The Physiology of the Testis

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The Physiology of the Testis
Alessandro Ilacqua, Davide Francomano, and Antonio Aversa
Contents
Functional Organization of the Testis . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . 2
Interstitial Compartment .. . ....................................................................... 4
Tubular Compartment .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 4
Germ Cells (See ▶Hormonal Regulation of Spermatogenesis) .. . .............................. 6
Hormonal Control of Testicular Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 6
Functional Organization of the Hypothalamic–Pituitary System . . . . . ........................... 7
The Kisspeptin–GPR54 System .................................................................. 8
GnRH ................................. ......................................... ................... 9
Structure of GnRH ............................................. ............................... 9
Secretion of GnRH .. .......................................................................... 11
Mechanism of GnRH Action .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . 12
Structure of Gonadotropins .. . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . 13
Secretion of Gonadotropins ................................................................... 14
Mechanism of Action of Gonadotropins .. . .................................................. 16
Endocrine Regulation and Relative Importance of LH and FSH for Spermatogenesis . .... 17
Insulin-Like Factor 3 (INSL3) .................................................................... 19
Steroid Hormones ................................................................................. 20
Testicular Descent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . 21
Vascularization, Temperature, and Regulation of Spermatogenesis . . . . . . . . . . . . . . . . ............. 22
Testicular Androgens . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . 23
Testosterone and Blood Transport ................................................................ 25
A. Ilacqua
Free Lance, Rome, Italy
e-mail: alessandro_ilacqua@alice.it
D. Francomano
Department Experimental Medicine, Sapienza University of Rome, Rome, Italy
e-mail: davide.francomano@uniroma1.it
A. Aversa (*)
Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro,
Catanzaro, CZ, Italy
e-mail: aversa@unicz.it
#Springer International Publishing AG 2016
A. Belfiore, D. LeRoith (eds.), Principles of Endocrinology and Hormone Action,
Endocrinology, DOI 10.1007/978-3-319-27318-1_17-1
1

Extratesticular Metabolism of Testosterone . . . . . . ................................................ 25
Mechanism of Androgen Action .............................................................. 26
Biological Actions of Androgens ............................................................. 28
Cross Talk Between Testis, Bone Marrow, and Pancreas . . . . . . . . . . . . ........................... 31
Aging .............................................................................................. 33
Cross-References .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
References .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 35
Summary
The testis is the most important organ for reproductive and sexual function. Male
fetal sexual differentiation of the genitalia is driven by Leydig cell-secreted
androgens and Sertoli cell (SC)-secreted anti-Müllerian hormone. The hormone
insulin-like factor 3 (INSL3) is produced by testicular Leydig cells (LCs)
depending on the state of LC differentiation and is stimulated by the long-term
trophic effects of luteinizing hormone (see testicular descent). INSL3 is, along
with the other major Leydig cell hormone testosterone (Te), essential for testicular
descent, which in humans should be completed before birth. The absence of
androgen receptor expression in SCs underlies a physiological stage of androgen
insensitivity within the male gonad in the fetal and early postnatal periods. From
fetal life to adulthood, the testis evolves through maturational phases showing
specific morphologic and functional features in its different compartments. The
seminiferous cords contain Sertoli and germ cells, surrounded by peritubular
cells, and the interstitial tissue contains LCs and connective tissue. During
infancy and childhood, LCs regress and Te secretion declines dramatically. SCs
remain immature and spermatogenesis is arrested at the premeiotic stage. At
puberty, LCs differentiate again, and Te concentration increases and provokes
SC maturation and germ cells undergo meiosis, the hallmark of adult spermato-
genesis driving to sperm production (see Interstitial compartment). During adult-
hood androgen receptors became expressed and spermatogenesis occurs, while in
aging, despite that sperm cell production remains partially affected, the secretion
rate of Te declines depending on the presence of comorbidities and drugs
affecting its production by LC (see aging).
Keywords
Leydig cell •Sertoli cell •Testosterone •Sperm cell
Functional Organization of the Testis
The male reproductive system (Fig. 1) is essential for the maintenance of the species
through two essential functions: gametogenesis and sexual function. The testes
produce the male gametes and the male sexual hormones (androgens). The term
spermatogenesis describes and includes all the processes involved in the production
of gametes, whereas steroidogenesis refers to the enzymatic reactions leading to the
production of male steroid hormones. Spermatogenesis and steroidogenesis take
2 A. Ilacqua et al.

place in two compartments morphologically and functionally distinguishable from
each other. These are the tubular compartment, consisting of the seminiferous
tubules and the interstitial compartment between the seminiferous tubules (Fig. 2).
Although anatomically separate, both compartments are closely connected with each
other. The integrity of both compartments is necessary for quantitative and qualita-
tive production of sperm cells. The function of the testis and thereby also the
function of its compartments are regulated by the hypothalamus and the pituitary
gland (endocrine regulation). These endocrine effects are mediated and modulated at
the testicular level by local control mechanisms (paracrine and autocrine factors)
(Basciani et al. 2010).
Seminiferous
tubules
Tunica albuginea
Efferent ducts
Epididymal duct
Vas d e f e r e n s
Rete testis
Fig. 1 Essentials of anatomy
of the Testis
LUMEN
Late spermatide
Early spermatid
Sertoli cell
Spermatocyte
Spermatogonia
Tight Junctional
complex
Basal compartment
Leydig cell
Arteriole
INTERSTITIUM
Adluminal
compartment
Fig. 2 The Testis
The Physiology of the Testis 3

Interstitial Compartment
The most important cells of this compartment are the Leydig cells (LCs). These cells
are the source of testicular testosterone (Te) and of insulin-like factor 3 (INSL3).
Aside from LCs, the interstitial compartment also contains immune cells, blood and
lymph vessels, nerves, fibroblasts, and loose connective tissue. In experimental
animals, this compartment comprises about 2.6% of the total testicular volume. In
the human testis, the interstitial compartment represents about 12–15% of the total
testicular volume, 10–20% of which is occupied by LCs and it counts approximately
200 10
6
cells. LCs produce and secrete the most important male sexual hormone,
that is, testosterone. From the developmental, morphological, and functional
viewpoint, different types of LCs can be distinguished: stem cells, progenitor
committed stem cell, fetal differentiated cells in the fetus, and adult differentiated
cells (Ge and Hardy 2007). Fetal LCs become neonatal LCs at birth and degenerate
thereafter or regress into immature LCs (Prince 2007). Fetal LCs produce
Te. Immature LCs that mainly produce androstane-3-α,17-β-diol instead of T have
also been described.
Adult LCs are rich in smooth endoplasmic reticulum and mitochondria with
tubular cristae. Other important cytoplasmic components are lipofuscin granules,
the final product of endocytosis and lysosomal degradation, and lipid droplets in
which the preliminary stages of Te synthesis take place and special formations,
called Reinke’s crystals, are often found in the adult LCs. These are probably
subunits of globular proteins whose functional meaning is not known. The prolifer-
ation rate of the LCs in the adult testis is rather low and is influenced by LH. The
ontogeny of LCs is not entirely clear and mesonephros, neural crest, and coelomic
sources have been involved. In the adult testis, LCs develop from perivascular and
peritubular mesenchymal-like cells, and the differentiation of these cells into LCs is
induced by LH but also by growth and differentiation factors derived from Sertoli
cell (SC).
Tubular Compartment
Spermatogenesis takes place in the tubular compartment. This compartment repre-
sents about 60–80% of the total testicular volume. It contains the germ cells and two
different types of somatic cells, the peritubular cells and the SC. The testis is divided
by septa of connective tissue into about 250–300 lobules, each one containing one to
three highly convoluted seminiferous tubules. Overall, the human testis contains
about 600 seminiferous tubules. The length of individual seminiferous tubules is
about 30–80 cm. Considering an average number of about 600 seminiferous tubules
per testis and an average length of the tubuli seminiferi of about 60 cm each, the total
length of the tubuli seminiferi is about 360 m per testis, i.e., 720 m of seminiferous
epithelium per man. The seminiferous tubules are covered by a lamina propria,
which consists of a basal membrane, a layer of collagen, and the peritubular cells
(myofibroblasts). These cells are stratified around the tubulus and form concentrical
4 A. Ilacqua et al.

layers that are separated by collagen layers. These characteristics differentiate the
human testicle from the majority of the other mammals, whose seminiferous tubules
are surrounded only by two to four layers of myofibroblasts. Mature sperm cells are
transported toward the exit of the seminiferous tubules by spontaneous contraction
of myofibroblast, and several regulators of cell contractions are reported, e.g.,
oxytocin, oxytocin-like substances, prostaglandins, androgenic steroids, and endo-
thelins. Peritubular contractility is mediated by endothelin and this effect is modu-
lated by the relaxant peptide adrenomedullin produced by SCs. Androgens may
regulate contractility of peritubular cells toward their action on contractility-related
genes, e.g., endothelin-1 and endothelin receptors A and B, adrenomedullin receptor,
and vasopressin receptor 1a (Zhang et al. 2006). In adulthood testis, SCs are somatic
cells, mitotically inactive, located within the germinal epithelium, on the basal
membrane, extend to the lumen of the tubulus seminiferous, and, in a broad sense,
can be considered as the supporting structure of the germinal epithelium; then they
are responsible for final testicular volume and sperm production in the adult. The SC
has several distinct functions that facilitate the maturation of the germ cells. First, it
provides a physical scaffold upon which the germ cells develop and migrate toward
the lumen of the tubule. Second, the SCs form the bloodtestis barrier with
specialized tight junctions that exist between these cells. Third, SCs create the
focused microenvironment essential for germ cell maturation. These distinctive
functions also encompass phagocytosis, fluid secretion, and production of a variety
of molecules. Through the production and secretion of 90% of tubular fluid, SCs
create and maintain the patency of the tubulus lumen. Special structural elements of
the blood-testis barrier prevent reabsorption of the secreted fluid, resulting in
pressurized compartment that maintains the patency of the lumen. The tubular
fluid contains a higher concentration of potassium ions and a lower concentration
of sodium ions. Other constituents are bicarbonate, magnesium and chloride ions,
inositol, glucose, carnitine, glycerophosphorylcholine, amino acids, and several
proteins. Therefore, the germ cells are immersed in a fluid of unique composition.
The basolateral aspect of neighboring SCs comprises membrane specializations
forming a band sealing the cells to each other and obliterating the intracellular
space (occluding tight junctions). Through the blood–testis barrier, the seminiferous
epithelium is divided into two regions, which are anatomically and functionally
completely different from each other. Early germ cells (preleptotene, zygotene) are
located in the basal region and the later stages of maturing germ cells in the
adluminal region. During their development, germ cells are displaced from the
basal to the adluminal compartment. This is accomplished by a synchronized
dissolution and reassembly of the tight junctions above and below the migrating
germ cells. Two important functions are postulated for the blood–testis barrier: the
physical isolation of haploid and thereby antigenic germ cells to prevent recognition
by the immune system (prevention of autoimmune orchitis) and the preparation of a
special milieu for the meiotic process and sperm development. Along the cell body,
extending over the entire height of the germinal epithelium, all morphological and
physiological differentiation and maturation of the germinal cell up to the mature
sperm cell take place.
The Physiology of the Testis 5

