Sperm Biology from Production
Damayanthi Durairajanayagam, Anil K. Rengan,
Rakesh K. Sharma and Ashok Agarwal
D. Durairajanayagam () · A. K. Rengan · R. K. Sharma · A. Agarwal
Center for Reproductive Medicine, Cleveland Clinic,
Cleveland, OH, USA
7 Setter Place, Kendall Park, NJ 08824, USA
Introduction to the Male Reproductive System
The male reproductive system is a complex and intricate
system that produces spermatozoa or sex cells to carry the
genetic material of the male. The components of the male
reproductive system include the hypothalamic–pituitary–
gonadal (HPG) axis, and both the external and internal
sexual organs. The male reproductive system forms during
the early stages of embryonic development, becomes fertile
during puberty and maintains the masculinity of the adult
male. The external genitalia include the scrotum, testes, and
penis whereas the internal genitalia include the epididymis,
seminal ducts, spermatic cords, seminal vesicles, ejaculatory
ducts, bulbourethral or Cowper’s glands, and the prostate
gland. The testes produce the male gametes (spermatozoa).
The excurrent duct system matures, stores, and transports the
gametes to the penis for expulsion, and the accessory glands
produce and modify the contents of the semen.
The Scrotum and the Regulation of Testicular
The testes are the only organs in the human body located
externally. Each testis is individually housed in a sac-like
structure called the scrotum. The temperature of the under-
lying testes is reflected by the temperature of the scrotum.
The process of spermatogenesis is optimal at temperatures
2–4 °C lower than that of core body temperature . In order
to maintain a hypothermic testis, the scrotum has several in-
tegral properties that facilitate the dissipation of heat. These
include thin scrotal skin, minimal subcutaneous fat, sparse
distribution of hair, and a large number of sweat glands. In
addition, the scrotal skin hangs loose and wrinkled with a
large, total surface area that adjusts according to the ambient
The cremaster and dartos muscles in the testis also help
to regulate testicular temperature. The cremaster muscle
is a thin layer of skeletal muscle that surrounds each testis
and spermatic cord. When this muscle contracts, the testes
rise closer to the abdomen, keeping them warm when am-
bient temperature is low. The dartos muscle is a thin layer
of smooth muscle fiber beneath the scrotal skin. When con-
tracted, the dartos muscle causes the exposed scrotal skin
surface area to decrease and heat to be conserved. Converse-
ly, when both these muscles are in a relaxed state, the testis
hangs further from the abdomen, enveloped by the scrotal
skin. This aids in keeping the temperature of the testes lower
than that of the core body. Furthermore, rising external tem-
peratures activate the cutaneous receptors on the scrotal skin
to initiate sweat secretion and active heat loss through the
evaporation of sweat .
The human testes are a pair of ovoid (ellipsoid) structures
measuring approximately 4.5–5 cm in length by 2.5–4 cm
in width and about 15–25 mL in volume (Fig. 5.1). The tu-
nica albuginea, the outer capsule of the testes, is composed
of a thick and flexible (though not stretchable) fibrous layer
of connective tissue . The parenchyma of the testis is di-
vided by the septa (connective tissue) into 250–300 conical
lobules. Each of these lobules consists of masses of highly
convoluted seminiferous tubules. Both ends of the seminif-
erous tubules connect at the hilus to form the rete testis .
The seminiferous tubules secrete fluid that flows into the rete
testis to be collected and delivered to the excurrent ductal
system of the epididymis .
Each testis is composed of two distinct compartments:
(1) the tubular compartment that contains the seminifer-
ous tubules and (2) the intertubular compartment that lies
G. L. Schattman et al. (eds.), Unexplained Infertility, DOI 10.1007/978-1-4939-2140-9_5,
© Springer Science+Business Media, LLC 2015
30 D. Durairajanayagam et al.
between the seminiferous tubules and contains the interstitial
tissue. Each of these compartments is anatomically separate
but remains closely linked together. Within the seminiferous
tubules are the spermatogonial germ and Sertoli cells. The
Sertoli cells provide a hormonally active environment for the
evolution of primitive germ cells into mature male gametes
The bulk (90 %) of the testicular volume is made up of
the seminiferous tubules and the germ cells that lie within
the invaginations of the Sertoli cells, which make up the
germinal epithelium. The seminiferous tubules also consist
of peritubular tissue or lamina propria . The peritubular
tissue contains myofibroblasts that cause peristaltic contrac-
tions of the seminiferous tubules. This movement helps to
transport the developing, immotile germ cells to the rete
testis . The intertubular spaces within the lobules contain
clusters of Leydig or interstitial cells that make up the en-
docrine portion of the testis. The interstitial tissue consists
primarily of blood and lymph vessels, nerve and collagenous
fibers, macrophages, and a variety of connective tissue cells.
The spermatogenic process is dependent on intra- and extra-
testicular hormonal regulatory processes, the functions of the
intertubular microvasculature, Leydig cells, and other cel-
lular components in the interstitium (intertubular space) .
The testis is responsible for synthesizing (steroidogen-
esis) and secreting androgens (i.e., testosterone), which is
directly interrelated to its second function, producing sper-
matozoa (spermatogenesis). These functions are under hor-
monal control via the pituitary gonadotropins—luteinizing
hormone (LH), and follicle-stimulating hormone (FSH).
Hormonal Control of Spermatogenesis
The hormonal regulation of spermatogenesis is under the
control of the hypothalamus–pituitary–gonadal (HPG) axis.
