ISOLATION OF GRANULOSAL-LIKE CELLS FROM THE BOVINE SECRETORY CORPUS-LUTEUM AND THEIR CHARACTERIZATION IN LONG-TERM CULTURE

Background: The isolation of cells termed type 5 from the bovine corpu s luteum was recently reported. Since these cells were reminiscent of immature granulosa cells, their morphological and functional relations hip requires further investigation in view of the novel concept of cor pus luteum growth. It suggests that putative stem cells of unknown ori gin supply the pool of small luteal cells. Methods: Bovine corpora lut ea were mechanically dispersed, cell suspensions separated over a Perc oll(R) density gradient, and type 5 cells purified by colony transfer. Granulosa cells were harvested from small-sized antral follicles. Obs ervations were carried out at the light and electron microscopical lev el. 3 beta-Hydroxy-steroid-dehydrogenase was localized histochemically in addition to intracellular lipid droplets stained with nile red. Im munolocalization was used to study Factor VIII antigen presence, the a rchitecture of the cytoskeleton, as well as the occurrence of neuronal cell adhesion molecules, and of neuronal cadherin-like molecules. The uptake of acetylated low density lipoprotein was examined. As for pro gesterone concentration, cells were seeded at low density on day zero. Cell numbers and progesterone levels of supernatants were determined on day 10 in culture. Results and Conclusions: Type 5 cells behaved mo rphologically Like immature granulosa cells, yet the total cell number and the progesterone concentration differed for type 5 cells compared to granulosa cells. The addition of LH had no influence on the proges terone concentration as seen for either type 5 cells or for granulosa cells. It is concluded that type 5 cells, which were originally mistak en for microvascular endothelial cells, display similarities with imma ture granulosa cells. Type 5 cells may play a role in renewal of lutea l cells. (C) 1994 Wiley-Liss, Inc.