Expression and function of Twitch-2B in the granulosa cells of mouse ovarian follicles

Briefly, antral follicles were dissected from 23- to 26-d-old RR8; Bact_Cre mice. Follicles were cultured for 24–30 h on organotypic membranes (Millipore; cat. No. PICMORG50) in the presence of follicle-stimulating hormone. The follicle was held in a perfusion slide consisting of a plastic slide (ibidi) and a glass coverslip and assembled using silicon grease. The slide was constructed such that medium containing ovine LH (National Hormone and Peptide Program; 10 μg/mL) could be perfused through a 200-μm-deep channel holding the follicle. Temperature was maintained at 30–34 °C, by use of a warm air blower (Nevtek). Preovulatory Follicles were imaged using a Zeiss Pascal confocal microscope with a 40X/1.2 NA objective. Images were collected every 10 seconds. Measurements were corrected for autofluorescence and for spectral bleed-through of CFP into the YFP channel. Ratios were calculated by dividing the mean CFP intensity in each region of interest by the mean YFP intensity. Data analysis was done using ImageJ and Excel software. Data is representative of 4 follicles.