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The mRNA-destabilizing protein Tristetraprolin targets
“meiosis arrester”Nppc mRNA in mammalian
preovulatory follicles
Guangyin Xi
a,1
, Lei An
a,1
, Wenjing Wang
a,1
, Jing Hao
a
, Qianying Yang
a
, Lizhu Ma
a
, Jinlun Lu
a
,
Yue Wang
a
, Wenjuan Wang
a
, Wei Zhao
a
, Juan Liu
a
, Mingyao Yang
a
, Xiaodong Wang
a
, Zhenni Zhang
a
,
Chao Zhang
a
, Meiqiang Chu
a
, Yuan Yue
a
, Fusheng Yao
a
, Meijia Zhang
a
, and Jianhui Tian
a,2
a
Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture and Rural Affairs, National Engineering Laboratory for
Animal Breeding, College of Animal Science and Technology, China Agricultural University, 100193 Beijing, P. R. China
Edited by R. Michael Roberts, University of Missouri, Columbia, MO, and approved April 13, 2021 (received for review August 31, 2020)
C-natriuretic peptide (CNP) and its receptor guanylyl cyclase, natri-
uretic peptide receptor 2 (NPR2), are key regulators of cyclic gua-
nosine monophosphate (cGMP) homeostasis. The CNP-NPR2-cGMP
signaling cascade plays an important role in the progression of
oocyte meiosis, which is essential for fertility in female mammals.
In preovulatory ovarian follicles, the luteinizing hormone (LH)-in-
duced decrease in CNP and its encoding messenger RNA (mRNA)
natriuretic peptide precursor C (Nppc) are a prerequisite for oocyte
meiotic resumption. However, it has never been determined how
LH decreases CNP/Nppc. In the present study, we identified that
tristetraprolin (TTP), also known as zinc finger protein 36 (ZFP36), a
ubiquitously expressed mRNA-destabilizing protein, is the critical
mechanism that underlies the LH-induced decrease in Nppc mRNA.
Zfp36 mRNA was transiently up-regulated in mural granulosa cells
(MGCs) in response to the LH surge. Loss- and gain-of-function anal-
yses indicated that TTP is required for Nppc mRNA degradation in
preovulatory MGCs by targeting the rare noncanonical AU-rich ele-
ment harbored in the Nppc 3′UTR. Moreover, MGC-specific knockout
of Zfp36, as well as lentivirus-mediated knockdown in vivo, impaired
the LH/hCG-induced Nppc mRNA decline and oocyte meiotic re-
sumption. Furthermore, we found that LH/hCG activates Zfp36/
TTP expression through the EGFR-ERK1/2–dependent pathway. Our
findings reveal a functional role of TTP-induced mRNA degradation, a
global posttranscriptional regulation mechanism, in orchestrating the
progression of oocyte meiosis. We also provided a mechanism for
understanding CNP-dependent cGMP homeostasis in diverse cellular
processes.
TTP/Zfp36
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CNP/Nppc
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mRNA degradation
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oocyte meiosis
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mural granulosa cells
Cyclic guanosine monophosphate (cGMP) is a ubiquitous sec-
ond messenger that regulates myriads functions, such as the
cell cycle, differentiation, apoptosis, and metabolism (1). During
recent decades, C-natriuretic peptide (CNP), encoded by natriuretic
peptide precursor C (Nppc), has emerged as a critical regulator of
cGMP homeostasis by activating its transmembrane guanylyl cyclase
receptor, natriuretic peptide receptor 2 (NPR2), which increases the
intracellular cGMP concentration (2, 3). Increasing evidence from
mouse models of genetic depletion or mutation of Nppc or Npr2
and related clinical assays have indicated that the CNP-NPR2-
cGMP signaling cascade is critical for the development and function
of the cardiovascular (4, 5), skeletal (6, 7), nervous (8, 9), and female
reproductive systems (10). In particular, CNP-NPR2-cGMP signal-
ing is the central regulator of the progression of oocyte meiosis (11),
the female germ cell division that is essential for haploid oocyte
formation.
In mammals, oocytes are arrested at the diplotene stage of meiosis
until the surge of luteinizing hormone (LH) restarts meiosis and
initiates the ovulatory process. In preovulatory follicles, LH-induced
CNP-NPR2-cGMP signaling inhibition is the most prominent
hallmark for meiotic resumption. Although LH also activates cGMP
hydrolysis (12) and decreases guanylyl cyclase activity (13, 14) and
NPR2 affinity for CNP in ovarian follicles (15), mouse models that
prevent cGMP hydrolysis or/and NPR2 inactivation can reinitiate
meiosis (12, 16), suggesting that these alterations are not indis-
pensable for LH-induced meiotic resumption. These facts suggest a
notable change upstream of the CNP-NPR2-cGMP signaling cas-
cade; indeed, remarkable decreases in CNP and Nppc messenger
RNA (mRNA) have been reported in mouse (14, 17–19), rat (13),
pig (20), and human (17) follicles, and the time course of the Nppc/
CNP decrease almost coincides with the resumption of meiosis
(14). Previous studies have established the critical role of low-level
CNP in decreasing cGMP and restarting meiotic progression. The
incubation of isolated cumulus-oocyte complexes (COCs) with
low-level CNP that recapitulates the follicular concentration after
LH administration resumes meiosis; however, high levels of CNP
maintain meiotic arrest (11, 20, 21). Thus, the LH-induced decrease
in CNP levels is thought to be important for fine-tuning meiotic
resumption (14, 22, 23). However, the mechanism underlying
decreased CNP is unknown and thus represents an unanswered
question in our understanding of the mechanism that controls
Significance
The fertility of female mammals depends upon the well-
orchestrated progression of oocyte meiosis, the female germ
cell division that is essential for haploid oocyte formation. In
preovulatory follicles, how LH induces a decrease in CNP and its
encoding mRNA Nppc, a prerequisite for oocyte meiotic re-
sumption, remains an outstanding question to understand the
mechanism that controls oocyte meiosis. The present study
shows that TTP, an mRNA-destabilizing protein, is one of the
regulatory components responsible for the LH-induced rapid
decrease in Nppc mRNA and oocyte meiotic resumption. This
finding not only highlights the importance of posttranscrip-
tional regulation in the follicle periphery to fine-tune oocyte
meiosis but also provides an insight into CNP-dependent cGMP
homeostasis in other physiological systems.
