The Implications of Insufficient Zinc on the Generation of Oxidative Stress Leading to Decreased Oocyte Quality

Abstract

Zinc is a transition metal that displays wide physiological implications ranging from participation in hundreds of enzymes and proteins to normal growth and development. In the reproductive tract of both sexes, zinc maintains a functional role in spermatogenesis, ovulation, fertilization, normal pregnancy, fetal development, and parturition. In this work, we review evidence to date regarding the importance of zinc in oocyte maturation and development, with emphasis on the role of key zinc-binding proteins, as well as examine the effects of zinc and reactive oxygen species (ROS) on oocyte quality and female fertility. We summarize our current knowledge about the participation of zinc in the developing oocyte bound to zinc finger proteins as well as loosely bound zinc ion in the intracellular and extracellular environments. These include aspects related to (1) the impact of zinc deficiency and overwhelming production of ROS under inflammatory conditions on the offset of the physiological antioxidant machinery disturbing biomolecules, proteins, and cellular processes, and their role in contributing to further oxidative stress; (2) the role of ROS in modulating damage to proteins containing zinc, such as zinc finger proteins and nitric oxide synthases (NOS), and expelling the zinc resulting in loss of protein function; and (3) clarify the different role of oxidative stress and zinc deficiency in the pathophysiology of infertility diseases with special emphasis on endometriosis-associated infertility.

Introduction

Zinc, a transition metal and the second most abundant trace element in the body, is required for the catalytic activity of hundreds of enzymes. An estimated 3000 proteins bind zinc to maintain their structural integrity and function [1]. Trace amounts of zinc are crucial for living organisms as they participate in normal growth and development, gene transcription, and protein, lipid, and nucleic acid metabolism [2]. Recent research has identified the detrimental role of zinc in the human body in immune function, wound repair, and reproduction [3–5]. Fluctuations of intracellular zinc are critical to create a fertilizable egg and the process of fertilization in mammals requires a fine-tuned and well-regulated process including progression of the developing oocyte through meiotic arrest and resumption. Several factors can impact this natural development process, leading to decreased oocyte quality, anovulation, spontaneous oocyte arrest, and apoptosis, namely chronological aging, inflammation, environmental factors, and gynecological disorders such as endometriosis and polycystic ovarian syndrome (PCOS) [6–8].

As sufficient levels of zinc are required in an abundance of physiological processes, the human body requires constant intake of zinc either through diet or supplementation; thus, assessment of zinc status is significantly hindered by the tight regulation of zinc homeostasis. Serum zinc is commonly utilized as a marker of zinc status but free zinc in the serum is found mainly bound to proteins such as albumin (low affinity), α2-macroglobulin (A2M, moderate affinity), and transferrin (high affinity) [9]. Although blood collection is a typically easy process, zinc levels are affected by several alternative factors including anemia and pregnancy [10]. It has been suggested that a more elusive method to measure zinc is through biomarkers such as the expression of metallothionein and/or zinc transporters in leukocytes [11].

Recent discussion has highlighted the importance of zinc as an antioxidant due to zinc deficiency being correlated with an increase in reactive oxygen species (ROS) and reactive nitrogen species (RNS), although the exact mechanism is poorly understood [12–15]. It is speculated that the link between low zinc and increased ROS activity is due to decreased activity of antioxidant enzymes that require zinc, such as Cu/Zn SOD [14]. Zinc participates in antioxidant activity, yet it is not redox active nor directly interacts with ROS; however, zinc-binding proteins can be targeted by ROS/RNS. In biological processes, zinc finger proteins play important roles in transcription and DNA repair, as their zinc finger motif mediates protein-DNA, protein-RNA, and protein–protein interactions [16, 17]. The method by which certain zinc finger proteins are specifically targeted by ROS is not understood, as there is an abundant number of these proteins and cysteine residues. Upon constant exposure to overwhelming ROS may result in irreversible loss of protein function, abnormal modification, and physiological dysfunction [18, 19].

