**1.4.1 Semen quality**

476 Toxicity and Drug Testing

However, recent studies show that the unique structural aspects of the BTB, such as the presence of focal adhesion protein FAK, also render the testis highly susceptible to damage from environmental toxicants. Third, during spermiogenesis when round spermatids differentiate into elongated spermatids, genetic material in the spermatid head condense to form the tightly packed nucleus with the formation of an acrosome above the head region and elongation of the spermatid tail. During this time, spermatids migrate towards the adluminal compartment of the seminiferous tubule until elongated spermatids are released into the tubule lumen via the disassembly of another ES, the apical ES, at spermiation. The apical ES anchors developing spermatids in the seminiferous epithelium until they are fully developed. Thus, disruption of the apical ES (e.g. by environmental toxicants) causes the premature release of spermatids that are structurally defective (e.g. lack of acrosome and/or

 Fig. 2. The process of normal mammalian spermatogenesis with three major phases: (1)

The immotile spermatozoa are transported from the lumen of the seminiferous tubules by peristaltic contractions of adjacent myoid cells. The spermatozoa are suspended in a fluid secreted by Sertoli cells and migrate through a series of ductules within the testis (rete testis), passing through the efferent ductules and eventually entering the epididymis. The efferent ductules concentrate the spermatozoa by reabsorbing fluid (O'Donnell et al., 2001). There is evidence from transgenic mice that this fluid resorption is regulated by estrogen (Hess et al., 1997). The segments of the epididymis, caput, corpus, and cauda secrete proteins, and endocytose secreted proteins from the epididymal lumen to contribute to the maturation of the spermatozoa (O'Donnell et al., 2001). It is within the epididymis that the spermatozoa gain motility machinery. However, these spermatozoa remain immotile as they are pushed through the rest of the reproductive tissues via peristaltic contractions. It is during this final passage that seminal fluid is produced by the seminal vesicles, which contributes about 70% to the semen, and the prostate gland, which contributes another 10–

spermatogonial phase, (2) spermatocyte phase, and (3) spermatid phase.

**1.2.2 Sperm maturation** 

tail) and which are incapable of fertilizing the ovum (Wong & Cheng, 2011).

Reports suggesting that sperm counts have declined in certain areas of industrialized countries throughout the world have contributed to concern about a possible worldwide decline in human semen quality (Swan et al., 1997). A meta-analysis by Carlsen et al.,(1992) reported a worldwide decline in sperm counts over the preceding 50 years, concluding that mean sperm concentrations had decreased by almost 50% from 1940 to 1990. Numerous researchers have attempted to determine whether this apparent decline is real or due to unrecognized biases in data collection and analysis. Confounding Variables may account for the observed findings. Potential confounders include increasing donor age, duration of abstinence, frequency of ejaculation, and even the season of sample collection, all of which influence sperm variables. Other suggested confounders include smoking, chemicals and radiation exposures, stress, ethnicity, and a variety of physical conditions including varicocele, infection, and genital abnormalities such as hypospadias and cryptorchidism. Theories explaining the apparent geographic disparities in sperm counts are currently only speculative, and include environmental, socioeconomic, racial, and methodologic differences (Swan et al., 1997). Fisch et al., (1996) reported yearly fluctuations in mean sperm counts and birth rates (Fisch et al., 1997), suggesting that this may be a more important variable than previously considered.

Environmental Toxicants Induced

Male Reproductive Disorders: Identification and Mechanism of Action 479

hypospadias. This will allow the monitoring of future trends and allow international comparisons on the incidences of these disorders. However, two male genital birth defects, hypospadias and cryptorchidism, both apparently representing mild degrees of feminization, have become important in the ongoing debate regarding the significance of endocrine disruptors or other environmental influences on male development (Sharpe & Skakkebaek, 1993). Several researchers have reported increases in each of these defects in the past three decades. To evaluate the hypothesis of common etiologies, pre- and peri-natal determinants of hypospadias, cryptorchidism, testicular cancer, and infertility are under investigation. Abnormal sex hormone exposure during critical periods of development has

