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

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. Electronic supplementary material The online version of this article (doi:10.1186/s13048-016-0269-0) contains supplementary material, which is available to authorized users.


Background
Reactive oxygen species (ROS) are generated in the ovary and are assumed to play roles in ovarian physiology as signaling molecules. They are however also involved in ovarian pathology and act as toxic factors [1,2].
Previous studies identified the neurotransmitters norepinephrine (NE) and dopamine (DA) in the human ovary [3]. Two studies in cultured human in vitro fertilization (IVF)-derived granulosa cells (GCs) showed that NE and DA stimulated the generation of ROS [4,5] and that the mechanisms involved intracellular uptake and cellular metabolism of the monoamines. The intracellular metabolic breakdown of monoamines generates aldehydes and hydrogen peroxide (H 2 O 2 ), which is an important member of the class of ROS.
Monoamine-induced ROS generation is among others linked to the levels of these factors [4,5] and thus higher levels of DA, for example, resulted in higher ROS levels. Both monoamines were detected in follicular fluid (FF), derived from IVF patients, in substantial levels. When samples from women suffering from PCOS were compared with samples from women without PCOS, a higher concentration of DA was apparent [4]. While NE is presumably mainly derived from the circulation and sympathetic fibers innervating the wall of follicles, the reason for the high concentrations of DA in the human ovary and specifically in PCOS patients remains unknown. It raises the question of local production in the ovary. DA is synthesized in several steps starting with the ubiquitously available amino acid L-tyrosine. Previous studies failed to reveal the presence of the ratelimiting enzyme tyrosine-hydroxylase (TH) other than in ovarian nerves [6,7]. Thus a substantial de novo synthesis in the ovary is unlikely.
DA can be generated from L-DOPA by dopamine decarboxylase (DDC), an enzyme expressed in many cells of the body. Generation of DA from exogenous L-DOPA is the basis for the standard treatment of Parkinson's disease patients [8][9][10]. Interestingly, DA likely contributes to neuronal cell death in Parkinson's disease by its ability to increase ROS [11]. In contrast, its precursor L-DOPA has antagonist actions and lowers ROS in neuronal cells [12]. The mechanisms of actions are not fully explored, but they include induction of cellular response, as detailed in recent studies [12,13]. These include changes in metabolic routes, cytoskeletal integrity, pCREB and CD39 expression, to name a few. In addition, ROS scavenging abilities of L-DOPA have been established, as well [14].
The presence of high amounts of DA in FF and the lack of knowledge about L-DOPA in FF prompted us to explore these points. We examined human FF, GCs and ovarian sections.

Methods
Human GC isolation, culture and treatment As described previously, human GCs were derived from FF aspirates of IVF patients stimulated according to routine protocols, in general the "long" stimulation protocol was used [4,5,[15][16][17][18]. The patients underwent IVF treatment primarily for male factor infertility. Age varied and ranged up to 39 years. In addition, from some of the IVF-patients blood samples were drawn and serum was kept at −20°C for later analyses. FF aspirates from two to five patients were pooled for GCs preparation, following a method previously described [19]. It involves a cell strainer (40 μm; BD, Franklin Lakes, NJ, USA) for filtration of the aspirates. GCs, which remained in cell strainer, were retrieved by washing with Dulbecco's modified Eagle's medium (DMEM)/Ham's F12 Medium (Gibco -Life Technologies, Carlsbad, CA, USA). The filtrate was centrifuged and the supernatant (i.e., cellfree FF) was frozen at −20°C until further use. Remaining cell aggregates in the acquired cell suspension were suspended mechanically by using a 0.9-mm cannula. Washed cells were re-suspended and cultured in DMEM/Ham's F12 medium supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml) and 10 % FCS (all from PAA) [15][16][17][18]. Primary GCs were cultured for up to 6 days. Cells were rinsed on day 1 of culture with fresh medium to remove non-adherent and dead cells. For all experiments, DMEM/Ham's F12 medium without supplements was used. DA (Sigma-Aldrich, St Louis, MO, USA) and L-DOPA (Sigma-Aldrich) were used in several experiments.

Confluence measurement
GCs were cultured up to 4 days and confluence of GCs was monitored for 24 h by taking time-lapse pictures every 10 min using a live cell analyzer (Peqlab, Erlangen, Germany). ELISA measurements of L-DOPA L-DOPA was determined in FF and serum using an enzyme-linked immunosorbent assay (ELISA) kit (Bioassay Technology Laboratory, Shanghai, China), following the instructions of the manufacturer. According to this information, the assay was validated for human serum, body fluids, tissue and cell culture media. Measurements were performed in microtiter plates, and reactions were monitored at 450 nm in a microplate reader (BMG labtech, Ortenberg, Germany). L-DOPA in the range of 5-3200 pg/ml can be detected with this assay. The samples were diluted 1/20 for measurements.

