MBoC | ARTICLE

The RNA-binding protein ELAVL1/HuR is

essential for mouse spermatogenesis, acting
both at meiotic and postmeiotic stages
Mai Nguyen Chia, Jacques Auriola, Bernard Jégoub, Dimitris L. Kontoyiannisc, James M.A. Turnerd,
Dirk G. de Rooije, and Dominique Morelloa
a
CBD, UMR5547, IFR 109, Université Paul Sabatier, 31062 Toulouse Cedex, France; bINSERM U625, GERHM, Institut
Fédératif de Recherche 140, F-35042 Rennes, France; cInstitute of Immunology, Biomedical Sciences Research Center
Alexander Fleming, 16672 Vari, Greece; dDivision of Stem Cell Biology and Developmental Genetics, Medical
Research Council, National Institute for Medical Research, London NW7 1AA, UK; eDepartment of Endocrinology and
Metabolism, Faculty of Science, Utrecht University, 3584 CH Utrecht, The Netherlands

ABSTRACT Posttranscriptional mechanisms are crucial to regulate spermatogenesis. Accu- Monitoring Editor
rate protein synthesis during germ cell development relies on RNA binding proteins that A. Gregory Matera
University of North Carolina
control the storage, stability, and translation of mRNAs in a tightly and temporally regulated
manner. Here, we focused on the RNA binding protein Embryonic Lethal Abnormal Vision Received: Mar 11, 2011
(ELAV) L1/Human antigen R (HuR) known to be a key regulator of posttranscriptional regula- Revised: May 16, 2011
tion in somatic cells but the function of which during gametogenesis has never been investi- Accepted: Jun 20, 2011
gated. In this study, we have used conditional loss- and gain-of-function approaches to ad-
dress this issue in mice. We show that targeted deletion of HuR specifically in germ cells leads
to male but not female sterility. Mutant males are azoospermic because of the extensive
death of spermatocytes at meiotic divisions and failure of spermatid elongation. The latter
defect is also observed upon HuR overexpression. To elucidate further the molecular mecha-
nisms underlying spermatogenesis defects in HuR-deleted and -overexpressing testes, we
undertook a target gene approach and discovered that heat shock protein (HSP)A2/HSP70-2,
a crucial regulator of spermatogenesis, was down-regulated in both situations. HuR specifi-
cally binds hspa2 mRNA and controls its expression at the translational level in germ cells.
Our study provides the first genetic evidence of HuR involvement during spermatogenesis
and reveals Hspa2 as a target for HuR.

INTRODUCTION
Spermatogenesis is a highly regulated and complex process through successive divisions, into haploid round spermatids. Subsequently,
which spermatozoa are produced. It involves the differentiation of dramatic morphological changes take place in those postmeiotic
diploid spermatogonia into spermatocytes and then, through two haploid germ cells that undergo an elongation phase during sper-
miogenesis, transforming them into mature spermatozoa. In partic-
This article was published online ahead of print in MBoC in Press (http://www ular, the chromatin progressively compacts while the spermatid dif-
.molbiolcell.org/cgi/doi/10.1091/mbc.E11-03-0212) on July 7, 2011. ferentiates, leading to transcriptional silencing before differentiation
Address correspondence to: Dominique Morello (morello@cict.fr). is completed (Kimmins and Sassone-Corsi, 2005). Thus the synthe-
Abbreviations used: ARE, AU-rich element; CB, chromatoid body; ELAV, Embry-
onic Lethal Abnormal Vision; H&E, hematoxylin and eosin; HSP, heat shock pro-
sis of proteins required for spermatozoa assembly and function is
tein; HuR, Human antigen R; IHC, immunohistochemistry; IP, immunoprecipitation; thought to rely on the appropriate storage and translational control
mRNP, messenger ribonucloeprotein complex; PGC, primordial germ cell; of mRNAs that have been transcribed at earlier meiotic or postmei-
qRT-PCR, quantitative RT-PCR; RBP, RNA-binding protein; RNP, ribonucleoprotein;
WT, wild type. otic steps (Steger, 1999, 2001). This hypothesis is strengthened by a
© 2011 Chi et al. This article is distributed by The American Society for Cell Biol- study showing that many mRNAs that are silent during early steps of
ogy under license from the author(s). Two months after publication it is available differentiation are stored in ribonucleoproteins (RNPs) and later on
to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported
Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
shift into polysomes where they are actively translated (Iguchi et al.,
“ASCB®,“ “The American Society for Cell Biology®,” and “Molecular Biology of 2006). The factors controlling mRNA fate during spermiogenesis are
the Cell®” are registered trademarks of The American Society of Cell Biology. beginning to be identified and include RNA-binding proteins (RBPs)

