- Email: [email protected]
Cryopreservation of boar sperm induces differential microRNAs expression
Accepted Manuscript Cryopreservation of boar sperm induces differential microRNAs expression Yan Zhang, Dinghui Dai, Yu Chang, Yuan Li, Ming Zhang, Guangbin Zhou, Zhanghua Peng, Changjun Zeng PII:
S0011-2240(16)30433-3
DOI:
10.1016/j.cryobiol.2017.04.013
Reference:
YCRYO 3843
To appear in:
Cryobiology
Received Date: 17 November 2016 Revised Date:
28 April 2017
Accepted Date: 28 April 2017
Please cite this article as: Y. Zhang, D. Dai, Y. Chang, Y. Li, M. Zhang, G. Zhou, Z. Peng, C. Zeng, Cryopreservation of boar sperm induces differential microRNAs expression, Cryobiology (2017), doi: 10.1016/j.cryobiol.2017.04.013. 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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Cryopreservation of boar sperm induces differential
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microRNAs expression
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Yan Zhang1§, Dinghui Dai1§, Yu Chang1, Yuan Li1, Ming Zhang1, Guangbin Zhou1,
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Zhanghua Peng2, Changjun Zeng1 *
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1. College of Animal Sciences and Technology, Sichuan Agricultural University, Chengdu,
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Sichuan, 611130, China
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2. Agriculture and Forestry Bureau, Luxian County, Sichuan Province, 646100, China
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§ These authors are contributed equally to this work.
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* Correspondence author. Email: [email protected], Tel/Fax: +86-28-86291010
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Abstract: Lower conception rates and litter sizes limit the wide use of artificial
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insemination with frozen-thawed boar sperm, due to a lack of understanding of the
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mechanisms that cause cryodamage and cryoinjury to sperm during cryopreservation.
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CryoMiRs, a family of freeze-related microRNAs (miRNAs), are associated with
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freeze tolerance, and regulate metabolism in mammalian hibernators and insects. Thus,
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we speculate that miRNAs maybe involved in the regulation of the freeze-thaw
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process and may affect boar sperm function. In this study, we studied the differential
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expression of 46 miRNAs that have roles in spermatogenesis, sperm maturation, and
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sperm quality in response to cryopreservation (with or without 3% glycerol). The
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results indicated that, in response to cryopreservation with 3% glycerol, 14 miRNAs
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were significantly up-regulated, but only two miRNAs (miR-22 and miR-450b-5p)
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were significantly down-regulated, relative to fresh sperm. Preservation with 3%
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glycerol caused up-regulation of 17 miRNAs, but only caused down-regulation of one
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miRNA (miR-24), relative to sperm cryopreserved without glycerol. Functional
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annotations of these differentially expressed miRNAs indicated that these miRNAs
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and their targets are mainly associated with metabolic and cellular processes.
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Therefore, our findings show that cryopreservation results in changes in miRNA
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expression, and suggest that the anti-freeze mechanisms of boar sperm need to be
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studied further.
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Keywords: Boar; Sperm; Cryopreservation; Freeze tolerance; miRNAs
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Introduction In the past few decades, the use of artificial insemination (AI) has significantly
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increased in the pork industry, and AI is now of great economic value to this industry.
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Nowadays, more than 99% of AIs are conducted using liquid-stored semen incubated
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at 15-20°C for 0~5 days. However, no more than 1% of AI procedures use
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frozen-thawed semen, due to the reduced conception rate and litter size associated
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with freeze-thawing semen, which is mainly attributed to sperm structural and
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functional cryoinjury during cryopreservation [29]. Numerous studies have
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demonstrated that cryopreservation of sperm decreases viability and motility, changes
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sperm cholesterol content，increases the number of sperm that undergo acrosome
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reaction [40], damages the plasma and mitochondrial membranes [41, 46], increases
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apoptosis [59], causes DNA fragmentation [46], and produces reactive oxygen species
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(ROS) that can cause lipid peroxidation [11]. Although substantial research has focused on
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boar spermatozoa cryopreservation, it remains unclear whether miRNAs are involved in the
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functional regulation of spermatozoa response to cryopreservation.
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Mammalian hibernators and insects can resist cold temperatures and survive
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under extreme environmental conditions by depressing their metabolic rate, a process
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called hypometabolism. During mammalian hibernation, transcription and translation
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of genes associated with energy consumption are inhibited [18, 45]. However, the
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over-expression of some genes involved in glycolysis, fatty acid metabolism,
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gluconeogenesis, amino acid metabolism, and other metabolic pathways contributes
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to the organism’s long-term survival at low temperature [7, 56]. A recently discovered
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ACCEPTED MANUSCRIPT family of miRNAs called CryomiRs are rapidly emerging as a potential modulators of
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cold tolerance in animals that can withstand freezing [34, 39]. In freeze-tolerant
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mammals and insects, miRNAs can rapidly regulate metabolism-related genes and
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signaling pathways, and inhibit ATP metabolism to help the animal adapt to extreme
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environmental conditions [4]. Changes in miR-21 and miR-16 expression in response
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to freeze stress indicate that these miRNAs play important roles in regulation of the
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cell-cycle and apoptosis [4]. A target gene of miR-195, fatty acid synthase (FAS),
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which is involved in fatty acid biosynthesis, was shown to be down-regulated in the
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liver of hibernating thirteen-lined squirrels [33]. Furthermore, miRNAs are also
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widely involved in the regulation of lipid and glucose metabolism, and in cholesterol
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homeostasis in mammalian hibernators [34]. Three miRNAs (miR-26a, miR-126, and
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miR-217) can regulate PTEN expression in the wood frog and could potentially
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contribute to Akt-mediated inhibition of apoptosis during freezing [62]. Therefore, the
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key mechanisms of freeze tolerance and metabolic regulator may be associated with
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the reversible phosphorylation control of metabolic enzymes and miRNAs control of
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gene transcript expression[50].
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One potential explanation for sperm cryoinjury is as follows: during sperm
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cryopreservation, loss of membrane integrity causes loss of ATP and Mg2+, which
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decreases the intracellular concentration of ATP and increases the AMP/ADP
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concentration [21]. In contrast, McLaughlin et al. reported that decreased ATP
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production does not contribute to the poor motility of cryopreserved spermatozoa [42].
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Additionally, decreased cell growth, differentiation, proliferation, and mRNA
ACCEPTED MANUSCRIPT translation during cryopreservation reduces consumption of ATP. Thus, miRNAs
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likely play a critical role in the regulation of sperm mRNA expression during
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cryopreservation. Although miRNAs are thought to be involved in metabolic
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depression and are differentially expressed in mammalian hibernators and insects, it is
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not known if miRNAs participate in freeze protection or freeze tolerance during boar
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sperm cryopreservation. Our previous study showed that some miRNAs are
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differentially expressed between fresh and frozen boar sperm [63]. Therefore, we
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speculated that freeze-tolerance related miRNAs (CryomiRs) may exist in boar sperm,
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and may regulate carbohydrate, lipid, and cholesterol metabolism during sperm
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cryopreservation. Here, 46 candidate miRNAs were selected to detect their expression
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changes in response to cryopreservation. Furthermore, the predicted target genes of
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differentially expressed miRNAs involved in DNA repair and apoptosis were further
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evaluated.
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Materials and methods
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Animal ethics statement
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Semen collection and treatment were conducted according to the Regulations of
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the Administration of Affairs Concerning Experimental Animals (Ministry of Science
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and Technology, China, revised in June 2004) and approved by the Institutional
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Animal Care and Use Committee in the College of Animal Science and Technology,
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Sichuan Agricultural University, Sichuan, China, under permit No. DKYB20081003.
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Furthermore, all experimental protocols were approved by the College of Animal
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Science and Technology, Sichuan Agricultural University.
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Sperm collection and cryopreservation The sperm-rich fractions of ejaculates were collected from Landrace boars (N=5)
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using a manual collection method [30]. Sperm quality parameters were measured with
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a sperm quality analyzer, SQA-V (MES, Israel). Only sperm with normal morphology,
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motility greater than 0.8, and a concentration higher than 1×108 were used.
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Firstly, all fresh ejaculates were divided into one of three treatment groups: (I)
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fresh sperm, directly used to extract total RNA; (II) cryopreserved sperm without
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glycerol, which underwent a freezing program; and (III) cryopreserved sperm with
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3% glycerol, which underwent the same freezing program. The boar sperm freezing
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program was performed according to a previous protocol [13, 61]. Briefly, the semen
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were diluted (1:1 v/v) with Beltsville Thawing Solution and cooled slowly to 15°C for
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2 hrs. Then, the sperm pellet was diluted to a concentration of 2×109 mL-1 using
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lactose-egg yolk (LEY) extender and slowly cooled to 4°C for 2 hrs in the refrigerator.
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At 4°C, sperm were further diluted with a second freezing extender (LEY
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supplemented with glycerol) to yield final concentration of 3% glycerol. Finally, the
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mixtures were packaged into 0.25 mL straws (FHK, Japan) and frozen using a
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controlled-rate freezing instrument (CryoMed Controlled-Rate Freezer, Thermo
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Fisher, USA) and then stored at -196°C.
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miRNA extraction and cDNA synthesis
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miRNAs were isolated using a mirVana miRNA Isolation Kit (Ambion, USA)
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based on a previous protocol [63]. First, to eliminate somatic cell contamination, the
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sperm pellet was suspended in a cold hypotonic solution with 0.5% Triton X-100
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(Roche,
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manufacturer's instructions. Finally, the concentration and quality of the miRNAs
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were determined using a NanoDrop ND1000 spectrophotometer (NanoDrop
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Technologies, USA).
