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F O R P H Y S I C I A N S – resources
Mitochondria as genetic forces in early human development
Mitochondrial transmission across generations is uniparental
It has long been known that the human mitochondrial
genome is 16 560 kb of double stranded DNA that
encodes 13 proteins in the respiratory chain, and 22
unique transfer RNAs and 2 ribosomal RNAs (Clayton
2000, Trounce 2000). Although not without some controversy
(Cummins 2004), mitochondria are inherited
through the maternal lineage with paternal mitochondria
arriving at fertilization targeted for destruction primarily
by ubiquitin-dependent proteolysis (Schwartz & Vissing
2002, 2003, Johns 2003, Sutovsky 2004). The maternal
transmission of mitochondria between generations is the
genetic basis for the inheritance of certain debilitating or
ultimately lethal metabolic disorders in the human (Chinnery
& Turnbull 1999, Christodoulou 2000, Leonard &
Schapira 2000, Chinnery 2004), and heterogeneity in
mtDNA is used in forensic medicine to assist in the identification
of individuals and in anthropology to trace the
origins and geographical dispersal of populations.
All of the mitochondria in the mature oocyte (metaphase
II, MII stage) arise from the clonal expansion of an
extremely small number of organelles present in each primordial
germ cell that after colonization of the forming
ovary, expands by mitosis to form numerous progeny that
can be identified as ‘nests’ of primordial oocytes (Makabe
& Van Blerkom 2004). By some estimates (Jansen 2000b),
,10 mitochondria may be the progenitors of the tens to
hundreds of thousands of organelles present in the human
oocyte at fertilization. That mitochondrial transmission
across generations is uniparental and that procreation
requires their significant numerical expansion presents
some potentially unique biological challenges. For
example, it has been argued that this uniparental replication
might be expected to follow the ultimately lethal consequences
of Muller’s ratchet hypothesis (Muller 1964,
and concisely discussed by Jansen 2000a), whereby
species extinction is an inevitable consequence of asexual
reproduction owing to the accumulation of deleterious
mutations by random genetic drift. Indeed, because of
maternal inheritance and high mutation frequency, the
human mtDNA should be prone to Muller’s ratchet. Counteracting
this natural entropic tendency to mutational
degradation and extinction is the severe reduction in
maternal germline mtDNA copy number in the primordial
germ cell, a phenomenon generally known as the ‘mitochondrial
bottleneck’ (Bergstrom & Pritchard 1998). As a
result of the reduction in progenitor organelles, the
accumulation of mtDNA mutations is diminished with
certain mutations, such as those that could adversely
affect replication or metabolic capacity being eliminated
by natural selection or ‘dying out’ through oogenesis
(Hoekstra 2000). However, others have argued that there
is not a single mitochondrial bottleneck at the outset of
oogenesis, but rather an active selection process that
occurs throughout oogenesis and early embryogenesis and
involves multiple stage-specific bottlenecks and differential
patterns of mitochondrial segregation (Howell et al.
2000). The occurrence of individuals with maternally
inherited metabolic diseases (OXPHOS diseases) resulting
from known mtDNA mutations demonstrates that (a) the
bottleneck is not an effective natural means of eliminating
oocytes carrying potentially lethal mitochondrial genetic
defects and (b) that developmental competence does not
require that the mitochondrial complement be genetically
normal or even capable of normal levels of respiration
(oxidative phosphorylation).
Mitochondrial transmission between generations is not necessarily monogenomic: homoplasmy and heteroplasmy
As maternally inherited organelles, the mtDNA genotype(s)
in the embryo is largely determined by what existed in the
few mitochondria contained within the primordial germ
cell and resulting primary oocyte. If the expansion of the
mitochondrial population during oogenesis involves an
identical genome, the MII oocyte and resulting embryo
would be expected to be homoplasmic. Heteroplasmy
occurs when two or more different mitochondrial genotypes
occur in the same cell, whether a primordial germ
cell, oogonia, oocyte or blastomere. Heteroplasmy per se
does not imply an adverse condition if the mtDNA
mutations are benign with respect to function, but can
become problematic and have cytopathological consequences
if the mutant form(s) has reduced respiratory
capacity and occurs at toxic levels (mutant load). While
heteroplasmy as a factor in human infertility or early
embryo demise is a current issue in reproductive medicine
(Brenner 2004, Cummins 2002, 2004), it is necessary to
note that adverse developmental consequences of mtDNA
mutations become relevant only when they affect mitochondrial
activities (e.g. replication and respiration) at
levels that are inconsistent with cell survival or normal
function (Christodoulou 2000). Indeed, the threshold levels
at which OXPHOS diseases become clinically significant
are usually quite high (Chinnery & Turnbull 1999, Leonard
& Schapira 2000, Trounce 2000).
Heteroplasmy, detected by highly sensitive analysis of
mtDNA in the oocyes of certain women, has been related
to infertility by virtue of the occurrence of certain mtDNA
genotypes such as the ‘common deletion’ mtDNA 4977,
which in one report was proposed to increase with
maternal age and negatively affect competence in women
>40 years old (Keefe et al. 1995). However, in a recent
review of the relationship between competence and the
various types of mtDNA mutations detected in human
oocytes obtained by ovarian hyperstimulation for IVF,
Brenner (2004) found no compelling evidence to suggest
that any occurred at loads which could compromise outcome.
