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Molecular Mechanisms of Trophoblast Survival:From Implantation to Birth

Andrea Jurisicova,* Jacqui Detmar and Isabella Caniggia

INTRODUCTIONProgrammed cell death (PCD) is an

evolutionarily conserved processthat has been documented to play a

vital role during development inmany diverse species including

mammals, amphibians, and in-sects. It is commonly known that

cell death is required for adjustingthe number of cells and for remov-

ing unnecessary organs such as thetadpole tail during amphibian

metamorphosis, disintegration oflarval organs during insect meta-

morphosis, elimination of sex-spe-cific organs such as the Mullerian

duct in males or the Wolffian duct infemales, as well as formation of the

digits in mammals (reviewed in Ja-cobson et al., 1997). Cell death is

also a driving force behind cellularsculpturing of embryonic struc-

tures, such as the creation of the

proamniotic cavity and the forma-

tion of tubular structures (Coucou-

vanis and Martin, 1995) including

neural tube closure (Weil et al.,

1997) and development of the earcanal (Leon et al., 2004), tooth re-

modeling (Matalova et al., 2004),

and selection of immune cells (Vaux

and Korsmeyer, 1999; Ranger et

al., 2001), as well as elimination of

misplaced, injured, or otherwise

dangerous cells such as primordial

germ cells (Molyneaux and Wylie,

2004). While this programmed cell

death is not always apoptotic in na-

ture, as both necrotic-like and au-

tophagic-like death have been ob-

served to occur in some organs

(Chautan et al., 1999; Baehrecke,2003), it is always tightly regulated

by conserved molecular pathways

that guard the decision-making and

execution of cell death machinery

(Assuncao Guimaraes and Linden,2004; Levine and Klionsky, 2004).

Thus, it is obvious that a misbal-ance in cell death can have profound

developmental consequences with awide spectrum of phenotypes, rang-

ing from mild developmental delay,retardation, induction of congenital

malformations, and defects or even

death of the embryo. In such cases,either too little or too much deathcan have detrimental consequences.

Developmental cell death also ap-

pears to be evolutionarily conserved,as embryos from invertebrates such

as C. elegans and Drosophila up tomammals express and utilize similar

molecules to activate, execute, andfinalize cell death, although the com-

plexity and redundancy in mamma-lian system is far more explicit (Meier

et al., 2000; Twomey and McCarthy,2005).

The core cell death pathways havebeen extensively reviewed else-

where (Danial and Korsmeyer,2004; Tran et al., 2004; Kroemer

and Martin, 2005; Strasser, 2005),and thus are not discussed here. In-

stead, this review focuses on our cur-rent understanding of these regula-

tory pathways in the blastocyst, witha focus on the trophoblast lineage of

the developing placenta both prior toand after implantation.

Cell Death Prior to

Implantation

Successful pre- and postimplanta-tion embryonic development de-

Fetal development depends upon a coordinated series of events in boththe embryo and in the supporting placenta. The initial event in placenta-tion is appropriate lineage allocation of stem cells followed by the forma-tion of a spheroidal trophoblastic shell surrounding the embryo, facilitat-ing implantation into the uterine stroma and exclusion of oxygenatedmaternal blood. In mammals, cellular proliferation, differentiation, anddeath accompany early placental development. Programmed cell death isa critical driving force behind organ sculpturing and eliminating abnormal,misplaced, nonfunctional, or harmful cells in the embryo proper, althoughvery little is known about its physiological function during placental de-

velopment. This review summarizes current knowledge of the cell deathpatterns and molecular pathways governing the survival of cells within theblastocyst, with a focus on the trophoblast lineage prior to and afterimplantation. Particular emphasis is given to human placental develop-ment in the context of normal and pathological conditions. As molecularpathways in humans are poorly elucidated, we have also included anoverview of pertinent genetic animal models displaying defects in tropho-blast survival. Birth Defects Research (Part C) 75:262280, 2005. 2006 Wiley-Liss, Inc.

Andrea JurisicovaandIsabella Caniggiaare from the Department of Obstetrics and Gynecology, University of Toronto, Mount SinaiHospital, and the Department of Physiology, University of Toronto, Toronto, Ontario, Canada.Jacqui Detmar is from the Institute of Medical Studies, University of Toronto, Toronto, Ontario, Canada.

*Correspondence to: A. Jurisicova, SLRI876, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada, M5G 1X5.E-mail: [email protected]

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bdrc.20053

REVIEW

Birth Defects Research (Part C) 75:262280 (2005)

2006 Wiley-Liss, Inc.


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pends on the sequential implemen-

tation of a certain genetic program,

which is profoundly influenced not

only by the genetic endowment ofthe gametes, but also by interac-

tions with the surrounding environ-

ment. For one in 10 couples, failure

to achieve a pregnancy remains amajor problem. Clinical results of

assisted reproductive technology(ART) procedures in North America

suggest that clinical pregnancy is

achieved in only 30% of patientsundergoing this treatment. This low

rate of reproductive fitness is not

restricted to infertility patients,since in the general population up

to 40% of conceptions may be lost

before they are clinically recog-

nized as pregnancy (Hertig et al.,

1959). The cellular and molecularreasons behind such a high rate of

failure are unclear; however, lowdevelopmental competence of con-

ceived embryos is believed to be

the most likely culprit. While it iswell recognized that a significant

proportion of human embryos dis-

play a variety of cellular defects, in-cluding embryo fragmentation

and/or cleavage arrest, abnormal

cavitation, depletion of cells, or ab-normal lineage allocation, the un-

derlying molecular mechanisms re-

sponsible for these deficiencies areunclear.

Successful development of mam-malian preimplantation embryos cul-

minates in the first major step to-ward embryonic differentiation, that

being the formation of the trophecto-

derm (TE) and the inner cell mass(ICM) at the blastocyst stage. The

polar trophectoderm, a fluid-trans-porting epithelium, is the precursor

of the majority of the extraembry-

onic placental tissues (Kunath et al.,

2004). On the other hand, cells of theICM, comprising approximately onefourth of the total cell count in the

human expanded blastocyst (Hardy

et al., 1989; Hardy, 1997), contrib-ute to the epiblast (e.g., embryo

proper) and are represented by em-bryonic stem cell population (ES).

The third embryonic lineage, estab-

lished in fully expanded blastocystsand located on the surface of the ICM

layer, called the primitive endoderm(PE), represents a population of cells

that will give rise to another ex-

traembryonic tissue called parietal

and visceral endoderm (Kunath et

al., 2005). Murine stem cells for allthese lineages have been success-

fully established and a subset of ge-

netic pathways, responsible for the

original allocation of these lineages,

has also been elucidated (Ralstonand Rossant, 2005). However, the

contribution of cell death to develop-ment at this stage has been poorly

explored.

There are two windows for celldeath susceptibility during preim-

plantation development: early cleav-

age stage, frequently described asembryonic arrest with or without cel-

lular fragmentation (reviewed in Ju-

risicova and Acton, 2004), and apop-totic-like death coinciding with

formation of the blastocyst (Hardy,1997; Pampfer and Donnay, 1999).

While cell death at the blastocyststage has been documented for

over 20 years, its regulation and

function in early developing em-bryos remain poorly understood.

Cells exhibiting characteristic fea-

tures of apoptosis have been ob-served in blastocysts of several

mammalian species (Handyside,

1986; Papaioannou and Ebert,1988; Knijn et al., 2003), including

human (Hardy et al., 1989); this

cellular phenomenon has been ob-served in embryos developing in

vivo, but appears to be elevated byconditions used during in vitro cul-

ture (Jurisicova et al., 1998; Gjor-ret et al., 2003).

