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Dicer1
Depletion in Male Germ Cells Leads to Infer tility
Due to Cumulative Meiotic and Spermiogenic Defects
Yannick Romero
1
, Oliver Meikar
2
, Marilena D. Papaioannou
1
,Be
´
atrice Conne
1
, Corinne Grey
3
, Manuel a
Weier
4
, Franc¸ois Pralong
5
, Bernard De Massy
3
, Henrik Kaessmann
4
, Jean-Dominique Vassalli
1
, Noora
Kotaja
2
, Serge Nef
1
*
1 Department of Genetic Medicine and Development, University of Geneva Medical School, University of Geneva, Geneva, Switzerland, 2 Department of Physiology,
Institute of Biomedicine, University of Turku, Turku, Finland, 3 Institut de Ge
´
ne
´
tique Humaine, IGH - CNRS, Montpellier, France, 4 Center for Integrative Genomics,
University of Lausanne, Lausanne, Switzerland, 5 Department of Internal Medicine, University Hospital, Lausanne, Switzerland
Abstract
Background:
Spermatogenesis is a complex biological process that requires a highly specialized control of gene expression.
In the past decade, small non-coding RNAs have emerged as critical regulators of gene expression both at the
transcriptional and post-transcriptional level. DICER1, an RNAse III endonuclease, is essential for the biogenesis of several
classes of small RNAs, including microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), but is also
critical for the degradation of toxic transposable elements. In this study, we investigated to which extent DICER1 is required
for germ cell development and the progress of spermatogenesis in mice.
Principal Findings:
We show that the selective ablation of Dicer1 at the early onset of male germ cell development leads to
infertility, due to multiple cumulative defects at the meiotic and post-meiotic stages culminating with the absence of
functional spermatozoa. Alterations were observed in the first spermatogenic wave and include delayed progression of
spermatocytes to prophase I and increased apoptosis, resulting in a reduced number of round spermatids. The transition
from round to mature spermatozoa was also severely affected, since the few spermatozoa formed in mutant animals were
immobile and misshapen, exhibiting morphological defects of the head and flagellum. We also found evidence that the
expression of transposable elements of the SINE family is up-regulated in Dicer1-depleted spermatocytes.
Conclusions/Significance:
Our findings indicate that DICER1 is dispensable for spermatogonial stem cell renewal and
mitotic proliferation, but is required for germ cell differentia tion thr ough the meiotic and haploid phases of
spermatogenesis.
Citation: Romero Y, Meikar O, Papaioannou MD, Conne B, Grey C, et al. (2011) Dicer1 Depletion in Male Germ Cells Leads to Infertility Due to Cumulative Meiotic
and Spermiogenic Defects. PLoS ONE 6( ): e25241. doi:10.1371/journal.pone.0025241
Editor: John J. Rossi, Beckman Research Institute of the City of Hope, United States of America
Received July 4, 2011; Accepted August 29, 2011; Published
Copyright: ß 2011 Romero et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This project is supported by grant no. 3100A0-119862 to SN from the Swiss National Science Foundation. CG and BdM are supported by grants from
the Centre National de la Recherche Scientifique and the Agence Nationale de la Recherche (ANR-09-BLAN-0269-01). NK and OM are supported by the Acade my
of Finland, Emil Aaltonen Foundation and Turku Graduate School of Biomedical Sciences. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: serge.nef@unige.ch
Introduction
Spermatogenesis is a complex biological process that involves
the proliferation and differentiation of diploid spermatogonia into
mature haploid spermatozoa. This process is divided into three
sequential phases: first, the mitotic proliferation of spermatogonia
gives rise to primary spermatocytes. These then undergo two
meiotic divisions to generate haploid spermatids. The final phase,
spermiogenesis, involves the morphological metamorphosis of
early round spermatids into mature spermatozoa. As might be
expected, the production of mature sperm requires a highly
specialized control program of gene expression at both the
transcriptional and post-transcriptional levels [1].
Small RNA molecules are important regulators of mRNA
transcription, stability, turnover, processing, storage and transla-
tion (for review see [2]). These small RNAs can be classified into
different categories based on their biogenesis, mechanism of action
and function, and include microRNAs (miRNAs), endogenous
small interfering RNAs (endo-siRNAs) and piwi-interacting RNAs
(piRNAs). There is increasing evidence that small RNA-directed
gene silencing pathways are essential for normal spermatogenesis
[3,4]. Not only have a large set of miRNAs [5,6] and piRNAs
[7,8,9] been identified in male germ cells, but all essential
members of the RNA interference (RNAi) machinery, including
DROSHA, DICER1, AGO2 (EIF2C2), PIWIL1(MIWI), PIWIL4
(MIWI2) and PIWIL2 (MILI) to name but a few, are expressed in
meiotic and post-meiotic germ cells [10,11].
DICER1 is a conserved RNAse III endonuclease that is essential
for the processing of several classes of small RNAs, including
miRNAs and endo-siRNAs, as well as for the degradation of toxic
transposable elements [12,13]. DICER1 has been found in nearly
all organisms, and multiple studies have shown its essential role in
animal development [14,15,16,17,18,19,20]. The functional rele-
vance of DICER1 and miRNAs in spermatogenesis is only just
PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e25241
October 5, 2011
10
beginning to be unravelled. Selective ablation of Dicer1 in Sertoli
cells leads to infertility, due to the complete absence of spermatozoa.
More precisely, progressive testicular degeneration results from the
defective maturation of Sertoli cells and their incapacity to properly
support meiosis and spermiogenesis [21,22]. However, the role of
DICER1 in the male germ cell lineage in general, and specifically
during spermatogenesis, is not as clear. Two groups have used Cre
recombinase-expressing mice, TNAP-Cre (Tissue Non-Specific
Alkaline Phosphatase) in an attempt to generate animals in which
Dicer1 is absent in germ cells [23,24]. Hayashi and colleagues found
that germ cell-specific deletion of Dicer1 causes a defect in
proliferation of male gonocytes and late adult infertility, likely due
to spermatogenic arrest [23]. Maatouk and colleagues found that
these mutant mice were subfertile, due to defects in both sperm
motility and the transition from round to elongating spermatids
[24]. Importantly, these results should be interpreted with caution as
the TNAP-Cre transgenic mouse is neither fully penetrant (only
,50% of germ cells express Cre), nor specific to germ cells. It should
also be emphasized that the expression of TNAP-Cre begins as early
as E10 [25], so that the primary effects appear in the primordial
germ cell (PGC) population. It is therefore difficult to precisely
interpret the mechanism and timing of any spermatogenesis defects
occurring in the adult.
To investigate the precise roles of germ-cell DICER1 during
spermatogenesis and to overcome the above-mentioned caveats of
previous studies, we developed a mouse model in which the Dicer1
gene was inactivated in a specific and fully penetrant manner in
the male germ cell lineage. Our results indicate that Dicer1 is
required for normal spermatogenesis, since the deletion of Dicer1 in
male germ cells led to multiple defects in meiosis and
spermiogenesis resulting in the absence of functional spermatozoa
and complete infertility.