Author contributions: G.X., L.A., M.Z., and J.T. designed research; G.X., Wenjing Wang,
J.H., Q.Y., L.M., J. Lu, Y.W., Wenjuan Wang, W.Z., J. Liu, M.Y., X.W., Z.Z., C.Z., M.C., Y.Y.,
and F.Y. performed research; G.X., L.A., and J.T. analyzed data; G.X., L.A., and J.T. wrote
the paper; and Wenjing Wang, X.W., Z.Z., and C.Z. were responsible for animal care and
management.
The authors declare no competing interest.
This article is a PNAS Direct Submission.
Published under the PNAS license.
1
G.X., L.A., and Wenjing Wang contributed equally to this work.
2
To whom correspondence may be addressed. Email: tianjh@cau.edu.cn.
This article contains supporting information online at https://www.pnas.org/lookup/suppl/
doi:10.1073/pnas.2018345118/-/DCSupplemental.
Published May 24, 2021.
PNAS 2021 Vol. 118 No. 22 e2018345118 https://doi.org/10.1073/pnas.2018345118
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oocyte meiosis (22). Given that the turnover of CNP is very rapid
(24), a decrease in Nppc mRNA could rapidly decrease the amount
of CNP (14). In addition, the LH-induced decrease in CNP is in-
dependent of the natriuretic peptide clearance receptor NPR3
(18). Therefore, we focused on the possible mechanism underlying
the LH-induced decrease in Nppc mRNA.
By reanalyzing a previously published transcriptome that pre-
sented rapid effects of LH on gene expression in the mural gran-
ulosa cells (MGCs) of mouse preovulatory follicles (25), we noticed
that zinc finger protein 36 homolog (Zfp36) that encodes triste-
traprolin (TTP) showed an expression pattern that was inversely
related to the LH-induced decrease in Nppc mRNA. TTP is an
RNA-binding protein that regulates mRNA stability by binding
to AU-rich elements (AREs) in the 3′untranslated regions (UTRs)
of target mRNAs and destabilizes mRNA via poly(A) tail removal or
deadenylation (26, 27). Furthermore, we noticed that Nppc mRNA
is a representative ARE-containing transcript. Based on these find-
ings, we hypothesized that TTP might target and degrade Nppc
mRNA and thus could be the key factor underlying the LH-induced
decrease in Nppc mRNA.
In the present study, we identified that TTP-induced Nppc mRNA
degradation is a regulatory component of meiotic progression in
preovulatory follicles. Notably, we reported that a rare noncanonical
ARE motif mediated the TTP-induced destabilization of Nppc
mRNA. Additionally, we found that LH transiently activated Zfp36
via the epidermal growth factor receptor (EGFR)-extracellular
regulated kinase (ERK)1/2-dependent pathway. Thus, focusing on
the upstream part of the signaling cascade, the present study de-
termined a mechanism by which CNP-NPR2-cGMP signaling is
inactivated during meiotic resumption and ovulation processes and
provides insight into CNP-dependent cGMP homeostasis in other
physiological systems.
Results
Zfp36/TTP Is Transiently Up-Regulated in Response to the LH/hCG
Surge and Targets the 3′UTR of the Nppc mRNA. To explore the mech-
anism underlying the LH-induced decrease in Nppc mRNA, we tested
the previous notion that theLH-induceddecreaseinNppc mRNA is a
universal switch for CNP-NPR2-cGMP signaling inhibition. Female
Institute of Cancer Research (ICR) mice were administered with
human chorionic gonadotropin (hCG) to mimic the endogenous
LH surge (14, 17–19), and then we detected the Nppc mRNA levels
in MGCs and ovaries at various time points. We found that the Nppc
mRNA levels decreased immediately in MGCs and ovaries to less
than 50% by 2 h post-hCG and further declined to 10% by 5 h post-
hCG (Fig. 1Aand SI Appendix,Fig.S1A). Our findings, together
with previous results obtained from mice with a different genetic
background (17–19) and from pigs (20), indicated that the rapid
decrease in the Nppc mRNA levels is a hallmark event common to
different genotypes and species.
