Abstract
YY super-males have rarely been detected in nature and only been
artificially created in some fish species including tilapia and yellow
catfish (Pelteobagrusfulvidraco), which provides a promising model for
testis development and spermatogenesis. In our previous study,
significant differences in morphology and miRNA expression were
detected between XY and YY testis of yellow catfish. Here, solexa
sequencing technology was further performed to compare mRNA expression
between XY and YY testis. Compared with unigenes expressed in XY
testis, 1146 and 1235 unigenes have significantly higher and lower
expression in YY testis, respectively. 605 differentially expressed
unigenes were annotated to 1604 GO terms with 319 and 286 genes having
relative higher expression in XY and YY testis. KEGG analysis suggested
different levels of PI3K-AKT and G protein-coupled receptor (GPCR)
signaling pathways between XY and YY testis. Down-regulation of
miR-141/429 in YY testis was speculated to promote testis development
and maturation, and several factors in PI3K-AKT and GPCR signaling
pathways were found as predicted targets of miR-141/429, several of
which were confirmed by dual-luciferase reporter assays. Our study
provides a comparative transcriptome analysis between XY and YY testis,
and reveals interactions between miRNAs and their target genes that are
possibly involved in regulating testis development and spermatogenesis.
Introduction
Sex determination and differentiation are the most significant
developmental events that direct the embryonic gonads into either
testes or ovaries [[35]1,[36]2]. The XY sex-determining system is the
most popular sex-determination system found in vertebrates. Sry and
dmrt1 are conserved sex-determining genes, triggering differentiation
of testes from bi-potential gonads in mammals and birds [[37]3,[38]4].
Until now, multiple sex-determining genes including dmy/dmrt1Y and
dmrt1 in Oryzias latipes [[39]5,[40]6], gsdf in Oryzias luzonensis
[[41]7], sox3 in Oryzias dancena [[42]8], amhy in Odontesthes hatchery
[[43]9], sdY in Oncorhynchus mykiss [[44]10], amhr2 in Takifugu
rubripes [[45]11] and dmrt1 in Cynoglossus semilaevis [[46]12] have
been identified to participate in the male sex determination in
different fish species. Moreover, several important genes such as
cyp19a1, foxl2, wnt4 for ovary differentiation and dmrt1, sox9, amh for
testicular differentiation have been revealed in teleosts
[[47]13–[48]15].
As an evolutionary link between invertebrates and higher vertebrates,
fish species have a very complex sex determination system with XX/XY
male heterogametic system as the main sex determination system
[[49]16]. Until now, YY super-males have only been artificially
produced in some fish species [[50]17–[51]19], but they could not
survive in case of higher vertebrates including human and mouse.
Previous studies on rainbow trout (Oncorhynchusmykiss) reported obvious
differences in gene expression and morphology between XY and YY testis
[[52]20,[53]21]. Significant differences of aromatase expression were
found in spermatogonia, spermatids and epithelial cells among XY and YY
testis [[54]21]. Moreover, a relative higher level of androgen receptor
expression was observed in efferent ducts of YY testis compared with XY
testis [[55]20]. However, the sperm quality and quantity of XY and YY
males were the similar in Nile tilapia (Oreochromisniloticus)[[56]22].
There are no comprehensive studies regarding genetic differences
between XY and YY males.
In our previous study, we observed larger spermatogenic cyst and more
spermatids in YY super-males than XY males by histological analysis,
suggesting a higher degree of testis maturity. Many miRNAs that are
potentially involved in testis development and spermatogenesis were
identified to be differentially expressed between XY and YY testis
[[57]23]. The establishment and maintenance of spermatogenesis in fish
requires specialized gene regulatory networks in the testis [[58]24].
Here, we utilized RNA-Seq approach to identify genes and pathways that
were differentially expressed between XY and YY testis and their
functional relationship with miRNAs. Hopefully, our findings would
provide a clue about the genetic mechanism of testicular differences
between XY and YY males.