The strongest evidence suggesting a link between these male reproductive tract disorders, aside from the (largely imperfect) data which suggests they are all increasing in incidence, is the fact that epidemiologically the occurrence of one disorder is a risk factor for the occurrence of another (Skakkebaek et al., 2001, Sharpe, 2003). This has led to the proposal that low sperm counts, hypospadias, cryptorchidism and testicular germ cell cancer are interrelated disorders comprising a 'testicular dysgenesis syndrome' (TDS; Skakkebaek et al., 2001, Sharpe, 2003, 2010; **Figure 3**). The disorders that comprise TDS all have their roots in fetal development, suggesting that a possible causal link lies in abnormal hormone synthesis or action during reproductive tract development. From the historical literature, it is well known that the administration of diethylstilboestrol (DES; a potent synthetic oestrogen) to pregnant humans and rodents causes reproductive tract abnormalities in the offspring (Stillman, 1982). In male rodents, neonatal administration of DES induces a reduction in the number of Sertoli cells (the major somatic cell type which supports spermatogenesis) (Sharpe et al., 2003). There is also data suggesting DES administration to humans induces an increase in the incidence of cryptorchidism, although it is less certain whether hypospadias and testicular cancer show any significant increase (Stillman, 1982). DES only induces male reproductive tract abnormalities after administration at very high doses, which are probably not relevant to environmental considerations. However, what is of more concern is that, when administered at high doses, DES and other potent oestrogens are capable of reducing androgen levels and expression of the androgen receptor protein relative to control rats (McKinnell et al., 2001, Rivas et al., 2002). This raises the important question of whether some of the genital tract abnormalities that arise from in utero administration of potent oestrogens are caused by lowered androgen levels and/or action.

There are a number of commonly used environmental chemicals that have been identified as having anti-androgenic properties. These chemicals have been administered to pregnant rodents during the period of reproductive tract development. When the male pups were examined, they displayed many of the abnormalities associated with flutamide administration. Some chemicals (vinclozolin, procymidone, linuron, p,p′DDE (1,1,1 dichloro-2,2-bis(p-chlorophenyl)ethane) act as androgen receptor antagonists, others (phthalate esters) reduce androgen synthesis, but it is likely that other modes of action are also involved in the toxicity induced by these compounds (Gray et al.,2001). The following sections provide information on a few well-characterised examples of anti-androgenic

been postulated as a likely shared pathologic mechanism (Toppari et al., 1996).

**1.4.4 There is a link between these male reproductive health issues** 

**1.5 Anti-androgenic compounds in the environment** 

#### **1.4.2 Testicular cancer**

Testicular cancer is often quoted as the commonest cancer of young men. The secular trends across Europe and the United States show that it is increasing in incidence in Caucasian men (SEER 2003). There is widespread geographical variation and the incidence of testicular cancer can vary up to 10-fold between countries. In Denmark in 1980, the age standardised incidence rate per 100 000 population was 7.8% whereas in Lithuania it was 0.9%, although in all countries where registry data has been analysed there was an annual increase of 2.3– 3.4% (Adami et al., 1994). The increase in testicular cancer has been linked to a birth cohort effect, suggesting that factors affecting in utero development may be important (Bergstrom et al., 1996). Testicular germ cell cancer arises from cells which have similar characteristics to fetal germ-cells; these pre-malignant cells are termed carcinoma-in situ (CIS) cells (Rajpert-De Meyts et al., 2003). How these cells persist during development and what causes them to proliferate after puberty is not well understood, although it is thought that the factors that promote normal germ cell division may also be important in promoting CIS proliferation. Abnormal intrauterine hormone levels i.e. decreased androgen and/or increased oestrogen levels are believed to be important in the occurrence of testicular cancer (Sharpe & Skakkebaek 1993). Similarly, decreased androgen and/or increased oestrogen levels have also been implicated in the occurrence of cryptorchidism, hypospadias and low sperm counts (Sharpe & Skakkebaek 1993). Although genetics almost certainly plays a major role in the etiology of the disease, other etiologies, including environmental factors, need to be elucidated to explain why, for example, major differences in testicular cancer rates exist among the relatively genetically homogenous Scandinavian countries. Increases in testicular cancer rates are not recent phenomena. A doubling in incidence was documented in Denmark within 25 years after the initiation of cancer registration in 1943 (Ekbom & Akre, 1998). Mortality data from Great Britain show an increase in mortality due to testicular cancer beginning in the 1920s (Davies, 1981). These mortality data raise an important distinction: if environmental risk factors play a role in testicular cancer incidence, relevant exposures must therefore have existed since the turn of the century. This would make it less likely that organochlorines such as DDT and other endocrine-disrupting chemicals are possible etiologic agents. Research is ongoing to explore new genetic markers for early detection of carcinoma *in situ* cells in semen, as well as to define the role of hormonal assays (e.g., inhibin-B) as screening tools for testicular cancer and carcinoma *in situ*.

