hormone 7. Earlier studies have demonstrated that TCS exposure decreases thyroid hormone concentrations in a dose-dependent manner 5,12. Evidence suggests that TCS upregulates phase II glucuronidation and sulfation of thyroid hormones in the liver, enhancing their catabolism that results in hypothyroxinemia 44,45. Thus, testicular alterations induced by TCS could be attributed, at least in part, to disruption of thyroid hormone homeostasis.

Pomegranate and its constituents have been consumed for centuries without adverse effects. In the present work, rats of group II that received PJ alone did not show any significant changes in serum testosterone levels, or in the histological or histomorphometric characteristics of the STs, compared with the control group. These results are consistent with previous studies that have postulated that pomegranate, in various forms, can be included as part of a healthy lifestyle with no risk of toxic reactions 46. In the USA, the Food and Drug Administration has approved pomegranate as Generally Recognized as Safe 47. Research in human volunteers has demonstrated the safety of pomegranate extract in amounts up to 1420 mg/day (870 mg gallic acid equivalents) for 28 days, with no adverse effects 48. Another study in patients with carotid artery stenosis demonstrated that PJ consumption (121 mg/l ellagic acid equivalents) for up to 3 years has no toxic effect 49. In addition, punicalagin has been proved to have no toxic effect in rats as confirmed by histological examination of rat organs 50.

Concomitant administration of PJ with TCS for 60 days was associated with preservation of the histological and histomorphometric characteristics of most of the STs that retained their normal epithelial stratification. Such prevailed improvement was further supported by the significant increase in serum testosterone levels as compared with TCS (group III)-treated rats, indicating the protection of Leydig cells as well.

Pomegranate has been revered as a symbol of fertility by several civilizations, and today modern science is validating this belief. Several researchers have pointed out an improvement in epididymal sperm concentration and motility, germ cell layer thickness, and diameter of STs in nonstressed healthy laboratory animals following pomegranate administration 23,51. In addition, in-vitro PJ supplementation to rooster’s semen improves sperm motility, viability, and acrosomal integrity during cool storage 52. Moreover, PJ and pomegranate extracts have been reported to ameliorate spermatogenic disruption and damage in sperm quality associated with OS, induced by lead acetate 53. Similarly, Abdou et al. 54 have demonstrated that pomegranate peels and seeds decrease carbon tetrachloride-induced sperm shape abnormalities.

A wealth of evidence indicates high antioxidant and free radical scavenging capacity of PJ attributable mainly to its protective effects 52,55. Its antioxidant activity is considered the highest among most of the plants, and this is mostly attributed to its rich polyphenolic content, mainly ellagic acid and punicalagin 53. In such a context, several studies have postulated a modulatory effect of ellagic acid on OS-induced male reproductive dysfunction in experimental animals, thus improving male fertility. This is indicated by increased epididymal sperm concentration, glutathione, glutathione peroxidase, and catalase activities along with improvement in the testicular histological view 56,57. In addition, quercetin, which has been shown to be a component in pomegranate, is reported to improve sperm quality 53.

The powerful antioxidants in pomegranate act by boosting the levels of glutathione, which helps to protect DNA in the cells from free radical damage. After oral intake of PJ, the antioxidant enzymatic activities (catalase and glutathione peroxidase) increase, whereas lipid peroxidation (malondialdehyde level) decreases 23. PJ further increases the enzymes that protect LDL and HDL from oxidation 23,58.

PJ is a rich source of vitamins A, C, and E 22. Several authors have reported the positive influence of these antioxidant vitamins on male reproductive functions. For instance, vitamin A enhances testosterone production and permits the maintenance of Sertoli cell tight junctions that contribute to the blood–testis barrier 52,59. It further plays indispensable roles in spermatogenesis by promoting differentiation of spermatogonia, adhesion of germ cells to Sertoli cells, and release of mature sperm into the lumen of STs 59,60. Pomegranate fruit also contains a significant amount of vitamin C 19. In the male reproductive system, vitamin C is known to protect spermatogenesis, and it plays a major role in semen integrity and fertility in both men and animals. It increases serum testosterone levels as well 61,62. Vitamin C is an important antioxidant, contributing up to 65% of the total antioxidant capacity of seminal plasma. Several authors have postulated the protective effect of vitamin C on testicular OS 63.

Evidence suggests that PJ and vitamin C increase the production of natural antioxidants in the sperm and blood of rats, which probably protects the vulnerable fatty acids against oxidation 23,64. Again, vitamin C is known to support spermatogenesis at least, in part, through its capacity to stimulate both sperm production and testosterone secretion 64.

The powerful antioxidant capacity of PJ might explain the relatively normal ultrastructure of mitochondria in group IV rats. PJ supplies the daily recommended dose of vitamin C and hence its protective effect on the mitochondria through stimulation of complex 1 enzyme of the mitochondrial respiratory chain 22,65. Inhibition of complex 1 is known to disrupt mitochondrial respiration and stimulates the mitochondrial production of ROS. Following OS, a 3–10-fold increase in mitochondrial DNA damage has been reported, compared with that of the nuclear DNA 66. Vitamin C is known to protect mitochondrial DNA against oxidative damage 67. In addition, vitamin E is known to maintain the structural integrity of sperm 68,69. Therefore, the above-mentioned multiple properties of PJ may explain its protective effects on the histological characteristics of the STs and on the serum testosterone levels in rats receiving it with TCS, indicating that PJ administration is safe and beneficial too.

Overall, the results of this work reinforce the notion that TCS poses a hazard to male reproductive health; therefore, it would be wise to restrict its use. Furthermore, the results recommend PJ as a good remedy and nontoxic adjunct against male reproductive dysfunction. Additional studies are needed to more fully characterize the effect of TCS on the hypothalamic–pituitary–gonadal axis, as well as the potential adverse health outcomes of these changes.

Acknowledgements

Conflicts of interest

There are no conflicts of interest.

Table
Table:
No title available.

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Keywords:

histology; pomegranate juice; seminiferous tubules; testosterone; triclosan; ultrastructure

© 2014 The Egyptian Journal of Histology