Volume 22 August 15, 2011 2875

that specifically control stabilization and translation of their target In view of the embryonic lethality of Elavl1−/– embryos (Ghosh et al.,
mRNAs. Numerous RBPs are synthesized solely in late phases of 2009; Katsanou et al., 2009), we used a conditionally defective HuR
spermatogenesis, ensuring a temporal regulation of their target mR- allele containing target sites for the Cre/loxP recombination system
NAs (Iguchi et al., 2006). RBPs such as Miwi, Ddx25, Msy2, Sam68, (Elavl1 floxed allele or Elavl1fl; Katsanou et al., 2009) and a battery
and CUGBP1, have been shown to play crucial roles in spermatid of Cre-expressing transgenic mice, including Sycp1-Cre (Vidal et al.,
differentiation as their knockout (KO) led to spermiogenic arrest and 1998), Vasa-Cre (Gallardo et al., 2007), Vav-Cre (de Boer et al., 2003),
subsequent male sterility (Deng and Lin, 2002; Tsai-Morris et al., and Nestin-Cre (http://jaxmice.jax.org/strain/002858.html), which
2004; Yang et al., 2005; Kress et al., 2007). have been shown to function exclusively (the first two) or less spe-
To gain further insight into the contribution of posttranscriptional cifically in reproductive tissues. Except for the Vasa-Cre mice (see
control during spermatogenesis, we focused on Embryonic Lethal later in the text), the efficiency of recombination was too low to
Abnormal Vision (ELAV) L1/Human antigen R (HuR), an RBP that be- permit a complete deletion of Elavl1 in all germ cells. Indeed, the
longs to the ELAV family of proteins (Ma et al., 1996; Myer et al., germ cells develop as a syncytium where cells stay connected to
1997). HuR was first identified in somatic cells for its ability to bind one another by intracellular bridges after cell division, allowing com-
an AU-rich element (ARE) contained in the 3’ UTR of c-fos and Il3 munication between cells. If recombination is not complete in one
mRNAs and then to increase the stability of many ARE-containing or a few of a clone, HuR expression will occur in adjacent haploid
mRNAs (reviewed in (Bevilacqua et al., 2003). RNA-immunoprecipi- HuR− cells, compromising further study on the consequence of HuR
tation (IP) assays in human colorectal carcinomas revealed that, in deletion. The passing through of the HuR protein from Elavl1+ to
addition, HuR could bind mRNAs containing a U-rich 17- to 20-nu- Elavl1− haploid daughter cells was well illustrated by immunofluo-
cleotide-long motif, most frequently located in their 3’ UTR (Lopez rescence analysis of Elavl1+/– testis showing that all round sperma-
de Silanes et al., 2004). Besides its protective role against mRNA tids expressed HuR, whereas only 50% were expected to do so
degradation, HuR was also shown to regulate the translation of var- (Supplemental Figure S1 Elavl1+/− testis). The same result was ob-
ious mRNAs (reviewed in Galban et al., 2008). tained when analyzing Elavl1fl/−; Sycp1-Cre testes, showing that the
Studies of HuR function in vivo have been compromised by the recombinase was not fully efficient (Supplemental Figure S1). Its in-
fact that its constitutive inactivation is lethal to embryos (Katsanou efficiency was further confirmed by crossing Elavl1fl/−; Sycp1-Cre
et al., 2009). Thus most of our knowledge comes from transformed males with wild-type (WT) females. Approximately 50% of the pups
cells or experimental situations in which the level of HuR is naturally were Elavl1+/− and 50% were Elavl1fl/+, a proportion significantly dif-
(cancer cells) or artificially increased through the use of transgenic ferent from the 100% Elavl1+/− expected if the Cre recombinase
mice. In this respect, we previously have reported that HuR overex- were fully efficient (see Supplemental Figure S1 for details).
pression in macrophages leads to translational silencing of specific In Vasa-Cre mice, the Cre recombinase is active in PGCs (Gallardo
cytokine mRNAs (Katsanou et al., 2005) and that fertility is compro- et al., 2007) and therefore guarantees the deletion of Elavl1 in the
mised in the HuR-overexpressing transgenic testis (Levadoux-Martin germ cells that all derive from these precursor cells (Figure 1A).
et al., 2003). Our recent data indicate that during normal spermato- To inactivate HuR specifically in PGC (genotyped as Vasa-Cre;
genesis HuR expression is tightly regulated both spatially and tem- Elavl1fl/−), we first crossed Elavl1+/− mice with Vasa-Cre heterozygous
porally (Nguyen Chi et al., 2009). In particular, we have shown that mice, then selected Elavl1+/−; Vasa-Cre males that were crossed with
HuR is a component of the mammalian germ cell nuage, also called Elavl1fl/fl females (Figure 1A). Surprisingly, the number of Vasa-Cre;
the chromatoid body (CB; Parvinen, 2005). This germ cell–specific Elavl1fl/− pups was dramatically low as only four of 400 mice with
perinuclear cytoplasmic structure contains polyadenylated mRNAs such a genotype were obtained. Similarly, the transmission of the
and various components of microRNA and RNA-processing path- Vasa-Cre allele was lower than expected (26% instead of 50%,
ways and is therefore proposed to act as a center of mRNA storage n = 400), whereas its transmission was at the expected Mendelian
and processing (Kotaja et al., 2006). Whereas HuR concentrates frequency in the previous (Vasa-Cre × Elavl1+/−) generation (48%,
within the CB of early spermatids, it subsequently transits to poly- n = 43). These results strongly suggest that, in some cases, Vasa
somes together with its target ARE-containing mRNAs, suggesting regulatory sequences are active in early embryogenesis, leading to
that HuR participates in the control of mRNA storage/translation in Vasa-Cre; Elavl1−/– embryos. As we previously reported, Elavl1−/–
spermatids (Nguyen Chi et al., 2009). embryos die in utero because HuR is required for placental branch-
We now have studied the role of HuR during spermatogenesis by ing morphogenesis (Katsanou et al., 2009). Hence, midgestational
using both conditional KO and HuR-overexpressing mice. We show embryo death led to reduced transmission of both Vasa-Cre trans-
that inactivation of HuR in primordial germ cells (PGCs) is incompati- gene and Elavl1− allele.
ble with proper postmeiotic cell formation and spermatid maturation. Among the four PGC-specific HuR-KO animals, we obtained one
In addition, HuR overexpression in round spermatids delays sperma- female and three males. To examine male fertility, two Vasa-Cre;
tid differentiation and therefore the production of fully competent Elavl1fl/– males were crossed with untreated or superovulated WT
transgenic spermatozoa. Further analysis based on a candidate gene females. Despite repeated matings, from 6 to 9 wk, no pregnant
approach revealed that HSPA2, a protein that belongs to the 70-kDa females were obtained, whereas control males (Elavl1fl/fl, both Elavl1
heat shock protein (HSP70) family that is essential for male germ cell alleles active or Elavl1fl/− or Elavl1+/−, a single allele active) were
differentiation, is misregulated both in HuR-overexpressing and -de- fully fertile (unpublished data), strongly suggesting that Vasa-Cre;
ficient germ cells. Our study demonstrates that HuR is required both Elavl1fl/– males were sterile. To confirm this hypothesis, these two
in meiotic and postmeiotic steps in mouse spermatogenesis and males were killed at 9 wk. Their testes and epididymides were
points to HSPA2 as a prominent molecular target of HuR. remarkably smaller than those of controls, and the ratio testis/body
weight was significantly different from that of control (WT or
RESULTS Elavl1fl/−) males (Figure 1B and unpublished data). Immunohis-
HuR is essential for normal male fertility tochemistry (IHC) using anti-HuR antibody on HuR mutant testis sec-
To explore the biological function of HuR during spermatogenesis, tions showed the absence of HuR protein in all types of germ cells,
we first analyzed the consequences of its inactivation in germ cells. in contrast to control (Elavl1fl/−, Elavl1fl/fl, or WT) testis sections

2876 | M. Nguyen Chi et al. Molecular Biology of the Cell

 (Figure 1C for Elavl1fl/−, Supplemental Fig-
A 1 2 3 4 5 6
Elavl1 +/-
X Vasa-Cre; Elavl1+/+ ure S1, and unpublished data). The only
positive HuR cells corresponded to the so-
Elavl1+
HH H
matic Sertoli cells in which the Cre recombi-
Elavl1 fl/fl X Vasa-Cre; Elavl1+/-

ATG
nase was not active. Comparative histologi-
cal analysis of sectioned epididymides from
Elavl1fl H H HH
fl/- WT, Elavl1+/−, and Elavl1−/– mutant mice re-

ATG
neo
Vasa-Cre; Elavl1 (tail) vealed a complete loss of spermatozoa in
X Vasa-Cre mutant epididymides (Figure 1D).
Elavl1 - H H
Vasa-Cre; Elavl1 -/- (testis) Interestingly, the Vasa-Cre; Elavl1fl/–
female we obtained showed no overt ovar-
B ian abnormalities (unpublished data); its fe-
cundity was comparable to that of control
(Elavl1fl/−) females, and deliveries were still
observed at the age of 12 mo (unpublished
data). In addition, upon successive mating
with WT males, none of the progeny carried
an Elavl1fl allele but were all heterozygous
(+/–). Even though only one Vasa-Cre;
Elavl1fl/– female was obtained, these results
clearly show that the Elavl1fl allele has effi-
ciently been recombined in each oocyte.
Thus HuR depletion in PGCs induces male
sterility but does not seem to compromise
female fertility.

C Testis Elavl1 fl/- Elavl1 -/-

HuR is required for the first meiotic
division progression
The loss of spermatozoa in HuR-KO males
suggests that germ cell differentiation is im-
paired. Thus we performed histological
analysis of testis sections from both juvenile
HuR + Topro

* and adult control or mutant mice. Whereas

* adult testes from WT mice at 9 wk of age
showed spermatogenic cells in all of the dif-
ferent stages of differentiation (spermatogo-
nia, spermatocytes, round and elongated
spermatids; see Figure 2, A, C, and G, for

50Mm 50Mm HuRfl/−; Vasa-Cre males were slightly lighter

than their control (Elavl1fl/+ or Elavl1fl/−)
brothers. (C) Immunoconfocal analysis of HuR
D Epididymis Elavl1 fl/- Elavl1 -/-
expression in control (Elavl1fl/−) and mutant
Control Elavl1 -/- (Elavl1fl/−; Vasa-Cre) tubule sections. Whereas
HuR (green) is expressed in spermatocytes
and all round spermatids of control Elavl1+/−
testis (dashed and solid arrows in the left
panel, respectively), its expression is
*** restricted to interstitial cells and to Sertoli
cells in mutant tubules (arrow in the right
panel). * indicates unspecific labeling of dead
cells. All nuclei are labeled with Topro (blue).
Scale bars, 50 μm. (D) Histological analysis of
control (Elavl1fl/−) and mutant (Elavl1fl/−;
FIGURE 1: HuR KO males are sterile. (A) Left, schematic of the complete exon–intron Vasa-Cre) epididymides. H&E-stained
orientation of the HuR locus and magnification of the region containing the ATG-containing exon sections from 9-wk-old males. Whereas
2 (gray box). In the targeted locus (Elavl1fl), exon 2 is flanked by two loxP sites, allowing exon 2 control epididymis tubules contain many
excision by the Cre recombinase (Katsanou et al., 2009). Right, crossing strategy to target HuR spermatozoa (arrow), no sperm was observed
gene deletion specifically in the germ cells. Using Vasa-cre mice expressing the Cre recombinase in the mutant ones. The weight (g) of mutant
in the PGCs, germ cells of Vasa-Cre; Elavl1fl/− males do not express HuR (Elavl1−/– testis), whereas epididymides (n = 4) is significantly different
somatic tissues do (Elavl1fl/– tail). (B) The size and weight of 9-wk-old HuR mutant testes (n = 4) from that of control (Elavl fl/+ or Elavl1fl/−)
were significantly reduced compared with control (Elavl1fl/+ or Elavl1fl/−) testes (n = 8) even though epididymides (n = 8).

