- Research article
- Open Access
Functional changes in mRNA expression and alternative pre-mRNA splicing associated with the effects of nutrition on apoptosis and spermatogenesis in the adult testis
© The Author(s). 2017
- Received: 30 April 2016
- Accepted: 7 December 2016
- Published: 10 January 2017
The effects of nutrition on testis mass in the sexually mature male have long been known, however, the cellular and molecular processes of the testis response to nutrition was not fully understood.
We tested whether the defects in spermatogenesis and increases in germ cell apoptosis in the testis that are induced by under-nutrition are associated with changes in mRNA expression and pre-mRNA alternative splicing using groups of 8 male sheep fed for a 10% increase or 10% decrease in body mass over 65 days.
We identified 2,243 mRNAs, including TP53 and Claudin 11, that were differentially expressed in testis from underfed and well-fed sheep (FDR < 0.1), and found that their expression changed in parallel with variations in germ cell numbers, testis size, and spermatogenesis. Furthermore, pairs of 269 mRNAs and 48 miRNAs were identified on the basis of target prediction. The regulatory effect of miRNAs on mRNA expression, in combination with functional analysis, suggests that these miRNAs are involved in abnormal reproductive morphology, apoptosis and male infertility. Nutrition did not affect the total number of alternative splicing events, but affected 206 alternative splicing events. A total of 159 genes, including CREM, SPATA6, and DDX4, were differentially spliced between dietary treatments, with functions related to RNA splicing and spermatogenesis. In addition, three gene modules were positively correlated with spermatogenesis-related phenotypic traits and negatively related to apoptosis-related phenotypic traits. Among these gene modules, seven (CFLAR, PTPRC, F2R, MAP3K1, EPHA7, APP, BCAP31) were also differentially expressed between nutritional treatments, indicating their potential as markers of spermatogenesis or apoptosis.
Our findings on significant changes in mRNAs and pre-mRNA alternative splicing under-nutrition suggest that they may partly explain the disruption of spermatogenesis and the increase germ cell apoptosis. However, more research is required to verify their causal effects in regulating spermatogenesis and germ cell apoptosis.
- Pre-mRNA alternative splicing
The development of mature haploid spermatozoa from diploid spermatogonial cells  can be affected by many factors, including photoperiod, hormones, temperature and nutrition. The effects of nutrition on testis mass in the sexually mature male have long been known, as has the direct relationship between testicular mass and sperm production . In addition, with change in testicular size, the efficiency of sperm production also changes . We have been investigating the cellular and molecular processes of the testis response to nutrition, and we have found that under-nutrition despaired spermatogenesis in adult sheep [4, 5].
Within the testis, spermatogenesis is a strictly regulated process, at both the transcriptional and the post-transcriptional level . In recent years, a novel mechanism of post-transcriptional control, mediated by microRNAs (miRNAs), has emerged as an important regulator of spermatogenesis [6, 7]. miRNAs are small (~22 nucleotides) endogenous RNAs that negatively regulate gene expression by targeting the 3′untranslated region (3′UTR)  and/or coding region  of mRNAs. We have recently found that the expression of a number of miRNAs is affected by nutrition in sexually mature male sheep, and most of the predicted targets of the differentially expressed miRNAs were mainly involved in reproductive system development and function . However, the regulatory relationship between these miRNAs and their corresponding mRNAs targets in testis remains to be verified. We therefore decided to profile mRNA expression in the testes of well-fed and underfed male sheep using RNA-seq so we could explore the relationships between the miRNAs we had identified and their putative targets.
In addition to the disruption of spermatogenesis, under-nutrition of sexually mature male sheep increased apoptosis in germ cells . Our recent study has revealed higher levels of expression of miR-98 in underfed sheep than in well-fed sheep  and this miRNA has been reported to play a critical role in apoptosis . Since the molecular mechanisms through which miRNAs regulate the expression of apoptosis-related genes are still controversial, we decided to explore these processes further using our nutritional model.
