Bcl-x and Bax Regulate Mouse Primordial Germ Cell Survival and Apoptosis during Embryogenesis
Edmund B. Rucker, III,
Patricia Dierisseau,
Kay-Uwe Wagner,
Lisa Garrett,
Anthony Wynshaw-Boris1,
Jodi A. Flaws and
Lothar Hennighausen
Laboratory of Genetics and Physiology (E.B.R., P.D., K.-U.W.,
L.H.) National Institute of Diabetes, Digestive and Kidney
Diseases National Institutes of Health Bethesda, Maryland
20892
Genetic Disease Research Branch (L.G., A.W.-B.)
National Human Genome Research Institute National Institutes of
Health Bethesda, Maryland 20892
Department of
Epidemiology and Preventive Medicine (J.A.F.) University of
Maryland School of Medicine Baltimore, Maryland 21201
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ABSTRACT
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Restricted germ cell loss through apoptosis is
initiated in the fetal gonad around embryonic day 13.5 (E13.5) as part
of normal germ cell development. The mechanism of this germ cell
attrition is unknown. We show that Bcl-x plays a crucial role in
maintaining the survival of mouse germ cells during gonadogenesis. A
bcl-x hypomorphic mouse was generated through the
introduction of a neomycin (neo) gene into the
promoter of the bcl-x gene by homologous recombination.
Mice that contained two copies of the hypomorphic allele had severe
reproductive defects attributed to compromised germ cell development.
Males with two mutant alleles lacked spermatogonia and were sterile;
females showed a severely reduced population of primordial and primary
follicles and exhibited greatly impaired fertility. Primordial germ
cells (PGCs) in bcl-x hypomorph mice migrated to the
genital ridge by E12.5 but were depleted by E15.5, a time when Bcl-x
and Bax were present. Two additional bcl-x transcripts were
identified in fetal germ cells more than 300 bp upstream of previously
reported start sites. Insertion of a neo cassette led to a
down-regulation of the bcl-x gene at E12.5 in the
hypomorph. Bax was detected by immunohistochemistry in germ cells from
bcl-x hypomorph and control testes at E12.5 and E13.5.
Bcl-x function was restored, and animals of both genders were fertile
after removal of the neo selection cassette using
Cre-mediated recombination. Alternatively, the loss of Bcl-x function
in the hypomorph was corrected by the deletion of both copies of the
bax gene, resulting in a restoration of germ cell survival.
These findings demonstrate that the balance of Bcl-x and Bax control
PGC survival and apoptosis.
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INTRODUCTION
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Formation of the mouse fetal gonad begins with the migration of
the primordial germ cells (PGCs) from the base of the allantois at
embryonic day 8.5 (E8.5) to the genital ridge at E11.5 (1 2 ). During
this migratory period, the number of PGCs increases from about 100 to
more than 3,000 and by E13.5, PGCs number greater than 22,000 in the
fetal gonads (3 ). After the colonization of the gonads, the gonocytes
continue to proliferate but also undergo a transient burst of apoptosis
around E13.5 (4 ). The majority of gonocytes in the testes enter mitotic
arrest as prospermatogonia by E15.5 (5 ), whereas the germ cells in the
ovaries enter prophase I of meiosis around E13.5 (6 ). A second round of
apoptosis is initiated in the postnatal testis concomitantly with the
initiation of spermatogenesis around postnatal day 8 (p8) (7 8 9 )
and in the prepubertal ovary between P7 and P14 (4 10 ).
Although the loss of rodent PGCs has been attributed to apoptosis as
determined by flow cytometry (4 ), electron microscopy (11 ), and 3'-end
labeling for DNA fragmentation (10 ), the genetic pathways controlling
fetal gonadal development are largely unknown. While c-kit
and its ligand mast cell growth factor (MGF) have been shown to control
the migration of PGCs to the genital ridge before E12.5 (12 13 ),
little is known about the molecular mechanisms controlling the survival
and restricted apoptosis that occur at subsequent fetal stages. In
contrast, members of the Bcl-2 family have been shown to govern
postpartum stages of gametogenesis. For example,
bax-deficient mice exhibit reduced cell death during
spermatogenesis and folliculogenesis. In males, this leads to an
increase in the number of spermatocytes (14 ) and in females to an
abundance of ovarian follicles in postnatal life (15 ). Mice deficient
in Bcl-w have defective spermatogenesis and spermiogenesis, which
results in a Sertoli-cell-only phenotype by 6 months of age (16 17 ).
Bcl-x, a protein controlling cell survival, is expressed widely during
development (18 ). Previous studies have shown that mice harboring an
inactivated bcl-x
gene2 die at E12.5
(19 ). Therefore, it was not possible to establish the role of Bcl-x in
the ontogeny of gonads and other organs at later stages of development.
To understand the role of Bcl-x in organ development, it is therefore
necessary to inactivate the gene exclusively in specific cell types
using the Cre-loxP recombination system (20 ). Toward this goal, we have
used homologous recombination to flank the promoter, exon 1, and the
major coding exon 2 of the bcl-x gene with loxP sites. This
targeted allele contained a loxP flanked (or floxed) neo
cassette in the bcl-x promoter and an additional loxP site
in intron 2. Mice that contained two neomycin-tagged targeted alleles
(bcl-x
flneo/flneo) had
a dramatically attenuated reproductive ability due to down-regulation
of the bcl-x gene (i.e. a hypomorphic allele).
The hypomorphic allele of bcl-x has allowed us to define its
function as a principal cell survival molecule in germ cell
development. Selective placement of loxP sites flanking the
neo cassette facilitated its removal by partial Cre-mediated
recombination. Mice obtained by this method contained a floxed
bcl-x gene (fl) with a single loxP site in the
promoter and a second loxP site in intron 2. The ablation of the
neo cassette restored bcl-x expression and
eliminated the male and female loss-of-germ-cell phenotype.
Because the mechanism behind normal germ cell death was unknown, we
have investigated whether a Bcl-x/Bax rheostat might explain the
dynamics involved in germ cell survival and death in the fetal gonad.
To test this hypothesis, we also have generated mice that contained two
copies of the bcl-x hypomorph allele and two bax
null alleles to quantitate surviving germ cells. Our model postulates
that the loss of Bcl-x (cell survival factor) during this period of
apoptosis would result in a relative increase in the amount of Bax
(cell death factor), thereby forcing the germ cells into premature cell
death. Concomitant loss of the bax alleles should restore
the balance of survival and death factors and result in an increase in
germ cell numbers.