#### **1.4.3 Congenital abnormalities (Cryptorchidism and hypospadias)**

Cryptorchidism and hypospadias are abnormalities normally detected at birth (congenital abnormalities). Cryptorchidism occurs when the testis does not descend into the scrotal sac; this is generally unilateral but can be bilateral. Hypospadias is a developmental abnormality of the penis in which the urethral opening is not located at the tip of the glans penis but can occur anywhere along the shaft. Determining whether there is a real increase in hypospadias and/or cryptorchidism is confounded by changes in diagnostic criteria and recording practices which make the registry data unreliable (Toppari et al., 1996). Despite this, cryptorchidism is the most common congenital abnormality of the newborn (2–4% incidence) and trends for hypospadias suggest a progressive increase; based on registry data, hypospadias is the second most common (0.3–0.7% at birth) congenital malformation (Sharpe, 2003). Prospective studies are underway, which employ standardised diagnostic criteria, to collect robust data about the current incidence of cryptorchidism and

Testicular cancer is often quoted as the commonest cancer of young men. The secular trends across Europe and the United States show that it is increasing in incidence in Caucasian men (SEER 2003). There is widespread geographical variation and the incidence of testicular cancer can vary up to 10-fold between countries. In Denmark in 1980, the age standardised incidence rate per 100 000 population was 7.8% whereas in Lithuania it was 0.9%, although in all countries where registry data has been analysed there was an annual increase of 2.3– 3.4% (Adami et al., 1994). The increase in testicular cancer has been linked to a birth cohort effect, suggesting that factors affecting in utero development may be important (Bergstrom et al., 1996). Testicular germ cell cancer arises from cells which have similar characteristics to fetal germ-cells; these pre-malignant cells are termed carcinoma-in situ (CIS) cells (Rajpert-De Meyts et al., 2003). How these cells persist during development and what causes them to proliferate after puberty is not well understood, although it is thought that the factors that promote normal germ cell division may also be important in promoting CIS proliferation. Abnormal intrauterine hormone levels i.e. decreased androgen and/or increased oestrogen levels are believed to be important in the occurrence of testicular cancer (Sharpe & Skakkebaek 1993). Similarly, decreased androgen and/or increased oestrogen levels have also been implicated in the occurrence of cryptorchidism, hypospadias and low sperm counts (Sharpe & Skakkebaek 1993). Although genetics almost certainly plays a major role in the etiology of the disease, other etiologies, including environmental factors, need to be elucidated to explain why, for example, major differences in testicular cancer rates exist among the relatively genetically homogenous Scandinavian countries. Increases in testicular cancer rates are not recent phenomena. A doubling in incidence was documented in Denmark within 25 years after the initiation of cancer registration in 1943 (Ekbom & Akre, 1998). Mortality data from Great Britain show an increase in mortality due to testicular cancer beginning in the 1920s (Davies, 1981). These mortality data raise an important distinction: if environmental risk factors play a role in testicular cancer incidence, relevant exposures must therefore have existed since the turn of the century. This would make it less likely that organochlorines such as DDT and other endocrine-disrupting chemicals are possible etiologic agents. Research is ongoing to explore new genetic markers for early detection of carcinoma *in situ* cells in semen, as well as to define the role of hormonal assays

(e.g., inhibin-B) as screening tools for testicular cancer and carcinoma *in situ*.