Germ Cells (See ▶Hormonal Regulation of Spermatogenesis)
Hormonal Control of Testicular Function
The endocrine regulation of testicular function, i.e., the production of sperm and of
androgens is well investigated. Understanding the hormonal interactions has impor-
tant clinical consequences, presented in the following paragraphs. Figure 3offers an
overview of the hormonal regulation of testicular function and physiological Te
actions in different target organs.
Cortex
Hypothalamus
GnRH
Pituitary
LH
Inhibin
Follistatin
Estradiol FSH Activin
Testosterone
Sertolicells
Dihydro-
testosterone
Testo-
sterone
Leydig cells
Spermatogonia
Primary
spermatocyte
Secondary
spermatocyte
Spermatide
Sperm
Testis
and
epididymis
Testosterone
Target organs
Fig. 3 Hormonal regulation of the testicular function and effects of androgens
6 A. Ilacqua et al.

Functional Organization of the Hypothalamic–Pituitary System
The gonadotropins luteinizing hormone (LH)and follicle-stimulating hormone
(FSH) are produced and secreted by the gonadotropic cells of the anterior pituitary.
Their designation is derived from the function exerted in females. In males, they
control steroidogenesis and gametogenesis in the testis. Pituitary gonadotropins are
the central structure controlling gonadal function and, in turn, are regulated by the
hypothalamic gonadotropin-releasing hormone (GnRH). Since GnRH secretion is
pulsatile, gonadotropin release also occurs in discrete peaks, more evident in the case
of LH, due to its shorter half-life in circulation compared to FSH. In turn, GnRH
secretion depends on the activation of the GPR54 receptor, located on the surface of
the GnRH neurons and stimulated by the peptide kisspeptin (Fig. 4). The pituitary
function is also under the control of gonadal steroids and peptides that influence its
activity both directly and through the hypothalamus. Due to their very strict ana-
tomical and functional connection, the hypothalamus and pituitary gland have to be
considered as a unique functional unit. The hypothalamus is the rostral extension of
the brain stem reticular formation. It contains the cellular bodies of neurons that
project their axon terminals toward the median eminence (ME), a specialized region
located at the floor of the third ventricle from which the pituitary stalk originates.
The hypothalamus is classically subdivided into three longitudinal zones: peri-
ventricular, medial, and lateral, the latter functioning as the connecting area between
limbic and brain stem regions, whereas the former two contain most of the nuclei
controlling neuroendocrine and visceral functions. The ME is the ventral bulge of the
hypothalamus and is the site where the axon terminals of the neurosecretory neurons
make contact with the capillary plexus, giving rise to the hypophyseal portal
circulation. The nerve terminals form buttons on the capillaries and release the
neurohormones into the portal blood by diffusion through the basal membrane.
The ME is outside the blood–brain barrier and thereby freely accessible to the
regulatory influences of hormones and substances present in the systemic circulation
and mediating the release of neurohormones in portal blood. The superior hypophy-
seal arteries provide the blood supply of the ME. The long portal hypophyseal
vessels originate from the confluence of capillary loops, which supply the anterior
pituitary gland with the highest blood flow of any organ in the body. In humans, the
arcuate nucleus
Steroids
Leptin
Other signs
kisspeptin
GnRH receptor
portal pituitary
circulation
GnRH
LH/FSH
Fig. 4 Current model of
regulation of gonadotropin
secretion. Role of Kisspeptin
The Physiology of the Testis 7

perikarya of neurons stained positive for GnRH are especially found in the ventral
part of the mediobasal hypothalamus, between the third ventricle and the ME,
scattered throughout the periventricular infundibular region.
The pituitary gland lies in the sella turcica, beneath the hypothalamus and optic
chiasm, covered with the sellar diaphragm. Thus, pituitary tumors can result in visual
impairment by exerting pressure on the optical nerves. Gonadotropic cells are
localized in the anterior pituitary, the most ventral part of the gland, of ectodermic
origin from Rathke’s pouch. The anterior pituitary consists of the anterior lobe
(or pars distalis, the anatomically and functionally most important part), the pars
intermedia, and pars tuberalis. The pars distalis is of pivotal importance for pituitary
function. Gonadotropin-producing cells constitute approximately 15% of the ade-
nohypophyseal cell population, are scattered in the posteromedial portion of the pars
distalis, and are basophilic and PAS positive. Although the secretion of LH and FSH
can be partially dissociated under certain circumstances, the same cell type is
believed to secrete both gonadotropins. About 80% of the gonadotropic cells in
men contain both LH and FSH. The cells have a very well-developed rough
endoplasmic reticulum and a large Golgi complex and are rich in secretory granules.
In normal men, the pituitary contains approximately 700 IU of LH and 200 IU of
FSH. Following gonadectomy or in primary hypogonadism, the cells become
vacuolated and large (castration cells). Finally, pituitary gonadotrophs are often
found in close connection with prolactin cells, suggesting a paracrine interaction
between the two cell types.
The Kisspeptin–GPR54 System
GnRH secretion is under the control of the kisspeptin–GPR54 system. Kisspeptin is
the product of the KISS1 gene, located on chromosome 1q32.1. The name of the
KISS1 gene derives from the chocolate “kisses”of Hershey, Pennsylvania, the city in
which the gene was identified. KISS1 was originally described as a human tumor
suppressor gene for its ability to inhibit the growth of melanoma and breast cancer
metastasis. Later on, it was shown that kisspeptin (also known as metastin) is the
natural ligand of the orphan receptor GPR54 and has an important role in initiating
GnRH secretion at puberty. The product of the KISS1 gene is a 145-amino-acid
peptide, which is cleaved into a 54-amino-acid peptide known as kisspeptin-54.
Shorter peptides, sharing a common C-terminal, RF-amidated motif with kisspeptin-
54, are probably degradation products. Kisspeptin-expressing neurons are located in
the anteroventral periventricular nucleus (AVPV), periventricular nucleus,ante-
rodorsal preoptic nucleus, and arcuate nucleus (ARC). Outside the nervous system,
the KISS1 gene is expressed in the placenta, testis, pancreas, liver, and intestine
(Popa et al. 2008a). When injected icv or iv to rodents and primates, kisspeptin
stimulates LH secretion, an effect mediated by the interaction with its receptor,
GPR54, an orphan G protein-coupled receptor, located on the surface of the
GnRH-secreting neurons. The GPR54 gene is located on chromosome 19p13.3. It
was discovered that loss-of-function mutations of GPR54 in the human cause failure
8 A. Ilacqua et al.

to progress through puberty and hypogonadotropic hypogonadism (De Roux et al.
2003). Therefore, the kisspeptin–GPR54 system is essential to initiate gonadotropin
secretion at puberty and to maintain normal androgenization in adulthood. In fact,
kisspeptin neurons located in ARC and AVPV send projections to the medial
preoptic area, a region rich in GnRH cell bodies, providing the anatomical evidence
of a direct relationship between kisspeptin fibers and GnRH neurons which, in turn,
express GPR54. The indispensable role of the kisppeptin–GPR54 system for gonad-
otropin secretion is proven also by the hypogonadotropic hypogonadal phenotype of
mice bearing targeted null mutations of the kiss1 or gpr54 gene (Seminara et al.
2003;d’Anglemont de Tassigny et al. 2007) (Fig. 4). GPR54 signals through a Gq
type of G protein. Experimentally, kisspeptin stimulates phosphatidylinositol
(PI) turnover, calcium mobilization, and arachidonic acid release in GPR54-
expressing cells and induces phosphorylation of mitogen-activated protein kinases
(MAPK). Continuous infusion of kisspeptin results in rapid increase in LH secretion
after 2 h, followed by a decrease to the basal levels by 12 h of infusion due to
desensitization of GPR54, since GnRH is still able to elicit LH secretion under these
conditions. This suggests that endogenous, pulsatile kisspeptin release is physiolog-
ically responsible for pulsatile GnRH and LH secretion.
Kisspeptin is sensitive to steroid levels within the circulation and is the mediator
of the negative and positive feedback regulation of gonadotropin secretion. In fact,
although androgens, estrogens, and progesterone suppress gonadotropin secretion
through androgen receptor (AR)-, estrogen receptor (ER)-α-, and progesterone
receptor (PR)-dependent mechanisms, respectively, none of these sex steroids affect
GnRH secretion by direct action on GnRH neurons. On the contrary, kisspeptin
neurons in the ARC are direct targets of sex steroids in all species and should be
viewed as the site of the negative feedback control of GnRH production. In addition,
kisspeptin produced by the AVPV, a sexually dimorphic nucleus rich in steroid-
sensitive neurons in the female, mediates the positive feedback effects of estrogen on
GnRH secretion (Popa et al. 2008b). Finally, kisspeptin neurons seem to be involved
in the regulation of the reproductive axis by metabolic signals sensing the energy
balance of the organism, e.g., leptin. Reproductive hormones are inhibited during
starvation, and kisspeptin mediates some of leptin’s effects on reproduction.
According to the current model, leptin and perhaps other adiposity and satiety factors
stimulate KISS1 expression, which results in stimulation of GnRH release. When
levels of adiposity and satiety factors decrease or when such factors are not detected,
the expression of KISS1 (and presumably its secretion) decreases, thus reducing
excitatory input to GnRH neurons (Chou 2014).
GnRH
Structure of GnRH
Two forms of GnRH, termed GnRH-I (or GnRH) and GnRH-II, encoded by separate
genes have been identified, and they are structurally very similar but show a
significantly different tissue distribution and regulation of gene expression (Cheng
The Physiology of the Testis 9

