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Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. Author contributions
  10. References
  11. Supporting Information

MicroRNA (miRNA) is a kind of small non-coding RNA molecules that function as important gene expression regulators by targeting messenger RNAs for post-transcriptional endonucleolytic cleavage or translational inhibition. In this study, small RNA libraries were constructed based on adult dairy goat testicular tissues and sequenced using the Illumina high-throughput sequencing technology. Blasted to miRNAs of cow and sheep in miRBase 19.0, 373 conserved miRNAs were identified in dairy goat testis and 91 novel paired-miRNAs were found. Expression of miRNAs in the dairy goat testis (miR-10b, miR-126-3p, miR-126-5p, miR-34c, miR-449b and miR-1468) was confirmed by qRT-PCR. In addition, the 128 conserved miRNAs were found by comparing the miRNA expression profiles in dairy goat testis with those in cow and mouse, which all might be involved in dairy goat testis development and meiosis. This study reveals the first miRNA profile related to the biology of testis in the dairy goat. The characterization of these miRNAs could contribute to a better understanding of the molecular mechanisms of reproductive physiology and development in the dairy goat.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. Author contributions
  10. References
  11. Supporting Information

MicroRNA is a member of the small RNA coding the length of approximately 22 nt, and it can pass and bind to the target 3′ end non-coding region (UTR) of mRNA, coding region or 5′ end non-coding region with inhibition mRNA translation or making its degradation, thus completing the mission of gene regulation transcription (Filipowicz et al. 2008). Appropriately, miRNA can regulate nearly 30% of coding protein genes in the human body, including the development, immune, heart function, tumorigenesis, cell proliferation, apoptosis, virus pathogenic and many physiological and pathological activities (Filipowicz et al. 2008; Kim et al. 2009). MicroRNAs exist in many eukaryotic organisms, including lower organisms and human body, and the main characteristics of their performance are conservative and specific expression (Kim et al. 2009). MicroRNA was first discovered in nematodes and then in other species have been found in the progress of the sequencing technologies, especially the second-generation sequencing technology, which also speed up to discover the new species' microRNA and new microRNAs (Li et al. 2010). MicroRNA deep sequencing technology is based on the principle of synthesis sequencing-by-synthesis with no nucleic acid hybridization issue. The current Illumina-Solexa high-throughput sequencing technology is highly efficient and sensitive. And the miRNAs with trace amount, even single molecule could be captured by this technology.

Studies have shown that the microRNAs involve in regulating multiple biological processes, including mammalian reproductive system development and gametogenesis process (Niu et al. 2011). Through the deep sequencing technology, the testicular tissue microRNA expression profiles of human, rat, mice, cow and pig have been fully analysed, respectively. One of the main characteristics of miRNAs is conservative, and some conservative miRNAs, such as miR-34c, miR-21, miR-221, miR-146 and miR-449, had been found important for mammalian spermatogenesis (Bouhallier et al. 2010; Huszar and Payne 2013; Li et al. 2013; Niu et al. 2011; Smorag et al. 2012; Yang et al. 2013; Zhang et al. 2012a,b). So, there may be a hypothesis that there are a number of conservative miRNAs involving in mammalian reproductive system development and gametogenesis process.

Saanen dairy goat, an important economic animal, has a relatively short period of gestation and provides fur, meat and milk and other kinds of valuable products. Goat's milk contains several nutrients and is suitable for drinking and for modern dairy industry as an important raw material (Zhu et al. 2013). Nowadays, dairy goats have been used as a transgenic animal model for peptide medicine research (Jung et al. 2000). Thus, the mechanisms of germ cells and reproductive organ development in dairy goat are worth further investigation.

Up to date, microRNA expression spectrum in dairy goat testicular tissue and reproductive cells has not been reported. Therefore, we used the second-generation sequencing technology to do in-depth analysis of the microRNA expression in dairy goat testicular tissue. As a result, a number of goat testicular microRNAs were identified, 128 of which were also conservative in mice and cows. These sequencing miRNA data are of great reference values for the further study of their function in male goat reproductive system development and gametogenesis.

