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Vitamin E modifies the ultrastructure of testis and epididymis in mice exposed to lead intoxication
Accepted Manuscript Title: Vitamin E modifies the ultrastructure of testis and epididymis in mice exposed to lead intoxication Authors: Mohamed A. Fahim, Saeed Tariq, Ernest Adeghate PII: DOI: Reference:
S0940-9602(12)00164-1 doi:10.1016/j.aanat.2012.11.001 AANAT 50746
To appear in: Received date: Revised date: Accepted date:
19-3-2012 3-11-2012 13-11-2012
Please cite this article as: Fahim, M.A., Tariq, S., Adeghate, E., Vitamin E modifies the ultrastructure of testis and epididymis in mice exposed to lead intoxication, Annals of Anatomy (2010), doi:10.1016/j.aanat.2012.11.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Vitamin E modifies the ultrastructure of testis and epididymis in mice exposed to lead intoxication Mohamed A. Fahim1, Saeed Tariq2, Ernest Adeghate2 *
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Departments of Physiology1 and Anatomy2, Faculty of Medicine & Health Sciences, United Arab Emirates University, P. O. Box 17666, Al Ain, United Arab Emirates
Address correspondence to: Ernest Adeghate MD, PhD Departments of Anatomy,
Faculty of Medicine & Health Sciences, United Arab Emirates University,
P. O. Box 17666, Al Ain, United Arab Emirates E mail: [email protected] Telephone: +971-3-7137496 Fax: +971-3-7672033 1 Page 1 of 22
Abstract Lead (Pb) is known to cause abnormal function of several systems including the male reproductive system, where it has been shown to reduce sperm count. In order to examine the
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morphological basis of the reduction in sperm count and a possible effect of vitamin E, lead acetate (1 mg/Kg body weight) was given to control and vitamin E-treated mice daily, intraperitoneally for 3 weeks. The testis and body of epididymis of the mice were subjected to
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electron microscopy study. Pb caused degenerative changes in spermatids inducing vacuolization and a reduction in the number of cytoplasmic organelles in Leydig cells. Pb also destroyed the
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stereocilia of epididymal epithelium. The addition of vitamin E ameliorated the severity of these morphological changes. In conclusion, Pb-induced reduction in sperm count may be due to
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changes in the ultrastructure of spermatids, epididymal epithelia and Leydig cells. These changes
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can be reduced by vitamin E.
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microscopy
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Key words: Vitamin E, antioxidant, lead, toxicology, testis, epididymis, spermatozoa, electron
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Introduction Lead has been in use by the human population for the past 5, 000 years. During this period lead
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production has been said to have increased from a mere 10 tons per annum to a staggering 1 million tons per year, a phenomenon that has accompanied industrial progress (Davidson and Rabinowitz M, 1992; Brown and Margolis, 2012). Lead contamination was reported to be high
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between 1900 and 1975 before changes in the United States of America (USA) laws were
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enacted (Brown and Margolis, 2012). During this period (1900 - 1975), lead was frequently in contact with the human population because it was part of the component of several industrial as well as domestic appliances, which include lead pipes, batteries, radiators and cosmetics. The
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USA federal law and those initiated worldwide to cut down the number of people with higher blood lead level started to bear results by the year 2008. The number of children (1-5 years of
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age) with a blood lead level ≥10 µg/dL was reduced from 13.5 million in 1978, to 250,000 by 2007-2008 (US Environmental Protection Agency, 2010). Although leaded gasoline has been phased out in many countries, leaded fuel is still used in some African (Algeria), Middle Eastern
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(Iraq, Yemen) and Asian (Afganistan, Myanmar and North Korea), countries (United Nations
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Environmental Program, 2012). The population of people in these countries is more than 194 million. As expected, the blood lead levels of people exposed to leaded gasoline in some of these
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countries was significantly higher than controls (Al-Rudainy, 2010) because large quantities of lead were released into the open air from the exhaust pipes of motor vehicles. While strict environmental regulations has led to a significant decrease in lead pollution over the last three decades, occupational lead contamination is still a great concern especially for workers associated with long-term exposure to lead pipe, metallurgical, lead-acid battery and lead wire factories. It is also a concern for people exposed to leaded gasoline (Al-Rudainy, 2010). In addition, children living in houses built before 1978 in the USA with lead-base water pipes or paint may have higher than normal blood lead levels (Brown and Margolis, 2012).
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Lead toxicity, whether occupational or otherwise, therefore, is a great concern because it has been implicated in a variety of health conditions including neurological (Perlstein and Attala, 1966, Han et al, 2007) and cancer (Roy, 1992; Patra et al., 2001) disorders in humans.
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Lead toxicity has been shown to impair sperm shape and reduce the number of cells in semen (Acharya et al., 1997; Eyden et al., 1978). This observation may explain some of the causes of
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idiopathic male infertility, which accounts for about 40-60% of all cases of male patients complaining of abnormal reproduction (Dohle et al, 2005). How does a heavy metal, such as lead
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cause sperm damage? The mechanism by which lead toxicity leads to impaired sperm morphology is far from clear. However, it has been shown that lead, an example of a heavy metal, is capable of inducing oxidative stress. Some organs withstand the burden of stress better
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than others. However, it has been shown that because testis in particular readily succumbs to oxidative stress (Tomascik-Cheeseman et al, 2004), it is probably not surprising that lead-
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induced oxidative stress is easily manifested in the testis. Lead, in particular, can accumulate in the reproductive system and has been implicated in the development of oxidative stress via
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induction of lipid peroxidation (Marchlewicz M, et al 2004; Mishra and Acharya, 2004). It is well known that lipid peroxidation results in the generation of reactive oxygen species. In view
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of this, many investigators have used a variety of antioxidants, including vitamin C (Hsu et al, 1998) and vitamin E (Patra et al, 2011) to prevent the occurrence and or subdue oxidative stress
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in tissues. Some of these investigations have reported beneficial effects of antioxidants on heavy metal-induced toxicity (Han et al, 2007), while others (Willems et al., 1982) have questioned the usefulness of these agents in the amelioration of heavy metal-induced lesions in the reproductive system of laboratory animals.
