Cytoplasmic Transfer

Cytoplasmic transfer in oocytes: biochemical aspects

Rachel Levy1,4, Kay Elder2 and Yves Menezo3

1Laboratoire de Biologie de la Reproduction, Hopital Nord, 42055 Saint Etienne, France, 2Bourn Hall Clinic, Bourn, Cambridge

CB3 7TR, UK and 3IRHLaboratoire Marcel Merieux, 1 rue Laborde, 69500 BRON, France

4To whom correspondence should be addressed. E-mail: [email protected]

Cytoplasmic control of preimplantation development is not a `new' concept; the rst cytoplasmic transfer

experiment was performed in the mouse during the early 1980s, as a means of overcoming cleavage arrest at the

2-cell stage, the `2-cell block'. Since the rst human pregnancy following the transfer of cytoplasm from donor

oocytes into the oocytes of a patient with a history of poor embryo development and recurrent implantation failure

in 1997, >30 children have been born after direct injection of ooplasm from fresh, mature or immature, or

cryopreservedthawed donor oocytes into recipient oocytes via a modied ICSI technique. Transfer of ooplasm was

thus applied with astonishing speed in humans, in the absence of extensive research to evaluate the efcacy and the

possible risks of the method. This review focuses on biochemical mechanisms by which transfer of ooplasm might

confer a benet: by correcting a putative imbalance between anti- and pro-apoptotic factors and/or correction of

defective mitochondrial membrane potential. We also emphasize the `empirical' state of this technique, and the

related risks.

Key words: apoptosis/epigenetic risk/ooplasm transfer

Introduction

Cytoplasmic control of preimplantation development is not a

`new' concept; the rst cytoplasmic transfer experiment was

performed in mouse during the early 1980s, as a means of

overcoming cleavage arrest at the 2-cell stage, the `2-cell block'.

Transfer of cytoplasm from embryos without cleavage arrest to

those from strains that did arrest alleviates this 2-cell block,

allowing the embryos to continue development in vitro

(Muggleton-Harris and Whittingham 1982). In 1997, Cohen et al.

announced the rst human pregnancy following the transfer of

cytoplasm from donor oocytes into the oocytes of a patient with a

history of poor embryo development and recurrent implantation

failure. The live birth of ve healthy children was reported by the

end of 1998 (Cohen et al., 1998), and this method was then

proposed as a means of restoring some unknown defects in oocytes

from patients with repeated implantation failure, using a bolus of

donor cytoplasm via the transfusion of unidentied factors (mRNA

or proteins) into the recipient defective oocyte. Since then, >30

children have been born after direct injection of ooplasm from

fresh, mature or immature, or cryopreservedthawed donor

oocytes into recipient oocytes via a modied ICSI technique

(Cohen et al., 1997, 1998; Lanzendorf et al., 1999; Huang et al.,

1999; Dale et al., 2001; Hwang et al., 2002). Transfer of ooplasm

was thus applied with astonishing speed in humans, in the absence

of extensive research to evaluate the efcacy and the possible risks

of the method. As a result, the potential dangers (De Rycke et al.,

2002; Templeton, 2002; Winston and Hardy, 2002) and unpre-

dictable outcomes (Cummins 2001, 2002) of this technique have

been highlighted in recent publications.

Following ovulation, zygote survival depends almost exclu-

sively on maternal mRNA and proteins that accumulate during

oocyte growth and maturation, until the stage where the new

zygote genome is activated, the maternal-to-zygotic transition

(MZT), which happens during the 48-cell stage in humans

(Braude et al., 1988). Maternal transcripts are thus responsible for

the rst few cleavage divisions and for transition of the maternally

controlled zygote into an activated embryonic genome (Latham,

1999). Therefore, the quality of the oocyte cytoplasm, whether or

not manipulated, will establish the future of the embryo. This

review focuses on biochemical mechanisms by which transfer of

ooplasm might confer a benet: by correcting a putative imbalance

between anti- and pro- apoptotic factors and/or correction of

defective mitochondrial membrane potential. We also emphasize

the `empirical' state of this technique, and the related risks, such as

mitochondrial heteroplasmy, mitochondrial disease and nuclear

mitochondrial interaction, epigenetic aspects, and potential inu-

ence on regulatory protein polarization and on aspects of mRNA

polyadenylation.

