Profiling of miRNAs in porcine germ cells during spermatogenesis

in Reproduction
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Xiaoxu Chen College of Animal Science and Technology, Northwest A&F University, Shaanxi, China

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Dongxue Che College of Life Science, Northwest A&F University, Shaanxi, China

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Pengfei Zhang College of Animal Science and Technology, Northwest A&F University, Shaanxi, China

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Xueliang Li College of Animal Science and Technology, Northwest A&F University, Shaanxi, China

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Qingqing Yuan State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

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Tiantian Liu College of Animal Science and Technology, Northwest A&F University, Shaanxi, China

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Jiayin Guo College of Animal Science and Technology, Northwest A&F University, Shaanxi, China

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Tongying Feng College of Animal Science and Technology, Northwest A&F University, Shaanxi, China

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Ligang Wu Shanghai Key Laboratory of Molecular Andrology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

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Minzhi Liao College of Life Science, Northwest A&F University, Shaanxi, China

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Zuping He State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

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Wenxian Zeng College of Animal Science and Technology, Northwest A&F University, Shaanxi, China

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Spermatogenesis includes mitosis of spermatogonia, meiosis of pachytene spermatocytes and spermiogenesis of round spermatids. MiRNAs as a ~22 nt small noncoding RNA are involved in regulating spermatogenesis at post-transcriptional level. However, the dynamic miRNAs expression in the developmental porcine male germ cells remains largely undefined. In this study, we purified porcine spermatogonia, pachytene spermatocytes and round spermatids using a STA-PUT apparatus. A small RNA deep sequencing and analysis were conducted to establish a miRNAs profiling in these male germ cells. We found that 19 miRNAs were differentially expressed between spermatogonia and pachytene spermatocytes, and 74 miRNAs differentially expressed between pachytene spermatocytes and round spermatids. Furthermore, 91 miRNAs were upregulated, while 108 miRNAs were downregulated in spermatozoa. We demonstrated that ssc-miR-10a-5p, ssc-miR-125b, ssc-let-7f and ssc-miR-186 were highly expressed in spermatogonia, pachytene spermatocytes, round spermatids and spermatozoa respectively. The findings could provide novel insights into roles of miRNAs in regulation of porcine spermatogenesis.

Abstract

Spermatogenesis includes mitosis of spermatogonia, meiosis of pachytene spermatocytes and spermiogenesis of round spermatids. MiRNAs as a ~22 nt small noncoding RNA are involved in regulating spermatogenesis at post-transcriptional level. However, the dynamic miRNAs expression in the developmental porcine male germ cells remains largely undefined. In this study, we purified porcine spermatogonia, pachytene spermatocytes and round spermatids using a STA-PUT apparatus. A small RNA deep sequencing and analysis were conducted to establish a miRNAs profiling in these male germ cells. We found that 19 miRNAs were differentially expressed between spermatogonia and pachytene spermatocytes, and 74 miRNAs differentially expressed between pachytene spermatocytes and round spermatids. Furthermore, 91 miRNAs were upregulated, while 108 miRNAs were downregulated in spermatozoa. We demonstrated that ssc-miR-10a-5p, ssc-miR-125b, ssc-let-7f and ssc-miR-186 were highly expressed in spermatogonia, pachytene spermatocytes, round spermatids and spermatozoa respectively. The findings could provide novel insights into roles of miRNAs in regulation of porcine spermatogenesis.

Introduction

Spermatogenesis is a complex process occurring in the epithelium of seminiferous tubules within testis (Aravin & Bourc’his 2008). Spermatognial stem cells (SSCs), a subpopulation of undifferentiated spermatogonia, are the foundation of spermatogenesis (Kanatsu-Shinohara & Shinohara 2013). Spermatogonia enter into meiotic process to become spermatocytes, which undergo a long-lasting meiosis and transform into haploid round spermatids (Miller et al. 2013). Subsequently, round spermatids accomplish spermiogenesis. Eventually spermatozoa are released into the lumen of seminiferous tubules and matured during transit along epididymis (Guyonnet et al. 2011, Rathke et al. 2014).

This highly organized process of spermatogenesis requires timely coordinated gene expression that is regulated in transcriptional and post-transcriptional level (Yao et al. 2015). Growing evidences have revealed that a large number small noncoding RNAs are involved in the regulation of spermatogenesis (Kimmins & Sassone-Corsi 2005, Papaioannou & Nef 2010, Chen et al. 2017b). miRNAs, as 22 nt noncoding RNA, regulate gene expression by inducing mRNA degradation or inhibiting translation (Martinez & Tuschl 2004). Increasing evidences suggest that miRNAs play important roles in regulating SSCs self-renewal and differentiation (Niu et al. 2011, He et al. 2013, Moritoki et al. 2014), meiosis (Yu et al. 2005, Novotny et al. 2007), spermiogenesis (Korhonen et al. 2011) and sperm functions (Liu et al. 2012) in mice. The dynamically expressed miRNAs of spermatogonia and primary spermatocytes have been analyzed in mice (Smorag et al. 2012). Importantly, miRNA signatures of human spermatogonia, pachytene spermatocytes and round spermatids were unveiled recently, which provided new targets for the treatment of male infertility (Liu et al. 2015).

It has been estimated that as little as 40% of research data obtained from rodents yield successful human clinical trials (Vandamme 2015). In terms of anatomy, physiology and function, swine is considered close to humans. As such, swine is steadily gaining importance as large animal models in the field of biomedical research (Swindle et al. 2012). In previous study, Luo and coworkers detected the miRNAs profiling in immature and mature porcine testis tissues (Luo et al. 2010). The authors further analyzed miRNA expressions in porcine testes, epididymis and ejaculated spermatozoa (Luo et al. 2015). Moreover, recently Chen and coworkers identified differentially expressed miRNAs in epididymal and ejaculated spermatozoa (Chen et al. 2017a). However, the expression pattern of miRNAs in porcine male germ cells remains largely unknown.

The aim of the present study was to isolate the different types of porcine male germ cells and to establish a comprehensive miRNAs profiling in these isolated cells via a small RNA deep sequencing and analysis. This study would provide novel insights into roles of miRNAs in the regulation of porcine spermatogenesis.

Materials and methods

Animal

All experimental procedures involving animals were approved by the Northwest A&F University’s Institutional Animal Care and Use Committee. The testes of puberty Landrace (5-month-old) were obtained from Besun farm, Yangling, China. After the castration, testes were put into pre-cooled PBS (4°C) contained 2% of penicillin and streptomycin. The samples were brought to the lab immediately.

