1 Laboratory of Signal Transduction, National Institute of Environmental Health
Science, National Institutes of Health, Department of Health and Human
Services, Research Triangle Park, NC 27709, USA
2 Duke Comprehensive Cancer Center, Duke University Medical Center, Durham, NC
27710, USA
3 The Office of Clinical Research, National Institute of Environmental Health
Science, National Institutes of Health, Department of Health and Human
Services, Research Triangle Park, NC 27709, USA
4 Department of Medicine, Duke University Medical Center, Durham, NC 27710,
USA
5 Department of Biochemistry, Duke University Medical Center, Durham, NC 27710,
USA
* Author for correspondence (e-mail: black009{at}niehs.nih.gov)
Accepted 28 June 2004
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SUMMARY |
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Key words: Female fertility, Early embryonic development, CCCH tandem zinc-finger proteins
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Introduction |
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Increased interest in these proteins came from the phenotype of TTP
knockout mice, a systemic inflammatory syndrome that was largely prevented by
administration of antibodies to mouse tumor necrosis factor
(TNF
) (Taylor et al.,
1996
). Macrophages from these mice exhibited increased production
of TNF
upon LPS stimulation, because of increased stability of the
TNF
mRNA (Carballo et al.,
1997
). Thus, a model has emerged in which TTP, and possibly its
related proteins, bind to the 3'UTR of mRNAs containing class II ARE
sequences, resulting in mRNA destabilization and decreased levels of
translated proteins (Carballo et al.,
1998
; Blackshear,
2002
).
The zinc finger domains of human ZFP36l2 and ZFP36l1 are 71-73% identical
to that of TTP, and they are also able to bind to and destabilize the same set
of cytokine mRNAs regulated by TTP (Lai et
al., 2000). For example, like TTP, Zfp36l2 can bind to mRNAs
containing class II ARE sequences in cell-free and cell transfection
experiments, and stimulate their degradation
(Lai et al., 2000
;
Lai et al., 2003
); it also
shuttles between nucleus and cytoplasm
(Phillips et al., 2002
).
However, virtually nothing is known about its biological function. To gain
insights into its physiological role, we disrupted the mouse Zfp36l2
gene.
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Materials and methods |
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PCR, Southern, northern and western blot analysis
To genotype the animals, 100 ng of genomic DNA from tails was used as a
template in a single PCR reaction containing a forward primer
(5'-caggaccccagaaaaatgtcg) and a reverse primer
(5'-gttcagattgaggtttgccaggg), resulting in an amplified fragment of
600 bp in the wild-type (WT) and
2.4 kb in the Zfp36l2 mutant
animals, reflecting the insertion of the Neo sequence in that region
(Fig. 1B). To confirm the mouse
genotyping, Southern blot analysis was performed using 10 µg of genomic DNA
digested overnight with Sst I. The digested products were loaded into
a 0.4% agarose gel and transferred to a nylon membrane. The membrane was
UV-crosslinked and probed with a Zfp36l2 exon 2 32P
random-labeled probe (Fig.
1C).
Primary cells or tissues isolated from mice were used as a source of mRNA.
The cells were directly disrupted into a lysis buffer, whereas the dissected
tissues were rapidly frozen and pulverized in liquid nitrogen, and processed
using the RNeasy kit from Qiagen (Valencia, CA, USA) to extract total cellular
RNA. Cytoplasmic RNA from bone marrow-derived macrophages [grown as described
in Carballo et al. (Carballo et al.,
1997)] was extracted using the same kit, except that the nuclei
were removed using a RLN lysis buffer [50 mM Tris-HCl, pH 8, 140 mM NaCl, 1.5
mM MgCl2, 0.5% (v/v) Nonidet P40], according to the directions of
the manufacturer. RNA samples (10 µg) were separated by electrophoresis on
1.2% agarose/formaldehyde gels and used for northern blot analysis
(Stumpo et al., 1989
) with
different mouse Zfp36l2 32P-labeled probes as described in
the figure legends.
