Selection of optimal reference genes for quantitative RT-PCR studies of boar spermatozoa cryopreservation☆
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 (qbaseplus 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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2018, Reproductive BioMedicine OnlineCitation 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.
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Statement of funding: This work was supported by a Grant from the National Natural Science Foundation of China (No. 30901028).
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These authors contributed equally to this work.