Results
Germ cell-specific deletion of Dicer1 and miRNAs in Ddx4-
Cre;Dcr1
fx/fx
testes
To investigate the functional relevance of DICER1 in spermato-
genesis, we disrupted Dicer1 in the male germ line by combining a
conditional Dicer1 allele (Dcr1
fx
) [15] with a Ddx4(MVH or Vasa)
promoter-driven transgenic Cre line [26]. This Ddx4-Cre transgene
has been reported to induce specific Cre-mediated recombination in
.90% and .97% of spermatogonia by embryonic day (E)18 and
postnatal day (P)3, respectively [26]. When Ddx4-Cre; Dcr1
fx/wt
males
were mated with Dcr1
fx/fx
females, we observed that only ,11% of
the offspring were Ddx4-Cre;Dcr1
fx/fx
mutant males, instead of the
expected ratio of 25% (for details see Table S1). This discrepancy
from the Mendelian ratio is a consequence of early recombination
events at the 1- or 2-cell stage, as previously described [26], leading
to global embryonic Cre-mediated deletion of Dicer1 and lethality
around E7.5 [15,27]. In the remaining viable Ddx4-Cre;Dcr1
fx/fx
males, Dcr1 exon 24, which encodes most of the second RNAse III
domain [15], was efficiently and specifically deleted in the germ cell
lineage: using elutriated fractions containing spermatocytes isolated
from P60 control and mutant Ddx4-Cre;Dcr1
fx/fx
mice, we found that
levels of Dcr1 transcripts that contained exon 24 were reduced by
,93%; a proportion corresponding to the purity of the spermato-
cyte fractions (Fig. 1A, B). Furthermore, qRT-PCR showed
complete loss of miR-34c and miR-184, two miRNAs expressed
specifically in spermatocytes/spermatids ([28], and H. Kaessmann,
personal communication; Fig. 1C). Together, these data show a
complete loss of Dicer1 and miRNA biogenesis in the male germ-cell
lineage.
Germ cell-s pecific deletion of Dicer1 resulted in reduced
testis size and sperm count, and complete male infertility
Ddx4-Cre;Dcr1
fx/fx
males were viable and grew normally displaying
normal external genitalia, when compared to control littermates. At
P60, testes in which Dicer1 was depleted in germ cells showed a
reduction in size (compare Fig. 2A and B with C)anda55%
decrease in weight compared to control Ddx4-Cre;Dcr1
fx/wt
and
Dcr1
fx/fx
littermates (10767mgversus23869 mg and 196611 mg
respectively, for details see Table S2 and Fig. 2G). Internal
reproductive organs (i.e. seminal vesicles) were normally developed
and, importantly, plasma testosterone levels were unchanged
between experimental and control groups (Table S2). While
control Ddx4-Cre;Dcr1
fx/wt
and Dcr1
fx/fx
males were fertile, Ddx4-
Cre;Dcr1
fx/fx
mutant males were found to be sterile, although they
were sexually active and produced vaginal plugs in female partners
(data not shown). Histological analysis and sperm count in mutant
individuals revealed that sperm production was drastically altered in
the mutant testis. Indeed, mature spermatozoa were rarely found in
Figure 1. Germ cell-specific deletion of
Dicer1
and miRNA depletion in
Ddx4-Cre;Dcr1
fx/fx
testes. (A) Diagram of exons 22 to 24 of the
Dcr1
fx/fx
(+/+) and Ddx4-Cre;Dcr1
fx/fx
(2/2) alleles; the protein domains encoded by each exon are also noted. Exon 24 is flanked with loxP sites (black
triangles), and excision occurs upon Ddx4-Cre recombinase expression. The primer pairs used in real-time RT-PCR for quantifying the excision of exon
24 (green) compared to exon 23 (red) are shown. (B) Histogram showing the exon 24 to exon 23 ratio of Dicer1 mRNA in elutriated P60 spermatocytes
isolated from +/+ (n = 3) and 2/2 (n = 3) testes; (C) Graph showing the expression of spermatocyte-specific miRNAs. Transcript abundance was
quantified by real-time RT-PCR and normalized to 18s rRNA (B), U6 (C). Results are mean6SEM, ***p,0.0001 versus controls.
doi:10.1371/journal.pone.0025241.g001
Dicer in Spermatogenesis
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the tubule’s lumen due to a near complete absence of elongated
spermatids (Fig. 2F). As a consequence, epididymal ducts were
almost completely devoid of mature spermatozoa, whereas
exfoliated germ cells were found in the lumen of mutant
epididymides (data not shown). Finally, epididymal sperm count
analysis revealed a ,99% decrease in the number of spermatozoa in
Ddx4-Cre;Dcr1
fx/fx
males compared to control Ddx4-Cre;Dcr1
fx/wt
and Dcr1
fx/fx
littermates (0.14160.041 *10
6
/ml versus 8.3960.5
*10
6
/ml and 10.5861.175 *10
6
/ml respectively; Fig. 2H and
Table S2).
Dicer1 mutant mice exhibit abnormal seminiferous
tubular cell association, germ cell apoptosis and
organization defects
Adult seminiferous tubules contain several types of germ cells;
each cell type is layered within the tubule in a centripetal manner
and germ cells are arranged in typical cellular associations within
the seminiferous tubules known as stages of the seminiferous
epithelium cycle. In mice, a spermatogenetic cellular association is
divided into 12 stages (from I to XII; [29]). An in depth analysis of
the mutant seminiferous epithelium histology and cytology
revealed multiple defects. First, the relative proportions of the
different populations of germ cells are clearly unbalanced
(Fig. 2M, N and O). Using propidium iodide incorporation
followed by flow cytometry analysis, we found that the proportion
of haploid (1n) cells was reduced by ,30%, while tetraploid (4n)
cells showed a 2.5-fold increase in Ddx4-Cre;Dcr1
fx/fx
males
(Fig. 2I). This result corroborates our analysis of the mutant
testis histology, in which almost no elongated spermatids were
found (Fig. 2N). Secondly, vacuoles (Fig. 2M, arrows) were
numerous in the mutant epithelium, likely due to germ cell
sloughing, as were the Sertoli cell cytoplasmic extensions in the
lumen (Fig. 2M, asterisk). As a possible consequence of these
multiple cellular defects, we observed a reduction in the small
diameter of mutant seminiferous tubules when compared to
Dcr1
fx/fx
littermates (12362 mm versus 16768 mm, Table S2).
Another important characteristic of mutant seminiferous tubules
was the disturbance of the synchronization of germ cell
associations (Fig. 2O). For instance, we observed leptotene
spermatocytes (Fig. 2O, black arrowheads), which are ordinarily
found in stage IX–X tubules, associated with round spermatids
(Fig. 2O, red arrowhead), normally found in stage I–VIII tubules.
These observations prevented the clear identification of specific
tubule stages in Dicer1 mutant testes. Interestingly, although global
germ cell organization was completely disturbed, Sertoli cell
nuclear organization and number appeared normal (Fig. S1;
Table S1).
The significant reduction of post-meiotic germ cells (Fig. 2I)in
mutant seminiferous tubules suggested that germ cell survival may
be affected in the absence of DICER1. A TUNEL assay revealed
that apoptosis was 8-fold higher in mutant testes compared to
control seminiferous tubules (11568 apoptotic cells/20 tubules
Figure 2. Reduction in testis size, apoptosis and near complete
absence of mature spermatozoa in
Ddx4-Cre;Dcr1
fx/fx
(
2
/
2
)
testes. At P60, testes from 2/2 (n = 16) mice (C) showed a 55%
reduction (G) in weight compared to control Dcr1
fx/fx
(+/+; n = 11) (A)
and Ddx4-Cre;Dcr1
fx/wt
(+/2; n = 6) (B) littermates. H&E staining of testes
sections (D–F) revealed near complete absence of mature spermatozoa
and elongated spermatids in 2/2 testes. (H) Epididymal sperm count
analysis showed a ,99% decreased in 2/2 epididymides. (I) DNA
content histogram of P60 +/+ (upper panel) and 2/2 (lower panel)
testes sorted by FACS using propidium iodide. (J–L) H&E staining of +/+
testes showing stage I–II (J), II–III (K) and XI (L) seminiferous tubules. (M–
O) 2/2 tubules are devoid of elongated spermatids and show
numerous seminiferous epithelium defe cts such as vacuolization
(arrows), and Sertoli cell cytoplasmic lumen extensions (asterisk). Sparse
(2–3) round spermatids (arrowhead in M), and zygotene spermatocytes
(arrowhead in N) were found in tubules. (O) Germ cell association is
disturbed in tubules as shown by the presence of leptotene
spermatocyte (black arrowhead) and (2–3) few round spermatids (red
arrowhead). Global TUNEL/eosin staining of P60 +/+ (P) and 2/2 (Q)
testes revealed massive apoptosis (brown nuclei) in some specific 2/2
seminiferous tubules. Histological analysis indicated that meiotic cells
are the most abundant apoptotic population found in 2/2 (S). (T) The
overall number of TUNEL-positive cells is increased ,8-fold in 2/2
compared to +/+ individuals. Results are mean 6SEM, *p,0.05,
**p,0.01, ***p,0.001 versus controls. ns: not significant. Scale bar:
50
mm (A–F).