Western blot analysis was performed using protein extracts containing
overexpressed and endogenous Zfp36l2. For the protein extracts containing
overexpressed Zfp36l2, HEK 293 cells were transiently transfected with the
mouse full-length Zfp36l2 cDNA
(Phillips et al., 2002) using
calcium-phosphate precipitation as described previously
(Lai et al., 1999
). Protein
extracts containing endogenous Zfp36l2 were extracted from bone marrow-derived
macrophages, spleen or ovaries, in a lysis buffer composed of
ß-glycerolphosphate (50 mM, pH 8.2), 0.25 mM sucrose, 1 mM EDTA, 1 mM
EGTA, 1 mM dithiothreitol (DTT), 50 mM NaF, 10 mM benzamide-HCl, 0.5 mM
phenylmethylsulphonyl fluoride (PMSF), 2 µM peptstatin and 2 µg/ml
leupeptin. Ten to 400 µg of protein from a cytosolic fraction of these
homogenates was boiled in sample buffer and separated by sodium dodecyl
sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analysis
was performed with antisera directed against either the amino-terminal or
carboxyl-terminal peptide of mouse Zfp36l2 (S.B.V.R. and P.J.B., unpublished).
The secondary antibody, a goat anti-rabbit polyclonal antibody conjugated with
horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA, USA) was diluted
to 1:50,000, and proteins were visualized with enhanced chemiluminescence
(SuperSignal West Pico, Pierce, Rockford, IL, USA).
Embryo transfer experiments
WT and Zfp36l2 mutant females were bred with vasectomized males to
induce pseudopregnancy. On day 1.5 post-coitum, WT two-cell stage embryos were
transferred into one oviduct of each pseudopregnant female
(Brinster et al., 1985).
Ovary transplantation procedure
Young females (4-6 weeks-old) were anesthetized; the left ovary from the
recipient animal was removed, and the transplanted ovary (either +/+ to a
/ recipient, or / to a +/+ recipient) was placed
inside the previously emptied capsule
(Jones and Krohn, 1960). The
right ovary was left intact, but its oviduct was cauterized. One week after
the surgery, the animals were mated with WT males. Vaginal plugs were checked
on a daily basis. Any pups produced were genotyped to confirm that the ova
came from the transplanted ovary. Animal care and all experiments were in
accordance with institutional guidelines for animal use at NIEHS.
Superovulation, in vivo fertilization and embryo culture
Adult females (5-8 weeks-old) were subjected to exogenous hormone
injections to induce superovulation, according to standard procedures
(Fraser and Drury, 1975), and
then mated with WT males. The next morning, vaginal plugs were checked and in
vivo fertilized ova were recovered from the swollen ampulla 17-19 hours after
hCG injection. The embryos were briefly treated with hyaluronidase (1 mg/ml)
and further cultured in microdrops of Whittens medium (M16) covered with
paraffin oil and incubated at 37°C in a humidified atmosphere of 5%
CO2 in air until the time of observation.
Microscopy
For the fluorescent microscopic analysis, the embryos were fixed in 4%
(v/v) paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4) for 30
minutes and then stained with 800 nM of Hoechst 33258 dye in a mounting medium
composed of PBS azide (25 mg/mL) and 50% glycerol
(Perreault and Mattson, 1993).
Observation was performed on a Zeiss LSM 510 confocal microscope (Zeiss,
Thornwood, NY, USA). The unfixed embryos were photographed using Nomarski
optics with 400 ASA black and white film.
