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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). 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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). 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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) 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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) 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 (E12.5–E14.5) 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, D–F and J–L).
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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). Classically, AP reactivity has
been used for primordial germ cell detection from E8.5–E15.5 (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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) (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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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 7–9) 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 7–9). 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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). 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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). 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 8–12 pups.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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
EcoRI–NotI 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 NotI–SmaI–SgfI
linker was introduced into the NotI site of the
EcoRV–EcoRV 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/6–129SvEv 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 XbaI–XbaI 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 XbaI–EcoRV
fragment, 3.5-kb EcoRV–HindIII fragment, and
2-kb HindIII–SpeI 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 Bouin’s 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
manufacturer’s 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 1–2 h
each. Using distilled water, embryos were rinsed three times, 10 min
each, and transferred to freshly prepared AP stain for 15–20 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 Bouin’s solution for 18–24 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 Weigert’s 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, Scheffe’s 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 |
|---|
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FOOTNOTES |
|---|
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 1119/1–1).
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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