The presence of dead and dying

cells has been confirmed in blasto-cysts from a number of mammalian

species through the use of severaltechniques, implicating apoptosis

as a mode of death during blasto-

cyst formation (reviewed in Hardy,

1997, 1999). Positive terminaltransferase mediated dUTP DNAEnd Labeling (TUNEL) labeling, in-

dicative of fragmented DNA as well

as condensed chromatin morphol-ogy, in a subset of cells in the

mouse blastocyst has confirmedthat peak levels of PCD in murine

embryos developing in vivo occur at

97 hr postcoitum (Handyside,1986). Culture of murine preim-

plantation embryos in vitro fromzygote until blastocyst stage in

mice elevates cell death indices

(percentage of dead cells), with

death restricted predominantly to

cells of the ICM lineage (Brison andSchultz, 1997; Hardy, 1997). This

is even more evident in embryos of

different mammalian species that

have been conceived, manipulated,

and cultured in vitro (Jurisicova etal., 1998; Gjorret et al., 2003; Hao

et al., 2004a). Under these condi-tions, death occurs in both the ICM

and in the TE lineage (reviewed in

Hardy, 1997). The susceptibility todeath of these two different lin-

eages (i.e., ICM and TE) are likely

influenced by the mode of fertiliza-tion, as both human and murine

blastocysts obtained byin vitrofer-

tilization (IVF) display trophecto-dermal cell death (Jurisicova et al.,

1998, 1999), while murine blasto-cysts conceived in vivo, but cul-

tured in vitro, have almost exclu-sive localization of dead cells to the

ICM (Hardy, 1997).

Based on cell counts of spare hu-man embryos donated to research,

we have observed that the cell

death index (CDI) contributes tothe loss of approximately 15% of

cells at the blastocyst stage (Jurisi-

cova et al., 1999). In most of theembryos, cell death appeared ran-

dom, affecting both the ICM and

trophectoderm; however, a sub-population of blastocysts exhibited

depletion of the majority of cellscomprising the ICM, as evidenced

by extensive chromatin condensa-tion and/or fragmented DNA (see

Fig. 1). This could likely result in the

total elimination of the embryoniclineage destined to form the fetus,

leaving only precursor cells for theplacenta. Such blastocysts may ini-

tiate implantation, but would prob-

ably create only extraembryonic

tissues with an empty embryonicsac, a condition clinically termed a

blighted ovum. Lack of an ICM

has been previously described in

human embryos cultured in vitro(Winston et al., 1991; Desai et al.,

1997), and was attributed to theabnormal allocation of cells to the

ICM and TE lineages. We propose

that in some embryos, cells of theICM become established but are

rapidly eliminated via PCD in theearly developing blastocyst. Since

cells of the TE have extremely well-

MOLECULAR MECHANISMS OF TROPHOBLAST SURVIVAL 263

Birth Defects Research (Part C) 75:262280, (2005)


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developed phagocytotic capabilities

(Drake and Rodger, 1987), apopto-tic bodies produced by dying cells of

the ICM are most likely engulfedand degraded very rapidly (Hardy,

1997). Therefore, the remainingcells of the failing blastocyst would

not be negatively affected by cellu-lar debris produced by the dead

cells following secondary necrosis.It is interesting to note that the lev-

els of cell death in human blasto-cysts were previously observed to

be elevated in the ICM of high-grade embryos in comparison to

low grade embryos, suggesting

that apoptosis may play a regula-tory role in the maintenance of cellnumber during normal healthy em-

bryo development (Hardy et al.,2003).

Further examination of CDI anddistribution of dead cells identified

an additional subgroup of human

blastocysts characterized by exces-sive activation of the cell death cas-

cade in both ICM and TE lineages(Fig. 1). These blastocysts would

likely be unable to initiate and/orsustain postimplantation develop-

ment and, therefore, would not be

clinically recognized as a preg-nancy. However, they may contrib-

ute to the pool of biochemicalpregnancies, which are frequently

observed after IVF (Simon et al.,1999). In these cases, patients

manifest mildly elevated humanchorionic gonadotrophin (hCG) and

have delayed menstrual period.However, the hCG, which reflects

the activity of cells from the tropho-blast lineage, declines within a few

days thereafter, followed by men-strual period, indicating loss of

pregnancy. The incidence of cell

death in the human blastocyst hasadditionally been shown to be di-rectly correlated with early embryo

quality (Hardy et al., 1989; Stoneet al., 2005), and deregulated cell

death very likely contributes to lowdevelopmental potential of human

embryos.

Growth Factors, Nutritional

Needs, and Cell Death

In contrast to the in vitro environ-ment, the in vivo microenviron-

ment provides additional growth

factors and cytokine mediated ef-fectors that positively influence

embryo development and growth.Thus, embryo development is con-

trolled not only by innate factors,but also through autocrine and

paracrine effectors (reviewed byBrison, 2000; Hardy and Spanos,

2002). Differences between cleav-age rates (Bowman and McLaren,

1970) and levels of apoptosis (Bri-son and Schultz, 1997) in in vivo

and in vitro cultured mouse em-bryos suggest that the maternal

environment is exerting a paracrine

effect on the preimplantation stageembryo. Studies of in vitro culturedembryos have further demon-

strated positive autocrine effects,either through reducing culture vol-

ume or by grouped embryo culture(Lane and Gardner, 1992; Brison

and Schultz, 1997). Supplementa-

tion of culture media with variousgrowth factors has resulted in ele-

vated rates of blastocyst formationaccompanied by increased cell

number, likely resulting from sup-pression of cell death. Transform-

Figure 1. Schematic drawing of mammalian embryodevelopment from morula to blastocyst (TE, trophectoderm; ICM, inner cell mass;PE, primitive endoderm). Three patterns of cell death in human blastocyst observed under in vitro culture aredescribed. Representativephotomicrographs are shown for expanded human blastocysts at day 7 that were analyzed for cell death by assessing chromatin status(DNA stain in blue) and fragmentation of DNA (TUNEL labeling in red). Normal nuclei have diffuse pale-blue staining and ovalmorphology, while nuclei with condensed chromatin, indicative of dead cells, exhibit bright white staining and disintegrate into smallround nuclear blebs. Cell death index (CDI) reflects the proportion of cells in the embryo displaying hallmarks of apoptosis.

264 JURISICOVA ET AL.



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ing growth factor-(TGF-) (Brison

and Schultz, 1997), platelet acti-

vating factor (PAF) (ONeill, 1998),

and insulin-like growth factor-1(IGF-1) (Brison, 2000) have been

shown to decrease the incidence of

apoptotic cells in mouse and human

blastocysts, while tumor necrosisfactor (TNF-) has been shown to

exert the opposite effect in rat(Pampfer et al., 1997) and mouse

embryos (Wuu et al., 1999; Fabian

et al., 2004). Animal model in vitrostudies demonstrated that addition

of IGF-1 to the culture medium re-

sulted in a significant decrease in

the number of apoptotic cells perembryo, mostly in cells of the ICM

lineage (Makarevich and Markkula,

2002), while supplementation with

growth hormone exerted similar ef-fects targeting the TE (Kolle et al.,

2002, 2003). We and others haveexplored the biological effects of

these growth factors in human pre-

implantation embryos with similaroutcomes, suggesting conservation

of these molecular pathways. The

percentage of apoptotic nuclei inhuman blastocysts was decreased

by approximately 50% upon addi-

tion of IGF-1 to culture (Spanos etal., 2000). Addition of granulocyte-

macrophage colony stimulating

factor (GM-CSF) to human embryocultures increased blastocyst for-

mation rates two-fold (Sjoblom etal., 2002) and decreased the per-

centage of apoptotic nuclei, whileincreasing the number of viable

ICM cells (Sjoblom et al., 2002).

Given these data, we focused ourefforts on analysis of the growth

hormone pathway. Human em-bryos express growth hormone re-

ceptor and respond to it in a similar

way as murine embryos, exhibiting

a higher number of cells allocatedto both ICM and TE lineages (Di Be-

rardino, 2004). Collectively, thesedata further support the important

role of autocrine and paracrine me-diators in human embryonic cell

death and development (Hardy and

Spanos, 2002).While the effects of cytokines on

embryo growth and developmenthave been known for several years,

the downstream effects on gene

expression in the context of embry-onic cell death remain relatively un-

explored. The inhibiting activity of

TGF- in murine blastocysts was re-

cently linked to the antiapoptotic

action of the inhibitor of apoptosis(IAP) molecule, survivin, and is me-

diated by activation of the phos-

phatidylinositol-3 kinase (PI3K)

signaling pathway (Kawamura etal., 2003, 2005). The PI3K/Akt sig-

nal transduction pathway is a well-known mediator of growth promot-

ing and cell survival signals (Gross

et al., 2005). Recent reports sug-

gest that both PI3K and Akt exhibitan apical staining pattern in tro-

phectoderm cells, and inhibition of

Akt activity resulted in reduced in-sulin-stimulated glucose uptake,

leading to a significant delay in

blastocyst hatching (Riley et al.,

2005). The site of expression ofthese prosurvival signaling mole-

cules appears to coincide with theexpression of Death receptor

5/Killer and its ligand Trail (Riley et

al., 2004), raising a possibility that

these molecules may work in onesignaling pathway.

