1 review of literature 1.1 description of male reproductive...

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1 REVIEW OF LITERATURE

1.1 Description of male reproductive system

In mammals, the male reproductive system consists of paired testes,

epididymides, ductus deferens, accessory sex glands and the penis. Testes execute two

important functions, spermatogenesis and steroidogenesis which are very vital for the

perpetuation of life. Spermatogenesis or the production of spermatozoa takes place

within the seminiferous tubules of the testis and steroidogenesis or the synthesis of

testosterone occurs within the interstitial compartment. In the seminiferous tubules,

spermatogenesis takes place within the stratified epithelium whereas testosterone

production occurs within the Leydig cells which are scattered in a vascular, loose

connective tissue in the interstitial compartment between the seminiferous tubules.

Testosterone, produced by the Leydig cells of the testis, plays an essential role in

determining male secondary sexual characteristics, production of spermatozoa and

fertility (Bremer, 1911; Heller and Clermont, 1963; for reviews see Kerr, 1992a; Hess

and de Franca, 2008; Huleihel and Lunenfeld, 2004).

Epididymis is a single, long and highly convoluted duct which connects the

testicular efferent ducts to the vas deferens, a coiled duct which connects epididymis to

the ejaculatory duct. Epididymis plays an important role in the transport and storage of

testicular spermatozoa. In most mammals, epididymis is classified into three distinct

regions based on its gross morphology; caput or head, corpus or body and cauda or tail

region. The corpus region is thinner and it joins the wider segments, caput and cauda.

Spermatozoa produced in the testis are functionally immature and they attain functional

maturity as they migrate through the epididymis. The absorptive and secretary activity

of epididymal epithelium helps to maintain a specific intraluminal environment which is

important for the maturation of spermatozoa (Kirchhoff et al., 1998; for reviews see

Aitken et al., 2007; Cooper, 2011). As spermatozoa mature, they move into the vas

deferens where it is stored until ejaculation.

1 Review of Literature

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Seminal vesicles are paired sac-like glands that are attached to the vas deferens

near the base of the bladder. Seminal vesicles are composed of tubular alveoli with

active secretary epithelium. The inner surface of the seminal vesicles consist of tubules

that are thrown into an intricate system of folds to form irregular diverticula. The

secretions of the seminal vesicles constitute the major portion of seminal fluid, the fluid

that carries spermatozoa (Davies and Mann, 1947). Seminal vesicles are highly

androgen-dependent and their secretions are alkaline and contain fructose, proteins,

citric acid, inorganic phosphorous, prostaglandins, and low-molecular-weight proteinase

inhibitors. Seminal vesicle secretions promote capacitation, increase stability of

spermatozoa and help to prevent immune response against spermatozoa in the female

reproductive tract (for reviews see Maxwell et al., 2007; Gonzales, 2001).

Prostate is an elastic, donut-shaped fibromuscular gland that surrounds the

urethra at neck of the urinary bladder. Prostate is encapsulated by a thin vascularized

fibroelastic tissue layer (Flickinger, 1972; Nemeth and Lee, 1996). The primary function

of prostate is to secrete milky fluid which contains proteins and hormones which form a

part of the seminal fluid produced by seminal vesicles. The prostatic fluid is rich in acid

phosphates, citric acid, fibrinolysin, prostate specific antigen, amylase, kallikreins, zinc

and calcium which are important for the normal functioning of spermatozoa. The

secretions of the prostate compose 30% of the seminal fluid volume. Prostate is an

androgen-sensitive organ and its growth and regression depends on the presence or

absence of circulating androgens (for review see Kumar and Majumder, 1995).

1.1.1 Testis

Testes are paired encapsulated ovoid organs that lie in the scrotum. Testes are

encapsulated by a tough fibrous capsule which consists of three layers- an outer tunica

vaginalis, the middle tunica albuginea and the innermost tunica vasculosa. The muscle

layers inside the tunica vasculosa contain arteries, veins and lymphatic vessels (Leeson,

1975). Capsular contractions of the testis are responsible for the transport of

spermatozoa from testis into the epididymis. The important functions of testis are


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spermatogenesis and steroidogenesis which takes place in two different compartments,

seminiferous tubules and interstitium, respectively (Lacy, 1962). Though these

compartments are anatomically divided, they are functionally connected to each other

and their integrity is essential for normal germ cell production.

The seminiferous tubules of the testis consist of two major cell types- the germ

cells and the supporting cells or Sertoli cells. The Sertoli cells are uniformly distributed

in the seminiferous epithelium along with developing germ cells and they nourish the

germ cells throughout their development. The seminiferous tubule is lined by a basal

lamina which contains peritubular myoid cells. The myoid cells constitute a partial

permeability barrier by preventing the entry of large molecules into the germinal

epithelium. However, the major exclusion barrier is formed by the tight and gap

junctions which exist between the adjacent Sertoli cells (Dym and Fawcett, 1970;

Holash et al., 1993; for review see Jégou, 1993). These inter-Sertoli cell junctions,

called the blood-testis barrier, divide the seminiferous epithelium into two distinct

compartments: the basal and the adluminal compartments. In the basal compartment

resides the spermatogonia and early spermatocytes and they are readily accessible to

systemic circulation. The adluminal compartment, which contains meiotic and post-

meiotic spermatocytes, is sequestered from systemic circulation and is exposed only to

the components transported by the Sertoli cells (for reviews see Pelletier and Byers,

1992; Mruk and Cheng, 2004; Ravel and Jaillard, 2011). During the process of

spermatogenesis, the undifferentiated spermatogonia which reside at the basal

compartment of the seminiferous epithelium undergo a series of mitotic divisions to

form primary spermatocytes. The primary spermatocyte then gets translocated to the

adluminal compartment and this requires extensive restructuring of the inter-Sertoli tight

junctions. (for reviews see Lui and Lee, 2006; Mruk and Cheng, 2010). In the adluminal

compartment, the spermatocytes undergo two consecutive rounds of meiosis to form

mature haploid spermatids. Apart from providing physical support to germ cells, Sertoli

cells offer unique environment in the adluminal compartment by providing specific


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growth factors and nutrients which are critical for germ cell survival (for review see

Petersen and Soder, 2006). On the other hand, germs cell factors also play an essential

role in controlling the Sertoli cell activity. The communications between germ cells and

Sertoli cells are vital for successful spermatogenesis (Grootegoed et al., 1989).

The interstitial compartment of the testis consists of steroid-secreting Leydig

cells, blood and lymphatic vessels, nerves, macrophages, fibroblasts and loose

connective tissues. However, the principal cells of this compartment are the Leydig

cells. Adult Leydig cells are rich in smooth endoplasmic reticulum and mitochondria

with tubular cristae (Fawcett et al., 1973). Leydig cells are the site of androgen

production and the most biologically important androgen produced by Leydig cells is

testosterone. The production of testosterone by Leydig cells is under the control of

pulsatile release of pituitary LH which, in turn, acts through LH receptors present on

Leydig cells. The LH-stimulated testosterone production plays an important role in the

development of male reproductive tract and maintenance of spermatogenesis (for review

see Luetjens et al., 2005). The intercellular communications between Sertoli, Leydig and

germ cells are crucial for the regulation of testicular spermatogenesis and

steroidogenesis.

