Characterisation of Caenorhabditis eleganssperm transcriptome and proteome
© Ma et al.; licensee BioMed Central Ltd. 2014
Received: 4 September 2013
Accepted: 13 February 2014
Published: 28 February 2014
Although sperm is transcriptionally and translationally quiescent, complex populations of RNAs, including mRNAs and non-coding RNAs, exist in sperm. Previous microarray analysis of germ cell mutants identified hundreds of sperm genes in Caenorhabditis elegans. To take a more comprehensive view on C. elegans sperm genes, here, we isolate highly pure sperm cells and employ high-throughput technologies to obtain sperm transcriptome and proteome.
First, sperm transcriptome consists of considerable amounts of non-coding RNAs, many of which have not been annotated and may play functional roles during spermatogenesis. Second, apart from kinases/phosphatases as previously reported, ion binding proteins are also enriched in sperm, underlying the crucial roles of intracellular ions in post-translational regulation in sperm. Third, while the majority of sperm genes/proteins have low abundance, a small number of sperm genes/proteins are hugely enriched in sperm, implying that sperm only rely on a small set of proteins for post-translational regulation. Lastly, by extensive RNAi screening of sperm enriched genes, we identified a few genes that control fertility. Our further analysis reveals a tight correlation between sperm transcriptome and sperm small RNAome, suggesting that the endogenous siRNAs strongly repress sperm genes. This leads to an idea that the inefficient RNAi screening of sperm genes, a phenomenon currently with unknown causes, might result from the competition between the endogenous RNAi pathway and the exogenous RNAi pathway.
Together, the obtained sperm transcriptome and proteome serve as valuable resources to systematically study spermatogenesis in C. elegans.
Spermatogenesis is a process during which undifferentiated germ cells develop into mature sperm cells. A sperm cell has a highly condensed nucleus and lacks many organelles, such as ribosomes and Golgi apparatus; thus, sperm is transcriptionally and translationally quiescent [1–7]. Early observations that RNAs exist in sperm were originally dismissed ; however, over the past decade, the view that sperm contains complex RNA populations, including mRNAs and non-coding RNAs, has become established [1–7]. These RNA populations contain not only non-functional remnant spermatogenesis-expressed transcripts, but also RNAs with potential functions during early embryogenesis [9–11]. Recently, the rapid development of microarray and high-throughput sequencing technologies has assisted the profiling of sperm RNAs in a broad range of species, including human [12, 13], mouse , invertebrates [14–17] and plants [18–22].
The germline of Caenorhabditis elegans is transparent, and is well suited for cellular characterization of spermatogenesis . The transcription of sperm genes is initiated during the pachytene stage; when spermatocytes enter the karyosome stage, global transcription ceases [24–26]. In C. elegans, approximately 60 genes that are essential for normal spermatogenesis have been characterized [23, 27]. By microarray analyses, Reinke et al. identified sets of genes that are spermatogenesis-enriched, oogenesis-enriched and sex-regulated [28, 29]. Notably, their analyses showed that the sperm-enriched genes encode considerable numbers of kinases and phosphatases, and are depleted from the X-chromosome, echoing the findings of Reuben et al. that the X-chromosome in males exhibits striking H3K9 methylation . These microarray analyses identified sperm genes on a large-scale; however, the analyses were based on comparisons between germ cell mutants, and purified sperm cells were not used. In addition, their microarray analyses only identified sperm-enriched genes, while omitted those sperm genes that are also abundant in oocyte.
Compared with sperm genes, the C. elegans sperm small RNAs have been investigated more extensively. A few studies reported deep sequencing of the small interfering RNAs (siRNAs) of spermatogenic cells [14–17]. It was demonstrated that the ERI-1/RRF-3/ALG-3 pathway is essential for production of the spermatogenesis-specific 26G endogenous siRNAs (endo-siRNAs) . Han et al. also showed that 26G endo-siRNAs regulate spermatogenic gene expressions .