The exact effector death pathway

used by IGF to suppress cell deathin ICM is less understood. IGF-I ex-

posure appears to change the ratioof Bax/Bcl-2, and this shift in bal-

ance between pro- and antiapop-

totic Bcl-2 family members wasproposed to contribute to de-

creased apoptosis (Kolle et al.,2002). However, preimplantation

embryos exposed to high concen-

trations of IGF-1 or insulin also un-dergo extensive apoptosis of the

ICM, and deficiency in Bax, or ex-posure to inhibitors of either

caspase or ceramide synthase ac-

tivity, prevents this event (Chi etal., 2000). Furthermore, it has

been proposed that the embryo-

toxic insult caused by high IGF-1levels may be responsible for the

high incidence of pregnancy lossseen in women with polycystic

ovary syndrome (Chi et al., 2000),as these patients exhibit insulin re-

sistance and show elevated levels

of IGF-1 due to insufficient produc-tion of IGF-1 binding protein

(Conover, 1992).On the other hand, imbalance in

glucose metabolism has been

linked to excessive activation ofseveral death pathways in preim-

plantation embryos. Cell death in-duced by elevated exposure to glu-

cose in vitro downregulates glucose

transporters, leading to a drop inintraembryonic free glucose (Chi et

al., 2002) and triggering massive

apoptosis in the ICM lineage (Pam-

pfer et al., 1997). This pathwayalso involves excessive accumula-

tion of Bax (Moley et al., 1998) andcleaved caspase 3/6 (Hinck et al.,

2001, 2003), as well as promotes

activation of the p53 dependent cell

death pathway (Keim et al., 2001).Disruption of any of these pathways

by gene knockout or biochemical

inhibition approaches partially res-cues cell death triggered by intra-

cellular glucose starvation (Keim et

al., 2001). Moreover, the diabetic

mouse

in vivo model caused by de-struction of islet cells by streptoco-

zin treatment results in recapitula-tion of these embryonic phenotypes

(Chi et al., 2000, 2002).

Given these data, hence the

question arises, what is the func-tion of cell death during early em-

bryo development? While several

speculations were put forward,very little experimental data exist

to support these theories. Mostlikely, it is a means to eliminate

cells with abnormal genetic com-

plements, reduced developmentalpotential (Hardy, 1999), and/or de-

fects in lineage allocation. The reg-ulation of cell death at these early

stages is further thought to be crit-

ical to later development of theconceptus (Moley, 2001). The exis-

tence of this link was confirmed by aseries of elegant experiments that

induced cell death during preimplan-

tation embryo development by dis-rupting glucose metabolism. Both in

vitro and in vivo exposure to hyper-

glycemic environment results in in-creased apoptosis observed in the

blastocyst, leading to an increasednumber of malformations and con-

genital abnormalities such as micro-gnathia, omphalocele, and cranial

and neural tube defects; these ab-

normalities could be prevented byablation of the cell death initiator,

Bax (Moley et al., 1998; Chi et al.,

2000). At least one report in humansalso indicates that transfer of em-

bryos with increased cellular frag-mentation, previously linked to the




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bryonic carcinoma cell lines of

solely embryonic or trophectoder-

mal developmental potential were

transplanted into the blastocelecavity of mouse blastocysts indi-

cated that cells capable of forming

TE are preferentially eliminated by

PCD (Pierce et al., 1989). Extracel-lular hydrogen peroxide generated

via polyamine oxidation was impli-cated as a mediator of PCD in the

blastocyst; however, genes re-

sponsible for the activation or exe-cution of this death are unknown

(Pierce et al., 1991). Thus, it is

tempting to speculate that PARP-1is a key regulator of cell death in

ICM cells that have potential to be-

come TE lineage. In wild-type em-

bryos, PARP-1 would be activated

in the response to DNA damagecaused by hydrogen peroxide in the

subset of ICM considering their TEfate due to their low antioxidant

status perhaps, which would lead to

their elimination. ICM cells of em-bryos lacking PARP-1 fail to die

when they switch the fate, leading

to formation of trophoblast lineagein vitro as well as in vivo produced

carcinoma tumors (Masutani et al.,

2001). This surveillance pathwaymay not be functional in humans,

as human ES cells readily differen-

tiate into the trophoblast lineageunder in vitro conditions without

any genetic manipulation (Xu et al.,2002).

Myeloid cell leukemia-1 (Mcl-1) isa prosurvival Bcl-2 family member

facilitating proper differentiation of

stem cells (Opferman et al., 2003,2005). Since Mcl-1 has been pro-

posed to work upstream of otherBcl-2 family members (Nijhawan et

al., 2003), it is not surprising that

embryonic disruption of this mole-

cule leads to preimplantation le-thality in mice, well before themidgestational death of embryos

caused by Bcl-x deletion (Mo-

toyama et al., 1995). While blasto-cysts lacking Mcl-1 could be de-

tected at day 4 in the reproductivetract in vivo, these embryos failed

to implant and were unable to pro-

duce viable blastocyst outgrowthsin vitro (Rinkenberger et al., 2000),

suggesting a compromised TE lin-eage viability. No overt increase in

apoptosis was observed in these

blastocysts and embryonic loss

could not be rescued by the disrup-tion of Bax or p53, indicating that

Mcl-1 may not act through theseapoptotic pathways and/or may

have other cellular function(s). Wehave previously observed that an-

other proapoptotic multichannelBcl-2 family member, Mtd/Bok, is

abundantly expressed in murine

blastocyst, particularly in the TElineage (Jurisicova et al., 2000), a

finding that will be discussed laterin the context of human placenta.

As the preferred Bcl-2 interactivepartner of Mtd is Mcl-1 (Hsu et al.,

1997), it is possible that deletion ofthis particular cell death gene may

be able to rescue Mcl-1 lethality inthe early developing embryo.

Defender against apoptosis-1(Dad-1) is an evolutionarily highly

conserved enzyme, which catalyzesthe transfer of pre-assembled high

mannose oligosaccharides ontospecific asparagine residues of nas-

cent polypeptides (Sanjay et al.,1998). This enzyme is capable of

interacting with and modifyingMcl-1 function (Makishima et al.,

2000). The functional importanceof this interaction can be indirectly

shown from results of gene target-ing experiments, as disruption of

this gene leads to embryonic lethal-ity at the blastocyst stage (Nishii et

al., 1999; Brewster et al., 2000),phenocopying the Mcl-1 knockout

mouse. Dad-1 mutants, however,display an increased rate of cell

death in the ICM both in vitro and in

vivo, suggesting that this enzymemay have additional modification

targets besides Mcl-1.

