TOPAZ1, a novel germ cell-specific expressed gene conserved during evolution across vertebrates

Abstract

Background: We had previously reported that the Suppression Subtractive Hybridization (SSH) approach was relevant for the isolation of new mammalian genes involved in oogenesis and early follicle development. Some of these transcripts might be potential new oocyte and granulosa cell markers. We have now characterized one of them, named TOPAZ1 for the Testis and Ovary-specific PAZ domain gene.

Principal findings: Sheep and mouse TOPAZ1 mRNA have 4,803 bp and 4,962 bp open reading frames (20 exons), respectively, and encode putative TOPAZ1 proteins containing 1,600 and 1653 amino acids. They possess PAZ and CCCH domains. In sheep, TOPAZ1 mRNA is preferentially expressed in females during fetal life with a peak during prophase I of meiosis, and in males during adulthood. In the mouse, Topaz1 is a germ cell-specific gene. TOPAZ1 protein is highly conserved in vertebrates and specifically expressed in mouse and sheep gonads. It is localized in the cytoplasm of germ cells from the sheep fetal ovary and mouse adult testis.

Conclusions: We have identified a novel PAZ-domain protein that is abundantly expressed in the gonads during germ cell meiosis. The expression pattern of TOPAZ1, and its high degree of conservation, suggests that it may play an important role in germ cell development. Further characterization of TOPAZ1 may elucidate the mechanisms involved in gametogenesis, and particularly in the RNA silencing process in the germ line.

Figure 1. Structure of the sheep TOPAZ1 gene.

Sheep TOPAZ1 cDNA derived from exons 1–20 and contained 4952 nucleotides. TOPAZ1 was composed of twenty exons in sheep, bovine, human and mouse. The PAZ domain is located in the second exon and the CCCH domain in exon 8. The nucleotide number of each exon of sheep TOPAZ1 is indicated under the Figure.

Figure 2. (A) Nucleotide sequence conservation of mammalian TOPAZ1 transcripts and (B) Conservation of PAZ and CCCH domains of TOPAZ1 protein in mammalian species.

(A) The percentage nucleotide identity between sheep TOPAZ1 cDNA and that of other mammalian species is indicated on the right-hand side. These alignments were obtained using the Vista Genome browser (http://genome.lbl.gov/vista/index.shtml). (Ba) The PAZ domain surround (in red) consisted of 58 amino acids. The percentages of identity were 96%, 78%, and 71% between sheep-cattle, sheep-human and sheep-mouse, respectively. (Bb) The CCCH domain is indicated in the green box, and consisted of 20 amino acids. Amino acid identity was 90%, 85%, and 80% between sheep-cattle, sheep-human and sheep-mouse, respectively. Identical residues are noted with an asterisk.

Figure 3. Molecular phylogeny of TOPAZ1.

Phylogenetic analysis was performed on a 620 amino-acid alignment extending from the CCCH zinc finger domain to the C-terminal end using the neighbor-joining method ; (1,000 bootstrap replicates). The tree was not rooted. Accession numbers: Homo sapiens (human) NP_001138502; Pan troglodytes (chimpanzee) XP_526186; Macaca mulatta (rhesus monkey) XP_001114967; Mus musculus (house mouse) EDL09106; Rattus norvegicus (rat) EDL76795; Bos taurus (cattle) NW_001494083.2 (genomic); Ovis aries (sheep) HM631979; Equus caballus (horse) NW_001867381.1 (genomic); Canis familiaris (dog) NW_876276.1 (genomic); Monodelphis domestica (gray short-tailed opossum) XP_001381211; Ornithorhynchus anatinus (duck-billed platypus) NW_001794448.1 (genomic); Gallus gallus (chicken) NW_001471633.1 (genomic); Anolis carolinensis (green anole, lizard) ENSEMBL scaffold_98 (genomic); Xenopus tropicalis (frog) ENSEMBL scaffold_320 (genomic); Ictalurus punctatus (catfish) ABD91555; Danio rerio (zebrafish) NW_001877999.1 (genomic); Tetraodon nigroviridis (pufferfish) ENSEMBL Chr. 21.

Figure 4. Expression of TOPAZ1 mRNA in different sheep (A) and mouse (B) tissues.

(A) The expression of sheep TOPAZ1 mRNA was tested using RT-PCR in four adult somatic tissues (heart, liver, lung and kidney) and in gonads (60 dpc ovary and adult testis). (B) The expression of the mouse Topaz1 gene was tested in seven adult somatic tissues (brain, heart, stomach, liver, lung spleen and kidney) and in adult testis. GAPDH (glyceraldehyde-3P-dehydrogenase) and Actb amplification served as loading controls.