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RESULTS
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Bcl-x
flneo/flneo Mice
Show a Loss of Spermatogonia and Oocytes
As part of the targeting strategy, exons 1 and 2 of the
bcl-x gene were floxed by loxP sites, and a neo
gene was introduced into the bcl-x promoter (Fig. 1A
). Chimeras were generated from
embryonic stem cells that were targeted as determined by Southern blot
(Fig. 1B
) and PCR analysis (Fig. 1C
). All homozygous males that carried
two targeted alleles
(flneo/flneo
mice) were sterile.

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Figure 1. Generation of flneo Mice
A, Schematic representation of the bcl-x endogenous (+)
locus and predicted targeted (flneo) allele.
Open boxes represent exons with corresponding exon
numbers. The PGK-neo cassette was cloned into the promoter
XhoI site in the antisense direction. Open
arrows denote 34-bp loxP sites; PCR primers are represented by
filled triangles in intron 2. The third loxP site was
cloned into the SpeI site. Predicted amplification
of the intron 2 region with primers should give an endogenous band of
approximately 160 bp and a targeted band of 195 bp. The bold
line shown in the targeted locus represents the
EcoRV-EcoRV region of homology used in
the targeting construct. Predicted fragment sizes for the endogenous
and targeted alleles from a XbaI digest using the shown
external probe are 9.5 kb and 6.5 kb, respectively. Restriction enzyme
sites shown are EV, EcoRV; Sp, SpeI; Xb,
XbaI; Xh, XhoI. B, Homologous
recombination at the bcl-x locus results in the decrease
of a 9.5-kb XbaI allele to a 6.5-kb allele. A Southern
blot was performed from XbaI-digested genomic tail DNA
from F2 generation mice using the 5'-external probe as
shown in panel A. Wild-type mice (+/+) contain the endogenous 9.5-kb
allele, whereas the heterozygous (flneo/+)
and homozygous
(flneo/flneo)
bcl-x mice contain either one or two targeted alleles of
6.5 kb. Lane 1, control; lanes 2 and 3, heterozygous floxed; lanes 4
and 5, homozygous floxed. C, PCR verifies the presence of the flanking
loxP site in targeted ES cells. Genomic DNA from ES cell clones was
amplified using above primers to reveal the endogenous and floxed
alleles as predicted from panel A. Lanes 16 (targeted ES clone) and 7
(nontargeted clone) show the PCR products with the predicted 165-bp and
195-bp bands. Products were subsequently isolated and sequenced for
verification. Note that clones 1 and 6 did not contain the loxP site in
intron 2, indicating that homologous recombination must have occurred
upstream of that site.
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Approximately 25% of the females were fertile (n > 20), but they
never gave birth to more than three pups. The testes from adult
flneo/flneo
mice were considerably smaller than those of wild-type controls (Fig. 2A
). HistoBank accession numbers for
all images areshown in Table 1
.
HistoBank can be accessed at http://HistoBank.nih.gov.
The small testes size in
flneo/flneo
mice was due to the absence of spermatogonia, spermatocytes, and
spermatids from the seminiferous tubules (Fig. 2B
). Seminiferous
tubules from control (wild-type or
flneo/+) mice contained the
expected mixed population of developing germ cells undergoing
spermatogenesis (Fig. 2C
). The accessory glands and seminal vesicles
from
flneo/flneo
mice did not reveal any overt morphological phenotypic differences. At
p9, testes did not have spermatogonia as determined by germ cell
nuclear antigen immunohistochemistry (GCNA IHC) (data not
shown).

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Figure 2. Reproductive Organs in Wild-Type and
flneo/flneo Mice
A, Comparison of genital tracts from
flneo/flneo and
wild-type 3.5-month-old males (HistoBank accession numbers 1456, 1457,
1458). Cross-sections of seminiferous tubules of
flneo/flneo (B)
(accession numbers 1342 and 1362) and control (C) (accession number
1363) testes shown in panel A. Ovaries from p9 reveal a decline in
primordial follicles in
flneo/flneo
ovaries (D) compared with controls (E). Removal of the
neo cassette reverted the loss-of-germ-cell phenotype as
shown in 2-month-old fl/fl testis (F) and ovary (G). H,
Schematic representation of the hypomorphic, targeted
bcl-x locus containing the loxP-flanked
neo cassette in the promoter. Block
arrows represent loxP sites. I, Schematic representation of the
floxed bcl-x locus after removal of the
neo cassette. Arrowhead, Sperm; lu,
lumen; sg, spermatogonial cell; st, Sertoli cell; ld, Leydig cell;
white arrowhead, primordial follicle;
arrow, primary follicle; white block
arrow, preantral and early antral stage follicles. Sections in
panels B and C were stained by H&E. Sections in panels D, E, F, and G
were visualized using Weigerts stain. Original magnification is 0.8x
(panel A) and 630x (panels B, C, D, E, F, and G).
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At p9,
flneo/flneo
ovaries (Fig. 2D
) contained 94% fewer primordial follicles than
control ovaries (Fig. 2E
). Normal ovaries at p9 consisted of primordial
follicles, each of which contained an intact oocyte surrounded by a
single layer of morphologically normal granulosa cells. The number of
primordial follicles in
flneo/flneo
ovaries decreased from p1 (33.9%) to p19 (6.4%) compared with
follicle counts from normal ovaries. At p19 and at 3 months, follicle
counts from control and
flneo/flneo
ovaries were even more distinct from each other (Fig. 3
, A and B). Although the primordial
follicle counts relative to controls did not change in
flneo/flneo
ovaries at p19 and 3 months (6.4% vs. 5%), there was a
decline in the populations of primary (32.7% vs. 14.5%)
and preantral staged follicles (73.1% vs. 23.4%) with
advancing age (Fig. 3
).

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Figure 3. Graphic Depiction of Follicle Numbers in
bcl-x
flneo/flneo
and Wild-Type Ovaries at p19 (A) and 3 Months (B)
Data represent the mean ± SEM. For p19, n = 3
for control, and n = 4 for fl/fl and
flneo/flneo. For
primordial follicles at p19, P < 0.002 for
flneo/flneo. For
3 months n = 3 for fl/fl, n = 5 for
flneo/flneo,
n = 7 for control. P = 0.001 for primordial
follicles; P = 0.14 for primary follicles;
P = 0.005 for antral staged follicles.
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To distinguish whether the observed phenotype was the result of
structural rearrangements of the bcl-x locus after targeting
or a hypomorphic allele, we modified the targeted allele (Fig. 2H
) by
breeding flneo/+ mice into the
EIIaCre line (21 ). Bcl-x
flneo/+; EIIaCre transgenic mice
exhibiting mosaic deletion patterns (i.e. incomplete
Cre-mediated deletion) and were assayed by PCR for the deletion of the
neo cassette (Fig. 2I
) and then backcrossed into wild-type mice to
remove the Cre transgene. Additionally, bcl-x null alleles
(-), which lacked exons 1 and 2, were also generated as described in
Materials and Methods. The loss-of-germ cell phenotype was
reverted upon removal of the neo gene (Fig. 2
, F and G and
Fig. 3A
). All fl/fl (no neo; Fig. 2F
) and fl/-
males (n > 20) were fertile with active spermatogenesis occurring
in the seminiferous tubules. Similarly, fl/fl and
fl/- females were fertile (n>20) and had normal litter
sizes and a normal distribution of follicles (Figs. 2G
and 3A
).