Materials and Methods

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. Author contributions
  10. References
  11. Supporting Information

Goat testicular cells and total RNA extraction

The dairy goat testicular cells were isolated in our laboratory as previously reported (Hua et al. 2011). Three adult Saanen testicular tissues were collected at 4°C in the laboratory and washed with PBS containing penicillin (500 IU/ml) and streptomycin (500 mg/ml) five to ten times. The tunica albuginea was peeled using forceps under the stereomicroscope. The testicular tissues were cut into 1- to 2-cm3 size tissues. Then, the tissues were incubated with CDD-mixed digestive enzyme (2 mg/ml collagenase + 2 µg/ml DNase + 2 mg/ml decomposition enzyme) for 15–20 min, and 4 ml 10% FBS DMEM medium was added, filtered by 400-mesh sieve and centrifuged by 1500 r/min for 5 min. Testicular cells were harvested, and the total RNA was extracted with RNAiso plus reagent (Takara), using RNA Bioanalyser (Agilent Technologies) determination of total RNA purity and concentration.

Small RNA library construction and deep sequencing

A small RNA library was generated from the customer sample using the Illumina Truseq Small RNA Preparation kit according to Illumina's TruSeq Small RNA Sample Preparation Guide. The purified cDNA library was used for cluster generation on Illumina's Cluster Station and then sequenced on Illumina GAIIx following vendor's instruction for running the instrument. Raw sequencing reads were obtained using Illumina's Sequencing Control Studio software version 2.8 (SCS v2.8, San Diego, CA, USA) following real-time sequencing image analysis and base-calling by Illumina's Real-Time Analysis version 1.8.70 (RTA v1.8.70, San Diego, CA, USA). The extracted sequencing reads were stored in file and were then used in the sequencing data analysis.

Sequencing data analysis

The sequencing data analysis was performed using the ACGT101-miR v4.2 packages (LC Sciences, Houston, TX, USA) and was briefly described as follows (Li et al. 2010). 1. Mappable sequences from raw sequencing data: after the raw sequence reads were extracted from image data, a series of digital filters (LC Sciences) were employed to remove various unmappable sequencing reads, which are ‘impurity' sequences due to sample preparation, sequencing chemistry and processes, and the optical digital resolution of the sequencer detector. Those remaining sequenced sequences (filtered sequenced sequences with lengths between 15 and 32 bases) were grouped by families (unique sequences) and were used to map with the reference database files. 2. Mapping miRNA-mappable unique sequences to mirs and genome: in this section, various remaining sequenced sequences were further blasted against pre-miRNA (mir) and mature miRNA (miR) sequences listed in the latest release of miRBase (miRBase, http://www.mirbase.org/index.shtml), or against genome based on the public releases of appropriate species. In this study, cattle and sheep were used as sequencing species for unique sequences to mirs, and goat was for unique sequences to genome (Dong et al. 2013). Sequences were matched as follows: (i) matching sequences were first mapped to the selected mirs in miRBase, (ii) these mirs were mapped to the genome and (iii) the mirs/miRs were known as the specific species.

Real-time fluorescent quantitative PCR (Q-PCR) detection

For quantitative PCR studies of validated miRNAs, the total RNAs from fresh testis tissues were reverse-transcribed to cDNAs using RT primers for miRNAs and 5S RNA (Chen et al. 2005), as listed in Table 1. The reaction mix was subjected to thermo cycler for 5 min at 60°C to anneal the primers. After cooling to room temperature, the remaining reagents (5 × buffer, 10 mm dNTPs, RNase inhibitor and M-MuLV reverse transcriptase) were added according to the experimental protocol (Thermo Scientific, Waltham, MA, USA). The reaction proceeded for 45 min at 60°C followed by a 5-min incubation at 70°C to inactivate the enzymes. The cDNAs were stored indefinitely at −20°C or −80°C.

Table 1. Primers for miRNA reverse transcription and real-time PCR
miRNAPrimersSequence
miR-10bReverse transcription primerCTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACAAATTC
Up primerACACTCCAGCTGGGTACCCTGTAGAACCGA
Down primerGTGCAGGGTCCGAGGT
miR-126-3pReverse transcription primerCTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGCATTATT
Up primerACACTCCAGCTGGGTCGTACCGTGAGTAA
Down primerGTGCAGGGTCCGAGGT
miR-126-5pReverse transcription primerCTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCGCGTAC
Up primerACACTCCAGCTGGGCATTATTACTTTTGGT
Down primerGTGCAGGGTCCGAGGT
miR-34cReverse transcription primerGTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCAATCA
Up primerGCGCTAGGCAGTGTAGTTAG
Down primerGTGCAGGGTCCGAGGT
miR-1468Reverse transcription primerCTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCAGCAAA
Up primerACACTCCAGCTGGGCTCCGTTTGCCTGTTT
Down primerGTGCAGGGTCCGAGGT
miR-449bReverse transcription primerCTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGCCAGCTA
Up primerACACTCCAGCTGGGTGGCAGTGTATTGTTA
Down primerGTGCAGGGTCCGAGGT
5SReverse transcription primerGTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCAGGCG
Up primerCTGGTTAGTACTTGGACGGGAGAC
Down primerGTGCAGGGTCCGAGGT