Volume 22 August 15, 2011 Crucial role of HuR for spermatogenesis | 2877
 Control Elavl1 -/- their respective localization in a schematic tubule section), dramatic
spermatogenic defects were observed in the testes of the two adult
A B mutant males (Figure 2, B and D, and Supplemental Figure S1). Nu-
merous vacuoles were observed in seminiferous tubules that con-
tained essentially spermatocytes but lacked spermatids (Figure 2, D
vs. C). Spermatocytes progressed until meiotic divisions, but mei-
otic divisions were compromised, and massive cell death was visible
in stage XII tubules where meiotic divisions normally take place
(Figure 2, B and D). At 4 wk of age, in the WT testes, all tubule cross-
adult

100µm
sections showed at least round spermatids and, in many tubules,
C D elongating spermatids were already present as the most advanced
II Scytes type of germ cells (Figure 2E). By contrast, in the third remaining
HuR-KO male that was killed at P29, approximately half of the tu-
bules contained numerous vacuoles and showed a nearly complete
ES
P* block of spermatogenic maturation at the spermatocyte stage
(Figure 2F) and numerous dead or dying cells (Figure 2H), akin to
Stage XII Stage XII what was observed in mutant adult testes. The remaining tubules
contained spermatids, as expected, but their differentiation was de-
E RS F V S layed (see later in the text; Figure 2F).
All together, these results suggest that HuR is initially required
4 weeks

for meiotic progression. To further characterize the stage of de-

ES fect in HuR mutant testes, we first thoroughly analyzed the kinet-
S
ics of HuR expression during meiosis of WT males. To substage
meiosis, we carried out dual immunocytochemical staining of
surface-spread germ cells or testis sections using anti-HuR anti-
body together with anti-SYCP3, which marks a lateral compo-
G G H A nent of the synaptonemal complex (Lammers et al., 1994), the
dynamics of which during meiosis has been well characterized
(Turner et al., 2001), or anti-phospho-H2AX histone (γH2AX) an-
*P tibody that marks sites of double-strand breaks (Mahadevaiah
RS
et al., 2001). HuR was not expressed in early prophase sperma-
tocytes (leptotene-zygotene), but accumulated as the pachytene
lumen stage progressed (Figure 3A and unpublished data). Although
present in diplotene spermatocytes, HuR staining disappeared
FIGURE 2: Meiotic divisions are compromised in Elavl1−/– testis. during the diplotene-metaphase I transition (Figure 3B). In post-
(A–D) Histological analysis of testis from 9-wk-old control
meiotic haploid cells, HuR accumulated in round spermatids but
(Elavl1fl/+; Vasa-cre) (A and C) and mutant (Elavl1fl/−; Vasa-cre)
was absent in elongated spermatids (Figure 3B and Nguyen Chi
(B and D) mice. Sections were stained with H&E. The low-
magnification views of control (A) and mutant (B) seminiferous et al., 2009).
tubules show that mutant tubules contain fewer germ cells and To know more precisely at which stage HuR absence could af-
numerous vacuoles. Whereas control tubules contain all types of fect meiosis in HuR mutant germ cells, we then analyzed meiosis
differentiating germ cells (A and C), as schematized in G, in progression in mutant testes by using the combination of anti–
mutant ones, germ cells differentiate normally until spermatocyte phospho-H2AX and anti-SYCP3 antibodies. Early spermatocyte dif-
stage but apparently cannot carry out meiotic divisions (B and D). ferentiation was not affected in mutant, as cells at leptotene and
The metaphase plates of the HuR-deficient spermatocytes are zygotene stages were present in normal number (Figure 3C). This
irregular, the chromosomes look thick, and the cytoplasm is result is not surprising because these cells normally do not express
starting to stain, indicating apoptosis (D). Dashed arrows indicate
HuR (Figure 3A, WT). Later on during differentiation, we observed
two secondary spermatocytes (II Scytes). P, pachytene
all the prophase stages from pachytene spermatocytes to the diaki-
spermatocytes; ES, elongated spermatids. Twelve stages of
spermatogenesis have been determined in mouse nesis step. Neither synapsis of homologous chromosomes nor sex
spermatogenesis on the basis of specific cell associations and key body formation was perturbed (Figure 3D and unpublished data).
morphological criteria (Kotaja et al., 2004) that were used to stage Compared with control germ cells, however, the relative number of
the different sections of seminiferous tubules shown in this study. midpachytene spermatocytes was increased twofold whereas that
(E, F, and H) Histological analysis of testis from 4-wk-old control of cells at diplotene and diakinesis stages was dramatically dimin-
(Elavl1fl/+; Vasa-cre) (E) and mutant (Elavl1fl/−; Vasa-cre) (F and H) ished (Figure 3E). This finding shows that HuR-deficient cells prog-
mice. Whereas control tubules (E) contain round (RS) and ress efficiently to midpachytene stage and suggests that cell death
elongated spermatids (ES), spermatid formation and takes place just before or during meiotic divisions, leading to a
differentiation are compromised in mutant tubules (F and H).
strong deficit in haploid cells as shown earlier in this article (Figure
Approximately 50% of mutant tubules contain numerous vacuoles
2). Collectively, our results indicate that HuR is dynamically ex-
(V), and germ cell differentiation is arrested before postmeiotic
stages (F). In a few apparently normal mutant tubules, round pressed during meiosis and is required for meiotic progression.
spermatids have begun elongation (S), but the process is delayed.
The tubule shown in H shows massive cell death among the HuR is required for spermatid differentiation
spermatocytes in meiotic divisions (A, arrows). (A, B, E, and Besides tubules in which spermatogenesis was arrested, juvenile HuR-
F: ×20; C, D, and H: ×60). deficient testes contained ∼50% of tubules in which spermatocytes

2878 | M. Nguyen Chi et al. Molecular Biology of the Cell

 A γH2AX HuR Topro B Pachytene Diplotene Metaphase I RS ES

Topro
ES
Elavl1 fl/- (control)

HuR
L

SCP3
Merge
Pachytene Diplotene Late Diplotene

γH2AX
SCP3

C γH2AX HuR Topro D Pachytene Diplotene Metaphase I

SCP3

P
L
Topro/γH2AX
Elavl1 -/-

Merge

E
STAGE Control (n=130) Elavl1 -/- (n=102)
Leptotene 3 1
Zygotene 4 2
Pachytene 15 27
Diplotene 11 1
Diakinesis 2 0
FIGURE 3: HuR expression in meiotic and postmeiotic stages. (A and C) Sections from adult (9-wk-old) control (Elavl1fl/−)
(A) or mutant (Elavl1fl/−; Vasa-cre) (C) testis were immunostained with anti-HuR (green) and anti-γH2AX (red) and
analyzed under a confocal microscope. Neither leptotene spermatocytes (L, large arrows) nor elongated spermatids
(S, dashed arrows) express HuR, whereas pachytene spermatocytes (P, thin arrows) do express HuR in the control testis.
γH2AX-stained sex bodies are normally observed in mutant pachytene spermatocytes (C). All nuclei are labeled with
Topro. (B, D, and E) Expression of HuR during germ cell differentiation was analyzed by confocal immunofluorescence
using surface-spread germ cells prepared from control (B) or mutant (D) 9-wk-old testes. In control germ cells, a
combination of anti-HuR and anti-Sycp3 (SCP3) or anti-Sycp3 and anti-γH2AX antibodies reveals the dynamism of HuR
expression during prophase I. In postmeiotic stages, HuR concentrates in round spermatids (RS), but its expression is
lost in elongating spermatids (ES) (B). Inactivation of HuR does not compromise early meiotic events as observed using
a combination of anti-Sycp3 (red) and anti-γH2AX (green) antibodies (D). Cells in meiotic divisions, however, are rarely
observed as revealed by counting cells labeled with a combination of anti-Sycp3 and -γH2AX antibodies: A significant
increase in the number of pachytene spermatocytes and a strong reduction of meiotic cells (diplotene or diakinesis
stage) are observed in mutant cells (E).

