Analysis of MicroRNA Expression in the Prepubertal
Gregory M. Buchold1,2., Cristian Coarfa4., Jong Kim5, Aleksandar Milosavljevic4", Preethi H.
Gunaratne1,5", Martin M. Matzuk1,2,3*"
1Department of Pathology, Baylor College of Medicine, Houston, Texas, United States of America, 2Department of Molecular and Cellular Biology, Baylor College of
Medicine, Houston, Texas, United States of America, 3Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States of
America, 4Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas, United States of America, 5Department of Biology and Biochemistry,
University of Houston, Houston, Texas, United States of America
Only thirteen microRNAs are conserved between D. melanogaster and the mouse; however, conditional loss of miRNA
function through mutation of Dicer causes defects in proliferation of premeiotic germ cells in both species. This highlights
the potentially important, but uncharacterized, role of miRNAs during early spermatogenesis. The goal of this study was to
characterize on postnatal day 7, 10, and 14 the content and editing of murine testicular miRNAs, which predominantly arise
from spermatogonia and spermatocytes, in contrast to prior descriptions of miRNAs in the adult mouse testis which largely
reflects the content of spermatids. Previous studies have shown miRNAs to be abundant in the mouse testis by postnatal
day 14; however, through Next Generation Sequencing of testes from a B6;129 background we found abundant earlier
expression of miRNAs and describe shifts in the miRNA signature during this period. We detected robust expression of
miRNAs encoded on the X chromosome in postnatal day 14 testes, consistent with prior studies showing their resistance to
meiotic sex chromosome inactivation. Unexpectedly, we also found a similar positional enrichment for most miRNAs on
chromosome 2 at postnatal day 14 and for those on chromosome 12 at postnatal day 7. We quantified in vivo
developmental changes in three types of miRNA variation including 59 heterogeneity, editing, and 39 nucleotide addition.
We identified eleven putative novel pubertal testis miRNAs whose developmental expression suggests a possible role in
early male germ cell development. These studies provide a foundation for interpretation of miRNA changes associated with
testicular pathology and identification of novel components of the miRNA editing machinery in the testis.
Citation: Buchold GM, Coarfa C, Kim J, Milosavljevic A, Gunaratne PH, et al. (2010) Analysis of MicroRNA Expression in the Prepubertal Testis. PLoS ONE 5(12):
Editor: Jo-Ann L. Stanton, University of Otago, New Zealand
Received August 5, 2010; Accepted November 6, 2010; Published December 29, 2010
Copyright: ? 2010 Buchold 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 research was supported in part by National Institutes of Health, National Institute of Child Health and Human Development (NIH-NICHD)
5R01HD057880 (to M.M.M); National Institutes of Health, Epigenomics Roadmap Initiative grant from National Institute on Drug Abuse (NIH-NIDA) 5U01DA025956
and National Human Genome Research Institute (NIH-NHGRI) 5R01HG4009 (to A.M.), and National Institutes of Health, National Institute of Child Health and
Human Development (NIH-NICHD) 5T32HD007165 (to G.M.B.). The authors also thank the Cullen Foundation for their generous support of the University of
Houston’s Institute for Molecular Design (IMD) Illumina/Solexa Sequencer. 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: email@example.com
. These authors contributed equally to this work.
"These authors also contributed equally to this work.
The expression and modification of miRNAs have been an area of
intense interest. In brief, miRNA biosynthesis involves primary
miRNA (pri-miRNA) transcription by RNA polymerase II and
folding of the pri-miRNA into a secondary structure that is
recognized and cleaved by the microprocessor complex, Drosha
and DGCR8, to yield a stem-loop or pre-miRNA. This pre-miRNA
is exported from the nucleus by exportin 5 and cleaved by Dicer
in the cytoplasm to yield a double-stranded RNA of 21–22 nts
containing both strands of the hairpin, designated 5p and 3p
[1,2,3,4]. Subsequently, the two strands are separated and generally
one of the two (the guide strand) is incorporated into the RISC
effector complex, containing Argonaute proteins, while the passenger
or star strand is degraded. However, some star strands may be stable
and functional.Using the specificity contained within nucleotides 2–7
(59seed) and 13–16 (anchor) of the guide strand, the RISC complex
targets mRNAs through complementary sequences in their 39 UTR
for cleavage or translational repression [5,6]. During miRNA
biosynthesis, RNA-binding proteins, such as LIN28, can associate
with the small RNA, preventing or altering its processing .
Genetic studies disrupting miRNA functions in mammals by
targeting Dicer, Drosha, DGCR8, or individual miRNAs have
revealed specific and global roles of miRNAs a variety of
developmental processes and pathologic states. Germ cell-specific
deletion of Dicer2/2shows that miRNAs are required for
regulation of male gonocyte proliferation . MicroRNAs have
also been implicated in the pathogenesis of human germ cell
tumors (e.g., mir-372 and mir-373)  or several cancers including
testicular cancer (e.g. let-7c) .
Localization studies of miRNAs and their associated enzymes
suggest that they may contribute to post-meiotic male germ cell
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function. Complexes of miRNAs and their targets as well as Dicer
accumulate in the chromatoid body of spermatids ; however,
their function and localization have not been described in earlier
spermatogenic cells. A number of mRNAs are associated with the
chromatoid body that are first transcribed in spermatocytes but
have no detectable protein expression until some days later .