This axis begins as the higher center sends signals to the hy-
pothalamus, which acts as the integrating center. The hypo-
thalamus releases gonadotropin releasing hormone (GnRH)
in discrete pulses that peak every 1.5 h. GnRH acts on the
anterior pituitary to stimulate gonadotropin production (LH
and FSH). A continuous production of GnRH will cause go-
nadotrophin desensitization, which will diminish LH and
FSH release. LH is released in a similar pulsatile pattern to
that of GnRH while FSH release is influenced by inhibin. LH
and FSH act on the testes to produce testosterone and inhib-
in, respectively. LH acts on the Leydig cells in the testes to
stimulate testosterone production through the conversion of
cholesterol. When testosterone levels accumulate, it exerts a
negative feedback effect at the pituitary (short loop) to sup-
press the release of LH and at the hypothalamus (long loop),
which ultimately suppresses GnRH production and thereby
regulates testosterone levels. FSH acts on the Sertoli cells to
stimulate inhibin and androgen-binding protein (ABP) secre-
tion. Accumulating inhibin levels exert a negative feedback
effect at the pituitary to suppress FSH release, thereby regu-
lating inhibin levels.
FSH is required at the onset of puberty to initiate sper-
matogenesis as its action on Sertoli cells is necessary for
germ cell maturation. Testosterone is essential for maintain-
ing the spermatogenic process. Its actions are facilitated by
the Sertoli cells. Spermatocytes have ABP receptors but not
Fig.5.1 The human spermato-
zoa, testis, and epididymis. To the
left is a mature human sperma-
tozoon showing the components
that make up the head, midpiece
and tail sections. To the right is
a view of the human testis and
the seminiferous tubules, as well
as the epididymis, showing the
corpus ( head) and caudal ( tail)
sections. (Reprinted with permis-
sion, Cleveland Clinic Center for
Medical Art & Photography ©
2010–2013. All rights reserved.)
5 Sperm Biology from Production to Ejaculation
androgen receptors whereas the Sertoli cells have androgen
receptors. The binding of ABP to testosterone may assist tes-
tosterone movement toward the lumen of the seminiferous
tubule onwards to the epididymis. FSH also induces the con-
version of testosterone to 5α-dihydrotestosterone (5α-DHT)
and 17β-estradiol. 5α-DHT is more active than testosterone
and along with 17β-estradiol, is involved in the development
and function of the penis, scrotum, accessory sex glands,
secondary sex characteristics, libido and potency.
Leydig cells are embedded in groups that surround the con-
nective tissue between seminiferous tubules in the testicle.
These endocrine cells are the principal source of testoster-
one, the production of which is stimulated by LH (Table 5.1).
In adults, testosterone in circulation is kept within the physi-
ological range of 300–1200 ng/dL while intratesticular levels
of testosterone are far higher. In the testes, testosterone levels
are highest at the basement membrane of the seminiferous
Testosterone, the major male androgen in circulation and in
the Leydig cells, is responsible for primary and secondary
sex characteristics. It is synthesized from cholesterol in the
Leydig cells. Primary sex characteristics are structures re-
sponsible for promoting the development, preservation, and
delivery of sperm cells while secondary sex characteristics
are structures and behavioral features that externally differ-
entiate men from women.
Sertoli cells, also known as sustentacular or nurse cells, are
highly specialized cells that regulate the development of
spermatogonia into spermatozoa (Table 5.1). They originate
from the tubular basement membrane and extend up toward
the lumen of the seminiferous tubules. The basement mem-
brane acts as a barrier that prevents large molecules in the in-
terstitial fluid from entering the tubule but allows the entry of
testosterone. Sertoli cells provide sustenance for developing
spermatogonia and are involved in germ cell phagocytosis.
The formation of lipid droplets in Sertoli cells is associat-
ed with this phagocytosis . The number of lipid droplets
found in Sertoli cells increases as the testes advance in age
. They also produce and secrete anti-Müllerian hormone
(AMH), inhibin, activin, growth factors, enzymes, and ABP.
AMH is involved in embryonic development and contrib-
utes to the regression of Müllerian ducts. Inhibin, another
hormone, helps to regulate FSH secretion from the anterior
pituitary. When FSH binds to high-affinity FSH receptors on
the Sertoli cells, ABP is secreted (by Sertoli cells) into the
lumen of the seminiferous tubule, where it binds to testos-
terone (secreted by Leydig cells). This causes testosterone
to become less lipophilic and more concentrated within the
Neighboring Sertoli cells have membrane specializations
at the basolateral side that forms a band, sealing the cells to-
gether and forming a tight junction. The blood–testis barrier
prevents molecules in the blood from moving past the tight
junctions toward the lumen of the seminiferous tubules. This
ensures that the germ cells in the later stages of development
remain inaccessible to any harmful molecules in circulation.
The Blood–Testis Barrier
In the mammalian testes, the blood–testis barrier is com-
posed of specialized junctions that are tightly bound between
adjacent Sertoli cells in the epithelium of the seminiferous
tubule. This barrier is also known as the Sertoli cell seminif-
erous epithelium barrier. The strong intercellular junctional
complexes that link two adjacent Sertoli cells in the tubule
form an additional barrier between the tubular lumen and the
interstitial fluid outside the tubule. This divides the seminif-
erous tubule space into two parts: the basal (basement mem-
brane) compartment that is in contact with blood and lymph
vessels and the adluminal (lumen) compartment that is iso-
lated from these fluids. The blood and lymph vessels and
Table 5.1 Functions of the Leydig and Sertoli cells
Functions of the Leydig cells Functions of the Sertoli cells
Initiation and maintenance of spermatogenesis Maintains the integrity of seminiferous tubules epithelium
Activation of the hypothalamus–pituitary–gonadal axis Secretion of hormones—inhibin and androgen-binding protein (ABP)
Production of testosterone—manifestation of male secondary sex
Secretes tubular fluid into the tubular lumen for transport of sperm within
Differentiation of male genital organs Delivery of nutrients to germ cells
Masculinization of the brain and sexual behavior Steroidogenesis and steroid metabolism
– Aids in process of phagocytosis and elimination of cytoplasm
– Regulates the spermatogenic cycle
– Acts as a hormonal target for LH, FSH, and testosterone
32 D. Durairajanayagam et al.
nerves are located in the interstitium between the tubules and
do not penetrate the seminiferous tubules . The Sertoli
cells are surrounded by closely aligned myoid or peritubular
cells. These arrangements collectively form the blood–testis
barrier, which provides an immunologically privileged site
for spermatogenesis to thrive.