successfully completed their meiotic divisions, giving rise to round adult HuR mutant testes, meiotic divisions were rarely successful, only
spermatids (Figure 2F). These spermatids failed, however, to elongate occasionally giving rise to round spermatids that failed to begin elon-
at the appropriate stage. Indeed, elongated spermatids appeared gation (Supplemental Figure S2). Therefore spermatogenic defects in
much later in the epithelial cycle and were malformed (Figure 4A). In HuR mutant testes occurred in a majority of spermatocytes that did

Volume 22 August 15, 2011 Crucial role of HuR for spermatogenesis | 2879
A ended with a restricted list of genes and fo-
Step XI cused on Hsp70-2/Hspa2 not only because
the meiotic arrest observed in HuR-deficient
-/-
Control Elavl1 germ cells resembles the one described in
HSPA2 mutant germ cells (Dix et al., 1996),
but also because of increasing evidence for
a role of HSPA2 in spermiogenesis (Govin
et al., 2006). HSPA2 is a member of the
HSP70 family that is expressed exclusively in
male germ cells (reviewed in Eddy, 1999). Its
depletion induces male but not female in-
fertility, spermatogenic cell development
being arrested in prophase of meiosis I at
the G2-M-phase (Eddy, 1999).
60Mm To test whether Hspa2 was involved in
the phenotype observed in Elavl1-mutant
B germ cells, we studied the expression pat-
Step VI Step XI tern of HSPA2 protein in Elavl1−/– testes.
HuR tg WT HuR tg Immunohistochemical analysis revealed a
global decrease compared with control
* testes (Figure 5, 4 wk). Noticeably, HSPA2
was located primarily in the nuclei of mu-
tant pachytene spermatocytes, whereas in
control testes it was abundant both in their
nucleus and cytoplasm, as previously re-
*
ported (Dix et al., 1996). We observed
similar results in mutant adult testes (un-
wt tg published data). In addition, round sper-
matids present in some juvenile mutant tu-
bules poorly expressed HSPA2 (Figure 5).
FIGURE 4: Delayed elongation of spermatids in Elav1−/– and HuRtg testes. (A and B) Sections Therefore Hspa2 is misregulated in HuR-
from 4-wk-old WT, mutant (Elavl1−/–) or HuRtg testis were stained with periodic acid–Schiff deficient germ cells.
reagent. (A) Pictures were taken at stage XI to illustrate the spermiogenic defects. Whereas
numerous elongated spermatids were observed in WT testis, elongation of round spermatids HuR overexpression impairs spermatid
was delayed in HuR mutant testis. Insets show that mutant elongating spermatids are at steps differentiation
9–10, but they should be at approximately step 14, as shown in WT. (B) Pictures were taken at
The scarcity of Elavl1-KO males compro-
step VI and XI of spermatid differentiation to illustrate the spermiogenic defects observed at all
mises the study of the molecular mechanisms
stages from step VI. In step VI, as expected, the B mitotic spermatogonia are present (arrow) as
well as midpachytene spermatocytes (asterisk). No elongating spermatids are present, however. underlying HuR-mediated Hspa2 misregula-
In addition, the number of round spermatids is reduced, and those present show a tion. To overcome this difficulty, we switched
developmental delay, as their acrosome is more typical of stage V (zoom in the inset) than stage to an HuR-overexpression system using
VI (inset showing WT step 6 spermatid). At step XI, spermatocytes in zygotene (arrow) and transgenic mice that express a Myc-tagged
diplotene (asterisk) phases of the meiotic prophase are present. Spermatids have initiated HuR transgene specifically in their germ cells
elongation, but show delay compared with age-matched WT. They appear much more like step and do not produce fully competent trans-
IX (see inset). genic gametes (Levadoux-Martin et al.,
2003). To further characterize the spermato-
not complete meiotic divisions or in the round spermatids derived genic defects, we compared the morphology and histology of WT
from the remaining spermatocytes the differentiation of which was and transgenic testes at various ages. We observed the first defects
blocked before the completion of elongation. at P28, when the transition between round and elongating sperma-
tids takes place (Supplemental Figure S4, D and I). Spermatid differ-
HSPA2 is down-regulated in HuR-deleted germ cells entiation started to be impaired at stage VI: The number of round
Because HuR is an RBP known to regulate the expression of numer- spermatids was reduced, and their acrosome development was de-
ous but specific genes, we searched for HuR targets the misexpres- layed, being typical of stage 5 (Figure 4B). A similar delay of sperma-
sion of which could be responsible for meiotic and postmeiotic de- tid differentiation was observed in tubules from stage VII to XI. As
fects observed in Elavl1-deleted germ cells and carefully analyzed exemplified in Figure 4B, at stage XI, spermatids from transgenic
the list of 443 genes recently published the mutations or deletions testes have initiated elongation, but were typical of step 9, whereas
of which cause reproductive defects (Matzuk and Lamb, 2008). In in WT testes they were fully elongated. Thus HuR overexpression
this list, we selected the genes the mutation of which produces a promotes a delay in the development of round spermatids, a pheno-
reproductive phenotype according to the following four criteria: type reminiscent of the one observed in Elavl1-deficient spermatids.
1) defects are selectively observed in males but not in females; 2) the
mutation triggers impairments at late-prophase I or meiotic division Mislocalization of Myc-HuR in transgenic spermatids
stage; 3) the cellular defects are similar to those observed in Elavl1- We previously reported that HuR subcellular localization was dy-
KO testis; and 4) the mutation leads to complete infertility. We thus namic during WT spermatid differentiation: HuR first accumulates in

2880 | M. Nguyen Chi et al. Molecular Biology of the Cell

 Control Elavl1 -/- together, cell fractionation and immunofluo-
rescence experiments show that, in contrast
to the WT situation, the transgenic HuR pro-
1 tein is weakly associated to polysomes but
accumulates in the CB and other cytosolic
2 structures in transgenic elongating sperma-
tids. Due to its mislocalization, we postulate
4 weeks

1:P that the transgenic protein may alter the me-

1:P tabolism of HuR target mRNAs.

