In Vitro Fertilization, 4th Edition

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 In-Vitro Fertilization
Fourth Edition

Kay Elder
Bourn Hall Clinic, Cambridge

Brian Dale
Centre for Assisted Reproduction, Naples

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Names: Elder, Kay, 1946–
Title: In-vitro fertilization / Kay Elder, Bourn Hall Clinic,
Cambridge, Brian Dale, Centre for Assisted Reproduction, Naples.
Description: Cambridge, United Kingdom ; New York, NY :
Cambridge University Press, 2020. | Includes bibliographical
references and index.
Identifiers: LCCN 2019009973 | ISBN 9781108441810
(paperback : alk. paper)
Subjects: LCSH: Fertilization in vitro, Human. | Fertilization (Biology)
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..................................................................
Every effort has been made in preparing this book to provide accurate and
up-to-date information that is in accord with accepted standards and prac-
tice at the time of publication. Although case histories are drawn from actual
cases, every effort has been made to disguise the identities of the individuals
involved. Nevertheless, the authors, editors, and publishers can make no
warranties that the information contained herein is totally free from error,
not least because clinical standards are constantly changing through research
and regulation. The authors, editors, and publishers therefore disclaim all
liability for direct or consequential damages resulting from the use of
material contained in this book. Readers are strongly advised to pay careful
attention to information provided by the manufacturer of any drugs or
equipment that they plan to use.
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 Contents
Preface vii
Acknowledgments x

1 Review of Cell and Molecular Biology 1 10 Sperm and ART 194

2 Endocrine Control of Reproduction: 11 Oocyte Retrieval and Embryo Culture 213
Controlled Ovarian Hyperstimulation
12 Cryopreservation of Gametes and
for ART 40
Embryos 254
3 Gametes and Gametogenesis 53
13 Micromanipulation Techniques 284
4 Gamete Interaction 77
14 Preimplantation Genetic Diagnosis 311
5 First Stages of Development 97
15 Epigenetics and Human Assisted
6 Implantation and Early Stages of Fetal Reproduction 331
Development 120 John Huntriss
7 Stem Cell Biology 135
8 The Clinical In-Vitro Fertilization
Laboratory 156 Index 358
9 Quality Management in the IVF The color plates appear between pages 166 and 167
Laboratory 180

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 Preface

The union of male and female gametes during the Half a century later, the creation of new life via
process of fertilization marks the creation of a com- human IVF continues to attract debate and discus-
pletely new individual, a unique event that ensures sion, prompting many governments to define “the
genetic immortality by transferring information from beginning of a human life” in formulating legislation
one generation to the next. It also creates variation, surrounding assisted reproductive technologies
which introduces the effects of evolutionary forces. (ART). Not surprisingly, these definitions vary from
During the first half of the nineteenth century, fertil- country to country and often reflect the theological
ization and the creation of early embryos were studied beliefs of the nations involved. Scientifically, a
in a variety of marine, amphibian and mammalian number of basic facts regarding fertilization and
species, and by the early 1960s had been successfully embryo development must be considered in defining
achieved in rabbits (Chang, 1959), the golden hamster the “beginning of life.” Both in vivo and in vitro,
(Yanagimachi and Chang, 1964) and mice (Whitting- gametes and preimplantation embryos are produced
ham, 1968). Following a decade of extensive research in great excess, with only a tiny proportion surviving
in mouse, rat and rabbit reproductive biology and to implant and produce offspring; human gametes are
genetics, Robert Edwards began to study in-vitro certainly error-prone, and the majority are never des-
maturation of human oocytes in the early 1960s tined to begin a new life. Some female gametes may
(Edwards, 1965). On February 15, 1969, the journal undergo fertilization, but subsequently fail to support
Nature published a paper authored by R.G. Edwards, further development due to deficiencies in the process
B.D. Bavister and P.C. Steptoe: “Early stages of fertil- of oogenesis. Once gametes are selected, their success-
ization in vitro of human oocytes matured in vitro” ful interaction is probably one of the most difficult
(Edwards et al., 1969). The paper scandalized the steps on the way to the formation of a new life. At this
international community; reporters and camera crews stage the two genomes have not yet mixed, and
from all around the world fought to gain entry to the numerous developmental errors can still occur, with
Physiological Laboratory in Cambridge, where failures in oocyte activation, sperm decondensation or
Edwards and his team were based. It drew fierce in the patterns of signals that are necessary for the
criticism from Nobel Laureates and much of the transition to early stages of embryo development.
scientific, medical and religious establishment in the A fertilized ovum is a totipotent cell that initially
United Kingdom and elsewhere, being regarded as divides into a few cells that are equally totipotent,
tampering with the beginning of a human life: reli- but for a brief period of time these cells can give rise
gious, ethical and moral implications were numerous. to one (a normal pregnancy), none (a blighted ovum
In-vitro fertilization (IVF) is now accepted completely or anembryonic vesicular mole) or even several
as a clinical procedure; in the quest for improvements (monozygotic twinning) individuals. Although fertil-
via new technology we should not be disheartened or ization is necessary for the life of a being, it is not the
surprised by irrational criticism, but draw courage only critical event, as preimplantation embryo devel-
from the pioneering work of Bob Edwards and his opment can be interrupted at any stage by lethal
colleagues, whose brave perseverance opened up an processes or simple mistakes in the developmental
entirely new field of interdisciplinary study that program. A series of elegantly programmed events
embraces science, medicine, ethics, the law and social begins at gametogenesis and continues through to
anthropology. parturition, involving a myriad of synchronized
vii