Measurement of H 2 O 2 generation
H 2 O 2 was measured by using an Amplex Red kit (Invitrogen -Life Technologies, Carlsbad, CA, USA) following the instructions of the manufacturer. Amplex Red was used in a final concentration of 2.5 μM. GCs were placed in 96-well plates and fluorescence levels were measured for 2 h at 544 nm excitation/590 nm emission in a microplate reader (BMG labtech). The blank for each value was subtracted.

Immunohistochemistry
Sections of human ovaries derived from a local collection at Anatomy III, Cell Biology (Munich, Germany) were used for immunohistochemistry. Immunohistochemistry was performed with a rabbit antiserum raised against human DDC (Sigma-Aldrich) and the corresponding blocking peptide (Sigma-Aldrich). Tissue samples and immunohistochemistry were described previously [5,18]. In brief, after removal of paraffin, antigen retrieval and blocking of endogenous peroxidase activity, the tissue was incubated in 5 % appropriate serum, diluted in phosphate-buffered saline. Antiserum incubation was done overnight at 4°C. The antiserum against human DDC was diluted 1:50. Afterwards, incubation with a biotinylated secondary antibody (1:500 dilution; Dianova, Hamburg, Germany) for 2 h at room temperature was performed. A Vectastain ABC Kit (Vector Laboratories, Burlingame, CA, USA) and a 3,3′-diaminobenzidine tablet set (Sigma-Aldrich) were used for the final staining procedure. Slides were covered with Entellan (Merck Millipore, Billerica, MA, USA). For control purposes, incubation with antigen peptide preadsorbed antiserum was employed.

Laser microdissection
Human ovarian tissue samples embedded in paraffin were cut into sections and mounted on a polyethylene naphthalene membrane, as previously described [20]. For dissection of the cellular compartments a laser microdissection (LMD) device (P.A.L.M. Microlaser Technologies, Bernried, Germany) was used. RNA isolation was performed by using a RNeasy Mini Kit (Qiagen, Hilden, Germany).

Statistics
Statistical analyses were done using Prism 5 (GraphPad Software, San Diego, CA, USA). For the confluence measurements an unpaired t-test was performed (P < 0.05). A one-way ANOVA followed by the Newman-Keuls post-test (P < 0.05) was performed for ROS (DCFH 2 -DA) measurements.

Results
We detected L-DOPA in IVF-derived FF by using ELISA measurements (Fig. 1a; n = 11 preparations). The levels varied (range 6.8-9.8 ng/ml), but on average a concentration of 8.2 ng/ml was quantified. L-DOPA was also detected in serum samples, obtained from a total of 11 different women (Fig. 1b). The levels varied and ranged from 7.4 to 40.9 ng/ml, with an average of 16.7 ng/ml.
Next, the role of L-DOPA was explored in cultured human GCs. L-DOPA in the medium enhanced the survival of cultured GCs (P < 0.05). This is concluded from automated confluence measurements of cultured GCs, treated with L-DOPA compared to control cells (Fig. 2a). When DA was used instead of L-DOPA, confluence was reduced (P < 0.05) indicating reduced cell survival (Fig. 2b).
Amplex red assays revealed that H 2 O 2 is a specific product of cultured human GCs under basal conditions. H 2 O 2 levels increased during a 2 h measurement. This was seen in several independent measurements (Fig. 3a). We speculated that the trophic role of L-DOPA may be related to an interference with H 2 O 2 and or its consequences in GCs. This mode of action of L-DOPA was identified in further experiments using the ROS indicator DCF and exogenous H 2 O 2 . H 2 O 2 -scavenging actions of L-DOPA were observed, which occurred rapidly and were depending on the concentration of L-DOPA ( Fig. 3b; Additional file 1). Concentration levels as low as 20 nM were sufficient for a significant effect.
DDC converts L-DOPA to DA and immunohistochemistry showed its presence in the theca cells (TCs) but not GCs of large human follicles and the cells of the CL in the human ovary. The pre-adsorption control supports specificity of the immunohistochemical staining ( Fig. 4a-d). Results of laser microdissection studies of the cells of the follicular wall followed by RT-PCR (Fig. 4e-h) also showed the presence of DDC in this tissue. In cultured human GCs, DDC mRNA was not detectable (Fig. 4i). DA was reported previously in FF in higher concentrations than in serum ( [4]), suggesting a local synthesis.

