Targeted Expression of Bcl-2 in Mouse Oocytes Inhibits Ovarian Follicle Atresia and Prevents Spontaneous and Chemotherapy-Induced Oocyte Apoptosis In Vitro

Yutaka Morita1, Gloria I. Perez, Daniel V. Maravei, Kim I. Tilly and Jonathan L. Tilly2

Vincent Center for Reproductive Biology Department of Obstetrics and Gynecology Massachusetts General Hospital/Harvard Medical School Boston, Massachusetts 02114


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Members of the Bcl-2 family serve as central checkpoints for cell death regulation, and overexpression of Bcl-2 is known to inhibit apoptosis in many cell types. To determine whether targeted expression of Bcl-2 could be used to protect female germ cells from apoptosis, we generated transgenic mice expressing fully functional human Bcl-2 protein only in oocytes. Transgenic mice were produced using a previously characterized 480-bp fragment of the mouse zona pellucida protein-3 (ZP3) gene 5'-flanking region to direct oocyte-specific expression of a human bcl-2 complementary DNA. Immunohistochemical analyses using a human Bcl-2-specific antibody showed that transgene expression was restricted to growing oocytes and was not observed in the surrounding ovarian somatic cells or in any other nonovarian tissues. Histomorphometric analyses revealed that ovaries collected from transgenic female mice possessed significantly fewer atretic small preantral follicles compared with wild-type sisters, resulting in a larger population of healthy maturing follicles per ovary. However, the number of oocytes ovulated in response to exogenous gonadotropin priming and the number of pups per litter were not significantly different among wild-type vs. transgenic female mice. Nonetheless, oocytes obtained from transgenic mice and cultured in vitro were found to be resistant to spontaneous and anticancer drug-induced apoptosis. We conclude that targeted expression of Bcl-2 only in oocytes can be achieved as a means to convey resistance of the female germ line to naturally occurring and chemotherapy-induced apoptosis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Among the increasing number of cell death regulatory molecules that have been identified to date, members of the bcl-2 (B-cell lymphoma/leukemia-2) gene family are considered principal players in the cascade of events that activate or inhibit apoptosis (1, 2). In the ovary, expression of many of the apoptosis regulatory proteins encoded by bcl-2 gene family members have been identified, and recent evidence strongly supports a fundamental role for Bcl-2 and related proteins in regulating ovarian cell death (3). For example, increased expression of the death susceptibility factor, Bax, is positively correlated with apoptosis in granulosa cells of the rat (4) and human (5) ovary and in luteal cells of the bovine (6) and rabbit (7, 8) ovary. Moreover, mice with a targeted disruption in the bax gene show a number of phenotypic abnormalities in the ovary, including apparent defects in the normal induction of apoptosis in granulosa cells during atresia (9), as well as a surfeit of oocyte-containing primordial follicles that leads to a dramatic extension of ovarian lifespan (10). By comparison, mice lacking functional Bcl-2 protein possess reduced numbers of primordial follicles relative to their wild-type (i.e., Bcl-2-intact) sisters (11). That the negative impact of Bcl-2 deficiency on primordial follicle endowment reflects an oocyte-intrinsic apoptosis defect is evidenced by the findings of numerous follicle-like structures possessing granulosa cells but devoid of oocytes in Bcl-2-null female mice (11) as well as the reported expression of approximately 50–75 copies of polyadenylated bcl-2 mRNA per mouse oocyte (12). These data together with those from more recent studies documenting resistance of Bax-deficient mouse oocytes to apoptosis induced by exposure to chemotherapy (13) collectively support a critical role for these proteins in controlling ovarian germ cell endowment and depletion. To further determine the functional role of Bcl-2 in regulating apoptosis in female germ cells, we designed the present study to generate transgenic mice expressing human Bcl-2 only in oocytes using a previously characterized fragment of the murine zona pellucida protein-3 (ZP3) gene promoter to drive transgene expression (14).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transgene Construction and Generation of the Mouse Lines
Transgenic mice expressing human Bcl-2 in female germ cells were created using a fragment of the mouse ZP3 gene promoter that has been fully-characterized to convey oocyte-specific expression of the gene (14) (Fig. 1AGo). After pronuclear injection of the linearized transgene and subsequent transfer of 60 microinjected embryos to surrogate pseudopregnant females, two founder mice (one male and one female) were identified from 21 offspring by Southern blot analysis of EcoRI-digested genomic DNA using a radiolabeled human bcl-2 cDNA as probe. Both founders transmitted the transgene through the germ line, as indicated by the finding that approximately half of the offspring produced from the subsequent mating of either founder mouse with a wild-type partner were transgene positive by Southern blot analysis (Fig. 1BGo). The integrated transgene copy number was essentially the same between the two lines established from each founder mouse, and the ratio of transgenic to wild-type offspring was not significantly different (~50:50; data not shown). By gross overall appearance, the transgenic mice exhibited normal growth with no obvious abnormalities. As identical results were obtained in preliminary studies of female mice from the two different founder lines (data not shown), all subsequent experiments employed both lines of mice, and the resultant data were pooled.