The fluid found in the tubular compartment of the testes
differs from that in found in the interstitium as the former
contains low concentrations of glucose and high concentra-
tions of potassium ions and steroid hormones. The tight junc-
tions of the blood–testis barrier break and reform around the
migrating cells to ensure that the barrier remains intact.
The process of spermatogenesis is also regulated indepen-
dently from within the testis. The Leydig cells secrete (1)
testosterone, (2) neuroendocrine substances that serve as
neurotransmitters, and (3) growth factors for neighboring
Leydig cells, blood vessels, lamina propria of the seminif-
erous tubules, and Sertoli cells [12–14]. Leydig cells also
contribute toward the nutrition of the Sertoli cells and help to
regulate blood flow in the intertubular microvasculature .
The cells of the peritubular tissue influence myofibroblast
contractility and regulate spermatozoa transportation via
peristaltic movements of the seminiferous tubules. The Ser-
toli cells deliver different growth factors, and various germ
cells participate in the development and regulation of other
Spermatogenesis is an extremely intricate process of cell
differentiation, starting with germ cell (spermatogonia)
development and culminating in the production of highly
specialized spermatozoa. This process produces the genetic
material required for species replication. Spermatogenesis
occurs in the lumen of the seminiferous tubules. It was clas-
sically believed that human spermatogenesis takes about 64
days in the testis (from spermatogonium to spermatid) with
an additional 10–14 days in the epididymis for maturation of
spermatozoa. Thus, the entire process took about 70 ± 4 days
to complete . However, a more recent report suggests
that the entire process from production to ejaculation of sper-
matozoa is completed within a shorter period: an average of
64 ± 8 days (with a range of 42–76 days) . Spermatogen-
esis begins at puberty and occurs continually throughout the
entire male adult life span in contrast to oogenesis, which
is finite in women. The baseline number of precursor cells
in the testes is regulated by FSH. Early in embryonic de-
velopment, the gonocytes, which precede the formation of
spermatogonial germ cells, undergo active mitotic replica-
Spermatogenesis involves a series of cellular events that
begin in the basal compartment and end in the apical com-
partment. The basal and the luminal compartments are kept
separate by tight junctions. In the seminiferous tubules, the
developing cells are arranged in a highly ordered sequence
from the basement membrane toward the lumen (Fig. 5.2).
Spermatogonia are positioned directly on the basement mem-
brane. Primary spermatocytes, secondary spermatocytes,
and spermatids lie closest to the lumen. Spermatogonia and
primary spermatocytes are found in the basal compartment
whereas secondary spermatocytes and spermatids are found
in the adluminal compartment.
During spermatogenesis, two events occur in the basal
compartment outside the blood–testis barrier: (1) the re-
newal and proliferation of spermatogonia via mitosis and
differentiation and (2) the cell cycle progression from type B
spermatogonia to preleptotene spermatocytes. The following
three events occur in the adluminal or apical compartment
behind the blood–testis barrier: (1) the cell cycle progression
from zygotene to pachytene and then to diplotene spermato-
cytes, followed by meiosis I and meiosis II; (2) spermiogen-
esis, during which the round spermatids develop into elon-
gated spermatids and eventually spermatozoa; and finally
(3) spermiation, which involves spermatozoa maturation and
subsequent release into the lumen (Table 5.2).
The following is an overview of the spermatogenic
events. First, the primary spermatocytes undergo two mei-
otic divisions. The first division gives rise to two haploid
secondary spermatocytes, which is followed by the second
division, which gives rise to four haploid spermatids (1n, 23
chromosomes). Two of these spermatids carry the X mater-
nal chromosome while the other two spermatids carry the
Y paternal chromosome. Each spermatid will subsequently
undergo spermiogenesis, a metamorphosis into spermato-
zoa. The spermatozoa are then released into the lumen of the
seminiferous tubule (Fig. 5.3).
Spermatogonia are a population of long-living primordial
germ cells that undergo mitosis to provide a renewing stem
cell population and meiosis for spermatozoa production.
Germ cells are named according to their morphological ap-
pearance and can be categorized into two classes: Type A and
Type B. In humans, Type A cells, the most rudimentary of
cells, can be further classified as “pale Type A (Ap)” and “dark
Type A (Ad)” spermatogonia. Ap spermatogonia can divide
mitotically into more Ap cells or Type B spermatogonia. Type
A spermatogonia comprise the stem cell pool whereas Type B
spermatogonia continue to develop into spermatids.
5 Sperm Biology from Production to Ejaculation
Ap spermatogonia remain attached to the basal mem-
brane and continue to replenish its numbers, allowing the
spermatogenic process to persist despite the aging process.
