الفهرس 
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Abstract
Declines in fertility have become a serious concern, and has been accompanied by increasing demands for infertility treatment. A male contributory factor is involved in approximately half of all cases. However, in a considerable proportion of men with subfertility, no medical or surgical factors are implicated, and the etiology remains unclear. There is an increasing awareness of the potential role of genetic and environmental factors in idiopathic male infertility.
Male appears more susceptible than the female to the effects of occupational or environmental exposures to reproductive toxicants, and it is not surprising that environmental agents have been postulated to be contributory to deteriorating semen quality and a decline in male reproductive health.
The oceanographic results which are manifested by of the present study elucidated that coastal ecosystems of Alexandria in Egypt are affected from industrial and anthropogenic activities such as wastes from multiple industrial activities such as metal works, petroleum, port activities and urban wastewaters.The resultsshowed that, the Eastern Harbor and El-Max Bay have Cd level in the mussel tissue more than the permissible limits suggested by Food and Agriculture Organization (FAO)(0.5 μg/g) and the Pb level in mussel tissue in Abou-Qir Bay and El-Max Bay higher than the permissible level suggested by(FAO)(0.5 μg/g). Accumulation of heavy metals such as Pb and Cd in living organisms such as mussels leads to concentrations several orders of magnitude higher than those of the surrounding water.
Consequently, the objective of the current study was endeavored to study the effects of Pb and Cd exposure on the male fertility through the determination of the Pb, Cd, GSH, MDA, Testosterone, LH, FSH, prolactin, seminal parameters and protamine P2. This study was conducted on 40 infertile men and 15 normal controls.
Metals may affect the male reproductive system directly, when they target specific reproductive organs, or indirectly, when they act on the neuroendocrine system. Since the 1950s, studies have shown that in vivo acute exposure to Cd caused BTB disruption, germ cell loss, testicular edema, hemorrhage, necrosis, and sterility in several mammalian species (e.g., rodents, rabbit, dog, calf, stallion), and in vitro studies have illustrated Cd-induced damage to testicular cells. Many studies have also associated reduced male fertility, such as reduced sperm count and poor semen quality, in men exposed to Cd and/or other environmental toxicants.
Initial studies that recognized Cd could induce profound and irreversible injury to mammalian testes described the disruption of endothelial cells of microvessels, edema and hemorrhage by morphological analysis, apparently as the result of a primaryIt was postulated that the mammalian testis was more sensitive than other organs because of its unique vasculature. Although the vascular system is indeed a major target of Cd toxicity.
Johnson (1969), suggested that the BTB could be the first target of Cd toxicity in guinea pigs testes. Afterwards, Setchell and Waites (1970), demonstrated that the BTB is more vulnerable to Cd toxicity than the tight junction (TJ) in the microvessels in adult rat testes, since the damage to the BTB occurred prior to the microvessels found in the interstitium.
In addition, a single low dose of Cd at 1 mg/kg b.w. disrupted TJ associated microfilaments in rat Sertoli cells and also induced spermiation failure without visible vascular lesion in the testes. Taken together, these observations are rather unusual since the BTB is known to be one of the tightest blood tissue barriers in mammals.
More importantly, the BTB is created by coexisting tight junction, desmosome-like junction, gap junction, and basal ectoplasmic specialization (basal ES). The basal ES is a testis-specific actin-based atypical adherens junction (AJ) type. The physiological significance for the coexistence of these different junction types at the BTB remains unknown.However, different studies have suggested that their coexistence is needed to permit the timely restructuring of the BTB to facilitate the transit of preleptotene spermatocytes at stage VIII of the epithelial cycle while maintaining the immunological barrier simultaneously. It was proposed that adaptors (e.g., α-catenin) are ‘switching’ between their association with the TJ- (e.g., occludin) or basal ES- (e.g., N-cadherin) based integral membrane proteins so that TJ can ‘open.
Meanwhile the basal ES continues to maintain the barrier function transiently via an ‘engagement’ and ‘disengagement’ mechanism. However, it is likely that because of this unique morphological layout of the BTB in the testis, which confers its unusual barrier function to facilitate the transit of preleptotene spermatocytes, this has also rendered this barrier vulnerable to environmental toxicants, such as Cd. For instance, other studies have shown that E-cadherin is one of the primary targets of Cd toxicity in epithelial cells since Cd interacts with the putative calcium-binding motif in Ecadherin, causing a disruption of the cadherin-based cell adhesion.