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Figure 1. Transgene Construction and Southern Blot Analysis of Genotype

A, A 480-bp portion of the mouse ZP3 promoter (14 ) was fused to a 1.9-kb DNA fragment containing the coding sequence and 3'-untranslated region (UTR) of human bcl-2. To maximize the efficiency of transgene expression, a rabbit ß-globin exon2/intron2/exon3-containing gene fragment was placed between the ZP3 promoter and the coding sequence of human bcl-2, and a rabbit ß-globin exon3/polyA-containing gene fragment was placed downstream of the 3'-UTR of the human bcl-2 cDNA. B, Transgenic (Tg) and wild-type (WT) mice were identified by Southern blot analysis of EcoRI-digested genomic DNA using the human bcl-2 cDNA as a probe. Representative results from analysis of eight offspring from the two transgenic lines arising from each founder mouse are shown. As anticipated, the size of the human bcl-2 transgene excised by EcoRI digestion is 1.9 kb.

 
Transgene Expression Pattern
Using a monoclonal antibody specific for human Bcl-2 protein, immunohistochemical analysis was performed to confirm and localize expression of the transgene product. As anticipated, human Bcl-2 protein was not detected in ovaries of wild-type female mice (Fig. 2Go, A and B), but was robustly expressed in growing oocytes within ovaries of transgenic sisters (Fig. 2Go, C–H). In addition to the intense levels of human Bcl-2 staining in oocytes of late primary and small preantral follicles (Fig. 2Go, C–G), human Bcl-2 immunoreactivity remained easily detectable in oocytes through the later stages of follicle development, including large antral (Graafian) follicles (Fig. 2HGo). By comparison, in transgenic mice human Bcl-2 protein was not detected in the surrounding granulosa cells (Fig. 2Go, C–H) or in any other ovarian or nonovarian cell type aside from growing oocytes, including cells within the uterus (Fig. 2IGo), spleen, liver, adrenal gland, and testis (data not shown). Furthermore, human Bcl-2 protein was not detected in resting oocytes of primordial follicles in transgenic females (Fig. 2GGo) and was not always detected in oocytes of primary follicles (Fig. 2GGo). Of further note in the transgenic females, human Bcl-2 protein accumulated to very high levels in the region corresponding to the zona pellucida of growing oocytes (Fig. 2Go, C–G). Although on occasion faint brown staining from the diaminobenzidine immunoreaction was observed in somatic cells of some of the sections analyzed using tissues from either wild-type or transgenic mice (for example, Fig. 2Go, D and E), this was considered nonspecific because it was not consistently observed (for example, see Fig. 2Go, F and G), and the 480-bp ZP3 gene promoter fragment used has been previously characterized as driving oocyte-specific expression (14).



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Figure 2. Human Bcl-2 Expression in Wild-Type and Transgenic Mice

Sections were analyzed by immunohistochemistry using a monoclonal antibody against human Bcl-2. Human Bcl-2 immunostaining (brownreaction product) was not detectable in the ovaries of wild-type mice (A and B), but was intense in growing oocytes of transgenic mice (C–H). In F, an atretic small preantral follicle is highlighted (arrow) to reconfirm that complete oocyte dissolution is the driving force behind atresia at these early stages of development (10 22 23 ). In G, a primordial (arrow) and an early primary (arrowhead) follicle are highlighted to show the relative absence of immunostaining vs. the robust Bcl-2 immunoreaction in the large growing oocyte of an adjacent large preantral follicle undergoing transition to the early antral stage of development. H shows that the transgene product persists at high levels in oocytes of large antral (Graafian) follicles. All other tissues examined in transgenic mice, including the uterus (I), spleen, liver, adrenal gland, and testis (data not shown), were negative. These data are representative of results obtained in at least three separate experiments. AF, Antral follicle; CL, corpus luteum. Original magnifications: A and D, x100; E, F, H, and I, x200; B, C, and G, x400.

 
Histomorphometric Analysis of Ovarian Follicle Numbers
Analysis of serial ovarian sections from wild-type and transgenic female mice shortly after puberty (day 42 postpartum) revealed no significant differences in the numbers of healthy or atretic primordial or primary follicles (Fig. 3Go, A and B), although transgenic females consistently possessed almost 30% fewer atretic primary follicles than wild-type counterparts (Fig. 3BGo). A parallel assessment of small preantral follicle numbers indicated that transgenic female mice possessed significantly fewer atretic small preantral follicles (Fig. 3BGo), with a corresponding significant elevation in the number of nonatretic small preantral follicles (Fig. 3AGo), per ovary compared with those in wild-type sisters.



