title: Norepinephrine stimulates progesterone production in highly estrogenic bovine granulosa cells cultured under serum-free, chemically defined conditions.
abstract: BACKGROUND
  Since noradrenergic innervation was described in the ovarian follicle, the actions of the intraovarian catecholaminergic system have been the focus of a variety of studies. We aimed to determine the gonadotropin-independent effects of the catecholamine norepinephrine (NE) in the steroid hormone profile of a serum-free granulosa cell (GC) culture system in the context of follicular development and dominance.
  
  
  METHODS
  Primary bovine GCs were cultivated in a serum-free, chemically defined culture system supplemented with 0.1% polyvinyl alcohol. The culture features were assessed by hormone measurements and ultrastructural characteristics of GCs.
  
  
  RESULTS
  GCs produced increasing amounts of estradiol and pregnenolone for 144h and maintained ultrastructural features of healthy steroidogenic cells. Progesterone production was also detected, although it significantly increased only after 96h of culture. There was a highly significant positive correlation between estradiol and pregnenolone production in high E2-producing cultures. The effects of NE were further evaluated in a dose-response study. The highest tested concentration of NE (10 (-7) M) resulted in a significant increase in progesterone production, but not in estradiol or pregnenolone production. The specificity of NE effects on progesterone production was further investigated by incubating GCs with propranolol (10 (-8) M), a non-selective beta-adrenergic antagonist.
  
  
  CONCLUSIONS
  The present culture system represents a robust model to study the impact of intrafollicular factors, such as catecholamines, in ovarian steroidogenesis and follicular development. The results of noradrenergic effects in the steroidogenesis of GC have implications on physiological follicular fate and on certain pathological ovarian conditions such as cyst formation and anovulation.
authors.affiliation: Hospital Israelita Albert Einstein, São Paulo, Brazil. cpiccinato@uwalumni.com
authors.forename: Carla A
authors.initials: CA
authors.lastname: Piccinato
authors.forename: Luis H
authors.initials: LH
authors.lastname: Montrezor
authors.forename: Cristhianna A V
authors.initials: CA
authors.lastname: Collares
authors.forename: Alessandra A
authors.initials: AA
authors.lastname: Vireque
authors.forename: Alzira A M
authors.initials: AA
authors.lastname: Rosa e Silva
mesh_terms: D000735:Androstenedione / Q000032:analysis; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002784:Cholesterol / Q000032:analysis; D016895:Culture Media, Serum-Free / Q000737:chemistry; D004958:Estradiol / Q000096:biosynthesis; D005260:Female; D006107:Granulosa Cells / Q000187:drug effects* / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D009638:Norepinephrine / Q000494:pharmacology*; D011284:Pregnenolone / Q000096:biosynthesis; D011374:Progesterone / Q000096:biosynthesis*
chemical_list: D016895:Culture Media, Serum-Free; D000735:Androstenedione; D011374:Progesterone; D004958:Estradiol; D011284:Pregnenolone; D002784:Cholesterol; D009638:Norepinephrine