In Situ DNA 3′-End-Labeling Analysis
At the end of the incubation period without or with the experimental treatments, oocytes were transferred into Ty-rode solution for 30 sec at 37°C to remove the zona pel-lucida, washed quickly in PBS, and then immediately fixed for 30 min in neutral-buffered 1% (wt/vol) paraformaldehyde prepared in PBS containing 0.1 mg/ml polyvinyl alcohol (average molecular weight of 30 000-70 000). After fixation, oocytes were washed once more with PBS, transferred to Superfrost-Plus slides (Fisher Scientific, Pittsburgh, PA) in small drops (10 oocytes/10 ^l drop), and air-dried. Slides were heated at 65°C for 4 h and then stored at 4°C until processed for in situ DNA 3′-end-labeling analysis (ISEL) as detailed elsewhere, with slight modifications.
As previously described, virgin female B6C3F1/ CrlBR mice at 7 wk of age (Charles River Laboratories, Wilmington, MA) were superovulated with 10 IU of eCG, followed by 10 IU of hCG 48 h later. Cumulus-oocyte complexes were collected from the oviducts 16 h after hCG injection. Oocytes were denuded of cumulus cells by a 1-min incubation in 80 IU/ml hyaluronidase and then washed three times with human tubal fluid (HTF) culture medium (Irvine Scientific, Santa Ana, CA) supplemented with 0.5% BSA (Fraction V, fatty-acid free). Oocytes were pooled into groups of 10, transferred into 0.1-ml drops of prewarmed culture medium (see below) under paraffin oil, and cultured for 24 h at 37°C in a humidified atmosphere of 5% CO2 and 95% air, without or with 200 nM dXr.
Equine CG was obtained from Professional Compounding Centers of America (Houston, TX; oocyte studies), Cal-biochem (La Jolla, CA; granulosa cell and follicle studies) or the National Hormone and Pituitary Program of the National Institute of Diabetes and Digestive and Kidney Diseases (A.F. Parlow; granulosa cell K+ studies); hCG was obtained from Serono Laboratories (Norwell, MA). The potassium-sensitive dye PBFI (the cell-permeant acetoxyl-methyl ester of potassium-binding benzofuran isophthalate) was purchased from Molecular Probes (Eugene, OR). Human recombinant caspase-1 and caspase-3 (generously provided by Dr W.W. Wong, BASF Bioresearch Corp., Worcester, MA) were prepared as N-His affinity-tagged proteins in Escherichia coli and activated prior to each experiment by dithiothreitol treatment.
Until very recently, however, it was unclear whether potassium efflux directly influenced the function or activity of different effectors of apoptosis, in particular the caspase family of cystein proteases and the endonucleases that are associated with most paradigms of cell death. Using glucocorticoid-treated thymocytes, Hughes et al. have reported that inhibition of potassium efflux by disruption of the normal potassium electrochemical gradient prevents caspase-3 activation as well as endogenous nuclease activity.
It is now generally accepted that apoptosis in most species and cell types is precisely regulated by the actions of a number of intracellular molecules, derived from both active gene transcription (i.e., proteins) and various metabolic events, including mitochondrial respiration (e.g., reactive oxygen species) and release of proapoptotic factors (e.g., cytochrome c, apoptosis-inducing factor), phospholipid turnover (e.g., ceramide, sphingosine-1-phosphate, diacyl-glycerol), and ion fluxes. Despite the diversity and complexity of the events surrounding the induction of cell death, many genetic and biochemical studies have provided evidence that there likely exists an ordered and evolutionarily conserved pathway by which cells activate, execute, and complete the process of self-destruction.
Apoptosis plays a fundamental role in the development of multicellular organisms and in the maintenance of homeostasis in many tissues. In all vertebrate species examined to date, this form of controlled cellular deletion is the underlying mechanism responsible for female germ cell depletion from the ovary under both normal and pathological conditions. For example, the tremendous levels of oogonium and oocyte attrition that occur in waves during development of the fetal ovary are a result of apoptosis, as defined by both morphological and biochemical criteria.