mitochondrial distribution in mammalian cells jiang lei
TRANSCRIPT
MITOCHONDRIAL DISTRIBUTION IN MAMMALIAN CELLS
Thesis
Submitted to
The College of Arts and Sciences of the
UNIVERSITY OF DAYTON
In Partial Fulfillment of the Requirements for
The Degree
Master of Science in Biology
by
Lei Jiang
UNIVERSITY OF DAYTON
Dayton, Ohio
December, 2009
MITOCHONDRIAL DISTRIBUTION IN MAMMALIAN CELLS
APPROVED BY:
Dr. Shirley Wright, Thesis Advisor
Dr. Mark Nielsen, Committee Member
Dr. Carl Friese, Committee Member
Dr. Jayne Robinson, Department Chair
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ABSTRACT
MITOCHONDRIAL DISTRIBUTION IN MAMMALIAN CELLS
Name: Jiang, Lei University of Dayton
Advisor: Dr. Shirley Wright
The mitochondrion is an important and essential cell organelle which
provides about 90% energy for the organism and plays a number of important
roles in cell functions. Recent research found that mitochondrial distribution in
somatic cells is homogenously scattered throughout the cytoplasm, while the
distribution in some stem cells and mature oocytes is perinuclear. These
findings suggested that the spatial distribution of mitochondria may be related to
their normal functions. In addition, Bavister has hypothesized that this periuclear
localization of mitochondria may indicate the pluripotency of stem cells. But
mitochondrial distribution has not been examined in embryonic stem cells to date.
My thesis investigates the distribution of mitochondria during development in
mammalian cells and proposes experiments to determine and compare the spatial
distribution of mitochondria in embryonic stem cells and differentiated cells using
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fluorescence staining of MitoTracker Green to test Bavister’s hypothesis. In
addition, research suggested that mitochondrial distribution is mediated by the
cytoskeleton in higher eukaryotes. Therefore, an additional goal of my research
was to address the effects of cytoskeletal disruptors on mitochondrial
arrangement.
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ACKNOWLEDGMENTS
I would like to thank the faculty, staff and my fellow graduate students of
the University of Dayton Department of Biology for being so supportive of me.
I would also like to thank my committee members, Dr. Friese and Dr.
Nielsen for their guidance and support.
I would like to express my appreciation to everyone who has helped me
with this work. This includes Dr. Krane and Venky, who kindly provided the
MLE-15 cells, and Matt and Tracy for their help with microscopy.
I would especially like to thank my advisor, Dr. Shirley Wright, for providing
the time, support and guidance for the work, and always being so patient and
encourage me all the time. I really learned so much from her besides science.
Finally, I would like to thank my family. I am so blessed to have you there
for me and love me and support me unconditionally.
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TABLE OF CONTENTS
ABSTRACT........................................................................................ iii
ACKNOWLEDGMENTS ..................................................................... v
TABLE OF CONTENTS..................................................................... vi
LIST OF ILLUSTRATIONS ................................................................ ix
LIST OF ABBREVIATIONS ................................................................ x
CHAPTER
I. INTRODUCTION...........................................................................1
A. Focus of Thesis....................................................................................... 2
B. Significance ............................................................................................ 4
II. INTRODUCTION TO GAMETOGENESIS
AND DEVELOPMENT ..................................................................6
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A. Spermatogenesis .................................................................................... 7
B. Oogenesis ............................................................................................... 8
C. Fertilization ............................................................................................. 9
D. Preimplantation Development............................................................... 12
E. Implantation .......................................................................................... 15
III. THE MITOCHONDRION AND ITS FUNCTIONS........................18
A. Structure ............................................................................................... 19
B. Origin .................................................................................................... 21
C. Biogenesis ............................................................................................ 22
D. Inheritance............................................................................................ 23
E. Biochemistry ......................................................................................... 23
F. Mitochondrial Polarity............................................................................ 25
G. Function................................................................................................ 26
H. Mitochondrial-Related Diseases and Disorders.................................... 27
I. Role in Gametogenesis and Development ........................................... 31
IV. MITOCHONDRIAL DISTRIBUTION ............................................38
A. Role of the Cytoskeleton....................................................................... 39
B. Distribution in Somatic Cells ................................................................. 40
C. Distribution during Oogenesis, Fertilization and Preimplantation
Development ......................................................................................... 41
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V. MITOCHONDRIAL DISTRIBUTION IN EMBRYONIC
STEM CELLS..............................................................................47
A. Embryonic Stem Cells........................................................................... 48
B. Bavister’s Hypothesis............................................................................ 49
C. Possible Reasons for Perinuclear Mitochondria in Stem Cells ............. 51
D. Hypothetical Experiments Designed to Test Bavister’s Hypothesis ...... 53
i. Research Objectives ......................................................................... 53
ii. Materials ........................................................................................... 53
iii. Research Method.............................................................................. 54
iv. Expected Results .............................................................................. 56
VI. CONCLUSIONS AND FUTURE DIRECTIONS ..........................60
A. Conclusions .......................................................................................... 61
B. Future Directions................................................................................... 62
LITERATURE CITED........................................................................64
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LIST OF ILLUSTRATIONS
Figure 1. From human oogenesis to implantation. ........................................... 16
Figure 2. Mammalian blastocyst. . ................................................................. 17
Figure 3. Schematic diagram of mitochondrial structure. ................................. 36
Figure 4. Schematic diagram of bioenergetic pathways in mitochondria. ........ 37
Figure 5. Diagram showing location of mitochondria in a typical somatic cell .. 45
Figure 6. Distribution of mitochondria during oogenesis and fertilization in
mammalian oocytes............................................................................................ 46
Figure 7. Preparation of cultured embryoinc stem (ES) cells. .......................... 57
Figure 8. Measurement of mitotracker fluorescence intensity.. ........................ 58
Figure 9. Mitochondrial distribution in mouse lung epithelial (MLE-15) cells. ... 59
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LIST OF ABBREVIATIONS
ATP: adenosine-5'-triphosphate
ATSC: adult rhesus macaque stromal cell line
BSA: bovine serum albumen
DAPI: 4',6-diamidino-2-phenylindole
DMEM: Dulbecco’s modified eagle medium
ER: endoplasmic reticulum
ES cells: Embryonic stem cells
FBS: fetal bovine serum albumen
GSH: glutathione
GV: germinal vesicle
GVBD: germinal vesicle breakdown
ICM: inner cell mass
IVF: in vitro fertilization
iPSCs: induced pluripotent stem cells
LIF: Leukemia inhibitory factor
MES: mouse embryonic stem cells
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Met I: metaphase I
Met II: metaphase II
MFs: microfilaments
MLE-15: mouse lung epithelial cells
MPF: mitosis-promoting factor or maturation-promoting factor
mtDNA: mitochondrial genome or DNA
MTs: microtubules
NEAA: non essential amino acids
NFAT: nuclear factor of activated T cells
NO: nitric oxide
OXPHOS: oxidative phosphorylation
PBS: phosphate-buffered saline
ROS: reactive oxygen species
TNF: tumor necrosis factor
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CHAPTER I
INTRODUCTION
1
A. Focus of Thesis
Mitochondria are known as “the power house of the cell,” because they
provide energy for the cell. Mitochondria generate adenosine-5'-triphosphate
(ATP) from carbohydrates, proteins and fats that are obtained by organisms in
their diet to provide energy for biological activities. Normal mitochondrial
distribution is critical for several cell functions including intracellular calcium
homeostasis, cell signaling and apoptosis (Hales et al., 2004; Katayama et al.,
2006; Cerveny et al., 2007).
As “the power house for cells,” mitochondria play critical roles during
gametogenesis, fertilization and embryonic development (Smith et al., 2005;
Katayama et al., 2006; Dumollard et al., 2009; Van Blerkom et al., 2009).
Recently, researchers found there were translocations of mitochondria during
early embryonic developmental stages, which implicate mitochondrial
distribution, in playing an important role during oogenesis and early embryonic
development (Barnett et al., 1996; Sun et al., 2001; Katayama et al., 2006;
Wang et al., 2009). These data lead to the questions: Is the spatial
distribution of mitochondria important for mitochondrial function? What role
does mitochondrial spatial distribution play in gametogenesis and
embryogenesis? My research will review the current literature to address
these questions. Data on mitochondrial distribution during gametogenesis
and development will be collected and analyzed.
In 2006, Bavister et al. hypothesized that mitochondrial distribution is
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different in stem cells and somatic cells, and that perinuclear localization of
mitochondria may be and indicator for “stemness.” Therefore, my research
objectives are to determine and compare the spatial distribution of
mitochondria in an undifferentiated cell type such as mouse embryonic stem
cells (MES cells), and a differentiated cell type such as mouse lung epithelial
cells (MLE-15). In addition, research suggests in most higher eukaryotes that
mitochondrial distribution is mediated by the cytoskeleton (Haggeness et al.,
1978). Interestingly, the cytoskeleton influences mitochondrial activity
including mitochondrial respiratory activity, fusion and fission (Boldogh and
Pon, 2007). While studies have focused on mitochondrial-cytoskeletal
interactions, less focus has been on their role in differentiation and
development of disease. Therefore, an additional goal of my research is to
review the literature to examine the effects of cytoskeletal disruptors on
mitochondrial arrangement. This study will help elucidate the behaviors of
mitochondrial distribution during differentiation; and demonstrate the impact of
the cytoskeleton on mitochondrial distribution.