THE PLACENTA AND FETAL

PROGRAMMING

In the last decade, substantial evi-dence has supported the idea that a

compromised in utero environmentcan influence health during postna-

tal life. Barker (1992) introduced

the concept of fetal programming,which proposes that a number of

organ structures and functions un-dergo programming during embry-

onic and fetal life. This develop-mental programming determines

the physiologic and metabolic setpoints, which will ultimately cue re-

sponses to the environment in the

adult. Adverse in utero conditions,

such as placental insufficiency, in-

adequate maternal nutrition, and

altered maternal stress hormone

profiles lead to developmental ad-

aptations by the embryo/fetus that

readjust these set points (reviewedby Lau and Rogers, 2004; Wobus

and Boheler, 2005). These adaptive

measures ostensibly have short-

term benefits to the embryo and fe-

tus, but these changes to the ge-

netically-determined body plan

may confer a discordant physiology

on the adult, leading to increased

risk of disease. It has been repeat-

edly shown in human populations

that growth-compromised fetuses

resulting from intrauterine growth

restriction (IUGR) following placen-tal insufficiency are at increased

risk for adverse short- and long-

term outcomes, such as hyperten-

sion, obesity, and type II diabetes,

that can extend into adult life

(Barker, 1992; Bernstein et al.,

2000; Clausson et al., 2001). There

is a rapidly-growing body of evi-

dence suggesting that the placenta

is involved in fetal programming

(reviewed by Godfrey, 2002; Bas-

chat, 2004), with particular empha-

sis on the impact of placental insuf-ficiency on cardiac development

(reviewed by Law and Shiell, 1996;

Huxley et al., 2000), neural devel-

opment (Blair and Stanley, 1988;

Mallard et al., 1999; Bui et al.,

2002) and endocrinology (reviewed

by Kanaka-Gantenbein et al.,

2003). Proper establishment of the

placenta at these times allows the

fetus to grow and attain its devel-

opmental potential in the third tri-

mester, in preparation for postnatal

life. Investigations into how normal

placental cell death assists in shap-

ing placental architecture and func-

tion will provide us with information

to better understand how aberrant

or insufficient cell death in this tran-

sient organ contributes to overall

fetal, infant, and adult health. Ad-

ditionally, the study of animal mod-

els of placentation provides useful

clues and allows us profound in-

sight into the regulation and pat-

terning of cell death in the mamma-

lian placenta.




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Human Placental

Development

The placenta is a life-sustaining

though transient organ, which medi-

ates the physiological exchange be-

tween mother and fetus. During

human placental development, un-

differentiated mononuclear cytotro-

phoblast (CT) cells reside in two dif-

ferent types of highly specialized

chorionic villi: floating and anchoring

villi. These highly proliferative, yet,

undifferentiated trophoblast cells fol-

low two separate pathways of differ-

entiation (Cross et al., 1994). In

floating villi, these cells have the

unique ability to fuse and form the

multinucleated syncytiotrophoblast

(ST) layer, which functions as the

natural physical barrier between

maternal and fetal cells, and be-

cause it is directly bathed in ma-

ternal blood, it mediates gas and

nutrient exchange for the devel-

oping fetus. This layer is subjected

to continuous renewal, as aged

nuclei together with small

amounts of cytoplasm are re-

leased into the maternal circula-

tion as membrane-sealed struc-

tures termed syncytial knots,

and are eventually believed to be

cleared by maternal lung macro-

phages. This dynamic process,

also known as trophoblast turn-

over, is necessary, as the multinu-

cleated ST cells are highly differ-

entiated and, as such, unable to

regenerate (Huppertz and King-

dom, 2004).

In anchoring villi, proliferative

CT cells differentiate and invade

deeply into the endometrium

reaching the first third of the ad-

jacent myometrium. This popula-

tion of migratory/invasive cells,also known as extravillous tropho-

blasts (EVT), have the unique fea-

ture of remodeling the pregnant

endometrium and its vasculature.

Endovascular invasion within the

uterine wall is necessary, since

the remodeling of the maternal

spiral arteries generates a low re-

sistance vascular system that per-

mits continuous and adequate

blood flow to the growing fetus

(Aplin, 1991).

Cell Death in the Human

Placenta

During normal placentation, a bal-

ance between proliferation, differen-

tiation, and apoptosis is mandatoryfor trophoblast cells homeostasis, fa-

cilitating maintenance of normalplacental villi functions. While pro-

liferation and differentiation of tro-phoblast cells have been exten-

sively examined, only in the past

decade have studies begun to ad-dress the importance of apoptosis

during normal and abnormal pla-

cental development.Numerous studies have reported

that during pregnancy, the rate of

cell death is low in the first trimes-ter of pregnancy and mostly re-

stricted to the chorionic villous cy-totrophoblasts, followed by anincrease with advancing gestation

and a shift to the syncytium (Smith

et al., 1997; Gruslin et al., 2001).

ST cells, and to a lesser extent CTcells, express the prosurvival Bcl-2

family members Bcl-2 and Mcl-1

(Huppertz et al., 1998), while theproapoptotic proteins Bax and p53

have been found in cytotropho-

blasts and mesenchymal cells ofthe chorionic villi throughout gesta-

tion (Ratts et al., 2000). This

unique spatial distribution of pro-and antiapoptotic molecules be-

tween the adjacent cell layers hasbeen postulated to confine the ap-

optotic events to a subset of spe-cific nuclei within the renewing syn-

cytium, thereby preventing the

death of the entire layer. Interest-ingly, a large number of apoptotic

nuclei in syncytial knots show de-creased expression of the anti-ap-

optotic Bcl-2 and Mcl-1 (Huppertz

et al., 1998). Downstream events

of death execution and syncytialknot formation appear to be medi-ated by Caspase 3 (Huppertz et al.,

1999). Thus, it is not surprising that

the expression of the X-linked in-hibitor of apoptosis (Xiap), a pro-

survival inhibitor of apoptotic pro-

tein known to interfere withexecutor caspase activation, was

found to decrease with advancinggestation, and it inversely corre-

lated with increased trophoblast

apoptosis found at term (Gruslin etal., 2001). Others have reported

abundant Bcl-2 expression in con-

junction with apoptotic nuclei in

syncytiotrophoblast overlying fi-brin-containing fibrinoid deposition

(Ratts et al., 2000). Since this ex-

tracellular matrix molecule is in-

volved in reparative and degenera-

tive processes within the humanplacenta, Bcl-2 may be involved in

these events (Marzioni et al.,1998). More recently, studies have

highlighted the importance of cell

death in regulating the events in-volved in trophoblast cell differenti-

ation towards both syncytial forma-

tion and extravillous trophoblastcells remodeling of the spiral arter-

ies. It is now established that apo-

ptotic cell death in the developing

placenta is associated with tropho-

blast cell turnover, enabling re-newal of syncytium (Huppertz et

al., 1999). It has been postulatedthat apoptosis is initiated in the vil-

lous trophoblasts facilitating fusion,

but is delayed in the ST layer, beingonly finalized prior to the extrusion

of apoptotic nuclei in the form of

syncytial knots (Huppertz et al.,1998). The balance between tro-

phoblast differentiation/fusion and

extrusion of aging material istightly regulated, i.e., fusion of un-

differentiated cells occurs within

two to four days, while syncytial fu-sion of nuclei and their extrusion

requires three to four weeks (Hup-pertz and Kingdom, 2004). This

constant process of cell fusion andturnover represents a unique bio-

logical example of fine tuning of the

apoptotic cascade. Recent in vitrostudies suggest that caspase 8

plays a major role in driving the firstevents of trophoblast turnover, as

its inhibition using antisense oligo-

nucleotides and/or caspase-spe-

cific inhibitors impairs cell fusion(Black et al., 2004).Emerging evidence suggests that

the events associated with tropho-

blast invasion into the spiral arter-ies are in part also mediated by ap-

optosis. Using an in vitro coculturesystem of trophoblast cells and spi-

ral arteries, it has been recently

shown that the expression ofcleaved PARP, an apoptotic marker,

localizes only to those arteries that

exhibit trophoblast remodeling fol-lowing loss of endothelial cells




8/19

(Cartwright et al., 2002). The na-

ture of the molecular cascade ofdeath activation in this model is

less clear; however, a recent report

suggests that the endothelial celldeath induced by trophoblasts dur-

ing the process of remodeling ap-

pears to be mediated via Fas/Fasligand interaction (Ashton et al.,2005).

Oxygen as a Regulator of

Trophoblast Fate

Early embryonic development takesplace in a relatively hypoxic environ-

ment. Oxygen electrode studieshave shown that at five to eight

weeks gestation, oxygen tension is

low (

20 mm Hg, equivalent to 3%O2), while at 1012 weeks, when theintervillous space opens to maternal

blood, oxygen levels increase to55

mm Hg (810% O2) (Rodesch et al.,1992; Burton et al., 1999). The steep

rise of oxygen tension experiencedby trophoblast cells has been found

to be associated with increased ex-pression of genes associated with

oxidative stress (Jauniaux et al.,2000). Increasing evidence suggests

that oxygen is a key regulator for thecellular events occurring during early

placentation. Low pO2levels supportproper trophoblast proliferation,

while the increase of pO2 after theintervillous space opens to maternal

blood is essential to finalize the pro-cess of trophoblast differentiation/

invasion (Jaffe et al., 1997).The precise mechanisms by which

oxygen modulates these develop-mental events are not fully under-

stood; however, hypoxia-induciblefactor-1 (HIF-1), a master regulator

of oxygen homeostasis (Semenza,

2001a, 2001b; Kaelin, 2002), seemsto play a key role in this cellular re-sponse (Caniggia et al., 2000).

