Ultrastructural Alterations of Midgut Epithelium, But Not Greater Wing Fluctuating Asymmetry, in Paper Wasps (Polistes dominula) from Urban Environments.

Polistes paper wasps can be used to monitor trace metal contaminants, but the effects of pollution on the health of these insects are still unknown. We evaluated, in a south-eastern area of Spain, whether workers of Polistes dominula collected at urban and rural sites differ in health of midgut tissue and in fluctuating asymmetry, an estimate of developmental noise. We found that wasps collected at the urban sites had abundant lead (Pb)-containing spherites, which were less visible in wasps from the rural sites. Evident ultrastructural alterations in the epithelium of the midgut of the wasps collected at the urban sites included broken and disorganized microvilli, a high amount and density of heterochromatin in the nucleus of epithelial cells, cytoplasmic vacuolization and mitochondrial disruptions. Altogether, these findings suggest a negative effect on the transmembrane transport and a less efficient transcription. On the contrary, a healthy epithelium was observed in wasps from the rural sites. These differences may be preliminarily linked with levels of lead pollution, given that wasps from urban sites had double the Pb concentrations of wasps from rural sites. Level of fluctuating asymmetry was unrelated to wasp origin, thus suggesting no link between developmental noise and Pb-driven pollution.

Effect of different oxidative stress degrees generated by hydrogen peroxide on motility and DNA fragmentation of zebrafish (Danio rerio) spermatozoa.

An increase in reactive oxygen species (ROS) or decrease in antioxidant barriers can provoke lipid peroxidation of the membranes or DNA damage of the spermatozoa. The aim of this work is to study the effect of the different degrees of oxidative stress generated by H2 O2 incubation on total motility, kinetics, and DNA fragmentation of zebrafish (Danio rerio) spermatozoa. For this process, experimental groups were incubated in 50 µM (Low; L) and 200 µM (High; H) H2 O2 , respectively, for 20 min at 4ºC. Sperm motility parameters were obtained with a computer-assisted sperm analysis (CASA) system. Sperm DNA fragmentation (SDF) was assessed using the sperm chromatin dispersion test. Both low and high H2 O2 concentration groups showed lower motility than control groups. Progressive motility of spermatozoa incubated in the H group dropped rapidly in comparison with other groups. Regarding SDF, the control and L groups had significantly lower values than the H group (25.0% and 31.6% vs. 48.1% fragmented sperm for C, L, and H groups, respectively; p < 0.05). Sperm motility, mostly progressive motility, decreased as H2 O2 concentration increased, mainly when time after sperm activation increased. SDF increased as the H2 O2 concentration increased. However, measurements of the halo area did not agree with the subjective SDF rate.

The phosphorylated pathway of serine biosynthesis is essential both for male gametophyte and embryo development and for root growth in Arabidopsis.

This study characterizes the phosphorylated pathway of Ser biosynthesis (PPSB) in Arabidopsis thaliana by targeting phosphoserine phosphatase (PSP1), the last enzyme of the pathway. Lack of PSP1 activity delayed embryo development, leading to aborted embryos that could be classified as early curled cotyledons. The embryo-lethal phenotype of psp1 mutants could be complemented with PSP1 cDNA under the control of Pro35S (Pro35S:PSP1). However, this construct, which was poorly expressed in the anther tapetum, did not complement mutant fertility. Microspore development in psp1.1/psp1.1 Pro35S:PSP1 arrested at the polarized stage. The tapetum from these lines displayed delayed and irregular development. The expression of PSP1 in the tapetum at critical stages of microspore development suggests that PSP1 activity in this cell layer is essential in pollen development. In addition to embryo death and male sterility, conditional psp1 mutants displayed a short-root phenotype, which was reverted in the presence of Ser. A metabolomic study demonstrated that the PPSB plays a crucial role in plant metabolism by affecting glycolysis, the tricarboxylic acid cycle, and the biosynthesis of amino acids. We provide evidence of the crucial role of the PPSB in embryo, pollen, and root development and suggest that this pathway is an important link connecting primary metabolism with development.

A method for resolving occlusions when multitracking individuals in a shoal.

