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

studyquestion: Is the neurotransmitter dopamine (DA) in the human ovary involved in the generation of reactive oxygen species (ROS)? summary answer: 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. what is known already: DAis an important neurotransmitter in the brain, and the metabolism ofDAresults in the generation ofROS. 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. study design, size and duration: 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. participants/materials, setting andmethods: DAin follicular fluid andGCswasdetermined 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 GCswere examined for the presence and abundance of transcripts of theDAtransporter (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. main results and role of chance: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 aDAreceptor-independent generation ofROSin GCs. Blockers ofDATandMAOA/ B, as well as an antioxidant, prevented the generation ofROS (P , 0.05). Agonists ofDAreceptors (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 ofDAT andMAO-A/B in GCs were 2-fold higher (P , 0.05) and the DA-inducedROS levels were found to be more than 4-fold increased (P , 0.05) compared with non-PCOS cells. Furthermore,DAat a high concentration induced apoptosis in PCOS-derived GCs. limitations, reasons for caution: While the results in IVF-derived follicular fluid and inGCsreveal 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. wider implicationsof thefindings: The results identifyDAas a factor in thehumanovary, which, viaROSgeneration, could play a role in ovarian physiology and pathology.The results obtained in samples fromwomenwithPCOSsuggest the involvement ofDA,acting viaROS, in this condition.