Chronological age enhances aging phenomena and protein nitration in oocyte.

Background: The average age of childbearing has increased over the years contributing to infertility, miscarriages, and chromosomal abnormalities largely invoked by an age-related decline in oocyte quality. In this study, we investigate the role of nitric oxide (NO) insufficiency and protein nitration in oocyte chronological aging. Methods: Mouse oocytes were retrieved from young breeders (YB, 8-14 weeks [w]), retired breeders (RB, 48-52w) and old animals (OA, 80-84w) at 13.5 and 17 hours after ovulation trigger. They were assessed for zona pellucida dissolution time (ZPDT); ooplasmic microtubule dynamics (OMD); cortical granule (CG) status and spindle morphology (SM), as markers of oocyte quality. Sibling oocytes from RB were exposed to NO supplementation and assessed for aging phenomena (AP). All oocyte cumulus complexes were subjected to fluorescence nitrotyrosine (NT) immunocytochemistry and confocal microscopy to assess morphology and protein nitration. Results: At 13.5 h from hCG trigger, oocytes from RB compared to YB had significantly increased ZPDT (37.8 ± 11.9 vs 22.1 ± 4.1 seconds [s]), OMD (46.9 vs 0%), CG loss (39.4 vs 0%), and decreased normal SM (30.3 vs 81.3%), indicating premature AP that worsened among oocytes from RB at 17 hours post-hCG trigger. When exposed to SNAP, RB AP significantly decreased (ZPDT: 35.1 ± 5.5 vs 46.3 ± 8.9s, OMD: 13.3 vs 75.0% and CG loss: 50.0 vs 93.3%) and SM improved (80.0 vs 14.3%). The incidence of NT positivity was significantly higher in cumulus cells (13.5 h, 46.7 ± 4.5 vs 3.4 ± 0.7%; 17 h, 82.2 ± 2.9 vs 23.3 ± 3.6%) and oocytes (13.5 h, 57.1 vs 0%; 17 h, 100.0 vs 55.5%) from RB compared to YB. Oocytes retrieved decreased with advancing age (29.8 ± 4.1 per animal in the YB group compared to 10.2 ± 2.1 in RB and 4.0 ± 1.6 in OA). Oocytes from OA displayed increased ZPDT, major CG loss, increased OMD and spindle abnormalities, as well as pronuclear formation, confirming spontaneous meiosis to interphase transition. Conclusions: Oocytes undergo zona pellucida hardening, altered spindle and ooplasmic microtubules, and premature cortical granule release, indicative of spontaneous meiosis-interphase transition, as a function of chronological aging. These changes are also associated with NO insufficiency and protein nitration and may be alleviated through supplementation with an NO-donor.

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

OBJECTIVE: 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), because they aid in electron transfer and dimeric stability, and their bioavailability is crucial in regulating NOS coupling. We have previously shown that sufficient levels of nitric oxide (NO) are essential for maintaining 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 reactive oxygen species from the monomeric form can further disrupt oocyte quality and NO bioavailability. DESIGN: Experimental laboratory study. SETTING: Laboratory. ANIMALS: B6D2F1 mice. INTERVENTION(S): Sibling oocytes were retrieved from super-ovulated B6D2F1 mice from 3 age groups: 8-14 weeks (young breeders [YBs]), 48-52 weeks (retired breeders [RBs]), and 80-84 weeks (old animals [OAs]). MAIN OUTCOME MEASURE(S): Oocytes were scored for ooplasmic/spindle microtubule (MT) morphology, chromosomal alignment, and cortical granule (CG) intactness using immunofluorescence and confocal microscopy with 3 dimension image reconstruction and subjected to an high-performance liquid chromatography assay to measure the concentrations of H4B and its metabolites, as well as the zinc measurement using atomic absorption spectrophotometry. RESULT(S): Oocyte scoring showed a reduction in "good" quality oocyte percentage as age increases, with YB having the highest percentage of quality oocytes followed by RB and OA. The high-performance liquid chromatography analysis showed a significant progressive decrease in total H4B in RB and OA (0.00098 picogram (pg)/oocyte and 0.00069 pg/oocyte, respectively) compared with YB (0.00125 pg/oocyte). Atomic absorbance spectrophotometry revealed a significant progressive decrease in zinc concentration in RB and OA compared with YB (8.45 pg/oocyte and 5.82 pg/oocyte vs. 10.05 pg/oocyte, respectively). CONCLUSION(S): Age-related diminution in oocyte quality is paralleled by a decline in the levels of H4B and zinc. The resultant deficiency in the oocytes can lead to the inability of NOS to maintain dimerization. Consequent uncoupling of NOS generates superoxide instead of 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 reactive oxygen species generation and oocyte quality deterioration related to the chronological age.

