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Khan S.N.,Wayne State University | Shaeib F.,Wayne State University | Najafi T.,Wayne State University | Najafi T.,Wayne State University | And 6 more authors.
PLoS ONE | Year: 2015

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 Lshaped 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. © 2015 Khan et al. Source


Khan S.N.,Wayne State University | Shaeib F.,Wayne State University | Thakur M.,Wayne State University | Jeelani R.,Wayne State University | And 4 more authors.
Free Radical Biology and Medicine | Year: 2016

Previous theoretical studies have suggested that utilization of 3-D imaging to acquire morphologic parameters of meiotic spindles may be useful in infertility related procedures as an assessment of oocyte quality. However, our results show that treatment of oocytes with increasing concentrations of peroxynitrite (ONOO-) caused a dramatic alteration in spindle shape in which morphologic parameters are not measurable or are uninformative in terms of oocyte quality. Metaphase II mouse oocytes (n=520) were treated with increasing concentrations of ONOO-, after which all oocytes were fixed and subjected to indirect immunofluorescence. Oocyte quality was assessed by alterations in the microtubule-organizing center (MTOC), pericentrin location, microtubule morphology, and chromosomal alignment. In untreated oocytes, pericentrin is primarily assembled utilizing the acentrosomal MTOC, which appears as a condensation at both spindle poles. The spindle has a symmetrical pointed barrel shape, assembled around the chromosomal plate at the spindle equator. Oocytes treated with low concentrations of ONOO- (<2.5 μM) showed shortening of the spindle apparatus, while pericentrin scatters from a tight condensation to a dispersed cluster around each spindle pole. At higher ONOO- concentrations (>2.5 μM) the central attachments between microtubules are strained and bend or unevenly break, and the MTOC proteins are further dispersed or undetectable. Peroxynitrite mediated MTOC damage, which deranges the chromosomal scaffold at the time of assembly and separation, caused the deterioration in oocyte quality. These results provide a link between reactive oxygen species and poor reproductive outcomes and elucidate the underlying etiology, which could be used as a superior biomarker for oocyte quality compared to existing assessment tools. © 2016 Elsevier Inc. All rights reserved. Source


Goud P.T.,Wayne State University | Goud P.T.,University of California at Davis | Goud P.T.,California Fertility Center | Goud A.P.,Wayne State University | And 6 more authors.
Fertility and Sterility | Year: 2014

Objective To study follicular microenvironment in terms of free radical dynamics, oocyte quality, and assisted reproductive technology (ART) outcomes among women with (group A) and without (group B) endometriosis. Design Prospective cohort study. Setting University ART center. Patient(s) Women with and without endometriosis undergoing ART (n = 28). Intervention(s) Follicular fluid (FF), granulosa cells (GCs), immature oocytes (IOs), and ART data on sibling cohort oocytes in groups A and B were compared. Main Outcome Measure(s) ART live birth outcomes, maturation, and aging among in vitro matured (IVM) oocytes, nitrate levels in FF, and nitrotyrosine (NT) footprints and apoptosis in the GCs. Result(s) Clinical characteristics and ART live birth outcomes were no different between groups A and B. Women from group A had significantly lower peak serum E2 (2,068.8 ± 244.6 pg/mL vs. 2,756.2 ± 205.0 pg/mL) and higher apoptosis (80.0% vs. 22.2%) and NT staining (70.0% vs. 22.2%) in GCs compared with group B. Fewer IOs underwent IVM to MII (0.6 ± 0.3) in group A compared with group B (1.4 ± 0.2). IVM oocytes had significantly higher incidence of cortical granule loss (83.3% vs. 24.0%) and spindle disruption (66.7% vs. 16.0%) and higher zona pellucida dissolution timing (133.8 ± 9.4 s vs. 90.5 ± 5.8 s) in group A compared with group B. FF nitrate levels were significantly higher in women who failed to conceive in group A (478.2 ± 43.1 nmol/L) compared with those that did conceive (173.3 ± 19.0 nmol/L). Conclusion(s) Increased protein nitration, GC apoptosis, resistance to IVM, and oocyte aging indicate the involvement of oxidative dysregulation of NO in the pathophysiology of altered follicular milieu and poor oocyte quality in women with endometriosis. © 2014 by American Society for Reproductive Medicine. Source


Goud P.T.,Wayne State University | Goud P.T.,University of California at Davis | Goud P.T.,California Fertility Center | Goud A.P.,University of California at Davis | And 7 more authors.
PLoS ONE | Year: 2014

Nitric oxide (NO) is reported to play significant a role in oocyte activation and maturation, implantation, and early embryonic development. Previously we have shown that NO forms an important component of the oocyte microenvironment, and functions effectively to delay oocyte aging. Thus, precise information about intra-oocyte NO concentrations [NO] will result in designing more accurate treatment plans in assisted reproduction. In this work, the direct, real-time and quantitative intra-oocyte [NO] was measured utilizing an L-shaped amperometric integrated NO-selective electrode. This method not only provides an elegant and convenient approach to real-time the measurement of NO in physiological environments, but also mimics the loss of NO caused by rapid NO diffusion combined with its reactivity in the biological milieu. This experiment suggests that the NO levels of oocytes obtained from young animals are significantly higher than those of oocytes obtained from old animals. Additionally the NO levels stay constant during the measurements; however, the intraoocyte [NO] is reduced significantly (70-75% reduction) in response to L-NAME incubation, suggesting that NO measurements are truly NOS based rather than caused by an unknown interfering substance in our system. We believe this first demonstration of the direct quantitative measurement of [NO] in situ in an intact cellular complex should be useful in tracking real-time and rapid changes at nanomolar levels. Moreover, this finding confirms and extends our previous work showing that supplementation with NO delays the oocyte aging process. © 2014 Goud et al. Source

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