1.1.1.1 Spermatogenesis

Spermatogenesis is a complex process by which functional haploid

spermatozoa are formed from an interdependent population of undifferentiated germ

cells. Spermatogenesis comprises following phases: (a) spermatogoniogenesis (b)

meiosis (c) spermiogenesis and (d) spermiation. The primary phase or

spermatogoniogenesis consists of mitotic division of spermatogonial cells. This is

followed by two rounds of meiosis to form primary and secondary spermatocytes.

Spermiogenesis constitutes the final phase of spermatogenesis where immature

spermatids develop into mature spermatozoa. During the process of spermiation, mature

spermatozoa from the Sertoli cells are released into the lumen of the seminiferous

tubule. Production of spermatozoa begins at puberty and continues throughout the life of


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a male (Oakberg, 1956; for reviews see Clermont, 1972; Grootegoed et al., 2000).

Endocrine regulation by testosterone and the architecture of the Sertoli cells and

seminiferous tubules are important decisive factors in spermatogenesis (Steinberger et

al., 1973; for review see Griswold, 1998).

Spermatogonial stem cells constitute the most immature cells and they are

located at the base of the seminiferous epithelium. Spermatogonial stem cells proliferate

by mitotic division and they repeatedly multiply to replenish the germinal epithelium

(for review see Kolasa et al., 2012). There are two types of spermatogonia- type A

spermatogonia and type B spermatogonia. Type A and B spermatogonia could be

distinguished based on the absence and presence of heterochromatin, respectively. The

type A spermatogonia undergo a series of division to form A single (As), A paired (Ap)

and A aligned (Aal) spermatogonia. As type of spermatogonia divides and constitutes

the stem cell population for continued spermatogenesis (Rowley et al., 1971; for review

see de Rooij, 1998). Ap spermatogonia are connected through intercellular cytoplasmic

bridges and undergo division to form 4, 8 and 16 Aal spermatogonia. The Aal

The preleptotene primary spermatocytes then migrate upwards from the

basement membrane by traversing the Sertoli-Sertoli tight junctions and undergo

reduction-division by meiosis. DNA synthesis takes place in the preleptotene

spermatocytes. The preleptotene primary spermatocytes then enter into the leptotene

stage of the prophase I of meiosis where the chromatin reorganizes to form thread-like

structures. This is followed by pairing of homologous chromosomes and interchange of

genetic segments through formation of synaptonemal complexes at the zygotene stage.

At the pachytene stage, the nuclei enlarge and the chromosome becomes thicker and

spermatogonia undergo five successive divisions and gives rise to A2, A3, A4,

intermediate and type B spermatogonia. Type B spermatogonia further undergo mitotic

division to form preleptotene primary spermatocytes and this marks the end of mitotic

divisions taking place during spermatogenesis (Dym and and Cavicchia, 1978; Clermont

and Leblond, 1953).


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shorter. The synaptonemal complexes and the homologous chromosomes separate from

one another at the diplotene stage and the nuclear envelope disappears and the

chromosome condenses during diakinesis (Monesi, 1965; Oakberg, 1956). Meiosis I

produces small secondary spermatocytes which rapidly undergo meiosis II to form very

small haploid round spermatids. The haploid round spermatids then undergo a final

phase of differentiation through a process called spermiogenesis (de Rooij, 1983).

During spermiogenesis, the haploid round spermatids undergo complex

morphological and biochemical events resulting in the formation of mature

spermatozoa. The process of spermiogenesis is divided into a number of morphological

events which include golgi phase, cap phase, acrosomal phase and maturation.

Formation of several granules within golgi apparatus marks the first sign of

differentiation of spermatozoa (de Krester and Kerr, 1988; Leblond and Clermont,

1952). These granules coalesce to form acrosome vesicle. The centrioles migrate in the

direction opposite to the acrosome vesicles thereby providing polarity to the cell. The

nucleus becomes denser and smaller. The cytoplasmic tubules give rise to transient

sleeve-like structures called manchette (for review see Kierszenbaum and Tres, 2004).

Redistribution of mitochondria also takes place during the golgi phase. The golgi phase

is followed by the cap phase where the acrosome vesicles move distally and covers half

of the nuclear surface. The centrioles elongate to become tail portion of the spermatozoa

and the manchette assists in centriole elongation. In the acrosomal phase, the nucleus

still condenses. The cell gets elongated and a mature flagellum is formed. During the

final phase of maturation, tail of the spermatozoa gets lined with the mitochondria in the

proximal region. Spermatozoa discard excess cytoplasm into the lumen of the tubule or

it is pagocytized by the Sertoli cells as the residual body. Elongated spermatids and their

residual bodies influence the secretary functions of the Sertoli cell (Syed et al., 1995).

Finally, mature spermatozoa are released into the lumen of the seminiferous tubule and

this process is called spermiation. Spermatozoa, which are not released, are

phagocytosed by Sertoli cells. The process of spermiation is known to be influenced by


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various factors such as hormones, temperature and toxins. In rats, spermatogenesis is

organized into 14 stages. The overall duration of spermatogenesis is calculated to be

around 50 days in rats and 64 days in men (Clermont and Perey, 1957; for review see

Hess and de Franca, 2008). The process of spermatogenesis is under the control of

testosterone which is produced by the Leydig cells of the testis via steroidogenesis.

1.1.1.2 Steroidogenesis

Testosterone, the principal secretory androgen, is produced exclusively by the

Leydig cells of the testis. Biosynthesis of steroid hormones takes place through a series

of biochemical reactions catalyzed by specific enzymes located in the mitochondria and

smooth endoplasmic reticulum of the Leydig cells (Dufau et al., 1987; Wiebe, 1976).

Cholesterol, the essential precursor for the biosynthesis of all steroid hormones, is

incorporated into the Leydig cell from low density lipoproteins by receptor-mediated

endocytosis or is synthesized de novo within the cell from acetate. Cholesterol is stored

in an ester form in cytoplasmic lipid droplets and the number of droplets in Leydig cells

is considered to be inversely proportional to the rate to androgen synthesis (Freeman and

Ascoli, 1982; for review see Chang et al., 2006). During steroidogenesis, LH-induced

activation of cholesterol ester hydrolase hydrolyzes cholesterol ester which gets

transported into the mitochondria of the Leydig cells. The transport of cholesterol from

outer to inner mitochondrial membrane is achieved by StAR protein (Clark et al., 1994;

for review see Stocco, 2001). However, the exact mechanism by which StAR protein

transports cholesterol to the mitochondria remains unclear. In the inner mitochondrial

membrane, cholesterol is converted to pregnenolone by side chain cleavage enzyme,

cytochrome P450scc, which belongs to the family of monooxygenases. This step

involves three successive monooxygenations- a 22-hydroxylation, 20-hydroxylation and

the cleavage of C20-C22 bonds (Sugano et al., 1990; for review see Pikuleva, 2006).

Subsequently, pregnenolone diffuses across mitochondrial membrane and gets

translocated to endoplasmic reticulum where it undergoes a series of biochemical

reactions to form testosterone.


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Conversion of pregnenolone to testosterone takes place through two different

pathways- Δ4 pathway and Δ5 pathway (Weusten et al., 1987; Yanaihara and Troen,

1972). In Δ4 pathway, pregnenolone is metabolized to progesterone by 3β-HSD.

