Cross-species hybridizations on a multi-species cDNA microarray to identify evolutionarily conserved genes expressed in oocytes

Abstract

Background: Comparative genomic analysis using cDNA microarray is a new approach and a useful tool to identify important genetic sequences or genes that are conserved throughout evolution. Identification of these conserved sequences will help elucidate important molecular mechanisms or pathways common to many species. For example, the stockpiled transcripts in the oocyte necessary for successful fertilization and early embryonic development still remain relatively unknown. The objective of this study was to identify genes expressed in oocytes and conserved in three evolutionarily distant species.

Results: In this study we report the construction of a multi-species cDNA microarray containing 3,456 transcripts from three distinct oocyte-libraries from bovine, mouse and Xenopus laevis. Following the cross-species hybridizations, data analysis revealed that 1,541 positive hybridization signals were generated by oocytes of all three species, and 268 of these are preferentially expressed in the oocyte. Data reproducibility analyses comparing same-species to cross-species hybridization indicates that cross-species hybridizations are highly reproducible, thus increasing the confidence level in their specificity. A validation by RT-PCR using gene- and species-specific primers confirmed that cross-species hybridization allows the production of specific and reliable data. Finally, a second validation step through gene-specific microarray hybridizations further supported the validity of our cross-species microarray results. Results from these cross-species hybridizations on our multi-species cDNA microarray revealed that SMFN (Small fragment nuclease), Spin (Spindlin), and PRMT1 (Protein arginine methyltransferase 1) are transcripts present in oocytes and conserved in three evolutionarily distant species.

Conclusion: Cross-species hybridization using a multi-species cDNA microarray is a powerful tool for the discovery of genes involved in evolutionarily conserved molecular mechanisms. The present study identified conserved genes in the oocytes of three distant species that will help understand the unique role of maternal transcripts in early embryonic development.

Figure 1

Venn diagram representing clones present in oocytes of one, two or all three species. Clones are considered as present when their signal intensities are above threshold in every replicates (16/16), they are considered as ambiguous when their signal intensities are above threshold in 8 to 15 replicates, and finally are considered absent when their signal intensities are above threshold in less than 7 replicates. A) All the clones on the microarray slides are considered, B) only the clones preferentially expressed in the oocyte are considered. For the clones present in all three species, the number of clones derived from each oocyte library is given in table 1.

Figure 2

TIGR Multiexperiment Viewer representation of microarray data. Transcripts were ordered horizontally by clone number and grouped by species. Average log signal intensities are presented for clones hybridized with probes corresponding to one of the three species. The expressed clones that are conserved across all three species are represented in (A) and those conserved across species and preferentially expressed in the oocyte are represented in (B).

Figure 3

Same-species versus cross-species hybridization. The hybridization pattern of one member of the Zp gene family is represented. Average log signal intensities for two different clones corresponding to the same Zp genes from each species for A) same-species hybridization and B) cross-species hybridization. Probe species are indicated inside each bar. Bt; Bos taurus, Mm; Mus musculus, Xl; Xenopus laevis.

Figure 4

Average log signal intensity for all 3,456 clones. Shown here are average global log signal intensities values for same-species and cross-species hybridization. Results are separated according to the respective species of the clone in order to compare same-species versus cross-species hybridization. Probe species are indicated inside each bar. Bt; Bos taurus, Mm; Mus musculus, Xl; Xenopus laevis.

Figure 5

Validation of microarray result by simple detection analysis using RT-PCR for three candidate transcripts. Amplifications are performed in oocytes of two species with gene- and species-specific primers designed according to the known sequences in respective species. A) Amplification in mouse and Xenopus laevis oocytes of SMFN transcript originally found in the bovine oocyte-library. B) Amplification in bovine and Xenopus laevis oocytes of Spin transcript originally found in the mouse oocyte-library. C) Amplification in bovine and mouse oocytes of Prmt1 transcript originally found in the Xenopus laevis oocyte-library. Bt; Bos Taurus, Mm; Mus musculus, Xl; Xenopus laevis, Smfn; Small fragment nuclease, Spin; Spindlin, Prmt1; Protein arginine methyltransferase 1, Oo; oocyte, N; negative control.

Figure 6

Gene-specific cross-species hybridization on our multi-species array. Example of a representative result for average log signal intensities is shown for all the 3,456 transcripts on the array. The red dotted line represents background intensity. The three candidate genes and our positive control GAPD are identified. Note that Spin transcript is present 3 times on the multi-species microarray. Smfn; Small fragment nuclease, Spin; Spindlin, Prmt1; Protein arginine methyltransferase 1.