Yet in all other epithelia and endothelia, the anchoring junctions are restricted behind the tight junctions forming the adherens plaque, as a result, E-cadherin, an integral membrane protein at the AJ, is being sealed from Cd. But since E-cadherin coexists with TJ-proteins (e.g., occludin, claudins, JAM-A) at the BTB, Cd would have immediate access to E-cadherin, making the testis more susceptible to Cd toxicity. The Cd-induced BTB damage has also been studied in vitro by using a model in which primary Sertoli cells isolated from 20-day-old rat testes are plated at a cell density at 0.5-1.2 × 106 cells/ cm2 on a reconstituted basement membrane (e.g., Matrigel). These cells establish functional TJ, basal ES, and desmosome, which have been confirmed by both biochemical and morphological studies.
Cadmium is easily absorbable, relatively long accumulated in the tissues conducting essential functions in the organism, and represents a special risk for human health. cd toxicity impairs the reproductive functions of living organisms.
The elevated levels of Cd were found in azoospermic subjects, and the negative impact of Cd was shown in all examined biophysical semen characteristics except for the sperm volume. at the same time, positive correlations were observed between Cd and sperm motility, and cd and both linear velocity and curvilinear velocity (analysed by computer-assisted videomicrography).
The studies by massányi et al. (2004) suggested that Cd has a direct negative effect on spermatozoa quality, especially on the sperm morphology. The same authors reported the occurrence of separated flagellum of spermatozoa caused by Cd, in oligoasthenozoospermic subjects, negative correlations between Cd concentration in the sperm and sperm motility and sperm concentration (density) were found.
The most frequent causes of male infertility are associated with spermatogenesis. Because it is relatively easy to conduct, non-invasive and inexpensive to perform, semen analysis (sperm count, semen volume, sperm morphology and assessments of functional parameters) is one of the first laboratory tests commonly performed for infertile couples.
Studies on occupationally lead-exposed men have shown multiple sperm parameters being affected as seminal plasma or blood lead concentrations rise, usually at levels of> 40μg/dl, but sometimes even at levels of <10μg/dl. For instance, reductions in sperm count and sperm concentration or density, decreased volume of ejaculation, as well as correlations with asthenospermia, hypospermia, and teratospermia (53μg/dl) have been reported in male workers. Furthermore, higher percentages of immature and abnormal spermatozoa such as wide, round, and short sperm in lead exposed workers have been reported at both high (40μg/dl) and low (<15μg/dl) blood lead levels.
Many studies on reproductive system of male animals have documented lead as a toxicant for testicular tissue and functions such as significant reductions in the number of spermatozoa within the epididymis in mice administered lead acetate (0.25% and 0.50%) in drinking waterand halted spermatogenesis in rats. Many studies suggest spermatogenesis problems caused by lead, although, some researchers have failed to demonstrate correlations between lead and semen volume, pathologic sperm and sperm concentration among workers exposed to high lead levels, or abnormalities in sperm count and/or the sperm morphology in rabbits.
Macroscopic changes in accessory sex organs such as diminished weight of testes, seminal vesicles, epididymis, and ventral prostate have been demonstrated in various studies using experimental animals. Microscopic changes, histological as well as macroscopic ones, have been induced by increasing lead levels in lead exposed male rats including changes in the testicular tissues morphology, and decreased germ cells layer population.
In addition, two studies conducted on lead exposed mice demonstrated seminiferous tubule degeneration, and seminal abnormal cytology. Similarly, electron microscopic analysis has revealed that lead-exposed monkeys, when exposed during infancy, can induce testicular alterations, which persist in later life even when blood lead concentrations had decreased considerably.
It was reported that, the animals exposed to Pb showed a significant decrease in daily sperm production, epididymal sperm reserves and number of motile sperm, viable sperm and HOS tail coiled sperm, indicating decreased sperm production and deterioration in sperm quality in Pb-exposed rats. Earlier, it has been reported that Pb has the ability to accumulate in the testis and epididymis leading to anti-spermatogenic effects and also affects sperm functions, respectively.
Rafique et al. (2009) suggested that spermatozoa in epididymis absorb Pb more rapidly and thereby affects the motility of sperm. This may be one of the factors responsible for the observed increase in nonmotile and dead spermatozoa in Pb-exposed rats.
The mechanisms involved in the impairment of spermatozoa motility by metals might be due to an impairment of sperm ATPases, which play a critical role in sperm motility. The protective effect of Zn on spermatotoxic effects of Pb may be attributed to com-petition between Pb and Zn, or reduction of available Pb-binding sites in the spermatozoa.