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Figure 3. Histomorphometric Analysis of Follicle Development in Wild-Type and Transgenic Female Mice

Serial ovarian sections from age-matched wild-type (WT) and transgenic (Tg) female mice (day 42 postpartum) were processed, and estimation of the numbers of healthy (nonatretic; A) or atretic (B) primordial, primary, and small preantral follicles was made. These data are the mean ± SEM of combined results from an analysis of three mice per genotype (*, P < 0.05).

 
Ovulation Rates and Litter Size
Despite the reduced incidence of atresia of growing follicles in transgenic females (Fig. 3BGo), there was no significant difference (P > 0.05) in the numbers of oocytes retrieved from wild-type vs. transgenic female mice after superovulation with exogenous gonadotropins (26 ± 5 vs. 26 ± 4, respectively; mean ± SEM; n = 5 mice/genotype). Although, on the average, there were one or two more pups per litter from transgenic females, this did not convey a significant difference (P > 0.05) in litter size between wild-type and transgenic female mice (Fig. 4Go).



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Figure 4. Comparison of Litter Size in Wild-Type vs. Transgenic Female Mice

The number of pups per litter was determined in age-matched adult wild-type (WT) and transgenic (Tg) female mice after mating with adult wild-type male mice. These data represent the mean ± SEM of combined results from five independent experiments.

 
Incidence of Spontaneous and Chemotherapy-induced Oocyte Apoptosis in Vitro
Approximately 10% of the mature oocytes harvested from superovulated adult female wild-type mice and maintained in vitro in human tubal fluid initiated apoptosis within 15 h (Fig. 5AGo), and the incidence of oocyte apoptosis was dramatically increased to over 70% by addition of the chemotherapeutic drug, doxorubicin (DXR), at the start of culture (Fig. 5AGo). However, Bcl-2-expressing oocytes from transgenic sisters exhibited significantly lower rates of apoptosis both under basal conditions and in response to anticancer drug treatment (Fig. 5AGo). The incidence of spontaneous and DXR-induced apoptosis in oocytes from transgenic vs. wild-type female mice remained significantly lower as the culture period was extended to 18 or 24 h (Fig. 5BGo); however, by 48 h in culture the incidence of apoptosis, either spontaneous or drug induced, was comparable in oocytes harvested from wild-type vs. transgenic females (Fig. 5BGo).



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Figure 5. Incidence of Spontaneous and Chemotherapy-Induced Apoptosis in Oocytes from Wild-Type and TransgenicFemale Mice

Oocytes harvested by superovulation of age-matched adult wild-type (WT) and transgenic (Tg) female mice were cultured in vitro in the absence or presence of 200 nM DXR for 15 h (A) or for 18, 24, or 48 h (B). After culture, the incidence of apoptosis in each group was assessed as detailed previously (13 27 ). The total number of oocytes cultured under each experimental condition is given in parentheses above the respective bar (mean ± SEM; n = 3–7 independent experiments; *, P < 0.05 vs. respective WT group). N.D., None detected.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Apoptosis plays a fundamental role in normal germ cell endowment and follicular dynamics in the ovary, and cell fate in this organ is probably dependent on the actions of several proteins recently identified as key components of a program of cell death conserved through evolution (1, 2, 3, 16, 17, 18). Importantly, several studies concerned with elucidating the molecular mechanisms underlying germ cell and follicle loss from the ovary have provided information that may one day lead to significant advances in improving women’s health (18). For example, recent data derived from analysis of mice lacking expression of the proapoptotic Bcl-2 family member, Bax (9), indicate that ovarian lifespan can be dramatically extended by disrupting Bax-mediated apoptosis in the ovary (10), arguing that at least the mouse equivalent of the menopause can be delayed if not entirely prevented. Moreover, disruption of Bax expression has also been shown to provide murine oocytes with resistance to apoptosis induced by exposure to the anticancer drug, DXR, in vivo and in vitro (13). With premature ovarian failure documented as an unfortunate side-effect of chemotherapy in young girls and women (19, 20), such findings indicate that germ cell destruction in female cancer patients may not necessarily have to be inevitable. Consequently, devising methods of selectively regulating apoptotic pathways in oocytes holds great promise for the development of future therapeutic compounds to combat ovarian failure and possibly infertility.