doi:10.1371/journal.pone.0025241.g002
Dicer in Spermatogenesis
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versus 1561 apoptotic cells/20 tubules and Fig. 2T). Interestingly,
apoptotic cells were not distributed evenly but usually localized in
specific seminiferous tubules (Fig. 2Q and S). Histological analyses
involving double immunostaining for TUNEL and a subset of germ
cell markers (cH2AX (phosphorylated H2AX), H3K9me3 (tri-
methylated Lysine9 Histone 3), alpha tubulin or pH 3 (phosphor-
ylated-Ser10 Histone 3)), suggested that apoptotic cells mainly
comprise pachytene spermatocytes (Fig. S2 and data not shown).
Furthermore, aging mutant testes, at P180, showed high apoptotic
cell numbers with extensive tubular defects, although Sertoli Cell
Only (SCO) tubules were not observed (Fig. 3D and H). In short,
our data indicate that depletion of DICER1 RNAse activity in the
germinal compartment of the testis leads to significant defects in
spermatogenesis, affecting both the survival and differentiation of
male post-mitotic germ cells.
Ddx4-Cre;Dcr1
fx/fx
males show meiotic defects during
spermatogenesis
To further characterize the events that lead to infertility in
Ddx4-Cre;Dcr1
fx/fx
mutant mice, we compared the development of
control and mutant testes during the early phases of the first
spermatogenic wave, from P5 to P21. No discernible abnormality
was found in mutant testes during the mitotic phase of
spermatogonia A (P5), when spermatogonia B enter meiosis I
(P10) (Fig. S3), or when spermatocytes are in the zygotene/
pachytene transition (P12) (Fig. 3A and E). The first histological
abnormalities began to appear at P15 with the presence of germ
cell sloughing and Sertoli cell cytoplasmic extensions (Fig. 3B and
F). At this stage, apoptosis also increased by ,2.5-fold in mutant
testes in comparison with control individuals (416 1 apoptotic
cells/20 tubules versus 1663 apoptotic cells/20 tubules respec-
tively, Fig. 3J). By P21, in wild-type testis, meiosis I is complete
and round spermatids begin to differentiate, which was not the
case is in mutant testis. A difference in testis weight also became
apparent (23.7563.1 mg versus 51.1166 mg respectively,
Fig. 3I), which reflects seminiferous tubule deterioration and
increased apoptosis (2163 apoptotic cells/20tubules versus 862
apoptotic cells/20 tubules, Fig. 3J).
In order to better characterize the apoptotic cell population
present within the developing tubules, we performed electronic
microscopy (EM) of control and mutant testes at the end of
prophase I (P21; Fig. 4A, B, C, D). In Dicer1-depleted germ cell
individuals, most of the pachytene spermatocytes showed apo-
ptotic features (e.g. misshapen nuclear membrane and fragmented
heterochromatin, arrows and arrowheads in Fig. 4D, respective-
ly), and those that survived prophase I rarely reached the
metaphase step. In addition, metaphase plates appeared abnormal
(data not shown). Zygotene (not shown) and pachytene spermato-
cyte nuclei in Ddx4-Cre;Dcr1
fx/fx
mutant tubules seemed to contain
abnormally large perinuclear heterochromatin areas (arrowheads
Figure 3. Tubular defects appear as early as P15 in
Ddx4-Cre;Dcr1
fx/fx
mutant testes. H&E staining of control (+/+) (A, B, C, D) and mutant
(2/2) (E, F, G, H) testes at P12 (A&E), P15 (B&F) ,P21 (C&G) and P180 (D&H). The first anatomical defects including vacuolization (asterisk), apoptosis
and germ cell disorganization appeared at P15 (F) and worsened by P21 (G). In aging individuals, at P180 (D&H), tubular histology is strongly affected,
with numerous vacuoles and few germ cells remaining. Reduced testis weight ratio (I) correlated with an increase in apoptotic rate (J) during the first
spermatogenic wave. For I and J, numbers of animals were n = 5 minimum for each genotype. Results are mean 6SEM, *p,0.05, **p,0.01,
***p,0.001 versus controls. ns: not significant.
doi:10.1371/journal.pone.0025241.g003
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PLoS ONE | www.plosone.org 4 ber 2011 | Volume 6 | Issue | e25241Octo 10
in Fig. 4B and C). We used ser-10 phosphorylation of histone 3
as a specific marker of cells in metaphase (i.e. spermatogonia and
late meiotic cells; [11]) to compare mutant individuals to control
littermates at P60. This revealed an increased number of cells in
meiotic metaphase in the tubules of the mutant group (Fig. S4).
These findings indicate that spermatocytes may be partially
blocked in the late prophase I phase in Ddx4-Cre; Dcr1
fx/fx
mice.
Since pachytene spermatocytes seemed to be affected by the
deletion of Dicer1, we analysed the expression of cH2AX at
P12, during the zygotene to pachytene transition. During the
normal zygotene phase, cH2AX localizes to the whole nucleus,
but in the subsequent pachytene stage remains only in the XY
body (or sex body) where X and Y-linked genes undergo transcrip-
tional silencing [30]. At this stage, we found that the number of
pachytene spermatocyte-containing tubules was reduced by ,2
-fold (3.8860.4 versus 8.1660.1 XY body-positive tubules/20
tubules respectively; Fig. 4G) in mutants compared to control
littermates (arrowheads in Fig. 4E and F). Chromosomal spread
preparations from germ cells collected at P12 confirmed the
reduced number of pachytene spermatocytes in mutant testes (Fig. 4
H, I, J), whereas, pre-leptotene and leptotene cells were more
numerous compared to controls. No difference in the meiotic
process was observed, such as synapsis (SYCP3 staining) or repair
(DMC1 punctuate staining). Taken together, these data show
that the loss of Dicer1 in germ cells severely impairs the first
spermatogenic wave, by delaying the transition from the lepto-
tene to the zygotene/pachytene stages of the first meiotic pro-
phase, and by increasing apoptosis in mid/late pachytene stage
spermatocytes.