Apoptosis

During the human life span, >99.9% of our cells undergo a

physiological suicide process known as apoptosis (Vaux et al.,

Human Reproduction Update, Vol.10, No.3 pp. 241250, 2004 DOI: 10.1093/humupd/dmh016

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1999). This cell death is dened as a process that follows an

intrinsic genetic program, activated at a controlled time. Apoptosis

is commonly used to control cell number and maintain home-

ostasis, acting as a defensive strategy by removing mutated,

damaged, superuous, unwanted or useless cells. Recently, its role

in gamete maturation and early embryogenesis has been high-

lighted (Tilly, 2001, 2003; Levy, 2001; Russell et al., 2002), and

morphological and biochemical markers of apoptosis have been

described in the mammalian pre-embryo (Jurisicova et al., 1995,

1996; Levy et al., 1998; Brison, 2000; Hardy et al., 2001). Unlike

cell death due to necrosis, apoptosis strictly affects individual

cells, with morphological features that include condensation of

chromatin at the nuclear membrane, membrane blebbing (without

loss of integrity), shrinking of cytoplasm leading to the loss of

intercellular connections, and nally the formation of membrane-

bound vesicles (apoptotic bodies) (Wyllie et al., 1980; Sanders and

Wride, 1995). Caspase substrates can regulate the key morpho-

logical changes in apoptosis. Several caspase substrates also act as

transducers and ampliers that determine the apoptotic threshold

and cell fate (Fischer et al., 2003). Mitochondria exert a decisive

role in cell death control through the release of pro-apoptotic

factors in the cytoplasm, cytochrome C and apoptosis-inducing

factor (AIF), leading to the activation of a caspase cascade

together with changes in membrane asymmetry and exposure of

phosphatidylserine (PS) on the membrane. In considering a

potential benet of cytoplasmic transfer, the impact on the

recipient oocyte should be considered in terms of balance between

pro- and anti-apoptotic factors (for review, see Levy, 2001). This

balance is regulated by both intrinsic and extrinsic factors.

Intrinsic factors

DNA fragmentation has been reported in mouse gonadotrophin-

stimulated atretic follicles and oocytes (Perez et al., 1999). Similar

results were observed in human ovarian follicles obtained after

oocyte retrieval from patients undergoing IVF. Controlled ovarian

stimulation is routinely used in clinical practice in order to obtain a

large number of oocytes, and this can sometimes lead to an

excessive follicular response to administered gonadotrophins. This

can potentially result in inadequate biochemical maturation of

oocyte cytoplasm. Oocytes with such a deciency could have an

unbalanced apoptotic system that might jeopardize the processes

of fertilization and subsequent development. In support of this

hypothesis, an increasing number of highly variable apoptosis-

related transcripts has been detected during different stages of

murine and human oocyte development (Jurisicova et al., 1998,

2003; Exley et al., 1999; Kawamura et al., 2003). The quantity,

and in particular the quality, of these transcripts is clearly a crucial

element for successful fertilization and embryo development.

Although it is not routinely possible to test each oocyte for

apoptotic transcripts before IVF, the different pathways that

regulate control of apoptosis in the oocyte have been extensively

studied.

Oral antioxidants

A recent study analysed the effect of oral antioxidants on both the

number and the quality of oocytes retrieved from aged mice after

exogenous ovarian stimulation (Tarin et al., 2002). Interestingly,

the negative effects of female ageing in oocytes retrieved from

oviducts and ovaries were counteracted by both early and late

administration of oral antioxidants, as assessed by number of

oocytes and percentage with normal metaphase II chromosome

distribution and/or morphological evidence of apoptosis. Despite

this apparently benecial effect, oral antioxidant administration is

a non-specic approach to regulation of apoptosis in oocytes.

Application of these data in clinical IVF should be made with

caution, but it might be worthy of consideration prior to the more

aggressive approach of cytoplasmic transfer. A more physiological

approach might be via manipulation of culture systems for in vitro

oocyte maturation (IVM) but so far this technique has had a

relatively low success rate compared with conventional IVF/ICSI.

Growth hormone

Recent evidence suggests that growth hormone (GH) is involved in

ovarian regulation and may act as a survival factor during in vitro

culture, reducing apoptosis by altering the bax:bcl-2 ratio during

early embryogenesis (Kolle et al., 2002). These authors analysed

the mechanisms of GH action during IVM of bovine cumulus

oocyte complexes (COC) (Kolle et al., 2003): COC matured

in vitro in the presence of GH revealed a signicantly (P < 0.05)

higher proportion of proliferating cumulus cells, and GH treatment

signicantly reduced (P < 0.05) apoptosis in the cumulus cells as

determined by terminal deoxynucleotidyl transferase-mediated

dUDP nick-end labelling (TUNEL) assay. These data suggest that

GH increases cumulus expansion by promoting cell proliferation

and inhibiting apoptosis. We have recently detected the GH

receptor in naked oocytes as well as in COC (Menezo et al., 2003).

Thompson et al. (2000) demonstrated that GH can increase the

capacity of liver cells to repair DNA, and by analogy we

hypothesize that a similar mechanism might occur in the female

gamete: GH could upgrade oocyte quality by increasing its DNA

repair capacity. This is clearly an important process at the time of

fertilization and during early embryogenesis (especially in ICSI).

