2. Literature Review
2.1 Male reproduction
Reproduction is the process to maintain one’s species by the production of offspring.
With the reproductive system, the individual can produce, nourish and transport the
gamete (female oocyte or male sperm), then create a new individual following the union
of two kinds of gametes. Though both two sexual reproductive systems are involved in
the same physiological purpose, they are different in shape, structure and, needless to say,
in function. Becoming a male, it requires the presence of testicular determining factor
(TDF) to develop the male reproductive system (Berta et al., 1990). TDF is controlled by
the Y chromosome. When TDF is synthesized within the bipotential gonad, the
development and differentiation of the testes and other organs within the male
reproductive system is stimulated (Svingen and Koopman, 2013).
2.1.1 Testicular constitution
The testes are considered the primary reproductive organs in the male because they
produce testosterone and spermatozoa. In addition, they produce other substances such as
inhibin, estrogen and a variety of proteins believed to be important to spermatozoal
function. There are other organs belong to the reproductive system, such as spermatic
cord, epididymis, ductus deferens, prostate, seminal vesicles, penis and so on. All of them
contribute to a successful fertile ability.
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The testes occur as a pair of oval-shaped organs originally in abdomen. They
descended into the scrotum late in gestation or when a male individual is going through
the puberty, depending on the species. The testis is generally divided into two
compartments: one is interstitium in which Leydig cells located; the other is seminiferous
tubules consisting of Sertoli cells and germ cells. Although being anatomically separated,
both compartments are closely connected with each other.
The interstitium consists of all cells and materials outside the seminiferous tubules
such as blood vessels, connective tissue, lymphatics, nerves and Leydig cells. This
compartment comprises about 2.6% of the total testicular volume in experimental animals.
Nevertheless, the interstitium in the human testis resides about 12–15% of the total
testicular volume, and 10–20% of which is occupied by Leydig cells (Nieschlag et al.,
2010).
Leydig cells clustering between the seminiferous tubules were first described in 1850
by German anatomist Franz von Leydig (1821–1908). However, scientists spent almost a century to identify an endocrine role for Leydig cells. A 3β-HSD (a critical dehydrogenase
in the biosynthesis of all steroid hormones) histochemical localization in cells of the
interstitial compartment was illustrated in the end of 1950s (Wattenberg, 1958). In 1965,
Christensen and Mason further indicated that androgen biosynthesis is much more active
in isolated interstitium than in isolated seminiferous tubules of the rat testis (Christensen
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and Mason, 1965). With these two striking evidences, the fact that androgen production
by Leydig cells had been fairly confirmed and accepted. Not only testosterone but also
insulin-like factor 3 (INSL3) is the major secretory product of Leydig cells in the testis.
Although INSL3 was discovered only recently, it have been known so far that INSL3 is
essential for the process testicular descent (Nef and Parada, 1999) and is also involved in
germ cell survival (Kawamura et al., 2004). Due to the great relevance to testicular
function, testosterone and INSL3 are considered the indispensable hormones for male
reproduction.
According to discrete phases of testosterone secretion during the life cycle, separate
populations of Leydig cells could be distinguished. In the mammalian, there are two
different populations of Leydig cells existing in the testis at different periods, fetal Leydig
cells (FLCs) and adult Leydig cells (ALCs). FLCs are terminally differentiated cells in
the fetal testis. After birth, FLCs start to regress and ALCs are formed during pubertal
development, processing through a series of Leydig cells lineages (precursor stem cells,
progenitors, immature and mature adult Leydig cells) (Benton et al., 1995). Both FLCs
and ALCs express abundant 3β-HSD in their membranes of SER and secrete testosterone
for male sexual differentiation in the fetus and secondary sexual maturation and fertility
after birth.
The seminiferous tubules in which spermatogenesis takes place represents about 60–
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80% of total testicular volume. It contains the germ cells and two types of somatic cells,
the peritubular cells and the Sertoli cells. The Sertoli cell occupies about 37% of the
epithelium in human and about 15-20% in the rodent species (Nieschlag et al., 2010).