Spermatogonia continuously increase in number via succes-
sive, but usually incomplete, mitosis. On the other hand, Ad
cells seldom divide, potentially serving as a dormant reserve
or nonproliferative stem cells that give rise to Ap spermato-
Type B spermatogonia have more chromatin within the
inner nuclear envelope than to the intermediate or type A
Fig.5.2 Seminiferous tubule.
A cross section of the germinal
epithelium in the seminiferous
tubule. The germinal epithelium
is divided by the Sertoli cell into
two compartments, i.e., the basal
and adluminal compartments.
Fully formed spermatozoa are re-
leased into the lumen. (Reprinted
with permission, Cleveland Clinic
Center for Medical Art & Pho-
tography © 2010–2013. All rights
Table 5.2 Terminology in spermatogenesis
Spermatogoniogenesis Process of producing spermatogonia through multiple mitoses to amass a large population of stem cells, most of
which undergo meiosis to produce spermatozoa
Spermatogenesis Process of differentiation of a spermatogonium into a spermatid
Purpose: to produce (via mitosis and meiosis) the necessary genetic material for species replication
Spermatocytogenesis Process of producing spermatocytes that occurs in the basal compartment of the seminiferous tubules
Spermiogenesis A complex metamorphosis that transforms round spermatids (from the final division of meiosis) into a complex
Spermiation Process whereby a mature spermatid frees itself from the Sertoli cell and enters the tubular lumen
34 D. Durairajanayagam et al.
spermatogonia. Type B spermatogonia divide mitotically
to produce primary spermatocytes, operating as differential
precursors to the preleptotene spermatocytes. Spermatogo-
nia remain joined by intercellular bridges but dissolve in the
advanced phases of spermatid development. The synchrony
of germ cell maturation is thus maintained , which is
likely to aid in its biochemical interactions.
Spermatocytogenesis involves the formation of spermato-
cytes and takes place in the basal compartment of the
seminiferous tubule. The process begins with the primary
spermatocytes undergoing meiosis I to form secondary sper-
matocytes. The prophase of the first meiotic division is very
long and thus, the primary spermatocyte has the longest
lifespan. Secondary spermatocytes then undergo the meiosis
II to produce spermatids. Secondary spermatocytes have a
comparably shorter lifespan of 1.1–1.7 days.
Spermatogenesis, from spermatogonium division to
spermatozoa release into the tubule, takes about 64 days to
complete. Sperm released into the lumen of the seminiferous
tubules are immature and incapable of moving on their own.
They are pushed through the lumen both by other developing
sperm cells moving toward the lumen and by the bulk flow
of fluid secreted by Sertoli cells. Sperm cells entering the
epididymis complete maturation after 10–14 days of transit,
aided by protein secretions from epididymal cells.
Disruption of Spermatogenesis
Type A spermatogonia are necessary for spermatogenesis,
and in cases of reduced spermatogenesis, it is likely that
Ad spermatogonia are absent . When Type A or Type B
spermatogonia are absent and the germinal epithelium is
made up only of Sertoli cells, then spermatogenesis will
not occur. This “Sertoli Cell Only Syndrome” may be
congenital ( absence of spermatogonia from birth) or ac-
quired (spermatogonia destroyed by exposure to radiation,
etc.). Spermatogenic arrest at the spermatogonial stage oc-
curs when Ap spermatogonia fail to develop into Type B
Fig.5.3 Spermatogenesis. Major
events in the life of a sperm
spermiogenesis, and spermia-
tion. (Reprinted with permission,
Cleveland Clinic Center for
Medical Art & Photography ©
2010–2013. All rights reserved.)
5 Sperm Biology from Production to Ejaculation
Mitosis (Cytodifferentiation of Spermatids)
Mitosis involves nuclear division and separation of dupli-
cated chromosomes to form two daughter cells with genetic
content exactly identical to its parent cell (diploid, n = 46).
Mitosis is vital for proliferation and maintenance of sper-
matogonial cells. Meiosis involves an intricate series of
events that encompass the duplication of chromosomes,
nuclear envelope breakdown, and equal division of chro-
mosomes and cytoplasm that leads to the formation of two
daughter cells. Specific regulatory proteins interact on DNA
loop domains during cellular replication [20, 21]. The germ
cells involved in the mitotic phase are the Type A spermato-
gonia, which first form the Type B spermatogonia and later
the primary spermatocytes. Through a series of mitotic divi-
sions, developing germ cells, which are interconnected by
intracellular bridges, produce primary spermatocytes—the
largest germ cell of the germinal epithelium. The base-
line number of spermatogonia is established after puberty.
Mitosis then supplies the precursor cells and initiates the dif-
ferentiation and maturation processes.
Meiosis is a complex process during which chromosomal
exchange of genetic material occurs to form four daughter
cells with half the number of chromosomes (haploid, n = 23)
compared to their parent cells. The purpose of meiosis is
to ensure genetic diversity. The germ cells involved in the
meiotic phase are the primary spermatocytes, secondary
spermatocytes, and spermatids. Meiosis occurs twice in suc-
cession as meiosis I and meiosis II; each meiotic process
consists of prophase, metaphase, anaphase, and telophase.
Prophase itself is made up of four stages: leptotene, zygo-
tene, pachytene, and diplotene. Leptotene takes place in the
basal compartment while the remaining three take place in
the adluminal compartment. Meiosis I is the reducing divi-
sion in which the number of chromosomes are halved (i.e.,
the replicated chromosomes in one cell is split between two
diploid cells). Meiosis II is the division in which there is no
DNA replication and the sister chromatids are split, resulting
in four halpoid cells.