Progesterone is then hydroxylated at C17 to form 17α-hydroxyprogesterone which is,

then, cleaved between C17 and C20 bonds to form androstenedione. Both these

reactions are catalyzed by cytochrome P450 17α-hydroxylase/ C17, 20 lyase. The C17

keto group of androstenedione then gets reduced to a hydroxyl group to form

testosterone and this step is catalyzed by 17β-HSD (for reviews see Hanukoglu, 1992;

Payne and Hales, 2004; Miller, 2008). Androstenedione formed in the Δ4 pathway is an

important precursor for the production of extratesticular estrogens. Estradiol is produced

by the extratesticular aromatization of androstenedione to estrone which, subsequently,

gets reduced to estradiol in the peripheral tissues. In Δ5

1.1.1.3 Hormonal control of spermatogenesis and steroidogenesis

pathway, pregnenolone

undergoes C17 hydroxylation to form 17α-hydroxypregnenolone which, in turn, is

cleaved between C17 and C20 bonds to form DHEA (Fluck et al., 2003). These

reactions are catalyzed by cytochrome P450 17α-hydroxylase/ C17, 20 lyase. DHEA

could be converted to androstenedione by the action of 3β-HSD and then to testosterone

by 17β-HSD. Testosterone is transported into the circulation by spermatic vein (Ando et

al., 1985). Testosterone synthesis in Leydig cells is regulated by LH. Other factors such

as FSH, insulin-like growth factor-1 and cytokines also regulate the biosynthesis of

testosterone (for reviews see Herrmann et al., 2002; Huhtaniemi and Toppari, 1995;

Sofikitis et al., 2008). FSH also controls spermatogenesis through paracrine regulation

of testicular functions.

The endocrine regulation of spermatogenesis and steroidogenesis is

accomplished through a classical feedback loop which involves interactions between

hypothalamus, pituitary and testis, also called the hypothalamo-pituitary-gonadal axis.

The production of spermatozoa is dependent on pituitary gonadotropins, LH and FSH

(for review see Franchimont et al., 1975), which are released in response to the pulsatile


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release of hypothalamic GnRH. A high pulse rate of GnRH results in LH production

while a low pulse rate results in the production of FSH (Molter-Gerard et al., 1999). In

testis, LH binds to the receptors located on the Leydig cells and cause testosterone

synthesis which, in turn, could negatively influence the release of hormones from

hypothalamus and pituitary (Plant et al., 1978; Resko et al., 1977). FSH targets the

receptors located on the Sertoli cells and induces the production of androgen-binding

protein which helps in the transport of testosterone through the tight junction complexes

of the Sertoli cells (Fakunding et al., 1976). FSH also stimulates Sertoli cells to secrete

inhibin and activin, both of which have negative influence on hormone release from

hypothalamus and pituitary (Vliegen et al., 1993; for review see Weinbauer and

Nieschlag, 1995).

FSH is the primary endocrine hormone involved in the regulation of testicular

functions (Nieschlag et al., 1999). FSH exerts its effects on testis through FSH receptors

located on the Sertoli cells. FSH has a key role to play in controlling the Sertoli cell

populations which, in turn, modulates the number of germ cells proceeding through the

mitotic and meiotic phases of spermatogenesis (Meroni et al., 2002; for review see

Griswold, 1998). FSH controls DNA synthesis in mitotic and meiotic spermatogonia

and also prevents induction of apoptosis in round spermatids (Henriksen et al., 1996;

Shetty et al., 1996). The responsiveness of Sertoli cells to FSH diminishes as they stop

proliferating and start differentiating, and it has been demonstrated that FSH regulates

the expression of Sertoli cell genes that are involved in controlling responsiveness to

androgens (Johnston et al., 2004; for review see Means et al., 1980). Through targeted

disruption of FSH receptor gene, it has been demonstrated that FSH signaling is

essential for maintaining sperm motility and viability (Dierich et al., 1998). FSH also

plays an important role in germ cell development by enhancing germ cell survival and

proliferation. On the contrary, it has also been reported that FSH-deficient males,

despite having small testis, are fertile (Kumar et al., 1997). Using an in vitro system it

was demonstrated that suppression of FSH and/ or testosterone impairs final stages of


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spermatogenesis and spermiation suggesting the synergistic role FSH and testosterone

on spermiation process (Vigier et al., 2004). Though it is apparent that FSH and

testosterone have distinct role on spermatogenesis, they act co-operatively to promote

maximum spermatogenic output and also in the maintenance of Sertoli-germ cell

interactions (Kerr et al., 1992b). Apart from this, FSH receptor transgenic mice showed

marked reduction in Leydig cell population and steroidogenesis implicating the role of

Sertoli cells in the regulation of Leydig cell functions (Baker et al., 2003). FSH has been

shown to stimulate the release of various Sertoli cell products (Mather et al., 1983). The

products of Sertoli cell has been reported to play a role in regulating Leydig cell

functions (for review see Lejeune et al., 1992).

The ability of LH to act on LH receptors present on Leydig cells is important

for successful spermatogenesis and steroidogenesis (for reviews see Dufau et al., 1984;

Hansson et al., 1976; McLachlan et al., 1995). LH-receptor belongs to the family of G-

protein-coupled receptors and mediates the actions of LH on Leydig cells. LH regulates

Leydig cell development, Leydig cell number, testosterone biosynthesis and its secretion

(for review see Dufau, 1998). Binding of LH to LH receptor initiates cAMP production

and adenylate cyclase-protein kinase enzymatic pathway. Other pathways such as

phospholipase C and mitogen-activated protein kinase pathways are also known to be

involved in LH-receptor dependent proliferation and differentiation. The acute action of

cAMP includes mobilization and transport of cholesterol into the steroidogenic pathway

and StAR protein synthesis (for reviews see Stocco, 2000 and Stocco et al., 2005). The

chronic action involves the transcriptional and post-transcriptional stimulation of gene

expression of steroidogenic enzymes thereby upregulating steroidogenesis (for review

see Miller, 2007).

Two-thirds of the testosterone synthesized by Leydig cells freely diffuses into

the adluminal compartment of the seminiferous tubule while others are tightly bound to

steroid hormone binding protein or ABP (Rommerts et al., 1976). Testosterone

withdrawal has been shown to cause detachment of spermatids from Sertoli cells


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resulting in complete stoppage of spermatogenesis (Sharpe et al., 1994). Within the

seminiferous tubule, Sertoli cells express receptors for testosterone (for review see

Walker and Cheng, 2005). Testosterone acts on Sertoli cells through classical and non-

classical pathways. The classical action of testosterone begins when testosterone

transfuses through the plasma membrane and bind to androgen receptors present in the

cytoplasm of the Sertoli cells (Tsai et al., 1980). The non-classical action of testosterone

takes place through calcium influx (for review see Walker, 2003). Both classical and

non-classical pathways play an important role in maintaining spermatogenesis.

Testosterone acts synergistically with FSH to initiate, maintain and restore

spermatogenesis. Specifically, testosterone helps in the formation of blood-testis barrier,

maintenance of Sertoli and germ cell connections and release of mature sperm from the

Sertoli cells (Sharpe, 1987). In the absence of testosterone, formation of blood-testis

barrier is compromised and germ cells are prematurely released from the Sertoli cells

(Yan et al., 2008).

