Isolation and ultrastructure of rabbit ovarian mesothelium (surface epithelium)

Abstract

This study evaluated the efficacy of various dissociating procedures for isolating mesothelial or surface epithelial cells from rabbit ovaries. The procedure which provided the highest and most homogenous cell yield involved the following sequence: (a) incubation of whole ovaries for 60 min in Clostridium histolyticum collagenase, type I (300 U/ml); (b) vortexing of ovaries at 30 rpm for 60 s in medium 199; (c) removal of partially detached surface epithelium by gentle scraping with a microdissecting blade; and (d) unit gravity enrichment of isolated epithelial organoids (surface layers and villous processes). As shown by light and ultrastructural microscopy, this procedure cleaved the surface epithelium from over two-thirds of the underlying basement membrane and tunica albuginea. As estimated by further dispersal of epithelial organoids with trypsin-EDTA, 0.8 to 1.5 X 10(6) surface cells were routinely obtained from a single ovary. This isolation procedure is simple and provides an experimental tool for investigating in vitro the regulation and developmental behavior of ovarian surface epithelium.