Impaired Hspa2 expression

in HuR-overexpressing germ cells
To test this hypothesis, we analyzed HSPA2
expression in HuRtg testes and observed
2:RS 2:RS that HSPA2 expression was indeed de-
2 1
creased upon HuR overexpression, as shown
FIGURE 5: Impaired expression of HSPA2 in HuR mutant testes. Immunohistochemical analysis by Western blot analysis of whole-cell ex-
of HSPA2 expression in 4-wk-old control (Elavl1+/−) and Elavl1−/– testes. HSPA2 expression in the tracts (Figure 7A). Although reproducible,
KO spermatocytes is mainly nuclear as shown in the enlarged view (1P, for pachytene the decrease was moderate, suggesting that
spermatocyte, right inset), whereas it is both cytoplasmic and nuclear in the WT pachytene
the defect might take place in a restricted
spermatocytes (left panel, 1P inset). In addition, HSPA2 is weakly expressed in the round
spermatids (2: RS) contained in some mutant tubules (right), whereas it is highly expressed in WT
population of germ cells. Analysis at the cel-
round spermatids (2: RS, left panel). lular level using IHC confirmed this hypoth-
esis, showing a particularly strong reduction
of HSPA2 in elongating spermatids in adult
the CB and then, at steps 4 and 5, exits the CB and associates with testes (Figure 7B). The decrease at the protein level was not corre-
polysomes (Nguyen Chi et al., 2009). As the first sign of spermatid lated with a decreased level of Hspa2 mRNA, as similar levels of
differentiation delay observed in HuRtg testis coincided with the step Hspa2 mRNA were found in WT and transgenic testes both by quan-
at which HuR exits the CB, we hypothesized that the Myc-HuR trans- titative RT-PCR (qRT-PCR) and microarray experiments (Figure 7C
genic protein could be mislocalized in transgenic spermatids. To test and unpublished data), but was due to its decreased translation. In-
this hypothesis, we subjected adult (P40) testicular extracts to su- deed, analysis of Hspa2 mRNA relative abundance in the RNP and
crose density gradient fractionation and compared the profile of polysome fractions of the sucrose density gradients described ear-
Myc-HuR sedimentation to that of the endogenous HuR protein in lier in this article revealed an increased amount of Hspa2 mRNA in
WT extract. Comparison of RNA absorbance profiles between WT RNP fractions and a reduced (≈15%) association with polysomes in
and transgenic cytoplasmic extracts showed a slight but reproduc- transgenic testicular extracts (Figure 7D). By contrast, the relative
ible decrease in the amount of polysomes in extracts of transgenic abundance in the RNP and polysome fractions of two control mR-
cells (Figure 6A). Western blot analysis revealed a similar distribution NAs that do not bind HuR, PGK2 and the longest GCNF transcript
of the S6 small ribosomal subunit protein between the two gradients (Yang et al., 2003), was similar in transgenic and control extracts
(Figure 6B), indicating that the global translation profile is not al- (Supplemental Figure S6).
tered in HuRtg testes. The endogenous HuR protein distributed These data suggested that HuR binds Hspa2 mRNA and regu-
throughout the gradient, including the high-molecular-weight frac- lates its expression. To test this hypothesis, we performed a RNA-IP
tions that are enriched in polysomes (Figure 6B). By contrast, the assay using cytoplasmic testicular extracts from P17 or 6-wk-old WT
Myc-HuR transgenic protein failed to sediment with polysomes but males that did not yet contain haploid cells or were enriched in sper-
was instead predominantly concentrated in low-molecular-weight matids, respectively. We observed that Hspa2 mRNA was specifi-
fractions (Figure 6, B and C, for quantification). Those fractions in- cally retained by anti-HuR antibody in both types of cytoplasmic
clude nontranslating mRNA-containing protein complexes (mRNPs) extracts. Interestingly, the enrichment of the Hspa2 mRNA in HuR IP
and several components of the CB, such as MVH (Figure 6B), DCP1a was more important (≈20-fold) in adult than in P17 samples (Figure 7E
(Nguyen Chi et al., 2009), or MIWI and GW182, as described previ- and Supplemental Figure S7B), whereas HuR level was unchanged
ously (Grivna et al., 2006), suggesting that the transgenic protein (Nguyen Chi et al., 2009), suggesting that HuR affinity for Hspa2
might accumulate in the CB or other cytosolic structures that sedi- mRNAs increased between meiotic and postmeiotic stages. Simi-
ment in the RNP fractions. To test this hypothesis, we used immuno- larly, Hspa2 mRNA was specifically retained by anti-Myc antibody in
fluorescence microscopy on seminiferous tubule squash prepara- transgenic extracts (Figure 7E). All together, these results show that
tions and analyzed endogenous and Myc-HuR localizations during HuR and HuR transgenic proteins bind Hspa2 mRNA and strongly
spermatid differentiation. Similarly to the endogenous HuR protein suggest that HuR up-regulates Hspa2 mRNA translation during
in WT early round spermatids (steps 1–3) (Figure 6D and Nguyen spermatogenesis in a specific and direct manner.
et al., 2009), the transgenic protein accumulated predominantly in
the nucleus but was also detected in a perinuclear structure that DISCUSSION
contains polyadenylated mRNAs and MVH (Figure 6D and Supple- Spermatogenesis is a complex process that relies on extensive
mental Figure S5A) and thus corresponds to the CB, as previously regulation of mRNA storage and translation. In this study, we
reported (Nguyen Chi et al., 2009). In contrast to the WT situation, have investigated the role of the RBP HuR in mammalian sper-
however, transgenic HuR persisted in the CB of more mature sper- matogenesis by using both germ cell–specific loss- and gain-
matids (83% at steps 4–5) and also accumulated in other cytosolic of-function strategies. We have provided evidence that HuR is
structures, some of which also contained MVH (Figure 6E). Taken essential for male germ cell differentiation. In addition, at the

Volume 22 August 15, 2011 Crucial role of HuR for spermatogenesis | 2881
 A B end of prophase I stages, does not affect
the normal progression of meiotic prophase,
LMW HMW
1 2 3 4 5 6 7 8 9 10 indicating that HuR most probably does not
control expression of genes involved in
WT
S6 chromosome pairing, double-strand breaks,
tg
HuR and/or DNA repair the mutations of which
provoke a halt during the meiotic prophase I

germ cell extracts

WT (Kuznetsov et al., 2007). HuR mutant germ
tg HuR cells, however, fail to progress further on.
HuR
Spermatocytes die during meiotic divisions,
C WT myc-
and spermatids are lacking in nearly all tu-
*** tg HuR bules of adult testes, showing that HuR is
*
% of prot eins in each fract ion

HuR
required for the completion of meiosis. In
* contrast, HuRtg spermatocytes divide cor-
WT rectly, most probably because HuRtg is not
* MVH
* *** ** HuR
tg highly overexpressed in these cells. Indeed,
we estimated that the amount of HuR mRNA
(endogenous plus transgenic) in transgenic
spermatocytes was only fivefold higher than
in WT spermatocytes, whereas transgenic
HMW
postmeiotic cells that exhibit differentiation
LMW
defects (see below) express nearly 20-fold
D Step 1 to 3
E Step 6 to 8
more HuR than the WT ones (Supplemental
Figure S3).
HuR MVH Merge HuR MVH Merge
HuR is required for spermiogenesis
WT

completion
In adults, despite a nearly complete failure
of germ cells to progress through meiotic
Myc- Myc- divisions, some spermatocytes manage to
tg