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 Preface

interdependent mechanisms, choreographed such Further scientific knowledge gained from the use
that each must function at the right time during of sophisticated technology has expanded our under-
embryogenesis. Combinations of both physiological standing of clinical and laboratory variables that can
and chromosomal factors result in a continuous influence human preimplantation development; man-
reduction, or “selection” of conception products agement of patients and treatment cycles has also
throughout the stages that lead to the potential been influenced by commercial pressures as well as
implantation of an embryo in the uterus. Preimplan- legislative issues. The rapid expansion in both
tation embryogenesis might be described as a type of numbers of cycles and range of treatments offered
Darwinian filter where only the fittest embryos sur- has introduced a need for more rigorous control
vive, and the survival of these is initially determined and discipline in the IVF laboratory routine, and it
during gametogenesis. is especially important that IVF laboratory personnel
It may be argued that the task of elaborating and have a good basic understanding of the science that
defining the concept of a “new individual” belongs to underpins our attempts to create the potential begin-
philosophers and moralists. For some, the beginning ning of a new life.
of human life coincides with the formation of a dip- A vast and comprehensive collection of published
loid body in which the male and female chromosomes literature covers clinical and scientific procedures and
are brought together. For others, true human life only protocols, as well as information gained from modern
occurs after implantation of the embryo in the uterine molecular biology techniques. Many books are now
mucosa. Many believe that a new individual is formed available that cover single chapters (and in some cases
only after differentiation of the neural tube, whilst individual paragraphs) of this edition. The range and
others believe that life begins when a fetus can live variety of media, equipment and supplies available
outside the uterus. In its most extreme form, some specifically for use in human IVF continues to
philosophers consider the acquisition of self- aware- expand, and the commercial companies involved have
ness of the newborn to define a new life. Most scien- evolved and merged to provide an ever-increasing
tists would probably agree that life is a continuous range of products. For this reason, we have tried to
cyclical process, with the gametes merely bridging the avoid mention of specific equipment and supplies.
gap between adult stages. Science, one of the bases of IVF is successfully carried out with numerous adap-
human intellect and curiosity, is generally impartial tations in individual labs, and specific detailed proto-
and often embraces international and religious cols are no longer appropriate; protocols and
boundaries; ethicists, philosophers and theologians procedures supplied by individual companies should
cannot proceed without taking into account the new be carefully studied and followed, in the perspective of
information and realities that are continuously gener- the relevant basic scientific principles provided in this
ated in the fields of biology and embryology. book. As with previous editions, our aim in preparing
Advances in the expanding range and sensitivity of this fourth edition was to try to distill large bodies of
molecular biology techniques, in particular genomics, information relevant to human IVF into a compre-
epigenomics and proteomics, continue to further our hensive background of physiological, biochemical and
understanding of reproductive biology, at the same physical principles that provide the scientific founda-
time adding further levels of complexity to this tion for well-established protocols in current use,
remarkable process of creating a new life. together with an extensively updated list of references.
The first edition of this book was written two This book is dedicated to Bob Edwards, who
decades ago; the field of human IVF has undergone embraced and inspired all who were blessed with the
significant transformation in many different ways, experience of knowing him . . . we salute and honor
with accelerated expansion since publication of the his infinite vision and endless optimism:
third edition in 2010. IVF is now practiced in most There wasn’t any limit, no boundary at all to the
countries of the world, and the number of babies born future . . . and it would be so that a man wouldn’t have
is estimated as approaching 8–10 million. room to store such happiness. . .
(James Dickey, American poet and novelist, 1923–1997)