Successful fertilization of an ovum by spermatozoa depends not only on sperm count and morphology but is also relevant to functional parameters. Lead has been shown to incur detectable negative effects on blood, semen and/or spermatozoa quality in workers, such as inducing prolonged liquefaction time and decreasing sperm motility(165). It has been negatively associated with sperm motility and viability (blood lead levels ≤ 10 µg/dl), and a reduction in the functional maturity of sperm among men with mean blood lead levels of 45µg/dl.
The aforementioned findings cast light and confirmed the results of the present study which illustrated that the infertile men group exposed to Pb and Cd showed striking significant decreases in sperm count, motility and quality. This may due to cytotoxic effect of Pb and Cd.
Reproductive hormones play an important and complicated role in the regulation of spermatogenesis and sperm development. The results of experimental studies in rats have shown that the effects of lead involve multiple sites on male reproductive hormones although the most important part of these disorders probably occurs in the hypothalamic-pituitary-testosterone (HPT) axis.
Furthermore, due to imbalances in the HPT hormonal axis induced by lead exposure, pituitary cells release inappropriate levels of LH and change the steroid negative feedback loop, usually at the hypothalamus level. Increased concentrations of other reproductive hormones, such as follicle stimulation hormone (FSH), secreted from the pituitary gland, have been observed following lead exposure in men and in lead treated rats.
On the other hand, inappropriate inhibin B overproduction in excessively lead exposed subjects may be induced by a Cell of Sertoli dysfunction, which suggests spermatogenesis impairment. Meanwhile, research on male monkeys has shown that alterations in Sertoli cell function may occur due to decreases in inhibin/FSH, rather than by a direct effect on the cells
Such findings are consistent with a failure to find significant microscopic alterations in rat’s Sertoli cells, except for increased lysosomal size, verified by ultrastructural examination on the rats’ cells. Thus, the Sertoli cells may be not a direct target of lead toxicity and lead’s effects on FSH disruption is the more likely cause of reproductive dysfunction rather than by a direct effect on the cells.
The heavy metal cadmium (Cd) is one of the most toxic industrial and environmental metals and acts as an endocrine disruptor in humans and rodents. Cd is recognized as an endocrine disruptor that modifies, among others, prolactin (PRL) secretion in a number of species, including humans.
In male rodents, it is well established that Cd significantly alters the circulating levels of several hormones (e.g., testosterone, LH, FSH). Previous studies have shown that Cd impairs the testosterone production in isolated Leydig cells without affecting their viability, demonstrating that steroidogenic disruption in Leydig cells is likely to be an initial target of Cd toxicity as an endocrine modulator. Cd also decreased steroidogenic acute regulatory protein (StAR), LH receptor and cAMP levels in the testis.
Cd can also modify hormone levels by affecting the hypothalamic-pituitary-testicular axis in different aspects, not only via its effects on Leydig cells. For instance, Cd affected the circadian pattern release of noradrenaline, a regulator of hypothalamus hormone secretion, which resulted in changes in the daily pattern of plasma testosterone and LH levels. In short, it is important to note that the endocrine disruption induced by Cd is likely to be multi-factorial, mediated via its effects on Leydig cells and/or the hypothalamic-pituitary-testicular axis.
Moreover, Norman Barwin, (1978) found that any chemical agent suppressing the secretion of pituitary gonadotropins will produce antispermatogenic and antifertility effects; impaired secretion of luteinizing hormone by the pituitary results in deficient androgen secretion by the testes, cessation of spermatogenesis and loss of libido. Steroidal hormones may inhibit the secretion of pituitary gonadotropins via negative feedback to the hypothalamus, which results in inhibition of spermatogenesis and testosterone secretion.
Hyperprolactinemia causes infertility in around 11% of oligospermic males. Hyperprolactinemia inhibits the pulsatile secretion of the gonadotrophin releasing hormone, which causes decreased pulsatile release of follicle stimulating hormone, luteinizing hormone, and testosterone, which in turn causes spermatogenic arrest, impaired sperm motility, and altered sperm quality. It later produces secondary hypogonadism and infertility.
Hyperprolactinemia also directly influences spermatogenesis and steroidogenesis by acting on prolactin receptors present in Sertoli cells and Leydig cells in testes, and produces primary hypogonadism and infertility. There are many studies suggesting that hyperprolactinemia has a definite role in the male infertility, and is one of the reversible causes of infertility.