Real-time quantitative PCR was performed on a CFX96 real-time PCR detection system (Bio-Rad, USA) according to the manual for BioEasy SYBR Green I Real-Time PCR kit (Bioer, China). Briefly, 0.5 μl of cDNA was added to 7.5 µl of the 2 × SYBR green PCR master mix with 0.1 µl of Taq polymerase enzyme (Bioer, China), 300 nm of each primer and ddH2O to a final volume of 15 µl. The reactions were amplified for 15 s at 95°C and 30 s at 58°C for 40 cycles. The thermal denaturation protocol was run at the end of the PCR to determine the number of products that were presented in the reaction mix. Reactions were typically run in duplicate. miRNA relative expression quantity was detected and calculated using relative quantitative standard curve method (Li et al. 2013). The primers for the validated miRNAs are listed in Table 1.

Conservative testicular miRNA target gene prediction and pathway analysis

The conservative testicular miRNAs in the dairy goat testis were identified by comparing with testis miRNA expression profiles in cow and mouse in previous reports (Huang et al. 2011; Aguilar et al. 2010). Target genes in relation to the conservative testicular miRNAs were searched in miRWalk database using mouse miRNA name (Harsh et al. 2011). GO categories and pathway analysis were mainly based on the DAVID Bioinformatics Resources 6.7 (NIAID/NIH) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The target genes of conservative miRNA were organized into a functional classification based on the molecular function (MF), cell component (CC) or biological process (BP) (Ashburner et al. 2000), and the identified pathways were shown with a P value. The miRNA–pathways interaction network was constructed using Cytoscape v2.8.3 software (http://www.cytoscape.org/).

Results

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. Author contributions
  10. References
  11. Supporting Information

Small RNA Reads in Capra hircus Testis

Total RNA from three Saanen dairy goats for small RNA library was analysed using RNA Bioanalyser (Fig. 1). A total of 18 216 057 raw reads were obtained by sequencing short RNAs from Capra hircus testis. Only reads between 15 and 31 nucleotides, corresponding to conventionally accepted miRNA length and mapping perfectly to the available goat genome scaffolds, were included in the data set. All identified sequences were able to fold into the hairpin-loop structure: characteristic of a folded pre-miRNA.

image

Figure 1. RNA bioanalyser of total RNA from dairy goat testis. The total RNA from 3 goat testicular tissues. The gellike (Ladder, 4000 Ladder; goat A, B, C, total RNA from 3 goat testicular tissues) image showed clear separation of total RNA to 18S and 28S subunits and degradation products

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A total of 373 conservative miRNAs ranged from 15 to 31 nucleotides mapped to mature miRNAs and pre-miRNAs of cattle and sheep in the version 19.0 of miRBase, and pre-miRNAs further mapped to goat genome (Table S1), 91 novel paired-miRNAs were found in dairy goat testis (Table S2). The length distribution and read number percentage of sequences are presented in Fig. 2, and the chromosomal distribution of conserved miRNAs is shown in Fig. 3. Compared with cattle and sheep miRNAs in miRBase 19.0, it was found that 327 miRNAs in goat testis were conserved to cattle miRNAs, 28 miRNAs were conserved to sheep miRNAs, and 18 miRNAs were conserved to both species' miRNAs (Fig. 4).

image

Figure 2. Length distribution and read number percentage of conserved miRNAs sequences in Capra hircus testis. All the reads were ranged from 15 to 31 nucleotides in length. Approximately 98.78% of sequences are distributed in 20–24 nt range. nt, nucleotides

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image

Figure 3. Chromosomal distribution of conserved miRNAs in Capra hircus testis. According to miRNA number, the top 3 chromosomes with miRNA mapping number are Chr 19, 21 and X, while the top 3 chromosomes with miRNA read numbers are Chr 1, 7 and 15 according to reads. UN, uncertain chromosome