In vitro studies have indicatedthat, hypoxia, one of the most phys-

iologically relevant stimuli is able toinitiate apoptosis of term tropho-

blasts (Nelson et al., 1999; Levy et

al., 2000). Increased cell death interm primary isolated cells exposed

to severe hypoxic conditions (15mm Hg) is accompanied by in-

creased expression of the proapop-totic Bax and p53 and decreased ex-

pression of Bcl-2 (Levy et al., 2000).

Interestingly, addition of epidermalgrowth factor inhibits the hypoxia-

induced trophoblast cell death (Levyet al., 2000). In the human placenta,

the low oxygen-stimulatory effect oncell death appears to entail intrinsic

(mitochondrial) rather than extrinsic(death receptor) pathways. This isconsistent with observations that re-

active oxygen species do not medi-ate trophoblast cell apoptosis in-

duced by TNF- (Smith et al., 1999).Recent evidence indicates that

the effect of oxygen on trophoblastcell death is exquisitely dependent

upon the pO2 levels. While tropho-blast cells exposed to both severe

hypoxia (10 mm Hg) and to highoxygen levels (140 mm Hg) un-

dergo apoptosis readily, cells ex-posed to oxygen levels ranging be-

tween 15 and 40 mm Hg show a lowfrequency of apoptosis (Mackova et

al., 2003). This is not surprising, ifone considers that during normal

placental development the physio-logical oxygen levels that permit

proper trophoblast differentiationrange between 15 and 55 mm Hg.

In vitro studies using villous ex-plant cultures have demonstrated

that severe hypoxia favors ne-

crotic, as opposed to apoptotic,

shedding of syncytiotrophoblasts(Huppertz et al., 2003). Hung et al.(2002) have proposed that inter-

mittent placental perfusion leads toincreased oxidative stress, and

have reported that hypoxia-reoxy-genation is a powerful inducer of

classical events associated with ap-optosis, including mitochondrial cy-

tochrome C release, caspase 3 ac-tivation, and PARP cleavage. Thus,

rather than hypoxia, it is the localhypoxia-reoxygenation injury that

may be more relevant to activationof cell death pathways in develop-

ing placenta, particularly in theconditions associated with placen-

tal insufficiencies.

Cell Death in Physiological

and Pathological

Conditions of Placental

Hypoxia

Preeclampsia is an unpredictable

and devastating disorder that man-ifests abruptly, and remains a ma-

jor cause of maternal and perinatal

morbidity and mortality complicat-

ing 57% of pregnancies. This dis-

ease, unique to humans, is charac-terized by a general maternal

inflammatory response likely due to

the release of placental factors,

leading to the damage of variousorgans, including kidney, lungs,

and heart, often resulting in life-long complications. A key feature

that characterizes this disease is

the excessive shedding and depor-tation of placental debris into the

maternal circulation due to exces-

sive placental trophoblast cell

death (Knight et al., 1998; Redmanand Sargent, 2000). This is be-

lieved to be the culprit of the ma-

ternal endothelial injury leading to

the onset of the clinical manifesta-tions including hypertension, pro-

teinuria, and edema (Roberts et al.,1989; Broughton Pipkin and Rubin,

1994). Although preeclampsia is

the most intensively studied clinicalcondition in pregnancy, its cause

remains elusive and numerous hy-

potheses on its origin abound. It is,however, accepted that the pla-

centa plays a key role in its patho-

genesis, as its removal at deliveryresults in prompt resolution of this

disease. A typical hallmark of pre-

eclampsia is the lack of remodelingof the maternal spiral arteries by

trophoblast cells, and this is be-lieved to be the cause for insuffi-

cient uteroplacental circulationleading to placental hypoxia (Hung

et al., 2002). Both relative and di-

rect measures of utero-placentalblood flow (Lunell et al., 1979), as

well as Doppler studies (Kingdomand Kaufmann, 1997), support the

notion that this serious disease is

associated with insufficient blood

flow and placental hypoxia. The in-sufficient perfusion of the placenta

often causes fetal growth restric-tion. Recent studies suggest that

the rate of apoptosis is increased inpreeclampsia due to placental oxi-

dative stress (Hung et al., 2002;

Myatt and Cui, 2004). Thus, it isplausible that hypoxic stress may

be responsible for the increased ap-optotic rates of syncytiotropho-

blasts observed in preeclamptic

placentae (Allaire et al., 2000). Thedeportation of placental debris into




9/19

the maternal circulation is believed

to be the culprit of the maternal en-

dothelial injury. These fragments

are normally present in the plasmaof normal pregnant women; how-

ever, the release of this placental

debris is increased in preeclampsia,

a likely consequence of excessiveplacental cell death.

While it is now widely acceptedthat preeclampsia is associated

with increased trophoblast cell

death, the molecular pathways be-hind this deregulated process are

unclear. Elevated cell death in pre-

eclampsia has been detected both

in villous trophoblast and in extra-villous trophoblast cells within an-

choring villi at the fetal-maternal

interface. Widespread cell death

measured by TUNEL is detected inextravillous trophoblast cells within

the uterine wall, and is accompa-nied by decreased expression of the

prosurvival factor Bcl-2 (Di Be-

rardino, 2004). More recently, ithas been proposed that excess of

macrophages found in the placental

bed of preeclamptic patients maycurtail extravillous trophoblast in-

vasion by an apoptotic mechanism

that involves TNF and tryptophandepletion (Reister et al., 2001).

While no differences in the expres-

sion of Bcl-2 family proteins, suchas Bax, Bcl-2, Bak, and Bcl-XL, are

found between preeclampsia/IUGRand control placentae, elevated ex-

pression of p53 and Fas, but notFasL, has been detected in villous

trophoblast of preeclamptic placen-

tae (Hsu et al., 2001; Levy et al.,2002). Only Ishihara et al. (2002)

have reported decreased expres-

sion of the pro-survival factor Bcl-2in syncytiotrophoblast of severe

preeclamptic and IUGR placentae.

Our recently published observa-tions have highlighted the impor-

tance of Mtd/Bok, a proapoptoticBcl-2 family member, during both

normal and abnormal placentation.Mtd/Bok (Mtd: Matador/Bok: Bcl-2

ovarian killer) belongs to the mul-

tidomain pore-forming subfamily ofproapoptotic Bcl-2 family mem-

bers, including Bax and Bak (Hsu etal., 1997; Inohara et al., 1998).

Mtd transcript is alternatively

spliced and encodes for two proteinisoforms: Mtd-L that primarily in-

teracts with Mcl-1, and Mtd-S re-

sulting from fusion of BH3 and BH1

domains, the function of which can-

not be antagonized by any knownantiapoptotic Bcl-2 family member

(Hsu and Hsueh, 1998). We identi-

fied a third Mtd splice variant,

Mtd-P, with a unique developmen-tal expression profile in normal hu-

man placenta. The unique expres-sion and distribution of Mtd-P is a

result of its response to oxygen, a

master regulator of trophoblast dif-ferentiation (Soleymanlou et al.,

2005b). Mtd-P, a C-terminal trun-

cated protein resulting from exon II

skipping, is a proapoptotic mole-cule involved in trophoblast cell

death, regulating mitochondrial

membrane potential. We deter-

mined that the elevated expressionof this novel splice variant is unique

to pregnancies complicated by se-vere early onset preeclampsia

(Soleymanlou et al., 2005b). In-

creased Mtd-P expression in pre-eclampsia may be causative of in-

creased trophoblast cell death

leading to excessive shedding ofplacenta debris observed in this

disease. Interestingly, placentae

obtained from normotensive con-trol subjects, as well as late-onset

(term) preeclampsia, IUGR, or es-

sential hypertensive subjects, didnot show any alteration in Mtd-P

expression. The high Mtd-P expres-sion found in early severe, but not

late, onset preeclampsia raises thepossibility that the classical defini-

tion of preeclampsia might be too

broad and might encompass differ-ent diseases. In support of this

idea, Redman and Sargent (2000)have recently classified preeclamp-

sia into two very distinct catego-

ries: the placental and the maternal

preeclampsia. They define the firsttype as being linked to placental al-teration following hypoxia insult,

whereas the second type is associ-

ated with a defect in the maternalvasculature and not in the placenta.