My thesis investigates the distribution of mitochondria during
development in mammalian cells. To provide background information for my
thesis so that the mitochondrial distribution during stages of gametogenesis,
fertilization and preimplantation development could be understood better, first
a literature review of gametogenesis and development will be presented in
Chapter II. Next a summary of the mitochondrion including its structure,
biochemistry and function will be discussed in Chapter III. Chapter IV
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addresses how the distribution of mitochondria changes during oogenesis and
development. Chapter V discusses Dr. Barry Bavister’s hypothesis that
mitochondrial distribution may be an indicator of "stemness," and proposes
experiments that could be used to address this hypothesis. Lastly, Chapter VI
addresses conclusions and future directions.
B. Significance
Embryonic stem cells (ES cells) are unique cell populations with the
ability to undergo both self-renewal and differentiation (Odorico et al., 2001).
They have the potential to be used to treat a variety of diseases (Levy et al.,
2004; Faulkner and Keirstead, 2005). Although mitochondria play important
roles in normal cell function, their distribution, interaction with the cytoskeleton
as well as their role during differentiation have not been examined in ES cells.
Therefore, my research to understand the spatial distribution of mitochondria in
gametes, undifferentiated and differentiated cells may reveal specific
differences among these cell types that will allow us a better understanding in
stem cell biology, and may provide novel targets for stem cell therapy.
Besides addressing the distribution of mitochondria during differentiation, this
study will also demonstrate the impact of the cytoskeleton on mitochondrial
spatial arrangement. This study may also lead to a better understanding of
mitochondria in development, so that a suitable therapy for
mitochondria-related disorders can be developed. Finally yet importantly, this
study tests Bavister’s hypothesis that the arrangement of mitochondria
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changes after fertilization to meet the energy needs of cells as development
progresses (Lonergan et al., 2007). If Bavister’s hypothesis holds true,
mitochondrial distribution can be an exciting, brand new indicator for
“stemness” of cells.
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CHAPTER II
INTRODUCTION TO GAMETOGENESIS AND
DEVELOPMENT
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Gametogenesis is the process in which primordial germ cells undergo
meiosis and differentiate to become mature gametes. Spermatogenesis is
the formation of the male gamete, the sperm. Oogenesis is the formation of
the female gamete, the ovum or egg cell (Gilbert, 2006). Both of these
processes will be described in detail below.
A. Spermatogenesis
In mammals, sperm develop within seminiferous tubules in the testes
and are stored in the epididymis (Heller and Clermont, 1963). Several steps
occur during spermatogenesis (Gilbert, 2006). First, diploid male germ cells
called spermatogonia (a type of stem cell) undergo mitosis during
spermatocytogenesis to become primary spermatocytes. Then each primary
spermatocyte undergoes two rounds of meiosis during spermatidogenesis to
form four spermatids. After that, the spermatids undergo spermiogenesis.
During this process, each spermatid produces a flagellum, condenses the
nucleus, develops a mid-piece, and differentiates into a mature sperm.
Mitochondria translocate during spermatogenesis. In germ cells and
spermatocytes of mammals, mitochondria are scattered all through out the
cytoplasm. However, along with formation of the flagellum, the mitochondria
begin to accumulate around the flagellum in the mid-piece of the sperm.
Finally, a mature sperm has formed with a flagellum that has grown from the
centriole pair in the neck, a mid-piece containing mitochondria that form a ring
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around the base of the axoneme, and a head containing a highly condensed
nucleus with an acrosome at the anterior end. The mitochondria located in
the mid-piece of the sperm provide ATP needed to whip the flagellum and
propel the sperm (Gilbert, 2006).
During spermiogenesis in Drosophila, mitochondria in early spermatids
aggregate and fuse into two large organelles that wrap around each other to
form a giant elaborate structure called the Nebenkern (Benard and Karbowski,
2009). The protein mitofusin (Fzo1) is required for mitochondrial fusion
during Nebenkern formation. Mutations in the fzo gene prevent mitochondrial
fusion during Nebenkern formation and the mutant lies are sterile.
B. Oogenesis
In mammals, oocytes develop within follicles in the ovary (Fig. 1; Eppig
and O’Brien, 1996). Primary oocytes are formed before birth in the fetus by
mitosis of primordial germ cells called oogonia (Gilbert, 2006). Each diploid
primary oocyte is contained in the primary follicle and arrests in prophase I of
meiosis. During this period, the primary follicle enlarges due to synthesis of
RNA and organelles for later oocyte growth, fertilization and preimplantation
development. The large nucleus of the primary oocyte is known as the
germinal vesicle (GV). When the follicle cells grow and divide to from a larger
follicle, the primary oocyte also grows larger. Then germinal vesicle
breakdown (GVBD) occurs during first meiosis (Meiosis I). The primary
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oocyte divides into a haploid secondary oocyte and a haploid first polar body,
which are both contained within a mature follicle. During ovulation, the
secondary oocyte is released from the ovary and enters the oviduct (Fig. 1).
The secondary oocyte is arrested in metaphase II (Met II) until fertilization.
After fertilization, the secondary oocyte undergoes a second meiotic division
(Meiosis II) to create an ovum and a second polar body (Fig. 1).
Meiotic competence is the oocytes’ ability to complete meiosis (Motlik et
al., 1984). It will determine whether oocytes can mature. Meiotic
competence may depend on the spatial arrangement of mitochondria.
Abnormal distribution of mitochondria was found to lead to arrest of oocyte
development (Wang et al., 2009). Therefore, proper mitochondrial
distribution during oocyte maturation is necessary for further development and
is a determinant for oocyte quality (Wang et al., 2009). The first and second
polar bodies disintegrate later to discard the extra chromosomes. As in
spermatogenesis, both processes of mitosis of primordial germ cells and
follicular cells, and meiosis of oocytes require ATP. Besides that,
mitochondria also provide ATP for glutathione (GSH) production, which helps
detoxify the cell during oocyte maturation (Dumollard et al., 2009).
C. Fertilization
Fertilization is the fusion of haploid gametes to reconstitute a diploid cell
with the potential to become a new individual (Gilbert, 2006). Fertilization is
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not a moment or an event, but a series of events from contact of gametes to
the activation of development (Gilbert, 2006). When the oocytes are mature,
they will be ovulated from the ovary and enter the oviduct (Fig. 1). The sperm
also travel from the vagina to the oviduct to meet the oocytes. During this trek,
capacitation of sperm happens so the sperm gain the capacity to fertilize the
egg. The major driving force for the trip of sperm from the vagina to the
oviduct is the muscular activity of the uterus (Gilbert, 2006). Sperm motility is
important once sperm arrive within the oviduct (Gilbert, 2006). ATP provided
by mitochondria are critical for sperm motility. Recent research found that
mtDNA mutation can lead to impaired spermatogenesis and impaired sperm
motility (Shamsi et al., 2008).
Fertilization occurs in the upper third of the oviduct (Fig. 1). The
fertilized egg cell is called a zygote (Gilbert, 2006). Fertilization begins from
the contact of sperm and egg. Sperm have a cap-like structure at their
anterior part of their head called the acrosome. Once the sperm reaches the
zona pellucida an extracellular matrix which surrounds the oocyte, it will trigger
the acrosome reaction. Acrosomal enzymes like acrosin and hyaluronidase
are released to digest the zona pellucida so that the sperm can penetrate the
zona pellucida. After that, part of the sperm’s plasma membrane (at the
equatorial segment) fuses with the oocyte’s plasma membrane, and the
contents of the sperm including the sperm nucleus and sperm mitochondria
are delivered into the ooplasm. An exception is the Chinese hamster: the
tail and mid-piece of the sperm remain outside the oocyte after fertilization
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(Ankel-Simons and Cummins, 1996). In most species, the nuclei in mature
sperm are highly condensed and genetically inactive. During fertilization, the
sperm nucleus decondenses and is reactivated (Wright, 1999). After
fertilization, the activated sperm nucleus undergoes a series of morphological
and biochemical transformations and becomes the male pronucleus, while the
oocyte chromosomes develop into the female pronucleus These
transformations including decondensation of the sperm nucleus and assembly
of the pronuclear envelope are ATP-dependent (Raskin et al., 1997; Collas and
Poccia, 1998). Once decondensed, the sperm DNA can begin transcription
and replication immediately (Gilbert, 2006). By 15 hours after fertilization,
the two pronuclei migrate together; the pronuclear envelopes gradually
disappear and the chromosomes from the sperm and oocyte intermix (Gilbert,
2006). Pronuclear migration and apposition require participation of the
cytoskeleton including microtubules and actin (Maro, 1985; Branzini et al.,
2007).
This whole process of fertilization, from ovulation, the movement of the
sperm, the transformations of the sperm nucleus and pronuclear DNA
replication require a considerable amount of ATP, which is supplied by
mitochondria. It has not been determined whether sperm mitochondria
produce ATP when in the ooplasm. This is an issue because sperm
mitochondria are thought to degrade in the ooplasm, and thus do not
contribute their mtDNA to the embryo.