Cryptorchidism and hypospadias are abnormalities normally detected at birth (congenital abnormalities). Cryptorchidism occurs when the testis does not descend into the scrotal sac; this is generally unilateral but can be bilateral. Hypospadias is a developmental abnormality of the penis in which the urethral opening is not located at the tip of the glans penis but can occur anywhere along the shaft. Determining whether there is a real increase in hypospadias and/or cryptorchidism is confounded by changes in diagnostic criteria and recording practices which make the registry data unreliable (Toppari et al., 1996). Despite this, cryptorchidism is the most common congenital abnormality of the newborn (2–4% incidence) and trends for hypospadias suggest a progressive increase; based on registry data, hypospadias is the second most common (0.3–0.7% at birth) congenital malformation (Sharpe, 2003). Prospective studies are underway, which employ standardised diagnostic criteria, to collect robust data about the current incidence of cryptorchidism and

**1.4.3 Congenital abnormalities (Cryptorchidism and hypospadias)** 

**1.4.2 Testicular cancer** 

hypospadias. This will allow the monitoring of future trends and allow international comparisons on the incidences of these disorders. However, two male genital birth defects, hypospadias and cryptorchidism, both apparently representing mild degrees of feminization, have become important in the ongoing debate regarding the significance of endocrine disruptors or other environmental influences on male development (Sharpe & Skakkebaek, 1993). Several researchers have reported increases in each of these defects in the past three decades. To evaluate the hypothesis of common etiologies, pre- and peri-natal determinants of hypospadias, cryptorchidism, testicular cancer, and infertility are under investigation. Abnormal sex hormone exposure during critical periods of development has been postulated as a likely shared pathologic mechanism (Toppari et al., 1996).

#### **1.4.4 There is a link between these male reproductive health issues**

The strongest evidence suggesting a link between these male reproductive tract disorders, aside from the (largely imperfect) data which suggests they are all increasing in incidence, is the fact that epidemiologically the occurrence of one disorder is a risk factor for the occurrence of another (Skakkebaek et al., 2001, Sharpe, 2003). This has led to the proposal that low sperm counts, hypospadias, cryptorchidism and testicular germ cell cancer are interrelated disorders comprising a 'testicular dysgenesis syndrome' (TDS; Skakkebaek et al., 2001, Sharpe, 2003, 2010; **Figure 3**). The disorders that comprise TDS all have their roots in fetal development, suggesting that a possible causal link lies in abnormal hormone synthesis or action during reproductive tract development. From the historical literature, it is well known that the administration of diethylstilboestrol (DES; a potent synthetic oestrogen) to pregnant humans and rodents causes reproductive tract abnormalities in the offspring (Stillman, 1982). In male rodents, neonatal administration of DES induces a reduction in the number of Sertoli cells (the major somatic cell type which supports spermatogenesis) (Sharpe et al., 2003). There is also data suggesting DES administration to humans induces an increase in the incidence of cryptorchidism, although it is less certain whether hypospadias and testicular cancer show any significant increase (Stillman, 1982). DES only induces male reproductive tract abnormalities after administration at very high doses, which are probably not relevant to environmental considerations. However, what is of more concern is that, when administered at high doses, DES and other potent oestrogens are capable of reducing androgen levels and expression of the androgen receptor protein relative to control rats (McKinnell et al., 2001, Rivas et al., 2002). This raises the important question of whether some of the genital tract abnormalities that arise from in utero administration of potent oestrogens are caused by lowered androgen levels and/or action.

#### **1.5 Anti-androgenic compounds in the environment**

There are a number of commonly used environmental chemicals that have been identified as having anti-androgenic properties. These chemicals have been administered to pregnant rodents during the period of reproductive tract development. When the male pups were examined, they displayed many of the abnormalities associated with flutamide administration. Some chemicals (vinclozolin, procymidone, linuron, p,p′DDE (1,1,1 dichloro-2,2-bis(p-chlorophenyl)ethane) act as androgen receptor antagonists, others (phthalate esters) reduce androgen synthesis, but it is likely that other modes of action are also involved in the toxicity induced by these compounds (Gray et al.,2001). The following sections provide information on a few well-characterised examples of anti-androgenic

