**2.3 Testicular dysfunction due to EDC exposure**

*Male Reproductive Health*

were not significantly affected.

testes and penis size [28].

on human testes [29].

**2.2 Alterations in testicular morphology**

weights of testes and epididymis of rats treated with 10, 50 or 200 mg/kg of TCS

Rodents were exposed to BPA by the oral route or subcutaneous injections [24, 25]. A dose of 2 ng/g body weight induced a decrease in epididymal weight and an increase in prostate weight. Bisphenol S (BPS), considered a safe substitute for BPA, has chemical similarities with BPA and may act as an EDC. Thus, a recent work compared the effects of BPA and BPS on the morphology and physiology of the ventral prostate of adult gerbils [26]. Animals treated with BPA and BPS showed no alterations in prostate weight. Regarding histopathology, BPS-treated animals showed intense prostatic hyperplasia; increased relative frequency of epithelium, muscular stroma and non-muscular stroma; and decreased luminal compartment, and BPA-treated animals showed increased occurrence of hyperplastic growth. But, in general the authors found that BPS

Exposure to metals also induced effects on testes size. A dose of 5 mg/kg body weight of cadmium chloride (CdCl2) administered to rats by oral gavage caused a significant decrease in testes and epididymis weight [19]. Moreover, Hg and zinc (Zn) significantly decreased the absolute and relative testicular weights in murine, with Hg producing the highest reduction in weight [27]. Similar results were obtained by Narayana et al. [22] and Geng et al. [23] that showed a decrease in the

Rats exposed to phthalates demonstrated reduced testicular weights and histologic changes in the seminiferous tubules [20, 21]. Moreover, rats exposed to phthalates during the prenatal period developed reproductive anomalies, namely, smaller

Human studies related to the effects of exposure to EDCs on testicular volume/ weight are limited but in accordance with animal studies. For instance, in a study in Croatian men, no occupational exposures were exposed to metals, and blood Cd was negatively correlated with testes size, suggesting that this metal exerts toxicity

Experimental studies showed that exposure to EDCs had adverse effects on testes, resulting in testicular damage at structural and consequently functional level. Male rats treated with 20 mg/(kg day) of TCS exhibited several histopathological malformations in the testes and sex accessory tissues [18]. Lumen of vas deferens from the treated rats exhibited the presence of stereocilia detached from the epithelium and the presence of eosinophilic bodies. Moreover, the stereocilia were found to be thin, few or absent in the epithelium of TCS-treated rats. Rats treated with a high dose of TCS (200 mg/kg) showed changes in the cauda epididymis and in the testis compared with the control group [4]. In the cauda epididymis, the alterations included vacuolated and exfoliated epithelial cells. Moreover, these authors identified the absence of sperm tails in the seminiferous tubules in the TCS-treated groups.

Mice exposed to BPA showed the formation of morphologically multinucleated giant cells in testicular seminiferous tubules [30], disruption of the blood-testis barrier (BTB) and impaired spermatogenesis [31, 32]. Similar results were obtained by other study using pesticides that induced severe degenerative changes in seminiferous tubules [23]. Metals, such as Cd and Hg, also induced structural alterations in the testis structure, including damage in the vascular endothelium and in the BTB integrity and necrosis and disintegration of spermatocytes [27, 33]. In general, these animal studies showed that EDCs induced changes in testicular morphology, which may be a reason for the decline of male fertility. For instance, damage in epididymis

promoted more structural and histopathological changes than BPA.

weights of reproductive organs of rats exposed to pesticides.

**24**

The two main functions of the testes are spermatogenesis (exocrine function) and steroidogenesis (endocrine function). In normal conditions the gonadotrophinreleasing hormone (GnRH) is secreted by the hypothalamus, stimulating the synthesis of LH and the follicle-stimulating hormone (FSH) [34]. LH is recognized by LH receptors in LC stimulating T biosynthesis (steroidogenesis). FSH is recognized by FSH receptors in SC having an important role in spermatozoa production (spermatogenesis). Several studies showed that these functions are affected by exposure to EDCs (**Figure 1**) [10, 18, 35–39]. Prenatal exposure to EDCs was associated with testicular anomalies later in life, which includes reduced semen volume and quality, increased incidence of cryptorchidism and hypospadias and increased incidence of testicular cancer [40]. EDCs reduced SC number and impaired LC development, inducing testicular anomalies at morphological and functional level [39]. This section presents the studies that assessed the relationship between animal and human exposure to EDCs and testicular dysfunction, including alterations in reproductive hormone levels.

