Hong et al.

BMC Genomics (2018) 19:539

https://doi.org/10.1186/s12864-018-4931-3

RESEARCH ARTICLE Open Access

Profiling of testis-specific long noncoding

RNAs in mice
Seong Hyeon Hong, Jun Tae Kwon, Jihye Kim, Juri Jeong, Jaehwan Kim, Seonhee Lee and Chunghee Cho*

Abstract
Background: Spermatogenesis, which is the complex and highly regulated process of producing haploid
spermatozoa, involves testis-specific transcripts. Recent studies have discovered that long noncoding RNAs
(lncRNAs) are novel regulatory molecules that play important roles in various biological processes. However, there
has been no report on the comprehensive identification of testis-specific lncRNAs in mice.
Results: We performed microarray analysis of transcripts from mouse brain, heart, kidney, liver and testis. We found
that testis harbored the highest proportion of tissue-specific lncRNAs (11%; 1607 of 14,256). Testis also harbored the
largest number of tissue-specific mRNAs among the examined tissues, but the proportion was lower than that of
lncRNAs (7%; 1090 of 16,587). We categorized the testis-specific lncRNAs and found that a large portion
corresponded to long intergenic ncRNAs (lincRNAs). Genomic analysis identified 250 protein-coding genes located
near (≤ 10 kb) 194 of the loci encoding testis-specific lincRNAs. Gene ontology (GO) analysis showed that these
protein-coding genes were enriched for transcriptional regulation-related terms. Analysis of male germ cell-related
cell lines (F9, GC-1 and GC-2) revealed that some of the testis-specific lncRNAs were expressed in each of these cell
lines. Finally, we arbitrarily selected 26 testis-specific lncRNAs and performed in vitro expression analysis. Our results
revealed that all of them were expressed exclusively in the testis, and 23 of the 26 showed germ cell-specific
expression.
Conclusion: This study provides a catalog of testis-specific lncRNAs and a basis for future investigation of the
lncRNAs involved in spermatogenesis and testicular functions.
Keywords: cis-acting, Long noncoding RNA, Spermatogenesis, Testis, Testis-specific lncRNAs

Background piwi-interacting RNAs (piRNAs) [4], in the mouse testis.

Spermatogenesis is the complex and tightly regulated dif- However, a recent transcriptomic analysis revealed that
ferentiation process through which haploid spermatozoa there is pervasive transcription of long noncoding RNAs
are produced in the seminiferous tubules of the testis. (lncRNAs) in the mouse testis [5].
This process can be divided into three successive phases: LncRNAs are arbitrarily defined as noncoding RNAs lon-
the mitotic division of spermatogonia, the meiosis of sper- ger than 200 nucleotides. Compared with protein-coding
matocytes and the morphological change of spermatids transcripts, they tend to be shorter and have lower expres-
during spermiogenesis [1]. It is expected that a highly or- sion levels, fewer exons, less sequence conservation
ganized intrinsic genetic network is responsible for con- and more tissue and cell type-specific expression pat-
trolling spermatogenesis in the testis, and that the terns [6–11]. Studies have shown that lncRNAs are
elucidation of the underlying molecular mechanism will novel regulatory molecules involved in processes of
help us further understand male germ cell development. genetic regulation, including transcription [12, 13],
The previous studies have largely focused on identifying epigenetic modification [14, 15], alternative splicing of
and characterizing protein-coding genes [2] and small pre-mRNAs [16, 17] and mRNA stabilization or decoy
noncoding RNAs, such as microRNAs (miRNAs) [3] and functions [18, 19]. lncRNAs have also been shown to
play important roles in other biological processes, in-
* Correspondence: [email protected] cluding cell differentiation [19–21] and tissue devel-
School of Life Sciences, Gwangju Institute of Science and Technology,
Gwangju 61005, Korea opment [21, 22].

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Hong et al. BMC Genomics (2018) 19:539 Page 2 of 12

Recent studies have identified lncRNAs in the mouse (Fig. 1). The expression profiles obtained from this ana-
testis and examined their biological roles during male lysis are summarized in Table 1 and Additional file 1.
germ cell development [23–27]. However, although nu- The maximal numbers of both lncRNAs (14,256) and
merous studies have investigated the protein-coding genes mRNAs (16,587) were observed in testis compared to the
that show tissue-specific expression and play important other tissues, suggesting that testis is characterized by a
roles during spermatogenesis, no previous study has com- high level of transcriptomic diversity and complexity. To
prehensively identified and characterized the mouse perform tissue-specific expression profiling, we compared
testis-specific lncRNAs. the expression levels of transcripts among the five tissues
Here, we used microarray analysis to profile the previ- across three microarray experiments and classified the
ously annotated lncRNAs and mRNAs expressed in vari- transcripts as present (P), marginal (M) or absent (A) of
ous mouse tissues, including testis. By comparing the which expression level was lower than the background
lncRNA and mRNA expression profiles among the tissues, level. We regarded a transcript as a tissue-specific lncRNA
we identified lncRNAs and mRNAs that showed differen- or mRNA with the expression showing a fold-change ≥5
tial expression in each tissue. We found that testis has the and P-value ≤0.05 for a tissue relative to the others and
largest number of tissue-specific lncRNAs. We also pro- with three independent P or M only in the tissue. We
filed lncRNA and mRNA expression in three mouse male found that testis exhibited the largest numbers of
germ cell-related cell lines (F9, GC-1 and GC-2). To the tissue-specific lncRNAs (1607) and mRNAs (1090) among
best of our knowledge, this is the first study to identify the analyzed tissues (Table 1, Fig. 2a and Additional file 2).
mouse testis-specific lncRNAs and analyze their charac- The testis-specific lncRNAs were more abundant than
teristics. Our data provide a valuable resource for future the testis-specific mRNAs, even though there were
investigation of the lncRNAs that are involved in male fewer total lncRNAs than mRNAs in this tissue
germ cell development and testicular functions. (Fig. 2b). We speculated that these newly identified
testis-specific lncRNAs could be involved in regulat-
ing testicular gene expression and/or functions.
Results To validate our microarray-based selection of
Identification of lncRNAs and mRNAs in mouse tissues tissue-specific lncRNAs, we performed quantitative
To identify lncRNAs and mRNAs expressed in major real-time PCR (qRT-PCR) on several arbitrarily selected
tissues (brain, heart, kidney, liver and testis) in ICR mice tissue-specific lncRNAs (Fig. 3). As controls, we used
and germ cell-related cell lines (F9, GC-1 and GC-2) in ENSMUST00000161380, an lncRNA that is predicted
mice, we performed a microarray analysis using the to be ubiquitously expressed and the transition pro-
Arraystar mouse lncRNA microarray V3.0, which con- tein 1 (Tnp1) gene, which encodes a testis-specific
tains probes for 35,923 lncRNAs and 24,881 mRNAs mRNA [28]. The tissue-specific lncRNAs subjected to