Most of the studies published on the effect of lead-induced toxicity on the reproductive system of laboratory animals have focused on the structure of sperm and sperm count but less on the ultrastructure of the male reproductive system. Reports of studies that examine the effect of lead toxicity are contradictory, while many have addressed only the changes observed by light microscopy (Godowicz and Galas, 1992; Ait Hamadouche et al, 2009; Suradkar et al., 2010) rather than the ultrastructural changes that can only be assessed by transmission electron microscopy. Literature reports on the ultrastructure of the male reproductive organs are even 4 Page 4 of 22
more confusing with some studies indicating that lead exposure causes no ultrastructural changes in the testis and Leydig cells (Wenda-Różewicka et al, 1996). The aim of the present work, therefore, was to investigate the toxic effect of exposure to lead on
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effect of Vitamin E on lead-induced toxicity in these organs.
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TO mice seminiferous tubules of testes, epididymis, Leydig cells and the possible protective
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Materials and Methods
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Experimental protocol
The present study was designed to observe the changes in the testes, epididymis and Leydig cells after intraperitoneal (i.p.) administration of lead acetate (1 mg/kg body weight). TO mice were
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used in this study. Mice were acclimated to laboratory conditions with regular temperature control ranging from 23±2 °C and with a balanced laboratory chow and water ad libitum. A total
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of 24 healthy male TO mice ranging from 32-35 g body weight were selected and randomly divided into 4 groups. The first group of mice (n =6) received 5% glucose solution and served as
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vehicle control group. The second group of mice was injected with lead acetate (1 mg/kg body
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weight, i.p.) and the third group was given vitamin E (Tocopherol diluted in soya oil 100 mg/kg body weight). The fourth group was treated with lead acetate (1 mg/kg body weight) and vitamin E (100 mg/kg body weight). Dilution of chemicals was made so that the volume of each injection was maintained at a volume of 0.2 ml/mouse.
Electron microscopy
After 3 weeks of 5 days per week of lead and/or vitamin E administration, all the groups were sacrificed and the testes and body of epididymis were dissected out from the same morphological area, free from other accessory tissues cut, into small pieces and washed with 0.1 M phosphate buffer and then immersed in a modified McDowell and Trump (1976) fixative for 3 hours at room temperature. After rinsing with phosphate buffer, the tissues were postfixed with 1% osmium tetroxide for 1 hour. The tissue samples were later washed with distilled water, dehydrated in a series of graded ethanol and propylene oxide and then infiltrated and embedded 5 Page 5 of 22
in Agar100 epoxy resin and polymerized at 65! C for 24 hours. Blocks were trimmed and semithin and ultrathin sections were cut with Reichert Ultracuts, ultramicrotome. Semithin sections (0.5 ! m thickness) were stained with 1% aqueous toluidine blue on glass slides and ultrathin sections (95 nm) on 200 mesh copper grids were then contrasted with uranyl acetate
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followed by lead citrate according to a previously described method (Draper et al., 1999). The
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grids were examined and photographed with a Philips CM10 Transmission Electron Microscope.
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Morphometric analysis
Electron micrographs of 20-30 sections of either seminiferous tubules, epididymis or Leydig
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cells were taken per mouse (n=6). The number of spermatozoa or cytoplasmic organelles was counted manually and blindly on the electron micrographs of 20 different sections per mouse.
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The average of the count was taken and compared with those of other groups.
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Statistical analysis
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All data were presented as mean + standard deviation. Analyses were tabulated using Microsoft Excel Sheet. The four different groups were compared using Students t-test.
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Values less or equal to 0.05 were considered to be statistically significant.
Results
Figure 1 shows the luminal region of the seminiferous tubules of control (a), mice treated with vitamin E only (b), mice treated with Pb only (c) and mice treated with both Pb and vitamin E (d). Pb caused a significant decrease in the density of spermatozoa when compared to control and or mice treated with vitamin E only. The administration of vitamin E prevented a decrease in the density of spermatozoa observed in the testis of lead-intoxicated mice. The structure of the nucleus of the spermatid of Pb-treated mice appeared disorganized. The addition of vitamin E reduced the nuclear disorganization of spermatids.
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Morphometric analysis showed lower sperm count in the lumen of seminiferous tubules of the testis of Pb-treated mice compared to controls (Fig. 2). The administration of vitamin E significantly improved the sperm count.
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The ultrastructure of Leydig cells of normal (a), mice treated with vitamin E only (b), mice treated with Pb only (c) and mice treated with both Pb and vitamin E (d) is displayed in figure 3.
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The ultrastructure of Leydig cells of normal mice and those treated with vitamin E alone showed intact cytoplasmic granules, mitochondria and endoplasmic reticuli. The ultrastructure of Leydig
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cells of mice treated with vitamin E only did not significantly differ from that of control. In contrast, Pb-treated mice showed a significant reduction in the density of cytoplasmic granules (Fig. 4) and vacuolarization in the cytoplasm of Leydig cells. In addition, the cytoplasm of the
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Leydig cells of Pb treated mice showed increased cytoplasmic processes and caveolae compared
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to those from controls and vitamin E-treated mice.
Addition of vitamin E to Pb-treated mice resulted in a marked effort by Leydig cells to
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reassemble large number of cytoplasmic organelles. However, the cytoplasmic processes of
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Leydig cell of Pb-treated mice did not disappear after the addition of vitamin E. Figure 5 shows the luminal surface of the epididymis of control (a), mice treated with vitamin E
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only (b), mice treated with Pb only (c) and mice treated with both Pb and vitamin E (d). The lumen of the epididymis of mice treated with Pb contains much fewer spermatozoa compared to those of control or mice treated with vitamin E alone. The density of spermatozoa in the lumen of the epididymis of mice treated with vitamin E only was similar to that of normal control. The lumen of the epididymis of Pb treated mice given vitamin E have large number of spermatozoa comparable to that of control or those treated with vitamin E alone. The stereocilia of the epithelial cells of the epididymis of Pb-treated mice appear to be fewer compared to those of control or vitamin E treated mice, because some of the epithelial cells have lost their stereocilia.