It has also been reported that spermatogenesis and a large number of apoptotic factors are regulated by alternative pre-mRNA splicing that generates multiple transcript species from a common mRNA precursor and thus raises protein diversity and allows the system to cope with the increasingly broad spectrum of functional and behavioural complexity [12, 13]. To date, eight types of alternative splicing have been reported: cassette exon, alternative 5′ splice site, alternative 3′ splice site, mutually exclusive exon, coordinates cassette exons, alternative first exon, alternative last exon and intron retention . We therefore also tested the hypothesis that nutritional treatment will induce differences in alternative splicing, and these changes will be related to the regulation of spermatogenesis and germ cell apoptosis in the testis.
Overall, this study used testicular tissue from under-fed and well-fed sexually mature sheep to pursue four objectives: 1) To investigate the differences of the expression of mRNAs; 2) To evaluate the influence of miRNAs on spermatogenesis and the expression of apoptosis-related genes; 3) To explore the relationships between alternative pre-mRNA splicing and spermatogenesis and apoptosis; 4) To investigate the relationships between the gene modules and spermatogenesis and apoptosis related phenotypic traits.
The experimental protocol was approved by the Animal Ethics Committee of the CSIRO Centre for Environment and Life Sciences, Floreat, Western Australia (Project No.1202).
Animals and treatments
From May to July (autumn-winter), 24 Merino male sheep (age 24 months, body mass 65.7 ± 4.7 kg, scrotal circumference 31.8 ± 2.5 cm) were housed in individual pens in a building with windows that allowed good penetration of natural light at Floreat, Western Australia. During the 3-week acclimatization period, all sheep were fed daily with 750 g oaten chaff (8.4% crude protein; 8.0 MJ/Kg metabolisable energy) and 200 g lupin grain (35.8% crude protein; 13.0 MJ/Kg metabolisable energy). At the start of the treatment period (end of May; mid-autumn), the animals were allocated into three dietary treatment groups (high, maintenance and low) balanced for training success to semen collection, body mass, scrotal circumference, temperament, poll-horn type, and sperm quality (the percentage of live and motile sperm, sperm concentration). Animals fed their maintenance requirements were expected to maintain constant body mass. The high diet was designed to allow the animals to gain 10% live weight over 65 days whereas the low diet was designed to allow 10% loss in weight. At the start of the treatment period, individual daily allowance was 1.2 kg oaten chaff plus 0.3 kg lupin grain for the rams in the high-diet group, 0.7 kg chaff and 0.18 kg lupin grain for the maintenance group, and 0.51 kg chaff and 0.13 kg lupin grain for the low-diet group. Every week, the animals were weighed and the amount of feed offered to each individual was adjusted to ensure achievement of target live weight.
The data on the effects of nutritional treatments on body mass, scrotal circumference, semen quality and spermatozoal quality, Sertoli cell number and quality were published before [4, 5]. Our previous work showed significant differences between High diet and Low diet in terms of the above parameters, with the values for maintenance-fed rams being generally similar to those for the High diet. For purposes of the current study, we decided to perform RNA-Seq only with the two extreme groups (testis growing versus testis shrinking) to determine the factors that contribute to the huge differences in phenotype.
Tissue Collection and preservation
After 65 days, all male sheep were killed with intravenous overdose of sodium pentobarbitone, and the testes were immediately removed, dissected and weighed. Three samples were chosen from top, middle and bottom parts of both testes (~1 cm3 for each); those from the right testis were snap-frozen in liquid nitrogen and stored at −80 °C for RNA isolation.
Isolation of RNA
About 1 cm3 tissues from top, middle and bottom parts of the right testes were mixed and grinded to powder in liquid nitrogen for RNA isolation. The trizol method was used to isolate total RNA  from testis samples. The quality and quantity of the RNA were determined by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA). Only RNA with an integrity number (RIN) greater than 7.0 was used for further analysis.
Small RNA library sequencing and Identification of miRNAs
The methodology details and outcomes for miRNAs have been reported elsewhere .
Construction and sequencing of the RNA-seq library
Total RNA (1.0 μg each) from each sample was used to construct RNA-seq libraries with a unique index using the TruSeq mRNA Sample Preparation kit (Illumina, San Diego, CA) according to the manufacturer’s instruction. Quantitative real time PCR (qPCR) was performed for library quantification using the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Carlsbad, CA) and KAPA SYBR Fast ABI Prism qPCR kit (Kapa Biosystems, Woburn, MA). Individual libraries were then pooled for sequencing at Génome Québec (Montréal, Canada) using the HiSeq 2000 system (Illumina). Sequencing was performed as 100 bp paired-end reads. All reads were de-multiplexed according to their index sequences with CASAVA version 1.8 (Illumina) and reads that did not pass the Illumina chastity filter were discarded.