Therefore, this translational delay may result from the action of
miRNAs localized in other germ cell RNA granules such as
intermitochondrial cement, MIWI2 pi-P bodies, chromatoid
bodies, etc. . This correlates with the dramatic increase in
overall miRNAs at postnatal day 14 (P14) when pachytene cells
are first abundant in the testis. Several groups have described the
complement of miRNAs present in the adult mouse testis
[14,15,16] or human testicular tumors [10,17,18] using low-
throughput assays and more recently Next Generation Sequencing
methods . Next Generation Sequencing of female tissues has
uncovered novel small RNAs missed by prior analyses and
allows the identification of sequence differences reflective of
potential post-transcriptional modification relevant to target
Editing of a variety of RNAs occurs frequently in mammals with
the majority of modifications caused by A-to-I editing and 39
terminal A and U additions with C-to-U editing occurring less
frequently [21,22]. Sixteen percent of miRNAs are also modified
[23,24,25], predominantly by adenosine deamination of precursor
miRNAs by ADARs [26,27,28,29,30]. ADAR-dependent editing
can control targeting specificity and the stability and processing of
miRNA precursor transcripts [30,31,32]. The editing of nucleo-
tides in the vicinity of Dicer or Drosha processing sites can prevent
the further maturationand
[31,33,34,35]. In the rat, the highest degree of ADAR-dependent
editing occurs in brain followed by testis . The second major
class of miRNA editing events (C-to-U) depends on the APOBEC
(apolipoprotein B mRNA editing enzyme, catalytic polypeptide)
family of cytidine deaminases which can prevent translational
inhibition of miRNA targets . Additional types of editing
yielding variant miRNAs, sometimes called isomiRs , have
been described in the adult ovaries and testes including variations
in the 59 end cleavage by Drosha or Dicer and nontemplated
nucleotide addition to the mature miRNA [19,20,39,40]. Varia-
tions in the miRNA 59 end alter their 59seed sequence, lowering
the affinity of miRNA to target mRNAs. Uridylation of the
miRNA 39 end results in their destabilization. LIN28-dependent
uridylation oflet-7 miRNAs
ZCCHC11 is the most notable example of this modification
[41,42,43]. Thus, editing events act to oppose or modulate the
action of miRNAs.
Previously, we reported the presence of miRNAs in the testis
prior to P14  but did not describe the profile in detail. Our
current studies demonstrate that the miRNAs most enriched at P7
and P14 derive predominantly from chromosome 12, and then
chromosomes 2 and X, in contrast to those from the adult that are
expressed from more diverse chromosomal locations. To date, the
description of miRNA editing from deep sequencing in a
developmental context has only been described in vitro ; our
current study is the first to analyze editing in the in vivo context of
pubertal spermatogenesis, preceding the high degree of editing
described in the adult testis. All types of editing events are
modestly higher at P7, overlapping with only a fraction of the
editing events in the adult testis. We believe that profiling miRNA
changes during normal testicular development will aid in the
interpretation of significant changes in miRNAs occurring in
pathologic states (i.e., infertility and cancer) and may suggest novel
regulation of miRNAs in male germ cells.
Animal husbandry and tissue isolation
Mice were generated from our mating colony bearing the Gasz
mutant allele, Gasztm1Zuk, by intercrossing heterozygous sires and
homozygous null dams of B6;129 mixed background (129S7/
AB2.26C57BL6/J) . Breeders were housed as trios (one male
and two females) with pups in microisolator cages on a 12 hr light/
dark cycle (7am-7pm) at 70uF62uF. Mice were provided Harland
Teklad 2919 (breeder chow), acidified water; and nestlets for
environmental enrichment. Testes were collected under inhaled
anesthesia from two GASZ+/2mice with litter-matched GASZ
null controls from two different litters on postnatal day 7, 10, and
14. Additional details of the animal husbandry and experimental
design can be found in the ARRIVE checklist (Checklist S1).
These studies were carried out in accordance with the NIH Guide
for the Care and Use of Laboratory Animals under Baylor College
of Medicine IACUC approved protocol AN-716.
Small RNA isolation and sequencing
Testicular small RNAs were isolated using the mirVana kit
(Ambion, Austin, TX) and sequenced by Illumina-Solexa sequenc-
ing as described previously . RNA quality and the presence of
small RNAs were evaluated on a 2100 Bioanalyzer (Agilent). After
passing the quality controls, 15 ug of total RNA was used in the
Illumina DGE small RNA sample prep kit to synthesize a small
RNA library. Small RNA populations were sequenced on the
Illumina 1 G Genome Analyzer (University of Houston).
Identification of alternatively processed miRNAs
Reads which differed from the mature miRNA sequences as
described in microrna.org by 1 nucleotide at the 59 end and
mapped to the pre-miRNA sequence were identified as alternative
59 isoforms. Those reads which matched 100% to the mature
miRNA sequence but contained additional nucleotides A(n) or
U(n) that did not match the pre-miRNA sequence were identified
as alternative 39 adenylated or uridylated miRNAs. Those reads
which contained a single A to G mismatch within the mature
miRNA sequence were identified as candidate ADAR-edited
miRNAs. Finally those reads which contained additional uridines,
but the surrounding sequence matched 100% to the mature
miRNA (similar to an insertional polymorphism) were identified as
miRNAs affected by internal uridylation. The ratio of alternative
reads to canonical mature miRNA reads were analyzed for
Identification of putative novel testicular miRNAs
Reads which do not match to mature miRNAs or other
ncRNAs including snoRNAs, and tRNAs were mapped and
contigs were assembled through the annotation pipeline described
previously . Criteria, including the presence of a stem-loop,
reads indicating the presence of a miRNA star strand, and
consistent 59 end processing, were used to rank candidate
miRNAs. We initially evaluated candidates independent of size;
however, to achieve high confidence miRNAs we ultimately chose
to subject candidates to a size cutoff of ,25 nt present in Gasz2/2
controls to exclude possible novel piRNAs.