DNA probes and accession number
The mouse Zfp36l2 RefSeq currently in GenBank, NP_031591.1, represents a
partial protein that lacks the amino terminal contribution of the first exon
and is also truncated at the carboxyl-terminal when compared with the current
human protein RefSeq NP_008818 (Blackshear
et al., 2003). The Zfp36l2 exon 2 probe corresponds to
nucleotides 10176476-10176912 (reverse complement) of the mouse genomic contig
represented by GenBank accession number NT_039658.2 and was derived from the
EST clone accession number AA021952; this contained approximately 72 bp of
unspliced 3' intron sequence as well as approximately 365 bp of 5'
exon 2 sequence. The Zfp36l2 exon 1 and intron probes correspond,
respectively, to nucleotides 1140-1528 and 1576-2075 of the mouse genomic
clone represented by GenBank accession number M97165; the intron probe
contained 14 bp of 5' exon 2 sequence.
Nomenclature
The approved nomenclature for the human gene encoding the third TTP family
member is ZFP36l2 (no OMIM number), whereas the mouse gene is
Zfp36l2. As mentioned in the text, the encoded protein is also known
as TIS11D or 11D, ERF2 and BRF2. Approved gene symbols were obtained from the
HUGO Gene Nomenclature Committee and the Mouse Genomic Nomenclature Committee
(Blackshear et al., 2003).
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Results |
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Expression of the Zfp36l2 intron in mature transcript from mutant mice
In transcripts from mutant mice, exon 2 appeared to be fused with at least
part of the single intron present in Zfp36l2, but not with the
Neo transcript (Fig.
2). Expression of transcripts hybridizing to the Zfp36l2
intron was markedly increased in total RNA derived from cells and tissues of
the N-Zfp36l2 mutant mice as compared with WT
(Fig. 2A). Because unprocessed
RNA is generally nuclear, we investigated whether the Zfp36l2 intron
was expressed in cytosolic RNA derived from WT and
N-Zfp36l2 mice. As
shown in Fig. 2B, the
Zfp36l2 intron was not detected in cytosolic RNA preparations from WT
mice, but abundant expression was observed in samples from the
N-Zfp36l2 mice, demonstrating that at least a portion of the
Zfp36l2 intron is part of the mature transcript present in the mutant
mice. Direct sequencing of this new transcript confirmed that 403 bp
corresponding to the 3'-end of the intron was fused with the second exon
of Zfp36l2 (not shown). The Zfp36l2 intron is very rich in
GC nucleotides (
70%), which has hampered our efforts to sequence the
extreme 5'-end of the mutant transcript.
|
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To date, biochemical assays with the WT and N-Zfp36l2 proteins
expressed in 293 cells have shown no differences in three assays of tandem
CCCH zinc finger protein function: binding to ARE-containing RNA probes in a
gel shift assay; deadenylation of a modified TNF transcript in cell-based
co-transfection assays (Lai et al.,
1999
); and stimulated deadenylation of ARE-containing,
polyadenylated RNA probes in a cell-free assay
(Lai et al., 2003
) (S.B.V.R.,
W. S. Lai and P.J.B., unpublished). In addition, the cytosolic localizations
of
N-Zfp36l2-GFP and WT-Zfp36l2-GFP expressed in 293 cells were
indistinguishable (S.B.V.R. and P.J.B., unpublished).
Homozygous N-Zfp36l2 females are sterile despite normal estrous cycles and sexual behavior
Heterozygous matings from two independent mouse colonies on the C57BL/6NTac
background generated 305 offspring, with genotypes of 79 (26%) WT, 153 (50%)
heterozygous, and 73 (24%) N-Zfp36l2/
homozygous. The percentages of males (27:49:24) and females (24:51:24) of each
genotype followed the same distribution. This typical Mendelian inheritance
pattern indicated that the targeted disruption of the open reading frame of
the Zfp36l2 gene at exon 1 had no deleterious effect on viability
when both eggs and sperm came from heterozygous animals. The
N-Zfp36l2
mice exhibited apparently normal lifespans. In contrast to the TTP-deficient
mice (Taylor et al., 1996
;
Carballo et al., 1998
), no
evidence of systemic inflammation has been detected under normal husbandry
conditions, and macrophages from
N-Zfp36l2 mice express similar levels
of TNF
mRNA as the WT cells when exposed to LPS (not shown). The
observed Mendelian frequency of
N-Zfp36l2 mice is also in contrast to
mice deficient in another family member, Zfp36l1, which die in mid gestation
(Stumpo et al., 2004
).