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There are a couple of cases of maternal inheritance of mtDNA found in
mammals (Hutchison et al., 1974). The typical mammalian sperm mid-piece
contains about 50-75 mitochondria with one copy of mtDNA in each. In
contrast, the oocyte contains around 10 to 10 mitochondria which exceed
that of sperm by a factor of at least 10 . Therefore, a simple explanation of
maternal inheritance is the paternal contribution of mtDNA is diluted by that of
the maternal (Ankel-Simons and Cummins, 1996).
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D. Preimplantation Development
After fertilization, the zygote enters preimplantation development which
includes formation of the zygote, cleavage, activation of the embryonic
genome, and the beginning of cellular differentiation (Kanka, 2003). This
stage takes 3 to 4 days in mice, 5 to 7 days in humans (Lanza, 2006). Upon
activation of mitosis-promoting factor (MPF), cleavage is initialized in the
zygote (Gilbert, 2006). The zygote undergoes first mitosis and divides into a
2-cell stage, 4-cell stage, 8-cell stage and 16-cell stage embryo which is called
morula (Fig. 1). The morula is a solid ball of cells. The embryos undergo
cleavage stages in the oviduct (Fig. 1). As the zygote divides, the cells
become smaller. The early embryo does not increase in size during these
first few cleavages. Therefore, the morula is the same size as the zygote
(Gilbert, 2006).
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An important event called compaction occurs at the 8-cell stage (Maro
et al., 1990; Gilbert, 2006). During compaction, cells increase their adhesion
by forming tight junctions, adherent junctions and gap junctions between the
individual blastomeres. As a result, the blastomeres flatten upon each other.
The cytoskeleton plays a role during compaction. Microfilaments facilitate
microtubule redistribution during compaction and the organization of the actin
also changes during preimplantation development (Albertini et al., 1987; Maro
et al., 1990). After compaction, the embryo emerges as the morula (Fig. 1).
The next stage is the blastula which in mammals is called a blastocyst
(Gilbert, 2006). During the blastocyst stage, a process named cavitation
happens, during which fluid is transferred across the outer blastomeres to form
a fluid filled cavity called the blastocoel. The blastocyst is the stage at which
differentiation of the embryo starts. Two cell types are present in the
blastocyst: inner cell mass (future embryo) and trophoblast (future placenta)
(Fig. 2).
Expression of several genes including Cdx2, eomesodermin, Oct4, and
Nanog leads to differentiation of the two cell types in the blastocyst (Gilbert,
2006). Oct4 stimulates the morula cells to become inner cell mass and not
trophoblast, while Nanog works at the next differentiation event, promoting
the ICM cells to become the pluripotent embryonic epiblast and preventing
the ICM cells from differentiating into hypoblast (Gilbert, 2006). In addition,
Oct4, Nanog and Sox2 are key factors in maintaining pluripotency in ES cells
(Pan and Thomson, 2007). Oct4 expression is initiated at the late 4-cell
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stage and becomes restricted to the inner cell mass (ICM) of the blastocyst.
In contrast, Nanog transcripts are first detected in the compacted morula and
become restricted to the ICM. Homozygous Nanog mutant embryos give
rise to an ICM, but they fail to maintain pluritpotency in the cells of the epiblast
which instead differentiate into primitive endoderm cells causing embryo
death (Facucho-Oliveria and St. John, 2009). These data suggest that
Nanog cooperates with Oct4 and Sox2 during late preimplantation and early
post-implantation embryogenesis to maintain the pluripotency of the epiblast
cells (Facucho-Oliveria and St. John, 2009).
Cdx2 and eomesodermin are involved in trophoblast formation (Gilbert,
2006). At the 8-cell stage, Oct4, eomesodermin and Cdx2 are expressed in
each blastomere. At the blastocyst stage, Cdx2 and eomesodermin
expression is restricted to the trophoblast cells. Eomesodermin stimulates
expression of genes that form trophoblast (Gilbert, 2006). Cdx2 blocks Oct4
and Nanog expression in the trophoblast, thereby maintaining the trophoblast
cells.
Developmental competence is the capacity of the zygote to undergo
normal development. The activation of the embryonic genome called zygotic
gene activation is an important event that must occur following fertilization.
Zygotic gene activation occurs at different developmental stages in various
species (Schultz et al., 1995). For example, it occurs at the 2-cell stage in the
mouse and 4-cell stage in the human (Gilbert, 2006). Microarray analysis
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identified 4,562 genes that were differentially expressed in the preimplantation
embryo, such as Cdk2, Cap1 and Ube2j2 (Jeong et al., 2006).
Developmental competence depends on a normal mitochondrial
distribution. Research studies with mice have found a slight but significant
decrease in ATP content during development from the 1-cell or 2-cell stage to
4-cell and 8-cell embryos. The ATP content remained constant in morulae
and early blastocysts, but was reduced significantly in late blastocysts just
before implantation (Spielmann et al., 1984). These results suggest a need
for ATP during preimplantation development, which is not surprising to see
since mitochondria provide energy for nearly all cell activities in eukaryotes
(Smith et al., 2005).
E. Implantation
After about 4-5 days of development, the embryo emerges from the
oviduct at the blastocyst stage (Fig. 1). After the blastocyst hatches from the
zona pellucida, it is ready to implant in the endometrium of the uterus (Fig. 1;
Gilbert, 2006). Gastrulation occurs during later stages of implantation.
During gastrulation, the three embryonic germ layers are formed by
differentiation of the ICM and cell migration. Implantation and gastrulation
need actively functioning mitochondria since they require a lot of energy in the
form of ATP.
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Figure 1. From human oogenesis to implantation. In mammals, oocytes
develop within follicles in the ovary. When the oocytes are mature, they will
be ovulated from the ovary and enter the oviduct. During ovulation, the
secondary oocyte is released from the ovary and enters the oviduct. The
secondary oocyte is arrested in metaphase II (Met II) until fertilization.
Fertilization occurs in the upper third of the oviduct. After fertilization, the
zygote enters preimplantation development. The zygote undergoes first
mitosis and divides into a 2-cell stage, 4-cell stage, 8-cell stage and 16-cell
stage embryo which is called morula. The next stage is the blastula which in
mammals is called a blastocyst. After about 4-5 days of development, the
embryo emerges from the oviduct at the blastocyst stage. The blastocyst is
now ready to implant in the endometrium of the uterus.
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Figure 2. Mammalian blastocyst. Two cell types are present in the
blastocyst: inner cell mass (future embryo) and trophoblast (future placenta).
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CHAPTER III
THE MITOCHONDRION AND ITS FUNCTIONS
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A. Structure
Mitochondria are important and essential organelles in eukaryotic cells.
There can be hundreds to thousands of mitochondria per cell (Freitas, 1999).
For example, there are 1000-2000 mitochondria per cell in liver cells (Alberts et
al., 1994). Some cells such as sea urchin sperm have a single mitochondrion
(Ardon et al., 2009). Mitochondria range from 1–10 micrometers in diameter
(Freitas, 1999; Campbell and Reece, 2005). They are surrounded by a
double-membrane system consisting of inner and outer membranes (Fig. 3).
These two membranes are composed of a phospholipid bilayer and proteins
similar to that of the eukaryotic plasma membrane (Alberts et al., 1994). The
outer membrane contains porin proteins which form channels that are
permeable to water-soluble molecules that are 5 kDa or less (Ha et al., 1993).
The inner membrane has a high protein content and contains the phospolipid,
cardiolipin, but lacks porins (Alberts et al., 1994; Cooper and Hausman, 2006).
The inner and outer membranes are separated by an intermembrane space.
It has a similar ionic concentration to the cytosol (Alberts et al., 1994). The
space enclosed by the inner membrane is called the matrix. The matrix
contains the mitochondrial genome (mtDNA) and enzymes responsible for
oxidative phosphorylation (OXPHOS). It is rich in protein. The inner
membrane is folded inward to the matrix cristae. This increases the surface
area of the inner mitochondrial membrane enhancing the function of this
organelle. The cristae are numerous and prominent when mitochondria are
active (Alberts et al., 1994).
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Mitochondria are unique among the cytoplasmic organelles because
they contain their own DNA (Cooper and Hausman, 2006). The fission and
fusion of mitochondria are under the control of both the nuclear genome and
their own genome (Benard and Karbowski, 2009). Mitochondria are
semi-autonomous and self-producing organelles.
The mtDNA has several characteristics. First, it is circular like bacteria,
which could be evidence for the origin of mitochondria (Cooper and Hausman,
2006). Secondly, it has a few genetic code variants. Mitochondria use a
slightly different genetic code from the “universal” genetic code (Cooper and
Hausman, 2006). For example, AGA and AGG are universal for arginine in
mammals; but in mtDNA, they are for a stop condon. In another example,
UGA is standard for a stop condon, but encodes for tryptophan in mitochondria.
Third, there are multiple copies of mtDNA (2-10) per mitochondrion (Wiesner
et al., 1992).