Mapping and annotation of RNA-seq reads
RNA-seq reads were aligned to the ovine genome (OAR 3.1) using Tophat 2.0.10 with default parameters . Each BAM file obtained from TopHat2 and the GTF file obtained from ENSEMBL (v75.30) were used in the htseq-count (http://www-huber.embl.de/users/anders/HTSeq/) to determine the number of reads mapped to each gene.
Identification of differentially expressed (DE) mRNAs
Differentially expressed (DE) mRNAs were investigated with the bioinformatics tool, edgeR that utilizes a negative binomial distribution to model sequencing data . The expression of mRNAs in each library was normalized to counts per million reads (CPM) with the formula: CPM = (reads number/total reads number per library) × 1,000,000. mRNAs with CPM > 5 in at least 50% of the samples were subjected to DE analysis. Fold changes were defined as ratios of arithmetic means of CPM within each comparison group. Significant DE mRNAs were determined by an adjusted P value (False discovery rate, FDR < 0.1) based on Benjamini and Hochberg multiple testing correction  as well as fold change > 1.5 .
Validation of mRNA expression using RT-qPCR
Details of primers used for RT-qPCR
Product length (bp)
Piwi-Like RNA-Mediated Gene Silencing 1
Spermatogenesis Associated 4
Inhibin beta A
Forkhead box O3
Phosphatase and tensin homolog
Cytochrome P450, Family 51, Subfamily A, Polypeptide 1
Construction of miRNA-mRNA regulatory relationships
The results for miRNAs were all obtained from a previous study using the same samples . The predicted regulatory relationships between differentially expressed miRNAs and differentially expressed mRNAs were identified on the basis of two criteria as suggested by previous studies [20, 21]: negative correlation and computational target prediction. Genes targeted by miRNAs were predicted by TargetScan Release 6.0 (http://www.targetscan.org/) and miRanda (http://www.microrna.org/microrna/home.do). Target genes predicted by both TargetScan (default parameters) and miRanda (total score > 145, total energy < −10 kcal/mol) were used for further analysis . Pairwise Pearson correlation coefficient (R) was computed for each miRNA and their predicted target genes, and multiple testing corrections were done by calculating FDR. Significant miRNA-mRNA pairs were defined as R < −0.9 and FDR < 0.1.
Functional analysis for DE mRNAs
The identities of the DE mRNAs in the miRNA-mRNA regulatory relationships were uploaded into IPA software (Ingenuity Systems, www.ingenuity.com) to detect the top functions. A threshold of P < 0.01 was applied to enrich significant biological functions. The IPA regulation z-score algorithm was used to predict the direction of change for a given function (increase or decrease), with a z-score > 2 suggesting a significant increase whereas a z-score < −2 suggesting a significant decrease. The GO terms were defined and the KEGG pathways were enriched using Database for Annotation, Visualization and Integrated Discovery (DAVID, http://david.abcc.ncifcrf.gov) . For each analysis, the functional annotation clustering option was used and significant GO terms and KEGG pathways were declared at P < 0.05 and molecule number > 2.
Identification and annotation of alternative splicing (AS) events
TopHat2 was used to predict the splice junctions (options: −a 19, −g 1, −-max-intron-length 17325, and --min-intron-length 81). A total of 15 million reads were randomly selected from each sample for analysis to make sure that the comparison was at the same level . JuncBASE was used to annotate all AS events (cassette exons, alternative 5′ splice site, alternative 3′ splice site, mutually exclusive exons, coordinate cassette exons, alternative first exons, alternative last exons, and intron retention) and calculate the Percentage Spliced Index (PSI) . Splicing analysis was performed for events that had at least 20 reads and the PSI differences (ΔPSI) are higher than 10% .
