Nobiletin alleviates heat stress-induced meiotic arrest via restoring CDK1 and MAPK activity in porcine oocytes

Abstract

Heat stress is a critical environmental factor that disrupts porcine oocyte maturation and impairs embryonic development. This study investigated the protective role of Nobiletin under heat stress using an in vitro maturation model. Porcine cumulus-oocyte complexes (COCs) were cultured under control conditions (38.5 °C), exposed to heat stress at 41 °C from 15 to 24 h (HS group), or treated with Nobiletin (1-10 μM) during heat exposure (HS + Nob group). Heat stress significantly reduced polar body extrusion and increased metaphase I arrest. Treatment with 10 μM Nobiletin restored meiotic progression to near-control levels. Heat stress elevated reactive oxygen species (ROS), disrupted spindle morphology, and increased γH2A.X expression, indicating oxidative damage and chromosomal instability. This was accompanied by suppressed activation of CDK1 and ERK1/2, essential kinases for meiotic resumption and spindle formation. Nobiletin treatment reduced ROS, preserved spindle integrity, and restored phosphorylation and activity of CDK1 and ERK1/2, promoting normal meiotic progression. Notably, this recovery was independent of the heat shock response, as HSP90 and HSF1 levels remained unchanged. Following parthenogenetic activation, oocytes in the HS + Nob group showed improved developmental competence, with higher blastocyst formation rates, increased total cell numbers, and reduced apoptotic cell ratios compared to the HS group. These findings suggest that Nobiletin mitigates heat stress-induced meiotic arrest by reducing oxidative stress and restoring CDK1 and MAPK pathway activity, ultimately restoring porcine oocyte quality and developmental potential under thermal stress.