The Impact Of Myeloperoxidase And Its Related Oxidants On Metaphase Ii Mouse Oocyte Quality

Inflammatory reactions mediated by oxidative stress (OS) have been implicated in the deterioration of oocyte quality, which may lead to subfertility. Oxidative stress generated from enhancement of activated macrophages secondary to an inflammatory response are the major source of reactive oxygen species (ROS) such as superoxide (O2•−), hydrogen peroxide (H2O2), hydroxyl radical (•OH), and hypochlorous acid (HOCl), as well as, the pro-inflammatory enzyme myeloperoxidase (MPO). Previously, it has been shown that these ROS have deleterious effect on oocytes; however the link between inflammation through macrophage activity and oocyte quality remains unclear. In this work, we investigated: 1) the mechanism through which direct exposure of ROS and MPO, or through their generation by activated macrophages, deteriorate oocyte quality and whether melatonin (MLT), a potent MPO inhibitor and ROS scavenger, can protect oocyte quality; and 2) the mechanism through which MLT inhibits MPO catalytic activity. Our results indicated that ROS differentially deteriorate oocyte quality in a dose dependent manner possibly secondary to the overwhelming of the defense antioxidant capacity of the cumulus oocyte complex (COC). Cumulus cells demonstrated protection against H2O2 and •OH insult at low concentrations, but this protection was lost at higher concentrations and all concentrations of HOCl as judged by changes in the organized compact cumulus cell mass into a dispersed mass of cells with decreased cumulus cell number and viability. Therefore, increasing ROS concentration overpowered the antioxidant machinery provided by the oocyte and /or cumulus cells, through loss of cumulus cells, or the lack of scavengers for specific ROS. This mechanism of damage may be associated with infertility related to COC dysfunction and thus deterioration in oocyte quality. Myeloperoxidase as well as activated macrophages negatively affected oocyte quality in a time dependent fashion. In all circumstances cumulus cells did not offer protection to the oocyte; however significant protection was offered by MLT. Kinetic studies have shown that MLT inhibits the MPO chlorinating (generation of HOCl) activity through its ability to compete with Cl-, the natural substrate of MPO, and serve as a one electron substrate of MPO Compounds I and II. Thus, MLT preserves the MPO peroxidation activity (by consuming H2O2 at slower rates) without the generation of HOCl through a two-step one-electron (1e−) oxidation pathway. This study is the first to link activated macrophages, a major source of MPO and ROS, and oocyte quality deterioration, highlighting the effects of activated macrophages in infertility caused by inflammation. MLT has beneficial therapeutic effects in preserving oocyte quality, thus improving reproductive outcomes in patients with chronic inflammation

Galactose and its Metabolites Deteriorate Metaphase II Mouse Oocyte Quality and Subsequent Embryo Development by Disrupting the Spindle Structure

Premature ovarian insufficiency (POI) is a frequent long-term complication of classic galactosemia. The majority of women with this disorder develop POI, however rare spontaneous pregnancies have been reported. Here, we evaluate the effect of D-galactose and its metabolites, galactitol and galactose 1-phosphate, on oocyte quality as well as embryo development to elucidate the mechanism through which these compounds mediate oocyte deterioration. Metaphase II mouse oocytes (n=240), with and without cumulus cells (CCs), were exposed for 4hours to D-galactose (2μM), galactitol (11μM) and galactose 1-phosphate (0.1mM), (corresponding to plasma concentrations in patients on galactoserestricted diet) and compared to controls. The treated oocytes showed decreased quality as a function of significant enhancement in production of reactive oxygen species (ROS) when compared to controls. The presence of CCs offered no protection, as elevated ROS was accompanied by increased apoptosis of CCs. Our results suggested that D-galactose and its metabolites disturbed the spindle structure and chromosomal alignment, which was associated with significant decline in oocyte cleavage and blastocyst development after in-vitro fertilization. The results provide insight into prevention and treatment strategies that may be used to extend the window of fertility in these patients

Kinetic studies on the reaction between dicyanocobinamide and hypochlorous acid.