viii

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 Preface

Further Reading Edwards RG (1989) Life Before Birth:

Reflections on the Embryo Debate.
Chromosome abnormalities in 1255
cleavage-stage human embryos.
Braude P, Bolton V, Moore S (1988) Hutchinson, London. Reproductive Biomedicine Online 1:
Human gene expression first occurs 17–27.
between the four- and eight-cell Edwards RG, Hansis C (2005) Initial
stages of preimplantation differentiation of blastomeres in 4- Munné S, Cohen J (1998)
development. Nature 333: 459–461. cell human embryos and its Chromosome abnormalities in
significance for early embryogenesis human embryos. Human
Carp H, Toder V, Aviram A, Daniely and implantation. Reproductive Reproduction Update 4: 842–855.
M, Mashiach S, Barkai G (2001) Biomedicine Online 2: 206–218.
Karyotype of the abortus in Nothias JY, Majumder S, Kaneko KJ,
recurrent miscarriages. Fertility and Edwards RG, Bavister BD, Steptoe PC et al. (1995) Regulation of gene
Sterility 75: 678–682. (1969) Early stages of fertilization in expression at the beginning of
vitro of human oocytes matured in mammalian development. Journal
Chang MC (1959) Fertilization of vitro. Nature 221: 632–635. of Biological Chemistry 270:
rabbit ova in vitro. Nature 184: 406. 22077–22080.
Hassold T, Chiu D (1985) Maternal
Edwards RG (1965) Maturation in age-specific rates of numerical Steptoe PC, Edwards RG (1978) Birth
vitro of human ovarian oocytes. chromosome abnormalities with after the re-implantation of a
Lancet ii: 926–929. special reference to trisomy. Human human embryo. [Letter] Lancet 2:
Edwards RG (1965) Meiosis in ovarian Genetics 70: 11–17. 366.
oocytes of adult mammals. Nature Hassold T, Chen N, Funkhouser J, et al. Warner C (2007) Immunological
196: 446–450. (1980) A cytogenetic study of aspects of embryo development. In:
Edwards RG (1965) Maturation in 1000 spontaneous abortuses. Annals Elder K, Cohen J (eds.) Human
vitro of mouse, sheep, cow, pig, of Human Genetics 44: 151–178. Preimplantation Embryo Evaluation
rhesus monkey and human ovarian Jacobs PA, Hassold TJ (1987) and Selection. Informa Healthcare,
oocytes. Nature 208: 349–351. Chromosome abnormalities: origin London, pp. 155–168.
Edwards RG (1972) Control of human and etiology in abortions and live Whittingham DG (1968) Fertilization
development. In: Austin CR, Short births. In: Vogal F, Sperling K (eds.) of mouse eggs in vitro. Nature 200:
RV (eds.) Artificial Control of Human Genetics. Springer-Verlag, 281–282.
Reproduction, Reproduction in Berlin, pp. 233–244. Yanagimachi R, Chang MC (1964) IVF
Mammals, Book 5, Cambridge Márquez C, Sandalinas M, Bahçe M, of golden hamster ova. Journal of
University Press. Cambridge, Alikani M, Munné S (2000) Experimental Zoology 156: 361–376.
pp. 87–113.