As an initial step toward this goal, we report herein the creation of the first transgenic mouse line of which we are aware that displays selective expression of a classic antiapoptotic molecule (i.e., Bcl-2) only in oocytes. To accomplish this, we used a previously characterized fragment of the mouse ZP3 promoter that conveys a high basal level of oocyte-specific gene expression (14). Under normal conditions, the protein encoded by the ZP3 gene is one of three glycoproteins that constitute the oocyte extracellular coat or zona pellucida (15). Using molecular probes to examine ZP3 expression during murine oocyte development (21), it has been established that ZP3 is expressed only in growing oocytes, and that ZP3 mRNA transcripts accumulate in oocytes to unusually high steady state levels. The distribution pattern of human Bcl-2 protein in our transgenic mice is fully consistent with these previous studies (21), supporting the fidelity of the ZP3 gene promoter fragment used to drive expression of the human bcl-2 cDNA in oocytes. Of note, however, in transgenic mice intense human Bcl-2 immunoreactivity was observed in the outer boundaries of the growing oocytes adjacent to or overlapping the zona pellucida. The reason(s) for this remains unknown, and unfortunately will prove difficult to assess because the relatively sparse number of oocytes available for biochemical analyses will make follow-up studies, such as transgene product trafficking, unfeasible.

We have recently shown that Bcl-2-null female mice possess a significantly smaller pool of primordial follicles (11), whereas bax gene disruption in mice (9) prevents atresia of primordial and primary follicles (10). As Bcl-2 is known to heterodimerize with and inactivate Bax (1, 2), we next tested whether atresia rates were affected in Bcl-2 transgenic females. Histomorphometric analyses of follicle numbers in serial ovarian sections indicated that wild-type and transgenic female mice possessed roughly equivalent numbers of primordial follicles. This observation is in keeping with the fact the ZP3 gene (and hence the transgene) is not actively transcribed until oocytes begin growth at the primary stage of follicle development (15). Somewhat surprisingly, we also noted no significant differences in the numbers of atretic (or healthy) primary follicles in wild-type vs. transgenic females despite the fact that Bcl-2 expression had been initiated by this point in follicle development. However, it is important to note that although not statistically different from the wild-type values, Bcl-2 transgenic females showed a 30% reduction in the incidence of primary follicle atresia. One possible explanation for these findings is that the human Bcl-2 protein produced from the transgene, at least in some pools of primary follicle oocytes, had not yet accumulated to a level required to fully protect from cell death activation. In support of this hypothesis and the possibility that the numerical decline in atresia rates of primary follicles is biologically significant, transgenic female mice did exhibit a significant reduction in the number of atretic small preantral follicles compared with that in their wild-type sisters, resulting in a larger cohort of healthy maturing follicles per ovary.

These findings provide the first unequivocal evidence that preservation of oocyte viability through germ cell-intrinsic mechanisms directly impacts on the process of follicular atresia, at least at this stage of follicle development. These data support and extend previous studies that atresia of immature (primordial, primary, and small preantral) follicles is driven by oocyte, as opposed to granulosa cell, apoptosis (10, 22, 23). Interestingly, this larger population of healthy small preantral follicles did not lead to differences in superovulation rates or litter size among wild-type vs. transgenic females. The reasons for this remain to be defined, although it is possible that increased atresia at latter stages of follicle development (i.e. antral), driven by granulosa cell demise (3, 17, 18), compensates for the excess number of maturing follicles in the transgenic females to reestablish a normal ovulatory quota for fertilization and implantation. Moreover, our current efforts to examine natural cycle ovulation rates in wild-type and transgenic females may help to further clarify the impact, if any, of reduced atresia rates in the immature follicle population on the number of mature follicles ultimately ovulated per cycle. Whatever the case, these findings together with previous data obtained from analysis of Bcl-2-deficient and Bax-deficient female mice (9, 10, 11, 13) support the hypothesis that Bcl-2 family members are indeed central regulatory components in the ovarian cell death pathway (3).