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Morphometric analysis showed a lower sperm count in the lumen of the epididymis of Pb-treated mice compared to controls (Fig. 6). The administration of vitamin E significantly improved the
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sperm count.
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Discussion
This study showed that Pb, as a potential occupational hazard, possesses a significant health
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hazard to the reproductive capacity of male mice. The number and density of the spermatozoa in the lumen of the seminiferous tubules and epididymis of mice treated with Pb was significantly
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lower compared to that of control or those treated with vitamin E alone. The observation that Pbtreated mice have a lower sperm count has been well documented (Acharya et al, 2003, Ait
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Hamadouche et al, 2009). In a similar study, cells of the seminiferous tubules show signs of degeneration (heterochromatic nuclei, irregular basal lamina, vacuolization) in albino rats given
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25 mg/kg body weight of lead acetate per os (El Shafai et al., 2011).
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The results of this study confirmed that lead exposure does indeed reduce the number of spermatozoa in the testis and epididymis at all developmental stages. What then are the
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morphological explanations of the lower sperm count in lead-treated mice? Several studies, at the light microscopy level, have shown that the weight of the testis of animals treated with Pb is reduced with a concomitant increase in abnormal morphological changes (Vigeh et al, 2011). The observation of this study showed that spermatids underwent degenerative changes, which may lead to a reduced number of spermatozoa. In addition, Pb also induced epididymal epithelial cells to lose their stereocilia. The loss of stereocilia may inhibit the ability of the epididymis to transport spermatozoa toward the vas (ductus) deferens. The increased morphological changes in the spermatids coupled with the inability of the epididymis to carry available spermatozoa could explain the infertility associated with lead exposure.
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The infertility observed in Pb-intoxication is probably due to the morphological changes seen in the seminiferous tubules. Pb treatment also caused vacuolization and a decrease in the number of cytoplasmic organelles of Leydig cells, the cells responsible for the production of testosterone. Testosterone, in turn, is required for the maturation of sperm. This “double jeopardy” of loss of
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viable cells within the seminiferous tubules and the impairment of the function of Leydig cells
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may add a tremendous burden to the ability of the male genitals to perform their function.
What then is the cause of the degenerative changes observed in the cells of the testis and the
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epididymis? It has been shown that Pb can cause oxidative stress (Ercal et al, 1996, Acharya et al, 2003), resulting in increased lipid peroxidation. Lead exposure has also been shown to reduce the tissue levels of testicular levels of endogenous antioxidants such as glutathione, catalase and
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superoxide dismutase in rats (2010; Abdel Moniem et al, 2010) and mice (Sharma et al (2010). These antioxidants are important markers of oxidative stress.
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Since it has been shown that oxidative stress can induce faulty signal transduction and apoptosis in cells (Adeghate, 2004), the oxidative stress induced by Pb may therefore result in the
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epididymis.
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morphological changes observed in Leydig and spermatid cells of the testis and epithelium of the
It has also been shown that Pb is capable of decreasing lactate utilization and respiration in
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Sertoli cells of the rat testis (Batarseh, 1986). This observation may be a consequence of the effect of oxidative stress.
In view of the fact that Pb exposure has been shown to induce oxidative stress in the testis of rodents (Ercal et al, 1996, Acharya et al, 2003), it was decided to examine whether vitamin E, an antioxidant, would prevent oxidative stress-induced morphological changes in the testicular, epididymal and Leydig cells of mice subjected to Pb exposure. Several studies have examined the role of antioxidants on Pb-induced oxidative stress. For example it has been shown that flaxseed oil improved the antioxidant pool in the testis of albino rats subjected to Pb intoxication (Abdel Moniem et al, 2010). Other studies examining the effect of vitamin C on chronic Pb toxicity have shown the beneficial effect of vitamin C, an antioxidant 9 Page 9 of 22
on Pb-induced changes in the testis of Wistar rats (Shaban El-Neweshy and Said El-Sayed, 2011). The results of this study showed that vitamin E increased sperm count in the testicular as well as epididymal lumina and reduced the occurrence of morphological changes in these organs. The
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observation of this study has shown that vitamin E as an antioxidant is capable of reducing the deleterious impact of Pb-induced oxidative stress in mice male reproductive organs, in
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accordance with literature reports. Mishra and Acharya (2004) showed that vitamin E reduced the impact of Pb-induced reduction in sperm count in Swiss mice. The fact that Pb can reduce the showed a strong association of both Pb and vitamin E.
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level of vitamin E in wild animals residing in Pb mining regions (Rodríguez-Estival et al, 2011) In conclusion, Pb in its acetate form caused degenerative changes in the spermatids of the
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seminiferous tubules of mice testis. It also reduced the quantity of stereocilia in the epithelial cells of the epididymis and caused cellular abnormalities in testosterone-producing Leydig cells.
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These morphological alterations may explain why Pb induces infertility in male subjects. Our
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result also showed that vitamin E can reduce the impact of Pb toxicity in the male genital organs.
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Al-Rudainy , L.A., 2010. Blood Lead Level Among Fuel Station Workers. Oman Med. J. 25(3): 208–211. American Academy of Pediatrics Committee on Environmental Health., 2005. Lead exposure in children: prevention, detection, and management. Pediatrics. 116,1036-1046. Batarseh, L.I., Welsh, M.J., Brabec, M.J., 1986. Effect of lead acetate on Sertoli cell lactate production and protein synthesis in vitro. Cell Biol Toxicol. 2, 283-92. Brown, M.J., Margolis, S., 2012. Lead in drinking water and human blood lead levels in the United States. MMWR Surveill Summ. 61 Suppl, 1-9.