Fig. 1 Schematic diagram of the expression profiles of lncRNAs and mRNAs in mouse major tissues and germ cell-related cell lines
Hong et al. BMC Genomics (2018) 19:539 Page 3 of 12

Table 1 Summary of the microarray results obtained in five qRT-PCR analysis were ENSMUT00000125184 (brain),
mouse tissues and three mouse cell lines ENSMUST00000125187 (heart), NR_102276 (kidney),
lncRNAs mRNAs lncRNAs mRNAs ENSMUST00000120145 (liver), NR_038002 (testis)
Tissues Total expressed Specifically expressed and AK018904 (testis). Our qRT-PCR analysis demon-
Brain 12,832 15,579 595 553 strated that these lncRNAs were specifically or pre-
dominantly expressed in the corresponding tissues
Heart 10,086 13,354 104 89
(Fig. 3), confirming our microarray data.
Kidney 9915 13,319 102 106
Liver 6290 9431 48 76 Chromosomal distribution of testicular lncRNAs
Testis 14,256 16,487 1607 1090 To characterize the genomic nature of the identified tes-
Germ cell-related cell lines Total expressed Testis-specific ticular lncRNAs and mRNAs, we examined the chromo-
F9 9065 12,512 128 187 somal localization of the loci that expressed these
transcripts. Our analysis revealed that they were widely
GC-1 10,571 13,492 143 167
distributed throughout the mouse chromosomes.
GC-2 10,652 13,443 126 175
Chromosome 5 and the X chromosome contain the
highest numbers of loci for testis-specific lncRNAs and
mRNAs, respectively (Fig. 4a and Additional file 3). To
further examine whether one or more chromosomes
were enriched with testis-specific lncRNAs and mRNAs,
we compared the ratio of testis-specific transcripts to

Fig. 2 Characteristics of the identified tissue-specific lncRNAs and mRNAs. a Heat map showing the relative expression levels of the
lncRNAs that exhibited differential expression in five major mouse tissues. Red and green colors indicate higher and lower expression
levels, respectively. b The percentage of tissue-specific lncRNAs and mRNAs in each mouse tissue
Hong et al. BMC Genomics (2018) 19:539 Page 4 of 12

Fig. 3 qRT-PCR-based validation of six arbitrarily selected tissue-specific lncRNAs. Data show the mean ± SEM of triplicates for each sample

a b

c d

Fig. 4 Chromosomal distribution and classification of testicular lncRNAs. a The number of testis-specific transcripts (lncRNAs and mRNAs found to
be specifically expressed in testis through the microarray analysis) on each chromosome. b The ratio of testis-specific transcripts to testicular
transcripts on each chromosome. The ratio was obtained by dividing the number of testis-specific transcripts by the number of total testicular
transcripts. c lncRNAs were classified into six subtypes (exonic sense, exonic antisense, intronic sense, intronic antisense, intergenic, and
bidirectional) based on the positional and directional relationship between their genomic regions and those of nearby mRNAs. d The
percentages of the six subtypes among the testicular lncRNAs
Hong et al. BMC Genomics (2018) 19:539 Page 5 of 12