Results
Illumina sequencing, sequence assembly and functional annotation
In order to identify differentially expressed mRNA in testes of male
and super-male yellow catfish, two solexa libraries were constructed by
XY and YY testes respectively. After removing adaptors and low quality
reads, a total of 87,149,414 and 75,448,188 reads were obtained in each
profile respectively. After de novo assembly, 78148 unigenes were
obtained by paired-end method of Trinity and TGICL clustering with mean
length of 944bps ([59]S1 Fig).
The unigenes of de novo assembly were searched against the NCBI
non-redundant (nr), SWISS-PROT, KEGG, GO and KOG protein databases by
using BLASTX with a cut-off E-value of 1e^-5 ([60]S1 Table). Finally, a
total of 19,795 (25.33%) unigenes were significantly matched with nr
database. Among these BlastX-hit unigenes ([61]Fig 1), 11591 (58.56%)
hits were assigned to Daniorerio while only 776 (3.92%) hits overlapped
with Ictalurus punctatus (the close species to Pelteobagrus
fulvidraco), perhaps due to the limited amount of genomic data on
GeneBank about the species of Siluriformes. In addition, there were a
number of unigenes without hitting results, which may contain
non-coding fragments or some undetected genes.
Fig 1. Species distribution of the top BlastX hits using the assembled
unigenes of yellow catfish.
Fig 1
[62]Open in a new tab
The unigenes were searched against the NCBI Nr database with a cut-off
E-value<1e-5.
Analysis of differentially expressed genes (DEGs)
To identity differentially expressed unigenes between XY male and YY
super-male, the expression of assembled unigenes were counted by RPKM
method. After comparison (XYvsYY testis) with fold change threshold
value = 2 and FDR test (P< 0.05), 4458 unigenes were found expressed
with significant difference. Among them 1006 were detected from testis
of YY super-male and 1072 were found from testis of XY male. Besides
this, 1146 unigenes from YY and 1235 from XY testis were detected with
significantly higher expression. The MA scatter plots comparison reveal
the differential expressed genes existed between testes of male and
super-male in yellow catfish ([63]Fig 2).
Fig 2. Difference of unigene expression between male (XY) and super-male
(YY).
[64]Fig 2
[65]Open in a new tab
The X-axis represents the value of normalized expression counts, and
the Y-axis represents the level of differential expression.
Significantly differential expressions are marked in red and other in
grey.
GO annotation and analysis of DEGs enriched in YY testis
The 4458 differentially expressed unigenes (DEGs) were searched against
the Gene Ontology database ([66]www.geneontology.org) to determine
which kinds of GO term DEGs mainly participated in. Finally, 605 DEGs
were annotated to 1604 GO terms ([67]S2 Table) accompanied by 319 and
286 genes with relative higher expression in XY and YY testes,
respectively. The functions of 286 DEGs with higher expression in YY
than XY testis were assigned to biological process, cellular component
and molecular function ([68]Fig 3). In biological process, proteolysis
(58 DEGs, GO:0006508), RNA-dependent DNA replication (38 DEGs, GO:
0006278) and DNA integration (33DEGs, GO:0015074) were the most
prominent terms. Integral component of membrane (112 DEGs,GO:0016021)
was the most prominent within cellular component followed by nucleus
(67DEGs,GO:0005634), cytoplasm (48DEGs,GO:0005737) and plasma membrane
(40DEGs,GO:0005886). In molecular function, most of the annotated
unique sequences were assigned to zinc ion binding (74DEGs,GO:0008270),
ATP binding (51DEGs,GO: 0005524), metal ion binding
(53DEGs,GO:0046872), RNA binding (46 sequences,GO:0003723) and
RNA-directed DNA polymerase activity (38DEGs,GO:0003964)([69]Fig 3).
Fig 3. Functional classification of the DEGs that has higher expression in YY
than XY based on gene ontology (GO) terms: biological process (red), cellular
component (blue),molecular function (green).
[70]Fig 3
[71]Open in a new tab
The X-axis is number of unigenes.