HuR HuR
divide and give rise to round spermatids that
HuR

fail to elongate. In juvenile mutant testes,

some tubules contain round spermatids, but
their differentiation is delayed, leading to an
FIGURE 6: HuR exit from the CB is compromised in HuRtg spermatids. (A–C) Germ cell absence of elongated spermatids or mal-
cytoplasmic extracts from a pool of 10 P40 WT or transgenic (HuRtg) testes were fractionated on formed ones. A similar differentiation delay
15–50% sucrose density gradients. RNA absorbance profiles at 260 nm are shown. Low-
is observed in HuRtg spermatids. The major-
molecular-weight (LMW) fractions (fractions 1–5) contain RNP complexes, components of the CB
and ribosome subunits. High-molecular-weight (HMW) fractions (6–10) include polysomes
ity of HuRtg males, however, are fertile most
(A). Proteins were extracted from each fraction and analyzed by Western blot using anti-S6, probably because WT haploid cells that do
anti-endogenous HuR, anti-Myc, or anti-MVH antibodies (B). The distribution of endogenous not accumulate the HuR transgenic protein
HuR (HuR) in both WT and transgenic (HuRtg) extracts is different from that of the transgenic at a detrimental level normally differentiate
HuR protein (myc-HuR) as revealed by quantification of three independent gradients. Their level into sperm (Levadoux-Martin et al., 2003).
of expression in a given fraction is given as the percentage of the level found in all fractions Thus beside its role during meiotic phase,
(C). *p < 0.05, **p < 0.01, and ***p < 0.005. (D and E) Round spermatids at the indicated steps HuR is also required for spermatid differen-
of differentiation were prepared from tubule squashes of WT and HuRtg testes. Cells were tiation, during which it may play a role in
doubly stained with anti-HuR (or anti-Myc that specifically recognizes the transgenic protein) chromatin condensation and/or cell elonga-
(green) and anti-MVH (red) antibodies. In WT, both MVH and HuR localize within the CB of early
tion, two major events of spermiogenesis.
(steps 1–3) round spermatids (arrow), and HuR exits the CB at further steps of differentiation. In
transgenic spermatids, HuR stays in the CB of more mature spermatids (steps 6–8) (arrow).
HuR controls Hspa2 mRNA translation
As mentioned earlier in this article, in the list
molecular level, we have identified Hspa2/Hsp70-2, an essen- of genes the mutations or deletions of which cause reproductive
tial regulator of spermatogenesis, as a direct downstream tar- defects (Matzuk and Lamb, 2008), we selected Hspa2/Hsp70-2 be-
get of HuR. cause 1) absence of HSPA2 leads to complete male sterility and
meiotic disorders that resemble those observed in HuR-deficient
HuR is a key regulator of meiotic division in males germ cells (Dix et al., 1996) and 2) Hspa2/Hsp70-2 may represent a
Specific deletion of HuR in germ cells leads to male sterility, associ- target of HuR not only in meiosis but also in spermiogenesis, during
ated with dramatically reduced testis size, spermatogenic defects, which HSPA2 has been proposed to play a decisive role in genome-
and absence of spermatozoa in the epididymides. In sharp contrast wide reorganization occurring during postmeiotic stages (Quenet
with hematopoietic and intestinal systems (Ghosh et al., 2009), HuR et al., 2009). We have observed that HSPA2 expression is down-
is not essential for progenitor germ cell survival in gametogenesis regulated in HuR-deleted and -overexpressing haploid germ cells
because it starts to accumulate in midpachytene spermatocytes. and that endogenous and transgenic HuR proteins bind Hspa2
Therefore its deletion at earlier stages, from the leptotene to the mRNA in germ cells. Further studies will be needed to know whether

2882 | M. Nguyen Chi et al. Molecular Biology of the Cell

 HuR directly binds Hspa2 mRNA and to de-
A
WT HuRtg WT termine the sequences involved. The analy-
1 2
sis of the Hspa2 3’UTR, however, revealed
1 2 3 3
HuRtg

HSPA2 / Tubulin
two AUUUA pentamers and four U-rich se-
myc-HuR * quences that are conserved in mammals
(Supplemental Figure S7A), and represent
HSPA2 potential binding sites for HuR (Mukherjee et
al., 2009). The association of Hspa2 mRNA
Tubulin with translating ribosomes was decreased in
HuRtg germ cells, leading to a reduced level
ratio 1 0.9 0.8 0.6 0.7 0.8 of HSPA2 protein, particularly in elongating
spermatids. Decreased Hspa2 mRNA trans-
lation was correlated with a strong associa-
B WT HuRtg tion of Myc-HuR with mRNPs and a concom-
itant failure to accumulate in polysomes, a
behavior that sharply contrasts with HuR in a
WT context, suggesting that the transgenic
protein behaves as a dominant- negative
2 months

form of HuR. Our data led us to propose the

following model (Figure 8): In WT sperma-
tids, HuR binds to Hspa2 mRNA and allows
HSPA2 synthesis during spermatid differen-
tiation (Eddy, 1999). By contrast in transgenic
spermatids, the transgenic protein binds to
Hspa2 mRNAs but fails to associate with
translating polysomes, preventing Hspa2
translation. Similarly, the absence of HuR re-
sults in Hspa2 translation inhibition in
C WT tg D WT tg E Elavl1−/– spermatids. The decrease of HSPA2
expression in both situations might be re-
*** sponsible for spermatid development arrest.
NS *
Relative hspa2 mRNA

In addition, HSPA2 expression is down-regu-

* lated in HuR-deficient spermatocytes, and
HuR binds Hspa2 mRNA at this stage. There-
fore we propose that HuR also controls
Hspa2 mRNA translation during meiosis. We
have noticed a decrease, however, in the cy-
toplasmic level of HSPA2 both in adult and
antibodies IgG HuR myc-HuR juvenile spermatocytes, suggesting that ad-
Total RNPs Polysomes germ cell WT WT tg ditional HuR-mediated regulatory mecha-
extracts nisms, such as nucleocytoplasmic trafficking
FIGURE 7: Impaired expression of HSPA2 in HuRtg testes. (A) Western blot analysis of Myc-HuR or stability of HSPA2 protein, might be in-
transgene and HSPA2 expression in WT (1–3) and transgenic (4–6) testes. Quantification volved.
of HSPA2 signals and normalization to tubulin signals reveals a slight but reproducible Although related, there are nevertheless
and significant decrease of HSPA2 expression in transgenic tubules (*p < 0.05). some differences between the phenotypes
(B) Immunohistochemical analysis of HSPA2 expression in 2-mo-old WT and HuRtg testes. HSPA2
of HSPA2- and HuR-deficient mouse testes.
expression is particularly decreased in the haploid cells of transgenic tubules. (C) Gene
Globally, HuR deficiency is slightly less severe
expression profiles from P28 WT and HuRtg testes were analyzed using the Affymetrix
microarrays approach (unpublished data). The relative Hspa2 mRNA expression after than HSPA2 deficiency, possibly because
normalization indicates no significant difference between WT and transgenic testes (p = 0.66). other members of the Hu family might com-
(D) qRT-PCR detection of Hspa2 mRNA in mRNP and polysomal fractions from control and HuRtg pensate for HuR absence. Besides Hspa2,
testes. Data are derived from measurements in pooled fractions (1–5, mRNPs, 6–10, polysomes) the list of candidate genes contained four
normalized to 18S rRNA and presented as percentages of total cytoplasmic Hspa2 mRNA in other genes that retained our attention: cy-
each condition. The values were obtained after analysis of three independent sucrose gradient clin A1 (Liu et al., 1998; Nickerson et al.,
experiments, each one using a pool of 6–10 testes from P40 WT or HuRtg males (p < 0.05). 2007), Gal3st1 (Honke et al., 2002), Dmrt7
(E) HuR endogenous and transgenic proteins bind Hspa2 mRNA. Cytoplasmic extracts from (Kawamata and Nishimori, 2006), and Parp2
testes of 6-wk-old WT males were prepared and immunoprecipitated using anti-HuR antibody.
(Quenet et al., 2009) because they encode
Control immunoglobulin was used in parallel (left panel). Similarly, cytoplasmic germ cell extracts
mRNAs containing putative HuR binding
were prepared from a pool of P28 WT or HuRtg testes (right panel). RNA-IP was performed
using anti-Myc antibody (9E10) using the same amount of WT and HuRtg protein extracts. RNAs sites. Should their binding to HuR be demon-
were extracted. The amount of Hspa2 mRNA retained in the immunoprecipitates was analyzed strated in germ cells, expression of those
by qRT-PCR. Amplification of contaminating traces of 18S rRNA was performed as an internal genes might be modified together with
control that serves for normalization. Three independent experiments using different pools of HSPA2 in HuR-deficient germ cells, contrib-
WT and HuRtg germ cells were performed. SEMs are shown; ***p < 0.005. uting to the observed phenotype. Clearly

