**1.1 The male reproductive system: Environmental influence**

Global changes in semen quality are suggested to be produced by the enhanced exposure to environmental chemicals contained in pesticides, food sources, cosmetics, plastics, electronics, and other synthetic materials (Carlsen et al., 1992). The biological basis for this hypothesis is the action of certain chemical compounds, both naturally occurring and anthropogenic (man-made), on endogenous hormone receptors and hormone-dependent pathways. These chemicals are termed hormonally active agents, environmental estrogens, hormone mimics, and endocrine disrupters/disruptors (US. EPA, 1998a; 1998b; National Research Council [NRC], 1989). A wide range of mechanisms of action are described for endocrine disrupters, including agonists of the estrogen receptor (ER) genistein, diethylstilbestrol (DES; Roy et al., 1997), and bisphenol A (BPA; Kuiper et al., 1998); androgen receptor (AR) antagonists such as vinclozolin ( Wong et al., 1995), linuron, procymidone (Gray et al., 1999), phthalates (Foster et al., 2001), and *p,p*′-dichlorodiphenyl dichloroethylene (*p,p*′-DDE; Kelce et al., 1995) and aryl hydrocarbon receptor (AhR) agonists, which include dioxins (Toyoshiba et al., 2004), polychlorinated biphenyls (PCB), polycyclic aromatic hydrocarbons (PAH), and polychlorinated dibenzofurans (PCDF; Peterson et al., 1993). Exposure to endocrine-disrupting chemicals may occur through environmental routes (air, soil, water, food) or via occupational exposures (**Figure 1**) (Sharpe & Irvine, 2004).

#### **1.2 The testis: Male reproductive organ**

The testis is both an endocrine gland and a reproductive organ, responsible for the production of hormones and male gametes and an important target for endocrine disruption. The testis consists of two types of tissues: seminiferous tubules, supported by Sertoli cells, and the interstitial compartment, comprised of Leydig cells (Fisher, 2004; Akingbemi, 2005). Testicular functions (spermatogenesis steroidogenesis) are regulated by the hypothalamic-pituitary-testicular (HPT) axis which involves the pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH; Jana et al., 2006). Testicular functions are proposed to be regulated by a number of hormones and growth factors in addition to FSH, LH, and androgens, including insulin-like growth factor, oxytocin, and transforming growth factor-α and estrogens (Pryor et al., 2000).

#### **1.2.1 Spermatogenesis**

Spermatogenesis is the formation of the male gamete or spermatozoa. Spermatogenesis is dependent on the integrity of the architecture of the seminiferous tubules and Sertoli cells and endocrine regulation and is regulated by testosterone and FSH. In response to LH, Leydig cells produce androgens, including testosterone, which along with FSH bind to their respective Sertoli cell receptors to regulate spermatogenesis. Spermatogenesis requires unique associations between Sertoli cells and developing male germ cells such that the seminiferous tubules are lined by Sertoli cells and joined by tight junctions forming the

focused on the possibility that exposures to hormonally active compounds, particularly during childhood and *in utero*, are to blame, at least in part, for changes in semen quality, increasing rates of testicular cancer, and malformations of the male urogenital tract (Sharpe, 2010). The ability to investigate environmental determinants of these indicators of male

Global changes in semen quality are suggested to be produced by the enhanced exposure to environmental chemicals contained in pesticides, food sources, cosmetics, plastics, electronics, and other synthetic materials (Carlsen et al., 1992). The biological basis for this hypothesis is the action of certain chemical compounds, both naturally occurring and anthropogenic (man-made), on endogenous hormone receptors and hormone-dependent pathways. These chemicals are termed hormonally active agents, environmental estrogens, hormone mimics, and endocrine disrupters/disruptors (US. EPA, 1998a; 1998b; National Research Council [NRC], 1989). A wide range of mechanisms of action are described for endocrine disrupters, including agonists of the estrogen receptor (ER) genistein, diethylstilbestrol (DES; Roy et al., 1997), and bisphenol A (BPA; Kuiper et al., 1998); androgen receptor (AR) antagonists such as vinclozolin ( Wong et al., 1995), linuron, procymidone (Gray et al., 1999), phthalates (Foster et al., 2001), and *p,p*′-dichlorodiphenyl dichloroethylene (*p,p*′-DDE; Kelce et al., 1995) and aryl hydrocarbon receptor (AhR) agonists, which include dioxins (Toyoshiba et al., 2004), polychlorinated biphenyls (PCB), polycyclic aromatic hydrocarbons (PAH), and polychlorinated dibenzofurans (PCDF; Peterson et al., 1993). Exposure to endocrine-disrupting chemicals may occur through environmental routes (air, soil, water, food) or via occupational exposures (**Figure 1**)

The testis is both an endocrine gland and a reproductive organ, responsible for the production of hormones and male gametes and an important target for endocrine disruption. The testis consists of two types of tissues: seminiferous tubules, supported by Sertoli cells, and the interstitial compartment, comprised of Leydig cells (Fisher, 2004; Akingbemi, 2005). Testicular functions (spermatogenesis steroidogenesis) are regulated by the hypothalamic-pituitary-testicular (HPT) axis which involves the pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH; Jana et al., 2006). Testicular functions are proposed to be regulated by a number of hormones and growth factors in addition to FSH, LH, and androgens, including insulin-like growth factor,

Spermatogenesis is the formation of the male gamete or spermatozoa. Spermatogenesis is dependent on the integrity of the architecture of the seminiferous tubules and Sertoli cells and endocrine regulation and is regulated by testosterone and FSH. In response to LH, Leydig cells produce androgens, including testosterone, which along with FSH bind to their respective Sertoli cell receptors to regulate spermatogenesis. Spermatogenesis requires unique associations between Sertoli cells and developing male germ cells such that the seminiferous tubules are lined by Sertoli cells and joined by tight junctions forming the

oxytocin, and transforming growth factor-α and estrogens (Pryor et al., 2000).

reproductive health is currently limited by available methodologies and data.

