Fernanda Amicarelli

Cellular and molecular aspects of ovarian follicle ageing

It is well established that age-related decline of the biological capacity of a woman to reproduce is primarily related to the poor developmental potential of her gametes. This renders female ageing the most significant determinant of success in IVF. Starting with a reference picture of the main molecular and cellular failures of aged oocytes, granulosa cells and follicular microenvironment, this review focuses on age-related biochemical mechanisms underlying these changes. According to the most relevant concept of ageing, age-associated malfuction results from physiological accumulation of irreparable damage to biomolecules as an unavoidable side effect of normal metabolism. More than a decade after the free radical theory of ovarian ageing, biological and clinical research supporting the involvement of oxidative injuries in follicle ageing is discussed. Looking for the aetiology of oxidative stress, we consider the effect of ageing on ovarian and follicular vascularization. Then, we propose a potential role of advanced glycation end-products known to be involved in the physiological ageing of most tissues and organs. We conclude that future investigation of age-related molecular damage in the different ovarian components will be imperative in order to evaluate the possibility to save or rescue the developmental potential of aged oocytes.

Scavenging system efficiency is crucial for cell resistance to ROS-mediated methylglyoxal injury.

Methylglyoxal is a reactive dicarbonyl compound endogenously produced mainly from glycolytic intermediates. Recent research indicates that methylglyoxal is a potent growth inhibitor and genotoxic agent. The antiproliferative activity of methylglyoxal has been investigated for pharmacological application in cancer chemotherapy. However, various cells are not equally sensitive to methylglyoxal toxicity. Therefore, it would be important to establish the cellular factors responsible for the different cell-type specific response to methylglyoxal injury, in order to avoid the risk of failure of a therapy based on increasing the intracellular level of methylglyoxal. To this purpose, we comparatively evaluated the signaling transduction pathway elicited by methylglyoxal in human glioblastoma (ADF) and neuroblastoma (SH-SY 5Y) cells. Results show that methylglyoxal causes early and extensive reactive oxygen species generation in both cell lines. However, SH-SY 5Y cells show higher sensitivity to methylglyoxal challenge due to a defective antioxidant and detoxifying ability that, preventing these cells from an efficient scavenging action, elicits extensive caspase-9 dependent apoptosis. These data emphasize the pivotal role of antioxidant and detoxifying systems in determining the grade of sensitivity of cells to methylglyoxal.

Age-Associated Changes in Mouse Oocytes During Postovulatory In Vitro Culture: Possible Role for Meiotic Kinases and Survival Factor BCL2

Abstract To elucidate molecular mechanisms underlying oocyte senescence, we investigated whether oocytes from female mice of advanced reproductive age exhibit a precocious postovulatory aging that, in turn, may be responsible for the precocious activation of an apoptotic program. During a 9-h in vitro culture, the frequency of oocytes showing MII aberrations, spontaneous activation, and cellular fragmentation increased in old oocytes (P < 0.05), whereas it did not change in the young group. In old oocytes, the activities of MPF (a complex of the cyclin-dependent kinase cdc2 and cyclin B1) and MAPK (mitogen-activated protein kinase) decreased precociously, showing a first drop as early as 3 h after the beginning of in vitro culture (P < 0.05). Immunoblotting and immunocytochemical analysis revealed that, in oocytes of the old group, reduction of BCL2 expression at protein level occurred earlier than in the young group (P < 0.05) and was not associated to the loss of BCL2 transcripts detected by RT-PCR. These changes are followed by an abrupt increase of the rate of TUNEL-positive oocytes after 24 h of culture to a value of 67% ± 6%. Exposure of young oocytes to 20 μM roscovitine or 20 μM U0126, specific inhibitors of MPF and MAPK, resulted in the decreased percentage of oocytes showing positive immunostaining for BCL2 and in an increased rate of DNA fragmentation. Present results suggest that the developmental competence of oocytes ovulated by aging mice may be negatively influenced by a downregulation of MPF and MAPK activities that in turn induces the activation of a proapoptotic signaling pathway.

