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      Long-term apoptosis-related protein expression in the diabetic mouse ovary

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          Abstract

          Emerging evidence has shown that oocytes from diabetic ovaries exhibit delayed maturation, mitochondrial dysfunction and meiotic defects, which are related increased apoptosis. The main objective of the present study was to analyze the apoptosis pathways activated during follicular loss at multiple time points in a diabetic mouse model. Twenty BALB/c mice were used in this study, and diabetes mellitus was induced by streptozotocin injection. Three diabetic and two control animals were sacrificed on days 15, 20, 70 and 80 posttreatment. The ovaries were then removed; one was used for follicular counting, TUNEL, immunohistochemistry and immunofluorescence, while the other was used for Western blot analysis. The proteins studied were BAX, BCL2, t-BID, FAS, FASL, active caspase 8, active caspase 9 and active caspase 3. Follicular apoptosis decreased over time, with the highest values observed at 15 days posttreatment. Granulosa cells were positive for active caspase 3, which showed constant expression levels at all time points. FAS, FASL, t-BID and active caspase 8 showed strong cytoplasmic immunostaining in the oocytes and granulosa cells of the diabetic mice, with significant increases observed at 15, 20 and 70 days posttreatment. BAX expression was slightly higher in the diabetic mouse ovaries than in the control ovaries at 15, 20 and 70 days posttreatment, whereas the highest active caspase 9 expression was at observed 20 days posttreatment. Low BCL2 protein levels were detected in the diabetic mouse ovaries at all time points. This study describes for the first time the behavior of apoptosis-related proteins in the diabetic mouse ovary and shows not only that the FAS/FASL pathway contributes to follicular loss but also that antral follicles are the most affected.

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          Most cited references32

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          Global prevalence of diabetes: estimates for the year 2000 and projections for 2030.

          The goal of this study was to estimate the prevalence of diabetes and the number of people of all ages with diabetes for years 2000 and 2030. Data on diabetes prevalence by age and sex from a limited number of countries were extrapolated to all 191 World Health Organization member states and applied to United Nations' population estimates for 2000 and 2030. Urban and rural populations were considered separately for developing countries. The prevalence of diabetes for all age-groups worldwide was estimated to be 2.8% in 2000 and 4.4% in 2030. The total number of people with diabetes is projected to rise from 171 million in 2000 to 366 million in 2030. The prevalence of diabetes is higher in men than women, but there are more women with diabetes than men. The urban population in developing countries is projected to double between 2000 and 2030. The most important demographic change to diabetes prevalence across the world appears to be the increase in the proportion of people >65 years of age. These findings indicate that the "diabetes epidemic" will continue even if levels of obesity remain constant. Given the increasing prevalence of obesity, it is likely that these figures provide an underestimate of future diabetes prevalence.
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            Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation

            Programmed cell death (PCD) plays a key role in developmental biology and in maintenance of the steady state in continuously renewing tissues. Currently, its existence is inferred mainly from gel electrophoresis of a pooled DNA extract as PCD was shown to be associated with DNA fragmentation. Based on this observation, we describe here the development of a method for the in situ visualization of PCD at the single-cell level, while preserving tissue architecture. Conventional histological sections, pretreated with protease, were nick end labeled with biotinylated poly dU, introduced by terminal deoxy- transferase, and then stained using avidin-conjugated peroxidase. The reaction is specific, only nuclei located at positions where PCD is expected are stained. The initial screening includes: small and large intestine, epidermis, lymphoid tissues, ovary, and other organs. A detailed analysis revealed that the process is initiated at the nuclear periphery, it is relatively short (1-3 h from initiation to cell elimination) and that PCD appears in tissues in clusters. The extent of tissue-PCD revealed by this method is considerably greater than apoptosis detected by nuclear morphology, and thus opens the way for a variety of studies.
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              Role of reactive oxygen species (ROS) in apoptosis induction.

              Reactive oxygen species (ROS) and mitochondria play an important role in apoptosis induction under both physiologic and pathologic conditions. Interestingly, mitochondria are both source and target of ROS. Cytochrome c release from mitochondria, that triggers caspase activation, appears to be largely mediated by direct or indirect ROS action. On the other hand, ROS have also anti-apoptotic effects. This review focuses on the role of ROS in the regulation of apoptosis, especially in inflammatory cells.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: InvestigationRole: MethodologyRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Visualization
                Role: InvestigationRole: MethodologyRole: Visualization
                Role: InvestigationRole: MethodologyRole: Visualization
                Role: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: InvestigationRole: Project administrationRole: ResourcesRole: SupervisionRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                7 September 2018
                2018
                : 13
                : 9
                : e0203268
                Affiliations
                [1 ] Facultad de Ciencias de la Salud, Carrera de Medicina, Universidad Maimónides, Ciudad Autónoma de Buenos Aires, Buenos Aires, Argentina
                [2 ] Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Ciudad Autónoma de Buenos Aires, Buenos Aires, Argentina
                [3 ] Centro de Estudios Farmacológicos y Botánicos-CONICET (CEFYBO), Buenos Aires, Argentina
                Universite Paris-Sud, FRANCE
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0003-1561-2563
                Article
                PONE-D-17-39250
                10.1371/journal.pone.0203268
                6128485
                30192809
                f1b466f9-5e87-48c4-b7da-2d02f9996e19
                © 2018 Fraunhoffer et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 4 November 2017
                : 19 August 2018
                Page count
                Figures: 5, Tables: 1, Pages: 14
                Funding
                Funded by: Fundación Científica Felipe Fiorellino - Universidad Maimónides
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100002923, Consejo Nacional de Investigaciones Científicas y Técnicas;
                Award Recipient :
                This research was supported by the Fundación Cientifica Felipe Fiorellino - Universidad Maimónides, Buenos Aires, Argentina. NAF is a recipient of a postdoctoral fellowship from the National Research Council, CONICET-Argentina. AMA is a doctoral fellow from Universidad Maimónides. MAB, KVC and MFO are medical students of the Universidad Maimónides. ECH is a Principal Researcher of the Centro de Estudios Farmacológicos y Botánicos-CONICET (CEFYBO), MB is the director of the School of Medicine at Universidad Maimónides. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Medicine and Health Sciences
                Endocrinology
                Endocrine Disorders
                Diabetes Mellitus
                Medicine and Health Sciences
                Metabolic Disorders
                Diabetes Mellitus
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Cell Death
                Apoptosis
                Biology and Life Sciences
                Anatomy
                Reproductive System
                Ovaries
                Medicine and Health Sciences
                Anatomy
                Reproductive System
                Ovaries
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Epithelial Cells
                Granulosa Cells
                Biology and Life Sciences
                Anatomy
                Biological Tissue
                Epithelium
                Epithelial Cells
                Granulosa Cells
                Medicine and Health Sciences
                Anatomy
                Biological Tissue
                Epithelium
                Epithelial Cells
                Granulosa Cells
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Lutein Cells
                Granulosa Cells
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Germ Cells
                OVA
                Oocytes
                Biology and Life Sciences
                Biotechnology
                Biology and Life Sciences
                Anatomy
                Histology
                Medicine and Health Sciences
                Anatomy
                Histology
                Research and Analysis Methods
                Experimental Organism Systems
                Model Organisms
                Mouse Models
                Research and Analysis Methods
                Model Organisms
                Mouse Models
                Research and Analysis Methods
                Experimental Organism Systems
                Animal Models
                Mouse Models
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                All relevant data are within the paper and its Supporting Information files.

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