- Email: [email protected]
Dysregulation of long noncoding RNAs in mouse testes and spermatozoa after exposure to cadmium
Accepted Manuscript Dysregulation of long noncoding RNAs in mouse testes and spermatozoa after exposure to cadmium Fengxin Gao, Peng Zhang, Hongyan Zhang, Yunhui Zhang, Yunwen Zhang, Qingyun Hao, Xiaoning Zhang PII:
S0006-291X(17)30141-9
DOI:
10.1016/j.bbrc.2017.01.091
Reference:
YBBRC 37153
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 11 January 2017 Accepted Date: 18 January 2017
Please cite this article as: F. Gao, P. Zhang, H. Zhang, Y. Zhang, Y. Zhang, Q. Hao, X. Zhang, Dysregulation of long noncoding RNAs in mouse testes and spermatozoa after exposure to cadmium, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.01.091. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Dysregulation of long noncoding RNAs in mouse testes and spermatozoa after
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exposure to cadmium
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Fengxin Gao1, Peng Zhang1, Hongyan Zhang, Yunhui Zhang, Yunwen Zhang, Qingyun Hao,
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Xiaoning Zhang*
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Institute of Life Science and School of Life Science, Nanchang University, Nanchang 330031,
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P. R. China
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*Corresponding Author:
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Xiaoning Zhang, Ph.D.
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These authors contributed equally to this work.
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Institute of Life Science and School of Life Science, Nanchang University
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999 Xuefu R.D., Nanchang 330031, P. R. China
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Tel: +86 15879024862
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E-mail address: [email protected]
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ACCEPTED MANUSCRIPT ABSTRACT
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There is increasing evidence that cadmium (Cd) exposure can cause male subfertility and
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even complete infertility in mammals. Long noncoding (lnc) RNAs are critical for
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spermatogenesis, and their dysregulation might lead to male infertility. However, whether
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they are involved in Cd-induced subfertility is unknown. Here we found that intraperitoneal
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exposure to Cd in mice led to male subfertility indicated by reductions in testicular sperm
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production and motility, and by abnormal morphology. Testicular and sperm RNAs were used
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to investigate lncRNA expression profiles by strand-specific RNA sequencing at the
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transcriptome level to help determine any RNA-related mechanisms in Cd-induced
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subfertility. The Cd-treated testes and spermatozoa exhibited aberrant expression profiles for
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lncRNAs and mRNAs. Of the lncRNAs, there were 139 with upregulated expression and 174
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with downregulated expression in testes; in contrast, 685 were upregulated and 375 were
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downregulated in spermatozoa. For mRNA expression, 214 were upregulated and 226 were
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downregulated in testes; 272 were upregulated and 111 were downregulated in spermatozoa.
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Gene ontology and pathway analyses showed that the functions of differentially expressed
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lncRNA targets and mRNAs were closely linked with many processes involved in
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spermatogenesis. Additionally, many newly identified lncRNAs showed inducible expression,
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suggesting that they might be good candidate markers for Cd-induced male reproductive
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toxicity. This study provides a preliminary database for further exploring lncRNA-related
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mechnisms in male infertility induced by Cd.
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Keywords:
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Cadmium; Long noncoding RNA; Male infertility; RNA sequencing; Spermatozoa.
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1. Introduction Cadmium (Cd), a transition metal, is found widely in the environment because it is used
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in smelting, battery manufacturing, pigment and plastic production, and also in alloys, solders
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and electroplating [1]. Cd can be absorbed into the human body via food chains or through
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acute or chronic occupational and environmental exposures, resulting in the occurrence of
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many diseases, even cancers [2, 3]. As a heavy metal, Cd is not biodegradable and is
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characterized by long environmental persistence and biological half-life. Hence, it easily
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accumulates in a variety of mammalian tissues, especially the liver and kidneys. Cd can also
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be detected in the testis and epididymis [4, 5]. Thus, Cd can cause reproductive toxicity such
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as abnormal morphology of testes and spermatozoa, decreased sperm output and vitality, and
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even complete male infertility [6-8]. However, the mechanisms by which Cd causes infertility
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have not been elucidated in mammals.
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There is increasing evidence that mature spermatozoa retain many functional RNAs. Thus, sperm transfer-RNA-derived small RNAs, a class of noncoding (nc)RNAs, contribute
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to the paternal inheritance of diet-induced obesity and metabolic disorders [9, 10], and to the
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development of early mammalian embryos [11, 12]. These studies suggested that RNAs
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including ncRNAs of mature spermatozoa have important biological functions rather than
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being “useless residues”. Thus, sperm RNAs might be used to understand the history of the
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spermatogenic process, present sperm status, and predict future successful fertilization and
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embryo development.
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Long non-coding (lnc)RNAs, which are commonly defined as ncRNAs > 200 bp, are
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regulatory molecules that modulate a wide variety of functions and are involved in various
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lncRNAs in spermatogenesis have been reported, establishing the testis and spermatogenic
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cells as having high levels [13, 14]. Expression analysis of purified spermatogenic cells
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confirmed the highly regulated expression of lncRNAs during spermatogenesis [15]. Further
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studies have proved that lncRNAs are critical for spermatogenesis, and that their
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dysregulation might lead to male infertility [16, 17]. They have the potential to be surrogate
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indicators of chemical stress responses, including those to Cd [18]. Moreover, lncRNAs
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might serve as signatures for DNA damage and repair related to the epigenetic mechanisms
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underlying Cd toxicity and prove to be biomarkers for this pathology [19]. However, the
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abnormal expression and/or regulation of lncRNAs that might be involved in the reduced
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fertility caused by Cd exposure is unknown.
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Here we aimed to explore the lncRNA-related mechanisms of Cd-induced male
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subfertility by investigating and analyzing lncRNA expression profiles at the transcriptome
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level by strand-specific RNA sequencing.
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2. Materials and Methods
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2.1. Chemicals
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Cadmium chloride (CdCl2, 99% purity) was purchased from Tianjin Chemical Reagent
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Plant (Tianjin, P. R. China). All other chemicals obtained from local companies were of
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analytical purity.
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2.2. Animals and treatments
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Eight-week-old male C57BL/6J mice (body weight 20.0 ± 1.5 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, P. R. China). All mice were allowed to
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2 °C) and relative humidity (40–60%) environment with a 12/12-h light/dark cycle. They
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were divided randomly into four groups. Three treatment groups (n = 8 each) were
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administered with a single intraperitoneal injection of 1, 3, or 5 mg of CdCl2/kg body weight
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(bw). The control group (n = 8) was injected with saline (0.9% NaCl). Water and food were
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provided ad libitum. Mice were euthanized by cervical dislocation at 35 days after treatment.
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Testes and mature epididymal spermatozoa were quickly harvested until further study. All
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experiments were carried out according to the guidelines of the Institutional Animal Ethics
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Committee (IAEC) of Nanchang University (Nanchang, P. R. China).