Then,
miRNAs
were
extracted
according
to
the
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Germany)[47].
MiRNA first-strand cDNAs were synthesized from 0.06 µg of miRNA from each
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sample using the PrimeScript miRNA qPCR Starter Kit version 2.0 (Takara Biotech,
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China) according to the manufacturer's instructions. Subsequently, the cDNA was
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stored at -20°C till use.
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Primer design and RT-qPCR analysis
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All primers of selected 46 miRNAs were designed according to previous reports
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(Table 1), and were selected for study based on reported associations with
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spermatogenesis, sperm maturation, and sperm quality parameters [15, 16, 22, 36, 37].
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Relative miRNA expression was evaluated using Quantitative RT-PCR (RT-qPCR).
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Sperm miRNA expression levels were normalized to endogenous ssc-miR-27a-3p
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[63]. RT-qPCR was performed using SYBR PrimeScript miRNA RT-PCR Kit (Takara
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Biotech, China) on a StepOnePlus real-time PCR system (Applied BioSystems, USA)
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using an annealing temperature of 60°C, according to our laboratory’s protocols [63].
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Target prediction of differentially expressed miRNAs
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To date, a relatively small number of porcine miRNAs and predicted target
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mRNAs have been identified [15]. In order to further understand the functions of
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differentially expressed miRNAs between all treatment conditions, putative targets
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were
predicted
using
miRWalk
database
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miRWalk combines several independent target prediction tools, including TargetScan
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[35], miRanda [28], picTar [31], and miRWalk [16]. Then, PANTHER
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(http://www.pantherdb.org) and DAVID (https://david.ncifcrf.org) were used to
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explore the gene ontology and KEGG pathways of predicted target mRNAs.
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Seven of the predicted mRNA targets (Fas, Bcl-2, BRCA1, H2AFX, PTEN, API5,
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TP53) that are known to be involved in sperm DNA repair and apoptosis were
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selected for further study with RT-qPCR (Table 2, 3). All primers were designed with
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homologous counterparts in the GenBank Database using Premier Primer 5.0 software
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and cited from previous literature. GAPDH was used as a reference gene [61].
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Total RNA isolation and RT-qPCR analysis
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Total RNA from frozen-thawed boar sperm was extracted using a TRIzol LS
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Reagent kit (Invitrogen, USA). Briefly, the straws were thawed rapidly in a water bath
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at 37°C for 40 seconds. The sperm were resuspended in 1 mL of cold hypotonic
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solution with 0.5% Triton X-100 to remove somatic contaminations [47]. The quality
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of the total RNA was assessed using a NanoDrop ND1000 spectrophotometer
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(NanoDrop Technologies, USA).
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cDNA was synthesized from 1 µg of total RNA from each sample using a
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PrimeScript RT-PCR kit (Takara Biotech, Dalian, China) according to the
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manufacturer’s protocol. RT-qPCR was performed using SYBR Premix Ex Taq II
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(TaRaKa Biotech, China) on a StepOnePlus real-time PCR system (Applied
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BioSystems, USA) using an annealing temperature of 61.6°C according to our
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laboratory’s protocols [61].
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Statistical analysis The average cycle threshold (Ct) value for each triplicate of RT-qPCR was used to
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calculate relative mRNA expression levels using the 2-∆∆Ct method [38]. Statistical
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analysis of the relative expression levels of miRNAs and predicted target mRNAs was
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performed using Tukey's HSD (honest significant difference) test using SAS9.0
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software. Each experiment was independently conducted three times. Differences of P
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< 0.05 were regarded as statistically significant.
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Results
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Differential miRNA expression between fresh and cryopreserved sperm
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The relative expression levels of 46 miRNAs across the three treatment conditions
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(fresh sperm, sperm cryopreserved without glycerol, and sperm cryopreserved with
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3% glycerol) are shown in Fig. 1. The results indicated that 23 miRNAs were
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differentially expressed across the three treatments (P<0.05) (Fig. 2). Seven miRNAs
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(miR-34c, miR-124, miR-181a, miR-186, miR-224, miR-450b-5p, and miR-450c-5p)
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were down-regulated and two miRNAs (miR-98 and miR-374a) were significantly
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up-regulated in sperm cryopreserved without glycerol relative to fresh sperm.
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Fourteen miRNAs (let-7a, let-7c, let-7d, let-7e, let-7f-5p, let-7i, miR-9-5p, miR-26a,
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miR-98,
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significantly up-regulated, but only two miRNAs (miR-22 and miR-450b-5p) were
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significantly down-regulated in sperm cryopreserved with 3% glycerol relative to
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fresh sperm (P<0.05). Interestingly, seventeen miRNAs were significantly
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miR-181a,
miR-186,
miR-212,
miR-374a-5p,
miR-374b-5p)
were
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up-regulated, but only one miR (miR-24) was significantly down-regulated between
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cryopreserved sperm with or without 3% glycerol (P<0.05). GO and KEGG pathway analysis of these differentially expressed miRNAs’
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predicted mRNA targets indicated that these target mRNAs play important roles in
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biological processes (metabolic processes, cell processes, and biological regulation),
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molecular functions (binding and catalytic activity), and cell components (cell part,
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organelles, and membranes). These genes are mainly involved in the following
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pathways: cancer, MAPK signaling pathways, focal adhesion, and regulation of the
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actin cytoskeleton (Fig. 5).
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Relationship between differentially expressed miRNAs and their mRNA
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targets known to be involved in sperm DNA repair and apoptosis
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RT-qPCR results indicated that, except for PTEN, the relative expression levels
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of six genes (Fas, Bcl-2, BRCA1, H2AFX, API5, and TP53) were significantly
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different between fresh sperm and sperm cryopreserved with 3% glycerol (P<0.05).
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There was no significant difference in the relative expression levels of Fas, Bcl-2,
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BRCA1, API5, or TP53 between sperm cryopreserved without glycerol and sperm
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cryopreserved with 3% glycerol. Only H2AFX expression differed between all three
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treatments (P<0.05). Furthermore, no significant difference in PTEN expression was
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found among the three treatments (Fig. 3).
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Based on previous studies and target gene prediction tools, we decided to further
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evaluate the relationship between miRNAs and their predicted target genes which are
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involved in sperm apoptosis and DNA repair (Fig. 4). Our results showed that the
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cryopreserved with 3% glycerol relative to the other two treatments (P<0.05). The
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expression of miR-98 is significantly higher in both cryopreserved treatments relative
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to fresh sperm (P<0.05). Interestingly, the expression of their common target gene,
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Fas, was significantly higher in fresh sperm than in the cryopreserved treatment
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conditions (Fig. 4A). The expression of PTEN is higher in the cryopreserved sperm
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than in fresh sperm (Fig. 4B). Meanwhile, the expression pattern of miR-22 in fresh
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and cryopreserved sperm is the opposite of PTEN’s expression pattern. These results
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are consistent with the fact that miRNAs degrade or translationally silence their target
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mRNAs. Similarly, the expression level of Bcl-2, an anti-apoptotic gene, is also
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consistent with the changes we observe in miR-98 expression, but not miR-34c (Fig.
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4C). There were no significant differences in the expression levels of miR-182,
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miR-34c, miR-504-5p, and miR-244 and their target genes (BRCA1, API and TP53) in
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the cryopreserved sperm, with or without 3% glycerol (Fig. 4D-4F). The expression
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level of miR-24 and its target gene, H2AFX, was significantly higher in the sperm
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cryopreserved without glycerol than in 3% glycerol condition (Fig. 4G).
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Discussion
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It is generally accepted that nuclear-encoded protein translation is unlikely to
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occur in mature sperm cells and translational repression is essential for spermatid
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differentiation. However, Gur and Breitbart [23] have demonstrated that nuclear genes
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are expressed as proteins in sperm. Although ejaculated sperm are capable of using
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mRNAs transcripts for protein translation during the final maturation steps before
ACCEPTED MANUSCRIPT fertilization, the mechanism is presently still unknown [24]. During sperm
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cryopreservation, some epigenetic-related mRNA (Dnmt3a, Dnmt3b, Jhdm2a, Kat8,
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Prm1, Prm2 and IGF2) and low protein levels were typically found in sperm after
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freeze-thawing [60]. miRNAs are critical regulators of mRNA expression, which
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inhibit translation of target mRNA or induce mRNA degradation [25]. The process of
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cryopreservation not only induces expression of apoptosis-related mRNAs [59], but
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also changes miRNA expression [63]. In the present study, we found miR-98 and
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miR-374a to be up-regulated, and seven miRNAs (miR-34c, miR-124, miR-181a,
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miR-186, miR-224, miR-450b-5p, and miR-450c-5p) to be down-regulated in
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cryopreserved sperm (without glycerol) relative to fresh sperm. This further
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demonstrated that the freeze-thaw process changes miRNA expression. Seventeen
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miRNAs were significantly up-regulated, but only miR-24 was significantly
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down-regulated, with the addition of cryoprotectants (3% glycerol) relative to
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freezing without cryoprotectants. Recently, 83 miRNAs were differentially expressed
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in high and low motile post-thaw bovine sperm, and let-7a and miR-26a are
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up-regulated in high motile sperm [10]. Likewise, let-7a and miR-26a are also
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significantly up-regulated in cryopreserved boar sperm with 3% glycerol compared
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with cryopreserved boar sperm without 3% glycerol.