total testicular transcripts among chromosomes (Fig. 4b). the expression of genes that encode proteins involved in
The ratio was similar among the chromosomes, except transcriptional regulation.
for the sex chromosomes: the ratios of testis-specific
lncRNAs and mRNAs were both remarkably high for Conservation of mouse testis-specific lncRNAs in human
the Y chromosome, even though relatively few loci on Although lncRNAs generally show poor sequence con-
this chromosome expressed testis-specific transcripts (21 servation among species [8], we utilized the BLASTN
testis-specific lncRNAs and 13 testis-specific mRNAs). program [32] to examine the sequence conservation be-
Based on this finding, we speculate that the expressions tween human lncRNAs and the mouse testis-specific
of testis-specific lncRNAs and mRNAs may be related lncRNAs identified in this study. Starting from the 1607
on the Y chromosome, perhaps through cis-regulation mouse testis-specific lncRNAs, we obtained genomic se-
between the two groups of testis-specific transcripts. quence information for 1508 (93.8%) from diverse gen-
ome databases (e.g., UCSC genome browser, NCBI and
Ensemble). When we used an E-value ≤10− 5 as a cut off,
Classification of testicular lncRNAs we found that only 79 (5.2%) of the mouse testis-specific
We categorized all of the identified lncRNAs based on the lncRNAs were partially conserved in the human genome
position and direction of their genomic regions relative to (Additional file 7). This indicates that testis-specific
nearby mRNAs. The lncRNAs were classified into six sub- lncRNAs have low primary sequence conservation, and
types: exonic sense, exonic antisense, intronic sense, in- are thus likely to experience rapid evolution.
tronic antisense, intergenic and bidirectional (Fig. 4c and
Additional file 4). If an lncRNA overlapped with the exon Expression of testis-specific lncRNAs in germ cell lines
or intron of a coding gene with the same or opposite dir- To further characterize the testis-specific lncRNAs, we
ection, it was called exonic sense, exonic antisense, in- used microarray analysis to investigate their expression
tronic sense or intronic antisense, respectively. A profiles in three relevant cell lines: F9, GC-1 and GC-2
bidirectional lncRNA was defined as a transcript whose (Fig. 1 and Additional file 8). F9 embryonal carcinoma
locus was less than 1 kb from an mRNA-encoding region, cells are stem cells of testicular carcinoma, and have the
regardless of its direction. Finally, a genomic locus more ability to differentiate into three embryonic layers [33].
than 1 kb from any mRNA-encoding genomic region was Although F9 cells are considered to be similar to embry-
considered to be a long intergenic ncRNA (lincRNA). onic cells, the former can transcribe germ cell-specific
Using this classification scheme, we found that the major- genes and possess the characteristics of male germ cells
ity of testis-specific (51.6%) and testicular (41.2%) [34]. Immortalized GC-1 cells exhibit the characteristics
lncRNAs were lincRNAs (Fig. 4d). of a stage between type B spermatogonia and primary
Because many lincRNAs are known to act in a cis-regula- spermatocytes [35], while GC-2 cells were derived from
tory manner [29, 30], we investigated the potential cis-regu- cells arrested at a premeiotic stage and exhibit the char-
latory targets of 829 testis-specific lincRNAs by searching acteristics of spermatocytes [36]. Our analysis revealed
for protein-coding genes within 10 kb up- and downstream that some of the testis-specific lncRNAs (14%; 228 of
of their encoding loci. We found that 23.4% (194 of 829) of 1607) and mRNAs (23%; 260 of 1090) were detectable in
the lincRNAs co-localized with 250 protein-coding genes one or more of the cell lines. When assessed individu-
(Additional file 5). Interestingly, some of these genes are ally, the various cell lines were found to express 128
testis-specific or predominant genes (e.g., Tnp1, Glt6d1, (F9), 143 (GC-1) and 126 (GC-2) of the lncRNAs and
1700018C11Rik, Speer4e, Spata31d1d, Tmco5, Spata17, 187 (F9), 167 (GC-1) and 175 (GC-2) of the mRNAs
1700080E11Rik, Rcc1, Agbl3 and 1700057G04Rik), suggest- (Fig. 5 and Additional file 9). Thus, these cell lines
ing the expression regulation of these genes by the lincR- should prove useful for the further characterization of
NAs through transcriptional or post-transcriptional certain testis-specific lncRNAs and mRNAs.
mechanisms. Gene Ontology (GO) [31] analysis of all of
the identified nearby protein-coding genes was performed In vitro expression of testis-specific lncRNAs
to explore their functions. We found that 40 diverse GO As we initially identified our tissue-specific transcripts
terms were significantly enriched (P-value ≤0.05), including using only five tissues in our microarray analysis, we
regulation of transcription DNA-templated (GO:0006355), assessed whether the selected lncRNAs were indeed
transcription DNA-templated (GO:0006351), positive regu- testis-specific by selecting 26 of them predicted to have
lation of transcription from RNA polymerase II promoter relatively abundant expression, and using reverse
(GO:0045944), negative regulation of transcription from transcription-polymerase chain reaction (RT-PCR) to
RNA polymerase II promoter (GO:0000122) and cell differ- examine their distribution in 10 mouse tissues. Our re-
entiation (GO:0030154) (Additional file 6). This suggests sults showed that all 26 of the testis-specific lncRNAs
that the testis-specific lincRNAs could potentially modulate were transcribed only in the mouse testis (Fig. 6).
Hong et al. BMC Genomics (2018) 19:539 Page 6 of 12

a b

Fig. 5 Venn diagram showing the number of testis-specific lncRNAs and mRNAs in three mouse germ cell-related cell lines. a Two hundred
twenty eight of the testis-specific lncRNAs were identified across the three cell lines. b Two hundred sixty testis-specific mRNAs were identified
across the three cell lines