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2.3. Sperm collection and purification
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Purified sperm suspensions were obtained as described [20] with some modifications. Briefly, the vas deferens and cauda epididymis were isolated and perforated, and then the
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sperm were allowed to swim out into phosphate buffered saline in a 5% CO2/air incubator
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(Heal Force, Hong Kong) at 37 °C for 15 min. The suspensions were then filtered with a
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500-mesh sieve to remove any tissue debris. The sperm were then treated with somatic cell
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lysis buffer (0.1% SDS, 0.5% Triton X-100 in DEPC-treated H2O) for 30 min on ice to
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eliminate somatic cell contamination. Non-sperm cell contamination (< 0.2%) was checked
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by microscopy.
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2.4. Sperm functional parameters analysis
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Sperm density was assessed by counting in a hemocytometer. The number of sperm
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cells in at least eight of the large corner squares (1 mm2) was counted, ignoring the cells on
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the upper and right square boundaries and the mean value was calculated. Sperm motility was
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analyzed using a computer-assisted semen analysis (CASA) system (WLJY-9000, WeiLi Co.,
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Ltd., Beijing, P. R. China). At least 200 spermatozoa were counted for each assay.
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2.5. RNA isolation and quality control Total RNAs were extracted from the purified sperm suspensions (Contorl, n = 4;
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Cd-treated, n = 2 ) and testes (Contorl, n = 4; Cd-treated, n =2) samples for each group using
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RNeasy® Plus Micro (Qiagen, Düsseldorf, Germany) according to the manufacturer’s
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instructions with some modifications. After the spermatozoa had been lysed with RLT buffer
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(a guanidine–thiocyanate lysis buffer supplemented with 10 mM D-L-dithiothreitol), we
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homogenized the samples by passing them through a 20-G needle attached to a 1-ml syringe
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10–15 times. Additionally, on-column DNase I digestion was performed to eliminate trace
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amounts of DNA contamination for all samples. Finally, the concentration and purity of the
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total RNA samples were evaluated using a NanoPhotometer (IMPLEN, Munich, Germany)
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with optical density measurements at 1.8 < A260/A280 < 2.2. The integrity of RNA was always
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checked by electrophoresis in 1.5% agarose gels.
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2.6. Strand-specific transcriptome sequencing
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Strand-specific transcriptome sequencing was conducted using an Illumina HiSeq™
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4000 system with a PE100 strategy (Illumina, San Diego, CA, USA) according to the
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manufacturer’s instructions, as developed by BGI Wuhan Pharmaceutical Co. Ltd. (Wuhan, P.
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R. China). Briefly, the RNase H protocol was performed to removal ribosomal RNAs as
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described [21].
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2.7. Bioinformatic analysis
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The raw data produced by the Illumina HiSeq™ 4000 platform was subjected to quality
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low-quality reads. The clean reads were aligned to reference sequences using the
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SOAPaligner/SOAP2 software (http://soap.genomics.org.cn/soapaligner.html). The gene
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expression level was calculated using the fragments per kilobaseof exon per million
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fragments mapped (FPKM) method [22]. Differentially expressed genes were analyzed using
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DEGseq software [23].
Long ncRNA-targeted genes were predicted based on cis and trans regulatory
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principles. For each lncRNA, we calculated the Pearson and Spearman correlation
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coefficients of its expression value with that of each mRNA. The mRNAs that were
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coexpressed with the lncRNA of interest were defined as having Pearson and Spearman
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correlations > 0.8 and P < 0.05. The mRNA loci within 10 kb upstream or 20 kb downstream
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of lncRNA were defined as cis-regulated target genes. RNAplex analysis [24] was conducted
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on lncRNA trans-targeted genes with free energies of less than –30 kJ·mol-1.
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Gene Ontology (GO; http://www.geneontology.org) and Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/) pathway analyses were applied to
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determine the functional roles of the differentially expressed lncRNA targets and mRNAs.
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GO terms and KEGG pathways with corrected P values < 0.05 were considered as being
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significantly enriched by the differentially expressed genes.
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2.8. Real-time reverse transcription (RT) quantitative polymerase chain reaction (qPCR)
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Total RNA was reverse-transcribed to cDNA using PrimeScript™ RT reagent kits with
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gDNA Eraser (TaKaRa Bio Inc., Otsu, Japan). qPCR was performed with the StepOnePlus™
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Real-Time PCR Systems (Applied Biosystems, CA, USA) using the SYBR Premix
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procedure was carried out with denaturation at 98 °C for 30 s, followed by 40 cycles of
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denaturation at 98 °C for 5 s, annealing at 60 °C for 15 s, and extension at 72 °C for 20 s.
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Expression levels of the Rplp1 and β-actin genes were used as internal controls to normalize
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the related gene expression levels. Samples were run three times with a good reproducibility.
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Melting curves were constructed to check the specificity of PCR products. The primer
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sequences used for qPCR are shown in Table 1. All primers were obtained from GENEWIZ,
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Inc. (Suzhou, P. R. China).
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2.9. Statistical analysis
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Results are expressed as the mean ± standard deviation (SD) of the mean. All statistical
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analyses were performed using SPSS v. 19.0 (IBM Corp., Armonk, NY, USA). Comparisons
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between control and CdCl2 treated groups were performed using one-way analysis of
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variance, followed by Fisher’s least significant difference post hoc test; P < 0.05 was
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regarded as statistically significant, and P < 0.01 was considered extremely statistically
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significant.
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3. Results
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3.1. Effects of CdCl2 on testis and sperm morphology and physiological sperm parameters
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Thirty-five days after CdCl2 exposure, no significant difference was observed in the body weights of mice in all groups (Fig. 1A). However, the testicular weight decreased
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significantly (P < 0.05) in the 3 and 5 mg/kg bw groups compared with the control group (Fig.
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1B). Exposure to 1 and 3 mg CdCl2/kg bw did not affect sperm morphology, but 5 mg
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CdCl2/kg bw caused some sperm head and tail abnormalities (12.5%) after 35 days. CdCl2 (3
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and 5 mg/kg bw) significantly reduced sperm motility and density (Fig. 1C and 1D).
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3.2. LncRNA and mRNA expression profiles
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Differentially expressed lncRNAs of testes and spermatozoa were found at 35 days after mice had been exposed to Cd, compared with untreated mice. In the testes, there were
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42,399 detectable lncRNAs. Of these, there were 139 with upregulated expression and 174
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with downregulated expression (≥2.0 fold-change, P < 0.05). In contrast, 20,231 lncRNAs
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were identified in mature spermatozoa; 685 were upregulated and 375 lncRNAs were
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downregulated by Cd. These Differentially expressed lncRNAs might be good candidate
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markers for Cd-induced male reproductive toxicity.
