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.
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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).
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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.,
1 Review of Literature
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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.
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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
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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
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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
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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
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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
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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
1 Review of Literature
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
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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
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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).
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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
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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
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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).