ix

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 Acknowledgments

We are deeply indebted to the colleagues who Elisabetta Tosti and Alessandra Gallo (sperm–
patiently advised, reviewed and revised new material oocyte interaction, including new figure)
for this fourth edition of the book. Rusty Poole gave Marc van den Bergh (the IVF laboratory and
us a starting point by offering invaluable suggestions quality management)
for updates required for every chapter, and we are
Charles Cornwell (micromanipulation)
immensely grateful for the expert advice, review and
revision on specific topics offered by: Tereza Cindrova-Davies (implantation and early
placental development)
Yves Ménézo (overall basic biochemistry,
metabolism, metabolic pathways, oxidative stress Special and very sincere thanks to Zuzana Holubcova
and DNA methylation) for help in obtaining images for the cover, to Alejan-
Melina Schuh and Bianka Seres (meiosis in human dra Elder Ontiveros for her help in creating new
oocytes, including figures; new oocyte images illustrations, and to Mike Macnamee and all of the
supplied by Julia Uraji) friends and colleagues at Bourn Hall Clinic who con-
tinue to offer endless encouragement and support.
Kathy Niakan and Helen O’Neill (genome editing)

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 Chapter
Review of Cell and Molecular Biology

1
Gametogenesis, embryo development, implantation is around 20 µm; the oocyte is the largest cell in the
and in-vitro culture involve numerous complex body, with a diameter of approximately 120 µm in its
pathways and interactions at the cellular and molecu- final stages of growth (Figure 1.1). The basic elements
lar level; a true understanding of their significance and organelles in an individual cell vary in distribu-
requires fundamental knowledge of the underlying tion and number according to the cell type. Bacterial
principles. This chapter therefore provides a con- cells differ from mammalian cells in that they have no
densed overview and review of basic terminology and distinct nucleus, mitochondria or endoplasmic reticu-
definitions, with particular emphasis on aspects rele- lum. Their cell membrane has numerous attachments,
vant to reproductive biology and in-vitro fertilization. and their ribosomes are scattered throughout the
cytoplasm.
Cell membranes are made up of a bimolecular
Mammalian Cell Biology layer of polar lipids, coated on both sides with protein
In 1839, two German scientists, Matthias Jakob films. Some proteins are buried in the matrix, others
Schleiden and Theodor Schwann, introduced the ‘cell float independently of each other in or on the mem-
theory,’ the proposal that all higher organisms are brane surface, forming a fluid mosaic of different
made up of a single fundamental unit as a building functional units that are highly selective and special-
block. In 1855, Rudolf Virchow extended this cell ized in different cells. Cells contain many different
theory with a suggestion that was highly controversial
at the time: ‘Omnis cellulae e celula’ (all living cells
arise from pre-existing cells). This statement has N
become known as the ‘biogenic law.’ The cell theory rER

is now accepted to include a number of principles: PB1 GJ FC

1. All known living things are made up of cells. MT

MV Ch
2. The cell is the structural and functional unit of all TZP
living things. SP
3. All cells come from pre-existing cells by division
G
(spontaneous generation does not occur).
4. Cells contain hereditary information that is
CG
transmitted from cell to cell during cell division. M
5. The chemical composition of all cells is basically
the same.
6. The energy flow (metabolism and biochemistry) PVS
ZP
of life occurs within cells.
Although these features are common to all cells, the Figure 1.1 Schematic diagram of oocyte ultrastructure showing
expression and repression of genes dictate individual the zona pellucida (ZP) and the perivitelline space (PVS), first polar
variation, resulting in a large number of different body (PB1), microvilli (MV), rough endoplasmic reticulum (rER),
chromosomes (Ch) on the spindle (SP), Golgi complex (G), cortical
types of variegated but highly organized cells, with granules (CG), two follicle cells (FC) attached to the oocyte and to
convoluted intracellular structures and intercon- each other via gap junctions (GJ). TZP = transzonal process, MT = 1
nected elements. The average size of a somatic cell microtubules, M = mitochondria, N = nucleus.

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 Chapter 1: Review of Cell and Molecular Biology

