258 EXPRESSION PATTERN OF THE MATERNAL FACTOR ZYGOTE ARREST 1 (ZAR1) GENE IN OVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS

Abstract

Zygote arrest 1 (ZAR1) is an ovary-specific maternal factor that plays essential roles during the oocyte-to-embryo transition. It is evolutionary conserved in vertebrates, and the protein is characterized by the presence of a homeobox zing finger domain, suggesting its role in transcription regulation. Discovered in mice (Wu et al. 2003 Nat. Genet. 33, 187-191), its expression pattern has been analyzed in oocytes and pre-implantation embryos in mice, cattle, and pig (Pennetier et al. 2004 Biol. Reprod. 71, 1359-1366; Brevini et al. 2004 Mol. Reprod. Dev. 69, 375-380; Reprod. Biol. Endocrinol. 21, 4-12). Because no information on Zar1 ortholog in the ovine species is available, the aim of this study was to assess its presence and to analyze its expression pattern in oocytes and pre-implantation embryos. Reverse transcription-polymerase chain reaction (RT-PCR) was performed in germinal vesicle (GV) and IVM metaphase II (MII) oocytes, and, following IVFC, in 2- (2C), 4- (4C), 8- (8C), and 12-16- (12C-16C) cell embryos, morulae, and blastocysts. Primers were designed on the basis of bovine and swine conserved sequences and contained an intron-spanning region. The PCR product was sequenced and the alignment, performed with BLAST (www.ncbi.nlm.nih.gov/BLAST/), confirmed its homology with the Zar1 orthologous genes present in public databases, sharing 98%, 92%, and 89% nucleotide identity with the bovine, porcine, and human sequences, respectively. Real-time PCR revealed that Zar1 is present at its highest level in the GV oocyte, has a major decrease at the MII stage (around 10-fold), remains constant in the embryo first cleavage steps (2C-4C), drops again at the 8C stage (around 30-fold), and disappears in morulae and blastocysts. Our data demonstrate that a Zar1 ortholog is present in the ovine species. Results are also in accordance with the expression patterns characterized in other mammalian species and suggest that Zar1 transcript in the ovine oocyte may be necessary for normal female reproduction in the transition from oocyte to embryonic life.