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Davidson, C.I., Rabinowitz, M., 1992. Lead in the environment: from sources to human receptors. In: Needleman, H. (Ed.) Human lead exposure. Boca Raton, CRC Press, Florida, pp. 65-86.
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albino rats and the possible protective effect of ascorbic acid. Food Chem. Toxicol. 49, 734-743. Ercal, N., Treratphan, P., Hammond, T.C., Mathews, R.H., Grannemann, N.H., Spitz, D.R.,
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Han, J.M., Chang, B.J., Li, T.Z., Choe, N.H., Quan, F.S., Jang, B.J., Cho, I.H., Hong, H.N., Lee, J.H., 2007. Protective effects of ascorbic acid against lead-induced apoptotic neurodegeneration in the developing rat hippocampus in vivo. Brain Res. 1185, 68-74. Hsu, P.C., Liu, M.Y., Chen, L.Y., Guo, Y.L., 1998. Effects of vitamin E and/or C on reactive oxygen species-related lead toxicity in rat sperm. Toxicology. 128, 169 - 179. 12 Page 12 of 22
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Figure legend Figure 1 shows the luminal region of the seminiferous tubules of control (a), mice treated with
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vitamin E only (b), mice treated with Pb only (c) and mice treated with Pb and vitamin E (d). Note the significant decrease in the density of spermatozoa in the lumen (**) of seminiferous
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tubules of Pb-treated rats (c) compared to control (a) and those of mice treated with vitamin E only (b). The spermatids of Pb-treated mice show signs of degeneration (arrow). Scale bar = 2
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µm
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Figure 2 shows the average number of spermatozoa in the lumen of 20 sections of seminiferous tubules per mouse (n=6) in the testis of normal mice (a), mice treated with vitamin E only (b), mice treated with Pb only (c) and mice treated with both Pb and vitamin E (d). Note a significant
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reduction in the number of spermatozoa in lead treated mice compared to control. There is a significant improvement in the sperm count of mice treated with vitamin E after lead
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administration. Control vs control treated with Vit E (p > 0.2): not significant; Control vs Control treated with Pb (***p < 0.0000006): highly significant; Control treated with Vit E vs Control
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treated with Pb (p < 0.0001) highly significant; Control treated with Pb vs Control treated with
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Pb and Vit E (*p < 0.00002): highly significant. Figure 3 shows the ultrastructure of Leydig cells of normal (a), mice treated with vitamin E only (b), mice treated with Pb only (c) and mice treated with both Pb and vitamin E (d). Note that the Leydig cells of Pb-treated mice (c) showed a significant reduction in the density of cytoplasmic granules, increased vacuolarization (thick arrow) and pinching of the plasma membrane (thin arrow) compared to controls. Electron lucent secretory granules (arrow head) were observed in the cytoplasm of the Leydig cells of mice treated with both Pb and vitamin E. Scale bar = 2 µm Figure 4 shows the average number of endocrine granules in 20 sections of Leydig cells per mouse (n=6) in the testis of normal (a), mice treated with vitamin E only (b), mice treated with Pb only (c) and mice treated with both Pb and vitamin E (d). Note a significant reduction in the number of endocrine granules in lead treated mice compared to control. There is a significant 15 Page 15 of 22
improvement in the number of Leydig granules in mice treated with vitamin E after lead administration. Control vs control treated with Vit E (p > 0.5): not significant; Control vs Control treated with Pb (***p < 0.00001): highly significant; Control treated with Vit E vs Control treated with Pb (p < 0.0001) highly significant; Control treated with Pb vs Control treated with
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both Pb and Vit E (*p < 0.00005): highly significant.
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Figure 5 shows the luminal surface of the epididymis of control (a), mice treated with vitamin E only (b), mice treated with Pb only (c) and mice treated with Pb and vitamin E (d). Note that the
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lumen of the epididymis of mice treated with Pb contains much fewer spermatozoa (arrow) compared to those of control or mice treated with vitamin E alone. Some epithelial cells of
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epididymis of Pb-treated rats are devoid of stereocilia (arrow head). Scale bar = 2 µm
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Figure 6 shows the average number of spermatozoa in the lumen of 20 sections of epididymis per mouse (n=6) of control (a), mice treated with vitamin E only (b), mice treated with Pb only (c) and mice treated with both Pb and vitamin E (d). Note a significant reduction in the number of
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spermatozoa in lead treated mice compared to control. There is a significant improvement in the
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sperm count of mice treated with vitamin E after lead administration. Control vs Control treated with Vit E (p > 0.7): not significant; Control vs Control treated with Pb (***p < 0.0000002):
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highly significant; Control treated with Vit E vs Control treated with Pb (p < 0.0001): highly significant; Control treated with Pb vs Control treated with both Pb and Vit E (*p < 0.000006): highly significant.
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S0940-9602(12)00164-1 doi:10.1016/j.aanat.2012.11.001 AANAT 50746
To appear in: Received date: Revised date: Accepted date:
19-3-2012 3-11-2012 13-11-2012
Please cite this article as: Fahim, M.A., Tariq, S., Adeghate, E., Vitamin E modifies the ultrastructure of testis and epididymis in mice exposed to lead intoxication, Annals of Anatomy (2010), doi:10.1016/j.aanat.2012.11.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Vitamin E modifies the ultrastructure of testis and epididymis in mice exposed to lead intoxication Mohamed A. Fahim1, Saeed Tariq2, Ernest Adeghate2 *
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Departments of Physiology1 and Anatomy2, Faculty of Medicine & Health Sciences, United Arab Emirates University, P. O. Box 17666, Al Ain, United Arab Emirates
Address correspondence to: Ernest Adeghate MD, PhD Departments of Anatomy,
Faculty of Medicine & Health Sciences, United Arab Emirates University,
P. O. Box 17666, Al Ain, United Arab Emirates E mail: [email protected] Telephone: +971-3-7137496 Fax: +971-3-7672033 1 Page 1 of 22
Abstract Lead (Pb) is known to cause abnormal function of several systems including the male reproductive system, where it has been shown to reduce sperm count. In order to examine the
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morphological basis of the reduction in sperm count and a possible effect of vitamin E, lead acetate (1 mg/Kg body weight) was given to control and vitamin E-treated mice daily, intraperitoneally for 3 weeks. The testis and body of epididymis of the mice were subjected to
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electron microscopy study. Pb caused degenerative changes in spermatids inducing vacuolization and a reduction in the number of cytoplasmic organelles in Leydig cells. Pb also destroyed the
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stereocilia of epididymal epithelium. The addition of vitamin E ameliorated the severity of these morphological changes. In conclusion, Pb-induced reduction in sperm count may be due to
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changes in the ultrastructure of spermatids, epididymal epithelia and Leydig cells. These changes
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can be reduced by vitamin E.