Spermiogenesis is impaired in Dicer1 mutant mice
To characterize the morphological defects in the developing
germ cells after meiosis, we performed an EM study on testis
sections at P60. The few spermatids found in Ddx4-Cre;Dcr1
fx/fx
mutant mice displayed various defects; the acrosome of round
spermatids was fragmented in Ddx4-Cre;Dcr1
fx/fx
with multiple
acrosomal granules tethering the nucleus ( Fig. 5C, arrowheads)
instead of a single granule found in control testes (Fig. 5 A,
arrowhead). Interestingly, we found no apparent defects in the
overall morphology and position of the chromatoid body, a cloud-
like structure thought to be the site of RNA processing and/or
storage (white arrows in Fig. 5A and B). The few remaining
Figure 4. Meiotic progression defects in
Ddx4-Cre;Dcr1
fx/fx
(
2
/
2
) mutant testes. Representative transmission electron micrographs from P21
control (A) and mutant (B–D) testes. Note the enlarged perinuclear heterochromatin areas (arrowheads) and the irregular and abnormal nucleus
shape (arrows) in mutant pachytene spermatocytes typical of apoptotic cells. (E, F) Anti-cH2AX staining (red), present in the whole nucleus in early
meiotic stages and restricted to the XY body from pachytene phase, revealed a reduction in the number of tubules containing XY body positive cells
(arrowheads, punctual red staining) within 2/2 testis (F) compared to wild-type (E). DAPI (blue) was used for nuclear staining. (G) shows the
quantification of XY body positive tubules per 20 tubules. Anti-SYCP3 (red), DMC1 (green) and cH2AX (blue) staining of chromosomal spread
preparations from control (H), and mutant (I) P12 testes was used to quantify meiotic prophase I cells. (J) Note the higher number of early meiotic
cells (i.e., Preleptotene and leptotene) and the reduced number of late meiotic cells (i.e. mid/Late zygotene and pachytene) in 2/2 cell preparations
suggesting a delayed progression of germ cells into meiosis. Results are mean 6SEM, ns = not significant, *p,0.05, **p,0.01, ***p,0.001 versus
controls. PL: Pre-leptotene, EL: Early leptotene, M/L L: mid/late leptotene, EZ: early zygotene, M/L Z: mid/late zygotene, P: Pachytene.
doi:10.1371/journal.pone.0025241.g004
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elongated spermatids of mutant animals showed abnormal head
shape and chromatin condensation (red arrows in Fig. 5E and F).
Acrosomes were also misshapen and fragmented (compare
arrowheads in Fig. 5 E and F with D). Finally, mitochondria
displayed abnormal shape and dilated cristae or intermembrane
space, which appeared as a dense structure, compared to control
spermatids (blue arrows, Fig. 5 D, E, F).
Since round and elongated spermatids showed significant
defects in acrosome formation and nuclear condensation we
analysed further the morphology of the remaining epididymal
sperm (Fig. 2 H). All of the few spermatozoon-like structures
found in the epididymides of P60 mutant individuals displayed
drastic morphological defects of the head and flagellum (Fig. 5G,
H, I, J). Using a dye specifically staining mitochondria, we
evidenced a heterogeneous and aberrant localisation of these
organelles in the mutant spermatozoa, with mitochondria
surrounding the nucleus (arrows in Fig. 5L and M)or
mislocalized within the flagella (arrowheads in Fig. 5M). In
addition to the misshapen but normally-sized spermatozoa present
in the mutant sperm, we were able to see what resembled small
pin-head spermatozoa (Fig. 5N; a-tubulin red staining). Never-
theless, these structures were devoid of nuclei and harbour a faint
and sparse mitochondrial staining. None of the mutant sperma-
tozoa were motile when examined by visual inspection with a light
microscope (data not shown). Our results indicate that functional
DICER1 is required for normal spermiogenesis, since its ablation
in germ cells leads to a reduced number of haploid germ cells and
extensive defects in global sperm morphology.
Expression of transposable elements increases in
spermatocytes lacking DICER1 activity
In addition to its essential role in miRNA biogenesis, DICER1
has been implicated in the control of repetitive elements. The
recent discovery that DICER1 is critical for cell survival by
degrading toxic retrotransposon transcripts in retinal pigment
epithelium [12] raised the possibility that DICER1 might fulfil the
same function in the germ cell lineage. To investigate this
possibility, we examined the expression levels of IAP (intracisternal
Figure 5. Spermiogenic and sperm alterations in
Ddx4-Cre;Dcr1
fx/fx
(
2
/
2
) testes. Representative transmission electron micrographs from P60
control (A&D) and mutant (B, C, E & F). In round spermatids (A–C), chromatoid body is normally shaped and located near the nucleus (red arrows),
whereas acrosome is fragmented in mutant (arrowheads). In elongated spermatids (D–F), nuclear shape (red arrows), chromatin condensation, and
acrosome (arrowheads) are abnormal in 2/2 (E and F) mice. Mitochondria showed hyperplasia of the intermembrane space or cristae (blue arrows,
E). Scale bar: 2
mm. H&E staining of epididymal sperm spreads of +/+ (G) and 2/2 (H, I and J) adult mice. In contrast to +/+ animals (G), spermatozoa
of 2/2 mice exhibited multiple defects such as head morphology, mid-piece defects (H and I) and reduced overall size (J). Black arrowhead shows
the ectopic localization of the mid-piece in (I). Scale bars: 10
mm. Immunostaining of epididymal sperm spreads of control +/+ (K) and 2/2 (L, M and
N) mice using DAPI staining to label nuclear DNA (blue), Mitotracker as a mitochondrial marker (green) and anti-a-tubulin to label the flagellum (red).
Spermatozoa of 2/2 mice exhibited head morphology defects (L, M and N). White arrows show that some mutant spermatozoa display abnormal
shaped nuclei and co-localized ectopic mitochondrial staining. White arrowhead in (M) indicates the ectopic localization of the mid-piece
mitochondria compared to control individuals (K). Images were taken with a 406 objective.
doi:10.1371/journal.pone.0025241.g005
Dicer in Spermatogenesis
PLoS ONE | www.plosone.org 6 ber 2011 | Volume 6 | Issue | e25241Octo 10
A particle element), LINE1 (long interspersed nuclear element 1)
and SINE B1 and B2 (short interspersed nuclear element B1 and
B2) by quantitative RT-PCR in two groups of P60 elutriated germ
cells (Fig. 6). While transcript levels for transposable elements
LINE1 and IAP were unaffected, we observed a significant up-
regulation of SINE B1 and SINE B2 transcripts in enriched
populations of leptotene/zygotene/mid-pachytene spermatocytes
(1.8-fold increase for SINE B1 and 1.5- fold for SINE B2) and late-
pachytene/diplotene spermatocytes (1.8-fold increase for SINE B1
and 1.6- fold for SINE B2) prepared from mutant testes. Whether
the ,1.5–2 fold increase in SINE transcripts affect germ cell
survival and/or spermatogenesis remains an open question.
Discussion
DICER1 is essential for the processing of several classes of small
non-coding RNAs, including miRNAs and endo-siRNAs, and for
the degradation of retrotransposon transcripts. In this study we
used the Cre/Lox system to conditionally inactivate Dicer1 in male
germ cells. We found that DICER1 is not required for
spermatogonia stem cell renewal and mitotic proliferation, but is
essential for the meiotic and haploid phases of spermatogenesis.
The first defects appeared as early as P12 when we observed a
delayed progression of prophase I, followed at P15 by an increase
in apoptosis of mid/late pachytene stage spermatocytes. Not all
spermatocytes died by apoptosis, as a small fraction of these
completed meiosis. The transition from round spermatids to
mature spermatozoa was also severely affected since the few
spermatozoa that did form in mutant animals (at 1% of control
levels) were immobile and misshapen, exhibiting irreversible
morphological defects due to disturbance in acrosome formation,
nuclear condensation in the few remaining round and elongated
spermatids.
The loss of Dicer1 in male germ cells thus led to a complex and
severe phenotype, which likely represents the sum of low impact
defects that accumulate at various meiotic and post-meiotic stages.