and Leung 2005). GnRH-I, the peptide involved in gonadotropin regulation, is a
decapeptide produced in the GnRH neurons of the hypothalamus. They originate
from olfactory neurons and during embryonic development migrate toward the basal
forebrain along branches of the terminal and vomeronasal nerves, across the nasal
septum. This event is regulated by a number of factors that influence the migration of
different portions of the GnRH neuronal population at different steps along the route
and the formation of the olfactory bulb (Tobet and Schwarting 2006). The impor-
tance of such factors is demonstrated by mutations in the respective coding gene in
patients with Kallmann syndrome (KS). In about 10% of patients with KS and
anosmia due to a hypoplasia of the bulbus olfactorius, mutations or deletions of
the KAL1 gene on the X chromosome were detected. This gene was first implicated
in KS and encodes for anosmin-1 which is produced in the bulbus and in other
tissues and which is transiently expressed as an extracellular matrix and basal
membrane protein during organogenesis and interacts with heparan sulfate. Other
genes implicated in GnRH neuron migration and KS are those encoding the fibro-
blast growth factor receptor 1 (FGFR1) and its ligand fibroblast growth factor
8(FGF8), as well as prokineticin 2 (PK2) and its receptor (PKR2) (Kim et al. 2008).
In primates, the main locations of GnRH neurons are the mediobasal hypothal-
amus and the ARC, but they are found also in the anterior hypothalamus, preoptic
area, septum, and other parts of the forebrain. GnRH neurons are synaptically
connected with terminals stained positive for pro-opiomelanocortin-related peptides
and enzymes involved in the metabolism of catecholamines and gamma-
aminobutyric acid (GABA). Furthermore, GnRH-positive neurons of the ARC are
connected to neuropeptide Y (NPY) neurons in the preoptical area and in the
eminentia mediana. All these substances are known to influence GnRH secretion.
The gene encoding GnRH is localized at the chromosomal site 8p21-p11.2. GnRH is
produced by successive cleavage stages from a longer precursor, called prepro-
GnRH, transported along the axons to the ME and there released into portal blood.
In the precursor with a length of 92 amino acids, GnRH is preceded by a signal
peptide consisting of 24 amino acids and followed by a stretch of 56 amino acids
forming the GnRH-associated peptide (GAP). Prepro-GnRH is processed in the
rough endoplasmic reticulum and in the Golgi complex, the first step being the
removal of the signal peptide and the cyclization of the aminoterminal Gln residue to
pyroGlu. At the junction between GnRH and GAP, a Gly-Lys-Arg sequence pro-
vides a processing signal important for the cleavage of GAP and C-terminal
amidation of the last Pro residue. Mature GnRH is therefore a single chain
decapeptide cyclized at the N terminus and amidated at the C terminus and assumes
a folded conformation as the result of a ß-II type bend involving the central
Tyr-Gly-Leu-Arg residues that brings the N- and C termini in close proximity (Millar
et al. 2008). GnRH has a very short half-life (<10 min) and is mostly retained and
degraded in the pituitary gland immediately after secretion by several peptidase
systems. As the generation of synthetic analogs of GnRH has shown, the amino acids
in position 6–10 are important for high-affinity binding of the neuropeptide, whereas
positions 1–3 are critical for biological activity and positions 5–6 and 9–10 are
involved in enzymatic degradation. The discovery of the amino acid sequence of
10 A. Ilacqua et al.

GnRH permitted the design of GnRH analogs exerting agonistic or antagonistic
action relative to the endogenous GnRH.
Secretion of GnRH
GnRH is released into the portal blood in discrete pulses (Fig. 5). The frequency of
GnRH pulses and the amplitude of its secretory episodes determine the quality of LH
and FSH secretion from the pituitary gland. GnRH is the sole releasing factor for
both gonadotropins, but modulating its frequency results in preferential release of
LH or FSH (Hayes and Crowley 1998). The pulsatile nature of GnRH secretion is
partly an intrinsic characteristic of the GnRH neurons, since isolated immortalized
GnRH neurons have a spontaneous pulsatile secretory activity in vitro. However,
in vivo GnRH is under the control of the kisspeptin/GPR54 system, which mediates
the effects of the peripheral steroids on GnRH secretion and is involved in the
control of GnRH pulsatility. The pulse generator is under the continuous tonic
inhibition of peripheral steroids and, e.g., gonadectomy results in an immediate
increase of frequency and amplitude of gonadotropin secretion. Thus, in the absence
of steroids, the pulse generator becomes free-running (Lopez et al. 1998). In humans,
continuous
Pulsatile
GnRH
Gonadotropic
cells GnRH analogues
(receptor blockade)
Gonadotropin
secretion
TESTIS
Pulsatile
LH LH
Physiological
pattern
GnRH GnRH
Non-Physiological
pattern
Fig. 5 Importance of the pulsatile pattern of GnRH secretion for gonadotropin secretion and
testicular function
The Physiology of the Testis 11

the major hormone controlling GnRH secretion is testosterone (Te), which inhibits
gonadotropin secretion via negative feedback both at the hypothalamic and pituitary
level. Te can act as such or after metabolism to DHT or estradiol. The effects of Te
and its metabolites vary depending on the experimental model, but in general, we
can assume that both Te and DHT act mainly at the hypothalamic level by decreasing
the frequency of GnRH pulsatility, whereas estrogens suppress gonadotropin secre-
tion by reducing the amplitude of their peaks at the pituitary level. Progesterone
inhibits gonadotropin release at least in part via ARC dopaminergic and NPY
neurons. The negative feedback action of androgens and progestins is most impor-
tant for the development of a male fertility control regimen. Among the neurotrans-
mitters and neuromodulators that might influence GnRH secretion, the noradrenergic
system and NPY show stimulatory activity, whereas interleukin-1, dopaminergic,
serotoninergic and GABAergic systems are inhibitory. Opioid peptides seem to
modulate the negative feedback of gonadal steroids. Finally, also leptin has been
shown to stimulate gonadotropin secretion, as well.
Mechanism of GnRH Action
GnRH acts through interaction with a specific G-coupled receptors linked by the
typical seven-membrane domain structure. With 328 amino acids, it is the smallest G
protein-coupled receptor known up to now and possesses a rather short extracellular
domain. The intracellular C terminus is practically absent, and the signal transduc-
tion is probably carried out by the intracytoplasmic loops connecting the seven
membrane-spanning segments, especially the third one which is unusually long.
The receptor contains two glycosylation sites, projecting into the extracellular space,
of uncertain function. The conformation of the binding site is unknown. The gene
encoding the human GnRH receptor includes three exons separated by two introns.
The 50flanking region contains multiple TATA transcription initiation sites and
several cis-acting regulatory sequences, which confer responsiveness to cAMP,
glucocorticoids, progesterone, thyroxin, PEA-3, AP-1, AP-2, and Pit-1-sensitive
sequences. The GnRH receptor is specifically expressed in the gonadotropic cells
within the pituitary. An orphan receptor, the steroidogenic factor-1 (SF-1), is
involved in the expression of human GnRH. Transcription factors such as SF-1,
Pit-1, and Pro-Pit-1 are generally needed for the development and maturation of the
hypothalamic–hypophyseal–gonadal axis. SF-1-deficient mice and patients bearing
a mutation in the Pro-Pit-1 gene exhibit pronounced alterations of gonadotropin
secretion. Recently, a second GnRH receptor gene has been identified in nonhuman
primates (type II GnRH receptor) which is structurally and functionally distinct from
the classical, type I receptor. The GnRH type II receptor, however, is not functional
in humans and many other species and its role is yet unknown. Both GnRH-I and
GnRH-II were shown to signal through the GnRH type I receptor (ligand-selective
signaling). While GnRH-I regulates gonadotropins, GnRH-II appears to be a
neuromodulator and stimulates sexual behavior. Following GnRH receptor interac-
tion, a hormone-receptor complex is formed. This results in the interaction with Gq
12 A. Ilacqua et al.

protein, hydrolysis of PI, and production of diacylglycerol and inositol tri-
sphosphate, which leads to calcium mobilization from the intracellular stores and
influx of extracellular calcium into the cell. Diacylglycerol and calcium then activate
protein kinase C (PKC), inducing protein phosphorylation and further activation of
calcium channels. The increase in intracellular calcium results in prompt gonado-
tropin release by exocytosis and, with time, more sustained gonadotropin synthesis
and secretion. Thereafter, the hormone-receptor complex is internalized by endocy-
tosis and undergoes degradation in lysosomes. GnRH is capable of modulating
number and activity of its own receptors, and the effects depend on the secretory
pattern and dose of neurohormone. The receptor expression is higher when GnRH is
given in a pulsatile manner, and the withdrawal of GnRH during the interpulse
intervals leads to increase of GnRH-binding sites just before the next pulse occurs
(self priming). Conversely, continuous exposure to GnRH results in an initial rise in
response followed by desensitization. This property of the GnRH receptor is
exploited in therapy with GnRH agonists that, owing to their prolonged and
sustained stimulatory activity, cause slow receptor desensitization and decrease of
gonadotropin secretion. The molecular mechanism of receptor desensitization is not
completely understood. Owing to the lack of an intracellular domain, GnRH agonists
cannot induce phosphorylation and rapid desensitization of the receptor (Kim et al.
2008).
Gonadotropins
Structure of Gonadotropins
LH and FSH are glycoprotein hormones secreted by the pituitary gland that control
development, maturation, and function of the gonads. Like the related thyroid-
stimulating hormone (TSH) and human chorionic gonadotropin (hCG), they consist
of two polypeptide chains, αand β, bearing carbohydrate moieties N-linked to
asparagine (Asn) residues. The α-subunit is common to all members of the glyco-
protein hormone family, whereas the β-subunit, although structurally very similar,
differs in each hormone and confers specificity of action.
Separate but structurally related genes, localized on different chromosomes,
encode for the subunits. The gene encoding for the α-subunit is composed of four
exons and three introns, whereas the βgenes consist of three exons separated by two
introns. The FSH-βgene is located on chromosome 11 and differs from the other
glycoprotein hormone β-subunit genes in possessing a rather long 30untranslated
region probably involved in RNA stability. The LH-βgene belongs to an extraordi-
nary complex cluster of genes also including at least seven nonallelic hCGβ-like
genes arranged in tandem on chromosome 19. The regulation of gene expression of
LH and FSH has been extensively studied in experimental animals, especially
rodents, and involves a complex interplay between hypothalamic GnRH and gonadal
steroids and peptides acting at the hypothalamic and pituitary level (Burger et al.
2004). The common α-subunit contains two glycosylation sites, at position 52 and
78. The glycosylation sites of FSH-βare 7 and 24, whereas LH is glycosylated only
The Physiology of the Testis 13