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image

Figure 4. Identified conserved miRNAs in capra hircus testis compared with bos taurus and ovis aries miRNAs in miRBase 19.0. Compared with cattle and sheep miRNAs in miRBase 19.0, 373 conservative miRNAs were found in dairy goat testis. In which, 327 miRNAs in goat testis were conserved to cattle miRNAs, 28 miRNAs were conserved to sheep, and 18 miRNAs were conserved to both species' miRNAs. Blue circle, bos miRNAs; red circle, capra miRNAs; green circle, ovis miRNAs

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Identification and characterization of targets of conserved miRNAs in dairy goat testicle

Conserved miRNAs are found in many animal species and have important functions in mammalian development and physiological processes (Ji et al. 2012). To identify conserved miRNAs in dairy goat testicle, the miRNAs in dairy goat testis were compared with testicular miRNAs in cow and mouse (Aguilar et al. 2010; Huang et al. 2011); then 128 conserved miRNAs in testis were found among goat, cow and mouse (Fig. 5) and are shown in Fig. 6 and Table S3. In these miRNAs, miR-34c and miR-21 have a higher level of reads, which were important for spermatogonial stem cells' self-renewal and spermatogenesis (Bouhallier et al. 2010; Niu et al. 2011). And miR-146 and miR-221 were also conserved miRNAs in testis, which modulate the undifferentiated and differentiated state in mammalian male germ cells (Huszar and Payne 2013; Yang et al. 2013).

image

Figure 5. Identified conserved miRNAs in capra hircus testis compared with bos taurus and mus musculus miRNAs in testicular tissues (Aguilar et al. 2010; Huang et al. 2011). 128 conserved miRNAs in dairy goat testis were found among goat, cow and mouse, compared with testicular miRNAs in bos taurus and mus musculus. Blue circle, cow miRNAs; red circle, goat miRNAs; black circle, mouse miRNAs

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image

Figure 6. The reads of 128 conservative miRNAs in dairy goat testis. The 128 conserved expression miRNAs in goat, cow and mouse testicle were plotted. The highlighted miRNAs with arrows, miR-34c, miR-21, miR-146 and miR-221, were reported important for mammalian male germ cells

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To further understand the physiological functions and biology processes involved these miRNAs during dairy goat testis development and meiosis, target genes in relation to the conservative testicular miRNAs were obtained from miRWalk database by the same mouse miRNA name. A total of 1187 annotated mRNA transcripts were found as target genes for 128 conserved miRNAs (Table S4).

GO categories is an international standardized classification system for gene annotations that provides insights into the molecular functions of genes in various biological processes. The GO enrichment analysis from biological processes showed that the top 4 most enriched GO categories were involved in the regulation of transcription and 16.3% of the genes were involved in the regulation of cell proliferation. The analysis of cellular components showed that 22.6% and 14.1% of the genes were clustered into plasma membrane, and most of them were intracellular. The analysis of molecular function showed that all genes were assigned different functions, and the functions were divided into two kinds: binding activity and transcription activity. The 10 most enriched GO categories for target genes are listed in Tables 2-4. The target genes were classified according to KEGG function annotations to identify the pathways that were actively regulated by miRNAs in dairy goat testis (Table 5). The KEGG Pathway annotation showed that most of target genes were involved in cancer, signalling pathway and actin cytoskeleton. The most enriched pathway annotated was the pathway in cancer, with 10.8% annotated genes, followed by MAPK signalling pathway (6.6%), focal adhesion (5.1%) and cytokine–cytokine receptor interaction (4.7%). Among these, cellular signalling pathways regulated by let-7s, miR-146b, miR-21, miR-221 and miR-34c of testis are presented in Fig. 7.

