Laboratory of Reproductive Biology, Graduate School of Agricultural Science, Kobe University, Kobe, Japan.
PLoS One. 2013;8(2):e57296. doi: 10.1371/journal.pone.0057296. Epub 2013 Feb 26.
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.
世界范围内家畜雄性生殖性能下降是一个严重的问题。我们提出假设，mRNA 剪接错误导致的异常精子运动相关蛋白的衍生可能是其因果因素之一。据认为，新鲜的睾丸组织对于检测 mRNA 的剪接错误是必要的。然而，通过手术方法从许多农业上重要的公牛中获得睾丸组织是困难的，因为这样的程序可能对公牛的生殖性能产生有害影响。本研究的目的是检验从射出的精子中收集的 mRNA 片段作为替代分析样本检测剪接错误的有用性。在第一个实验中，我们对编码精子运动调节剂“cAMP”合成酶的公牛睾丸 ADCY10mRNA 的选择性剪接和剪接错误进行了特征描述。在睾丸中，通过选择性剪接产生了缺少外显子 11 的变异体，编码截断的 ADCY10。然而，在缺少外显子 11 的变异体中偶尔观察到伴随着第二个环化酶结构域移码的剪接错误。这种异常的变异体由于剪接错误保留了连接外显子 10 起始部分的内含子核苷酸（4 个碱基，CCAG），并因此产生了识别核苷酸序列（GCNNGC）的限制性内切酶（Cac8I）的切割位点。在第二个实验中，我们从射出的精子中回收了残留的睾丸 mRNA 片段，并观察到 ADCY10 的剪接错误导致的异常变异体的衍生。射出的精子保留了编码外显子 9、10、12 和 13 的缺少外显子 11 的变异体的 mRNA 片段。此外，使用精子 mRNA，可以通过 Cac8I 消化处理检测到上述 ADCY10mRNA 片段的异常变异体。这些结果表明精子 mRNA 片段可用于检测公牛睾丸 mRNA 中的剪接错误。
