Nicotinamide mononucleotide biosynthesis and the F-actin cytoskeleton regulate spindle assembly and oocyte maturation quality in post-ovulatory aged porcine oocytes.

Background: Post-ovulatory aging (POA) is associated with reduced fertilization rates and poor embryo quality both in vivo and in vitro. However, the relationship between nicotinamide adenine dinucleotide (NAD⁺) and the filamentous actin (F-actin) cytoskeleton in POA-induced oocytes remains unknown. Here, we investigated the mechanisms by which the NAD⁺ salvage pathways function in poor oocyte maturation upon POA through the F-actin cytoskeleton.

Methods: Porcine oocytes were aged by extending in vitro maturation (IVM) for an additional 24 h to create a POA model. F-actin and adducin 1 (ADD1)-related spindle assembly were analyzed using immunofluorescence, western blotting, and RNA sequencing to identify key gene categories in the POA and IVM groups. To assess NAD⁺ function in restoring oocyte maturation, nicotinamide mononucleotide (NMN) was added and the maturation efficiency was evaluated. Expression of spindle assembly factors, F-actin cytoskeleton factors, aging markers, and NAD⁺-related genes was analyzed via quantitative polymerase chain reaction, immunofluorescence, and western blotting.

Results: We revealed unique interactions between the F-actin/ADD1-related cytoskeleton and aging factors (clusterin (CLU) and FAM111 trypsin-like peptidase A (FAM111A)) in poor-quality oocytes. POA oocytes were established with an extension of 24 h based on 44 h of IVM. They exhibited actin collapses and abnormal cortical F-actin, ADD1, and acetyl(Ac)-α-tubulin protein levels, which resulted in defective spindle assembly. RNA sequencing analysis was performed to identify differentially expressed genes involved in the oocyte viability response to aging, the cytoskeleton, and NAD metabolic processes using IVM and/or POA oocytes. This showed that NAD-binding genes were differentially expressed after POA induction, eight of which were downregulated compared with IVM oocytes. Importantly, activation of NAD⁺ pathways upon addition of NMN to the medium at 24 h after IVM rescued the maturation capability of POA oocytes with perturbations of spindle assembly and cortical F-actin.

Conclusion: F-actin polymerization through NAD⁺ generated from NMN is an essential factor in determining oocyte quality. This effect is mediated by microtubules related to spindle assembly in POA oocytes.