Estrogens also play an important role in the regulation of spermatogenesis (for

reviews see Carreau et al., 2012; O'Donnell et al., 2001). Estrogen receptors are

localized in Leydig and Sertoli cells of testis, efferent ductules and epididymis (Zhou et

al., 2002). Estrogens biosynthesis is catalyzed by a microsomal member of the

cytochrome P450 superfamily, aromatase cytochrome P450 (for review see Simpson et

al., 1994). Evidence shows that germ cells secrete estrogens into the seminiferous

tubular fluid which may be important for the functions of the efferent ductules and

epididymis. Estrogens are reported to have both stimulatory and inhibitory effect on

germ cell proliferation and differentiation (Hess et al., 1997; for review see Sierens et

al., 2005). Administration of aromatase inhibitors to male monkeys have been shown to

cause reduced spermatogenesis and sperm concentrations indicating the crucial role of

estrogens in maintaining spermatogenesis (Shetty et al., 1998). High levels of estrogens

are present in proliferating Sertoli cells and their levels decline as Sertoli cells stop

differentiation and start maturation. Estrogens regulate the expression of cell adhesion


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molecule, neural cadherins, which are involved in the maintenance of germ cell-Sertoli

cell adhesion (Newton et al., 1993). However, increased exposure to estrogens has been

shown to impair the proliferation, differentiation and steroidogenic activity of Leydig

cells. Environmental estrogens have deleterious effects on male fertility (for review see

Saradha and Mathur, 2006) and neonatal exposure to exogenous estrogens has been

shown to cause permanent change to reproductive tract gene expression (for review see

Akingbemi and Hardy, 2001). Neonatal exposure to diethylstilbestrol, a synthetic

estrogen, has been shown to impair testicular steroidogenesis in adulthood (Fielden et

al., 2002). 17β-estradiol administration to adult rats has been shown to cause 33-48 %

decrease in basal and stimulated testosterone production (Keel and Abney, 1982). Adult

male rats when administrated with estradiol showed a significant decrease in circulating

concentrations of FSH and LH, which subsequently lead to reduction in serum and

testicular testosterone levels (Jong et al., 1975). Apart from hormones, several other

factors have also been shown to influence testicular functions.

1.1.2 Factors influencing male reproduction

Testis performs high energy demanding functions, spermatogenesis and

testosterone biosynthesis, whose proper implementation is very essential for the

perpetuation of life. Successful execution of spermatogenesis and steroidogenesis

depends on several factors. Studies have demonstrated the importance of various growth

factors on male fertility. Differential expression of fibroblast growth factors and their

receptors in testis, epididymis, seminal vesicles and prostate have been reported which

indicates the importance of FGF signaling in the development and maturation of

spermatozoa (for review see Cotton et al., 2008). Sertoli cells and spermatocytes

differentially express ligands of TGF superfamily which play a key role in regulating

testis development and spermatogenesis (Itman et al., 2011). Dietary supplements like

vitamins and minerals also play a vital role in maintaining male reproductive functions.

Supplementation of vitamin E and ascorbic acid in drinking water has been reported to

increase sperm concentration, motility, ejaculate volume and reduce the production of


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free radicals in the testis of rabbits (Yousef et al., 2003). It has also been demonstrated

that vitamin A is important for the initiation of meiosis and spermatogenic wave in testis

(for review see Hogarth and Griswold, 2010). Other vitamins such as folic acid,

tocopherol and essential minerals such as zinc and iron have been shown to ameliorate

the toxic effects caused by various external agents on testis (Gunn et al., 1961;

Latchoumycandane and Mathur, 2002; El-Demerdash et al., 2006). Apart from these

factors, glucose has also been shown to be important for proper functioning of testis.

1.1.2.1 Insulin signaling and glucose transport

For many years it has been known that glucose has an important role in the

maintenance of normal reproductive functions. Glucose is very essential for the

successful accomplishment of high energy demanding testicular spermatogenesis and

steroidogenesis (Amrolia et al, 1988). It has been demonstrated that cytochalasin B, an

inhibitor of glucose transport, competitively binds to proteins which are involved in the

facilitated uptake of glucose by Leydig cells, and inhibits LH-stimulated testosterone

synthesis (Murono et al., 1982). High testosterone production has been observed in the

presence of glucose indicating the necessity of this compound, in addition to LH, for

testosterone production (Rommerts et al., 1973) and it was also shown that in the

absence of glucose there is no testosterone production (Amrolia et al., 1988). Transport

of glucose across the plasma membranes is accomplished by the family of facilitative

glucose transporter (GLUT) proteins (for reviews see Gould and Holman, 1993; Joost

and Thorens, 2001). There are 13 families of GLUT proteins which have been identified

till date. Expression of glucose transporter-1 to -3 in various rat testicular cell types has

been demonstrated (Kokk et al., 2007). Glucose transporters are also expressed by

mature spermatozoa as they require glucose for basic cell activity as well as for specific

functions such as motility and fertilizing ability (for review see Bucci et al., 2011).

GLUT-8 is one of the recently cloned members of the GLUT family and is

considered to be the chief glucose transporter in testis. GLUT-8 is expressed in heart,

skeletal muscles, brain, spleen, prostate and intestine but the expression of which was


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found to be highest in testis when compared to all other tissues (Ibberson et al., 2000;

2002). GLUT 8 mRNA was shown to be very high in Leydig cells of testis suggesting

the involvement of GLUT 8 in the transport of glucose for Leydig cell steroidogenesis

(Chen et al., 2003). GLUT-8 expression and translocation in response to insulin have

been shown to be very important for murine blastocyst survival (Pinto et al., 2002).

Differential expression of GLUT-8 protein during mouse spermatogenesis has also been

demonstrated (Kim and Moley, 2007; for review see Schmidt et al., 2009). The

expression begins when the round spermatids are formed on postnatal day 24 and persist

throughout spermatogenesis. GLUT-8 expression has been detected in the acrosome

region of the mature spermatozoa (Schurmann et al., 2002) but not in the immature

germ or Sertoli cells (Gomez et al., 2006). The expression of GLUT-8 protein in

spermatozoa suggests the importance of this transporter in regulating sperm functions.

Moreover, GLUT-2 has also been shown to be abundantly expressed in testicular cell

types (Kokk et al., 2005). The high expression of insulin signaling molecules and

glucose transporters in testis indicates the high energy expenditure of testicular

contractile cells and dependence on glucose as energy source (Kokk et al., 2007).

In testis, insulin receptor family also plays a crucial role in the formation of

gonads. Testis differentiation is induced by the expression of sex-determining region Y

(SRY) present in somatic progenitor cells which are destined to become Sertoli cells

(for reviews see Oh and Lau, 2006; Park and Jameson, 2005). Therefore, Sertoli cell

functions as organizing centers for testis differentiation. Mice which are mutant for

insulin receptors developed ovary and exhibited a completely feminine phenotype

indicating the importance of insulin signaling pathway in sex determination and testis

development (Nef et al., 2003). In cells, actions of insulin are initiated when insulin

binds to its receptor which, in turn, activates the intrinsic tyrosine kinase activity of the

receptor. This event leads to the phosphorylation of the tyrosine residues of various

docking proteins, collectively called the insulin signaling substrate (IRS) molecules

(for review see White and Kahn, 1994). The insulin signaling substrate molecules-1 to -


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6 are the key mediators of insulin signaling pathway. Three important pathways are

propagated in response to insulin action: phosphotidylinositol 3-kinase (PI3K), MAP

kinase and CbI/CAP pathways (for reviews see Boura-Halfson and Zick, 2009; Pessin

and Saltiel, 2000). The MAP kinase pathway is involved in cell growth while the

CbI/CAP complex mediates glucose transport. The PI3K cascade brings about the

metabolic functions of insulin. IRS-1 and IRS-2 have been shown to be expressed in

Sertoli, Leydig, interstitial and myoid cells indicating the dependency of these cell types

on insulin (Kokk et al., 2005, Kokk et al., 2007). Insulin and leptin have been reported

to increase total motility, progressive motility and acrosome reaction of human

spermatozoa thereby enhancing their fertilizing capacity (Lampiao and Plessis, 2008).