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microscopy
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Key words: Vitamin E, antioxidant, lead, toxicology, testis, epididymis, spermatozoa, electron
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Introduction Lead has been in use by the human population for the past 5, 000 years. During this period lead
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production has been said to have increased from a mere 10 tons per annum to a staggering 1 million tons per year, a phenomenon that has accompanied industrial progress (Davidson and Rabinowitz M, 1992; Brown and Margolis, 2012). Lead contamination was reported to be high
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between 1900 and 1975 before changes in the United States of America (USA) laws were
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enacted (Brown and Margolis, 2012). During this period (1900 - 1975), lead was frequently in contact with the human population because it was part of the component of several industrial as well as domestic appliances, which include lead pipes, batteries, radiators and cosmetics. The
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USA federal law and those initiated worldwide to cut down the number of people with higher blood lead level started to bear results by the year 2008. The number of children (1-5 years of
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age) with a blood lead level ≥10 µg/dL was reduced from 13.5 million in 1978, to 250,000 by 2007-2008 (US Environmental Protection Agency, 2010). Although leaded gasoline has been phased out in many countries, leaded fuel is still used in some African (Algeria), Middle Eastern
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(Iraq, Yemen) and Asian (Afganistan, Myanmar and North Korea), countries (United Nations
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Environmental Program, 2012). The population of people in these countries is more than 194 million. As expected, the blood lead levels of people exposed to leaded gasoline in some of these
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countries was significantly higher than controls (Al-Rudainy, 2010) because large quantities of lead were released into the open air from the exhaust pipes of motor vehicles. While strict environmental regulations has led to a significant decrease in lead pollution over the last three decades, occupational lead contamination is still a great concern especially for workers associated with long-term exposure to lead pipe, metallurgical, lead-acid battery and lead wire factories. It is also a concern for people exposed to leaded gasoline (Al-Rudainy, 2010). In addition, children living in houses built before 1978 in the USA with lead-base water pipes or paint may have higher than normal blood lead levels (Brown and Margolis, 2012).
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Lead toxicity, whether occupational or otherwise, therefore, is a great concern because it has been implicated in a variety of health conditions including neurological (Perlstein and Attala, 1966, Han et al, 2007) and cancer (Roy, 1992; Patra et al., 2001) disorders in humans.
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Lead toxicity has been shown to impair sperm shape and reduce the number of cells in semen (Acharya et al., 1997; Eyden et al., 1978). This observation may explain some of the causes of
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idiopathic male infertility, which accounts for about 40-60% of all cases of male patients complaining of abnormal reproduction (Dohle et al, 2005). How does a heavy metal, such as lead
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cause sperm damage? The mechanism by which lead toxicity leads to impaired sperm morphology is far from clear. However, it has been shown that lead, an example of a heavy metal, is capable of inducing oxidative stress. Some organs withstand the burden of stress better
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than others. However, it has been shown that because testis in particular readily succumbs to oxidative stress (Tomascik-Cheeseman et al, 2004), it is probably not surprising that lead-
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induced oxidative stress is easily manifested in the testis. Lead, in particular, can accumulate in the reproductive system and has been implicated in the development of oxidative stress via
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induction of lipid peroxidation (Marchlewicz M, et al 2004; Mishra and Acharya, 2004). It is well known that lipid peroxidation results in the generation of reactive oxygen species. In view
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of this, many investigators have used a variety of antioxidants, including vitamin C (Hsu et al, 1998) and vitamin E (Patra et al, 2011) to prevent the occurrence and or subdue oxidative stress
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in tissues. Some of these investigations have reported beneficial effects of antioxidants on heavy metal-induced toxicity (Han et al, 2007), while others (Willems et al., 1982) have questioned the usefulness of these agents in the amelioration of heavy metal-induced lesions in the reproductive system of laboratory animals.
Most of the studies published on the effect of lead-induced toxicity on the reproductive system of laboratory animals have focused on the structure of sperm and sperm count but less on the ultrastructure of the male reproductive system. Reports of studies that examine the effect of lead toxicity are contradictory, while many have addressed only the changes observed by light microscopy (Godowicz and Galas, 1992; Ait Hamadouche et al, 2009; Suradkar et al., 2010) rather than the ultrastructural changes that can only be assessed by transmission electron microscopy. Literature reports on the ultrastructure of the male reproductive organs are even 4 Page 4 of 22
more confusing with some studies indicating that lead exposure causes no ultrastructural changes in the testis and Leydig cells (Wenda-Różewicka et al, 1996). The aim of the present work, therefore, was to investigate the toxic effect of exposure to lead on
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effect of Vitamin E on lead-induced toxicity in these organs.
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TO mice seminiferous tubules of testes, epididymis, Leydig cells and the possible protective
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Materials and Methods
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Experimental protocol
The present study was designed to observe the changes in the testes, epididymis and Leydig cells after intraperitoneal (i.p.) administration of lead acetate (1 mg/kg body weight). TO mice were
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used in this study. Mice were acclimated to laboratory conditions with regular temperature control ranging from 23±2 °C and with a balanced laboratory chow and water ad libitum. A total
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of 24 healthy male TO mice ranging from 32-35 g body weight were selected and randomly divided into 4 groups. The first group of mice (n =6) received 5% glucose solution and served as
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vehicle control group. The second group of mice was injected with lead acetate (1 mg/kg body
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weight, i.p.) and the third group was given vitamin E (Tocopherol diluted in soya oil 100 mg/kg body weight). The fourth group was treated with lead acetate (1 mg/kg body weight) and vitamin E (100 mg/kg body weight). Dilution of chemicals was made so that the volume of each injection was maintained at a volume of 0.2 ml/mouse.