This is not to say that mtDNA is unimportant in the
establishment of competence or as an etiology of infertility
but rather, that additional investigation is needed to validate
such interpretations, especially if proactive therapies
(e.g. cytoplasmic transfer) are contemplated in an IVF
treatment cycle. In this respect, Brenner (2004) described
some promising leads related to point mutations in the
control region of the mitochondrial genome responsible
for replication. These mutations seemed to increase in frequency
in the oocytes of certain women, especially those
of advanced reproductive age and, if confirmed, could be
an important and unrecognized factor in outcome because
mitochondrial replication does not begin until after
implantation. Therefore, replication defects would not be
expected to compromise preimplantation embryogenesis,
but depending upon mutant load, could manifest as postimplantation
demises described as chemical (transient
elevation of human chorionic gonadotropin levels) or
anembryonic pregnancies (no fetal pole detected by ultrasonography).
Experimental approaches to the question of whether
specific mtDNA defects in human oocytes cause postimplantation
demise require some formidable challenges to
be overcome. For example, are unused blastocysts from
IVF programs, even if available, suitable material to screen
for mtDNA defects and should the trophoblast and inner
cell mass (ICM) be analyzed separately? For patients with
a history of repeated chemical or anembryonic pregnancies,
is it ethical to use IVF protocols to generate multiple
blastocysts such that some could be transferred or cryopreserved
while others are used for mtDNA analysis? If
specific mtDNA mutations that affect competence during
the pre- and postimplantation stages are clearly identified,
screening and ethical issues become moot as it would be
expected that the same protocols used for embryo biopsy
and preimplantation genetic diagnosis as applied to
chromosomes and nuclear DNA (Verlinsky & Kuliev 2000)
would be applicable to mtDNA. However, an assessment
of whether a particular mtDNA mutation could influence
competence and outcome requires the ability to accurately
quantify the mutant load. This is especially evident
when it is considered that some mtDNA-related OXPHOS
diseases clinically manifest only when a genetic defect
occurs at high load (Christodoulou 2000), while for others
the severity of the clinical symptoms is proportional to the
mutant load (Dahl et al. 2000). Whether the finding that
mtDNA copy numbers that can vary by over an order
of magnitude between MII oocytes in the same cohort
(see below) presents another challenge for the application
of mitochondrial analysis in clinical IVF, remains to
be determined.
Mitochondrial fine structure and metabolic activity
It has long been known from transmission electron
microscopy (TEM; Sotelo & Porter 1959, Baca & Zamboni
1967) that mitochondria in mammalian oocytes and early
embryos have a unique fine structure in which a spherical
profile, dense matrix and relatively few cristae are indicative
of an undeveloped state (for reviews see Van Blerkom
& Motta 1979, Makabe & Van Blerkom 2004). What
makes recent TEM studies of human oocytes and embryos
clinically relevant is the possibility that structural abnormalities
detected in certain infertile women could be associated
with mitochondrial dysfunctions that reduce their
metabolic activity and may, therefore, be an important
etiology of oocyte or embryo incompetence (Motta et al.
2000, for review). This may be especially relevant in
women of advanced reproductive age as reported by Muller-
Hocker et al. (1996).
Similar to other mammals (Van Blerkom & Motta 1979),
mitochondria in fully-grown human oocytes are the most
abundant organelles detected by electron microscopy
(Fig. 1A) and occur as spherical/ovoid elements <0.5mm
in diameter (Dvorak et al.. 1987). Typically, these mitochondria
contain only a few short cristae that rarely
penetrate an electron-dense matrix (Fig. 1B and C). This
phenotype persists through the cleavage and late morulae
stages of human embryogenesis in vitro before a gradual
transition to an elongated form with a matrix of low-tomoderate
electron density is observed (arrows, Fig. 1D
and E). An increased number of lamellar cristae that completely
traverse the inner mitochondrial matrix is generally
characteristic of mitochondria actively engaged in ATP
production by oxidative metabolism, and this profile represents
the predominant form seen at the blastocyst stage
in most mammals. For the human preimplantation embryo
developing in vitro, serial section TEM analysis has shown
that at the blastocyst stage virtually all cells contain (albeit
in different proportions) both undeveloped and welldeveloped
mitochondria (Fig. 1D). Unlike the situation
that prevails in other mammals such as the mouse and
rabbit (Van Blerkom & Motta 1979), for the human blastocyst
the fully developed mitochondrial phenotype shown
in Fig. 1E may predominate in some cells and be comparatively
scarce in others (Sathananthan et al. 1993, Van
Blerkom 1993). It is not known whether the apparent cellspecific
differences in the state of mitochondrial differentiation
observed in human blastocysts are related to the
conditions of culture and therefore not representative
of the in vivo situation. Alternatively, they could be a
normal aspect of early human development and represent
developmentally significant differences in mitochondrial
activity within the embryo, perhaps related to differential
cell function, as discussed below for the mouse blastocyst.