Hypochlorous acid (HOCl) is a potent oxidant generated by myeloperoxidase (MPO), which is an abundant enzyme used for defense against microbes. We examined the potential role of HOCl in corrin ring destruction and subsequent formation of cyanogen chloride (CNCl) from dicyanocobinamide ((CN)2-Cbi). Stopped-flow analysis revealed that the reaction consists of at least three observable steps, including at least two sequential transient intermediates prior to corrin ring destruction. The first two steps were attributed to sequential replacement of the two cyanide ligands with hypochlorite, while the third step was the destruction of the corrin ring. The formation of (OCl)(CN)-Cbi and its conversion to (OCl)2-Cbi was fitted to a first order rate equation with second order rate constants of 0.002 and 0.0002 µM(-1) s(-1), respectively. The significantly lower rate of the second step compared to the first suggests that the replacement of the first cyanide molecule by hypochlorite causes an alteration in the ligand trans effects changing the affinity and/or accessibility of Co toward hypochlorite. Plots of the apparent rate constants as a function of HOCl concentration for all the three steps were linear with Y-intercepts close to zero, indicating that HOCl binds in an irreversible one-step mechanism. Collectively, these results illustrate functional differences in the corrin ring environments toward binding of diatomic ligands

The Impact of Myeloperoxidase and Activated Macrophages on Metaphase II Mouse Oocyte Quality.

Myeloperoxidase (MPO), an abundant heme-containing enzyme present in neutrophils, monocytes, and macrophages, is produced in high levels during inflammation, and associated with poor reproductive outcomes. MPO is known to generate hypochlorous acid (HOCl), a damaging reactive oxygen species (ROS) utilizing hydrogen peroxide (H2O2) and chloride (Cl-). Here we investigate the effect of activated immune cells and MPO on oocyte quality. Mouse metaphase II oocytes were divided into the following groups: 1) Incubation with a catalytic amount of MPO (40 nM) for different incubation periods in the presence of 100 mM Cl- with and without H2O2 and with and without melatonin (100 μM), at 37°C (n = 648/648 total number of oocytes in each group for oocytes with and without cumulus cells); 2) Co-cultured with activated mouse peritoneal macrophage and neutrophils cells (1.0 x 106 cells/ml) in the absence and presence of melatonin (200 μM), an MPO inhibitor/ROS scavenger, for different incubation periods in HTF media, at 37°C (n = 200/200); 3) Untreated oocytes incubated for 4 hrs as controls (n = 73/64). Oocytes were then fixed, stained and scored based on the microtubule morphology and chromosomal alignment. All treatments were found to negatively affect oocyte quality in a time dependent fashion as compared to controls. In all cases the presence of cumulus cells offered no protection; however significant protection was offered by melatonin. Similar results were obtained with oocytes treated with neutrophils. This work provides a direct link between MPO and decreased oocyte quality. Therefore, strategies to decrease MPO mediated inflammation may influence reproductive outcomes

Diffused Intra-Oocyte Hydrogen Peroxide Activates Myeloperoxidase and Deteriorates Oocyte Quality.

Hydrogen peroxide (H2O2) is a relatively long-lived signaling molecule that plays an essential role in oocyte maturation, implantation, as well as early embryonic development. Exposure to relatively high levels of H2O2 functions efficiently to accelerate oocyte aging and deteriorate oocyte quality. However, little precise information exists regarding intra-oocyte H2O2 concentrations, and its diffusion to the oocyte milieu. In this work, we utilized an L-shaped amperometric integrated H2O2-selective probe to directly and quantitatively measure the real-time intra-oocyte H2O2 concentration. This investigation provides an exact measurement of H2O2 in situ by reducing the possible loss of H2O2 caused by diffusion or reactivity with other biological systems. This experiment suggests that the intra-oocyte H2O2 levels of oocytes obtained from young animals are reasonably high and remained constant during the procedure measurements. However, the intra-oocyte H2O2 concentration dropped significantly (40-50% reduction) in response to catalase pre-incubation, suggesting that the measurements are truly H2O2 based. To further confirm the extracellular diffusion of H2O2, oocytes were incubated with myeloperoxidase (MPO), and the diffused H2O2 triggered MPO chlorinating activity. Our results show that the generated hypochlorous acid (HOCl) facilitated the deterioration in oocyte quality, a process that could be prevented by pre-incubating the oocytes with melatonin, which was experimentally proven to be oxidized utilizing HPLC methods. This study is the first to demonstrate direct quantitative measurement of intracellular H2O2, and its extracellular diffusion and activation of MPO as well as its impact on oocyte quality. These results may help in designing more accurate treatment plans in assisted reproduction under inflammatory conditions

Melatonin prevents myeloperoxidase heme destruction and the generation of free iron mediated by self-generated hypochlorous acid.