Developmental analysis of testicular miRNAs by deep
Using Illumina-Solexa deep sequencing, we analyzed the small
RNA populations in testes of mice on postnatal day 7 (P7), 10
Prepubertal Testis miRNAs
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(P10), and 14 (P14) to assess piRNA populations in GASZ null
mice compared to controls . In the course of the analyses of
our piRNA findings we recognized that analysis of the miRNA
population in controls might provide an insight into their roles in
pubertal spermatogenesis. We identified reads belonging to the
682 mature miRNAs (608 pre-miRNAs) present in miRBase 13.0
version (http://www.mirbase.org/) and used these to describe
miRNA signatures for these ages, similar to our characterization of
human ovarian miRNAs . Since biologically effective changes
in miRNA suppression of respective mRNA targets appears to
have some minimal threshold, we initially focused on the top 50
miRNAs by read abundance at these three time-points (Figure 1A-
B, Table S1). We initially hypothesized that meiotic initiation at
P10 may be correlated with a unique miRNA signature; however,
we found a lack of evidence for such an enrichment arguing
against possible miRNA-mediated initiation of meiosis at P10.
Therefore, we focused our subsequent analysis on a comparison
between P7 and P14, identifying those miRNAs that showed
greater than a two-fold enrichment at P7 or P14 as potential
candidate miRNAs important for spermatogonial and spermato-
Several abundant miRNAs showed greater than a two-fold
enrichment at P7 (i.e., let-7e, 127, 181b-2, 503, and 181b-1), while
those showing the greatest fold enrichment (i.e., mir-122, 370, 770,
383, 410, 335, 615, 543, 665) were generally expressed at modest
levels (Figure 2A). By contrast, more than half of the miRNAs
most enriched at P14 (i.e., 449c, 34b, 34c, 743b, 471, 204, 878,
880, 883a, 743a, 881, 375, 760, 741, 470, 871, 465b-1, 465b-2,
883b, 465c-1, 465c2, 465a, 467a) were abundantly expressed
(Figure 2B). It is also notable that those miRNAs with the most
dramatic enrichment at P14 were clustered on a single region of
the X chromosome (discussed in greater detail below). MicroRNAs
are often enriched either early (in stem cells/early transit
amplifying cells) or late (in differentiating cells) in the differenti-
ation process. Consistent with the latter, many of the miRNAs
abundant at P14 were also previously reported as abundant at P21
and adult testes by low throughput methods  although the
Figure 1. The MicroRNA signature of the testis differs over
prepubertal development. MicroRNA signature of postnatal day 7
(P7) (A) and 14 (P14) (B) testes. The top 25 miRNAs (by percent read
abundance) at each age were plotted. Representative miRNAs are
labeled with those with greater abundance at P7 (putative spermato-
gonial role) in green and at P14 (putative spermatocyte role) in red.
Levels at P10 were intermediate between P7 and P14 indicating a lack
of miRNAs specific to meiotic initiation. ,80% of all miRNAs in the testis
at all three time-points were let-7 family miRNAs.
Figure 2. MicroRNAs with the most dramatic enrichment are
associated with chromosomes 2, 12, and X. Roughly 2% of
miRNAs were enriched more than five-fold at P7 (left). Of these miR-122
was the most enriched (43-fold). By contrast, nearly 10% of miRNAs
were enriched to the same degree at P14 (right). The most enriched was
miR-449c (41-fold). A number of miRNAs most induced at P7 are located
on chromsome 12 (boxed in green) while those most induced at P14
cluster on chromosomes 2 and X (boxed in red).
Prepubertal Testis miRNAs
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profiles were distinct. Thus the miRNA pools within pre-meiotic
and meiotic are likely to be different, and a subset of miRNAs
arising from the X chromosome during meiosis may continue to
remain prominent in post-meiotic spermatogenesis.
X chromosome miRNAs escape meiotic sex chromosome
Part of the dosage compensation that occurs between X- and Y-
bearing male germ cells is a spermatocyte-specific alteration of sex
chromatin through a process similar to X chromosome inactiva-
tion. The loss of mRNA transcription on the X chromsome during
male meiosis is often compensated by intronless retrogene
autosomal paralogs . Prior reports by Yan and colleagues
demonstrated that nearly all miRNAs on the X chromosome
display continuous expression during meiosis in contrast to most
protein-coding genes on the X chromosome . We performed
similar analysis using deep sequencing and found abundant reads
for many X chromosome miRNAs. Intriguingly, when their
position was mapped on the X chromosome, domains of
developmental expression patterns were seen in which transcrip-
tion predominated at either P7 or P14. The intergenic distances
for most are large enough (.5 kb) to indicate that they are not
transcribed from a common primary transcript unlike the miR-
17,92 cluster. In our prior analysis of piRNAs , we had
observed a number of miRNA variants, previously missanotated as
piRNAs, derived from this cluster on Xq. This region (including
mir-743a to mir-547) represents a discontinuity in the synteny
between rodents and primates. However, the primate X
chromosome contains an analogous cluster of clade-specific
miRNAs (hsa-mir-890 to hsa-mir-514-3). In addition to the X
chromosome, we also found enrichment for miRNAs derived from
chromosome 2 at P14 (Figure 3B) and for chromosome 12 at P7
(Figure 3C). In the mouse, miRNAs are non-randomly distributed
over the genome with 40% deriving from these three chromo-
somes (Table 1). During mouse pubertal spermatogenesis the
miRNA complement comes predominantly from limited chromo-
somal domains, shifting from expression of chromosome 12 at P7
to chromosomes 2 and X at P14.