KEGG analysis of DEGs between XY and YY testis
KEGG annotation is the process of mapping interested genes to the
metabolic pathways. In our study, 312 DEGs were mapped to 252 KEGG
pathways. Enrichment analysis shows that 143 YY highly expressed
unigenes were enriched in 192 pathways, and most of the DEGs were
assigned to pathways in cancer (16 DEGs, ko05200), PI3K-Akt signaling
pathway (12 DEGs, ko04151), phagosome (12 DEGs, ko04145), neuroactive
ligand-receptor interaction (11 DEGs, ko04080), cytokine-cytokine
receptor interaction (10 DEGs, ko046760), tuberculosis (10 DEGs,
ko05152) and focal adhesion (10 DEGs, ko4510). The XY highly expressed
unigenes were enriched in pancreatic secretion (23 DEGs, ko04972),
protein digestion and absorption (20 DEGs, ko04974) and neuroactive
ligand-receptor interaction (13 DEGs, ko04080) ([72]Fig 4).
Fig 4. KEGG classification of the DEGs between transcriptomes of YY and XY
testis.
[73]Fig 4
[74]Open in a new tab
The green and red columns represent the signaling pathways enriched in
YY and XY testes, respectively.
The PI3K-AKT signaling pathway is involved in many fundamental
functions including testis development and spermatogenesis, and
stimulated by many kind of growth factors that specifically binds to
receptor tyrosine kinase (RTK) or G protein-coupled receptors (GPCR)
[[75]25,[76]26]. As members of RTK, the spleen tyrosine kinase (Syk),
colony stimulating factor 1 receptor (Csf1r), and prolactin receptor
(Prlr) were expressed about 3.68, 3.71 and 1.73 fold higher in YY
testis than in XY, respectively. In addition, β1-Integrin (Itgb1) and
β2-Integrin (Itgb2) were also expressed 2.71 and 3.15-fold higher in YY
testis than in XY ([77]Fig 5). In the “neuroactive ligand–receptor
interaction” signaling pathway, expression of multiple genes associated
with G protein signaling were significantly up-regulated in YY testis
([78]Fig 6), like Kiss1r (GPR54) and somatostatin receptors (Sstr) in
Class A of GPCR signaling and metabotropic glutamate receptor 5 (GRM5)
in Class C of GPCR signaling. Meanwhile, the mRNA levels of glutamate
receptor AMPA 2b (gria2b), glutamate receptor AMPA 4a (gria4a) and
prolactin receptor (PRLR) were also higher in YY than in XY. In
contrast, the expression levels of histamine receptor H1 (Hrh1),
neuropeptide FF receptor 2 (Npffr2), leukotriene B4 receptor 1 (Ltb4r1)
and nicotinic acetylcholine receptor α1 (Chrnα1) were higher in XY than
in YY testis.
Fig 5. DEGs involved in the PI3K-AKT signaling pathway.
[79]Fig 5
[80]Open in a new tab
YY highly expressed unigenes are shown in green and XY highly expressed
unigenes are shown in red. The numbers in parentheses indicate the
value of fold-change (RPKM value of XY/ RPKM value of YY).
Fig 6. DEGs involved in the GPCR signaling pathway.
[81]Fig 6
[82]Open in a new tab
YY highly expressed unigenes are shown in green and XY highly expressed
unigenes are shown in red. The numbers in parentheses indicate the
value of fold-change (RPKM value of XY/ RPKM value of YY).
qRT-PCR confirmation of DEGs between XY and YY testis
To verify the accuracy of the sequencing data, twelve DEGs related to
RTK and G protein signaling pathway were arbitrarily selected and
validated by quantitative real-Time PCR (qRT-PCR). The twelve DEGs
includes four genes (Hrh1, Npffr2, Ltb4r1, Chrnα1) relatively high
expressed in XY and eight genes (Prlr, Csf1r, Itgb1, Kiss1r, Sstr,
GRM5, Gria2b, Gria4a) relatively high expressed in YY testis in the
transcriptome data ([83]Fig 7A). As in the qRT-PCR results, the
relative expression levels of twelve differentially expressed genes
were completely consistent with the Solexa sequencing ([84]Fig 7B).
Fig 7. Concordance of solexa sequencing data with qRT-PCR data.
[85]Fig 7
[86]Open in a new tab
(A) Profile of solexa sequencing value for selected genes with
normalized expression data. (B) Profile of relative expression of
selected genes by qRT-PCR. *p<0.05 indicates the significant difference
in gene expression between male (XY) and super-male (YY).