Figure 1 A–E and I are transmission electron micrographs that have been colorized to enhance the visualization of certain subcellular components.
(A) Mitochondria (M, blue) and complexes of smooth-surfaced endoplasmic reticulum (SER, red) in a metaphase human oocyte. The
association between mitochondria and the SER is shown at high magnification for a single complex in I. Differences in mitochondrial fine structure
seen in human oocytes (C) and cleavage (B) and blastocyst stage embryos (D,E) indicate a progressive transformation to forms presumed to
be more active in respiration. c, cristae, MII, metaphase II spindle. These pictures are adapted from Makabe & Van Blerkom (2004). (F, G) Scanning
laser confocal microscopic images of a human pronuclear (F,G) and a normal appearance 8-cell embryo (H) stained with mitochondrialspecific
fluorescent probes (H1,2). A symmetrical distribution of peri-pronuclear (PN) mitochondria (M) is shown in a fully complied image (F)
and 5m section (G). Differences in the mitochondrial segregation between blastomeres during cleavage present as differential intensities of fluorescence
(H1,2) and can be traced back to asymmetric peri-pronuclear aggregation at the one-cell stage. The arrow in H1 denotes the second
polar body. These pictures are adapted from Van Blerkom et al. (2002). (J) A compiled 15 m scanning laser confocal microscopic image showing
spherical complexes of SER in a living MII human oocyte stained with an SER-specific probe. An arrow indicates the MII chromosomes. (K-O)
Conventional epifluorescent microscopic images showing red J-aggregate fluorescence in a human pronuclear (PN, arrows, K) and blastocyststage
(arrows, L) embryo, and in a peri-implantation, day 5.5 (M) mouse blastocyst observed in the FITC (N) and RITC (O) channels after staining
with JC-1. The potential developmental relevance of pericortical J-aggregate fluorescence to high polarized mitochondria in the oocyte and
early embryo, and differential J-aggregate fluorescence between the mural (mTR) and polar trophectoderm (pTR) and inner cell mass (ICM) are
discussed in the text. Amy Jones provided image K (see also Jones et al. 2004). Green fluorescence in this image is derived from JC-1 monomeric
staining in low polarized mitochondria. Yellow fluorescence in N results from signal cross-over from the RITC channel.
The undeveloped morphology of oocyte and early
embryo mitochondria has been traditionally interpreted in
the context of low respiratory activity. However, there are
two important factors to consider for the human oocyte:
(a) if the number of mitochondria and mtDNA copies are
synonymous (Cummins 2002), the apparent mitochondrial
complement in human oocytes may number in the hundreds
of thousands and (b) levels of mitochondrial ATP
generation are likely to be demand-related and in part
determined by stage-specific exogenous conditions. For
example, owing to the absence of an intrafollicular
vasculature, the oocyte may reside in a near anoxic
environment for most of its life and energy demands may
be minimal or supplemented with ATP produced by glycolysis
or imported from exogenous sources such as the
associated granulosa cells (Albertini 2004), whose cytoplasmic
extensions penetrate deeply into the ooplasm and
communicate directly with the oolemma by means of gap
junctions (Motta et al. 2003, Makabe & Van Blerkom
2004). Changes in the level of oocyte metabolism could
occur during follicular growth as the fluid-filled antrum
develops and perifollicular blood flow rates increase significantly,
which may increase the intrafollicular concentration
of dissolved oxygen available to the oocyte during
the preovulatory period (see review by Van Blerkom
2002). If the cumulus and coronal cells are significant
sources of ATP for the oocyte, increased mitochondrial
ATP generation may occur during meiotic maturation to
compensate for the loss of contact with the surrounding
somatic cells that occurs at the resumption of meiosis.
Stage-specific spatial remodeling of the maturing oocyte
cytoplasm is also associated with mitochondrial redistributions
that suggest that ATP production may occur at different
levels within the ooplasm. Transient and focal
upregulation of mitochondrial respiration may be an
important mechanism by which differential intracytoplasmic
energy demands in the maturing oocyte and early
embryo are met without involving the entire mitochondrial
complement (Van Blerkom & Runner 1984). Indeed,
Dumollard et al. (2004) demonstrated that ATP supply and
demand are tightly coupled in the MII mouse oocyte and
newly fertilized egg, suggesting that up- or down-regulated
mitochondrial activity is likely stage-specific and differentially
localized within the cytoplasm during early
development. According to Dumollard et al. (2004), the
underdeveloped morphology of oocyte and early embryo
mitochondria in the mouse may serve to limit oxidative
phosphorylation capacity/organelle and consequently
reduce the potential for generating reactive oxidative
species at levels where oxidative stresses could either
compromise mitochondrial function or perhaps initiate
apoptosis (Liu & Keefe 2000, Liu et al. 2000). The question
of how mitochondrial activity is regulated may be of
particular interest in clinical IVF if oocytes with very
different numerical complements occur within cohorts
(see below). For example, could premature arrest of preovulatory
meiotic maturation or of early embryogenesis be
associated with a mitochondrial complement that is insufficient
to supply nascent ATP demands, especially if stagespecific
demands are differentially located within the
cytoplasm? At the other extreme, could oocytes with unusually
high numbers of mitochondria be unable to tightly
regulate focal supply and demand and, as a result, generate
reactive oxidative species at levels that become developmentally
toxic after fertilization (Van Blerkom 2004)?