In the present study, serum levels of LH and testosterone were strikingly significant reduced in infertile patient group consist withconsist with high significant elevation of FSH and prolactin. The present observation was running parallel with finding of (Gunnarrsson et al 2003) and (Hassanin and safwat 2014). Therefore, it may be suggested that the exposure to Pb and Cd induced hormonal disruption.
Most studies during the last decade have implicated oxidative stress as a mediator of sperm dysfunction. In the etiology of male infertility, there is growing evidence that damage inflicted to spermatozoa by reactive oxygen species (ROS) plays a key role. The spermatozoa have a high content of polyunsatured fatty acids within the plasma membrane and a low concentration of scavenging enzymes within the cytoplasm and they are susceptible to the peroxidation in the presence of elevated seminal reactive oxygen species levels.
Increased levels of seminal OS have been correlated with sperm dysfunction through different mechanisms that include lipid peroxidation of sperm plasma membrane and impairment of sperm metabolism, motility, and fertilizing capacity. In addition, OS has been shown to affect the integrity of the sperm chromatin and to cause high frequencies of single and double DNA strand breaks. Previous studies have been indicated that increased sperm nuclear DNA damage strongly and negatively affects natural fertility.
Mammalian spermatozoa are very susceptible to attack by reactive oxygen species (ROS) attack since they are rich in polyunsaturated fatty acids, which results in decreased sperm motility, presumably by a rapid loss of intracellular ATP leading to decreased sperm viability, and increased morphology defects with deleterious effects on sperm capacitation and acrosome reaction. Lipid peroxidation of sperm membrane is considered to be the key mechanism of ROS-induced sperm damage leading to infertility.
Increasing evidence suggests that an induction of oxidative stress in the testis represents another common response after exposure to environmental toxicants. Increase in oxidative stress can be seen in up to 80% of clinically proven infertile men, and exposure to environmental toxicants is a major factor contributing to such increase.
It has been reported that environmental toxicant-induced oxidative stress can cause male infertility by disrupting the cell junctions and adhesion between Sertoli-Sertoli cells and/or Sertoli-germ cells via the phosphatidylinositol 3-kinase (PI3K)/c-Src/focal adhesion kinase (FAK) signaling pathway. These findings are significant because human exposure to environmental toxicants is often below toxic dose levels that cause cell death.
The result of the current study elucidated that the infertile patient group were exposed to Cd and Pb and they were suffering from severe oxidative stress which manifested by the presence of high significant level of MDA associated with panic significant decrease in the level of GSH in the blood of infertile patient. Therefore, Cd and Pb could be a possible causative agent for low sperm count among infertile patients beside sperm concentration, and sperm motility is also severely affected by Cd and Pb. Sperm motility is recognized to be more sensitive to this trace element and reduced sperm motility has been observed at a dose for below the dose affecting sperm production.
The decrease in sperm count is correlated with decrease in testosterone levels and increase in ROS as evident from suppressed antioxidant enzyme activities. The consequences of such oxidative damage could include loss of motility due to LPO in the testis and epididymis. Body has also evolved a protection mechanism against toxic metals, through metallothionein. A study also reported that Cd induces MT mRNA without increase in MT protein in interstitial cells of testis. The inability to induce metal-detoxicating MT protein, in response to Cd, might account for the higher susceptibility of testis to metal toxicity. These changes may be one of the possible mechanisms of inhibition of steroidogenic enzyme activity, testosterone levels and sperm count/ sperm motility after lead and cadmium co-exposure.
Zinc and lead compete for similar binding sites on the metallothionein-like transport protein in the gastrointestinal tract. The competition between zinc and lead might decrease the absorption of lead, thus reducing lead toxicity. Dietary supplementation with zinc and in combination with ascorbic acid and thiamine reduces lead toxicity in humans. Zinc has an important role in spermatogenesis in the male reproductive system, and the most probable site of action is the primary spermatocyte.
Zinc supplementation competes for and effectively reduces the availability of binding sites for lead uptake. In another study, zinc was administrated to lead-exposed rats along with chelating agents CaNa2 EDTA, succimer, and D-penicillamine. Zinc enhanced the efficacy of lead chelation by reducing the blood, hepatic and renal lead level, and overturning the inhibited activity on blood aminolevulinic acid dehydratase (ALAD). It has been reported that the prevention of δ-ALAD inhibition and increased cellular SOD in the testis of lead-exposed rats following zinc supplementation. The protective effects of zinc against testicular damage caused by lead might be due to competition between lead and zinc.
