Background: H 2 O 2 oxidizes peroxiredoxins (Prxs) to sulfenic acid intermediates which form disulfides or become hyperoxidized. Results: Rate constants for hyperoxidation and disulfide formation were obtained for Prx2 and Prx3. Conclusions: Prx2 is more susceptible than Prx3 to hyperoxidation due to slower disulfide formation. Significance: H 2 O 2 reacts with Prx sulfenic acid faster than with most reduced thiols.

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BackgroundWe have shown recently that maternal undernutrition (UN) advanced female pubertal onset in a manner that is dependent upon the timing of UN. The long-term consequence of this accelerated puberty on ovarian function is unknown. Recent findings suggest that oxidative stress may be one mechanism whereby early life events impact on later physiological functioning. Therefore, using an established rodent model of maternal UN at critical windows of development, we examined maternal UN-induced changes in offspring ovarian function and determined whether these changes were underpinned by ovarian oxidative stress.Methodology/Principal FindingsOur study is the first to show that maternal UN significantly reduced primordial and secondary follicle number in offspring in a manner that was dependent upon the timing of maternal UN. Specifically, a reduction in these early stage follicles was observed in offspring born to mothers undernourished throughout both pregnancy and lactation. Additionally, antral follicle number was reduced in offspring born to all mothers that were UN regardless of whether the period of UN was restricted to pregnancy or lactation or both. These reductions were associated with decreased mRNA levels of genes critical for follicle maturation and ovulation. Increased ovarian protein carbonyls were observed in offspring born to mothers UN during pregnancy and/or lactation and this was associated with peroxiredoxin 3 hyperoxidation and reduced mRNA levels; suggesting compromised antioxidant defence. This was not observed in offspring of mothers UN during lactation alone.ConclusionsWe propose that maternal UN, particularly at a time-point that includes pregnancy, results in reduced offspring ovarian follicle numbers and mRNA levels of regulatory genes and may be mediated by increased ovarian oxidative stress coupled with a decreased ability to repair the resultant oxidative damage. Together these data are suggestive of maternal UN potentially contributing to premature ovarian ageing in offspring.

Mammalian 2-cysteine peroxiredoxins (Prxs) are susceptible to hyperoxidation by excess H2O2. The cytoplasmic family member Prx2 hyperoxidizes more readily than mitochondrial Prx3 due to slower dimerization of the sulfenic acid (SpOH) intermediate. Four variant amino acids near the C-terminus have been shown to contribute to this difference. We have performed kinetic analysis of the relationship between hyperoxidation and disulfide formation, using whole-protein MS and comparing wild-type (WT) Prx2 and Prx3 with tail-swap mutants in which the four amino acids were reversed. These changes make Prx3 more sensitive and Prx2 less sensitive to hyperoxidation and accounted for ~70% of the difference between the two proteins. The tail swap mutant of Prx3 was also more susceptible when expressed in the mitochondria of HeLa cells. The hyperoxidized product at lower excesses of H2O2 was a semi-hyperoxidized dimer with one active site disulfide and the other a sulfinic acid. For Prx2, increasing the H2O2 concentration resulted in complete hyperoxidation. In contrast, only approximately half the Prx3 active sites underwent hyperoxidation and, even with high H2O2, the predominant product was the hyperoxidized dimer. Size exclusion chromatography (SEC) showed that the oligomeric forms of all redox states of Prx3 dissociated more readily into dimeric units than their Prx2 counterparts. Notably the species with one disulfide and one hyperoxidized active site was decameric for Prx2 and dimeric for Prx3. Reduction and re-oxidation of the hyperoxidized dimer of Prx3 produced hyperoxidized monomers, implying dissociation and rearrangement of the subunits of the functional homodimer.

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