Electron microscopy
After 3 weeks of 5 days per week of lead and/or vitamin E administration, all the groups were sacrificed and the testes and body of epididymis were dissected out from the same morphological area, free from other accessory tissues cut, into small pieces and washed with 0.1 M phosphate buffer and then immersed in a modified McDowell and Trump (1976) fixative for 3 hours at room temperature. After rinsing with phosphate buffer, the tissues were postfixed with 1% osmium tetroxide for 1 hour. The tissue samples were later washed with distilled water, dehydrated in a series of graded ethanol and propylene oxide and then infiltrated and embedded 5 Page 5 of 22
in Agar100 epoxy resin and polymerized at 65! C for 24 hours. Blocks were trimmed and semithin and ultrathin sections were cut with Reichert Ultracuts, ultramicrotome. Semithin sections (0.5 ! m thickness) were stained with 1% aqueous toluidine blue on glass slides and ultrathin sections (95 nm) on 200 mesh copper grids were then contrasted with uranyl acetate
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followed by lead citrate according to a previously described method (Draper et al., 1999). The
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grids were examined and photographed with a Philips CM10 Transmission Electron Microscope.
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Morphometric analysis
Electron micrographs of 20-30 sections of either seminiferous tubules, epididymis or Leydig
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cells were taken per mouse (n=6). The number of spermatozoa or cytoplasmic organelles was counted manually and blindly on the electron micrographs of 20 different sections per mouse.
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The average of the count was taken and compared with those of other groups.
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Statistical analysis
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All data were presented as mean + standard deviation. Analyses were tabulated using Microsoft Excel Sheet. The four different groups were compared using Students t-test.
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Values less or equal to 0.05 were considered to be statistically significant.
Results
Figure 1 shows the luminal region of the seminiferous tubules of control (a), mice treated with vitamin E only (b), mice treated with Pb only (c) and mice treated with both Pb and vitamin E (d). Pb caused a significant decrease in the density of spermatozoa when compared to control and or mice treated with vitamin E only. The administration of vitamin E prevented a decrease in the density of spermatozoa observed in the testis of lead-intoxicated mice. The structure of the nucleus of the spermatid of Pb-treated mice appeared disorganized. The addition of vitamin E reduced the nuclear disorganization of spermatids.
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Morphometric analysis showed lower sperm count in the lumen of seminiferous tubules of the testis of Pb-treated mice compared to controls (Fig. 2). The administration of vitamin E significantly improved the sperm count.
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The ultrastructure of Leydig cells of normal (a), mice treated with vitamin E only (b), mice treated with Pb only (c) and mice treated with both Pb and vitamin E (d) is displayed in figure 3.
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The ultrastructure of Leydig cells of normal mice and those treated with vitamin E alone showed intact cytoplasmic granules, mitochondria and endoplasmic reticuli. The ultrastructure of Leydig
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cells of mice treated with vitamin E only did not significantly differ from that of control. In contrast, Pb-treated mice showed a significant reduction in the density of cytoplasmic granules (Fig. 4) and vacuolarization in the cytoplasm of Leydig cells. In addition, the cytoplasm of the
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Leydig cells of Pb treated mice showed increased cytoplasmic processes and caveolae compared
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to those from controls and vitamin E-treated mice.
Addition of vitamin E to Pb-treated mice resulted in a marked effort by Leydig cells to
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reassemble large number of cytoplasmic organelles. However, the cytoplasmic processes of
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Leydig cell of Pb-treated mice did not disappear after the addition of vitamin E. Figure 5 shows the luminal surface of the epididymis of control (a), mice treated with vitamin E
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only (b), mice treated with Pb only (c) and mice treated with both Pb and vitamin E (d). The lumen of the epididymis of mice treated with Pb contains much fewer spermatozoa compared to those of control or mice treated with vitamin E alone. The density of spermatozoa in the lumen of the epididymis of mice treated with vitamin E only was similar to that of normal control. The lumen of the epididymis of Pb treated mice given vitamin E have large number of spermatozoa comparable to that of control or those treated with vitamin E alone. The stereocilia of the epithelial cells of the epididymis of Pb-treated mice appear to be fewer compared to those of control or vitamin E treated mice, because some of the epithelial cells have lost their stereocilia.
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Morphometric analysis showed a lower sperm count in the lumen of the epididymis of Pb-treated mice compared to controls (Fig. 6). The administration of vitamin E significantly improved the
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sperm count.
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Discussion
This study showed that Pb, as a potential occupational hazard, possesses a significant health
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hazard to the reproductive capacity of male mice. The number and density of the spermatozoa in the lumen of the seminiferous tubules and epididymis of mice treated with Pb was significantly
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lower compared to that of control or those treated with vitamin E alone. The observation that Pbtreated mice have a lower sperm count has been well documented (Acharya et al, 2003, Ait
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Hamadouche et al, 2009). In a similar study, cells of the seminiferous tubules show signs of degeneration (heterochromatic nuclei, irregular basal lamina, vacuolization) in albino rats given
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25 mg/kg body weight of lead acetate per os (El Shafai et al., 2011).
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The results of this study confirmed that lead exposure does indeed reduce the number of spermatozoa in the testis and epididymis at all developmental stages. What then are the
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morphological explanations of the lower sperm count in lead-treated mice? Several studies, at the light microscopy level, have shown that the weight of the testis of animals treated with Pb is reduced with a concomitant increase in abnormal morphological changes (Vigeh et al, 2011). The observation of this study showed that spermatids underwent degenerative changes, which may lead to a reduced number of spermatozoa. In addition, Pb also induced epididymal epithelial cells to lose their stereocilia. The loss of stereocilia may inhibit the ability of the epididymis to transport spermatozoa toward the vas (ductus) deferens. The increased morphological changes in the spermatids coupled with the inability of the epididymis to carry available spermatozoa could explain the infertility associated with lead exposure.