The meiotic process is regulated by its own specific
mechanisms . In the seminiferous tubules, meiosis be-
gins with the detachment of Type B spermatogonia from the
basement membrane to form preleptotene primary spermato-
cytes. In theory, each primary spermatocyte yields four sper-
matids, but the actual yield is lower as some of these germ
cells are lost in the process. After meiosis I, each daughter
cell (secondary spermatocyte) contains one half of the ho-
mologous chromosome pair. The secondary spermatocytes
then quickly undergo meiosis II, during which time the chro-
matids separate at the centromere, yielding early round sper-
matids with haploid chromosomes “22X” or “22Y.” During
the entire meiotic phase, homologous chromosomes pair up,
cross over, and exchange genetical material to form an en-
tirely new genome. Defects during meiosis include apoptotic
spermatocytes and spermatogenic arrest of primary sper-
matocytes. These germ cells bordering the seminiferous tu-
bules cease to develop further and disintegrate .
In spermiogenesis, haploid spermatids undergo complete
differentiation or morphogenesis to form highly specialized
spermatozoa with fully compacted chromatin. These mor-
phological changes begin after meioses I and II. In humans,
there are eight different stages (Sa-1, Sa-2, Sb-1, Sb-2, Sc-1,Sc-2,
Sd-1,and Sd-2) involved in the maturation of spermatids
to spermatozoa. Each stage is identifiable by the matur-
ing cell’s morphological characteristics. In the postmeiotic
phase, there is progressive condensation of the nuclear chro-
matin (to about 1/10 the volume of an immature spermatid)
with the inactivation of the genome. In addition, the Golgi
apparatus forms the acrosome cap, and the flagellum struc-
tures begin to develop . Histones—alkaline proteins that
condense the DNA—are converted into transitional proteins,
and protamines are converted into well-developed disulfide
bonds. Defects during spermiogenesis include acrosomal
and flagellar defects, absence of the acrosome or the mid-
peice of the flagellum, and impaired nuclear condensation in
malformed spermatids .
The nucleus and its contents undergo several changes dur-
ing spermatogenesis. During the first eight steps of spermio-
genesis , the nucleus elongates and flattens, giving the
head its characteristic oval shape. This nuclear compaction
is believed to facilitate oocyte penetration and help to op-
timize spermatozoa swimming capacity . This nuclear
compaction includes chromatin remodeling. During the last
postmeiotic phase of spermiogenesis, histone molecules,
around which DNA is organized, are converted to transla-
tional proteins that are then converted to protamines .
Protamines contain large amounts of cysteine, which aids
in disulfide bond formation as the sperm cells mature in
the epididymis [26–28]. Protamines in the chromatin of the
spermatozoa are replaced by histones from the oocyte with-
in 2–4 h of fertilization.
36 D. Durairajanayagam et al.
During spermiation, the mature sperm cell releases itself
from the Sertoli cell and moves into the lumen of the semi-
niferous tubule . Spermatids originating from the same
spermatogonia remain attached to each other by bridges,
facilitating the transfer of cytoplasmic products. Spermia-
tion may also involve the movement of spermatids as they
progress toward the lumen of the seminiferous tubules .
Mature spermatids close their intracellular bridges and dis-
connect from the germinal epithelium, becoming free cells
(spermatozoa). At this stage, portions of the sperm cell cy-
toplasm, known as the cytoplasmic droplet, are eliminated.
However, the cytoplasmic droplet may remain in immature
spermatozoa during the process of spermiation, becoming
“excess residual cytoplasm” .
The Cycle or Wave of Seminiferous Epithelium
Spermatogenesis involves the division of primitive sper-
matogonial cells into germ cell types through the process of
meiosis. At any given time, groups of cells in different devel-
opmental phases are present within the germinal epithelium
of the seminiferous tubule. Germ cells are localized in spatial
units known as stages, designated by Roman numerals. Each
stage is distinguished by (1) acrosome development, (2) mei-
otic phase, (3) nucleus shape, and (4) spermatozoa release
into the lumen of the seminiferous tubule  (Fig. 5.4). The
same typical aspects of germ cell epithelium appear every
16 days . The time it takes for Type A spermatogonial to
divide is shorter than that required for the entire process of
spermatogenesis. The development of Type A spermatogonia
into mature spermatids followed by the delivery of mature
spermatozoa through the epididymal duct system takes any-
where between 42 and 76 days .
Rete Testis and Epididymis
Spermatozoa in the lumen of the seminiferous tubules leave
the testis through the rete testis and several vasa (ductuli)
efferentia. The ductuli combine to form a single, highly con-
voluted duct at the head of the epididymis. The epididymis,
located along the dorsolateral edge of each testis, allows for
post-testicular maturation and storage of spermatozoa during
their passage from the testis to the vas deferens. It is divided
into three unique segments: the caput epididymis (head) for
spermatozoa concentration, corpus epididymis (body) for
spermatozoa maturation, and cauda epididymis (tail) for
spermatozoa storage. As they pass through the epididymis,
spermatozoa attain their full maturity, fertilizing ability, and
motility, although they typically do not move under their own
control until after ejaculation. The epididymal epithelium is
sensitive to androgen stimulation and possesses both absorp-
tive and secretory abilities. As they journey through the epi-
didymis, spermatozoa undergo changes in membrane protein
composition, phospholipid and fatty acid content, net surface
charge, immunoreactivity, and adenylate cyclase activity. At
the caput, a significant amount of the fluid that carries sper-
matozoa from the seminiferous tubules is reabsorbed, greatly
increasing spermatozoa concentration. Spermatozoa remain
motionless in the male genital tract and are transported by
the flow of fluid in the testes, and thereafter by contraction
of the organs.