Sample collection

The isolation of porcine germ cells was described in previous study (Zheng et al. 2014). Briefly, the testis was cut into small pieces and incubated in collagenase Type IV (0.2% w/v; Invitrogen) at 37°C for 30 min to obtained seminiferous tubules. The seminiferous tubules were digested by 0.25% trypsin-EDTA (Hyclone, Logan, UT, USA) at 37°C for 15 min. After filtration, the single cell was collected by centrifugation and subjected to differential plating to remove the adherent cells.

The STA-PUT apparatus via the velocity sedimentation was utilized to enrich spermatogonia (A), pachytene spermatocytes (PS) and round spermatids (RS) from cell suspension. The process was strictly carried out as described by Liu et al. 2015. The different cell types were collected and rigorously examined under a phase-contrast microscope to identify cellular morphology and cell types. The cells of each type were pooled and re-suspended in TRIzol (Invitrogen) and stored at −80°C until usage. The samples from each type of germ cells were prepared for immunocytochemistry and qRT-PCR analysis.

The fresh ejaculated spermatozoa (SP) were collected from healthy adult Landrace pig using the gloved hand technique. The semen was incubated in pre-warmed water (37°C) and delivered to laboratory immediately. The upper fraction of swim-up spermatozoa was collected followed by centrifuging at 600 g for 5 min. After the removing of supernatant, the sperm pellets were re-suspended in TRIzol and stored at −80°C.

All samples were collected from two individuals and considered as two biological replicates.

Quantitative RT-PCR (qRT-PCR)

RNA was extracted from the isolated spermatogonia, pachytene spermatocytes, round spermatids, spermatozoa with TRIzol (Invitrogen) according to the manufacturer’s protocol. For each sample, total RNA was used for reverse transcription using PrimeScript RT regent kit with gDNA Eraser (Takara). The quality of the resultant cDNAs was verified by PCR analysis of β-actin expression. The primer information is shown in the Supplementary Table 1 (see section on supplementary data given at the end of this article). qRT-PCR was performed with SYBR Green II PCR Mix (Takara) using an IQ-5 (Bio-Rad). The relative gene expression level was normalized to β-actin and analyzed using the 2–ΔΔCT method. For real-time PCR of miRNA, stem-looped primers (Supplementary Table 2) were used for reverse transcription as described previously (Tang et al. 2006). The relative expression level of miRNAs was normalized to 5s RNA and calculated using the comparative Ct method (2–ΔΔCT).

Immunocytochemistry and meiotic spread assay

To identify the cells, the freshly isolated spermatogonia, round spermatids were fixed with 0.4% paraformaldehyde (PFA) for 30 min at room temperature and washed with cold PBS for three times. The cells were further permeabilized for 5 min using 0.4% Triton-X 100 (Sigma-Aldrich) followed by washing with PBS. After blocked nonspecific reaction in 1% BSA for 30 min at room temperature, the cells were incubated with primary antibodies, including GPR125 (G protein-coupled receptor 125, Abcam), GFRA1 (GDNF family receptor A1, Abcam), UCHL1 (ubiquitin C-terminal hydrolase L1, AbD Serotec, Germany), SCP3 (synaptonemal complex protein 3, Abcam), Acrosin (Santa Cruz, USA) and PRM2 (Protamine 2, Santa Cruz, USA) at a dilution with 1:200 overnight at 4°C. Next day, the cells were washed with PBS for three times and incubated with rhodamine-conjugated secondary antibody IgG (Sigma-Aldrich) at a dilution with 1:200 for 1 h at room temperature. For labeling nucleus, cells were washed and counterstained with DAPI (CWBIO, Beijing, China). A fluorescence microscope (Leica) was used for fluorescence observation and photographing.

For identification of the isolated pachytene spermatocytes, meiotic spread assays were conducted strictly based on previous study (Liu et al. 2015). Briefly, a hypotonic solution was used for preparing cell lysis which was spread evenly over slides layered with 1% PFA and 0.15% Triton-X-100. After drying for 24 h at room temperature in a humid chamber, the cells on the slides were treated with 0.04% photoflo for 5 min and blocked with 4% normal serum. The cells were incubated with primary antibodies including SCP3 (Abcam) and CREST (nBAF chromatin remodeling complex subunit, Immunovision, USA) overnight at 37°C in a humid chamber. The secondary antibody Alexa 555 donkey anti-rabbit (Molecular Probes) and Alexa 350 goat anti-mouse (Molecular Probes) were used to incubate for 90 min at 37°C. After washing with TBS, the cells were treated with antifade and fluorescence images were taken by a fluorescence microscope (Leica).

RNAseq, data processing and analysis

Small RNA deep sequencing was carried out by Shanghai Institute of Biochemistry and Cell Biology, which provided an improved deep sequencing method (Yang et al. 2016). In brief, cDNA libraries of small RNAs were constructed using 10 ng of total RNA. After the adaptor ligation, first-strand cDNA synthesis and PCR amplification, the 130 bp–160 bp DNA fragments corresponding to the small RNA fraction were acquired. Subsequently, an Illumina Hiseq 2000 sequencer was used for the sequencing of small RNA libraries. For data analysis, small RNA sequence adaptors from the Illumina sequencing were first removed and other contaminants were filtering out using software developed by BGI. Then, read sequences longer than 18 nt were used for mapping to the libraries of miRNAs, tRNAs, rRNAs, sn/snoRNAs (small nuclear/small nucleolar RNA) and repeat elements. The small RNA tags were mapped to reference sequence (Sus_scrofa.Sscrofa10.2) by Bowtie to analyze their expression and distribution on the reference. Mapped small RNA tags were used to look for known miRNAs. miRBase was used as reference, modified software mirdeep2 was used to obtain the potential known miRNAs. The percentage and length distribution of small RNAs was based on the acquired read counts. The differential expressed miRNAs between spermatogenesis stages were analyzed with R DEGseq package. Then, acquired miRNA expression level was normalized by the equation (read counts × 1,000,000)/libsize (total miRNA read counts) and presented as TPM. And the volcano, PCA and heatmap were all drew by R according to the differential expressed miRNAs TPM. miRNA targets were predicted by miRanda. Alignment score of 160 or greater and an energy threshold of −10.0 kcal/mol or less targets were considered as candidates. And alignment score of 170 or greater and an energy threshold of −20.0 kcal/mol or less targets were used for functional annotation and further verification.

Luciferase reporter assay

Reverse transcription of RNA from porcine testis tissue was performed according to manufacturer’s instruction of Transcriptor First-Strand cDNA synthesis kit (Roche). Gene 3′ UTR was amplified and cloned into the dual-luciferase reporter vector psiCHECK2 at the site digested with NotI and XhoI. Primers are listed in the Supplementary Table 1.

psiCHECK2 with targeted gene 3′UTR were cotransfected with miRNA mimics/NC into Hela cells using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturing protocols. After 48 h, all target validation assays were performed with the Dual-Luciferase Assay System (Promega). Firefly luciferase activity was normalized to renilla luciferase activity.