The relationship between gene modules with sheep phenotypic traits
A weighted gene co-expression network was constructed for all samples using the WGCNA package in R to analyse the expressed mRNAs (CPM > 1 in at least 8 samples) . Briefly, a matrix of pairwise correlations between all pairs of genes across all samples was constructed. An adjacency matrix was then calculated, using the correlation matrix of the expression sets, and transformed into a topological overlap matrix that was then used to derive a distance matrix of hierarchical clustering. Finally, the mRNAs were assigned into different modules based on hierarchical clustering. Modules with eigengenes (defined as the first principal component of each module and considered as a representative of the gene expression profiles in that module) that were highly correlated were then merged. All these steps were performed using the ‘blockwiseModules’ function in the WGCNA package, using the major parameters described previously . The correlations were calculated for the relationships between module eigengenes and phenotypes (testis weight, sperm number per testis, tubule diameter, seminiferous epithelium volume, change of scrotal circumference, apoptotic cells/tubule; Additional file 1: Table S1).
High Quality RNA-seq data were obtained from all samples
Profile of mRNAs in sheep testis
The 15 functions most commonly related to the most highly expressed 3000 genes (as determined by DAVID software). P value indicates the relevance of the function (lower value means greater relevance)
cell cycle process
modification-dependent macromolecule catabolic process
modification-dependent protein catabolic process
proteolysis involved in cellular protein catabolic process
cellular protein catabolic process
protein catabolic process
male gamete generation
cellular macromolecule catabolic process
cell cycle phase
macromolecule catabolic process
Effects of nutrition on mRNA expression
RT-qPCR validation of differentially expressed genes
Six mRNAs were selected from the DE mRNAs and the RT-qPCR results were consistent with the sequencing data for all of them. For example, both the sequencing data and the RT-qPCR results showed that PIWIL1 was expressed at a lower level in underfed males than in well-fed males (Additional file 5: Figure S2). In addition, the expression of INHBA was down regulated in well-fed male sheep (Additional file 5: Figure S2).
The relationship between gene modules with sheep phenotypic traits
Functional analysis for the genes in Modules 7, 9 and 10
Functional analysis suggested that genes in Modules 7 and 9 were associated with both spermatogenesis and apoptosis, but genes in Module 10 were only related to apoptosis. Specifically, in Module 7, 35 genes were related to spermatogenesis whereas 46 genes were associated with apoptosis. Six genes (NFKBIL1, XRCC5, ERCC1, APP, BCAP31, RRAGA) were related to both spermatogenesis and apoptosis. For Module 9, eight genes (RXFP1, ITCH, ITGB1, XHD, TAF7L, WNT2, SPIN1, LNPEP) were associated with spermatogenesis, whereas 16 genes (CFLAR, TAF9B, CCK, VAV3, NR3C1, CDH13, CROP, CDKN1B, PTPRC, ATP7A, RTN3, HSPD1, ITM2B, F2R, MAP3K1, RAD21) were associated with apoptosis, but no genes were common to spermatogenesis and apoptosis. For Module 10, four genes (EPHA7, SCIN, NGFRAP1 and MAP3K7) were related to apoptosis. Interestingly, of all the genes mentioned above, seven (APP, BCAP31, CFLAR, PTPRC, F2R, MAP3K1and EPHA7) were differentially expressed between nutritional treatments, indicating their pivotal roles in reproduction or apoptosis.
miRNA-mRNA regulatory relationships
Identification of alternative splicing events
We initially obtained 940,607 junctions from the 16 RNA-seq libraries with the Tophat2 software. Totally, 6376 alternative splicing events (∆PSI > 10% in at least one library and at least 20 reads mapped) were identified from these junctions (Additional file 8: Table S5). Among these alternative splicing events, 4,820 (75.6%) were previously annotated as known alternative splicing events in the Ensembl Database, which can be mapped to 2288 unique genes. Eight types of alternative splicing events were identified, including 1,131 cassette exons, 645 alternative 5′ splice sites, 578 alternative 3′ splice sites, 17 mutually exclusive exons, 86 coordinate cassette exons, 578 alternative first exons, 247 alternative last exons, and 2,810 intron retentions.
