After incubation, follicles were collected in 12- X 75mm polypropylene tubes, snap-frozen, and stored at -80°C until processed for analysis of low-molecular weight DNA integrity by 3′-end radiolabeling and conventional agarose gel electrophoresis (CAGE; see below) or were embedded in 0.5% agarose plugs for high-molecular weight DNA analysis after pulsed-field gel electrophoresis (PFGE; see below). For the PFGE and CAGE analyses, genomic DNA present in Time 0 follicles (no incubation) served as control data points for levels of background DNA cleavage present before the experimental manipulations in vitro.
Additionally, some follicles were also fixed, embedded in paraffin, sectioned, and mounted to glass slides for fluorescence microscopy of nuclear morphology (see below). All studies involving rats described herein were approved by and performed in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committees at the Massachusetts General Hospital, the University of North Carolina-Charlotte, or the NIEHS.
Conventional Agarose Gel Electrophoretic Analysis of Internucleosomal DNA Cleavage
Genomic DNA was prepared from intact follicles or isolated nuclei as described elsewhere. The quantity and purity of the DNA preparations were estimated by spectro-photometric measurements of the optical density of each sample at 260 versus 280 nm. Following isolation and quantitation, DNA samples (1 ^g/reaction) were 3′-end labeled with [a32P]dideoxy-ATP (3000 Ci/mmol; Amersham, Arlington Heights, IL) by using the terminal transferase reaction (Boehringer-Mannheim), fractionated through conventional 2% agarose gels, and then analyzed by autoradiography and (3-counting of low-molecular weight (<10 kb) DNA fragments, as described elsewhere.