The maintenance of zinc concentrations at the cellular level can be accomplished through binding to proteins/enzymes or as labile zinc in intracellular compartments [1]. At the cellular level, zinc is mostly found in zinc-storing vesicles known as zincosomes, the nucleus, and distributed between the cytoplasm (bound to metallothioneins) and organelles [20]. Metallothioneins are zinc-chelating proteins that complex roughly 20% of intracellular zinc helping to protect against high cytotoxic metal concentrations and acting as mechanisms of zinc compartmentalization and zinc sequestration [20]. The distribution of zinc between the cytosol and organelles is accomplished through membrane channels or by zinc transporting proteins, keeping the overall cytosolic free zinc concentration to picomolar or nanomolar levels [9, 20]. The coordination of zinc release within the cell increasing cytosolic concentrations can impact gene expression, enzymatic activity, and cell signaling.

The disturbance of key zinc-binding proteins, including zinc finger proteins and nitric oxide synthases (NOSs), due to overwhelming ROS in the oocyte microenvironment may adversely impact oocyte quality through diminishing the ability of the oocyte to develop and be fertilized as described in Fig. 1 [21, 22]. The disrupted oxidant to antioxidant ratio in inflammatory diseases such as endometriosis or with the introduction of zinc-chelating agents by environmental toxins such as glyphosate may interfere with zinc bioavailability and protein functionality, thereby increasing oxidative stress and decreasing oocyte quality [8, 22–24]. As there are gaps within the field to understand the source, contribution, and importance of zinc in the oocyte, this review aims to summarize the current understanding of zinc in oocyte development and connect research regarding oxidative stress and zinc-binding proteins and enzymes (i.e., zinc finger proteins and NOS) to oocyte quality and fertility in inflammatory disorders.

Fig. 1.

The implications of oxidative stress on zinc availability contributing to decreased oocyte quality. In the presence of sufficient levels of zinc and antioxidants, inflammation and the production of oxidative stress are decreased allowing for (1) appropriate levels of nitric oxide (NO) from nitric oxide synthase (NOS) activity and (2) appropriate levels of zinc to bind to zinc finger proteins and participate in physiological processes requiring zinc such as zinc sparks. When this pathway is followed, oocyte quality is maintained through metaphase II arrest allowing for fertilization. In the event of overwhelming oxidative stress where zinc is deficient and antioxidant function goes down, i.e., in SOD where zinc is required for activity, resulting in destruction of NOS and NO consumption, inadequate zinc for zinc finger proteins, and disruption in zinc ion concentration ([Zn²⁺]) and homeostasis. In this detrimental pathway, the oocyte may not develop properly and/or prematurely exits MII arrest resulting in decreased maturation competence and inability to be fertilized

Methodology

An extensive literature review was conducted through the online databases PubMed, Science Direct, and Springer Link using the keywords zinc, oocyte development, oocyte maturation, control of meiotic arrest, zinc finger proteins/zinc-binding proteins, EMI2, reactive oxygen species/ROS, infertility, and antioxidants. Of the results found, included references are peer reviewed articles written in the English language. Individual hand searches of the references of retrieved literature was conducted. Ninety-two original articles presenting an overview of oocyte meiotic development, reactive oxygen species, inflammatory conditions and infertility, and zinc in the female reproductive process were included in this review.

Overview of Oocyte Development and the Role of Zinc

Humans are born with all the eggs they will ever have arrested in the diplotene stage of prophase I of the cell cycle, characterized by a large nucleus covered by a nuclear envelope known as the germinal vesicle (GV) [25]. A select group of follicles are stimulated each ovarian cycle by a surge in follicle-stimulating hormone (FSH) in which one of the recruited follicles acquires an increasing number of granulosa cells with enough FSH receptors to respond to stimulation from the falling FSH levels, becoming the dominant follicle. The oocyte in the dominant follicle is stimulated by a rise in luteinizing hormone (LH) before ovulation to resume meiosis. During this time, a rise in intracellular zinc is crucial to allow for the progression from prophase I to metaphase II without an intervening interphase [1, 25–27].