Another clinically relevant issue when considering apoptosis in female germ cells is the spontaneous fragmentation of oocytes known to occur as a result of in vitro culture used in assisted reproductive technology programs (24, 25, 26). Having recently established that spontaneous oocyte fragmentation is unquestionably an example of apoptosis (27), we next examined the impact of Bcl-2 overexpression on culture-induced oocyte death. These experiments revealed that spontaneous oocyte death occurring in vitro could be markedly delayed by targeted expression of Bcl-2, with a complete suppression of oocyte fragmentation at the earliest time point evaluated. Using this same in vitro culture system combined with addition of an anticancer drug to trigger massive oocyte death as a model to explore chemotherapy-induced ovarian failure (13), we further observed that overexpression of Bcl-2 blocked the apoptotic response of oocytes to doxorubicin treatment. Again, the most pronounced effects were achieved in the short term cultures, suggesting that human Bcl-2 protein arising from the ZP3 promoter-driven transgene is degraded over time so that the protective effects of declining Bcl-2 levels are lost in the extended cultures. This hypothesis would be in agreement with the fact that ZP3 expression is absent in ovulated oocytes (15) combined with the predicted transgene product turnover rate based on the half-life of human bcl-2 mRNA (~3 h) and protein (>=10 h but <20 h) estimated from studies of somatic cell lineages (28). Alternatively, it may be that oocytes, even in the presence of high Bcl-2, default into apoptosis upon prolonged exposure to a lethal stimulus. Although more studies are needed to delineate these and other possibilities, the data presented provide a strong impetus to further pursue technologies for achieving cell-specific expression of apoptosis molecules in the ovary as a novel means to combat ovarian failure and infertility in women.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transgene Construction and Generation of Transgenic Mice
The pBS/pKCR3{Delta}RI vector (29), containing portions of the rabbit ß-globin gene (end of exon 2, intron 2, and exon 3 plus 3'-flanking sequence) including the polyadenylation sequence for efficient transgene expression, was a gift from Dr. Jolene Windle (University of Texas Health Science Center, San Antonio, TX). A full-length human bcl-2 cDNA in pBluescript II KS (plasmid 3087) was a gift from Dr. Stan Korsmeyer (Dana-Farber Cancer Institute, Boston, MA). The bcl-2 cDNA, obtained by digestion of the plasmid with EcoRI, was isolated and subcloned in the EcoRI site of exon 3 in pBS/pKCR3{Delta}RI. After large scale preparation, the 3.145-kb ß-globin-bcl-2 minigene was excised with SalI and XhoI, and then reinserted into the SalI and XhoI sites of pBluescript II SK+ (Stratagene, La Jolla, CA) to expand the restriction enzyme choices in the cloning cassette over that present in pBS/pKCR3{Delta}RI. This plasmid was designated pBSK-ß-globin-bcl2.

A 480-bp fragment of the murine ZP3 gene 5'-flanking region that conveys oocyte-specific expression of the gene (14, 15, 30, 31) was generated by PCR using primers with 5'-flanking SalI (forward primer, 5'-GTCGACGATCCTGGTGTGGTGAC-3') and ClaI (reverse primer, 5'-ATCGATCTGGGCTCAGAATGAGAGG3') restriction sites (underlined in the primer sequences). The amplified product (corresponding to bp -470 to +10), obtained using mouse liver genomic DNA as template, was digested with SalI and ClaI and then subcloned into these sites of pBluescript II SK+ for large scale plasmid preparation (pBSK-ZP3) and automated sequence analysis (ABI PRISM 377, version 3.0, Perkin Elmer, Foster City, CA). Once confirmed, the ZP3 gene promoter was excised from pBSK-ZP3 using SalI and SmaI, and the SalI site was filled in using Klenow enzyme and deoxynucleotides. The promoter was then subcloned into the EcoRV site of pBSK-ß-globin-bcl2 and sequenced to obtain a single clone with the ZP3 promoter in the correct orientation. This final plasmid (designated pBSK-ZP3-ß-globin-bcl2) was digested with XhoI and XbaI to excise vector sequence and fractionated on agarose gels, and the 3.725-kb ZP3-ß-globin-bcl2 minigene was purified and microinjected into the pronucleus of each of 110 FVB strain one-cell zygotes (~1 pl of a 2 ng/µl stock) using standard protocols (32). Microinjected embryos were transferred to the oviducts of three foster pseudopregnant female ICR mice (20 embryos/surrogate female, 60 embryos total), and 21 offspring were obtained (two of which were positive for transgene incorporation).

All studies involving animals described herein were approved by and performed in strict accordance with the guidelines of the Massachusetts General Hospital institutional animal care and use committee and the NIH Guide for the Care and Use of Laboratory Animals.

Southern Blot Analysis of Transgene Expression
For Southern blot analysis, 5 µg of genomic DNA, extracted from tail snips (33) and digested with EcoRI (5 µg/reaction), were resolved by conventional agarose gel electrophoresis and transferred onto nylon membranes (Schleicher & Schuell, Inc., Keene, NH). The membranes were then hybridized with the full-length (1.9-kb) human bcl-2 cDNA (see above) after radiolabeling with [{alpha}-32P]deoxy-ATP (3000 Ci/mmol; Amersham-Pharmacia, Piscataway, NJ) by random priming (34) and purification by column chromatography (NucTrap Push Columns, Stratagene), essentially as previously described (4).

