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


    ABSTRACT
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 ABSTRACT
 THE GENETICS OF PROGRAMMED...
 THE MANY FACES OF...
 PCD GENE KNOCKOUTS AND...
 TRANSGENIC EXPRESSION OF PCD...
 CRE RECOMBINASE-LOXP AND...
 DOUBLE-STRANDED RNA INTERFERENCE
 CONCLUDING REMARKS AND FUTURE...
 REFERENCES
 
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.


    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. 1Go) 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. 1Go) (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.

 

    THE MANY FACES OF CELL DEATH IN THE OVARY
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 THE GENETICS OF PROGRAMMED...
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 DOUBLE-STRANDED RNA INTERFERENCE
 CONCLUDING REMARKS AND FUTURE...
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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.


    PCD GENE KNOCKOUTS AND OVARIAN FUNCTION
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 DOUBLE-STRANDED RNA INTERFERENCE
 CONCLUDING REMARKS AND FUTURE...
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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. 1Go). Furthermore, studies of mutant mice lacking expression of various components of the core PCD machinery (e.g. see Table 1Go) 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 1Go). 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

 
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.


    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-{alpha} 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-{alpha}/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.


    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 1Go), 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-{alpha} 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.


    DOUBLE-STRANDED RNA INTERFERENCE
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 THE MANY FACES OF...
 PCD GENE KNOCKOUTS AND...
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 CRE RECOMBINASE-LOXP AND...
 DOUBLE-STRANDED RNA INTERFERENCE
 CONCLUDING REMARKS AND FUTURE...
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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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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.


    FOOTNOTES
 
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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