Moreover, it has been shown that human spermatozoa releases insulin in pulsatile

fashion which gets regulated in an autocrine manner and it was hypothesized that sperm-

derived insulin may play a role in the capacitation of spermatozoa (Aquila et al., 2005).

Thus, insulin plays an important role in proper functioning of the testis and maintaining

the fertilizing ability of spermatozoa. Several factors are known to influence insulin

signaling and glucose transport in the body. Of the various factors, reactive oxygen

species (ROS) are implicated as one of the key regulators of glucose homeostasis in the

body. Although low levels of ROS are essential for insulin signaling, increased ROS

could have a negative impact on glucose homeostasis.

1.1.2.2 Oxidative stress and antioxidant system

ROS are free radicals involving oxygen with one or more unpaired electrons in

the outer shell. They are highly reactive and they attain stability by acquiring electrons

from lipids, proteins, carbohydrates or any nearby molecule thereby causing a cascade

of damage. The most common ROS include superoxide anion (O2-), hydrogen peroxide

(H2O2), singlet oxygen (1O2) and hydroxyl radicals (OH-). ROS has been associated

with various diseases and also play an important physiological and pathological role in

male fertility (for reviews see Aitken and Krausz, 2001; Betteridge, 2000; Sies, 1997).


16

Spermatozoa are source and target of ROS. Though ROS are known for their

damaging effects on spermatozoa, evidence suggests that spermatozoa generate

superoxide anions which have beneficial effects on sperm functions (for reviews see

Agarwal and Allamaneni, 2011; Aitken, 1995). Low and controlled generation of ROS

by spermatozoa is involved in tyrosine phosphorylation and signal transduction for

sperm capacitation and acrosome reaction (Aitken et al., 1998; for review see

de Lamirande and O'Flaherty, 2008). Spermatozoa exposed to superoxide anion have

been shown to have higher capacitation and acrosome reaction and these effects were

reverted when spermatozoa were exposed to antioxidant enzyme, superoxide dismutase

(SOD) (de Lamirande and Gagnon, 1993). Other antioxidants such as catalase, which

reduce the required levels of ROS, could also impair sperm activation and acrosome

reaction (Ecroyd et al., 2003). However, increased levels of ROS can have damaging

effects on spermatozoa. Spermatozoa are particularly vulnerable to ROS attack because

they are richly endowed with polyunsaturated fatty acids (for review see Griveau and

Lannou, 1997). Limited volume of cytoplasmic space for the availability of intracellular

antioxidant enzymes also results in the high generation of reactive oxygen species by

defective spermatozoa (Koppers et al., 2008). Therefore, the fine balance between ROS

production and the scavenging mechanism are essential for acquisition of the fertilizing

ability of the spermatozoa.

There is cell-to-cell variation in ROS production by spermatozoa at various

stages of maturation. ROS generation was high in immature spermatozoa with

cytoplasmic retention while low levels of ROS were generated from mature sperm cells

(Gil-Guzman, 2001). During spermiation, the spermatid cytoplasm is shunted into a

lobule which is phagocytosed by the Sertoli cells. Aberrant retention of cytoplasm is

associated with increased generation of reactive oxygen species which results in

infertility (Gomez et al., 1996). The mitochondria are also a significant source of

reactive oxygen species in defective sperm cells. The internal source of sperm ROS

constitutes mitochondria electron transport chain (which generates H2O2) and the sperm

plasma membrane NADPH oxidase system (Aitken and Clarkson, 1987; Vernet et al.,


17

2001). A relationship has been observed between ROS generation and germ cell

development during various stages of spermatogenesis. Male germ cells, at various

stages of differentiation (pachytene spermatocytes, round and elongated spermatids)

have been shown to produce low levels of ROS (Gil-Guzman, 2001). Moreover, the

process of steroidogenesis in Leydig cells is also a source of ROS. The products formed

during normal steroidogenesis could act as pseudosubstrates and interact with P450

enzymes, resulting in the formation of pseudosubstrate-P-450-O2

Testis has an elaborate array of antioxidant enzymes which protects it from the

damaging effects caused by ROS (Bauche et al., 1994; for reviews see Agarwal et al.,

2006; Aitken and Roman, 2008). Superoxide dismutase catalyzes the dismutation of

superoxide anion radical to hydrogen peroxide which, in turn, is metabolized by catalase

and glutathione peroxidase (for review see Drevet, 2006). Hydrogen peroxide, as such,

is not reactive product but it may get reduced to highly reactive hydroxyl radical or

singlet oxygen. These reactive radicals can cause formation of lipid peroxides from

polyunsaturated fatty acids of biomembranes leading to the deterioration of membrane

structure and integrity (for reviews see Machlin and Bendich, 1987; Marnett, 1999).

Testicular membranes are rich in polyunsaturated fatty acids and are particularly

susceptible to peroxidation injury. Considerable changes in the developmental profile of

antioxidant enzymes in maturing testis have been reported. High levels of SOD were

detected at the age between 6 and 10 in rats suggesting the protective effect against

peroxidative factors (Peltola et al., 1992). In addition to the major antioxidant enzymes,

testis also relies on small molecular weight antioxidant factors for protection against

oxidative damage. Zinc, which is a core constituent of SOD plays a central role in

protecting the testis from oxidative damage (for review see Tapiero and Tew, 2003).

complex. This

complex is a source of damaging free radicals because of the inability of the

pseudosubstrate to be hydroxylated (Quinn and Payne, 1985; for review see Hanukoglu,

2006). The ROS generated by various testicular cells are scavenged by the powerful

antioxidant defense system of the testis and epididymis.


18

Vitamin E, a powerful lipophilic antioxidant, deficiency in rats have been shown to

impair spermatogenesis indicating its importance in maintaining viable spermatid

population (Bensoussan et al., 1998). Ascorbic acid administration to normal rats has

been shown to stimulate sperm production and testosterone secretion (Sonmez et al.,

2005). Melatonin, the pineal hormone, also plays a crucial role in protecting the testis

from oxidative damage caused by external agents (Ozen et al., 2008; for review see

Rodriguez et al., 2004). Several exogenous and endogenous factors have been shown to

suppress the antioxidant defense system and induce oxidative stress in testis. It has

become evident from various studies that testis is highly dependent on oxygen to drive

spermatogenesis and at the same time vulnerable to attack by reactive oxygen species

(for review see Aitken and Roman, 2008).

The ROS produced during normal spermatogenesis have been reported to be

involved in the regulation of apoptosis in the testis (Erkkila et al., 1999). Testicular

apoptosis occurs continuously throughout spermatogenesis and the intrinsic and

extrinsic apoptotic pathways have been shown to play a role in regulating testicular

apoptosis. The intrinsic or the mitochondrial pathway involves various pro-apoptotic

and anti-apoptotic proteins which recruit and activate the caspase cascade to induce

apoptosis. The extrinsic pathway has been shown to be mediated through Fas and Fas

ligand along with caspase proteins. Sertoli cells have been shown to express Fas ligand

which signals the killing of Fas expressing germ cells thereby limiting the number of

germ cells (Lee et al., 1997). Various factors such as withdrawal of growth factors,

radiation and oxidative stress are known to trigger apoptosis in the testis.