Next, to identify the potential factor controlling Nppc expression
levels, we reanalyzed previously published transcriptome data that
identified early response genes in MGCs at 1 h post-hCG. We
noticed that the expression of the Zfp36 gene, whose product, TTP,
is responsible for degrading mRNAs that have AU-rich 3′UTRs,
was up-regulated by 3.3-fold within 1 h after LH/hCG adminis-
tration (25). Given that the up-regulation of Zfp36 coincided with
thedecreaseinNppc levels and that the Nppc 3′UTR is consid-
erably AU-rich, we hypothesized that Zfp36/TTP might be impli-
cated in the decrease in Nppc mRNA. To this end, we isolated
MGCs from preovulatory follicles at various times after injection
with hCG and detected the expression dynamics of Zfp36 using
real-time reverse transcription quantitative PCR (RT-qPCR). In
agreement with the results of previous transcriptome data (25),
we found a transient and significant increase in Zfp36 mRNA, by
approximately sevenfold and tenfold at 1 and 2 h post-hCG,
respectively, in MGCs and ovaries, followed by a rapid decline to
the basal level (Fig. 1Band SI Appendix, Fig. S1B). In addition,
the transient activation of Zfp36 mRNA in MGCs was accom-
panied by a transiently increased abundance of TTP at 2 to 4 h in
response to hCG injection (Fig. 1C). Collectively, these results
suggested that Zfp36/TTP is activated in response to hCG during
a specific time window in preovulatory follicles. Moreover, by
analyzing the previously published transcriptome data of ovarian
MGCs pre- and post-LH/hCG administration from different
species (28–31), we found that activation of Zfp36/TTP in pre-
ovulatory MGCs in response to the LH/hCG surge appears to be
common in these species (SI Appendix, Fig. S2).
Next, immunohistochemical detection indicated that the LH/
hCG-induced TTP is mainly localized in MGCs and cumulus
cells (Fig. 1D), which overlaps with the distribution of Nppc mRNA
in the preovulatory follicle (11). The spatiotemporally associated
patterns, together with the negatively correlated expression dynamics
between Nppc and Zfp36, suggested that Zfp36-encoded TTP might
be an inducible repressor of Nppc mRNA in response to the LH/
hCG surge. Therefore, we determined whether TTP could target the
3′UTR of Nppc mRNA. Given that the high level of TTP protein
appeared by 2 and 3 h post-hCG and Nppc mRNA was significantly
decreased at 2 h post-hCG, we isolated preovulatory MGCs at 2 and
3 h post-hCG and used RNA immunoprecipitation (RIP) to test
the binding of TTP to the Nppc mRNA. In a TTP-specific pulldown,
Nppc mRNA, but not nontargeted beta actin (Actb) mRNA, was
significantly enriched compared with the Immunoglobulin G (IgG)
controls (Fig. 1 Eand Fand SI Appendix,Fig.S3A). The specificity of
the interaction was confirmed by sequencing the RIP-qPCR amplicon.
Having confirmed the binding of TTP to the Nppc 3′UTR, we
next examined whether the Nppc 3′UTR can mediate the TTP-
induced mRNA destabilization. We generated luciferase constructs
containing the full-length Nppc 3′UTR, the tumor necrosis factor
(Tnf)3′UTR, or the Actb 3′UTR. The Tnf 3′UTR was used as the
targeted control because Tnf mRNA is a well-accepted target of
TTP (32), whereas the Actb 3′UTR was used as the nontargeted
control (Fig. 1G). Humanembryonic kidney (HEK293) cells were
cotransfected with the psiCHECK2 luciferase vector containing
Nppc 3′UTR or the control 3′UTRs, together with a Zfp36 over-
expression vector or empty vector. Compared with cells transfected
with the empty vector, overexpression of Zfp36 significantly reduced
the luciferase activity of the reporter containing the full-length Nppc
3′UTR to the level comparable to that of the reporter containing
full-length Tnf 3′UTR but did not alter the luciferase activity of the
reporter containing full-length Actb 3′UTR (Fig. 1G). To further
determine the role of Nppc 3′UTR in mediating TTP-induced de-
stabilization of target mRNA, we measured the luciferase mRNA
stability after blocking transcription using actinomycin D (ActD),
which is frequently used to detect mRNA stability by inhibiting
de novo mRNA synthesis, thus facilitating the measurement of the
remaining mRNA (33–35). The results showed that TTP over-
expression significantly accelerated the destabilization of luciferase
mRNA containing the full-length Nppc 3′UTR to approximately a
quarter of that of the control vector after 3 h (Fig. 1H), which is
comparable to the rate of decline of Nppc mRNA in MGCs fol-
lowing the LH/hCG surge. The reliability of the mRNA stability
assay was supported by the fact that TTP overexpression significantly
degraded luciferase mRNA containing the Tnf 3′UTR but not that
containing the Actb 3′UTR (SI Appendix,Fig.S3Band C). These
results indicated that TTP can destabilize the Nppc mRNA via its
3′UTR. Similar results were also obtained using the bovine NPPC
3′UTR (SI Appendix,Fig.S3Dand E). Collectively, these experi-
ments demonstrated that Zfp36/TTP can be activated in preovulatory
follicles in response to the LH/hCG surge and then targets the Nppc
3′UTR, which might contribute to hCG-induced destabilization of
Nppc mRNA.