Statistical analysis

SPSS, V17.0 statistical software was used for statistical analysis. All values were presented as mean ± s.e.m., and statistically significant was consider when P value less than 0.05(*), 0.01(**).

Results

Identification of male germ cells

Porcine male germ cells at different stages during spermatogenesis were isolated by STA-PUT apparatus via the velocity sedimentation. The cell types were verified under a phase-contrast microscope based on the cell morphological and phenotypic characteristics. The diameters of spermatogonia, pachytene spermatocytes and round spermatids were around 10, 15 and 5 μm, respectively (Fig. 1A). Immunocytochemical analysis further revealed that the isolated spermatogonia were positive for GPR125, GFRA1 and UCHL1 (Fig. 1B), pachytene spermatocytes positive for SCP3 and round spermatids positive for Acrosin and PRM2. Meiosis spread assay indicated that SCP3 and CREST were co-expressed in the isolated pachytene spermatocytes. Moreover, the purity of isolated germ cells was more than 88% (Fig. 1C). To further verify the purity of the isolated cells, expression of marker genes (SOHLH1 for spermatogonia, SCP3 for pachytene spermatocytes, and PRM1 for round spermatids) was detected (Gan et al. 2011). Analysis showed that SOHLH1 (spermatogenesis and oogenesis-specific basic helix-loop-helix 1), SCP3 and PRM1 (protamine 1) were significant highly expressed in the isolated spermatogonia, pachytene spermatocytes and round spermatids, respectively (Fig. 1C). Taken together, these results suggest that spermatogonia, pachytene spermatocytes and round spermatids were successfully isolated from the mixed germ cells.

Figure 1
Figure 1

Identification of porcine male germ cells. (A) The morphological characteristics of freshly isolated porcine spermatogonia (A), pachytene spermatocytes (PS) and round spermatids (RS) under a phase-contrast microscope. (B) Immunocytochemistry revealed the expression of GFRA1, GPR125 and UCHL1 in spermatogonia. Meiosis spread assays displayed the expression of SCP3 and CREST in the freshly isolated pachytene spermatocytes. Acrosin and PRM2 were identified to express in the round spermatids. (C) Percentage of positive staining cell was determined. qRT-PCR was conducted to determine the specific marker genes for male germ cells (SOHLH1 for spermatogonia, SCP3 for pachytene spermatocytes and PRM1 for spermatids). Data are presented as the mean ± s.e.m., **P < 0.01. Scale bars in A and B = 10 μm.

Citation: Reproduction 154, 6; 10.1530/REP-17-0441

Profiling of small RNAs

In total, 7 samples were successfully sequenced with the Illumina Hiseq 2000 sequencer and used for the study. Total acquired reads varied from 8.8 million to 14.2 million. After adaptor sequences were trimmed, the quality reads were mapped to the porcine genome. And the mapped ratio of filter reads were 95.4, 95.4, 95.1, 93.9, 94.2, 83.3 and 83.9% for samples of spermatogonia (A), pachytene spermatocytes 1 (PS1), pachytene spermatocytes 2 (PS2), round spermatids 1 (RS1), round spermatids 2 (RS2), spermatozoa 1 (SP1), spermatozoa 2 (SP2), respectively (Table 1). The double peak of small RNA was detected in all cell types, whose peaks were distribution at the length of 20–25 nt and ~30 nt. The first peak around 20–25 nt was lower than the second peak located on ~30 nt in spermatogonia, pachytene spermatocytes and round spermatids (Fig. 2B, C, D, E and F). Conversely, the first peak of spermatozoa was higher than the second peak in spermatozoa (Fig. 2G and H). As shown in Fig. 2A, miRNAs were highly abundant in spermatozoa, which can also be reflected by the distribution of small RNA categories according to the length (Fig. 2G and H). In addition, the high peak of small RNA around 30 nt means the expression of PIWI-interacting RNAs (piRNAs) in spermatogonia, pachytene spermatocytes and round spermatids. These observations indicate the dynamic expression level of small RNAs in spermatogenesis.

Figure 2
Figure 2

Categories and length distribution of small RNAs. (A) Percentage of different categories of small RNAs mapped to the porcine genome in each developmental stage. (B, C, D, E, F, G and H) Composition of small RNA categories according to length distribution in each sample. Spermatogonia (A), pachytene spermatocytes (PS), round spermatids (RS), spermatozoa (SP).

Citation: Reproduction 154, 6; 10.1530/REP-17-0441

Table 1

Data processing metrics for small RNA reads for specific development stages.

Sample Total reads Quality reads Reads for mapping Mapped reads Mapped ratio
A 14,025,211 13,505,759 13,186,169 12,573,691 95.40%
PS1 14,794,199 14,207,264 13,879,331 13,241,856 95.40%
PS2 11,051,084 10,603,406 10,370,530 9,865,042 95.10%
RS1 13,936,919 13,327,024 12,841,061 12,055,271 93.90%
RS2 10,763,730 10,345,374 10,089,406 9,507,414 94.20%
SP1 11,936,011 11,411,295 10,845,902 9,029,783 83.30%
SP2 8,880,017 8,476,409 8,007,996 6,717,602 83.90%

A, Spermatogonia; PS, Pachytene Spermatocytes; RS, Round Spermatids; SP, Sperm.

Dynamic expression of miRNA during porcine spermatogenesis

In order to identify the differential expressed miRNA between contiguous stages, R DEGseq package was used for calculating. Scatter plot was used to show the differentially expressed miRNA between continually stages during spermatogenesis (Fig. 3A, B and C, Supplementary Table 3). Six upregulated miRNAs and 13 downregulated miRNAs were identified between pachytene spermatocytes and spermatogonia (Table 2). In addition, 70 miRNAs were upregulated and 4 miRNAs were downregulated in round spermatids compared with pachytene spermatocytes (Table 2). Finally, 199 miRNAs were differentially expressed between spermatozoa and round spermatids, reflecting that these differences may in part derive from epididymal secretion during spermatozoa transit from caput to caudal region (Table 2). Functional annotation showed that miRNAs differentially expressed between pachytene spermatocytes and spermatogonia may be involved in cell differentiation, development, cell signal transduction (Supplementary Fig. 1A). The target genes between round spermatids and pachytene spermatocytes were involved in cell localization, migration and response to stress (Supplementary Fig. 1B). Furthermore, target gene clusters of differentially expressed miRNAs between spermatozoa and round spermatids participate in regulation of metabolic process, development process and programmed cell death (Supplementary Fig. 1C). Principal component analysis (PCA) suggested that pachytene spermatocytes were similar to round spermatids (Fig. 3D). Meanwhile, miRNAs expression in spermatogonia and spermatozoa was different with that in pachytene spermatocytes and round spermatids (Fig. 3D). The minor differences between pachytene spermatocytes biological replicates or round spermatids biological replicates were due to the differences of sample derived individual animals.