In the present study, we purified highly pure C. elegans sperm cells and applied high-throughput approaches to obtain sperm transcriptome and proteome. A large number of long or intermediate-sized non-coding RNAs (lncRNAs hereafter) are found in sperm transcriptome, implying the important roles of lncRNAs during spermatogenesis. We showed that sperm proteome/transcriptome is enriched in not only kinase and phosphatase proteins/genes as previously reported [28, 29], but also ion binding proteins/corresponding genes, underlying the crucial roles of intracellular ions in sperm. Our extensive RNAi screening of sperm genes did not produce many defective phenotypes. Our further analysis revealed a tight correlation between sperm transcriptome and sperm small RNAome, which leads to a view that the phenomenon of inefficient RNAi screening might result from the endogenous RNAi pahway that acts strongly during spermatogenesis and is to compete over the exogenous RNAi pathway.
Sequencing C. eleganssperm transcriptome and proteome
We further assessed the level of possible contaminations for sperm transcriptome by examining 15 somatic marker genes (Additional file 1: Table S2) for their abundances in the trancriptome dataset. Only five (wrt-2, ges-1, hlh-17, myo-2 and myo-3) were detected in sperm transcriptome, and the number of reads for these five genes ranged only from 1 to 3, thus, somatic cell contamination is much low in the prepared sperm cells. The RPKM (Reads Per Kilobase of exon model per Million mapped reads) values for these five genes range from 3.42e-4 to 3.27e-3. To obtain a higher confidence transcriptome dataset, we set the RPKM cut-off to 3.27e-3. The resultant sperm transcriptome dataset contains 3,760 genes (Additional file 4) and our following analyses are based on these 3,760 genes.
In parallel with sperm mRNA sequencing, sperm protein extract was prepared and separated on SDS-polyacrylamide gel prior to removal of the MSP fraction. Subsequently, the protein extract was processed for shotgun LC-MS/MS analysis. LC-MS/MS analysis identified 27,667 peptides, corresponding to 2,994 proteins in the C. elegans protein dataset (WS229), with a probability >95% using percolator scores (data available from http://184.108.40.206/miaolab/C.elegans%20data.htm). None of the above-mentioned somatic cell markers were detected in this proteome dataset. 207 proteins (6.9%) were degenerative (i.e., they cannot be distinguished among homologous proteins).
Validation of novel lncRNAs
Recently, Nam et al. identified ~170 polyadenylated lncRNAs in C. elegans, and many of these lncRNAs are predicted to be associated with male identity and sperm functions . Thus, we compared these lncRNAs with the non-coding portion of our sperm transcriptome dataset by BLAST (E-value = 9e-13, this E-value cutoff allows alignment to have ~60 bp contiguous perfect match). 32 lncRNAs were found in sperm transcriptome, and linc-40 was shown to be male-specific (Figure 4b). Together, these results suggest that apart from coding genes, substantial numbers of lncRNAs are expressed and may play functional roles during spermatogenesis.
Sperm are enriched in ion binding proteins
Sperm gene/protein abundance exhibits a skewed distribution
RNAi analysis of sperm enriched genes
We further tested the RNAi of 486 genes that have read counts above 20 using the strain pie-1::gfp::H2B at 25°C (25°C is a challenging condition for sperm fertility, e.g., fer-1, spe-9 and rrf-3 all control fertility in a temperature-sensitive manner). RNAi was conducted over three generations . However, our RNAi assay did not produce as many defective phenotypes as those collected from the Wormbase RNAi dataset. We describe five unreported genes that control fertility as below. First, RNAi for the gene ogdh-1 or asb-2 resulted in complete sterility. OGDH-1 is a mitochondrial 2-oxoglutarate dehydrogenase. ogdh-1 RNAi worms had severe germline defects without germ cells being produced (Figure 8a). ASB-2 is the subunit of a mitochondrial ATPase. asb-2 RNAi worms failed to produce living embryos and its germline growth was retarded when compared with the RNAi control (Figure 8b,c show the germlines at ~60 h after egg stage). The dissected germline of some asb-2 RNAi worms evidently lacked sperm (Additional file 1: Figure S4). Moreover, RNAi for the genes nspd-4, F22B5.4 and K09E4.1 led to production of unfertilized oocytes (Figure 8d,e and Additional file 1: Figure S5). The brood sizes of the RNAi worms for these three genes were significantly reduced when compared with that of RNAi control (t-test, P < 0.01) (Figure 8h). The observation of unfertilized oocytes in the uterus of RNAi worms as well as reduced brood sizes hints that these genes are involved in spermatogenesis.