Finally, the size of mtDNA can vary considerably between different
species. For instance, the size of mtDNA is 6 kb to 77 kb in protists, 19 kb to
100 kb in fungi, 187 kb to 570 kb in plants, usually more than 200 kb in humans,
and less than 40 kb (usually 13 kb to 22 kb) in most other animals (Bullerwell
and Gray, 2004). Despite enormous variations in genome size, the coding
function of the mtDNA has remained relatively stable. Plants have larger
mtDNA. An example is the largest sequenced plant mtDNA to date:
Arabidopsis thaliana mtDNA, which is 367 kb and encodes for 57 proteins
(Unseld et al., 1997). However, they do not appear to contain significantly
20
more genetic information. The genome expansion is accounted for primarily
by large intergenic regions, repeated segments, introns, as well as by
incorporation of foreign DNA (plastid, nuclear and plasmid DNAs). In general,
mtDNA codes only for genes involved in the mitochondrial translation
apparatus, electron transport and OXPHOS (Bullerwell and Gray, 2004). For
example, human mtDNA is relatively small and has 16,569 bp. It has 37
genes with no introns and encodes 13 peptides (Anderson et al., 1981). The
13 peptides localize to the inner mitochondrial membrane. Human mtDNA
also encodes for two rRNA (16S and 12S rRNA) and 22 tRNAs which are
required for translation of the proteins encoded by mtDNA (Pérez-Martínez et
al., 2008). The D loop contains a transcriptional promoter sequence. The
rest of the proteins found in mitochondria are encoded by the nuclear genome
which has about 1,500 mitochondrial-related genes (Lonergan et al., 2007).
B. Origin
The origin and evolution of mitochondria remains controversial.
However, the most popular hypothesis is the endosymbiotic theory of Lynn
Margulis (Sagan, 1967). It is thought that primordial eukaryotic cells were
unable to metabolically use oxygen. At some point, they were colonized by
primitive aerobic bacteria that provided oxidative metabolism to the primordial
eukaryotic cells. In return, the primitive eukaryotic cells provided for the
bacteria which eventually evolved into mitochondria. Endosymbiotic theory
posits mitochondria are the direct descendants of a bacterial endosymbiont
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(alpha-Proteobacteria) that became established at an early stage in a
nucleus-containing host cell (Gray et al., 1999). This symbiotic relationship is
believed to have developed 1.7-2 billion years ago (Feng et al., 1997). The
ability of these bacteria to conduct respiration benefited the host cells, so it is
considered as an evolutionary advantage.
Several pieces of evidence support the endosymbiotic theory (Gray et
al., 1999). First, mtDNA is circular which is different from nuclear DNA and is
similar to DNA of bacteria. Besides that, the mitochondrion is about the same
size as a bacterium. Also, mitochondria carry several enzymes and transport
systems similar to those of prokaryotes. The last piece of evidence is from
the genome. In recent years, the genomes of a large variety of mitochondria
and bacteria were sequenced. DNA sequence analysis and phylogenetic
construction data support a monophyletic origin of the mitochondria from an
alpha-proteobacteria ancestor (Bullerwell and Gray, 2004).
C. Biogenesis
Binary fission is the process by which mitochondria reproduce (Benard
and Karbowski, 2009). Their reproduction is not necessarily timed with the
cell cycle. Instead, energy demands of the cell appear to dictate
mitochondrial replication in that more mitochondria are present when energy
needs are high such as during exercise. New mitochondria can also form by
22
fusing together (Benard and Karbowski, 2009). The significance of this not
understood.
D. Inheritance
Mitochondrial inheritance is unusual and unlike that of the nuclear
genome. The nuclear genome exhibits biparental inheritance in which each
daughter cell receives a copy of the nuclear genome. During fertilization, the
ovum and sperm contribute to the genome equally. In contrast, mitochondria
exhibit uniparental inheritance in most organisms. Although the sperm
contributes one or more mitochondria to the zygote, they are ubiquitinated and
actively degraded. Therefore, sperm mitochondria do not contribute genetic
information to the embryo (Sutovsky et al., 1999). Instead, mitochondria are
maternally inherited.
E. Biochemistry
The primary function of mitochondria is to generate useful energy in the
form of ATP for cellular activities (Fig. 4). They do this by the process of
OXPHOS in which carbohydrates, proteins and fats that are obtained by
organisms in their diet are broken down and converted into ATP (Cooper and
Hausman, 2006). About 90% of the energy the organism uses for various
activities is produced by mitochondria (Pike and Brown, 1984).
23
Mitochondria produce ATP by a chemiosmotic mechanism (Cooper and
Hausman, 2006). In this process, carbohydrates and fats obtained by the
organism are used for energy production. The carbohydrates first split into
pyruvate in the cytosol through glycolysis (Fig. 4). Then the pyruvates are
transported into the mitochondrial matrix where they are oxidized into
acetyl-CoA and enter the citric acid cycle (also known as the TCA cycle).
Fats are converted to fatty-acyl-CoA and transported into the matrix where
they are oxidized to Acetyl-CoA and enter the TCA cycle.
The enzymes for the citric acid cycle in the matrix oxidize the
acetyl-CoA to carbon dioxide, and produce three molecules of NADH and one
molecule of FADH2, which are used as a source of electrons for the electron
transport chain. The electrons released by NADH are transferred through the
electron transport chain by complex I, cytochrome c, and complexes III and IV.
Electrons released by FADH2 are transferred through the electron transport
chain by complexes II and III, cytochrome c, and complex IV. A proton
gradient is created in the intermembrane space which is pH 7 compared to pH
8 in the matrix. The energy stored in the proton gradient then drives the
synthesis of ATP when the protons flow back to the matrix through complex V
that is also called ATP synthase. This coupling of ATP synthesis and proton
flow is called chemiosmosis (Cooper and Hausman, 2006).
24
F. Mitochondrial Polarity
Mitochondrial polarity (ΔΨm) refers to the potential difference across
the inner mitochondrial membrane (Van Blerkom et al., 2002). The
magnitude of mitochondrial polarity is a determinant of several mitochondrial
functions, such as regulation of ionic fluxes and ATP liberation, and therefore
reflects mitochondrial activity (Van Blerkom et al., 2002; Wang et al., 2009). It
is also the driving force for other activities, including mitochondrial protein
translocation and modification, material transport, energy transition, and
intercellular contact and communication (Huang et al, 2002; Van Blerkom et al.,
2006). Mitochondria that look morphologically homogenous in differentiated
cells and in mouse and human oocytes can be distinguished based on ΔΨm
as visualized with mitochondria-specific potentiometric fluorescent probes
such as JC-1 (Van Blerkom et al., 2002, 2009). The mitochondria with high
ΔΨm are often pericortical (Van Blerkom et al., 2002). Therefore, although
morphologically homogenous, mitochondria can be functionally
heterogeneous. The significance of changes in mitochondrial ΔΨm is not
well understood; however, it may relate to the presence of contact points
between cells, or the need for high activating mitochondria in the cortex in
preparation for cytokinesis.
25
G. Function
Besides this conventional function of energy production as described
above, recent research suggests that mitochondria are involved in calcium
homeostasis, cell signaling, oxygen sensing and apoptosis (Duchen, 1999;
Chandel and Schumacker, 2000). For instance, mitochondria also participate
in calcium storage and the transport of Ca2+ (Berridge et al., 1998;
Hanjnoczky et al., 2007). When mitochondria are near the plasma membrane,
they can regulate Ca2+ entry. When mitochondria are near the endoplasmic
reticulum (ER) which is the major site of calcium storage, the mitochondria are
involved in Ca2+ propagation. Recent research revealed that mitochondria
regulate calcium signaling and the Ca2+ -dependent nuclear factor of activated
T cells (NFAT) pathway whose target genes are essential for embryonic heart
development (Cao and Chen, 2009).
Mitochondria also play a role in programmed cell death – apoptosis
(Duchen, 1999). When the Bcl-2 proteins on mitochondrial membranes
detect DNA damage, they activate Bax proteins, which cause the
mitochondrion to release cytochrome C, and other proteins, which trigger
downstream apoptosis signals like caspases. This finally leads to destruction
of the cell. Thus mitochondrion – mitochondrion interactions are important for
apoptotic signals. In addition, mitochondria are found to translocate during
the tumor necrosis factor (TNF)-induced apoptosis in HeLa cells (Domnina et
al., 2002). This suggests mitochondrial location may play a role in the
process of apoptosis.
26
H. Mitochondrial-Related Diseases and Disorders
Mitochondrial diseases and disorders of the mitochondrial respiratory
chain can be caused by mutations of either mtDNA or the nuclear genome
(DiMauro, 2004). There are various symptoms of mitochondrial diseases with
various causes (Wallace, 1999). They can be present in various body regions
and the disease varies from person to person. Many organs could be
affected in mitochondrial diseases such as the cells of the brain, nerves,
muscles, kidneys, heart, liver, eyes, ears, or pancreas. One or more organs
could be affected in different patients, so the disorder can range in severity
from mild to fatal. The symptoms might include poor growth, muscle
weakness, visual or hearing loss, mental retardation, heart, liver or kidney
disease, diabetes, respiratory or gastrointestinal disorders, obesity, Leber’s
hereditary optic neuropathy, Leigh syndrome and even infertility (Wallace,
1999; Schapira, 2006).