1.1.2.3 Apoptosis

Germ cell death is recognized as a significant feature of mammalian

spermatogenesis. Various stages of spermatogenesis are vulnerable to apoptotic cell

death (for reviews see Pentikainen et al., 2003; Sasagawa et al., 2001). Apoptosis has

been reported to occur during the differentiation of germ cells in order to adjust their

number in testis. Apoptosis occurs mostly during the spermatogonial division of type A


19

spermatogonia while maturational division of spermatocytes and spermatids are less

susceptible to apoptotic cell death (Blanco-Rodriguez and Martinez-Garcia, 1996; for

review see Print and Loveland, 2000). The triggering factors for spontaneous germ cell

apoptosis during spermatogenesis are not completely understood. Apoptosis has also

been reported to take place in Leydig cells during its development (for review see

Haider, 2004). During the prepubertal and pubertal stages of development, there is an

increase in the Leydig cell number due to the differentiation of mesenchymal stem cells

into Leydig cells and the mitotic division of newly formed Leydig cells. Although the

cellular mechanisms involved in maintaining a constant population of Leydig cells is not

well established, apoptosis is thought to play an important role in the regulation of

Leydig cell number (for review see Yuan and Xu, 2003).

Apoptosis is characterized by chromatin condensation, membrane blebbing,

cell volume shrinkage, cytoplasmic vacuolization and disassembly of cells into

membrane-bound apoptotic bodies. The biochemical features of apoptosis include

exposure of phosphatidylserine from the inner leaflet to the external leaflet of the

plasma membrane, activation of caspase proteins and DNA cleavage (for review see

Kiechle and Zhang, 2002). Apoptotic process is particularly important for germ cells

because errors happening during mitosis and meiosis of germ cell development require

apoptotic machinery to eliminate the cells with genetic defects. When germ cells

differentiate into spermatogonia, there occurs an increased phase of apoptosis, called the

first wave of apoptosis (Jahnukainen et al., 2004; Rodriguez et al., 1997). A high level

of caspase expression is seen in testis during the first wave of apoptosis (Moreno et al.,

2006). Hormones such as FSH, LH and testosterone have been reported to regulate

induction of apoptosis in testis (for review see Kiess and Gallaher, 1998).

Apoptosis is a complex mechanism which acts in a cascade-like fashion.

Generally, apoptosis occurs in cells through two major pathways: intrinsic or

mitochondria-dependent pathway and extrinsic or death receptor pathways. Caspases, a

family of aspartate-specific cysteine proteases, play an important role in the execution of


20

these pathways (for review see Fan et al., 2005). Caspases are synthesized as inactive

proenzymes which are cleaved at aspartate residues to form active enzymes with large

and small subunits. Caspases are of two types depending on structure and function:

initiator caspases such as caspases-8, -9 and -10 and effector caspases such as caspases-

3, -6 and -7. Initiator caspases have specific protein-protein interaction domains whereas

effector caspases do not have pro-domains and also lacks the ability to autoactivate.

Initiator caspases are crucial for the induction of apoptosis. Initiator caspases are

involved in the cleavage of inactive pro-form of effector caspases leading to its

activation. The activated effector caspases are responsible for proteolytic degradation of

various cellular targets which ultimately leads to cell death (for reviews see Feinstein-

Rotkopf and Arama, 2009; Said et al., 2004; Wang et al., 2005).

The intrinsic apoptotic pathway is initiated during conditions of mitochondrial

stress. Upon receiving the signal, the proapoptotic proteins present in the cytoplasm, bax

and bid, gets translocated to the outer mitochondrial membrane which results in the

release of cytochrome C and the internal content of the mitochondria (Luo et al., 1998).

Another proapoptotic protein, bak, which resides within the mitochondria, also plays an

important role in the release of cytochrome C from the mitochondria to the cytosol (for

reviews see Tsujimoto, 2003; Wei et al., 2001). Cytochrome C, then, forms a complex

in the cytoplasm by binding with ATP and Apaf-1 which, in turn, activates caspase-9,

the initiator protein. Activated caspase-9 binds with cytochrome C-ATP-Apaf-1

complex and activates caspase-3, the effector caspase, which initiates protein

degradation and DNA fragmentation (for review see Crompton, 1999; Saelens et al.,

2004).

The binding of Fas to its ligand FasL on the target cell triggers the extrinsic

apoptotic pathway. The aggregation of these proteins results in the activation of an

adaptor protein known as FADD on the cytoplasmic side of the receptors. FADD then

recruits caspase-8, an initiator protein, to form DISC (Chinnaiyan et al., 1995; for

review see Nagata and Golstein, 1995). Binding of caspase-8 to DISC recruits the


21

effector caspase protein, caspase-3 to initiate the degradation of the cell. Activated

caspase-8 has the ability to cleave bid which also acts as signal to facilitate the release

of cytochrome C from mitochondria to initiate the intrinsic apoptotic pathway.

The ratio of germ cells to Sertoli cells number remains constant in mammalian

spermatogenesis and this is achieved by the early apoptotic wave in testis. Temporary

high expression of apoptosis-promoting bax protein also plays a crucial role in

balancing early and massive wave of apoptosis among germ cells during the first round

of spermatogenesis (Rodriguez et al., 1997). Sertoli cells tightly regulate germ cell

proliferation and differentiation by expressing FasL. FasL expression by Sertoli cells

results in the apoptotic cell death of germ cells expressing Fas, thereby limiting the

number of germ cells they can support. Upregulation of Fas in germ cells also occurs as

a part of self-elimination process due to inadequate support by Sertoli cells (Lee et al.,

1997 and 1999). Several external and internal factors are known to trigger intrinsic and

extrinsic apoptotic pathways in testis. Heat, radiation, temperature changes, hormonal

factors, testicular diseases and environmental contaminants have been reported to

activate germ cell apoptosis in testis.

1.1.2.4 Environmental contaminants

In the recent years, there has been much concern regarding the adverse effects

of various environmental contaminants on male reproduction. With the advent of

industrialization, economic development and urbanization drastic changes occurred in

the lifestyle and surroundings of humans, which resulted in the extensive production,

and use of substances that could facilitate life. As a result, many potentially hazardous

chemicals got released into the environment at an alarming rate and exposure to these

chemicals has become inevitable. These chemicals released into the environment turned

out to be one of the leading causative factors for the high incidence of various

pathological conditions including cancers and reproductive abnormalities (for reviews

Clapp et al., 2008; Irigaray et al., 2007). Several man-made chemical compounds

released into the environment have been shown to adversely affect the reproductive


22

health of wild life. Several populations of American alligator living in Lake Apopka, the

site of spill of the pesticide dicofol, exhibited altered plasma hormone concentrations,

hepatic functioning and reproductive tract anomalies (for review see Guillette, 2000).

Evidence have accumulated demonstrating the toxic effects of DDT on eggshell

thinning and decreased hatching success of avian species (Elliott et al., 1988; for review

see Colborn et al., 1993). Other abnormalities such as sex reversal in birds and fishes,

abnormal thyroid function, and decreased fertility were also reported following exposure

to various environmental toxicants (for review see Guillette and Guillette, 1996).

Simultaneously, a declining trend in male reproductive heath of humans was observed in

many industrialized nations. A meta-analysis report on a 50% worldwide decline in

sperm density between 1940 and 1990 aroused enormous scientific and public concern

about the imminent threat of synthetic chemicals to male reproductive heath (for review

see Carlsen et al., 1992). Since then, several reports have indicated the role of

environmental contaminants on the negative impact on the male reproductive health

(Fisher et al., 1999; for review see Toppari et al., 1996). Several epidemiological data

indicate a relationship between exposure to various environmental contaminants and

increasing trend in male infertility (Duty et al., 2003; for review see Jensen et al., 2006).