Effects of nutrition on alternative splicing
This study appears to be the first to profile the whole transcriptome in sheep testis, to construct the regulatory relationships between miRNAs and mRNAs in testis, to explore the relationships between pre-mRNA alternative splicing and testis function, and to link the gene modules with phenotypic traits related to spermatogenesis and apoptosis. In the context of an experimental model of reversible testis growth in the sexually mature male, we have been able to identify mRNAs that are associated with testis function and, more importantly, apoptosis in germ cells. These findings strongly support the hypothesis that the decline in spermatogenesis and increase in germ cell apoptosis induced by under-nutrition in the sexually mature male sheep are, at least, partially due to changes in mRNA expression and pre-mRNA alternative splicing.
We found over 2,000 mRNAs that were differentially expressed between treatments, with over 1,000 mRNAs, including TP53 and Claudin 11, that were more highly expressed in underfed than in well-fed sheep. This result supports our previous observations based on qPCR . A high level of TP53 indicates more cells going through apoptosis , and this result is in agreement with our previous finding-more TUNEL positive cells were observed in testes from underfed than in well-fed sheep , so we conclude that under-nutrition increases apoptosis in germ cells. On the other hand, Claudin-11 is a tight junction protein expressed in Sertoli cells and rarely in other cell types in the testis  and plays a central role in the formation of tight junctions [30, 31]. In testicular tissue from underfed sheep, increased expression of Claudin 11 and disorganization of Claudin 11 protein strongly indicate the impairment of tight junctions . In addition, in the present study, over 1000 mRNAs showed lower expression in underfed than in well-fed sheep, including CYP51A1 and SPATA4. CYP51A1 is a member of the cytochrome P450 family and is expressed strongly by germ cells , illustrating its crucial role in spermatogenesis. Therefore, the lower level of CYP51A1 expression in underfed sheep is coherent with the decrease in numbers of germ cells and defective spermatogenesis caused by undernutrition . SPATA4 has also been reported to be testis-specific and associated with spermatogenesis, and involved in maintaining spermatogenesis . Therefore, the reduced expression of SPATA4 in underfed sheep is also consistent with compromised spermatogenesis with under-nutrition.
The functional analysis using IPA revealed that, for mRNAs that are differentially expressed between nutritional treatments, the most common functions are quantity of germ cells, testis size, quantity of Sertoli cells and quantity of connective tissue cells. Thus, the differentially expressed transcriptomes are consistent with the reductions of testis mass and sperm production in underfed rams . Among all the genes that were related to these functions, we considered the genes that were related to more than one function may be more important, such as IGF1R, INHBA, TP53. In the testes of adult mice lacking IGF1R in their Sertoli cells, there is a reduction in testis size and daily sperm production , indicating a role for IGF1R in control of sperm production by Sertoli cells. A protein product of the INHBA gene, activin A, is an important regulator of testicular cell proliferation . As indicated above, TP53 regulates spermatogenesis by inducing apoptosis . We propose that, the expression of IGF1R, INHBA and TP53 may be used as biomarkers of sperm production.
To further investigate the crucial genes in spermatogenesis and apoptosis, we looked at the relationships between these phenotypes and the gene modules that we discovered in the testis. We found five genes (CFLAR, PTPRC, F2R, MAP3K1, EPHA7) that appear to be crucial for apoptosis and two genes (APP and BCAP31) related to both apoptosis and spermatogenesis. More importantly, all seven of these genes were differentially expressed between nutritional treatments, suggesting that they played pivotal roles in the control of testis function. These conclusions are supported by previous studies. For example, CFLAR is involved in inhibition of the death receptor-activated pathway ; MAP3K1 has both anti- and pro-apoptotic functions ; EPHRINA5-EPHA7 complex induces apoptosis through TNFR1 . Therefore, these seven genes are potential biomarkers of spermatogenesis and apoptosis. Interestingly, the changes in these genes were associated with change in testis mass, raising the possibility that factors associated with change in testicular tissue, rather than direct effects of nutritional treatments on testicular tissue, are responsible for changes in spermatogenesis and apoptosis [5, 10]. If this were to be the case, then the relationships between gene modules and phenotypes observed in the present study could be applied more generally to other factors that can cause changes in the testis mass, such as photoperiod, stress and temperament, or physical fitness.