Germ Cell Depletion in
flneo/flneo
Embryos
Hematoxylin-eosin (H&E) staining was used to identify whether germ
cells were lost during migration or depleted during development.
Analyses of fetal testes (E12.5E14.5) from
flneo/flneo
embryos established that although the germ cells had entered the
developing genital ridge, there was a decline in gonocytes between
E12.5 (Fig. 4
, A and G) and E13.5 (Fig. 4
, B and H). By E14.5, the loss of spermatogonia was reflected by the
formation of a lumen in the developing testis cords (Fig. 4
, C and I).
GCNA antibodies can be used for germ cell detection from E11.5 to the
diplotene/dictyate stage of the first meiotic division (22 ). In males,
GCNA is found in spermatogonia, spermatocytes, and round spermatids; in
females, it is present until oocytes arrest at the dictyate stage and
gain a layer of granulosa cells (22 ). GCNA IHC confirmed the decrease
in germ cell numbers between E12.5 and E14.5 (Fig. 4
, DF and JL).
Morphometric analyses revealed that
flneo/flneo
testis contained similar numbers of germ cells as wild-type controls at
E12.5, and that the numbers decreased to 68% of controls at E13.5 and
15% at E14.5 (Fig. 5
).

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Figure 4. The Loss of Gonocytes in
flneo/flneo
Embryos Occurs in the Embryonic Testis after Colonization by the
Primordial Germ Cells
Testes from wild-type (AF) and
flneo/flneo
embryos (GL) were compared by H&E staining (AC, GI) and GCNA IHC
(DF, JL) at different developmental stages. Germ cells are found in
E12.5 (A and D) and E13.5 (B and E) control testes as well as E12.5 (G
and J) and E13.5 (H and K)
flneo/flneo
testes. At E14.5, mutant testes (I and L) show a depletion in gonocytes
compared with controls (C and F) within the developing seminiferous
tubules. lu, Lumen; arrowhead, seminal cord;
white arrowhead, GCNA-positive cells;
arrow, normal gonocyte; large arrow,
apoptotic cell; asterisk, developing lumen from loss of
gonocytes. Original magnification is 630x.
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Figure 5. Graphic Depiction of Gonocyte Numbers in
bcl-x
flneo/flneo
and Wild-Type Fetal Gonads
Gonocyte numbers in wild-type and bcl-x
flneo/flneo and
testes between E12.5 and E14.5. Data represents the mean ±
SEM. n = 3 for all samples.
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Whole-mount alkaline phosphatase (AP) staining and GCNA IHC of
flneo/flneo
ovaries also showed a decrease in germ cell numbers from E13.5E15.5
(Fig. 6
). Classically, AP reactivity has
been used for primordial germ cell detection from E8.5E15.5 (23 ). AP
staining was decreased in E12.5 and E13.5
flneo/flneo
ovaries (Fig. 6
, B and D) compared with wild-type controls (Fig. 6
, A
and C). GCNA IHC on E14.5 and E15.5 wild-type ovaries (Fig. 6
, E and G)
showed normal germ cells within the developing organ. However, E14.5
flneo/flneo
ovaries contained germ cells with fragmented nuclei (Fig. 6F
), and by
E15.5 the number of germ cells present had greatly diminished (Fig. 6H
). Histological evaluation of embryonic ovaries indicated that
flneo/flneo
ovaries did not contain fewer gonocytes at E12.5 than wild-type
controls. However, by E13.5, the number of germ cells had decreased to
42% (not shown). Apoptotic germ cells were characterized by condensed,
darkly staining pyknotic nuclei that often appear fragmented. E13.5
testes showed higher levels of apoptotic germ cells in
flneo/flneo
(18.6% ± 6.7; range, 10.9%22.9%) vs. control (4.5% ±
1.8; range, 2.5%6.7%) (Fig. 4G
). Even though both
flneo/flneo
and wild-type ovaries contained apoptotic germ cells, the
flneo/flneo
ovaries had a greater percentage of gonocytes undergoing apoptosis than
the wild-type ovaries. At E13.5, the percentage of apoptotic gonocytes
ranged from 28%42.7% in the
flneo/flneo
ovaries, whereas it only ranged from 11.1%19.1% in the wild-type
ovaries (data not shown).

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Figure 6. Oocytes in
flneo/flneo and
Wild-Type Fetal Ovaries
AP levels were determined in mutant E13.5 (B) and E14.5 (D) ovaries and
same stage controls at E13.5 (A) and E14.5 (C) as described in
Materials and Methods. Visualization of germ cells in
E14.5 control (E), E14.5
flneo/flneo (F),
E15.5 control (G), and
flneo/flneo
ovaries (H), as determined by GCNA staining. Arrow,
Normal germ cell; arrowhead, apoptotic germ cell;
asterisk, mesonephros. Original magnification is 100x
(AD), 200x (G and H), 630x (E and F).
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Transcriptional Initiation Sites in the bcl-x Gene and
Expression of Bcl-x in Gonads
The neo gene was integrated 488 bp 5' of the previously
identified transcriptional start sites in the bcl-x gene
(Fig. 7
) (24 ). To test whether the
tissue-specific hypomorphic allele was the result of more distal
transcription start sites in gonads that are juxtaposed to the neo
gene, we performed 5'-RACE (rapid amplification of cDNA ends) to
identify bcl-x mRNA start sites in gonads. After having
sequenced the products, we identified two additional
bcl-x transcripts in the adult testis, ovary, and fetal
gonad at -168 and -314 relative to the published start site. These
sites were more distal from those previously reported in brain (+1) and
thymus (+73) (Fig. 7
). Transcriptional start sites in the adult and
fetal gonads were confirmed by RT-PCR (data not shown).

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Figure 7. 5'-RACE Shows That Gonads Have Additional Upstream
Initiation Sites for mRNA Transcription of the bcl-x Gene
Bold, boxed sequence denotes the XhoI
site into which the neo gene was cloned. Bold
bases designate the transcription start sites for testis (two
sites; determined in this study), brain (24 ), or thymus (24 ).
White arrowheads, Gonad I and gonad II sites;
solid arrowhead, brain site; arrow,
thymus site. The numbering of the transcription start sites for bcl-x
is relative to the initiation site in brain at (+1) (24 ). GenBank
accession number AF088904.
