Novel Approach for the Detection of the Vestiges of Testicular mRNA Splicing Errors in Mature Spermatozoa of Japanese Black Bulls

Taichi Noda,Mitsuhiro Sakase,Moriyuki Fukushima,Hiroshi Harayama,Stefan Schlatt

doi:10.1371/journal.pone.0057296

Taichi Noda, Mitsuhiro Sakase + Show 3 more

Open Access

https://doi.org/10.1371/journal.pone.0057296

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Abstract

There is a serious problem with the reduction of male reproductive performance of the livestock in the world. We have a hypothesis that the splicing error-caused derivation of aberrant sperm motility-related proteins may be one of its causal factors. It is thought that fresh testicular tissues are necessary for the detection of splicing errors of the mRNA. However, it is difficult to obtain testicular tissues from a number of agriculturally important bulls by surgical methods, because such procedures may have deleterious effects on bulls’ reproductive performance. The aim of this study was to examine the usefulness of mRNA fragments collected from ejaculated spermatozoa as alternative analytical samples for detection of the splicing errors. In the first experiment, we characterized the alternative splicing and splicing error of bull testicular ADCY10 mRNA which coded the synthase of the regulatory molecule for sperm motility “cAMP”. In testes, the exon 11-lacking variant coding the truncated ADCY10 was derived by alternative splicing. However, splicing errors, which accompanied the frame shift in the second cyclase domain, were occasionally observed in the exon 11-lacking variant. This aberrant variant retained intronic nucleotides (4 bases, CCAG) connecting the initial part of exon 10 due to splicing errors and consequently yielded the cleavage site for a restriction enzyme (Cac8I) which recognized the nucleotide sequences (GCNNGC). In the second experiment, we recovered residual testicular mRNA fragments from ejaculated spermatozoa and observed the splicing error-caused derivation of the aberrant variant of ADCY 10. Ejaculated spermatozoa conserved mRNA fragments of the exon 11-lacking variant coding exons 9, 10, 12 and 13. Moreover, the above-mentioned aberrant variant of ADCY10 mRNA fragment was detectable by Cac8I digestion treatment using the sperm mRNAs. These results indicate the utility of sperm mRNA fragments for the detection of splicing errors in bull testicular mRNAs.

Highlights

RNA splicing is carried out by the spliceosome, which is composed of seven types (e.g., U1, U2) of small nuclear ribonucleoprotein particles and a large number of auxiliary proteins [e.g., U2 auxiliary factor (U2AF), splicing factor (SF) 1] [1]
These results indicate the utility of sperm mRNA fragments for the detection of splicing errors in bull testicular mRNAs
All PCR products were amplified more efficiently in testes than in livers. These results were supported by Northern blotting using a cRNA probe with affinity to the nucleotide sequences coding the first cyclase domain (Figure 2)

Summary

Introduction

RNA splicing is carried out by the spliceosome, which is composed of seven types (e.g., U1, U2) of small nuclear ribonucleoprotein particles (snRNPs) and a large number of auxiliary proteins [e.g., U2 auxiliary factor (U2AF), splicing factor (SF) 1] [1]. Three types of intronic sequences (splicing signals) are important in the recognition of the borderline between the exon and the intron. Splicing errors occur occasionally due to incorrect selection of the cryptic splicing signals, which are similar to the true splicing signals [2]. Such splicing errors are involved in splicing flexibility [2]. The splicing flexibility is a critical factor in the derivation of various transcripts from a single gene in the eukaryote, and it plays important roles in genetic evolution and biological variation [2]

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