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

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 h 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 gallotannin and nicotinamide mononucleotide together showed improvements in spindle width, while treating oocytes with 2-HNA and nicotinic acid combined showed an improvement in both spindle length and width. 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.     

Nicotinic acid supplementation at a supraphysiological dose increases the bioavailability of NAD+ precursors in mares

NAD⁺ deficiency has recently been linked with increased occurrences of congenital abnormalities and embryonic death in human and animal subjects. Early embryonic death is a major component of pregnancy loss in mares and very little is known regarding the requirement for NAD⁺ in horses. The aim of this study was to quantify NAD⁺ and its metabolites in the plasma and urine of mares after orally administering an acute dose of nicotinic acid and determine the absorption, metabolism and excretion of this essential precursor for NAD⁺ biosynthesis. Nicotinic acid (5 g per os) was administered to four mares via a dosing syringe. Blood samples were collected at 0, 0.25, 0.5, 1, 2, 4, 6 and 22 h, and urine samples were collected at 0, 3, 6 and 22 h. The samples were processed and analysed by mass spectrometry. A general additive model was applied to all metabolite concentration values followed by a post-hoc multiple comparisons test. Nicotinic acid was rapidly absorbed into peripheral blood within 15 min of administration and the concentrations of nicotinic acid, nicotinamide (NAM), nicotinuric acid, nicotinic acid mononucleotide and nicotinic acid adenine dinucleotide (NaAD) increased significantly in plasma at 30 min. The concentrations of NAM, nicotinic acid riboside and NaAD increased significantly in urine at 3 h. The levels of NAM and NaAD remained significantly elevated in plasma at 22 h, sixfold and ninefold greater, respectively, than the basal levels at 0 h. While the extracellular levels of NAD⁺ in the samples remained undetected, the large, sustained elevation of NaAD levels in plasma indicates that the NAD⁺ levels were boosted within the cellular compartments. The results show that nicotinic acid supplementation increases the bioavailability of NAD⁺ precursors in mares, which is proposed to be beneficial during periods of peak NAD⁺ demand, such as during early embryo development.     

Supplementing media with NAD+ precursors enhances the in vitro maturation of porcine oocytes

In vitro maturation (IVM) is an important reproductive technology used to produce embryos in vitro. However, the developmental potential of oocytes sourced for IVM is markedly lower than those matured in vivo. Previously, NAD⁺-elevating treatments have improved oocyte quality and embryo development in cattle and mice, suggesting that NAD⁺ is important during oocyte maturation. The aim of this study was to examine the effects of nicotinic acid (NA), nicotinamide (NAM) and nicotinamide mononucleotide (NMN) on oocyte maturation and subsequent embryo development. Porcine oocytes from small antral follicles were matured for 44 h in a defined maturation medium supplemented with NA, NAM and resveratrol or NMN. Mature oocytes were artificially activated and presumptive zygotes cultured for 7 days. Additionally, oocytes were matured without treatment then cultured for 7 days with NMN. Supplementing the IVM medium with NA improved maturation and blastocyst formation while NAM supplementation improved cleavage rates compared with untreated controls. Supplementing the IVM or embryo culture media with NMN had no effect on maturation or embryo development. The results show that supplementing the maturation medium with NA and NAM improved maturation and developmental potential of porcine oocytes.