Analysis of miRNA hairpin cleavage products
Dicer cleavage of the pre-miRNA yields two products, the 59
and 39 portions of the base-paired regions of the stem-loop.
Initially, it was believed that only one strand (guide) is
incorporated into the RISC effector complex while the other
strand (star) was nonfunctional and degraded. Now both mature
miRNAs derived from the pre-miRNA are believed may have
activity against targets. Due to speculation that there might be
differential processing during testicular development or differences
in the relative stability of the two strands in the testis, we analyzed
the 5p and 3p miRNA strands during prepubertal testis
development. The representative 5p (blue) and 3p (red) sequences
from mouse miR-125 are shown (Figure 4A). We found that 359 of
the 465 mouse pre-miRNAs, produced reads from one or both
strands during P7-P14 of testicular development. After calculating
the ratio of 5p to 3p reads from each processed pre-miRNA, we
determined that the majority (.86%, Figure 4B and Table S2)
showed a ratio that was significantly different from one at all three
ages (range 0.0001–780,000). This indicates that the testis is not
distinct from other organs with respect to pre-miRNA processing,
showing differential abundance of the hairpin cleavage products
(5p or 3p) for nearly all pre-miRNAs. Most reads derive from the
5p half of their respective pre-miRNAs. Since the majority of reads
in the testis at the ages assessed are composed of let-7 family
miRNAs, predominantly represented by their 5p reads, this
further inflates this bias. In very rare cases (,1%), the strand
preference changed over development, but most showed the same
preference at all three time-points. We further assessed the
developmental differences (P7 vs. P14) in the abundance of 5p and
3p miRNAs individually. This demonstrated that roughly an equal
number of 5p miRNAs were increasing with age (32.8%) as
decreasing (37.8%) with a small amount (7.6%) remaining
constant over time (Figure 4C). A similar distribution of
developmental patterns was seen on 3p miRNAs. We also detected
Figure 3. Chromosomes 2, 12, and X predominant source of
pubertal-associated testis miRNAs. (A–C) Plots of miRNA expres-
sion ratios (P14/P7). (A) A large cluster miRNAs on chromosome X (ChrX)
and most miRNAs on chromosome 2 (Chr2) are enriched for expression
at P14 (ratio .2, red line). While those from chromosome 12 (Chr12) are
enriched for expression at P7 (ratio ,0.5, green line). Those on the
distal arms of the X chromosome show enrichment at P7, as expected
for genes showing the classical meiotic sex chromosome inactivation
(MSCI) pattern of repression on P14. The large number of miRNAs on
the mid-arm of chromosome X that escape this repressive process,
displaying a greater abundance at P14, is in agreement with prior
reports. We found a number of miRNAs falling within this cluster were
ones that we had previously determined were misidentified as piRNAs
in public databases (red dashed box).
Prepubertal Testis miRNAs
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24 examples (7%) in which the developmental pattern of 5p and
3p miRNAs from the same pre-miRNA were in discordance. Thus
we reject the hypotheses that a) the selection of the more abundant
strand (5p vs. 3p) may shift and b) the stability of all miRNAs are
coordinately regulated over pubertal testicular development.
Alternatively processed miRNAs
We determined to capture developmental miRNA variation in
multiple locations relative to the mature miRNA including 59 end
variation, internal editing, and nontemplated nucleotide addition to
the 39 end. We summarize the major events in which .5% of the
reads are of the edited form (Table S3). To address 59 end variation,
we took an approach similar to the discovery of genomic insertions
or deletions. The reads mapping with up to two nucleotide
mismatches to mature miRNAs, but which matched the pre-
miRNA (and genomic sequence), resulting from the utilization of
distinct 59 end cleavage sites by Dicer or Drosha were identified.
Prior reports of adult mammalian tissues, including testis, describe
59 end heterogeneity as affecting 8% of all miRNA reads, with
greater variability on star strands . We found that (Table S4)
alternative 59 end processing was rare with the exception of twenty
miRNAs in whichthe 59 cleavage variant waselevated at P7 relative
to P14. Reads which matched the miRNA precursor with 1–3
mismatches were designated internally edited miRNA isoforms
(Table S5). Most high abundance (104reads) miRNAs such as let-7
family member let-7b-5p showed very little internal editing.
