Insights into the NAD⁺ biosynthesis pathways involved during meiotic maturation and spindle formation in porcine oocytes

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Treatments that elevate NAD⁺ levels have been found to improve oocyte quality in mice, cattle, and pigs, suggesting that NAD⁺ is vital during oocyte maturation. This study aimed to examine the influence of different NAD⁺ biosynthetic pathways on oocyte quality by inhibiting key enzymes. Porcine oocytes from small antral follicles were matured for 44 h in a defined maturation system supplemented with 2-hydroxynicotinic acid (2-HNA, nicotinic acid phosphoribosyltransferase; NAPRT inhibitor), FK866 (nicotinamide phosphoribosyltransferase; NAMPT inhibitor), or gallotannin (nicotinamide mononucleotide adenylyltransferase; NMNAT inhibitor) and their respective NAD⁺ pathway modulators (nicotinic acid, nicotinamide, and nicotinamide mononucleotide, respectively). Cumulus expansion was assessed after 22 hr of maturation. At 44 h, maturation rates were determined and mature oocytes were fixed and stained to assess spindle formation. Each enzyme inhibitor reduced oocyte maturation rate and adversely affected spindle formation, indicating that NAD⁺ is required for meiotic spindle assembly. Furthermore, NAMPT and NMNAT inhibition reduced cumulus expansion, whereas NAPRT inhibition affected chromosomal segregation. Treating oocytes with NAD⁺ pathway inhibitors in combination with nicotinamide mononucleotide or nicotinic acid improved spindle parameters compared with the inhibitors alone. These results indicate that the salvage pathway plays a vital role in promoting oocyte meiotic progression, while the Preiss-Handler pathway is essential for spindle assembly.

Graphical Abstract