title: Evidence for the maintenance of macrophage-like cells in long-term bovine granulosa cell cultures.
abstract: Ovarian macrophages may be involved in the degradation of the basal membrane after rupture of a preovulatory follicle, or in angiogenesis during follicle and corpus luteum development, or may support steroidogenesis. The present study describes easy access to macrophage-like cells in the ovary and characterizes them. Cells from bovine antral follicles with 1-4 ml of their naturally occurring fluid were plated on day zero. On day 10, the granulosa cell culture showed macrophage-like cells. They developed as non-adherent cells and also adhered to the monolayer as single cells or as hemopoietic-like clones. The macrophage-like cells contained strong activity of non-specific esterase, acid phosphatase or 3beta-hydroxy-steroid dehydrogenase (3beta-HSD). Only the steroid enzyme was absent on day 19. As detected by immunolocalization, macrophage-like cells of various sizes showed a response for the CD18 surface molecule of leukocytes, for the CD45 molecule related to hemopoietic progenitor cells, and for the CD14 molecule selectively expressed on cells of the monocyte-macrophage lineage. The macrophage-like cells exhibited a granular-like actin structure as revealed by fluorochrome-labeled phalloidin. The ultrastructure of small, intermediate or large macrophage-like cells was similar to that of monocytes or of macrophages. In situ immunolabeling of intact or regressing follicles revealed single cells positive for CD14, CD18 or CD45. In order to establish that in vitro proliferation of the macrophage-like cells had occurred, their number was assessed between days 1 to 19 of cultivation. The cultures derived from 23 follicles were classified into groups A, B, or C according to the number of non-adherent cells (<5000, <12 500, or >/=12 500 per 16-mm well) on day 12 of cultivation. The total number of macrophage-like cells (non-adherent and adherent) increased approximately fourfold in all groups between days 1 to 12. The total number decreased to day 5 levels between days 15 to 19. Thus, the macrophage-like cells probably represent a subpopulation of the macrophage family. They proliferate in co-culture with granulosa cells.
authors.affiliation: Department of Anatomy, University of Basel, Pestalozzistrasse 20, CH-4056 Basel, Switzerland.
authors.forename: K
authors.initials: K
authors.lastname: Spanel-Borowski
authors.forename: A M
authors.initials: AM
authors.lastname: Ricken
mesh_terms: D015096:3-Hydroxysteroid Dehydrogenases / Q000032:analysis; D000135:Acid Phosphatase / Q000032:analysis; D000818:Animals; D018821:CD18 Antigens / Q000032:analysis; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000032:analysis; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D005260:Female; D006107:Granulosa Cells / Q000166:cytology*; D017493:Leukocyte Common Antigens / Q000032:analysis; D018950:Lipopolysaccharide Receptors / Q000032:analysis; D008264:Macrophages / Q000166:cytology* / Q000201:enzymology / Q000276:immunology; D006080:Ovarian Follicle / Q000166:cytology*; D013997:Time Factors
chemical_list: D018821:CD18 Antigens; D018950:Lipopolysaccharide Receptors; D015096:3-Hydroxysteroid Dehydrogenases; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; D000135:Acid Phosphatase; D017493:Leukocyte Common Antigens