A T > G Mutation in the NR5A2 Gene Is Associated With Litter Size in Hu Sheep Through Upregulation of Promoter Activity by Transcription Factor MTF-1

Abstract

Nuclear receptor subfamily 5 group A member 2 (NR5A2), also referred to as LRH-1 or FTF, is an orphan nuclear hormone receptor that is involved in regulating embryonic development, ovarian granulosa cell differentiation, gonadal sex differentiation, and steroidogenesis in mammals. However, little is known about how <i>NR5A2</i> regulates reproduction in sheep. In this study, we amplified the promoter sequence of <i>NR5A2</i> and determined that its core promoter region ranged from -721 nt to -281 nt. A T > G polymorphism at -700 nt was detected in the core promoter region. Association analysis found that the litter sizes of Hu ewes at their second and average parities with genotype GG (2.20 ± 0.20 and 1.97 ± 0.06, respectively) were significantly higher than those of ewes with genotype TG (1.68 ± 0.10 and 1.74 ± 0.05, respectively) (<i>p</i> < 0.05) and TT (1.67 ± 0.10 and 1.62 ± 0.06, respectively) (<i>p</i> < 0.05). The litter size of Hu ewes at their third parity with genotype GG (2.10 ± 0.10) was significantly higher than that of ewes with genotype TT (1.56 ± 0.12) (<i>p</i> < 0.05). A luciferase assay showed that the -700G allele increased the luciferase activity relative to the -700T allele. Furthermore, the -700T > G polymorphism created a novel binding site for metal-regulatory transcription factor 1 (MTF-1). A competitive electrophoretic mobility shift assay confirmed that MTF-1 specifically bound with the G-type promoter of <i>NR5A2</i>. An overexpression experiment demonstrated that MTF-1 was involved in the alteration of NR5A2 transcription activity and further increased <i>NR5A2</i> gene mRNA expression. Our findings revealed that the -700T > G polymorphism promoted <i>NR5A2</i> expression due to the positive effects on <i>NR5A2</i> gene transcription activity by MTF-1 and thereby increased fecundity in Hu sheep.