However its less abundant (103reads) star strand let-7b-3p, showed
a developmental shift in editing at position 15 (A.G), likely to
represent an ADAR-dependent event. At P7 the levels were 26% of
the total let-7e-3preads, butthis declined on P14 to 15%.The effect
of this editing event would be to enhance the inhibition of target at
P14 relative to P7. Since Let-7 family members are believed to
restrain proliferation reduced effectiveness of let-7 against its targets
due to editing may be beneficial in the predominantly mitotically
active testis at P7. In contrast to the editing of let-7b-3p, 34b-5p and
376b-3p, four of eighteen miRNAs showed a peak of editing at P10
and the eleven others showed a developmental increase from P7 to
P14. Reads matching the mature miRNA sequence with additional
39 (A)n or (U)n that did not match the pre-miRNA sequence were
designated as 39 edited miRNAs (Table S6). Slightly more than half
(61%) of the thirty four miRNAs in this category showed a greater
proportion of their edited forms at P14. Overall variant miRNAs
compose a greater proportion of the miRNA pool at later
developmental stages, suggesting that miRNA effects may weaken
with increasing pubertal testicular development.
Table 1. Positional analysis of pubertal miRNA expression.
ChrTotal % Total
1 21 4%2 3%7 7%
2 5912%1 2%29 29%
3 204%0 0%5 5%
422 5%00%6 6%
5 153%1 2%1 1%
6 214%1 2%3 3%
7 26 5%5 8%5 5%
8 15 3%3 5%0 0%
1128 6%2 3%3 3%
12 6113% 3761%0 0%
13 204%0 0%5 5%
14 21 4%1 2%3 3%
17 11 3%1 2%1 1%
18 112%1 2%11%
MicroRNAs are distributed nonrandomly over the mouse genome with
predominant enrichment on chromosomes 2, 12, and X (in bold). Most miRNAs
on chromosome 12 were enriched at P7, while those on chromosomes 2 and X
are enriched at P14.
Figure 4. Mature miRNAs and star strands are differentially
expressed in the testis. (A) Example miRNA identifying the 5p (red)
and 3p (blue) miRNAs that will result from Dicer cleavage of the mmu-
mir-125a pre-miRNA hairpin. Levels of 5p and 3p often are dramatically
different in abundance in non-reproductive tissues, producing a 5p/3p
ratio different from 1. (B) Shown are representative miRNAs in which
reads were detected for both mature 5p and 3p sequences. Contrary to
one report arguing for equivalent levels of both strands in the testis, we
found that the pubertal testis was similar to other non-reproductive
tissues. 73–87% of miRNAs displayed a 5p/3p ratio ,0.5 or .2 (range
0.0001–240,000), consistent with differential stability of most 5p/3p
miRNA pairs. (C) Most miRNAs showed predominant expression of
either 5p or 3p reads. In 87% of miRNAs the 5p miRNA was more
abundant compared to 3p (13%). Among the 5p miRNAs roughly one
third were enriched at P7 (blue), P14 (teal), or unchanged (gray). Equal
amounts of the 3p miRNAs were enriched at P7 (red) and P14 (pink).
Prepubertal Testis miRNAs
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Discovery of novel testicular miRNAs
We subjected our reads that mapped to pre-miRNA sequences
(putative star strands) as well as those that did not map to a known
mature miRNA to a miRNA discovery pipeline described in
Creighton et al. 2009 that identified those reads whose
surrounding 200 nucleotides had some capacity to fold into a
double-stranded RNA using the Vienna secondary structure
prediction package. We screened for those hairpins that captured
a number of reads typically mapping to one side of the stem,
rejecting those which did not cluster tightly, overlapped with the
loop, or mapped to known non-coding RNAs. We identified 198
putative novel miRNAs and determined their developmental
abundance (Tables S7, S8, S9). These were assigned to several
categories based on a) their ability to be produced by predicted
Drosha and Dicer cut sites and b) the presence of a putative star
strand. We found 107 high confidence novel miRNAs, 104 lacking
star strands, and three (76, 131, 143) with a corresponding star
strand. Thirteen of 107 (12%) of the high confidence class mapped
to repetitive elements including SINEs, LINEs, LTRs, and DNA
transposons. These candidates likely represent endo-siRNAs,
piRNAs, or SINE-associated small RNAs rather than true
miRNAs. Since 17 of the 52 (32%) that met fewer criteria -
‘‘potential miRNAs’’ - were also mapped to repetitive elements,
this provides some validation for the cut site criteria. Most of the
candidates were expressed at very low levels while a significant
fraction of those expressed at a higher level were associated with
repeats (Table S9). Greater than 75% of all high confidence
candidates were expressed at P7 or P10 alone or P7-P10, which
would have been most likely undetectable in the prior sequencing
study involving adult testis . The least differentiated cells in the
testis may be the source of undiscovered miRNAs just as
embryonic stem cells possess miRNAs distinct from those in adult
tissues . An additional 143 pre-miRNAs have been described
in the latest miRBase 15.0 version but only candidate 185 has been
identified as mir-3099-3p, first isolated in newborn ovaries .
We assessed evolutionary conservation of the 59seed sequence of
our candidates to rat and human syntenic regions using the UCSC
genome browser and found relatively few were conserved to
human (26/198 or 13%) although nearly half (45%) were
conserved to rat. Most candidates had 59seed sequences distinct
from known miRNAs; however the following candidates, con-
served with humans, were similar to those in parentheses: 5 (miR-
190/190b), 19 (miR-29b-2), 92 (miR-1195), 93 (miR-134), 132
(miR-486), and 183 (miR-345-3p). Those that were abundant but
not conserved with humans (34, 69, 76, 88, 94, 100, 105, 110, 144,
176) had novel 59seeds with the exception of 110, which was
similar to miR-411. Another criteria that may increase our
confidence in candidates representing miRNAs rather than other
types of ncRNA could include close linkage to known miRNAs
(,5000 bp). Two of our candidates show this association (85 to
miR-700 and 90 to miR-805). Nine pairs of candidate reads
displayed close linkage to other candidates, all mapped to mRNA
exons. We assigned these candidates overlapping with exons as
gene-associated piRNAs rather than miRNAs, which are more
commonly contained within introns when they overlap an mRNA.
