Dynamics of the Mammalian Sperm Head Modifications and
Maturation Events From Spermatogenesis to Egg Activation
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204
Advances in Anatomy,
Embryology
and Cell Biology
Co-ordinating Editor
H.-W. Korf, Frankfurt
Editors
F. Beck, Melbourne . F. Clascá, Madrid
M. Frotscher, Freiburg . D.E. Haines, Jackson
N. Hirokawa, Tokyo . Z. Kmiec, Gdansk
E. Marani, Enschede . R. Putz, München
J.-P. Timmermans, Antwerpen
Kiyotaka Toshimori
Dynamics
of the Mammalian
Sperm Head
Modifications and Maturation
Events From Spermatogenesis
to Egg Activation
With 36 Figures
Kiyotaka Toshimori
Department of Anatomy and Developmental Biology
Chiba University Graduate School of Medicine
1-8-1 Inohana, Chiba
260-8670 Japan
e-mail: [email protected]
ISSN 0301-5556
ISBN 978-3-540-89978-5 e-ISBN 978-3-540-89979-2
Library of Congress Control Number: 2008943974
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List of Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2 Mammalian Sperm Head . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.1 Structures, Domains, and Related Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.2 Cytoplasmic Layers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.2.1 Periacrosomal and Subacrosomal Layers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.2.2 Postacrosomal Layer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.3 Membrane System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3 Sperm-Head Formation and Factors Affecting It . . . . . . . . . . . . . . . . . . . . . . . . . 17
3.1 Cellular Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.1.1 Stem-Cell Factor, Steel Factor, and the c-kit Receptor . . . . . . . . . . . . . . . . . . . . 18
3.1.2 Intercellular Bridges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.1.3 Ectoplasmic Specialization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.2 IgSF Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.2.1 IgSF Proteins Expressed on Sertoli Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.2.2 IgSF Proteins Expressed on Germ Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
4 Post-testicular Modifications and Maturation Events Occurring
in the Sperm Head . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
4.1 In the Epididymis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
4.2 In the Female Reproductive Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
5 Dynamics of the Acrosome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
5.1 Acrosome Biogenesis During Spermatogenesis . . . . . . . . . . . . . . . . . . . . . . . . . 31
5.1.1 Golgi Phase (Steps 1–3 in Mice, Sa in Humans) . . . . . . . . . . . . . . . . . . . . . . . . . 32
5.1.2 Cap Phase (Steps 4–7 in Mice, Sb1–2 in Humans) . . . . . . . . . . . . . . . . . . . . . . . 32
5.1.3 Elongation Phase or Acrosome Phase (Steps 8–12 in Mice,
Sc in Humans) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
5.1.4 Maturation Phase (Steps 13–16 in Mice; Sd1–2 In Humans) . . . . . . . . . . . . . . 34
5.2 Pathways for the Transport of Acrosomal Molecules . . . . . . . . . . . . . . . . . . . . 34
5.3 Microenvironment of the Acrosome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
5.4 Post-testicular Modifications and Maturation Events . . . . . . . . . . . . . . . . . . . . 36
5.4.1 Acrosomal Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
5.4.2 Acrosomal Matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
5.4.3 Subcompartments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
5.5 Acrosomal Enzymes and Other Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
5.6 Failure of Acrosome Biogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
vi List of Contents
6 Dynamics of the Perinuclear Theca . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
6.1 Biogenesis During Spermatogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
6.2 Functions of Perinuclear Theca-Associating Molecules . . . . . . . . . . . . . . . . . . 44
7 Dynamics of the Sperm Nucleus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
7.1 Formation and Maturation of the Nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
7.2 Abnormal Nucleus Formation in Gene-KO Mice . . . . . . . . . . . . . . . . . . . . . . . . 48
7.3 DNA Fragmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
7.3.1 DNA Damage in Germ Cells and Mature Spermatozoa . . . . . . . . . . . . . . . . . . 49
7.3.2 Evaluation of DNA Fragmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
8 Dynamics of the Membrane System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
8.1 Modifications in Sperm Plasma-Membrane Proteins . . . . . . . . . . . . . . . . . . . . 53
8.2 Zona Pellucida Binding and the Acrosome Reaction . . . . . . . . . . . . . . . . . . . . 55
8.3 Primary Binding of Sperm to the Zona Pellucida . . . . . . . . . . . . . . . . . . . . . . . 56
8.4 The Acrosome Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
8.5 Rapid and Slow Release of Acrosomal Molecules . . . . . . . . . . . . . . . . . . . . . . . 60
8.6 Secondary Binding to the Zona Pellucida and the Candidate
Molecules Involved . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
8.7 Priming for Gamete Membrane Fusion and the Candidate
Molecules Involved . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
8.8 Gamete Membrane Fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
9 Dynamics of Egg (Oocyte) Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
9.1 Sperm (Cytoplasmic) Factor and Candidate Molecules Involved
in Egg Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
9.2 Sperm Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
9.3 After Egg Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
10 Status of Sperm-Head Components During Normal Fertilization,
IVF, ICSI, and Round Spermatid Injection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
11 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Summary
Mammalian spermatozoa have complex structures. The structure–function
relationship of sperm has been studied from various viewpoints. Accumulated
evidence has shown that the sperm components undergo sequential changes from
the beginning of spermatogenesis to the time of fertilization/embryogenesis.