Volume 22 August 15, 2011 Crucial role of HuR for spermatogenesis | 2883
 Collection of tissues, purification of
WT testes HuR tg testes Elavl1 -/- testes
spermatogenic cells, tubule squashes, in
prophase I meiotic prophase I meiotic prophase I meiotic situ hybridization on tubule squashes, and
division division division sucrose density fractionation were per-
round elongated round elongated round elongated formed as described previously (Nguyen
spermatid spermatid spermatid spermatid spermatid spermatid Chi et al., 2009) with the following antibod-
ies: rabbit anti-SYCP3 and anti-γH2AX
myc- (Novus Biologicals, Littleton, CO), anti-
HuR HuR HuR MVH (Abcam, Cambridge, MA), anti-HuR
mRNPs myc- (19F12; Clonegene, Hartford, CT), Myc
myc-
HuR Hspa2 mRNA
(Chromatoid body) HuR (9E10; Santa Cruz Biotechnology, Santa
Hspa2 mRNA
Cruz, CA). Anti-HSPA2 antibody was
polysomes
mRNPs mRNPs provided by E.M. Eddy (NIEHS, NIH).
HuR (Chromatoid body) (Chromatoid body) Nuclei were labeled with TO-PRO3 or DAPI
Hs
(Molecular Probes, Eugene, OR). Polyade-
pa polysomes polysomes
2 mRNA nylated mRNAs were detected by in situ
hybridization on tubule squash prepara-
tions, using a biotinylated DNA oligo(dT)
probe, as described (Nguyen Chi et al.,
Hspa2 translation Hspa2 translation Hspa2 translation
2009). Images were obtained with a Leica
SP2 or SP5 confocal microscope equipped
FIGURE 8: A model for the role of HuR in the translational control of its Hspa 2 target mRNA in
with helium-neon lasers and appropriate
WT or HuR-overexpressing or -deficient germ cells. Effects of manipulating HuR level of
filter combinations.
expression on spermatogenesis progression are shown. Absence of HuR leads to meiotic
defects whereas mild HuR overexpression does not alter early spermatogenesis. In
spermiogenesis, both HuR deletion and strong overexpression result in spermatid differentiation Histological and IHC analyses. Testes and
arrest. The cytoplasm of spermatids has been schematically divided into two compartments, epididymides were stored in aqueous
mRNPs (including the CB) corresponding to untranslating mRNAs, and polysomes, where the Bouin’s solution for 48 h and in ethanol 70%
translation is active. In WT spermatids, HuR binds to Hspa2 mRNAs in mRNPs and polysomes, before embedding in paraffin wax. Sections
ensuring their regulated translation. In HuRtg spermatids, HuR overexpression results in the (5 μm thick) were stained with either
nearly complete loss of HuR association to polysomes, leading to translational inhibition of hematoxylin and eosin (H&E) or by the
Hspa2 mRNA, a situation similar to the one observed in Elav1−/– germ cells. periodic acid–Schiff technique. Analysis of
HSPA2 on testis sections was performed by
further studies are required to characterize all the mRNAs the trans- IHC as described (Dix et al., 1997). Sections were counterstained
lation of which is impaired following HuR deletion and to reinforce with hematoxylin.
the conclusions we have drawn from the limited number of animals.
A first step toward this challenging goal relies on Cre-expressing RNA-IP experiment. Briefly, tunica albuginea was removed from
lines that should fulfill two requirements: 1) the recombination 10 to 20 P17 testes that were subsequently minced in phosphate-
should be fully efficient in all germ cells to avoid HuR trafficking buffered saline. Cells were collected without debris after sedimentation
through cytoplasmic bridges from cells in which recombination has (5 min, 4°C) and centrifuged for 5 min at 4°C and 1500 rpm. Germ
not taken place to Elavl1− cells; and 2) the Cre recombinase should cell suspension preparation for P28 old testes and the IP experiment
not be active during embryonic development because HuR activity were performed as described previously (Nguyen Chi et al., 2009).
is essential for life (Ghosh et al., 2009; Katsanou et al., 2009). Total RNA from cytoplasmic extracts or immunoprecipitated materials
To conclude, we have shown that manipulating HuR expression was extracted with TRIzol reagent, reverse transcribed, and qRT-PCR
results in dramatic spermatogenic defects leading to male infertility. amplified using a Bio-Rad (MyiQ; Hercules, CA) instrument and
Our study provides the first genetic evidence that HuR is crucial for Hspa2 primers (forward: CAG-TCA-GGA-TGT-CTG-CCC-GCG;
spermatogenesis and highlights the key role of RBPs in controlling reverse: GTC-GCC-GAT-GAG-ACG-CTC-GG) and 18S rRNA primers
this process. (forward: GTA-ACC-CGT-TGA-ACC-CCA-TT; reverse: CCA-TCC-
AAT-CGG-TAG-TAG-CG) to normalize RNA levels.
MATERIALS AND METHODS
Production of HuR-deleted or -overexpressing mice Statistical analyses. mRNA was extracted from the testes of three
Mice were maintained in accordance with institutional guidelines P28 WT and three HuRtg mice. Transcript expression analysis was
(French National Center for Scientific Research; CNRS). Their use performed using Affymetrix microarrays. Hspa2 mRNA expression
followed the French laws and was in accordance with the Euro- was normalized using the GC-RMA algorithm. Mean and standard
pean Directive (86/609/EEC). The transgenic mice overexpress- errors of the mean were determined with at least three independent
ing HuR were produced and genotyped as described previously experiments. Values of p were calculated using the two-tailed
(Gouble et al., 2002). The strategy to generate and genotype unpaired t test.
Elavl1fl/fl, Elavl1fl/–, or Elavl1+/– mice has been described (Kat-
sanou et al., 2009). The different strains expressing the Cre re- ACKNOWLEDGMENTS
combinase were first mated with Elavl1+/– mice to produce We thank V. Vallet-Erdtmann for her priceless help with histological
Elavl1+/–. Cre males were then crossed with Elavl1fl/fl following analysis of HuRtg transgenic mice and M. Fawal for his expertise with
the scheme described in Figure 1A to obtain HuR-deleted germ sucrose gradients. We deeply thank F. Le Masson for his suggestion
cells. to consider HSPA2 as a potential HuR target mRNA and E.M. Eddy