After excluding those candidates mapping to exons or repetitive
elements, 75 candidates remained. Of these, 12 were 24 nt or less
while the remainder were 25–30 nt in length. A length assessment
of all mouse mature miRNAs identifies only 1.3% of miRBase
miRNAs larger than 24 nts (4.5%,20 nt and 94.2% 20–24 nt).
Since we have observed that the number of miRNA reads is
proportionally increased in GASZ null testes lacking piRNAs ,
we tested the value of excluding those candidates which are
absent or not increased in GASZ null testes as probable piRNAs.
Eighty-three percent (10/13) of the candidates ,25 nt long were
increased in GASZ null testes relative to controls, while of the
larger size category only candidates 5 and 90 (3%) showed a
similar increase. Forty percent of the candidates in the larger size
were determined to partially overlap with known piRNAs or map
within piRNA clusters. Thus, we identified 11 putative novel testis
miRNAs with high confidence and detected miR-3099-3p
previously described in newborn ovary (Table 2). We believe that
the remaining 64 are likely piRNAs. Using the 59seed sequence
target prediction analysis in TargetScan 5.1, we analyzed the
targets of the 11 putative novel candidates. Most showed a very
restricted target repertoire between 4–100 targets. A small fraction
of targets overlapped between the novels, but was not correlated
with similar developmental pattern.
The mouse testis displays a distinct microRNA profile during
prepubertal development with miRNAs enriched putatively in
spermatogonia (e.g., miR-122) and spermatocytes (e.g., miR-409c).
MiRNAs from particular chromosomes are active at specific times
during spermatogenesis (e.g., chromosome 12 at P7 and chromo-
somes 2 and X at P14). We have identified 11 putative novel testis-
expressed miRNAs in addition to the miRNA variants on the X
chromosome described previously . Compared to adult testis
sequencing results, the complexity of the juvenile miRNA profiles
are much lower. Let-7 family members contribute 80% of juvenile
miRNA reads but compose only 11% of adult testis reads. While
the top 50 miRNAs by abundance in the adult testis include let-7
family members and many X chromosomally encoded miRNAs,
167 miRNAs are .5-fold enriched in adult testes compared to P14
testes. Many of these miRNAs are also enriched at P14 compared
to P7, but a number of miRNAs are specifically enriched in the
adult including the mir-17 to -92a-1 cluster on chromosome 14,
mir-135a, mir-135b, mir-190, and mir-215.
It is intriguing that newborn ovaries, predominantly composed
of meiotic oocytes, show a similar enrichment for expression of
miRNAs from chromosomes 2 and X, potentially indicating some
common regulation of meiotic miRNAs in both sexes .
However, the particular cluster on the X chromosome highly
expressed in ovaries (miR-450b to miR-322) is centromeric to a
similar cluster in testes (miR-743a to miR-465a). Furthermore, the
majority of the ovarian cluster is conserved with human, with the
exception of mir-322, nearest to the male cluster, whereas, the
male cluster is almost entirely rodent-specific miRNAs. One might
be tempted to speculate that the enrichment of miRNAs on mouse
chromosomes 2 and X may suggest a possible role in spermatocyte
Although i12p, duplication, or increased expression of pluripo-
tency factors encoded on the p arm of human chromosome 12 is
common to seminomas [49,50], the enrichment of mouse
chromosome 12 miRNAs at P7 does not have an obvious
importance to testicular cancer since this mouse chromosome is
syntenic to human chromosome 14. The only gene mapped to
mouse chromosome 12 and human chromosome 14 with
polymorphisms associated with azoospermia is MLH3, which
causes a meiotic arrest . However, we speculate that miRNAs
from human chromosome 14 may be abundant in differentiated
spermatogonia and deficient in testicular tumors arrested at an
earlier stage of germ cell development. Assessment of possible
mechanistic action of miRNAs abundant in human testicular
cancer would be greatly benefited by comparison of the miRNA
complement from newborn mouse testis or isolated gonocytes with
their corresponding mouse testicular cancer models.
Prepubertal Testis miRNAs
PLoS ONE | www.plosone.org6December 2010 | Volume 5 | Issue 12 | e15317
Differential processing of some pre-miRNAs, leading to tissue-
specific differences in the relative abundance of their 5p and 3p
mature miRNAs, has been described by deep sequencing .
These differences could theoretically reflect differential pre-
miRNA processing in dividing versus post-mitotic cells or cell
cycle-specific processing, the abundance of which differs by tissue.
Such regulation could derive from regulation of pre-miRNA-
binding proteins. The relatively synchronous development of the
first wave of spermatogenesis should allow detection of such
regulation during the shift in the germ cell compartment from a
mitotic to meiotic state and be favorable to purification of the
processing regulator. However, we found that the mature miRNA
strand (5p versus 3p) that was predominant does not appear to
differ during mitotic or meiotic testicular development, nor does
destabilization of microRNA star strands appear to be develop-
mentally regulated in the testis during this interval. By contrast,
about 20% of miRNAs expressed in the adult testis switch from the
strand predominant at P14 (Table S10). Seventy-five percent of
the shifts favor the increase of the star strand in the adult testis,
independent of the predominant strand (5p or 3p) at P14.