Most mitochondrial diseases are inherited and the inheritance patterns
vary from Mendelian to maternal inheritance as well as a combination of the
two (Wallace, 1999). The major causes of mitochondrial diseases are
nuclear DNA defects, mtDNA defects, or a combination of both defects. For
example, deletions in mtDNA and tRNA point mutations cause
cardiomyopathies (Arbustini et al., 1998) and diabetes (Suzuki et al., 1997;
Liou et al., 2003). The genotype-to-phenotype correlations in mitochondrial
diseases are complex since the same mutation can result in multiple
27
phenotypes, and the same phenotype can result from several different
mutations. In addition, the distribution of mtDNA mutations varies from being
present in all tissues to only being found in specific cells or tissues (Schapira,
2006).
Besides DNA defects, mitochondrial ΔΨm declination can also lead to
mitochondrial-related disorders (Wang et al., 2009). In Alzheimer's disease,
cyclical mitochondrial ΔΨm fluctuation linked to electron transport, F0F1
ATP-synthase and mitochondrial Na+/Ca+2 exchange are found to be reduced
(Thiffault and Bennett, 2005). In some circumstances, mitochondrial
diseases can be triggered by medicines or toxic substances (Schapira, 2006).
The mitochondrion is such a crucial organelle. Defects in mtDNA or
dysfunction of OXPHOS or other functions of mitochondria prevent the cell
from functioning normally. For example, if one of the multi-subunit complexes
on the electron transportation chain such as ATP synthase has defects in
structure, chemiosmosis cannot happen and ATP is not produced. A recent
study found that defects in human complex I result in energy generation
disorders, and also lead to neurodegenerative disease (Lazarou et al., 2009).
The cells suffer from energy crisis. What is more, the incompletely burned
food might accumulate inside the cell as poison to harm the body even further.
For example, free radicals such as reactive oxygen species (ROS) might be
produced and cause oxidative stress (Schapira, 2006; Dumollard et al., 2009).
The principal location for ROS generation is complex III (Chen, 2003).
However, since a great number of genes are involved, the biochemical
28
mechanisms of mitochondrial diseases are diverse.
Mitochondrial dysfunction is also involved in mammalian aging and
carcinogenesis (Wallace, 1999). Several types of cancers have mtDNA
mutations. The progressive accumulation of mtDNA mutations during a
lifetime has been suggested to contribute to aging and carcinogenesis
(Chinnery et al., 2002; Trifunovic and Larsson, 2008). At least 271 cancer
mutations were observed in conserved positions of mtDNA, and 70 of them
appeared in more than one tumour (Santos et al., 2008). As mentioned
earlier, excessive ROS generation can cause oxidative stress, which is one of
the important reasons for DNA damage. Therefore, it is easy to understand
the progressive accumulation of mtDNA mutations in mitochondrial disorder
patients which can be even worse. Besides causing aging and cancer, the
progressive accumulation of mtDNA mutations also cause some age-related
dysfunctions which decrease oocyte quality so that mitochondrial function can
be seen as a parameter to evaluate oocyte quality (Wang et al., 2009).
One notable characteristic in mitochondrial diseases caused by mtDNA
mutations is that they can cause ultrastructural changes of mitochondria, like
reduction of the amount of cristae membranes caused by depletion of mtDNA
(Gilkerson et al., 2000) and swelling mitochondria with sparse cristae in
transmission electron microscopic studies of cultivated human skin fibroblasts
harboring three different pathologic mtDNA point mutations (Brantová et al.,
2006). When the clinical characteristics and ultrastructural features of
skeletal muscle in mitochondrial cytopathies were examined with a
29
transmission electron microscope, there was an excess proliferation and
abnormal shape of mitochondria. Some mitochondria were enlarged or
contained multiple granules or paracrystalline structures (Zhang et al., 2009).
These ultrastructural changes are valuable in the diagnosis of mitochondrial
disorders. Additionally, these results also revealed the tight relationship
between mitochondrial structure and function.
In addition to the tight relationship of mitochondrial structure and
mitochondrial diseases, recent studies found mitochondrial diseases are also
related to mitochondrial dynamics. The balance of mitochondrial fusion and
fission which are opposing forces are critical for cell survival (Benard and
Karbowski, 2009). Mutations in two mitochondrial fusion genes MFN2 and
OPA1 cause prevalent neurodegenerative diseases. In addition, impaired
MFN2 expression also causes other diseases such as type 2 diabetes or
vascular proliferative disorders (Liesa et al., 2009).
Currently, mitochondrial-related diseases cannot be cured and the
treatment for these diseases is still very limited. The treatment for
mitochondrial diseases is highly individualized due to the complexity of various
causes. Certain vitamin and enzyme therapies could be helpful for some
patients like Coenzyme Q10, vitamin B family, vitamin C, biotin, vitamin E and
other antioxidants (Przyrembel, 1987). However, the use of antioxidants in
mitochondrial diseases has not been tested in clinical trials yet (Schapira,
2006). Because many mitochondrial disorders are maternally inherited, a
recently proposed treatment for inherited mitochondrial disease is embryonic
30
mitochondrial transplantation and gene therapy (Kyriakouli et al., 2008).
These approaches have only been attempted in cell cultures and have showed
promise. However, they are still far from clinical application.
I. Role in Gametogenesis and Development
Mitochondrial fusion is important for formation of the Nebenkern and
mutations which inhibit mitochondrial fusion lead to sterility of mutant flies
(Benard and Karbowski, 2009). ATP production is important for sperm motility.
As described in Chapter II, sperm carry mitochondria in their midpiece to
provide energy for fertilization. A recent research study found that mtDNA
mutation can lead to impaired spermatogenesis and impaired sperm motility
(Shamsi et al., 2008).
Mitochondria play a central role in oogenesis and early embryonic
development since they provide ATP for the processes from oogenesis to
implantation. In addition, mitochondria also provide ATP for the processes of
mitosis and meiosis in the events of DNA replication, breakdown and formation
of the nuclear membrane, and mitotic spindle movements. Mitochondria
appear to be required for oocyte and embryo maintenance and development
(Wang et al., 2009). For example, the mitochondrial fusion proteins, Mfn1
and Mfn2 are vital for embryo survival in Mfn1-/- and Mfn2-/- knockout mice
(Benard and Karbowski, 2009). There are several examples that
mitochondrial dysfunction causes infertility. In mouse oocytes, mitochondrial
31
dysfunction can result in preimplantation embryo arrest after in vitro fertilization
(Thouas
et al., 2004).
The number of mitochondria changes during development from the
oocyte stage to implantation (Van Blerkom, 2009). The actual number of
mitochondria per oocyte or blastomere is highly controversial, and to date has
not been established so that there is a consensus among researchers (Van
Blerkom, 2009). Estimates of mitochondrial number based on mtDNA copy
number per mitochondrion (which also appear to vary with meiotic competence
and development) do not agree with estimates based on morphometry using
tissue sections and transmission electron microscopy (Van Blerkom, 2009).
What is consistent, however, is that mitochondrial numbers decline in
unhealthy oocytes, and oocytes from women of advanced maternal age. This
may relate to the maternal-age associated decline in meiotic and
developmental competence (Van Blerkom, 2009). The assisted reproductive
technique of cytoplasmic transfer has been shown to restore oocyte health
(Von Blerkom, 2009). In this technique, cytoplasm containing mitochondria
from the oocyte of a younger, healthy individual is transferred to an oocyte of
advanced maternal age, and oocyte health is restored (Wang et al., 2009).
This indicates that there is an age-related difference in cytoplasm, which may
be based on mitochondrial activity.
Facucho-Oliveira et al. (2007) claim that there is no mtDNA replication
until postimplantation. This implies that mtDNA copy number does not
increase, but could decline due to mtDNA mutation or damage. This would
32
leave oocytes highly vulnerable to mitochondrial dysfunction (Facucho-Oliveira
et. al., 2007; Wang et al., 2009).
In addition to a threshold number of mitochondria being critical for
meiotic competence and developmental competence, defects in actual
mitochondrial structure result in a decline in meiotic competence. For
example, defects in mitochondrial structure including swelling or disrupted
cristae cause a decrease in meiotic competence and increase in infertility in
women of advanced maternal age (Wang et al., 2009). Moreover,
mitochondria change shape during development and this is important for
developmental competence (Van Blerkom, 2009).
The relative level of mitochondrial ΔΨm is important for normal
fertilization and early embryonic development (Van Blerkom et al., 2006, 2007).
Mitochondria in oocytes and preimplantation embryos may have different
mitochondrial ΔΨm. High-polarized pericortical mitochondria may have a role
in the acquisition of oocyte competence and the regulation of early
developmental processes (Van Blerkom et al., 2002), while low-polarized
mitochondria are associated with mitochondrial dysfunction and abnormal
embryos (Wilding et al., 2001). A recent study found mitochondrial ΔΨm in
human and mouse oocytes can be regulated by competition between oxygen
and nitric oxide (NO) (Van Blerkom et al., 2008). It has been suggested that
NO from cumulus cells depresses ATP production in mitochondria, and this is
important for ovulation to occur (Wang et al., 2009).