Table 2. The 10 most enriched GO categories for the target genes of 128 conserved testicular miRNAs(biological process)
GO AccessionGO termsCluster frequency(%)p value
GO:0045449Regulation of transcription29.44.7E-37
GO:0051252Regulation of RNA metabolic process24.37E-46
GO:0006355Regulation of transcription, DNA-dependent23.94.7E-45
GO:0006350Transcription22.65E-25
GO:0006357Regulation of transcription from RNA polymerase II promoter16.41.6E-60
GO:0042127Regulation of cell proliferation16.31.4E-69
GO:0010604Positive regulation of macromolecule metabolic process15.97.5E-55
GO:0031328Positive regulation of cellular biosynthetic process14.37.6E-51
GO:0009891Positive regulation of biosynthetic process14.32.7E-50
GO:0051173Positive regulation of nitrogen compound metabolic process14.25E-53
Table 3. The 10 most enriched GO categories for the target genes of 128 conserved testicular miRNAs (cellular component)
GO AccessionGO termsCluster frequency(%)p value
GO:0005886Plasma membrane22.65.06E-08
GO:0005576Extracellular region16.29.56E-13
GO:0044459Plasma membrane part14.11.67E-07
GO:0043228Non-membrane-bounded organelle12.90.019878118
GO:0043232Intracellular non-membrane-bounded organelle12.90.019878118
GO:0031974Membrane-enclosed lumen12.87.79E-14
GO:0043233Organelle lumen12.57.16E-14
GO:0070013Intracellular organelle lumen12.41.35E-13
GO:0031981Nuclear lumen11.42.92E-18
GO:0044421Extracellular region part10.81.33845E-19
Table 4. The 10 most enriched GO categories for the target genes of 128 conserved testicular miRNAs (molecular function)
GO AccessionGO termsCluster frequency(%)p value
GO:0003677DNA binding25.22.96E-39
GO:0030528Transcription regulator activity21.44.83E-49
GO:0000166Nucleotide binding17.91.97E-04
GO:0003700Transcription factor activity17.63.36E-56
GO:0017076Purine nucleotide binding14.80.003874324
GO:0032555Purine ribonucleotide binding14.30.003434685
GO:0032553Ribonucleotide binding14.30.003434685
GO:0001882Nucleoside binding13.51.16E-04
GO:0001883Purine nucleoside binding13.41.27E-04
GO:0030554Adenyl nucleotide binding13.20.000186018
Table 5. The 10 most enriched KEGG pathways for the target genes of 128 conserved testicular miRNAs
Pathway IDPathway nameTarget genes with pathway annotation(%)p value
mmu05200Pathways in cancer10.81.78E-41
mmu04010MAPK signalling pathway6.64.40E-17
mmu04510Focal adhesion5.19.75E-14
mmu04060Cytokine-cytokine receptor interaction4.74.15E-08
mmu05215Prostate cancer3.83.00E-19
mmu04810Regulation of actin cytoskeleton3.87.38E-06
mmu04722Neurotrophin signalling pathway3.72.09E-11
mmu04350TGF-beta signalling pathway3.64.11E-17
mmu05220Chronic myeloid leukaemia3.31.22E-16
mmu05218Melanoma3.28.3E-17
image

Figure 7. The network of miRNA pathways related to let-7s, miR-146b, miR-21, miR-221 and miR-34c in testis. In the network, only the relationship between miRNAs and cellular signalling pathways is shown, and the target genes of let-7s, miR-146b, miR-21, miR-221 and miR-34c involved in these pathways are displayed in Table S4

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Validation of sequencing miRNAs expression with Q-PCR

To validate the existence of the sequencing Saanen dairy goat miRNAs, the same RNA preparations used for the deep sequencing were subjected to Q-PCR assay. The expression levels of selected miRNAs were determined, including 4 miRNAs with higher read numbers (miR-10b, miR-126-3p, miR-126-5p and miR-34c) and 2 miRNAs with lower read numbers (miR-449b and miR-1468), the Q-PCR results are shown in Fig. 8. The expression levels of miRNAs were represented using the Normalized Fold Expression. The expression pattern of miRNAs was consistent with the deep sequencing results; this confirmed that the sequencing data were credible.

image

Figure 8. Q-PCR validation of the identified miRNAs by using deep sequencing technology. The expression level of miR-10b, miR-126-3p, miR-126-5p, miR-34c, miR-449b and miR-1468 were detected using real-time PCR analysis. 5S RNA was used as real-time PCR normalization. Data are presented as the mean ± SEM (n = 3)

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Discussion

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. Author contributions
  10. References
  11. Supporting Information