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To demonstrate that the loss of germ cells is attributed to a temporal
specific down-regulation of the bcl-x gene, quantitative
RT-PCR using TaqMan (Perkin-Elmer Corp., Norwalk, CT)
was performed to obtain a relative expression profile of
bcl-x to an endogenous control, glyceraldehyde 3-phosphate
dehydrogenase (GAPDH). Approximately 150 germ cells were isolated from
E12.5 gonads, and mRNA from lysed cells was used directly for cDNA
synthesis. The first strand synthesis product was then used for
quantitative RT-PCR analysis. Normalization of bcl-x mRNA
obtained from germ cells was performed by comparison to GAPDH mRNA.
Based on this internal standardization, there was an approximate
15-fold decrease in relative levels of bcl-x transcript in
the gonocytes isolated from
flneo/flneo
gonads compared with control gonads (n = 4).
Ribonuclease (RNase) protection assays (RPAs) revealed a decline of
bcl-x and other bcl-2 family members from mRNA
isolated from
flneo/flneo
(Fig. 8
, lane 10) as compared with
control (Fig. 8
, lanes 79) testes, with the caveat that the mutant
testes lacked spermatogonial cells. Bcl-w expression was not reduced in
flneo/flneo
testes, probably due to its localization also in Sertoli cells (16 17 ). Variability of Bcl-w expression was found in wild-type controls
(compare lanes 79). RPAs showed that the inserted neo gene
had no effect on bcl-x gene expression in a variety of
tissues from adult mice, including heart, kidney, and seminal vesicles
(Fig. 8
). Additional, nonreproductive tissues (e.g. brain,
lung, and spleen) did not demonstrate a reduction in bcl-x
message (data not shown). Histological examination of spleen, thymus,
and liver in these mice did not reveal any differences compared with
controls (not shown).

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Figure 8. RPA Shows That the Loss of bcl-x Is
Tissue Specific
Total RNA was isolated from fresh mouse tissues by a modified
guanidinium thiocyanate-phenol-chloroform procedure (48 ). Purified RNA
(10 µg) was used with the m-Apo2 riboprobe template set
(PharMingen, San Diego, CA) according to manufacturers
specifications with 32P-UTP labeling (Amersham Pharmacia Biotech, Arlington Heights, IL). L32 and GAPDH were
included as internal controls. Protected fragments were resolved using
6% polyacrylamide gels from Long Ranger gel solutions (J.T. Baker) at
120 V for 2 h in 0.5 x TBE. Lane 1, Wild-type heart; lane 2,
flneo/flneo
heart; lane 3, wild-type kidney; lane 4,
flneo/flneo
kidney; lane 5, wild-type seminal vesicle; lane 6,
flneo/flneo
seminal vesicle; lanes 79, wild-type testis; lane 10,
flneo/flneo
testis. mRNA was harvested from adult males approximately 3 months of
age. Note that bcl-x is reduced in the mutant testis
sample (lane 10) in comparison to control testis (lanes 79).
Expression in the seminal vesicle remains equivalent between control
(lane 5) and mutant (lane 6). Nonreproductive tissues similarly
reflected endogenous expression profiles of bcl-x in
mutant mice.
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Expression of Bcl-x and Bax in the Fetal Testis
Bcl-x IHC established its expression at E13.5, the time when
gonocytes are lost in
flneo/flneo
testis (Fig. 9
). Expression in wild-type
testes was confined to the PGCs (Fig. 9B
), and expression was reduced
in E13.5
flneo/flneo
testis (Fig. 9A
). Similarly, whole mount in situ
hybridization demonstrated lower Bcl-x expression in
flneo/flneo
seminiferous tubules (data not shown). The loss of germ cells in
flneo/flneo
testes due to apoptosis was first observed at E13.5. To determine
whether Bax could be a controlling factor in the apoptotic event, we
performed IHC and established the expression of Bax at E13.5 (Fig. 9
).
Bax was expressed in wild-type testes at E13.5 and E14.5 in PGCs and,
to a lesser degree, in somatic cells of the mesonephros (Fig. 9
, D and
E). The same staining pattern was observed in E13.5
flneo/flneo
testes (Fig. 9C
). No Bax expression was observed in wild-type testes at
day E15.5. The Bax negative control (bax-null E14.5 testis)
showed a low level of nonspecific product primarily restricted to the
interstitial cells (Fig. 9F
). RT-PCR confirmed that Bax is expressed in
wild-type and
flneo/flneo
germ cells (Fig. 9G
).

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Figure 9. IHC Localization of Bcl-x and Bax in Wild-Type and
flneo/flneo Fetal
Testes
Bcl-x was localized in E13.5 fetal testes as described in
Materials and Methods. Panels A and B show Bcl-x
staining in E13.5 seminiferous cords of
flneo/flneo (A)
and wild-type (B) mice. Arrow, Seminiferous cord; gc,
germ cells; ms, mesonephros; arrowhead, apoptotic cells.
Bax was localized in E13.515.5 fetal testes as described in
Materials and Methods. Panels C and D show Bax staining
in E13.5 seminiferous cords of
flneo/flneo (C)
and wild-type (D) mice. Panels E and F show Bax staining in E14.5
wild-type (E) and E14.5 bax-null (F) testes.
Arrow, Seminiferous cord; gc, germ cells; ms,
mesonephros; arrowhead, apoptotic cells; white
arrowhead, Bax peri-nuclear staining. Original magnification is
630x. G, RT-PCR of bax expression in fetal testes. Lane
1, No DNA control; lanes 24, wild-type testes; lanes 56,
flneo/flneo
testes; lanes 78, bax -/- testes.
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Genetic Rescue of the Loss-of-Germ-Cell Phenotype through the
Deletion of Bax
Since Bax is expressed in PGCs, we investigated whether Bax
induced the apoptosis and attrition of PGCs in
flneo/flneo
gonads. Bax-null alleles were bred into the bcl-x
hypomorph [bcl-x
flneo/flneo] to
generate bax-null bcl-x hypomorph [bax
-/-; bcl-x
flneo/flneo] mice.