This type of phenotype, resulting from the cumulative impairment
of several developmental cellular processes and increased cell
death, has in fact been observed in numerous conditional Dicer1
knockouts [15,16,17,31]. Conditional removal of Dicer1 in the
male germ cell lineage led to various phenotypes whose severity
depends on the efficiency of Cre recombination and/or the stage
at which DICER1 has been inactivated. Initial studies using a
Tnap-Cre transgene has been problematic due to early inactivation
(E10) as well as the partial (50%) and non-specific Cre-mediated
expression [25]. As a result of inefficient Cre recombination, Tnap-
Cre;Dicer
fx/fx
mice were found either fertile or subfertile and it was a
rather difficult task to evaluate precisely the impact of DICER1 on
sperm production [23,24]. The use of a specific, fully penetrant
Cre line such as the Ddx4-Cre transgene was instrumental to
investigate in depth the function of DICER1 in spermatogenesis.
Interestingly, ablation of DICER1 in germ cells at different
development stages using two different spermatogonia-specific Cre
mice (the Ddx4-Cre and Ngn3-Cre) revealed a gradation in severity
of the reproductive phenotype. Ddx4-Cre and Ngn3-Cre transgenes
are both efficient at deleting DICER1 in gonocytes at ,P0 and in
spermatogonia at ,P5–P7, respectively (Korhonenet al, co-
submitted manuscript). However, germ cell-specific removal of
DICER1 using a Ngn3-Cre transgenic resulted in a phenotype
slightly milder than the Ddx4-Cre;Dicer1
fx/fx
described here. Both
Figure 6. Deregulation of transposable elements in
Ddx4-Cre;Dcr1
fx/fx
(
2
/
2
) mutant spermatocytes. Quantitative real-time PCR performed
on elutriated germ cell fractions enriched either in leptotene/zygotene/early pachytene spermatocytes (A) or late pachytene/diplotene
spermatocytes (B) originating from +/+ and 2/2 testes at P60. Results are mean 6SEM, ns = not significant, *p,0.05, **p,0.01, ***p,0.001
versus controls.
doi:10.1371/journal.pone.0025241.g006
Dicer in Spermatogenesis
PLoS ONE | www.plosone.org 7 ber 2011 | Volume 6 | Issue | e25241Octo 10
mutant males were infertile and exhibited similar defects in
spermato- and spermiogenesis but the meiotic phenotype was
more severe and precocious in Ddx4-Cre;Dicer1
fx/fx
animals with
defects in prophase I progression and a lower number of
remaining spermatozoa. Since small RNAs are stable molecules,
we hypothesize that ablation of DICER1 in gonocytes at ,P0 will
deplete the pool of the remaining miRNAs or endo-siRNAs more
extensively than in P5–P7 spermatogonia thus affecting spermato-
genesis and sperm production more severely.
There is no doubt that DICER1 is essential for spermatogenesis
and sperm production. However, emerging from these findings is
the essential question of which DICER1 activity is crucial for germ
cell function. In other words, the relative contribution of miRNAs
versus other DICER1-dependent non-coding RNAs to the
phenotype remains unclear. DICER1 is essential for both endo-
siRNA and canonical miRNA biogenesis, but not for piRNA
production (for a review see [2]). Recent expression profiling
analyses using RNA sequencing technology revealed that male
spermatogenic cells expressed numerous classes of small non-
coding RNAs including miRNAs and endo-siRNAs [5,6].
Whether the infertility phenotype observed in our Ddx4-
Cre;Dcr1
fx/fx
is consecutive either to the loss of endo-siRNAs
and/or miRNAs is still uncertain. While revising this manuscript,
it has just been reported that germ cell inactivation of Dicer1 or
Drosha, an RNase III enzyme required for canonical miRNA
biogenesis but not for endo-siRNA processing, both resulted in
male infertility due to oligozoopsermia or azoospermia caused by
constant depletion of phachytene spermatocytes and spermatids
[6]. The similar phenotype observed in Drosha knockout testes
when compared with the Dicer mutants suggests a role for miRNAs
in regulating spermatogenesis. These results are different to those
found in female germ cells, where both miRNAs and endo-siRNAs
are also present in the developing oocytes [32,33]. While mouse
oocytes lacking Dicer1 arrest in meiosis I due to disorganized
spindles and defects in chromosomal alignment [34,35], oocytes
deficient for Dgcr8, an RNA-binding protein that assists DROSHA
in the processing of microRNAs, mature normally and do not
impair early embryonic development [36]. This striking difference
in phenotype between Dicer1- and Drosha-deficient oocytes suggests
that endo-siRNAs, rather than miRNAs, are essential for oocyte
maturation and the early stages of mammalian development [6].
Another important difference between male and female germ cells
is the timing of defects. In oocytes lacking Dicer1 a specific defect is
observed at metaphase, affecting either the spindle assembly
checkpoint (SAC) and/or the anaphase-promoting complex
(APC/C). Indeed, it was demonstrated that DICER1 is not
required for female germ cell development but only for meiotic
maturation [37]. Our results clearly show that unlike oocytes,
defects in spermatogenesis appear earlier during prophase I.
The chromatoid body (CB) is a dynamic, cloud-like, dense
structure located in the cytoplasm of round spermatids, and is
thought to be the site of RNA processing and/or storage [38]. This
cytoplasmic perinuclear organelle contains various types of RNAs,
including piRNAs, mRNAs and miRNAs as well as various RNA-
binding proteins or proteins involved in RNA processing pathway
(e.g. DDX4, PIWIL1, PIWIL2, DICER1, tudor domain proteins
such as TDRD6 and TDRD7; [39]). The potential role of the CB
in the mRNA regulation [38], together with the multiple
cumulative meiotic and post-meiotic defects found in Dicer1-
depleted germ cells, suggested the hypothesis that the CB structure
and/or its function may be affected in Dicer1-depleted germ cells.
EM studies of testes at P60 revealed that the CB was present, and
normally shaped in round spermatids lacking Dicer1, this suggests
that DICER1 is not essential for the formation of the CB. Whether
its molecular composition and/or its function in RNA regulation is
affected in mutant germ cells still remains unclear.
The mouse and human genomes are crowded with transposable
elements that account for 30 to 40% of their total size [40].
Different silencing mechanisms have been developed in germ cells
to suppress their activities, including DNA methylation [41] and
RNAi-triggered silencing (for a review see [42]). The large set of
piRNAs expressed in spermatocytes highlights the importance of
these small RNAs in protecting male germ cells from transposable
elements [43]. While piRNA biogenesis requires neither
DROSHA nor DICER1, there is substantial evidence that
regulation of transposable element expression includes DICER1-
dependent mechanisms [44,45,46]. Several retrotransposons from
the MT and SINE families were shown to be significantly up-
regulated in mouse oocytes lacking Dicer1 [34]. Recently, DICER1
was demonstrated to be involved in retrotransposon transcript
degradation. Dicer1 ablation in retinal pigmented epithelium (RPE)
induced accumulation of alu/SINE RNAs in both human and
mouse cells, resulting in cytotoxicity and RPE degeneration [12].
Our genetic analysis suggests that the SINE family of retro-
transposons is up-regulated in spermatocytes lacking Dicer1.
Whether this 1.5- to 2-fold increase in SINE expression is
sufficient to affect survival and/or specific stages of spermatogen-
esis remains unclear and requires further analysis. In this
perspective, high throughput RNA sequencing combined with
an in-depth differential proteomic analysis of Dicer1- and Dgcr8-
depleted germ cells would be instrumental for a better compre-
hension of the roles and impact of small non-coding RNAs in the
production of mature sperm.