It is also likely that different molec-

ular pathways will be driving patho-physiological defects in preeclamp-

tic placental tissue in these twodifferent subtypes.

Placentae from high altitude

pregnancies represent a uniquenatural model of chronic placental

hypoxia (Fig. 2). Because of the

reduction in maternal arterial ox-

ygen pressure, these placentae

are exposed to reduced uteropla-cental oxygenation (Zamudio,

2003). In pregnancies at high al-

titude (2700 m), intrauterine

growth is restricted and the inci-dence of preeclampsia is elevated

two- to four-fold (Palmer et al.,1999; Zamudio, 2003), support-

ing the widespread belief that pla-

cental hypoxia is associated withpreeclampsia. As such, the high

altitude model represents a natu-

ral laboratory to study preeclamp-sia. Using high-throughput func-

tional genomics, we have recently

reported a striking similarity in

global gene expression between

low oxygen-treated explants, highaltitude placentae, and placentae

from preeclamptic pregnancies,indicating that changes in global

gene expression in preeclamptic

placentae are due to reduced ox-ygenation (Soleymanlou et al.,

2005a).

Pregnancies at high altitude alsoshow incomplete remodeling of the

spiral arteries, similar to that re-

ported in preeclampsia (Tissot vanPatot et al., 2003). Histomorpho-

logical studies have shown that

high altitude placentae are sub-jected to unique changes including

increased villous vascularization,increased capillary density, and

thinning of villous membrane,events believed to ensure adequate

oxygen delivery to the fetus. More-

over, high altitude placentae alsoexhibit reduced perisyncytial fibrin-

type fibrinoid deposition when com-pared to placentae from lower alti-

tude pregnancies (Mayhew et al.,

2002; Zamudio, 2003). The signif-

icance of reduced fibrin depositionis unknown. It is yet unclearwhether this change is related to an

altered trophoblast apoptotic rheo-

stat affecting trophoblast turnover.Although studies of cell death in in

vitro and in vivo pathological mod-els of placental hypoxia are avail-

able, little is known about the apo-

ptotic adaptation/changes thatoccur in high-altitude placentae.

We have recently found that alti-tude-induced chronic hypoxia leads

to increased expression of the pro-




10/19

tector Mcl-1L and decreased ex-

pression of the proapoptotic Mtd-Pand Mcl1-S. In addition, high alti-

tude placentae appear to have adecreased rate of trophoblast turn-

over, as identified by decreasedsyncytin expression (Caniggia et

al., 2004). Hence, in pregnanciesfrom high altitude-induced chronic

hypoxia, a molecular shift towardsinhibition of trophoblast cell death

occurs, and this may protect/slow

down trophoblast cell turnover anddeath.

THE MOUSE PLACENTA AS

A MODEL

Recent comparative studies be-

tween mouse and human placentaehave revealed striking similarities

in cellular mechanisms and tissue

framework, providing justificationfor using the mouse as a suitable

animal model for studies of humanplacentation. At day 6 of mouse de-

velopment (two days after implan-tation), two diploid populations of

trophoblast derivatives are estab-lished: the proximal extraembry-

onic ectoderm and the distal ecto-

placental cone. Between days 7.5

and 9.5, the placenta undergoes

major morphological remodeling,resulting in the formation of three

distinct cellular layers, each withunique morphology and function

(Rossant, 2001). The fusion of the

allantois with the chorion and sub-

sequent branching establishes anetwork of small canals collectively

referred to as the labyrinth. This

layer is responsible for gas and nu-trient exchange, and thus bears a

similarity to floating chorionic villi in

humans (Cross, 2000). The spon-

giotrophoblast layer is distal to thelabyrinth and consists of variable-sized, hormone-producing cells

with the capability of differentiating

into glycogen cells. Due to the ex-pression of several gene markers,

as well as to their spatial distribu-

tion, the cells of this layer are con-sidered to be analogous to human

column CT cells (Hemberger andCross, 2001). The two most distal

trophoblast cells in the mouse pla-

centa are glycogen cells, a cell typeof distinct morphology but un-

known function, and giant cells.

Both are invasive cell types and are

in direct contact with the maternal

decidua; as such, these cells areanalogous to human EVT cells

(Cross, 2000). Giant cells have dis-tinctively large, polyploid nuclei,

which very efficiently produce sev-

eral key regulatory, luteotropic,and lactogenic hormones, as well

as angiogenic factors (Cross,

2000). To further support the use of

the mouse as a model organism forhuman placentation, recent reports

have established that a subset of

specialized murine giant cells is in-

vasive and capable of remodelingmaternal spiral arteries (Cross etal., 2002). This is identical to the

events that occur during human

placentation, where spiral arteriesof the placental bed are invaded by

EVT cells. During invasion of thehuman spiral arteries, the endothe-

lial and smooth muscle layers are

dislodged and replaced by EVTcells. The presence of homologous

cell types and cellular behaviorshighlight the utility of the mouse

placenta as a suitable model for

Figure 2. Putative model of trophoblast cell death in physiological and pathological models of human placental hypoxia. In pregnanciesfrom high altitude, chronic hypoxia regulates the placental Mcl-1/Mtd rheostat via alteration of splicing, favoring trophoblast cellsurvival in contrast to preeclampsia, where increased trophoblast demise and turnover is observed.




11/19

study of normal, human placenta-

tion. Such a model is highly attrac-tive, as it allows for genetic manip-

ulation and induction of variouspathological conditions.

While it is now clear that apopto-sis plays a functional role in placen-

tal tissue morphogenesis, the un-derlying mechanisms coordinating

cell death have not been studied inthis context. The localization and

extent of cell death in the rodent

placenta and how this correlates tocell death patterns in human pla-centa remain largely unknown;

however, some sporadic in vivo(Zybina et al., 2000; Mu et al.,

2002) and in vitro (Goncalves et al.,2003) observations have surfaced

as supplementary data while inves-

tigating other events during pla-centation. In order to overcome

this dearth of information regardingcell death during placental develop-

ment, we are currently finalizingsystematic analyses of the tempo-

ral and spatial distribution of dying

cells throughout gestation in mouseplacentae obtained after natural

matings, and these results aresummarized in Figure 3. Prelimi-

nary analyses of these tissues indi-cate the presence of sporadic cell

death in both the labyrinth andspongiotrophoblast layers, which

likely reflects normal cell turnover(Detmar et al., 2004), similar to

that reported for ST turnover in hu-

mans (Huppertz et al., 1998). Inaddition, more extensive, orga-nized death of giant cells is ob-

served over gestation, resulting inalmost entire elimination of this cell

type at term. Furthermore, startingat approximately embryonic day

15.5, fetal glycogen cells and ma-ternal decidual cells undergo orga-

nized cell death at the maternal-fetal interface (Detmar et al.,

2004), creating focal perforations

that may facilitate delivery of theconceptus. Additionally, we have

observed that the extent of cell

death in murine placentae is influ-enced by genetic background, sug-

gesting either the presence of ge-netic modifiers that regulate cell

death susceptibility, or that regu-late other physiological factors,

such as nutrition and hypoxia, influ-encing the delicate balance be-

tween anti- and proapoptotic path-ways (Detmar et al., 2004). Similar

variability in the severity and pen-

etrance of embryonic phenotypescaused by the disruption of celldeath-associated genes have been

described for caspase-3 (Kuida etal., 1996; Woo et al., 1998) Rho

(Humphries et al., 2001), and Daxx(Michaelson et al., 1999).