types of membrane, and each one encloses a space fibers in the spindle are contractile, and they pull
that defines an organelle, or a part of an organelle. the chromosomes apart during cell division.
The function of each organelle is determined largely The nucleus of each cell is surrounded by a layered
by the types of protein in the membranes and the membrane, with a thickness of 7.5 nm. The outer layer
contents of the enclosed space. Membranes are of this membrane is connected to the ER, and the outer
important in the control of selective permeability, and inner layers are connected by ‘press studs,’ creating
active and passive transport of ions and nutrients, pores in the nuclear membrane that allow the passage
contractile properties of the cell, and recognition of/ of ions, RNA and other macromolecules between the
association with other cells. nucleus and the cell cytoplasm. These pores have an
Cellular membranes always arise from pre- active role in the regulation of DNA synthesis, since
existing membranes, and the process of assembling they control the passage of DNA precursors and thus
new membranes is carried out by the endoplasmic allow only a single duplication of the pre-existing DNA
reticulum (ER, see below). The synthesis and metab- during each cell cycle. The inner surface of the mem-
olism of fatty acids and cholesterol is important in brane has nuclear lamina, a regular network of three
membrane composition, and fatty acid oxidation proteins that separate the membrane from peripheral
(e.g., by the action of reactive oxygen species, ROS) chromatin. DNA is distributed throughout the nucleo-
can cause the membranes to lose their fluidity, as well plasm wound around spherical clusters of histones to
as have an effect on transport mechanisms. form nucleosomes, which are strung along the DNA
Microvilli are extensions of the plasma membrane like beads. These are then further aggregated into
that increase the cell surface area; they are abundant in the chromatin fibers of approximately 30 nm diameter.
cells with a highly absorptive capacity, such as the The nucleosomes are supercoiled within the fibers in a
brush border of the intestinal lumen. Microvilli are cylindrical or solenoidal structure to form chromatin,
present on the surface of oocytes, zygotes and early and the nuclear lamina provide anchoring points for
cleavage stage embryos in many species, and in some chromosomes during interphase (Figure 1.2):
species (but not humans) their distribution is thought

Active chromatin = euchromatin – less condensed
to be important in determining the site of sperm entry.

Inactive (turned off ) = heterochromatin – more
Cell cytoplasm is a fluid space, containing water,
condensed
ions, enzymes, nutrients and macromolecules; the
cytoplasm is permeated by the cell’s architectural
Before and during cell division, chromatin
becomes organized into chromosomes.
support, the cytoskeleton.
Microtubules are hollow polymer tubes made up Three types of cell lose their nuclei as part of normal
of alpha–beta dimers of the protein tubulin. They are differentiation, and their nuclear contents are broken
part of the cytoskeletal structure and are involved in down and recycled:
intracellular transport, for example, the movement of

Red blood cells (RBCs)
mitochondria. Specialized structures such as centri-

Squamous epithelial cells
oles, basal bodies, cilia and flagella are made up of
microtubules. During prophase of mitosis or meiosis,
Platelets.
microtubules form the spindle for chromosome Other cells may be multinuclear: syncytia in muscle
attachment and movement. and giant cells (macrophages), syncytiotrophoblast.
Microfilaments are threads of actin protein, usu- Nuclear RNA is concentrated in nucleoli, which
ally found in bundles just beneath the cell surface; form dense, spherical particles within the nucleoplasm
they play a role in cell motility, ionic regulation and in (Figure 1.3); these are the sites where ribosome sub-
endo- and exocytosis. units, ribosomal RNA and transfer RNA are manufac-
Centrioles are a pair of hollow tubes at right tured. RNA polymerase I rapidly transcribes the genes
angles to each other, just outside the nucleus. These for ribosomal RNA from large loops of DNA, and the
structures organize the nuclear spindle in preparation product is packed in situ with ribosomal proteins to
for the separation of chromatids during nuclear generate new ribosomes (RNP: ribonucleoprotein
division. When the cell is about to divide by mitosis, particles).
2 one of the centrioles migrates to the antipode of the Mitochondria are the site of aerobic respiration.
nucleus so that one lies at each end. The microtubule Each cell contains 40–1000 mitochondria, and they

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 Chapter 1: Review of Cell and Molecular Biology

2 nm

11 nm

30 nm

300 nm Figure 1.3 Human oocyte at germinal vesicle stage, showing

prominent nucleolus.