Studying the collective behavior of fishes often requires tracking a great number of individuals. When many fishes move together, it is common for individuals to move so close to each other that some fishes superimpose themselves on others during one or several units of time, which impacts on tracking accuracy (i.e., loss of fish trajectories, interchange of fish identities). Type 1 occlusions arise when two fishes swim so near each other that they look like one long fish, whereas type 2 occlusions occur when the fishes' trajectories cross to create a T- or X-shaped individual. We propose an image processing method for resolving these types of occlusions when multitracking shoals in two dimensions. We assessed processing effectiveness after videorecording shoals of 20 and 40 individuals of two species that exhibit different shoal styles: zebrafish (Danio rerio) and black neon tetras (Hyphessobrycon herbertaxelrodi). Results show that, although the number of occlusions depended on both the number of individuals and the species, the method is able to effectively resolve a great deal of occlusions, irrespective of the species and the number of individuals. It also produces images that can be used in a multitracking system to detect individual fish trajectories. Compared to other methods, our approach makes it possible to study shoals with water depths similar to those seen in the natural conditions of the two species studied.

A minimally invasive methodology based on morphometric parameters for day 2 embryo quality assessment.

The risk of multiple pregnancy to maternal-fetal health can be minimized by reducing the number of embryos transferred. New tools for selecting embryos with the highest implantation potential should be developed. The aim of this study was to evaluate the ability of morphological and morphometric variables to predict implantation by analysing images of embryos. This was a retrospective study of 135 embryo photographs from 112 IVF-ICSI cycles carried out between January and March 2011. The embryos were photographed immediately before transfer using Cronus 3 software. Their images were analysed using the public program ImageJ. Significant effects (P < 0.05), and higher discriminant power to predict implantation were observed for the morphometric embryo variables compared with morphological ones. The features for successfully implanted embryos were as follows: four cells on day 2 of development; all blastomeres with circular shape (roundness factor greater than 0.9), an average zona pellucida thickness of 13 µm and an average of 17695.1 µm² for the embryo area. Embryo size, which is described by its area and the average roundness factor for each cell, provides two objective variables to consider when predicting implantation. This approach should be further investigated for its potential ability to improve embryo scoring.

Main Steps in Image Processing and Quantification: The Analysis Workflow.

In the last decades, the variety of programs, algorithms, and strategies that researchers have at their disposal to process and analyze image files has grown extensively. However, these are only pointless tools if not applied with the careful planning required to achieve a succesful image analysis. In order to do so, the analyst must establish a meaningful and effective sequence of orderly operations that is able to (1) overcome all the problems derived from the image manipulation and (2) successfully resolve the question that was originally posed. In this chapter, the authors suggest a set of strategies and present a reflection on the main milestones that compose the image processing workflow, to help guide the way to obtaining unbiased quantitative data.

Cell Proliferation High-Content Screening on Adherent Cell Cultures.

Pulse-chase experiments using 5-bromo-2'-deoxyuridine (BrdU), or the more recent EdU (5-etynil-2'-deoxyuridine), enable the identification of cells going through S phase. This chapter describes a high-content proliferation assay pipeline for adherent cell cultures. High-throughput imaging is followed by high-content data analysis using a non-supervised ImageJ macroinstruction that segments the individual nuclei, determines the nucleoside analogue absence/presence, and measures the signal of up to two additional nuclear markers. Based upon the specific combination with proliferation-specific protein immunostaining, the percentage of cells undergoing different phases of the cell cycle (G0, G1, S, G2, and M) might be established. The method can be also used to estimate the proliferation (S phase) rate of particular cell subpopulations identified through labelling with specific nuclear markers.

Zn2+ chelation by serum albumin improves hexameric Zn2+-insulin dissociation into monomers after exocytosis.

ß-cells release hexameric Zn2+-insulin into the extracellular space, but monomeric Zn2+-free insulin appears to be the only biologically active form. The mechanisms implicated in dissociation of the hexamer remain unclear, but they seem to be Zn2+ concentration-dependent. In this study, we investigate the influence of albumin binding to Zn2+ on Zn2+-insulin dissociation into Zn2+-free insulin and its physiological, methodological and therapeutic relevance. Glucose and K+-induced insulin release were analyzed in isolated mouse islets by static incubation and perifusion experiments in the presence and absence of albumin and Zn2+ chelators. Insulin tolerance tests were performed in rats using different insulin solutions with and without Zn2+ and/or albumin. Albumin-free buffer does not alter quantification by RIA of Zn2+-free insulin but strongly affects RIA measurements of Zn2+-insulin. In contrast, accurate determination of Zn2+-insulin was obtained only when bovine serum albumin or Zn2+ chelators were present in the assay buffer solution. Albumin and Zn2+ chelators do not modify insulin release but do affect insulin determination. Preincubation with albumin or Zn2+ chelators promotes the conversion of "slow" Zn2+-insulin into "fast" insulin. Consequently, insulin diffusion from large islets is ameliorated in the presence of Zn2+ chelators. These observations support the notion that the Zn2+-binding properties of albumin improve the dissociation of Zn2+-insulin into subunits after exocytosis, which may be useful in insulin determination, insulin pharmacokinetic assays and islet transplantation.