The peritubular cells form the contractile, basal membrane around the seminiferous
tubules. Inside the tubules, the various germ cells arrange from marginal (basal) to central
(adluminal) tubules as the spermatogenic process. The process of spermatogenesis can be
subdivided into three phases. It begins with the proliferation of spermatogonia. Then,
spermatogonia go through the meiosis to generate spermatocytes and spermatids. Finally,
spermatids undergo a remarkable transformation that creates fully differentiated
spermatozoa. Among the spermatogonia of the seminiferous tubules are the Sertoli cells,
they support the structure of the germinal epithelium, convert testosterone to estradiol and
secrete numerous factors to coordinate the spermatogenesis (Walker and Cheng, 2005).
2.1.2 Regulation of HPG axis
Puberty is the process of acquiring reproductive competence, namely the ability to
accomplish reproduction successfully in a male or female. The onset of puberty occurs
when the activity of HPG axis increases, and results in the production of gonadal steroids
and other growth-associated hormones.
HPG axis is one of the central neuroendocrine system relying on the dynamic
interaction of three endocrine glands, hypothalamus, pituitary and gonad (male testis or
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female ovary). In male, HPG axis operate as a three-tiered functional hierarchy. The first
level is that the gonadotropin-releasing hormone GnRH is secreted from the neurons
which reside almost exclusively in the preoptic area and hypothalamus and terminate in
the median eminence (Colledge, 2009; King and Anthony, 1984). Unlike a preovulatory
surge of GnRH every few weeks in the female, the release of GnRH in the male occurs
in frequent, intermittent bursts that appear throughout the day and night. Generally, these
bursts of GnRH last for a few minutes. At the second level, released GnRH is transported
to the anterior pituitary, where it can bind and activate GnRH receptors on gonadotropes,
thereafter stimulating synthesis and secretion of the gonadotropins, lutenizing hormone
(LH) and follicle-stimulating hormone (FSH). The episodes of LH occur between four to
more than eight times every 24 hours and each time last from 10 to 20 minutes. In contrast
to the pattern of LH secretion, the pulses of FSH are longer duration, but lower
concentrations than that of LH. Then, the activation of LH and FSH on Leydig cells and
Sertoli cells respectively is considered as the last level. Through the circulation system,
LH induces steroidogenesis and testosterone secretion from Leydig cells for
spermatogenesis and maturation of peripheral reproductive tissues, and FSH supports the
development and spermatogenic activities of Sertoli cells. Leydig cells synthesize and
secrete testosterone less than 30 minutes after an appearance of LH episode, and this
response usually lasts for 20 to 60 minutes. Testosterone from Leydig cells and estradiol
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and inhibin from Sertoli cells then negatively feedback to the hypothalamus and pituitary,
resulting in a decrease in GnRH secretion (Knobil et al., 2006).
2.1.3 GnRH pulse generators for the pubertal onset
In early studies, the pituitary, testes and ovaries of immature animals are all
potentially functional as they become active when transplanted into adult animals or when
stimulated by GnRH (Mason et al., 1986; Wildt et al., 1980). Moreover, other
observations also revealed that a sustained increase in pulsatile GnRH released from
GnRH neurons is the dominant signal required for heralding the puberty (Ebling, 2005).
Therefore, neuroendocrinologists believed that the hypothalamus, rather than the pituitary
or gonads, is the major site that launches the preliminary signal for the onset of puberty.
In past few decades, many strong cases have been built for afferent synaptic
neurotransmitters, glial cell products, growth factors, and prostaglandins as contributing
the cascade of signals that trigger the GnRH pulse in pubertal animals (Ojeda et al., 2010).
Multiple components of these regulatory systems have been identified.
Neurotransmitters, like glutamate and the peptide kisspeptin, are secreted from the
specific neurons which compose a complex network and integrate neuronal excitatory or
inhibitory afferents to GnRH neurons. For example, kisspeptin from Kiss1 neurons fires
the GnRH neurons by intracellular calcium pathways (Oakley et al., 2009). Kiss1 neurons
also produce neurokinin B, a peptide recently implicated in the control of human puberty
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(Topaloglu et al., 2009), and dynorphin, an opioid peptide which inhibits GnRH secretion
(Navarro et al., 2009). Other inhibitory counterpart of this neuronal circuit for the GnRH
pulse is provided by both GABAergic neurons and opiatergic neurons (Terasawa and
Fernandez, 2001). In addition, cell-cell signalling molecules produced from glial cells
stimulate GnRH release, and have been shown to be critical for the correct timing of the
pubertal process (Ojeda et al., 2008).