Chronic exposure to 50 Hz magnetic fields causes a significant weakening of antioxidant defence systems in aged rat brain

Several studies suggest that extremely low-frequency magnetic fields (ELF-MFs) may enhance the free radical endogenous production. It is also well known that one of the unavoidable consequences of ageing is an overall oxidative stress-based decline in several physiological functions and in the general resistance to stressors. On the basis of these assumptions, the aim of this study was to establish whether the ageing process can increase susceptibility towards widely present ELF-MF-mediated pro-oxidative challenges. To this end, female Sprague-Dawley rats were continuously exposed to a sinusoidal 50 Hz, 0.1 mT magnetic field for 10 days. Treatment-induced changes in the major antioxidant protection systems and in the neurotrophic support were investigated, as a function of the age of the subjects. All analyses were performed in brain cortices, due to the high susceptibility of neuronal cells to oxidative injury. Our results indicated that ELF-MF exposure significantly affects anti-oxidative capability, both in young and aged animals, although in opposite ways. Indeed, exposed young individuals enhanced their neurotrophic signalling and anti-oxidative enzymatic defence against a possible ELF-MF-mediated increase in oxygen radical species. In contrast, aged subjects were not capable of increasing their defences in response to ELF-MF treatment but, on the contrary, they underwent a significant decrease in the major antioxidant enzymatic activities. In conclusion, our data seem to suggest that the exposure to ELF-MFs may act as a risk factor for the occurrence of oxidative stress-based nervous system pathologies associated with ageing.

Methylglyoxal induces oxidative stress-dependent cell injury and up-regulation of interleukin-1β and nerve growth factor in cultured hippocampal neuronal cells

Methylglyoxal (MG) is one of the most powerful glycating agents of proteins and other important cellular components and has been shown to be toxic to cultured cells. Under hyperglycaemic conditions, an increase in the concentration of MG has been observed in human body fluids and tissues that seems to be responsible for diabetic complications. Recent data suggest that diabetes may cause impairment of cognitive processes, according to a mechanism involving both oxidative stress and advanced glycation end product (AGE) formation. In this work, we explored the molecular mechanism underlying MG toxicity in neural cells, by investigating the effect of MG on both the interleukin-1beta (IL-1beta), as the major inducer of the acute phase response, and the nervous growth factor (NGF) expression. Experiments were performed on cultured neural cells from rat hippocampus, being this brain region mostly involved in cognitive processes and, therefore, possible target of diabetes-mediated impairment of cognitive abilities. Results show that MG treatment causes in hippocampal neural cells extensive, oxidative stress-mediated cell death, in consequence of a strong catalase enzymatic activity and protein inhibition. MG also causes a very significant increase in both transcript and protein expression of the NGF as well as of the pro-inflammatory cytokine IL-1beta. MG co-treatment with the antioxidant N-acetylcysteine (NAC) completely abrogates the observed effects. Taken together, these data demonstrate that hippocampal neurons are strongly susceptible to MG-mediated oxidative stress.

Cerium Oxide Nanoparticles Trigger Neuronal Survival in a Human Alzheimer Disease Model By Modulating BDNF Pathway

In engineering and materials science, nanotechnology has made significant advances in the reduction of free radical damage. Despite such advances, there has been little application to biomedical problems. Cross-disciplinary interactions and the application of this technology to biological systems has led to the elucidation of novel nanoparticle antioxidants. Oxidative stress and free radical produc- tion are associated with neurodegenerative conditions, including aging, trauma, Alzheimers and Parkinsons diseases, etc. The antioxi- dant properties of cerium oxide nanoparticles show promise in the treatment of such diseases. Recent reports suggest that CeO2 and other nanoparticles are potent, and probably regenerative, free radical scavengers in vitro and in vivo. In this work, the effects of CeO2 nanopar- ticles on an in vitro human AD model are investigated. The validation of new therapeutic agents implies the understanding of their mechanisms of action, therefore the following parameters were investigated under nanoparticles treatment: cell viability, cell death, neu- rite atrophy, neuronal marker localization and the expression of factors, i.e. PPARs, BDNF, TrkB, involved in the signal transduction pathways of neuronal survival. The data obtained, demonstrate that CeO2 nanoparticles do not act as mere anti-oxidant agents, but they seems to affect, directly or indirectly, signal transduction pathways involved in neuronal death and neuroprotection, raising the possibility of their use as therapeutic tools for neurodegenerative diseases.

The aging ovary--the poor granulosa cells.