The only observed phenotype in the homozygous N-Zfp36l2 females was
complete infertility, whereas the males were fertile. This was observed in two
independent mouse colonies derived from distinct ES cell clones, through seven
backcrosses into the C57BL/6NTac strain; it was also seen in the 129S6/SvEvTac
background, in mouse colonies derived from both ES cell clones. These data
strongly suggest that the female infertility phenotype was caused by the
disruption of Zfp36l2.
To gain insights into the etiology of this infertility, N-Zfp36l2
female mice were subjected to a continuous mating study with stud males for
six months, and evaluated for cycling, behavioral estrous and mating. The
N-Zfp36l2 female mice (n=14) bred on multiple occasions, as
determined by the presence of vaginal plugs every 5-6 days; however, no
pregnancies were observed. This cyclical presence of vaginal plugs suggested
that the mutant females exhibited typical estrous cycles and normal sexual
behavior. The anatomy of their reproductive tracts also appeared normal
(Fig. 4A-F), even though their
ovaries expressed
N-Zfp36l2 protein at lower levels than the
full-length Zfp36l2 in WT mice (Fig.
4G). Thus, homozygosity for
N-Zfp36l2 had no
obvious deleterious effects on the development of the female reproductive
tract.
|
Rescue of N-Zfp36l2 female sterility by transplantation of WT ovaries
A potential ovarian etiology for the infertility of the N-Zfp36l2
females was investigated by classical ovary transplant experiments
(Jones and Krohn, 1960
). Donor
ovaries from
N-Zfp36l2 animals were transplanted into WT females, from
which the left ovary had been removed, leaving the right ovary in situ but
with a cauterized oviduct. Although all five WT females transplanted with
N-Zfp36l2 ovaries displayed copulatory plugs, none carried a successful
pregnancy (Table 1). In
contrast, three out of five
N-Zfp36l2 females transplanted with WT
ovaries carried successful pregnancies; that the offspring were derived from
WT oocytes was confirmed by genotyping. Transplantation of WT ovaries into
heterozygous females, known to have no fertility defect, showed a similar rate
of successful pregnancies (68%), reflecting the efficacy of the surgical
procedure.
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Discussion |
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As noted in the Results section, two lines of mice derived from distinct ES
cell clones, each in two distinct genetic backgrounds, C57BL/6NTac and
129S6/SvEvTac, exhibited complete female infertility as the only apparent
phenotype. This was in the setting of apparently normal female reproductive
behavior, reproductive tract anatomy, uterine function and maternal behavior,
and the ability of the ovary to release oocytes and form corpus lutea. Ova
derived from the N-Zfp36l2 females could also be fertilized, but the
zygotes then divided only once and ceased cell division. There was also a
modest decrease in the number of oocytes released after superovulation,
suggesting the possibility of an abnormal ovarian environment for oocyte
maturation and/or release.
The physiological function of TTP, the prototype of this family of CCCH
tandem zinc finger proteins, is to destabilize certain ARE-containing mRNAs,
of which GM-CSF and TNF are known to be affected in cells derived from TTP
knockout mice (Blackshear,
2002). In early development, degradation of maternal mRNAs is
coupled to activation of transcription by the newly formed zygotic nucleus
(zygotic gene activation) (Bachvarova and
De Leon, 1980
; Clegg and
Pikó, 1983
; Schultz,
1993
). It seems possible that Zfp36l2 could mediate the
destabilization of specific maternal transcripts in early embryonic
development. In the mouse, the initiation of zygotic gene activation occurs at
the transition from the initial cleavage step to the two-cell stage. This
process has been linked to a massive degradation of maternal mRNAs
remarkably, approximately 40% of the maternal RNA pool is lost during embryo
development from the one- to two-cell stage
(Bachvarova and De Leon, 1980
),
by mechanisms that are presently unknown.