In addition, this study further identified miRNA-RNA relationships which may regulate the above altered expression events. Of particular importance are oar-novel-miR-33 and oar-novel-miR-31 because they paired with the greatest number of mRNAs, indicating a crucial role in testis function. Novel-miR-33 is homologous to miR-296 that is specific to embryonic stem cells and has been reported to be highly conserved between species . So far, there is no direct evidence for a role for miR-296 in testis function. However, one study proved that miR-296 was more highly expressed in mature testis than in immature testis, indicating a pivotal role in spermatogenesis in the adult. In addition, miR-296 was also defined as anti-apoptotic . Therefore, the reduced expression of novel-miR-33 (miR-296) in underfed sheep  illustrates decreased testis function and increased cell apoptosis. By contrast, novel-miR-31 is homologous with miR-34 which has been shown to enhance germ cell phenotype during the late stages of spermatogenesis in other species . In the current study, therefore, the lower level of novel-miR-31 in underfed sheep is coherent with the loss of germ cell function .
Nutritional treatment did not affect the total number of alternative splicing junctions, in contrast with some previous reports of nutritional effects on other biological processes [43, 44]. The lack of effect of nutritional treatment on the total number of alternative splicing junctions suggests that alternative splicing is a fine-tuner in the testis that stabilizes testis function, as suggested for other tissues [45, 46]. However, with respect to specific genes, we found 159 that were differentially spliced between high diet and low diet groups. Functional analysis of these genes indicated roles in RNA splicing and spermatogenesis, and suggests that nutrition affects spermatogenesis by changing pre-mRNA splicing. For example, the alternative splicing event for CREM (cAMP response element modulator) is alternative last exon. It has been reported that CREM mRNA exhibits a remarkable array of alternative splice variants . For example, during male meiosis, the inactive CREM variant is switched to active CREM variant (by incorporation of transactivating domains) directed by alternative splicing. Therefore, in the mature male sheep, it is possible that nutrition affects the number of active CREM isoforms, potentially explaining the disruption of spermatogenesis in underfed sheep. Future studies on verification of alternative splicing events genes related to spermatogenesis detected by RNA-seq using qPCR and how such changes affect the activity, possibly involving construction of a shortened ‘minigene’ containing the regulated exons and splicing signals , will be required to better understand this process. In addition, it is essential to determine whether the changes in splicing can affect protein expression.
In conclusion, we have identified two molecular mechanisms that could explain the effect of nutrition on spermatogenesis and germ cell apoptosis in the adult male: 1) nutrition-induced changes in the expression of mRNAs in sheep testis, the functions of the differentially expressed mRNAs are mainly related to spermatogenesis and germ cell apoptosis, an important regulator in these processes are the regulatory relationships between miRNAs and mRNAs; 2) nutritional treatment causes differences in pre-mRNA alternative splicing, and these changes are closely involved in spermatogenesis and germ cell apoptosis in testis. Some differentially spliced genes (CREM and DDX4), and testis phenotype related genes (CFLAR, PTPRC, F2R, MAP3K1, EPHA7, APP and BCAP31) should be able to work as potential biomarkers for spermatogenesis and apoptosis. To make the study move forward, confirming the predicted functions of these genes using in vivo and in vitro experiments are required.
The authors are very grateful to Dr John Milton (School of Animal Biology, University of Western Australia) for designing the nutrition treatments, and to Mr Matt Wilmot (CSIRO, Perth, Western Australia), Dr César Rosales Nieto and Mr Gary Cass (School of Animal Biology, University of Western Australia) for their assistance in handling the sheep. Mr Sheng Zhang (Zhejiang University, China), Mrs Margaret Blackberry, Dr Trina Jorre de St Jorre and Mr Fahad Almohsen (School of Animal Biology, University of Western Australia) provided valuable assistance with the collection and preservation of tissue.
This project was funded by Canadian government and UWA School of Animal Biology, and Yongjuan Guan was financially supported by a Scholarship for International Research Fees from the University of Western Australia.
YG and GL performed the molecular analysis, data analysis and draft writing; YG and GBM contributed to animal study; LLG contributed to interpretation of the molecular data. All authors contributed to manuscript writing. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
All the sequencing data were deposited in the publicly available NCBI’s Gene Expression Omnibus Database. The data are accessible through GEO Series accession number GSE68274 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE68274).
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