TTP Targets the Noncanonical ARE Motif in the Nppc 3′UTR. Lucif-
erase assays demonstrated that the full-length Nppc 3′UTR
contains functional repressive motifs that respond to TTP. AREs
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are essential for recruiting TTP to the 3′UTR of target mRNA;
therefore, we attempted to determine the potent ARE(s) that
direct TTP-induced Nppc mRNA degradation. As expected, we
found four scattered AUUUA motifs, the canonical ARE sequence,
locatedintheNppc 3′UTR (Fig. 1Iand SI Appendix,Fig.S4A). We
then constructed a range of luciferase reporters that contained dif-
ferent mutant ARE motifs (Fig. 2A); however, none of the detected
canonical ARE motifs were essential for mediating TTP-induced
repression of any reporters (Fig. 2B). To exclude possible synergis-
tic or additive effects of these canonical ARE motifs, we further
constructed a reporter containing the 3′UTR in which all putative
ARE motifs were mutated (Fig. 2A). Unexpectedly, in line with the
results of each single mutation, the tetrad mutation also led to a
significant decrease in luciferase activity (Fig. 2B), indicating that the
reporter still retains significant AU richness in the 3′UTR, and
implying that other unidentified motif(s) might underlie the TTP-
induced degradation of Nppc mRNA. This unexpected observation
was reminiscent of the presence of noncanonical, but AU-rich
3′UTR motifs, also known as non-AUUUA AREs. Previous stud-
ies have shown that canonical ARE motifs are dispensable for rapid
mRNA degradation; however, noncanonical ARE motifs can func-
tionally mediate the rapid destabilization of target mRNA (36, 37).
Thus, we screened the Nppc 3′UTR and characterized multiple non-
AUUUA but AU-rich motifs. We hypothesized that the Nppc
3′UTR contained repressive noncanonical AREs that could medi-
ate TTP-induced destabilization of Nppc mRNA.
To confirm the presence and location of repressive noncanonical
ARE motifs, we next constructed a range of luciferase reporters
that contained different truncated fragments from the proximal to
distal portions of the Nppc 3′UTR (Fig. 2C). Similar to the results
using the full-length Nppc 3′UTR, overexpression of TTP signifi-
cantly decreased the luciferase activity of the reporter constructs
containing longer proximal regions (1 to 365, 1 to 275), even
though the truncated region contained canonical ARE sites (Fig. 2D).
However, when shorter fragments (1 to 160, 1 to 97) were retained,
the luciferase activity of these reporters were not repressed by TTP
overexpression, which was comparable to the full-length Nppc
3′UTR deletion reporter construct (Fig. 2D). This result suggested
that the uncharacterized repressive motifs were located in the 160 to
275 region of the Nppc 3′UTR. Additionally, we observed an in-
creased trend of luciferase activity when distal fragments (down-
stream from 275) were deleted (Fig. 2D), implying that weak
repressive AU-rich motifs were located in this region.
Next, we performed a more detailed analysis of the 160 to 275
region of the Nppc 3′UTR and confirmed the location of the non-
canonical repressive ARE, which was characterized by UU/UA di-
nucleotide clusters (231 to 257) (Fig. 2E). More importantly, we
provided direct evidence that this non-AUUUA ARE motif could
functionally mediate TTP-induced destabilization of target mRNA
(Fig. 2F). Interestingly, by screening the Nppc 3′UTR of different
mammalian species, we found that the UU/UA dinucleotide clusters
are highly conserved (SI Appendix,Fig.S4B). Collectively, these
Fig. 1. TTP is up-regulated in response to the LH/hCG surge and targets the Nppc 3′UTR. (Aand B) RT-qPCR detection of the expression dynamics of Nppc (A)
and Zfp36 (B) mRNA in preovulatory MGCs in response to hCG administration. (C) Western blotting analysis for TTP in preovulatory MGCs at different time
points after hCG administration. The upper and lower bands indicate phosphorylated (inactive) and unphosphorylated (active) forms of TTP, respectively. (D)
Immunohistochemical staining of TTP in preovulatory follicles before and after hCG administration. (Right) Higher magnification of boxed regions. (Scale bar,
100 μm.) (E) RIP analysis for the direct binding of TTP to the Nppc 3′UTR. The RIP sample was subjected to RT-PCR, and the amplified products were visualized
using agarose gel electrophoresis. (F) Quantitative detection of the interaction between TTP and Nppc 3′UTR by RIP–RT-qPCR. (G) Schematic diagram of the
luciferase constructs containing full-length Nppc 3′UTR, Tnf 3′UTR (targeted control), or Actb 3′UTR (nontargeted control). Relative luciferase activity of
different luciferase vectors, which were cotransfected with a Zfp36 overexpression vector or empty vector in HEK293 cells. (H) Measurement of the stability of
the luciferase mRNA that contained the Nppc 3′UTR. The luciferase vector was cotransfected with the Zfp36 expression vector or empty vectors in HEK293
cells, which were treated with ActD to terminate transcription. Total RNAs were prepared at the indicated time points to detect the remaining luciferase
mRNA levels after ActD treatment. (I) Schematic diagram depicting the location of canonical AU-rich elements (AREs, presented as red triangles) in the Nppc 3′
UTR. All data are presented as the mean ±SEM of three independent experiments. *P<0.05, **P<0.01, ***P<0.001. In A,B, and C, * indicates a significant
difference compared with data at 0 h post-hCG.
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The mRNA-destabilizing protein Tristetraprolin targets “meiosis arrester”Nppc mRNA in
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observations showed that TTP can bind to the Nppc 3′UTR via
the noncanonical non-AUUUA ARE motif to destabilize its
target mRNA.
Nppc mRNA in Preovulatory MGCs Is Degraded by Up-Regulated TTP.
Having established the correlation between TTP and the non-
canonical ARE motif in the Nppc 3′UTR, we next aimed to test
whether TTP can regulate Nppc expression in preovulatory MGCs.