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As shown in Figure 2, there were fewer differentially expressed mRNAs in
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spermatozoa compared with lncRNAs. In testes, 214 mRNAs were upregulated and 226 were
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downregulated by Cd treatment; 272 were upregulated and 111 were downregulated in
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spermatozoa. These dysregulated lncRNA or mRNA might be good candidate markers for
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Cd-induced male reproductive toxicity. All detailed information of differentially expressed
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mRNAs and lncRNAs are provided in Supplementary Tables S1–S4.
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3.3. GO analysis and KEGG pathway analysis In the testes, GO analysis showed that the genes with aberrant mRNA expression
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mainly took part in the following biological processes: cell cycle, cytosolic calcium ion
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transport, macromolecule metabolic processes, cell division, and regulation of
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phosphatidylinositol 3-kinase activity. Differentially expressed lncRNA target genes mainly
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took part in protein serine/threonine phosphatase activity. KEGG pathway analysis of
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differentially expressed mRNAs showed they were involved in the cell cycle,
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ubiquitin-mediated proteolysis, nuclear factor (NF) kappa B signaling pathway, protein
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processing in endoplasmic reticulum and the mRNA surveillance pathway.
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In spermatozoa, the significantly enriched GO terms of differentially expressed mRNAs included mRNA metabolic processes and macromolecule metabolic processes. The
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differentially expressed lncRNA target genes were mainly enriched in intermediate filament
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cytoskeleton organization and intermediate filament-based processes. The RNA transport,
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mRNA surveillance pathway, riboflavin metabolism and ribosomal pathways were affected
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by Cd exposure as shown by mapping the differentially expressed mRNA genes to the KEGG
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database.
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3.4. qPCR validation
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To confirm the reliability of our sequencing data, we randomly selected 10 lncRNAs
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from the pool of lncRNAs with fold changes > 2.0, and analyzed their expression levels by
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qPCR in testes and sperm samples. As shown in Figure 4, the qPCR results were consistent
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with the sequencing data and showed the same trend of dysregulation for each lncRNA.
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4. Discussion At 35 days after treating mice with a single subcutaneous injection of Cd, sperm
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motility and density in the epididymis and vas deferens were impaired dramatically. Similar
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results have been found following subcutaneous and oral treatments with Cd [5, 25]. These
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findings suggested that male infertility brought about by Cd exposure results from abnormal
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spermatogenesis (decreases in sperm production and increases in abnormal morphology) and
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dysfunction of mature spermatozoa (motility impairment).
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In our study, altered lncRNA expression profiles were detected in Cd-treated testes and
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spermatozoa by a strand-specific RNA-seq approach. Apart from mRNAs, numerous lncRNAs were abnormally expressed after Cd exposure. Of the differentially expressed
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lncRNAs, 139 were upregulated, whereas 147 were downregulated in the Cd-treated testes.
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Simultaneously, 685 lncRNAs were up-regulated and 375 were downregulated by Cd in
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spermatozoa. The expression of 10 of the dysregulated lncRNAs was further validated by
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qPCR. To our knowledge, this is the first report on the profiles and potential roles of
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lncRNAs in Cd-induced reproductive toxicity, especially in terms of mature sperm
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dysfunction. We proposed that the dysregulated lncRNA and/or mRNA might be good
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candidate markers for Cd-induced male reproductive toxicity.
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Sperm RNAs including ncRNAs have important biological functions [9, 10]. However,
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the RNA content in spermatozoa is only about 1–2% of that in somatic cells and because the
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relative quantity of DNA in spermatozoa is much higher than RNA, preventing any
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contamination by somatic cell RNAs and sperm DNA was essential for our study. However,
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we were able to obtain highly pure sperm RNA as indicated by the inability of qPCR to
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amplify genetic biomarkers of leukocytes (CD4), testicular germ cells (c-kit), epithelial cells
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(E-cadherin) and genomic DNA products of Prm2 (data shown in Fig. S1). These findings
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guaranteed that our data were reliable. The total amounts of lncRNAs and differentially
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expressed lncRNAs caused by Cd treatment were more than the mRNA contents in
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spermatozoa. A previous study reported that sperm lncRNAs displayed unusual expression
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profiles in mice with diabetes-related low fertility [26]. Therefore, we propose that sperm
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lncRNAs might be good biomarkers of past spermatogenic processes, present sperm status
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and future successful fertilization potential under stressed conditions.
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Regulating the expression of adjacent genes by Cis-regulatory elements or distant gene targets by Trans-regulatory elements are mechanisms associated with lncRNAs. Here, the
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lncRNA target genes and their functions were predicted based on the GO and KEGG
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databases. When the expression profiles associated with both the lncRNA transcripts and the
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lncRNA targeted mRNA genes changed significantly between control samples and Cd-treated
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samples, this was taken to indicate the existence of interactions between lncRNAs and the
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genes associated with mRNA transcripts.
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Protein phosphatases are involved in spermatogenesis [27, 28]. Our GO analysis
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showed that the differentially expressed lncRNA target genes mainly participate in protein
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serine/threonine phosphatase activity in the testis, implying that these lncRNAs might
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modulate these enzymes to cause subfertility after Cd exposure. However, the differentially
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expressed lncRNA target genes were mainly enriched in intermediate filament cytoskeleton
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organization and intermediate filament-based process in spermatozoa. Interestingly,
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cytoskeleton organization is very important for spermatogenesis [29] and stress factors could
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disturb it. Osmotic stress-induced morphological defects and decreased sperm motility result
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from reorganization of the actin cytoskeleton [30], and F5-peptide causes aspermatogenesis
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by disrupting the organization of actin- and microtubule-based cytoskeletal elements [31].
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Hence, Cd serving as a chemical stress factor appears to block normal cytoskeletal
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organization, thereby contributing to its reproductive toxicity.
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RNA metabolism plays a crucial role in sperm formation and maturation, during which most RNAs are degraded or eliminated from the sperm cytoplasm to ensure genomic stability
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and the cessation of translational activity [32]. In our study, both in testes and mature
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spermatozoa, RNA metabolism-related and differentially expressed mRNA gene GO terms
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(macromolecular metabolic processes, mRNA metabolic processes) and pathways (RNA
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transport, mRNA surveillance pathways and ribosomal activity) were disturbed by Cd. In
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addition, GO terms for the differentially expressed mRNAs were also enriched in those
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concerning the cell cycle, cytosolic calcium ion transport, cell division, regulation of
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phosphatidylinositol 3-kinase activity. Moreover, the differentially expressed testicular
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mRNAs were concerned with the KEGG pathways involving the cell cycle,
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ubiquitin-mediated proteolysis, the NF-kappa B signaling pathway, and protein processing in
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the endoplasmic reticulum after Cd exposure. Together, many interacting pathways appeared
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to contribute to the subfertility caused by Cd.
