Programmed Cell Death in the Ovary: Insights and Future Prospects Using Genetic Technologies
James K. Pru and
Jonathan L. Tilly
Vincent Center for Reproductive Biology Department of
Obstetrics and Gynecology Massachusetts General Hospital/Harvard
Medical School Boston, Massachusetts 02114
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ABSTRACT
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Programmed cell death (PCD) plays a prominent role
in development of the fetal ovaries and in the postnatal ovarian cycle.
As is the case with other major organ systems, an evolutionarily
conserved framework of genes and signaling pathways has been implicated
in determining whether or not ovarian germ cells and somatic cells will
die in response to either developmental cues or pathological insults.
However, the identification of increasing numbers of potential ovarian
cell death regulatory factors over the past several years has
underscored the need for studies to now separate correlation
(e.g. endogenous gene expression) from function
(e.g. requirement of the gene product for the execution of
PCD). In this regard, genetic technologies have recently been used to
examine the functional significance of specific proteins and
signaling molecules to the regulation of PCD in the female gonad
in vivo. In addition to the more classic approaches, such
as the use of genetic null and transgenic mice, methods that achieve
cell lineage-selective and/or developmentally timed gene targeting are
on the horizon for use by reproductive biologists to more accurately
dissect the mechanisms by which PCD is controlled in the ovary. This
minireview will highlight some of the advances that have already been
made using gene knockout and transgenic mice, as well as provide an
overview of the current and future status of cell lineage-selective
gene disruption, in the context of PCD and ovarian function.
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THE GENETICS OF PROGRAMMED CELL DEATH (PCD) VIA APOPTOSIS
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In cells of both invertebrate (Caenorhabditis elegans,
Drosophila melanogaster) and vertebrate animal species, the
core cell death machinery is composed of three families of proteins
that are classified based on structural and functional similarity
(reviewed in Refs. 1, 2, 3). In C. elegans, PCD is controlled,
for the most part, by the actions and interactions of the
egl-1 (proapoptotic)/ced-9 (antiapoptotic),
ced-4 (proapoptotic), and ced-3 (proapoptotic)
gene products (reviewed in Ref. 4). A similar situation has emerged in
Drosophila, with orthologs of EGL-1/CED-9
(Decbl/dBorg-1/Drob-1/DBok, one in the genome database as yet unnamed),
CED-4 (Dark/HAC-1/Dapaf-1), and CED-3 (Dcp-1, Dcp-2/Dredd, drICE,
Dronc, Decay, two in the genome database as yet unnamed) having been
documented as critical components of fly cell death (reviewed in Refs.
1, 2). Quite strikingly, vertebrates have retained, and expanded on,
this complex molecular framework for the control of PCD through
evolution (reviewed in Ref. 1). At present, at least 19 CED-9 orthologs
(collectively referred to as Bcl-2 family members) have been identified
in vertebrates (reviewed in Refs. 1, 5, 6). Each member of this
family has been subclassified based on its reported function in cell
death regulation (antiapoptotic: Bcl-2, Bcl-xL,
Mcl-1, A1/Bfl-1, Bcl-w, Bcl-B, NR-13; proapoptotic: Bcl-xS,
Bax, Bak, Bad, Hrk/DP5, Bid, Bik/Blk, Bim, Bok/Mtd, Noxa, Bcl-rambo,
Bcl-GL, Bcl-GS), with one
member (Diva/Boo) remaining controversial in terms of function (7, 8).
Three CED-4 orthologs are known to be expressed in vertebrates (Apaf-1,
Flash, Nod1/Card4), although only one (Apaf-1) has been extensively
characterized with respect to its function in apoptosis in
vivo and in vitro (9, 10, 11, 12, 13). Lastly, at least 14 CED-3
orthologs (collectively referred to as cysteine aspartic acid-specific
proteases or caspases) have been described in vertebrate species,
although some appear more critical than others in the control of
apoptosis (reviewed in Refs. 14, 15, 16). Of further note, caspases have
also been subclassified based on whether the enzyme functions primarily
as an initiator or an effector of PCD (reviewed in Refs. 14, 15, 16).
Data derived from both subcellular localization studies and biochemical
assays have strongly implicated many members of the bcl-2
gene family as modulators of mitochondrial function or stability
(reviewed in Refs. 5, 6, 17, 18). In fact, the ability of Bcl-2 and
related proteins to modulate release of apoptogenic factors, such as
cytochrome c, apoptosis-inducing factor, and Smac/Diablo, from
mitochondria into the cytosol (Fig. 1
) is
now believed to be one of the most important determinants of whether or
not PCD will proceed (reviewed in Ref. 3). Indeed, the release of
cytochrome c from mitochondria has been established as a critical
signal for many cells to commit to the next stage of PCD,
i.e. the recruitment and activation of Apaf-1, a cytoplasmic
adaptor protein (reviewed in Ref. 19). In the presence of ATP/dATP,
cytochrome c causes a conformational change in Apaf-1, facilitating
heterodimeric interaction of the protein with the proform of caspase-9.
Generation of this apoptosome then leads to cleavage activation of
caspase-9, probably through an induced proximity model (reviewed in
Ref. 20). Once activated, caspase-9 functions as the apical enzyme in a
proteolytic cascade, involving sequential cleavage activation of
several effector or downstream caspases, which rapidly disables and
dismantles the cell now destined to die (Fig. 1
) (reviewed in Refs.
14, 15, 16, 19). The final step in PCD involves phagocytic clearance of
the cell corpse, thus eliminating the generation of a local
inflammatory response.

