IL-6 and Mouse Oocyte Spindle

Interleukin 6 (IL-6) is considered a major indicator of the acute-phase inflammatory response. Endometriosis and pelvic inflammation, diseases that manifest elevated levels of IL-6, are commonly associated with higher infertility. However, the mechanistic link between elevated levels of IL-6 and poor oocyte quality is still unclear. In this work, we explored the direct role of this cytokine as a possible mediator for impaired oocyte spindle and chromosomal structure, which is a critical hurdle in the management of infertility. Metaphase-II mouse oocytes were exposed to recombinant mouse IL-6 (50, 100 and 200 ng/mL) for 30 minutes and subjected to indirect immunofluorescent staining to identify alterations in the microtubule and chromosomal alignment compared to untreated controls. The deterioration in microtubule and chromosomal alignment were evaluated utilizing both fluorescence and confocal microscopy, and were quantitated with a previously reported scoring system. Our results showed that IL-6 caused a dose-dependent deterioration in microtubule and chromosomal alignment in the treated oocytes as compared to the untreated group. Indeed, IL-6 at a concentration as low as 50 ng/mL caused deterioration in the spindle structure in 60% of the oocytes, which increased significantly (P<0.0001) as IL-6 concentration was increased. In conclusion, elevated levels of IL-6 associated with endometriosis and pelvic inflammation may reduce the fertilizing capacity of human oocyte through a mechanism that involves impairment of the microtubule and chromosomal structure

Hypochlorous Acid-Induced Heme Degradation from Lactoperoxidase as a Novel Mechanism of Free Iron Release and Tissue Injury in Inflammatory Diseases

Lactoperoxidase (LPO) is the major consumer of hydrogen peroxide (H2O2) in the airways through its ability to oxidize thiocyanate (SCN−) to produce hypothiocyanous acid, an antimicrobial agent. In nasal inflammatory diseases, such as cystic fibrosis, both LPO and myeloperoxidase (MPO), another mammalian peroxidase secreted by neutrophils, are known to co-localize. The aim of this study was to assess the interaction of LPO and hypochlorous acid (HOCl), the final product of MPO. Our rapid kinetic measurements revealed that HOCl binds rapidly and reversibly to LPO-Fe(III) to form the LPO-Fe(III)-OCl complex, which in turn decayed irreversibly to LPO Compound II through the formation of Compound I. The decay rate constant of Compound II decreased with increasing HOCl concentration with an inflection point at 100 µM HOCl, after which the decay rate increased. This point of inflection is the critical concentration of HOCl beyond which HOCl switches its role, from mediating destabilization of LPO Compound II to LPO heme destruction. Lactoperoxidase heme destruction was associated with protein aggregation, free iron release, and formation of a number of fluorescent heme degradation products. Similar results were obtained when LPO-Fe(II)-O2, Compound III, was exposed to HOCl. Heme destruction can be partially or completely prevented in the presence of SCN−. On the basis of the present results we concluded that a complex bi-directional relationship exists between LPO activity and HOCl levels at sites of inflammation; LPO serve as a catalytic sink for HOCl, while HOCl serves to modulate LPO catalytic activity, bioavailability, and function

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

A review of nitric oxide and oxidative stress in typical ovulatory women and in the pathogenesis of ovulatory dysfunction in PCOS

Abstract Polycystic ovary syndrome (PCOS) is a heterogeneous functional endocrine disorder associated with a low-grade, chronic inflammatory state. Patients with PCOS present an increased risk of metabolic comorbidities and often menstrual dysregulation and infertility due to anovulation and/or poor oocyte quality. Multiple mechanisms including oxidative stress and low-grade inflammation are believed to be responsible for oocyte deterioration; however, the influence of nitric oxide (NO) insufficiency in oocyte quality and ovulatory dysfunction in PCOS is still a matter for debate. Higher production of superoxide (O2 •−) mediated DNA damage and impaired antioxidant defense have been implicated as contributory factors for the development of PCOS, with reported alteration in superoxide dismutase (SOD) function, an imbalanced zinc/copper ratio, and increased catalase activity. These events may result in decreased hydrogen peroxide (H2O2) accumulation with increased lipid peroxidation events. A decrease in NO, potentially due to increased activity of NO synthase (NOS) inhibitors such as asymmetric dimethylarginine (ADMA), and imbalance in the distribution of reactive oxygen species (ROS), such as decreased H2O2 and increased O2 •−, may offset the physiological processes surrounding follicular development, oocyte maturation, and ovulation contributing to the reproductive dysfunction in patients with PCOS. Thus, this proposal aims to evaluate the specific roles of NO, oxidative stress, ROS, and enzymatic and nonenzymatic elements in the pathogenesis of PCOS ovarian dysfunction, including oligo- anovulation and oocyte quality, with the intent to inspire better application of therapeutic options. The authors believe more consideration into the specific roles of oxidative stress, ROS, and enzymatic and nonenzymatic elements may allow for a more thorough understanding of PCOS. Future efforts elaborating on the role of NO in the preoptic nucleus to determine its influence on GnRH firing and follicle-stimulating hormone/Luteinizing hormone (FSH/LH) production with ovulation would be of benefit in PCOS. Consequently, treatment with an ADMA inhibitor or NO donor may prove beneficial to PCOS patients experiencing reproductive dysfunction and infertility