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In conclusion, our study for the first time has determined genome-wide lncRNAs
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expression profiles in mice with Cd-induced subfertility using strand-specific RNA
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sequencing in testes and mature spermatozoa. Thus, lncRNAs might serve as signatures and
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candidates for further comprehensive understanding and investigation of the toxic effect of
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Cd on male infertility.
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Conflict of interest
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The authors declare that there is no conflict of interest.
Acknowledgments
This work was supported by the National Basic Research Program of China
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(2015CB943000), the Doctoral Scientific Research Foundation of Nanchang University
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(06301190) and the Foundation of Gansu Key Laboratory of Biomonitoring and
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Bioremediation for Environmental Pollution (GBBL2015002).
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Data access
The RNA-seq data sets generated in this study have been submitted to the NCBI Gene
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Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo) under accession numbers
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GSE88739. (The following link has been created a private access link that be distributed to
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journal reviewers:
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https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=glwvwgiwjhkbtiv&acc=GSE88739)
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TopHat and Cufflinks, Nat. Protoc. 7 (2012) 562-578.
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expressed genes from RNA-seq data, Bioinformatics 26 (2010) 136-138. [24] H. Tafer, I.L. Hofacker, RNAplex: a fast tool for RNA-RNA interaction search, Bioinformatics 24 (2008) 2657-2663.
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[23] L. Wang, Z. Feng, X. Wang, X. Wang, X. Zhang, DEGseq: an R package for identifying differentially
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S.H. Gao, Differential Expression of Long Noncoding RNAs between Sperm Samples from Diabetic
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and Non-Diabetic Mice, PLoS One 11 (2016) e0154028.
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[27] Y. Kitagawa, K. Sasaki, H. Shima, M. Shibuya, T. Sugimura, M. Nagao, Protein phosphatases
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possibly involved in rat spermatogenesis, Biochem. Biophys. Res. Commun. 171 (1990) 230-235.
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[28] J.V. Silva, M.J. Freitas, M. Fardilha, Phosphoprotein phosphatase 1 complexes in spermatogenesis, Curr. Mol. Pharmacol. 7 (2014) 136-146. [29] R.D. Moreno, J. Palomino, G. Schatten, Assembly of spermatid acrosome depends on microtubule organization during mammalian spermiogenesis, Dev. Bio. 293 (2006) 218-227.
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[30] L.M. Correa, A. Thomas, S.A. Meyers, The macaque sperm actin cytoskeleton reorganizes in response
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to osmotic stress and contributes to morphological defects and decreased motility, Biol. Reprod. 77
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(2007) 942-953.
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[31] Y. Gao, D.D. Mruk, W.Y. Lui, W.M. Lee, C. Yan Cheng, F5-peptide induces aspermatogenesis by
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disrupting organization of actin- and microtubule-based cytoskeletons in the testis, Oncotarget (2016).
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[32] G.D. Johnson, E. Sendler, C. Lalancette, R. Hauser, M.P. Diamond, S.A. Krawetz, Cleavage of rRNA
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ensures translational cessation in sperm at fertilization, Mol. Hum. Reprod. 17 (2011) 721-726.
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Fig. 1. Effect of Cd on body weight, testis weight, mature sperm motility and density. The
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mice received either physiological saline solution (control, Con) or a single intraperitoneal
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injection of Cd (1, 3, or 5 mg/kg). Mice were euthanized by cervical dislocation at 35 days
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after treatment. Testes and mature spermatozoa from the epididymis and vas deferens were
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harvested quickly. Body (A) and testicular weights (B) were measured using an electronic
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balance. Sperm density (C) was assessed by hemocytometer counts. Sperm motility (D) was
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analyzed using a CASA system. Al least 200 spermatozoa were counted for each assay. Data
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are presented as the mean ± SD, (n = 8). **P < 0.01 compared with control.
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Fig. 2. Long ncRNA and mRNA expression profiles after Cd exposure. The differentially
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expressed genes were analyzed using DEGseq software based on the FPKM method (≥ 2.0
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fold-change with P < 0.05). Differentially expressed lncRNAs (A) and mRNAs (B) in
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spermatozoa. Differentially expressed lncRNAs (C) and mRNAs (D) in testes.
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Fig. 3. Validation of sequencing data using qPCR. Total RNA was isolated from testes and
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spermatozoa after exposure of mice to Cd for 35 days, and then qPCR was performed to
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detect the RNA expression levels. Rplp1 and β-actin genes were used as loading controls to
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normalize RNA expression levels. Data are expressed as the mean ± standard deviation (n =
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3).
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Supplementary Fig. S1. Purification of sperm RNA. (A) Morphology of collected
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spermatozoa under phase contrast microscopy. Non-sperm cell contamination was < 0.2%. (B)
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Biomarkers of leukocytes (CD4), testicular germ cells (c-kit) and epithelial cells (E-cadherin)
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could be amplified easily from the RNA extracted from unpurified sperm samples. (C) After
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purification, these non-sperm cell markers could not be amplified from the extracted RNA,
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while positive markers of spermatozoa (e.g., Prm2) were detected easily.
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Supplementary files
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Table S1. Differentially expressed lncRNAs in spermatozoa.
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Table S2. Differentially expressed mRNAs in spermatozoa.
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Table S3. Differentially expressed lncRNAs in testes.
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Table S4. Differentially expressed mRNAs in testes.
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ACCEPTED MANUSCRIPT Table 1. Primers of Examined LncRNA Genes Product Length
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NONMMUG076598.1 (Lnc3)
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NONMMUG059135.1 (Lnc4)
82
NONMMUG035621.2 (Lnc5)
121
NONMMUG027897.2 (Lnc6)
142
NONMMUG055873.1 (Lnc7)
156
NONMMUG060363.1 (Lnc8)
105
NONMMUG076521.1 (Lnc9)
155
NONMMUG086668.1 (Lnc10)
235
RPLP1 (NM_018853.3)
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147
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β-actin (NM_007393)
F: 5’-ACTGGTACTCTCGTTCCCCA 60 R: 5’-CGGGTCGCGGTAATAAAGGT F: 5′-GACCGGTTAGCTGTAGCGAT -3′ 60 R: 5′-TACGGAAAAGACCCACCTGT -3′ F: 5′-TAGGGAATCCTGTAGGGCCA-3′ 60 R: 5′-CCATGCTTTCTGCTTCGGTC-3′ F: 5’-TGGTGTCAACATCATACAGTGTC-3’ 60 R: 5’-ACAGTCCTTCTTCTCCCATTGAC-3’ F: 5′-GCAGCATGGAAGTGTAAGCC-3′ 60 R: 5′-AGGAATACCCCACTCCTGGT-3′ F: 5′-AGTCAGTGCTCTTAACCGC-3′ 60 R: 5′-TAGAGAGGCGAGGATCGG-3′ F: 5′-TGTGTCATGGGAGAGACACC -3′ 60 R: 5′-ACTTGCTCCATGTCAGCCTT-3′ F: 5’-ATCCATTCAGCAACGCCTCT-3’, 60 R: 5’-AGGTCTCAGAATGGTGTGCG-3’ F: 5’-ACAGAATCGAGCCTGCACTC 60 R: 5’-TGCAGCTTTCCGAGTCACTT F: 5’-GACCCTGGAATAGCTGTCTCG 60 R: 5’-GCCGACTAGGCCATCTTCTG F: 5’- AACATTGGGAGCCTCATCTGC 60 R: 5’- R:CCTCGGACTCTTCCTTCTTTGC
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NONMMUG085507.1 (Lnc2)
Tm (°C)
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NONMMUG014147.2 (Lnc1)
Primers
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F: 5′-CCCATCTACGAGGGCTAT-3′ R: 5′-GTCACGCACGATTTCC-3′
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ACCEPTED MANUSCRIPT Highlights: 1.