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CryomiRs (cold-modulated miRNAs), as key regulators of freeze tolerance in
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cold-tolerant insects and hibernating mammals, control the reversible phosphorylation
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of metabolic enzymes, thereby reducing energetic consumption, and gene transcript
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expression [39, 49, 50]. Fatty acid synthase (FAS), an enzyme involved in fatty acid
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biosynthesis and a putative miR-195 target, is down-regulated in hibernating
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squirrels’ livers [33]. Furthermore, miR-29a and miR-233 and their target mRNAs
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could also regulate glycolysis or glucose metabolism in these cold-tolerant vertebrates
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[4, 34]. Under freezing stress conditions, three miRNAs (miR-26a, miR-126, and
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miR-217) can regulate PTEN expression via activation of AKT pathways to inhibit
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apoptosis [62]. Based on these enlightening findings, we postulated that miRNAs were probably
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associated with anti-freeze, anti-apoptosis, and energy consumption regulation
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mechanisms during sperm cryopreservation. It is well known that the cellular
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metabolic activity decreases during cryopreservation, followed by irreversible
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cryoinjury [58]. During cryopreservation, intracellular ice crystal formation leads to
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cryodamage of the sperm plasma membrane and mitochondrial or nuclear DNA,
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which causes apoptotic changes and a severe decline in sperm quality [27, 58].
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Furthermore, gene transcription and protein translation associated with energy
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consumption are inhibited at low temperature [18, 45]. In this study, gene ontology
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analysis of differentially expressed miRNAs’ predicted mRNA targets showed that
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these targets are mainly involved in metabolic processes, cell processes, and
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biological regulation, and play important roles in binding and catalytic activities (Fig.
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5). It was reported that the PTEN/PI3K/Akt signaling pathway is involved in cellular
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processes such as glucose metabolism, cell cycle regulation, and protein translation
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under conditions of environmental stresses [1, 17]. For instance, miR-29a and
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miR-233 are involved in glucose metabolism in wood frogs and hibernating
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thirteen-lined ground squirrels [4, 33]. MiR-195 is associated with lipid metabolism in
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mammalian hibernators [34]. Via targeting of IDH1, miR-181a decreases expression
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of lipid synthesis-related genes and increases expression of genes involved in
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β-oxidation [12]. In the present study, we found that miR-181a expression was
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up-regulated in sperm cryopreserved with 3% glycerol, but down-regulated in sperm
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cryopreserved without glycerol, which is consistent with the superior cryoprotective
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effects of glycerol. Furthermore, miR-22, miR-24, and miR-182 targets are thought to
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be associated with sperm structure and motility [2, 26, 52]. Thus, the process of
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freeze-thaw influences miRNA expression and results in abnormal sperm morphology,
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motility, and fertility. Furthermore, it was suggested that glycerol provides better
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cryoprotective effects due to the different miRNA expression profile of sperm
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cryopreserved with or without 3% glycerol. However, the molecular mechanism of
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cryoprotection during sperm cryopreservation requires further study. The association between miRNAs’ predicted target mRNAs and spermatozoa
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apoptosis and DNA damage was explored further. Apoptosis (programed cell death) is
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a physiological mechanism required for organic function and tissue development and
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is also necessary to dismantle and engulf damaged cells that represent a threat to the
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integrity of the organism. Previous studies have revealed that sperm also undergo
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apoptotic changes during cryopreservation [9, 59]. In this study, we used RT-qPCR to
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assay the expression of seven DNA repair and apoptosis-related genes (Fas, Bcl-2,
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BRCA1, H2AFX, API5, TP53, PTEN) that are thought to be target genes of the
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freeze-related miRNAs that we identified. The Fas-Fas ligand (FasL) pathway is a
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major death receptor-signaling pathway. Binding of FasL to Fas induces activation of
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caspase-8 and triggers apoptosis [59]. Fas is a direct target of the let-7/miR-98 family.
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Let-7 and miR-98 expression levels are significantly lower in fresh sperm than in
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sperm cryopreserved with 3% glycerol, while the Fas expression profile is opposite to
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let-7/miR-98 (P<0.05). Let-7/miR-98 expression is reduced during activation-induced
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cell death, which is accompanied by increased Fas expression [53]. The anti-apoptotic
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protein Bcl-2 is located mainly on the outer membrane of mitochondria. Bcl-2 can
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prevent apoptosis by inhibiting the release of cytochrome C [57]. Bcl-2 is a target of
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miR-34 and miR-98, and can be down-regulated by direct binding of miR-34 [5, 55].
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BRCA1 is an E3 ubiquitin ligase that plays an essential role in DNA repair [6].
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BRCA1 is directly targeted by miR-182, and miR-182-mediated down-regulation of
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ACCEPTED MANUSCRIPT BRCA1 impedes DNA repair [44]. H2AFX, a key double-stranded DNA break repair
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protein, was identified as a target of miR-24. MiR-24 binds to the 3’UTR of H2AX
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mRNA and decreases H2AFX mRNA and protein levels, which inhibits the DNA
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damage response and increases cell death after DNA damage [32]. Our results show
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that miR-24 and miR-182 may impact DNA damage repair via regulation of their
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target genes, H2AFX and BRCA1. Apoptosis inhibitor 5 (API-5), which prevents
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apoptosis after growth factor deprivation, is targeted by miR-224, and plays a role in
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sensitizing cells to apoptosis in hepatocellular carcinoma patients [54]. Freeze
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extension with 3% glycerol attenuates apoptosis better than sperm cryopreservation
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without glycerol. In this study, the expression level of API-5 in sperm cryopreserved
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with 3% glycerol is lower than that in sperm cryopreserved without glycerol. This
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result is consistent with the higher expression of miR-224 that we observed in sperm
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cryopreserved with 3% glycerol relative to sperm preserved without glycerol. Studies
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suggest that members of the miR-34 family are direct p53 targets, which regulates
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apoptosis [48]. MiR-34a is a direct p53-target gene and p53-dependent induction of
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miR-34a expression occurs after DNA damage [51]. Notably, inactivation of miR-34a
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strongly attenuates p53-mediated apoptosis in cells exposed to genotoxic stress,
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whereas over expression of miR-34a mildly increases apoptosis [48]. Our results
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show that TP53 expression is significant lower in cryopreserved sperm than in fresh
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sperm, which is not consistent with the changes in miR-34c and miR-504-5p
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expression in the three treatments. Furthermore, PTEN is targeted by miR-22 and
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miR-26 through a specific miR-binding site in the PTEN 3’ UTR [3, 19]. However, in
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ACCEPTED MANUSCRIPT our study, we observed no significant difference in PTEN expression among the three
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treatments, which suggests that PTEN may be regulated by miR-22 and miR-26a in
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the boar sperm. Likewise, The result of cryopreserved bull high- and low-motile
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sperm also showed that PTEN could be targeted by the simultaneous action of
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miR-17-5p, miR-26a-5p, miR-486-5p [64].
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Conclusion
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In this study, 23 miRNAs associated with processes of energy metabolism,
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sperm structure, motility, and apoptosis were differentially expressed during boar
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sperm cryopreservation. Putative target transcripts of differentially expressed
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miRNAs encode proteins that are critical for spermatozoa metabolic processes and
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cellular process. Together, our findings suggest miRNAs are widely associated with
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the process of boar sperm freeze-thaw. These findings will further our understanding
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of cryoinjury, freeze tolerance, and antifreeze mechanisms of sperm cryopreservation.
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Acknowledgements
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This work is supported by a grant from National Natural Science Foundation of China (NO. 31570533).
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Figure Legends
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Figure-1: Heat map of the differential expression levels of 46 selected miRNAs
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between fresh ejaculate, sperm cryopreserved without glycerol, and sperm
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cryopreserved with 3% glycerol.
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Figure-2: RT-qPCR assays show that the expression of twenty-three of forty-six
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selected miRNAs was significantly different among the three different treatments.
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Different letters indicate statistical significance (P < 0.05).
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Figure-3: Relative expression levels (as determined by RT-qPCR) across the three
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treatments for seven target genes (Fas, Bcl-2, PTEN, API5, BRCA1, H2AFX and
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TP53), which are possibly involved in sperm apoptosis and DNA repair. Different
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letters indicate statistical significance (P < 0.05).
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Figure-4: Comparison of the relative expression levels of miRNAs’ predicted target
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genes in sperm across three different treatments (as determined by RT-qPCR). (A)
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Fas, (B) PTEN, (C) Bcl-2, (D) BRCA1, (E) TP53, (F) API5 and (G) H2AFX. Different
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letters indicate statistical significance (P < 0.05).
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Figure-5: Categorization of predicted mRNA targets according to their GO terms and
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KEGG pathways following analysis with PANTHER and DAVID online tools. (A)
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KEGG pathways, (B) Biological process, (C) Cellular component, and (D) Molecular
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function.
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Table 2. Predicted target mRNAs of differentially expressed miRNAs involved in
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sperm apoptosis.
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Table 3. Primers for quantification of target mRNAs that are differentially expressed
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in boar sperm and associated with apoptosis.
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Supplemental data 1: Relative expression level of all selected 46 miRNAs and
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mRNAs.
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Supplemental data 2: Melt-curve of all selected 46 miRNAs and mRNAs.