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The infertility observed in Pb-intoxication is probably due to the morphological changes seen in the seminiferous tubules. Pb treatment also caused vacuolization and a decrease in the number of cytoplasmic organelles of Leydig cells, the cells responsible for the production of testosterone. Testosterone, in turn, is required for the maturation of sperm. This “double jeopardy” of loss of
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viable cells within the seminiferous tubules and the impairment of the function of Leydig cells
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may add a tremendous burden to the ability of the male genitals to perform their function.
What then is the cause of the degenerative changes observed in the cells of the testis and the
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epididymis? It has been shown that Pb can cause oxidative stress (Ercal et al, 1996, Acharya et al, 2003), resulting in increased lipid peroxidation. Lead exposure has also been shown to reduce the tissue levels of testicular levels of endogenous antioxidants such as glutathione, catalase and
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superoxide dismutase in rats (2010; Abdel Moniem et al, 2010) and mice (Sharma et al (2010). These antioxidants are important markers of oxidative stress.
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Since it has been shown that oxidative stress can induce faulty signal transduction and apoptosis in cells (Adeghate, 2004), the oxidative stress induced by Pb may therefore result in the
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epididymis.
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morphological changes observed in Leydig and spermatid cells of the testis and epithelium of the
It has also been shown that Pb is capable of decreasing lactate utilization and respiration in
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Sertoli cells of the rat testis (Batarseh, 1986). This observation may be a consequence of the effect of oxidative stress.
In view of the fact that Pb exposure has been shown to induce oxidative stress in the testis of rodents (Ercal et al, 1996, Acharya et al, 2003), it was decided to examine whether vitamin E, an antioxidant, would prevent oxidative stress-induced morphological changes in the testicular, epididymal and Leydig cells of mice subjected to Pb exposure. Several studies have examined the role of antioxidants on Pb-induced oxidative stress. For example it has been shown that flaxseed oil improved the antioxidant pool in the testis of albino rats subjected to Pb intoxication (Abdel Moniem et al, 2010). Other studies examining the effect of vitamin C on chronic Pb toxicity have shown the beneficial effect of vitamin C, an antioxidant 9 Page 9 of 22
on Pb-induced changes in the testis of Wistar rats (Shaban El-Neweshy and Said El-Sayed, 2011). The results of this study showed that vitamin E increased sperm count in the testicular as well as epididymal lumina and reduced the occurrence of morphological changes in these organs. The
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observation of this study has shown that vitamin E as an antioxidant is capable of reducing the deleterious impact of Pb-induced oxidative stress in mice male reproductive organs, in
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accordance with literature reports. Mishra and Acharya (2004) showed that vitamin E reduced the impact of Pb-induced reduction in sperm count in Swiss mice. The fact that Pb can reduce the showed a strong association of both Pb and vitamin E.
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level of vitamin E in wild animals residing in Pb mining regions (Rodríguez-Estival et al, 2011) In conclusion, Pb in its acetate form caused degenerative changes in the spermatids of the
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seminiferous tubules of mice testis. It also reduced the quantity of stereocilia in the epithelial cells of the epididymis and caused cellular abnormalities in testosterone-producing Leydig cells.
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These morphological alterations may explain why Pb induces infertility in male subjects. Our
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result also showed that vitamin E can reduce the impact of Pb toxicity in the male genital organs.
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lead acetate induced oxidative stress in testes of adult rats. Afr. J. Biotechnol. 9, 7216-7223. Acharya, U.R., Mishra, N., Acharya, S., 1997. Effect of lead acetate on male germinal
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Acharya, U.R., Acharya, S., Mishra M., 2003. Lead acetate induced cytotoxicity in male
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Adeghate, E., 2004. Molecular and cellular basis of the aetiology and management of diabetic
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cardiomyopathy: A short review. Mol. Cell. Biochem. 261, 187-191. Ait Hamadouche, N., Slimani, M., Merad-Boudia, B., Zaoui, C., 2009. Reproductive toxicity of
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lead acetate in adult male rats. Am. J. Sci. Res. 3, 38-50.
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Al-Rudainy , L.A., 2010. Blood Lead Level Among Fuel Station Workers. Oman Med. J. 25(3): 208–211. American Academy of Pediatrics Committee on Environmental Health., 2005. Lead exposure in children: prevention, detection, and management. Pediatrics. 116,1036-1046. Batarseh, L.I., Welsh, M.J., Brabec, M.J., 1986. Effect of lead acetate on Sertoli cell lactate production and protein synthesis in vitro. Cell Biol Toxicol. 2, 283-92. Brown, M.J., Margolis, S., 2012. Lead in drinking water and human blood lead levels in the United States. MMWR Surveill Summ. 61 Suppl, 1-9.
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Davidson, C.I., Rabinowitz, M., 1992. Lead in the environment: from sources to human receptors. In: Needleman, H. (Ed.) Human lead exposure. Boca Raton, CRC Press, Florida, pp. 65-86.
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Dohle, G.R., Colpi, G.M., Hargreave, T.B., Papp, G.K., Jungwirth, A., Weidner, W., 2005. EAU
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Working Group on Male Infertility, EAU guidelines on male infertility. Eur. Urol. 48, 703-711. Draper, C.E., Adeghate, E.A, Singh, J., Pallot, D.J., 1999. Evidence to suggest morphological
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and physiological alterations of lacrimal gland acini with ageing. Exp. Eye Res. 68, 265-276. El Shafai, A., Zohdy, N., El Mulla, K., Hassan, M., Morad, N., 2011. Light and electron
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albino rats and the possible protective effect of ascorbic acid. Food Chem. Toxicol. 49, 734-743. Ercal, N., Treratphan, P., Hammond, T.C., Mathews, R.H., Grannemann, N.H., Spitz, D.R.,
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1996. In vivo indices of oxidative stress in lead-exposed C57BL/6 mice are reduced by treatment
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with meso-2,3-dimercaptosuccinic acid or N-acetyl cysteine. Free Rad. Biol. Med. 21, 157-161. Eyden, B.P., Maisin, J.R., Mattelin, G., 1978. Long-term effects of dietary lead acetate on 272.
