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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

ii
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

iii
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 

viii
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

2
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

3
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

4
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.

5
CHAPTER II

INTRODUCTION TO GAMETOGENESIS AND

DEVELOPMENT

6
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

7
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

8
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

9
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

10
(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.

11
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 5 to 10 8 mitochondria which exceed

that of sperm by a factor of at least 10 3 . 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).

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).

12
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

13
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

14
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.

15
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.

16
 

Figure 2. Mammalian blastocyst. Two cell types are present in the

blastocyst: inner cell mass (future embryo) and trophoblast (future placenta).

17
CHAPTER III

THE MITOCHONDRION AND ITS FUNCTIONS

18
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).

19
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

21
(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

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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