Direct real-time measurement of intra-oocyte nitric oxide concentration in vivo.

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 intra-oocyte [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.

Dynamics of nitric oxide, altered follicular microenvironment, and oocyte quality in women with endometriosis.

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.

Nitric oxide extends the oocyte temporal window for optimal fertilization.

Deteriorating oocyte quality is a critical hurdle in the management of infertility, especially one associated with advancing age. In this study, we explore the role of nitric oxide (NO) on the sustenance of oocyte quality postovulation. Sibling oocytes from superovulated mice were subjected to intracytoplasmic sperm injection (ICSI) with cauda-epididymal spermatozoa following exposure to either the NO donor, S-nitroso-N-acetylpenicillamine (SNAP, 0.23 microM/min), an NO synthase (NOS) inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME, 1 mM), or an inhibitor of soluble guanylyl cyclase (sGC), 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ, 100 microM); while their sibling oocytes were subjected to ICSI either before (young) or after culture for the corresponding period of time (old). Outcomes of normal fertilization, cleavage, and development to the morula and blastocyst stages were compared. Embryos from each subgroup were also subjected to TUNEL assay for apoptosis. A significant deterioration in the ability of the oocytes to undergo normal fertilization and development to morula and blastocyst stages occurred among oocytes aged in culture medium compared to their sibling cohorts subjected to ICSI immediately after ovulation (P<0.05). This deterioration was prevented in oocytes exposed to SNAP. In contrast, exposure to L-NAME or ODQ resulted in a significant compromise in fertilization and development to the morula and blastocyst stages (P<0.05). Finally, apoptosis was noted in embryos derived from aged oocytes and those exposed to L-NAME or ODQ, but not in embryos derived from young oocytes or oocytes exposed to SNAP. Thus, NO is essential for sustenance of oocyte quality postovulation.

Reactive oxygen species and oocyte aging: role of superoxide, hydrogen peroxide, and hypochlorous acid.

Aging of the unfertilized oocyte inevitably occurs following ovulation, limiting its fertilizable life span. However, the mechanisms that regulate oocyte aging are still unclear. We hypothesize that reactive oxygen species such as superoxide (O2-), hydrogen peroxide (H2O2), and hypochlorous acid (HOCl) are likely candidates that may initiate these changes in the oocyte. In order to test this hypothesis, we investigated direct effects of O2- [hypoxanthine/xanthine oxidase system generating 0.12 (n=42) and 0.25 (n=45) microM O2-/min], H2O2 (20 or 100 microM, n=60), and HOCl, (1, 10, and 100 microM, n=50) on freshly ovulated or relatively old mouse oocytes, while their sibling oocytes were fixed immediately or cultured under physiological conditions (n=96). The aging process was assessed by the zona pellucida dissolution time (ZPDT), ooplasm microtubule dynamics (OMD), and cortical granule (CG) status. The ZPDT increased 2-fold in relatively old, compared to young, untreated oocytes (P<0.0001). Exposure to O2- increased it even further (P<0.0001). Similarly, more O2- exposed oocytes exhibited increased OMD and major CG loss, with fewer having normal OMD and intact CG compared to untreated controls. Interestingly, young oocytes resisted "aging," when exposed to 20 microM H2O2, while the same enhanced the aging phenomena in relatively old oocytes (P<0.05). Exposure to even very low levels of HOCl induced the aging phenomena in young and relatively old oocytes, and higher concentrations of HOCl compromised oocyte viability. Overall, O2-, H2O2, and HOCl each augment oocyte aging, more so in relatively old oocytes, suggesting compromised antioxidant capacity in aging oocytes.