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Table 1. Primers used for miRNA qRT-PCR in boar sperm.
MIMAT0013865 MIMAT0002151 MIMAT0025356 MIMAT0013866 MIMAT0002152 MIMAT0002153 MIMAT0002168 MIMAT0002125 MIMAT0007754 MIMAT0013950 MIMAT0002165 MIMAT0015709 MIMAT0002134 MIMAT0002135 MIMAT0002148 MIMAT0013870 MIMAT0013916 MIMAT0013908 MIMAT0013905 MIMAT0002116 MIMAT0002117 MIMAT0002156
TGAGGTAGTAGGTTGTATAGTT TGAGGTAGTAGGTTGTATGGTT AGAGGTAGTAGGTTGCATAGTT TGAGGTAGGAGGTTGTATAGTT TGAGGTAGTAGATTGTATAGTT TGAGGTAGTAGTTTGTGCT TCTTTGGTTATCTAGCTGTATGA TAGCAGCACATCATGGTTTACA TAGCAGCACGTAAATATTGGCG TGTGCAAATCCATGCAAAACTGA TAGCTTATCAGACTGATGTTGA AGTTCTTCAGTGGCAAGCTTTA TGGCTCAGTTCAGCAGGAACAG TTCAAGTAATCCAGGATAGGCT TTCACAGTGGCTAAGTTCCGC CTAGCACCATCTGAAATCGGTTA AGGCAGTGTAGTTAGCTGATTGC TATTGCACTTGTCCCGGCCTGT TGAGGTAGTAAGTTGTATTGTT TCAAATGCTCAGACTCCTGT TCAAATGCTCAGACTCCTTG TTAAGGCACGCGGTGAATGCCA
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ssc-let-7a ssc-let-7c ssc-let-7d-5p ssc-let-7e ssc-let-7f ssc-let-7i ssc-miR-9-1 ssc-miR-15b ssc-miR-16 ssc-miR-19b ssc-miR-21 ssc-miR-22-5p ssc-miR-24-3p ssc-miR-26a ssc-miR-27a ssc-miR-29a ssc-miR-34c ssc-miR-92a ssc-miR-98 ssc-miR-105-1 ssc-miR-105-2 ssc-miR-124a
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Primer sequence (5’-3’)
Amplification efficiency (%) 98.3 99.5 97.3 97.6 99.6 97.1 99.7 97.0 100.2 99.9 99.4 95.1 99.1 99.9 98.2 104.2 95.8 100.2 98.4 99.5 99.4 101.3
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Gene name
miRBase accession
R2
0.999 0.999 1.000 1.000 1.000 0.999 0.999 0.999 0.999 0.999 0.998 1.000 1.000 0.997 1.000 0.997 0.999 0.999 1.000 0.999 0.998 1.000
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1.000 0.999 1.000 0.999 1.000 1.000 1.000 1.000 0.997 0.999 1.000 0.999 0.999 0.999 0.994 0.995 1.000 1.000 0.999 0.999 1.000 0.998 1.000 1.000 1.000
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96.8 99.3 98.5 99.1 99.3 99.2 99.1 99.2 100.3 100.6 96.3 100.1 97.8 100.5 95.9 98.6 101.6 95.5 96.2 98.9 98.1 100.9 98.2 95.0 97.9
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TAACAGTCTACAGCCATGGTCG TGAGATGAAGCACTGTAGCTC AACATTCAACGCTGTCGGTGAGTT TTTGGCAATGGTAGAACTCACACT CAAAGAATTCTCCTTTTGGGCTT CTGACCTATGAATTGACAGCC TGGAATGTAAGGAAGTGTGTGA ACCTTGGCTCTAGACTGCTTACT AGCTACATCTGGCTACTGGGTCTC CAAGTCACTAGTGGTTCCGTTTA AAAAGCTGGGTTGAGAGGGCGA CCTAGTAGGTGTTCAGTAAGTGT TTATCAGAATCTCCAGGGGTAC AATTGCACGGTATCCATCTGTAA TTATAATACAACCTGATAAGTG ATATAATACAACCTGCTAAGTG CAGCAGCAATTCATGTTTTGAA TTTTGCGATGTGTTCCTAATAT TTTTGCAATATGTTCCTGAATA TTTTGCGATGTGTTCCTAATAC TAGCAGCGGGAACAGTACTGCAG AGACCCTGGTCTGCACTCTATCT TCAACACTTGCTGGTTTCCTCT TCGGGGATCATCATGTCACGA CCGTCCTAAGGTTGTTGAGTT
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MIMAT0025361 MIMAT0013879 MIMAT0010191 MIMAT0025366 MIMAT0002162 MIMAT0013910 MIMAT0013864 MIMAT0025370 MIMAT0013942 MIMAT0002132 MIMAT0013878 MIMAT0002150 MIMAT0013933 MIMAT0015711 MIMAT0013913 MIMAT0013915 MIMAT0013920 MIMAT0010188 MIMAT0013927 MIMAT0015717 MIMAT0010189 MIMAT0013931 MIMAT0013961 MIMAT0013924 MIMAT0013943
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ssc-miR-132 ssc-miR-143-3p ssc-miR-181a ssc-miR-182 ssc-miR-186 ssc-miR-192 ssc-miR-206 ssc-miR-212 ssc-miR-222 ssc-miR-224 ssc-miR-320 ssc-miR-325 ssc-miR-361-5p ssc-miR-363 ssc-miR-374a-5p ssc-miR-374b-5p ssc-miR-424-5p ssc-miR-450a ssc-miR-450b-5p ssc-miR-450c-5p ssc-miR-503 ssc-miR-504 ssc-miR-505 ssc-miR-542-5p ssc-miR-676-3p
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Table 2. Predicted target mRNAs of differentially expressed miRNAs involved in sperm apoptosis. Predicted target
let-7a/7c/7d/7e/7f/7i, miR-98 miR-22, miR-26 miR-24 miR-182
Fas cell surface death receptor Phosphatase and tensin homolog H2A histone family, member X Breast cancer 1, early onset
FAS PTEN H2AFX BRCA1
apoptosis tumor suppressor DNA damage response DNA damage response
miR-224 miR-34c, miR-504
Apoptosis inhibitor 5 Tumor protein p53
API5 TP53
apoptosis inhibitor apoptosis
miR-34c, miR-98
B-cell CLL/lymphoma 2
BCL2
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miRNA
Official symbol
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Encoded protein function
apoptosis inhibitor
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Table 3. Primers for quantification of target mRNAs that are differentially expressed in boar sperm and associated with apoptosis. Product Amplification Gene Primer (5’-3’) GenBank accession R2 length (bp) efficiency (%) F: ACTCACTCTTCTACCTTTGATGCT GAPDH 100 AF017079 93.9 1.000 R: TGTTGCTGTAGCCAAATTCA F: CGTGAGGGTCAATTCTGCTGT Fas 123 NM_213839 101.5 0.995 R: CTTGTCTGTGTAATCCTCCCCC F: GGCAACCCATCCTGGCACCT Bcl-2 134 XM_003121700 100.6 0.997 R: AACTCATCGCCCGCCTCCCT F: CGAGGTCCAAAGCGAGCAA BRCA1 171 XM_013989938.1 98.3 1.000 R: ACCCTGGCGGAAGGTGAAT F: ACAACAAGAAGACGCGGATCA H2AFX 121 XM_003129950.4 100.5 0.997 R: TGGATATTGGGCAGGACGC F: GCAGAGTTGCACAGTATCCTTTCG PTEN 151 NM_001143696 96.0 0.999 R: ACACCAGTTCGTCCCTTTCCAG F: GAGGAGCTTTACCGCAACTATGG API5 102 XM_003480707 100.8 0.999 R: ACCACCTTTGACACCATCCAGTA F: TTGAGGTGCGTGTTTGTGCC TP53 120 NM_213824 100.3 1.000 R: TGGGCAGTGCTCGCTTAGTG
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Fig.1. Heat map of the differential expression levels of 46 selected miRNAs between fresh ejaculate, sperm cryopreserved without glycerol, and sperm cryopreserved with 3% glycerol.
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Fig.2. RT-qPCR assays show that the expression of twenty-three of forty-six selected miRNAs was significantly different among the three different treatments. Different letters indicate statistical significance (P < 0.05).
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Fig.3. Relative expression levels (as determined by RT-qPCR) across the three treatments for seven target genes (Fas, Bcl-2, PTEN, API5, BRCA1, H2AFX and TP53), which are possibly involved in sperm apoptosis and DNA repair. Different letters indicate statistical significance (P < 0.05).
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Fig.4. Comparison of the relative expression levels of miRNAs’ predicted target genes in sperm across three different treatments (as determined by RT-qPCR). (A) Fas, (B) PTEN, (C) Bcl-2, (D) BRCA1, (E) TP53, (F) API5 and (G) H2AFX. Different letters indicate statistical significance (P < 0.05).
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Fig.5. Categorization of predicted mRNA targets according to their GO terms and KEGG pathways following analysis with PANTHER and DAVID online tools. (A) KEGG pathways, (B) Biological process, (C) Cellular component, and (D) Molecular function.