Although there is no evidence for a global shift of processing (i.e.,
favoring the 5p at earlier and the 3p at later time-points), the same
pre-miRNA may be processed distinctly in early and late
spermatogenesis. Were transcription of the pre-miRNA to remain
constant, preferential accumulation of the star strand could favor
translational de-repression due to loss of the corresponding strand
or inhibition of a distinct set of targets.
Analysis of miRNA editing in the testis showed that most types
of editing events were higher at P14 than P7. Questions have been
raised about the possibility that apparent editing of miRNAs
outside the 59 and 39 ends could derive from Dicer-dependent
processing of other ncRNAs, such as tRNAs, followed by 39
uridylation or adenylation . We were able to detect several
miRNAs, which showed evidence of significant internal editing
(.5%) in juvenile mice. Presumptive ADAR- or APOBEC-
dependent affects were detected affecting adenosines and cyti-
dines, respectively, but nearly half of internal editing events in
juvenile testis miRNAs affects uridines and occurs at sites near the
39 end. The necessity of ADAR-dependent editing is unclear since
no reproductive defects have been described in ADAR1- or
ADAR2-deficient mice, but SPNR (spermatid perinuclear RNA-
binding protein) has similarity to ADARs outside the deaminase
domain and is essential to spermatid function [52,53].
Twenty juvenile mouse testis miRNAs display variation in 59
end cleavage including let-7a-1-5p. However, we did not identify
those from adult testis such as miR-133, -223, or -155 .
Confounding our analysis of 39 variation, poly(A) or poly(U) tracts
in the pre-miRNA follow the canonical 39 cleavage site in 17%
and 35% of mouse miRNAs. 39 end cleavage resulting in longer
miRNAs may mimic post-transcriptional adenylation or uridyla-
tion, conferring inherent instability upon 39 cleavage variants. For
those miRNAs in which 39 end adenylation or uridylation could be
distinguished from differential cleavage site selection, we found a
relative increase in this modification from P7 to P14. Ninety-one
miRNAs in the adult testis display 39 nucleotide addition (A or U,
range 10%–100%) . The majority of miRNAs uridylated in
juvenile testes were distinct from those modified in adult testis, but
some were affected in both including mir-24-1, -24-2, -103-1, 103-
2, -199a, and -342 . Abundant P14 X-chromosomally encoded
miRNAs do not display 39 uridylation with the exception of let-7f-
2. The increase in uridylation at P14, decreasing target specificity
and miRNA stability, argue for a relatively lower effectiveness of
these miRNAs against target at this time. However, differences in
miRNA uridylation between P7 and P14 are modest compared to
the high degree of uridylation of specific miRNAs in the adult
testis. Since the majority of cells in the adult testis are spermatids,
these modifications may be targeted to pre-miRNAs by spermatid
proteins that bind to the loop domain of pre-miRNAs similar to
LIN28 binding to let-7 pre-miRNAs. The semi-synchronous
nature of the first wave of spermatogenesis may facilitate in the
identification of possible testicular cofactors for miRNA modifying
enzymes and such factors may be associated with the chromatoid
By utilizing Next Generation Sequencing, our studies charac-
terized the complete miRNAome and its editing in vivo during
prepubertal testicular development. The peculiar behaviors of a
large number of miRNAs expressed in the germline (i.e., resistance
to meiotic sex chromosome inactivation and strong chromosomal
Table 2. Identification of putative novel testis-expressed miRNAs.
Candidate miRNAMature sequence lengthchr:start-stop (strand)Intronic to mRNA
22 chr4:133904334-133904355 (+)Slc30a2
22 chr7:6756349-6756370 (+)Usp29
24 chr8:112363200-112363223 (+)Ap1g1
22 chr11:117863593-117863614 (+)Pgs1
After excluding known miRNAs, clusters of Solexa reads showing evidence of hairpin formation were identified (203 candidates). We set strong inclusion criteria for
maximal length (,24 nt) and enrichment in GASZ2/2testes due to piRNA depletion. Seventeen putative novel miRNAs were identified, 12 of which displayed
enrichment in GASZ null testes. Most are expressed at relatively low level, have limited conservation, and map to introns of autosomal genes.
Prepubertal Testis miRNAs
PLoS ONE | www.plosone.org7December 2010 | Volume 5 | Issue 12 | e15317
association) argues for the need to identify additional miRNA
transcriptional regulators capable of acting in cis on chromosomes
2, 12, and X. We expect that the developmental associations with
these three chromosomes in this study will frame future
investigations of translational control of spermatogonial or
spermatocyte mRNAs. The very low copy number and poor
evolutionary conservation of the novel miRNAs identified in this
study is consistent with other attempts to identify tissue-specific
miRNAs, suggesting that few additional abundant conserved novel
miRNAs remain undiscovered in the mouse genome. While our
miRNA catalog did not identify a class of miRNAs that regulates
meiosis initiation, they do provide a normative control by which to
evaluate miRNA changes in murine and human testicular cancers.