Reconstruction of ooplasm recipient oocytes with frozen-thawed donor microcytoplasts and influence on the microtubular spindle.

OBJECTIVE(S): To cryopreserve micromanipulated ooplast segments (microcytoplasts) from mouse oocytes, compare microcytoplast and parent or recipient oocyte fusion performed within or without the zona pellucida, compare electrofusion of fresh or frozen oocyte with frozen-thawed microcytoplasts, and assess spindle integrity after reconstruction of oocytes. DESIGN: Prospective experimental study. SETTING: University-based experimental laboratory. ANIMAL(S): Mouse (MII) oocytes obtained after superovulation (n = 363). INTERVENTION(S): Micromanipulation of oocytes (n = 363) into microcytoplasts (n = 181), cryopreservation of microcytoplasts along with parent and sibling control oocytes (n = 182), reconstruction by electrofusion of microcytoplast and parent or recipient oocyte performed with (group A, n = 35) or without a zona pellucida (group B, n = 32), comparison of electrofusion of fresh oocyte (group C, n = 40) or frozen oocyte (group D, n = 36) with frozen-thawed microcytoplasts fused within zona, and assessment of spindle morphology of reconstructed oocyte. MAIN OUTCOME MEASURE(S): Post-thaw survival, success of fusion, and spindle integrity as assessed by immunostaining. RESULT(S): Higher success of post-thaw fusion was seen in group A (91.4%) compared with group B (56.2%). The post-thaw fusion of microcytoplasts with either fresh or frozen oocytes was not significantly different. Spindle integrity was 82.5% in group C as compared with 47.2% in group D. CONCLUSION(S): Microcytoplasts created from oocytes can be successfully cryopreserved, thawed, and used to reconstruct oocytes with intact spindles.

Activation of the cGMP signaling pathway is essential in delaying oocyte aging in diabetes mellitus.

Uncontrolled diabetes mellitus (DM) adversely affects oocyte maturation and embryo development via mechanisms that are yet unclear. Nonetheless, DM may cause uncoupling of nitric oxide synthases (NOSs) with reduction in the bioavailability of nitric oxide (NO), which is critical to maintain oocyte viability and prevent aging. The current study investigates the role of NO-mediated signaling related to oocyte aging in diabetic and nondiabetic mice. Age-related alterations in the oocytes, including ooplasmic microtubule dynamics (OMD), cortical granule (CG) status, and zona pellucida (ZP) hardening as well as the integrity of the spindle/chromatin were studied using confocal microscopy. Oocytes obtained from diabetic mice exhibited accelerated aging compared to that from nondiabetic mice. Moreover, oocytes from diabetic animals were exquisitely sensitive to NOS and guanylate cyclase (GC) inhibitors (L-NAME, ODQ), which induced aging and relatively resistant to its delay by the cGMP derivative (8-Br-cGMP). Oocytes from nondiabetic control mice displayed similar sensitivity to L-NAME in older oocytes, although to a significantly lower extent than that of DM (P < 0.04-0.0001). Despite the differences in response between DM and nonDM mice, the activation of cGMP pathway is essential to maintain the integrity of oocytes and delay oocyte aging. These findings not only indicate the role of NO signaling in the prevention of oocyte aging but also suggest enhanced aging and NO insufficiency in oocytes from diabetic mice. A comprehensive model incorporating our current findings with NOS, GC, and G kinase cycles is presented.

Nitric oxide delays oocyte aging.

