Abstract
Simple SummaryGrowth differentiation factor 9 (GDF9) is an important gene for ovine fertility. GDF9 is highly expressed in the ovary as opposed to other tissues, but the reason for this is unknown. Our study found this can be caused by the methylation level of the promoter CpG island mC-4 site. This finding contributes to the understanding of the regulatory mechanism of GDF9 gene in reproduction.Growth differentiation factor 9 (GDF9) plays an important role in the early folliculogenesis of sheep. This study investigated the mRNA expression of ovine GDF9 in different tissues by real-time PCR. GDF9 exhibits significantly higher levels of expression (p < 0.01) in the ovary, relative to other tissues, indicating that its expression is tissue specific. To explore the regulatory mechanism of this tissue-specific expression, the methylation level of one CpG island (−1453 to −1854) of GDF9 promoter in ovary and heart was determined. In this region (−1987 to −1750), only the mC-4 site was present in the Sp4 binding site showed differential methylation between the heart and ovary; with increased (p < 0.01) methylation being observed in the heart. Additionally, the methylation level was negatively correlated with GDF9 mRNA expression (R = −0.75, p = 0.012), indicating that the methylation of this site plays an important role in transcriptional regulation of GDF9. The methylation effect of the mC-4 site was confirmed by using dual-luciferase. Site-directed mutation (methylation) of mC-4 site significantly reduced (p < 0.05) basal transcriptional activity of GDF9 promoter in oocytes. These results imply that methylation of GDF9 promoter CpG island mC-4 site may affect the binding of the Sp4 transcription factor to the GDF9 promoter region in sheep, thereby regulating GDF9 expression and resulting in a tissue-specific expression.
Highlights
Growth differentiation factor 9 (GDF9) is a member of the transforming growth factor beta superfamily and was first identified in the human ovary [1]
The tissue expression pattern of GDF9 has been examined in many animals, with high expression levels in ovary [14,32,33]
Ovine GDF9 expression was explored by real-time PCR, and GDF9 expression was significantly higher in ovary than in other tissues
Summary
Growth differentiation factor 9 (GDF9) is a member of the transforming growth factor beta superfamily and was first identified in the human ovary [1]. GDF9 mutations (FecGH , FecGT , FecGE , FecGF , and FecGV ) resulted in hyperprolificacy in heterozygotes. A recent study discovered highly heritable markers within GDF9, which are important in determining prolificacy traits in sheep [9]. GDF9 is an important candidate gene in ovine fertility. GDF9 is highly expressed in the ovaries of many species [10,11,12,13,14]. GDF9 expression was higher in both fetal and adult ovaries when compared to expression in other tissues, indicating that
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