S0011-2240(16)30433-3
DOI:
10.1016/j.cryobiol.2017.04.013
Reference:
YCRYO 3843
To appear in:
Cryobiology
Received Date: 17 November 2016 Revised Date:
28 April 2017
Accepted Date: 28 April 2017
Please cite this article as: Y. Zhang, D. Dai, Y. Chang, Y. Li, M. Zhang, G. Zhou, Z. Peng, C. Zeng, Cryopreservation of boar sperm induces differential microRNAs expression, Cryobiology (2017), doi: 10.1016/j.cryobiol.2017.04.013. 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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Cryopreservation of boar sperm induces differential
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microRNAs expression
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Yan Zhang1§, Dinghui Dai1§, Yu Chang1, Yuan Li1, Ming Zhang1, Guangbin Zhou1,
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Zhanghua Peng2, Changjun Zeng1 *
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1. College of Animal Sciences and Technology, Sichuan Agricultural University, Chengdu,
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Sichuan, 611130, China
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2. Agriculture and Forestry Bureau, Luxian County, Sichuan Province, 646100, China
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§ These authors are contributed equally to this work.
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* Correspondence author. Email: [email protected], Tel/Fax: +86-28-86291010
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Abstract: Lower conception rates and litter sizes limit the wide use of artificial
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insemination with frozen-thawed boar sperm, due to a lack of understanding of the
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mechanisms that cause cryodamage and cryoinjury to sperm during cryopreservation.
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CryoMiRs, a family of freeze-related microRNAs (miRNAs), are associated with
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freeze tolerance, and regulate metabolism in mammalian hibernators and insects. Thus,
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we speculate that miRNAs maybe involved in the regulation of the freeze-thaw
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process and may affect boar sperm function. In this study, we studied the differential
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expression of 46 miRNAs that have roles in spermatogenesis, sperm maturation, and
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sperm quality in response to cryopreservation (with or without 3% glycerol). The
25
results indicated that, in response to cryopreservation with 3% glycerol, 14 miRNAs
26
were significantly up-regulated, but only two miRNAs (miR-22 and miR-450b-5p)
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were significantly down-regulated, relative to fresh sperm. Preservation with 3%
28
glycerol caused up-regulation of 17 miRNAs, but only caused down-regulation of one
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miRNA (miR-24), relative to sperm cryopreserved without glycerol. Functional
30
annotations of these differentially expressed miRNAs indicated that these miRNAs
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and their targets are mainly associated with metabolic and cellular processes.
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Therefore, our findings show that cryopreservation results in changes in miRNA
33
expression, and suggest that the anti-freeze mechanisms of boar sperm need to be
34
studied further.
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Keywords: Boar; Sperm; Cryopreservation; Freeze tolerance; miRNAs
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Introduction In the past few decades, the use of artificial insemination (AI) has significantly
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increased in the pork industry, and AI is now of great economic value to this industry.
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Nowadays, more than 99% of AIs are conducted using liquid-stored semen incubated
41
at 15-20°C for 0~5 days. However, no more than 1% of AI procedures use
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frozen-thawed semen, due to the reduced conception rate and litter size associated
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with freeze-thawing semen, which is mainly attributed to sperm structural and
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functional cryoinjury during cryopreservation [29]. Numerous studies have
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demonstrated that cryopreservation of sperm decreases viability and motility, changes
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sperm cholesterol content，increases the number of sperm that undergo acrosome
47
reaction [40], damages the plasma and mitochondrial membranes [41, 46], increases
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apoptosis [59], causes DNA fragmentation [46], and produces reactive oxygen species
49
(ROS) that can cause lipid peroxidation [11]. Although substantial research has focused on
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boar spermatozoa cryopreservation, it remains unclear whether miRNAs are involved in the
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functional regulation of spermatozoa response to cryopreservation.
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Mammalian hibernators and insects can resist cold temperatures and survive
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under extreme environmental conditions by depressing their metabolic rate, a process
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called hypometabolism. During mammalian hibernation, transcription and translation
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of genes associated with energy consumption are inhibited [18, 45]. However, the
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over-expression of some genes involved in glycolysis, fatty acid metabolism,
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gluconeogenesis, amino acid metabolism, and other metabolic pathways contributes
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to the organism’s long-term survival at low temperature [7, 56]. A recently discovered
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cold tolerance in animals that can withstand freezing [34, 39]. In freeze-tolerant
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mammals and insects, miRNAs can rapidly regulate metabolism-related genes and
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signaling pathways, and inhibit ATP metabolism to help the animal adapt to extreme
63
environmental conditions [4]. Changes in miR-21 and miR-16 expression in response
64
to freeze stress indicate that these miRNAs play important roles in regulation of the
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cell-cycle and apoptosis [4]. A target gene of miR-195, fatty acid synthase (FAS),
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which is involved in fatty acid biosynthesis, was shown to be down-regulated in the
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liver of hibernating thirteen-lined squirrels [33]. Furthermore, miRNAs are also
68
widely involved in the regulation of lipid and glucose metabolism, and in cholesterol
69
homeostasis in mammalian hibernators [34]. Three miRNAs (miR-26a, miR-126, and
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miR-217) can regulate PTEN expression in the wood frog and could potentially
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contribute to Akt-mediated inhibition of apoptosis during freezing [62]. Therefore, the
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key mechanisms of freeze tolerance and metabolic regulator may be associated with
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the reversible phosphorylation control of metabolic enzymes and miRNAs control of
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gene transcript expression[50].
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One potential explanation for sperm cryoinjury is as follows: during sperm
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cryopreservation, loss of membrane integrity causes loss of ATP and Mg2+, which
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decreases the intracellular concentration of ATP and increases the AMP/ADP
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concentration [21]. In contrast, McLaughlin et al. reported that decreased ATP
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production does not contribute to the poor motility of cryopreserved spermatozoa [42].
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Additionally, decreased cell growth, differentiation, proliferation, and mRNA
ACCEPTED MANUSCRIPT translation during cryopreservation reduces consumption of ATP. Thus, miRNAs
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likely play a critical role in the regulation of sperm mRNA expression during
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cryopreservation. Although miRNAs are thought to be involved in metabolic
84
depression and are differentially expressed in mammalian hibernators and insects, it is
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not known if miRNAs participate in freeze protection or freeze tolerance during boar
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sperm cryopreservation. Our previous study showed that some miRNAs are
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differentially expressed between fresh and frozen boar sperm [63]. Therefore, we
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speculated that freeze-tolerance related miRNAs (CryomiRs) may exist in boar sperm,
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and may regulate carbohydrate, lipid, and cholesterol metabolism during sperm
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cryopreservation. Here, 46 candidate miRNAs were selected to detect their expression
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changes in response to cryopreservation. Furthermore, the predicted target genes of
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differentially expressed miRNAs involved in DNA repair and apoptosis were further
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evaluated.
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Materials and methods
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Animal ethics statement
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Semen collection and treatment were conducted according to the Regulations of
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the Administration of Affairs Concerning Experimental Animals (Ministry of Science
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and Technology, China, revised in June 2004) and approved by the Institutional
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Animal Care and Use Committee in the College of Animal Science and Technology,
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Sichuan Agricultural University, Sichuan, China, under permit No. DKYB20081003.
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Furthermore, all experimental protocols were approved by the College of Animal
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Science and Technology, Sichuan Agricultural University.
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Sperm collection and cryopreservation The sperm-rich fractions of ejaculates were collected from Landrace boars (N=5)
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using a manual collection method [30]. Sperm quality parameters were measured with
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a sperm quality analyzer, SQA-V (MES, Israel). Only sperm with normal morphology,
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motility greater than 0.8, and a concentration higher than 1×108 were used.
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Firstly, all fresh ejaculates were divided into one of three treatment groups: (I)
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fresh sperm, directly used to extract total RNA; (II) cryopreserved sperm without
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glycerol, which underwent a freezing program; and (III) cryopreserved sperm with
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3% glycerol, which underwent the same freezing program. The boar sperm freezing
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program was performed according to a previous protocol [13, 61]. Briefly, the semen
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were diluted (1:1 v/v) with Beltsville Thawing Solution and cooled slowly to 15°C for
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2 hrs. Then, the sperm pellet was diluted to a concentration of 2×109 mL-1 using
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lactose-egg yolk (LEY) extender and slowly cooled to 4°C for 2 hrs in the refrigerator.
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At 4°C, sperm were further diluted with a second freezing extender (LEY
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supplemented with glycerol) to yield final concentration of 3% glycerol. Finally, the
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mixtures were packaged into 0.25 mL straws (FHK, Japan) and frozen using a
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controlled-rate freezing instrument (CryoMed Controlled-Rate Freezer, Thermo
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Fisher, USA) and then stored at -196°C.
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miRNA extraction and cDNA synthesis
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miRNAs were isolated using a mirVana miRNA Isolation Kit (Ambion, USA)
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based on a previous protocol [63]. First, to eliminate somatic cell contamination, the
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sperm pellet was suspended in a cold hypotonic solution with 0.5% Triton X-100
ACCEPTED MANUSCRIPT 125
(Roche,
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manufacturer's instructions. Finally, the concentration and quality of the miRNAs
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were determined using a NanoDrop ND1000 spectrophotometer (NanoDrop
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Technologies, USA).
Then,
miRNAs
were
extracted
according
to
the
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Germany)[47].
MiRNA first-strand cDNAs were synthesized from 0.06 µg of miRNA from each
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sample using the PrimeScript miRNA qPCR Starter Kit version 2.0 (Takara Biotech,
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China) according to the manufacturer's instructions. Subsequently, the cDNA was
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stored at -20°C till use.