Figure 3
Figure 3

The relationship among different phases according to differentially expressed genes and PCA. (A) Scatter plot comparison revealed the patterns of genes between A and PS. (B) Scatter plot comparison revealed the patterns of genes between PS and RS. (C) Scatter plot comparison revealed the patterns of genes between RS and SP. Standard selection criteria to identify differentially expressed genes was established at P value <0.01; the log2 Fold change >0.5 were expressed as up genes (red dots), log2 Fold change <−0.5 were expressed as down genes (green dots). (D) Two main principal components explain the correlation among A, PS, RS, SP.

Citation: Reproduction 154, 6; 10.1530/REP-17-0441

Table 2

The number of differentially expressed miRNAs.

Item Comparison Upregulated Downregulated
1 PS vs A 6 13
2 RS vs PS 70 4
3 SP vs RS 91 108

Differentially expressed miRNAs between continual stages during spermatogenesis were considered as dynamically expressed miRNAs and used for hierarchical clustering analysis (Supplementary Fig. 2). In order to further verify miRNAs expression in specific germ cells, miRNAs with high read counts and differentially expressed among different stages were chosen for further determination. Analysis revealed that the expression of ssc-miR-10a-5p in spermatogonia was significantly higher than that in pachytene spermatocytes, round spermatids, but was similar to that in spermatozoa (Fig. 4A). Furthermore, the expression of ssc-miR-125b in pachytene spermatocytes was significantly higher than that in round spermatids and spermatozoa, but comparable with that in spermatogonia (Fig. 4B). In addition, ssc-let-7f and ssc-miR-186 exhibited the highest abundance in round spermatids and spermatozoa, respectively (Fig. 4C and D). Together, these results further confirmed the quality and authenticity of the RNA sequencing data obtained in this study.

Figure 4
Figure 4

Verification of the expression level of miRNAs at each developmental stage. (A) The expression level of ssc-miR-10a-5p during spermatogenesis. (B) The expression level of ssc-miR-125b during spermatogenesis. (C) The expression level of ssc-let-7f during spermatogenesis. (D) The expression level of ssc-miR-186 during spermatogenesis.

Citation: Reproduction 154, 6; 10.1530/REP-17-0441

Prediction and verification of miRNA targets

According to the miRanda prediction, alignment score more than 170 and energy threshold less than −20 kcal/mol candidates were selected for target verification. As shown in Fig. 5, MAP2K6 (mitogen-activated protein kinase kinase 6), LRRC8A (leucine-rich repeat-containing 8 family member A), CDC6 (cell division cycle 6) and CDC42 (cell division cycle 42) were considered as candidate targets of ssc-miR-10a-5p, ssc-miR-125b, ssc-let-7f and ssc-miR-186 respectively. To identify the function of miRNAs, genes 3′UTR was cloned into psi-check2 downstream from the renilla luciferase coding region. Each reporter construct was separately co-transfected with the miRNA mimics or negative control RNA into Hela cells. For ssc-miR-10a-5p, the luciferase activity was downregulated 19.2% by transfecting with ssc-miR-10a-5p mimics compared to negative control RNA (Fig. 6A). Similarly, the declined percentage of luciferase activity for ssc-miR-125b, ssc-let-7f and ssc-miR-186 were 33.2, 50 and 63.5% respectively (Fig. 6B, C and D). These results demonstrate that MAP2K6, LRRC8A, CDC6 and CDC42 were the target of ssc-miR-10a-5p, ssc-miR-125b, ssc-let-7f and ssc-miR-186 respectively.

Figure 5
Figure 5

Predicted targets and binding sites of differentially expressed miRNAs. (A) The predicted binding sites of ssc-miR-10a-5p in MAP2K6 3′UTR. (B) The predicted binding sites of ssc-miR-125b in LRRC8A 3′UTR. (C) The predicted binding sites of ssc-let-7f in CDC6 3′UTR. (D) The predicted binding sites of ssc-miR-186 in CDC42 3′UTR.

Citation: Reproduction 154, 6; 10.1530/REP-17-0441

Figure 6
Figure 6

Targets validation of differentially expressed miRNAs using dual-luciferase assay. (A) MAP2K6 was a target of ssc-miR-10a-5p. (B) LRRC8A was a target of ssc-miR-125b. (C) CDC6 was a target of ssc-let-7f. (D) CDC42 was a target of ssc-miR-186. Data shown are the mean ± s.e.m., **P < 0.01.

Citation: Reproduction 154, 6; 10.1530/REP-17-0441

Discussion

Fluorescent-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS), based on cell surface markers, are most frequently used methods for cell purification. Several markers have been identified for isolating gonocytes or undifferentiated spermatogonia (Tokuda et al. 2007, Zheng et al. 2009, Aloisio et al. 2014). Although CD90 (Ryu et al. 2004, Zheng et al. 2014), α6-integrin and β1-integrin (Shinohara et al. 1999) have been proven as useful surface markers for isolating male germ stem cells, lacking of appropriate surface markers hindered the application of FACS and MACS for isolation of pachytene spermatocytes and round spermatids. Alternatively, STA-PUT has been successfully used for purification of spermatogonia, pachytene spermatocytes and round spermatids in mice and humans (Gan et al. 2011, Liang et al. 2014, Liu et al. 2015). However, it has not been applied to male germ cells in livestock animals. In this study, we applied STA-PUT to isolate porcine male germ cells. The isolated germ cells displayed normal morphology with a proper diameter and were positive for specific cell markers, indicating that spermatogonia, pachytene spermatocytes and round spermatids were successfully isolated in pigs. This study would provide as a reference for the isolation of male germ cells in other livestock species.