In parallel, new insights have emerged over the past few years

about the role of ceramide and sphingosine-1-phosphate (S1P) as

mediators and suppressors, respectively, of cancer therapy-

induced oocyte apoptosis. These studies have allowed the

development of novel lipid-based strategies to combat apoptosis-

related infertility and premature menopause in female cancer

patients (Casper and Jurisicova, 2000; Morita et al, 2000; Spiegel

and Kolesnick, 2002; Tilly and Kolesnick, 2002). Such aetiolo-

gical strategies for restoring apoptotic balance might provide a

more elegant approach to improving oocyte cytoplasm quality than

oocyte microsurgery.

Apoptotic gene expression

An understanding of the genes responsible for regulating apoptosis

is crucial in order to elucidate the mechanisms involved and the

reasons why it occurs. Recent data report that mammalian oocytes

and preimplantation embryos possess abundant levels of tran-

scripts encoding cell death suppressor and inducer genes, and

caspase genes were also detected during all developmental stages.

Signicant differences in apoptosis-related mRNA between non-

fragmented and fragmented mouse and human embryos were also

demonstrated, suggesting that cellular fragmentation in a subset of

human preimplantation embryos could be regulated by certain

components of a genetically programmed cell death (Van Blerkom

et al., 2001; Jurisicova et al., 2003). However, Martinez et al.

(2002) also suggest that caspases in preimplantation human

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embryos are involved in developmental processes unrelated to cell

death, and therefore data regarding specic gene expression should

be interpreted/extrapolated with caution.

Apoptosis plays an active and crucial role in the developing

embryo through the removal of decient cells (Hardy, 1999), and

must be considered as a normal feature in both in vivo and in vitro

preimplantation development. However, apoptosis may also have

detrimental effects if either the number of apoptotic cells or ratio

of these cells to the normal cells exceeds a critical threshold. This

could occur both in suboptimal culture conditions in vitro and

during in vivo compromised development. Jurisicova et al. (1998)

suggest a model in which embryo development is regulated by a

balance of pro- (i.e. Bax) and anti- (i.e. Bcl-2) apoptotic gene

expression with successive checkpoints, from the time of

oogenesis throughout the preimplantation period. Mature oocytes

with appropriate anti-apoptotic mRNA will succeed through

maturation and fertilization, whereas poor quality oocytes with

high levels of cell death inducers or low levels of cell death

antagonists will fail to fertilize, or fragment and/or arrest.

An elegant mathematical model demonstrates that this apoptotic

ratio seems to be programmed at an early stage in the zygote, and

suggests that once apoptosis has been triggered, the process cannot

be disrupted or reversed. This underlines the importance of healthy

gametes as the basis for fertilization and successful healthy

embryo development (Hardy et al., 2001), and also implies that

addition of a 1015% transfer of ooplasm is unlikely to adequately

correct the balance between death promoters and death inhibitors.

Extrinsic factors

Culture conditions

Levels of cell death in several species in vitro were found to be

signicantly higher than in vivo (Papaioannou and Ebert, 1986;

Brison and Schultz, 1997; Jurisicova et al., 1998). In vitro

production also disturbs the chronology of apoptosis in bovine

embryos (Gjorret et al., 2003); this is in full agreement with

observations that gene activation can be inuenced by culture

conditions (Niemann and Wrenzycki, 2000). There is controversy

surrounding the effects of fetal calf serum supplementation on the

development of bovine embryos (Byrne et al., 1999); supplemen-

tation of culture media with amino acids has been shown to benet

preimplantation embryo development in several species (Devreker

et al., 2001), and restriction of sulphur-free amino acids induces

apoptosis (Kang et al., 2002; Lu et al., 2003). Glucose and insulin

concentrations adversely affect rat blastocyst development both

in vivo and in vitro (Pampfer et al., 1997; Pampfer, 2000; Jimenez,

2003), particularly at the blastocyst stage, where there was an

association with a higher degree of apoptosis in the inner cell mass

in vitro.

Growth factors

Growth factors are known to regulate cell proliferation and

differentiation during mammalian preimplantation development,

some also acting as survival factors (Hardy and Spanos, 2002).

Supplementation of culture medium with exogenous growth

factors such as transforming growth factor-b (TGF-b) partiallyreduced excessive in vitro apoptotic cell death without accelerat-

ing development or increasing nal cell number (Brison and

Schultz, 1997, 1998). Insulin-like growth factor I (IGF-I) was

recently conrmed to have a clear anti-apoptotic action during

blastocyst development (Herrler et al., 1998; Spanos et al., 2000).

Platelet-activating factor (PAF; 1-o-alkyl-2-acetyl-sn-glycero-3-

phosphocholine), a potent ether phospholipid, also acts as an

autocrine growth/survival factor for the mammalian preimplanta-

tion embryo (O'Neill, 1997; Stojanov and O'Neill, 1999).

Air pollution and smoking

Germinal cells are vulnerable to genetic damage from smoking,

apparently by altering the meiotic spindle of oocytes and sperm,

leading to chromosome errors that affect reproductive outcomes

(Zenzes, 2000). An understanding of the mechanisms involved

could help in the development of potential corrective intervention.