33
Mitochondria also regulate intracellular calcium and proapoptotic factors
during these processes (Wang et al., 2009). A local transient increase in free
Ca2+ can lead to an increase or decrease in mitochondrial respiration locally in
mouse oocytes (Van Blerkom, 2009). Intracellualr Ca2+ (in addition to ATP)
is important for nuclear envelope breakdown during meiotic maturation in
oocytes (Wang et al., 2009). This is consistent with a role of mitochondria in
maintaining intracellular calcium homeostasis (Duchen, 1999).
Another example that mitochondria play a central role during early
embryonic development is that mammalian embryonic cells have a low
glucose metabolism at the earliest stages of development. Both glycolysis
and the pentose phosphate pathway are suppressed, so the citric acid cycle of
mitochondria is the major source to reduce equivalents (or electron donors,
such as NADH) that are used in antioxidant defense (Dumollard et al., 2009).
Therefore, mitochondrial dysfunction in embryos is very likely to cause
developmental retardation.
The functions of mitochondria decline as the organism ages. It has
been proposed that ROS may cause oxidative stress in mitochondria
(Schapira, 2006). This may result in mutations in mtDNA, resulting in a
reduction of copies of mtDNA, and the reproduction and increase of mtDNA
damage and misfolded enzymes. When the ratio of normal mitochondria to
dysfunctional mutant mitochondria falls below the threshold, the cell cannot get
enough energy from mitochondria and programmed cell death will be activated.
34
In support of this idea, ROS can make the oocyte unhealthy (Wang et al.,
2009).
35
Figure 3. Schematic diagram of mitochondrial structure.
36
Figure 4. Schematic diagram of bioenergetic pathways in mitochondria.
37
CHAPTER IV
MITOCHONDRIAL DISTRIBUTION
38
A. Role of the Cytoskeleton
The cytoskeleton is involved in the distribution of mitochondria
(Katayama et al., 2006; Kabashima, 2007). The cytoskeleton is a network of
protein filaments extending throughout the cytoplasm, and is found in all
eukaryotic cells. The cytoskeleton provides a structural framework for the
cells, maintains cell shape, and positions of organelles (Pollack and
Kopelovich, 1979). In addition, it is involved in a variety of cellular activities
such as cell movements, transportation, exchange of energy, cell division, cell
differentiation, and even signal transduction (Liu et al., 2002).
The cytoskeleton has three principal types of protein filaments:
microfilaments (MFs), intermediated filaments, and microtubules (MTs). MFs,
which are also called actin filaments, are made by polymerization of actin
monomers to form F actin. In contrast, MTs are made by polymerization of
proteins called tubulin. MTs are assembled from reversible polymerization of
dimers of two types of tublin proteins, α-tubulin and β-tubulin. MTs in most
cells extend outward from a microtubule-organizing center called centrosome.
MTs play a role in a variety of cellular processes. Mitochondrial movements
and morphology are regulated through MTs (Linden et al., 1989). During
mitosis, MTs form the mitotic spindle for chromosome separation (Cooper and
Hausman, 2006). Both MFs and MTs are required for completion of
cytokinesis (Larkin and Danilchik, 1999).
39
Actin and tubulin polymerization have been extensively studied in vitro.
There are specific drugs to block their assembly. For example, colchicine and
nocodazole lead to disassembly of MTs by binding to tubulin thereby inducing
depolymerization (Leach et al., 2005), whereas cytochalasin B and
cytochalasin D disrupt MFs by binding to the barbed end of an actin filament
thereby preventing monomer addition and slow the rate of filament
polymerization (Bonder and Mooseker, 1986). These drugs have been used
to gain insight into the functions of the cytoskeleton.
B. Distribution in Somatic Cells
Mitochondria are dynamically distributed in cells and utilize cytoskeletal
tracks and motor proteins for their movements (Frederick and Shaw, 2007).
Appropriate mitochondrial distribution is essential for cell survival and
developmental competence of embryos (Katayama et al., 2006; Nagai et al.,
2006). The spatial organization of mitochondria has been shown to depend
on MTs in neurons and pig embryos (Sun et al., 2001). Several cell functions
need accurate mitochondrial distribution. For example, during cytokinesis,
equal mitochondrial distribution is critical to ensure viability of daughter cells,
since they are essential organelles that provide energy for all cellular activities
(Hales, 2004). On the other hand, mitochondrial distribution is restricted by
the arrangement of the cytoskeleton. These interactions of mitochondria with
the cytoskeleton are crucial for normal mitochondrial function (Boldogh and
Pon, 2007).
40
In differentiated somatic cells, mitochondria are distributed
homogeneously. For example, mitochondria are scattered throughout the
cytoplasm of these differentiated cells (Figure 5). In porcine fetal fibroblast
cells, mitochondria are not scattered throughout the cytoplasm, but form a
meshwork filling the entire cell. In addition, this localization depends on
microtubules, since nocodazole treatment leaves mitochondria tightly located
around the nucleus in a dot-like pattern (Katayama et al., 2006). Using
different cytoskeletal modulators, other studies have shown mitochondrial
distribution depends on microfilaments. In two-cell embryos of golden
hamsters, microtubles and microfilaments were found to control mitochondrial
distribution mutually (Kabashima, 2007). In two-cell embryos of golden
hamsters, normal mitochondrial distribution is perinuclear with a few
mitochondria at the cell cortex. After nocodazole treatment, mitochondria
were found to extend into the subcortical region and aggregated in patches.
After cytochalasin D treatment, there were less mitochondria at the cell cortex,
suggesting that mitochondria moved back around the nucleus. After a
treatment of both inhibitors, mitochondrial distribution was found to be similar
to the pattern after cytochalasin D treatment (Kabashima, 2007).
C. Distribution during Oogenesis, Fertilization and
Preimplantation Development
Several studies have tried to clarify the relationship between
mitochondrial distribution and oogenesis, fertilization and preimplantation
41
development. The studies found the distribution patterns of mitochondria are
stage- and cell-cycle-specific (Fig. 6). Homogeneous and heterogeneous
spatial arrangements are two major mitochondrial distribution patterns in
oocytes (Wang et al., 2009).
Research on mitochondrial distribution in pig oocytes using MitoTracker
Green FM stain showed that during oogenesis, GV and Met II oocytes have
scattered mitochondrial distribution. However, during fertilization and
preimplantation development, pronuclear embryos, 4-cell embryos and
blastocysts have a perinuclear mitochondrial arrangement (Sun et al., 2001).
Therefore, mitochondria translocate during normal development. The
dramatic translocation of mitochondria upon fertilization suggests that the
change may be triggered by the fertilization event.
Even primary oocytes have a different mitochondrial distribution than
mature oocytes. A recent research study on mitochondrial distribution in
canine oocytes found that the primary oocyte in the GV stage has a different
mitochondrial distribution than Met II stage oocytes (Valentini et al., 2009).
The GV stage oocytes have three types of mitochondrial distribution. The
first type of distribution is small aggregates diffused throughout the cytoplasm;
the second type is diffused tubular networks; the third type is pericortical
tubular networks. However, most Met II stage oocytes showed a diffused
tubular mitochondria network. Besides the mitochondrial distribution pattern
difference, they provided a possible explanation for it: the changes in the
42
mitochondrial distribution pattern in immature canine oocytes is related to the
reproductive cycle stage (Valentini et al., 2009).
Similar to pig and canine oocytes, evidence of stage- and cell
cycle-specific mitochondrial distribution were also found in mouse and human
oocytes (Van Blerkom, 2009). In GV stage mouse oocytes, mitochondria are
largely uniformly distributed throughout the cytoplasm. However, from GVBD
to metaphase I (Met I), the mitochondria begin to surround the newly
condensed chromosomes as a sphere and form a perinuclear distribution. In
Met II oocytes mitochondria are randomly and uniformly distributed. The
perinuclear distribution returns after fertilization. It becomes more obvious
after fertilization in two-cell embryos (Van Blerkom, 2009).
In addition to changes in density of mitochondria, mitochondrial
membrane potential can also vary. Recent studies showed the transition of
mitochondria from homogeneous to heterogeneous is correlated with the
cumulus apoptosis during oogenesis and different developmental stages
(Wang et al., 2009).
Mitochondria are even involved with developmental competence. An
example is abnormal mitochondrial distribution in Met II mouse oocytes leads
to reduced developmental competence. In a recent study, a treated group of
Met II oocytes of mtGFP-tg mice were frozen in liquid nitrogen for 5 minutes
and thawed (Nagai et al., 2006). This treated oocytes showed a significantly
lower developmental competence when they were compared with normal,
43
untreated control oocytes. The control oocytes showed perinuclear
mitochondrial staining, and these oocytes when fertilized had a high
developmental competence. The treated oocytes had randomly clumped
mitochondria in the cytoplasm, and poor developmental competence. The
meiotic arrest in the oocytes with abnormal mitochondrial distribution may be
due to abnormal distribution of ATP (Nagai et al., 2006). Moreover, another
study found the loss of developmental competence due to oocyte
mitochondrial dysfunction. This might be overcome by cytoplasmic transfer
of normal mitochondria into the oocyte (Nagai et al., 2004). In the same
manner, abnormal mitochondrial distribution also causes loss of meiotic
competence in human oocytes. During in vitro fertilization (IVF), abnormal
mitochondrial distribution that had dense clusters of mitochondria in human
oocytes arrested meiosis prematurely (Van Blerkom, 2009).