Increasing incidence of testicular cancer and cryptorchidism were reported among the

people living in various industrialized nations (Hansen, 1999a; Meeks et al., 2012;

Paulozzi, 1999). Studies conducted on pesticide manufacturers and agricultural workers

have demonstrated prevalence of testicular dysfunctions and increased male fertility

suggesting the deleterious effects of environmental contaminants on male reproduction

(Oliva et al., 2001). Maternal exposure to various pesticides during critical stages of

development have been shown to cause disturbances in organ differentiation, still birth,

genetic defects, urogenital malformation in male pups and testicular dysfunctions

(Aydogan and Barlas, 2006; Gray et al., 2000).

Most of the environmental chemicals are hormonally active compounds and

target the endocrine system to cause reproductive anomalies. Lindane, an


23

organochlorine pesticide, when administered to rats at a dose of 4 and 8 mg/ kg body

weight caused changes in the histological architecture of the testis and accumulation of

testicular lipid components (Chowdhury et al., 1990). Induction of oxidative stress, and

transient increase in apoptosis-related proteins were also observed in the testis following

exposure to a single dose (5mg/ kg bw) of lindane (Saradha et al., 2008a and 2009).

Endosulfan, an organochlorine insecticide, administration at 2.5, 5 or 10 mg/ kg body

weight for 70 days caused decreased sperm count in cauda epididymis, reduced

intratesticular spermatid counts and impaired spermatogenesis (Sinha et al., 1995).

Decrease in the weights of testis and accessory sex glands, impaired steroidogenesis,

and reduced DNA and RNA concentrations were reported when endosulfan was orally

administered to rats for 30 days (Chitra et al., 1999). Administration of TCDD, a

polychlorinated dibenzo-p-dioxin, at a dose of 0.2 and 2 ng/ ml has been shown to

suppress hCG-induced testosterone production in purified Leydig cells (Lai et al., 2005).

Short term and long term exposure to TCDD has been reported to induce oxidative

stress and decrease the levels of antioxidant enzymes in the testis and epididymis of rats

(Latchoumycandane and Mathur, 2002; Latchoumycandane et al., 2003). Linuron, a

urea based herbicide, at doses of 50 or 75 mg/ kg body weight when administered orally

to pregnant rats from gestational day 13-18 caused an ex vivo reduction in testosterone

secretion (Wilson et al., 2009). Aldrin, an organochlorine insecticide administration for

13 and 26 days impaired steroidogenesis by suppressing the activities 3β−HSD and 17β-

HSD through pituitary release of gonadotrophin (Chatterjee et al., 1988). Single

exposure to 50 mg/ kg body weight of methoxychlor has been reported to transiently

increase the levels of apoptotic proteins such as pro- and cleaved caspase-3, cytochrome

C, Fas and Fas-L in the peritubular germ cells which implies the activation of

mitochondrial and Fas-L-mediated death pathways on exposure to methoxychlor

(Vaithinathan et al., 2010). Nonylphenol, the final biodegradation product of

nonylphenol polyethoxylates, at 10-40 µM caused intracellular accumulation of reactive

oxygen species, increased lipid peroxidation and loss of mitochondrial membrane

potential in Sertoli cells (Gong and Han, 2006). Bisphenol A (BPA), a plasticizer, has


24

also been reported to cause male reproductive abnormalities (Chitra et al., 2003a; Izumi

et al., 2011; Yang et al., 2010). Though a few studies have demonstrated the ability of

BPA to impair testicular functions at doses below the NOAEL dose (vom Saal et al.,

1998), controversies still exist regarding the low dose effects of BPA on male

reproductive health.

1.2 Bisphenol A

BPA (4-[2-(4-hydroxyphenyl) propane; CAS 80-05-7), a plastic monomer, is

used in the manufacture of polycarbonate plastics and epoxy resins. Polycarbonate

plastics are lightweight, tough and optically clear plastics which are used to make

various consumer products such as baby bottles, water bottles, toys and medical

equipments whereas epoxy resins finds application as protective coatings in dental

sealants, food and beverage containers. BPA is one of the highest volume chemicals

produced and it is estimated that 8 billion pounds of BPA is produced each year with 6-

10 % growth in demand per year (for review see Vandenberg et al., 2009).

BPA was first synthesized by A. P. Dianin in 1891. BPA is produced by acid

catalyzed condensation of phenol and acetone. The ester bonds that link BPA to one

another is not stable and therefore, heating or being in contact with acidic or basic

substances results in the hydrolysis of ester bonds linking BPA molecules resulting in its

release into the materials which comes in contact with them (Krishnan et al., 1993).

Specifically, heating of metallic cans to sterilize food, presence of acidic or basic

Structure of Bisphenol A


25

substances in food and repeated washing of polycarbonate bottles have been shown to

increase the leaching of BPA (Howdeshell et al., 2003; Kang et al., 2003). BPA

contamination is also widespread in the environment which makes BPA, a ubiquitous

environmental contaminant (for review see Staples et al., 1998). BPA may be released

into the environment during manufacturing processes such as fugitive emission during

processing and from unreacted products and it is estimated that more than one billion

pounds of BPA is released into the environment per year. There is a widespread human

exposure to this chemical through various sources (for review Groff, 2010) and FDA

has expressed much concern over infant exposure to BPA. Though a few countries have

banned the usage of BPA in consumer products, the compound is still being used in

developing countries like India.

The primary route of exposure of humans to BPA is through diet (for review

see Vandenberg et al., 2007) although air, dust and water are considered as possible

sources of exposure (Fromme et al., 2002). Exposure to BPA can also occur following

application of dental sealants made with BPA-derived materials such as BPA

dimethacrylate (Olea et al., 1996). A wide range of paper products has been reported to

contain BPA which also constitutes an important source of exposure (Ozaki et al.,

2004). BPA has been detected in urine, breast milk, amniotic fluid, placental tissue,

umbilical cord, saliva and other body fluids and tissues of various populations (Calafat

et al., 2005; Ikezuki et al., 2002; Schonfelder et al., 2002). Highest exposure of BPA

occurs to infants and children. Pharmacokinetic studies have demonstrated that BPA is

absorbed into the blood and gets metabolized. Following ingestion, BPA binds to

glucuronic acid to form BPA-glucuronide (Knaak and Sullivan, 1966). The process of

glucuronidation makes BPA, water soluble and helps in its elimination through urine.

Evidence shows that neonatal animals have less ability to handle BPA than adults due to

their underdeveloped glucuronidation ability in early life (Domoradzki et al., 2004). In

rodents, BPA-glucuronide is excreted from the liver into the gut in bile. In the gut, BPA

is cleaved into BPA and glucuronic acid which is reabsorbed into the blood stream. This


26

enterohepatic recirculation results in the slow elimination of BPA in rodents (Doerge et

al., 2010).

In the recent years, BPA has gained much attention due to its ability to act like

estrogen. The estrogenic nature of BPA was first discovered in 1930's and its use as

estrogenic chemical was shelved due to the discovery of diethylstilbestrol as a more

powerful estrogen. Based on a three generation reproductive toxicity study, the

Scientific Committee on Food has considered the NOAEL dose of BPA to be 5 mg/ kg/

day and based on this a tolerable daily intake of 10 µg/ kg/ day of BPA was estimated

(EFSA, 2010). However, various studies have demonstrated the potential health effects

of BPA at or doses lower than the current acceptable NOAEL dose for the compound

(for review see vom Saal et al., 2005).