oxidation, peroxidation, electron transport chains

and oxidative phosphorylation.
They also act as a Ca2+ store and are important
700 nm in calcium regulation. Mitochondria contain their
own double-stranded DNA that can replicate inde-
pendently of the cell, but the information for their
assembly is coded for by nuclear genes that direct
Centromere
the synthesis of mitochondrial constituents in the
cytoplasm. These are transported into the mitochon-
1400 nm dria for integration into its structures.
A number of rare diseases are caused by mutations
in mitochondrial DNA, and the tissues primarily
Sister chromatids
affected are those that most rely on respiration, i.e.,
Figure 1.2 Levels of chromatin packaging. From the top: DNA the brain and nervous system, muscles, kidneys and
double helix, nucleosome ‘beads on a string,’ chromatin fiber of
packed nucleosomes, section of extended chromosome, condensed the liver. All the mitochondria in the developing
chromosome and finally the entire chromosome. human embryo come from the oocyte, and therefore
all mitochondrial diseases are maternally inherited,
are most abundant in cells that are physically and transmitted exclusively from mother to child. In the
metabolically active. They are elliptical, 0.5–1 µm in sperm, mitochondria are located in the midpiece, pro-
size, with a smooth outer membrane, an intermem- viding the metabolic energy required for motility;
branous space, and a highly organized inner mem- there are no mitochondria in the sperm head.
brane that forms cristae (crests) with elementary
Oocytes contain 100 000–1 000 000 mitochondria.
particles attached to them, ‘F1-F0 lollipops,’ which
Sperm contain 70–100 mitochondria, in the
act as molecular dynamos. The cristae are packed with midpiece of each sperm. These are incorporated
proteins, some in large complexes: the more active the into the oocyte cytoplasm, but do not contribute
tissue, the more cristae in the mitochondria. Cristae to the zygote mitochondrial population – they are
are the site of intracellular energy production and eliminated at the four- to eight-cell stage.
transduction, via the Krebs (TCA) cycle, as well as
All of the mitochondria of an individual are 3
processes of oxidation, dehydrogenation, fatty acid descendants of the mitochondria of the zygote,

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 Chapter 1: Review of Cell and Molecular Biology

which contains mainly oocyte mitochondria, The endoplasmic reticulum (ER) is an intercon-
i.e. mitochondria are maternal in origin. nected lipoprotein membrane network of tubules,
: Paternal mtDNA has been identified in a few vesicles and flattened sacs that extends from the
exceptional cases, transmitted with an unusual nuclear membrane outwards to the plasma mem-
autosomal dominant-like inheritance (Luo brane, held together by the cytoskeleton. The ER itself
et al., 2018). is a membrane-enclosed organelle that carries out
complex biosynthetic processes, producing proteins,
lipids and polysaccharides. As new lipids and proteins
The Human Mitochondrial Genome are made, they are inserted into the existing ER mem-
The sequence of human mitochondrial DNA was brane and the space enclosed by it. Smooth ER (sER)
published by Fred Sanger in 1981, who shared the is involved in metabolic processes, including synthesis
1980 Nobel Prize in Chemistry with Paul Berg and and metabolism of lipids, steroids and carbohydrates,
Walter Gilbert, ‘for their contributions concerning the
as well as regulation of calcium levels. The surface of
determination of base sequences in nucleic acids.’
The mitochondrial genome has:
rough ER (rER) is studded with ribosomes, the units
of protein synthesis machinery. Membrane-bound

Small double-stranded circular DNA molecule
vesicles shuttle proteins between the rER and the
(mtDNA) 16 568 bp in length.
Golgi apparatus, another part of the membrane

37 genes that code for:
system. The Golgi apparatus is important in modify-
2 ribosomal RNAs ing, sorting and packaging macromolecules for secre-
22–23 tRNAs tion from the cell; it is also involved in transporting
10–13 proteins associated with the inner lipids around the cell, and in making lysosomes.
mitochondrial membrane, involved in energy
production.
Other mitochondrial proteins are encoded by
Rough Endoplasmic Reticulum (rER)
nuclear DNA and specifically transported to the
Has attached 80S ribonucleoprotein particles, the
mitochondria. ribosomes (bacterial ribosomes are 70S), which
Mitochondrial DNA is much less tightly packed are made in the nucleus and then travel out to the
and protected than nuclear DNA and is therefore cytoplasm through nuclear pores.
more susceptible to ROS damage that can cause
Ribosomes are composed of two subunits: 40 s
mutations. and 60 s (bacteria: 30S and 50S); the association
As it is inherited only through the maternal line, between the subunits is controlled by Mg2+
mutations can be clearly followed through gener-
concentration.
ations and are used as ‘markers’ in forensic science
and archaeology, as well as in tracking different
Polysomes are several ribosomes which move
human populations and ethnic groups. along a single strand of mRNA creating several
copies of the same protein.
Mitochondria can be seen in different distribu-
tions during early development (Figure 1.4); they Smooth Endoplasmic Reticulum (sER)
do not begin to replicate until the blastocyst stage,
A series of flattened sacs and sheets, site of lipid
and therefore an adequate store of active mitochon- and steroid synthesis.
dria in the mature oocyte is a prerequisite for early
Cells that make large amounts of steroids have
development. extensive sER.

