Selection of optimal reference genes for quantitative RT-PCR studies of boar spermatozoa cryopreservation

doi:10.1016/j.cryobiol.2014.01.004

Cryobiology

Volume 68, Issue 1, February 2014, Pages 113-121

https://doi.org/10.1016/j.cryobiol.2014.01.004 Get rights and content

Abstract

Reference genes can be used to normalize mRNA levels across different samples for the exact comparison of the mRNA expression level. It is important to select reference genes with high quality for the accurate interpretation of qRT-PCR data. Although several studies have attempted to validate reference genes in pigs, no validation studies have been performed on spermatozoa samples frozen with different cryoprotectants. In this study, 11 commonly used reference genes (ACTB, B2M, GAPDH, HPRT1, RPL4, SDHA, YWHAZ, PPIA, PGK1, S18, and BLM) were investigated in boar spermatozoa frozen with six different cryoprotectants using qRT-PCR. The expression stability of these reference genes in different samples was evaluated using geNorm (qbase^plus software), NormFinder, and BestKeeper. The geNorm results revealed that PGK1, ACTB, and RPL4 exhibit high expression stability in all of the samples, and the NormFinder results indicated that GAPDH is the most stable gene. Furthermore, the BestKeeper results indicated that the three most stable genes are PPIA, GAPDH, and RPL4 and that S18, B2M and BLM are the three least stable genes. There are a number of differences in the ranking order of the reference genes obtained using the different algorithms. In conclusion, GAPDH, RPL4, and PPIA were the three most stable genes in frozen boar spermatozoa, as determined based on the cycle threshold coefficient of variation (Ct CV%) and the comprehensive ranking order, and this finding is consistent with the BestKeeper results

Introduction

Artificial insemination (AI) is widely used in the modern pig breeding industry. However, AI with cryopreservation or frozen-thawed (FT) semen is limited to no more than 1% of total inseminations due to low conception rate and litter size [11]. During cryopreservation, physical and chemical factors, including a rapid change in temperature or thermal stress, oxidative stress, and osmotic stress, lead to damage of not only the spermatozoa plasma membrane [33] but also the mitochondrial membrane [40], [42], which triggers molecular changes that interfere with fertilization and regulate molecular signals of apoptosis [3]. Although substantial advances have been made toward boar spermatozoa cryopreservation, the cryopreservation conditions are still considered suboptimal, and the factors that influence boar spermatozoa cryopreservation remain unknown. In general, viability, motility, and fertility are often used to assess the quality of frozen spermatozoa. Additionally, the injuries resulting from cryopreservation on the structure of mammalian spermatozoa can also result in frozen spermatozoa with poor quality. Therefore, the analysis of the expression of different genes will aid our understanding of cryotolerance mechanisms and the apoptotic changes that occur during cryopreservation.

It is well documented that spermatozoa contain a complex and specific population of RNAs [18], [34], [39], [41]. These transcripts may play an important role in spermatozoa development, chromatin packaging, genomic imprinting, several paternal contributions, and the delivery of paternal RNAs to oocytes after fertilization [8], [17], [25]. Many studies have demonstrated that the caspase (cysteine-aspartic proteases) gene family is related to spermatozoa apoptosis and the low viability and fertility caused by cryoinjury during cryopreservation [21], [22]. Therefore, it is important to identify the changes in gene expression involved in the cryopreservation of spermatozoa.

Real-time reverse transcription polymerase chain reaction (RT-PCR) is sensitive and specific for mRNA detection and quantitative gene expression analysis [2], [6], [54], [55]. To identify specific variations of a gene, one compulsory step is the selection of housekeeping genes (HKGs) or reference genes for normalization that are thought to be stably expressed across different samples, treatments, and physiological states [36]. However, numerous studies demonstrate that HKGs or reference genes are regulated and that their expression levels vary under certain experimental conditions [28], [50]. Furthermore, due to the low yield of mRNA in spermatozoa, the requirements for proper reference genes or HKGs as an internal control have become increasingly stringent. In this study, we conducted an extensive evaluation of 11 commonly used reference genes, namely ACTB, B2M, GAPDH, HPRT1, RPL4, SDHA, YWHAZ, PPIA, PGK1, S18, and BLM, in boar spermatozoa frozen with different freezing extenders. Furthermore, we used geNorm (qbase^plus software), NormFinder, and BestKeeper to evaluate the stability of these 11 reference genes.