BPA has been shown to cause extensive damage to multiple organ systems

including kidney, liver, lungs, pancreas, nervous system, cardiovascular system,

endocrine and reproductive system. BPA plays a role in altering the functions of

nervous system through multiple pathways. Behavioral defects such as hyperactivity at

30 µg/ kg/ day (Ishido et al., 2004), an increase in aggressiveness at 2–40 µg/ kg/ day

(Farabollini et al., 2002; Kawai et al., 2003) and altered reactivity to painful or fear-

provoking stimuli at 40 µg/ kg/ day (Aloisi et al., 2002) were reported following BPA

exposure. Exposure to low doses of BPA has been shown to induce oxidative stress in

liver by decreasing the activities of antioxidant enzymes (Bindhumol et al., 2003).

Administration of low doses of BPA to mice showed de novo fatty acid synthesis

through increased expression of lipogenic genes contributing to hepatic steatosis

(Marmugi et al., 2012). High levels of BPA in urine and circulation has been related to

cardiovascular diseases (Lind and Lind, 2011; Melzer et al., 2010). Recently, BPA has

also been shown to induce pancreatic dysfunctions and cause hyperglycemia,

hyperinsulinemia and is considered to be a potential diabetogenic agent (Adachi et al.,

2005; Alonso-Magdalena et al., 2006, 2010 and 2011).


27

BPA has also been shown to have profound effects on female reproductive

system. In utero exposure to BPA at low doses has been reported to cause early vaginal

opening in female offspring (Honma et al., 2002). Dose-dependent increase in meiotic

abnormality called congression failure was observed in human oocytes indicating that

BPA can induce chromosomal aberrations (Brienco-Enriquez et al., 2011). Prenatal

exposure to BPA caused formation of cystic ovaries, adenomatous hyperplasia with

cystic endometrial hyperplasia and atypical hyperplasia indicating high incidence of

endometriosis and associated infertility in female animals following BPA exposure

(Signorile et al., 2010). Decreased uterine responsiveness to progesterone and estradiol

were observed when BPA was administered at a dose of 0.05 and 20 mg/ kg/ day on

postnatal day 1, 3, 5 and 7 (Varayoud et al., 2008). BPA exerts its effects on various

organs owing to its ability to act like estrogen. Due to the hormone-like property of

BPA, the male reproductive system is considered to be an extremely sensitive target for

BPA toxicity.

1.2.1 Effect of BPA on male reproduction

The toxic effects of BPA on male reproduction have been demonstrated.

Exposure to BPA at critical stages of development has been shown to cause various

adverse effects in male offspring. Perinatal exposure to BPA has been reported to

significantly impair spermatogenesis and fertility in F1 and F2 generation male

offspring (Salian et al., 2009). Decline in sperm quality and increased sperm DNA

damage was observed in infertile patients with high urinary concentrations of BPA

(Meeker et al., 2010). Maternal exposure to BPA at a dose of 20 ng has been reported to

cause a significant decrease in the efficiency of spermatogenesis (daily sperm

production per gram of testis) and testicular weight (Sakaue et al., 2001). Exposure of

male mice to BPA (480 and 960 mg/ kg) from postnatal day 35 to 49 has been reported

to activate mitochondrial and Fas mediated death pathways, increase the TUNEL

positive germ cells in stage VII-VIII and increase the levels of caspase 3, -8, -9, Bax,

Fas and FasL (Li et al., 2009b; Wang et al., 2010). The disruption of the Sertoli cell


28

barrier and change in the redistribution of connexin 43, a gap junctional protein, was

observed when BPA was administered to rats. BPA has been reported to induce loss of

gap junction function of Sertoli cells through redistribution of occluding/ zona occludin-

1/ focal adhesion kinase complex proteins at the blood testis barrier and by activating

the mitogen activated protein kinase pathway (Li et al., 2009a). Docking of BPA with

gap junctional protein, connexin 26, showed the interaction of BPA with the pore lining

residues of N-terminal helix and first transmembrane helix of connexin-26 protein

(Cheng et al., 2011). Hypermethylation of the promoter region of ER-α and ER-β in the

testis of rats neonatally exposed to BPA has also been demonstrated (Doshi et al., 2011).

BPA has also been shown to have an impact on testicular steroidogenesis.

Subcutaneous administration of BPA (100 and 200 mg/ kg/ day) and estradiol decreased

the plasma and testicular levels of estradiol, steroidogenic enzymes and cholesterol

carrier proteins in Leydig cells. A decrease in the number of Leydig cell number and the

expression of estrogen receptor-α mRNA were also observed on administration with

BPA (Nakamura et al., 2010). BPA has been shown to induce Nur77 gene expression,

an orphan nuclear receptor involved in steroidogenesis, and thereby alter steroidogenesis

in testicular Leydig cells (Song et al., 2002). BPA has also been shown to increase

aromatase activity in rat testicular Leydig cells through increased mRNA expression of

Cox-2 and proteins involved in MAP kinase signaling pathway (Kim et al., 2010). It has

been demonstrated that BPA impaired hCG-stimulated AMP production and

steroidogenesis by preventing the coupling between LH receptor and adenylate cyclase

in Leydig tumor cell lines in vitro (Nikula et al., 1999). Various mechanism(s) have

been speculated for the observed effects of BPA on rodents.

1.2.2 Mechanism of action of BPA

Diverse biological effects have been observed following exposure to low doses

of BPA and various molecular mechanisms have been proposed with the help of in vitro

assay systems. Accumulation of BPA was found to be three times higher in fat when

compared to kidney, muscles and other tissues (Csanady et al., 2002). The primary


29

mechanism by which BPA impairs various functions in the body is through disruption

of endocrine system. Due to its estrogenic activities, BPA enhances or inhibits the

actions of endogenous estrogens and disrupts estrogenic nuclear hormone receptor

action. BPA can bind to ER-α and ER-β and induce signals that could alter estrogen-

responsive gene expression (Routledge et al., 2000). However, the involvement of non-

classical estrogen receptors in exerting the effects of BPA has also been identified

(Alonso-Magdelena et al., 2006). BPA has been shown to upregulate the mRNA

expression of GnRH and impair the feedback regulatory circuits in HPG-axis thereby

leading to reproductive dysfunctions (Xi et al., 2011). Prenatal exposure to BPA

increased the expression of Hsp90 causing altered gonocyte development (Wang et al.,

2004). The estrogenic potency of BPA can modulate hypothalamo-pituitary axis or can

directly induce change in the expression of estrogen receptors in various tissues

including testis. BPA can also impair the signal transduction pathways through

mechanisms independent of transcriptional activity of nuclear hormone receptors. The

G-protein coupled seven-transmembrane estrogen receptor that binds to estradiol is also

thought to be involved in mediating the actions of BPA (Thomas and Dong, 2006).

Bisphenol has been reported to induce cytotoxicity by disrupting the

intracsellular energy status of mitochondria. The inhibition of the activities of human

hepatic cytochromes, CYP2C8 and CYP2C19 has been observed following incubation

with BPA. Loss of activity of cytochrome P450 in xenobiotics metabolizing system,

initiates peroxidation of free radicals which results in loss of protein synthesis, reduction

in the capacity of liver to excrete various low density lipoproteins and finally leads to

induction of oxidative stress and cell death (Comporti, 1985).

1 review of literature 1.1 description of male reproductive...

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