Growth factors and beta cell replication.

Recent studies have demonstrated that human islet allograft transplantation can be a successful therapeutic option in the treatment of patients with Type I diabetes. However, this impressive recent advance is accompanied by a very important constraint. There is a critical paucity of pancreatic islets or pancreatic beta cells for islet transplantation to become a large-scale therapeutic option in patients with diabetes. This has prompted many laboratories around the world to invigorate their efforts in finding ways for increasing the availability of beta cells or beta cell surrogates that potentially could be transplanted into patients with diabetes. The number of studies analyzing the mechanisms that govern beta cell proliferation and growth in physiological and pathological conditions has increased exponentially during the last decade. These studies exploring the role of growth factors, intracellular signaling molecules and cell cycle regulators constitute the substrate for future strategies aimed at expanding human beta cells in vitro and/or in vivo after transplantation. In this review, we describe the current knowledge on the effects of several beta cell growth factors that have been shown to increase beta cell proliferation and expand beta cell mass in vitro and/or in vivo and that they could be potentially deployed in an effort to increase the number of patients transplanted with islets. Furthermore, we also analyze in this review recent studies deciphering the relevance of these specific islet growth factors as physiological and pathophysiological regulators of beta cell proliferation and islet growth.

Targeted inactivation of hepatocyte growth factor receptor c-met in beta-cells leads to defective insulin secretion and GLUT-2 downregulation without alteration of beta-cell mass.

Overexpression of hepatocyte growth factor (HGF) in the beta-cell of transgenic mice enhances beta-cell proliferation, survival, and function. In the current studies, we have used conditional ablation of the c-met gene to uncover the physiological role of HGF in beta-cell growth and function. Mice in which c-met is inactivated in the beta-cell (MetCKO mice) display normal body weight, blood glucose, and plasma insulin compared with control littermates. In contrast, MetCKO mice displayed significantly diminished glucose tolerance and reduced plasma insulin after a glucose challenge in vivo. This impaired glucose tolerance in MetCKO mice was not caused by insulin resistance because sensitivity to exogenous insulin was similar in both groups. Importantly, in vitro glucose-stimulated insulin secretion in MetCKO islets was decreased by approximately 50% at high glucose concentrations compared with control islets. Furthermore, whereas insulin and glucokinase expression in MetCKO islets were normal, GLUT-2 expression was decreased by approximately 50%. These changes in beta-cell function in MetCKO mice were not accompanied by changes in total beta-cell mass, islet morphology, islet cell composition, and beta-cell proliferation. Interestingly, however, MetCKO mice display an increased number of small islets, mainly single and doublet beta-cells. We conclude that HGF/c-met signaling in the beta-cell is not essential for beta-cell growth, but it is essential for normal glucose-dependent insulin secretion.

Novel players in pancreatic islet signaling: from membrane receptors to nuclear channels.

Glucose and other nutrients regulate many aspects of pancreatic islet physiology. This includes not only insulin release, but also insulin synthesis and storage and other aspects of beta-cell biology, including cell proliferation, apoptosis, differentiation, and gene expression. This implies that in addition to the well-described signals for insulin release, other intracellular signaling mechanisms are needed. Here we describe the role of global and local Ca(2+) signals in insulin release, the regulation of these signals by new membrane receptors, and the generation of nuclear Ca(2+) signals involved in gene expression. An integrated view of these pathways should improve the present description of the beta-cell biology and provide new targets for novel drugs.

Increased glucose sensitivity of stimulus-secretion coupling in islets from Psammomys obesus after diet induction of diabetes.