The development of a competent oocyte intimately depends on the maintenance of energetic homeostasis in the ovarian and follicular microenvironment. On this basis, it is very likely that the oocyte ages as the ovary ages. Starting from the molecular evidence for energy perturbations in the whole ovary, we review current knowledge on the involvement of endogenous highly reactive metabolites in follicle aging. The first part provides an update of recent findings that confirm the key role of oxidative stress in aged granulosa cells. The second part focuses on studies providing evidence for the implication of advanced glycation end product (AGE) in aging reproductive dysfunction. With their prolonged half-life and ability to act as signaling molecules AGEs may gradually accumulate in the ovary and potentiate the wide spatiotemporal spread of oxidative stress. Clinical evidence for this view supports the hypothesis that AGE is a good candidate as a predictive marker and therapeutic target in new strategies for improving reproductive counseling in aging women.

AGE-DEPENDENT ULTRASTRUCTURAL ALTERATIONS AND BIOCHEMICAL RESPONSE OF RAT SKELETAL MUSCLE AFTER HYPOXIC OR HYPEROXIC TREATMENTS

This work deals with the antioxidant enzymatic response and the ultrastructural aspects of the skeletal muscle of young and aged rats kept under hypoxic or hyperoxic normobaric conditions. It is in fact well known that the supply of oxygen at concentrations higher or lower than those occurring under normal conditions can promote oxidative processes that can cause tissue damage. The enzymes investigated were both those directly involved in reactive oxygen species (ROS) scavenging (superoxide dismutase, catalase and selenium-dependent glutathione peroxidase), and those challenged with the detoxication of cytotoxic compounds produced by the action of ROS on biological molecules (glutathione transferase, glyoxalase I, glutathione reductase), in order to obtain a comparative view of the defence strategies used with respect to aging. Our results support the hypothesis that one of the major contributors to the aging process is the oxidative damage produced at least in part by an impairment of the antioxidant enzymatic system. This makes the aged organism particularly susceptible to oxidative stress injury and to the related degenerative diseases, especially in those tissues with high demand for oxidative metabolism.

DEVELOPMENTAL ASPECTS OF DETOXIFYING ENZYMES IN FISH (SALMO IRIDAEUS)

The activities of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, glutathione transferase and glyoxalase I have been studied during the embryologic development of rainbow trout (Salmo iridaeus) and in several other trout tissues to investigate the protective development metabolism. A gradual increase of superoxide dismutase, catalase, glutathione reductase, glyoxalase I and glutathione transferase activities was noted throughout embryo development. In all trout tissues investigated glutathione peroxidase was found to be extremely low compared to catalase activity. The highest activity of superoxide dismutase, glyoxalase I and glutathione reductase was found in liver followed by kidney. No change in the number of GST subunits was noted with the transition from the embryonic to the adult stages of life according to the SDS/PAGE and HPLC analyses performed on the GSH-affinity purified fractions.

PPARγ-dependent effects of conjugated linoleic acid on the human glioblastoma cell line (ADF)

Conjugated linoleic acid (CLA) has been shown to exert beneficial effects against carcinogenesis, atherosclerosis and diabetes. It has been demonstrated that CLA modulates lipid metabolism through the activation of peroxisome proliferator‐activated receptors (PPARs). The PPAR family comprises 3 closely related gene products, PPAR α, β/δ and γ, differing for tissue distribution, developmental expression and ligand specificity. It has also been demonstrated that activated PPARγ results in growth inhibition and differentiation of transformed cells. These observations stimulated a great interest toward PPARγ ligands as potential anticancer drugs to be used in a differentiation therapy. Glioblastomas are the most commonly diagnosed primary tumors of the brain in humans. The prognosis of patients with high‐grade gliomas is poor and only marginally improved by chemotherapy. The aim of this work was to study the effects of CLA and of a specific synthetic PPARγ ligand on cell growth, differentiation and death of a human glioblastoma cell line as well as on parameters responsible for the metastatic behavior of this tumor. We demonstrate here that CLA and PPARγ agonist strongly inhibit cell growth and proliferation rate and induce apoptosis. Moreover, both treatments decrease cell migration and invasiveness. The results obtained show that CLA acts, directly or indirectly, as a PPARγ activator, strongly suggesting that this naturally occurring fatty acid may be used as brain antitumor drug and as a chemopreventive agent. Moreover, the γ‐agonist, once experimented and validated on man, may represent a useful coadjuvant in glioblastoma therapy and in the prevention of recurrences.