Collectively, our RNAi screen identified a few genes that regulate germline development and sperm fertility. It is worth noting that our study, consistent with prior studies, showed that downregulation of sperm genes by RNAi feeding approach is inefficient to produce defective phenotypes [28, 47].
Endo-RNAi pathway represses sperm gene expression
We also examined the expressions of the 53 sperm specific/enriched genes (shown in earlier result) between him-5 and him-5;rrf-3 adult worms (him-5;rrf-3 worms produces offspring at 20°C, whereas are sterile at 25°C) by RT-PCR (25 amplification cycles). Notably, of the 53 genes, 32 genes were expressed at elevated levels in the him-5;rrf-3 as compared with him-5 (Figure 9b). This expression elevation is particularly evident for worms grown at restrictive temperature (25°C). This result demonstrated that RRF-3, as a vital component of the endo-siRNA pathway, negatively regulates the expression of sperm genes. Together, we propose that the inefficient RNAi of sperm genes might result from the competition between exogenous RNAi and endogenous RNAi pathways during spermatogenesis (see Discussion).
Discussion and conclusion
The sperm cell has highly condensed nucleus packed with protamines instead of histones and lacks many essential organelles, such as ribosomes and Golgi apparatus . Therefore, sperm cell is devoid of transcriptional and translational activities. The long assumed view was that, at fertilization, sperm transmits solely its DNA to the oocyte, which has all the components necessary for early embryonic development. However, this notion has been changed by recent advancement of high-throughput technologies that made the unravelling of the sperm transcriptome and epigenome possible. It was discovered that sperm not only retains the histones that may play significant roles during early embryogenesis [1, 5, 7, 48–50], but also contains complex RNA species, including mRNAs and non-coding RNAs, some of which are vital for early embryonic development [4, 11]. C. elegans is an easily manipulated model organism, and is ideal to study sperm biology. By microarray analysis, Reinke et al. identified spermatogenesis, oogenesis and sex-regulated genes in C. elegans. To improve study of spermatogenesis, here, we isolated highly pure sperm cells and obtained sperm transcriptome and proteome by high-throughput approaches.
First, sequencing sperm transcriptome showed that considerable numbers of lncRNAs are present in sperm. lncRNAs are a major group of ncRNAs whose functions are poorly understood. In Drosophila, some lncRNAs were thought to be involved in male-specific processes related to sexual dimorphism . Recently, Nam et al. showed that, in C. elegans, many lncRNAs are associated with processes such as dauer formation, male identity, sperm formation, and interaction with sperm-specific mRNAs . In our small-scale screen, eight novel lncRNAs were isolated, one of which was specifically expressed in males.
We showed that sperm gene sets are significantly enriched in not only kinase/phosphatase genes as previously reported [28, 29], but also genes encoding ion binding proteins. Sperm development is regulated posttranslationally. The Cl-, Na+, K+ and Ca2+ ion channels have been implicated to respond to extracellular signals and mediate sperm activation [36–41]. Therefore, our identification of the sperm genes encoding ion binding proteins will help uncover the intracellular ion regulated pathways associated with sperm function.
We showed that sperm genes or proteins have skewed abundance, i.e., a small set of genes/proteins are greatly enriched in sperm. This supports previous observations that male-enriched genes have biased expressions than female-enriched genes in zebrafish , fly  and C. elegans. This result may suggest that compared with somatic cells, sperm only require a small number of proteins to complete their post-translational regulations, e.g., signalling factors for sperm activation, MSP dynamics and interaction with oocyte.