Next, we examined the germ cell-specific expression Discussion

patterns of these 26 testis-specific lncRNAs using the Recent studies have shown that lncRNAs play important
germ cell-less testis of W/Wv (c-kit) mutant mice [37]. roles in regulating testicular development and spermato-
This analysis showed that 23 of the testis-specific genesis [26, 27]. Testis lncRNAs have been identified in
lncRNAs were absent from the testis of W/Wv mutant mammalian species, including mouse [23–25], rat [39], pig
mice (Fig. 7b), suggesting that they are germ cell-specific [40] and human [5]. In mice, several studies have investi-
lncRNAs. The other three testis-specific lncRNAs may gated the expression profiles of testis lncRNAs during post-
also be expressed in somatic cells of the testis. natal development [23], during specific developmental
Finally, we examined the developmental expression of stages of spermatogenesis [24] and at critical time points
these 26 testis-specific lncRNAs in mouse testis at differ- during male germ cell development [25]. In order to under-
ent postnatal days. Spermatogonial stem cells gradually stand the unique and complicated features of the transcrip-
proliferate and differentiate to produce spermatogonia, tome in testis, we need to identify lncRNAs that are
spermatocytes and round spermatids during the first differentially expressed in testis relative to other tissues [5].
round of spermatogenesis in the prepubertal mouse [38]. However, no previous study had set out to identify
Meiosis begins in the mouse testis at postnatal day 10. lncRNAs that are specifically or predominantly expressed
At postnatal day 14, pachytene spermatocytes are highly the testis of mouse. To address this gap, we herein used
enriched to about one-third of the total cells in the sem- microarray analysis to identify tissue-specific lncRNA tran-
iniferous tubules. Round spermatids appear in the sem- scripts in mouse brain, heart, kidney, liver and testis. We
iniferous tubules at postnatal day 18 (Fig. 7a). If the identified 14,256 lncRNAs and 16,487 mRNAs that were
testis-specific lncRNAs are expressed only in germ cells significantly expressed in mouse testis. Of them, 1607
during the first round of spermatogenesis, they will first lncRNAs and 1090 mRNAs were testis-specific. Among the
appear in the testis at a postnatal time that corresponds studied tissues, testis had the most tissue-specific lncRNAs
to a specific stage of spermatogenesis. Indeed, we found (11%) and mRNAs (7%). Notably, lncRNAs exhibited more
that four of the testis-specific lncRNAs were first testis specificity than mRNAs, suggesting that lncRNAs
expressed at postnatal day 14, suggesting that they are may be involved in male germ cell development.
expressed in early-stage spermatocytes; three were first We observed that the testis-specific lncRNAs and
expressed at postnatal day 16, suggesting that they are mRNAs were enriched on the Y chromosome relative to
expressed from pachytene spermatocytes; and 16 were the other mouse chromosomes. Genes of the mammalian
first expressed at postnatal day 21, suggesting that they Y chromosome are known to play important roles in sex
were expressed from round spermatids (Fig. 7b). These determination (e.g., Sry) [41] and spermatogenesis (e.g.,
results suggest that most of the identified testis-specific Eif2s3y) [42], suggesting that the testis-specific lncRNAs of
lncRNAs are expressed specifically in spermatogenic the Y chromosome deserve further investigation. The func-
cells, and their transcription appears to be regulated in a tions of lncRNAs cannot be predicted due to their poor se-
stage-dependent manner. quence conservation and our lack of knowledge regarding
Hong et al. BMC Genomics (2018) 19:539 Page 7 of 12

Fig. 6 Tissue distribution of 26 arbitrarily selected novel testis-specific lncRNAs in 10 mouse tissues, as assessed by RT-PCR. The results were
normalized with respect to the band intensity of glyceraldehyde-3-phosphate dehydrogenase (Gapdh). B, Brain; E, Epididymis; H, Heart; K, Kidney;
Li, Liver; Lu, Lung; O, Ovary; Sk, Skeletal muscle; Sp, Spleen; and T, Testis

relevant functional motifs and domains in protein-coding protein-coding genes within the 10-kb regions up- and
genes. However, a number of studies have inferred the pu- downstream of 194 testis-specific lincRNAs. Our GO ana-
tative functions of lncRNAs by examining their genomic re- lyses revealed that these genes were mainly involved in regu-
lationships with protein-coding genes, and some lncRNAs lating transcription. It seems possible that the testis-specific
have been shown to regulate the expression of their over- lincRNAs could contribute to spermatogenesis through such
lapping or neighboring protein-coding genes [43]. We function. Interestingly, some of the identified cis-acting tar-
herein identified several subtypes of lncRNAs, and found get protein-coding genes are known to be involved in
that lincRNAs constituted the largest proportion of both spermatogenesis (e.g., Tnp1 and Spata17), raising the possi-
testis and testis-specific lncRNAs [10]. We hypothesized bility that the testis-specific lincRNAs could play direct
that the testis-specific lincRNAs could act through a cis-re- regulatory roles in male germ cell development. Alterna-
gulatory mechanism, and thus identified and examined 250 tively, they may act in trans by targeting protein-coding
Hong et al. BMC Genomics (2018) 19:539 Page 8 of 12

Fig. 7 Developmental expression patterns of the 26 novel testis-specific lncRNAs during the first round of spermatogenesis. a Schematic diagram
showing the first round of spermatogenesis, which is composed of the mitotic, meiotic and postmeiotic phases. The meiotic phase consists of
preleptotene (PL), leptotene (L), zygotene (Z), pachytene (P), diplotene (D), meiotic division I (MI) and meiotic division II (MII). b Developmental
expression patterns of testis-specific lncRNAs in germ cell-lacking testes (W/Wv mutant mice) and wild-type mouse testes were assessed by RT-
PCR of samples taken at different postnatal days

genes located on the other mouse chromosomes. To answer conservation of the mouse testis-specific lncRNAs with
whether testis-specific lincRNAs indeed regulates the ex- human lncRNAs. However, relatively few mouse
pression of these protein-coding genes requires further testis-specific lncRNAs (5.2%) were found to be con-
investigation. served in the human lncRNAs. We speculate that the
Although it was previously reported that lncRNAs testis-specific lncRNAs may not require sequence con-
have a very low sequence conservation among species servation to maintain their functionality. Therefore, in
[8], we used BLAST analysis to investigate the sequence the future it will be important to examine the short
Hong et al. BMC Genomics (2018) 19:539 Page 9 of 12