The complete loss of Bax rescued the hypomorph loss-of-germ-cell
phenotype as postnatal testes contain germ cells. At p1,
bax-null bcl-x hypomorph testes (n = 3) had
a 22-fold increase in the number of GCNA-positive cells per
seminiferous tubule compared with the bcl-x hypomorph
(n = 3) testis (Fig. 10B
). GCNA
IHC also showed the population of seminiferous tubules with germ cells
in the bax-null bcl-x hypomorph testes (n =
3) testis (Fig. 11
). At p22, control
testes (Fig. 12
, A and B) showed
spermatogonial descendants within the bax +/- and
bax -/- seminiferous tubules. However, bax
+/-;
flneo/flneo
(Fig. 12C
) males did not have spermatogonia and bax-null
bcl-x hypomorph testes (Fig. 12D
) males had excess numbers
of spermatogonia and spermatocytes resembling the bax -/-
phenotype (14 ). Consistent with this, the bax-null
bcl-x hypomorph and bax-null males were sterile
(n > 6). Likewise, removal of both bax alleles from
bcl-x hypomorph females resulted in higher follicle numbers
compared with the bcl-x hypomorph females. At p1, there is a
4-fold increase in the number of follicles in bax-null
bcl-x hypomorph ovaries vs. bcl-x
hypomorph (n > 4) (Fig. 10A
). Follicle counts in
bax-null bcl-x hypomorph ovaries were actually
higher than control ovary at the same stage. These females (n = 6)
also had a normal reproductive lifespan and litter sizes that
varied between 812 pups.

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Figure 10. Graphical Depiction of Germ Cell Numbers in p1
Gonads
Gonads were obtained from bax hemizygous or
homozygous-null mice containing 1 or 2 hypomorphic bcl-x
alleles. Gonads were collected and counted from female (A) and male (B)
mice. For testes, n = 3; for ovaries, n = 9 for
flneo/+; bax +/-; n = 7
for flneo/+; bax-/-; n
= 5 for
flneo/flneo;
bax +/- and
flneo/flneo;
bax--/-. For ovaries, P = 0.03 for
flneo/flneo;
bax +/- vs.
flneo/+; bax +/- and
P < 0.003 for
flneo/flneo;
bax +/- vs.
flneo/+; bax-/- and
flneo/flneo;
bax -/-. For testes, P < 0.003.
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Figure 11. Rescue of the bcl-x Hypomorph Male
Phenotype through the Deletion of Bax
Cross-sections of p1 testes of (A) bcl-x
flneo/+; bax +/-, (B)
bcl-x flneo/+; bax -/-, (C)
bcl-x
flneo/flneo; bax
+/- and (D) bcl-x
flneo/flneo;
bax -/- mice. Removal of one bax allele
did not reverse the bcl-x hypomorph phenotype, whereas
the loss of both bax alleles resulted in an increase in
germ cells in the testis. GCNA IHC was performed on all testes with
eosin counterstaining. Original magnification is 100x. HistoBank
accession numbers 1354, 1355, 1356, 1357.
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Figure 12. Dominance of the bax-Null Phenotype
over the bcl-x Hypomorph
Cross-sections of seminiferous tubules from p22 testes of bax
+/- (A) and bax -/- (B) mice. C, Removal of
one bax allele in bax -/+; bcl-x
flneo/flneo mice did
not reverse the loss-of-germ-cell phenotype. D, The loss of both
bax alleles results in the presence of spermatogenesis
in bax -/-;bcl-xflneo/flneotestis. H&E staining was performed on all sections. lu, Lumen;
*, Leydig cells; arrow, seminiferous tubule. Original
magnification is 200x.
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 |
DISCUSSION
|
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During embryonic life, germ cells migrate through the dorsal
mesentery of the hindgut and arrive at the genital ridge around E11.5
to colonize the indifferent gonad (1 2 ). In the male and female,
mitotically active germ cells become quiescent at E15.5 and E13.5,
respectively, with female germ cells immediately entering meiosis (5 6 25 ). Coincident with the loss of mitotic activity, many germ cells
undergo attrition through apoptosis, which is suggested to be necessary
for the future maintenance of supportive cells and germ cells in a
distinct ratio (4 ). We now demonstrate that accurate regulation of
Bcl-x concentration in PGCs is required for their survival during
normal fetal gonad development, and that Bax is a counterbalance in
this developmental process. This finding proves the concept that the
balance of different Bcl-2 family members can determine cell survival
and death in vivo (26 ).
A priori, cell survival imposed by Bcl-x should affect male and
female germ cells similarly as determined by the levels of apoptosis.
However, given the discrepancy between germ cell attrition in males and
females in the bcl-x hypomorph, it is apparent that germ
cell survival in the embryonic testis is more dependent upon Bcl-x. At
p1, germ cells in the
flneo/flneo
male are decreased to 4% compared with wild-type, whereas female germ
cells have declined to 34% of controls. Since female germ cells exit
the mitotic cell cycle and enter meiosis about 1 day earlier than male
germ cells, it is possible that the population of female germ cells
that enter meiosis early are the ones that survive. The presence of a
small percentage of oocytes after birth suggests that Bcl-x is
important, but not essential, for female germ cell survival. Based on
the presence of additional bcl-x transcripts with more
distal start sites in germ cells, the bcl-x gene expression
might be influenced by the presence of the neo gene at
postnatal stages of development. This hypothesis is supported by
comparison of follicle numbers in
flneo/flneo
and control ovaries at p1, p19, and 3 months. At p1, there is a 3-fold
reduction in follicle numbers in
flneo/flneo
vs. control ovaries, compared with a 15-fold reduction in
follicle numbers at p19. Likewise, there is a decrease in the primary
and preantral follicle populations between p19 and 3 months in the
flneo/flneo
ovaries compared with control ovaries. The increased atresia in this
model compliments the reduced atresia and increased reproductive
lifespan found with bax-null females (15 ).
The bcl-x Hypomorph
The insertion of the neo gene in the promoter of the
bcl-x gene resulted in a hypomorph allele that linked Bcl-x
to germ cell survival. Although the time and effort is more
considerable in pursuing the conditional gene targeting approach, the
wealth of information from generating multiple alleles outweighs this
concern. The altered alleles might display an altered expression
profile and/or transcript that could lead to an analyzable phenotype
(27 28 ). Therefore, a single targeting event can be used to examine a
combination of subtle and complex phenotypes in the mouse. We have used
this approach to demonstrate the role of Bcl-x in the ontogeny of germ
cell development. We suggest that the tissue specificity of the
bcl-x hypomorph allele is a result of cloning the
neo gene within 170 bp to newly discovered, gonad-specific
transcriptional start sites in the bcl-x gene promoter.
Reversal of the phenotype upon removal of the neo gene also
demonstrated that the remaining loxP site is not embedded within a
critical cis element. Bcl-x IHC, TaqMan quantitative RT-PCR,
and in situ hybridization demonstrate that there is a
reduction of Bcl-x protein and mRNA in
flneo/flneo
fetal gonads compared with wild-type fetal gonads. The RPA
validated the tissue specificity of the hypomorph, as bcl-x
mRNA levels are equivalent in other
flneo/flneo
and wild-type tissues examined. We suggest that the neo gene located in
the bcl-x promoter prevents the full utilization of
transcriptional elements required for a normal developmental expression
profile in the fetal gonads.