MiRNAs are small non-coding RNA that plays important roles in regulating spermatogenesis (McIver et al. 2012, Kotaja 2014, Yao et al. 2015). Analysis of miRNA transcriptome indicated that 224 unique miRNAs were differentially expressed between porcine ovary and testis. Meanwhile, miRNA located on the X chromosome exhibited a testis-preferential or testis-specific pattern (Li et al. 2011). In addition, there were 51 upregulated miRNAs and 78 downregulated miRNAs when comparing immature and mature testis (Luo et al. 2010). Recently, the miRNAs expression pattern in testis, epididymis and ejaculated spermatozoa were revealed. The co-expressed miRNAs were involved in the cell cycle process, metal ion binding, plasma membrane modification and p53 signal pathway (Luo et al. 2015). Furthermore, the variety of small RNAs in pig epididymal spermatozoa, ejaculated spermatozoa and seminal plasma was also analyzed (Chen et al. 2017a). In order to further understand the miRNAs expression pattern during porcine spermatogenesis, we conducted a small RNA deep sequencing of porcine-specific stages germ cells during spermatogenesis. Meanwhile, we systematically analyzed the characteristics of miRNAs expression. The analysis revealed that miRNAs were differentially expressed among porcine spermatogonia, pachytene spermatocytes, round spermatids and spermatozoa. In addition, 212 miRNAs were differentially expressed in the different types of male germ cells, suggesting that these miRNAs might be involved in regulating porcine spermatogenesis. Recent reports revealed the expression pattern of miRNAs during mouse (Smorag et al. 2012) or human spermatogenesis (Liu et al. 2015). Through profiling microarray data, Smorag and coworkers identified 45 miRNAs that were dynamically expressed in mouse SSCs, premeiotic cells and primary spermatocytes (Smorag et al. 2012). Similarly, Liu and coworkers found that 56 miRNAs were differentially expressed in human spermatogonia, pachytene spermatocytes and round spermatids (Liu et al. 2015). In the present study, using a small RNA deep sequencing analysis, we identified 212 miRNAs that were dynamically expressed in porcine spermatogonia, pachytene spermatocytes, round spermatids and spermatozoa (Supplementary Fig. 2). Particularly, several miRNAs (miR-125b, -10a, -99a, -99b) in porcine spermatogonia and spermatocytes exhibited a similar expression pattern as in human and mouse (Smorag et al. 2012, Liu et al. 2015), suggesting that these miRNAs may play a conserved roles in regulating spermatogonial proliferation and meiotic initiation. Conversely, let7 family miRNA (let-7a, 7c, 7f) was highly expressed in porcine pachytene spermatocytes and round spermatids, whereas this family was exceedingly expressed in human and mouse spermatogonia (Smorag et al. 2012, Liu et al. 2015). In addition, miR-34c was differentially expressed in porcine spermatozoa. In previous studies, miR-34c was detectable in pig ejaculated spermatozoa, epididymal spermatozoa and seminal plasma (Chen et al. 2017a). However, it was highly expressed in human spermatogonia (Liu et al. 2015), mouse SSCs (Niu et al. 2011), mouse pachytene spermatocytes and round spermatids (Smorag et al. 2012). Moreover, miR-34b/c and miR-449a/b/c knockout mouse model showed miR-34c played important roles in spermatogenesis (Yuan et al. 2015). These findings suggested that miRNAs play conserved and significant role in spermatogenesis.

Previous study has shown that a few miRNAs play vital roles in regulating SSC self-renewal and differentiation (Gou et al. 2014). miR-10a has been demonstrated to regulate differentiation (Foley et al. 2011, Okamoto et al. 2012), to modulate cell senescence (Zhu et al. 2013), to inhibit cell proliferation and promote cell apoptosis (Fan et al. 2016). Additionally, miR-10a is involved in retinoic acid induced the sequential programs of spermatogonial differentiation and the entry in meiosis (Huang et al. 2010, Meseguer et al. 2011, Busada & Geyer 2016). However, miR-10a functions in regulating spermatogenesis remain largely unclear. In the present study, we found that ssc-miR-10a-5p was highly expressed in porcine spermatogonia and spermatozoa, and that MAP2K6 was a target of ssc-miR-10a-5p. The function of ssc-miR-10a-5p in regulating spermatogonial proliferation and differentiation in pigs is worthy of study.

Evidence showed that miR-125b was significantly upregulated with testicular RA (retinoic acid) intervention when dogs were administrated with CYP26B1 (cytochrome P450 family 26 subfamily B member 1) inhibitor and all-trans-RA (Kasimanickam et al. 2014). In line with previous findings in human and mouse (Smorag et al. 2012, Liu et al. 2015), we observed that ssc-miR-125b was highly expressed in porcine spermatogonia and pachytene spermatocytes. We also identified that ssc-miR-125b can target LRRC8A, which has been demonstrated to activate AKT pathway and played essential roles in development, survival of T cells (Kumar et al. 2014). Meanwhile, our unpublished data have revealed that PI3K/AKT pathway was involved in regulation of survival of spermatogonial stem cells. Taken together, we speculate that ssc-miR-125b- LRRC8A-AKT acts as a candidate pathway in regulating survival of porcine spermatogonia and pachytene spermatocytes.

Additionally, we demonstrated that ssc-let-7f was highly expressed in round spermatids and targeted 3′UTR of CDC6. Previous study revealed that CDC6 was essential for meiotic maturation in oocytes (Lemaitre et al. 2002, Eward et al. 2004) and to avoid unscheduled DNA replication during meiotic maturation (Daldello et al. 2015). Comparably, the upregulation of ssc-let-7f may participate in the accomplishment of spermatocytes meiosis by regulating CDC6 expression.

Remarkably, ssc-miR-186 can be used as a reference for qRT-PCR in spermatozoa because of its constant expression level (Zhang et al. 2015). In the present study, we found that ssc-miR-186 was presented with high abundance in porcine spermatozoa and could target CDC42. Interestingly, CDC42 has been demonstrated to be expressed higher in asthenozoospermic group spermatozoa than normospermic samples (Salvolini et al. 2013). In addition, CDC42 could arrest capacitation and subsequently inhibit the acrosome reaction (Baltierrez-Hoyos et al. 2012). Therefore, it is worth to elucidate whether ssc-miR-186 regulates capacitation and acrosome reaction via CDC42 in pigs.

Lastly, round spermatids undergo spermiogenesis including acrosomal biogenesis, flagellum development, chromatin condensation, cytoplasmic reorganization and exclusion, and eventually transform into spermatozoa, which are released into the lumen of seminiferous tubules and further become mature in epididymis (Rathke et al. 2014). During the maturation in epididymis, spermatozoa plasma membrane is modified by exosomes that are secreted from epididymal epithelia (Nixon et al. 2015). The exosomes derived from the ampulla of ductus deferens and sexual glands (seminal vesicles, prostate and bulbourethral glands) further decorate the plasma membranes (Aalberts et al. 2014) during ejaculation. The miRNAs could be delivered to spermatozoa mediated with the exosomes during sperm maturation and ejaculation process. Therefore, it is not surprising that only 36.81% transcripts were common between bovine spermatids and mature spermatozoa (Gilbert et al. 2007). Recently, Chen and coworkers conducted a comparative analysis of small RNA profiles in porcine ejaculated spermatozoa, epididymal spermatozoa and seminal plasma (Chen et al. 2017a). The authors found that over 70% of small RNAs detected from ejaculated spermatozoa are the ejaculated spermatozoa specific. Compared with epididymal spermatozoa, 117 miRNAs were downregulated, 26 were upregulated and 102 were unchanged in ejaculated spermatozoa (Chen et al. 2017a). These upregulated sperm-borne small RNAs may originate from the exosomes. Importantly, the lack of comparison between porcine round spermatids and ejaculated spermatozoa was replenished in the present study. We found that 7 of the top 10 highly expressed miRNAs across the three libraries reported previously by Chen and coworkers (Chen et al. 2017a) were differentially expressed between round spermatids and spermatozoa. In the current study, spermatogonia, pachytene spermatocytes and round spermatids were collected from the same individual, and ejaculated spermatozoa was collected from another individual. Whether a proportion of the observed variance between spermatozoa and the other germ cells are the resulting from individual difference deserves further validation.

