title: Spheroids of granulosa cells provide an in vitro model for programmed cell death coupled to steroidogenesis.
abstract: We describe the use of rotary cultures (72 rpm) as an excellent method for generating spheroids from dispersed bovine granulosa cells (GC). The GC spheroids were symmetrical (diameter between 100 and 200 microm), easily accessible, and could be obtained at high yields. On day one, the spheroids showed a two-layered outer zone of cells that stained lighter than the inner zone in semi-thin sections. Bromodeoxyuridine (BrdU) uptake was frequent and randomly distributed. By day two, a striking decrease in BrdU uptake was noted. Apoptotic bodies appeared up to day four, as did TUNEL and propidium iodide labelled dead cells. At that time, the inner zone contained cells with large-sized vacuoles and the core was amorphous. The large-sized vacuoles were identified at the ultrastructural level and represented autophagosomes and autophagolysosomes that were in different stages of development. Surprisingly, conspicuous signs of cell death were accompanied by an increase in spontaneous luteinization compared to conventional stationary cultures. We detected high levels of progesterone (immunoassay) accompanied by high levels of the proteins and enzymes relevant for steroidogenesis (StAR, P450scc, 3beta-HSD by immunoblot and immunohistochemistry, respectively).
  
  
  CONCLUSIONS
  Concomitant to cell death, GC spheroids augment progesterone synthesis. The GC spheroids provide an ideal model for studying steroidogenesis coupled to programmed cell death at the level of the mitochondria.
authors.affiliation: Department of Anatomy, University of Leipzig, D-04103 Leipzig, Germany.
authors.forename: Katja
authors.initials: K
authors.lastname: Hummitzsch
authors.forename: Albert M
authors.initials: AM
authors.lastname: Ricken
authors.forename: Daniel
authors.initials: D
authors.lastname: Kloss
authors.forename: Sabine
authors.initials: S
authors.lastname: Erdmann
authors.forename: Marcin S
authors.initials: MS
authors.lastname: Nowicki
authors.forename: Andrée
authors.initials: A
authors.lastname: Rothermel
authors.forename: Andrea A
authors.initials: AA
authors.lastname: Robitzki
authors.forename: Katharina
authors.initials: K
authors.lastname: Spanel-Borowski
mesh_terms: D000818:Animals; D017209:Apoptosis; D002417:Cattle; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism; D005260:Female; D006107:Granulosa Cells / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D011374:Progesterone / Q000096:biosynthesis*; D014617:Vacuoles / Q000378:metabolism
chemical_list: D011374:Progesterone; D002784:Cholesterol; D002786:Cholesterol Side-Chain Cleavage Enzyme