Discussion
To our knowledge neither presence nor functions of L-DOPA in the human ovary, in FF and human GCs have been examined. We readily detected L-DOPA in IVF-derived FF and found that the concentration range is somewhat lower compared to serum. Serum values of L-DOPA in women undergoing IVF are not published to our knowledge, but the reported serum/ plasma levels in humans vary substantially, when determined by HPLC [21]. In comparison, our results obtained by ELISA measurements indicate somewhat higher levels than described [22]. This observation may be conditioned by the IVF treatment, since L-DOPA may to be linked to gonadotropin levels [23,24], but also be due to inter-individual variation due to metabolism and nutrition. Thus further studies will require extensive recruitment of patients.
L-DOPA has anti-oxidative abilities [12,13]. The mechanisms are not fully known, but include a ROS scavenger action, as well as induction of gene expression. The nature of L-DOPA to specifically antagonize H 2 O 2 was previously shown [12,14,25]. In GCs the rapid onset of effects, which were observed in the ROS measurements within minutes (Additional file 1), support the scavenger abilities of L-DOPA. Other actions of L-DOPA in GCs remain to be shown.
We found that GCs produce H 2 O 2 , but the reasons remain to be fully elucidated. Several enzymes, namely steroidogenic enzymes, oxidases (specifically the NOX 4 and 5 enzymes [26]) may be responsible for ROS generation.
DA, formed by DCC from L-DOPA, is a factor described previously in high concentrations in FF and it increases the generation of H 2 O 2 in GCs. This is due to uptake and cellular metabolism of DA [4]. In GCs this may lead to oxidative stress and our studies using an automated evaluation of cell confluence indeed revealed a deleterious action of DA. In contrast L-DOPA increased cell confluence. Cell confluence is a measure for cell viability, cell number and/or size and increases seen in the L-DOPA group can be interpreted as overall trophic action [4,5,26]. Results of cell counting after exposure to L-DOPA confirm this assumption (data not shown). Clearly, a detailed evaluation of the cellular events, which may also include cell death events, are required to decipher the actions of L-DOPA.
If transferable to the in vivo situation, DA and L-DOPA thus may act antagonistically on the ROS environment of the follicle. The present study also indicates    that L-DOPA levels in FF are somewhat lower than the ones in serum, at least in samples available to us. However, FF and serum samples do not stem from the same women, hence this point must be evaluated further.
The samples from human CLs available to us were positive for DDC protein. They likely stem from the mid phase of the life span of the CL. Unfortunately the expression of DDC in other phases of the CL (formation, regression) could not be studied due to a lack of samples. We did not detect DDC in IVF-derived cultured GCs. They were cultured under basal conditions for a few days and may represent cells comparable to the young, forming CL. This may in part explain the discrepancy but, clearly, a more detailed study on DDCexpression during the life span of the CL will be required.
Expression of DDC in TCs and in the CL imply that DA can be generated in the ovary from its immediate precursor, rather than from L-tyrosine, since TH, the rate limiting enzyme, was reported in neuronal elements but not in TCs and GCs of the human ovary [7]. In brain expression of different catecholamine-synthesizing enzymes in different neurons were reported [27], implying that synthesis of catecholamines not necessarily depends on de novo synthesis from L-tyrosine in all cells.
With regard to the ovarian pathophysiology, DCC expression in TCs may imply that their number and the presence of a CL could be correlated with higher DA levels within the ovary. The relevance for PCOS, in which hyperthecosis is reported, remains to be shown. However we have previously shown higher DA in FF of PCOS women undergoing IVF [4], a result that could be related to more TCs (hyperthecosis) expressing DDC.
In summary, L-DOPA could be an antioxidant factor of relevance for the human ovary. This conclusion from our studies raises the question whether an enhanced exogenous supply of L-DOPA may be a novel therapeutic approach to treat ovarian conditions related to oxidative stress. Furthermore, to our knowledge positive actions of systemic L-DOPA in mice were reported. They include a small increase in fertility, namely increased litter size [28]. Reports in human are however to our knowledge almost missing, a fact related to the onset of Parkinson's disease after childbearing years [29]. Only one brief study stemming from 1980 reported that L-DOPA positively affects female fertility [30].

Conclusions
Taken together our results indicate that L-DOPA is an antioxidant factor in the human ovarian system. In contrast to DA it shows positive effects on granulosa cells. Since L-DOPA is used in therapy of Parkinson disease, it may be relevant for treatment of ovarian diseases as well.