MicroRNA is a kind of important non-coding small molecules, involved in the regulation of cell proliferation, differentiation, apoptosis and the development of biology. Mature miRNAs are generated from endogenous hairpin-shaped transcripts through a series of nuclease shear processing. The function of miRNA is as guide molecules in post-transcriptional gene regulation by base-pairing with the target mRNAs, usually in the 3′ untranslated region (UTR). Binding of a miRNA to the target mRNA typically leads to translational repression and exonucleolytic mRNA decay, although highly complementary targets can be cleaved endonucleolytically (Kim et al. 2009). In the research of the function of multiple species microRNA, scientists had found that miRNA expression had sequential sex and tissue specificity. This suggests that their specific expression may play important roles in the regulation of development and differentiation of germ cells (Mciver et al. 2012). Extensive high-throughput sequencing studies of miRNAs have been performed in several animal models. However, little is known about the diversities of these regulatory RNAs in goat (Capra hircus), which is one of the most important agricultural animals and the oldest domesticated species in worldwide (Li et al. 2012). So, it is worth to investigate the microRNAs expression of germ cells or reproductive organ in dairy goat.

Illumina/Solexa high-throughput sequencing has special advantages for small RNA sequencing and is used as a powerful tool for the identification of miRNAs. The sequencing results are typically validated using real-time quantitative RT-PCR, Northern blot or microarray analysis. In our study, using the Illumina high-throughput sequencing technology, 373 conserved miRNAs were first identified in the dairy goat testis by blasting to miRNAs of cow and sheep in miRBase 19.0, and 91 novel paired-miRNAs. The expression of sequencing miRNAs was confirmed by qRT-PCR analysis, 6 conserved miRNAs were selected including 4 miRNAs with higher read numbers (miR-10b, miR-126-3p, miR-126-5p and miR-34c) and 2 miRNAs with lower read numbers (miR-449b and miR-1468). The results of qRT-PCR analysis indicated that the high-throughput sequencing data were available and reliable. It is well known that conserved miRNAs were found in many animal species and had important functions in mammalian development and physiological processes (Ji et al. 2012). In this study, we found that of the 373 sequencing miRNAs in dairy goat testis, 327 miRNAs were conserved to cattle miRNAs, 28 miRNAs were conserved to sheep, and 18 miRNAs were conserved to both species' miRNAs. Especially, 128 testicular miRNAs were found conserved among goat, cow (the closest animal relative of dairy goat) and mouse (the most important animal model), suggesting that there might be a number of conserved miRNAs important for testis development and spermatogenesis. Previous studies had shown that there were some specific germ cell miRNAs associated with spermatogenesis, such as miR-34c, miR-21, miR-221, miR-146 and miR-449 (Bouhallier et al. 2010; Niu et al. 2011; Smorag et al. 2012; Zhang et al. 2012a; Huszar and Payne 2013; Li et al. 2013; Yang et al. 2013). Importantly, all of these germ cell miRNAs were conserved and also found in Saanen dairy goat testis.

The analysis of conserved expression miRNAs in goat testis showed that the top 10 abundant miRNAs were miR-99a, miR-143, miR-100, miR-10b, miR-34c, miR-21, miR-26a and let-7 family (let-7t, let-7g and let-7a), which also were the top 10 abundant miRNAs in testis among goat, cattle and mouse. This suggests that the top 10 abundant miRNAs may play some important roles in the development or physiology of testicular tissue. In which, miR-99a and miR-100, belong to microRNA-99 family, have been reported to exhibit abnormal expression in various malignant tumours and regulate the DNA damage responses (Mueller et al. 2012; Sun et al. 2013); miR-143 has been shown to possess antitumorigenic activity with the involvement in various cancer-related events such as proliferation, invasion and migration (Bauer and Hummon 2012); miR-10b also involves in the regulation of cancer cell (Tsukerman et al. 2012; Guessous et al. 2013); miR-26a regulates cell cycle and differentiation (Dey et al. 2012; Zhang et al. 2012b); miR-34c and miR-21 were reported important for self-renewal of spermatogonial stem cell and spermatogenesis (Bouhallier et al. 2010; Niu et al. 2011); and the let-7 family is highly expressed and conserved across animal species, including mammals, flies, worms and plants, and it is one of the most important miRNA regulators of fundamental biological processes (Roush and Slack 2008). In these conserved miRNAs, it had been reported that there were some conserved miRNAs in testis involved in self-renewal of spermatogonial stem cell or spermatogenesis, such as miR-34c, miR-21, miR-221, miR-146 and miR-449 (Bouhallier et al. 2010; Niu et al. 2011; Smorag et al. 2012; Zhang et al. 2012a; Huszar and Payne 2013; Li et al. 2013; Yang et al. 2013). Remarkably, miR-34c, miR-21, miR-221 and miR-146 are with higher read numbers. This suggests that the conserved miRNAs with high expression level could play important roles in self-renewal of spermatogonial stem cell or spermatogenesis. And the previous reports in our laboratory had demonstrated that the miR-34c regulated mouse embryonic stem cells differentiation into male germlike cells through RARg (Zhang et al. 2012a) and worked downstream of p53 leading to dairy goat male germ line stem cell (mGSCs) apoptosis (Li et al. 2013). So, it is quite possible that these conserved miRNAs with higher read numbers in testis are also functional in dairy goat testis cells, even other animals. And the conserved miRNAs with higher read numbers could be candidate miRNAs for our future study on the molecular mechanisms of spermatogenesis in dairy goat.