Materials and Methods
Animals
Dcr1
flox
(Dcr1
fx
) and Ddx4:Cre (Mvh-Cre) mice were kindly
provided by B.D. Harfe and D.H. Castrillon respectively, and
were genotyped as described [15,26]. To achieve selective
inactivation of Dicer1 in germ cells, we mated transgenic male
mice expressing Cre recombinase under the control of the Ddx4
promoter with female mice carrying two floxed Dicer1 alleles in
order to generate 50% Ddx4-Cre;Dcr1
fx/wt
and 50% Dcr1
fx/wt
mice.
Ddx4-Cre;Dcr1
fx/wt
males were mated with Dcr1
fx/fx
females in order
to produce Ddx4-Cre;Dcr1
fx/fx
as well as Dcr1
fx/fx
and Ddx4-
Cre;Dcr1
fx/wt
control littermates. The genetic background of these
mice is mixed C57Bl/6J and SV129. Protocols for the use of
animals were approved by the commission d’Ethique de
l’Expe´rimentation Animale of the University of Geneva Medical
School and the Geneva Veterinarian Office.
Isolation of testicular cells
Male germ cells were obtained from P60 adult mice (n = 3, each
pool of germ cells was isolated from either four pairs of Ddx4-
Cre;Dcr1
fx/fx
mutant testes or two pairs of Dcr1
fx/fx
control testes) by
mechanical disruption and liberase treatment (Roche Applied
Science). Cells were elutriated and separated into multiple
fractions according to [47] and subsequently frozen for further
analysis.
RNA extraction and Quantitative Real-Time PCR
Total RNAs from elutriated germ cells were extracted using
Trizol (Sigma-Aldrich) according to the manufacturer’s instruc-
tions. For further quantification of repetitive elements associated
RNAs, extracted RNA was subjected to DNAse treatment using
the Turbofree DNA kit (Ambion). Quantification of transcript
levels was performed using the Kapa Sybr Fast kit (Kapa
Dicer in Spermatogenesis
PLoS ONE | www.plosone.org 8 ber 2011 | Volume 6 | Issue | e25241Octo 10
Biosystems) with primers for Dicer1 already described in [48],
SINE B1, B2 and LINE L1 RNA primers described in [49] and
IAP primers sequences came from [50]. Taqman miRNA kit
(Applied Biosystems) was used for quantification of miRNAs
according to the manufacturer’s instructions using primers for
miR-34c and miR-184 (for probe sequences see Applied
Biosystems website). Data were analyzed using the 2
2DDCt
method
as described in [51]; normalization was performed with 18s rRNA
levels for repetitive elements and mRNA transcripts, whereas U6
small RNA was used for miRNA level normalization. Each assay
was performed at least in three independent technical and
biological replicates.
Measurement of hormonal plasma levels
Blood was collected by cardiac puncture from P60 mice. Plasma
samples were stored at-20uC and used subsequently to assess the
levels of LH, FSH and testosterone. Hormone levels were
measured in individual adult plasma samples (n = 3–9) from P60
adult mice. LH was measured by RIA, using a commercially
available kit supplied by IDS (LH RIA CT # AHR002, Lie`ge,
Belgium), whereas FSH was measured by IRMA, using a kit from
the same supplier (FSH IRMA CT # AHR004). Testosterone
concentrations were assayed using a kit from MP Biomedicals
(Testo DA kit, CT number 07-189102, Eschwege, Germany).
Intra- and inter-assay coefficients of variation (CVs) of all three
assays were less than 5% and 10%, respectively.
Sperm analysis and Immunofluorescence assays
Epididymal sperm count was performed with sperm extracted
from the caudal epididymis and ductus deferens of adult (P60)
male mice and was analyzed for its concentration as previously
described [52]. Ploidy was assessed on Ddx4-Cre;Dcr1
fx/fx
and
Dcr1
fx/fx
P60 testis using Propidium Iodide incorporation as
described in [53]. Epididymal sperm from Ddx4-Cre;Dcr1
fx/fx
and
Dcr1
fx/fx
was spread on SuperFrost Plus glass slides, fixed
10 minutes at room temperature (RT) with 4% paraformaldehyde
(PFA), then permeabilized in PBS/triton-X100 0.5% and
incubated for 1 hour at RT with anti-alpha-Tubulin (Abcam
clone B-5-1-2, 1:500). For fluorescent staining an Alexa594
secondary antibody (Invitrogen) was used for signal revelation;
nuclei were counterstained using DAPI and Mitotracker (1
mM,
Invitrogen) was used for mitochondrial staining.
Histology and Immunochemistry
Tissues were fixed overnight either in 4% PFA or in Bouin’s
fixative and embedded in paraffin. Five-
mm sections were stained
with haematoxylin and eosin (H&E) or processed for immunohis-
tochemistry (IHC). For IHC analysis, PFA-fixed sections were
incubated overnight at 4uC with the following antibodies: anti-
GATA4 (Santa Cruz Biotechnology, sc-9053, 1:500), anti-
pH 3(Ser10) (Millipore, Cat#06-570, 1:500), anti-cH2AX (Cal-
biochem, Cat#dr-1017, 1:500) and anti-H3K9me3 (Millipore,
Cat#07-523, 1:500). For fluorescent staining, Alexa-conjugated
secondary antibodies (Invitrogen) were used for signal revelation
and sections were counterstained using DAPI. All images were
obtained either with a Zeiss Axioscope microscope and processed
using the AxioVision LE software.
Apoptosis Assays
Apoptotic assays were performed by TdTmediated X-dUTP
nicked labeling (TUNEL) reaction using the Apoptag kit
(Millipore) stained with either DAB chromogen and counter-
stained with eosin, or with Permanent Red or anti-Digoxygenin
coupled to fluorescein and counterstained with DAPI. The
percentage of apoptotic, TUNEL positive cells within seminiferous
tubules was expressed as the average number of apoptotic cells
within 20 seminiferous tubes. A minimum of 100 seminiferous
tubules were counted per testis (5 sections/testis) and at least 3
animals per genotype per age were assessed.
Chromosome Spread Preparation and Immunostaining
Spermatocytes nuclear spreads were prepared and stained as
previously described [54]. The primary antibodies used were:
guinea pig anti-SYCP3 (gift from C. Heyting, 1:25 000), mouse
anti-cH2AX (Upstate, 05-636, 1:25 000) and rabbit anti-DMC1
(Santa Cruz Biotechnology, H100, 1:200). Secondary antibodies
were AlexaFluor-488 and AlexaFluor594 conjugates (Molecular
Probes).
Digital images were obtained by using a cooled CCD camera,
Coolsnap HQ (Photometrics), coupled to a Leica DMRA2
microscope using the same exposure time for all acquisitions.
Each colour signal was acquired as a black-and-white image using
appropriate filter sets and was merged with Photoshop Imaging
software.
Electron microscopy
P60 and P21 testes from Ddx4-Cre;Dcr1
fx/fx
and Dcr1
fx/fx
individuals were fixed in 0.1 M Na Cacodylate with 4%
Glutaraldehyde in PBS. After fixation with 1% osmium tetroxide,
the testes were embedded in epoxy resin (Glycidether 100, Merck).
Selected areas were sectioned, stained with 5% uranyl acetate and
5% lead citrate, and visualized on a JEOL 1200 EX transmission
electron microscope.
Supporting Information
Figure S1 Sertoli cells organization and number are not
affected in testes lacking Dicer1 in the germinal com-
partment. Gata-4 immunostaining (red) revealed the presence of
Sertoli cells nuclei at the edge of tubules in Dcr1
fx/fx
(+/+) control
individuals (A) as well as in Ddx4-Cre;Dcr1
fx/fx
mutant mice (2/2)
(B), indicating that Sertoli cells organization is not affected by
Dicer1 depletion in germ cells. (C) The number of Sertoli cells
within seminiferous tubules is also unaffected (n = 3 animals per
genotype; a minimum of 20 tubules were analyzed per individual).