conserved sequences and/or secondary structures of guidelines. Protocols were reviewed and approved by the
testis-specific lncRNAs. Institutional Animal Care and Use Committees (IACUC)
We used microarray analysis to examine the expression of Gwangju Institute of Science and Technology (GIST)
profiles of the selected lncRNAs and mRNAs in the cell (permit number: GIST-2017-013). We used 8-week-old
lines, F9, GC-1 and GC-2 [], which exhibit germ cell char- ICR male mice (Damul Science, Daejeon, Korea) for our
acteristics. A total of 128, 143 and 126 testis-specific microarray analysis. Mouse brain, heart, kidney, liver and
lncRNAs and 187, 167 and 175 testis-specific mRNAs testis tissues were obtained and immediately frozen in li-
were found to be expressed in the F9, GC-1 and GC-2 cell quid N2. Three biological replicates were performed in all
lines, respectively. Fifty five lncRNAs and 105 mRNAs cases, and samples were stored at − 80 °C until use.
were expressed in an overlapping manner in all of the cell
lines. It is possible that these transcripts are expressed Cell culture
during the whole period of spermatogenic stages from F9 (CRL-1720), GC-1 (CRL-2053) and GC-2 (CRL-2196)
which the cell lines were derived. It should be noted that cells of mouse origin were obtained from the American
these cell lines do not possess all the characteristics and Type Culture Collection (ATCC, Manassas, VA, USA)
phenotypes of male germ cells, such as meiosis and sper- and maintained at 37 °C and 5% CO2 in Dulbecco’s
miogenesis, suggesting weak and/or no expression of modified Eagle’s medium (DMEM) (Life Technologies,
some of the testis- or germ cell-specific transcripts in the Carlsbad, CA, USA) supplemented with 10% fetal bovine
cell lines. Nonetheless, these cell lines could prove useful serum (HyClone, Logan, UT, USA). The culture dishes
for studying the expression and functions of certain for the F9 cells were pre-coated with 0.1% gelatin. Be-
testis-specific lncRNAs. cause retinoic acid (RA) is known to induce F9 cell dif-
We arbitrarily selected 26 of the novel testis-specific ferentiation [46], it was omitted from the medium. The
lncRNAs and examined their tissue distributions in 10 culture media were changed every 1–2 days, and the
mouse tissues. All of the examined lncRNAs were, indeed, cells were sub-cultured every 3–4 days. At passages 3 or
specifically expressed in the mouse testis. Additionally, 4, cultured cells were collected for RNA extraction.
both developmental expression analysis during the first
round of spermatogenesis and expression analysis in germ RNA extraction, quality control and labeling
cell-less mutant mice confirmed the germ cell-specificity Total RNA from five major mouse tissues and three
of these lncRNAs. Interestingly, 16 of the 26 selected mouse germ cell-related cell lines was isolated with an
lncRNAs were expressed between 16 and 21 days after RNeasy Plus Mini kit (Qiagen, Valencia, CA, USA) ac-
birth in mouse, suggesting that these lncRNAs undergo a cording to the manufacturer’s instructions. Total RNA
transcriptional change between the late spermatocyte and was quantified and checked for quality using an
haploid round spermatid stages. During this period, epi- ND-1000 spectrophotometer (Thermo Fisher Scientific,
genetic regulation (e.g., DNA methylation and histone Waltham, MA, USA). RNA degradation and DNA con-
modification) occurs actively in testis [44]. As lncRNAs tamination were examined by 1% denaturing agarose gel
have been implicated in regulating protein-coding genes electrophoresis. RNA samples with RNA Integrity Num-
at the epigenetic level [45], we speculate that these ber (RIN) scores > 8 were used for microarray analysis.
germ-specific lncRNAs may be involved in epigenetic Each 1 μg of total RNA was reverse-transcribed into
regulation during spermatogenesis. cDNA using a MMLV-RT kit (Life Technologies). The
cDNA was transcribed and labeled with Cyanine 3-CTP
Conclusions (Cy3) using a Quick-Amp labeling kit (Agilent, Santa
We herein used microarray analysis to identify and Clara, CA, USA) according to the manufacturer’s proto-
characterize lncRNAs in mice, and then focused on com- col. The labeled cRNAs were quantified using an
prehensively cataloging the lncRNAs specifically found in ND-1000 spectrophotometer.
the testis, which showed the highest proportion of
tissue-specific expression. Our findings provide a basis for Mouse lncRNA microarray analysis
further investigating the functions of testis-specific Total cRNA was hybridized to a Mouse LncRNAs Micro-
lncRNAs, which should improve our understanding of the array (8 × 60 K; Arraystar) using a hybridization oven
lncRNA-mediated regulatory mechanisms that may be as- (Agilent). The hybridized microarray chip was washed ac-
sociated with mouse germ cell development. cording to the manufacturer’s protocol, and the hybridized
images were scanned using a DNA microarray scanner
Methods and quantified with the Feature Extraction Software (both
Animals and tissue preparation from Agilent). Data normalization and selection of
All animal experiments were performed in accordance fold-changed transcripts were performed using Gene-
with Korean Food and Drug Administration (KFDA) Spring GX 7.3 (Agilent). Total lncRNAs and mRNAs were
Hong et al. BMC Genomics (2018) 19:539 Page 10 of 12