Section snippets

Spermatozoa collection and cryopreservation

Three ejaculates from Landrace boar were collected using a manual collection method [27]. Only the spermatozoa-rich fractions of the ejaculates that exhibited a spermatozoa motility higher than 0.8, normal morphology, and spermatozoa concentration higher than 1 × 10⁸ mL⁻¹ were used. After the spermatozoa were subjected to a quality examination, the sperm was diluted (1:1 v/v) with Beltsville Thawing Solution (BTS, 3.7 g of glucose, 0.3 g of Na₃ citrate, 0.125 g of NaHCO₃, 0.125 g of Na₂-EDTA, 0.075 g

RNA quality

The agarose gel electrophoresis and qRT-PCR results show that the CD45, c-kit, and E-cadherin genes were not detected, which indicates the lack of somatic cell contamination in the total RNA of boar spermatozoa. The RNA purity, which was determined using the 260/280 absorbance (A260/A280) ratios, ranged from 1.91 to 2.03. Thus, the quality of the total RNA of boar spermatozoa was satisfactory for the following studies.

Verification, amplification efficiency, and expression level of candidate reference genes

Using the published primer, SDHA generated more than a single peak and was

Discussion

It is crucial to choose an appropriate reference gene for an exact comparison of mRNA expression in different samples or tissues. The inappropriate choice of reference genes frequently results in a loss of accuracy and a decreased statistical significance, particularly in the case of genes with small expression differences. The optimal reference gene should be transcribed at a constant level in all types of cells at all times throughout the cell cycle and differentiation. Moreover, the

Conclusion

In summary, our study used three different statistical algorithms to analyze 11 potential reference genes in boar spermatozoa samples frozen with different cryoprotectants and identified GAPDH, RPL4, and PPIA as the most stable reference genes. Our results will benefit the study of the expression of genes in boar spermatozoa and will facilitate further exploration of the mechanism of boar spermatozoa cryopreservation.

Acknowledgments

We would like to give special thanks to the anonymous reviewers for their comments on the earlier version of this manuscript. We also thank Ying Ren and Jinyue Li for their kind help with the collection of the boar spermatozoa. This work was supported by a Grant from the National Natural Science Foundation of China (No. 30901028).