LncRNA profiles were changed by Cd exposure in mouse testes and spermatozoa.
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Differentially expressed lncRNA targets and mRNAs were linked with
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spermatogenesis. Providing a database for exploring lncRNA-related mechnisms in male infertility.
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LncRNA might be candidate markers for Cd-induced male reproductive toxicity.
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S0006-291X(17)30141-9
DOI:
10.1016/j.bbrc.2017.01.091
Reference:
YBBRC 37153
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 11 January 2017 Accepted Date: 18 January 2017
Please cite this article as: F. Gao, P. Zhang, H. Zhang, Y. Zhang, Y. Zhang, Q. Hao, X. Zhang, Dysregulation of long noncoding RNAs in mouse testes and spermatozoa after exposure to cadmium, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.01.091. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Dysregulation of long noncoding RNAs in mouse testes and spermatozoa after
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exposure to cadmium
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Fengxin Gao1, Peng Zhang1, Hongyan Zhang, Yunhui Zhang, Yunwen Zhang, Qingyun Hao,
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Xiaoning Zhang*
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Institute of Life Science and School of Life Science, Nanchang University, Nanchang 330031,
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P. R. China
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*Corresponding Author:
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Xiaoning Zhang, Ph.D.
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These authors contributed equally to this work.
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Institute of Life Science and School of Life Science, Nanchang University
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999 Xuefu R.D., Nanchang 330031, P. R. China
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Tel: +86 15879024862
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E-mail address: [email protected]
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ACCEPTED MANUSCRIPT ABSTRACT
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There is increasing evidence that cadmium (Cd) exposure can cause male subfertility and
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even complete infertility in mammals. Long noncoding (lnc) RNAs are critical for
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spermatogenesis, and their dysregulation might lead to male infertility. However, whether
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they are involved in Cd-induced subfertility is unknown. Here we found that intraperitoneal
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exposure to Cd in mice led to male subfertility indicated by reductions in testicular sperm
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production and motility, and by abnormal morphology. Testicular and sperm RNAs were used
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to investigate lncRNA expression profiles by strand-specific RNA sequencing at the
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transcriptome level to help determine any RNA-related mechanisms in Cd-induced
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subfertility. The Cd-treated testes and spermatozoa exhibited aberrant expression profiles for
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lncRNAs and mRNAs. Of the lncRNAs, there were 139 with upregulated expression and 174
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with downregulated expression in testes; in contrast, 685 were upregulated and 375 were
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downregulated in spermatozoa. For mRNA expression, 214 were upregulated and 226 were
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downregulated in testes; 272 were upregulated and 111 were downregulated in spermatozoa.
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Gene ontology and pathway analyses showed that the functions of differentially expressed
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lncRNA targets and mRNAs were closely linked with many processes involved in
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spermatogenesis. Additionally, many newly identified lncRNAs showed inducible expression,
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suggesting that they might be good candidate markers for Cd-induced male reproductive
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toxicity. This study provides a preliminary database for further exploring lncRNA-related
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mechnisms in male infertility induced by Cd.
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Keywords:
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Cadmium; Long noncoding RNA; Male infertility; RNA sequencing; Spermatozoa.
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1. Introduction Cadmium (Cd), a transition metal, is found widely in the environment because it is used
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in smelting, battery manufacturing, pigment and plastic production, and also in alloys, solders
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and electroplating [1]. Cd can be absorbed into the human body via food chains or through
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acute or chronic occupational and environmental exposures, resulting in the occurrence of
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many diseases, even cancers [2, 3]. As a heavy metal, Cd is not biodegradable and is
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characterized by long environmental persistence and biological half-life. Hence, it easily
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accumulates in a variety of mammalian tissues, especially the liver and kidneys. Cd can also
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be detected in the testis and epididymis [4, 5]. Thus, Cd can cause reproductive toxicity such
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as abnormal morphology of testes and spermatozoa, decreased sperm output and vitality, and
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even complete male infertility [6-8]. However, the mechanisms by which Cd causes infertility
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have not been elucidated in mammals.
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There is increasing evidence that mature spermatozoa retain many functional RNAs. Thus, sperm transfer-RNA-derived small RNAs, a class of noncoding (nc)RNAs, contribute
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to the paternal inheritance of diet-induced obesity and metabolic disorders [9, 10], and to the
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development of early mammalian embryos [11, 12]. These studies suggested that RNAs
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including ncRNAs of mature spermatozoa have important biological functions rather than
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being “useless residues”. Thus, sperm RNAs might be used to understand the history of the
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spermatogenic process, present sperm status, and predict future successful fertilization and
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embryo development.
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Long non-coding (lnc)RNAs, which are commonly defined as ncRNAs > 200 bp, are
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regulatory molecules that modulate a wide variety of functions and are involved in various
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lncRNAs in spermatogenesis have been reported, establishing the testis and spermatogenic
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cells as having high levels [13, 14]. Expression analysis of purified spermatogenic cells
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confirmed the highly regulated expression of lncRNAs during spermatogenesis [15]. Further
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studies have proved that lncRNAs are critical for spermatogenesis, and that their
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dysregulation might lead to male infertility [16, 17]. They have the potential to be surrogate
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indicators of chemical stress responses, including those to Cd [18]. Moreover, lncRNAs
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might serve as signatures for DNA damage and repair related to the epigenetic mechanisms
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underlying Cd toxicity and prove to be biomarkers for this pathology [19]. However, the
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abnormal expression and/or regulation of lncRNAs that might be involved in the reduced
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fertility caused by Cd exposure is unknown.
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Here we aimed to explore the lncRNA-related mechanisms of Cd-induced male
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subfertility by investigating and analyzing lncRNA expression profiles at the transcriptome
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level by strand-specific RNA sequencing.