Translational activation of masked maternal transcripts by addition of a
long poly(A) tail is also an important regulatory process triggered by
fertilization that is required for early embryonic development
(Wormington, 1994). Because
TTP and its family members can promote the deadenylation of ARE-containing
mRNAs (Lai et al., 1999
), it
is possible that a putative deadenylation function of
N-Zfp36l2 may
also affect the translation of proteins required for early embryonic
development.
After fertilization, the initial developmental program implements the
degradation of maternal proteins and maternal mRNAs. Studies in C.
elegans have shown that some invertebrate CCCH zinc finger proteins such
as PIE-1, POS-1, MEX-1, MEX-5 and MEX-6 are important for proper execution of
developmental programs (Mello et al.,
1996; Tabara et al.,
1999
; Guedes and Priess,
1997
; Schubert et al.,
2000
). In these models, the asymmetric segregation of CCCH
proteins between germ and somatic cells is important for the formation of the
so-called germ plasma and depends on the degradation of CCCH proteins in
somatic cells (DeRenzo et al.,
2003
). OMA-1 and OMA-2 have been likened to mammalian TTP-like
proteins; whereas single knockouts of their genes have no phenotype in C.
elegans, disruption of both leads to impairment of oocyte maturation and
female infertility (Detwiler et al.,
2001
). Interestingly, a single point mutation in OMA-1
(zu405) leads to an embryonic lethal phenotype because of increased
stability of the mutant protein (Lin,
2003
).
Although most of the C. elegans CCCH proteins do not exhibit the
spacing and other requirements that define the TTP family of tandem CCCH zinc
finger proteins in mammals, the amino acids that coordinate zinc are identical
(Detwiler et al., 2001).
However, none of the phenotypes described after disruption of these genes in
C. elegans approximates the two-cell arrest of embryonic development
of the
N-Zfp36l2 mouse. Because the phenotype we describe is only seen
when the
N-Zfp36l2 mutation originates with the females, our
findings implicate Zfp36l2 as a potential maternal effect gene.
Whether this phenotype is secondary to the continued presence of specific
mRNAs that fail to be degraded by
N-Zfp36l2, possibly resulting in
elevated levels of maternal proteins derived from these mRNAs, remains to be
determined.
Our results strongly suggest that the amino-terminus of Zfp36l2 plays a
critical biochemical role in early embryonic development, raising the
intriguing possibility that this sequence contains a potential functional
domain. Because the two previously characterized domains of Zfp36l2, the
tandem zinc finger RNA-binding domain (Lai
et al., 2000) and the carboxyl-terminal nuclear export sequence
(Phillips et al., 2002
) are
still present, it is possible that the amino-terminus of Zfp36l2 is involved
in regulating the interaction of the protein with other cellular proteins or
structures. This putative interaction might therefore represent an attractive
target for novel contraceptive strategies. The Zfp36l2 amino-terminus contains
a leucine-rich region that resembles a leucine-rich repeat present in the
Mater gene product (Tong et al.,
2000
), which, like Zfp36l2, is required for the progression of
mouse embryos beyond the two-cell stage.
By analogy with the mouse model, Zfp36l2 may also be involved in the
regulation of early embryonic development in humans. Abnormalities in its
expression or structure could be involved in some proportion of the 10% of
cases of human female infertility that are still without a known cause
(Greenhouse et al., 1998).
Recent resequencing of the gene encoding human ZFP36l2 in 72 humans
from diverse ethnic groups did not identify any gross splicing mutations that
might lead to amino terminal truncation of ZFP36l2 in humans
(Blackshear et al., 2003
).
Nonetheless, 9/23 human ESTs in GenBank on 11/20/03 that spanned the single
intron splice site contained retained intronic sequences, and the mRNAs from
which they were derived would probably encode the truncated protein. Future
resequencing studies will therefore focus on women with unexplained primary
infertility.
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ACKNOWLEDGMENTS |
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