The common biological theme in cell development and maturation
is that the loss of a proapoptotic factor leads to a decrease in
apoptosis, whereas the elimination of an antiapoptotic factor results
in increased apoptosis (26 29 ). The role of four proteins from the
Bcl-2 family (Bcl-x, Bax, Bcl-2, Bcl-w) in gonadal development has now
been addressed through gene deletion. Their essential functions appear
to be stage specific and nonoverlapping. Bcl-2 null mice
have a statistically significant reduced number of primordial follicles
and diminished levels of primary and preantral stage follicles (11 ).
Bax has been implicated in granulosa cell death, as ovaries of
bax-null mice have fewer atretic cells in atretic follicles
(14 ). Bax -/- and bcl-w -/- males are both
infertile and do not produce mature sperm. Germ cells in
bcl-w -/- males are generally depleted by 6 months of age
with subsequent reduction in the Sertoli cell and Leydig cell
populations (16 17 ). While Bax, Bcl-w, and Bcl-2 have been shown to
preferentially control postnatal stages of germ cell development and
maturation, Bcl-x is required at a very early stage, providing for germ
cell survival in the fetal gonad (Fig. 13
). The bcl-x gene encodes
two alternatively spliced transcripts (30 ), one encoding a cell
survival molecule (Bcl-xL) and one encoding a
protein (Bcl-xS) that can induce cell death
in vitro (31 ). Suppression of bcl-x transcripts in our
hypomorph would affect both splice forms. However, since
bcl-xS mRNA is far less abundant than
bcl-xL mRNA and can only be detected by
RT-PCR (32 33 ), its role in vivo remains unknown.

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Figure 13. Schematic Depicting Proposed Roles of Bcl-2 Family
Members in Germ Cell Survival and Apoptosis
The timeline above represents both embryonic (E) and postnatal (P)
stages of growth, in days.
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The Bcl-x/Bax Rheostat
Based on our results, we propose a genetic mechanism
controlling the balance of germ cell survival and the surge of
apoptosis during fetal development where Bcl-x and Bax are competing
factors in this determination. A Bcl-x/Bax rheostat model is in accord
with a Bcl-2/Bax rheostat model proposed by Korsmeyer and co-workers
(34 ). Although the rheostat model implies a direct interaction between
Bcl-x and Bax to elicit the cellular response, either
heterodimerization-dependent or -independent mechanisms for these
molecules could occur. Survival and death in vivo through
the balance of Bcl-x and Bax probably contains cell-specific components
since the absence of Bax can rescue germ cells (this study) as well as
neurons, but not hematopoietic cells in
bcl-x/bax-deficient embryos (35 ). We suggest that the
decline of Bcl-x during gonad development in the bcl-x
hypomorph allows Bax to proceed unabated in promoting cell death, as
Bax is clearly present in the time window of the apoptotic surge
between E12.5 and E14.5. Removal of both bax alleles from
the bcl-x hypomorph restores the survival of germ cells,
further demonstrating that the ratio of Bax and Bcl-x in the developing
PGCs is critical for the demarcation and regulation of pathways for
cell survival and cell death. Bcl-xL can inhibit
PGCs from undergoing apoptosis in vitro (36 ).
The Bcl-x/Bax rheostat model has also been demonstrated to
define cell fate in yeast as well as mammalian cells. Expression of Bax
in Saccharomyces pombe (37 ) or Saccharomyces
cerevisiae (38 ) leads to cell death. However, coexpression of Bax
and Bcl-x resulted in a suppression of apoptosis. Human
polymorphonuclear neutrophils have been shown to decrease
bcl-x mRNA upon induction of apoptosis or decrease
bax mRNA in response to apoptotic inhibitors (39 ). The
Bcl-x/Bax ratio might also be an important indicator in
1-adrenoceptor activation, which increases
myocardial resistance to ischemic injury, thereby reducing cell death
(40 ). Ovarian follicle development is also linked to maintenance of
this ratio. Follicle growth and survival in the rat are correlated with
an attenuation of bax mRNA while bcl-2 and
bcl-x mRNAs are relatively constant. In vitro
induction of apoptosis in antral staged follicles was associated with a
decrease in Bcl-x and an increase in Bax (41 ). Our genetic studies now
support these correlative studies on the Bcl-x/Bax rheostat.
The Bcl-2 Family in Germ Cell Development
Several Bcl-2 family members (Bax, Bcl-2, Bcl-w)
have been linked to germ cell development through gene deletion
experiments (Fig. 13
) (11 14 16 17 ). Gametogenesis requires a
balance of these members, although spermatogenesis appears to be less
refractory to their altered expression than does folliculogenesis.
Generally, gene disruption (Bcl-w, Bax) leads to male sterility at
postnatal stages of development. In the ovary, removal of Bax or Bcl-2
leads to a surfeit or reduction in the number of primordial follicles,
respectively (11 14 ); however, neither bax-null nor
bcl-2-null females are sterile. Bax is important for ovarian
function as bax-deficient females contain an excess of
primordial follicles during postnatal life and exhibit a prolonged
ovarian life span into advanced chronological age (15 ). Likewise, the
balance of these members has been shown to be important because ectopic
expression of Bcl-x and Bcl-2 in the germ cells under the PGK promoter
leads to male sterility (9 ). However, Bcl-x appears to be more
important than Bcl-2 during spermatogenesis since Bcl-2 is expressed at
very low levels in normal mouse testis. While Bax, Bcl-w, and Bcl-2
have been shown to control preferentially postnatal stages of germ cell
development and maturation, Bcl-x is required at an early stage for
germ cell survival in the fetal gonad. The availability of the
bcl-x hypomorph in combination with the bax-null
mice has now established a balancing role for these molecules in fetal
germ cell development. Whether Bcl-x, like Bax, also modulates
postnatal gametogenesis remains unknown and awaits the conditional
inactivation of the floxed bcl-x gene at distinct stages of
development using current Cre transgenic mice.