at position 30. In mammals the α-subunit is also produced by the placenta, and
conversely, the pituitary gland has been shown to contain and secrete trace amounts
of hCG. α- and β-subunits are non-covalently linked, and the probable tertiary
structure of pituitary gonadotropins can be approximated and deduced by analogy
with its cognate hCG, whose crystal structure has been resolved. LH and hCGβ
subunits are structurally very similar and, in fact, LH and hCG act on the same
receptor. A peculiar feature of hCGβis a carboxyl-terminal extension containing
four O-linked sugar residues that remarkably reduces the rate of metabolism and
increases the half-life of the hormone. This peculiarity of hCGß has been recently
exploited for the production of a synthetic gonadotropin hybrid containing a similar
C-terminal extension in the β-subunit, which resulted in a conspicuous increase of
the gonadotropin half-life. The oligosaccharide structure consists of a central man-
nose core, bound to an Asn residue through two residues of N-acetylglucosamine,
and terminal extensions of tetrasaccharide branches, bi- or tri-antennary, terminating
with sialic acid (FSH) or sulfate (LH) residues. These carbohydrate structures can be
more or less extended in length and are rich in sugar terminals, constituting the
molecular basis of the gonadotropin heterogeneity evident after chromatographic
separation. Having a different terminal glycosylation, LH and FSH also have a
different half-life. LH is rich in N-acetylglucosamine sulfate and is quickly removed
from the circulation after interaction with specific liver receptors that recognize
sulfate terminals. This rapid removal of sulfate LH from the blood results in rapid
clearance of a relevant amount of the LH discharged in each secretory episode and
“amplifies”the pulsatile features of LH in circulation. Conversely, FSH is predom-
inantly sialylated and thereby protected from immediate capture and metabolism
within the liver. As a result, LH and FSH half-lives are about 20 min and 2 h,
respectively. Therefore, although both gonadotropins are secreted simultaneously
from the pituitary gland following a GnRH pulse, LH appears to be highly pulsatile
and FSH much less so (Moyle and Campbell 1995). Glycosylation is fundamental
for gonadotropin secretion and bioactivity and strongly influences the half-life in
circulation and in vivo biopotency. It is well known that gonadotropin activity
depends on glycosylation, which is not critical for receptor binding but is important
for receptor activation. Furthermore, isoforms completely devoid of carbohydrates
cannot be secreted by the producing cells and behave as competitive antagonists of
the wild type. Polymorphic variants of the FSH-βgene exist as well and are
associated with serum FSH levels in men (Grigorova et al. 2008). Such a variant
was found to be associated with a significant reduction in free Te and testes volume,
but, on the contrary, in an increase of semen volume, sex hormone-binding globulin,
serum Te, and estradiol.
Secretion of Gonadotropins
After the synthetic process is completed, LH and FSH are stored in different
secretion granules, ready to be released upon stimulation with GnRH. A portion of
molecules, however, is not stored in
14 A. Ilacqua et al.

secretory granules, i.e., does not enter the regulated pathway of secretion and is,
instead, constitutively secreted. FSH especially follows the latter route. Storage in
separate granules and the natural propensity to follow one of the two secretory
pathways are the main reasons why the same GnRH stimulus can, under certain
conditions, preferentially release one of the two gonadotropins. Low GnRH pulse
frequency causes preferential release of FSH probably due to differential expression
of the FSH receptor (Ferris and Shupnik 2006).
LH and FSH are measurable in the pituitary gland as early as the 10th week of
gestation and during the 12th week in peripheral blood. In fetal life and in infancy,
FSH is predominant over LH and the FSH/LH ratio is higher in females than in
males. The relative abundance of the two gonadotropins undergoes changes during
development. Te drives the initial phase of testicular migration and the development
of male external genitalia. The fetal testicle already produces Te during the 10th
week of gestation, under the stimulation of fetal LH and maternal hCG. The role of
maternal hCG in this crucial phase of gonadal development is suggested by the fact
that a mutation of the LH βchain leading to a biologically inactive gonadotropin is
associated with normal sexual differentiation. Conversely, inactivating mutations of
the LH receptor produce a clinical syndrome resembling complete androgen insen-
sitivity, with a phenotype of female external genitalia (Huhtaniemi et al. 1999;
Themmen et al. 1998).
During infancy gonadotropins in serum are very low. The pulsatile secretion of
gonadotropins becomes evident at the time of puberty, when LH and FSH pulses in
serum are detected first during nighttime and then progressively also during the day.
Before puberty, gonadotropin levels are very low and GnRH secretion appears to be
extremely limited, even in the presence of negligible steroid production by the
gonads. High sensitivity of the hypothalamus to negative steroid feedback is
believed to suppress GnRH production before puberty, but certainly other factors
such as body mass, leptin, and signals from the central nervous system are important
to maintain the hypothalamic–pituitary–gonadal axis silent before the
programmed time.
The steroid regulation of gonadotropin gene expression, synthesis, and secretion
is rather complex and shows many facets depending on the experimental model. In
general, however, it is currently accepted that gonadal steroids exert their negative
control on gonadotropins mainly at the hypothalamic level, depressing the release of
GnRH most probably via the kisspeptin/GPR54 system. The steroid effect at the
pituitary level is more complex, but there is considerable evidence that estrogens
inhibit GnRH-stimulated gonadotropin synthesis and secretion at this level. Te is the
main testicular product suppressing FSH and LH secretion in men. FSH secretion is,
obviously, also under the control of some other factor(s) related to the efficiency of
spermatogenesis, since oligozoospermia is often accompanied by selective increase
of serum FSH in the presence of normal Te levels. The mutual relationship between
FSH and inhibin secretion in man is well established: a pronounced inverse corre-
lation was found between serum concentrations of inhibin B and serum levels of
FSH, testis size, and sperm numbers. Clearly, inhibin B is the physiologically
relevant form of inhibin in men. It appears at present that the serum levels of inhibin
The Physiology of the Testis 15

B directly reflect the integrity of the germinal epithelium and of the Sertoli cells
(Boepple et al. 2008).
Mechanism of Action of Gonadotropins
LH and FSH exert their function via specific receptors. The gonadotropin receptors
also belong to the family of the G protein-coupled receptors and are characterized by
a very large extracellular domain to which the hormone binds, specifically, the usual
membrane-spanning domain including seven hydrophobic segments connected to
each other through three extracellular and three intracellular tracts and an intracel-
lular carboxyl-terminal domain (Simoni and Casarini 2014).
The genes for LH and FSH receptors are localized on chromosome 2 and consist
of 11 and 10 exons, respectively. The last exon encodes a small portion of the
extracellular domain, the entire transmembrane domain, and the intracellular C
terminus. The extracellular domain contains the high-affinity hormone-binding site
and is rich in leucine repeats. The 50-flanking region of the two genes contains no
conventional promoter and has multiple transcriptions start sites. In addition, the
human LH receptor was recently shown to contain a cryptic exon (denominated exon
6A) which plays a very important role in the intracellular processing of the mature
receptor protein and can be mutated in rare cases of LH resistance. Several alterna-
tively spliced transcripts of LH and FSH receptors have been described, lacking one
or more exons, but presently it is not known whether these RNA isoforms are
translated into proteins of any physiological function. Single nucleotide polymor-
phisms give rise to allelic variants with different biological activity in vitro and/or
in vivo.
The mature receptor proteins are glycosylated at several points, a process that
does not seem to be involved in receptor activation and signal transduction but
probably necessary for receptor folding and transport to the cell membrane.
Recently, the partially deglycosylated complex of human FSH bound to the extra-
cellular hormone-binding domain of its receptor was crystallized, showing that
binding specificity is mediated by key interaction sites involving both the common
α- and hormone-specificβ-subunits. On binding, FSH undergoes a concerted con-
formational change that affects protruding loops implicated in receptor activation.
The FSH–FSHR complexes form dimers in the crystal important for transmembrane
signal transduction resulting in activation of the G protein, cAMP production, and
activation of PKA. Gonadotropins act mainly through stimulation of intracellular
cAMP. More recently it has been shown that LH and FSH can also induce an
increase of calcium influx in target cells, but the physiological importance of this
mechanism is still unknown. cAMP remains, therefore, the main signal transducer
and calcium could possibly act as a signal amplification or modulating mechanism.
Following the hormone-receptor interaction, there is an increase in cAMP concen-
trations and subsequent activation of PKs which, in turn, phosphorylate existing
proteins such as enzymes, structural and transport proteins, and transcriptional
activators. Activating and inactivating mutations of the gonadotropin receptors
16 A. Ilacqua et al.

have been identified. Recent human data suggest that gonadotropins may act as cell
survival factors for spermatogonia rather than as stimulators of cell proliferation,
permitting differentiation of Ap spermatogonia into B-type spermatogonia (Simoni
and Casarini 2014; Piersma et al. 2007).
Endocrine Regulation and Relative Importance of LH and FSH
for Spermatogenesis
Spermatogenesis starts with the division of stem cells and ends with the formation of
mature sperm. The various germ cells are arranged in typical cellular associations
within the seminiferous tubules known as spermatogenic stages, and the entire
spermatogenic process can be divided into four phases and requires 16 days for
the development and differentiaton of an Ap spermatogonium into a mature sperm:
1. Mitotic proliferation and differentiation of diploid germ cells (spermatogonia)
(spermatogoniogenesis)
2. Meiotic division of tetraploid germ cells (spermatocytes) resulting in haploid
germ cells (spermatids)
3. Transformation of spermatids into testicular sperm (spermiogenesis)
4. Release of sperm from the germinal epithelium into the tubular lumen
(spermiation).
It must be pointed out, however, that a recent review recommends 74 days by
including time for spermatogonial renewal (Amann 2008).
The gamete development through several stages (from spermatogenesis to
spermiation) is regulated by the brain, e.g., the hypothalamus and pituitary via
GnRH and gonadotropins. Importantly, the hypothalamic–hypophyseal circuit is
subject to negative feedback regulation mediated by testicular factors. Te inhibits
the secretion of LH and FSH. For FSH, the protein hormone inhibin B plays an
important role.
For the interpretation of hormonal regulation and hormonal effects on spermato-
genesis, the following terminology should be remembered:
1. Initiation: First complete cycle of spermatogenesis during puberty.
2. Maintenance: Hormonal requirements of intact spermatogenesis in the adult.
3. Reinitiation: Hormonal requirements for the restimulation of gametogenesis after
transitory interruption.
4. Qualitatively normal spermatogenesis: All germ cells are present although in
subnormal numbers.
5. Quantitatively normal spermatogenesis: All germ cells are present in normal
numbers.
Considerable efforts were undertaken to unravel the relative importance of LH/Te
and FSH for qualitative and quantitative initiation, maintenance, and reinitiation of
The Physiology of the Testis 17