Identification of potential target genes for miR-141/429 from DEGs and
validation by dual-luciferase reporter assays
In our previous study, relative lower expression of miR-141/429 was
observed in YY testis indicating a higher degree of testis maturity
than XY testis. A high dose of 17α-ethinylestradiol (EE2) up-regulates
the expression of miR-141/429 [[87]23]. Here, we examined whether some
DEGs are potential target genes for miR-141-3p and miR-429b-3p.
Finally, 31 and 11 YY enriched DEGs were predicted to be targeted by
miR-141-3p and miR-429b-3p ([88]Table 1). For example, Itgb2, a factor
involved in the PI3K-AKT signaling pathway, was highly expressed in YY
and was a putative target of miR-141-3p. In addition, Gria4a is a
factor for the neuroactive ligand–receptor interaction signaling
pathway and also a putative target for miR-141-3p. AMH and Tgfβr1 were
potential targets for miR-141-3p and miR-429b-3p, respectively.
Table 1. The predicted targets of miR141/429 that displayed higher expression
in YY than XY.
miRNA name No. of tanscript Putative target Target gene annotation
pfu-miR-141-3p CL52176Contig1 Aacs acetoacetyl-CoA synthetase
pfu-miR-141-3p CL51317Contig1 Amh anti-mullerian hormone
pfu-miR-141-3p CL40242Contig1 Amph amphiphysin
pfu-miR-141-3p CL55509Contig1 Bcl6 B-cell lymphoma 6 protein
pfu-miR-141-3p CL12469Contig1 Cacnα2d4 voltage-dependent calcium
channel alpha-2/delta-4
pfu-miR-141-3p CL75180Contig1 Chrd chordin
pfu-miR-141-3p CL9871Contig1 Col6α collagen type VI alpha
pfu-miR-141-3p CL1267Contig1 Ctnna catenin alpha
pfu-miR-141-3p CL74732Contig1 Cxcl13 C-X-C motif chemokine 13
pfu-miR-141-3p CL52163Contig1 Dmbt1 deleted in malignant brain tumors 1
protein
pfu-miR-141-3p comp12586_c0_seq1 Dnah dynein heavy chain, axonemal
pfu-miR-141-3p CL54671Contig1 Nsdhl sterol-4alpha-carboxylate
3-dehydrogenase (decarboxylating)
pfu-miR-141-3p CL15376Contig1 Ext1 glucuronyl/N-acetylglucosaminyl
transferase EXT1
pfu-miR-141-3p CL49906Contig1 Fads2 fatty acid desaturase 2 (delta-6
desaturase)
pfu-miR-141-3p CL53372Contig1 Gli2 zinc finger protein GLI2
pfu-miR-141-3p CL72487Contig1 Gria4 glutamate receptor 4
pfu-miR-141-3p CL15787Contig1 Hya hyaluronoglucosaminidase
pfu-miR-141-3p CL53574Contig1 Itgb2 integrin beta 2
pfu-miR-141-3p CL14671Contig1 Lamα4 laminin alpha 4
pfu-miR-141-3p CL4177Contig1 Ldlrap1 low density lipoprotein receptor
adapter protein 1
pfu-miR-141-3p CL29924Contig1 Mep1b meprin B
pfu-miR-141-3p CL56196Contig1 Mx1 interferon-induced GTP-binding
protein Mx1
pfu-miR-141-3p CL7053Contig1 Myh myosin heavy chain
pfu-miR-141-3p CL54823Contig1 Nadph hydroxymethylglutaryl-CoA reductase
(NADPH)
pfu-miR-141-3p CL54540Contig1 Nod1 nucleotide-binding oligomerization
domain-containing protein 1
pfu-miR-141-3p CL27176Contig1 Ntrk3 neurotrophic tyrosine kinase,
receptor type 3
pfu-miR-141-3p CL53494Contig1 Oxtr oxytocin receptor
pfu-miR-141-3p CL37449Contig1 Prlr prolactin receptor
pfu-miR-141-3p CL5783Contig1 Sema3 semaphorin 3
pfu-miR-141-3p CL13367Contig1 Socs3 suppressor of cytokine signaling 3
pfu-miR-141-3p CL47996Contig1 Ttn titin
pfu-miR-429b-3p CL54671Contig1 Erg26 sterol-4alpha-carboxylate
3-dehydrogenase (decarboxylating)
pfu-miR-429b-3p CL15376Contig1 Ext1 glucuronyl/N-acetylglucosaminyl
transferase EXT1
pfu-miR-429b-3p CL49906Contig1 Fads2 fatty acid desaturase 2 (delta-6
desaturase)
pfu-miR-429b-3p CL55088Contig1 Fmn2 formin 2
pfu-miR-429b-3p CL15787Contig1 Hya hyaluronoglucosaminidase