When fed a high-energy (HE) diet, diabetes-prone (DP) Psammomys obesus develop type 2 diabetes with altered glucose-stimulated insulin secretion (GSIS). Beta-cell stimulus-secretion coupling was investigated in islets isolated from DP P. obesus fed a low-energy (LE) diet (DP-LE) and after 5 days on a HE diet (DP-HE). DP-LE islets cultured overnight in 5 mmol/l glucose displayed glucose dose-dependent increases in NAD(P)H, mitochondrial membrane potential, ATP/(ATP + ADP) ratio, cytosolic calcium concentration ([Ca(2+)](c)), and insulin secretion. In comparison, DP-HE islets cultured overnight in 10 mmol/l glucose were 80% degranulated and displayed an increased sensitivity to glucose at the level of glucose metabolism, [Ca(2+)](c), and insulin secretion. These changes in DP-HE islets were only marginally reversed after culture in 5 mmol/l glucose and were not reproduced in DP-LE islets cultured overnight in 10 mmol/l glucose, except for the 75% degranulation. Diabetes-resistant P. obesus remain normoglycemic on HE diet. Their beta-cell stimulus-secretion coupling was similar to that of DP-LE islets, irrespective of the type of diet. Thus, islets from diabetic P. obesus display an increased sensitivity to glucose at the level of glucose metabolism and a profound beta-cell degranulation, both of which may affect their in vivo GSIS.

SERCA3 ablation does not impair insulin secretion but suggests distinct roles of different sarcoendoplasmic reticulum Ca(2+) pumps for Ca(2+) homeostasis in pancreatic beta-cells.

Two sarcoendoplasmic reticulum Ca(2+)-ATPases, SERCA3 and SERCA2b, are expressed in pancreatic islets. Immunocytochemistry showed that SERCA3 is restricted to beta-cells in the mouse pancreas. Control and SERCA3-deficient mice were used to evaluate the role of SERCA3 in beta-cell cytosolic-free Ca(2+) concentration ([Ca(2+)](c)) regulation, insulin secretion, and glucose homeostasis. Basal [Ca(2+)](c) was not increased by SERCA3 ablation. Stimulation with glucose induced a transient drop in basal [Ca(2+)](c) that was suppressed by inhibition of all SERCAs with thapsigargin (TG) but unaffected by selective SERCA3 ablation. Ca(2+) mobilization by acetylcholine was normal in SERCA3-deficient beta-cells. In contrast, [Ca(2+)](c) oscillations resulting from intermittent glucose-stimulated Ca(2+) influx and [Ca(2+)](c) transients induced by pulses of high K(+) were similarly affected by SERCA3 ablation or TG pretreatment of control islets; their amplitude was increased and their slow descending phase suppressed. This suggests that, during the decay of each oscillation, the endoplasmic reticulum releases Ca(2+) that was pumped by SERCA3 during the upstroke phase. SERCA3 ablation increased the insulin response of islets to 15 mmol/l glucose. However, basal and postprandial plasma glucose and insulin concentrations in SERCA3-deficient mice were normal. In conclusion, SERCA2b, but not SERCA3, is involved in basal [Ca(2+)](c) regulation in beta-cells. SERCA3 becomes operative when [Ca(2+)](c) rises and is required for normal [Ca(2+)](c) oscillations in response to glucose. However, a lack of SERCA3 is insufficient in itself to alter glucose homeostasis or impair insulin secretion in mice.

Assessment of the implantation of day-2 human embryos by morphometric nonsubjective parameters.

OBJECTIVE: To demonstrate the usefulness of image analysis in designing objective embryonic morphometric variables. DESIGN: Retrospective study of 214 top-quality day-2 embryo photographs from 50 double-embryo transfers resulting in no pregnancy (group 0) and 57 resulting in twin pregnancy (group 1). SETTING: Human reproduction unit. PATIENT(S): Study of 107 in vitro fertilization-intracytoplasmic sperm injection (IVF-ICSI) cycles in women age <36 years with double-embryo transfer of top-quality embryos. Only the first cycle of IVF-ICSI was included. INTERVENTION(S): Standard IVF-ICSI protocols. MAIN OUTCOME MEASURE(S): The embryo photographs were analyzed using the ImageJ program. The effects of the embryo variables and the clinical variables on embryo implantation were evaluated using a stepwise dichotomous logistic regression. RESULT(S): Significant differences were observed, owing to the women's ages, internal perimeter, roundness factor, and zona pellucida thickness. Embryos with smaller internal perimeter, circular shape, and thinner zona pellucida were more likely to implant. CONCLUSION(S): Morphometric variables lower the subjectivity of the current embryo grading systems. These variables are nonsubjective factors to consider when predicting implantation. Embryo image analysis is an accurate tool that can improve IVF-ICSI outcomes and reduce the number of twin pregnancies.

Disruption of hepatocyte growth factor/c-Met signaling enhances pancreatic beta-cell death and accelerates the onset of diabetes.

