Expression and in vitro effect of phoenixin-14 on the porcine ovarian granulosa cells

Abstract

Phoenixin-14 (PNX-14) regulates energy metabolism via the G protein-coupled receptor 173 (GPR173); elevated plasma levels have been described in patients with polycystic ovary syndrome. The aims were to investigate the ovarian expression of PNX-14/GPR173 and the in vitro effect of PNX-14 on granulosa cells (Gc) function. Transcript and protein levels of PNX-14/GRP173 were analysed by real-time PCR, western blot and immunohistochemistry in the porcine ovarian follicles at days 2–3, 10–12 and 16–18 of the oestrous. For in vitro experiments, Gc were isolated from follicles at days 10–12 of the oestrous (4–6 mm) and PNX-14 at doses 1–1000 nM was added for 24–72 h to determine Gc proliferation. Cell cycle progression, E2 secretion, expression of proliferating cells nuclear antigen, cyclins, mitogen-activated kinase (MAP3/1; ERK1/2), protein kinase B (AKT) and signal transducer and activator of transcription 3 (STAT3) were studied. The involvement of these kinases in PNX-14 action on Gc proliferation was analysed using pharmacological inhibitors. Levels of GPR173 were increased in the ovarian follicles with oestrous progression, while only PNX-14 protein was the highest at days 10–12 of the oestrous. Immuno-signal of PNX-14 was detected in Gc and theca cells and oocyte, while GPR173 was mostly in theca. Interestingly, PNX-14 stimulated Gc proliferation, E2 secretion, cell cycle progression and cyclins expression and had a modulatory effect on MAP3/1, AKT and STAT3 activation. Our study suggests that PNX-14 could be an important factor for porcine reproduction by influencing ovarian follicle growth through direct action on Gc function.