Many studies, some beginning in the 1930s (Boeil &
Nicholas 1939) and continuing during the subsequent decades
(Fridhandler 1961, Brinster 1967, Stern et al. 1971,
Ginsberg & Hillman 1973, 1975a, Biggers & Borland
1976, Gott et al. 1990) up to the present time (for review
see Biggers 2004), have examined metabolic pathways of
ATP generation during early mammalian embryogenesis. It
is clear that mitochondrial oxidative metabolism is a major
contributor of ATP during the entire preimplantation stage,
with over 85% of all ATP produced in the mouse blastocyst
derived from mitochondria (Benos & Balaban 1983). Trimarchi
et al. (2000) estimated oxygen consumption by
individual mouse embryos and described a progressive
increase in uptake from the one-cell to the blastocyst stage
such that expanded mouse blastocysts consumed 60–70%
more oxygen for mitochondrial oxidative phosphorylation
than did cleavage stage embryos (30 %). The progression of
preimplantation development occurs against a background
of nonreplicating mitochondria whose numbers/cell would
be expected to be halved with each cell division. Stagespecific
changes in mitochondrial fine structure to forms
more consistent with high respiratory activity have traditionally
be assumed to compensate for the continual
decline in mitochondrial numbers at the blastomere level.
In the early embryo, these morphological changes largely
occur in concert with increased demands for ATP needed
to support stage-specific cellular biosynthetic activities,
plasma membrane production and developmentally critical
morphodynamic processes such as (a) formation of a fluidfilled
blastocoel cavity that begins with cavitation at the
morula stage and continues through the phase of blastocyst
expansion and (b) emergence of the embryo from within
the confines of the zona pellucida during the so-called
hatching stage that precedes implantation.
The extent to which fine structural defects in mitochondria
are associated with arrested or abnormal patterns
of human embryo development warrants investigation,
especially when it is considered that poor outcomes in
clinical IVF may be associated with certain cleavage-stage
embryo phenotypes (Veeck 1999). A similar situation
occurs at the end of the preimplantation stage as indicated
by the variety of blastocyst phenotypes that often show
significant defects in ICM or trophoblast development
(Van Blerkom 1993, Veeck & Zaninovic 2003). At present,
our understanding of mitochondrial fine structure in
human oocytes and preimplantation-stage embryos is limited
to a very few studies that present images from
selected thin sections. For the human, the relationship
between metabolism and competence is an emerging one,
and it may be timely to consider systematic fine structural
analysis of the arrested or seemingly abnormally developing
human embryos that are usually discarded by IVF programs,
as well as presumably normal embryos that may
be donated to research.
Is human oocyte and embryo competence related to the size of mitochondrial complement?
Traditional estimates of mitochondrial numbers in oocytes
by TEM have used representative sections whose selection
for numerical analysis is determined by a morphometric
algorithm. While laborious, this approach is capable of
offering reasonably accurate values assuming that mitochondria
are relatively uniformly distributed throughout
the cytoplasm. With this method, between 120 000
and 350 000 mitochondria have been estimated to occur
in MII human oocytes (Jansen 2000b, Cummins 2002).
However, when mtDNA copy numbers are determined by
polymerase chain reaction methodology using probes for
specific genes such as ATPase 6 (Van Blerkom 2004) or
directed to specific sequences (Brenner 2004), the number
of human mitochondrial genomes in MII oocytes from the
same or different cohort(s) has been reported to differ by
well over an order of magnitude, ranging from a low of
approximately 20 000 to well over 800 000 (Chen et al.
1995, Steuerwald et al. 2000, Reynier et al. 2001, Barrit
et al. 2002, Van Blerkom 2004). If the current consensus
that each oocyte mitochondrion contains a single genome
is accurate (Cummins 2002), this rather astonishing and
unexpected variation in mtDNA numbers between similarly
appearing oocytes raises some fundamental questions
about how competence may be determined well before
fertilization.
Reynier et al. (2001) proposed that premature arrest of
preovulatory meiotic maturation and fertilization failure
after conventional IVF may be directly related to low
mtDNA numbers, especially in the 20 000 to 60 000
range, if low copy numbers are associated with a reduced
metabolic capacity. Van Blerkom et al. (1995) measured
net cytoplasmic ATP levels in cohorts of unfertilized and
uninseminated MII human oocytes obtained from women
undergoing IVF and GIFT procedures and reported that
the ATP content of equivalently appearing oocytes could
differ by an order of magnitude. Whether a developmentally
significant relationship exists between ATP content
and mitochondrial or mtDNA copy numbers remains to
be determined, and the simplest interpretation of very
high mtDNA copy numbers is that each mitochondrion
contains more than a single genome. However, it is difficult
to determine the actual number of organelles by electron
microscopy and mtDNA copy number by PCR in the
same oocyte because these procedures use mutually
exclusive protocols. One approach currently under investigation
in our laboratory uses quantitative mitochondriaspecific
fluorescence (Van Blerkom et al. 2000) to provide
an initial distinction between oocytes that can be used to
select specimens for electron microscopy and mtDNA
determination. If these studies confirm that the size of the
mitochondrial complement and mtDNA copy numbers
are related, it may be possible to establish a normal
complement size consistent with competence. While
incompetence may be understood in the context of subnormal
levels, the existence of supranormal complements
presents potentially different types of metabolic issues
and questions.