Immunohistochemistry
For analysis of human Bcl-2 expression, tissues from 6-week-old wild-type and transgenic mice were fixed overnight at 4 C in neutral-buffered 3.7% paraformaldehyde and embedded in paraffin. Longitudinal sections (6 µm) were cut, mounted on SuperFrost-Plus slides (Fisher Scientific, Springfield, MA), and analyzed by immunohistochemistry as detailed previously (5, 35). When comparing ovarian sections from wild-type and transgenic mice, the sections were mounted in tandem on the same slide so that the assessment of human Bcl-2 immunostaining was performed in parallel. Briefly, paraffin sections were rehydrated and subjected to high temperature antigen unmasking (36) before immunoanalysis with a 1:500 (vol/vol) dilution of a mouse monoclonal antibody against human Bcl-2 that does not cross-react with murine Bcl-2 protein (clone 124; DAKO Corp., Carpinteria, CA). Chromogenic detection of the sites of Bcl-2-primary antibody complexes was performed by incubating sections for 1 h with a 1:200 dilution of a biotinylated goat antimouse IgG antibody (Oncogene Research Products, Cambridge, MA), followed by addition of avidin-biotin horseradish peroxidase complex components (ABC kit, Vector Laboratories, Inc., Burlingame, CA) at 20 C for 45 min. Sections were then washed and incubated with 0.5 mg/ml 3,3'-diaminobenzidine and 0.03% hydrogen peroxide for 1 min at 20 C, and colorimetric reactions (generation of a brown reaction product) were terminated by placing the slides in a buffer consisting of 10 mM Tris-HCl and 1 mM EDTA (pH 8.0). Negative controls, conducted by omitting the primary antibody, yielded no reaction product (data not shown). As a further confirmation of the specificity of the immunostaining, ovarian sections from wild-type and transgenic females were assessed by immunohistochemistry using a different human Bcl-2-specific antiserum (35, 37) and identical results were obtained (data not shown). Slides were analyzed by conventional light microscopy after light counterstaining with hematoxylin.

Histomorphometric Analysis
Ovaries were collected from wild-type and transgenic female mice on day 42 postpartum, fixed (0.34 N glacial acetic acid, 10% formalin, and 28% ethanol), embedded in paraffin, and serially sectioned (8 µm). The serial sections from each ovary were aligned in order on glass microscope slides, stained with hematoxylin and picric methyl blue, and analyzed for the number of healthy (nonatretic) and atretic primordial, primary, and small preantral follicles per section in every fifth section through the entire ovary. Primordial follicles were identified as having a compact oocyte surrounded by a single layer of flattened (fusiform) granulosa cells, whereas primary follicles were identified as having an enlarged oocyte surrounded by a single layer of cuboidal granulosa cells. Intermediate stage follicles (compact or enlarged oocyte with a single layer of mixed fusiform and cuboidal granulosa cells) were scored as primary because the change in granulosa cell morphology from fusiform to cuboidal is a sign that the primordial follicle is no longer quiescent. Small preantral follicles were identified as having an enlarged oocyte surrounded by at least a partial or complete second layer of cuboidal granulosa cells but no more than four layers of cuboidal granulosa cells (23). Each ovary was given a numerical code so that all follicle counts were conducted without knowledge of genetic background. After all counts were completed, slides were decoded, and the total number of healthy and atretic follicles per ovary was calculated (10, 11, 13, 38). Follicles at the primordial, primary, and small preantral stages of development were deemed atretic if the oocyte was degenerating (convoluted and condensed or fragmented) or absent (10, 11, 22, 23). Unlike atresia of antral follicles, which is driven by granulosa cell apoptosis (3, 17, 18), atresia at these earlier stages of follicle development is driven by oocyte death that is in many cases associated with preservation of granulosa cells until complete germ cell dissolution (10, 22, 23) (see also Fig. 2FGo).

Superovulation Rates
Adult wild-type and transgenic female mice, between 6–8 weeks of age, were superovulated with 10 IU equine CG (Professional Compounding Centers of America, Houston, TX) followed by 10 IU hCG (Serono Laboratories, Inc., Norwell, MA) 48 h later. Cumulus-oocyte complexes were collected from the oviducts 16 h after hCG injection and counted.

In Vitro Oocyte Cultures and Analysis of Apoptosis
In vitro oocyte cultures and analysis of oocyte apoptosis were performed as recently detailed from our laboratory (13, 27). Briefly, adult wild-type and transgenic female mice were superovulated as described above (see Superovulation Rates). After collection of cumulus-oocyte complexes, oocytes were denuded of cumulus cells by a 1-min incubation in 80 IU/ml hyaluronidase (Sigma Chemical Co., St. Louis, MO) followed by three washes with culture medium. The culture medium used for all experiments was human tubal fluid (Irvine Scientific, Santa Ana, CA) supplemented with 0.5% BSA (fraction V; Life Technologies, Grand Island, NY). After isolation, oocytes were cultured in 0.1-ml drops of culture medium (8–14 oocytes/drop) under paraffin oil (Sigma Chemical Co.), and incubated without (controls, spontaneous fragmentation) or with 200 nM doxorubicin (Sigma Chemical Co.) for 15, 18, 24, or 48 h at 37 C in a humidified atmosphere of 5% CO2-95% air.