GO annotation and KEGG Pathway analysis could provide a better understanding the functions of target genes of conserved miRNAs. The GO enrichment analysis revealed that more than 50% of the genes of miRNA target in dairy goat were involved in the regulation of transcription, more than 30% of the genes were annotated to plasma membrane ontology, and nearly all of the genes had binding functions or transcription functions (Table 2-4). KEGG analysis showed that approximately 10% of the genes were committed to a cancer pathway (Table 5). These results indicated that some miRNAs might be involved in mammary testicular spermatogenic function and function in mammary testicle cell proliferation, apoptosis and differentiation. The validation of the relationship between miRNAs and target mRNAs transcripts needs further biological experimental evidences in dairy goat. As the first goat genome has been sequenced and reported, the goat genome will be useful for understanding the genomic features including miRNAs and its target mRNAs. This will also be useful for the understanding of molecular mechanisms of reproductive physiology and improving the utility of the goat as a biomedical model and bioreactor (Dong et al. 2013).

In conclusion, this study reveals the first miRNA profile related to the biology of testis in the goat. In our study, small RNA libraries were built from adult dairy goat testicular tissues and sequenced using the Illumina high-throughput sequencing technology. Finally, 373 conserved miRNAs of dairy goat testis were identified by blasting miRNAs of cow and sheep in miRBase 19.0, and 91 novel paired-miRNAs were found in dairy goat testis. In addition, 128 conserved testicular miRNAs were found by comparing the miRNA expression profiles among dairy goat, cow and mouse, which might be involved in dairy goat testis development, spermatogenesis and meiosis. The characterization of these conserved miRNAs could contribute to a better understanding of the molecular mechanisms of reproductive physiology and development in the dairy goat.

Acknowledgements

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. Author contributions
  10. References
  11. Supporting Information

We are grateful to Prof. Jun Luo for biopsy sampling. This work was supported by the grants from the Program (31272518) of National Natural Science Foundation of China, the Key Project of Chinese Ministry of Education(2013CB947902), Doctoral Fund of Ministry of Education of China (RFDP, 20120204110030), the Program for New Century Excellent Talents of State Ministry of Education (NCET-09-0654), the Fundamental Research Funds for the Central Universities (QN2011012).

Author contributions

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. Author contributions
  10. References
  11. Supporting Information

Jiang Wu and Jinlian Hua designed the experiment and writed the manuscript. Jiang Wu perfomed the experiment. Jiang Wu,Haijing Zhu, Wencong Song, Mingzhao Li, Chao Liu, Na Li, Furong Tang, Hailong Mu, Mingzhi Liao, Xiangchen Li, and Weijun Guan analysed the data.

References

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. Author contributions
  10. References
  11. Supporting Information
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Supporting Information

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. Author contributions
  10. References
  11. Supporting Information
FilenameFormatSizeDescription
rda12217-sup-0001-TableS1.xlsapplication/msexcel348KTable S1 The conservative microRNA in Saanen dairy goat testis.
rda12217-sup-0002-TableS2.xlsapplication/msexcel52KTable S2 Paired-microRNAs(miRNAs) cloned in Saanen dairy goat testis.
rda12217-sup-0003-TableS3.xlsapplication/msexcel17KTable S3 The reads of 128 conserative testicular miRNAs among dairy goat, cow and mouse.
rda12217-sup-0004-TableS4.xlsapplication/msexcel3252KTable S4 Conserative testicular miRNAs target results.

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