Structural analyses have been performed using various new techniques of light and
electron microscopy as well as immunohistochemistry and immunocytochemistry
in combination with specific probes such as antibodies against sperm components.
Recently developed gene-manipulation techniques have accelerated investigations
on the events that govern the relationship between the structure and the molecular
components of sperm. In addition, animal models with gene manipulations have
been shown to exhibit various morphological and functional abnormalities that
lead to infertility.
In humans, male infertility is caused by a number of factors, such as the
external environment, nutrient changes, genotoxins, and mutagens. These factors
affect not only the development of germ cells during spermatogenesis but also the
functions of mature sperm, ultimately impairing fertilization or embryogenesis.
Typical phenotypes of impaired fertilization or embryogenesis are visible in
conditions such as azoospermia, oligozoospermia, and teratozoospermia, which
have been induced in model animals with gene deficiencies. Thus, comparative
analyses of the phenotypes expressed by model animals carrying gene mutations
and infertile human patients should be performed in relation to the normal (natural)
fertilization process to clarify the etiology of infertility due to male factors.
In this book, I discuss the events that occur in the normal sperm head and
govern the structure–function relationship from the time of spermatogenesis to that
of fertilization or egg activation. In this regard, I describe dynamic modifications
and maturation events that occur in sperm-head components and compare the
outcomes of these events with the outcomes of their failure.
Abbreviations Used in the Figure Legends
A acrosome
AA anterior acrosome
AF axial filaments
AP acrosome plate
BP basal plate
CD cytoplasmic droplet
CG cortical granule
CP connecting piece
DIC differential interference contrast
EF E-face
EP end piece
ER endoplasmic reticulum
ES equatorial segment
ES–PM plasma membrane covering the equatorial segment
FFR freeze-fracture replica
FS fibrous sheath
H head
IAM inner acrosomal membrane
IGS immunogold staining
IIF indirect immunofluorescence
KO knock-out
M microvilli
MP middle piece
MS mitochondrial sheath
N nucleus
Ne neck
NE nuclear envelope
NP nuclear plate
O oocyte
OAM outer acrosomal membrane
ODF outer dense fiber
P perforatorium
PA posterior acrosome
x Abbreviations Used in the Figure Legends
PAR postacrosomal region
PAS postacrosomal sheath or paracrystalline sheet
PC proximal centriole
PF P-face
PM plasma membrane
PP principal piece
PR posterior ring
PT perinuclear theca
PVS perivitelline space
RNE redundant nuclear envelope
TEM transmission electron microscope
Z zona pellucida
Dynamics of the Mammalian Sperm Head: Modifications
and Maturation Events from Spermatogenesis to Egg Activation
Kiyotaka Toshimori
Summary
Mammalian spermatozoa have complex structures. The structure–function
relationship of sperm has been studied from various viewpoints. Accumulated
evidence has shown that the sperm components undergo sequential changes
from the beginning of spermatogenesis to the time of fertilization/embryogen-
esis. Structural analyses have been performed using various new techniques of
light and electron microscopy as well as immunohistochemistry and immuno-
cytochemistry in combination with specific probes such as antibodies against
sperm components. Recently developed gene-manipulation techniques have
accelerated investigations on the events that govern the relationship between the
structure and the molecular components of sperm. In addition, animal models
with gene manipulations have been shown to exhibit various morphological and
functional abnormalities that lead to infertility.
In humans, male infertility is caused by a number of factors, such as the external
environment, nutrient changes, genotoxins, and mutagens. These factors affect not
only the development of germ cells during spermatogenesis but also the functions
of mature sperm, ultimately impairing fertilization or embryogenesis. Typical phe-
notypes of impaired fertilization or embryogenesis are visible in conditions such
as azoospermia, oligozoospermia, and teratozoospermia, which have been induced
in model animals with gene deficiencies. Thus, comparative analyses of the pheno-
types expressed by model animals carrying gene mutations and infertile human
patients should be performed in relation to the normal (natural) fertilization process
to clarify the etiology of infertility due to male factors.
In this book, I discuss the events that occur in the normal sperm head and govern
the structure–function relationship from the time of spermatogenesis to that of
fertilization or egg activation. In this regard, I describe dynamic modifications and
maturation events that occur in sperm-head components and compare the out-
comes of these events with the outcomes of their failure.