detected in three independent sets of the samples in each cells of testis [37]. To investigate the developmental ex-
tissue; those above background were flagged as present or pression patterns of the selected lncRNAs during the first
marginal (P or M, respectively). For each tissue, specific- round of spermatogenesis, total RNA from testes of pre-
ally expressed lncRNAs and mRNAs were chosen using pubertal and adult mice (7d, 8d, 14d, 16d, 21d, 24d, 28d,
the following criteria: 1) in three independent samples, 35d, 42d, 49d and 56d) was subjected to RT-PCR. Total
they were flagged as P or M in a single given tissue but ab- RNA was extracted using the TRIzol reagent (Invitrogen,
sent (A) in the other tissues; 2) fold-change ≥5; and 3) Carlsbad, CA, USA) according to the manufacturer’s
P-value ≤0.05. The raw and processed microarray data protocol, and cDNA was synthesized using random hex-
have been deposited in the Gene Expression Omnibus amers, oligo(dT) primers and Omniscript reverse tran-
(GEO) database at the National Center for Biotechnology scriptase (Qiagen). lncRNA-specific primers were
Information (NCBI), under GEO accession number designed using Primer-BLAST and are listed in Add-
GSE105024. itional file 11. Amplification was performed for 35–40 cy-
cles of 95 °C for 30 s, 50 °C or 55 °C for 30 s and 72 °C for
Validation of tissue-specific lncRNAs by qRT-PCR 30 s. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh)
Total RNA was extracted from five mouse tissues and were used as a loading control.
three germ cell-related cell lines, and reverse-transcribed
using RNeasy Plus Mini and Omniscript RT kits (Qia- Statistical analyses
gen). qRT-PCR was carried out using the TOPreal™ All experiments were performed in triplicate for each sam-
qPCR 2X premix (Enzynomics, Daejeon, Korea). The re- ple. Statistical analysis was performed using the Student’s
action volume contained 10 μl of TOPreal™ qPCR 2X t-test, and P ≤ 0.05 was taken as indicating a significant dif-
PreMix, 1 μl of 10 μM forward and reverse primers, 50– ference. Data are presented as the mean ± standard error of
100 ng of template cDNA and dH2O to a final volume the mean (mean ± SEM).
of 20 μl. The reactions were performed on a StepOne-
Plus Real-Time PCR System (Thermo Fisher Scientific)
as follows: 95 °C for 12 min 30 s, followed by 40 cycles Additional files
of 95 °C for 10 s, 60 °C for 15 s and 72 °C for 23 s. The
Additional file 1: Table S1. LncRNAs and mRNAs in the mouse brain,
melting curve analysis was performed from 65 °C to 95 °
heart, kidney, liver and testis. (XLSX 18613 kb)
C with increments of 0.3 °C. The primer sets used to Additional file 2: Table S2. Tissue-specific lncRNAs and mRNAs in the
amplify the selected lncRNAs and control genes are mouse brain, heart, kidney, liver and testis. (XLSX 4020 kb)
listed in Additional file 10. Amplifications were per- Additional file 3: Table S3. The numbers and ratios of testicular
formed in triplicate for each sample. Relative gene ex- lncRNAs and mRNAs on each mouse chromosome. (XLSX 11 kb)
pression levels were evaluated using the 2-ΔΔCt method Additional file 4: Table S4. The numbers and ratios of six types of
testicular lncRNA. (XLSX 10 kb)
[47], and normalized with respect to the level of the en-
Additional file 5: Table S5. Nearby protein-coding genes located
dogenous Gapdh mRNA. within genomic regions 10 kb up- and downstream of the testis-specific
lincRNAs. (XLSX 24 kb)
Searching for nearby protein-coding genes and gene Additional file 6: Table S6. GO terms of the nearby protein-coding
ontology analysis genes. (XLSX 19 kb)
As several lincRNAs act as through a cis-regulatory mech- Additional file 7: Table S7. Mouse testis-specific lncRNAs that show
conservation in human. (XLSX 26 kb)
anism [29, 30], we searched for protein-coding genes lo-
Additional file 8: Table S8. LncRNAs and mRNAs in three mouse germ
cated in the genomic regions 10 kb up- and downstream of cell-related cell lines (F9, GC-1 and GC-2). (XLSX 16402 kb)
the testis-specific lincRNAs. We then predicted their func-
Additional file 9: Table S9. Testis-specific lncRNAs and mRNAs in three
tional roles by GO analysis using the DAVID software [48]. mouse germ cell-related cell lines (F9, GC-1 and GC-2). (XLSX 712 kb)
GO terms with P-value ≤0.05 were considered significantly Additional file 10: Table S10. Primers used for qRT-PCR. (XLSX 10 kb)
enriched. Additional file 11: Table S11. Primers used for RT-PCR. (XLSX 11 kb)

RT-PCR analysis of tissue distribution and developmental

Abbreviations
expression patterns Agbl3: ATP/GTP binding protein-like 3; Eif2s3y: Eukaryotic translation initiation
To determine the tissue distribution patterns of 26 factor 2, subunit 3, structural gene Y-linked; Glt6d1: Glycosyltransferase 6
testis-specific lncRNAs arbitrarily selected from our micro- domain containing 1; GO: Gene ontology; lincRNA: Long intergenic ncRNA;
lncRNA: Long noncoding RNA; miRNA: microRNAs; piRNA: Piwi-interacting RNA;
array analysis, we performed RT-PCR using cDNA from 10 qRT-PCR: Quantitative real-time PCR; Rcc1: Regulator of chromosome
mouse tissues (brain, epididymis, heart, kidney, liver, lung, condensation 1; Spata17: Spermatogenesis associated 17;
ovary, skeletal muscle and testis). We also used cDNA from Spata31d1d: Spermatogenesis associated 31 subfamily D, member 1D;
Speer4e: Spermatogenesis associated glutamate (E)-rich protein 4e; Sry: Sex
germ cell-lacking testes of W/Wv mutant mice to investi- determining region of Chr Y; Tmco5: Transmembrane and coiled-coil domains
gate whether these lncRNAs were expressed in somatic 5; Tnp1: Transition protein 1
Hong et al. BMC Genomics (2018) 19:539 Page 11 of 12