Germinal vesicle oocyte: homogeneous clusters The Golgi apparatus was first observed by Camillo
associated with ER Golgi in 1898, using a novel silver staining technique

Metaphase I oocyte: polarized toward the spindle to observe cellular structures under the light micro-

Metaphase II oocyte: perinuclear ring and scope; he was awarded the 1906 Nobel Prize in Physi-
polar body ology or Medicine for his studies on the structure of

Embryos at 1c, 2c, 4c stages: perinuclear ring the nervous system. The Golgi apparatus consists of a
4
Cytoplasmic fragments in cleavage stage embryos fine, compact network of tubules near the cell nucleus,
contain large amounts of active mitochondria a collection of closely associated compartments with

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 Chapter 1: Review of Cell and Molecular Biology

Figure 1.4 Mitochondrial aggregation patterns in a

germinal vesicle (GV) oocyte (top), an MI oocyte (center)
and an MII oocyte (bottom). Frames to the left are in
fluorescence using the potential sensitive dye JC-1 to
show the mitochondria; frames on the right are
transmitted light images. The two mitochondrial patterns,
A (granular-clumped) and B (smooth), are shown. From
Wilding et al., 2001. See color plate section.

stacked arrays of smooth sacs and variable numbers of Transport occurs via vesicles, which bud from one
cisternae, vesicles or vacuoles. It is connected to rER, compartment and fuse with the next. The Golgi
linked to vacuoles that can develop into secretory apparatus will move to different parts of the cell
granules, which contain and store the proteins pro- according to the ongoing metabolic processes at
duced by the rER. All of the proteins exported from the time; it is very well developed in secretory cells
the ER are funneled through the Golgi apparatus, and (e.g., in the pancreas).
every protein passes in a strict sequence through each The Golgi apparatus also makes lysosomes,
of the compartments (cis, medial, trans). This process which contain hydrolytic enzymes that digest worn-
consists of three stages: out organelles and foreign particles, acting as ‘rub-
1. ‘Misdirected mail’ – sends back misdirected bish bins’ and providing a recycling apparatus for
proteins (cis). intracellular digestion; they contain at least 50 differ-
2. ‘Addressing’ – (medial) stacks of cisternae that ent enzymes, and ‘leaky’ lysosomes can damage and
modify lipid and sugar moieties, giving them ‘tags’ kill cells. Macromolecules inside the cell are trans-
for subsequent sorting. ported to lysosomes, those from outside the cell
3. ‘Sorting and delivering’ (trans): proteins and lipids reach them by pinocytosis or phagocytosis; phago-
are identified, sorted and sent to their proper cytosis only occurs in specialized cells (e.g., white
blood cells). 5
destination.

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 Chapter 1: Review of Cell and Molecular Biology

Peroxisomes are microbody vesicles that contain DNA and RNA

oxidative enzymes such as catalase; they dispose of toxic DNA RNA
hydrogen peroxide and are important in cell aging. Purines Adenine (A) Adenine (A)
(double ring) Guanine (G) Guanine (G)
Fundamental Principles of Molecular Pyrimidines Cytosine (C) Cytosine (C)
Biology (single ring) Thymine Uracil (U)
(T = methylated U)
Nucleic Acids
The nucleic acids, DNA (deoxyribose nucleic acid;
Figure 1.5) and RNA (ribose nucleic acid), are made
up of:
Methylation of cytosine is important in gene silen-
1. Nucleotides: organic compounds containing a
cing and imprinting processes.
nitrogenous base
Nucleotides also function as important cofactors
2. Sugar: deoxyribose in DNA, ribose in RNA
in cell signaling and metabolism: coenzyme A (CoA),
3. Phosphate group. flavin adenine dinucleotide (FAD), flavin mononu-
Nucleotides are purines and pyrimidines, determined cleotide, adenosine triphosphate (ATP), nicotinamide
by the structure of the nitrogenous base. adenine dinucleotide phosphate (NADP).
Nucleotides Deoxyribonucleic Acid (DNA)