In summary, we successfully isolated porcine spermatogonia, pachytene spermatocytes and round spermatids from adult testis tissues. Meanwhile, we systematically analyzed the miRNAs in different male germ cells. The findings would provide a new insight into regulation of porcine spermatogenesis.

supplementary data

This is linked to the online version of the paper at http://dx.doi.org/10.1530/REP-17-0441.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This study was supported in part by National Basic Research Program of China (973 program; 2014CB943100), the National Natural Science Foundation of China (Grant No. 31272439, 31230048), and the Doctoral Program Foundation of Higher Education of China (Grant No. 20130204110017) to W Z.

Acknowledgements

The authors thank Dr Wuzi Dong for suggestions and comments. The authors thank Yangling Besun farm for providing porcine testis tissues. The authors thank Dr Bin Wu at Arizona Center for Reproductive Endocrinology and Infertility, USA, for editing the revised manuscript.

References

  • Aalberts M, Stout TA & Stoorvogel W 2014 Prostasomes: extracellular vesicles from the prostate. Reproduction 147 R1R14. (doi:10.1530/REP-13-0358)

  • Aloisio GM, Nakada Y, Saatcioglu HD, Pena CG, Baker MD, Tarnawa ED, Mukherjee J, Manjunath H, Bugde A & Sengupta AL et al. 2014 PAX7 expression defines germline stem cells in the adult testis. Journal of Clinical Investigation 124 39293944. (doi:10.1172/JCI75943)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Aravin AA & Bourc’his D 2008 Small RNA guides for de novo DNA methylation in mammalian germ cells. Genes and Development 22 970975. (doi:10.1101/gad.1669408)

  • Baltierrez-Hoyos R, Roa-Espitia AL & Hernandez-Gonzalez EO 2012 The association between CDC42 and caveolin-1 is involved in the regulation of capacitation and acrosome reaction of guinea pig and mouse sperm. Reproduction 144 123134. (doi:10.1530/REP-11-0433)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Busada JT & Geyer CB 2016 The role of retinoic acid (RA) in spermatogonial differentiation. Biology of Reproduction 94 10. (doi:10.1095/biolreprod.115.135145)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Chen C, Wu H, Shen D, Wang S, Zhang L, Wang X, Gao B, Wu T, Li B & Li K et al. 2017a Comparative profiling of small RNAs of pig seminal plasma and ejaculated and epididymal sperm. Reproduction 153 785796. (doi:10.1530/REP-17-0014)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Chen X, Li X, Guo J, Zhang P & Zeng W 2017b The roles of microRNAs in regulation of mammalian spermatogenesis. Journal of Animal Science and Biotechnology 8 35. (doi:10.1186/s40104-017-0166-4)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Daldello EM, Le T, Poulhe R, Jessus C, Haccard O & Dupre A 2015 Control of Cdc6 accumulation by Cdk1 and MAPK is essential for completion of oocyte meiotic divisions in Xenopus. Journal of Cell Science 128 24822496. (doi:10.1242/jcs.166553)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Eward KL, Obermann EC, Shreeram S, Loddo M, Fanshawe T, Williams C, Jung HI, Prevost AT, Blow JJ & Stoeber K et al. 2004 DNA replication licensing in somatic and germ cells. Journal of Cell Science 117 58755886. (doi:10.1242/jcs.01503)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Fan Q, Meng XR, Liang HW, Zhang HL, Liu XM, Li LF, Li W, Sun W, Zhang HY & Zen K et al. 2016 miR-10a inhibits cell proliferation and promotes cell apoptosis by targeting BCL6 in diffuse large B-cell lymphoma. Protein and Cell 7 899912. (doi:10.1007/s13238-016-0316-z.)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Foley NH, Bray I, Watters KM, Das S, Bryan K, Bernas T, Prehn JHM & Stallings RL 2011 MicroRNAs 10a and 10b are potent inducers of neuroblastoma cell differentiation through targeting of nuclear receptor corepressor 2. Cell Death and Differentiation 18 10891098. (doi:10.1038/cdd.2010.172)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Gan H, Lin X, Zhang Z, Zhang W, Liao S, Wang L & Han C 2011 piRNA profiling during specific stages of mouse spermatogenesis. RNA 17 11911203. (doi:10.1261/rna.2648411)

  • Gilbert I, Bissonnette N, Boissonneault G, Vallee M & Robert C 2007 A molecular analysis of the population of mRNA in bovine spermatozoa. Reproduction 133 10731086. (doi:10.1530/REP-06-0292)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Gou LT, Dai P & Liu MF 2014 Small noncoding RNAs and male infertility. Wiley Interdisciplinary Reviews: RNA 5 733745. (doi:10.1002/wrna.1252)

  • Guyonnet B, Dacheux F, Dacheux JL & Gatti JL 2011 The epididymal transcriptome and proteome provide some insights into new epididymal regulations. Journal of Andrology 32 651664. (doi:10.2164/jandrol.111.013086)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • He ZP, Jiang JJ, Kokkinaki M, Tang L, Zeng WX, Gallicano I, Dobrinski I & Dym M 2013 MiRNA-20 and mirna-106a regulate spermatogonial stem cell renewal at the post-transcriptional level via targeting STAT3 and Ccnd1. Stem Cells 31 22052217. (doi:10.1002/stem.1474)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Huang HR, Xie CQ, Sun X, Ritchie RP, Zhang JF & Chen YE 2010 miR-10a contributes to retinoid acid-induced smooth muscle cell differentiation. Journal of Biological Chemistry 285 93839389. (doi:10.1074/jbc.M109.095612)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kanatsu-Shinohara M & Shinohara T 2013 Spermatogonial stem cell self-renewal and development. Annual Review of Cell and Developmental Biology 29 163187. (doi:10.1146/annurev-cellbio-101512-122353)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kasimanickam VR, Kasimanickam RK & Dernell WS 2014 Dysregulated microRNA clusters in response to retinoic acid and CYP26B1 inhibitor induced testicular function in dogs. PLoS ONE 9 e99433. (doi:10.1371/journal.pone.0099433)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kimmins S & Sassone-Corsi P 2005 Chromatin remodelling and epigenetic features of germ cells. Nature 434 583589. (doi:10.1038/nature03368)