(TIF)
Figure S2 Apoptotic cell population display pachytene
spermatocytes localisation in Ddx4-Cre;Dcr1
fx/fx
(
2
/
2
)
tubules. At P60, TUNEL-positive cells (green) are found near
late prophase I spermatocytes in Dicer1 mutant as shown by
cH2AX staining of the XY body (red foci, white arrowheads in A
and B). C and D show the localization of prophase I cells at the
edge of the tubule (arrows) and the XY body in round spermatids
(red foci, arrowheads) according the H3K9me3 immunostaining.
TUNEL-positive cells localize between those two populations. E
and F show the presence of few elongating spermatids stained
using anti-alpha-tubulin antibody (red) which are reduced and
disorganized (asterisks) in Dicer1-depleted germ cells tubules.
Cross-sections were counterstained with DAPI.
(TIF)
Figure S3 The early spermatogenic phase is not affect-
ed in Ddx4-Cre;Dcr1
fx/fx
(
2
/
2
) individuals. H&E staining
of P5 (A&B) and P10 (C&D) cross-sections from control (A&C) and
2/2 (B&D) seminiferous tubules. At these stages, no histological
differences were observed between mutant and control testes.
(TIF)
Dicer in Spermatogenesis
PLoS ONE | www.plosone.org 9 ber 2011 | Volume 6 | Issue | e25241Octo 10
Figure S4 Accumulation of metaphasic-like cell popula-
tion in Ddx4-Cre;Dcr1
fx/fx
(
2
/
2
)tubules. At P60, under-
going metaphasic cells are more abundant in Dicer1 mutant as
shown by Histone H3Ser-10 phosphorylated (pH 3) positive cells
(green, A and B). (C) shows a 2.5-fold increase of pH 3 positive
tubules in 2/2 versus +/+ individuals. Cross-sections were
counterstained with DAPI. Results are mean6SEM, *p,0.05,
versus controls.
(TIF)
Table S1 Frequency of Ddx4-Cre;Dcr1
fx/fx
obtained is
lower than expected. Ddx4-Cre;Dcr1
fx/wt
males were mated
with Dcr1
fx/fx
females in order to produce Ddx4-Cre;Dcr1
fx/fx
(2/2)
as well as Dcr1
fx/fx
(+/+) and Ddx4-Cre;Dcr1
fx/wt
(+/2) control
littermates. The genetic background of these mice is mixed. The
expected Mendelian ratio should be 25% for each genotype. Here,
we obtained ,11% of Ddx4-Cre;Dcr1
fx/fx
, probably due to an early
recombination event in some mutant embryos resulting in
embryonic death.
(TIF)
Table S2 Table comparing reproductive and endocrine
measurements between Ddx4-Cre;Dcr1
fx/fx
(
2
/
2
) indi-
viduals compared to Dcr1
fx/fx
(
+
/
+
) and Ddx4-
Cre;Dcr1
fx/wt
(
+
/
2
) control littermates.
(TIF)
Acknowledgments
We would like to thank Chantal Combe´pine, Nicolas Veillard and Mara
Rodrigues for excellent technical assistance, Junior Reynoird for histology
advice and Pierre Calvel for critical reading of the manuscript. We are
grateful to D. Castrillon for the Ddx4:Cre transgenic mice and B.D. Harfe
and M.T. McManus for the Dcr1
fx
mice.
Author Contributions
Conceived and designed the experiments: YR JDV NK SN. Performed the
experiments: YR OM MDP BC CG MW. Analyzed the data: YR OM
MDP CG FP BdM JDV NK SN. Contributed reagents/materials/analysis
tools: MW FP HK. Wrote the paper: YR SN.
References
1. Eddy EM (2002) Male germ cell gene expression. Recent Prog Horm Res 57:
103–128.
2. Kim VN, Han J, Siomi MC (2009) Biogenesis of small RNAs in animals. Nat
Rev Mol Cell Biol 10: 126–139.
3. He Z, Kokkinaki M, Pant D, Gallicano GI, Dym M (2009) Small RNA
molecules in the regulation of spermatogenesis. Reproduction 137: 901–911.
4. Papaioannou MD, Nef S (2010) microRNAs in the testis: building up male
fertility. J Androl 31: 26–33.
5. Buchold GM, Coarfa C, Kim J, Milosavljevic A, Gunaratne PH, et al. (2010)
Analysis of microRNA expression in the prepubertal testis. PLoS One 5: e15317.
6. Song R, Hennig GW, Wu Q, Jose C, Zheng H, et al. (2011) Male germ cells
express abundant endogenous siRNAs. Proc Natl Acad Sci U S A 108:
13159–13164.
7. Aravin A, Gaidatzis D, Pfeffer S, Lagos-Quintana M, La ndgraf P, et al. (2006) A
novel class of small RNAs bind to MILI protein in mouse testes. Nature 442:
203–207.
8. Girard A, Sachidanandam R, Hannon GJ, Carmell MA (2006) A germline-specific
class of small RNAs binds mammalian Piwi proteins. Nature 442: 199–202.
9. Grivna ST, Beyret E, Wang Z, Lin H (2006) A novel class of small RNAs in
mouse spermatogenic cells. Genes Dev 20: 1709–1714.
10. Gonzalez-Gonzalez E, Lopez-Casas PP, Del Mazo J (2008) The expression
patterns of genes involved in the RNAi pathways are tissue-dependent and differ
in the germ and somatic cells of mouse testis. Biochim Biophys Acta.
11. Kimmins S, Crosio C, Kotaja N, Hirayama J, Monaco L, et al. (2007)
Differential functions of the Aurora-B and Aurora-C kinases in mammalian
spermatogenesis. Molecular endocrinology 21: 726–739.
12. Kaneko H, Dridi S, Tarallo V, Gelfand BD, Fowler BJ, et al. (2011) DICER1
deficit induces Alu RNA toxicity in age-related macular degeneration. Nature
471: 325–330.
13. Luense LJ, Carletti MZ, Christenson LK (2009) Role of Dicer in female fertility.
Trends Endocrinol Metab 20: 265–272.
14. Gonzalez G, Behringer RR (2009) Dicer is required for female reproductive
tract development and fertility in the mouse. Mol Reprod Dev 76: 678–688.
15. Harfe BD, McManus MT, Mansfield JH, Hornstein E, Tabin CJ (2005) The
RNaseIII enzyme Dicer is required for morphogenesis but not patterning of the
vertebrate limb. Proc Natl Acad Sci U S A 102: 10898–10903.
16. Harris KS, Zhang Z, McManus MT, Harfe BD, Sun X (2006) Dicer function is
essential for lung epithelium morphogenesis. Proc Natl Acad Sci U S A 103:
2208–2213.
17. Huang CC, Yao HH (2010) Inactivation of Dicer1 in Steroidogenic factor 1-
positive cells reveals tissue-specific requirement for Dicer1 in adrenal, testis, and
ovary. BMC Dev Biol 10: 66.
18. Kobayashi T, Lu J, Cobb BS, Rodda SJ, McMahon AP, et al. (2008) Dicer-
dependent pathways regulate chondrocyte proliferation and differentiation. Proc
Natl Acad Sci U S A.
19. O’Rourke JR, Georges SA, Seay HR, Tapscott SJ, McManus MT, et al. (2007)
Essential role for Dicer during skeletal muscle development. Dev Biol 311:
359–368.
20. Suarez Y, Fernandez-Hernando C, Yu J, Gerber SA, Harrison KD, et al. (2008)
Dicer-dependent endothelial microRNAs are necessary for postnatal angiogen-
esis. Proc Natl Acad Sci U S A 105: 14082–14087.