2 Research Methodologies for Adult Neurogenesis
Abbreviations Used in the Figure Legends
A: acrosome
AA: anterior acrosome
AF: axial filaments
AP: acrosome plate
BP: basal plate
CD: cytoplasmic droplet
CG: cortical granule
CP: connecting piece
DIC: differential interference contrast
EF: E-face
EP: end piece
ER: endoplasmic reticulum
ES: equatorial segment
ES–PM: plasma membrane covering the equatorial segment
FFR: freeze-fracture replica
FS: fibrous sheath
H: head
IAM: inner acrosomal membrane
IGS: immunogold staining
IIF: indirect immunofluorescence
KO: knock-out
M: microvilli
MP: middle piece
MS: mitochondrial sheath
N: nucleus
Ne: neck
NE: nuclear envelope
NP: nuclear plate
O: oocyte
OAM: outer acrosomal membrane
ODF: outer dense fiber
P: perforatorium
PA: posterior acrosome
PAR: postacrosomal region
PAS: postacrosomal sheath or paracrystalline sheet
PC: proximal centriole
PF: P-face
PM: plasma membrane
PP: principal piece
Research Methodologies for Adult Neurogenesis 3
PR: posterior ring
PT: perinuclear theca
PVS: perivitelline space
RNE: redundant nuclear envelope
TEM: transmission electron microscope
Z: zona pellucida
Chapter 1
Introduction
I have been studying mammalian spermatozoa, fertilization, and early embryonic
development for more than 30 years (i.e., from 1975 to the present), and the scope
of my studies has ranged from basic research to its clinical applications. Recently,
I have been involved in combination studies involving gene manipulation and
ultrastructural, biochemical, and physiological approaches and have conducted
these studies on both laboratory animals and clinical materials. My research has
been focused not only on the continuously induced modifications in the sperm-
head components that lead to sperm maturation but also on fertilization failure
and infertility that can occur because of inappropriate sperm-component modi-
fications.
A mature spermatozoon is a highly differentiated haploid cell with a head and
a flagellum (tail). Although the shape of the sperm head varies among species, it
can generally be classified into two major categories: (1) the sickle-shaped (fal-
ciform) head, as seen in rodents, and (2) the paddle-shaped (spatulate, fan-like)
head, as seen in many species, including humans (Fig. 1.1). In most animals, the
head appears flat in the ventrodorsal view. The sperm head comprises a nucleus, an
acrosome, and a small volume of cytoplasm in the form of cytoplasmic layers (Fig.
1.2). The nucleus contains the paternal genome, consisting of DNA strands that
are uniquely organized, along with the nuclear proteins protamines 1 and 2. The
mature sperm acrosome is a cap-like saccule that covers the proximal region of the
head; it is entirely covered by an acrosomal membrane that encloses hydrolyzing
enzymes and matrix proteins. The cytoplasm between the nucleus and overlying
plasma membrane becomes thin with narrow layers (spaces) toward the plasma
membrane (Fig. 1.3).
The tail contains motility-related apparatus, mitochondria, an axoneme, and
cytoskeletal structures such as outer dense fibers (ODFs) and fibrous sheaths (FSs)
(Fig. 1.2). These components have recently been found to associate with unique
cytoplasmic substances. Structurally, the tail is divided into four major regions
(domains); the neck, middle piece (midpiece), principal piece, and end piece. The
neck region is structurally complex: its major components are the basal plate, a
redundant nuclear envelope, 2–3 mitochondria (neck mitochondria), and a con-
necting piece (comprising the capitulum, centrosome, and segmented columns
connected to the ODFs). Centrosomes contain the proximal centriole and various
6 Introduction
Fig. 1.1a–b Light micrographs showing mammalian sperm head and tail. DIC image. a Mouse.
b Human
MS Longitudinal
ODF column
ODF FS
Central pair
AF Rib
Peripheral
OAM microtubules
A (tubulin)
IAM Cross section
FS
P arms
NE
AA (dynin) rib of FS
Cross section
ES(PA)
AF
Principal
piece
BP ODF
N (50µm)
Head RNE
AF
(5µm) PC MS
CP
ODF
Neck
End piece
Middle piece (5µm)
(5µm)
Fig. 1.2 Scheme of sperm head and tail components
pericentriolar matrix proteins. They are involved in microtubule formation when
the sperm reaches the oocyte, and they morphologically develop into the sperm
aster during fertilization in nonrodent mammals, including humans. The distal
centriole is transformed into the proximal part of the axoneme.
In this book, I will primarily discuss the events related to sperm-head modifi-
cations and I will also discuss the sperm-tail structures and their involvement in
these events. Events involving the tail components have previously been discussed
in many comprehensive reviews (e.g., Eddy 2007).