pfu-miR-429b-3p CL53522Contig1 Lamα1 laminin, alpha 1
pfu-miR-429b-3p CL2394Contig1 Lamα3 laminin, alpha 3
pfu-miR-429b-3p CL55132Contig1 Mmp9 matrix metalloproteinase-9
(gelatinase B)
pfu-miR-429b-3p CL61104Contig1 Mylk myosin-light-chain kinase
pfu-miR-429b-3p CL15079Contig1 Sec61α protein transport protein SEC61
subunit alpha
pfu-miR-429b-3p comp105559_c1_seq2 Tgfβr1 TGF-beta receptor type-1
[89]Open in a new tab
To determine whether there are direct interactions between miR-141-3p
and PI3K-AKT or GPCR signaling pathway, we used dual-luciferase
reporter assays to measure the inhibitory effect of this miRNA on Itgb2
and Gria4a. There is one binding site for miR-141-3p in either Itgb2 or
Gria4a 3’UTR ([90]Fig 8A). We sub-cloned the 3’UTR of Itgb2 or Gria4a
into the pmirGLO vector, and co-transfected each construct with miR-141
mimic or its appropriate control into HEK293 cells. The results showed
that miR-141-3p down-regulated luciferase activity by 69% (±3%) in
Gria4a 3’UTR and 26% (±4%) in Itgb2 3’UTR, respectively ([91]Fig 8B).
These results strongly support the prediction of Itgb2 or Gria4a as
direct targets of miR-141-3p.
Fig 8. Itgb2 and Gria4a are targets of miR-141.
[92]Fig 8
[93]Open in a new tab
(A) Sequence alignment of Itgb2 and Gria4a 3’UTR to miR-141/-429b. Red
color letters represent seed sequences of miR-141/-429b and their
binding positions. (B) The activities of pmirGLO reporters that is
linked to the 3’UTR of Itgb2 and Gria4a were suppressed by miR-141 when
compared to its control oligonucleotide. *p<0.05 indicates the
significant difference.
Discussion
In vertebrates with XY sex-determining system, the expression of
sex-determining gene on Y chromosome leads to the development of male
phenotypes and testis. Yellow catfish, an important aquaculture fish in
China has XY sex-determining system and displays sexual size dimorphism
as male grows 2–3 times faster than female. 454 pyrosequencing and
illumina sequencing studies have been performed to compare
differentially expressed genes and pathways between XX ovary and XY
testis and provide a valuable genomic resource for studying fish
reproduction, sex determination and differentiation [[94]27,[95]28].
However, there are limited studies regarding the gene expression
difference between XY and YY fish. Therefore, we used solexa sequencing
technology to compare mRNA expression between XY and YY testis of
yellow catfish.
In fully mature 18 month-old Nile tilapia, the sperm quality and
quantity of XY and YY males were the similar [[96]22]. However, Herrera
et al. found that YY tilapia has superior reproductive capacity than XY
fish, since the primordial germ cells and spermatogenic cells in YY
were larger than XY fish during gonad development and the lobules,
blastemal of the reproductive duct and mature sperm cells appeared
earlier in YY than XY fish. Moreover, YY tilapia has bigger testis,
thicker somatic tissues and more spermatogenic cells, as well as
matures earlier than XY fish [[97]29]. In yellow catfish, we also found
that YY matures earlier and has superior reproductive capacity than XY
fish, as larger spermatogenic cyst and more spermatids were observed in
YY fish [[98]23]. Interestingly, androgen receptor has a relative
higher level of expression in efferent ducts of YY testis compared to
XY testis in rainbow trout [[99]20]. All these studies suggest that YY
testis has substantial difference in histology, structure and gene
expression compare to XY testis.