Prophase I Through Fertilization

Cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) have been determined as critical for maintaining meiotic arrest [28]. Before the LH-surge occurs, cGMP from granulosa cells is transferred through gap junctions to the oocyte and antagonizes the activity of phosphodiesterase 3A (PDE3A) [28, 29]. The surge in LH results in a decrease in follicular cGMP induced by LH with increased phosphorylation of connexin 43 in the granulosa cells [30, 31]. Connexin 43 has been determined to be involved in the permeability of the gap junctions between the granulosa cells and oocytes, and phosphorylation of connexin 43 decreases the permeability [31]. It is thus speculated a closure in the gap junctions, in part, may contribute to the decrease in cGMP. The decrease in cGMP results in an increase in PDE3A activity [32, 33] which, when active, hydrolyzes cAMP allowing for decreased cAMP activity and progression through meiosis [27]. The degradation of cAMP signals the breakdown of the germinal vesicle seen in prophase I, known as germinal vesicle breakdown (GVBD), and formation of the first meiotic spindle in metaphase I [27]. From there the oocyte resumes meiosis I, asymmetrically divides giving off the first polar body, and enters meiosis II where it is again arrested until fertilization [25, 34].

Metaphase II (MII) arrest depends on the “cytostatic factor” (CSF) activity referring to not a single molecule or gene, but a series of mechanisms that arrest the oocyte in metaphase [35, 36]. Notably, the maturation-promoting factor (MPF), a heterodimer made up of cyclin B1 and CDK1, sustains MII arrest. MPF activity is eventually inactivated through the anaphase-promoting complex/cyclosome (APC/C) that facilitates the degradation of cyclin B1 through ubiquitin-mediated proteolysis [35]. Additionally, Wee1, a tyrosine kinase, destabilizes MPF through phosphorylation of Cdk1, triggering the dissociation of cyclin B1 that allows for degradation by APC/C [36]. APC/C activity is inhibited by early meiotic-inhibitor 1-related protein 1 (Emi2), a zinc finger protein that was first identified in xenopus laevis oocytes [26, 35, 37]. At its zinc-binding region (ZBR), Emi2 binds APC/C thereby inhibiting it from degrading MPF keeping the oocyte arrested. Emi2 therefore preserves CDK activity during the metaphase arrest.

At fertilization, the oocyte undergoes egg activation triggered by the spermatozoa, followed by an observed rapid efflux of zinc ions, termed “zinc sparks,” regulated by calcium oscillations are observed in oocytes from several species including mouse, bovine, human, and even in the roundworm Caenorhabditis elegans and are associated with progression of the cell cycle [38–44]. The release of zinc from the oocyte is mediated by exocytosis of zinc on loaded vesicles containing around 10⁶ zinc atoms [45]. Emi2 is degraded through the rise in intracellular calcium (Ca²⁺) that activates calmodulin kinase II (CaMKII) to phosphorylate Emi2, destabilizing the complex and releasing APC/C [37]. The now activated APC/C then is free to promote anaphase by degrading many substrates including cyclin B1, decreasing MPF activity and allowing the oocyte to complete the meiotic division [36]. Completion of meiosis involves sequential events including extrusion of a second polar body, formation of pronuclei (male and female), and the transition to mitotic embryonic divisions [36].

The Importance of Zinc

The process of meiotic maturation from prophase I to MII (after meiotic resumption) in the mouse model occurs in about 12–14 h, with the total cellular content of zinc increasing by more than 50% [26, 46, 47]. The oocyte remains arrested in prophase I in the antral follicle due in part to high cAMP/PKA preventing MPF activity, and, in vitro, oocytes will mature spontaneously without treatment with a PDE3A inhibitor. It was found in a study by Tian and Diaz that treatment of mouse oocytes with a zinc-specific chelator, TPEN, resulted in GVBD even in the presence of a PDE3A inhibitor in more than 90% of oocytes treated [48]. Moreover, another study found that chelation of zinc at prophase I resulted in premature arrest in telophase I, signifying the importance of zinc as an inorganic signal for oocyte maturation [38]. Recently, our lab has shown that zinc decreases in the oocyte as a function of increasing maternal age, which is correlated with decreasing oocyte quality (Camp et. al., in press). In this study, we reported that MII oocytes from young mice contained around 10.05 picograms (pg) of zinc/oocyte with around 100% good quality oocytes while old animals on average had around 5.82 pg of zinc/oocyte and produced no good quality oocytes as shown through cortical granule status and microtubule dynamics. Similarly, a study by Kim et al. investigated intraoocyte zinc concentrations required for meiotic progression and found MII mouse oocytes have approximately 6.0 × 10¹⁰ atoms of Zn²⁺/oocyte, which is equivalent to a concentration of 6.5 pg/oocyte [46]. The authors of this study determined zinc-insufficient oocytes treated with TPEN all showed the spindle in a telophase configuration signifying an unusual block in meiosis [46].