spermatogenesis (Fig. 6). It is generally assumed that either Te or FSH alone is able
to initiate, maintain, and reinitiate spermatogenesis but only to a qualitative extent.
In order to achieve quantitative effects on germ cell production and sperm numbers,
at least under physiological conditions, both LH and FSH activities are needed
(Weinbauer et al. 2004).
These assertions are based upon controlled studies in nonhuman primate models
and volunteers and on studies in patient populations and case reports. The latter has
provided interesting information but can also confound interpretation of findings
owing to the variable endocrine and medical history of the patients. Complete
spermatogenesis is seen in the vicinity of Te-producing LC tumors and in patients
with activating mutations of the LH receptor, suggesting that pharmacologically high
local Te concentrations induce sperm formation. The aim of treatment is to obtain
sufficiently high intratesticular Te concentrations, which are crucial. This is normally
pursued clinically by giving hCG, which contains high LH activity, together with
FSH. On the other hand, patients bearing a defective FSH-βsubunit, presented with
azoospermia. One of these patients was normally virilized, suggesting the need of
FSH for complete initiation of spermatogenesis in man (Lindstedt et al. 1998);
conversely, patients with selective LH deficiency can have complete spermatogen-
esis, indicating the ability of FSH to initiate the entire male germ cell development
cascade.
Exogenous provision of supranormal doses of Te or of gestagenic compounds
suppresses gonadotropin secretion through the negative feedback mechanism and
leads to a drastic decrease of sperm numbers in the ejaculate. In primates it is
essential that complete suppression of FSH secretion be achieved despite inhibition
of LH secretion. In the latter study, albeit LH bioactivity had been completely
eliminated, a slight and transient rebound of FSH secretion provoked an escape of
Testosterone ? / FSH?
Testosterone ? /FSH
Spermiogenesis
Testosterone
FSH
Meiosis
Spermatogoniogenesis
Testosterone
FSH?
Ad
Ap
BPL LZ
EP
MP
LM
II
Sa1/2 Spermatids
Sb1 Sb2 Sc Sd1 Spermatozoa
Sd2
SPERMIATION
Ad = A-dark spermatogonium (testicular stem cells, divides rarely), Ap = A-pale spermatogonium (self-renewing a nd progenitor cell for spermatogenesis), B = B
spermatogonium, Pl = preleptotene spermatocytes, L = leptotene spermatocytes, EP = early pachytene spermatocytes, MP = mid pachytene spermatocytes, LP = late
pachytene spermatocytes, II = 2nd meiotic division, RB = residual body, Sa1–Sd2 = developmental stages ofspermatid maturation
Fig. 6 Sites of action of testosterone and FSH on the spermatogenic process in primates
18 A. Ilacqua et al.

spermatogenic suppression. In gonadotropin-suppressed men, either FSH or LH
maintained spermatogenesis. The importance of FSH is also evident from a hypoph-
ysectomized patient in whom an activating mutation of the FSH receptor coexisted
with normal spermatogenesis in the absence of LH. Conversely, inactivating muta-
tions of FSH action do not necessarily lead to a complete block of spermatogenesis.
Although either hormone on its own has the potential to elicit the entire spermato-
genic process, this is not always the case in patients receiving androgen/hCG
therapies. In case of failure of hCG, however, the addition of FSH has been shown
to permit completion of spermatogenesis in hypogonadotropic men with azoosper-
mia. Finally, in primates, both gonadotropins are necessary for spermatogenesis. The
biological meaning of this dual regulation system is not clear yet. From a clinical
viewpoint it is concluded that the synergistic action of LH/Te and FSH is necessary
for the initiation, maintenance and also for reinitiation of normal spermatogenesis
(Tobet and Schwarting 2006). The regulation of testicular function is primarily
controlled by central structures, but the complexity of the testicular cell types and
architecture also mandates a variety of local control and regulatory mechanisms. The
categories of local interactions and communication can be classified as paracrine,
autocrine,and intracrine. In addition, the interplay between the different testicular
compartments is also subsumed under local interactions. It is evident that the
endocrine mechanisms play the central role in the regulation of testicular function
and factors produced locally are important for the modulation of hormone activity
and local factors could thus be seen as mediators of hormone action and intra-/
intercellular communication. Moreover, it can be reasonably assumed that other still
unidentified protein factors mediate the communication between interstitial and
tubular compartments, between SC and germ cells and between germ cells
(Weinbauer and Wessels 1999). Growth factors bind to surface receptors and induce
cell-specific differentiation events via specific signal transduction cascades. Among
those factors participating in the local regulation of spermatogenesis are trans-
forming growth factor (TGF)-αand TGF-β, inhibin and activin, nerve growth factor
(NGF), insulin-like growth factor I (IGF-I), fibroblast growth factor (FGF), and
epidermal growth factor (EGF) (Laron and Klinger 1998).
Insulin-Like Factor 3 (INSL3)
INSL3 is a member of the relaxin-like peptide family, and it is emerging as a key
factor in the regulation of a variety of developmental processes related to reproduc-
tion (Foresta et al. 2004). The binding of the hormone to its specific receptor LGR8
(also known as leucine-rich repeat-containing G protein-coupled receptor) activates
adenylate cyclase and cAMP production through Gs proteins, although alternative
mechanisms have been proposed (Kumagai et al. 2002). INSL3 is expressed in pre-
and postnatal LC of the testis. The major known endocrine role of INSL3 is related to
the regulation of the transabdominal phase of testicular descent by action on the
gubernaculum. As a consequence, Insl3 and Rxfp2 knockout mice have bilateral
cryptorchid testes, and mutations in the INSL3 and RXFP2 genes have been
The Physiology of the Testis 19

associated with testis maldescent also in humans (Ferlin et al. 2003). In addition to
the prenatal role for INSL3, further possible endocrine and paracrine actions in adult
males have recently gained particular attention based on several observations. First,
in adults, INSL3 is produced constitutively but in a differentiation-dependent man-
ner by the LC under the effect of luteinizing hormone (LH), and substantial circu-
lating INSL3 levels are present in adult men. Reduced plasma concentrations are
seen in situations of undifferentiated or altered LC status (such as hypogonadism),
and INSL3 has been suggested to be even more sensitive than Te to impaired LC
function (Ferlin et al. 2006). Second, RXFP2 is expressed in many tissues besides
the gubernaculum, including the kidney, skeletal muscle, thyroid, pituitary gland,
brain, and bone marrow, and paracrine roles for INSL3 have been suggested in the
testis, ovary, thyroid, and mammary gland (Fig. 7).
Steroid Hormones
Te is the main secretory product of the testis, along with 5α-dihydrotestosterone
(DHT), androsterone, androstenedione, 17-hydroxyprogesterone (17-OH-Pg), pro-
gesterone (Pg), and pregnenolone. The role of androsterone, 17-OH-Pg, and Pg in
the testis is unknown, but progesterone receptors have been found in some peri-
tubular cells and on spermatozoa. For Te, a classic endocrine factor, compelling
evidence is available as a pivotal local regulator of spermatogenesis. Rodent data
demonstrated that selective elimination of LC, interruption of testicular Te transport,
Fig. 7 Hormonal control of testicular descent
20 A. Ilacqua et al.

and specific SC androgen receptor knockout models provoked profound alterations
of germ cell maturation (Takaimya et al. 1998). Selective peritubular cell androgen
receptor knockout mice exhibited specific SC and peritubular cell defects. Sper-
matogenesis was present in boys with Te-producing LC tumors but only in seminif-
erous tubules adjacent to the tumor and not in tumor-free areas. Similarly, activating
mutations of the LH receptor prematurely induced qualitatively normal
spermatogenesis.
In fertile men, testicular Te concentrations exceed that of SHBG/ABP by about
200-fold, indicating a substantial surplus of Te in the testis. Testicular Te concen-
trations are >80-fold higher than those in plasma (Coviello et al. 2005). Te is
metabolized to DHT by testicular 5α-reductase activity and to estradiol by testicular
aromatase activity. To what extent these metabolic activities are essential for sper-
matogenesis besides Te itself is not entirely clear.
Although it is established beyond doubt that Te is an essential local regulator of
spermatogenesis, it has been surprisingly difficult to demonstrate a clear-cut rela-
tionship between testicular Te concentrations and germ cell production. In non-
human primates, no correlation between testicular androgen levels and germ cell
production/spermatozoa number was observed. Similarly, contraceptive studies in
volunteers failed to demonstrate a correlation between intratesticular steroids and
germ cell numbers (Matthiesson et al. 2005b). In the nonhuman primate, Te induces
the formation of smooth muscle actin in the peritubular cells during prepubertal
testicular maturation. Peritubular cells express the androgen receptor. Te effect is
significantly reinforced by FSH. Since FSHR are found only in SC, it follows that
FSH influences androgen action indirectly through factors arising in the SC. This
indicates that as an endocrine factor, FSH can also induce the formation of physi-
ologically relevant, locally acting factors in the primate testis. Interestingly, recom-
binant FSH stimulates Te production in men (Levalle et al. 1998) and in patients with
selective FSH deficiency, lending further support to the importance of local interac-
tions between SCs, LCs, and peritubular cells in connection with the actions of
androgens and gonadotropins (Lofrano-Porto et al. 2008).
Testicular Descent
The incidence of positional anomalies of the testis is over 3% and ranges among the
most common congenital defects. These defects are associated with spermatogenic
disturbances such as fewer spermatogonial stem cells at birth compared with normal
boys and increased risk of testicular tumor development. Testicular descent is
multifactorial with two distinct phases. The first is descending phase from the
lower kidney pole to the pelvic cavity (transabdominal phase of descent) controlled
by the swelling of the gubernaculum. The shortening of the gubernacular cord and
the outgrowth of the gubernacular bulb controlled by the genitofemoral nerve are
independent of androgens. The gubernaculum deposits extracellular matrix, rich in
glycosaminoglycans and hyaluronic acid, and forms a cone-like structure at the
caudal end of the gonad, anchoring the developing testis close to the inguinal region
The Physiology of the Testis 21