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The purpose of this study was to identify proteins associated with differences in the freezing tolerance of sheep sperm and to analyze their functions. Qualified fresh semen from four breeds of rams, the Australian White, white-head Dorper, Black-head Dorper, and Hu sheep breeds, were selected for cryopreservation. The sperm freezing tolerance was investigated by evaluation of the overall vitality, progressive vitality, and rapidly advance vitality of the sperm. A differential model of sperm freezing tolerance was constructed for sheep breeds showing significant differences. Differentially expressed proteins associated with sperm freezing tolerance were identified using iTRAQ and the protein functions were analyzed. It was found that sperm freezing tolerance was best in Hu sheep and worst in white-head Dorper sheep. These two breeds were used for the construction of a model based on differences in freezing tolerance and the identification of sperm proteins expressed differentially before freezing and after thawing. A total of 128 differentially expressed proteins (88 up-regulated and 40 down-regulated) were identified before freezing and after thawing in Hu sheep sperm (fresh/frozen Hu sheep sperm referred to as HL vs. HF), while 219 differentially expressed proteins (106 up-regulated and 113 down-regulated) were identified in white-head Dorper sheep (fresh/frozen white-head Dorper sheep sperm referred to as WL vs. WF). A comparison of these differentially expressed proteins showed that 57 proteins overlapped between the two breeds while 71 were only expressed in Hu sheep and 162 were only expressed in white-head Dorper sheep. Functional annotation and enrichment analyses of differentially expressed proteins down-regulated in Hu sheep involved in phosphorylation of phosphatidylinositol phosphate kinases, regulation of GTPase activity and glycolysis/gluconeogenesis signaling pathway. Up-regulated proteins of Hu sheep participated in oxidoreductase activity and oxidative phosphorylation process of sperm freezing. Furthermore, down-regulated in white-head Dorper sheep involved in the metabolic regulation of carbohydrate and nuclear sugar metabolism. Up-regulated proteins of white-head Dorper sheep involved in the ferroptosis and oxidative phosphorylation pathways. Collectively, These proteins were found to participate mainly in oxidative phosphorylation as well as phosphorylation and metabolic processes in the mitochondria to affect the freezing tolerance of sheep sperm.
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Semen freezability is associated with genetic markers, and there is a diverse set of sperm transcripts that have been attributed to various cellular functions. RNA-Seq was performed to compare the transcript profiles of spermatozoa from boars with different semen freezability. We examined ejaculates from the Polish large white (PLW) boars that were classified as having good and poor semen freezability (GSF and PSF, respectively; n = 3 boars per group) by assessing post-thaw motility characteristics, mitochondrial membrane potential, plasma membrane and acrosome integrity. Total RNA was isolated from fresh spermatozoa from boars of the GSF and PSF groups and subjected to RNA-Seq (Illumina NextSeq 500 platform). Transcript abundance was assessed with the DESeq2, DESeq, and EdgeR Bioconductor R packages, and varying numbers of differentially expressed gene (DEG) transcripts were detected in the spermatozoa of each boar. Using RNA-Seq, we identified several genes associated with inflammation and apoptosis (FOS, NFATC3, ITGAL, EAF2 and ZDHHC14), spermatogenesis (FGF-14 and BAMBI), autophagy (RAB33B), protein phosphorylation (PTPRU and PTPN2) and energy metabolism (ND6 and ACADM) that were predominantly up-regulated in poor freezability ejaculates. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) validated the transcript expression levels detected by RNA-Seq and thus confirmed the reliability of this technique. Subsequent validation with western blotting showed that the expression of three proteins was in accordance with the transcript abundance. Overall, we demonstrated that the up-regulation of the DEG transcripts in spermatozoa was associated with poor semen freezability. We suggest that spermatozoa transcriptome profiling provides a foundation to further elucidate the relevance of sperm-related transcripts on cryo-survival. The sperm-related transcripts, namely FOS, NFATC3, EAF2, BAMBI, PTPRU, PTPN2, ND6 and ACADM, are potential markers for predicting the freezability of boar semen.
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The present study was conducted to characterise rabbit sperm proteins focusing on the influence of the genetic origin. Six samples were recovered during two months from five males from genotype A (New Zealand White origin) and five from genotype R (California origin). Sperm proteins were extracted and subjected to in-gel digestion nano LC-MS/MS and bioinformatics analysis. The resulting library included 487 identified proteins validated with ≥95% Confidence (unused Score ≥ 1.3). All the identified proteins belonged to Oryctolagus cuniculus taxonomy. These data are available via ProteomeXchange with identifier PXD007989. Only 7 proteins were specifically implicated in reproductive processes according to Gene Ontology annotation. Regarding the comparison of the sperm proteins abundance between genotypes, forty proteins were differentially expressed. Among them, 25 proteins were over-expressed in genotype A, while 15 proteins were over-expressed in genotype R. In conclusion, this study characterizes for the first time rabbit sperm proteins and provides evidence that genotype is related to a specific abundance of spermatozoa proteins.
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Citation Excerpt :
In a study by Riesco and Robles (2013), cryopreservation was observed to alter several transcripts (i.e. eif2s1 and lhcgr), while several others (i.e. human HOXB1 and ACTB) remained stable in spermatozoa. Transcript analysis of frozen boar spermatozoa showed that the transcripts B2M, BLM, HPRT1, PGK1, S18, SDHA, YWHAZ, PPIA, RPL4, DNMT3A, DNMT3B, JHDM2A, KAT8 and PRM1 differed after cryopreservation (Zeng et al., 2014a). In the latter study, several micro-RNA (i.e. let-7c, ssc-miR-26a and ssc-miR-186) also underwent changes.
The cryopreservation of spermatozoa was introduced in the 1960s as a route to fertility preservation. Despite the extensive progress that has been made in this field, the biological and biochemical mechanisms involved in cryopreservation have not been thoroughly elucidated to date. Various factors during the freezing process, including sudden temperature changes, ice formation and osmotic stress, have been proposed as reasons for poor sperm quality post-thaw. Little is known regarding the new aspects of sperm cryobiology, such as epigenetic and proteomic modulation of sperm and trans-generational effects of sperm freezing. This article reviews recent reports on molecular and cellular modifications of spermatozoa during cryopreservation in order to collate the existing understanding in this field. The aim is to discuss current freezing techniques and novel strategies that have been developed for sperm protection against cryo-damage, as well as evaluating the probable effects of sperm freezing on offspring health.
Cryopreservation of boar sperm induces differential microRNAs expression
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The Fas-Fas ligand (FasL) pathway is a major death receptor-signaling pathway. Binding of FasL to Fas induces activation of caspase-8 and triggers apoptosis [59]. Fas is a direct target of the let-7/miR-98 family.
Lower conception rates and litter sizes limit the wide use of artificial insemination with frozen-thawed boar sperm, due to a lack of understanding of the mechanisms that cause cryodamage and cryoinjury to sperm during cryopreservation. CryoMiRs, a family of freeze-related microRNAs (miRNAs), are associated with freeze tolerance, and regulate metabolism in mammalian hibernators and insects. Thus, we speculate that miRNAs maybe involved in the regulation of the freeze-thaw process and may affect boar sperm function. In this study, we studied the differential expression of 46 miRNAs that have roles in spermatogenesis, sperm maturation, and sperm quality in response to cryopreservation (with or without 3% glycerol). The results indicated that, in response to cryopreservation with 3% glycerol, 14 miRNAs were significantly up-regulated, but only two miRNAs (miR-22 and miR-450b-5p) were significantly down-regulated, relative to fresh sperm. Preservation with 3% glycerol caused up-regulation of 17 miRNAs, but only caused down-regulation of one miRNA (miR-24), relative to sperm cryopreserved without glycerol. Functional annotations of these differentially expressed miRNAs indicated that these miRNAs and their targets are mainly associated with metabolic and cellular processes. Therefore, our findings show that cryopreservation results in changes in miRNA expression, and suggest that the anti-freeze mechanisms of boar sperm need to be studied further.
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The quantitative real time PCR (qRT-PCR) has become an important tool for gene-expression analysis for a selected number of genes in life science. Although large dynamic range, sensitivity and reproducibility of qRT-PCR is good, the reliability majorly depend on the selection of proper reference genes (RGs) employed for normalization. Although, RGs expression has been reported to vary considerably within same cell type with different experimental treatments. No systematic study has been conducted to identify and evaluate the appropriate RGs in spermatozoa of domestic animals. Therefore, this study was conducted to analyze suitable stable RGs in fresh and frozen-thawed spermatozoa. We have assessed 13 candidate RGs (BACT, RPS18s, RPS15A, ATP5F1, HMBS, ATP2B4, RPL13, EEF2, TBP, EIF2B2, MDH1, B2M and GLUT5) of different functions and pathways using five algorithms. Regardless of the approach, the ranking of the most and the least candidate RGs remained almost same. The comprehensive ranking by RefFinder showed GLUT5, ATP2B4 and B2M, MDH1 as the top two stable and least stable RGs, respectively. The expression levels of four heat shock proteins (HSP) were employed as a target gene to evaluate RGs efficiency for normalization. The results demonstrated an exponential difference in expression levels of the four HSP genes upon normalization of the data with the most stable and the least stable RGs. Our study, provides a convenient RGs for normalization of gene-expression of key metabolic pathways effected during freezing and thawing of spermatozoa of buffalo and other closely related bovines.