P
G Guanine C Cytosine P A T

NH2 S
O
S

N
HN N P
G C

N S
H2N N N O S
H
H
P P
T A
A Adenine T Thymine
S
S
NH2 O
P P
N H3C A T
N NH
S
S
N N O
N
H P
P
H C G
S
S

P
Figure 1.5 Structure of DNA. Complementary base pairs form the DNA double helix; two hydrogen bonds form between A and T,
three hydrogen bonds form between G and C. The two polynucleotide chains must be antiparallel to each other to allow pairing. S= sugar,
P= phosphate group.
6

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 Chapter 1: Review of Cell and Molecular Biology

DNA Methylation of protamines and histones is a

Double-stranded helix with paired bases to form crucial component of imprinting processes: an asso-
complementary strands. ciation has been found between Beckwith–

G=C or A=T. Wiedemann syndrome and epigenetic alterations of
LitI and H19 during in-vitro fertilization (DeBaum

Pentose deoxyribose – phosphate backbone.
et al., 2003).

Stabilized by H bonds between purines and
Each mammalian cell contains around 1.8 m of
pyrimidines, on the inside of the helix.
DNA, of which only 10% is converted into specific

Each pitch of the double helix has 10 base pairs.
proteins; the noncoding part of the DNA still carries
genetic information and probably functions in regu-
DNA Replication latory control mechanisms.
DNA copies itself by semi-conservative replication:
each strand acts as a template for synthesis of a
complementary strand. The Genetic Code: The Biochemical Basis
1. Free nucleotides are made in the cytoplasm and of Heredity
are present in the nucleoplasm before replication In 1968, the Nobel Prize in Physiology or Medicine
begins. was awarded to Robert Holland, Ghobind
2. The double helix unwinds and hydrogen bonds, Khorana and Marshall Nirenberg ‘for their interpret-
holding the two DNA strands together, break. This ation of the genetic code and its function in protein
leaves unpaired bases exposed on each strand. synthesis’:
3. The sequence of unpaired bases serves as a
DNA transfers information to mRNA in the form
template on which to arrange the free nucleotides of a code defined by a sequence of
from the nucleoplasm. nucleotide bases.

The code is triplet, unpunctuated and
4. DNA polymerase moves along the unwound parts of
nonoverlapping.
the DNA, pairing complementary nucleotides from
Three bases are required to specify each amino
the nucleoplasm with each exposed base. acid, there are no gaps between codons and
5. The same enzyme connects the nucleotides codons do not overlap.
together to form a new strand of DNA, hydrogen
Since RNA is made up of four types of
bonded to the old strand: nucleotides (A, C, G, U), the number of
triplet sequences (codons) that are possible is

DNA polymerase forms new hydrogen bonds
4  4  4 = 64; three of these are ‘stop codons’
on the 50 30 strand. that signal the termination of a

DNA ligase acts on the 30 50 strand. polypeptide chain.

Several replication points appear along the
The remaining 61 codons can specify 20 different
strand, which eventually join. amino acids, and more than one codon can
specify the same amino acid. (Only methionine
6. DNA is then mounted on ‘scaffolding proteins,’
[Met] and tryptophan [Trp] are specified by a
histones – and this is then wrapped around non-
single codon).
histones to form chromatin. Histones are basic
Since the genetic code thus has more
proteins that bind to nuclear DNA and package information than it needs, it is said to be
it into nucleosomes; the regulation of gene ‘degenerate.’
expression involves histone acetylation and
A mutation in a single base can alter the coding
deacetylation. There are two ATP-dependent for an amino acid, resulting in an error in protein
remodeling complexes and acetyltransferases that synthesis: translated RNA will incorporate a
preferentially bind activated states and fix different amino acid into the protein, which may
chromatin configurations: then be defective in function. (Sickle cell anemia
and phenylketonuria are examples of single-gene

Histone acetyltransferase coactivator complex defects.)

Histone deacetylase corepressor complex.

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