Polycyclic aromatic hydrocarbons (PAH), found not only in

cigarette smoke but also in polluted air, interact with the aryl

hydrocarbon receptor (Ahr) and induce toxicity mediated by a Bax

pathway (Zenzes, 2000; Matzuk, 2001). Cadmium nicotine and its

metabolite, cotinine, are detectable in gonadal tissues and uids in

association with smoking; cotinine is incorporated into ovarian

granulosa-lutein cells, with the potential to compromise the

follicle's developmental potential. Smoking-related effects are

detectable in ovarian granulosa-lutein cells, oocytes, sperm and

preimplantation embryos. The damage caused by these factors

might be repaired during meiosis in the germinal cells, but

ejaculated sperm have a very limited capacity for repairing

damage: smoking is associated with low sperm quality.

Transmission of altered DNA by sperm (but not yet by oocytes)

was demonstrated in preimplantation embryos, and paternal

preconception smoking has been linked to a signicantly elevated

risk of childhood cancers, particularly acute leukaemia and

lymphoma (Ji et al., 1997). If damage has already occurred in

the ejaculated sperm, addition of external cytoplasm to an oocyte

is unlikely to change the poor prognosis for embryo development

caused by the DNA-damaged sperm. Finally, several studies

suggest that cotinine can inhibit fragmentation (and apoptosis) in

developing embryos of smokers, and this acquisition of resistance

to fragmentation via smoking may confer a dangerous survival

advantage to genetically altered embryos (Zenzes, 1999, 2000). In

this case, external addition of healthy cytoplasm might confer an

increased capacity for DNA repair, if the transplanted bolus

contains the necessary repair enzymes, such as polymerase beta or

the multifunctional DNA repair enzyme APEX. Murdoch and Van

Kirk (2001) have demonstrated that polymerase beta is upgraded

by estrogens, and we were also able to demonstrate the presence of

APEX mRNA in human oocytes. Since a high capacity for DNA

repair is clearly necessary during the rst cycle of DNA

replication, when pronuclei are formed, the presence of mRNA

coding for DNA repair enzymes could be considered as a marker

of oocyte quality. In borderline `aged' oocytes, the addition of

extra `good' cytoplasm could thus enhance the prognosis for

obtaining an ongoing pregnancy.

Oxidative stress

The generation of reactive oxygen species (ROS) is a factor of

major importance: ROS are generated by several different

metabolic pathways, including oxidative phosphorylation,

NADPH oxidase and xanthine oxidase systems. ROS generation

is enhanced during in vitro culture, especially at the time

of genomic activation (Nasr-Esfahani et al., 1990; Johnson and

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Nasr-Esfahani MH, 1994). There are at least two key substrates

that exert a major inuence on metabolism in in vitro culture

conditions, glucose and sulphur amino acids, including methio-

nine. Excessive glucose levels induce apoptosis, by increasing the

level of Bax, a pro-apoptotic factor, and by inuencing the

expression of caspase 3 and caspase-activated deoxyribonuclease

(CAD) (Pampfer, 2000; Pampfer et al., 2001; Leunda-Casi et al.,

2002). In murine and bovine systems, hyperglycaemia affects sex

ratio through apoptosis, related to the production of X-linked

inhibitor of apoptosis protein (XIAP) (Jimenez et al., 2003).

An imbalance in oxidative stress can also induce apoptosis

(Korsmeyer et al., 1993, 1995). In preimplantation human

embryos, both H2O2 concentration and DNA fragmentation levels

are higher in fragmented than in non-fragmented embryos (Yang

et al., 1998). We recently demonstrated (Oger et al 2003) that

sperm DNA fragmentation is also linked to ROS. Over a certain

limit of fragmentation, DNA cannot be repaired or will be repaired

with a high level of aberrations, thus leading to possible mutations.

In the oocyte, the oxidation product of sperm DNA, 8 oxo-guanine,

may be removed from oxidatively damaged DNA by classical base

excision repair (Bessho et al., 1993). When base excision repair is

absent or drops below a critical threshold, mistakes are introduced,

with G:C replaced by T:A. This mutation may lead to

carcinogenesis (Boiteux and Radicella 1999, 2000). On this

basis, addition of exogenous high quality cytoplasm that boosts

DNA repair systems could provide an advantage for further

embryonic development

Can apoptosis in the preimplantation embryo be prevented?

There is increasing evidence that human development is regulated

before implantation by factors derived from both the oocyte and

the embryo, including growth factors and apoptosis-related genes.