We used a well-established in vitro model using MGCs isolated
from preovulatory follicles, which maintain high levels of Nppc
mRNA via supplementation with estrogen (18, 38). Given that LH
failed to decrease Nppc mRNA in vitro because of the dilution of
LH-induced epidermal growth factor (EGF)-like peptides in the
culture medium (18), we recapitulated the LH-induced rapid de-
crease of Nppc mRNA by modulating EGFR signaling, as reported
in previous studies (18, 39). In addition, we found that the ad-
ministration of the EGFR kinase inhibitor, AG1478, completely
blocked the hCG-induced rapid up-regulation of TTP (Fig. 3A),
implying that the activation and function of TTP largely depends
on LH/hCG-activated EGFR signaling. Moreover, our results
showed that Zfp36 was rapidly and transiently up-regulated and
that Nppc mRNA was decreased in cultured mouse MGCs in
response to EGF supplementation (Fig. 3B), thus recapitulating
the LH/hCG-induced changes in ovaries and MGCs (Fig. 1 A
and Band SI Appendix, Fig. S1 Aand B). These findings support
the feasibility of the model to study the role of TTP in controlling
endogenous Nppc expression. Furthermore, the in vitro model has
the unique advantages of MGC-specific genetic manipulation of
Zfp36 and efficient transcriptional termination to assess mRNA
stability.
Using the in vitro model, we next determined if TTP up-regulation
was essential and sufficient to decrease Nppc mRNA levels via loss-
and gain-of-function studies. As expected, when the expression of
Zfp36/TTP was reduced to basal level via small interfering (siRNA)-
mediated knockdown (Fig. 3Cand SI Appendix,Fig.S5A), EGF
supplementation failed to induce the rapid decrease in Nppc mRNA
but maintained the steady-state expression levels in preovulatory
MGCs, which contrasted with the groups transfected with the blank-
control and negative-control siRNAs (Fig. 3C). In contrast, over-
expression of TTP was sufficient to maintain Nppc mRNA at low
levels in preovulatory MGCs (Fig. 3Dand SI Appendix,Fig.S5B).
This result was consistent with the observation from the in vivo
Fig. 2. TTP targets the noncanonical ARE motifs in the Nppc 3′UTR. (A) Schematic diagram of the luciferase reporters that contained single or all mutant
canonical ARE motifs in the Nppc 3′UTR. (B) Relative luciferase activity of luciferase reporters containing different mutant ARE motifs, which were
cotransfected with the Zfp36 overexpression vector (pCAGGS-Zfp36) or empty vector (pCAGGS) in HEK293 cells. The reporter containing the Actb 3′UTR was
detected as a nontargeted control. (C) Schematic diagram of the luciferase reporters that contained different truncated regions from the proximal to distal
portions of the Nppc 3′UTR. Canonical ARE sites located in the Nppc 3′UTR are indicated by blue arrows. (D) Relative luciferase activity of luciferase reporters
containing different Nppc 3′UTR truncations, which were cotransfected with the Zfp36 overexpression vector or empty vectors in HEK293 cells. The reporters
that contained full-length or deleted Nppc 3′UTRs were used as positive or negative control, respectively. (E) The sequence of the Nppc 3′UTR containing
putative noncanonical ARE motifs (highlighted in red). (F) Relative luciferase activity of luciferase reporters containing further truncated Nppc 3′UTR
fragments. All data are presented as the mean ±SEM of at least three independent experiments. **P<0.01, ***P<0.001.
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experiment that down-regulated Zfp36 was associated with failure
to decrease the Nppc mRNA level (Fig. 4C). Next, we attempted
to confirm that TTP-induced rapid decrease in Nppc mRNA is
dependent on mRNA destabilization. We found that Nppc mRNA
maintained a relatively stable level until 4 h after ActD treatment;
however, Zfp36 overexpression significantly accelerated the desta-
bilization of Nppc mRNA, similar to that of the targeted control Tnf
mRNA (Fig. 3Eand SI Appendix,Fig.S5C). By contrast, accelerated
destabilization of nontargeted control Actb mRNA was not ob-
served following Zfp36 overexpression (SI Appendix, Fig. S5D).
Using cultured bovine MGCs as the model, our results also
suggest that the physiological role of EGF-induced TTP in degrading
NPPC mRNA might be common to different species (SI Appendix,
Fig. S5 E–I).