**1.1 The male reproductive system: Environmental influence** 

(Sharpe & Irvine, 2004).

**1.2.1 Spermatogenesis** 

**1.2 The testis: Male reproductive organ** 

Fig. 1. Routes of human exposure to some common environmental chemicals. DDE= 1, 1 dichloro-2, 2-bis (p- chlorophenyl) ethylene; DDT= dichlorodiphenyltrichloroethane; PAHs= polycyclic aromatic hydrocarbons; PCBs= polychlorinated biphenyls. (Modified from Sharpe & Irvine, BMJ, 2004).

blood–testis barrier (BTB; Walker & Cheng, 2005). There are three major phases of spermatogenesis: (1) spermatogonial phase, (2) spermatocyte phase, and (3) spermatid phase (**Figure 2**). In the first phase the diploid spermatogonia undergo mitosis and create stem cells and diploid primary spermatocytes. During the second phase the primary spermatocytes undergo two rounds of meiosis, producing haploid spermatids. Finally, the spermatids begin a differentiation phase, sometimes referred to as spermiogenesis, during which the immature gametes develop into mature spermatozoa (O'Donnell et al., 2001). Spermatids continue their differentiation (spermiogenesis) while physically associated with the Sertoli cells. Spermiogenesis includes polarization of the spermatid, formation of the acrosome cap and flagellum, condensation, elongation of the nucleus, and cytoplasmic remodelling to produce the characteristic appearance of the mature spermatozoa. Spermatozoa are morphologically mature but immotile and are then released into the lumen of the seminiferous tubules (spermiation). At this stage these immotile testicular spermatozoa are not yet capable of fertilization (O'Donnell et al., 2001). The BTB between Sertoli cells comprises a co-existing tight junction (TJ), desmosome, gap junction and a testisspecific adherens junction (AJ) called the basal ectoplasmic specialization (ES). The basal ES is typified by the presence of actin filament bundles 'sandwiched' between the plasma membrane and the cisternae of endoplasmic reticulum in two neighbouring Sertoli cells.

Environmental Toxicants Induced

Male Reproductive Disorders: Identification and Mechanism of Action 477

30%. Seminal fluid is comprised of proteins, enzymes, fructose, mucus, vitamin-C, flavins, phosphorylcholine, and prostaglandins (Purvis et al., 1986). Decreases in seminal fluid

Male reproductive tract development is a dynamic process requiring the interaction of many factors and hormones. One of the major factors essential for the development of the male internal and external male reproductive tract are the androgens, testosterone and dihydrotestosterone (DHT) (Phillips & Tanphaichitr, 2008). Androgens are produced by the testes during fetal and neonatal development and are essential for the maintenance of the Wolffian duct that differentiates into the epididymis, vas deferens and the seminal vesicles. The masculinization of these reproductive structures is mediated by testosterone. The masculinization of the external genitalia and prostate is largely mediated by DHT which is a more potent metabolite of testosterone and is produced by the action of the enzyme 5αreductase. The central role of androgens in driving these developmental processes illustrates why chemicals that can interfere with the synthesis or action of androgens can have deleterious consequences for the developing male genital tract. Administration of the antiandrogen, flutamide (an androgen receptor antagonist), during male reproductive tract development resulted in abnormalities in the formation of the external genitalia hypospadias and cryptorchidism; internally, agenesis of the epididymis, vas deferens and prostate (Mylchreest et al., 2000). Within the testis, degeneration of the seminiferous epithelium and Leydig cell hyperplasia were common (although this may be a consequence of the cryptorchidism rather than an anti-androgenic effect). The male pups also displayed retained thoracic nipples and a reduced anogenital distance (feminised) which are both indicative of reduced androgen action in fetal life (Mylchreest et al., 2000). In summary, both testosterone- and DHT-mediated male reproductive tract development is impaired by

volume may therefore indicate diminished seminal vesicle or prostate functions.

flutamide when administered over the period of reproductive tract differentiation.

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

**1.4 Problems with male reproductive health** 

variable than previously considered.

**1.4.1 Semen quality** 

**1.3 The role of androgens in male reproductive tract development** 

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

Fig. 2. The process of normal mammalian spermatogenesis with three major phases: (1) spermatogonial phase, (2) spermatocyte phase, and (3) spermatid phase.

### **1.2.2 Sperm maturation**

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–

30%. Seminal fluid is comprised of proteins, enzymes, fructose, mucus, vitamin-C, flavins, phosphorylcholine, and prostaglandins (Purvis et al., 1986). Decreases in seminal fluid volume may therefore indicate diminished seminal vesicle or prostate functions.