 |
MATERIALS AND METHODS
|
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Construction of the bcl-x Targeting Plasmid
A 10.5-kb EcoRV fragment of the bcl-x gene
was isolated from a mouse 129/SvJ
genomic DNA library clone and
ligated into Bluescript IISK (pBS, Stratagene, La Jolla,
CA). This clone contained 3.5 kb of the promoter, exon 1, intron 1,
exon 2, and 5.5 kb of intron 2. The targeting construct pBXFL was
prepared by cloning a floxed neo gene into the Xho site in
the promoter, a downstream loxP site within intron 2 at the
SpeI site, and a thymidine kinase (TK) gene at the
bcl-x-pBS junction. The loxP site was generated by annealing
two primers to give SpeI overhangs: 5'-CTA GAT AAC TTC GTA
TAA TGT ATG CTA TAC GAA GTT AT-3' and 5'-CTA GAT AAC TTC GTA TAG CAT
ACA TTA TAC GAA GTT AT-3'. Both the neo and TK genes were regulated by
the PGK-I promoter and polyadenylation signal and were obtained from
plasmid pPNT (42 ). The TK cassette was used to enrich for colonies that
had undergone homologous recombination vs. random
integration. The floxed neomycin plasmid pNeop was prepared by ligating
a Klenow filled-in XhoI-BamHI fragment from pPNT into a
BamHI digested and Klenow filled-in double loxP plasmid
pBS246 (Life Technologies, Inc., Gaithersburg, MD). An
EcoRINotI fragment from pNeop was Klenow
filled-in and ligated into a filled-in XhoI site of the
bcl-x/loxP plasmid to generate plasmid pNpbx. For cloning of
the TK cassette, a NotISmaISgfI
linker was introduced into the NotI site of the
EcoRVEcoRV bcl-x clone using the following
oligonucleotides: 5'-GGC CGC GCC CGG GCG CGA ATC GCA T-3'; 5'-GGC CAT
GCG ATC GCG CCC GGG CGC-3'. The TK cassette was directionally cloned
into pNpbx as an SgfI-NotI fragment from pBS-TK;
pBS-TK contained an engineered SgfI site at the
SalI site and the BamHI-HindIII TK
fragment from pPNT. The functionality of the loxP sites was determined
before electroporation by transfecting pBXFL into bacterial AM-1 cells
and by DNA sequencing (43 ).
Embryonic Stem (ES) Cell Targeting at the bcl-x Locus
and Generation of flneo Mice
Linearized vector (25 µg) was electroporated (600 V/25
µFarads) into 2 x 107 TCI 129SvEv ES
cells and maintained as described previously (44 ). Colonies resistant
to G418 (280 µg/ml) and FIAU (0.2 µM) selection were
isolated 7 days after electroporation. Clones verified as targeted by
Southern hybridization were checked for the downstream loxP site by PCR
using forward (5'-GCC ACC TCA TCA GTC GGG-3') and reverse (5'-TCA GAA
GCC GCA ATA TCC CC-3') primer. The reaction conditions were 4 min at 94
C (1 cycle), 30 sec at 94 C/30 sec at 56 C/30 sec at 72 C (30 cycles),
5 min at 72 C. The endogenous allele gave a 160-bp band while the
targeted allele was 195 bp in size. Positive ES cell clones were
injected into 3.5-day blastocysts harvested from superovulated C57BL/6
donors (2.5 U PMSG, Calbiochem, La Jolla, CA ; 2.5U hCG,
Organon, West Orange, NJ ). Germline transmission
was evaluated by backcrossing into a C57BL/6 background. Offspring
carrying a floxed allele with a neo cassette were designated as
flneo and represent a mixed
C57BL/6129SvEv background.
Generation and Genotyping of bcl-x Null (-),
bcl-x Floxed (flneo),
and bax Null Mice
Heterozygous bcl-x floxed mice
(flneo/+) were mated with
MMTV-Cre mice that exhibited Cre expression in the female
germline (45 ). Cre transgenic
flneo/+ dams were bred with
wild-type males, and the progeny was checked for the recombined allele.
Null alleles having a complete deletion of the floxed neomycin gene as
well as exons 1 and 2 were confirmed by PCR. Reaction conditions were 4
min at 94 C (1 cycle), 30 sec at 94 C/30 sec at 58 C/30 sec at 72 C (30
cycles), 5 min at 72 C using forward (5'-CGG TTG CCT AGC AAC GGG GC-3')
and reverse primers (5'-TCA GAA GCC GCA ATA TCC CC-3'). The floxed neo
cassette was deleted by crossing
flneo/+ mice with
EIIa-Cre mice (46 ). PCR reaction conditions were identical
to the null allele verification using the following primers: forward,
5'-CGG TTG CCT AGC AAC GGG GC-3'; reverse, 5' CTC CCA CAG TGG AGA CCT
CG-3'. Bax knockout mice were a kind gift of Dr. Stanley
Korsmeyer and were genotyped using the following primers and reaction
conditions. For the wild-type allele, forward primer 5'-GTT GAC CAG AGT
GGC GTA GG-3' and reverse primer 5'-GAG CTG ATC AGA ACC ATC ATG-3' were
used under the same condition profile as described above. For the
bax knockout allele, the same forward primer and PCR
reaction profile as for the wild-type were used with a different
reverse primer, 5'-CCG CTT CCA TTG CTC AGC GG-3'. The wild-type allele
gave a 300-bp band while the bax knockout allele gave a
506-bp band.
Sequence Analysis of the bcl-x Promoter Region
A 9.5-kb XbaIXbaI fragment was sequenced
with the AmpliTaq Dye Terminator Cycle Sequencing Kits
(Perkin-Elmer Corp., Branchburg, NJ) on an ABI
Prism 310 Genetic Analyzer (ABI Advanced Biotechnologies, Inc.,
Columbia, MD). Subclones were generated to facilitate sequencing
using M13 forward and reverse primers: a 4-kb XbaIEcoRV
fragment, 3.5-kb EcoRVHindIII fragment, and
2-kb HindIIISpeI fragment were subcloned into
pBS. Additional oligonucleotide sequencing primers were designed using
the MacVector 4.0 program and synthesized by Lofstrand Oligos
(Gaithersburg, MD). Sequences were compiled using the Sequencher 3.0
program (Gene Codes Corp., Ann Arbor, MI). GenBank accession number
AF088904.
5'-RACE for Determination of Testis-Specific Transcripts
One microgram of total RNA isolated from wild-type mouse testis
was used for the 5'-RACE procedure (Life Technologies, Inc.) as described by the manufacturer. The
bcl-x-specific primer 1 used for the first strand cDNA
synthesis is as follows: 5'-TGT GTT TAG CGA TTC TC-3'. Amplification of
the 5'-transcripts was performed using an abridged universal
amplification primer (provided in kit) and a bcl-x-specific
primer 2 (5'-TAA GGT TAT TCA AAT CTA TCT CC-3'). Amplification was
performed using Platinum Taq (0.25U, Life Technologies, Inc.) under the standard conditions described in
the kit. Amplicons were cloned into pCRII vector
(Invitrogen, San Diego, CA) and sequenced using M13
forward and reverse primers.