Environmental Toxicants Induced

**1.5.2 DDT and** *p,p* **′-DDE** 

Male Reproductive Disorders: Identification and Mechanism of Action 481

Taiwan 1978–79) was followed to determine effects on male reproductive health. Median serum PCB levels measured in Yu-Cheng mothers was 26.8 ng/ml: a relatively high PCB exposure that would be expected to affect fetal development. Sexual development and semen quality were evaluated in Yu-Cheng sons, aged 16 yr and older. Seminal volume and sperm concentration were not different between exposed and control boys. However, proportions of sperm with normal morphology and motility were reduced in exposed boys (Guo et al., 2000). The effects of PCB exposure on semen quality in men from the general population appear to affect differentiation of spermatids (spermiogenesis) and posttesticular development (sperm maturation), which would manifest as decreased sperm morphology and motility, respectively. Further studies of PCB exposure—both individual

The persistent pesticide, DDT, is broken down in the environment, and one of its metabolites is p,p′-DDE, which has been shown to act as an androgen receptor antagonist both in vivo and in vitro (Kelce et al., 1995). Studies in which p,p′-DDE was administered to rats during development (gestational day (GD)14–18; 100 mg/kg/day) affected androgendependent aspects of male development such that it reduced anogenital distance, caused nipple retention and, depending on the rat strain, induced hypospadias (You et al., 1998). Another DDT derivative, methoxychlor and its metabolites, have been shown to interact with both oestrogen receptors and the androgen receptor (AR). The methoxychlor metabolite, 1,1-Trichloro-2,2-bis (4 hydroxyphenyl) ethane, is an oestrogen receptor (ER)- α agonist, an ER-β antagonist and an androgen receptor (AR) antagonist (Gray et al., 2001). This illustrates that these chemicals may act by more than one mechanism to induce effects on the exposed population. Moreover, several population studies conducted to examine the effects of DDT and metabolite exposure on male reproductive health support the hypothesis that DDT exposure is related to reduced semen quality. Significantly higher seminal concentrations of *p,p*′-DDE were also reported in infertile patients compared to a fertile control group in India. Seminal fluid levels of fructose, γ-glutamyl transpeptidase, and acid phosphatase were positively correlated with *p,p*′-DDE concentrations in infertile men. The high concentration of fructose, a marker for seminal vesicle function and an important energy source for sperm, may indicate non-utilization of fructose by sperm. DDT exposure may be associated with abnormal metabolism in sperm, including decreased fuel utilization, in infertile men (Pant et al., 2004). Mexican men living in the areas where DDT was used for malaria control, but without occupational exposure to DDT, exhibited serum levels of *p,p*′- DDE approximately 350-fold greater than Canadian men exposed to background environmental levels. *p,p*′-DDE concentrations in the Mexican men were correlated with increases in SHBG concentration and negatively correlated with testosterone levels. Both semen volume and total sperm number were inversely correlated to *p,p'*-DDE levels. Thus, androgen levels and semen quality are adversely affected by high *p,p*′-DDE body burden (Ayotte et al., 2001). The studies examining DDT exposure and semen quality report consistent effects on sperm motility and sperm morphology, similar to the PCB studies. The increased SHBG concentrations associated with serum *p,p*′-DDE described by Ayotte et al.,(2001) provides a possible mechanism for observed reductions in plasma testosterone and sperm number. By inducing SHBG synthesis, *p,p*′-DDE may exert its antiandrogenic effects by reducing the amount of bioavailable testosterone, thereby impairing spermatogenesis.

congeners and PCB mixtures—and sperm parameters are required.

Fig. 3. Testicular dysgenesis syndrome. Both genetic and environmental factors affect testicular development and functions. Damage of the testicular cells (Leydig cells and Sertoli cells), disrupts androgen production from Leydig cells and secretion of paracrine factors from sertoli cells, leading to birth defects (hypospadias, cryptorchidism) and impaired germ cell differentiation, apparent later as reduced semen quality or in the worst cases as carcinoma *in situ* (CIS) of the testis and consequent testicular cancer. (Modified from Skakkeback et al. Human Reproduction, 2001).
