Potential role of COUP-TF in regulating major yolk protein gene expression in sea urchin

Major yolk protein (MYP) serves as a critical nutrient reservoir for gonadal development in sea urchins, but the transcriptional regulation of its encoding gene remains largely uncharacterized. Here, we cloned and analyzed the MYP promoter from Mesocentrotus nudus and found two conserved elements with putative binding sites for the orphan nuclear receptor COUP-TF (chicken ovalbumin upstream promoter-transcription factor). We cloned the full-length cDNA encoding COUP-TF from M. nudus (MnCOUP-TF). The cDNA encoded a 472-amino acid protein highly homologous to other COUP-TF/SVP subfamily members. RT-PCR and qPCR analyses revealed that transcripts of both MnCOUP-TF and MYP were present in several tissues, including gonads, and their levels exhibited a significant positive correlation during gonadal maturation (R² = 0.6764, p < 0.01). Electrophoretic mobility shift assays demonstrated specific binding of MnCOUP-TF to the MYP promoter, and transactivation assays showed that MnCOUP-TF activates transcription through these sites. Furthermore, total lipids extracted from gonadal tissue enhanced MnCOUP-TF-dependent reporter activity in a dose-dependent manner. This enhancement was comparable to the effect of 9-cis-retinoic acid, a compound known to activate COUP-TF at high concentrations, although it is not considered to be a physiological COUP-TF ligand. These results suggest that MnCOUP-TF regulates MYP expression in a ligand-dependent manner and that endogenous activators may be present in gonadal lipids. To our knowledge, this is the first molecular evidence implicating COUP-TF in yolk protein gene regulation in sea urchins, providing new insights into nuclear receptor-mediated control of reproduction in marine invertebrates.