Ooplasm donated from a healthy oocyte is presumed to provide

specic endogenous survival growth factors, mRNA and/or

appropriate anti apoptotic mRNA to restore the balance of

apoptosis-related mRNA. Recurrent high levels of fragmentation

and poor embryo quality and development have been an indication

for cytoplasm donation, and it is of interest to note that the

pregnancies reported after cytoplasmic transfer (CT) were asso-

ciated with a signicant reduction in embryo fragmentation

(Lanzendorf et al., 1999; Dale et al., 2001). However, CT did

not enhance success in women with advanced reproductive age or

poor ovarian reserve; in these cases there was no improvement in

embryo quality, and pregnancies resulted in biochemical losses

and aneuploid clinical loss (Opsahl et al., 2002). Transfer of

ooplasm can also introduce hazardous elements, by increasing the

level of apoptosis in the embryo. Since the process of apoptosis in

the preimplantation embryo is not fully elucidated and the anti- or

pro- apoptotic factors have not yet been fully identied, CT is

applied on a solely empirical basis. Progress in molecular

technology has greatly increased our knowledge about the number

of known related apoptosis genes expressed in the oocyte and

during embryonic development. Quantitative and real-time

RTPCR or a microarray methodology applied to mRNA

phenotyping could provide helpful tools to investigate the nature

of the transferred cytoplasmic elements. Survivin is a member of

the IAP (inhibitors of apoptosis) family that can bind to caspases

and modulate their function. This factor is present at the mRNA

and protein level in the preimplantation embryo, and is a potential

candidate as one of the anti-apoptotic factors (Kawamura et al.,

2003).

Although preventing apoptosis might appear to be benecial,

the potential effect of its prevention on further embryo develop-

ment must also be considered. For example, apoptosis is the

developmentally acquired phenomenon that occurs in embryos

exposed to elevated temperature. Experimental inhibition of group

II caspases mediating heat-induced apoptosis in bovine embryos

had a clear detrimental effect on embryonic resistance to heat

shock. Apoptosis after heat shock can be viewed as an adaptative

mechanism, benecial in allowing embryonic survival and devel-

opment following stress (Paula-Lopes and Hansen, 2002a,b;

Paula-Lopes et al., 2003).

Mitochondria

Correction of defective mitochondrial membrane potential

Healthy mitochondria are essential for accurate chromatid segre-

gation at the time of fertilization and during subsequent mitotic

division. Mitochondria in the cytoplasm are responsible for

respiratory processes and ATP production (OXPHOS). They also

generate ROS, which are deleterious for biological material,

including DNA. It has been proposed that women with a history of

IVF failure produce poor quality oocytes that may contain ageing

mitochondria, resulting in decreased energy production. Chaotic

mosacism has also been related to low mitochondrial potential

(Wilding et al., 2003). Since aged or defective mitochondria might

result in aberrant chromosomal segregation or developmental

arrest, injection of a bolus of cytoplasm from healthy donor

oocytes could potentially restore global mitochondrial activity and

rejuvenate recipient oocytes from women with recurrent IVF

failure (Wilding et al., 2001).

In addition to their function in cellular respiration, mitochondria

also have a central role in the apoptotic-signalling pathway.

Cellular anomalies in function at any level are eventually

translated into the release of apoptogenic factors from the

mitochondrial intermembrane space, resulting in the demise of

the cell. These pro-apoptotic mitochondrial factors may contribute

to both caspase-dependent and caspase-independent processes in

apoptotic cell death (Ravagnan et al., 2002; van Gurp et al., 2003).

Mild oxidative treatment has been shown to induce a decline in

mitochondrial membrane potential in mouse zygotes, suggesting

that oxidative stress induces programmed cell death (PCD), and

that mitochondria are involved in the early phase of oxidative

stress-induced PCD. Mitochondrial malfunction may thus con-

tribute to cell cycle arrest, triggering mild oxidative stress that is

followed by cell death (Liu and Keefe, 2000; Liu et al., 2000). Liu

et al. tested this hypothesis by using reconstructed zygotes with

nuclei and cytoplasm from H2O2-treated or untreated zygotes, and

transfusing healthy mitochondria in order to correct decient

organelles and suppress PCD. Arrested, reconstituted zygotes

displayed TUNEL staining indicative of apoptosis at a rate similar

to that of H2O2-treated controls, suggesting that apoptotic potential

could be transferred cytoplasmically. Reconstituted zygotes

derived from H2O2-treated pronuclei mixed with untreated

cytoplasm showed signicantly increased rates of cleavage and

development to morula and blastocyst, indicating that healthy

cytoplasm could partly rescue pronuclei from oxidative stress.


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Although oxidation had an effect on both nuclei and cytoplasm,

cytoplasm was more sensitive to the oxidative stress. This

evidence suggests that cytoplasm, and most probably its

mitochondrial component, plays a central role in mediating

development as well as apoptotic cell death induced by oxidative

stress in mouse zygotes.