At the end of the incubation period, oocytes were fixed in 1% paraformaldehyde containing 0.1 mg/ml polyvinyl alcohol (average mol wt, 30,000–70,000; Sigma Chemical Co.) for 30 min at room temperature in the dark. After fixation, oocytes were washed once with PBS and transferred to SuperFrost-Plus slides in a small volume of PBS and then mixed with Hoechst 33342 (Sigma Chemical Co.) (30 µl of a 1 mg/ml stock solution prepared in sterile water combined with 750 µl 2.3% sodium citrate and 250 µl 95% ethanol) at a final concentration of 30 µg/ml. Hoechst staining was carried out in the dark for 3 min at 37 C, after which the solution was carefully removed and replaced with mounting medium. The slides were coverslipped, and oocytes were analyzed by light (morphology) and UV fluorescence (chromatin) microscopy to determine the occurrence of apoptosis (cellular condensation, budding and fragmentation, and chromatin segregation into apoptotic bodies). The percentage of oocytes that underwent apoptosis of the total number of oocytes cultured per drop in each experiment was then determined, and all experiments were independently repeated three to seven times with different mice.

Data Presentation and Statistical Analysis
The combined data from the replicate experiments were subjected to a one-way ANOVA followed by Scheffe’s F test, and statistical significance was assigned at P < 0.05. Graphs represent the mean ± SEM of combined data from the replicate experiments, whereas representative photomicrographs of autoradiograms (Southern blot) or immunostaining are presented for qualitative analysis.


    ACKNOWLEDGMENTS
 
We thank Dr. Mason Freeman of the MGH Transgenic Mouse Core Facility for technical assistance with creation of the mouse lines reported herein. We also thank Dr. Jolene Windle for providing the pBS/pKCR3{Delta}RI vector, Dr. Stan Korsmeyer for his generous gift of human bcl-2 cDNA, and Mr. Sam Riley for outstanding technical assistance with the image analysis and data presentation.


    FOOTNOTES
 
Address requests for reprints to: Jonathan L. Tilly, Ph.D., Massachusetts General Hospital, VBK137E-GYN, 55 Fruit Street, Boston, Massachusetts 02114. E-mail: jtilly{at}partners.org

This work was supported by NIH Grants R01-AG-12279, R01-ES-08430, and R01-HD-34226 (to J.L.T.) and by Vincent Memorial Research Funds.

1 On leave from the Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo (Tokyo, Japan) and supported by the Japanese Society for the Promotion of Science. Back

2 Investigator in the Massachusetts General Hospital Reproductive Endocrine Sciences Center, supported by NIH Grant P30-HD-28138. Back

