5 CLONINGAND CHARACTERIZATION OF NEWBORN OVARY HOMEOBOX GENE (NOBOX) IN CATTLE

Abstract

Newborn ovary homeobox (NOBOX) is a homeobox gene that is preferentially expressed in the oocytes and is essential for folliculogenesis in mice. NOBOX knockout mice are infertile, and lack of NOBOX perturbs the expression of many germ-cell-specific genes and microRNAs. Furthermore, mutations in the NOBOX gene associated with premature ovarian failure have been described in humans. However, the temporal and cell-specific expression of NOBOX in bovine oocytes and the potential function of NOBOX in early embryogenesis have not been described previously. The objectives of this study were to clone the complementary (c)DNA encoding for bovine NOBOX, analyze the expression of NOBOX mRNA in bovine tissues including fetal ovaries of different developmental stages, and characterize the temporal expression patterns of NOBOX mRNA during oocyte maturation and early embryogenesis. Based on the sequence of a predicted cDNA for bovine NOBOX, we successfully amplified, using RT-PCR, a cDNA fragment representing the coding region of bovine NOBOX from bovine fetal ovary cDNA. Additional 5′ and 3′ sequences were obtained using rapid amplification of cDNA ends (RACE) procedures. The assembled full-length NOBOX cDNA is 2275 bp with an open reading frame encoding a protein of 500 amino acids with a conserved homeodomain and typical nuclear localization signal. The predicted NOBOX protein shares 61% and 49% amino acid sequence identity with its human and mouse counterparts, respectively. A BLAST search of the bovine genome database at the National Center for Biotechnology Information (NCBI) revealed that the bovine NOBOX gene is located on chromosome 4, spans approximately 5.5 kb, and is encoded by 7 exons. Northern blot analysis revealed an approximately 2.3-kb bovine NOBOX RNA transcript. RT-PCR analysis of RNA samples from a panel of 14 different bovine tissues revealed that expression of NOBOX mRNA is restricted to ovarian samples and can be detected in fetal ovaries harvested as early as 105 days of gestation, when primary follicles start to form. Further RT-PCR analysis using RNA isolated from oocytes and granulosa and cumulus cells of antral follicles indicates that bovine NOBOX is expressed in oocytes but not in other follicular cells. Real-time PCR analysis demonstrated that NOBOX mRNA is abundant in germinal vesicle and metaphase II stage oocytes, as well as from pronuclear to 8-cell stage embryos, but barely detectable in embryos collected at the morula and blastocyst stages, suggesting that NOBOX might be a maternal effect gene. Collectively, our results demonstrate that bovine NOBOX is specifically expressed in oocytes and may play a role in early embryonic development in addition to its known function in folliculogenesis.