2884 | M. Nguyen Chi et al. Molecular Biology of the Cell

for his help with analysis of HSPA2 pattern of expression. We thank Kotaja N, Kimmins S, Brancorsini S, Hentsch D, Vonesch JL, Davidson I,
N. Vanzo for critical reading of the manuscript. We are grateful Parvinen M, Sassone-Corsi P (2004). Preparation, isolation and character-
ization of stage-specific spermatogenic cells for cellular and molecular
to the Histopathology (Rangueil) and Imagery (IFR109) Platforms. analysis. Nat Methods 1, 249–254.
This work was supported by the Association pour la Recherche con- Kress C, Gautier-Courteille C, Osborne HB, Babinet C, Paillard L (2007).
tre le Cancer (ARC) contract No. 3823 and the Fondation pour la Inactivation of CUG-BP1/CELF1 causes growth, viability, and spermato-
Recherche Médicale (PhD fellowship to M.N.C.). genesis defects in mice. Mol Cell Biol 27, 1146–1157.
Kuznetsov S et al. (2007). RAD51C deficiency in mice results in early pro-
REFERENCES phase I arrest in males and sister chromatid separation at metaphase II
Bevilacqua A, Ceriani MC, Capaccioli S, Nicolin A (2003). Post-transcrip- in females. J Cell Biol 176, 581–592.
tional regulation of gene expression by degradation of messenger Lammers JH, Offenberg HH, van Aalderen M, Vink AC, Dietrich AJ,
RNAs. J Cell Physiol 195, 356–372. Heyting C (1994). The gene encoding a major component of the lateral
de Boer J et al. (2003). Transgenic mice with hematopoietic and lymphoid elements of synaptonemal complexes of the rat is related to X-linked
specific expression of Cre. Eur J Immunol 33, 314–325. lymphocyte-regulated genes. Mol Cell Biol 14, 1137–1146.
Deng W, Lin H (2002). miwi, a murine homolog of piwi, encodes a cytoplas- Levadoux-Martin M, Gouble A, Jegou B, Vallet-Erdtmann V, Auriol J,
mic protein essential for spermatogenesis. Dev Cell 2, 819–830. Mercier P, Morello D (2003). Impaired gametogenesis in mice that over-
Dix DJ, Allen JW, Collins BW, Mori C, Nakamura N, Poorman-Allen P, express the RNA-binding protein HuR. EMBO Rep 4, 394–399.
Goulding EH, Eddy EM (1996). Targeted gene disruption of Hsp70-2 Liu D, Matzuk MM, Sung WK, Guo Q, Wang P, Wolgemuth DJ (1998). Cyclin
results in failed meiosis, germ cell apoptosis, and male infertility. Proc A1 is required for meiosis in the male mouse. Nat Genet 20, 377–380.
Natl Acad Sci USA 93, 3264–3268. Lopez de Silanes I, Zhan M, Lal A, Yang X, Gorospe M (2004). Identification
Dix DJ, Allen JW, Collins BW, Poorman-Allen P, Mori C, Blizard DR, of a target RNA motif for RNA-binding protein HuR. Proc Natl Acad Sci
Brown PR, Goulding EH, Strong BD, Eddy EM (1997). HSP70-2 is USA 101, 2987–2992.
required for desynapsis of synaptonemal complexes during meiotic Ma WJ, Cheng S, Campbell C, Wright A, Furneaux H (1996). Cloning and
prophase in juvenile and adult mouse spermatocytes. Development characterization of HuR, a ubiquitously expressed Elav-like protein. J
124, 4595–4603. Biol Chem 5, 8144–8151.
Eddy EM (1999). Role of heat shock protein HSP70-2 in spermatogenesis. Mahadevaiah SK, Turner JM, Baudat F, Rogakou EP, de Boer P,
Rev Reprod 4, 23–30. Blanco-Rodriguez J, Jasin M, Keeney S, Bonner WM, Burgoyne PS
Galban S et al. (2008). RNA-binding proteins HuR and PTB promote (2001). Recombinational DNA double-strand breaks in mice precede
the translation of hypoxia-inducible factor 1alpha. Mol Cell Biol 28, synapsis. Nat Genet 27, 271–276.
93–107. Matzuk MM, Lamb DJ (2008). The biology of infertility: research advances
Gallardo T, Shirley L, John GB, Castrillon DH (2007). Generation of a and clinical challenges. Nat Med 14, 1197–1213.
germ cell-specific mouse transgenic Cre line, Vasa-Cre. Genesis 45, Mukherjee N, Lager PJ, Friedersdorf MB, Thompson MA, Keene JD (2009).
413–417. Coordinated posttranscriptional mRNA population dynamics during
Ghosh M, Aguila HL, Michaud J, Ai Y, Wu MT, Hemmes A, Ristimaki A, T-cell activation. Mol Syst Biol 5, 288.
Guo C, Furneaux H, Hla T (2009). Essential role of the RNA-binding Myer VE, Fan XC, Steitz JA (1997). Identification of HuR as a protein impli-
protein HuR in progenitor cell survival in mice. J Clin Invest 119, cated in AUUUA-mediated mRNA decay. EMBO J 16, 2130–2139.
3530–3543. Nguyen Chi M, Chalmel F, Agius E, Vanzo N, Khabar KS, Jegou B, Morello
Gouble A, Grazide S, Meggetto F, Mercier P, Delsol G, Morello D (2002). D (2009). Temporally regulated traffic of HuR and its associated ARE-
A new player in oncogenesis: AUF1/hnRNPD overexpression leads to containing mRNAs from the chromatoid body to polysomes during
tumorigenesis in transgenic mice. Cancer Res 62, 1489–1495. mouse spermatogenesis. PLoS One 4, e4900.
Govin J, Caron C, Escoffier E, Ferro M, Kuhn L, Rousseaux S, Eddy EM, Nickerson HD, Joshi A, Wolgemuth DJ (2007). Cyclin A1-deficient mice
Garin J, Khochbin S (2006). Post-meiotic shifts in HSPA2/HSP70.2 lack histone H3 serine 10 phosphorylation and exhibit altered aurora B
chaperone activity during mouse spermatogenesis. J Biol Chem 281, dynamics in late prophase of male meiosis. Dev Biol 306, 725–735.
37888–37892. Parvinen M (2005). The chromatoid body in spermatogenesis. Int J Androl
Grivna ST, Pyhtila B, Lin H (2006). MIWI associates with translational machin- 28, 189–201.
ery and PIWI-interacting RNAs (piRNAs) in regulating spermatogenesis. Quenet D, Mark M, Govin J, van Dorsselear A, Schreiber V, Khochbin S,
Proc Natl Acad Sci USA 103, 13415–13420. Dantzer F (2009). Parp2 is required for the differentiation of post-meiotic
Honke K et al. (2002). Paranodal junction formation and spermatogenesis germ cells: identification of a spermatid-specific complex containing
require sulfoglycolipids. Proc Natl Acad Sci USA 99, 4227–4232. Parp1, Parp2, TP2 and HSPA2. Exp Cell Res 315, 2824–2834.
Iguchi N, Tobias JW, Hecht NB (2006). Expression profiling reveals meiotic Steger K (1999). Transcriptional and translational regulation of gene expres-
male germ cell mRNAs that are translationally up- and down-regulated. sion in haploid spermatids. Anat Embryol (Berl) 199, 471–487.
Proc Natl Acad Sci USA 103, 7712–7717. Steger K (2001). Haploid spermatids exhibit translationally repressed
Katsanou V, Milatos S, Yiakouvaki A, Sgantzis N, Kotsoni A, Alexiou M, mRNAs. Anat Embryol (Berl) 203, 323–334.
Harokopos V, Aidinis V, Hemberger M, Kontoyiannis DL (2009). The Tsai-Morris CH, Sheng Y, Lee E, Lei KJ, Dufau ML (2004). Gonadotropin-reg-
RNA-binding protein Elavl1/HuR is essential for placental branch- ulated testicular RNA helicase (GRTH/Ddx25) is essential for spermatid
ing morphogenesis and embryonic development. Mol Cell Biol 29, development and completion of spermatogenesis. Proc Natl Acad Sci
2762–2776. USA 101, 6373–6378.
Katsanou V, Papadaki O, Milatos S, Blackshear PJ, Anderson P, Kollias G, Turner JM, Burgoyne PS, Singh PB (2001). M31 and macroH2A1.2 colocalise
Kontoyiannis DL (2005). HuR as a negative posttranscriptional modulator at the pseudoautosomal region during mouse meiosis. J Cell Sci 114,
in inflammation. Mol Cell 19, 777–789. 3367–3375.
Kawamata M, Nishimori K (2006). Mice deficient in Dmrt7 show infertil- Vidal F, Sage J, Cuzin F, Rassoulzadegan M (1998). Cre expression in
ity with spermatogenic arrest at pachytene stage. FEBS Lett 580, primary spermatocytes: a tool for genetic engineering of the germ line.
6442–6446. Mol Reprod Dev 51, 274–280.
Kimmins S, Sassone-Corsi P (2005). Chromatin remodelling and epigenetic Yang G, Zhang YL, Buchold GM, Jetten AM, O’Brien DA (2003). Analysis
features of germ cells. Nature 434, 583–589. of germ cell nuclear factor transcripts and protein expression during
Kotaja N, Bhattacharyya SN, Jaskiewicz L, Kimmins S, Parvinen M, Filipowicz spermatogenesis. Biol Reprod 68, 1620–1630.
W, Sassone-Corsi P (2006). The chromatoid body of male germ cells: Yang J, Medvedev S, Yu J, Tang LC, Agno JE, Matzuk MM, Schultz RM,
similarity with processing bodies and presence of Dicer and microRNA Hecht NB (2005). Absence of the DNA-/RNA-binding protein MSY2 results
pathway components. Proc Natl Acad Sci USA 103, 2647–2652. in male and female infertility. Proc Natl Acad Sci USA 102, 5755–5760.

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