Spermatozoa mature outside the testes, leaving those
within the testes with poor motility and fertilization abil-
ity. The cauda epididymis stores mature spermatozoa,
permitting repetitive, rich ejaculations. Sperm cell stor-
age capacity diminishes distally. At the cauda epididymis,
spermatozoa lose fertilizing potential first, motility second,
and vitality last. These cells, along with nearly half of all
spermatozoa released from the testes, will disintegrate and
undergo reabsorption by the epididymal epithelium. This
includes older gametes that must be eliminated from the
male reproductive tract regularly to ensure high quality of
The vas deferens is a muscular tube adjacent to the pros-
tate that extends from the epididymis, passing through the
inguinal canal into the peritoneal cavity and opening into
the urethra. Near the prostate end, the vas deferens en-
larges and forms a gland called the ampulla. This portion,
along with excretory canals of the seminal vesicles, forms
the ejaculatory ducts and joins the urethra. The ampulla is
where the majority of sperm cells are stored for ejaculation.
Few spermatozoa find their way from the caudal epididy-
mis into the seminal vesicle where they will then degener-
ate. These cells are generally found in the terminal portion
of the ejaculate.
Accessory Sex Glands
The seminal vesicles, prostate gland, and Cowper’s
( bulbourethral) gland are collectively known as the accesso-
ry sex glands. These glands secrete fluids that act as the me-
dium for sperm transport and sustenance (Table 5.3). These
secretions make up the seminal plasma of the ejaculate. The
seminal vesicles join the ampullary portion of the vas defer-
ens and produce fructose and coagulating proteins. The pros-
tate gland is located at the junction of the vas deferens and
the urethra. Fluid produced by the prostate contains zinc, cit-
ric acid, and acid phosphatase, which give semen its typical
odor. In addition, the prostate secretes enzymes that liquefy
5 Sperm Biology from Production to Ejaculation
the seminal coagulum. The Cowper’s gland is situated distal
to the prostate gland and empties into the bulbous urethra.
Fluid from the Cowper’s gland lubricates the urethra prior
Structure of Spermatozoon
A morphologically normal sperm cell is about 45–50 µm in
length and consists of a head and tail.
Table 5.3 Composition of semen
Component Function Source
Sperm Carries the paternal genetic material Seminiferous tubules
Mucus Acts as a lubricant Bulbourethral glands
Water Provides a liquid medium All accessory glands
Buffers Neutralizes the acidic environment of the vagina Prostate, bulbourethral glands
L-carnitine Nourishes the spermatozoa Epididymis
Citric acid Prostate
Enzymes Forms coagulum in vagina, then liquefies Seminal vesicles and prostate
Prostaglandins Smooth muscle contraction; aids sperm transport within both the male and female
Fig.5.4 Stages in spermato-
genesis. The sequential stages of
differentiation during spermato-
genesis: from a diploid germ cell
into a fully functional spermato-
zoon. (Reprinted with permis-
sion, Cleveland Clinic Center for
Medical Art & Photography ©
2010–2013. All rights reserved.)
38 D. Durairajanayagam et al.
According to Kruger’s strict criteria , a morphologically
normal head should be smooth and symmetrically oval in
shape with a broad base and tapering apex. The sperm head
measures between 4.0–5.5 µm in length and 2.5–3.5 µm in
width, with a length-to-width ratio of between 1.50 and 1.70
[32, 33]. The head is the most important part of the mature
male gamete as it contains a nucleus, which is composed
of packed chromosomal paternal genetic material (mostly
DNA) containing 23 chromosomes. The nucleus comprises
about 65 % of the head, but like most somatic cells, lacks a
large cytoplasm to match .
The head also contains a well-defined acrosome region, a
cap-like covering of the anterior two thirds of the head (40–
70 % of the apex) . The acrosome is represented by the
Golgi complex [35, 36]. The acrosome contains a number
of hydrolytic enzymes, such as hyaluronidase and acrosin,
which are required for fertilization . During fertilization,
the acrosomal membrane fuses with the oocyte plasma mem-
brane oocyte at numerous sites. This is followed by the acro-
some reaction, an event characterized by acrosomal enzyme
release from the head tip.
Among the common abnormalities of the sperm head are
defective shape or size and the presence of numerous vacu-
oles (> 20 %) within the head surface. Shape defects include
large, small, tapering, pyriform, amorphous, double heads,
and various other combinations .
The neck is formed by the fragile junction between the head
and tail portion.
The tail measures 40–50 μm in length (nearly ten times the
length of the head) and provides motility for the cell. The
sperm cell’s entire motility apparatus is contained in the tail,
propelling the sperm body via waves generated in the neck
region that pass along distally in a whiplash manner.
The tail can be divided into the midpiece (anterior por-
tion), principal piece, and endpiece (posterior portion). Ide-
ally, the midpiece supports the head at exactly the center
position. It should be slender as well (maximum width of
1 µm), yet thicker than the rest of the tail and between 7.0
and 8.0 µm in length. The tail diameter should be between
0.4 and 0.5 µm, measuring about 50 µm in length. The tail
should have a well-defined endpiece, without any coiling
or abnormal bending (over 90°). The midpiece consists of
tightly packed mitochondria surrounded by a sheath. The mi-
tochondria in the midpiece supply energy in the form of ATP
for tail movement. The principal piece is the longest part of
the tail and comprises most of the propellant machinery. Mo-
tility plays a very important role in sperm transport through
the cervix; the sperm cells need to maintain motility despite
being suspended in fluid secreted by the female reproductive
organs. Moreover, motility is required to avoid phagocytosis
by polymorphonucleocytes found in female body fluid.