Received for publication February 2, 1999. Revision received March 19, 1999. Accepted for publication March 23, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Chao DT, Korsmeyer SJ 1998 BCL-2 family: regulators of cell death. Annu Rev Immunol 16:395–419[CrossRef][Medline]
  2. Adams JM, Cory S 1998 The Bcl-2 protein family: arbiters of cell survival. Science 281:1322–1326[Abstract/Free Full Text]
  3. Tilly JL, Tilly KI, Perez GI 1997 The genes of cell death and cellular susceptibility to apoptosis in the ovary: a hypothesis. Cell Death Differ 4:180–187[CrossRef]
  4. 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 136:232–241[Abstract]
  5. 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 5:67–76[CrossRef][Medline]
  6. Rueda BR, Tilly KI, Botros I, Jolly PD, Hansen TR, Hoyer PB, Tilly JL 1997 Increased bax and interleukin-1ß-converting enzyme (Ice) messenger RNA levels coincide with apoptosis in the bovine corpus luteum during structural regression. Biol Reprod 56:186–193[Abstract]
  7. Goodman SB, Kugu K, Chen SH, Preutthipan S, Tilly KI, Tilly JL, Dharmarajan AM 1998 Estradiol-mediated suppression of apoptosis in the rabbit corpus luteum is associated with a shift in expression of bcl-2 family members favoring cellular survival. Biol Reprod 59:820–827[Abstract/Free Full Text]
  8. 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 140:2555–2561[Abstract/Free Full Text]
  9. Knudson CM, Tung KSK, Tourtellotte WG, Brown GAJ, Korsmeyer SJ 1995 Bax-deficient mice with lymphoid hyperplasia and male germ cell death. Science 270:96–99[Abstract]
  10. 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 21:200–203[CrossRef][Medline]
  11. Ratts VS, Flaws JA, Kolp R, Sorenson CM, Tilly JL 1995 Ablation of bcl-2 gene expression decreases the number of oocytes and primordial follicles established in the post-natal female mouse gonad. Endocrinology 136:3665–3668[Abstract]
  12. 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 51:243–253[CrossRef][Medline]
  13. 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 3:1228–1332[Medline]
  14. Schickler M, Lira SA, Kinloch RA, Wassarman PM 1992 A mouse oocyte-specific protein that binds to a region of mZP3 promoter responsible for oocyte-specific mZP3 gene expression. Mol Cell Biol 12:120–127[Abstract]
  15. Wassarman PM 1988 Zona pellucida glycoproteins. Annu Rev Biochem 57:415–442[CrossRef][Medline]
  16. Hengartner MO 1996 Programmed cell death in invertebrates. Curr Opin Genet Dev 6:34–38[Medline]
  17. Tilly JL 1998 Cell death and species propagation: molecular and genetic aspects of apoptosis in the vertebrate female gonad. In: Lockshin RA, Zakeri Z, Tilly JL (eds) When Cells Die. A Comprehensive Evaluation of Apoptosis and Programmed Cell Death. Wiley-Liss, New York, pp 431–452
  18. 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 79–101
  19. Waxman, J 1983 Chemotherapy and the adult gonad: a review. J R Soc Med 76:144–148[Medline]
  20. Reichman BS, Green KB 1994 Breast cancer in young women: effect of chemotherapy on ovarian function, fertility and birth defects. Monogr Natl Cancer Inst 16:125–129[Medline]
  21. Epifano O, Liang L-f, Familari M, Moos Jr MC, Dean J 1995 Coordinate expression of the three zona pellucida genes during mouse oogenesis. Development 121:1947–1956[Abstract/Free Full Text]
  22. Baker TG 1963 A quantitative and cytological study of germ cells in human ovaries. Proc R Soc Lond B Biol Sci 158:417–433
  23. Gougeon A 1996 Regulation of ovarian follicular development in primates: facts and hypotheses. Endocr Rev 17:121–155[Medline]
  24. Takase K, Ishikawa M, Hoshiai H 1995 Apoptosis in the degeneration process of unfertilized mouse ova. Tohoku J Exp Med 175:69–76[Medline]
  25. Perez GI, Tilly JL 1997 Cumulus cells are required for the increased apoptotic potential in oocytes of aged mice. Hum Reprod 12:2781–2783[Abstract]
  26. Van Blerkom J, Davis PW 1998 DNA strand breaks and phosphatidylserine redistribution in newly ovulated and cultured mouse and human oocytes: occurrence and relationship to apoptosis. Hum Reprod 13:1317–1324[Abstract]
  27. Perez GI, Tao X-J, Tilly JL 1999 Fragmentation and death (apoptosis) of ovulated oocytes. Mol Hum Reprod, in press
  28. Reed JC 1996 A day in the life of the Bcl-2 protein: does the turnover rate of Bcl-2 serve as a biological clock for cellular lifespan regulation? Leuk Res 20:109–111[CrossRef][Medline]
  29. Howes KA, Ransom N, Papermaster DS, Lasudry JGH, Albert DM, Windle JJ 1994 Apoptosis or retinoblastoma: alternative fates of photoreceptors expressing the HPV-16 E7 gene in the presence or absence of p53. Genes Dev 8:1300–1310[Abstract]
  30. Lira SA, Schickler M, Wassarman PM 1993 cis-Acting DNA elements involved in oocyte-specific expression of mouse sperm receptor gene mZP3 are located close to the gene’s transcription start site. Mol Reprod Dev 36:494–499[Medline]
  31. Lira A, Kinloch RA, Mortillo S, Wassarman PM An upstream region of the mouse ZP 3 gene directs expression of firefly luciferase specifically to growing oocytes in transgenic mice. Proc Natl Acad Sci USA 87:7215–7219
  32. Hogan B, Beddington R, Costantini F, Lucy E 1994 Manipulating the Mouse Embryo. A Laboratory Manual, ed. 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  33. Gross-Bellard M, Oudet P, Chambon P 1973 Isolation of high-molecular-weight DNA from mammalian cells. Eur J Biochem 36:32–38[Medline]
  34. Feinberg AP, Vogelstein B 1983 A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13[Medline]
  35. Tao X-J, Tilly KI, Maravei DV, Shifren JL, Krajewski S, Reed JC, Tilly JL, Isaacson KB 1997 Differential expression of members of the bcl-2 gene family in proliferative and secretory human endometrium: glandular epithelial cell apoptosis is associated with increased expression of bax. J Clin Endocrinol Metab 82:2738–2746[Abstract/Free Full Text]
  36. Shi SR, Key ME, Kalra KL 1991 Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39:741–748[Abstract]
  37. Krajewski S, Bodrug S, Gascoyne R, Berean K, Krajewska M, Reed JC 1994 Immunohistochemical analysis of Mcl-1 and Bcl-2 proteins in normal and neoplastic lymph nodes. Am J Pathol 145:515–525[Abstract]
  38. Bergeron L, Perez GI, Macdonald G, Shi L, Sun Y, Jurisicova A, Varmuza S, Latham KE, Flaws JA, Salter JCM, Hara H, Moskowitz MA, Li E, Greenberg A, Tilly JL, Yuan J 1998 Defects in regulation of apoptosis in caspase-2-deficient mice. Genes Dev 12:1304–1314[Abstract/Free Full Text]