RESUMO
BACKGROUND: A previous study showed that dopamine (DA), which is contained in follicular fluid (FF) from IVF patients, strongly increased the production of reactive oxygen species (ROS) by cultured human granulosa cells (GCs). ROS, including H2O2, are assumed to play roles in ovarian physiology and pathology. Ovarian DA could be derived from the circulation, ovarian innervation and/or unknown ovarian sources. L-DOPA is the direct precursor of DA in its synthetic pathway. It was not yet described in FF. We examined L-DOPA levels in FF from IVF patients. As it may exert anti-oxidative and ROS-scavenging functions, we studied whether it exerts such actions in human GCs and whether DOPA-decarboxylase (DDC), the enzyme converting L-DOPA to DA, is expressed in the human ovary. RESULTS: ELISA measurements revealed that human IVF-derived FF contains L-DOPA. In cultured human GCs automated confluence analyses showed that L-DOPA enhanced their survival. This is in contrast to the actions of DA, which reduced cell survival. A dose-dependent mode of action of L-DOPA was identified using a fluorescent ROS indicator. The results showed that it antagonized intracellular ROS accumulation induced by exogenous H2O2. DDC was absent in follicular GCs, but immunohistochemistry identified it in theca cells (TCs) of large follicles in the human ovary. Laser micro-dissection followed by RT-PCR corroborated the expression. DDC was also identified in the steroidogenic cells of the corpus luteum. CONCLUSIONS: L-DOPA in FF is an antioxidant factor and exerts positive influences on GCs. Ovarian DA is derived from L-DOPA and has opposite actions. Exogenous L-DOPA is a standard therapy for Parkinson's disease, and the results raise the possibility that it may be able to exert positive actions as an antioxidant in ovarian conditions, as well.
RESUMO
Proliferation, differentiation and death of ovarian cells ensure orderly functioning of the female gonad during the reproductive phase, which ultimately ends with menopause in women. These processes are regulated by several mechanisms, including local signaling via neurotransmitters. Previous studies showed that ovarian non-neuronal endocrine cells produce acetylcholine (ACh), which likely acts as a trophic factor within the ovarian follicle and the corpus luteum via muscarinic ACh receptors. How its actions are restricted was unknown. We identified enzymatically active acetylcholinesterase (AChE) in human ovarian follicular fluid as a product of human granulosa cells. AChE breaks down ACh and thereby attenuates its trophic functions. Blockage of AChE by huperzine A increased the trophic actions as seen in granulosa cells studies. Among ovarian AChE variants, the readthrough isoform AChE-R was identified, which has further, non-enzymatic roles. AChE-R was found in follicular fluid, granulosa and theca cells, as well as luteal cells, implying that such functions occur in vivo. A synthetic AChE-R peptide (ARP) was used to explore such actions and induced in primary, cultured human granulosa cells a caspase-independent form of cell death with a distinct balloon-like morphology and the release of lactate dehydrogenase. The RIPK1 inhibitor necrostatin-1 and the MLKL-blocker necrosulfonamide significantly reduced this form of cell death. Thus a novel non-enzymatic function of AChE-R is to stimulate RIPK1/MLKL-dependent regulated necrosis (necroptosis). The latter complements a cholinergic system in the ovary, which determines life and death of ovarian cells. Necroptosis likely occurs in the primate ovary, as granulosa and luteal cells were immunopositive for phospho-MLKL, and hence necroptosis may contribute to follicular atresia and luteolysis. The results suggest that interference with the enzymatic activities of AChE and/or interference with necroptosis may be novel approaches to influence ovarian functions.