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Primer design and RT-qPCR analysis
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All primers of selected 46 miRNAs were designed according to previous reports
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(Table 1), and were selected for study based on reported associations with
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spermatogenesis, sperm maturation, and sperm quality parameters [15, 16, 22, 36, 37].
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Relative miRNA expression was evaluated using Quantitative RT-PCR (RT-qPCR).
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Sperm miRNA expression levels were normalized to endogenous ssc-miR-27a-3p
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[63]. RT-qPCR was performed using SYBR PrimeScript miRNA RT-PCR Kit (Takara
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Biotech, China) on a StepOnePlus real-time PCR system (Applied BioSystems, USA)
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using an annealing temperature of 60°C, according to our laboratory’s protocols [63].
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Target prediction of differentially expressed miRNAs
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To date, a relatively small number of porcine miRNAs and predicted target
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mRNAs have been identified [15]. In order to further understand the functions of
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differentially expressed miRNAs between all treatment conditions, putative targets
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were
predicted
using
miRWalk
database
ACCEPTED MANUSCRIPT (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk/micrornapredictedtarget.html).
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miRWalk combines several independent target prediction tools, including TargetScan
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[35], miRanda [28], picTar [31], and miRWalk [16]. Then, PANTHER
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(http://www.pantherdb.org) and DAVID (https://david.ncifcrf.org) were used to
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explore the gene ontology and KEGG pathways of predicted target mRNAs.
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Seven of the predicted mRNA targets (Fas, Bcl-2, BRCA1, H2AFX, PTEN, API5,
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TP53) that are known to be involved in sperm DNA repair and apoptosis were
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selected for further study with RT-qPCR (Table 2, 3). All primers were designed with
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homologous counterparts in the GenBank Database using Premier Primer 5.0 software
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and cited from previous literature. GAPDH was used as a reference gene [61].
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Total RNA isolation and RT-qPCR analysis
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Total RNA from frozen-thawed boar sperm was extracted using a TRIzol LS
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Reagent kit (Invitrogen, USA). Briefly, the straws were thawed rapidly in a water bath
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at 37°C for 40 seconds. The sperm were resuspended in 1 mL of cold hypotonic
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solution with 0.5% Triton X-100 to remove somatic contaminations [47]. The quality
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of the total RNA was assessed using a NanoDrop ND1000 spectrophotometer
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(NanoDrop Technologies, USA).
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cDNA was synthesized from 1 µg of total RNA from each sample using a
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PrimeScript RT-PCR kit (Takara Biotech, Dalian, China) according to the
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manufacturer’s protocol. RT-qPCR was performed using SYBR Premix Ex Taq II
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(TaRaKa Biotech, China) on a StepOnePlus real-time PCR system (Applied
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BioSystems, USA) using an annealing temperature of 61.6°C according to our
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laboratory’s protocols [61].
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Statistical analysis The average cycle threshold (Ct) value for each triplicate of RT-qPCR was used to
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calculate relative mRNA expression levels using the 2-∆∆Ct method [38]. Statistical
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analysis of the relative expression levels of miRNAs and predicted target mRNAs was
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performed using Tukey's HSD (honest significant difference) test using SAS9.0
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software. Each experiment was independently conducted three times. Differences of P
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< 0.05 were regarded as statistically significant.
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Results
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Differential miRNA expression between fresh and cryopreserved sperm
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The relative expression levels of 46 miRNAs across the three treatment conditions
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(fresh sperm, sperm cryopreserved without glycerol, and sperm cryopreserved with
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3% glycerol) are shown in Fig. 1. The results indicated that 23 miRNAs were
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differentially expressed across the three treatments (P<0.05) (Fig. 2). Seven miRNAs
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(miR-34c, miR-124, miR-181a, miR-186, miR-224, miR-450b-5p, and miR-450c-5p)
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were down-regulated and two miRNAs (miR-98 and miR-374a) were significantly
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up-regulated in sperm cryopreserved without glycerol relative to fresh sperm.
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Fourteen miRNAs (let-7a, let-7c, let-7d, let-7e, let-7f-5p, let-7i, miR-9-5p, miR-26a,
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miR-98,
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significantly up-regulated, but only two miRNAs (miR-22 and miR-450b-5p) were
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significantly down-regulated in sperm cryopreserved with 3% glycerol relative to
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fresh sperm (P<0.05). Interestingly, seventeen miRNAs were significantly
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miR-181a,
miR-186,
miR-212,
miR-374a-5p,
miR-374b-5p)
were
ACCEPTED MANUSCRIPT 191
up-regulated, but only one miR (miR-24) was significantly down-regulated between
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cryopreserved sperm with or without 3% glycerol (P<0.05). GO and KEGG pathway analysis of these differentially expressed miRNAs’
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predicted mRNA targets indicated that these target mRNAs play important roles in
195
biological processes (metabolic processes, cell processes, and biological regulation),
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molecular functions (binding and catalytic activity), and cell components (cell part,
197
organelles, and membranes). These genes are mainly involved in the following
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pathways: cancer, MAPK signaling pathways, focal adhesion, and regulation of the
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actin cytoskeleton (Fig. 5).
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Relationship between differentially expressed miRNAs and their mRNA
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targets known to be involved in sperm DNA repair and apoptosis
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RT-qPCR results indicated that, except for PTEN, the relative expression levels
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of six genes (Fas, Bcl-2, BRCA1, H2AFX, API5, and TP53) were significantly
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different between fresh sperm and sperm cryopreserved with 3% glycerol (P<0.05).
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There was no significant difference in the relative expression levels of Fas, Bcl-2,
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BRCA1, API5, or TP53 between sperm cryopreserved without glycerol and sperm
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cryopreserved with 3% glycerol. Only H2AFX expression differed between all three
208
treatments (P<0.05). Furthermore, no significant difference in PTEN expression was
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found among the three treatments (Fig. 3).
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Based on previous studies and target gene prediction tools, we decided to further
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evaluate the relationship between miRNAs and their predicted target genes which are
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involved in sperm apoptosis and DNA repair (Fig. 4). Our results showed that the
ACCEPTED MANUSCRIPT expression levels of let-7 family members are significantly higher in sperm
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cryopreserved with 3% glycerol relative to the other two treatments (P<0.05). The
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expression of miR-98 is significantly higher in both cryopreserved treatments relative
216
to fresh sperm (P<0.05). Interestingly, the expression of their common target gene,
217
Fas, was significantly higher in fresh sperm than in the cryopreserved treatment
218
conditions (Fig. 4A). The expression of PTEN is higher in the cryopreserved sperm
219
than in fresh sperm (Fig. 4B). Meanwhile, the expression pattern of miR-22 in fresh
220
and cryopreserved sperm is the opposite of PTEN’s expression pattern. These results
221
are consistent with the fact that miRNAs degrade or translationally silence their target
222
mRNAs. Similarly, the expression level of Bcl-2, an anti-apoptotic gene, is also
223
consistent with the changes we observe in miR-98 expression, but not miR-34c (Fig.
224
4C). There were no significant differences in the expression levels of miR-182,
225
miR-34c, miR-504-5p, and miR-244 and their target genes (BRCA1, API and TP53) in
226
the cryopreserved sperm, with or without 3% glycerol (Fig. 4D-4F). The expression
227
level of miR-24 and its target gene, H2AFX, was significantly higher in the sperm
228
cryopreserved without glycerol than in 3% glycerol condition (Fig. 4G).
229
Discussion
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It is generally accepted that nuclear-encoded protein translation is unlikely to
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occur in mature sperm cells and translational repression is essential for spermatid
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differentiation. However, Gur and Breitbart [23] have demonstrated that nuclear genes
233
are expressed as proteins in sperm. Although ejaculated sperm are capable of using
234
mRNAs transcripts for protein translation during the final maturation steps before
ACCEPTED MANUSCRIPT fertilization, the mechanism is presently still unknown [24]. During sperm
236
cryopreservation, some epigenetic-related mRNA (Dnmt3a, Dnmt3b, Jhdm2a, Kat8,
237
Prm1, Prm2 and IGF2) and low protein levels were typically found in sperm after
238
freeze-thawing [60]. miRNAs are critical regulators of mRNA expression, which
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inhibit translation of target mRNA or induce mRNA degradation [25]. The process of
240
cryopreservation not only induces expression of apoptosis-related mRNAs [59], but
241
also changes miRNA expression [63]. In the present study, we found miR-98 and
242
miR-374a to be up-regulated, and seven miRNAs (miR-34c, miR-124, miR-181a,
243
miR-186, miR-224, miR-450b-5p, and miR-450c-5p) to be down-regulated in
244
cryopreserved sperm (without glycerol) relative to fresh sperm. This further
245
demonstrated that the freeze-thaw process changes miRNA expression. Seventeen
246
miRNAs were significantly up-regulated, but only miR-24 was significantly
247
down-regulated, with the addition of cryoprotectants (3% glycerol) relative to
248
freezing without cryoprotectants. Recently, 83 miRNAs were differentially expressed
249
in high and low motile post-thaw bovine sperm, and let-7a and miR-26a are
250
up-regulated in high motile sperm [10]. Likewise, let-7a and miR-26a are also
251
significantly up-regulated in cryopreserved boar sperm with 3% glycerol compared
252
with cryopreserved boar sperm without 3% glycerol.