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2. Materials and Methods
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2.1. Chemicals
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Cadmium chloride (CdCl2, 99% purity) was purchased from Tianjin Chemical Reagent
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Plant (Tianjin, P. R. China). All other chemicals obtained from local companies were of
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analytical purity.
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2.2. Animals and treatments
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Eight-week-old male C57BL/6J mice (body weight 20.0 ± 1.5 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, P. R. China). All mice were allowed to
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2 °C) and relative humidity (40–60%) environment with a 12/12-h light/dark cycle. They
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were divided randomly into four groups. Three treatment groups (n = 8 each) were
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administered with a single intraperitoneal injection of 1, 3, or 5 mg of CdCl2/kg body weight
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(bw). The control group (n = 8) was injected with saline (0.9% NaCl). Water and food were
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provided ad libitum. Mice were euthanized by cervical dislocation at 35 days after treatment.
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Testes and mature epididymal spermatozoa were quickly harvested until further study. All
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experiments were carried out according to the guidelines of the Institutional Animal Ethics
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Committee (IAEC) of Nanchang University (Nanchang, P. R. China).
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Purified sperm suspensions were obtained as described [20] with some modifications. Briefly, the vas deferens and cauda epididymis were isolated and perforated, and then the
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sperm were allowed to swim out into phosphate buffered saline in a 5% CO2/air incubator
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(Heal Force, Hong Kong) at 37 °C for 15 min. The suspensions were then filtered with a
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500-mesh sieve to remove any tissue debris. The sperm were then treated with somatic cell
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lysis buffer (0.1% SDS, 0.5% Triton X-100 in DEPC-treated H2O) for 30 min on ice to
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eliminate somatic cell contamination. Non-sperm cell contamination (< 0.2%) was checked
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by microscopy.
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2.4. Sperm functional parameters analysis
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Sperm density was assessed by counting in a hemocytometer. The number of sperm
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cells in at least eight of the large corner squares (1 mm2) was counted, ignoring the cells on
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the upper and right square boundaries and the mean value was calculated. Sperm motility was
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analyzed using a computer-assisted semen analysis (CASA) system (WLJY-9000, WeiLi Co.,
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Ltd., Beijing, P. R. China). At least 200 spermatozoa were counted for each assay.
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2.5. RNA isolation and quality control Total RNAs were extracted from the purified sperm suspensions (Contorl, n = 4;
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Cd-treated, n = 2 ) and testes (Contorl, n = 4; Cd-treated, n =2) samples for each group using
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RNeasy® Plus Micro (Qiagen, Düsseldorf, Germany) according to the manufacturer’s
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instructions with some modifications. After the spermatozoa had been lysed with RLT buffer
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(a guanidine–thiocyanate lysis buffer supplemented with 10 mM D-L-dithiothreitol), we
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homogenized the samples by passing them through a 20-G needle attached to a 1-ml syringe
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10–15 times. Additionally, on-column DNase I digestion was performed to eliminate trace
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amounts of DNA contamination for all samples. Finally, the concentration and purity of the
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total RNA samples were evaluated using a NanoPhotometer (IMPLEN, Munich, Germany)
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with optical density measurements at 1.8 < A260/A280 < 2.2. The integrity of RNA was always
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checked by electrophoresis in 1.5% agarose gels.
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2.6. Strand-specific transcriptome sequencing
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4000 system with a PE100 strategy (Illumina, San Diego, CA, USA) according to the
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manufacturer’s instructions, as developed by BGI Wuhan Pharmaceutical Co. Ltd. (Wuhan, P.
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R. China). Briefly, the RNase H protocol was performed to removal ribosomal RNAs as
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described [21].
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2.7. Bioinformatic analysis
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The raw data produced by the Illumina HiSeq™ 4000 platform was subjected to quality
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low-quality reads. The clean reads were aligned to reference sequences using the
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SOAPaligner/SOAP2 software (http://soap.genomics.org.cn/soapaligner.html). The gene
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expression level was calculated using the fragments per kilobaseof exon per million
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fragments mapped (FPKM) method [22]. Differentially expressed genes were analyzed using
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DEGseq software [23].
Long ncRNA-targeted genes were predicted based on cis and trans regulatory
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principles. For each lncRNA, we calculated the Pearson and Spearman correlation
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coefficients of its expression value with that of each mRNA. The mRNAs that were
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coexpressed with the lncRNA of interest were defined as having Pearson and Spearman
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correlations > 0.8 and P < 0.05. The mRNA loci within 10 kb upstream or 20 kb downstream
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of lncRNA were defined as cis-regulated target genes. RNAplex analysis [24] was conducted
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on lncRNA trans-targeted genes with free energies of less than –30 kJ·mol-1.
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Gene Ontology (GO; http://www.geneontology.org) and Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/) pathway analyses were applied to
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determine the functional roles of the differentially expressed lncRNA targets and mRNAs.
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GO terms and KEGG pathways with corrected P values < 0.05 were considered as being
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significantly enriched by the differentially expressed genes.
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2.8. Real-time reverse transcription (RT) quantitative polymerase chain reaction (qPCR)
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Total RNA was reverse-transcribed to cDNA using PrimeScript™ RT reagent kits with
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gDNA Eraser (TaKaRa Bio Inc., Otsu, Japan). qPCR was performed with the StepOnePlus™
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Real-Time PCR Systems (Applied Biosystems, CA, USA) using the SYBR Premix
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procedure was carried out with denaturation at 98 °C for 30 s, followed by 40 cycles of
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denaturation at 98 °C for 5 s, annealing at 60 °C for 15 s, and extension at 72 °C for 20 s.
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Expression levels of the Rplp1 and β-actin genes were used as internal controls to normalize
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the related gene expression levels. Samples were run three times with a good reproducibility.
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Melting curves were constructed to check the specificity of PCR products. The primer
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sequences used for qPCR are shown in Table 1. All primers were obtained from GENEWIZ,
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Inc. (Suzhou, P. R. China).
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2.9. Statistical analysis
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Results are expressed as the mean ± standard deviation (SD) of the mean. All statistical
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analyses were performed using SPSS v. 19.0 (IBM Corp., Armonk, NY, USA). Comparisons
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between control and CdCl2 treated groups were performed using one-way analysis of
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variance, followed by Fisher’s least significant difference post hoc test; P < 0.05 was
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regarded as statistically significant, and P < 0.01 was considered extremely statistically
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significant.
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3. Results
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3.1. Effects of CdCl2 on testis and sperm morphology and physiological sperm parameters
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Thirty-five days after CdCl2 exposure, no significant difference was observed in the body weights of mice in all groups (Fig. 1A). However, the testicular weight decreased
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significantly (P < 0.05) in the 3 and 5 mg/kg bw groups compared with the control group (Fig.