Finally, to determine the physiological consequences of TTP
deficiency in vivo, we crossed Zfp36
flox/flox
mice and Fshr-Cre mice
to knock out Zfp36 specifically in MGCs. We detected that in the
resulting Zfp36
gc−/−
mice, Zfp36 expression was significantly de-
creased in preovulatory MGCs before and after hCG administra-
tion (Fig. 3F). The reduction in TTP protein levels in MGCs was
Fig. 3. TTP degrades endogenous Nppc mRNA in preovulatory MGCs. (A) Western blotting analysis for TTP, as well as phosphorylated and total EGFR, in
preovulatory MGCs in response to administration of hCG and AG1478, a specific EGFR inhibitor. MGCs were isolated from in vitro cultured preovulatory
follicles. (B) Expression dynamics of Nppc and Zfp36 in in vitro cultured MGCs in response to EGF administration. * indicates significant difference compared
with data at 0 h post-hCG. (C) Effect of Zfp36 knockdown on the EGF-induced decrease in Nppc mRNA in in vitro cultured MGCs. Total RNAs were prepared at
the indicated time points after EGF administration to detect Zfp36 (Upper) and Nppc (Lower) mRNA levels using RT-qPCR. (D) Effect of Zfp36 overexpression
on the Nppc mRNA levels in in vitro cultured MGCs. Nppc expression was maintained at a high level before overexpressing Zfp36. Total RNAs were prepared
at the indicated time points after transfection to detect Zfp36 (Upper) and Nppc (Lower) mRNA levels. (E) Measurement of the stability of Nppc mRNA in
MGCs following transfection with the Zfp36 overexpression vector (pCAGGS-Zfp36) or empty vector. Total RNAs were prepared at the indicated time points to
detect the remaining Nppc mRNA levels after ActD treatment. (F–H) The detection of Zfp36 knockout efficiency in MGCs at 2 h post-hCG, at which point Zfp36
should be fully activated in response to hCG administration. The expression levels of Zfp36/TTP in MGCs were detected at 0 h and 2 h post-hCG using RT-qPCR
(F), immunohistochemistry (G), and Western blotting (H). (G, Lower) Higher magnification of boxed regions. (Scale bar, 100 μm.) (I) Effects of MGC-specific
depletion of Zfp36 on Nppc mRNA levels at 0 h and 2 h post-hCG. (Jand K) Evaluation of oocyte meiotic resumption scored as GVBD percentage at 6 h (J)and
9h(K) post-hCG. Representative images show the morphology of oocytes isolated from preovulatory follicles of wild-type (WT) and Zfp36
gc−/−
mice (Right),
five to eight mice were used at each time point. (Scale bar, 100 μm.) Data show the means ±SEMs of three independent experiments. *P<0.05, **P<0.01,
***P<0.001.
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The mRNA-destabilizing protein Tristetraprolin targets “meiosis arrester”Nppc mRNA in
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further confirmed by immunohistochemical and Western blot-
ting detection at 2 h post-hCG (Fig. 3 Gand H), at which point
the high-level expression of Zfp36 should appear in preovulatory
follicles (Fig. 1Band SI Appendix,Fig.S1B). Incomplete up-
regulation of TTP/Zfp36, in turn, impaired the hCG-induced rapid
decrease in Nppc mRNA (Fig. 3I). When oocyte meiotic resump-
tion, scored as percent germinal vesicle breakdown (GVBD), was
evaluated, Zfp36
gc−/−
mice showed impaired meiotic resumption
compared with that in the wild-type controls (Fig. 3 Jand K). These
findings were also supported by results of lentivirus-mediated short
hairpin RNA (shRNA) interference of Zfp36 in preovulatory
follicles (SI Appendix, Fig. S6 A–F). Collectively, our results from
in vitro and in vivo models demonstrated that TTP-dependent
mRNA degradation underlies the rapid decrease in Nppc mRNA
in preovulatory follicles, which plays a functional role in fine-tuning
the progression of oocyte meiosis.
LH/hCG Activates Zfp36 through the EGFR-ERK1/2–Dependent Pathway.
Having confirmed the functional role of TTP-mediated mRNA
degradation of Nppc mRNA in preovulatory MGCs, we next
attempted to understand the mechanism underlying the transient
up-regulation of Zfp36 in response to the LH/hCG surge. Multiple
Fig. 4. LH/hCG activates Zfp36/TTP through the EGFR-ERK1/2–dependent pathway. (A) Effect of the EGFR-ERK1/2 pathway on the expression of Zfp36 and its
associated transcription factors. mRNA expression levels of Zfp36,Nppc,Elk1,andEgr1 in in vitro cultured MGCs that were treated with either EGF or AG1478 (a
specific EGFR inhibitor), U0126 (a specific ERK inhibitor), or their various combinations were detected. AG1478 or U0126 was administered 1 h before EGF sup-
plementation. (B) Western blotting analysis for TTP, EGR1, and phosphorylated and total ERK1/2 or ELK1 in in vitro cultured MGCs that were treated with either
EGF, AG1478, U0126, or their various combinations for 20 min and 1 h. (C) Expression levels of Zfp36,Nppc,Elk1,andEgr1 mRNA in isolated preovulatory MGCs
from mice that were intraperitoneally administrated with hCG, with or without PD0325901 (a specific ERK inhibitor). MGCs were collected at 3 h after hCG
administration, and PD0325901 was injected at 4 h before hCG. (D) Western blotting analysis for TTP, EGR1, and phosphorylated and total EGFR, ERK1/2, or ELK1
in MGCs from in vitro cultured preovulatory follicles that were treated with either hCG, AG1478, U0126, or their various combinations. (E) Western blotting
analysis for TTP, EGR1, and phosphorylated and total EGFR, ERK1/2, or ELK1 in isolated preovulatory MGCs from mice that were injected intraperitoneally with
either PD0325901, AG1478, or their various combinations at 4 h before hCG administration. All data are presented as the mean ±SEM of at least three inde-
pendent expe riments. *P<0.05, **P<0.01, ***P<0.001. (F) Working model of the signaling pathways that regulate the CNP-NPR2-cGMP signaling cascade and
meiotic resumption in preovulatory follicles. Pathways reported previously and those identified in the present study are indicated in black and blue, respectively.
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https://doi.org/10.1073/pnas.2018345118 The mRNA-destabilizing protein Tristetraprolin targets “meiosis arrester”Nppc mRNA in
mammalian preovulatory follicles
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molecular and cellular evidence indicated that Zfp36 transcrip-
tion is induced by E26 transformation-specific (ETS) transcrip-
tion factor ELK1 (ELK-1) and early growth response 1 (EGR-1)
in the EGFR-ERK1/2–dependent pathway in somatic cells (40–43).