Mitochondrial heteroplasmy, mitochondrial disease and

nuclearmitochondrial interaction

The presence of donor mitochondria was detected in the cells of

two out of 15 children at 1 year of age (Barritt et al., 2000, 2001a;

Brenner et al., 2000) following the clinical use of cytoplasmic

transfer from donor oocytes during IVF treatment. This hetero-

plasmy has been described as a possible therapeutic effect (Malter,

2002a,b). It has also raised signicant concerns regarding the

safety of the technique (Brenner et al., 2000). Symptomatic

mitochondrial disease due to mtDNA mutation has a very low

population frequency (1:8000), and therefore a theoretical risk of

mitochondrial disease transmission associated with ooplasmic

transfer seems unlikely. It could even be argued that ooplasmic

transplantation represents a methodology that might reduce the

random transmission of such mitochondrial diseases compared

with standard oocyte donation (Malter, 2002a). There is a high

degree of mitochondrial homoplasmy throughout the body, and

mitochondrial function is controlled by a complex combination of

nuclear and mitochondrial genes. Proteins required for mitochon-

drial function are synthesized in the cytoplasm and imported into

the mitochondrial matrix, and survival of mitochondrial genotypes

is critically dependent on the nuclear background. The dual nature

of this control means that the introduction of `foreign' mitochon-

drial DNA via ooplasmic transfer can introduce a conict between

the control mechanisms for nuclear DNA, recipient mtDNA and

donor mtDNA, leading to unpredictable outcomes (Cummins,

2001). The potential conict caused by mitochondrial hetero-

plasmy after fertilization is thought to be one of the evolutionary

reasons behind their uniparental inheritance. The elimination of

one set of mitochondrial genes at fertilization avoids the possi-

bility of lethal conict with the nuclear genes, and thus

mitochondria pass through a bottleneck during oogenesis or

early embryogenesis, with clonal expansion from one or a few of

these organelles resulting in potential restoration of homoplasmy.

Mitochondria are maternally inherited, so that if the child is

female, it is possible that both donor and recipient mitochondria

may be transmitted to future generations.

In conclusion, mitochondria are probably the main organelle

transferred during injection of cytoplasm, but the manner in which

they positively affect embryo rescue, especially their central role

in the apoptotic signalling pathway, needs further investigation.

The same conclusion may be applied in considering potentially

deleterious effects of mitochondrial inheritance after ooplasm

transfer in humans.

Chromosomal abnormalities following ooplasmictransplantation in humans

Multinucleation is a frequently observed phenomenon in early

human preimplantation embryos cultured in vitro, commonly

associated with impaired cleavage, poor embryo quality and

increased fragmentation, all of which may compromise their

implantation potential (Balakier and Cadesky, 1997; Royen et al.,

2003). This abnormality in human embryos has been strongly (but

not exclusively) correlated with embryo chromosomal abnormal-

ities (Kligman et al., 1996). Multinucleated embryos have a

substantially reduced viability, expressed as early cleavage arrest

and the formation of abortive blastocysts during in vitro culture;

this indicates that the phenomenon is highly lethal. Transfer of

cytoplasm from donor oocytes has been proposed as a strategy to

rescue these morphologically abnormal embryos.

Follow-up reports of pregnancies and infants born after

ooplasmic transplantation reveal that two out of 17 fetuses had

an abnormal 45,XO karyotype (Barritt et al., 2000, 2001b). One

singleton pregnancy ended in a spontaneous miscarriage in the rst

trimester with a fetal karyotype of 45,X0, and ultrasonography at

15 weeks gestation conrmed a twin gestation in the second, with

one fetus developing abnormally. Amniocentesis performed on the

abnormally developing twin revealed a 45,X0 karyotype, and this

twin was electively reduced. The authors hypothesize that there

may be a link between the chromosomal anomalies and oocyte

manipulation.

In addition to the two X0 fetuses, one of the babies born was

diagnosed at 18 months with `pervasive developmental disorder',

one of a spectrum of sex-linked autism-related diagnoses with a

high incidence, 1 in 250 children. Whether or not these sex-linked

chromosomal anomalies and the mechanism of appearance are

linked specically to cytoplasmic transfer is a matter of contro-

versy.

Epigenetic aspects

The possibility that `foreign' cytoplasm might exert an epigenetic

effect upon maternal and paternal genomes must also be

considered. Different genotypes of mice inuence maternal and

paternal genomes differently (Baldacci et al., 1992; Reik et al.,

1993; Renard et al., 1994; Roemer et al., 1997; Latham and

Sapienza, 1998; Pardo-Manuel de Villena et al., 2000; Pickard

et al., 2001). Specic manipulations of early mouse embryos result

in altered patterns of gene expression and induce phenotypic

alterations at later stages of development. Repression and DNA

methylation of genes encoding major urinary proteins (MUP),

repression of the gene encoding olfactory marker protein, and,

interestingly, reduced body weight, can be experimentally induced

by nuclear transplantation. Disturbingly, these acquired pheno-

types can be transmitted to most of the offspring of manipulated

parent mice (Roemer et al., 1997). Recent studies demonstrated

that blastomere fragmentation at the 2-cell stage in mouse embryos

is under the control of multiple genetic loci, affected by both

parental genotypes, possibly as a result of either genomic

imprinting or differences in mitochondrial origin (Hawes et al.,

2001, 2002). Transfer of cytoplasm could therefore also have

immediate adverse effects on early embryo development through

blastomere fragmentation and/or apoptosis.