By RNAi of hundreds of sperm-enriched genes, we identified a few genes, whose knockdown leads to production of unfertilized oocytes and significantly reduced brood size. It is worth noting that previous study showed that RNAi of sperm genes is inefficient for producing defective phenotypes [28, 43]. To our knowledge, the mechanism behind this phenomenon has been unclear. It could be caused by the low penetrance of RNAi to germline tissue. Because endo-RNAi pathway strongly acts during spermatogenesis [15, 17], here, we presume that this phenomenon might result from the competition between endogenous and exogenous RNAi pathways. The endogenous RNAi pathway may compete over the exogenous RNAi pathway, leading to the failure of RNAi of sperm genes.
C. elegansstrains and growth conditions
The following strains were used in this study: wild-type Bristol strain N2, spe-8(hc40)I, rrf-3(pk1426)II, dpy-4(e1166)IV, fem-1(hc17)IV, fem-3(q23)IV, fog-2(q71)V, him-5(e1490)V, pie-1p::gfp::H2B. All the strains were maintained at 16°C or 20°C. The strains fem-1 and fem-3 are temperature-sensitive. At 25°C, fem-1 only produces oocytes, while fem-3 overproduces sperm.
Large-scale culture of C. elegansand purification of mature sperm
Large-scale culture of C. elegans strain him-5 and purification of mature sperm cells were performed using a modified protocol based on published methods [51–53] (detailed protocols in Additional file 1).
cDNA synthesis and 454 pyrosequencing
The total RNA of C. elegans sperm was prepared using a mirVana miRNA Isolation kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Trace genomic DNA was removed using RNase-free DNase I (Promega, Madison, WI, USA). The DNase I-treated RNA was reverse transcribed to cDNA using a SMARTer PCR cDNA Synthesis kit (Clontech, Mountain View, CA, USA) and then amplified for 20 cycles using an Advantage 2 PCR kit (Clontech). Amplified cDNA was purified using a QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and 0.5 μg of the cDNA was subjected to 454 pyrosequencing using a GS FLX Titanium General Library Preparation Kit (Roche 454 Company, CT, USA).
Sperm protein preparation and mass spectrometry analysis
C. elegans sperm was suspended in PBS solution, disrupted by sonication for 8 s (repeated three times at intervals of 8 s), mixed with SDS loading buffer and heated for 10 min. After centrifugation, the supernatant was loaded on a 12% SDS-PAGE gel for separation. The resulting gel was silver stained using a Fast Silver Stain kit (Beyotime, Shanghai, China). The major sperm protein (MSP) band was removed prior to tryptic in-gel digestion, according to standard protocols. The tryptic peptides were extracted using 60% acetonitrile in 0.1% formic acid, dried in vacuum and then resuspended in 0.1% formaic acid for mass spectrometry analysis. LC-MS/MS analysis was performed in a LTQ-orbitrap XL (ThermoFinnigan, San Jose, CA) coupled online with an Eksgent Nano 2D LC system. Peptides mixture was first loaded on a trap column (300SB-C18, 5 × 0.3 mm, 5 μm particle) (Agilent Technologies, Santa Clara, CA), and then analysed using a self-packed capillary C18 column (75 μm i.d. × 150 mm, 3 μm particle, C18 resin), and eluted with a gradient of 4-35% of Buffer B (0.5% formic acid in acetonitrile) in Buffer A (0.5% formic acid in water) at a flow rate of ~300 nl/min for 120 min. Data-dependent scanning was incorporated to select the 10 most abundant ions from a full-scan mass spectrum (mass range 200–1800 Da) for fragmentation by collision-induced dissociation.
Mass spectrometry data processing
MS data were analyzed using Mascot (Matrix Science, version 2.3.02) against the C. elegans protein dataset (release WS229) with the following parameters: only tryptic peptides with up to two missed cleavage sites were allowed; 20 ppm mass tolerances for MS; and 0.8 Da for MS/MS fragment ions. Peptides with Mascot Percolator posterior error probability values lower than 0.05 were considered to be potential candidates. Decoy database searches in Mascot revealed a false positive rate of 1.25% at the peptide level. Proteins with ≥2 unique peptides were accepted as matches.
