Intraperitoneal ZP123 improves aged oocyte quality by restoring granulosa cell gap junctions and improving mitochondrial function.

<h4>Background</h4>Female reproductive aging is the most critical determinant of decreased fertility because the quality of oocytes gradually deteriorates with age, leading to poor embryo development, increased miscarriage rates, and reduced live births. Oocyte growth and maturation depend on bidirectional communication with surrounding granulosa cells through gap junctions, which are composed mainly of connexin 43 (CX43). Although intercellular communication mediated by CX43 is essential for folliculogenesis, how it becomes altered in the context of ovarian aging and drives oocyte deterioration remains largely unclear. This study aimed to clarify age-related alterations in gap junction communication and investigate whether augmenting CX43-mediated coupling via the peptide ZP123 might restore oocyte competence and enhance fertility outcomes in older females.<h4>Results</h4>Our data demonstrated that CX43 expression in mouse ovaries and granulosa cells markedly decreased in an age-dependent manner, and this reduction was evident at both the protein and mRNA levels. The level of phosphorylated CX43 at Ser373 decreased markedly, indicating that total CX43 loss was the primary contributor to impaired gap junction communication. In vitro, our study revealed that ZP123 treatment significantly enhanced dye transfer between KGN cells but did not increase CX43 expression, suggesting an improvement in gap junction intercellular communication without increasing CX43 protein abundance. In vivo, chronic ZP123 administration to aged mice restored the normal gap junction between granulosa cells, improved follicle development. Functionally, ZP123 enhanced oocyte maturation (GVBD and MII rates), corrected spindle abnormalities, improved mitochondrial distribution, and increased ATP production while reducing ROS accumulation. Moreover, the mitochondrial membrane potential and Ca²⁺ homeostasis were restored, and excessive opening of the mitochondrial permeability transition pore was prevented. Consistent with these cellular improvements, ZP123-treated aged mice presented increased ovulation numbers and enhanced preimplantation embryo development, including increased blastocyst formation rates.<h4>Conclusions</h4>This study revealed that disrupted CX43-mediated communication is a key mechanism underlying the decrease in oocyte quality during ovarian aging. Pharmacological enhancement of gap junctions with ZP123 effectively restored ovarian function, oocyte competence, and developmental potential, highlighting gap junction modulation as a novel and promising therapeutic avenue to mitigate age-related fertility decline in women of advanced maternal age.