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Figure 1. Schematic Model of the PCD Machinery in Vertebrates
Extracellular stimuli, such as cytokines (Fas ligand or
FasL), survival factors, stress, and genotoxicants, utilize
a number of signal transduction molecules, including sphingolipids
(ceramide, sphingosine-1-phosphate or S1P) and protein kinases
(phosphatidylinositol 3'kinase or PI3K, c-Akt), to relay information to
a central PCD rheostat governed by Bcl-2 family members (Bcl-2, Bcl-w,
Bax, Bak, Bid). If the stimulus is lethal, proapoptotic Bcl-2 family
member function prevails, causing release of apoptogenic factors, such
as cytochrome c and Smac/Diablo, into the cytosol. Cytochrome c is
essential for generation of the apoptosome containing Apaf-1 and
procaspase-9. Once formed, the apoptosome initiates the caspase cascade
after auto- or transcatalytic activation of the most apical enzyme,
caspase-9. Since caspase activation is believed to be the
point-of-no-return in the PCD pathway, a checkpoint composed of a
family of gene products referred to as inhibitor-of-apoptosis (IAP)
proteins serves to prevent premature or unwanted activation of
apoptosis by repressing caspase activation/activity. In the case of
FasL-initiated death, two distinct intracellular pathways
are believed to exist, one that can utilize mitochondria (via cleavage
activation of the proapoptotic Bcl-2 family member, Bid) and one that
can proceed directly to the caspase cascade (81 ). Proapoptotic pathways
are highlighted by red text and arrows, whereas
antiapoptotic pathways are highlighted by blue text and
arrows.
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THE MANY FACES OF CELL DEATH IN THE OVARY
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Apoptosis has been implicated in a spectrum of processes
associated with normal ovarian development and function, including
prenatal germ cell attrition (oocyte death; reviewed in Ref. 21),
postnatal follicular atresia (granulosa cell death; reviewed in Ref.
22), ovulation (ovarian surface epithelial cell death; reviewed in Ref.
23), and luteolysis (luteal cell death; reviewed in Ref. 24).
Additionally, premature ovarian failure, caused by exposure of females
to pathological stimuli in the environment (biohazardous chemicals) or
in the clinic (cancer therapies), is probably the result of
inappropriate activation of PCD in oocytes (reviewed in Ref. 21). Many
studies have now been published with respect to the identification of
apoptosis in each of the processes indicated above. In addition, work
from a number of laboratories has been directed toward characterizing
the expression and, in some cases, the possible function of gene
products that regulate PCD in oocytes, granulosa cells, and corpora
lutea. However, since these topics have been recently reviewed in
detail elsewhere (see above), the remainder of this minireview will be
devoted to a discussion of how genetic technologies have, or in some
cases hopefully will, become central to establishing a causal
vs. casual relationship between expression of a given
apoptosis-regulatory protein and the functional significance of that
protein in the control of PCD in the ovary.
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PCD GENE KNOCKOUTS AND OVARIAN FUNCTION
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At least 60 different proteins and signaling molecules have been
identified thus far as constituents of the intracellular framework that
governs apoptosis in mammals (Fig. 1
). Furthermore, studies of mutant
mice lacking expression of various components of the core PCD machinery
(e.g. see Table 1
) have
revealed redundancies in the process in that many organs are unaffected
by loss-of-function of what are considered some of the most basic
regulators of apoptosis. Therefore, efforts to assemble a molecular
blueprint of how PCD is regulated on an organ- or cell lineage-specific
basis are now a priority. In this regard, several laboratories,
including our own, have used mutant mice lacking proteins involved in
the control of PCD as a means to achieve this goal in both germ cell
and somatic cell lineages of the ovary (Table 1
). Unfortunately, a
detailed discussion of the results from each of these studies cannot be
accomplished within the page limits of this minireview. Therefore, we
will briefly overview observations that have been made from analysis of
three specific genetic null mouse lines, which have been fairly well
characterized with respect to ovarian phenotypes.
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Table 1. Genetic Null Mouse Lines, Lacking Various
Apoptosis Regulatory Proteins, in Which Female Reproductive Function
Has Been, or Is Being,
Evaluated1
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The first mutant mouse line, originally generated by the Korsmeyer
laboratory (25), harbors a targeted disruption in the gene encoding
Bax, a proapoptotic member of the Bcl-2 family. Expression of the
bax gene has been identified in oocytes (26, 27), granulosa
cells (28, 29), and luteal cells (30, 31) of various species, and
levels of bax expression appear to be positively correlated
with apoptosis in each of these cell lineages. In the first publication
describing the generation of these mice, Knudson and colleagues (25)
noted "a marked accumulation of unusual atretic follicles"
containing "numerous atrophic granulosa cells that presumably failed
to undergo apoptosis." Two years later, Perez et al. (32)
reported that primordial oocytes within the ovaries of bax
null mice were completely resistant to apoptosis induced by exposure to
a widely used chemotherapeutic drug (i.e. doxorubicin)
in vivo. That this defect is cell autonomous
(i.e. germ cell intrinsic) in nature was evidenced by the
finding that isolated Bax-deficient oocytes cultured in
vitro were similarly resistant to the proapoptotic effects of
doxorubicin (32). This study was then followed by an in-depth
evaluation of ovarian follicular dynamics in Bax-deficient mice (33).
Although the number of oocytes endowed in the ovaries of neonatal
bax null females is similar to that of their wild-type
sisters, Bax-deficient female mice exhibit a significant defect in
primordial and primary follicle atresia rates due to a marked reduction
in the incidence of postnatal oocyte death. Importantly, this defect in
oocyte death leads to a dramatic prolongation of ovarian life span in
aged bax mutant females (33). The involvement of Bax in germ
cell apoptosis has been further solidified by recent observations that
bax gene inactivation can suppress fetal oocyte death
resulting in loss of either Bcl-x (34) or Bcl-w (35) function. These
data, taken with findings that microinjection of recombinant Bax
protein into oocytes is sufficient in itself to trigger apoptosis (36),
collectively support a fundamental role for Bax in mediating both germ
cell and granulosa cell apoptosis.