Common abnormalities of the neck and midpiece region
are the absence of the regions themselves, thickened neck,
distended or irregular/bent midpiece, abnormally thin mid-
piece (no mitochondrial sheath), or a combination of these
abnormalities . The presence of excess residual cyto-
plasm (i.e., a cytoplasmic droplet greater than one third the
area of normal sperm head) at the posterior portion of the
midpiece is another common abnormality. The cytoplasmic
droplet is released during ejaculation as long as the sperm
has sufficiently matured in the epididymis. Common tail de-
fects include short or multiple hairpin broken tails, irregular
widths, coiled tails with terminal droplets, or a combination
of these defects .
Erection and Emission
An erection is caused by sexually related psychic and/
or physical stimulation. Before an erection occurs, visual,
auditory, olfactory, and tactile stimulation triggers acetylcho-
line release by the parasympathetic nervous system. Acetyl-
choline causes vasodilation of the pudendal arteries, which
leads to increased blood flow to the corpus cavernosum and
corpus spongiosum of the penis. As the venous outflow is
compressed, the penis becomes engorged with blood and
grows more turgid, leading to an erection. Penile erection
is required for penetration into the vagina for sperm deposi-
tion. Erectile dysfunction is the repeated inability to achieve
or maintain an erection rigid enough for sexual intercourse.
Semen, the mixture of sperm and fluids, is expelled via a
neuromuscular reflex in two sequential phases: emission and
ejaculation. At the start of emission, a series of coordinated
sequential contractions begins in the testis efferent ducts, the
cauda epididymis and the convoluted portion of the vas def-
erens. The contractions advance in an assimilated manner,
propelling the sperm from the cauda epididymis forward into
the prostatic urethra. Here, the prostatic fluid, the sperm-rich
fraction from the ampulla, and the fluid from the seminal
vesicle are deposited into the prostatic urethra. This action
propels sperm from the efferent ducts, through the ejaculato-
ry ducts, and into the urethra. The filling of the urethra with
5 Sperm Biology from Production to Ejaculation
sperm initiates sensory signals that travel to the sacrospinal
region of cord. The internal urethral sphincter is closed by
sympathetic discharge to prevent retrograde ejaculation into
the urinary bladder. During the emission phase, when sper-
matozoa pass into the urethra, sympathetic stimulations re-
lease adrenaline and initiate contraction of the smooth mus-
cles surrounding the ampulla, deferens ducts, and the cauda
Ejaculation is initiated after emission, and the process expels
semen from the penile urethra. It includes external sphincter
relaxation and rhythmic prostate contractions. The bulbos-
pongiosus muscle propels the semen in an antigrade manner
out of the external urethral meatus. Sperm that is not ejacu-
lated will gradually die and undergoes cytolysis. Ejaculation
involves both the sympathetic and parasympathetic nervous
systems. Parasympathetic fibers initiate the contraction of
the bulbospongiosus muscle, which leads to forcible expul-
sion of the semen from the urethra. Ascending impulses con-
tribute simultaneously toward the sensation of orgasm.
The ejaculate, or semen, is freshly produced at the time
of ejaculation. Ejaculation normally occurs in a definite
sequence. First, a small amount of Cowper’s gland fluid
is extruded followed by prostatic fluid and the sperm-rich
fraction from the ampulla, and finally secretions from the
seminal vesicle (Table 5.3). These secretions form the semi-
nal coagulum, a gel-like substance that normally liquefies in
about 20 min. Sperm cells are trapped within the gel matrix
of the coagulum and remain immotile until activation upon
Once the ejaculate is expelled, detumescence of the penis
begins under sympathetic control. Noradrenaline is secreted,
causing dilation of the penile vasculature and penile flaccidity.
Capacitation and Acrosome Reaction
The spermatozoa undergoes several chemical changes during
capacitation, which occurs in the cervix, uterine cavity, and
the fallopian tubes during the estrogenic phase. Capacitation
allows the acrosome reaction to occur as the sperm and oo-
cyte come into contact with one another. As with epididymal
maturation, capacitation is also required before fertilization
can occur. Capacitation takes place after ejaculation into the
female reproductive tract. During capacitation, spermatozoa
undergo a sequence of biochemical changes that ultimately
enable them to fertilize an ovum. The sperm plasmalemma
is reorganized to support the subsequent acrosome reaction;
seminal plasma factors are removed and modifications are
made to the sperm membrane, sterols, lipids, glycoproteins,
outer acrosomal membrane, and surface charge. The concen-
tration of intracellular free Ca2 + increases as well . In par-
ticular, it is the removal of cholesterol from the surface mem-
brane that allows for the acrosome reaction to occur .
The acrosome reaction is a form of exocytosis that gives
spermatozoa the ability to advance through the zona pellu-
cida and prepares them for fusion with the ovum membrane.
This process helps to dispel the contents of the acrosome,
including surface antigens and enzymes, for successful fer-
tilization. D-mannose binding lectins on the sperm surface,
for example, have been shown to help bind spermatozoa to
the zona pellucida [39, 40]. The acrosomal enzymes digest
the outer acrosomal membrane and the plasma membrane to
which it is attached. At this point, the head is covered only by
the inner acrosomal membrane. The posterior region of the
head is enclosed by a single membrane known as the post-
nuclear cap. The acrosome and postnuclear cap overlap to
form the equatorial segment, which does not take part in the
acrosome reaction. The spermatozoon progresses forward
and rapidly penetrate the three layers of the oocyte, mov-
ing through the cumulus oophorus, corona radiate, and zona
pellucida, respectively. Once inside the perivitelline space,
the cortical reaction is induced, triggering the completion of
meiosis II in the oocyte. Next, the spermatozoon attaches to
the vitelline membrane at the postnuclear cap area and fuses
with the oocyte membrane. Consequently, its tail breaks off
at the midpiece, detaching from the head, and is followed
by axoneme and head decondensation to free the male chro-
matids. Epididymal maturation, capacitation, and the acro-
some reaction induce cellular and chromatin modifications
in germ cells for their transformation into fully functional
spermatozoa (Fig. 5.5).