Early cleavage arrest in the homozygous embryos of
certain mutant mouse strains (Ginsberg & Hillman
1975b) has been related to unusually high levels of mitochondrial
oxidative phosphorylation and correspondingly
high cytoplasmic ATP levels. A situation where mitochondrial
ATP supply exceeds cellular demand could be
toxic if associated with elevated levels of oxidative free
radical production which could cause irreversible
nuclear and mtDNA damage leading to cytoplasmic
deterioration, mitochondrial disruption and eventually
death by degenerative or apoptotic processes (Liu &
Keefe 2000, Liu et al. 2000). Whether a similar situation
can occur in the human owing to high levels of ATP
generation (Van Blerkom et al. 1995) that may be associated
with unusually high mitochondria/mtDNA copy
numbers warrants further study. The importance of this
research would be demonstrated if meiotic spindle
defects and errors in chromosomal segregation that result
in lethal aneuploidies and other chromosomal disorders
common in human oocytes and early embryos could be
directly associated with differences in mitochondrial
complement size, mtDNA copy numbers and cytoplasmic
mechanisms that regulate mitochondrial activity
(Schon et al. 2000, Dumollard et al. 2004, Eichenlaub-
Ritter et al. 2004).
Spatial remodeling of mitochondria is a common aspect of early mammalian development
Studies of several mammalian species have shown that
mitochondria undergo stage-specific changes in distribution
during oocyte maturation and early embryogenesis.
During maturation of the mouse oocyte in vitro, mitochondria
translocate to the perinuclear region along
microtubular arrays extending from perinuclear microtubular
organizing centers (Van Blerkom 1991) to form a
sphere of organelles that encloses the condensing bivalent
chromosomes and, later, the nascent metaphase I and II
spindles (Van Blerkom & Runner 1984, Tokura et al.
1993). After fertilization in the mouse (Van Blerkom &
Runner 1984), hamster (Bavister & Squirrell 2000) and
human (Van Blerkom et al. 2000), mitochondria migrate
to the perinuclear region, again by a microtubulemediated
process, to form a condensed aggregate surrounding
the opposed pronuclei. A similar transient perinuclear
accumulation occurs in each blastomere during
the early cleavage stages. It is generally thought that
spatial remodeling of mitochondria may allow higher
ambient levels of ATP to occur in regions of the cytoplasm
where stage-specific activities may have higher energy
demands (Van Blerkom & Runner 1984, Barnett et al.
1996).
Evidence that mitochondrial inheritance between cells
may be a critical determinant of human embryo viability
was reported by Van Blerkom et al. (2000), who
described the consequences of disproportionate mitochondrial
segregation during cleavage. These authors
found that the pattern of mitochondrial inheritance
between the 2- and 12-cell stages could be related to
the geometry and symmetry of the perinuclear mitochon-
drial accumulation at the one-cell stage. An examination
of the developmental progression of individual embryos
with cleavage divisions producing blastomeres of uniform
size showed that different patterns of mitochondrial
segregation had different consequences for the resulting
blastomere(s) and for the ability of the embryo to
develop progressively in vitro. Digital imaging and quantitative
analysis of mitochondria stained with organellespecific
fluorescent probes by scanning laser confocal
microscopy enabled (a) the geometry of pronuclear mitochondrial
aggregation to be determined in fully-compiled
(symmetrical distribution, Fig. 1F) and individual
(Fig. 1G) images and (b) related to the intensity of
mitochondrial fluorescence in each blastomere during
cleavage. For example, the normal appearing 8-cell
embryo shown in Fig. 1H had an asymmetric peri-pronuclear
distribution of mitochondria at the pronuclear
stage. Differences in the relative intensity of mitochondrial
fluorescence between blastomeres were detected in
the intact embryo (Fig. 1H1 and 2) and confirmed by
disaggregation into individual blastomeres and reanalysis
(Van Blerkom et al. 2002). These authors measured the
ATP contents of each blastomere and related the levels
to previously quantified mitochondrial fluorescence and
reported that disproportionate mitochondrial inheritance
resulted in blastomeres between the 2- and 12-cell
stages with very different capacities to generate ATP. Sibling
embryos with a symmetrical pronuclear aggregation
(e.g. Fig. 1F,G) showed a uniform pattern of mitochondrial
segregation and relative metabolic equivalence
between blastomeres. At the blastomere level, the consequences
of disproportionate mitochondrial segregation
ranged from arrested cell division to abrupt blastomere
swelling and lysis which these investigators suggested
were directly related to mitochondrial content and ATP
generating capacity.