Given that LH-activated EGFR-ERK1/2 signaling in preovulatory
follicles is essential to initiate meiotic resumption (22, 44), we hy-
pothesized that the rapid induction of Zfp36/TTP in response to
LH/hCG might depend on the EGFR-ERK1/2 pathway. Therefore,
we examined the hypothesis using both in vitro and in vivo models.
In in vitro cultured MGCs, accompanied by the rapidly increased
Zfp36 and decreased Nppc mRNA levels, we observed that the
transcriptional activation of Egr1, but not Elk1, responded rapidly to
EGF stimulation through the EGFR-ERK1/2–dependent pathway
(Fig. 4A). Furthermore, Western blotting and immunofluorescence
assays showed that ERK1/2, as well as transcription factor ELK1,
could be phosphorylated (activated) in response to EGF stimulation
within 20 min (Fig. 4Band SI Appendix,Fig.S7Aand B). Subse-
quently, a marked increase in EGR1 was observed after 1 h (Fig. 4B
and SI Appendix,Fig.S7C), along with significantly increased TTP
levels (Fig. 4B). All these changes in response to EGFR signaling
activation could be attenuated by AG1478 (an EGFR signaling-
specific inhibitor) or U0126 (an ERK1/2-specific inhibitor) (Fig. 4 A
and B). Moreover, to further confirm the dependence of TTP in-
duction on the ERK1/2 pathway, mice were intraperitoneally in-
jected with the ERK1/2 inhibitor 4 h before hCG administration.
Consistent with the results from the in vitro model, in vivo inhibition
of the ERK1/2 pathway significantly blocked the induction of Egr1
and Zfp36 expression, and the decrease in Nppc mRNA levels
(Fig. 4C). Thus, the results of in vitro and in vivo assays indicated
that the ERK1/2 pathway is essential for rapidly activating the
transcriptional induction system of Zfp36.
Given that EGFR is not the only mediator of LH/hCG-in-
duced ERK1/2 activation (45–47), we next examined if EGFR
was indispensable in mediating the LH/hCG-induced activation
of the ERK1/2 pathway and downstream transcriptional induction
system of Zfp36. To this end, isolated mouse preovulatory follicles
were cultured and treated with hCG, which resulted in the hCG-
induced activation/expression of the ERK1/2 pathway, as well as
TTP (Fig. 4D). Additionally, the changes in transcription factor
levels could be completely reversed by blocking EGFR signaling
(Fig. 4D). ERK inhibition did not attenuate EGFR phosphory-
lation/activation (Fig. 4D). Notably, EGFR inhibition and ERK
inhibition, alone or in combination, showed a similar effect of
blocking hCG-induced TTP expression (Fig. 4D), suggesting that
the ERK1/2 pathway acts downstream of EGFR signaling. These
findings were further confirmed using in vivo models (Fig. 4E).
Collectively, these results demonstrated that the preovulatory LH
surge induces transient expression of Zfp36/TTP and its transcrip-
tional induction system via the EGFR-ERK1/2 signaling pathway.
Discussion
The progression of gonadotropin-controlled oocyte meiosis is
tightly regulated by the activity of the CNP-NPR2-cGMP signaling
cascade. Before the LH surge, CNP-NPR2-cGMP signaling main-
tains the active state. The “arrester,”CNP, is primarily synthesized
by MGCs and binds to NPR2 throughout the follicle to stimulate
cGMP production, thus maintaining meiotic arrest. Following the
LH surge, CNP-NPR2-cGMP signaling inhibition occurs consecu-
tively on multiple levels; however, it remains unclear what mecha-
nism maintains the inhibition of the cascade, achieving sustained
cGMP at low levels before and during meiotic resumption. Earlier
studies proposed that the LH-induced rapid dephosphorylation
(inactivation) of NPR2 guanylyl cyclase might contribute to
CNP-NPR2-cGMP signaling inhibition, thus causing the rapid
decrease in cGMP. However, further studies have indicated that
the NPR2 dephosphorylation-dependent rapid decrease in cGMP (13,
14) is dispensable for LH-induced meiotic resumption because the
mouse model that prevents NPR2 dephosphorylation (inactivation)
only attenuated part of the cGMP decrease, which resulted in
delayed, but not failed, meiotic resumption (16). Similarly, LH-
induced phosphodiesterase 5 (PDE5), which directly hydrolyzes
cGMP, only partially contributes to the rapid cGMP decrease
and does not affect the timing of meiotic resumption. More im-
portantly, mice carrying both active NPR2 and mutated PDE5
failed to completely block cGMP decrease (12). Therefore, these
results are reminiscent of upstream changes to the signaling cascade
(i.e., the LH/hCG-induced decrease in follicular ligand CNP and its
encoding mRNA Nppc), which was documented as an important
fail-safe mechanism that may ensure LH-induced CNP-NPR2-
cGMP signaling inhibition and meiotic resumption (22).
Our data further emphasized the importance of ovarian follicular
somatic cells in regulating cGMP homeostasis. To maintain cGMP at
high levels and sustain meiotic arrest, somatic cells not only produce
the ligand (CNP) that activates its guanylyl cyclase (NPR2) (11) but
also provide guanylyl substrate for de novo synthesis of cGMP (48).