The second mutant mouse line, originally generated by the Yuan
laboratory (37), lacks expression of one of the final executioners of
PCD, caspase-2. Like bax, the gene encoding caspase-2 is
expressed in multiple cell lineages of the ovary, including oocytes
(37, 38). However, general histological surveys of young adult
caspase-2 deficient females did not reveal the presence of the aberrant
atretic follicles noted in bax mutant female mice (discussed
in Ref. 39), suggesting that this specific caspase family member is not
essential for granulosa cell death to proceed. In contrast,
caspase-2 null female mice are born with a significantly
larger reserve of primordial oocytes (37), a phenotype recently
confirmed to be a result of defective apoptosis in the developing fetal
oocyte pool (40). Moreover, oocytes isolated from the ovaries of young
adult caspase-2-deficient females are, like bax null
oocytes, resistant to apoptosis induced by exposure to doxorubicin
(37). It should be emphasized, however, that while caspase-2 is clearly
important for apoptosis to occur in oocytes under some situations, the
enzyme does not appear to be required for the execution of PCD in
oocytes under all conditions. In fact, through the generation of
several double-mutant mouse lines, Morita and colleagues (40) recently
demonstrated that prenatal oocyte loss resulting from cytokine
insufficiency, but not that caused by meiotic defects, can be prevented
by inactivation of the caspase-2 gene. Therefore, even in
the same cell lineage, different stimuli for PCD can apparently recruit
into action different components of the core cell death machinery.
The third and final genetic null mouse line to be discussed in
this section is the caspase-3 deficient mouse, originally generated by
the Flavell laboratory (41). A tremendous amount of evidence has been
provided to suggest that caspase-3 is a principal executioner of PCD in
the ovary. For example, an inverse correlation exists between caspase-3
expression, at both the mRNA and protein level, and apoptosis in
granulosa cells of the rat ovary (38, 42). This work has been followed
by studies that have established the presence of processed
("active") caspase-3 in granulosa cells of adult murine and human
ovaries during the early stages of follicular atresia (39).
Furthermore, an induction of procaspase-3 processing and/or
caspase-3-like enzymatic activity occurs in murine (43) and avian (44)
granulosa cells during apoptosis in vitro, and peptide
inhibitors selective for caspase-3 suppress granulosa cell death in
murine ovarian follicles cultured in vitro without hormonal
support (45). Similarly, apoptotic death of both ovine (46) and bovine
(47) luteal cells is correlated with an induction of caspase-3
expression and/or activity. Regarding the female germ line, oocytes are
also known to express caspase-3 (48), and studies utilizing
caspase-3-selective inhibitors and substrates have implicated this
specific caspase family member in mediating oocyte apoptosis (32, 49).
With this information in mind, recent evaluations of ovarian
development and function in caspase-3 null female mice have
provided both expected and unexpected findings. Despite the fact that
caspase-3 is expressed in murine oocytes, loss of caspase-3 function
affects neither developmental nor anti-cancer therapy-induced oocyte
apoptosis (39). These results reemphasize the importance of studies to
separate correlation (i.e. expression of a PCD-regulatory
gene) from function (i.e. requirement of the gene product
for cellular survival or for apoptosis to proceed) in attempts to
construct a molecular blueprint of how PCD is initiated and executed in
specific cell lineages. By comparison, ovaries of young adult
caspase-3 mutant female mice possess numerous aberrant
atretic follicles containing granulosa cells that have failed to
complete the program of apoptosis (39). Moreover, in vitro
experiments were used to confirm that the defect in granulosa cell
death execution is cell autonomous in nature (39). Therefore,
caspase-3, while dispensable for oocyte apoptosis, is clearly required
for the normal progression of PCD in granulosa cells during follicular
atresia. Since preliminary studies indicate that female mice lacking
caspase-9 (50) exhibit defects in apoptosis in both oocytes and
granulosa cells (51), these studies collectively indicate that a
divergence in how ovarian germ cells vs. somatic cells
execute PCD occurs at a site downstream of caspase-9.
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TRANSGENIC EXPRESSION OF PCD-REGULATORY GENES IN THE OVARY
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An alternative approach to assessing the potential function of
core components of the PCD machinery in the ovary is to utilize
transgenic technology to direct expression of apoptosis-regulatory
genes to specific cell types. While this technology is useful for
testing the functional consequences of a specific gene product in cells
in their normal in vivo environment, it should be stressed
that transgenic overexpression suffers from the same principal drawback
as the use of cellular transfections in vitro. Ectopic
overexpression of a gene in vivo (or in vitro,
for that matter) reflects the potential, rather than the genuine,
function of the respective protein in a cell. Nonetheless, at present
there exist three published reports using such an approach, all of
which overexpressed the antiapoptotic protein, Bcl-2.
The first used a 6-kbp fragment of the 5'-flanking region of the murine
inhibin-
gene to direct expression of human Bcl-2 (52).
Analysis of transgene expression in these mice revealed the presence of
human Bcl-2 in the adrenal cortex, testicular Sertoli cells, and
multiple ovarian somatic cell lineages (stromal, granulosa,
theca-interstitial, luteal). Overexpression of Bcl-2 in the ovaries was
shown to reduce the incidence of granulosa cell apoptosis in immature
mice primed 4 days earlier with a low dose of equine CG (eCG), to
increase spontaneous ovulation rates in immature mice primed with a
high dose of eCG, and to increase the mean litter size in adult
animals. Interestingly, 20% of the
inhibin-
/bcl-2 transgenic female mice develop
well differentiated cystic teratomas with age, although oocytes do not
show evidence of transgene expression. Thus, it was concluded that
Bcl-2 overexpression in ovarian somatic cells has a secondary or
indirect effect on oocyte differentiation, leading to germ cell
transformation (52).
The second transgenic mouse line used a 480-bp fragment of the murine
zona pellucida protein-3 (ZP3) gene promoter to
target expression of human Bcl-2 only to developing oocytes of the
postnatal ovary (53). Consistent with the expression patterns of the
endogenous ZP3 gene in the mouse, human Bcl-2 was only
detected in oocytes of follicles that had initiated growth.