If aberrant segregation patterns were confirmed to be a
critical determinant of human embryo competence, the
development of noninvasive methods for the clinical IVF
laboratory would be warranted. Such methods would
need to be noninvasive, take advantage of the ability of
mitochondria to autofluoresce at particular wavelengths
(infrared) and be of sufficient sensitivity to detect differences
between blastomeres that are developmentally
significant. From a basic science viewpoint, it would
be relevant to investigate how different patterns of
perinuclear mitochondrial aggregation are established,
especially in light of the findings by Van Blerkom et al.
(2000) that perinuclear mitochondrial geometry may be
mediated by the distribution of corresponding arrays of
microtubules which, in turn, may be influenced by intrinsic
(ATP production, pHi; Van Blerkom 2002) or extrinsic
(pH; Gaulden 1992) factors.
Mitochondria as regulatory forces in early development
In addition to ATP production, mitochondria in somatic
cells (Pozzan et al. 2000) are directly involved in the
regulation of intracellular free Ca2+ by virtue of their
ability to sequester and release this cation in response
to a variety of signals including (a) electrical fluxes
(Ichas et al. 1997), (b) Ca2+ itself, by means of Ca2+-induced
Ca2+ release (CICR) pathways (Duchen 2000)
and (c) molecular signals associated with the activation
of the apoptotic pathway (Berridge et al. 1998). The signal
for CICR can originate from storage sites such as the
smooth surfaced endoplasmic reticulum (SER), specialized
storage granules or from Ca2+ released by other
mitochondria (mCICR; Rizzuto et al. 1994, Babcock
et al. 1997, Duchen 2000, Hajnoczky et al. 2000).
Focal changes in cell physiology, including localized
changes in ambient Ca2+ levels, have been shown to
up- or down-regulate respiratory activity in the corresponding
mitochondria. This is an important means by
which somatic cells undergoing spatial remodeling or
morphodynamic alterations involving mitochondrial
redistribution can keep ATP supply and demand in balance
at different locations within the cytoplasm
(Aw 2000). There is growing evidence that mitochondria
in oocytes and early embryos are involved in and subject
to the same regulatory forces that operate in
somatic cells (Rutter & Rizzuto 2000), especially with
respect to levels of intracellular Ca2+ and their respective
storage elements such as SER (Sousa et al. 1996, Liu
et al. 2001, Tesarik 2002, Van Blerkom et al. 2002,
2003, Dumollard et al. 2003, 2004).
TEM analysis of normal MII human oocytes demonstrates
that mitochondria are never far from interconnected
networks of SER (Fig. 1A). Indeed, these networks
exist as spherical domains of cisternae in which mitochondria
surround or are contained within their matrix (Fig.
1I), or both (Van Blerkom 2002, Makabe & Van Blerkom
2004). For the entire oocyte, TEM methodology cannot
efficiently determine the distribution and geometry of the
SER system. However, studies of human oocytes using ERspecific
fluorescent probes derived from the ‘Dil family’ of
lipophilic carbocyanine analogs (Molecular Probes,
Eugene, OR, USA) demonstrate that virtually every discrete
SER complex can be imaged by scanning laser confocal
microscopy, and that their relative density and
distribution within the ooplasm can be determined. In this
respect, it is reassuring that the spherical SER complexes
detected in sections of Dil-stained oocytes (15 mm; Fig. 1J)
have the same relative geometry and distribution
as those identified in TEM images that are colorized in
order to enhance the visualization of these elements
(Fig. 1A and I).
Several studies have investigated the relationship
between mitochondrial respiration and levels of intracellular-
free Ca2+ in MII oocytes and newly fertilized eggs. A direct role for mitochondria in the regulation of
intracellular free Ca2+ was indicated in the study of Liu
et al. (2001) who showed that by segregating mouse
oocyte mitochondria and SER into separate compartments,
the former was involved in the clearance of Ca2+ released
by the latter, thus mediating the establishment of Ca2+
oscillations during activation. These authors also proposed
that mitochondrial Ca2+ sequestration and release were
related to their metabolic activity. More recently, Dumollard
et al. (2003, 2004) reported the following for unfertilized
MII and newly penetrated mouse oocytes: (a)
mitochondrial oxidative metabolism is a major and developmentally
critical source of ATP at these stages, (b) high
ATP turnover rates at MII suggest that mitochondrial ATP
production and utilization are closely balanced and (c)
sperm-triggered Ca2+ oscillations are transmitted to mitochondria
and directly stimulate (up-regulate) mitochondrial
respiration in concert with these ionic transients. The
importance of these findings is related to the suggestion
that by limiting the up-regulation of Ca2+ to specific cytoplasmic
regions in which developmental activities occur
that have transiently higher ATP demands, a relatively
constant net level of cytoplasmic mitochondrial oxidative
metabolism can be maintained in the oocyte that meets
differential energy demands while reducing the potential
for generating damaging or toxic levels of reactive oxygen
species (Dumollard et al. 2004).