Figure 5. Expression of TOPAZ1 and DMC1 mRNA in sheep gonads at different developmental stages.

Quantification of TOPAZ1 (A) and DMC1 (B) genes expression using quantitative real-time RT-PCR analysis at several fetal stages (44, 55, 60, 65, 75, 82, 90, 114 dpc) and in adult ovary (red histograms) and testis (blue histograms). The HPRT1 (hypoxanthine phosphoribosyltransferase) gene was used as a reporter gene. Values indicated on the graph are means ± SEM of two independent RT experiments at each stage.

Figure 6. Expression of Topaz1 mRNA in mouse gonads at different developmental stages.

Quantification of Topaz1 gene expression using quantitative real-time RT-PCR analysis at several fetal stages (12.5, 13.5, 14.5, 16.5 dpc), after birth (1, 5, 10, 15 dpp) and in adult ovary (pink histograms) and testis (light blue histograms). The Actb gene was used as a reporter gene. Values indicated on the graph are means ± SEM of three independent RT experiments at each stage.

Figure 7. Expression of the Topaz1 transcript in mouse enriched germ cell fractions.

Germ cells were isolated from 13 dpc gonads by means of SSEA1+ immunomagnetic isolation. Quantification of Topaz1 gene expression using quantitative real-time RT-PCR analysis in whole ovaries (pink histogram) and testes (light blue histogram) and in germ cell fractions (hatched histograms). Values indicated on the graph are means ± SEM of three independent samples.

Figure 8. Expression of Topaz1, Mvh and Wt1 transcripts in mouse WlacZ/+ or WlacZ/WlacZ gonads.

(A) Expression of Mvh, a gene only expressed in germ cells, was used to verify the absence of germ cells from WlacZ/WlacZ gonads (F−/− and M−/−, hatched histograms) in contrast to WlacZ/+ gonads (F+/− and M+/−, plain histograms). (B) Expression of the Wt1 gene in somatic cells was used to verify the integrity of somatic cells. (C) The expression of Topaz1 was determined by qRT-PCR analysis in WlacZ/+ or WlacZ/WlacZ ovary and testis. The Hprt1 gene was used as a reporter gene. Values indicated on the graph are means ± SEM of two independent samples.

Figure 9. Effect of retinoic acid (RA) on Topaz1 expression in mouse fetal testis.

Mouse testes at 11 dpc were cultured for 3 days with (RA+, hatched histograms) or without (RA−, plain histograms) retinoic acid (10⁻⁶ M). At the end of the culture period, total RNA was extracted and Rec8 (A) and Topaz1 (B) mRNA expressions were measured by real-time quantitative RT-PCR. Values indicated on the graph are means ± SEM of three independent samples.

Figure 10. Specificity of anti-TOPAZ1 antibody by Western blot.

Western-blotting experiments were performed with cytosolic extracts from sheep adult testis, fetal ovary and liver. Anti-TOPAZ1 antibody recognized a 199 kDa protein in sheep adult testis and 60 dpc ovary, but no band was detected in sheep liver. As a positive control, ACTB expression was detected in each sample.

Figure 11. Immunodetection of TOPAZ1 protein in sheep fetal ovary.

Immunofluorescence was performed on transversal sections of sheep 60 dpc ovaries to detect TOPAZ1 (A) and MVH (B) proteins. Pre-immune serum (PI) was also tested (C). For each section, DAPI was performed to detect nuclei (D-E-F). TOPAZ1 showed similar cellular localization as MVH (A, B). The immunolabeling of TOPAZ1 and MVH was merged with DAPI (G, H). TOPAZ1 can be detected in the cytoplasm of germ cells in ovine fetal ovary. PI serum of TOPAZ1 did not reveal any staining (C). Scale bars = 200 µm in H (applies to A–G).

Figure 12. Immunodetection of TOPAZ1 protein in mouse adult testis.

Immunofluorescence was performed on sections of mouse adult testis to detect TOPAZ1 (A) and MVH (B) proteins. Anti-TOPAZ1 antibody raised against sheep protein was used to detect mouse Topaz1. For each section, DAPI was performed to detect nuclei (C–D). Mouse TOPAZ1 displayed a localization similar to that of MVH (A, B). The immunostaining of TOPAZ1 and MVH was merged with DAPI (E, F). TOPAZ1 can be detected in the cytoplasm of germ cell in mouse testis. Scale bars = 200 µm in H (applies to A–F).