Evidences from animal studies suggest that TCS reduces the production of T in LC and disturbs the function of major steroidogenic enzymes [41, 42]. Male rats treated with TCS or pesticides showed a significant decrease in the levels of serum LH, FSH, cholesterol, pregnenolone and T compared to control [18, 23]. Regarding human studies, a case-control study showed that urinary levels of phthalates and TCS were negatively associated with inhibin B and positively with LH [39]. Additionally, an inverse association was found between urinary levels of phthalates or BPA and testosterone and estradiol (E2) [38, 39]. Similar results were obtained by Meeker et al. [35] that showed an inverse association between BPA concentrations in urine and serum levels of inhibin B and E2:T ratio in men recruited through an infertility clinic. Moreover, a positive association between BPA concentrations in urine and FSH and FSH:inhibin B ratio was found. Hanoaka et al. [36] did not found an association between exposure to BPA and free T and LH concentrations in men. However, a significant decrease in FSH concentrations was found in the BPA exposed men. Urinary levels of BPA were not associated with sperm quality in fertile men but were associated with markers of androgenic action [37]. A significant inverse association was found between urinary levels of BPA and free androgen index (FAI) levels and the FAI:LH ratio. Further, a significant positive association between BPA and sex hormone-binding globulin (SHBG) was found in fertile men. Recently, Lassen et al. [10] examined associations between urinary BPA concentration and reproductive hormones in young men from the general population. The authors found positive associations between urinary BPA concentrations and T, E2, LH and free T levels. BPA and BPS induced significant changes in T and estradiol [26].

Meeker et al. [38] demonstrated that exposure to phthalates may be associated with altered male endocrine function. Urinary concentrations of some phthalates were inversely associated with T, E2 and FAI.

Metals, namely, Cd, also affect the development of the male reproductive system and testis function. Mice prenatal exposed to Cd showed defects on the development of gonads, depletion of germ cells and impairment of spermatozoa maturation [43]. Cd also induces testicular dysfunction, which results of the functional impairment of SC and LC. Regarding human studies, the effect of Cd exposure to male endocrine function was assessed by several authors (as reviewed by de Angelis et al. [33]).

The results obtained are controversial; some authors found that Cd concentrations were positively correlated with FSH, T, E2, LH and inhibin B and negatively correlated with prolactin [29, 44]. However, other authors did not find significant correlations between Cd concentrations and serum hormone levels [45, 46]. In general, these results suggest that exposure to EDCs may be associated with alterations in circulating hormone levels in men. Additionally, Yang et al. [47] showed that levels of GnRH and LH were significantly higher in occupationally manganese (Mn) exposed group compared with the non-exposed men. The levels of T were lower in the exposed group. However, this study demonstrated that there was no association between exposure to Mn and E2 and FSH and prolactin levels.

### **2.4 Molecular effects of EDCs**

The effects of EDCs on the morphology and function of the male reproductive system may be attributed to the interactions of these chemicals with several molecules. Male rats treated with 20 mg/(kg day) of TCS showed a significant reduction in the testicular levels of mRNA for cholesterol side-chain cleavage enzyme (*Cyp11a1*), 25-hydroxyvitamin D-1 alpha hydroxylase (*Cyp27b1*), 3β-hydroxysteroid dehydrogenase (*Hsd3b1*), 17β-hydroxysteroid dehydrogenase (*Hsd17b6*), steroidogenic acute regulatory protein (*Star*) and androgen receptor (*Ar*) as compared to control [18]. Moreover, the authors found that there was a decreased localization of StAR protein in testicular LC as determined by immunolocalization indicating a reduced expression of this protein in animals treated with TCS as compared to control. These results could be correlated to the reduction in LC number.