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1B). Exposure to 1 and 3 mg CdCl2/kg bw did not affect sperm morphology, but 5 mg
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CdCl2/kg bw caused some sperm head and tail abnormalities (12.5%) after 35 days. CdCl2 (3
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and 5 mg/kg bw) significantly reduced sperm motility and density (Fig. 1C and 1D).
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3.2. LncRNA and mRNA expression profiles
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Differentially expressed lncRNAs of testes and spermatozoa were found at 35 days after mice had been exposed to Cd, compared with untreated mice. In the testes, there were
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42,399 detectable lncRNAs. Of these, there were 139 with upregulated expression and 174
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with downregulated expression (≥2.0 fold-change, P < 0.05). In contrast, 20,231 lncRNAs
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were identified in mature spermatozoa; 685 were upregulated and 375 lncRNAs were
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downregulated by Cd. These Differentially expressed lncRNAs might be good candidate
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markers for Cd-induced male reproductive toxicity.
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As shown in Figure 2, there were fewer differentially expressed mRNAs in
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spermatozoa compared with lncRNAs. In testes, 214 mRNAs were upregulated and 226 were
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downregulated by Cd treatment; 272 were upregulated and 111 were downregulated in
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spermatozoa. These dysregulated lncRNA or mRNA might be good candidate markers for
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Cd-induced male reproductive toxicity. All detailed information of differentially expressed
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mRNAs and lncRNAs are provided in Supplementary Tables S1–S4.
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3.3. GO analysis and KEGG pathway analysis In the testes, GO analysis showed that the genes with aberrant mRNA expression
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mainly took part in the following biological processes: cell cycle, cytosolic calcium ion
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transport, macromolecule metabolic processes, cell division, and regulation of
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phosphatidylinositol 3-kinase activity. Differentially expressed lncRNA target genes mainly
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took part in protein serine/threonine phosphatase activity. KEGG pathway analysis of
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differentially expressed mRNAs showed they were involved in the cell cycle,
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ubiquitin-mediated proteolysis, nuclear factor (NF) kappa B signaling pathway, protein
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processing in endoplasmic reticulum and the mRNA surveillance pathway.
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In spermatozoa, the significantly enriched GO terms of differentially expressed mRNAs included mRNA metabolic processes and macromolecule metabolic processes. The
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differentially expressed lncRNA target genes were mainly enriched in intermediate filament
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cytoskeleton organization and intermediate filament-based processes. The RNA transport,
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mRNA surveillance pathway, riboflavin metabolism and ribosomal pathways were affected
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by Cd exposure as shown by mapping the differentially expressed mRNA genes to the KEGG
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database.
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3.4. qPCR validation
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To confirm the reliability of our sequencing data, we randomly selected 10 lncRNAs
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from the pool of lncRNAs with fold changes > 2.0, and analyzed their expression levels by
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qPCR in testes and sperm samples. As shown in Figure 4, the qPCR results were consistent
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with the sequencing data and showed the same trend of dysregulation for each lncRNA.
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4. Discussion At 35 days after treating mice with a single subcutaneous injection of Cd, sperm
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motility and density in the epididymis and vas deferens were impaired dramatically. Similar
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results have been found following subcutaneous and oral treatments with Cd [5, 25]. These
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findings suggested that male infertility brought about by Cd exposure results from abnormal
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spermatogenesis (decreases in sperm production and increases in abnormal morphology) and
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dysfunction of mature spermatozoa (motility impairment).
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In our study, altered lncRNA expression profiles were detected in Cd-treated testes and
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spermatozoa by a strand-specific RNA-seq approach. Apart from mRNAs, numerous lncRNAs were abnormally expressed after Cd exposure. Of the differentially expressed
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lncRNAs, 139 were upregulated, whereas 147 were downregulated in the Cd-treated testes.
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Simultaneously, 685 lncRNAs were up-regulated and 375 were downregulated by Cd in
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spermatozoa. The expression of 10 of the dysregulated lncRNAs was further validated by
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qPCR. To our knowledge, this is the first report on the profiles and potential roles of
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lncRNAs in Cd-induced reproductive toxicity, especially in terms of mature sperm
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dysfunction. We proposed that the dysregulated lncRNA and/or mRNA might be good
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candidate markers for Cd-induced male reproductive toxicity.
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Sperm RNAs including ncRNAs have important biological functions [9, 10]. However,
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the RNA content in spermatozoa is only about 1–2% of that in somatic cells and because the
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relative quantity of DNA in spermatozoa is much higher than RNA, preventing any
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contamination by somatic cell RNAs and sperm DNA was essential for our study. However,
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we were able to obtain highly pure sperm RNA as indicated by the inability of qPCR to
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amplify genetic biomarkers of leukocytes (CD4), testicular germ cells (c-kit), epithelial cells
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(E-cadherin) and genomic DNA products of Prm2 (data shown in Fig. S1). These findings
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guaranteed that our data were reliable. The total amounts of lncRNAs and differentially
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expressed lncRNAs caused by Cd treatment were more than the mRNA contents in
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spermatozoa. A previous study reported that sperm lncRNAs displayed unusual expression
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profiles in mice with diabetes-related low fertility [26]. Therefore, we propose that sperm
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lncRNAs might be good biomarkers of past spermatogenic processes, present sperm status
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and future successful fertilization potential under stressed conditions.
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Regulating the expression of adjacent genes by Cis-regulatory elements or distant gene targets by Trans-regulatory elements are mechanisms associated with lncRNAs. Here, the
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lncRNA target genes and their functions were predicted based on the GO and KEGG
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databases. When the expression profiles associated with both the lncRNA transcripts and the
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lncRNA targeted mRNA genes changed significantly between control samples and Cd-treated
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samples, this was taken to indicate the existence of interactions between lncRNAs and the
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genes associated with mRNA transcripts.
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Protein phosphatases are involved in spermatogenesis [27, 28]. Our GO analysis
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showed that the differentially expressed lncRNA target genes mainly participate in protein
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serine/threonine phosphatase activity in the testis, implying that these lncRNAs might
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modulate these enzymes to cause subfertility after Cd exposure. However, the differentially
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expressed lncRNA target genes were mainly enriched in intermediate filament cytoskeleton
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organization and intermediate filament-based process in spermatozoa. Interestingly,
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cytoskeleton organization is very important for spermatogenesis [29] and stress factors could
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disturb it. Osmotic stress-induced morphological defects and decreased sperm motility result
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from reorganization of the actin cytoskeleton [30], and F5-peptide causes aspermatogenesis
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by disrupting the organization of actin- and microtubule-based cytoskeletal elements [31].
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Hence, Cd serving as a chemical stress factor appears to block normal cytoskeletal
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organization, thereby contributing to its reproductive toxicity.