Funding 10. Cabili MN, Trapnell C, Goff L, Koziol M, Tazon-Vega B, Regev A, Rinn JL.
This work was supported by Mid-career Researcher Program through Na- Integrative annotation of human large intergenic noncoding RNAs reveals
tional Research Foundation of Korea funded by the Ministry of Science, ICT & global properties and specific subclasses. Genes Dev. 2011;25(18):1915–27.
Future Planning (NRF-2015R1A2A2A01005300) in the design of the study and 11. Mercer TR, Dinger ME, Sunkin SM, Mehler MF, Mattick JS. Specific expression
writing the manuscript, the Bio & Medical Technology Development Program of long noncoding RNAs in the mouse brain. Proc Natl Acad Sci U S A.
of the National Research Foundation of Korea funded by the Ministry of Sci- 2008;105(2):716–21.
ence, ICT & Future Planning (NRF 2013M3A9A7046297) in the design of the 12. Vance KW, Ponting CP. Transcriptional regulatory functions of nuclear long
study and writing the manuscript and GIST Research Institute (GRI), Korea in noncoding RNAs. Trends Genet. 2014;30(8):348–55.
the design of the study and collection, analysis, and interpretation of data 13. Rinn JL, Chang HY. Genome regulation by long noncoding RNAs. Annu Rev
and in writing the manuscript. Biochem. 2012;81:145–66.
14. Holoch D, Moazed D. RNA-mediated epigenetic regulation of gene
Availability of data and materials expression. Nat Rev Genet. 2015;16(2):71–84.
The microarray data can be found in the Gene Expression Omnibus (GEO) 15. Mercer TR, Mattick JS. Structure and function of long noncoding RNAs in
database at the National Center for Biotechnology Information (NCBI), under epigenetic regulation. Nat Struct Mol Biol. 2013;20(3):300–7.
GEO accession number GSE105024. 16. Gonzalez I, Munita R, Agirre E, Dittmer TA, Gysling K, Misteli T, Luco RF. A
lncRNA regulates alternative splicing via establishment of a splicing-specific
Authors’ contributions chromatin signature. Nat Struct Mol Biol. 2015;22(5):370–6.
CC conceived the study. SHH and CC designed the experiments. SHH 17. Bardou F, Ariel F, Simpson CG, Romero-Barrios N, Laporte P, Balzergue S,
performed the RNA preparation, cell culture and RT-PCR experiments, and Brown JW, Crespi M. Long noncoding RNA modulates alternative splicing
analyzed and interpreted the RT-PCR and microarray data. JTK, JiK, JJ, JaK, regulators in Arabidopsis. Dev Cell. 2014;30(2):166–76.
and SL contributed reagents, materials and analysis tools, and to the inter- 18. Gong C, Maquat LE. lncRNAs transactivate STAU1-mediated mRNA decay by
pretation of the RT-PCR and microarray data. SHH and CC wrote the manu- duplexing with 3' UTRs via Alu elements. Nature. 2011;470(7333):284–8.
script. All authors read and approved the final manuscript. 19. Kretz M, Siprashvili Z, Chu C, Webster DE, Zehnder A, Qu K, Lee CS,
Flockhart RJ, Groff AF, Chow J, et al. Control of somatic tissue differentiation
Ethics approval by the long non-coding RNA TINCR. Nature. 2013;493(7431):231–5.
All animal experiments were performed in accordance with Korean Food 20. Fatica A, Bozzoni I. Long non-coding RNAs: new players in cell
and Drug Administration (KFDA) guidelines. Protocols were reviewed and differentiation and development. Nat Rev Genet. 2014;15(1):7–21.
approved by the Institutional Animal Care and Use Committees (IACUC) of 21. Grote P, Wittler L, Hendrix D, Koch F, Wahrisch S, Beisaw A, Macura K, Blass G, Kellis
Gwangju Institute of Science and Technology (GIST) (permit number: GIST- M, Werber M, et al. The tissue-specific lncRNA Fendrr is an essential regulator of
2017-013). heart and body wall development in the mouse. Dev Cell. 2013;24(2):206–14.
22. Mathieu EL, Belhocine M, Dao LT, Puthier D, Spicuglia S. Functions of
Consent for publication lncRNA in development and diseases. Med Sci (Paris). 2014;30(8–9):790–6.
Not applicable. 23. Sun J, Lin Y, Wu J. Long non-coding RNA expression profiling of mouse
testis during postnatal development. PLoS One. 2013;8(10):e75750.
Competing interests 24. Liang M, Li W, Tian H, Hu T, Wang L, Lin Y, Li Y, Huang H, Sun F. Sequential
The authors declare that they have no competing interests. expression of long noncoding RNA as mRNA gene expression in specific
stages of mouse spermatogenesis. Sci Rep. 2014;4:5966.
Publisher’s Note 25. Bao J, Wu J, Schuster AS, Hennig GW, Yan W. Expression profiling reveals
Springer Nature remains neutral with regard to jurisdictional claims in developmentally regulated lncRNA repertoire in the mouse male germline.
published maps and institutional affiliations. Biol Reprod. 2013;89(5):107.
26. Anguera MC, Ma W, Clift D, Namekawa S, Kelleher RJ 3rd, Lee JT. Tsx
Received: 17 October 2017 Accepted: 5 July 2018 produces a long noncoding RNA and has general functions in the germline,
stem cells, and brain. PLoS Genet. 2011;7(9):e1002248.
27. Akhade VS, Dighe SN, Kataruka S, Rao MR. Mechanism of Wnt signaling
References induced down regulation of mrhl long non-coding RNA in mouse
1. Eddy EM. Male germ cell gene expression. Recent Prog Horm Res. 2002;57:103–28. spermatogonial cells. Nucleic Acids Res. 2016;44(1):387–401.
2. Sha J, Zhou Z, Li J, Yin L, Yang H, Hu G, Luo M, Chan HC, Zhou K, 28. Yu YE, Zhang Y, Unni E, Shirley CR, Deng JM, Russell LD, Weil MM,
Spermatogenesis study g. Identification of testis development and Behringer RR, Meistrich ML. Abnormal spermatogenesis and reduced
spermatogenesis-related genes in human and mouse testes using cDNA fertility in transition nuclear protein 1-deficient mice. Proc Natl Acad Sci
arrays. Mol Hum Reprod. 2002;8(6):511–7. U S A. 2000;97(9):4683–8.
3. Hayashi K, Chuva de Sousa Lopes SM, Kaneda M, Tang F, Hajkova P, Lao K, 29. Trimarchi T, Bilal E, Ntziachristos P, Fabbri G, Dalla-Favera R, Tsirigos A,
O'Carroll D, Das PP, Tarakhovsky A, Miska EA, et al. MicroRNA biogenesis is Aifantis I. Genome-wide mapping and characterization of notch-regulated
required for mouse primordial germ cell development and long noncoding RNAs in acute leukemia. Cell. 2014;158(3):593–606.
spermatogenesis. PLoS One. 2008;3(3):e1738. 30. Yap KL, Li S, Munoz-Cabello AM, Raguz S, Zeng L, Mujtaba S, Gil J, Walsh MJ,
4. Gan H, Lin X, Zhang Z, Zhang W, Liao S, Wang L, Han C. piRNA profiling Zhou MM. Molecular interplay of the noncoding RNA ANRIL and
during specific stages of mouse spermatogenesis. RNA. 2011;17(7):1191–203. methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional
5. Soumillon M, Necsulea A, Weier M, Brawand D, Zhang X, Gu H, Barthes P, silencing of INK4a. Mol Cell. 2010;38(5):662–74.
Kokkinaki M, Nef S, Gnirke A, et al. Cellular source and mechanisms of high 31. Huang d W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large
transcriptome complexity in the mammalian testis. Cell Rep. 2013;3(6):2179–90. gene lists using DAVID bioinformatics resources. Nat Protoc. 2009;4(1):44–57.
6. Dinger ME, Amaral PP, Mercer TR, Pang KC, Bruce SJ, Gardiner BB, Askarian- 32. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment
Amiri ME, Ru K, Solda G, Simons C, et al. Long noncoding RNAs in mouse search tool. J Mol Biol. 1990;215(3):403–10.
embryonic stem cell pluripotency and differentiation. Genome Res. 2008; 33. Alonso A, Breuer B, Steuer B, Fischer J. The F9-EC cell line as a model for the
18(9):1433–45. analysis of differentiation. Int J Dev Biol. 1991;35(4):389–97.
7. Pauli A, Valen E, Lin MF, Garber M, Vastenhouw NL, Levin JZ, Fan L, Sandelin 34. Kwon JT, Jin S, Choi H, Kim J, Jeong J, Kim J, Kim Y, Cho BN, Cho C.
A, Rinn JL, Regev A, et al. Systematic identification of long noncoding RNAs Identification and characterization of germ cell genes expressed in the F9
expressed during zebrafish embryogenesis. Genome Res. 2012;22(3):577–91. testicular teratoma stem cell line. PLoS One. 2014;9(8):e103837.
8. Kutter C, Watt S, Stefflova K, Wilson MD, Goncalves A, Ponting CP, Odom 35. Hofmann MC, Narisawa S, Hess RA, Millan JL. Immortalization of germ cells
DT, Marques AC. Rapid turnover of long noncoding RNAs and the evolution and somatic testicular cells using the SV40 large T antigen. Exp Cell Res.
of gene expression. PLoS Genet. 2012;8(7):e1002841. 1992;201(2):417–35.
9. Ulitsky I, Bartel DP. lincRNAs: genomics, evolution, and mechanisms. 36. Hofmann MC, Hess RA, Goldberg E, Millan JL. Immortalized germ cells
Cell. 2013;154(1):26–46. undergo meiosis in vitro. Proc Natl Acad Sci U S A. 1994;91(12):5533–7.
Hong et al. BMC Genomics (2018) 19:539 Page 12 of 12