Introduction 7
Fig. 1.3 Scheme of sperm-head subdomains and cytoplasmic layers
The function of mammalian spermatozoa is not only to carry the paternal
genome to the oocyte but also to activate the oocyte arrested at the metaphase
of the second meiosis (MII). For these purposes, elaborate structures develop in the
sperm head during spermatogenesis.
Spermatogenesis is conserved throughout evolution and is maintained
throughout the adult lives of most mammals. It is a highly programmed differ-
entiation process involving developmental regulation with the stage-specific
expression of genes and their transcript variants. Sertoli cells structurally and func-
tionally support germ cells in the seminiferous epithelium under the control of
testosterone (androgen) secreted by the Leydig cells and the follicle-stimulating
hormone (FSH) secreted by the pituitary gland. The function of the Leydig cells is
further controlled by a pituitary hormone—the luteinizing hormone or interstitial
cell-stimulating hormone (ICSH)—in the hypothalamic–pituitary–gonadal axis.
Spermatogenesis is completed in about 35 days in mice (approximately 11 days
for the mitotic phase, 10 for the meiotic phase, and 14 for the postmeiotic phase),
and about 74 days in humans (Clermont and Trott 1969). Mature spermatozoa
produced by spermatogenesis are released from the Sertoli cells. The testicular
spermatozoa are mature at the genomic level. In fact, spermatozoa can produce
embryos when injected directly into an oocyte in the form of a testicular sperm
extraction–intracytoplasmic sperm injection (TESE–ICSI).TESE–ICSI is an artificial
reproductive technique (ART) that has recently been developed for the treatment
of infertility.
Under natural (normal) conditions, spermiated spermatozoa pass through the
rete testis, conduit canals, and the ductus efferentes, en route to the epididymis.
8 Introduction
Spermatozoa that enter the caput epididymidis are functionally immature; they do
not possess the properties of forward motility and zona pellucida recognition. On
leaving the testes, spermatozoa mature by undergoing functional and morphologi-
cal modifications during their passage through the epididymis. With these modifi-
cations, the sperm components not only gain several functional properties such as
forward motility and zona pellucida recognition but they also develop the ability
to protect the spermatozoa; collectively, these modifications are called "maturation
in the epididymis" (Bedford 1967; Cooper 1998; Jones 1991; Orgebin-Crist 1967;
Orgebin-Crist et al. 1987; Toshimori 1998; Tulsiani 2006; Turner 1979; Yanagimachi
1994). Spermatozoa are modified under the influence of products secreted by the
epithelial cells lining the male reproductive tract. In most mammals, spermatozoa
take 2–10 days to pass through the epididymis, regardless of the total length of the
epididymal duct; in humans, this process takes 2–3 days. Spermatozoa that have
achieved structural and functional maturation are stored in the cauda epididymi-
dis and vas deferens until ejaculation; the cauda epididymidis is poorly developed
in humans as compared to other mammals.
During ejaculation, spermatozoa are further coated with materials secreted
by the seminal vesicle and prostate gland. In general, human semen contains 108
spermatozoa per milliliter (approximately 1% of the ejaculate in terms of weight),
along with components of the prostate fluid (approximately 20% by weight; zinc,
acid phosphatase, fibrinolysin precursors, polyamines, etc.) and products of the
seminal vesicle (40–80%; fructose, ascorbic acid, fibrinogen, prostaglandin, cho-
lesterol, etc.). These components contain many biological elements that sperma-
tozoa require to execute various functions.
When ejaculated spermatozoa enter the female reproductive tract, they
encounter several obstacles. First, they must pass through the viscous mucous at
the junction between the vagina and the uterus (i.e., the cervix), and that between
the uterus and the fallopian tube (oviduct; UT junction). At the UT junction, sper-
matozoa temporarily associate with the oviductal epithelium (Oh-Oka et al. 2003;
Yanagimachi 1994) and are considered to undergo further unknown modifica-
tions; the duration of this association varies depending on the estrous cycle (e.g.,
the association is retained for six months in the case of bats). After leaving the
UT junction, but before reaching the ampullary region, spermatozoa execute a
major part of their capacitation function; the physiological (functional) changes
that render ascending spermatozoa competent to penetrate the eggs (oocytes)
are collectively termed "capacitation."
Spermatozoa that have undergone capacitation exhibit vigorous (whiplash)
tail movement, i.e., hyperactivation. Hyperactivation is a visible sign that the
spermatozoa have undergone all the physiological changes necessary for sperm–
egg interaction. Under normal conditions, fully capacitated spermatozoa suc-
cessfully pass through the cumulus matrix to bind to the zona pellucida. The
binding of the spermatozoa to the zona pellucida is classified into two steps—
primary binding and secondary binding. After primary binding, but before sec-
ondary binding, the spermatozoa undergo the acrosome reaction. The acrosome