Comparison of these findings in the pubertal mouse testis to
published studies of adult mouse testis highlights the need to focus
future studies of the regulatory consequences of miRNA editing
during the terminal differentiation of spermatids and their
potential physical connection to the chromatoid body and its
findings of miRNA regulation in spermatogenesis may be
generalized to other somatic cell types, most notably to neurons.
Analogous to the translational regulation required to assemble
structures accessory to the sperm tail axoneme, the brain displays a
similar temporal-spatial compartmentalization of translation
associated with neuronal axons and an even greater fraction of
edited miRNAs. Future efforts to identify the mechanism for
selectivity in editing of miRNAs during testicular development
may ultimately offer potential new avenues to therapeutic
intervention in human infertility and neurologic disorders.
in the testis (P7-P14). Excel table containing the number of
reads, percent of total miRNA reads, chromosomal position, and
quantification of modified reads at internal and 39 positions. The
number of reads at each age were normalized by calculation of
their percentage in the miRNA pool at each age. Ratios were
calculated to compare the relative expression. Those miRNAs with
significant effects (.2-fold enrichment at P7 or at P14) are bolded.
Str, strand; Chr, chrosomosome.
Developmental analysis of miRNA expression
in the testis (P7-P14). Excel table containing the number of
reads, percent of total miRNA reads, chromosomal position, and
predicted secondary structures of candidate novel testicular
miRNAs. The number of reads at each age were normalized by
calculation of their percentage in the miRNA pool at each age.
Ratios were calculated to determine the bias of pre-miRNA
processing (5p/3p) at each age. The predominant miRNA (i.e. the
non-star strand) was identified. Developmental change of both 5p
and 3p mature miRNAs was also calculated using ratio calculation
and annotated as increasing (i) or decreasing (d) from P7 to P14.
Developmental analysis of 5p vs. 3p miRNAs
ular development. Four types of editing were evaluated by
assigning reads that were not an exact match to the mature
miRNA sequence: a) alteration of 59 end cleavage, b) A to G
transitions representing putative editing by ADARs, c) internal
insertions of uridine by an unknown process, and d) 39 addition of
A(n) or (U)n. [Indel, insertion/deletion]
Editing of miRNAs during prepubertal testic-
prepubertal testicular development. 59 variants are gener-
ally highest at P7 in the juvenile testis but represent a small
fraction of total reads.
59 cleavage variants of miRNAs during
tal testicular development. Most internal editing events are
highest at P14 in the juvenile testis, but other patterns are detected
less frequently. Increased editing is associated with declining levels
of the miRNA in 55% of cases. Uridine was the most common
base affected. The affected positions are bolded within the mature
Internal editing of miRNAs during prepuber-
prepubertal testicular development. 39 nucleotide addition
increased over juvenile testis development (61% of cases), but the
portion of modified reads represent a small fraction of total reads.
39 nuclotide addition to miRNAs during
RNA reads not identified as known miRNAs were analyzed for 1)
their ability to form a stem loop structure, 2) minimum free energy
less than -20 kcal/mol, 3) strong clean signal in the specific region
of the 15–25 nt reference hairpin, 4) signals should not fall in the
loop, 5) predicted Drosha and Dicer cut sites must be able to yield
a mature miR sequence that matches the read, 6) stable 59 end
(61 nt), 7) highly variable 39 end, 8) presence of star sequence at
lower copy number and matching miR with 39 2 nucleotide
overhang, 9) does not map to rRNA, tRNA, snoRNA, or snRNA.
Those matching all nine criteria were designated high-confidence
novel miRNAs with star sequences. Those reads that passed all but
criteria 6–8 were designated high-confidence miRNA without star
sequence. Those that also did not pass criteria 5 were designated
as potential miRNAs and the reads that failed criteria 1–4 were
identified as non-candidates. Manually curated Dicer and Drosha
cleavage sites on the hairpins were marked in blue and red lines.
Hairpin candidate evaluation. All testicular small
miRNAs. Hairpin candidates were mapped to the mouse genome
(mm9) using the UCSC Blat tool. Those that overlapped with other
genomic elements were identified including repetitive elements
(LINE, SINE, LTR). Conservation of the SEED sequence with
syntenic sequences on the rat (R.n.) and human (H.s.) was assessed
through the UCSC genome browser (Y, yes; N, no; M, mutated).
Description of novel testicular candidate
NAs. The number of reads from two testes of each age and
genotype (GASZ+/2or GASZ2/2) are shown with the reads
assigned to the 5p and 3p sections of the hairpin. A summary of
their developmental pattern is given. The majority of those
expressed at 50 reads or greater had some similarity to repetitive
elements, suggestive they represent piRNAs, endosiRNAs, or
SINE-associated small RNAs.
Developmental expression of candidate miR-
between P14 and adult testes. The expression of 5p versus 3p
mature miRNAs were compared between this study and adult testes.
Those miRNAs which shifted from a high to low 5p/3p ratio or vice-
Comparison of predominance of 5p vs. 3p
Prepubertal Testis miRNAs
PLoS ONE | www.plosone.org8December 2010 | Volume 5 | Issue 12 | e15317
The ARRIVE Checklist.
We thank Ashley Benham for her assistance in evaluating candidate novel
Conceived and designed the experiments: GMB CC JK AM PHG MMM.
Performed the experiments: GMB CC JK. Analyzed the data: GMB CC
JK. Contributed reagents/materials/analysis tools: CC JK AM PHG.
Wrote the paper: GMB MMM.
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