Fig. 3: MEF2A-dependent regulation of OXT receptor target genes after cOXT treatment.

A PCR array: Expression of MEF2A-regulated candidate genes after 10 ng/h cOXT treatment. Data calculated by ΔΔCT and presented as fold change vs. VEH. Fold changes > 1, upregulated mRNA expression, fold changes < 1, downregulated mRNA expression. n(VEH) = 5, n(10 ng/h cOXT) = 6. Pax3 *p = 0.021, Crfr2α *p = 0.02, sCrfr2α ^#p = 0.07, Pax2 *p = 0.05. B Upper panel: Representative example of 3 male rat PVN cDNA samples showing mCrfr2α (upper band, 400 bp) in VEH-treated animals, and mCrfr2α / sCrfr2α (lower band, 300 bp) expression in the cOXT treatment groups. Lower panel: Western Blot of mCRFR2α (~50 kDa) and sCRFR2α (~30 kDa) levels after VEH, 1 or 10 ng/h cOXT infusion and mild stress, using a polyclonal pan-CRFR2 antibody. C Ratio of mCRFR2α and sCRFR2α protein expression after cOXT and mild stress in male rats, shifting in the favor of sCRFR2α following treatment. Data are represented as mean ± SEM. F(2;15) = 5.311, p = 0.021; Holm-Sidak p = 0.019 10 ng/h vs VEH; n(VEH) = 6, n(1 ng/h) = 5, n(10 ng/h) = 6. D Treatment with acOXT had no effects on the ratio of mCRFR2α/sCRFR2α protein expression in % in the PVN of male rats. Data are represented as mean ± SEM. t = 0.784 with 11 degrees of freedom, two-tailed p value = 0.450; n(VEH) = 6, n(OXT) = 7. E siRNA-mediated knockdown of MEF2A in H32 cells incubated with 100 nM OXT decreased mCRFR2α and sCRFR2α protein levels, indicating a central role for MEF2A in the transcription of the CRFR2 gene. Data represented as mean ± SEM. mCRFR2α: Mann–Whitney Rank Sum Test <0.001, *p = 0.002; sCRFR2α: t = 2.574 with 10 degrees of freedom; one-tailed *p value = 0.0138, both vs. respective scr (scrambled) control; n(scr) = 6, n(MEF2A kd) = 6. F Representative stainings showing co-localization of OXT-neurophysin I (green), sCRFR2α (magenta), and OXTR-Venus (yellow) in the PVN (indicated by white dotted line) of male OXTR-Venus reporter mice. G Representative staining showing colocalization of OXT (green), sCRFR2α (magenta), and V1bR (yellow) in the PVN of male Wistar rats. H Pearson and Manders overlap coefficient of OXTR, OXT, and sCRFR2α positive neurons reveal substantial (45–64%) overlap between the OXTR, OXT, and sCRFR2α. I Pearson and Manders coefficient reveal partial (24–37%) overlap of V1a/b and sCRFR2α in the PVN. OXT-neurophysin and sCRFR2α show similar co-expression (~60%) in rats and mice, indicating a comparable expression pattern of the sCRFR2α in both species.