Genetic Models of Cell

Death in Mouse Placenta

Tumor suppressor genes encode

for proteins that are involved in cellcycle regulation and cellular differ-

Figure 3.Schematic and histological representations of cell death patterns in murine placenta. Diagrammatic transverse sections ofday 15.5 (A) and day 18.5 (B) murine placentae at midline, after in situ TUNEL staining, showing sporadic cell death patterns in mostplacental layers except the maternal decidua, where regionalized pockets of dying maternal decidual and fetal glycogen cells can befound, and in the giant cell layer, which exhibits organized cell death over gestation, resulting in diminished numbers of this cell typeat day 18.5. Accompanying histomicrographs represent 5-m sections of mouse placentae after TUNEL staining, showing isolatedtrophoblast giant cell (C) and labyrinthine trophoblast cell death (D) in day 15.5 placenta, and whole regions of maternal decidual andfetal glycogen cell death in day 18.5 placenta (E). TUNEL-positive cells are stained brown and sections are counterstained withhematoxylin.




12/19

entiation; such proteins are often

referred to as gatekeepers,inhib-

iting or permitting cell cycle pro-

gression, depending on the internaland external status of the cell. A

number of these genessuch as

p53have been implicated in pro-

moting apoptosis in various celltypes, as this outcome, or cell se-

nescence, is usually consideredpreferable to the propagation of a

cell with uncontrolled proliferative

potential. The mouse placenta ex-presses a number of these tumor

suppressors, including p53, retino-

blastoma (Rb), and phosphatase

and tensin homolog (PTEN).The p53 tumor suppressor is a

ubiquitously-expressed transcrip-

tion factor that is activated by DNA

damage (DSa-Eipper et al., 2001;Katayama et al., 2002) and other

cellular stressors, such as hypoxia(Long et al., 1997; Lee et al.,

2005). The primary function of this

protein is to induce cells to undergoG1 arrest (Xiong et al., 1993; el-

Deiry et al., 1994) or apoptosis by

activating the appropriate targetgenes. In addition to its transcrip-

tional activity, a novel p53-medi-

ated apoptotic pathway was re-cently described, suggesting that

p53 translocates to the mitochon-

dria, where it induces permeabiliza-tion of mitochondria via interaction

with Bcl-2 and BH3-only familymembers (Chipuk et al., 2004,

2005). The transcriptional and ap-optotic potential of p53 in placental

cells have been demonstrated in

both rodent and human models.p53 has been shown to upregulate

Bax and downregulate Bcl-2 in hu-

man trophoblast cultured under hy-poxic conditions (Levy et al., 2000).

In addition, p53 has exhibited in-

volvement in triggering the apopto-tic pathway in early, proliferating

trophoblast cells with compromisedgenomic stability (Lim and Hasty,

1996; Weiss et al., 2000). In vivostudies of the effects of DNA-dam-

age-inducing agents on rat placen-

tation revealed that cells within thelabyrinth appear most susceptible

to p53-mediated apoptosis, as evi-denced by increased levels of active

caspase-3 (Katayama et al., 2002;

Yamauchi et al., 2004). Lastly, a re-cent report demonstrates that the

loss of p53 in differentiating murineTS cells renders them not only re-

sistant to DNA-damage-induced

apoptosis, but also allows them tobypass the critical G1 checkpoint

(Soloveva and Linzer, 2004). This is

supported by the report that ex-

traembryonic cells, but not embry-onic cells, are resistant to p53-de-

pendent cell death after DNAdamage (Heyer et al., 2000). p53-

deficient mice are viable, but cells

derived from these animals demon-strate higher proliferation rates in

culture (Tsukada et al., 1993);

however, the growth dynamics of

trophoblast-derived cells were notassessed in this study. In light of

the above reports, it may be worth-

while to revisit the p53 knockout

placentae for overall trophoblastresistance to hypoxia- and DNA-

damage-induced apoptosis, and toassess p53-mediated apoptosis

patterns in differentiating tropho-

blast cell subpopulations.The Rb protein is also a tumor

suppressor, with a prominent role

in cell cycle regulation. The phos-phorylation state of Rb correlates

with its functional capacity: phos-

phorylation is maximal at the startof S phase (i.e., in proliferating

cells) and lowest after mitosis and

entry in G1(i.e., in quiescent cells);hence, the hypophosphorylated

form of Rb suppresses cell prolifer-ation. It was recently demonstrated

that hypophosphorylated Rb pre-dominates in murine TS cells de-

prived of Fgf-4 (a required growth

factor for TS cells) and hyperphos-phorylated Rb is found in actively

growing TS cells (Soloveva and Lin-

zer, 2004); however, all forms of Rbprotein were observed to decrease in

differentiating giant cells in vitro (So-

loveva and Linzer, 2004). Initialstudies producing Rb knockout mice

revealed that these mice die be-tween embryonic days 13.5 and

15.5, displaying abnormalities inerythropoiesis and central nervous

system due to excessive apoptosis,

failed differentiation, and disruptionof the cell cycle (Clarke et al., 1992;

Jacks et al., 1992; Lee et al., 1992).Additionally, it was later reported

that Rb mutant placentae exhibit in-

creased numbers of trophoblast cellsin the labyrinth, leading to disrupted

architecture of this placental layer

and resulting in decreased placental

vascularization and transport (Wu et

al., 2003). These data indicate thatRb mutant placentae appear to have

an unprogrammed proliferation de-

fect within the labyrinthine layer, off-

setting the balance between the celldeath and proliferation pathways. In

order to determine whether the em-bryonic phenotype was secondary to

a placental defect, tetraploid aggre-

gation and conditional knockout ap-proaches were employed to provide

Rb-deficient embryos with wild-type

placentae. Under these conditions,

the knockout embryos were carriedto term, but died shortly after birth

(Wu et al., 2003), suggesting that

the majority of embryonic pheno-

types in Rb-deficient embryos arecaused by a dysfunctional placenta

and are therefore, not due to a cell-autonomous Rb requirement by the

embryo. Rb family members bridge

the cell death and cycle pathways bydirectly repressing E2F/Dp transcrip-

tion factors (Trimarchi and Lees,

2002). Recently, it was reported thatDp1 knockout embryos die by em-

bryonic day 12.5 and demonstrate

increased rates of apoptosis in earlyextraembryonic cells (Kohn et al.,

2003). Dp1 deficiency might effect

this increased cell death by prevent-ing Rb-mediated repression of

p19ARF (a p53 stabilizer) or p73 (ap53 homolog with apoptotic func-

tions).PTEN is a tumor suppressor that

has been shown to inhibit cell mi-

gration, cell spreading, and focaladhesion formation through inter-

actions with focal adhesion kinase(Tamura et al., 1998; Gu et al.,

1999). Additionally, PTEN has been

shown to negatively regulate phos-

phoinositide-dependent kinase 1(PDK1), which phosphorylates andactivates protein kinase B (PKB/

Akt1), triggering a well-established

survival pathway (Staal and Hart-ley, 1988; Dudek et al., 1997).

PTEN has been shown to be ubiqui-

tously expressed in embryonic day7.5 mouse embryos, and its inacti-

vation in the mouse causes embry-onic lethality at day 9.5 (Stambolic

et al., 1998; Suzuki et al., 1998).

Knockout embryos demonstrate re-gions of increased proliferation, in-




13/19

cluding the allantois, the expansion

of which is hypothesized to inhibit

chorioallantoic fusion and there-

fore, causes embryonic death (Su-zuki et al., 1998). Interestingly,

murine trophoblast and endothelial

cells have been demonstrated to

express PKB/Akt1, and the conse-quences of PKB/Akt1-deficiency are

placental hypotrophy, with reduceddecidual basalis, glycogen-contain-

ing spongiotrophoblast, and vascu-

larization (Yang et al., 2003). Thesedefects indicate a deficiency in pla-

centation, leading to the observed

fetal growth reduction, neonatal le-

thality, and diminished life span af-ter exposure to genotoxic stress

(Yang et al., 2003). Lastly, it has

also been shown that PKB/Akt1 sig-

naling is involved in differentiationof murine TS cells to giant cells in

vitro (Kamei et al., 2002). Since gi-ant cells in the mouse placenta are

involved in the production of nu-

merous protein and steroid hor-

mones, it is possible that reducedplacental and fetal growth may be

due to the impaired differentiation

of this cell type.Murine Bruce/Apollon (baculovi-

rus inhibitor of apoptosis repeat-containing (BIR) ubiquitin-conju-

gating enzyme) is a large

(528-kDa) protein that has both anN-terminal BIR domain and a C-ter-

minal ubiquitin-conjugating do-main (UBC); these regions provide

Bruce with both antiapoptotic

(Hauser et al., 1998; Chen et al.,1999; Bartke et al., 2004) and ubiq-

uitinoylation capabilities (Bartke etal., 2004; Hao et al., 2004b). There

are several inhibitors of apoptosis

(IAP) proteins, including Bruce, andthese proteins function as cell death

antagonists by suppressing pro-

apoptotic proteins such as Smac/Diablo and active caspase-9 (Bartke

et al., 2004; Hao et al., 2004b). TheBruce gene is highly conserved, as

human APOLLON shares 92% iden-tity with Bruce, and has been dem-

onstrated to confer chemotherapeu-

tic resistance to certain cancer cells(Chen et al., 1999). Lotz et al.