As noted previously, stage-specific changes in mitochondrial
distribution occur during oocyte maturation,
pronuclear formation and early cleavage. If the SER
co-translocates with mitochondria, a metabolic regulatory
pathway may exist in which transient domains of differential
ionic signaling could upregulate mitochondrial
metabolism in order to support location-specific developmental
processes. Developmental processes that may
have higher energy demands and involve regional
domains of SER–mitochondrial complexes could include
germinal vesicle breakdown, formation of the first and
second metaphase spindles, pronuclear evolution and
movement, and the establishment of the first mitotic
spindle. Another possible consequence of establishing
differential ATP generating domains is that clusters of
mitochondria with different levels of oxidative phosphorylation
can locally change physiological characteristics
such as intracellular pH (Aw 2000). These focal
changes could, in turn, influence other cytoplasmic
activities, such as the capacity to promote microtubular
polymerization (Van Blerkom et al. 2000). This notion is
currently under investigation in our laboratory and, if
supported experimentally, could offer insight into how
certain developmental processes are compartmentalized
and focally regulated in a stage-specific manner during
early mammalian development.
Is mitochondrial polarity ( ) related to competence?
Mitochondrial polarity is a measure of the inner mitochondrial
membrane potential () and differences in
magnitude have been related to levels of respiration and
the ability of these organelles to participate in the regulation
of Ca2+ homeostasis. Mitochondrial respiration
involves outward proton pumping across the inner mitochondrial
membrane that creates a proton gradient that
has two components, a and a pH gradient, with the
energy stored in either component driving the conversion
of ADP to ATP by respiratory chain enzymes. The
relationship between and oxidative metabolism
first described by Mitchell & Moyle (1967) has been studied
in somatic cells with -sensitive fluorescent
reporter probes. One particular molecule, 5,5'6,6'-tetrachloro-
1,1,3,3'-tetraethylbenzimidazolycarbocyanine iodide
(JC-1), has become widely used to assess the relative
magnitude of since its specificity as a polarity reporter
was first described by Reers et al. (1991) and shown by
Smiley et al. (1991) to be able to detect domains of high
and low polarity within individual mitochondria. At
relatively low potentials (<100 mv), JC-1 usually exists as
a monomer with green fluorescence detected in the fluorescein
isothiocynate (FITC) channel. However, as the
potential increases (>140 mv) JC-1 monomers multimerize
to form metastable stacks or arrays termed J-aggregates
(Reers et al. 1995), named after Jelley (1937) who first
described the physical and chemical properties of these
unusual crystal-like carbocyanine multimers. JC-1 multimerization
shifts the fluorescence emission maxima to
longer wavelengths such that high or hyperpolarized
mitochondria in cultured somatic cells appear in the
rhodamine isothiocynate (RITC) channel as intense
orange-to-red fluorescent rods.
The unique capacity of JC-1 to report differences in
has been used in a variety of studies of somatic cells
under normal and experimental conditions, and more
recently, for mammalian oocytes and embryos to examine
the association between developmental competence and
mitochondrial activity (Wilding et al. 2001, 2002, 2003,
Van Blerkom et al. 2002, 2003, Acton et al. 2004, Jones
et al. 2004). In MII mouse and human oocytes and
embryos (Ahn et al. 2002, Van Blerkom et al. 2002, 2003,
Jones et al. 2004), the highest intensity of J-aggregate fluorescence
is reported to occur in the pericortical/subplasmalemmal
cytoplasm (arrows, Fig. 1K). If mitochondria
with different polarities are compartmentalized within the
oocyte, the apparent pericortical distribution of highpolarized
mitochondria is of particular interest if related
to metabolic or ionic activities, because it suggests a
mechanism by which domains of differential function
could be established. Although this is an appealing notion
from a development viewpoint, whether the distinct pericortical
complexes of SER and putative high-polarized
mitochondria detected in human oocytes (Van Blerkom
et al. 2002) are functional in this regard remains to be
determined. However, studies designed to test this hypothesis
need to consider the findings of Van Blerkom et al.
(2003) and Dumollard et al. (2004), who demonstrated
that owing to reversal of ATP synthase activity under conditions
of electron transport inhibition, a sufficient to
maintain J-aggregate formation can persist in mouse
oocytes, despite a significant reduction in mitochondrial
ATP generating capacity. Therefore, high levels of mitochondrial
respiratory activity and hyperpolarization
detected by J-aggregate formation are not necessarily
synonymous. Indeed, it has been suggested that mitochondria
which are actively involved in ATP synthesis undergo
continuous dissipation, which should be reflected by
an average low- rather than high-polarized condition
(Diaz et al. 1999).
While it is unclear whether high mitochondrial polarity
and elevated respiratory activity are related during early
development, there is some evidence that differences in
may be related to competence. Ahn et al. (2002)
reported that thawed 2-cell mouse embryos that failed to
divide or that developed abnormally showed a cryopreservation-
associated loss of mitochondrial hyperpolarization
in the subplasmalemmal domain. Jones et al. (2004) found
that cryopreservation of MII human oocytes was
accompanied by loss of the hyperpolarized pericortical
mitochondrial domain that characterized the fresh oocyte.
