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
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ABSTRACT
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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.
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INTRODUCTION
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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 5075 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).
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RESULTS
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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. 1A
). 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. 1B
). 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.
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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. 2
, A and B), but was robustly expressed
in growing oocytes within ovaries of transgenic sisters (Fig. 2
, CH).
In addition to the intense levels of human Bcl-2 staining in oocytes of
late primary and small preantral follicles (Fig. 2
, CG), human Bcl-2
immunoreactivity remained easily detectable in oocytes through the
later stages of follicle development, including large antral (Graafian)
follicles (Fig. 2H
). By comparison, in transgenic mice human Bcl-2
protein was not detected in the surrounding granulosa cells (Fig. 2
, CH) or in any other ovarian or nonovarian cell type aside from
growing oocytes, including cells within the uterus (Fig. 2I
), 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. 2G
) and was not always detected
in oocytes of primary follicles (Fig. 2G
). 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. 2
, CG). 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. 2
, D and E), this was considered
nonspecific because it was not consistently observed (for example, see
Fig. 2
, 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 (CH). 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.
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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. 3
, A and B),
although transgenic females consistently possessed almost 30% fewer
atretic primary follicles than wild-type counterparts (Fig. 3B
). A
parallel assessment of small preantral follicle numbers indicated that
transgenic female mice possessed significantly fewer atretic small
preantral follicles (Fig. 3B
), with a corresponding significant
elevation in the number of nonatretic small preantral follicles (Fig. 3A
), 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).
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Ovulation Rates and Litter Size
Despite the reduced incidence of atresia of growing follicles in
transgenic females (Fig. 3B
), 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. 4
).

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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.
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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. 5A
), 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. 5A
). 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. 5A
). 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. 5B
);
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. 5B
).

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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 = 37 independent experiments;
*, P < 0.05 vs. respective WT
group). N.D., None detected.
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DISCUSSION
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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 womens 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.
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MATERIALS AND METHODS
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Transgene Construction and Generation of Transgenic Mice
The pBS/pKCR3
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
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
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 [
-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. 2F
).
Superovulation Rates
Adult wild-type and transgenic female mice, between 68 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 (814 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, 30,00070,000; 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 Scheffes 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
|
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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
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
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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. 
2 Investigator in the Massachusetts General Hospital Reproductive
Endocrine Sciences Center, supported by NIH Grant
P30-HD-28138. 
Received for publication February 2, 1999.
Revision received March 19, 1999.
Accepted for publication March 23, 1999.
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