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CryomiRs (cold-modulated miRNAs), as key regulators of freeze tolerance in
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cold-tolerant insects and hibernating mammals, control the reversible phosphorylation
255
of metabolic enzymes, thereby reducing energetic consumption, and gene transcript
256
expression [39, 49, 50]. Fatty acid synthase (FAS), an enzyme involved in fatty acid
257
biosynthesis and a putative miR-195 target, is down-regulated in hibernating
258
squirrels’ livers [33]. Furthermore, miR-29a and miR-233 and their target mRNAs
ACCEPTED MANUSCRIPT 259
could also regulate glycolysis or glucose metabolism in these cold-tolerant vertebrates
260
[4, 34]. Under freezing stress conditions, three miRNAs (miR-26a, miR-126, and
261
miR-217) can regulate PTEN expression via activation of AKT pathways to inhibit
262
apoptosis [62]. Based on these enlightening findings, we postulated that miRNAs were probably
264
associated with anti-freeze, anti-apoptosis, and energy consumption regulation
265
mechanisms during sperm cryopreservation. It is well known that the cellular
266
metabolic activity decreases during cryopreservation, followed by irreversible
267
cryoinjury [58]. During cryopreservation, intracellular ice crystal formation leads to
268
cryodamage of the sperm plasma membrane and mitochondrial or nuclear DNA,
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which causes apoptotic changes and a severe decline in sperm quality [27, 58].
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Furthermore, gene transcription and protein translation associated with energy
271
consumption are inhibited at low temperature [18, 45]. In this study, gene ontology
272
analysis of differentially expressed miRNAs’ predicted mRNA targets showed that
273
these targets are mainly involved in metabolic processes, cell processes, and
274
biological regulation, and play important roles in binding and catalytic activities (Fig.
275
5). It was reported that the PTEN/PI3K/Akt signaling pathway is involved in cellular
276
processes such as glucose metabolism, cell cycle regulation, and protein translation
277
under conditions of environmental stresses [1, 17]. For instance, miR-29a and
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miR-233 are involved in glucose metabolism in wood frogs and hibernating
279
thirteen-lined ground squirrels [4, 33]. MiR-195 is associated with lipid metabolism in
280
mammalian hibernators [34]. Via targeting of IDH1, miR-181a decreases expression
281
of lipid synthesis-related genes and increases expression of genes involved in
282
β-oxidation [12]. In the present study, we found that miR-181a expression was
283
up-regulated in sperm cryopreserved with 3% glycerol, but down-regulated in sperm
284
cryopreserved without glycerol, which is consistent with the superior cryoprotective
285
effects of glycerol. Furthermore, miR-22, miR-24, and miR-182 targets are thought to
286
be associated with sperm structure and motility [2, 26, 52]. Thus, the process of
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freeze-thaw influences miRNA expression and results in abnormal sperm morphology,
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motility, and fertility. Furthermore, it was suggested that glycerol provides better
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ACCEPTED MANUSCRIPT 289
cryoprotective effects due to the different miRNA expression profile of sperm
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cryopreserved with or without 3% glycerol. However, the molecular mechanism of
291
cryoprotection during sperm cryopreservation requires further study. The association between miRNAs’ predicted target mRNAs and spermatozoa
293
apoptosis and DNA damage was explored further. Apoptosis (programed cell death) is
294
a physiological mechanism required for organic function and tissue development and
295
is also necessary to dismantle and engulf damaged cells that represent a threat to the
296
integrity of the organism. Previous studies have revealed that sperm also undergo
297
apoptotic changes during cryopreservation [9, 59]. In this study, we used RT-qPCR to
298
assay the expression of seven DNA repair and apoptosis-related genes (Fas, Bcl-2,
299
BRCA1, H2AFX, API5, TP53, PTEN) that are thought to be target genes of the
300
freeze-related miRNAs that we identified. The Fas-Fas ligand (FasL) pathway is a
301
major death receptor-signaling pathway. Binding of FasL to Fas induces activation of
302
caspase-8 and triggers apoptosis [59]. Fas is a direct target of the let-7/miR-98 family.
303
Let-7 and miR-98 expression levels are significantly lower in fresh sperm than in
304
sperm cryopreserved with 3% glycerol, while the Fas expression profile is opposite to
305
let-7/miR-98 (P<0.05). Let-7/miR-98 expression is reduced during activation-induced
306
cell death, which is accompanied by increased Fas expression [53]. The anti-apoptotic
307
protein Bcl-2 is located mainly on the outer membrane of mitochondria. Bcl-2 can
308
prevent apoptosis by inhibiting the release of cytochrome C [57]. Bcl-2 is a target of
309
miR-34 and miR-98, and can be down-regulated by direct binding of miR-34 [5, 55].
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BRCA1 is an E3 ubiquitin ligase that plays an essential role in DNA repair [6].
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BRCA1 is directly targeted by miR-182, and miR-182-mediated down-regulation of
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ACCEPTED MANUSCRIPT BRCA1 impedes DNA repair [44]. H2AFX, a key double-stranded DNA break repair
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protein, was identified as a target of miR-24. MiR-24 binds to the 3’UTR of H2AX
314
mRNA and decreases H2AFX mRNA and protein levels, which inhibits the DNA
315
damage response and increases cell death after DNA damage [32]. Our results show
316
that miR-24 and miR-182 may impact DNA damage repair via regulation of their
317
target genes, H2AFX and BRCA1. Apoptosis inhibitor 5 (API-5), which prevents
318
apoptosis after growth factor deprivation, is targeted by miR-224, and plays a role in
319
sensitizing cells to apoptosis in hepatocellular carcinoma patients [54]. Freeze
320
extension with 3% glycerol attenuates apoptosis better than sperm cryopreservation
321
without glycerol. In this study, the expression level of API-5 in sperm cryopreserved
322
with 3% glycerol is lower than that in sperm cryopreserved without glycerol. This
323
result is consistent with the higher expression of miR-224 that we observed in sperm
324
cryopreserved with 3% glycerol relative to sperm preserved without glycerol. Studies
325
suggest that members of the miR-34 family are direct p53 targets, which regulates
326
apoptosis [48]. MiR-34a is a direct p53-target gene and p53-dependent induction of
327
miR-34a expression occurs after DNA damage [51]. Notably, inactivation of miR-34a
328
strongly attenuates p53-mediated apoptosis in cells exposed to genotoxic stress,
329
whereas over expression of miR-34a mildly increases apoptosis [48]. Our results
330
show that TP53 expression is significant lower in cryopreserved sperm than in fresh
331
sperm, which is not consistent with the changes in miR-34c and miR-504-5p
332
expression in the three treatments. Furthermore, PTEN is targeted by miR-22 and
333
miR-26 through a specific miR-binding site in the PTEN 3’ UTR [3, 19]. However, in
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ACCEPTED MANUSCRIPT our study, we observed no significant difference in PTEN expression among the three
335
treatments, which suggests that PTEN may be regulated by miR-22 and miR-26a in
336
the boar sperm. Likewise, The result of cryopreserved bull high- and low-motile
337
sperm also showed that PTEN could be targeted by the simultaneous action of
338
miR-17-5p, miR-26a-5p, miR-486-5p [64].
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Conclusion
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In this study, 23 miRNAs associated with processes of energy metabolism,
341
sperm structure, motility, and apoptosis were differentially expressed during boar
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sperm cryopreservation. Putative target transcripts of differentially expressed
343
miRNAs encode proteins that are critical for spermatozoa metabolic processes and
344
cellular process. Together, our findings suggest miRNAs are widely associated with
345
the process of boar sperm freeze-thaw. These findings will further our understanding
346
of cryoinjury, freeze tolerance, and antifreeze mechanisms of sperm cryopreservation.
347
Acknowledgements
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This work is supported by a grant from National Natural Science Foundation of China (NO. 31570533).
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microRNA genes for quantitative reverse transcription polymerase chain reaction studies of boar spermatozoa cryopreservation, Theriogenology, 83 (2015) 634-641. [64] Y. Zhou, M. Zheng, Q. Shi, L. Zhang, W. Zhen, W. Chen, Y. Zhang, An epididymis-specific secretory protein HongrES1 critically regulates sperm capacitation and male fertility, PLoS One, 3 (2008) e4106.
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Figure-1: Heat map of the differential expression levels of 46 selected miRNAs
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between fresh ejaculate, sperm cryopreserved without glycerol, and sperm
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cryopreserved with 3% glycerol.
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Figure-2: RT-qPCR assays show that the expression of twenty-three of forty-six
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selected miRNAs was significantly different among the three different treatments.
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Different letters indicate statistical significance (P < 0.05).
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Figure-3: Relative expression levels (as determined by RT-qPCR) across the three
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treatments for seven target genes (Fas, Bcl-2, PTEN, API5, BRCA1, H2AFX and
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TP53), which are possibly involved in sperm apoptosis and DNA repair. Different
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letters indicate statistical significance (P < 0.05).
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Figure-4: Comparison of the relative expression levels of miRNAs’ predicted target
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genes in sperm across three different treatments (as determined by RT-qPCR). (A)
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Fas, (B) PTEN, (C) Bcl-2, (D) BRCA1, (E) TP53, (F) API5 and (G) H2AFX. Different
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letters indicate statistical significance (P < 0.05).
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Figure-5: Categorization of predicted mRNA targets according to their GO terms and
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KEGG pathways following analysis with PANTHER and DAVID online tools. (A)
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KEGG pathways, (B) Biological process, (C) Cellular component, and (D) Molecular
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function.
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Table 2. Predicted target mRNAs of differentially expressed miRNAs involved in
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sperm apoptosis.