In vitro studies investigated the effect of BPA on steroidogenesis [48, 49]. The authors found that BPA inhibited the production of testosterone in a concentrationdependent manner over the course of the 24 h incubation [48]. Moreover, the concentrations of E2 were greater in the presence of BPA. The decrease in the concentrations of T is related with the inhibition of activities of some enzymes, such as 3β-hydroxysteroid dehydrogenase (*HSD3B1*) and 17α-hydroxylase (*CYP17A*). However, the activity of aromatase was not altered by BPA treatment. More recently, additional results in MA-10 Leydig cell line showed that BPA affects steroidogenic genes, for instance, induces the upregulation of *CYP11A1* and *CYP19* genes [49]. Moreover, the authors found that BPA treatment induced the phosphorylation levels of c-Jun and the levels of protein expression of SF-1, suggesting that the JNK/c-Jun pathway may be involved in BPA toxicity. Similar results were observed in an animal study [49].

The testes from male Sprague-Dawley rats treated with CdCl2 showed a significant increase in the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) [19]. Geng et al. [23] found that pesticides altered the testicular protein expression of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax). Moreover, these authors showed that the activities of testicular enzymes including acyl carrier protein (ACP), lactate dehydrogenase (LDH) and gamma-glutamyltransferase (γ-GT) were significantly altered by exposure to pesticides.

### **3. Spermatozoa**

Sperm motility, together with concentration and morphology, is considered as one of the important predictors of male fertility in vivo. Declining human sperm quality has been demonstrated in several recent studies. Age, lifestyle, environmental pollutants and nutritional factors can affect semen quality [14, 50–52].

**27**

impact on semen quality.

concentration, motility and morphology.

*The Role of Endocrine-Disrupting Chemicals in Male Fertility Decline*

The present section focuses on studies of environmental exposure to EDCs and male reproductive function, as measured by declines in semen quality parameters

**3.1 Effects of EDCs on sperm production, morphology, motility and velocity**

semen parameters, such as semen volume or sperm morphology [8, 54].

TCS has been shown to decrease sperm density probably due to reduced testicular spermatogenesis [18]. A reduced sperm density was observed in the lumina of epididymal tubule from the treated rats. Rats treated with high doses of TCS (50 and 200 mg/kg) showed a significant decrease in the daily sperm production and an increase in the percentage of sperm abnormalities, which included elevated ratios of abnormal sperm head and tails [4]. Zhu et al. [56] performed a cross-sectional study to evaluate the association between exposure to TCS measured by urinary TCS concentration and semen quality in humans. The authors found an association between urinary TCS concentrations and poor semen quality parameters; namely, the authors found an inverse association between urinary TCS concentrations and percentage of sperm motility, sperm count, sperm concentration and percentage of normal morphology, suggesting that environmental exposure to TCS may have

Regarding exposure to PCBs, several studies showed an inverse association between exposure to PCB 153 and sperm motility, while relationships with sperm concentration or total sperm count were inconsistent [57–59]. Additionally, Hauser et al. [60] found an inverse dose–response relationship between PCB 138 and sperm

The correlation between exposure to metals and adverse consequences for human and animal fertility is not completely established. Several studies determined the effects of exposure to metals on male gametes. In vitro studies, using bovine sperm, determined the effect of direct exposure to Hg on male gametes [61, 62]. Arabi et al. [61] showed that exposure to Hg (50, 100, 200, and 300 μmol/l) induced LPO (lipid peroxidation), decreased the glutathione (GSH) content and decreased the percentage of viable spermatozoa. Additionally, a more recent study showed that bovine sperm exposed to Hg at 8 nM and 8 μM have less motility and have impaired sperm