Renard et al. (1994) reported a maternal effect on blastocyst

formation. DDK males bred with DDK females are relatively

fertile; DDK females cross-bred with males of other strains exhibit

reduced fertility, whereas the reciprocal crosses between non-

DDK females and DDK males are fertile. The reduction in fertility

in the former case results from an incompatibility between the

DDK maternal cytoplasm and the non-DDK paternal genome,


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expressed during morula-to-blastocyst transition. The active

molecule required is transmitted as mRNA that interacts with

the paternal genome. Transfer of ooplasm or oocyte RNA from the

inbred mouse DDK strain to non-DDK oocytes converts the

oocytes to a DDK phenotype, with subsequent post-zygotic arrest.

Although it is difcult to speculate on the possibility of a similar

situation in humans, it cannot be completely excluded. In such a

case, injection of healthy mRNA could not provide any form of

rescue for the embryos.

Finally, Pickard et al. (2001) demonstrated that the genotype of

the mother can inuence the epigenotype of the offspring by a

novel epigenetic marking process at fertilization which targets

DNA for later methylation in the fetus.

In vitro culture conditions can also alter the DNA methylation

imprinting process, mediated by S-adenosyl methionine (SAM).

The embryo has an SAM synthase, and SAM is synthesized in the

oocyte and in mouse and human embryos before genomic

activation (Menezo et al., 1989). The level of SAM can be

disturbed by perturbation of the endogenous methionine pool

(Menezo et al., 1989). Inhibitors of SAM synthesis, as well as

deciency in the endogenous methionine pool, signicantly affect

embryo development and may even induce apoptosis through

deprivation of sulphur amino acids (Kang et al., 2002; Lu et al.,

2003). This induces programmed cell death independently of

caspase activation, thus increasing apoptosis in the embryo (Son

et al., 2001; Kang et al., 2002). Methionine is incorporated by

mouse, bovine and human embryos (Menezo et al, 1989; Guyader-

Joly et al., 1997), and both methionine (Guyader-Joly et al., 1996)

and cysteine affect glutathione levels and glutathione-related

enzyme activities via trans-sulphuration pathways. It is important

to note that glutathione is one of the most important factors in

preventing the negative effects of ROS, thus reducing apoptosis.

Finally, methionine restriction alone may induce cell death

through caspase-dependent and -independent pathways (Lu et al.,

2003). All of this evidence illustrates that a misunderstanding of

embryo metabolism with respect to a single class of amino acids

may have a devastating effect on embryo development, especially

when oocytes initially have a borderline endogenous pool.

Borderline culture conditions can increase apoptosis in the

embryo, and this can be avoided by a careful understanding of

the biochemical pathways involved. If addition of `high quality

cytoplasm' helps to correct the biochemistry, it may be that

simpler, more direct approaches might also be effective.

Polarization of regulatory proteins

Developmental signicance of regulatory protein polarization in

early preimplantation embryos

Recent evidence suggests that the differentiation of individual

blastomeres may be initiated during very early steps of embryo

development. The regulatory protein leptin and the transcription

factor STAT 3 displayed polarized distribution in both mouse and

human oocytes. After fertilization, these polarized domains

become differentially distributed between blastomeres in the

cleavage stage embryo and between the inner cell mass and

trophoblast of the blastocyst (Antczak and Van Blerkom, 1997).

The pattern of inheritance of the polarized domains in daughter

blastomeres appears to be associated with the manner in which

successive equatorial or meridional planes of cell division interact

with the polarized protein domains (Edwards and Beard, 1997).

Similarly, considerable variation in the concentration of b-actinand IL-1 mRNA between individual blastomeres originating from

the same 68-cell stage embryo were noted (Krussel et al., 1998).

This phenomenon of regulatory protein polarization during early

human development also exists for transforming growth factor b2(TGFb2) and vascular endothelial growth factor (VEGF), theapoptosis-associated proteins Bcl-x and Bax, and the growth factor

receptors c-kit and epidermal growth factor receptor (EGF-R)

(Antczak and Van Blerkom, 1999).

It has been postulated that the localization of the polarized

protein domains to the plasma membrane and subjacent cytoplasm

exposes them to membrane extrusions which may occur during

fragmentation or biopsy. Loss of non-polarized proteins, such as

E-cadherin, has no developmental consequences, whereas spa-

tially limited blastomere fragmentation may deeply alter embryo

development if apoptosis proteins (Bcl-x and Bax) exist in a

polarized domain.