The rise in intracellular zinc and meiotic progression are dependent on maternally derived ZIP6 and ZIP10 genes that encode SLC39 family zinc transporters that import zinc ions across the plasma or luminal membrane into the cytosol [49]. At fertilization, the zinc spark begins with thousands of cortically enriched, zinc-loaded secretory compartments releasing around 15% of the oocytes total zinc content at activation [45]. At the time of activation, these vesicles undergo dynamic movement allowing for the rapid exocytosis of zinc. Recently discovered phenomenon of zinc sparks or zinc exocytosis at fertilization also results in about 300% increase in zinc in the zona pellucida and the resultant “Zn²⁺ shield” prevents penetration of supernumerary spermatozoa [50]. Tian and Diaz demonstrated that acute zinc deficiency in mice before conception led to a dramatic disruption of oocyte and global DNA methylation and decreased in transcription of critical factors involved in oocyte development such as Gdf9, ZP3, and Figla [51]. On the other hand, Jeon et al. showed improved development of preimplantation embryos and parthenotes derived from in vitro matured oocytes supplemented with zinc during maturation [52]. This was accompanied with increased intracellular GSH and transcription factor expression concomitant with decreased ROS [52]. Additionally, Zn²⁺ can also “decapacitate” and prevent additional spermatozoa from undergoing hyperactivation, and thus prevent polyspermy. The spatiotemporal characteristics of fertilization-induced zinc spark profile is also recently proposed as parameter for the assessment of oocyte/embryo quality, as the zinc spark profile was positively correlated with total number of cells in the resulting morulae and blastocysts [50].

Nitric Oxide, Zinc, and the Oocyte

Virtually every step of oocyte development involves nitric oxide (NO), including meiotic maturation, fertilization, embryonic cleavage, and implantation, as biological concentrations are important for maintaining oocyte quality and delaying oocyte aging [53–57]. NO is generated through NOS which requires both Zn²⁺ and Ca²⁺ to regulate activity [58, 59]. Zinc is required for the stability of the enzyme’s active dimeric form in all three isoforms: inducible, iNOS; endothelial, eNOS; and neuronal, nNOS. The zinc atom is coordinated in a tetrahedral arrangement between two Cys-Cys motifs from each monomer [59]. Here, Zn²⁺ serves a structural function rather than catalytic. NOSs require adequate amounts of the substrate L-arginine (L-Arg) and the cofactor tetrahydrobiopterin (H₄B) for heme coupling and O₂ reduction in NOS dimers to produce NO and L-citrulline [60]. When these are not present in sufficient amounts, the enzyme utilizes O₂ to instead produce O₂^•−.

NO can improve oocyte quality and delay aging through NO-mediated activation of soluble guanylyl cyclase and cGMP-dependent protein kinase [61, 62]. NO is a ligand of soluble guanylyl cyclase, which catalyzes the conversion of GTP to cGMP; therefore, sufficient amounts of NO are required to allow adequate production of cGMP and maintenance of oocyte arrest [57, 61, 63, 64]. Both ONOO⁻ and HOCl can downregulate NO production through destruction of NOS, as these molecules may target cysteine and histidine residues binding zinc and flavins to NOS [65]. This disturbance of zinc binding to proteins is of particular importance in the context of the developing oocyte, decreasing the production of NO and altering oocyte quality. The loss of NO production at the MII stage will alter the production of cGMP, leading to decreased cAMP levels, dysregulated calcium channels (i.e., InsP3 and RYR), and oocyte activation.

