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

<div><p>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 <i>ADCY10</i> 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 (<i>Cac8I</i>) 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 <i>ADCY 10</i>. 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 <i>ADCY10</i> mRNA fragment was detectable by <i>Cac8I</i> 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.</p></div

Detection of <i>ADCY10</i> variant mRNAs in bull testes by Northern blotting.

<p>Total RNAs from testes (1.0 µg), kidneys (1.1 µg) and livers (1.2 µg) were separated by electrophoresis in gels containing formaldehyde and subsequently transferred onto nylon membranes. The obtained membranes were hybridized with either an antisense or sense DIG-labeled cRNA probe which was synthesized with the recombinant DNA into which the product amplified by RT-PCR using primer set #11 was inserted. In addition, the bands of the ribosomal RNAs on the membranes were visualized with a transilluminator. (The panel is representative of three replicates).</p

Detection of <i>ADCY10</i> variant mRNAs in testes and determination of the site of splicing errors.

<p>A: The products amplified by RT-PCR, using ten sets of primers and tissular RNAs, were separated in 1% agarose gels containing 0.01% ethidium bromide. <i>Glycerol-3-phosphate dehydrogenase</i> (<i>G3PDH</i>) was used as the control. (The panel is representative of three replicates). <b>B:</b> Three recombinants (a)–(c) into which the DNA fragments amplified by RT-PCR using primer set #4 were inserted were subjected to the colony PCR with a forward primer for <i>pSPT19</i> and a reverse primer of primer set #4. <b>C:</b> The numbers surrounded by boxes indicate the numbers of exons of bull <i>ADCY10</i>. The variants I, II and III contain the nucleotide sequences of inserted parts of recombinants (a), (b) and (c), respectively. The black-colored boxes show the exons containing the stop codons. The question marks indicate that the exon containing the stop codon remains to be revealed. The parts surrounded by the broken line indicate the exons coding the cyclase domains.</p

Detection of vestiges of splicing errors using residuals of testicular mRNAs in bull spermatozoa.

<p>A: The products amplified by RT-PCR with total RNA from spermatozoa and primer set #4 were visualized with a transilluminator. <b>B:</b> Twenty-six recombinants into which the products amplified by RT-PCR with primer set #4 and RNA from frozen-thawed ejaculated spermatozoa were inserted were subjected to colony PCR using a forward primer for a <i>pSPT19</i> plasmid and a reverse primer of a primer set #4, in order to check the successful ligation. The molecular size of colony PCR products is larger by approximately 50 bases than that of PCR products (see panel A), because the colony PCR products contain the nucleotides coded by plasmids. The nucleotide sequences of the inserted parts of obtained recombinants were revealed by sequential analyses. Consequently, the recombinants coded variant II [the representative of 16 recombinants: (d)] or variant III [the representative of 10 recombinants: (e)]. <b>C:</b> Inserted DNAs of the 26 recombinants were treated with a restriction enzyme (<i>Cac8I</i>). Consequently, 16 recombinants coding variant II without the cleavage site for <i>Cac8I</i> were detected as the single band [representative: (d)]. Ten recombinants coding variant III with the cleavage site for <i>Cac8I</i> were detected as two bands [representative: (e)].</p

Multiple alignment of nucleotide sequences and amino acid sequences coded by <i>ADCY10</i> variants I – III.

<p>A part of sequences of nucleotides and amino acids coded by each variant are shown in the upper lanes and lower lanes, respectively. The asterisks mark identical nucleotides between the bull genome sequence (NW_001494712), and bull <i>ADCY10</i> variants I, II or III, and the hyphens are the lacking parts of the corresponding nucleotides between these sequences. The nucleotides surrounded by a box show the intronic sequences. “Stop” indicates the nucleotide triplet coding the stop codon. The underlined amino acids indicate the second cyclase domain.</p

Examination of the relationship between the splicing accuracy in <i>ADCY10</i> transcription process and sperm motility.

<p>The percentages of variant II mRNAs in frozen-thawed ejaculated spermatozoa positively correlated with those of motile spermatozoa immediately after thawing (n = 6, R = 0.87, p = 0.025).</p

A schematic drawing showing a model of the repair for TMZ and ACNU induced DNA damage.

<p>A schematic drawing showing a model of the repair for TMZ and ACNU induced DNA damage.</p

siRNA silencing of <i>FANCD1/BRCA2</i> in glioblastoma A172 cells after ACNU and TMZ treatments.

<p><b>a</b>, expression analysis of FANCD1/BRCA2 in cells transfected with siRNA for human <i>FANCD1/BRCA2</i> or the negative control siRNA using Western blots. Actin was used as a loading control, and the relative ratios of protein were normalized using actin levels. <b>b</b>, effect of siRNA silencing of <i>FANCD1/BRCA2</i> on cellular sensitivity to ACNU and TMZ. <b>c</b>, effect of siRNA silencing of <i>FANCD1/BRCA2</i> on cell growth after ACNU or TMZ treatment. Open columns, negative control siRNA; closed columns, <i>FANCD1/BRCA2</i> siRNA. Columns show the mean of three independent experiments; the bars indicate the SD. An asterisk (*) indicates significant difference (<i>P</i><0.05). Two asterisks (**), <i>P</i><0.01; three asterisks (***), <i>P</i><0.001.</p

Rad51 foci formation in glioblastoma A172 cells after ACNU and TMZ treatments.

<p><b>a</b>, typical photo. <b>b</b>, open columns, negative control siRNA; closed columns, <i>FANCD1/BRCA2</i> siRNA. Columns show the mean of two independent experiments; bars indicate the SD.</p

Sensitivity to TMZ.

<p><b>a</b>, <i>FANCwt</i> cells (open circles), <i>FANCA^−/−C^−/−</i> cells (closed triangles), <i>FANCC^−/−</i> cells (closed squares), and <i>FANCA^−/−</i> cells (closed circles); <b>b</b>, <i>FANCwt</i> cells (open circles) and <i>FANCD2^−/−</i> cells (closed circles); <b>c</b>, <i>FANCGwt</i> cells (open circles), <i>FANCGrev</i> cells (closed triangles), and <i>FANCG^−/−</i> cells (closed circles); <b>d</b>, <i>FANCD1wt</i> cells (open circles), <i>FANCD1rev</i> cells (closed triangles), and <i>FANCD1mt</i> cells (closed circles). Each point represents the mean of three independent experiments; bars indicate the SD.</p