Cotreatment with Cd and Zn prevents damage to the testes from Cd exposure. This suggests Cd interference in Zn-related metabolic functions. The competitive mechanism of interaction and Zn-induced metallothionein induction are the plausible mechanism behind protective effects of Zn against Cd toxicity. This is substantiated by the findings that Cd treatment decreases the testicular Zn concentration and elevates the levels of hepatic and renal metallothioneins.
Zn pretreatment can prevent of cadmium-induced testicular tumors whichmay be attributed to the ability of Zn to reduce the cytotoxicity of Cd in interstitial cells by enhancing efflux of Cd and decreasing accumulation of Cd in the nuclei of this target cell population in the rat testis. So, Cd altered testicular function mediated through induction of oxidative stress could be reversed by administration of Zn.
During the later stages of mammalian spermatogenesis, chromatin structure is completely reorganized through aseries of sequential steps that remove the nucleosomal histones and replace them with small, arginine-rich protamines. The tyrosine-containing protamine (P1) is present in sperm nuclei fall mammalian species, while the histidine containing protamine (P2) has been found in only a few species, including humans (511).
HumanP2 (HP2) has been proposed to be a zinc- finger protein, and may serve an important role in sperm chromatin Organization and fertility. Some cases of human male infertility are associated with altered normal lower percent of HP2. Also, a complete absence of HP2, or an increase in the amount of HP2 precursors have been reported, suggesting in complete processing during spermatogenesis. Moreover, it appears that HP2 affinity to DNA can be decreased considerably in infertile men. Further stabilization of sperm chromatin continues with Disulfide bond formation between the cysteine residues of protamines as spermatozoa pass through the epididymis.
Final stability of condensed Sperm chromatin is normally maintained during ejaculation By the secretion of prostatic zinc, which is sequestered by spermatozoa and binds to free thiols of cysteine. The degree of sperm chromatin condensation appears to be a sensitive and functional indicator of male reproductive function.
A study indicated that lead not only binds to HP2, but also affects HP2 binding to DNA. The interaction of lead with zinc-dependent proteins, particularly those rich in Sulfhydryll groups, may represent a fundamental mechanism underlying lead toxicity.
The role of other additional binding sites, such as histidine, remains to be established, since lead also has affinity for this amino acid. The presence of additional binding sites for Pb2+ is further supported by the finding that the UV/VIS spectra changes in HP2 reached saturation at 2 mol equivalents of Zn2+, but were not saturated at 5 mol Equivalents of Pb2+. These results suggest that Pb2+may Compete with or replace for Zn2+ at one or more sites in HP2. It has been reported that zinc supplementation protects testicular damage induced by lead exposure either by a competition between lead and zinc or by a displacement of lead by zinc. HP2 binding to other metals has been reported for Cd2+ , and for Ni2+and Cu2+ which are able to bind to the N-terminal motif of HP2. HP2 may be a common target for toxic metals. The conformational change of HP2 by binding Pb2+ probably through cross-linking of protamines could directly affect the content of S-S bonds within the nucleus.
The timing of sperm nuclear decondensation and pronucleus formation depends in part on the S-S bond content of the sperm chromatin. Micromolar concentrations of lead decreased HP2- DNA binding in a dose-response fashion. The conformational change of HP2 could directly affect the binding of HP2 to DNA.
Chemical alteration of DNA - protamine binding may lead to chromatin alterations, and the changes in the chromatin structure may eventually produce or permit DNA damage, similar to that shown by chemical binding to sulfhydryl groups in protamines.
Apoptosis is required for normal spermatogenesis in mammals and is believed to ensure cellular homeostasis and maintain the delicate balance between germ cells and Sertoli cells. Its second role is for the depletion of abnormal spermatozoa.
There is an established consensus on the implication of apoptosis in male infertility and poor WHO semen parameter; however, the exact mechanisms of its involvement remain to be elucidated. Relatively high rates of apoptosis have been reported in testicular biopsies from infertile men with different degrees of testicular insufficiency.
The proportions of apoptotic sperm is reported to be higher in ejaculated semen samples from infertile men compared to healthy men. Moreover, sperm caspases become more activated in patients with infertility than in healthy donors during cryopreservation.
Although apoptosis is considered a mechanism to ensure selection of sperm cells with undamaged DNA, sperm with DNA damage that are not eliminated by apoptosis may fertilize an ovum. Poor chromatin packaging and/or damaged DNA have been implicated in the failure of sperm decondensation after intracytoplasmic sperm injection, resulting in fertilization failure.