The Porcine TSPY Gene Is Tricopy but Not a Copy Number Variant

<div><p>The testis-specific protein Y-encoded (TSPY) gene is situated on the mammalian Y-chromosome and exhibits some remarkable biological characteristics. It has the highest known copy number (CN) of all protein coding genes in the human and bovine genomes (up to 74 and 200, respectively) and also shows high individual variability. Although the biological function of TSPY has not yet been elucidated, its specific expression in the testis and several identified binding domains within the protein suggests roles in male reproduction. Here we describe the porcine TSPY, as a multicopy gene with three copies located on the short arm of the Y-chromosome with no variation at three exon loci among 20 animals of normal reproductive health from four breeds of domestic pigs (Piétrain, Landrace, Duroc and Yorkshire). To further investigate the speculation that porcine TSPY is not a copy number variant, we have included five Low-fertility boars and five boars with exceptional High-fertility records. Interestingly, there was no difference between the High- and Low-fertile groups, but we detected slightly lower TSPY CN at all three exons (2.56-2.85) in both groups, as compared to normal animals, which could be attributed to technical variability or somatic mosaicism. The results are based on both relative quantitative real-time PCR (qPCR) and droplet digital PCR (ddPCR). Chromosomal localization of the porcine TSPY was done using fluorescence in situ hybridization (FISH) with gene specific PCR probes.</p></div

Y-chromosome specific fluorescence in situ hybridization signal of FISH probe#1–5 with Tyramide signal detection on a boar metaphase.

<p>DAPI stained metaphase chromosomes are converted grayscale and AF594 hybridization signal on the short arm of the Y-chromosome is red.</p

TSPY CN relative to the single copy AR gene, as determined by qPCR.

<p>CN was measured at three loci (Exon 1, 3, 5) in four breeds. Columns represent the average value (±SD) of 5 animals from the same breed. Different letters within efficiency calculation methods (STD or LinRegPCR) represent statistical significance according to two-way ANOVA (p<0.05).</p

Schematic view of the porcine TSPY gene (exons 1–5 in grey) and location of primers (green), TaqMan probes (red), PflMI enzyme cut positions (blue) and FISH probes (purple).

<p>Schematic view of the porcine TSPY gene (exons 1–5 in grey) and location of primers (green), TaqMan probes (red), PflMI enzyme cut positions (blue) and FISH probes (purple).</p

TSPY CN by ddPCR at exons 1, 3 and 5 in normal animals from four breeds, High-fertile and Low-fertile animals.

<p>Columns represent the average value (±SD) of 5 animals. Letters mark values that are statistically different (p<0.05).</p

Primer sequences used for real time PCR experiments and FISH probe preparation.

a<p>PCR product size in base pairs,</p>b<p>primer used in combination with HSFY-E1F,</p>c<p>with HSFY-E2R.</p><p>The table shows the primer name, primer sequences, the predicted PCR product size (base pairs, bp), GenBank accession number of the sequence that was used as the target for primer design, gene name and symbol, and the unique annealing temperature (°C) that was used for the PCR amplification.</p

Gel electrophoresis of hybridization probes (A) and flourescence <i>in situ</i> hybridization (FISH) analysis (B–G).

<p>A) The largest intense band in lane 1 shows 2072 bp fragment of the 100 bp DNA ladder (Invitrogen Canada Inc.). PCR products amplified by primer pairs HSFY-E1F/HSFY-E2R, HSFY-E1F/HSFY-E1R, HSFY-E2F/HSFY-E2R were loaded into lanes 2, 3, 4, respectively. FISH images show the hybridization of the same products in the same order, thus using the whole gene (E), exon 1 (F) or exon 2 (G) specific probes. Images in the upper row (B, C, D) show the metaphase chromosomes with only 4′,6-diamidino-2-phenylindole (DAPI) counterstaining. Bar represents 10 µm.</p

<i>HSFY</i> copy number in the calibrator sample measured by real time PCR with three different primers.

<p>There is no significant difference in mean <i>HSFY</i> copy number as measured by three different primer sets (HSFY8: HSFY8F/HSFY8R; HSFY10: HSFY10F/HSFY10R, and HSFY16: HSFY16F/HSFY16R) which target different regions of the gene. <i>HSFY</i> copy numbers for primer sets HSFY8, HSFY10 and HSFY16 were based on 20, 4 and 3 separate real time PCR runs, respectively.</p

Sequence alignment of the conserved DNA-binding domain in various species.

<p>The conserved heat shock factor (HSF) type DNA binding-domain shows a high degree of sequence homology among different species. Amino acids conserved between at least 5 proteins are marked with shaded boxes. The accession numbers for the proteins deposited in the sequence database on the NCBI website are as follows: hHSFY1 NP_149099.2; hHSFY2 NP_714927.1; mHSFYL NP_081937.1; rat NP_001012132.1; rhesus macaque ACL51668.1; cat NP_001035212.1; bovine (Hereford) NP_001070474.1.</p

Gel electrophoresis of the real time PCR product of both male and female DNA samples for <i>HSFY</i> and <i>ZAR1</i>.

<p><i>HSFY</i> shows male-specific amplification indicating that the target genes are on the Y chromosome. Zygote arrest 1 (<i>ZAR1</i>) is the autosomal control gene which shows amplification in both the male and female sample. N) Negative control M) 100 bp ladder – the top and bottom band represent 200 and 100 bp, respectively (Invitrogen Canada Inc.).</p