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RNA metabolism plays a crucial role in sperm formation and maturation, during which most RNAs are degraded or eliminated from the sperm cytoplasm to ensure genomic stability
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and the cessation of translational activity [32]. In our study, both in testes and mature
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spermatozoa, RNA metabolism-related and differentially expressed mRNA gene GO terms
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(macromolecular metabolic processes, mRNA metabolic processes) and pathways (RNA
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transport, mRNA surveillance pathways and ribosomal activity) were disturbed by Cd. In
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addition, GO terms for the differentially expressed mRNAs were also enriched in those
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concerning the cell cycle, cytosolic calcium ion transport, cell division, regulation of
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phosphatidylinositol 3-kinase activity. Moreover, the differentially expressed testicular
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mRNAs were concerned with the KEGG pathways involving the cell cycle,
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ubiquitin-mediated proteolysis, the NF-kappa B signaling pathway, and protein processing in
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the endoplasmic reticulum after Cd exposure. Together, many interacting pathways appeared
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to contribute to the subfertility caused by Cd.
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In conclusion, our study for the first time has determined genome-wide lncRNAs
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expression profiles in mice with Cd-induced subfertility using strand-specific RNA
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sequencing in testes and mature spermatozoa. Thus, lncRNAs might serve as signatures and
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candidates for further comprehensive understanding and investigation of the toxic effect of
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Cd on male infertility.
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Conflict of interest
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The authors declare that there is no conflict of interest.
Acknowledgments
This work was supported by the National Basic Research Program of China
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(2015CB943000), the Doctoral Scientific Research Foundation of Nanchang University
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(06301190) and the Foundation of Gansu Key Laboratory of Biomonitoring and
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Bioremediation for Environmental Pollution (GBBL2015002).
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Data access
The RNA-seq data sets generated in this study have been submitted to the NCBI Gene
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Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo) under accession numbers
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GSE88739. (The following link has been created a private access link that be distributed to
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journal reviewers:
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https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=glwvwgiwjhkbtiv&acc=GSE88739)
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Fig. 1. Effect of Cd on body weight, testis weight, mature sperm motility and density. The
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mice received either physiological saline solution (control, Con) or a single intraperitoneal
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injection of Cd (1, 3, or 5 mg/kg). Mice were euthanized by cervical dislocation at 35 days
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after treatment. Testes and mature spermatozoa from the epididymis and vas deferens were
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harvested quickly. Body (A) and testicular weights (B) were measured using an electronic
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balance. Sperm density (C) was assessed by hemocytometer counts. Sperm motility (D) was
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analyzed using a CASA system. Al least 200 spermatozoa were counted for each assay. Data
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are presented as the mean ± SD, (n = 8). **P < 0.01 compared with control.
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Fig. 2. Long ncRNA and mRNA expression profiles after Cd exposure. The differentially
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expressed genes were analyzed using DEGseq software based on the FPKM method (≥ 2.0
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fold-change with P < 0.05). Differentially expressed lncRNAs (A) and mRNAs (B) in
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spermatozoa. Differentially expressed lncRNAs (C) and mRNAs (D) in testes.
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Fig. 3. Validation of sequencing data using qPCR. Total RNA was isolated from testes and
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spermatozoa after exposure of mice to Cd for 35 days, and then qPCR was performed to
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detect the RNA expression levels. Rplp1 and β-actin genes were used as loading controls to
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normalize RNA expression levels. Data are expressed as the mean ± standard deviation (n =
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3).
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Supplementary Fig. S1. Purification of sperm RNA. (A) Morphology of collected
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spermatozoa under phase contrast microscopy. Non-sperm cell contamination was < 0.2%. (B)
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Biomarkers of leukocytes (CD4), testicular germ cells (c-kit) and epithelial cells (E-cadherin)
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could be amplified easily from the RNA extracted from unpurified sperm samples. (C) After
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purification, these non-sperm cell markers could not be amplified from the extracted RNA,
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while positive markers of spermatozoa (e.g., Prm2) were detected easily.
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Supplementary files
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Table S1. Differentially expressed lncRNAs in spermatozoa.
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Table S2. Differentially expressed mRNAs in spermatozoa.
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Table S3. Differentially expressed lncRNAs in testes.
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Table S4. Differentially expressed mRNAs in testes.
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ACCEPTED MANUSCRIPT Table 1. Primers of Examined LncRNA Genes Product Length
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NONMMUG076598.1 (Lnc3)
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NONMMUG059135.1 (Lnc4)
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NONMMUG035621.2 (Lnc5)
121
NONMMUG027897.2 (Lnc6)
142
NONMMUG055873.1 (Lnc7)
156
NONMMUG060363.1 (Lnc8)
105
NONMMUG076521.1 (Lnc9)
155
NONMMUG086668.1 (Lnc10)
235
RPLP1 (NM_018853.3)
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β-actin (NM_007393)
F: 5’-ACTGGTACTCTCGTTCCCCA 60 R: 5’-CGGGTCGCGGTAATAAAGGT F: 5′-GACCGGTTAGCTGTAGCGAT -3′ 60 R: 5′-TACGGAAAAGACCCACCTGT -3′ F: 5′-TAGGGAATCCTGTAGGGCCA-3′ 60 R: 5′-CCATGCTTTCTGCTTCGGTC-3′ F: 5’-TGGTGTCAACATCATACAGTGTC-3’ 60 R: 5’-ACAGTCCTTCTTCTCCCATTGAC-3’ F: 5′-GCAGCATGGAAGTGTAAGCC-3′ 60 R: 5′-AGGAATACCCCACTCCTGGT-3′ F: 5′-AGTCAGTGCTCTTAACCGC-3′ 60 R: 5′-TAGAGAGGCGAGGATCGG-3′ F: 5′-TGTGTCATGGGAGAGACACC -3′ 60 R: 5′-ACTTGCTCCATGTCAGCCTT-3′ F: 5’-ATCCATTCAGCAACGCCTCT-3’, 60 R: 5’-AGGTCTCAGAATGGTGTGCG-3’ F: 5’-ACAGAATCGAGCCTGCACTC 60 R: 5’-TGCAGCTTTCCGAGTCACTT F: 5’-GACCCTGGAATAGCTGTCTCG 60 R: 5’-GCCGACTAGGCCATCTTCTG F: 5’- AACATTGGGAGCCTCATCTGC 60 R: 5’- R:CCTCGGACTCTTCCTTCTTTGC
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NONMMUG085507.1 (Lnc2)
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NONMMUG014147.2 (Lnc1)
Primers
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F: 5′-CCCATCTACGAGGGCTAT-3′ R: 5′-GTCACGCACGATTTCC-3′
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ACCEPTED MANUSCRIPT Highlights: 1.
LncRNA profiles were changed by Cd exposure in mouse testes and spermatozoa.
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Differentially expressed lncRNA targets and mRNAs were linked with
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spermatogenesis. Providing a database for exploring lncRNA-related mechnisms in male infertility.
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LncRNA might be candidate markers for Cd-induced male reproductive toxicity.
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3.