A Large Expansion of the HSFY Gene Family in Cattle Shows Dispersion across Yq and Testis-Specific Expression

Heat shock transcription factor, Y-linked (HSFY) is a member of the heat shock transcriptional factor (HSF) family that is found in multiple copies on the Y chromosome and conserved in a number of species. Its function still remains unknown but in humans it is thought to play a role in spermatogenesis. Through real time polymerase chain reaction (PCR) analyses we determined that the HSFY family is largely expanded in cattle (∼70 copies) compared with human (2 functional copies, 4 HSFY-similar copies). Unexpectedly, we found that it does not vary among individual bulls as a copy number variant (CNV). Using fluorescence in situ hybridization (FISH) we found that the copies are dispersed along the long arm of the Y chromosome (Yq). HSFY expression in cattle appears restricted to the testis and its mRNA correlates positively with mRNA markers of spermatogonial and spermatocyte cells (UCHL1 and TRPC2, respectively) which suggests that HSFY is expressed (at least in part) in early germ cells

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Schematic bovine <i>HSFY</i> gene and targeted regions of the primers used for the PCR experiments.

<p>The numbered boxes represent the gene exons and the adjoining line represents the intron. Primer sets HSFY8F/HSFY8R (111 bp), HSFY10F/HSFY10R (113 bp), and HSFY16F/HSFY16R (102 bp) were used to measure <i>HSFY</i> copy number with real time PCR and target individual exons. Primer set HSFYRNAF/HSFYRNAR (215 bp) was designed specifically for mRNA amplification: HSFYRNAF targets exon 1 and HSFYRNAR targets exon 2 and together they span an intron to avoid co-amplification of residual DNA. The three primer sets shown below the schematic <i>HSFY</i> gene were used to create PCR probes for the fluorescence <i>in situ</i> hybridization (FISH) experiments and they target the full gene (including both exons and the intron: HSFY-E1F/HSFY-E2R, 1686 bp), exon 1 (HSFY-E1F/HSFY-E1R, 515 bp) and exon 2 (HSFY-E2F/HSFY-E2R, 752 bp). Primer sequences are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017790#pone-0017790-t001" target="_blank">Table 1</a>.</p

<i>HSFY</i> expression in different tissues.

<p><i>HSFY</i> was analyzed in a variety of tissues including testis (T), lung (Lu), heart (H), liver (Li), kidney (K) and ovary (O) using real time PCR and two different <i>HSFY</i> primer sets to amplify the gene as well as the reference gene, <i>GAPDH</i>. The results show that both HSFYRNA (HSFYRNAF/HSFYRNAR) and HSFY8 (HSFY8F/HSFY8R) primer sets amplify <i>HSFY</i> only in the testis and no other tissue. All tissues show expression of <i>GAPDH</i>. M) 100 bp ladder (Invitrogen Canada Inc.).</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

Comparison of the percent homologies (%) of the complete <i>HSFY</i> protein (A) and HSF-type DNA-binding domain (B), for all species analyzed.

<p>In all cases, the percent homology was highest at the conserved HSF-type DNA-binding domain with the exception of an analysis of the two human HSFY copies which showed that both the complete protein sequence and conserved region were identical (100% homology). Bovine showed 51% and 72% homology with human HSFY for the complete protein and conserved domain, respectively. Bovine HSFY was most closely related to cat HSFY with a complete protein homology and conserved domain homology of 58% and 81%, respectively. The accession numbers for the proteins found in the NCBI database are as follows: human hHSFY1: NP_149099.2; hHSFY2: NP_714927.1, mouse NP_081937.1, rat NP_001012132.1, rhesus macaque ACL51668.1, cat NP_001035212.1, bovine NP_001070474.1.</p

<i>HSFY</i> copy number (n = 24), <i>HSFY</i> mRNA levels (n = 22) and percent 56-day non-return rate (NRR) for all bulls.

<p>N/A = testis sample not available.</p><p>There were no significant differences in mean HSFY copy number among bulls and the average was 73.3±0.8 copies. HSFY mRNA levels did vary significantly among bulls (p<0.0001) with an average of 0.489±0.038 relative to GAPDH. The 56-day non-return rates, a measure of field fertility, ranged from 49.6%–77.3%, with an overall average of 65.9%.</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

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

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