Myeloperoxidase (MPO) generated hypochlorous acid (HOCl) formed during catalysis is able to destroy the MPO heme moiety through a feedback mechanism, resulting in the accumulation of free iron. Here we show that the presence of melatonin (MLT) can prevent HOCl-mediated MPO heme destruction using a combination of UV-visible photometry, hydrogen peroxide (H2O2)-specific electrode, and ferrozine assay techniques. High performance liquid chromatography (HPLC) analysis showed that MPO heme protection was at the expense of MLT oxidation. The full protection of the MPO heme requires the presence of a 1:2 MLT to H2O2 ratio. Melatonin prevents HOCl-mediated MPO heme destruction through multiple pathways. These include competition with chloride, the natural co-substrate; switching the MPO activity from a two electron oxidation to a one electron pathway causing the buildup of the inactive Compound II, and its subsequent decay to MPO-Fe(III) instead of generating HOCl; binding to MPO above the heme iron, thereby preventing the access of H2O2 to the catalytic site of the enzyme; and direct scavenging of HOCl. Collectively, in addition to acting as an antioxidant and MPO inhibitor, MLT can exert its protective effect by preventing the release of free iron mediated by self-generated HOCl. Our work may establish a direct mechanistic link by which MLT exerts its antioxidant protective effect in chronic inflammatory diseases with MPO elevation

Effect of catalase, an H₂O₂ scavenger, on intra-oocyte H₂O₂ concentration (n = 20).

<p>The error bars represent the standard errors of mean.</p

The effect of HOCl concentration on the formation, duration of (OCl) (CN)-Cbi and its conversion to (OCl)₂-Cbi.

<p>A solution containing sodium phosphate buffer (200 mM, pH 7.0) supplemented with 5 µm (final) dicyanocobiamide was rapidly mixed with an equal volume of buffer containing increasing concentrations of HOCl (200, 300, 600, 800, and 1200 µM, final) at 25°C. Replacement of the first CN^- molecule by OCl^-, duration, and its decay to (OCl)₂-Cbl were monitored as a function of time by observing spectral changes at 613 nm. The final concentration of HOCl in mixtures is indicated.</p

Diode array rapid scanning spectra for the intermediates and corrin ring destruction by reacting (CN)₂-Cbi with HOCl at three sequential time frames.

<p>Panel A, spectra traces collected at 0.0, 0.4, 0.8, 1.2, 1.6, and 2.4 s and was attributed to the replacement of the first molecule of CN^- with OCl in (CN)₂-Cbi. Panel B, spectra traces collected at 2.4, 5.0, 7.4, and 12.0 and were attributed to the replacement of the second molecule of CN^- with OCl in (CN)₂-Cbi. Panel C, spectra collected at 12.0, 22.0, 40.0, and 120.0 s and was attributed to corrin ring destruction. Experiments were carried out by rapid mixing a phosphate buffer solution (200 mM, pH 7.0), at 25°C, supplemented with 20 µM (CN)₂-Cbi with a same volume of a buffer solution supplemented with 80-fold excess of HOCl. Arrows indicate the direction of spectral change over time as each intermediate advanced to the next. These data are representative of three independent experiments.</p

Rate constants for the formation and decay of the intermediates that formed upon mixing (CN)₂-Cbi with HOCl.

<p>(CN)₂-Cbi was rapid-mixed with buffer containing HOCl at various concentrations. Rates of complex formation and decay were determined at three different pHs (6.5, 7.4, and 9.0) by following absorbance change at 613 or 493 nm, at 10°C, and the rate constants determined as described in the text. These data are representative of three independent experiments and the standard error for each individual rate constant has been estimated to be less than 3%.</p