The spatial translocation of mitochondria during development leads to
the question: Is the spatial distribution of mitochondria important for their
function? This is not well understood so far. Several possible explanations
were proposed based on the results from research, which will be addressed in
more detail in Chapter V.
44
Figure 5. Diagram showing location of mitochondria in a typical somatic cell.
A bovine pulmonary artery endothelial cell was triple-labeled for the nucleus
(blue) with 4',6-diamidino-2-phenylindole (DAPI), microtubules (green) with
anti–α-tubulin mouse IgG2b monoclonal antibody prelabeled with the Zenon®
Alexa Fluor® 488 Mouse IgG2b labeling kit and the mitochondria (red) with
anti-OxPhos Complex V subunit a, mouse IgG2b monoclonal antibody (A21350)
prelabeled with the Zenon® Alexa Fluor® 555 Mouse IgG2b labeling kit.
Mitochondria are scattered throughout the cytoplasm of this differentiated cell.
Diagram based on fluorescence micrographs from www.invitrogen.com.
45
Figure 6. Distribution of mitochondria during oogenesis and fertilization in
mammalian oocytes. The black spots represent mitochondria. GV,
germinal vesicle stage; HPM, high potential (polarized) mitochondria; GVBD,
germinal vesicle breakdown stage; M I, metaphase I; M II, metaphase II; PN,
pronuclear stage. Based on Sun et al., 2001; Valentini et al., 2009; and Van
Blerkom, 2009.
46
CHAPTER V
MITOCHONDRIAL DISTRIBUTION IN EMBRYONIC
STEM CELLS
47
A. Embryonic Stem Cells
ES cells are cultures of cells derived from the inner cell mass of a
blastocyst or earliest morula stage embryos (Wright, 1999; Fig. 7). They
have the ability to undergo self-renewal and differentiation, and have the
potential to give rise to an entire organism and to all cell lineages (Abbondanzo
et al., 1993; Odorico et al., 2001). Thanks to the characteristic of immortality,
ES cells are capable of unlimited, undifferentiated proliferation in vitro and can
be maintained for years in continuous culture (Thomson et al., 1998; Hoffman
and Carpenter, 2005).
ES cells have their specific gene expression. The key pluripotent
factors are Oct4 and Sox2. Oct4 is the master regulator and needs to be
maintained at a specific expression level in order to maintain pluripotency
(Niwa, 2000). Besides the two fundamental key factors, Nanog is also an
important transcription factor which is regulated by Oct4 and P53, and at the
same time works together with Oct4 and Sox2 to control the downstream gene
regulation and maintain pluripotency. These key factors form a regulatory
network to limit each other’s expression level, which is essential to maintain
the properties of ES cells (Pan and Thomson, 2007). Therefore, these
pluripotent factors Oct4, Sox2 and Nanog are often used as marker genes in
charactering ES cells.
Besides their specific gene expression, the ability to form a teratoma,
which is a tumor made of cells from all three germ layers, is another
48
characteristic of ES cells (Thomson et al., 1998). Recent studies suggest
perinuclear mitochondrial distribution could be another characteristic of ES
cells (Lonergan et al., 2006; Facucho-Oliveria and St. John, 2009). This
interesting finding will be discussed more in the next section.
ES cells have medical significance for they could potentially provide an
unlimited supply for tissue regeneration and tissue transplantation (Wright,
1999) and apply in cell replacement therapies (Hoffman, 2005). Conversely,
when a stem cell mutates, it could become a cancer cell sustaining its growth
and spreading rapidly (Reya et al., 2001).
B. Bavister’s Hypothesis
Although the spatial distribution of mitochondria with respect to the
cytoskeleton has been studied in several cell types, it has not been examined
in ES cells. The contribution of mitochondria to stem cell viability and
differentiation must be vitally important in view of their critical roles in all other
cell types. Recent research on translocation of mitochondria in the oocyte
during fertilization has shown that mitochondria cluster around the pronuclei
and can remain in a perinuclear pattern during embryonic development (Sun et
al., 2001). This strongly suggests a key role for mitochondria in ES cells,
because this clustering appears to be essential for normal embryonic
development. Moreover, ES cells are derived from blastocysts. Bavister
and associates have hypothesized that mitochondrial perinuclear clustering
49
persists through preimplantation embryonic development into the stem cells,
and that this localization is indicative of stem cell pluripotency (Lonergan et al.,
2006). My preliminary data using the protocols described below in Section D
showed that mitochondrial distribution in MLE-15 cells is a meshwork around
the nucleus. This is supportive of Bavister’s hypothesis. However, the
actual mitochondrial distribution in ES cells needs to be investigated. If the
results also support Bavister’s hypothesis, mitochondrial distribution could
possibly be an exciting, brand new marker for ES cells. Bavister’s group has
suggested that because zygotes and cleavage stage embryos including
blastocysts have perinuclear staining of mitochondria, ES cells may also have
perinuclear staining, indicating that the perinuclear localization of mitochondria
may be an indicator of “stemness.”
When they examined mitochondrial arrangement in an adult stem cell
line, Bavister et al. found perinuclear staining, indicating that the hypothesis
holds true for adult stem cells (Fig. 8; Lonergan et al., 2006). They also said
that since only two stem cell lines Rhesus & human have been examined,
many more cell lines will need to be investigated to see if the hypothesis holds.
It is important to be able to detect good stem cells with confidence especially
since many are suboptimal since they are derived from “leftovers” from IVF
clinics, and human ES cells may one day be used therapeutically.
Recently, studies agree that pluripotency seems to be associated with
anaerobic metabolism (Facucho-Oliveria and St. John, 2009). In addition,
small and immature mitochondria with a perinuclear distribution is an important
50
characteristic of ES cells (Facucho-Oliveria and St. John, 2009). This
characteristic can even apply for the novel method of generating human and
mouse induced pluripotent stem cells (iPSCs). Results of Oct4, Sox2 and
Nanog expression might not be sufficient to conclude they are pluripotent.
The mitochondrial properties should be investigated before concluding iPSCs
are pluripotent (Facucho-Oliveria and St. John, 2009).
C. Possible Reasons for Perinuclear Mitochondria in Stem
Cells
There are a number of potential reasons for the stem cells to have a
perinuclear distribution of mitochondria. First, perinuclear distribution
matches the OXPHOS demands of differentiating cells. The ATP content in
human ES cells and an adult rhesus macaque stromal cell line (ATSC) were
lower when cells were stem-like, but increased four-fold in human ES cell line
and five-fold in the monkey ATSC cell line upon differentiation. Another
benefit of perinuclear mitochondrial distribution at the stem cell stage is that
the mitochondrion and nucleus interaction could be easier (e.g., the nuclear
genome codes approximately 1500 mitochondrial-related genes). Moreover,
import and export of macromolecules across the nuclear pores involve the
energy-dependent Ran monomeric G protein transport system (Cooper and
Hausman, 2006). Positioning mitochondria near the nucleus might provide
the energy for this process in an efficient manner. Finally, perinuclear
51
arrangement of mitochondria might buffer the nucleus from fluctuations in
Ca2+ levels occurring in the cytoplasm (Lonergan et al., 2007).
Since the main functions of mitochondria are ATP synthesis and
calcium supply, the mitochondrial distribution in the oocyte may result from the
high demand of ATP and calcium during cytoplasmic maturation (Wang et al.,
2009). The perinuclear arrangement of mitochondria during embryonic
development is very likely to due to the high energy demand around nucleus
(Wang et al., 2009). Therefore, it is highly possible that the redistribution of
mitochondria from perinuclear in stem cells to scattered thoughout the
cytoplasm in somatic cells is due to the localized change of energy demand.
The iPSCs generated by defined factors from mouse or human
fibroblast cells were verified to have ES cell morphology, specific gene
expression such as Oct4, Sox2 and Nanog and were capable of teratoma
formation (Takahashi and Yamanaka, 2006; Yu et al., 2007). However,
another important characteristic of pluripotency has not been tested yet --
whether iPSCs have immature mitochondria with a perinuclear distribution.
For the considerable amount of data mentioned above, there are reasons to
believe that iPSCs indeed are pluripotent. Therefore, if we carry out the
described experiments in iPSCs, the staining of mitochondria should exhibit
perinuclear distribution just as ES cells do.
52
D. Hypothetical Experiments Designed to Test Bavister’s
Hypothesis
Given the need to identify healthy ES cells, it is important to know the
mitochondrial arrangement in healthy ES cells, and whether this is an indicator
of “stemness.” The following experiments are proposed to test Bavister’s
hypothesis.
Research Objectives
1. Determine and compare the spatial distribution of mitochondria with
respect to the nucleus and cytoskeleton in MES cells and a differentiated
cell line, MLE-15 cells. To investigate pluripotency of iPSCs with
regards to mitochondrial properties, mouse iPSCs generated from
fibroblast cells as described in Takahashi and Yamanaka (2006) could be
used.
2. Understand the spatial arrangement of mitochondria during
differentiation by staining of mitochondria in MES cells and MLE-15 cells.
