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- Identification of Potassium-Dependent and -Independent Components: MATERIALS AND METHODS(2)

MATERIALS AND METHODS(2)

Oocyte Incubations

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.

To examine potassium efflux, oocytes were cultured for 20 h without or with 200 nM DXR, and PBFI was added to these cultures (at final concentration of 5 ^M) 1 h before analysis by fluorescence microscopy (excitation at 340 nm; emission at 505 nm). For those experiments that involved disrupting the potassium gradient, oocyte incubations were carried out in a modified (‘‘ion-deficient’’) HTF medium. This medium was prepared at the National Institute of Environmental Health Sciences (NIEHS) Cell Culture Media Facility according to the composition and characteristics of HTF (Irvine Scientific), with the exception that NaCl (normally 101.6 mM) and KCl (normally 4.69 mM) were not added, so that effects of ‘‘experimental’’ KCl, NaCl, or LiCl (each at a final concentration of 150 mM) could be directly assessed. In those cultures lacking inclusion of exogenous KCl, NaCl, or LiCl, mannitol was added to correct for deficiencies in medium osmolality.

At the end of the incubation period, the oocytes were either examined alive (PBFI experiments) or fixed for 30 min in neutral-buffered 1% (wt/vol) paraformaldehyde prepared in 1X Dulbecco PBS and checked by differential interference contrast (DIC) microscopy for morphological changes characteristic of apoptosis. The percentage of oocytes that underwent cellular fragmentation out of the total number of oocytes cultured per drop in each experiment was determined. In some experiments, oocytes were further analyzed for the occurrence of DNA cleavage using terminal transferase-mediated DNA 3′-end labeling in situ or the comet assay (see below). All studies involving mice described herein were approved by and performed in accordance with the guidelines of the Massachusetts General Hospital Institutional Animal Care And Use Committee and the NIH Guide for the Care and Use of Laboratory Animals.

September 8, 2013 Cells
Tags: apoptosis follicle granulosa cells ovum