Although there is increased production, NO can be rapidly consumed by O₂^•− and myeloperoxidase (MPO). MPO, generated in inflammation from activated neutrophils and macrophages, compound I and II can consume NO as a physiological 1e⁻ substrate generating nitrosonium cation that then hydrolyzes into NO₂⁻. Under conditions of enhanced oxidative stress, the activation of inflammatory cytokines results in NO synthesis and subsequent accumulation of several NO and RNS species that can react with dioxygen producing nitrogen dioxide, nitrite, and nitrous acid. The NO intracellular carrier S-nitrosoglutathione (GSNO), formed during transnitrosylation of glutathione (GSH) with NO, can facilitate irreversible damage to tissues when produced at high levels [19, 66]. Also, GSNO can s-nitrosylate the zinc cysteine core, and it has been noted that the mechanism of zinc release under low concentrations of GSNO is reversible [19].

Oxidative Stress and Zinc

The delicate balance between ROS/RNS and antioxidants is important in the maintenance of homeostasis and regulation of physiological signaling. In the event that ROS production is increased contributing to enhanced oxidative stress, the present antioxidant/oxidant ratio is disrupted resulting in an imbalance and potential oxidative damage to the oocyte and its microenvironment. ROS can either directly damage the oocyte or deplete oocyte quality through disruption of cumulus cells, decreasing the ability of the oocyte to mature [67–70]. Cumulus cells synthesize glutathione (GSH) as well as other antioxidants to protect the oocyte against oxidative stress [71, 72]. ROS such as superoxide (O₂^•−), hydrogen peroxide (H₂O₂), hypochlorous acid (HOCl), peroxynitrite (ONOO⁻), and hydroxyl radical (^•OH) can disturb the cumulus cells and connexin proteins, leading to a disruption of cellular communication in the cumulus oocyte complex, potentially creating an imbalance in antioxidant machinery and oocyte activation due to depletion of cGMP [29, 68, 73].

Moreover, O₂•⁻ produced from overexpression of NADPH oxidase can result in increased production of ONOO⁻, the product of the reaction between O₂^•− and NO, that can further cause substantial damage through thiol oxidation, lipid peroxidation, inactivation of enzymes and ion channels via protein oxidation and nitration, and inhibition of mitochondrial respiration [74]. Enhanced NADPH oxidase activity and the subsequent production of ROS has been linked to zinc deficiency [75, 76]. NADPH oxidase generated O₂^•− overproduction and metabolism results in the activation of caspace-3 contributing to DNA breaks and damage and apoptosis [77]. Several studies have indicated zinc can inhibit caspase activity through binding to the cysteine-histidine catalytic dyad on the caspases [78, 79]. Specifically, zinc at biologically relevant low concentrations, as low as 1.7 nM, have been shown to inhibit caspase-3 activity, which can bind three zinc ions [80, 81]. Additionally, zinc functions to inhibit NADPH oxidases and is a cofactor for SOD which can catalyze the dismutation of O₂⁻ to H₂O₂, thus assisting the antioxidant defense. When H₂O₂ is generated, it can react with redox-active transition metals through the Fenton reaction producing ^•OH, which can induce lipid peroxidation events and is the most damaging ROS. Zinc can competitively antagonize the participating redox-active transition metals, such as iron, cobalt, copper, and nickel, and thus may aid in preventing against site-specific peroxidative injury [82–84]. In another way, zinc can protect select proteins and enzymes from oxidation by stabilizing sulfhydryl’s either through direct binding, nearby binding producing steric hindrance, or causing conformational changes to the protein [83]. However, ROS may play an important role in disturbing the binding of Zn²⁺ to essential proteins and enzymes, leading to decreased oocyte quality.