These ndings might have a clinical signicance in cytoplasmic

transfer. Is the transfused cytoplasm representative of the donor

oocyte as a whole? Does the injection procedure disturb recipient

oocyte polarization, with possible detrimental effects on subse-

quent development? The injection of foreign ooplasm into a

crucial polarized domain in the recipient can be hazardous for

subsequent embryo development if it alters regulatory protein

polarization in the early preimplantation embryos.

mRNA polyadenylation

The oocyte and the early preimplantation embryo inherit a stock of

mRNA synthesized during oogenesis and the nal stages of oocyte

maturation; these maternal mRNA drive the rst cleavage

divisions of the early embryo. At least two known mechanisms

are involved in the recruitment of mRNA for translation, the

stability of the message and its translation potential. Oocyte

maturation in the mouse is linked to a drop in the global quantity of

mRNA transcripts, and this drop continues after fertilization (Piko

and Clegg, 1982), with different messages being degraded at

widely diverse times. Some mRNA are rapidly degraded (ZP3),

and degradation is delayed for others.

Activation of oocyte mRNA translation is regulated via the

length of its poly (A) tail, involving cytoplasmic polyadenylation

element binding protein (CPEB), cleavage and polyadenylation

specic factor (CPSF) and poly(A) polymerase (PAP) (Dickson

et al., 2001; Hodgman et al., 2001).

Specic kinetic changes in polyadenylation contribute to gene

expression in embryos, and these changes inuence further

development (Brevini-Gandol et al., 1999; El Mouatassim

et al., 1999; Brevini et al., 2002). Inadequate cytoplasmic

maturation is linked to poor regulation of the mRNA polyadenyla-

tion process, and this leads to altered developmental competence

(Brevini-Gandol et al., 1999; El Mouatassim et al., 1999; Brevini

et al., 2002). The mRNA coding for Oct-4 (involved in early

differentiation and totipotency), connexin-32 and -34 (important

for blastocyst formation), for protection against free radicals and

for polyadenylate polymerase are involved in this process of ne

regulation. Injection of `good quality cytoplasm' might possibly

be benecial by assisting the process of polyadenylation via

injection of adenosine 3-5 biphosphate (PAP) and cytoplasmic


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polyadenylation element (CPE), and by elongating the polyA tail

of some mRNA. On the other hand, some of the polyadenylated

mRNA are submitted to a gradual reduction of their polyA tail

(connexin-43 and Oct-4), and in this case, injection of exogenous

factors could disturb the developmental process by introducing

asynchrony into the embryonic cell cycle. Extra foreign cytoplasm

might interfere more specically during the maternal-to-zygotic

transition cycle, with detrimental consequences. On the basis of

mRNA status in recipient and donor cytoplasm, cytoplasmic

transfer is of questionable value.

Conclusion

Based on the observations of Muggleton-Harris and Whittingham

(1982) in the mouse, the concept of ooplasmic transplantation is

currently very interesting. However, the indications for applying

this technology in human clinical practice need to be clearly

dened. Although there may be theoretical benets of providing

`appropriate' but unidentied factors and/or presumably healthy

mitochondria, the potential side-effects of these techniques must

not be neglected. Relevant research concerning the role of

apoptosis and the expression of apoptosis-related genes in oocyte

and preimplantation embryo, with particular attention to mito-

chondria, could certainly be of benet to infertile couples in the

near future. At present, in the absence of validation by proper cell

culture experiments or detailed animal research, the application of

such therapies in humans is difcult to justify.

Furthermore, the paternal contribution to successful embry-

ogenesis must not be ignored (Menezo and Dale, 1995). The

marked effect of sperm DNA fragmentation on embryo develop-

mental potential and unexplained recurrent pregnancy loss is now

well documented (Evenson et al., 2002; Carrell et al., 2003).

Several studies have provided evidence to support the conclusion

that sperm contain a complex repertoire of mRNA. Ostermeier

et al. (2002) have recently demonstrated a biochemical contribu-

tion of the male gamete and its complementary role in fertilization

and preimplantation embryogenesis. On this basis, it is difcult to

assume that faulty early preimplantation embryo development (i.e.

fragmentation) could be overcome by the addition of extra foreign

ooplasm alone.

The treatment of infertile patients based upon their aetiology

can be enhanced by an extensive understanding of the basic

physiologybiochemistry of the human oocyte and early embryo.

Before embarking upon `surgery and grafts' in preimplantation

embryos, it is now time to consider improving our knowledge

concerning embryo metabolism and its relation to culture condi-

tions based upon facts and evidence. This raises the question of

how suitable oocytes for donating ooplasm should be identied or

dened. It is obvious that criteria that will allow this evaluation are

not yet available for clinical IVF, and will only be elucidated

through fundamental basic research.

Aberrant methylation patterns at the 2-cell stage in mice have

recently been identied under conditions of `ordinary' in vitro

culture (Shi and Haaf, 2002). In humans, major birth defects have

been observed after assisted reproduction treatment by Hansen

et al. (2002), but more importantly, an intriguing excess of

imprinting anomalies in children conceived by IVF and/or ICSI

has been reported (Cox et al., 2002; DeBaun et al., 2003; Gicquel

et al., 2003; Maher et al., 2003; Orstavik et al., 2003; Powell,

2003). This reinforces the need for continuing evaluation of our

ongoing technologies, and further questions the justication for

more aggressive microsurgical techniques (Gosden et al., 2003).

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