Role of ZBED3 in PALD1/PIP2- dependent calcium homeostasis during oocyte maturation

Zinc Finger BED-Type Containing 3 (ZBED3) had been shown to be a novel component of the subcortical maternal complex (SCMC). In previous reports, ZBED3 depletion leads to asymmetric zygotic division and aberrant distribution of organelles in both oocytes and zygotes. However, the precise mechanism through which ZBED3 exerts its effects remains to be elucidated. To fill this gap, in this study, we generated Zbed3 gene knockout mice by using CRISPR/cas9 gene-editing technique to generate homozygous Zbed3^-/- female mice. A series of previously unreported phenotypes in oocytes were observed, including decreased fertility, abnormal spindle formation and migration, increased polyspermic fertilization, abnormal distribution of cortical granules (CGs), and disrupted calcium oscillations. To investigate the molecular mechanisms underlying the function of ZBED3 during oocyte maturation, we employed miniTurbo biotin ligase-based proximity labeling combined with mass spectrometry to identify protein interactomes in transfected HEK293 cells. OF the 187 ZBED3-interacting proteins, paladin 1 containing a phosphatase domain (PALD1) and E3 ubiquitin ligase makorin-1 (MKRN1) exhibited the highest fold changes and were subsequently validated. ZEBD3 suppressed PALD1 levels by enhancing its degradation via the ubiquitination-proteasome pathway. Depletion of Zbed3 results in an abnormal accumulation of PALD1. The ectopic overexpression of PALD1 recapitulates the phenotypic defects observed in Zbed3-deficient oocytes and early embryos. Moreover, knockdown of PALD1 partially rescued the oocyte maturation defects induced by Zbed3 depletion. Paladin is an endosomal phosphatidylinositol 4,5-bisphosphate (PIP2) phosphatase which directly modulates phosphoinositide metabolism by catalyzing the removal of phosphate groups from phosphoinositides. Furthermore, PALD1 overexpression reduced Ca²⁺ release from the endoplasmic reticulum (ER) by inhibiting its downstream target PIP2. Our study demonstrates that ZBED3 may regulate PIP2 protein levels by modulating the ubiquitin-proteasomal degradation of PALD1, thereby influencing oocyte maturation and providing a novel approach for assessing oocyte quality and developmental potential.