during fetal growth. In the second phase, the descent into the scrotum (inguino-
scrotal phase of descent) is controlled by androgen action. In the 26th gestational
week, the gubernaculum begins to grow through the inguinal canal and reaches the
scrotum by gestational week 35, pulling the testis in its path before the
gubernaculum shrinks to a fibrous remnant. The intra-abdominal pressure and the
shrinkage of the gubernaculum may force the testis through the inguinal canal. At
birth, the testes reach at the bottom of the scrotum, and in 97% of boys, testicular
descent is completed within another 12 weeks (Fig. 7). The physiological and
endocrine mechanisms that govern testicular descent are not known in detail.
INSL3 is a potential regulator of testicular descent as suggested by the fact that in
gene knockout mice, maldescended testes remain located in the abdominal cavity.
INSL3 produced by the LC together with androgen induces the gubernaculum
growth and is therefore needed in the early phase, and knockout mice for INSL3
gene have their testes high in the abdominal cavity. Estrogens or environmental
endocrine disruptors have also been suspected to induce a downregulated INSL3
expression and thus disturb testicular descent. Genetic analysis in men revealed
several functionally deleterious mutations in both INSL3 and its receptor GREAT/
LGR8 gene (Boepple et al. 2008).
Vascularization, Temperature, and Regulation
of Spermatogenesis
Vascularization of the testis has two main roles: transport and mobilization of
endocrine factors and metabolites, as well as regulation of testicular temperature.
The arterial supply of the testicular parenchyma follows the lobular division of the
seminiferous tubules. Each lobule is supplied by one artery from which segmental
arteries, supplying blood to the lateral regions of the lobuli. Segmental arteries and
capillaries become branched between the LC and finally give rise to the venous
system.
In men, testicular temperature is about 3–4C below core body temperature and
about 1.5–2.5 C above the temperature of scrotal skin. For the maintenance of a
physiologically lower temperature, the testis relies on two thermoregulatory systems.
Heat can be transferred to the external environment through the scrotal skin, as the
scrotal skin is very thin, possesses hardly any subcutaneous fat tissue, and has a very
large surface.
The second regulatory system is the pampiniform plexus. In this system, the
convoluted testicular artery is surrounded by several veins coiling around the artery
several times. Arterial blood arriving at the testis is thereby cooled down by the
surrounding venous blood. The usual explanation for the pampiniform plexus here is
to efficiently maintain the optimal temperature, which is below body temperature.
Recently, a new theory has been put forward hypothesizing that the process of
spermatogenesis results in a large amount of heat, which has to be regulated. Testes
22 A. Ilacqua et al.

are located in the scrotum in order to maintain lower body temperatures. Some
mammals’testes remain functional inside the body, e.g., elephants, but these animals
lack sweat glands and are closely related to aquatic ancestors which have to
compensate for the chilling effects of the heat-conducting environment, namely,
water. Human scrotal skin is devoid of subcutaneous fat and the presence of high
sweat gland density enables heat transmission. Upon exposure to cold temperatures,
the scrotal surface is minimized by contraction for preventing temperature loss, and
cremaster muscles retract the testes closer to the abdomen for temperature
maintenance.
Testicular Androgens
In men, testosterone is by far the most important and abundant androgen in blood.
More than 95% of the existing androgens derive from the testis, which synthesizes
about 6–7 mg Te per day. Apart the testes, the remaining contribution to androgen
production derives mainly from the adrenals. The site of androgen production in the
testis is the LC. Both synthesis and secretion are under regulation of pituitary LH and
local factors. The starting point for androgen synthesis is cholesterol and adult LCs
have additional requirements for cholesterol, because it is the essential precursor for
all steroid hormones. LH as the central regulatory factor controls both steroidogen-
esis and LC cholesterol homeostasis in vivo. Cholesterol can either be incorporated
by the cell through receptor-mediated endocytosis from low-density lipoproteins
(LDL) or can be synthesized de novo within the LCs starting from acetyl coenzyme
A (Sriraman et al. 2005). The conversion of cholesterol to Te goes through five
different enzymatic steps in which the side chain of cholesterol is shortened through
oxidation from 27-C to 19-C (Fig. 8). Te is the main secretory product of the testis,
along with DHT, androsterone, androstenedione, 17-OHPg, Pg, and pregnenolone.
The transformation of Te into DHT takes place principally into the target organs.
Androstenedione is important as a precursor for the production of extratesticular
estrogens. Biologically active estradiol can be produced as a result of extratesticular
aromatization of androstenedione to estrone that is subsequently reduced to estradiol
in peripheral tissues. Only a very small portion of the Te produced is stored in the
testis, and the androgen is mainly secreted in the blood.
Te concentrations in the testicular lymphatic circulation and in the venous blood
are very similar, but there are essential differences in the flow rate and velocity of
both systems. Therefore, transport of Te in the general blood circulation occurs
mainly through the spermatic vein. Androgens diffuse into interstitial fluid and then
enter testicular capillaries or enter capillaries directly from LC that is in direct
contact with the testicular microvasculature. The mechanism for Te transport from
the LC into the blood or lymphatics is not completely known. Probably lipophilic
steroids distributed within cells or small cell groups are released through passive
diffusion.
The Physiology of the Testis 23

Fig. 8 Biosynthesis of androgen
24 A. Ilacqua et al.

Testosterone and Blood Transport
SHBG concentration in men is about one third to one half of the concentration found
in women. During transport in plasma, Te is mainly bound to albumin or to sex
hormone-binding globulin (SHBG), which is produced by hepatocytes. A protein,
the androgen-binding protein (ABP), with similar steroid-binding characteristics is
produced in the testis. SHBG is an α-globulin consisting of different protein sub-
units. In rats it is expressed in SC and is secreted preferentially into the seminiferous
tubules and migrates into the caput epididymis where it is internalized by epithelial
cells that regulate androgen-dependent mechanisms of sperm maturation. Testicular
SHBG isoforms are found in sperm and released from these during the capacitation
reaction. Plasma SHBG is about 95 kDa in molecular weight, 30% of which is
represented by carbohydrate, and possesses one androgen-binding site per molecule.
Human testicular SHBG transcripts are expressed in the germ cells and contain an
alternative exon 1 sequence, appearing to encode an SHBG isoform that is 4–5 kDa
smaller than plasma SHBG. Te-binding capacity is also much lower compared to the
plasma SHBG. In normal men, only 2% of total Te circulates freely in the blood,
while 44% is bound to SHBG and 54% to albumin. The binding affinity of Te to
albumin is about 100 times lower compared to SHBG. However, since albumin
concentration is much higher than that of SHBG, the binding capacity of both
proteins for Te is about the same. The ratio of Te bound to SHBG over free SHBG
is proportional to SHBG concentration. A direct measurement of free Te is imprac-
tical in routine practice, so that several equations are used to estimate the free Te
concentration in serum (Selva et al. 2005). The main dissociation of Te from binding
proteins takes place in capillaries. The interaction of binding proteins with the
endothelial glycocalyx leads to a structural modification of the hormonal binding
site and thereby to a change in affinity. As a result Te is set free and can diffuse freely
into the target cell. SHBG not only binds Te but also estradiol. The type of binding is
influenced by the different SHBG isoforms, but generally Te binds threefold higher
than estradiol to SHBG. Its concentration in serum is under hormonal regulation and
primarily regulated through opposing actions of sex steroids on hepatocytes, estro-
gen stimulation, and androgen inhibiting it. Other hormones such as thyroid hor-
mones are also potent stimulators of SHBG production. In normal, healthy men with
an intact hypothalamic–pituitary–testicular axis, an increase in plasma concentra-
tions of SHBG leads to an acute decrease of free Te and simultaneous stimulation of
Te synthesis, persisting until achievement of normal concentrations. SHBG concen-
trations can be elevated in hypogonadal men (Rolf 1997).
Extratesticular Metabolism of Testosterone
Te is a precursor of two important hormones: through 5α-reduction, as it gives rise to
the highly biologically (three- to sixfold compared to Te) active hormone 5a-
dihydrotestosterone (DHT), and through aromatization to estradiol. The half-life of
Te in plasma is only about 12 min. Reduction of Te to DHT occurs in the
The Physiology of the Testis 25

endoplasmic reticulum through the enzyme 5α-reductase which is located in cellular
microsomes. Both Te and DHT bind to the same intracellular androgen receptor to
regulate gene expression in the target tissue. Although they interact with the same
androgen receptor, Te and DHT produce distinct biological responses and the
molecular mechanisms are still under debate. Two isoforms of 5α-reductase could
be identified in humans by NADPH-dependent enzymes reducing the double bond at
the four to five positions in C-19 as well as C-21 steroids. The gene for 5α-reductase
type I is located on chromosome 5 encoding for a protein with 259 amino acids,
while the gene for the 5α-reductase type II is on chromosome 2 encoding for a
shorter protein with 254 amino acids. The two isoforms are very similar to each
other, but show different biochemical properties. One works optimally at an alkaline
pH, the other at acidic pH. Also, the tissue distribution of the two forms is different.
Type I 5α-reductase has been localized in the non-genital skin, liver, brain, prostate,
ovary, and testis, while type II is mainly active in classical androgen-dependent
tissues, such as the epididymis, genital skin, seminal vesicle, testis, and prostate but
also in the liver, uterus, breast, hair follicles, and placenta. At the cellular level, DHT
sustains differentiation and growth and is particularly important for normal sexual
development and virilization in men. It also affects the muscle mass and the
deepening of the voice. Overall, Te effects result from influences of the hormone
itself and of its metabolites estradiol and DHT. Changes in the property of type II
5α-reductase due to mutation can result in complete androgen insensitivity syn-
drome (CAIS) or partial androgen insensitivity syndrome (PAIS) (Imperato-
McGinley and Zhu 2002). In human tissues five aldo-keto reductase isoforms
(AKR) exist with varying reductase activity on the 3-, 17-, and 20-ketosteroid
position with isoform (AKR1C2) predominately converting 5α-DHT to 3α-diol.
The inactivated metabolites are excreted in the urine (Penning et al. 2000). Some
androgen metabolites are excreted in free form; others are glucuronated by the liver
before excretion. The 17-glucuronidation of the DHT to metabolite androstane-
3α,17β-diol is directly correlated with increasing of the risk factors for metabolic
syndrome (total fat mass, its distribution, intrahepatic fat, disturbed lipid profile,
insulin resistance, and diabetes) (Vandenput et al. 2007).
Mechanism of Androgen Action
Te dissociates from SHBG at the target organ and diffuses into the cells. The
conversion of Te into DHT is organ dependent. The first step in androgen action is
binding to the androgen receptor, which belongs to the family of steroid hormone
receptors. The mechanism through which the androgen receptor and other nuclear
receptors act as transcriptional factors has a general mechanism in which they bind to
their ligand in the cytosol thus inducing conformational changes, loss of chaperones,
dimerization, and nuclear translocation. Into the nucleus both (ligand and nuclear
receptor) bind to specific sequences of genomic DNA and induce stimulation of
RNA synthesis. Chromatin remodeling such as modification of histones plays a role
in gene transcription, and many nuclear receptor-interacting co-regulators perform
26 A. Ilacqua et al.