Not only the neuroendocrine substrates but certainly other factors such as body mass,
leptin and environmental cues are important to regulate the beginning of puberty
(Falconer, 1984; Fernandez-Fernandez et al., 2006). Recently, novel regulatory elements,
such as epigenetics and miRNA pathways are also considered in the control of the
pubertal awakening (Lomniczi et al., 2013).
Although there are multiple factors influencing the GnRH secretion, it is not much
different that the timing of puberty within the same species. It is about 12.5 to 13 years
for the woman and 14 years for the man. With regard to the rodents, the puberty in both
female and male usually starts between postnatal fourth and sixth weeks (Falconer, 1984;
Ojeda et al., 1980).
2.1.4 Steroidogenesis
All steroid hormones are synthesized from cholesterol through a series of enzymatic
pathways. This process of conversions is so called steroidogenesis and usually takes place
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in adrenal glands and gonads. The end-product of steroidogenesis would be controlled by
the signals given from the upstream tropic hormones.
The production of testosterone in testicular Leydig cells is primarily under the
control of LH which is the tropic hormone released from the pituitary. In detail, LH binds
with its corresponding receptor (luteinizing hormone/choriogonadotropin receptor,
LHCGR) on the membrane surface. Then, the activated G-protein acts on a membrane
bound adenylyl cyclase to increase the concentration of intracellular second messenger
cAMP. cAMP thereafter activates a family of protein kinase, mostly protein kinase A
(PKA), to induce the activity of enzymes and the transcription of genes which are
necessary for steroidogenesis. No matter what the end-product is, steroidogenesis always
begins with two key pathways: cholesterol in the cytoplasm is transported into the
mitochondria via the steroidogenic acute regulatory protein (StAR) and is cleavage to
pregnenolone by cholesterol side chain enzyme system (CYP11A1). After these two
processes, pregnenolone is metabolized into various intermediates and active steroid
hormones by other specific enzymes (Ghayee and Auchus, 2007).
When testosterone is finally secreted from Leydig cells, it would respond to
spermatogenesis or be transferred into Sertoli cells for aromatase (CYP19A1)–induced
estradiol production. Combining testosterone, estradiol and other proteins, the testis could
complete the circuit of reproductive regulation.
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2.1.5 Spermatogenesis
While steroid hormones are secreted from the interstitium, the mature and haploid
spermatozoa essential for the fertility are produced within the seminiferous tubules with
the stimulation of diffused testosterone from the interstitium. The developmental process
during which spermatogonia enter the differentiation pathway and ultimately create
spermatozoa usually taking about 75 days in man and 35 days in mice is defined as
spermatogenesis (Hess and de Franca, 2008).
Spermatogenesis is generally divided into three phases. The first phase, namely the
proliferation or mitotic phase, is composed of all mitotic divisions of spermatogonia. The
division of spermatogonia gives rise to either the production of two new stem cells for
keeping the stem cell pool or undifferentiated spermatogonia that are destined to develop
into sperm. The second phase is termed the meiotic phase which involves primary and
secondary spermatocytes. During this phase, spermatocytes continue to mature as they go
through the sequential phases of meiosis. At the end of meiotic phase, haploid spermatids
are formed by the second (final) meiotic division. Subsequently, the spermatids head to
the differentiation phase. During the final phase, spermatids undergo spermiogenesis that
they are compacted and elongated into the mature spermatozoa containing a head (nuclear
materials) and a flagellum involving mitochondria (Jan et al., 2012). Under the viewing
of cross-section of a seminiferous tubule, these germ cells move from the basement
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membrane toward the lumen of a seminiferous tubule as they proliferate and mature.
Testosterone is the major androgen in testis that regulates spermatogenesis. With
androgen receptors in the nucleus and cytoplasm, testosterone can initiate the functional
responses required to support spermatogenesis in both Sertoli cells and peritubular myoid
cells. It is clear that Sertoli cells create a micro-environment that enables the sustained
generation of spermatozoa and peritubular myoid cells provide growth factors and assist the movement of fluid and sperm through the tubule lumen (Smith and Walker, 2014).