Comparison of the identity (%) of amino acid sequences of three <i>Sus scrofa</i> Piwi proteins with known Piwi proteins of other mammals.

<p>The table was based on ClustalW alignment of the amino acid sequences of Piwi proteins. The GenBank accession numbers of these sequences are: <b><i>Mus musculus</i></b> - NP_067286 (Piwil1), NP_067283 (Piwil2), NP_808573 (Piwil4); <b><i>Homo sapiens</i></b> - NP_004755 (Piwil1 isoform 1), NP_001129193 (Piwil2), NP_689644 (Piwil4); <b><i>Macaca mulatta</i></b> - NP_001182640 (Piwil1), NP_001182592 (Piwil2), NP_001182443 (Piwil4); <b><i>Pan troglodytes</i></b> - XP_001137815 (predicted Piwil1 isoform 1), XP_528083 (predicted Piwil2), XP_001143022 (predicted Piwil4 isoform 2); <b><i>Rattus norvegicus</i></b> - NP_001102323 (Piwil1), NP_001100746 (Piwil2), Q4G033 (Piwil4).</p

Size distribution of <i>Sus scrofa</i> piRNA isolated from testes (<b>A</b>) and ovaries (<b>B</b>).

<p>Size distribution of <i>Sus scrofa</i> piRNA isolated from testes (<b>A</b>) and ovaries (<b>B</b>).</p

Altered Expression of Porcine <em>Piwi</em> Genes and piRNA during Development

<div><p>Three <em>Sus scrofa Piwi</em> genes (<em>Piwil1</em>, <em>Piwil2</em> and <em>Piwil4</em>) encoding proteins of 861, 985 and 853 aminoacids, respectively, were cloned and sequenced. Alignment of the Piwi proteins showed the high identity between <em>Sus scrofa</em> and <em>Homo sapiens</em>. Relative transcript abundance of porcine <em>Piwil1</em>, <em>Piwil2</em> and <em>Piwil4</em> genes in testes, ovaries and oocytes derived from sexually immature and mature animals was examined using Real-Time PCR. Expression of the three <em>Piwi</em> mRNAs was proved to be tissue specific and restricted exclusively to the gonads. In testes of adult pigs the highest relative transcript abundance was observed for the <em>Sus scrofa Piwil1</em> gene. On the other hand, in testes of neonatal pigs the <em>Piwil1</em> transcript level was over 2–fold reduced while the level of <em>Piwil2</em> transcript was higher. As regards the expression of the <em>Piwil4</em> transcript, its level was 34-fold elevated in testes of neonatal piglet when compared to adult male. In ovaries of prepubertal and pubertal female pigs transcript abundance of the three <em>Piwi</em> genes was significantly reduced in comparison with testes. However, similarly to testes, in ovaries of neonatal pigs the <em>Piwil</em>2 gene was characterized by the highest relative transcript abundance among the three <em>Piwi</em> genes analysed. In prepubertal and pubertal oocytes <em>Piwil1</em> transcript was the most abundant whereas the expression of <em>Piwil4</em> was undetectable. We also demonstrated that expression of piRNA occurs preferentially in the gonads of adult male and female pigs. Moreover, a piRNA subset isolated from ovaries was 2–3 nucleotides longer than the piRNA from testes.</p> </div

Differences in aminoacid sequences of <i>Sus scrofa</i> Piwi proteins.

<p>Differences in aminoacid sequences of <i>Sus scrofa</i> Piwi proteins.</p

piRNA class of ∼30-nt small RNA identified in <i>Sus scrofa</i> testes and ovaries.

<p>RNA enriched with low-molecular-weight RNA was isolated from: <b>A</b> - testes of neonatal (1) and adult (2) male; <b>B</b> – ovaries of neonatal (1) and cyclic (2) females; <b>C</b> – ovaries of three-month-old (1), prebupertal (2) and pubertal (3) pigs. Approximately 8 µg of low-molecular-weight RNA was loaded on 15% acrylamide gel and stained using SYBR-gold. M – marker; set of synthetic RNA fragments available in the laboratory.</p

Sequence alignment showing the catalytic triad (DDH motif) of mouse Piwil1 and porcine Piwi family proteins.

<p>Catalytic aminoacids are shown in blue.</p

Expression levels of <i>Sus scrofa Piwi</i> family genes in testes of neonatal and adult animals.

<p>Total RNA extracted from the tissues was examined by real-time PCR. Relative transcript abundance of <i>Piwil1</i>, <i>Piwil2</i> and <i>Piwil4</i> genes was normalized to that of <i>β-actin</i> as a reference gene.</p

Expression levels of <i>Sus scrofa Piwi</i> family genes in ovaries of neonatal, prepubertal and pubertal females (A) and oocytes of prepubertal and pubertal females (B).

<p>Total RNA extracted from indicated tissues was examined by real-time PCR. Relative transcript abundance of <i>Piwil1</i>, <i>Piwil2</i> and <i>Piwil4</i> was normalized to that of <i>β-actin</i> as a reference gene.</p

Tissue specific expression of <i>Piwi</i> genes in testes of adult and neonatal pigs as well as in nine other somatic tissues of neonatal male.

<p>Expression of <i>Piwil1</i>, <i>Piwil2</i> and <i>Piwil4</i> was detected by reverse transcription PCR. Expression of <i>β-actin</i> was used as the inner control. NTC – non template control.</p