Furthermore, accumulation of the transgene product in oocytes was found
to convey resistance to both developmental and chemotherapy-induced
apoptosis (53). The third mouse line, generated shortly thereafter,
targeted expression of human Bcl-2 to developing fetal oocytes by using
a 4.8-kbp fragment of the murine c-kit gene promoter (54).
Evaluation of germ cell dynamics in these mice indicated that
overexpression of bcl-2 during fetal life results in an
increased endowment of primordial follicles in neonates, presumably due
to a reduced incidence of prenatal germ cell death during ovarian
development (54). The results from these latter two studies, when taken
with previous findings of reduced primordial oocyte numbers in
bcl-2 null female mice (55), collectively indicate that
oocyte fate is markedly affected by altering the levels of Bcl-2 in the
female germ line through various genetic approaches.
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CRE RECOMBINASE-LOXP AND CONDITIONAL GENE KNOCKOUTS
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While whole animal gene knockouts have thus far proved invaluable
for helping to define functionally relevant gene products that regulate
PCD in the ovary (Table 1
), limitations to the use of this technology
are evident (reviewed in Refs. 56, 57, 58). For example, disruption of some
genes encoding PCD regulators leads to embryonic or fetal lethality
(e.g. bcl-x, mcl-1, apaf-1,
caspase-7), thus precluding use of these mice for many
studies. Second, there is concern that the selection marker
(e.g. neomycin) in the expression cassette typically used to
disrupt the target gene can inadvertently influence expression of a
flanking gene(s), generating a phenotype that may not solely reflect
inactivation of the target gene. Third, one has to consider that a
phenotype observed in a given cell lineage can result from loss of gene
function in that cell type or in another cell lineage that influences
the function of the first cell type. In other words, does the gene
knockout phenotype in one cell lineage indirectly contribute to other
phenotypes in other cell lineages? The Cre recombinase-loxP
system circumvents many critical shortcomings of generalized knockouts
by incorporating two additional dimensions: the mouse genome can be
modified in a cell lineage-selective/specific fashion and in a
developmentally timed manner.
Generation of a conditional gene knockout in mice is relatively simple
in theory, with two different mouse lines required (reviewed in Refs.
56, 57, 58). Briefly, for mouse line 1 homologous recombination is used to
insert small DNA sequences, called loxP sites, into inactive
regions of DNA (e.g. introns) flanking a functionally
important part of the gene to be targeted. Each loxP site
corresponds to a 34-bp sequence, consisting of two 13-bp
palindromic sequences with an 8-bp central core. Once
accomplished for both alleles, such genes are said to be "floxed."
The P1 bacteriophage-derived enzyme, Cre recombinase, is then needed to
catalyze recombination between the two loxP sites, resulting
in excision of the intervening DNA sequence. However, since mammals do
not express Cre recombinase, a second mouse line is required to provide
expression of the enzyme. For the generation of this mouse line, a
promoter capable of directing cell lineage-specific/selective or
developmentally timed gene expression is ligated upstream of the gene
encoding Cre recombinase. This minigene is then used to produce
transgenic mice. When the two mouse lines are crossed, offspring will
be generated in which the floxed portion of the gene of interest is
excised when the promoter of the gene used to confer cell
lineage-specific/selective or developmentally timed expression is
normally activated. It is recommended that a reporter system be
initially employed to confirm the fidelity (level and specificity) of
Cre recombinase expression. Many such reporter mouse lines are now
available, including a sophisticated double-reporter line in which
lacZ is expressed before, and alkaline phosphatase is
expressed after, Cre recombinase-mediated excision (59).
Several laboratories have begun to generate the tools needed for the
use of conditional gene knockout technology in studying ovarian
development and function. Three relevant examples will be briefly
discussed here, although it should be noted that no published reports
yet exist in which the technology has been used to target
PCD-regulatory genes in the female gonads. The first example is a mouse
line generated to express Cre recombinase under the control of the
ZP3 gene promoter, thus providing a system to inactivate
specific genes only in growing oocytes (60). Another group has recently
followed suit with the independent generation of a second
ZP3 promoter-Cre recombinase mouse line to study the role of
specific maternal transcripts in completion of meiosis, activation of
the embryonic genome, and transformation of the highly differentiated
oocyte into a totipotent embryonic stem cell (61). Work from Lomeli and
colleagues (62) provides the second example, which describes the
generation of mice with primordial germ cell-specific expression of
Cre recombinase being achieved, in a somewhat atypical fashion, by
knocking the enzyme into the locus of the tissue nonspecific alkaline
phosphatase (TNAP) gene. Such a line will be useful for
defining those PCD-regulatory genes functionally relevant to
controlling the size of the germ line during embryogenesis. In the
final example, preliminary work of Zhang and colleagues (63) has
established several lines of mice expressing Cre recombinase under
control of the inhibin-
subunit promoter. Gonad-specific
expression of the enzyme was identified by RT-PCR, with Cre
recombinase-mediated excision of a reporter gene further confirming
expression of the enzyme in testicular Sertoli cells, Leydig cells, and
spermatogonia. These mouse lines, along with other transgenic lines
currently being generated, will certainly be valuable tools in future
studies to dissect the pathways by which PCD is regulated in specific
gonadal cell lineages.
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DOUBLE-STRANDED RNA INTERFERENCE
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A final technology that warrants some discussion is RNA
interference (RNAi), the epigenetic silencing of genes by introduction
of double-stranded (ds) RNA into cells. It should be emphasized that
RNAi is an evolutionarily conserved mechanism used by cells for
regulation of endogenous gene expression and protection against
dsRNA viruses (64). The process of RNAi, which involves the degradation
of dsRNA probably through the same enzymes involved with transposition
and nonsense-mediated RNA decay (65), was originally discovered in
C. elegans (66). However, RNAi has since been demonstrated
in other species (64), including vertebrates (67, 68). Experimental
adaptations of RNAi have recently been used quite successfully to study
gene function by causing the elimination of specific endogenous mRNA
transcripts. Furthermore, the dsRNA used to initiate RNAi can be
transmitted to the germ line and thus passed to subsequent generations.