OBJECTIVE: To determine the role of hepatocyte growth factor (HGF)/c-Met on ß-cell survival in diabetogenic conditions in vivo and in response to cytokines in vitro. RESEARCH DESIGN AND METHODS: We generated pancreas-specific c-Met-null (PancMet KO) mice and characterized their response to diabetes induced by multiple low-dose streptozotocin (MLDS) administration. We also analyzed the effect of HGF/c-Met signaling in vitro on cytokine-induced ß-cell death in mouse and human islets, specifically examining the role of nuclear factor (NF)-κB. RESULTS: Islets exposed in vitro to cytokines or from MLDS-treated mice displayed significantly increased HGF and c-Met levels, suggesting a potential role for HGF/c-Met in ß-cell survival against diabetogenic agents. Adult PancMet KO mice displayed normal glucose and ß-cell homeostasis, indicating that pancreatic c-Met loss is not detrimental for ß-cell growth and function under basal conditions. However, PancMet KO mice were more susceptible to MLDS-induced diabetes. They displayed higher blood glucose levels, marked hypoinsulinemia, and reduced ß-cell mass compared with wild-type littermates. PancMet KO mice showed enhanced intraislet infiltration, islet nitric oxide (NO) and chemokine production, and ß-cell apoptosis. c-Met-null ß-cells were more sensitive to cytokine-induced cell death in vitro, an effect mediated by NF-κB activation and NO production. Conversely, HGF treatment decreased p65/NF-κB activation and fully protected mouse and, more important, human ß-cells against cytokines. CONCLUSIONS: These results show that HGF/c-Met is critical for ß-cell survival by attenuating NF-κB signaling and suggest that activation of the HGF/c-Met signaling pathway represents a novel strategy for enhancing ß-cell protection.

Novel proapoptotic effect of hepatocyte growth factor: synergy with palmitate to cause pancreatic {beta}-cell apoptosis.

Increasing evidence suggests that elevation of plasma fatty acids that often accompanies insulin resistance contributes to beta-cell insufficiency in obesity-related type 2 diabetes. Circulating levels of hepatocyte growth factor (HGF) are increased in humans with metabolic syndrome and obesity. HGF is known to protect beta-cells against streptozotocin and during islet engraftment. However, whether HGF is a beta-cell prosurvival factor in situations of excessive lipid supply has not been deciphered. Mice overexpressing HGF in the beta-cell [rat insulin type II promoter (RIP)-HGF transgenic mice] fed with standard chow display improved glucose homeostasis and increased beta-cell mass and proliferation compared with normal littermates. However, after 15 wk of high-fat feeding, glucose homeostasis and beta-cell expansion and proliferation are indistinguishable between normal and transgenic mice. Interestingly, RIP-HGF transgenic mouse beta-cells and normal beta-cells treated with HGF display increased sensitivity to palmitate-mediated apoptosis in vitro. Palmitate completely eliminates Akt and Bad phosphorylation in RIP-HGF transgenic mouse islets. HGF-overexpressing islets also show significantly decreased AMP-activated protein kinase-alpha and acetyl-coenzyme A carboxylase phosphorylation, diminished fatty acid oxidation, increased serine palmitoyltransferase expression, and enhanced ceramide formation compared with normal islets. Importantly, human islets overexpressing HGF also display increased beta-cell apoptosis in the presence of palmitate. Treatment of both mouse and human islet cells with the de novo ceramide synthesis inhibitors myriocin and fumonisin B1 abrogates beta-cell apoptosis induced by HGF and palmitate. Collectively, these studies indicate that HGF can be detrimental for beta-cell survival in an environment with excessive fatty acid supply.

Delayed fatherhood in mice decreases reproductive fitness and longevity of offspring.

This study aims to analyze, in mice, the long-term effects of delayed fatherhood on reproductive fitness and longevity of offspring. Hybrid parental-generation (F(0)) males, at the age of 12, 70, 100, and 120 wk, were individually housed with a randomly selected 12-wk-old hybrid female. The reproductive fitness of first-generation (F(1)) females was tested from the age of 25 wk until the end of their reproductive life. In F(1) males, the testing period ranged from the age of 52 wk until death. Breeding F(1) females from the 120-wk group displayed interbirth intervals longer than females from the 12-, 70-, and 100-wk groups. Furthermore, F(2) pups begotten by F(1) studs exhibited weaning weights lower than pups from the 12- and 70-wk groups. Offspring from the 120-wk group exhibited shorter survival times associated with lower incidence of tumorigenesis and higher loss of body weight when approaching death when compared to F(1) offspring from younger age-groups. The results indicate that advanced paternal age at conception has negative long-term effects on reproductive fitness and longevity of offspring in the mouse model.