It is important to know the context under which specific signaling
pathways regulate sperm maturation as well as testis development in YY
that matures earlier than XY fish. The PI3K-AKT signaling pathway plays
essential roles in testis development and spermatogenesis, as loss of
p110beta subunit of phosphoinositide 3-OH kinase impaired
spermatogenesis and lead to defective fertility [[100]26]. Activation
of the PI3k/Akt pathway by membrane progestin receptor-alpha stimulated
sperms leads to hypermotility in Atlantic croaker [[101]25]. Lgr4, one
of the orphan GPCRs regulates sperm development and fertility
[[102]30]. Testosterone signaling is mediated by a G-protein-coupled
receptor and its interactors [[103]31]. We have found more PI3K-AKT and
GPCR signals in YY than XY yellow catfish, such as syk, prlr and
kiss1r, coincides with a higher degree for testis maturity and
advantageous spermatogenesis in YY than XY yellow catfish [[104]23]. As
one of the cytoskeletal component of spermatic flagella,
tyrosine-phosphorylated Syk could bind and phosphorylate to its
downstream part PLCγ1 and regulate the metabolic hyperactivated
motilityof spermatozoa [[105]32–[106]34].
In mammals, increased expression of miR-141/429 was associated with
defects of spermatogenesis [[107]35–[108]37]. High dose of
17α-ethinylestradiol (EE2) resulted in upregulation of miR-141/429 and
impairment of spermatogenesis [[109]23]. In our study, miR-141/429 was
predicted to target several factors in PI3K-AKT and GPCR signaling
pathways that were involved in testis development and spermatogenesis.
Further characterization of the interaction of miRNAs and their targets
could contribute to a better understanding about the molecular
mechanisms of testis develop and spermatogenesis.
Materials and Methods
Solexa library construction and sequencing
All experimental procedures involved in this study were permitted by
the Institutional Animal Care and Institute of Huazhong Agricultural
University. The total RNAs of XY (4 individuals) and YY (3 individuals)
testes were the same biological sample as described before [[110]23].
The RNA integrity analysis was performed by the Agilent 2100
Bioanalyzer (Agilent Technologies). An amount of 3μg total RNA was used
for libraries construction. All the following process was performed by
using the Illumina RNA Sample Preparation Kit, following the
manufacturer’s protocols. The mRNA was concentrated by oligo(dt)
magnetic beads and then made into short fragments (~200bp) to work as
templates for synthesizing the first strand cDNA. The double strand
cDNA libraries were synthesized and purified by agarose gel
purification, in which, the cDNA fragments were coupled by sequencing
adptorsat the 5’ and 3’ ends. The male (XY) and super-mal (YY)
libraries were sequenced on an IlluminaHiSeq 2000 platform. After
removing the adaptor, low quality bases (Length threshold value>35bp),
3’-end low quality bases (Quality threshold value>20) and the reads
containing the “N”, the clean reads were assembled into contigs with
the Trinity software [[111]30]. The generated contigs were clustered
into unigenes by performing TGICL software system [[112]31]. The raw
reads of yellow catfish gonad have been deposited to the NCBI database
(accession no: SRR1845493).
Quantification of differential expressed unigenes
To detect the differential expression of unigenes, DESeq software
package with RPKM (Reads Per Kb Million reads) method was performed to
quantify the expression of two expression profiles. The formula applied
was
[MATH:
RPKM=106CNL/103 :MATH]
, in which C is the number of reads mapper to merged transcripts, N and
L are the total mapped reads and base number of one unigene [[113]38].
The False Discovery Rate (FDR) method was applied in multiple
hypothesis of test to correct significant levels and eliminate
influence of random fluctuations and errors [[114]39]. After
calibration, the ratios of RPKMs were used to calculate fold-change
with threshold value cut-off of 2-fold and Negative binomial
distribution hypothesis-testing with P< 0.05 [[115]40].