For example, Quinn et al. (69) have reported in flies that
RNAi-mediated loss of Dronc (a Drosophila caspase family
member) function results in a dramatic decrease in cell death during
embryogenesis, thus confirming that Dronc is a key component of the
core PCD machinery in Drosophila. In a similar fashion, RNAi
was recently used to demonstrate that Debcl (dBorg-1/Drob-1/Dbok), the
first cloned member of the Drosophila Bcl-2 family, is
functionally required for developmental PCD during fly embryogenesis
(70).
In mammals, an interesting study by Svoboda et al. (68) used
RNAi to probe the functional significance of stored mRNA transcripts in
murine oocytes. It was demonstrated that dsRNA, microinjected into the
oocyte before fertilization, effectively and completely eliminated
maternal mRNA transcripts encoded for by specific genes (68). Almost in
parallel to this work, Wianny and Zernicka-Goetz (67) demonstrated that
RNAi could be used to target disruption of c-mos and
E-cadherin gene function in murine oocytes and
preimplantation embryos, producing phenotypes consistent with those
reported in the respective gene knockout mice. Therefore, these studies
collectively indicate that RNAi is indeed powerful new technology that
can be reliably used to fine tune the study of a given PCD gene product
in female germ cells and developing embryos, and potentially other cell
lineages involved in reproduction.
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CONCLUDING REMARKS AND FUTURE PROSPECTS
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In this minireview, we sought to briefly overview the genetic
control of apoptosis in the ovary, and provide some examples of recent
work showing that the framework of the core PCD machinery differs
between ovarian cell lineages. The challenge over the next several
years will be to continue to distinguish between regulators of PCD that
merely show correlative changes in gene expression with cell survival
or death from those gene products that actually contribute to
preventing or promoting apoptosis. In this regard, we currently have
the tools and technologies at our disposal to map the genetic blueprint
of each paradigm of apoptosis within the ovary. In addition to the
continued use of genetic null and transgenic mouse lines, there is
clearly a need to integrate newer genetic (Cre
recombinase-loxP) and biochemical (RNAi) strategies for gene
disruption into these investigations. However, like the ovary itself,
these technologies are continuously developing and evolving. The next
major hurdle for both the Cre recombinase-loxP and RNAi
systems will be the development of inducible gene disruption systems.
Indeed, tamoxifen- and RU486-inducible Cre recombinase-loxP
systems hold great promise (58, 71), and Lam and Thummel (72) have
provided hope that RNAi can also be incorporated into an inducible
system. It should also be stressed that there is a greater need then
ever to apply what is learned about PCD in invertebrate species to
mammalian models. The nematode and the fruit fly are primitive, from an
evolutionary standpoint, when compared with mammals, but it is the
simplistic nature of these invertebrate organisms that make C.
elegans and Drosophila so valuable for the study of
PCD. For example, Wu et al. (73) recently identified a
member of the AAA family of ATPases that associates with multiple
members of the apoptosome in C. elegans. Furthermore,
through the use of RNAi, it was shown that this gene is essential for
development. Such studies underscore one final point. Reproductive
biologists have come a long way, since the pioneering work of Flemming
in the nineteenth century (74), in understanding the roles and
regulation of apoptosis in the ovary; however, we have also just
begun.
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FOOTNOTES
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Address requests for reprints to: Jonathan L. Tilly, Ph.D., Massachusetts General Hospital, VBK137C-GYN, 55 Fruit Street, Boston, Massachusetts 02114. E-mail: jtilly{at}partners.org
Work conducted by the authors and discussed herein was supported by NIH
Grants R01-AG-12279, R01-ES-08430, and R01-HD-34226, by Department of
Defense Grant OC990138, and by Vincent Memorial Research Funds.
Received for publication December 27, 2000.
Revision received February 19, 2001.
Accepted for publication February 27, 2001.
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REFERENCES
|
---|
-
Tittel JN, Steller H 2000 A comparison of programmed cell
death between species. Genome Biol 1:3.13.6
-
Chen P, Abrams JM 2000 Drosophila apoptosis and
Bcl-2 genes: outliers fly in. J Cell Biol 148:625627[Abstract/Free Full Text]
-
Hengartner MO 2000 The biochemistry of apoptosis. Nature 407:770776[CrossRef][Medline]
-
Liu QA, Hengartner MO 1999 The molecular mechanism of
programmed cell death in C. elegans. Ann NY Acad Sci 887:92104[Abstract/Free Full Text]
-
Gross A, McDonnell JM, Korsmeyer SJ 1999 BCL-2 family members
and the mitochondria in apoptosis. Genes Dev 13:18991911[Free Full Text]
-
Antonsson B, Martinou JC 2000 The Bcl-2 protein family. Exp
Cell Res 256:5057[CrossRef][Medline]
-
Inohara N, Gourley TS, Carrio R, Muniz M, Merino J, Garcia I,
Koseki T, Hu Y, Chen S, Nuñez G 1998 Diva, a Bcl-2 homologue that
binds directly to Apaf-1 and induces BH3-independent cell death. J
Biol Chem 273:3247932486[Abstract/Free Full Text]
-
Song Q, Kuang Y, Dixit VM, Vincenz C 1999 Boo, a novel
negative regulator of cell death, interacts with Apaf-1. EMBO J 18:167178[Abstract/Free Full Text]
-
Zou H, Henzel WJ, Liu X, Lutschg A, Wang X 1997 Apaf-1, a
human protein homologous to C. elegans CED-4, participates
in cytochrome c-dependent activation of caspase-3. Cell 90:405413[Medline]
-
Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M,
Alnemri ES, Wang X 1997 Cytochrome c and dATP-dependent formation of
Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91:479489[Medline]
-
Cecconi F, Alvarez-Bolado G, Meyer BI, Roth KA, Gruss P 1998 Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian
development. Cell 94:727737[Medline]
-
Yoshida H, Kong Y-Y, Yoshida R, Elia AJ, Hakem A, Hakem R,
Penninger JM, Mak TW 1998 Apaf1 is required for mitochondrial pathways
of apoptosis and brain development. Cell 94:739750[Medline]
-
Honarpour N, Du C, Richardson JA, Hammer RE, Wang X, Herez J 2000 Adult Apaf-1-deficient mice exhibit male infertility. Dev Biol 218:248258[CrossRef][Medline]
-
Cryns V, Yuan J 1998 Proteases to die for. Genes Dev 12:15511570[Free Full Text]
-
Thornberry NA, Lazebnik Y 1998 Caspases: enemies within.