  • Korhonen HM, Meikar O, Yadav RP, Papaioannou MD, Romero Y, Da Ros M, Herrera PL, Toppari J, Nef S & Kotaja N 2011 Dicer is required for haploid male germ cell differentiation in mice. PLoS ONE 6. e24821. (doi:10.1371/journal.pone.0024821)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kotaja N 2014 MicroRNAs and spermatogenesis. Fertility and Sterility 101 15521562. (doi:10.1016/j.fertnstert.2014.04.025)

  • Kumar L, Chou J, Yee CSK, Borzutzky A, Vollmann EH, von Andrian UH, Park SY, Hollander G, Manis JP & Poliani PL et al. 2014 Leucine-rich repeat containing 8A (LRRC8A) is essential for T lymphocyte development and function. Journal of Experimental Medicine 211 929942. (doi:10.1084/jem.20131379)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lemaitre JM, Bocquet S & Mechali M 2002 Competence to replicate in the unfertilized egg is conferred by Cdc6 during meiotic maturation. Nature 419 718722. (doi:10.1038/nature01046)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Li M, Liu Y, Wang T, Guan J, Luo Z, Chen H, Wang X, Chen L, Ma J & Mu Z et al. 2011 Repertoire of porcine microRNAs in adult ovary and testis by deep sequencing. International Journal of Biological Sciences 7 10451055. (doi:10.7150/ijbs.7.1045)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Liang M, Li WQ, Tian H, Hu T, Wang L, Lin Y, Li YL, Huang HF & Sun F 2014 Sequential expression of long noncoding RNA as mRNA gene expression in specific stages of mouse spermatogenesis. Scientific Reports 4. (doi:10.1038/srep05966)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Liu WM, Pang RTK, Chiu PCN, Wong BPC, Lao KQ, Lee KF & Yeung WSB 2012 Sperm-borne microRNA-34c is required for the first cleavage division in mouse. PNAS 109 490494. (doi:10.1073/pnas.1110368109)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Liu Y, Niu MH, Yao CC, Hai YN, Yuan QQ, Liu Y, Guo Y, Li Z & He ZP 2015 Fractionation of human spermatogenic cells using STA-PUT gravity sedimentation and their miRNA profiling. Scientific Reports 5. (doi:10.1038/srep08084)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Luo L, Ye L, Liu G, Shao G, Zheng R, Ren Z, Zuo B, Xu D, Lei M & Jiang S et al. 2010 Microarray-based approach identifies differentially expressed microRNAs in porcine sexually immature and mature testes. PLoS ONE 5 e11744. (doi:10.1371/journal.pone.0011744)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Luo Z, Liu Y, Chen L, Ellis M, Li M, Wang J, Zhang Y, Fu P, Wang K & Li X et al. 2015 microRNA profiling in three main stages during porcine spermatogenesis. Journal of Assisted Reproduction and Genetics 32 451460. (doi:10.1007/s10815-014-0406-x)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Martinez J & Tuschl T 2004 RISC is a 5′ phosphomonoester-producing RNA endonuclease. Genes and Development 18 975980. (doi:10.1101/gad.1187904)

  • McIver SC, Roman SD, Nixon B & McLaughlin EA 2012 miRNA and mammalian male germ cells. Human Reproduction Update 18 4459. (doi:10.1093/humupd/dmr041)

  • Meseguer S, Mudduluru G, Escamilla JM, Allgayer H & Barettino D 2011 MicroRNAs-10a and -10b contribute to retinoic acid-induced differentiation of neuroblastoma cells and target the alternative splicing regulatory factor SFRS1 (SF2/ASF). Journal of Biological Chemistry 286 41504164. (doi:10.1074/jbc.M110.167817)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Miller MP, Amon A & Unal E 2013 Meiosis I: when chromosomes undergo extreme makeover. Current Opinion in Cell Biology 25 687696. (doi:10.1016/j.ceb.2013.07.009)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Moritoki Y, Hayashi Y, Mizuno K, Kamisawa H, Nishio H, Kurokawa S, Ugawa S, Kojima Y & Kohri K 2014 Expression profiling of microRNA in cryptorchid testes: miR-135a contributes to the maintenance of spermatogonial stem cells by regulating FoxO1. Journal of Urology 191 11741180. (doi:10.1016/j.juro.2013.10.137)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Niu Z, Goodyear SM, Rao S, Wu X, Tobias JW, Avarbock MR & Brinster RL 2011 MicroRNA-21 regulates the self-renewal of mouse spermatogonial stem cells. PNAS 108 1274012745. (doi:10.1073/pnas.1109987108)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Nixon B, Stanger SJ, Mihalas BP, Reilly JN, Anderson AL, Tyagi S, Holt JE & McLaughlin EA 2015 The microRNA signature of mouse spermatozoa is substantially modified during epididymal maturation. Biology of Reproduction 93. (doi:10.1095/biolreprod.115.132209)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Novotny GW, Sonne SB, Nielsen JE, Jonstrup SP, Hansen MA, Skakkebaek NE, Rajpert-De Meyts E, Kjems J & Leffers H 2007 Translational repression of E2F1 mRNA in carcinoma in situ and normal testis correlates with expression of the miR-17-92 cluster. Cell Death and Differentiation 14 879882. (doi:10.1038/sj.cdd.4402090)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Okamoto H, Matsumi Y, Hoshikawa Y, Takubo K, Ryoke K & Shiota G 2012 Involvement of microRNAs in regulation of osteoblastic differentiation in mouse induced pluripotent stem cells. PLoS ONE 7 e43800. (doi:10.1371/journal.pone.0043800)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Papaioannou MD & Nef S 2010 microRNAs in the testis: building up male fertility. Journal of Andrology 31 2633. (doi:10.2164/jandrol.109.008128)