Zinc-binding Proteins and ROS

As discussed, Emi2 is an important regulator of APC/C and miotic arrest at MII prior to fertilization, with the rise of intracellular calcium brought about by joining of a sperm cell leading to degeneration of Emi2 and thus resumption of meiosis [26, 85]. The structure of Emi2 is known and includes a destruction box (D-Box), zinc-binding region (ZBR), and an RL-tail at the C terminus [85]. A crystal structure of the ZBR fragment was determined by NMR spectroscopy showing the coordination of two zinc ions [85, 86]. The N-terminus contains two successive β-hairpins (β1 and β2 on the first and β3 and β4 on the second). The first zinc ion is coordinated between four cysteine residues (C4) at the tips of the first and second β-hairpins, which are tightly bound forming a scissor-like structure. The second zinc ion is coordinated at the C-terminal which forms a GAG-knuckle zinc-binding site with three cysteine residues and one histidine residue (C3H). The β4 strand associates with a β5 strand in an anti-parallel manner forming the C-terminal [85]. Interestingly, a study by Suzuki et al., investigated the role of zinc in the MII oocyte independent of calcium and found that Zn²⁺ chelation induces exit from MII with subsequent Cyclin B degradation without degradation of Emi2. A study by Bernhardt et al. determined that zinc insufficiency at the end of MI causes meiotic arrest, decreased CCNB1, and reduced MPF activity and concluded that a reduction in intracellular zinc availability is both necessary and sufficient to cause activation of the MII-arrested oocyte [19, 26]. Another report determined that reducing zinc content in the MII oocyte using the zinc-chelating agent TPEN resulted in release of CGs and spindle depolarization in MII porcine oocytes as well as a significant reduction in MPF activity, evidenced by p34cdc2 activity [87]. Together, these studies suggest the necessity of zinc in oocyte arrest and in Emi2 regulation of the APC/C allowing for meiotic arrest [88].

Zinc binding in zinc finger proteins and enzymes reduces the thiol oxidation potential which can be disturbed by ROS/RNS releasing zinc from the coordination sphere and generating loss of protein function [89]. Previously, our lab has shown that treating oocytes with agents capable of chelating zinc result in a decrease in oocyte zinc with an increase in ROS [22, 24]. Increased ROS can diminish MII oocyte quality either indirectly through disruption to the cumulus cells and connexin proteins or directly through direct disturbance to the oocyte. Moreover, this enhancement in ROS and decrease in zinc may promote mitochondrial damage subsequently increasing NADPH oxidase activity and the production of O₂^•−. O₂^•− can then react with existing NO to generate ONOO⁻; however, extracellularly generated ONOO⁻ can freely diffuse through the phospholipid membrane, as the calculated permeability constant of ONOO⁻ is ≈8.0 × 10⁻⁴ cm⋅s⁻¹, comparable to that of water [90]. ONOO⁻ may induce the destruction of zinc-binding domains through S-nitrosylation resulting in zinc dissociation. HOCl is produced during inflammation when MPO reacts with H₂O₂ in the presence of Cl⁻ [91], and can freely diffuse through the cell membrane facilitating oocyte deterioration and aging as evidenced by significant increases in zona pellucida dissolution time, altered spindle and chromosomal alignment, and cortical granule loss with the potential to oxidize zinc-binding domains [70, 92]. This resultant loss of function of zinc-binding proteins due to oxidation of the cysteinyl thiol residues, resulting in formation of a S–S disulfide bridge, may be reversible in the presence of a reducing agent [19]. Thus, the ability of ROS to disturb zinc binding essentially chelates zinc from the zinc-binding domain, rendering the protein or enzyme dysfunctional. This may mimic the results seen when using zinc-specific chelators, as described above, resulting in loss of MII arrest and oocyte aging phenomena.

