Female reproductive dysfunction during ageing: role of methylglyoxal in the formation of advanced glycation endproducts in ovaries of reproductively-aged mice.

Reproductive dysfunction with ageing has been so far extensively characterized in terms of depletion of ovarian follicles and reduced ability to produce gametes competent for fertilization. Nevertheless, molecular mechanisms underlying this process are still poorly understood. In the present study we addressed the hypothesis that methylglyoxal (MG), a major precursor of Advanced Glycation Endproducts (AGE), may contribute to molecular damage occurring during ovarian ageing. Our results showed that the biochemical activity of glyoxalase 1, the main component of the MG scavenging system, is significantly decreased in ovaries from reproductively-aged mice in comparison with the young group. This effect was associated with decreased expression at protein and RNA level of this enzyme and increased intraovarian level of MG. MG-arginine adducts argpyrimidine as detected with a specific antibody was found to accumulate with ageing in specific ovarian compartments. Separation of ovarian proteins by 2D gels and Western blotting revealed an approximate 30-fold increase in the extent of protein glycation in aged ovaries along with the appearance of eight argpyrimidine modified proteins exclusive for the aged group. In conclusion, the present results show that impaired MG detoxification causing relevant damage to the ovarian proteome might be one of the mechanisms underlying reproductive ageing and/or ageing-like ovarian diseases.

The Cosmic Silence experiment: on the putative adaptive role of environmental ionizing radiation.

Previously we reported that yeast and Chinese hamster V79 cells cultured under reduced levels of background environmental ionizing radiation show enhanced susceptibility to damage caused by acute doses of genotoxic agents. Reduction of environmental radiation dose rate was achieved by setting up an underground laboratory at Laboratori Nazionali del Gran Sasso, central Italy. We now report on the extension of our studies to a human cell line. Human lymphoblastoid TK6 cells were maintained under identical in vitro culture conditions for six continuous months, at different environmental ionizing radiation levels. Compared to "reference" environmental radiation conditions, we found that cells cultured in the underground laboratories were more sensitive to acute exposures to radiation, as measured both at the level of DNA damage and oxidative metabolism. Our results are compatible with the hypothesis that ultra-low dose rate ionizing radiation, i.e. environmental radiation, may act as a conditioning agent in the radiation-induced adaptive response.

Alterations in the protein kinase C signaling activated by a parthenogenetic agent in oocytes from reproductively old mice.

To investigate the effect of female age on oocyte developmental competence, we focused on protein kinase C (PKC), a major component of the signalling pathway involved in oocyte activation, and put forward the hypothesis that, as it occurs in many organs and tissues, aging affects PKC function in mouse oocytes. Biochemical activity of PKC along with the expression and subcellular distribution of some PKC isoforms were monitored in young and old mouse oocytes parthenogenetically activated by SrCl(2). We found that PKC activity increased reaching a level that was lower in old compared to young oocytes in association with an incomplete translocation of PKCbetaI to the plasma membrane. Moreover, old oocytes exhibited a reduced expression of PKCbeta1 and PKCalpha at the protein level, without significant effects on the expression of the Ca(2+)-independent PKCdelta. Detectable amounts of PKCbeta1 mRNA were observed in young and old oocytes at GV stage with no difference between the two groups of age. When meiotic progression to anaphase II up to first cleavage were analyzed, a delay in meiosis resumption and significantly lower rates of pronuclei formation and first cleavage were observed in old compared to young oocytes. Moreover, we found that, in contrast to SrCl(2), PMA (12-O-tetradecanoyl phorbol-13-acetate), a PKC agonist, was ineffective in activating old oocytes. Present findings provide evidence that aging affects the correct storage and activation of some PKCs, functional components of the machinery involved in oocyte activation, and suggest that these changes may negatively influence the activation competence of old oocytes.

Age-dependent changes in the expression of superoxide dismutases and catalase are associated with ultrastructural modifications in human granulosa cells.

Limited knowledge exists about changes in follicle quality associated with age. The aim of this work was to investigate whether ageing may cause oxidative stress-mediated alterations in human granulosa cells (GCs) from periovulatory follicles. GCs employed in this study were obtained from follicular aspirates of 20 younger women (range 27-32 years) and 20 older women (range 38-41 years) undergoing an IVF treatment. Results obtained from comparative RT-PCR analysis revealed that the mean relative levels of mRNAs coding for superoxide dismutases, Cu, ZnSOD (SOD1), MnSOD (SOD2) and catalase were significantly decreased in women > or =38 years (P < 0.05, Student's t-test). These changes were associated with a reduced expression of SOD1, SOD2 and catalase at the protein level. When examined at an ultrastructural level, most of the GCs from this group showed defective mitochondria and fewer lipid droplets than those observed in the younger group. These results indicate that GCs from older patients suffer from age-dependent oxidative stress injury and are taken as an evidence for reduced defence against reactive oxygen species (ROS) in GCs during reproductive ageing.

Body composition in coeliac disease adolescents on a gluten-free diet: a longitudinal study.

Our aim was to evaluate body composition in a group of coeliac disease adolescents on a gluten-free diet and to re-examine them at the end of the adolescence spurt. We studied 48 patients (group 1A), 30 age-matched healthy controls (group 2A), 11 group 1A patients after 4 years (group 1B) and 11 adolescents who were age- and sex-matched with group 1B (group 2B). Weight, height, bone mineral content, fat mass, fat-free mass (FFM) and bone mineral density were evaluated using dual-energy X-ray absorptiometry. All parameters were lower in group 1A than in group 2A subjects ( p<0.001). After 4 years, the body compartments of group 1B coeliac disease patients normalised, except for weight and FFM which remained lower than in group 2B subjects ( p<0.005). In conclusion, our study demonstrates that adolescence is a period where some parameters of body composition can still be recovered.

