L-DOPA in the hu man ovarian follicular fluid acts as an antioxidant factor on granulosa cells

Abstract

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.

Keywords: Granulosa cells; L-DOPA; Reactive oxygen species.

Fig. 1

L-DOPA levels in follicular fluid and serum. a L-DOPA levels in in vitro fertilization (IVF)-derived follicular fluid samples. Individual values of 11 FF are given, as well as the mean and SD. b Individual values of L-DOPA in sera from 11 patients are given, as well the mean and SD. Note that the samples shown in a and b do not stem from the same patients

Fig. 2

DA and L-DOPA effects on cultured human GCs. a Treatment with L-DOPA (200 nM) causes a significant increase of confluence in human GCs after 24 h compared to control group (P < 0.05, t-test). b DA (200 nM) stimulation decreases confluence in human GCs after 24 h compared to control group (P < 0.05, t-test). All values are shown as well as mean ± S.E.M. of n = 5 independent preparations of cells from two to five patients each

Fig. 3

Production of H₂O₂ and ROS in cultured human GCs. a Measurement of H₂O₂ using amplex red shows generation of H₂O₂ occurring over 2 h. Values are shown as mean ± S.E.M of n = 3 experiments. b L-DOPA (200 nM) significantly blocks H₂O₂ (1 mM) dependent DCF fluorescence intensity (P < 0.05, ANOVA, Newman-Keuls). Values are shown as mean ± S.E.M. of n = 3 independent preparations of cells from two to five patients each. Note that shown values are resulting from endpoint measurements after 2 h

Fig. 4

DDC in human ovarian tissue. a In human ovarian tissue TCs are positive for DDC in an immunohistochemical staining. b The pre-adsorption control is devoid of staining. c Cells of the human corpus luteum are positive for DDC using immunohistochemistry. d Pre-adsorption abolished staining of the corpus luteum. e–g Micrographs of human ovarian tissue before (e), during (f) and after (g) LMD. TCs and GCs of the follicle wall were excised. After RNA extraction a RT-PCR was performed. Bars indicate 50 μm (a and b) and 100 μm (c–g). h RT-PCR and sequencing showed, that DDC mRNA is present in samples of human TCs/GCs. All controls (input of H₂O instead of cDNA and RNA instead of cDNA) were negative. i DDC mRNA is absent in cultured human GCs (hGC; pool of seven preparations). Human liver cDNA was used as positive control. Controls (input of H₂O instead of cDNA) were negative