3. Determine whether mitochondrial distribution is cytoskeletal-dependent
after disruption of the cytoskeleton in MES cells and MLE-15 cells.
Materials
For undifferentiated cells, MES cells could be used. For differentiated
cells, MLE-15 (mouse lung epithelial) cells could be used. For iPSCs, mouse
53
iPSC-MEF could be used.
Research Method
To analyze the spatial arrangement of mitochondria during
differentiation and disease, the following techniques are proposed.
a) Cell culture. MLE-15 cells will be cultured in Dulbecco’s modified eagle
medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum
(FBS) and 100 U/mL penicillin-streptomycin. MES cells will be cultured in
DMEM containing 90 mL ES-qualified FBS, 6 mL non-essential amino acids
(NEAA), 6 mL GlutaMax, 6 mL 100 U/mL penicillin-streptomycin, 6 uL
β-mercaptoethanol, and 500 uL recombinant eukemia inhibitory factor (LIF) in
500 ml DMEM. Both cell types will be cultured at 37ºC in 5% CO2 in
humidified air.
b) Immunofluorescence microscopy. To visualize mitochondria, nuclei and
the cytoskeleton, fluorescence staining will be used. Cells attached to
microscope slides (in a thin glass bottom sterile culture dish) will be fixed in
70% ethanol for 10 minutes, and rehydrated in blocking solution containing
phosphate-buffered saline, (PBS, pH 7.2 ) and 0.3% bovine serum albumen
(BSA). Tubulin will be detected with a mouse monoclonal anti-tubulin
antibody (E7 antibody, Developmental Hybridoma Studies Bank, University of
Iowa), and actin will be detected using a mouse monoclonal anti-actin antibody
(Mab-5; LabVision Corporation Fremont, CA). Both antibodies will be diluted
54
1:100 in blocking solution and detected using a TRITC-conjugated
goat-anti-mouse secondary antibody. Fixed cells will be incubated for 1 hr at
37oC in primary antibody (either anti-tubulin or anti-actin antibody), washed
twice in PBS, incubated 1 hr at 37oC in secondary antibody, and mitochondria
stained for 30 min with 1 mM MitoTracker (Molecular Probes, Invitrogen) in
PBS followed by staining for 30 min in 5 ug/ml DAPI in PBS to reveal the
nucleus. Cells will be mounted in Vectashield (an anti-fade agent) and
viewed with a conventional fluorescence microscope using the UV, FITC and
Rhodamine filter sets, or an Olympus Fluoview FV1000 confocal laser
scanning microscope (Olympus America Inc., Melville, NY). The negative
control will include omission of the primary antibody from the protocol, and the
immunostaining data compared with the negative control to decide antigen
localization.
c) Mitochondrial localization during cytoskeletal disruption. To disrupt the
cytoskeleton MES and MLE-15 cells will be transferred into DMEM media
supplemented with 10-100 µM colchicine to disrupt microtubules (or
cytochalasin B to disrupt microfilaments), and cultured for 6 hr. After fixation
as above, samples will be stained as follows. For samples treated with
colchicine, anti-tubulin antibody will be used for first antibody. For samples
treated with cytochalasin B, anti-actin antibody will be the first antibody,
followed by secondary antibody as above, and MitoTacker and DAPI staining.
Cytoskeletal and mitochondrial patterns in treated and untreated cells will be
compared to reveal the role of the cytoskeleton in mitochondrial arrangement
55
during differentiation.
Expected Results
To demonstrate feasibility of using Mitotracker FM Green and DAPI to
localize mitochondria and nuclei, respectively, MLE-15 cells were
double-labeled with these dyes (Fig. 9). Mitochondria appear restricted to the
cytoplasm and are absent from the nucleus. This is consistent with Bavister’s
hypothesis. Thus the fluorescent dyes are feasible for use with MLE-15 cells.
Staining for mitochondria using MitoTracker FM Green is expected to
reveal differences in mitochondrial distribution in mouse undifferentiated and
differentiated cells. Treatment with the cytoskeletal disrupters (nocodazole,
colchicine, cytochalasin B) should show mitochondrial distribution is mediated
by the cytoskeleton. MES cells are expected to exhibit perinuclear
mitochondrial distribution, and cytoskeletal disrupters are expected to further
concentrate mitochondria around the nucleus. MLE-15 cells will exhibit
perinuclear or random, scattered mitochondrial distribution, and cytoskeletal
disrupters should concentrate mitochondria around the nucleus. The iPSCs
will exhibit perinuclear mitochondria distribution just as the MES cells do.
This would demonstrate a role of the cytoskeleton in maintaining the spatial
distribution of mitochondria.
56
Figure 7. Preparation of cultured embryonic stem (ES) cells. When inner
cell mass cells are removed from the blastocyst and placed in culture, the cells
survive and are known as ES cells.
57
Figure 8. Measurement of mitotracker fluorescence intensity. Passage 11
adult rhesus macaque stromal cells (ATSC) were stained with 50 nM
Mitotracker to show mitochondrial location. Cells were viewed with
fluorescence microscopy at 600 x magnification. The cell periphery is
outlined with a black line. The cell nucleus is shown in blue. The black dots
around the nucleus represent mitochondrial staining. The red lines are the
axes of the graph showing the extent of fluorescence intensity (green line)
across the cell. The intense mitochondrial fluorescence around the nucleus
drops precipitously toward the cell periphery, indicating the ATSC cell has a
perinuclear mitochondrial arrangement. (Recreated from Lonergan et al.,
2006)
58
A
B
C
Figure 9. Mitochondrial distribution in mouse lung epithelial (MLE-15) cells.
Cells stained with 1 mM MitoTracker to localize mitochondria (A) and 5 ug/ml
DAPI to visualize DNA (B) were viewed at 60x with conventional fluorescence
microscopy. C Shows mitochondria (green) and nuclei (blue) simultaneously.
Mitochondria appear restricted to the cytoplasm and are absent from nuclei.
59
CHAPTER VI
CONCLUSIONS AND FUTURE DIRECTIONS
60
A. Conclusions
Mitochondria play a vital role in oogenesis and development in
mammalians. This is because mitochondria not only provide ATP for these
developmental processes, but mitochondria also regulate calcium signaling
and apoptosis, which are critical in embryonic development. There is a
subcellular mitochondrial heterogeneity. Morphologically homogenous
mitochondria can be functionally heterogeneous, since they can still be
distinguished on the basis of mitochondrial ΔΨm. The significance of this is
not understood.
Patients with mitochondrial-related diseases have various symptoms
with various causes. Due to the complex causation of mitochondrial diseases,
there is no cure for them so far, and the treatments are highly individualized
and still very limited. Currently, embryonic mitochondrial transplantation and
gene therapy are the two proposed methods of treatment. However, they are
both experimental in vitro. In addition, mitochondrial transplantation is not
feasible in adults.
Mitochondrial distribution patterns in oocytes are stage- and
cell-cycle-specific. There are two major mitochondrial distribution patterns in
oocytes, homogeneous and heterogeneous based on mitochondrial spatial
distribution and polarity. Perinuclear mitochondrial distribution is suggested
to indicate the maturation of the oocyte. There is growing evidence in several
different mammalian species (mouse, dog, pig, human) supporting a
61
mitochondrial distribution change from scattered to perinuclear during
maturation of oocytes so that development can be proceed further, otherwise
there will be a meiotic arrest. Therefore, mitochondrial distribution plays a
central role in establishing meiotic competence. Due to the correlation of
mitochondrial distribution and oocyte meiotic competence, mitochondria can
be seen as signs for evaluation of oocyte quality in reproduction.
In summary, from the data of recent studies on mitochondrial
distribution, Bavister’s hypothesis is very likely to hold true. So the
perinuclear mitochondrial distribution could possibly be used as a brand new
marker for ”stemness.” My proposed research on comparison of
mitochondrial distribution in mouse MLE-15 and MES cells is designed to test
Bavister’s hypothesis and lead to a better understanding of mitochondrial
behavior in development. Understanding the role of the cytoskeleton will also
be important. This research will be of value in finding therapy for various
mitochondrial diseases and cancer, and will aid in understanding ES cell
function.
B. Future Directions
The spatial distribution of mitochondria in MES cells needs to be
determined and compared with the distribution of mitochondria in MLE-15 cells
to test if Bavister's hypothesis holds true. Experiments need to be carried out
to examine effects of the cytoskeletal modulators, nocodazole, colchicine or
62
cytochalasin on mitochondrial arrangement in order to determine the
interaction of mitochondrial distribution and the cytoskeleton.
After clarifying the mitochondrial distribution during development in
mammalian cells, the reasons for mitochondrial translocation during
development and the mechanisms regulating mitochondrial translocation
during differentiation deserve further investigation so that we can get a better
understanding of developmental processes with regards to mitochondria.
The more we understand about these mechanisms, the more likely for us to
find therapy or even a cure for mitochondrial disorders. Some research has
suggested that mitochondrial translocation is related to reproductive cycle
stages. Recent studies find that not only the mitochondrial distribution, but
the heterogeneity of shape and ΔΨm of mitochondria can be important for
mitochondria to function normally. These claims need further confirmation in
a variety of cell types.
63
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