Granulosa Cell Apoptosis in the Ovarian Follicle—A Changing View

N-Methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity may depend, in part, on the generation of nitric oxide (NO.) and superoxide anion (O2.-), which react to form peroxynitrite (OONO-). This form of neurotoxicity is thought to contribute to a final common pathway of injury in a wide variety of acute and chronic neurologic disorders, including focal ischemia, trauma, epilepsy, Huntington disease, Alzheimer disease, amyotrophic lateral scelerosis, AIDS dementia, and other neurodegenerative diseases. Here, we report that exposure of cortical neurons to relatively short durations or low concentrations of NMDA, S-nitrosocysteine, or 3-morpholinosydnonimine, which generate low levels of peroxynitrite, induces a delayed form of neurotoxicity predominated by apoptotic features. Pretreatment with superoxide dismutase and catalase to scavenge O2.- partially prevents the apoptotic process triggered by S-nitrosocysteine or 3-morpholinosydnonimine. In contrast, intense exposure to high concentrations of NMDA or peroxynitrite induces necrotic cell damage characterized by acute swelling and lysis, which cannot be ameliorated by superoxide dismutase and catalase. Thus, depending on the intensity of the initial insult, NMDA or nitric oxide/superoxide can result in either apoptotic or necrotic neuronal cell damage.

In the efforts aimed at improving the quality of in-vitro-matured human oocytes, the dynamic balance and roles of pro-/antioxidants merit further consideration. In-vitro maturation (IVM) is emerging as a popular technology at the forefront of fertility treatment and preservation. However, standard in-vitro culture conditions exert oxidative stress or an imbalance between oxidants and antioxidants. Reactive oxygen species (ROS) are oxygen-derived molecules formed as intermediary products of cellular metabolism. By acting as powerful oxidants, ROS can oxidatively modify any molecule, resulting in structural and functional alterations. ROS are neutralized by an elaborate defence system consisting of enzymatic and nonenzymatic antioxidants. This review captures the inherent and external factors that may modulate the oxidative stress status of oocytes. It discusses the suspected impacts of oxidative stress on the gamut of events associated with IVM, including prematuration arrest, meiotic progression, chromosomal segregation, cytoskeletal architecture and gene expression. In-vivo and in-vitro strategies that may overcome the potential influences of oxidative stress on oocyte IVM are presented. Future studies profiling the oxidative stress status of the oocyte may permit not only the formulation of a superior IVM medium that maintains an adequate pro-/antioxidant balance, but also the identification of predictors of oocyte quality.

Dramatic changes in the structure of cell membranes on apoptosis allow easy, sensitive and non-destructive analysis of this process with the application of fluorescence methods. The strong plasma membrane asymmetry is present in living cells, and its loss on apoptosis is commonly detected with the probes interacting strongly and specifically with phosphatidylserine (PS). This phospholipid becomes exposed to the cell surface, and the application of annexin V labeled with fluorescent dye is presently the most popular tool for its detection. Several methods have been suggested recently that offer important advantages over annexin V assay with the ability to study apoptosis by spectroscopy of cell suspensions, flow cytometry and confocal or two-photon microscopy. The PS exposure marks the integrated changes in the outer leaflet of cell membrane that involve electrostatic potential and hydration, and the attempts are being made to provide direct probing of these changes. This review describes the basic mechanisms underlying the loss of membrane asymmetry during apoptosis and discusses, in comparison with the annexin V-binding assay, the novel fluorescence techniques of detecting apoptosis on cellular membrane level. In more detail we describe the detection method based on smart fluorescent dye F2N12S incorporated into outer leaflet of cell membrane and reporting on apoptotic cell transformation by easily detectable change of the spectral distribution of fluorescent emission. It can be adapted to any assay format.