significant roles in gene transcription. Currently 48 nuclear receptors have been
identified in humans. These receptors share substantial functions and are thought to
have evolved from a single ancestral gene. Orphan nuclear receptors have also been
found for which no ligand has yet been identified. Members of this receptor family
possess an N-terminal domain, a DNA-binding domain, a hinge region, and a
hormone-binding domain (Kato et al. 2011).
Steroid receptors show high homology with the corresponding DNA-binding and
ligand-binding domains in the mineralocorticoid, glucocorticoid, and progesterone
receptors. In contrast, at the N-terminal domain, little similarity with these receptors
remains. The nuclear receptors are subdivided into two subfamilies depending on
their ligand partners’forming homodimers such as the androgen receptor and other
steroid receptors; another subfamily forms heterodimers with only one ligand, such
as the thyroid hormone receptor. An important characteristic of the N-terminal
domain of the androgen receptor is the presence of short tandem repeats (STRs)
CAG coding for polymorphic polyglutamine, TGG repeats coding for polyproline,
and GGC repeats for polyglycine. In normal men, about 17–29 glutamine repeats
and 13–17 glycine repeats are present. Alleles of small GGC size have been
associated with esophageal cancer, while in patients with Kennedy disease, a disease
with degenerating motoneurons, up to 72 such glutamine repeats are present.
Furthermore, in the androgen receptor, long CAG and GGC alleles are associated
with decreased transactivation function and have been associated with cancers in
women. In the androgen receptor, a low-size CAG (<19 repeats) and GGC (<15
repeats) alleles result in higher receptor activity and have been associated with earlier
age of onset and a higher grade and more advanced stage of prostate cancer at the
time of diagnosis (Francomano et al. 2013a; Tirabassi et al. 2015).
The number of glutamine repeats of the androgen receptor has been associated
with azoospermia or oligozoospermia, but no clear association was found. The
mentioned subtle differences in the number of repeats, e.g., CAG or TGG and
GGC of AR gene, have also been tested for spermatogenic effects. In spite of
many efforts to demonstrate that the number of CAG triplets influences the tran-
scriptional activity of the androgen receptor, no clear relationship to disturbances of
spermatogenesis has been found in a wide variety of human ethnics. Some
oligozoospermic and azoospermic men bearing mutations in the ligand-binding
domain have also been identified. Androgen receptor defects such as deletions or
inactivating mutations can profoundly alter receptor function. The resulting pheno-
type is highly variable ranging from poor virilization to testicular feminilization.
Inactivating mutations of the androgen receptor gene in a 46 XY male with testes
resulted in a female phenotype owing to the complete lack of all androgen activity.
However, there is no uterus and only a partially formed vagina, and during puberty
pubic and axillary hair is scant or absent. This syndrome of complete androgen
insensitivity (AIS) was earlier called testicular feminization. Similar clinical conse-
quences are also typical for mutations which severely damage the function of the
androgen receptor, such as those in the DNA-binding or androgen-binding domain.
Partial AIS (PAIS) is due to mutations in the androgen receptor gene and over
800 mutations have been reported (http://www.androgendb. mcgill.ca). Furthermore,
The Physiology of the Testis 27

mutations which involve co-activators or co-repressors can also lead to PAIS of
different severity. Moreover, mutations in the N-terminal domain of the androgen
receptor, which can lead to elimination of the androgen receptor function, play a
minor role in male idiopathic infertility (Zuccarello et al. 2008).
Biological Actions of Androgens
In primates, the androgen receptor can be found not only in the classical androgen-
dependent organs, such as the muscles, prostate, seminal vesicles, epididymis, and
testes, but also in almost every tissue, e.g., the hypothalamus, pituitary, kidney,
spleen, heart, and salivary glands. Hence, Te exerts a variety of actions on many
body targets. In the testis, the androgen receptor is expressed in SCs, peritubular
cells, and LCs, while the germ cells seem not to express it. Studies in cell-specific
androgen receptor knockout mice demonstrated that the SCs require androgen for the
maintenance of complete spermatogenesis and that spermatocyte and spermatid
development depends on androgens. The peritubular myoid cells maintain their
cell contractility, ensuring normal spermatogenesis and sperm output. A functional
androgen receptor in LCs is essential to maintain spermatogenesis and Te production
and is required for normal male fertility (Xu et al. 2007). Androgens are important in
every phase of human life. During the embryonal stage, Te determines the differen-
tiation of the sexual organs and, during puberty, the further development toward the
adult male phenotype, which is then maintained along with important anabolic
functions. DHT is the main androgen acting on the epididymis, vas deferens, seminal
vesicles, and prostate, originating from Te through 5α-reductase. These tissues are
particularly dependent on continuous androgen action. In addition, Te aromatization
to estrogens plays an important role in prostate growth. Estrogen concentrations in
prostate stromal tissue are clearly increased in case of benign prostatic hyperplasia
(BPH). Estrogens, acting in synergy with androgens and the estrogen receptor ß, are
required to regulate the proliferative and antiproliferative changes that occur during
normal prostate development and differentiation. In the epididymis, the seminal
vesicles, and the vas deferens, a lack of Te can result in regression of the secretory
epithelia, eventually leading to aspermia (ejaculation failure). The androgen effects
in these organs are mediated through Te, DHT, and estradiol.
Both Te and DHT are necessary for normal penis growth, which is positively
correlated with the increasing Te concentrations during puberty. The masculinization
of Wolffian ducts is primarily caused by Te, whereas the transformation of the
external genitalia, urethra, and prostate is primarily due to DHT. However, androgen
receptors are no longer expressed in the penis of adult men, and any androgen
deficiency after puberty results in only minor decrease of penis size. Similarly, Te
administration to adults is not capable of increasing penis size. Te is the main
androgen present in muscles, which have very low 5α-reductase activity. Skeletal
muscles are capable of converting circulating dehydroepiandrosterone (DHEA) to Te
and estrogen. Te has direct anabolic effects both on smooth and striated muscles with
an increase of muscular mass and hypertrophy of the fibers. In modern sports these
28 A. Ilacqua et al.

effects have led to an abuse of these steroids to increase the muscle mass in both
sexes, and loss of Te can lead to muscular atrophy. Also, androgens influence the
neuromuscular system (NM) throughout genomic and non-genomic pathways. The
genomic pathways are mainly involved in long-term effects of Te on muscle
structure and function, whereas the non-genomic pathways are responsible for
rapid effects of Te on NM metabolisms and functions and, probably, only as an
integrated step also on muscle structure. Even if scarce data exist in humans,
probably, steroids influence the nervous component of NM system (e.g., motor
behavior, neuronal activity, intracellular signaling) mainly through non-genomic
mechanisms (i.e., voltage-dependent K+ currents, Ca2+ channels, neurotransmitters,
etc.). Te replacement treatment would reduce excitability of the NM system, and it
would favor a predominant recruitment of slow-twitch motor units (Felici et al.
2016). Both androgens and estrogens induce an increase of bone density by stim-
ulating mineralization, while the lack of these steroids results in osteoporosis. The
skeleton develops distinctly in males and females, particularly at the periosteal
surface. Sex differences in skeletal morphology and physiology occur at or around
puberty, with little effect of gonadal steroids prior to puberty. At the beginning of
puberty, the increase in linear growth of bones is directly correlated with increasing
Te concentrations. It is known that gender differences, particularly with respect to
“bone quality”and architecture (i.e., predominantly bone width), are modulated by
the balance of the sex steroids estrogen and androgen. At the end of puberty,
depending on the presence of Te, epiphyseal closure occurs, an event that can be
consistently delayed in the presence of low Te concentrations. Low Te is associated
with increased risk of fracture, particularly with hip and nonvertebral fractures. It is
clear now that the androgen action on bone metabolism is mediated through estradiol
(Valimaki et al. 2004).
The effects of androgens on the skin and dependent organs vary in the different
cutaneous districts and are mediated by Te and, probably, DHT. Depending on Te,
the growth of sebaceous glands can be stimulated, and sebum production in the face,
upper part of the back, and in the skin of the chest can be induced. Te contributes to
the development of acne vulgaris, while estrogens can diminish sebum concentra-
tion. The effects of DHT and Te on the hair are influenced by the androgen sensitivity
of the hair follicle. While axillary hair and the lower part of pubic hair start growing
even in the presence of low androgen concentrations, much higher androgen levels
are necessary for the growth of the beard, upper part of the pubic hair, and chest hair.
The hairline is determined both by genetic factors and individual distribution of the
androgen receptor and depends on the androgen milieu. High 5α-reductase activity
has been observed in bald men, while in patients with 5α-reductase deficiency or
hypogonadism, there is no regression of the hair line. Since the growth of the scalp
hair is related to increased 5α-reductase activity, increased activity of this enzyme
with consequences for hair loss could be an expression of the precocious aging of the
hair follicles. Androgens stimulate hair follicles to alter hair color and size via the
hair growth cycle and seem to reduce alopecia (Randall et al. 2008).
During puberty, there is a Te-dependent growth of the length of the larynx of
about 1 cm. This size increase, together with the length and mass of the vocal cords,
The Physiology of the Testis 29