Antioxidant enzymatic defences in human follicular fluid: characterization and age-dependent changes.

The aim of this work was to study the antioxidant enzymatic defences in human follicular fluid and investigate their possible changes during reproductive ageing. To this end, we tested the specific activities and protein expression of enzymes involved in reactive oxygen species (ROS) scavenging and in detoxification of ROS byproducts in follicular fluid from young (range 27-32 years, n = 12) and older (range 39-45 years, n = 12) women participating in an IVF programme. Results show that all the tested enzymes [superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione transferase, glutathione reductase] were significantly expressed in human follicular fluid. However, when the two age groups were compared, we found that follicular fluid from older women exhibited a reduced level of glutathione transferase and catalase activities and a higher level of SOD activity. Immunoblot analysis revealed that ageing was associated with decreased protein expression of GST Pi isoform and did not affect SOD and catalase protein expression. Taken together, these findings indicate that reproductive ageing is accompanied by a change in the antioxidant enzymatic pattern that could impair ROS scavenging efficiency in the follicular environment.

Polymorphonuclear leukocyte myeloperoxidase levels in patients with Behçet's disease.

Myeloperoxidase was measured in the plasma and in the supernatants of polymorphonuclear granulocyte cultures from patients with Behçet's disease. High levels were found in both plasma and supernatants from patients with active disease. The addition of pentoxifylline to granulocyte cultures determined a significant decrease of myeloperoxidase levels in active patients only. Hyperactive neutrophils are present during the course of Behçet's disease and may be considered of importance in the pathogenesis of the vascular lesions.

Enhancement of endothelial cell E-selectin expression by sera from patients with active Behçet's disease: moderate correlation with anti-endothelial cell antibodies and serum myeloperoxidase levels.

We studied the in vitro E-selection expression of endothelial cells treated with sera from patients with Behçet's disease (BD) and factors (anti-endothelial cell antibodies, anti-neutrophil cytoplasmic antibodies, cytokines, and myeloperoxidase (MPO) that may contribute to adhesion molecule expression. A total of 21 patients with BD and 27 healthy controls were studied. In vitro E-selectin endothelial cell expression was investigated by ELISA after HUVEC incubation with sera or purified IgG from patients with BD and controls. Increased E-selectin expression was observed when endothelial cells were incubated with sera from patients with active disease or from patients with circulating anti-endothelial cell antibodies and high levels of plasma myeloperoxidase. Abnormalities of endothelial cell function have been suggested to play a role in the occurrence of vascular damage in BD. Our findings suggest that anti-endothelial cell antibodies and neutrophil hyperactivity, as inferred from the high plasma MPO levels in patients with active disease, may explain endothelial cell activation and neutrophil accumulation in vascular lesions.

IgG anti-endothelial cell antibodies (AECA) in type I diabetes mellitus; induction of adhesion molecule expression in cultured endothelial cells.

AECA were detected in 25 of 71 patients with type 1 diabetes mellitus and in two of 33 healthy subjects. Patients with diabetes of < 1 year duration and those with long-standing disease had the highest levels of these antibodies. Inhibition studies suggest that at least part of the AECA reactivity is due to cross-reactive anti-ssDNA antibodies. AECA-positive sera were able to increase intercellular adhesion molecule-1 (ICAM-1) and E-selectin on human umbilical vein endothelial cells (HUVEC). Increased binding of polymorphonuclear (PMN) cells was also found to accompany raised E-selectin expression. Soluble ICAM-1 and E-selectin were also found to be increased in the sera of AECA-positive patients. An effect of AECA on endothelial cell function is suggested in diabetes mellitus.

Anti-single-stranded DNA antibody in the sera of patients with type 2 diabetes mellitus. Relation to vascular complications.

Anti-single-stranded(ss)DNA antibodies were searched for by enzyme-linked immunosorbent assay (ELISA) in the serum of 202 outpatients with non-insulin-dependent diabetes mellitus and 135 healthy subjects to investigate their prevalence in the serum of patients with type 2 diabetes and their relationship with the presence of vascular complications. Of the 202 patients 128 had vascular complications. Anti-ssDNA antibodies were observed to be significantly more frequent in the serum of patients with vascular complications (33.6%) and in particular in patients with overt nephropathy (50%) than in patients without complications (6.7%) or controls (6.7%). Anti-ssDNA antibodies have been previously described in patients with type 1 diabetes before clinical evidence of vascular disease and their cross-reactivity with a variety of anionic biological molecules or cells, i.e. platelets and endothelial cells, assessed. It seems not unreasonable that these auto-antibodies detected in patients with type 2 diabetes could be of importance in the pathogenesis or progression of angiopathy.

Detection of anti-myeloperoxidase antibodies in the serum of patients with type 1 diabetes mellitus.

Anti-myeloperoxidase (anti-MPO) antibodies were detected in 34 of 88 (38%) patients with type 1 diabetes mellitus but in only 3 of 55 (5.7%) healthy subjects and in 4 of 20 patients with autoimmune disease. Specificity of anti-MPO antibodies was assessed by MPO inhibition studies. No relationship was found between the occurrence of anti-MPO and anti-thyroperoxidase antibodies. Levels of soluble intercellular adhesion molecule 1 (ICAM-1) were found to be higher in anti-MPO antibody-positive (n = 28, 508 +/- 126 ng/ml) than in anti-MPO antibody-negative (n = 58, 438 +/- 140 ng/ml: P < 0.05) patients. A state of chronic neutrophil activation has been described in diabetes mellitus. As anti-MPO antibodies can stimulate neutrophils to damage endothelial cells in systemic vasculitis, this suggests that a similar mechanism may be operative in the development of diabetic angiopathy.