Diminishing oocyte quality with advancing age is associated with deficiency of nitric oxide synthase cofactors, tetrahydrobiopterin and zinc, in mouse oocytes

To study the implications of decreased zinc and tetrahydrobiopterin (H4B) associated with chronological aging on oocyte quality using a mouse model. H4B and zinc are essential cofactors for nitric oxide synthase (NOS), as they aid in electron transfer and dimeric stability, and their bioavailability is crucial in regulating NOS coupling. We have previously shown sufficient levels of NO are essential for oocyte quality and activation, and NO levels decrease in the oocyte as a function of age. Thus, it is plausible that zinc and H4B may decrease as a function of age resulting in NOS dysfunction with subsequent depletion of NO. Additionally, increased production of ROS from the monomeric form can further disrupt oocyte quality and NO bioavailability.Experimental laboratory study.Laboratory.B6D2F1 mice.Sibling oocytes were retrieved from super-ovulated B6D2F1 mice from three age groups: 8-14 weeks [w] (young breeders, YB, n=112), 48-52w (retired breeders, RB, n=115) and 80-84w (old animals OA, n=75).Oocytes were: 1) scored for ooplasmic/spindle microtubule morphology, chromosomal alignment, and cortical granule (CG) intactness using immunofluorescence and confocal microscopy with 3-D image reconstruction (n= 62, 65 and 35 in YB, RB and OA respectively) and 2) subjected to an HPLC assay to measure concentrations of H4B and its metabolites, as well as zinc measurement using mass spectrophotometry (n= 50, 50 and 40 in YB, RB and OA respectively).1) Oocyte scoring showed a reduction in "good" quality oocyte percentage as age increased, with YB having the highest percent of quality oocytes followed by RB and OA. 2) HPLC analysis showed a significant progressive decrease in total H4B in RB and OA (0.098 ng/oocyte and 0.069 ng/oocyte, respectfully) compared to YB (0.125 ng/oocyte). Atomic absorbance spectrophotometry revealed a significant progressive decrease in zinc concentration in RB and OA compared to YB (0.805 ng/oocyte and 0.554 ng/oocyte versus 1.00 ng/oocyte, respectfully).Age related diminution in oocyte quality is paralleled by a decline in the levels of H4B and zinc. Resultant deficiency in the oocytes can lead to the inability of NOS to maintain dimerization. Consequent uncoupling of NOS generates superoxide (O2•-) instead NO, which participates in a multitude of reactions contributing to oxidative stress. Therefore, dysfunction of NOS secondary to zinc and H4B loss is a major mechanism involved in ROS generation and oocyte quality deterioration related to chronological age.