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^☆: Statement of funding: This work was supported by a Grant from the National Natural Science Foundation of China (No. 30901028).

¹: These authors contributed equally to this work.

View full text

Selection of optimal reference genes for quantitative RT-PCR studies of boar spermatozoa cryopreservation☆

Abstract

Introduction

Section snippets

Spermatozoa collection and cryopreservation

RNA quality

Verification, amplification efficiency, and expression level of candidate reference genes

Discussion

Conclusion

Acknowledgments

Theriogenology

Fertil. Steril.

J. Dairy Sci.

Mol. Aspects Med.

Anal. Biochem.

Biochem. Biophys. Res. Commun.

Theriogenology

Biochem. Biophys. Res. Commun.

Theriogenology

Meat Sci.

Biochem. Biophys. Res. Commun.

Mol. Cell. Probes

J. Biotechnol.

Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets

Cancer Res.

Basic principles of real-time quantitative PCR

Expert Rev. Mol. Diagn.

Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon minireview

J. Androl.

GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues

Phys. Genomics

Evaluation of suitable reference genes for gene expression studies in bronchoalveolar lavage cells from horses with inflammatory airway disease

BMC Mol. Biol.

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

J. Mol. Endocrinol.

AZ of Quantitative PCR

Quantitative real-time RT-PCR – a perspective

J. Mol. Endocrinol.

Highly purified spermatozoal RNA obtained by a novel method indicates an unusual 28S/S18 rRNA ratio and suggests impaired ribosome assembly

Mol. Hum. Reprod.

‘Desperate house genes’: the dramatic example of hypoxia

Br. J. Cancer.

Effects of centrifugation before freezing on boar sperm cryosurvival

J. Androl.

Evaluation and identification of reliable reference genes for pharmacogenomics, toxicogenomics, and small RNA expression analysis

J. Cell. Physiol.

Evaluation of suitable reference genes for gene expression studies in porcine alveolar macrophages in response to LPS and LTA

BMC Res. Notes

Evaluation of suitable reference genes for gene expression studies in porcine PBMCs in response to LPS and LTA

BMC Res. Notes

Selection of reference genes for qPCR in hairy root cultures of peanut

BMC Res. Notes