The Potential Role of Zinc and ROS in Endometriosis-associated Infertility

The role of zinc and the oxidant to antioxidant balance may serve as guidance for understanding inflammatory diseases and related sub-fertility. For example, the discussed interaction of oxidative stress and zinc can be applied to endometriosis. Endometriosis, an enigmatic disorder of the female reproductive tract, includes several biologically active proinflammatory mediators [8, 93]. Of these mediators, nuclear factor kappa B (NF-κB) has been noted to play a substantial role in endometriosis [94–97], as its signaling is activated in endometriotic cells by tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β), also noted to be prominent in endometriosis [98–100]. NF-κB activates transcriptional activity of proinflammatory cytokines/chemokines including IL1, IL,6, IL8, and TNF-α [95, 100–103]. Interestingly, it has been reported that inflammatory cytokines including TNF-α and IL-1β are associated with increased ROS and decreased zinc [104–106], while zinc has been shown to inhibit NF-κB activation in several different disorders [107–110]. Thus, the investigation of zinc in the inflammatory response of endometriosis as well as endometriotic cell proliferation and development may be of increasing interest.

Recently, we found immature oocytes from women with endometriosis had decreased maturation competence, increased cortical granule loss, zona pellucida hardening, and spindle/chromosome disruption signifying a decrease in oocyte quality [111]. The mechanistic action of endometriosis in alerting oocyte quality can be determined by examining the role of ROS on cumulus cells, NO, and zinc in the oocyte. First, we previously observed cumulus cells from patients with endometriosis presented an increased incidence of apoptosis and high protein nitration as evidence by high measured NO₂⁻ [111]. Second, in isolated oocytes without cumulus cells, there is high protein nitration, observed premature cortical granule loss accompanied by alteration in oocyte size, shape, and microtubule spindle formation [111]. These phenomena can be attributed to the above presented model, as studies have found alterations in mitochondrial function and cumulus cells [112]. Disruptions to the COC by ROS can alter the cGMP and cAMP levels contributing to disturbed maintenance of meiotic arrest explaining the zona pellucida hardening, granulosa cell apoptosis, and spindle/chromosome disruptions. Additionally, ROS-mediated destruction of NOSs can decrease the liable NO used in the guanylyl cyclase system, producing similar results as evidenced by our previous work reporting NO supplementation can delay oocyte aging [57, 61, 62]. In regard to the isolated oocyte, decreased zinc can generate an influx of Ca²⁺ into the oocyte activating PKC and increasing NADPH oxidase expression. In turn, this will increase the production of ONOO⁻ explaining the increased protein nitration [113]. Furthermore, this occurrence is supported by the finding of increased abnormal mitochondria and decreased mitochondrial mass in oocytes from patients with endometriosis [114].

The elevated production of ROS in endometriosis, independent of the calcium mediated mechanisms above, may also disturb zinc-binding regions of Emi2 and other zinc finger proteins in the oocyte explaining the disruption to the pericentrin, spindles, and chromosomal alignment. Thus, the effects of ROS on oocytes may be acting through removing zinc from the ZBR of Emi2, thereby inhibiting its functionality, releasing APC/C, and allowing for meiotic resumption.

Conclusion

The functional role of zinc as an antioxidant may be key to understanding oxidative stress in inflammatory diseases that impact fertility, with insufficiencies potentially resulting in dysfunctional antioxidant enzymes and increased ROS accumulation. The overproduction of ROS in this way may function to disrupt cumulus cell functioning, deplete NO production, and/or remove the bound zinc of critical zinc finger proteins such as Emi2 or those present in the microtubule organizing center producing similar effects as zinc-chelating agents, i.e., anomalous pericentrin formation, disappearance of spindle fibers, altered chromosomal alignment, and subsequent meiotic resumption without fertilization [22, 115, 116]. More research is needed to investigate the potential of zinc in improving the antioxidant defense and oocyte quality.

Abbreviations

LH

Luteinizing hormone

ZFP

Zinc finger protein

ROS

Reactive oxygen species

NO

Nitric oxide

iNOS

Inducible nitric oxide synthase

HOCl

Hypochlorous acid

O₂^•−

Superoxide

H₂O₂

Hydrogen peroxide

^•OH

Hydroxyl radical

ONOO⁻

Peroxynitrite

Cl⁻

Chloride

H₄B

Tetrahydrobiopterin

CAT

Catalase

RNS

Reactive nitrogen species