Long-term effects of delayed fatherhood in mice on postnatal development and behavioral traits of offspring.

This study aims to analyze, in mice, the long-term effects of delayed fatherhood on postnatal development, spontaneous motor activity, and learning capacity of offspring. Hybrid parental-generation (F(0)) males, at the age of 12, 70, 100, and 120 wk, were individually housed with a randomly selected 12-wk-old hybrid female. The resulting first-generation (F(1)) offspring were tested for several developmental and behavioral variables. Cumulative percentage of F(1) pups that attained immediate righting in the 120-wk group was lower than that found in the 12-, 70-, and 100-wk groups. Furthermore, the postnatal day of attaining immediate righting was higher in pups from the 120-wk group when compared to pups from the other age-groups. At the age of 20 wk, F(1) offspring from the 120-wk group displayed lower counts of motor activity than offspring from the 12-, 70-, and 100-wk groups. One week later, a higher percentage of offspring from the 100- and 120-wk groups entered the dark compartment during the retention trial of the passive-avoidance test when compared to offspring from the 12-wk group. Offspring from the 120-wk group exhibited also lower step-through latency in the retention trial than offspring from the 12-, 70-, and 100-wk groups. These results show that advanced paternal age at conception has long-term effects on preweaning development, spontaneous motor activity, and reduced passive-avoidance learning capacity of mouse offspring.

Beneficial effects of dithiothreitol on relative levels of glutathione S-transferase activity and thiols in oocytes, and cell number, DNA fragmentation and allocation at the blastocyst stage in the mouse.

We analyzed the effect of in vitro aging of mouse oocytes in the presence of dithiothreitol (DTT) on relative levels of glutathione S-transferase (GST) activity and thiols in oocytes, and cell number, DNA fragmentation and cellular allocation to the inner cell mass (ICM) and trophectoderm (TE) lineage at the blastocyst stage. Ovulated oocytes from gonadotropin primed hybrid female mice of 6-8 weeks of age were aged in vitro in the presence of 0, 5, 50, or 500 microM DTT for 6 hr prior to insemination. Relative levels of GST activity and thiols in oocytes were determined by confocal laser scanning microscopy, DNA fragmentation using a single-step TUNEL method, and cell allocation to the ICM and TE lineage by blastocyst staining with propidium iodide and Hoechst 33258. Non-aged oocytes exhibited higher relative levels of GST activity and thiols when compared to oocytes aged in the presence of 0, 5, and 50 microM DTT. Day 5 blastocysts from the 5, 50, and 500 microM DTT groups exhibited higher total number of cells, number of ICM cells, and ICM/TE ratio, but lower percentage of number of nuclei with DNA fragmentation/number of ICM cells than blastocyst from the 0 microM DTT group. These data show that DTT counteracts the negative effects of a post-ovulatory aging of mouse oocytes in vitro on relative levels of GST activity and thiols in oocytes, and percentage of number of nuclei with DNA fragmentation/number of ICM cells, total number of cells, number of ICM cells and ICM/TE ratio in Day 5 blastocysts.

Association of female aging with decreased parthenogenetic activation, raised MPF, and MAPKs activities and reduced levels of glutathione S-transferases activity and thiols in mouse oocytes.

This study aims to determine in the mouse whether oocytes from reproductively old females exhibit a different susceptibility to be parthenogenetically activated when compared to oocytes from young females. At the age of 10-12 (young-female group) or 60-62 (old-female group) weeks, hybrid female mice were superovulated using pregnant mare's serum gonadotropin (PMSG) followed by human chorionic gonadotropin (hCG) 48 hr later. After removing the cumulus cells, oocytes were exposed to any of two different activating protocols: (a) 6-min exposure to 8% ethanol; and (b) treatment with 200 microM thimerosal for 15 min followed by 8 mM dithiothreitol (DTT) for 30 min. Oocytes from old female mice displayed (1) lower total percentage of parthenogenetic activation and extrusion of the second polar body after treatment with either thimerosal + DTT or ethanol; (2) higher M-phase-promoting factor (MPF) and mitogen-activated protein kinases (MAPKs) activities; and (3) lower intracytoplasmic levels of glutathione S-transferases (GSTs) activity and thiols than oocytes from young females. These data show that female aging is associated with higher resistance of oocytes to be parthenogenetically activated, higher MPF and MAPKs activities and lower intracytoplasmic levels of GSTs activity and thiols.