  • Rathke C, Baarends WM, Awe S & Renkawitz-Pohl R 2014 Chromatin dynamics during spermiogenesis. Biochimica et Biophysica Acta 1839 155168. (doi:10.1016/j.bbagrm.2013.08.004)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ryu BY, Orwig KE, Kubota H, Avarbock MR & Brinster RL 2004 Phenotypic and functional characteristics of spermatogonial stem cells in rats. Developmental Biology 274 158170. (doi:10.1016/j.ydbio.2004.07.004)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Salvolini E, Buldreghini E, Lucarini G, Vignini A, Lenzi A, Di Primio R & Balercia G 2013 Involvement of sperm plasma membrane and cytoskeletal proteins in human male infertility. Fertility and Sterility 99 697704. (doi:10.1016/j.fertnstert.2012.10.042)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Shinohara T, Avarbock MR & Brinster RL 1999 beta1- and alpha6-integrin are surface markers on mouse spermatogonial stem cells. PNAS 96 55045509. (doi:10.1073/pnas.96.10.5504)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Smorag L, Zheng Y, Nolte J, Zechner U, Engel W & Pantakani DVK 2012 MicroRNA signature in various cell types of mouse spermatogenesis: evidence for stage-specifically expressed miRNA-221, -203 and -34b-5p mediated spermatogenesis regulation. Biology of the Cell 104 677692. (doi:10.1111/boc.201200014)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Swindle MM, Makin A, Herron AJ, Clubb FJ & Frazier KS 2012 Swine as models in biomedical research and toxicology testing. Veterinary Pathology 49 344356. (doi:10.1177/0300985811402846)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Tang FC, Hajkova P, Barton SC, O’Carroll D, Lee C, Lao KQ & Surani MA 2006 220-plex microRNA expression profile of a single cell. Nature Protocols 1 11541159. (doi:10.1038/nprot.2006.161)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Tokuda M, Kadokawa Y, Kurahashi H & Marunouchi T 2007 CDH1 is a specific marker for undifferentiated spermatogonia in mouse testes. Biology of Reproduction 76 130141. (doi:10.1095/biolreprod.106.053181)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Vandamme TF 2015 Rodent models for human diseases. European Journal of Pharmacology 759 8489. (doi:10.1016/j.ejphar.2015.03.046)

  • Yang Q, Lin J, Liu M, Li R, Tian B, Zhang X, Xu B, Liu M, Zhang X & Li Y et al. 2016 Highly sensitive sequencing reveals dynamic modifications and activities of small RNAs in mouse oocytes and early embryos. Science Advances 2 e1501482. (doi:10.1126/sciadv.1501482)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Yao CC, Liu Y, Sun M, Niu MH, Yuan QQ, Hai YA, Guo Y, Chen Z, Hou JM & Liu Y et al. 2015 MicroRNAs and DNA methylation as epigenetic regulators of mitosis, meiosis and spermiogenesis. Reproduction 150 R25R34. (doi:10.1530/REP-14-0643)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Yu Z, Raabe T & Hecht NB 2005 MicroRNA Mirn122a reduces expression of the posttranscriptionally regulated germ cell transition protein 2 (Tnp2) messenger RNA (mRNA) by mRNA cleavage. Biology of Reproduction 73 427433. (doi:10.1095/biolreprod.105.040998)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Yuan SQ, Tang C, Zhang Y, Wu JW, Bao JQ, Zheng HL, Xu C & Yan W 2015 mir-34b/c and mir-449a/b/c are required for spermatogenesis, but not for the first cleavage division in mice. Biology Open 4 212223. (doi:10.1242/bio.201410959)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Zhang Y, Zeng CJ, He L, Ding L, Tang KY & Peng WP 2015 Selection of endogenous reference microRNA genes for quantitative reverse transcription polymerase chain reaction studies of boar spermatozoa cryopreservation. Theriogenology 83 634641. (doi:10.1016/j.theriogenology.2014.10.027)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Zheng K, Wu X, Kaestner KH & Wang PJ 2009 The pluripotency factor LIN28 marks undifferentiated spermatogonia in mouse. BMC Developmental Biology 9 38. (doi:10.1186/1471-213X-9-38)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Zheng Y, He Y, An JH, Qin JZ, Wang YH, Zhang YQ, Tian XE & Zeng WX 2014 THY1 is a surface marker of porcine gonocytes. Reproduction Fertility and Development 26 533539. (doi:10.1071/RD13075)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • Zhu S, Deng S, Ma Q, Zhang T, Jia C, Zhuo D, Yang F, Wei J, Wang L & Dykxhoorn DM et al. 2013 MicroRNA-10A* and MicroRNA-21 modulate endothelial progenitor cell senescence via suppressing high-mobility group A2. Circulation Research 112 152164. (doi:10.1161/CIRCRESAHA.112.280016)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation

 

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  • Identification of porcine male germ cells. (A) The morphological characteristics of freshly isolated porcine spermatogonia (A), pachytene spermatocytes (PS) and round spermatids (RS) under a phase-contrast microscope. (B) Immunocytochemistry revealed the expression of GFRA1, GPR125 and UCHL1 in spermatogonia. Meiosis spread assays displayed the expression of SCP3 and CREST in the freshly isolated pachytene spermatocytes. Acrosin and PRM2 were identified to express in the round spermatids. (C) Percentage of positive staining cell was determined. qRT-PCR was conducted to determine the specific marker genes for male germ cells (SOHLH1 for spermatogonia, SCP3 for pachytene spermatocytes and PRM1 for spermatids). Data are presented as the mean ± s.e.m., **P < 0.01. Scale bars in A and B = 10 μm.

  • Categories and length distribution of small RNAs. (A) Percentage of different categories of small RNAs mapped to the porcine genome in each developmental stage. (B, C, D, E, F, G and H) Composition of small RNA categories according to length distribution in each sample. Spermatogonia (A), pachytene spermatocytes (PS), round spermatids (RS), spermatozoa (SP).

  • The relationship among different phases according to differentially expressed genes and PCA. (A) Scatter plot comparison revealed the patterns of genes between A and PS. (B) Scatter plot comparison revealed the patterns of genes between PS and RS. (C) Scatter plot comparison revealed the patterns of genes between RS and SP. Standard selection criteria to identify differentially expressed genes was established at P value <0.01; the log2 Fold change >0.5 were expressed as up genes (red dots), log2 Fold change <−0.5 were expressed as down genes (green dots). (D) Two main principal components explain the correlation among A, PS, RS, SP.

  • Verification of the expression level of miRNAs at each developmental stage. (A) The expression level of ssc-miR-10a-5p during spermatogenesis. (B) The expression level of ssc-miR-125b during spermatogenesis. (C) The expression level of ssc-let-7f during spermatogenesis. (D) The expression level of ssc-miR-186 during spermatogenesis.

  • Predicted targets and binding sites of differentially expressed miRNAs. (A) The predicted binding sites of ssc-miR-10a-5p in MAP2K6 3′UTR. (B) The predicted binding sites of ssc-miR-125b in LRRC8A 3′UTR. (C) The predicted binding sites of ssc-let-7f in CDC6 3′UTR. (D) The predicted binding sites of ssc-miR-186 in CDC42 3′UTR.

  • Targets validation of differentially expressed miRNAs using dual-luciferase assay. (A) MAP2K6 was a target of ssc-miR-10a-5p. (B) LRRC8A was a target of ssc-miR-125b. (C) CDC6 was a target of ssc-let-7f. (D) CDC42 was a target of ssc-miR-186. Data shown are the mean ± s.e.m., **P < 0.01.