leads to a lowering of vocal register. A deep voice is directly related to androgens so
that a lower register can also be induced in women by Te treatment. The depth of
voice in a man is correlated with the duration of the pubertal phase after which the
androgen receptors are lost. Once reached, register remains unchanged and no
modification of the voice can be obtained after puberty in hypogonadal patients.
The gender-specific change of the vocal register is correlated with the degree of
mineralization in human thyroid cartilage. Few chondrocytes near the mineralization
front are positive for the androgen receptor and also for alkaline phosphatase,
suggesting an involvement in androgen-mediated thyroid cartilage mineralization
(Claassen et al. 2006).
The influence of androgens on the hematopoietic system is twice. Through the
androgen-dependent, receptor-mediated erythropoietin synthesis, there is robust
stimulation of erythrocyte production. Androgens also directly affect the hemato-
poietic stem cells and lead to increased synthesis of hemoglobin. These effects can
also be demonstrated in vitro on the granulopoietic and thrombopoietic stem cells,
although the role of androgens in this field is still unclear.
In the central nervous system (CNS), Te can be either aromatized to E2 or reduced
to DHT. The individual activities of the different enzymes and distribution of
receptors are not homogeneous in the CNS, but rather vary according to the brain
region. Recent data show that neural stem/progenitor cells (NSPCs) from the
sub-ventricular zone of male and female mice respond to principal sex hormones;
therefore circulating estradiol and Te could be prime mediators of sexually dimor-
phic NSPC regulation (Ransome and Boon 2015). During the intrauterine period, a
boy’s brain develops in the male direction induced by Te and in a girl in the female
direction through its absence. The gender identity, the sexual orientation, and other
CNS-controlled behaviors are programmed at this early period of development.
Androgens are also important for other male characteristics such as aggressive
behavior, initiative, and concentration capacities. A connection with spatial orienta-
tion and mathematical and composition skills is still under discussion. There is a
close relationship between androgen milieu and normal corporeal and spiritual
performance and activity as well as good general mood and self-confidence. The
frequency and presence of sexual fantasies, morning erections, frequency of mas-
turbation or copulation, and sexual activity are related to blood Te concentrations in
the normal-to-subnormal range. Conversely, androgen deficiency is often accompa-
nied by loss of interest, lethargy, depressive mood, loss of libido, and sexual
inactivity. Furthermore in the adult, Te in the brain is neuroprotective and may
influence motor neuron regeneration in adulthood (Zitzmann 2006). Reduced Te
level is associated with depressive disorders and depends upon the androgen recep-
tor genotype. The role of Te in the CNS is still poorly understood, but evidence
suggests that Te could be helpful in the treatment of cognitive diseases, including
dementia. Te appears to activate a distributed cortical network and addition of Te
may improve spatial cognition in younger and older hypogonadal men (Isidori et al.
2015).
30 A. Ilacqua et al.

Te plays a key role in erectile function through coordinating and facilitating such
processes by androgen receptors localized within vascular endothelium and smooth
muscle cells. Thus, arterial functions may be directly subject to T influence, and most
likely, two independent pathways of T-induced effects within the vessel wall can be
assumed (i.e., genomic and non-genomic) (Fig. 9). Androgen sensitivity could be
also modulated by a functional polymorphism of the AR that influences the strength
of the genomic signal transduced from its interaction with an androgen as a bound
ligand. One such functional AR polymorphism is the exon 1 triplet CAG (poly-
glutamine) whereby the repeat length is inversely correlated with androgen sensi-
tivity. Thus, T may directly control the expression and activity of type
5 phosphodiesterases (PDE5) in human corpus cavernosum through the existence
of a single androgen-responsive element identified in human PDE5A gene promoter
(Francomano et al. 2013b). The effects of androgens on penile tissues in experimen-
tal models demonstrated that androgen deprivation induces:
1. Smooth muscle cell degeneration (apoptosis) and adipose tissue deposition with
associated fibrosis of corpus cavernosum
2. Reduction in the expression of nitric oxide synthase (eNOS and nNOS) and
decrease of arterial
3. Inflow and increase of venous outflow in the corpus cavernosum
4. Enhanced response to mediators of vasoconstriction and smooth muscle contrac-
tion such as a-adrenergic agents
5. Decrease of NO-mediated smooth muscle relaxation during sexual stimuli
6. Downregulation of expression of PDE5
Preclinical investigations provided evidence that PDE5i are less effective in
androgen-deficient animals and that the re-administration of androgen facilitates
PDE5i action, and this was confirmed in humans (Aversa et al. 2015).
Cross Talk Between Testis, Bone Marrow, and Pancreas
Emerging data suggest that bone mass, energy metabolism, and reproduction may be
coordinately regulated. Adiponectin has been proposed as a major player with its
strong association with impaired glucose tolerance, independently of adiposity.
Adiponectin and glucose homeostasis are both regulated by osteocalcin (OSCA),
an osteoblast hormone linked to vitamin D metabolism (Lee et al. 2007). Also, the
recent animal studies by Oury et al. revealed that the bone is a positive regulator of
male fertility and that this action may be mediated through OSCA, via its binding to
a specific G-coupled receptor, GPRC6A, present on Leydig cells that favors Te
biosynthesis. OSCA-deficient mice show a decrease in testicular, epididymal, and
seminal vesicles weights and sperm count, and Leydig cell maturation appears to be
halted in the absence of OSCA (Oury et al. 2011). OSCA-stimulated Te biosynthesis
The Physiology of the Testis 31

Fig. 9 Genomic and non genomic effects of testosterone on penile endothelial function
32 A. Ilacqua et al.

is positively regulated by insulin signaling in osteoblasts which in turn stimulates the
bioactivation of OSCA. In a feedback loop control, undercarboxylated active OSCA
then stimulates insulin secretion by the cells of the pancreatic islets, promotes insulin
sensitivity in peripheral organs, and favors Te biosynthesis in Leydig cells of the
testis.
Te in turn favors bone growth, maintenance, and maturation (Francomano et al.
2013a). Human data shows an association between visceral fat mass, insulin sensi-
tivity, OSCA, and T levels in humans, which significantly correlate with skeletal
health. In this view, OSCA results as an important marker of metabolic and gonadal
functionality, other than its well-established function as a marker of bone remodeling
(Migliaccio et al. 2013).
Aging
In aging men stochastic damage leads to gradual impairment of the hypo-
thalamic–pituitary–testicular axis, which is manifested as an age-related decrease
of total, free, and bioavailable Te. Primary testicular changes, including decreased
numbers of LCs, increased deposits of lipofuscin, and disrupted steroidogenesis,
reduce Te synthesis and its reserve capacities. Age-associated increases in SHBG
serum levels further aggravate decreases in bio-Te and free Te compared to total
Te. Although decreased, testicular functional reserve capacity is generally sufficient
for adequate GnRH/LH feed-forward signals, which should result in fully compen-
satory Te secretion. However, inadequate amplitude of GnRH and LH synchronous
pulses due to lower numbers and insufficient synchronization of hypothalamic
neurons with aging lower testicular Te output and availability. Thus, mild secondary
hypogonadism of the hypothalamic–pituitary unit in elderly men results in the
inability to compensate for mostly mild primary testicular hypogonadism/hypo-
androgenism. This secondary hypogonadism is observed, although there may be
reduced Te feedback inhibition, maintained LH secretory capacity of gonadotropins,
increased LH metabolic half-life, and increased efficacy of suboptimal effective
GnRH pulses. Men >80 years exhibit increasing LH levels, which may also occur
in middle-aged men due to the loss of elevated opioid tone (Kaufman and Vermeulen
2005). Te deficiency (TD) is now the preferred terminology by many experts over
the traditional term, hypogonadism or late-onset hypogonadism, due to its greater
specificity. Whereas hypogonadism refers to inadequate function of the testicles with
regard to both Te and sperm production, TD refers only to inadequate Te production.
As the large majority of affected men with TD are older and unconcerned with their
fertility, the term TD is more appropriate. The Massachusetts Male Aging Study
(MMAS) investigated the relationships between age-related transitions in Te and
health or lifestyle changes and reported that a 4–5 kg/m2 increase in BMI had a
comparable negative impact on Te as 10 years of aging (Travison et al. 2007).
MMAS also reported that weight gain was associated with subsequently lower levels
of Te than remaining nonobese. In obese diabetic patients, interventional weight loss
regimes resulted in increased Te. However, it is not known whether “unsupervised”
The Physiology of the Testis 33

weight loss in the general population would have a similar impact on Te (Camacho
et al. 2013). The diagnosis of TD requires the presence of characteristic symptoms
and/or signs together with confirmatory blood tests demonstrating low Te levels
(Table 1). It is important to note that individuals with low Te levels may not have any
symptoms or signs, and there is no good evidence at this point in time that these men
deserve treatment. Conversely, men with suggestive symptoms but with entirely
normal Te levels are not considered candidates for treatment.
The clinical diagnosis of TD requires double confirmation via demonstration of
low Te levels. The most frequently used test is total Te, and most professional
societies and experts recommend this test as their primary biochemical determinant.
However, it is critical to note that there is no universally accepted biochemical
definition of T deficiency. This is reflected by the different Te thresholds suggested
by different society guidelines and expert groups, as well (Table 2). TD is an
Table 1 Sign and symptoms of testosterone deficiency
Sexual symptoms Non-sexual symptoms Signs
Low sexual desire (libido) Decreased energy, vitality, well-
being and motivation
Enlarged waist
Erectile dysfunction Depressed mood, dysthymia,
feeling sad or blue
Obesity (increased
BMI)
Infrequent morning/nocturnal
erection
Poor concentration and memory Anemia
Difficulty achieving orgasm Increased sleepiness, fatigue Reduced testis and
prostate volumes
Diminished intensity of the
experience of orgasm
Diminished physical or work
performance
Reduced muscle bulk
and strength
Diminished sexual genital
sensation
Hot flushes, sweats Gynecomastia
Impaired cognition (“brain fog”) Reduced beard growth
and body hair
Low bone mineral
density
Table 2 Proposed cut-offs for plasma testosterone by different scientific societies
Total T
Free T (calculated
or EqD)
US Endocrine Society 2010 (Boepple et al. 2008;
Penning et al. 2000)
<300 ng/dL (<10.4
nmol/L)
<50–90 pg/mL
EAA, ISA, ISSAM 2009 (Vandenput et al. 2007)<3 50 ng/dL (12.1
nmol/L)
<65 pg/mL (<225
pmol/L)
EAU 2012 Kato et al. (2011)<3 50 ng/dL (12.1
nmol/L)
<84 pg/mL (<243
pmol/L )
80–100 pg/mL
Some experts (Francomano et al. 2013; Tirabassi
et al. 2015)
<400 ng/dL (13.9
nmol/L)
1.5 ng/dL (by RIA)
34 A. Ilacqua et al.

important clinical condition that affects men of all ages. The treatment with Te
provides symptom relief for many affected men and improves general health param-
eters. There is thus great value for the practicing clinician to be aware of the
condition and to diagnose and treat it at any age, when present (Aversa and
Morgentaler 2015).
Cross-References
▶Growth Hormones and Aging
▶Testosterone Deficiency Syndromes
▶The Endocrine Regulation of Spermatogenesis
▶The Endocrine Regulation of Sexual Hormones
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