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survival, body weight and semen cytology in mice. Bull. Environ. Contam. Toxicol. 19, 266 -
Godowicz, B., Galas, J., 1992. Toxic effect on fertility of inbred strain mice. Folia Biologica (Krakow). 40, 73-78.
Han, J.M., Chang, B.J., Li, T.Z., Choe, N.H., Quan, F.S., Jang, B.J., Cho, I.H., Hong, H.N., Lee, J.H., 2007. Protective effects of ascorbic acid against lead-induced apoptotic neurodegeneration in the developing rat hippocampus in vivo. Brain Res. 1185, 68-74. Hsu, P.C., Liu, M.Y., Chen, L.Y., Guo, Y.L., 1998. Effects of vitamin E and/or C on reactive oxygen species-related lead toxicity in rat sperm. Toxicology. 128, 169 - 179. 12 Page 12 of 22
Marchlewicz, M,. Michalska, T., Wiszniewska, B., 2004. Detection of lead-induced oxidative stress in the rat epididymis by chemiluminescence. Chemosphere. 57,15531562.
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Rodríguez-Estival, J., Taggart, M.A., Mateo, R., 2011. Alterations in vitamin A and E levels in liver and testis of wild ungulates from a lead mining area. Arch. Environ. Contam. Toxicol. 60, 361-371.
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Shaban El-Neweshy, M., Said El-Sayed, Y., 2011. Influence of vitamin C supplementation on lead-induced histopathological alterations in male rats. Exp. Toxicol. Pathol. 63, 221-227.
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Sharma V, Kansal L, Sharma A. Prophylactic efficacy of Coriandrum sativum (Coriander) on testis of lead-exposed mice. Biol Trace Elem Res. 2010 Sep; 136(3):337-54. Suradkar, S.G., Vihol, P.D., Patel, J.H., Ghodasara, D.J., Joshi, B.P., Prajapati, K.S., 2010.
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Figure legend Figure 1 shows the luminal region of the seminiferous tubules of control (a), mice treated with
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vitamin E only (b), mice treated with Pb only (c) and mice treated with Pb and vitamin E (d). Note the significant decrease in the density of spermatozoa in the lumen (**) of seminiferous
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tubules of Pb-treated rats (c) compared to control (a) and those of mice treated with vitamin E only (b). The spermatids of Pb-treated mice show signs of degeneration (arrow). Scale bar = 2
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µm
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Figure 2 shows the average number of spermatozoa in the lumen of 20 sections of seminiferous tubules per mouse (n=6) in the testis of normal mice (a), mice treated with vitamin E only (b), mice treated with Pb only (c) and mice treated with both Pb and vitamin E (d). Note a significant
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reduction in the number of spermatozoa in lead treated mice compared to control. There is a significant improvement in the sperm count of mice treated with vitamin E after lead
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administration. Control vs control treated with Vit E (p > 0.2): not significant; Control vs Control treated with Pb (***p < 0.0000006): highly significant; Control treated with Vit E vs Control
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treated with Pb (p < 0.0001) highly significant; Control treated with Pb vs Control treated with
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Pb and Vit E (*p < 0.00002): highly significant. Figure 3 shows the ultrastructure of Leydig cells of normal (a), mice treated with vitamin E only (b), mice treated with Pb only (c) and mice treated with both Pb and vitamin E (d). Note that the Leydig cells of Pb-treated mice (c) showed a significant reduction in the density of cytoplasmic granules, increased vacuolarization (thick arrow) and pinching of the plasma membrane (thin arrow) compared to controls. Electron lucent secretory granules (arrow head) were observed in the cytoplasm of the Leydig cells of mice treated with both Pb and vitamin E. Scale bar = 2 µm Figure 4 shows the average number of endocrine granules in 20 sections of Leydig cells per mouse (n=6) in the testis of normal (a), mice treated with vitamin E only (b), mice treated with Pb only (c) and mice treated with both Pb and vitamin E (d). Note a significant reduction in the number of endocrine granules in lead treated mice compared to control. There is a significant 15 Page 15 of 22
improvement in the number of Leydig granules in mice treated with vitamin E after lead administration. Control vs control treated with Vit E (p > 0.5): not significant; Control vs Control treated with Pb (***p < 0.00001): highly significant; Control treated with Vit E vs Control treated with Pb (p < 0.0001) highly significant; Control treated with Pb vs Control treated with
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both Pb and Vit E (*p < 0.00005): highly significant.
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Figure 5 shows the luminal surface of the epididymis of control (a), mice treated with vitamin E only (b), mice treated with Pb only (c) and mice treated with Pb and vitamin E (d). Note that the
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lumen of the epididymis of mice treated with Pb contains much fewer spermatozoa (arrow) compared to those of control or mice treated with vitamin E alone. Some epithelial cells of
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epididymis of Pb-treated rats are devoid of stereocilia (arrow head). Scale bar = 2 µm
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Figure 6 shows the average number of spermatozoa in the lumen of 20 sections of epididymis per mouse (n=6) of control (a), mice treated with vitamin E only (b), mice treated with Pb only (c) and mice treated with both Pb and vitamin E (d). Note a significant reduction in the number of
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spermatozoa in lead treated mice compared to control. There is a significant improvement in the
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sperm count of mice treated with vitamin E after lead administration. Control vs Control treated with Vit E (p > 0.7): not significant; Control vs Control treated with Pb (***p < 0.0000002):
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highly significant; Control treated with Vit E vs Control treated with Pb (p < 0.0001): highly significant; Control treated with Pb vs Control treated with both Pb and Vit E (*p < 0.000006): highly significant.
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