Science 281:13121316[Abstract/Free Full Text]
-
Zheng T, Hunot S, Kuida K, Flavell RA 1999 Caspase knockouts:
matters of life and death. Cell Death Differ 6:10431053[CrossRef][Medline]
-
Green DR, Reed JC 1998 Mitochondria and apoptosis. Science 281:13091312[Abstract/Free Full Text]
-
Kroemer G, Reed JC 2000 Mitochondrial control of cell death.
Nat Med 6:513519[CrossRef][Medline]
-
Budihardjo I, Oliver H, Lutter M, Luo X, Wang X 1999 Biochemical pathways of caspase activation during apoptosis. Annu
Rev Cell Dev Biol 15:269290[CrossRef][Medline]
-
Salvesen GS, Dixit VM 1999 Caspase activation: the
induced-proximity model. Proc Natl Acad Sci USA 96:1096410967[Abstract/Free Full Text]
-
Morita Y, Tilly JL 1999 Oocyte apoptosis: like sand through an
hourglass. Dev Biol 213:117[CrossRef][Medline]
-
Tilly JL, Robles R 1999 Apoptosis and its impact in clinical
reproductive medicine. In: Fauser BCJM, Rutherford AJ, Strauss III JF,
Van Steirteghem A (eds) Molecular Biology in Reproductive Medicine.
Parthenon, New York, pp 79101
-
Murdoch WJ 2000 Proteolytic and cellular death mechanisms in
ovulatory ovarian rupture. Biol Signals Recept 9:102114[CrossRef][Medline]
-
Rueda BR, Hoyer PB, Hamernik DL, Tilly JL 1997 Potential
regulators of physiological cell death in the corpus luteum. In: Tilly
JL, Struass JF, Tenniswood M (eds) Cell Death in Reproductive
Physiology. Springer-Verlag, New York, pp 161181
-
Knudson CM, Tung KSK, Tourtellotte WG, Brown GAJ, Korsmeyer SJ 1995 Bax-deficient mice with lymphoid hyperplasia and male germ cell
death. Science 270:9699[Abstract]
-
Jurisicova A, Latham K, Casper RF, Varmuza SL 1998 Expression
and regulation of genes associated with cell death during murine
preimplantation embryo development. Mol Reprod Dev 51:243253[CrossRef][Medline]
-
De Felici M, Di Carlo A, Pesce M, Iona S, Farrace MG,
Piacentini M 1999 Bcl-2 and Bax regulation of apoptosis in germ cells
during prenatal oogenesis in the mouse embryo. Cell Death Differ 6:908915[CrossRef][Medline]
-
Tilly JL, Tilly KI, Kenton ML, Johnson AL 1995 Expression of
members of the bcl-2 gene family in the immature rat ovary:
equine chorionic gonadotropin-mediated inhibition of granulosa cell
apoptosis is associated with decreased bax and constitutive
bcl-2 and bcl-xlong messenger
RNA levels. Endocrinology 136:232241[Abstract]
-
Kugu K, Ratts VS, Piquette GN, Tilly KI, Tao X-J, Martimbeau
S, Aberdeen GW, Krajewski S, Reed JC, Pepe GJ, Albrecht ED, Tilly JL 1998 Analysis of apoptosis and expression of bcl-2 gene
family members in the human and baboon ovary. Cell Death Differ 5:6776[CrossRef][Medline]
-
Rueda BR, Tilly KI, Botros I, Jolly PD, Hansen TR, Hoyer PB,
Tilly JL 1997 Increased bax and interleukin-1ß-converting
enzyme messenger RNA levels coincide with apoptosis in the bovine
corpus luteum during structural regression. Biol Reprod 56:186193[Abstract]
-
Dharmarajan AM, Hisheh S, Singh B, Parkinson S, Tilly KI,
Tilly JL 1999 Anti-oxidants mimic the ability of chorionic gonadotropin
to suppress apoptosis in the rabbit corpus luteum in vitro:
a novel role for superoxide dismutase in regulating bax
expression. Endocrinology 140:25552561[Abstract/Free Full Text]
-
Perez GI, Knudson CM, Leykin L, Korsmeyer SJ, Tilly JL 1997 Apoptosis-associated signaling pathways are required for
chemotherapy-mediated female germ cell destruction. Nat Med 3:12281332[Medline]
-
Perez GI, Robles R, Knudson CM, Flaws JA, Korsmeyer SJ, Tilly
JL 1999 Prolongation of ovarian lifespan into advanced chronological
age by Bax-deficiency. Nat Genet 21:200203[CrossRef][Medline]
-
Rucker EB, Dierisseau P, Wagner KU, Garrett L, Wynshaw-Boris
A, Flaws JA, Hennighausen L 2000 Bcl-x and Bax regulate mouse
primordial germ cell survival and apoptosis during embryogenesis. Mol
Endocrinol 14:10381052[Abstract/Free Full Text]
-
Maravei DV, Ross A, Waymire K, Morita Y, Robles R, Korsmeyer
SJ, MacGregor GR, Tilly JL, Bax gene inactivation rescues
gametogenic failure caused by Bcl-w-deficiency but not ataxia
telangiectasia mutated (Atm) gene knockout. Program of
the 82nd Annual Meeting of The Endocrine Society, Toronto, Ontario,
Canada, 2000, pp 317318
-
Morita Y, Perez GI, Paris F, Miranda S, Ehleiter D,
Haimovitz-Friedman A, Fuks Z, Xie Z, Reed JC, Schuchman EH, Kolesnick
RN, Tilly JL 2000 Oocyte apoptosis is suppressed by disruption of the
acid sphingomyelinase gene or by sphingosine-1-phosphate
therapy. Nat Med 6:11091114[CrossRef][Medline]
-
Bergeron L, Perez GI, Mcdonald G, Shi L, Sun Y, Jurisicova A,
Varmuza S, Latham KE, Flaws JA, Salter J, Hara H, Moskowitz MA, Li E,
Greenberg AH, Tilly JL, Yuan J 1998 Defects in regulation of apoptosis
in caspase-2-deficient mice. Genes Dev 12:13041314[Abstract/Free Full Text]
-
Flaws JA, Kugu K, Trbovich AM, DeSanti A, Tilly KI, Hirshfield
AN, Tilly JL 1995 Interleukin-1ß-converting enzyme-related
proteases (IRPs) and mammalian cell death: dissociation of IRP-induced
oligonucleosomal endonuclease activity from morphological apoptosis in
granulosa cells of the ovarian follicle. Endocrinology 136:50425053[Abstract]
-
Matikainen T, Perez GI, Zheng TS, Kluzak TR, Rueda BR, Flavell
RA, Tilly JL 2001 Caspase-3 gene knockout defines cell
lineage specificity for programmed cell death signaling in the ovary.