Several studies have been published regarding the association of exposure to phenols and human semen quality [53–55]. A case–control study was conducted to evaluate the association between exposure to phenols and idiopathic male infertility [55]. For that, the authors recruited idiopathic infertile men and fertile controls and measured urinary levels of BPA, benzophenone-3, pentachlorophenol, TCS, 4-*tert*octylphenol (4-*t*-OP), 4-*n*-octylphenol (4-*n*-OP) and 4-*n*-nonylphenol (4-*n*-NP) and semen parameters. The authors found that exposure to 4-*t*-OP, 4-*n*-OP and 4-*n*-NP was associated with idiopathic male infertility, and exposure to 4-*t*-OP and 4-*n*-NP was also associated with abnormal semen quality parameters. However, in this study the authors did not find more relationships between exposure to other phenols and idiopathic male infertility. In another study, urinary BPA concentrations were associated with declines in sperm concentration, motility and morphology [53]. An increasing urine BPA level was associated with lower semen concentration, lower total sperm count, lower sperm vitality and lower sperm motility [54]. Moreover, the authors demonstrated a dose–response relationship between increasing urine BPA level and reduction in semen quality. Lassen et al. [10] also found an inverse association between BPA concentrations and progressive motility, but in this study, BPA excretion was not associated with semen volume, sperm concentration, total sperm count or percentage morphologically normal forms. However, some authors did not find any association between urinary BPA concentrations and some

*DOI: http://dx.doi.org/10.5772/intechopen.88330*

or increased sperm DNA damage/fragmentation.

*Male Reproductive Health*

**2.4 Molecular effects of EDCs**

observed in an animal study [49].

The results obtained are controversial; some authors found that Cd concentrations were positively correlated with FSH, T, E2, LH and inhibin B and negatively correlated with prolactin [29, 44]. However, other authors did not find significant correlations between Cd concentrations and serum hormone levels [45, 46]. In general, these results suggest that exposure to EDCs may be associated with alterations in circulating hormone levels in men. Additionally, Yang et al. [47] showed that levels of GnRH and LH were significantly higher in occupationally manganese (Mn) exposed group compared with the non-exposed men. The levels of T were lower in the exposed group. However, this study demonstrated that there was no association

The effects of EDCs on the morphology and function of the male reproductive system may be attributed to the interactions of these chemicals with several molecules. Male rats treated with 20 mg/(kg day) of TCS showed a significant reduction in the testicular levels of mRNA for cholesterol side-chain cleavage enzyme (*Cyp11a1*), 25-hydroxyvitamin D-1 alpha hydroxylase (*Cyp27b1*), 3β-hydroxysteroid dehydrogenase (*Hsd3b1*), 17β-hydroxysteroid dehydrogenase (*Hsd17b6*), steroidogenic acute regulatory protein (*Star*) and androgen receptor (*Ar*) as compared to control [18]. Moreover, the authors found that there was a decreased localization of StAR protein in testicular LC as determined by immunolocalization indicating a reduced expression of this protein in animals treated with TCS as compared to

In vitro studies investigated the effect of BPA on steroidogenesis [48, 49]. The authors found that BPA inhibited the production of testosterone in a concentrationdependent manner over the course of the 24 h incubation [48]. Moreover, the concentrations of E2 were greater in the presence of BPA. The decrease in the concentrations of T is related with the inhibition of activities of some enzymes, such as 3β-hydroxysteroid dehydrogenase (*HSD3B1*) and 17α-hydroxylase (*CYP17A*). However, the activity of aromatase was not altered by BPA treatment. More recently, additional results in MA-10 Leydig cell line showed that BPA affects steroidogenic genes, for instance, induces the upregulation of *CYP11A1* and *CYP19* genes [49]. Moreover, the authors found that BPA treatment induced the phosphorylation levels of c-Jun and the levels of protein expression of SF-1, suggesting that the JNK/c-Jun pathway may be involved in BPA toxicity. Similar results were

The testes from male Sprague-Dawley rats treated with CdCl2 showed a significant increase in the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) [19]. Geng et al. [23] found that pesticides altered the testicular protein expression of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax). Moreover, these authors showed that the activities of testicular enzymes including acyl carrier protein (ACP), lactate dehydrogenase (LDH) and gamma-glutamyltransferase (γ-GT) were significantly altered by exposure to

Sperm motility, together with concentration and morphology, is considered as one of the important predictors of male fertility in vivo. Declining human sperm quality has been demonstrated in several recent studies. Age, lifestyle, environmental pollutants and nutritional factors can affect semen quality [14, 50–52].

control. These results could be correlated to the reduction in LC number.

between exposure to Mn and E2 and FSH and prolactin levels.

**26**

pesticides.

**3. Spermatozoa**

The present section focuses on studies of environmental exposure to EDCs and male reproductive function, as measured by declines in semen quality parameters or increased sperm DNA damage/fragmentation.
