title: Dopamine in human follicular fluid is associated with cellular uptake and metabolism-dependent generation of reactive oxygen species in granulosa cells: implications for physiology and pathology.
abstract: OBJECTIVE
  Is the neurotransmitter dopamine (DA) in the human ovary involved in the generation of reactive oxygen species (ROS)?
  
  
  CONCLUSIONS
  Human ovarian follicular fluid contains DA, which causes the generation of ROS in cultured human granulosa cells (GCs), and alterations of DA levels in follicular fluid and DA uptake/metabolism in GCs in patients with polycystic ovary syndrome (PCOS) are linked to increased levels of ROS.
  
  
  BACKGROUND
  DA is an important neurotransmitter in the brain, and the metabolism of DA results in the generation of ROS. DA was detected in human ovarian homogenates, but whether it is present in follicular fluid and plays a role in the follicle is not known.
  
  
  METHODS
  We used human follicular fluid from patients undergoing in vitro fertilization (IVF), GCs from patients with or without PCOS and also employed mathematical modeling to investigate the presence of DA and its effects on ROS.
  
  
  METHODS
  DA in follicular fluid and GCs was determined by enzyme-linked immunosorbent assay. GC viability, apoptosis and generation of ROS were monitored in GCs upon addition of DA. Inhibitors of DA uptake and metabolism, an antioxidant and DA receptor agonists, were used to study cellular uptake and the mechanism of DA-induced ROS generation. Human GCs were examined for the presence and abundance of transcripts of the DA transporter (DAT; SLC6A3), the DA-metabolizing enzymes monoamine oxidases A/B (MAO-A/B) and catechol-O-methyltransferase and the vesicular monoamine transporter. A computational model was developed to describe and predict DA-induced ROS generation in human GCs.
  
  
  RESULTS
  We found DA in follicular fluid of ovulatory follicles of the human ovary and in GCs. DAT and MAO-A/B, which are expressed by GCs, are prerequisites for a DA receptor-independent generation of ROS in GCs. Blockers of DAT and MAO-A/B, as well as an antioxidant, prevented the generation of ROS (P < 0.05). Agonists of DA receptors (D1 and D2) did not induce ROS. DA, in the concentration range found in follicular fluid, did not induce apoptosis of cultured GCs. Computational modeling suggested, however, that ROS levels in GCs depend on the concentrations of DA and on the cellular uptake and metabolism. In PCOS-derived follicular fluid, the levels of DA were higher (P < 0.05) in GCs, the transcript levels of DAT and MAO-A/B in GCs were 2-fold higher (P < 0.05) and the DA-induced ROS levels were found to be more than 4-fold increased (P < 0.05) compared with non-PCOS cells. Furthermore, DA at a high concentration induced apoptosis in PCOS-derived GCs.
  
  
  CONCLUSIONS
  While the results in IVF-derived follicular fluid and in GCs reveal for the first time the presence of DA in the human follicular compartment, functions of DA could only be studied in IVF-derived GCs, which can be viewed as a cellular model for the periovulatory follicular phase. The full functional importance of DA-induced ROS in small follicles and other compartments of the ovary, especially in PCOS samples, remains to be shown.
  
  
  CONCLUSIONS
  The results identify DA as a factor in the human ovary, which, via ROS generation, could play a role in ovarian physiology and pathology. The results obtained in samples from women with PCOS suggest the involvement of DA, acting via ROS, in this condition.
  
  
  BACKGROUND
  This work was supported by a grant from DFG MA1080/17-3 and in part MA1080/19-1. There are no competing interests.
authors.affiliation: Anatomy III-Cell Biology, Ludwig Maximilian University Munich, Munich, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Saller
authors.forename: L
authors.initials: L
authors.lastname: Kunz
authors.forename: D
authors.initials: D
authors.lastname: Berg
authors.forename: U
authors.initials: U
authors.lastname: Berg
authors.forename: H
authors.initials: H
authors.lastname: Lara
authors.forename: J
authors.initials: J
authors.lastname: Urra
authors.forename: S
authors.initials: S
authors.lastname: Hecht
authors.forename: R
authors.initials: R
authors.lastname: Pavlik
authors.forename: C J
authors.initials: CJ
authors.lastname: Thaler
authors.forename: A
authors.initials: A
authors.lastname: Mayerhofer
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D004298:Dopamine / Q000378:metabolism*; D018491:Dopamine Agonists / Q000494:pharmacology; D050483:Dopamine Plasma Membrane Transport Proteins / Q000096:biosynthesis; D005260:Female; D015571:Follicular Fluid / Q000378:metabolism*; D006107:Granulosa Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D011085:Polycystic Ovary Syndrome / Q000503:physiopathology; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D018491:Dopamine Agonists; D050483:Dopamine Plasma Membrane Transport Proteins; D017382:Reactive Oxygen Species; C493214:SLC6A3 protein, human; D004298:Dopamine
keywords: catecholamine; computational model; ovary; polycystic ovary syndrome; signaling