After the LH surge, cGMP begins to decrease rapidly in somatic
cells via the synergistic effect of decreased activity and affinity of
NPR2 guanylyl cyclase (14–16), direct hydrolysis of cGMP (12),
and decreased concentrations of CNP (17–19). Functional redun-
dancy also suggests that the timing of meiotic resumption and ovu-
lation is critical, and multiple pathways might have evolved as a fail-
safe system to ensure LH-induced meiotic resumption. Compared
with the rapid decrease of NPR2 activity and activated hydrolysis of
cGMP that occurs within 30 min, the decrease of CNP is a relatively
slow event (50% decrease within ∼2 h and further decrease until the
nuclear envelope is completely broken down) but is concurrent with
the progression of meiotic resumption (14). Thus, the decrease of
CNP/Nppc in the preovulatory follicle was thought to potentially
contribute to driving meiotic resumption, and the mechanism un-
derlying the decrease was considered as an important and long-
standing unanswered question (14, 22). Our study supported the
idea that the delayed decrease in Nppc mRNA could impair the
progression of meiotic resumption. These results, together with
previous works that focused on NPR2 inactivation and cGMP hy-
drolysis (12, 14, 16), allow us to presume that NPR2 inactivation and
cGMP hydrolysis may preferentially contribute to the rapid initiation
of meiotic resumption, while Nppc mRNA decline is primarily re-
sponsible for the later meiotic progression.
In the present study, we revealed that the preovulatory LH surge
can stimulate Zfp36 expression via activation of ELK-1 and EGR-1
transcription factors through the EGFR-ERK1/2 signaling path-
way. Up-regulated TTP subsequently targets noncanonical ARE
motifs in the 3′UTR of the Nppc mRNA, which induces its rapid
degradation (Fig. 4F). The turnover of CNP is rapid (24), and the
LH-induced decrease in the CNP peptide is independent of the na-
triuretic peptide clearance receptor NPR3 (18); therefore, the rapid
degradation of Nppc mRNA would lead to a decrease in CNP
peptide levels, thus maintaining CNP-NPR2-cGMP signaling inhibi-
tion during meiotic resumption.
We provided the functional link between TTP-mediated mRNA
degradation and oocyte meiotic progression. The involvement of
mRNA degradation-mediated posttranscriptional mechanism in
regulating CNP-NPR2-cGMP signaling has never been reported.
Furthermore, our results highlight the role of the rare noncanonical
AU-rich element in this process. Among the ARE-binding proteins
that regulate posttranscriptional mechanisms, TTP has the most
specific recognition and binding ARE sequences (i.e., canonical
AUUUA motifs) (26, 27). However, non-AUUUA, but AU-rich
motifs, can also destabilize target mRNA (36, 37, 49). The UU/UA
dinucleotide clusters observed in the Nppc 3′UTR, although gener-
ally rare in vertebrate genomes, were shown to correlate positively
with TTP-induced mRNA destabilization (49). Our results, together
with those of previous studies, supported the idea that noncanonical
ARE motifs efficiently mediate the mRNA-destabilizing function.
The present study identified that TTP-induced mRNA degrada-
tion, a global posttranscriptional regulatory mechanism, acts as a
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The mRNA-destabilizing protein Tristetraprolin targets “meiosis arrester”Nppc mRNA in
mammalian preovulatory follicles
https://doi.org/10.1073/pnas.2018345118
DEVELOPMENTAL
BIOLOGY
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regulatory component of orchestrating the signals for oocyte meiotic
maturation. We report that Nppc mRNA is the functional target of
TTP, via rare noncanonical ARE motifs. This finding might shed
light on the mechanism of CNP-dependent cGMP homeostasis in
regulating other physiological processes, considering the significant
regulatory role of CNP-NPR2-cGMP signaling in the development
and function of the cardiovascular, skeletal, and nervous systems.
Materials and Methods
Animal Studies and Ethical Approval. All female ICR mice used in this study were
from Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China).
Fshr-Cre mice and loxP-flanked Zfp36 mice (Zfp36
flox/flox
) were gifts from
Dr. Louis Dubeau (University of Southern California, Los Angeles, CA) and Dr. Perry
J. Blackshear (National Institute of Environmental Health Sciences, NIH), re-
spectively. All mice were maintained in a climate-controlled room on a 12 h
light/darkness cycle and allowed food and water ad libitum. The China Ag-
ricultural University Institutional Animal Care and Use Committee approved
this study, which was performed in accordance with the committee’s
guidelines. All efforts were made to minimize animal suffering.
Cell culture, isolation, and culture of follicles, Western blotting, immuno-
fluorescence and histologic analysis, RIP are described in SI Appendix,Materials
and Methods.
Data Availability. All study data are included in the article and/or SI Appendix.
ACKNOWLEDGMENTS. We thank Dr. Louis Dubeau (University of Southern
California) and Dr. Su Youqiang (Nanjing Medical University) for providing the
Fshr-Cre mice and breeding schemes. We also thank Dr. Perry J. Blackshear (Na-
tional Institute of Environmental Health Sciences, NIH) and Gu Ling (Nanjing
Agricultural University) for providing the Zfp36
flox/flox
mice and breeding
schemes. We thank Dr. Wang Chao (China Agricultural University) for his helpful
suggestions and for providing the lentiviral vectors. This work was supported by
grants from the National Natural Science Foundation of China (Grants 31972573
and 31672426), the National Key R&D Program (Grants 2017YFD0501901 and
2017YFD0501905), the Fundamental Research Funds for the Central Universities
(Grant 2020TC004), and the Beijing Innovation Consortium of Agriculture
Research System.
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