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. 2022 Jun 1;68(3):216-224.
doi: 10.1262/jrd.2021-130. Epub 2022 Mar 28.

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

Charley-Lea Pollard  1 , Ashleigh Younan  1 , Aleona Swegen  2   3 , Zamira Gibb  2 , Christopher G Grupen  1
Affiliations

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

Charley-Lea Pollard et al. J Reprod Dev. .

Abstract

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.

Keywords: In vitro maturation; Meiotic spindle; NAD+ precursors; Oocyte; Pig.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Fig. 1.
Fig. 1.
The Preiss-Handler and salvage pathways for NAD+ biosynthesis. Nicotinic acid (NA) is the initial metabolite in the Preiss-Handler pathway and is converted to NAD+ through a series of enzymatic reactions. The NAD+ synthesised is consumed by Sirtuins and poly-ADP-ribose polymerases, releasing nicotinamide (NAM) which can then be recycled back into NAD+ via the salvage pathway. Inhibitors for the two pathways utilised in this study are represented in red and the metabolites upstream of these inhibitors are utilised as NAD+ pathway modulators with the exception of nicotinic acid adenine dinucleotide (NaAD). NA, nicotinic acid; NaMN, nicotinic acid mononucleotide; NaAD, nicotinic acid adenine dinucleotide; NAM, nicotinamide; NMN, nicotinamide mononucleotide; NAD, nicotinamide adenine dinucleotide; NR, nicotinamide riboside; NaR, nicotinic acid riboside. NaPRT, nicotinic acid phosphoribosyltransferase; NMNAT, nicotinamide mononucleotide adenylyltransferase; NADS, nicotinamide nicotinamide phosphoribosyltransferase; SIRTs, Sirtuins; PARPs, poly ADP-ribose polymerases; NRK, nicotinamide riboside kinase
Fig. 2.
Fig. 2.
Representative images of spindle and metaphase plate measurements. Spindle length was calculated by the distance between spindle poles, the spindle width was calculated by the width of the microtubules across the metaphase plate, and metaphase plate width was calculated by the distance between the edges of the DNA along the spindle length axis. The α-tubulin is stained green with a FITC-conjugated antibody and the DNA is stained blue with Hoechst33342. Arrow head indicates M-Plate width, open arrow indicates spindle length and closed arrow indicates spindle width.
Fig. 3.
Fig. 3.
Representative images of spindle assembly and chromosome alignment in MII oocytes. A) oocytes displaying barrel-shaped spindles with well organised microtubule fibres and highly condensed DNA tightly aligned along the metaphase plate (normal spindles), B) barrel-shaped spindles with well organised microtubule fibres with highly condensed DNA loosely aligned along the metaphase plate (aberrant spindles), C) Disorganised spindles with well-defined microtubule fibres and chromosomes not aligned along the metaphase plate (aberrant spindles), D) incomplete spindle assembly with no discernible microtubule structure and uncondensed DNA with no discernible structure (aberrant spindles). The α-tubulin is stained green with a FITC-conjugated antibody and the DNA is stained blue with Hoechst33342.
Fig. 4.
Fig. 4.
The effects of NA (200 µM) and 2HNA (1.0 mM) alone and in combination on A) oocyte maturation, B) cumulus expansion, C) the proportion of oocytes with aberrant spindles, D) spindle length, E) spindle width, and F) M-plate width. Treatments labelled with different letters indicate statistical differences.
Fig. 5.
Fig. 5.
The effects of NAM (5 µM) and FK866 (100 nM) alone and in combination on A) oocyte maturation, B) cumulus expansion, C) the proportion of oocytes with aberrant spindles, D) spindle length, E) spindle width, and F) M-plate width. Treatments labelled with different letters indicate statistical differences.
Fig. 6.
Fig. 6.
The effects of NMN (100 µM) and gallotannin (55 µM) alone and in combination on A) oocyte maturation, B) cumulus expansion, C) the proportion of oocytes with aberrant spindles, D) spindle length, E) spindle width, and F) M-plate width. Treatments labelled with different letters indicate statistical differences.
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