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Table 3. Primers for quantification of target mRNAs that are differentially expressed
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in boar sperm and associated with apoptosis.
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Supplemental data 1: Relative expression level of all selected 46 miRNAs and
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mRNAs.
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Supplemental data 2: Melt-curve of all selected 46 miRNAs and mRNAs.
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Table 1. Primers used for miRNA qRT-PCR in boar sperm.
MIMAT0013865 MIMAT0002151 MIMAT0025356 MIMAT0013866 MIMAT0002152 MIMAT0002153 MIMAT0002168 MIMAT0002125 MIMAT0007754 MIMAT0013950 MIMAT0002165 MIMAT0015709 MIMAT0002134 MIMAT0002135 MIMAT0002148 MIMAT0013870 MIMAT0013916 MIMAT0013908 MIMAT0013905 MIMAT0002116 MIMAT0002117 MIMAT0002156
TGAGGTAGTAGGTTGTATAGTT TGAGGTAGTAGGTTGTATGGTT AGAGGTAGTAGGTTGCATAGTT TGAGGTAGGAGGTTGTATAGTT TGAGGTAGTAGATTGTATAGTT TGAGGTAGTAGTTTGTGCT TCTTTGGTTATCTAGCTGTATGA TAGCAGCACATCATGGTTTACA TAGCAGCACGTAAATATTGGCG TGTGCAAATCCATGCAAAACTGA TAGCTTATCAGACTGATGTTGA AGTTCTTCAGTGGCAAGCTTTA TGGCTCAGTTCAGCAGGAACAG TTCAAGTAATCCAGGATAGGCT TTCACAGTGGCTAAGTTCCGC CTAGCACCATCTGAAATCGGTTA AGGCAGTGTAGTTAGCTGATTGC TATTGCACTTGTCCCGGCCTGT TGAGGTAGTAAGTTGTATTGTT TCAAATGCTCAGACTCCTGT TCAAATGCTCAGACTCCTTG TTAAGGCACGCGGTGAATGCCA
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ssc-let-7a ssc-let-7c ssc-let-7d-5p ssc-let-7e ssc-let-7f ssc-let-7i ssc-miR-9-1 ssc-miR-15b ssc-miR-16 ssc-miR-19b ssc-miR-21 ssc-miR-22-5p ssc-miR-24-3p ssc-miR-26a ssc-miR-27a ssc-miR-29a ssc-miR-34c ssc-miR-92a ssc-miR-98 ssc-miR-105-1 ssc-miR-105-2 ssc-miR-124a
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Primer sequence (5’-3’)
Amplification efficiency (%) 98.3 99.5 97.3 97.6 99.6 97.1 99.7 97.0 100.2 99.9 99.4 95.1 99.1 99.9 98.2 104.2 95.8 100.2 98.4 99.5 99.4 101.3
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Gene name
miRBase accession
R2
0.999 0.999 1.000 1.000 1.000 0.999 0.999 0.999 0.999 0.999 0.998 1.000 1.000 0.997 1.000 0.997 0.999 0.999 1.000 0.999 0.998 1.000
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1.000 0.999 1.000 0.999 1.000 1.000 1.000 1.000 0.997 0.999 1.000 0.999 0.999 0.999 0.994 0.995 1.000 1.000 0.999 0.999 1.000 0.998 1.000 1.000 1.000
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96.8 99.3 98.5 99.1 99.3 99.2 99.1 99.2 100.3 100.6 96.3 100.1 97.8 100.5 95.9 98.6 101.6 95.5 96.2 98.9 98.1 100.9 98.2 95.0 97.9
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TAACAGTCTACAGCCATGGTCG TGAGATGAAGCACTGTAGCTC AACATTCAACGCTGTCGGTGAGTT TTTGGCAATGGTAGAACTCACACT CAAAGAATTCTCCTTTTGGGCTT CTGACCTATGAATTGACAGCC TGGAATGTAAGGAAGTGTGTGA ACCTTGGCTCTAGACTGCTTACT AGCTACATCTGGCTACTGGGTCTC CAAGTCACTAGTGGTTCCGTTTA AAAAGCTGGGTTGAGAGGGCGA CCTAGTAGGTGTTCAGTAAGTGT TTATCAGAATCTCCAGGGGTAC AATTGCACGGTATCCATCTGTAA TTATAATACAACCTGATAAGTG ATATAATACAACCTGCTAAGTG CAGCAGCAATTCATGTTTTGAA TTTTGCGATGTGTTCCTAATAT TTTTGCAATATGTTCCTGAATA TTTTGCGATGTGTTCCTAATAC TAGCAGCGGGAACAGTACTGCAG AGACCCTGGTCTGCACTCTATCT TCAACACTTGCTGGTTTCCTCT TCGGGGATCATCATGTCACGA CCGTCCTAAGGTTGTTGAGTT
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MIMAT0025361 MIMAT0013879 MIMAT0010191 MIMAT0025366 MIMAT0002162 MIMAT0013910 MIMAT0013864 MIMAT0025370 MIMAT0013942 MIMAT0002132 MIMAT0013878 MIMAT0002150 MIMAT0013933 MIMAT0015711 MIMAT0013913 MIMAT0013915 MIMAT0013920 MIMAT0010188 MIMAT0013927 MIMAT0015717 MIMAT0010189 MIMAT0013931 MIMAT0013961 MIMAT0013924 MIMAT0013943
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ssc-miR-132 ssc-miR-143-3p ssc-miR-181a ssc-miR-182 ssc-miR-186 ssc-miR-192 ssc-miR-206 ssc-miR-212 ssc-miR-222 ssc-miR-224 ssc-miR-320 ssc-miR-325 ssc-miR-361-5p ssc-miR-363 ssc-miR-374a-5p ssc-miR-374b-5p ssc-miR-424-5p ssc-miR-450a ssc-miR-450b-5p ssc-miR-450c-5p ssc-miR-503 ssc-miR-504 ssc-miR-505 ssc-miR-542-5p ssc-miR-676-3p
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Table 2. Predicted target mRNAs of differentially expressed miRNAs involved in sperm apoptosis. Predicted target
let-7a/7c/7d/7e/7f/7i, miR-98 miR-22, miR-26 miR-24 miR-182
Fas cell surface death receptor Phosphatase and tensin homolog H2A histone family, member X Breast cancer 1, early onset
FAS PTEN H2AFX BRCA1
apoptosis tumor suppressor DNA damage response DNA damage response
miR-224 miR-34c, miR-504
Apoptosis inhibitor 5 Tumor protein p53
API5 TP53
apoptosis inhibitor apoptosis
miR-34c, miR-98
B-cell CLL/lymphoma 2
BCL2
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apoptosis inhibitor
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Table 3. Primers for quantification of target mRNAs that are differentially expressed in boar sperm and associated with apoptosis. Product Amplification Gene Primer (5’-3’) GenBank accession R2 length (bp) efficiency (%) F: ACTCACTCTTCTACCTTTGATGCT GAPDH 100 AF017079 93.9 1.000 R: TGTTGCTGTAGCCAAATTCA F: CGTGAGGGTCAATTCTGCTGT Fas 123 NM_213839 101.5 0.995 R: CTTGTCTGTGTAATCCTCCCCC F: GGCAACCCATCCTGGCACCT Bcl-2 134 XM_003121700 100.6 0.997 R: AACTCATCGCCCGCCTCCCT F: CGAGGTCCAAAGCGAGCAA BRCA1 171 XM_013989938.1 98.3 1.000 R: ACCCTGGCGGAAGGTGAAT F: ACAACAAGAAGACGCGGATCA H2AFX 121 XM_003129950.4 100.5 0.997 R: TGGATATTGGGCAGGACGC F: GCAGAGTTGCACAGTATCCTTTCG PTEN 151 NM_001143696 96.0 0.999 R: ACACCAGTTCGTCCCTTTCCAG F: GAGGAGCTTTACCGCAACTATGG API5 102 XM_003480707 100.8 0.999 R: ACCACCTTTGACACCATCCAGTA F: TTGAGGTGCGTGTTTGTGCC TP53 120 NM_213824 100.3 1.000 R: TGGGCAGTGCTCGCTTAGTG
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Fig.1. Heat map of the differential expression levels of 46 selected miRNAs between fresh ejaculate, sperm cryopreserved without glycerol, and sperm cryopreserved with 3% glycerol.
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Fig.2. RT-qPCR assays show that the expression of twenty-three of forty-six selected miRNAs was significantly different among the three different treatments. Different letters indicate statistical significance (P < 0.05).
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Fig.3. Relative expression levels (as determined by RT-qPCR) across the three treatments for seven target genes (Fas, Bcl-2, PTEN, API5, BRCA1, H2AFX and TP53), which are possibly involved in sperm apoptosis and DNA repair. Different letters indicate statistical significance (P < 0.05).
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Fig.4. Comparison of the relative expression levels of miRNAs’ predicted target genes in sperm across three different treatments (as determined by RT-qPCR). (A) Fas, (B) PTEN, (C) Bcl-2, (D) BRCA1, (E) TP53, (F) API5 and (G) H2AFX. Different letters indicate statistical significance (P < 0.05).
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Fig.5. Categorization of predicted mRNA targets according to their GO terms and KEGG pathways following analysis with PANTHER and DAVID online tools. (A) KEGG pathways, (B) Biological process, (C) Cellular component, and (D) Molecular function.