Functional annotation and GO/KEGG enrichment analysis
The unigenes were searched against databases of NCBI nr, SWISS-PROT,
TrEMBL, Cdd, pfam and KOG by Blast X with a cut-off E-value of 10e^-5.
The results of BlastX annotation were uploaded on Blast 2 Go to
generate Gene Ontology annotations and mapped to the categories of GO
database (([116]geneontology.org/page/download-annotations)[[117]41].
The results of BlastX were also searched against the Kyto Encyclopedia
Genes and Genomes (KEGG) in Blast 2Go. All the differential expressed
unigenes were mapped to KEGG database with counted numbers of involved
interactive metabolic pathways (p<0.05). To investigate which GO item
and signaling pathways the DEGs participated in, all of the clustered
DEGs were mapped back to the GO and KEGG databases. Each enrichment
item was corresponded to a specific enrichment score that was
calculated by the formula, score value =
[MATH: mnMN
:MATH]
. The statistical significance of the GO enrichment was evaluated by
the hypergeometric distribution testing, P = 1-
[MATH: ∑i=0m-1<
mo
stretchy="false">(Mi)(N-M
n-i)(Nn) :MATH]
, where N is the number of unigenes with GO annotation, n is the number
of DEGs with GO annotation, M is the number of unigenes with one
specific GO annotation and m is the number of differently expressed
unigene with one specific GO annotation[[118]42].
Quantitative real-time PCR (qRT-PCR)
Briefly, 1μg of total RNA was reverse transcribed by using
PrimeScriptRT reagent Kit (Takara) according to the protocol. The
qRT-PCR reaction was performed in a 20μl reaction volume using the
Roche LightCycler 480 with SYBR Green PCR master mix(Roche) and three
biological replicates were conducted for each reaction. The β-actin was
selected as an internal reference to normalize the Ct values of each
reaction by using the 2^-ΔΔCt method [[119]43]. The ANOVA analysis was
used to perform differential expression analysis.
Identification of direct miRNA targets and validation by dual-luciferase
reporter assays
In our recent study, we found that miR-141-3p and miR-429b-3p have
higher expression level in XY than YY testis [[120]23]. To investigate
potential targets of miR-141-3p and miR-429b-3p, first the Open Reading
Frame (ORF) and 3’UTR of YY highly expressed unigenes were predicted,
by searching against the vertebrate genomic database in GENSCAN
([121]http://genes.mit.edu/GENSCAN.html). Perl scripts of both Target
Scan and miRanda were performed for searching the putative targets with
default parameters, including context score percentile ≥100 for Target
Scanand Max_Energy≤ −20 and for miRandabased on hybrid energy and
stability [[122]44,[123]45]
To characterize the interaction between miR-141-3p/-429b-3p and their
predicted target genes, the 3’-UTR of selected putative target genes
(Itgb2 and Gria4a) were inserted into the pmirGLO expression vector
(Promega, USA). Hek-293T cells were seeded in 96-well plates and
co-transfected with the constructed vectors and microRNA mimics or its
control oligonucleotide using DharmaFECT transfection reagent
(Dharmacon). 36h post transfection, the dual-luciferase reporter assay
system was used to detect reporter (Firefly and Renilla) activity as
described [[124]46]. The profile of relative luciferase activities were
normalized to Renilla luciferase activities.
Supporting Information
S1 Fig. Length distribution of the de novo assembled unignes.
(TIF)
[125]Click here for additional data file.^ (978.7KB, tif)
S1 Table. The annotation of unigenes in the transcriptomes.
(XLSX)
[126]Click here for additional data file.^ (8.7MB, xlsx)
S2 Table. The GO annotation of differently expressed unigenes.
(XLSX)
[127]Click here for additional data file.^ (149.1KB, xlsx)
Data Availability
All relevant data are within the paper and its Supporting Information
files.
Funding Statement
This work was supported by the National Natural Science Foundation of
China (31301931), the Fundamental Research Funds for the Central
Universities (52204-12018, 2013PY068), and State Key Laboratory of
Freshwater Ecology and Biotechnology (2015FB03).
References