(2004) reported that Bruce is ex-pressed predominantly in the diploid

trophoblast of the placenta, including

the labyrinthine layer and the spon-giotrophoblast; reduced Bruce ex-

pression was observed in trophoblastgiant cells. Analysis of earlier devel-

opmental stages revealed that Bruce

can also be detected in the chorion,within the cells of the ectoplacental

cone and in early trophoblast giant

cells, as well as in the late gastrula

stage embryo (Hitz et al., 2005; Renet al., 2005). Three separate groups

of investigators have producedBruce knockout mice, with two

groups observing a trophoblast pro-

liferation-related phenotype and no

alterations in cell death (Hitz et al.,2005; Lotz et al., 2004). The third

group also observed proliferation de-

fects but additionally reported in-creased rates of apoptosis in Bruce-

deficient placentae (Ren et al.,

2005). Loss of Bruce leads to embry-

onic and/or perinatal growth reduc-tion and lethality, which can likely be

attributed to the observed placentaldefects. Proliferation was severely

reduced in the spongiotrophoblast

layer (Hitz et al., 2005; Lotz et al.,2004), and was diminished in the

labyrinth (Lotz et al., 2004). Addi-

tionally, up regulation of Bax, Bak,and caspase-2 in mutant trophoblast

cells and activation of the mitochon-

drial cell death cascade in embryonicfibroblasts obtained from mutant

embryos was also detected (Ren et

al., 2005). Moreover, p53 expressionwas also demonstrated to be ele-

vated in Bruce-deficient placentae,particularly in spongiotrophoblast

cells, and silencing of p53 and Bruce

expression in cell lines resulted in im-proved cell viability (Ren et al.,

2005). Thus, p53 appears to acts asa downstream effector of Bruce, and

in the absence of Bruce, mitochon-

drially-mediated apoptosis ensues.The conflicting results of these stud-

ies is perhaps, not so unexpected in

hindsight. Bruce is a chimeric mole-cule, with both ubiquitinoylation and

apoptosis-inhibiting capabilities, un-derscoring the multifunctional na-

ture of the Bruce protein. Yeast ho-mologs of Bruce have likewise been

shown to be involved in cell division

(Uren et al., 1999; Silke and Vaux,2001). On the other hand, as previ-

ously stated, Bruce is also clearly as-sociated with the apoptotic pathway,

and further molecular analysis into

the nature of this enigmaticyet ex-citingmolecule in the right cellular

context is required, in order to fur-ther elucidate the function of Bruce

during mammalian placentation.

Daxx (Fas death domain-associ-ated protein) was initially re-

ported as a highly conserved pro-

tein that associated with the

intracellular domain of Fas andenhanced Fas-mediated apoptosis

in overexpression studies (Yang etal., 1997). As is the case with

Bruce, Daxx is a multifunctional

protein with seemingly contradic-

tory functions. It has been shownto be involved in both extrinsic

(TGF--mediated) and intrinsic

(p53-dependent DNA damage)apoptosis pathways (Gostissa et

al., 2004; Chang et al., 2005). In

addition to these cell death func-

tions, Daxx is also capable of tran-scriptionally repressing CRE,

E2F1, Sp1, NF-B, and the andro-gen receptor (Ecsedy et al., 2003;

Chang et al., 2005), demonstrat-

ing further potential in modulatingcellular behavior. Daxx is ex-

pressed in a number of murine (Su

et al., 2002) and human tissues,including placenta (Annotation:

GDS1096, GDS181, Geo profiles

at www.ncbi.nlm.nih.gov). Daxxdeficiency in mice leads to embry-

onic lethality by day 9.5 and both

embryonic and extraembryoniclineages are diminished in com-

parison to wild-type littermates,marked by increased rates of ap-

optosis by day 7.5 and day 8.5(Michaelson et al., 1999). This en-

hanced apoptosis was unex-

pected, since Daxx had, untilthen, only been associated with

promoting apoptotic events in the

cell; however, further investiga-tion revealed that Daxx mutant ES

cells also had elevated rates of ap-

optosis. It was later revealed thata similar Daxx deletion triggered

cell death by stimulating the JNK/p38-Bim-Bax pathway, leading to

the activation of caspase-9 andcaspase-3 (Song and Lee, 2004).

Lastly, Daxx has recently been re-

ported to play a role in viral pro-tection, as Daxx-null fibroblast

cell lines demonstrated enhancedviral gene expression compared to

Daxx-complemented cells (Greger

et al., 2005). Therefore, it is in-triguing to speculate that in addi-




14/19

tion to the role of Daxx as a regu-

lator of cell death in the placenta,

it may also be involved in protect-

ing the trophoblast and hence, the

fetus, from viral invasion.

Prostaglandin F2 receptor (FP)

is a G-protein-coupled receptor

that has been shown to induce theapoptosis cascade by activating

caspase-8 in luteal cells (Li et al.,

2001). Interestingly, FP lacks an

intracytoplasmic region possess-

ing any of the traditional death or

caspase-recruiting domains that

characterize other, typical death

associated receptors. Prostaglan-

din F2 receptor is highly ex-

pressed in the uterus (Sugimoto

et al., 1994), and has also been

shown to be expressed in the

mouse (Su et al., 2004) and hu-man (Su et al., 2002) placenta.

Homozygous deletion of FP re-

sulted in developmentally normal

and viable mice, but FP-deficient

females fail to deliver their fetuses

at term, which eventually died in

utero and were resorbed (Sugi-

moto et al., 1997). No changes

were detected in mutant placental

and decidual weights, nor were

there any disparities in placental

cell death patterns; however, ele-

vated decidual cell death overgestation was noted (Mu et al.,

2002). Further characterization of

phenotype revealed alterations of

decidual cell death patterns once

postterm fetuses were catego-

rized as either live or dead. Ele-

vated decidual cell death in dead

fetuses was associated with alter-

ations in the Bax/Bcl-2 ratio and

increased active caspase-3 levels

(Mu et al., 2003). Given these ob-

servations, it was hypothesized

that decidual cell death was nec-

essary for normal term delivery of

the conceptus, and that a Bax:

Bcl-2 rheostatis involved in reg-

ulating apoptosis in the postterm

placenta (Mu et al., 2003). On the

contrary, upregulation of Bcl-2

was reported in the decidua of

abortion-prone mice, perhaps

serving as a compensatory or pro-

tective mechanism (Bertoja et al.,

2005), especially considering the

premature deliveryof these pre-

term placentae.

As a well-spent day

brings happy sleep, so life

well used brings happy

death (Leonardo Da Vinci)

The functional meaning of cellulardeath in the context of tissue sur-

vival is beautifully reflected in thiswise observation. This is particu-

larly true for the life of the tropho-blast cells as the proper oxygen-

ated environment brings ameaningful death, while improper

oxygenation, characteristic featureof placental pathologies, leads to an

accelerated death which often re-sults in a harmful death. In sum-

mary, cell death plays a key role inthe appropriate maturation of the

placenta, and pro- and antiapop-

totic expression patterns can bemanipulated in the fetal-placental-maternal unit in order to optimize

the uterine environment for healthygestation. However, deregulation

of these pathways has clear nega-tive long-term developmental con-

sequences for both the mother and

the fetus, resulting in serioushealth threats that may ultimately

lead to death.

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