21. Papaioannou MD, Lagarrigue M, Vejnar CE, Rolland AD, Kuhne F, et al.
(2011) Loss of dicer in sertoli cells has a major impact on the testicular proteome
of mice. Mol Cell Proteomics 10: M900587MCP900200.
22. Papaioannou MD, Pitetti JL, Ro S, Park C, Aubry F, et al. (2009) Sertoli cell
Dicer is essential for spermatogenesis in mice. Dev Biol 326: 250–259.
23. Hayashi K, Chuva de Sousa Lopes SM, Kaneda M, Tang F, Hajkova P, et al.
(2008) MicroRNA biogenesis is required for mouse primordial germ cell
development and spermatogenesis. PLoS One 3: e1738.
24. Maatouk DM, Loveland KL, McManus MT, Moore K, Harfe BD (2008) Dicer1
is required for differentiation of the mouse male germline. Biol Reprod 79:
696–703.
25. Lomeli H, Ramos-Mejia V, Gertsenstein M, Lobe CG, Nagy A (2000) Targeted
insertion of Cre recombinase into the TNAP gene: excision in primordial germ
cells. Genesis 26: 116–117.
26. Gallardo T, Shirley L, John GB, Castrillon DH (2007) Generation of a germ
cell-specific mouse transgenic Cre line, Vasa-Cre. Genesis: the Journal of
Genetics & Development 45: 413–417.
27. Bernstein E, Kim SY, Carmell MA, Murchison EP, Alcorn H, et al. (2003) Dicer
is essential for mouse development. Nat Genet 35: 215–217.
28. Bouhallier F, Allioli N, Lavial F, Chalmel F, Perrard MH, et al. (2010) Role of
miR-34c microRNA in the late steps of spermatogenesis. Rna 16: 720–731.
29. Oakberg EF (1956) Duration of spermatogenesis in the mouse and timing of
stages of the cycle of the seminiferous epithelium. Am J Anat 99: 507–516.
30. Fernandez-Capetillo O, Mahadevaiah SK, Celeste A, Romanienko PJ,
Camerini-Otero RD, et al. (2003) H2AX is required for chromatin remodeling
and inactivation of sex chromosomes in male mouse meiosis. Dev Cell 4:
497–508.
31. Chen JF, Murchison EP, Tang R, Callis TE, Tatsuguchi M, et al. (2008)
Targeted deletion of Dicer in the heart leads to dilated cardiomyopathy and
heart failure. Proc Natl Acad Sci U S A 105: 2111–2116.
32. Tam W (2008) The emergent role of microRNAs in molecular diagnostics of
cancer. J Mol Diagn 10: 411–414.
33. Watanabe T, Totoki Y, Toyoda A, Kaneda M, Kuramochi-Miyagawa S, et al.
(2008) Endogenous siRNAs from naturally formed dsRNAs regulate transcripts
in mouse oocytes. Nature 453: 539–543.
34. Murchison EP, Stein P, Xuan Z, Pan H, Zhang MQ, et al. (2007) Critical roles
for Dicer in the female germline. Genes Dev 21: 682–693.
35. Tang F, Kaneda M, O’Carroll D, Hajkova P, Barton SC, et al. (2007) Maternal
microRNAs are essential for mouse zygotic development. Genes Dev 21:
644–648.
36. Suh N, Baehner L, Moltzahn F, Melton C, Shenoy A, et al. (2010) MicroRNA
Function Is Globally Suppressed in Mouse Oocytes and Early Embryos. Curr
Biol.
37. Mattiske DM, Han L, Mann JR (2009) Meiotic maturation failure induced by
DICER1 deficiency is derived from primary oocyte ooplasm. Reproduction 137:
625–632.
38. Kotaja N, Bhattacharyya SN, Jaskiewicz L, Kimmins S, Parvinen M, et al.
(2006) The chromatoid body of male germ cells: similarity with processing
bodies and presence of Dicer and microRNA pathway components. Proc Natl
Acad Sci U S A 103: 2647–2652.
39. Meikar O, Da Ros M, Liljenback H, Toppari J, Kotaja N (2010) Accumulation
of piRNAs in the chromatoid bodies purified by a novel isolation protocol. Exp
Cell Res 316: 1567–1575.
40. Mandal PK, Kazazian HH, Jr. (2008) SnapShot: Vertebrate transposons. Cell
135: 192–192 e191.
41. Bourc’his D, Bestor TH (2004) Meiotic catastrophe and retrotransposon
reactivation in male germ cells lacking Dnmt3L. Nature 431: 96–99.
Dicer in Spermatogenesis
PLoS ONE | www.plosone.org 10 ber 2011 | Volume 6 | Issue | e25241Octo 10
42. Siomi MC, Kuramochi-Miyagawa S (2009) RNA silencing in germlines–
exquisite collaboration of Argonaute proteins with small RNAs for germline
survival. Curr Opin Cell Biol 21: 426–434.
43. Kuramochi-Miyagawa S, Watanabe T, Gotoh K, Takamatsu K, Chuma S,
et al. (2010) MVH in piRNA processing and gene silencing of retrotransposons.
Genes Dev 24: 887–892.
44. Kanellopoulou C, Muljo SA, Kung AL, Ganesan S, Drapkin R, et al. (2005)
Dicer-deficient mouse embryonic stem cells are defective in differentiation and
centromeric silencing. Genes Dev 19: 489–501.
45. Svoboda P, Stein P, Anger M, Bernstein E, Hannon GJ, et al. (2004) RNAi and
expression of retrotransposo ns MuERV-L and IAP in preimplantation mouse
embryos. Dev Biol 269: 276–285.
46. Watanabe T, Takeda A, Tsukiyama T, Mise K, Okuno T, et al. (2006)
Identification and characterization of two novel classes of small RNAs in the
mouse germline: retrotransposon-derived siRNAs in oocytes and germline small
RNAs in testes. Genes Dev 20: 1732–1743.
47. Barchi M, Geremia R, Magliozzi R, Bianchi E (2009) Isolation and analyses of
enriched populations of male mouse germ cells by sedimentation velocity: the
centrifugal elutriation. Methods in molecular biology 558: 299–321.
48. Swetloff A, Conne B, Huarte J, Pitetti JL, Nef S, et al. (2009) Dcp1-bodies in
mouse oocytes. Mol Cell Biol in press.
49. Martens JH, O’Sullivan RJ, Braunschweig U, Opravil S, Radolf M, et al. (2005)
The profile of repeat-associated histone lysine methylation states in the mouse
epigenome. Embo J 24: 800–812.
50. Rowe HM, Jakobsson J, Mesnard D, Rougemont J, Reynard S, et al. (2010)
KAP1 controls endogenous retroviruses in embryonic stem cells. Nature 463:
237–240.
51. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using
real-time quantitative PCR and the 2(2Delta Delta C(T)) Method. Methods 25:
402–408.
52. Guerif F , Cado ret V, Plat M, Magistrini M, Lansac J, et al. (200 2)
Characterization of the fertility of Kit haplodeficient male mice. Int J Androl
25: 358–368.
53. Cederroth CR, Zimmermann C, Beny JL, Schaad O, Combepine C, et al.
(2010) Potential detrimental effects of a phytoestrogen-rich diet on male fertility
in mice. Mol Cell Endocrinol 321: 152–160.
54. Grad I, Cederroth CR, Walicki J, Grey C, Barlu enga S, et al. (2010) The
molecular chaperone Hsp90alpha is required for me iotic prog ression of
spermatocytes beyond pachytene in the mouse. PLoS One 5: e15770.
Dicer in Spermatogenesis
PLoS ONE | www.plosone.org 11 ber 2011 | Volume 6 | Issue | e25241Octo 10