Nitric oxide (NO) is a ubiquitous signaling molecule that plays a crucial role in oocyte maturation and embryo development. However, its role in oocyte aging is unclear. To examine how NO affects oocyte aging, we retrieved young and relatively old mouse oocytes and exposed them to increasing concentrations of NO donor S-nitroso acetyl penicillamine (SNAP). Aging related phenomena of ooplasmic microtubule dynamics (OMD), cortical granule (CG) exocytosis, zona pellucida (ZP) hardening, and spindle/chromatin integrity were studied at each SNAP concentration using fluorescence immunocytochemistry and confocal microscopy and compared with respective unexposed controls. Exposure of both young and old oocytes to NO resulted in a significant diminution in OMD and ZP dissolution time, whereas spontaneous CG loss decreased in old NO exposed oocytes compared to controls (P < 0.001 for all). Furthermore, NO exposure decreased the rate of spindle abnormalities in oocytes compared to unexposed controls. Interestingly, in old oocytes, the positive influence of NO was attenuated beyond 0.23 microM/min and disappeared at 0.46 microM/min NO. Overall, a significant dose-response relationship was noted between NO exposure and markers of aging with between 50 and 100 microM SNAP (0.11-0.23 microM/min NO, P < 0.0001). Collectively, our results demonstrate for the first time that exposure to NO delays oocyte aging and improves the integrity of the microtubular spindle apparatus in young and old oocytes.

Dynamic changes in microtubular cytoskeleton of human postmature oocytes revert after ooplasm transfer.

OBJECTIVE: Study of the influence of ooplasm transfer on the microtubule dynamics in human postmature oocytes. DESIGN: Prospective experimental study. SETTING: Academic hospital-based fertility center. MATERIALS(S): Human in vitro matured (IVM) oocytes (n = 65). Experimental groups: In set 1, sibling oocytes were processed either within 2-3 hours ("young"; n = 16) or at 12-14 hours after maturation ("presumably postmature," or PPM; n = 14). In set 2, young and PPM oocytes (n = 6 and 10, respectively) were assigned to be ooplasm donors and recipients, respectively. In set 3, PPM oocytes were used as ooplasm donors (n = 2) and recipients (n = 4). Control groups: Metaphase II oocytes from superovulated golden hamsters in set 1; sibling oocytes of ooplasm donor young (n = 4) and PPM oocytes (n = 7) in set 2; and sibling PPM oocytes in set 3 (n = 2). INTERVENTION(S): Immunocytochemistry for alphatubulin with or without treatment with taxol (Paclitaxel, a microtubule-enhancing agent) in set 1; aspiration and microinjection of approximately 20 picolitres ooplasm from donor young and PPM oocytes into recipient PPM oocytes in sets 2 and 3, respectively. Taxol treatment and tubulin immunocytochemistry on ooplasm recipients and control young and PPM sibling oocytes. MAIN OUTCOME MEASURE(S): Morphology and pattern of the microtubules in the spindle and ooplasm as evaluated by confocal microscopy and three-dimensional image reconstructions. RESULT(S): In set 1, taxol-untreated young oocytes had normal spindle morphology and orientation to the oolemma with no microtubules in the ooplasm. Taxol-treated young oocytes revealed markedly broadened spindle poles and minimal or absent ooplasmic microtubules. Taxol-untreated PPM oocytes had variable spindle morphology and a notable increase in cortical ooplasmic microtubules. Taxol treatment of PPM oocytes resulted in a marked increase in ooplasmic microtubules in addition to a broadening of spindle poles and formation of polar asters. In set 2, control young and PPM oocytes had the same findings as the corresponding oocytes in set 1. However, all ooplasm recipient PPM oocytes showed a striking diminution in ooplasmic microtubules, despite the taxol treatment, compared with their sibling PPM control oocytes in set 2 and PPM ooplasm-injected PPM oocytes in set 3. CONCLUSION(S): Postmature oocytes exhibit a dynamic increase in ooplasmic microtubules. However, these changes revert after transfer of ooplasm from young oocytes.