Endocrinology 142:24682480[Abstract/Free Full Text]
-
Morita Y, Maravei DV, Bergeron L, Wang S, Perez GI, Tsutsumi
O, Taketani Y, Asano M, Horai R, Korsmeyer SJ, Iwakura Y, Yuan J, Tilly
JL, Caspase-2 deficiency rescues female germ cells from death due to
cytokine insufficiency but not meiotic defects caused by ataxia
telangiectasia-mutated (Atm) gene inactivation. Cell
Death Differ, in press
-
Kuida K, Zheng TS, Na S, Kuan C, Yang D, Karasuyama H, Rakic
P, Flavell RA 1996 Decreased apoptosis in the brain and premature
lethality in CPP32-deficient mice. Nature 384:368372[CrossRef][Medline]
-
Boone DL, Tsang BK 1998 Caspase-3 in the rat ovary:
localization and possible role in follicular atresia and luteal
regression. Biol Reprod 58:15331539[Abstract]
-
Robles R, Tao X-J, Trbovich AM, Maravei DV, Nahum R, Perez GI,
Tilly KI, Tilly JL 1999 Localization, regulation and possible
consequences of apoptotic protease-activating factor-1 (Apaf-1)
expression in granulosa cells of the mouse ovary. Endocrinology 140:26412644[Abstract/Free Full Text]
-
Johnson AL, Bridgham JT 2000 Caspase-3 and -6 expression and
enzyme activity in hen granulosa cells. Biol Reprod 62:589598[Abstract/Free Full Text]
-
Maravei DV, Trbovich AM, Perez GI, Tilly KI, Talanian RV,
Banach D, Wong WW, Tilly JL 1997 Cleavage of cytoskeletal proteins by
caspases during ovarian cell death: evidence that cell-free systems do
not always mimic apoptotic events in intact cells. Cell Death Differ 4:707712[CrossRef]
-
Rueda BR, Hendry IR, Tilly JL, Hamernik DL 1999 Accumulation
of caspase-3 mRNA and induction of caspase activity in the
ovine corpus luteum following prostaglandin-F2
treatment in vivo. Biol Reprod 60:10871092[Abstract/Free Full Text]
-
Rueda BR, Hendry IR, Ndjountche L, Suter J, Davis JS 2000 Stress-induced mitogen-activated protein kinase signaling in the corpus
luteum. Mol Cell Endocrinol 164:5967[CrossRef][Medline]
-
Exley GE, Tang C, McElhinny AS, Warner CM 1999 Expression of
caspase and BCL-2 apoptotic family members in mouse preimplantation
embryos. Biol Reprod 61:231239[Abstract/Free Full Text]
-
Perez GI, Tao X-J, Tilly JL 1999 Fragmentation and death
(a.k.a. apoptosis) of ovulated oocytes. Mol Hum Reprod 5:414420[Abstract/Free Full Text]
-
Kuida K, Haydar TF, Kuan C-Y, Gu Y, Taya C, Karasuyama H, Su
MS-S, Rakic P, Flavell RA 1998 Reduced apoptosis and cytochrome
c-mediated caspase activation in mice lacking caspase-9. Cell 94:325337[Medline]
-
Maravei DV, Morita Y, Kuida K, Tilly JL 1999 Pre- and
postnatal ovarian apoptosis defects in caspase-9-deficient mice. Mol
Biol Cell 10[Suppl]:352 (abstract)
-
Hsu SY, Lai RJ-M, Finegold M, Hsueh AJW 1996 Targeted
expression of Bcl-2 in ovaries of transgenic mice leads to decreased
follicle apoptosis, enhanced folliculogenesis, and increased germ cell
tumorigenesis. Endocrinology 137:48374843[Abstract]
-
Morita Y, Perez GI, Maravei DV, Tilly KI, Tilly JL 1999 Targeted expression of Bcl-2 in mouse oocytes inhibits ovarian follicle
atresia and prevents spontaneous and chemotherapy-induced oocyte
apoptosis in vitro. Mol Endocrinol 13:841850[Abstract/Free Full Text]
-
Flaws JA, Hirshfield AN, Hewitt JA, Babus JK, Furth PA 2001 Effect of Bcl-2 on the primordial follicle endowment in the mouse
ovary. Biol Reprod 64:11531159[Abstract/Free Full Text]
-
Ratts VS, Flaws JA, Kolp R, Sorenson CM, Tilly JL 1995 Ablation of bcl-2 gene expression decreases the numbers of
oocytes and primordial follicles established in the post-natal female
mouse gonad. Endocrinology 136:36653668[Abstract]
-
Rossant J, Nagy A 1995 Genome engineering: the new mouse
genetics. Nat Med 1:592594[Medline]
-
Rossant J, McMahon A 1999 "Cre"-ating mouse mutantsa
meeting review on conditional mouse genetics. Genes Dev 13:142145[Free Full Text]
-
Nagy A 2000 Cre recombinase: the universal reagent for genome
tailoring. Genesis 26:99109[CrossRef][Medline]
-
Lobe CG, Koop KE, Kreppner W, Lomeli H, Gertsenstein M, Nagy A 1999 Z/AP, a double reporter for Cre-mediated recombination. Dev Biol 208:281292[CrossRef][Medline]
-
Lewandowski M, Wassarman KM, Martin GR 1997 Zp3-cre, a transgenic mouse line for the