The raw 454 sequencing reads were filtered by trimming off the adaptor sequences and removing short reads (<50 bp). Repeat sequences were masked, and tRNA, rRNA, snoRNA and snRNA sequences (WS228) were filtered before reads assembling using Newbler, version 2.3 (Roche). For sequence assembly, the C. elegans coding transcript data (WS228) was used as a reference; the unassembled reads were further assembled using the C. elegans genome as a reference. For the assembly we used the Newbler default parameters (overlap length >40 bp, identities >90%). Sequences in the non-coding portion were evaluated for coding potential using CPAT (http://lilab.research.bcm.edu/cpat/index.php). IGV genome browser (http://www.broadinstitute.org/igv/) was used to view reads assembling information for novel non-coding RNA transcripts. Sperm transcriptome was functionally annotated using the GO Slimmer tool (http://amigo.geneontology.org/cgi-bin/amigo/slimmer?session_id). Sperm primary siRNAs data described previously  were downloaded from the NCBI GEO database (http://www.ncbi.nlm.nih.gov/geo/). These siRNAs datasets were compared to our sperm transcriptome data using BLAST, and the siRNAs with a perfect antisense match were retained as the siRNAs targeting sperm genes. C. elegans polyadenylated lncRNA data was previously described . C. elegans genome data, cds transcript dataset and annotation data were all downloaded from Wormbase (ftp://ftp.wormbase.org/pub/wormbase/species/c_elegans/).
3′-rapid amplification of cDNA ends (3′RACE)
DNase I-treated sperm RNA was reverse transcribed into cDNA following the 3′RACE protocol in the FirstChoice RLM-RACE kit (Invitrogen). Nested PCR was conducted using the High Fidelity PCR SuperMix (TransGen, Beijing, China) and the amplified product was cloned into the pEASY-Blunt vector (TransGen) before sequencing to identify its 3′ sequence.
Reverse transcription PCR (RT-PCR)
The N2, fem-1(lf), fem-3(gf) and fog-2(lf) strains were synchronized and the adults were collected and frozen as starting materials. Total RNAs from these strains were isolated using a mirVana miRNA Isolation kit (Invitrogen). Total RNA (2 μg) was reverse transcribed into cDNA using the SuperScript III First-Strand Synthesis System (Invitrogen). The cDNA product was diluted 10-fold and used as the template for RT-PCR. The Act-1 and Cdc-42 genes were used as controls.
The RNAi treated pie-1p::gfp::H2B strain were anesthetized and mounted on agarose pads for visualization using a differential interference contrast (DIC) microscope (Zeiss, Axio Imager M2) as well as a confocal microscope (Zeiss LSM 510 Meta). The gonads of asb-2RNAi worms were dissected, and then fixed by cold methanol method (http://www.wormbook.org/toc_wormmethods.html), followed by DAPI staining and microscopic visualization.
RNA interference screen
The RNAi screen was performed as previously described [54, 55]. Briefly, the strain rrf-3;him-5 or pie-1p::gfp::H2B was fed with the RNAi bacteria obtained from the Ahringer RNAi library . Bacteria carrying the empty vector (L4440) were used as the control. The bacteria clones that affect fertility were sequenced to verify the gene sequences.
Brood size assay
The L4 stage hermaphrodites of the RNAi worms were individually picked onto plates, and transferred to new plates daily until no eggs were laid. Brood size was determined by counting the worms on all plates. 50 replications were performed. Worms fed with bacteria carrying L4440 were used as the control.
Availability of supporting data
The raw sperm transcriptome sequencing data has been deposited in NCBI SRA database with the accession number of SRA056374. Sperm mRNA-seq assembled data and sperm proteomics data can be downloaded from http://220.127.116.11/miaolab/C.elegans%20data.htm.
This work was supported by the National Basic Research Program of China (Grants 2012CB945022 and 2010CB912303 to L.M.; grant 2009CB825503 to Y.Z.; grants 2010CB833703, 2012CB966803 and 2011CB915501 to F.Y.) and the National Natural Science Foundation of China (Grants 31171337 and 30971648 to L.M., grant 90919047 to F.Y.). We thank Ping Huang, Zheng Cao, Zhiyu Liu, Wei Zhuang and Chao Sun for assistances with this work.
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