Thawed oocytes also exhibited a significantly reduced
capacity to up-regulate levels of intracellular free Ca2+ in
response to Ca2+ ionophore stimulation. The studies of
Wilding et al. (2001, 2002, 2003) on human oocyes and
embryos produced by IVF suggest that differences in the
ratio between high and low polarized mitochondria may
reflect aberrant mitochondrial distributions in the oocyte
and metabolic defects in the embryo that could result in
lethal chromosomal segregation errors. Acton et al. (2004)
reported that differences in mouse embryo competence
were related to the ratio of high-to-low polarized mitochondria
that, in turn, was related to whether fertilization
occurred in vivo or in vitro. Furthermore, these authors
reported that an increased ratio of high-to-low polarized
mitochondria was associated with increasing degrees of
fragmentation during cleavage of human embryos after
IVF. Van Blerkom et al. (2002) and Jones et al. (2004)
noted that within cohorts, some human oocytes exhibited
no detectable J-aggregate fluorescence while for others,
intense fluorescence occurred throughout the cytoplasm.
In the mouse, intense cytoplasmic J-aggregate fluorescence
was associated with abnormally elevated levels of
intracellular Ca2+ in activated oocytes (Van Blerkom et al.
2003). Whether the differences in are directly
related to competence or are consequences of other
developmentally lethal perturbations needs further investigation.
For example, Jones et al. (2004) suggested that
positive outcomes after cryopreservation may be derived
from those relatively few MII human oocytes in which cortical
domains of mitochondria remained hyperpolarized
after thawing, while loss of hyperpolarization in 2-cell
mouse embryos was coincident with changes in cell membrane
fluidity and subplasmalemmal actin microfilament
integrity (Ahn et al. 2002).
At present, the relationship between mitochondrial
function, intracellular Ca2+ and developmental competence
may be one that warrants detailed analysis with
respect to . Ozil & Huneau (2001) demonstrated that
experimentally reducing the mobilization of intracellular
free Ca2+ to levels slightly below those measured at
oocyte activation had lethal downstream consequences
first detected during organogenesis, i.e. after implantation.
Indeed, embryo performance in vitro during the preimplantation
stages was unremarkable with no evident morphological
indications that lethal defects would occur
several days after uterine transfer. The possibility that
differences in the state of mitochondrial polarization
between MII human oocytes is a factor in outcome after
embryo transfer, is an intriguing one, especially in view of
current pregnancy results with thawed oocytes that
demonstrate high frequencies of postimplantation failure
(Jones et al. 2004).
If spatial differences in within oocytes and blastomeres
are developmentally relevant, how do domains of
high and low polarized mitochondria develop? Diaz et al.
(1999) showed that whether mitochondria located beneath
the plasma membrane of cultured cell lines were high or
low polarized was directly influenced by the presence or
absence of intercellular contacts. Van Blerkom et al. (2002)
described very similar findings for mouse and human
oocytes and cleavage-stage embryos. For example, J-aggregate
fluorescence did not develop in pericortical regions in
experimentally manipulated oocytes where patches of
cumulus and corona cells remained intact, but did so after
their physical elimination. For cleavage-stage mouse
embryos, low polarized mitochondria predominated at
regions of intercellular contact. When embryos were disaggregated
and individual blastomeres repositioned, formerly
J-aggregate-negative cortical domains were J-aggregatepositive,
and vice versa. In the same study, these authors
reported that the ICM in both expanded human (Fig. 1L)
and mouse blastocysts contained low polarized mitochondria.
More recent studies with peri-implantation mouse
blastocysts (J Van Blerkom, H Cox & P Davis, unpublished
observations found that the highest levels of J-aggregate fluorescence
occurred in the abembryonic mural trophectoderm
and diminished significantly towards the polar
trophectoderm, where J-aggregate fluorescence was
undetectable or scant (Fig. 1M and O). The ICM and polar
trophectoderm both showed low polarized mitochondria
(Fig. 1N). These authors suggested that cell- and locationspecific
differences in seemed to be related to the
nature and extent of intercellular contact, with the abembryonic
mural trophoblast in the preimplantation mouse
embryo containing the only cells devoid of such contacts.
A possible developmentally significant relationship
between high DC and cell-specific function was indicated
in the study mentioned above (J Van Blerkom, H Cox & P
Davis, unpublished observations) by two dynamic processes
localized to the abembryonic trophectoderm: (a)
the elaboration of highly motile finger-like projections and
(b) the formation of enlarged cellular protrusions. The trophectodermal
projections are thought to be cytoplasmic
extensions involved in the initial stages of embryo contact
with and invasion into the endometrial epithelium (Bergstrom
& Nilsson 1976), and the enlarged cellular protrusions
are likely indications of transformation of
abembryonic mural trophoblast into nondividing, polyploid
giants cells (Maris et al. 1988). While these studies
suggest that differential mitochondrial polarity and cell
function/activity may be related during embryogenesis,
confirmation will require more critical studies in which
cell and embryo fate is determined when is
experimentally manipulated. Nevertheless, if additional
research continues to support the notion that mitochondrial
polarity, respiration and focal regulation of intracellular
Ca2+ are related in important ways during early
development, it may offer a new basis for understanding
how competence is established in the oocyte and maintained
in the embryo. For clinical IVF, it may be relevant
to ask whether intrafollicular factors or conditions that can
affect competence (Van Blerkom 2002) do so by influencing
these activities.
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