37. Geissler EN, Ryan MA, Housman DE. The dominant-white spotting (W) locus
of the mouse encodes the c-kit proto-oncogene. Cell. 1988;55(1):185–92.
38. Bellve AR, Cavicchia JC, Millette CF, O'Brien DA, Bhatnagar YM, Dym M.
Spermatogenic cells of the prepuberal mouse. Isolation and morphological
characterization. J Cell Biol. 1977;74(1):68–85.
39. Chalmel F, Lardenois A, Evrard B, Rolland AD, Sallou O, Dumargne MC,
Coiffec I, Collin O, Primig M, Jegou B. High-resolution profiling of novel
transcribed regions during rat spermatogenesis. Biol Reprod. 2014;91(1):5.
40. Ran M, Chen B, Li Z, Wu M, Liu X, He C, Zhang S, Li Z. Systematic
identification of long noncoding RNAs in immature and mature porcine
testes. Biol Reprod. 2016;94(4):77.
41. Koopman P, Gubbay J, Vivian N, Goodfellow P, Lovell-Badge R. Male
development of chromosomally female mice transgenic for Sry. Nature.
1991;351(6322):117–21.
42. Mazeyrat S, Saut N, Grigoriev V, Mahadevaiah SK, Ojarikre OA, Rattigan A,
Bishop C, Eicher EM, Mitchell MJ, Burgoyne PS. A Y-encoded subunit of the
translation initiation factor Eif2 is essential for mouse spermatogenesis. Nat
Genet. 2001;29(1):49–53.
43. Mercer TR, Dinger ME, Mattick JS. Long non-coding RNAs: insights into
functions. Nat Rev Genet. 2009;10(3):155–9.
44. Sasaki H, Matsui Y. Epigenetic events in mammalian germ-cell development:
reprogramming and beyond. Nat Rev Genet. 2008;9(2):129–40.
45. Cao J. The functional role of long non-coding RNAs and epigenetics. Biol
Proced Online. 2014;16:11.
46. Lehtonen E, Laasonen A, Tienari J. Teratocarcinoma stem cells as a model
for differentiation in the mouse embryo. Int J Dev Biol. 1989;33(1):105–15.
47. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using
real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods.
2001;25(4):402–8.
48. Huang DW, Sherman BT, Tan Q, Kir J, Liu D, Bryant D, Guo Y, Stephens R,
Baseler MW, Lane HC, et al. DAVID bioinformatics resources: expanded
annotation database and novel algorithms to better extract biology from
large gene lists. Nucleic Acids Res. 2007;35(Web Server issue):W169–75.