In humans, it takes a spermatogonium approximately 64
days to differentiate into four mature spermatids and into
mature spermatozoa . The daily production rate of sper-
matozoa is 3–4 million per gram of testicular tissue ,
which is meager in comparison to that of laboratory ani-
mals. More than 75 % of the developed sperm cells perish
due to apoptosis or degeneration, and more than 12.5 % of
the remaining cells are abnormal. In the end, the spermato-
genetic potential for reproduction amounts to approximately
12 % . Daily sperm production gradually decreases with
advancing age. This reduction could be attributed to the loss
of Sertoli cells, increase in germ cell degeneration during
prophase of meiosis, loss of primary spermatocytes, and the
loss of Leydig cells, non-Leydig interstitial cells, and myoid
Initial information regarding the success of spermato-
genesis is obtained by evaluating ejaculate under light
40 D. Durairajanayagam et al.
microscopy to assess the number, shape, and motility pat-
terns of spermatozoa and to assess other cellular components
present in the ejaculate .
Despite their biological necessity, spermatozoa are not
recognized by the immune system. While immune func-
tion is established shortly after birth, surface markers
found in late pachytene spermatocytes, spermatids, and
spermatozoa develop during puberty. The spermatozoa are
protected, however, by the blood–testis barrier, a micro-
environment in the seminiferous epithelium that renders
them free from immunological attack . Despite the
barrier, an immune monitoring system still exists in both
the testes and epididymis that defends against autoimmune
Disturbances to Spermatogenesis
Several factors can potentially disturb gamete proliferation
or differentiation and the intra- or extratesticular mecha-
nisms that regulate spermatogenesis. These include expo-
sure to physical agents such as heat or chemical substances,
poor nutrition, obesity, nicotine use, alcohol consumption,
ingestion of therapeutic and recreational drugs, bacterial
infections, hormonal imbalances, varicocele, cryptorchi-
dism, testicular cancer and radiation [45, 46]. Environmental
toxicants such as pesticides, phthlates, polychlorinated bi-
phenyls (PCBs) and endocrine disrupting chemicals (EDCs)
can also negatively impact the spermatogenic process [45,
Semen Parameters and Reference Range
A routine semen analysis is the “gold standard” for the initial
investigation of male fertility. The following factors are as-
sessed in the seminal ejaculate: physical characteristics (e.g.,
color, volume, pH, odor, viscosity, and liquefaction time),
sperm concentration, motility, progression, viability, and
morphology and leukocyte count. Semen parameters such
as sperm concentration, motility, and morphology can act as
markers of male fertility and may reflect testicular causes of
infertility. However, semen analysis must be performed on
two or three separate occasions (owing to its large individual
biological variability) before any conclusion can be made
. The World Health Organization (WHO) normal cutoff
values for semen characteristics are shown in Table 5.4.
Spermatogenesis is a highly organized, complex sequence of
differentiation events, both mitotic and meiotic, that yields
genetically distinct male gametes for fertilization with the
female ovum. In a broader scope, it helps to propagate a
species and contributes to genetic diversity. In human males,
spermatogenesis begins at puberty and persists throughout
life. Sperm production is a continuous process that occurs
in the seminiferous tubules within the blood–testis barrier of
the testis—an immune privileged site. Spermatogenesis in-
volves the transformation of spermatogonial germ cells into
spermatids via proliferation and cellular remodeling. The
process is regulated by various intrinsic and extrinsic factors.
Spermiogenesis converts the spermatids to motile spermato-
zoa, which are highly specialized haploid cells. Spermatozoa
are released along the seminiferous tubules into the epididy-
mis where post-testicular maturation and storage take place.
Fig.5.5 Sperm developmental
events. Changes that occur dur-
ing the development of a germ
cell into a spermatozoon leading
to its release and subsequent
maturation and storage in the
epididymis, prior to its journey
into the female reproductive
tract. (Reprinted with permis-
sion, Cleveland Clinic Center for
Medical Art & Photography ©
2010–2013. All rights reserved.)
5 Sperm Biology from Production to Ejaculation
Before fertilization can occur, spermatozoa must undergo
further biochemical changes via capacitation and the acro-
some reaction, both of which occur after ejaculation. The en-
tire sperm production process can be inhibited by numerous
factors, such as poor nutrition, hormonal imbalances, and
therapeutic drug side effects.
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Table 5.4 Reference values for semen characteristics according to the WHO, 4th (1999) and 5th (2010) edition
Parameter WHO 1999
(4th edition) 
(Lower reference limits obtained from lower 5th centile values)
Volume (mL) ≥ 2 1.5
Sperm concentration (106 per mL) ≥ 20 15
Total sperm count (106)≥ 40 39
Total motility (% motile) ≥ 50 40
Progressive motilitya(%) ≥ 25 % (grade a) 32 (grade a + b)
Vitality (% alive) ≥ 75 58
Morphology (% normal forms) 14 4
Peroxidase-positive leukocytes (106 per mL) < 1.0 < 1.0
a Grade a = rapid progressive motility (> 25 μm/s), grade b, slow/sluggish progressive motility (5–25 μm/s); normal, 50 % motility (grade a + b) or
25 % progressive motility (grade a) within 60 min of ejaculation
42 D. Durairajanayagam et al.
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