Quantitation of bcl-x Expression Levels Using the
TaqMan PCR System
Germ cells were isolated from appropriate staged embryos as
previously described using an EDTA treatment protocol (47 ). Yolk sacs
were collected for PCR verification of embryo genotype. Between 50 and
75 PGCs per gonad were transferred by mouth pipette into lysis buffer
consisting of 1% NP-40, 6 mM dithiothreitol, and 10 U/µl
of RNase inhibitor (Promega Corp., Madison, WI).
SuperScript II first strand synthesis kit (Life Technologies, Inc.) with random primers was used to generate the template for
PCR from the lysate. A 0.5-µl aliquot was used for PCR using the ABI
Prism 7700 Sequence Detection system (ABI Advanced Biotechnologies,
Inc.) and TaqMan PCR products (Perkin-Elmer Corp.).
Primers and probe for the bcl-x amplification were generated
using Primer Express (Perkin-Elmer Corp.): forward primer,
5'-ACC ACC TAG AGC CTT GGA TCC- 3'; reverse primer, 5'-TCT CGG CTG CTG
CAT TGT T-3'; bcl-x probe, 5'-6-FAM ACG GCG GCT GGG ACA CTT TTG
TG-TAMRA-3'. Reaction conditions consisted of 10 min at 95 C (1 cycle),
15 sec at 95 C/60 sec at 60 C (40 cycles) with 200
nM primers and probe. GAPDH and 18S control PCR
kits were used to normalize expression levels. Data were exported into
Microsoft Corp. Excel for analysis. Statistical
significance was determined by the Whitney-Mann U test to be at the
98% confidence level.
GCNA, Bcl-x, Bax IHC, and Bax RT-PCR
Gonads were fixed in Bouins solution
(Sigma, St. Louis, MO) overnight, transferred to 70%
ethanol, paraffin-embedded, and then sectioned at 5 µm. Sections were
deparaffinized through ethanol, rehydrated in water, and placed in
0.03% (vol/vol) hydrogen peroxide in methanol for 30 min at room
temperature. Sections were rinsed in 1x PBS, treated with 0.01
M citric acid, pH 6.0 at 90 C for 10 min, slow cooled an
additional 10 min, and finally rinsed in 1x PBS. Blocking was
performed with 10% serum at room temperature for 30 min followed by
the addition of diluted GCNA antibody (1:2), Bcl-x antibody (1:50;
Santa Cruz no. sc-1041; Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA) or Bax antibody (1:50; Santa Cruz no. sc-526;
Santa Cruz Biotechnology, Inc.) for overnight incubation
at 4 C. Upon washing in 1x Tris-buffered saline (TBS),
peroxidase-conjugated secondary antibody (1:300) was added for 1 h
at room temperature. After rinsing in 1x TBS, samples were incubated
in ABC solution and treated with 3',3-diaminobenzidine
(Vector Laboratories, Inc., Burlingame, CA) according to
manufacturers protocol). Slides were counterstained and mounted with
Permount (Sigma). For RT-PCR, RNA was isolated from germ
cells and used in a first-strand synthesis reaction as described in the
previous section. Bax forward (5'-ATG CGT CCA CCA AGA AGC TGA G-3') and
reverse primers (5'-CCC CAG TTG AAG TTG CCA TCA G-3') were used to
amplify a 162-bp diagnostic band. Reaction conditions consisted of 30
µM of each primer in the following profile: 4 min at 94 C
(1 cycle), 30 sec at 94 C/30 sec at 58 C/30 sec at 72 C (30 cycles), 5
min at 72 C.
AP Staining
Embryos were collected and fixed in absolute ethanol-glacial
acetic acid (7:1) for 1 h at 4 C and replaced twice within 24
h with absolute ethanol as described previously (23 ). Samples were then
placed in two changes of xylene after 12 h and rehydrated with two
changes of absolute ethanol and one change of 70% ethanol for 12 h
each. Using distilled water, embryos were rinsed three times, 10 min
each, and transferred to freshly prepared AP stain for 1520 min as
monitored under a dissecting stereo microscope. AP aqueous staining
solution contained 10 µg/ml
-naphthyl phosphate
(Sigma), 0.5 mg/ml Fast Red TR (Sigma),
0.06% (wt/vol) MgCl2, and 0.45% (wt/vol) Borax
(Sigma). Color development was stopped by placing embryos
in distilled water.
Histological Evaluation of Gonads
Gonads were fixed in Bouins solution for 1824 h and then
washed with 70% ethanol. After fixation, the tissues were dehydrated,
embedded in Paraplast (VWR Scientific, ), serially sectioned (8
µm, ovaries; 5 µm, testes), mounted on glass slides, and stained
with Weigerts hematoxylin-picric acid methylene blue (ovaries) or
hematoxylin-eosin (testes). Counting was performed on every tenth
section for embryonic and postnatal gonads. For ovaries, each section
was qualitatively evaluated for the appearance of the ovarian
follicles. Follicles were identified as normal if they contained an
intact oocyte, organized granulosa and thecal cell layers, and no
pyknotic bodies. Follicles were considered atretic if they contained
either a degenerating oocyte, disorganized granulosa cells, pyknotic
nuclei, shrunken granulosa cells, or apoptotic bodies. Apoptotic bodies
were identified as bodies in the granulosa cell layers or PGCs that
contained dark-staining masses of condensed chromatin.
Statistical Analysis
The mean number of germ cells or ovarian follicles was
calculated using ovaries from at least three different animals.
Differences in germ cell and follicles numbers were evaluated by
one-way ANOVA, with statistical significance assigned at
P < 0.05. When a significant P value was
obtained, Scheffes test was used in the post hoc analysis. The SPSS
(version 10) program (SPSS, Inc., Chicago, IL) was used to
compile statistics from the obtained data.
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to thank G. Enders for his kind gift of the
GCNA antibody and constructive criticism of the manuscript, S.
Korsmeyer for the bax-null mice, D. Loh and Nobura Motoyama for the
bcl-x clone, H. Westphal for the EIIa-Cre transgenic mice, J. Babus for
histology work, and K. Heermeier for the bcl-x subcloning. We are
grateful to C. Deng, G. Robinson, N. Strunnikova, M. Gallego, J.
Shillingford, and R. Humphreys for their technical advice and insight.
We would like to thank Ulrike Wagner for her computational assistance
with HistoBank.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Lothar Hennighausen, Laboratory of Genetics and Physiology, 9000 Rockville Pike Bethesda, Maryland 20892.
E.B.R. was supported by a NIH staff fellow award. Funds for the project
were obtained from NIH intramural resources. K.U.W. was supported by a
Deutsche Forschungsgemeinschaft fellowship (WA 1119/11).
1 Current address: University of California San Diego School of
Medicine, La Jolla, California 92093-0627. 
2 GenBank accession number AF088904. 
Received for publication July 26, 1999.
Revision received February 16, 2000.
Accepted for publication February 21, 2000.
 |
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