activation or inactivation of loxP-flanked target genes
specifically in the female germ line. Curr Biol 7:148151[Medline]
-
de Vries WN, Binns LT, Fancher KS, Dean J, Moore R, Kemler R,
Knowles BB 2000 Expression of Cre recombinase in mouse oocytes: a means
to study maternal effect genes. Genesis 26:110112[CrossRef][Medline]
-
Lomeli H, Ramos-Mejia V, Gertsenstein M, Lobe CG, Nagy A 2000 Targeted insertion of Cre recombinase into the TNAP gene:
excision in primordial germ cells. Genesis 26:116117[CrossRef][Medline]
-
Zhang F-P, Pakarainen T, Koskimies P, Huhtaniemi IT,
Gonad-specific Cre-recombinase expression and deletion of
loxP-flanked gene segments in transgenic mice utilizing the
murine 6-kb inhibin-
subunit promoter. Program of the
82nd Annual Meeting of The Endocrine Society, Toronto, Ontario, Canada,
2000, p 110
-
Plasterk RH, Ketting RF 2000 The silence of the genes. Curr
Opin Gen Dev 10:562567[CrossRef][Medline]
-
Domeier ME, Morse DP, Knight SW, Portereiko M, Bass BL, Mango
SE 2000 A link between RNA interference and nonsense-mediated decay in
Caenorhabditis elegans. Science 289:19281930[Abstract/Free Full Text]
-
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC 1998 Potent and specific genetic interference by double-stranded RNA in
Caenorhabditis elegans. Nature 391:806811[CrossRef][Medline]
-
Wianny F, Zernicka-Goetz M 2000 Specific interference with
gene function by double-stranded RNA in early mouse development. Nat
Cell Biol 2:7075[CrossRef][Medline]
-
Svoboda P, Stein P, Hayashi H, Schultz RM 2000 Selective
reduction of dormant maternal mRNAs in mouse oocytes by RNA
interference. Development 127:41474156[Abstract/Free Full Text]
-
Quinn LM, Dorstyn L, Mills K, Colussi PA, Chen P, Coombe M,
Abrams J, Kumar S, Richardson H 2000 An essential role for the caspase
Dronc in developmentally programmed cell death in
Drosophila. J Biol Chem 275:4041640424[Abstract/Free Full Text]
-
Colussi PA, Quinn LM, Huang DC, Coombe M, Read SH, Richardson
H, Kumar S 2000 Debcl, a proapoptotic Bcl-2 homologue, is a component
of the Drosophila melanogaster cell death machinery. J
Cell Biol 148:703714[Abstract/Free Full Text]
-
Muller U 1999 Ten years of gene targeting: targeted mouse
mutants, from vector design to phenotype analysis. Mech Dev 82:321[CrossRef][Medline]
-
Lam G, Thummel CS 2000 Inducible expression of
double-stranded RNA directs specific genetic interference in
Drosophila. Curr Biol 10:957563[CrossRef][Medline]
-
Wu D, Chen PJ, Chen S, Hu Y, Nuñez G, Ellis RE 1999 C. elegans MAC-1, an essential member of the AAA family of
ATPases, can bind CED-4 and prevent cell death. Development 126:20212031[Abstract/Free Full Text]
-
Flemming W 1885 Ueber die bildung von richtungsfiguren in
saugethiereiern beim untergang Graafscher follikel. Archiv Anatomie
Entwickelungsgeschichte (Archiv Anat Physiol), pp 221241
-
Sakamaki K, Yoshida H, Nishimura Y, Nishikawa S, Manabe N,
Yonehara S 1997 Involvement of Fas antigen in ovarian follicular
atresia and luteolysis. Mol Reprod Dev 47:1118[CrossRef][Medline]
-
Xu JP, Li X, Mori E, Guo MW, Mori T 1998 Aberrant expression
and dysfunction of Fas antigen in MRL/MpJ-lpr/lpr
murine ovary. Zygote 6:359367[CrossRef][Medline]
-
Robles R, Ross A, Mahar P, Perez GI, MacGregor GR, Tilly JL,
Excessive oocyte apoptosis and ovarian tumorigenesis in Bcl-w deficient
female mice. Program of the 81st Annual Meeting
of The Endocrine Society, San Diego, CA, 1999, p 243
-
Perez GI, Nakagawa T, Trbovich AM, Yuan J, Tilly JL 2001 Involvement of caspase-12 in doxorubicin-induced oocyte apoptosis. J
Soc Gynecol Invest 8[Suppl]:272A (Abstract)
-
Motoyama N, Wang F, Roth KA, Sawa H, Nakayama K, Nakayama K,
Negishi I, Senju S, Zhang Q, Fujii S, Loh DY 1995 Massive cell death of
immature hematopoietic cells and neurons in Bcl-x-deficient mice.
Science 267:15061509[Medline]
-
Wang S, Miura M, Jung YK, Zhu H, Li E, Yuan J 1998 Murine
caspase-11, an ICE-interacting protease, is essential for the
activation of ICE. Cell 92:501509[Medline]
-
Krammer PH 2000 CD95s deadly mission in the immune system.
Nature 407:789795[CrossRef][Medline]