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Endocrinology, doi:10.1210/en.2007-1661
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Endocrinology Vol. 149, No. 10 4988-4996
Copyright © 2008 by The Endocrine Society

Catecholamine Uptake, Storage, and Regulated Release by Ovarian Granulosa Cells

Monika Greiner, Alfonso Paredes, Verónica Rey-Ares, Sabine Saller, Artur Mayerhofer and Hernán E. Lara

Laboratory of Neurobiochemistry (M.G., A.P., H.E.L.), Department of Biochemistry and Molecular Biology, Faculty of Chemistry and Pharmaceutical Sciences, Universidad de Chile, Santiago 1, Chile; and Institute for Cell Biology (V.R.-A., S.S., A.M.), Ludwig-Maximilians University, 80539 Munich, Germany

Address all correspondence and requests for reprints to: Hernan E. Lara, Department of Biochemistry and Molecular Biology, Faculty of Chemistry and Pharmaceutical Sciences, Universidad de Chile, P.O. Box 233, Santiago-1, Chile. E-mail: hlara{at}ciq.uchile.cl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Catecholamines present in the mammalian ovary are involved in many normal aspects of ovarian functions, including initial follicle growth, steroidogenesis, and pathological states such as polycystic ovary syndrome. Sympathetic nerve fibers are the largest source of norepinephrine (NE), but not the only one. Surgical denervation of the rat ovary reduces, but does not eliminate, the ovarian content of NE. The aim of this work was to explore which intraovarian cells may participate in the ovarian NE homeostasis and the mechanisms involved. It was found that denervated rat ovaries can take up NE and cocaine considerably, decreased its uptake, suggesting involvement of catecholamine transporters. Granulosa cells of rat ovarian follicles present dopamine transporter and NE transporter. Their functionality was confirmed in isolated rat granulosa cells while cocaine blocked the uptake of NE. Furthermore, the presence of the vesicular monoamine transporter 2, together with the exocytotic protein (synaptosome-associated protein of 25 kDa) in granulosa cells, implies catecholamine storage and regulated release. Regulated calcium-dependent release of NE was shown after depolarization by potassium, implying all neuron-like cellular machinery in granulosa cells. These results in rats may be of relevance for the human ovary because dopamine transporter, NE transporter, vesicular monoamine transporter 2, and synaptosome-associated protein of 25-kDa protein and mRNA are found in human ovarian follicles and/or isolated granulosa cells. Thus, ovarian nonneuronal granulosa cells, after taking up catecholamines, can serve as an intraovarian catecholamine-storing compartment, releasing them in a regulated way. This suggests a more complex involvement of catecholamines in ovarian functions as is currently being recognized.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE SYMPATHETIC INNERVATION of the mammalian ovary has been involved in many aspects of the regulation of ovary functions, including steroidogenesis and early follicular development (1, 2). In rats, these actions are performed mainly by norepinephrine (NE) and vasoactive intestinal peptide (VIP), which are contained in and released from nerve fibers. NE acts on β2-adrenergic receptors present in theca and granulosa cells from rat ovary, and stimulates the production of progesterone and androgens, but not the secretion of estradiol (1, 2, 3, 4). In contrast to NE and acting on its own receptors, VIP stimulates estradiol and progesterone release from cultured granulosa cells and whole ovaries in vitro (5). Recent evidence has confirmed a role for VIP in the control of ovarian cyclical steroid production in adult cycling rats (6). These neurotransmitters may also facilitate the follicular development, as seen, for example, in the inhibition of follicular growth after ovarian denervation (7, 8, 9).

In the rat, a substantial fraction of the sympathetic innervation, targeting the ovary, is provided by neurons of the celiac ganglion and travels through the superior ovarian nerve (SON). Sympathetic innervation represents almost 90% of the NE present in the ovary, and yet, after surgical denervation, NE levels still remain over 10–15% (3), suggesting the existence of intraovarian cells participating in ovarian NE homeostasis. Furthermore, oocytes could be in part responsible for this because at least in monkeys they showed to take up dopamine (DA), through membrane DA transporter (DAT) and synthesize NE (via DA β-hydroxylase) (11). These mechanisms could have special relevance to pathological states, such as polycystic ovary syndrome (PCOS), the most frequent ovarian pathology in women during their fertile years. Using a rat model of PCOS, an increased ovarian sympathetic tone has been described, associated with the development and maintenance of PCOS (12). In addition, these findings have been recently extended to women (13), supporting a role for NE and catecholamines in maintaining the pathological condition. It is important to consider that sympathetic denervation or electrolytic lesion in the sympathetic pathway of the ovarian nerves in the rat reverses the formation of cysts (14, 15). However, the increased sympathetic tone in PCOS could also lie within the ovary itself and may include an increased activity of intraovarian neurons (found in some species and Wistar rats) or other unknown intraovarian compartment that could regulate NE homeostasis. The present study was designed to address these issues, and search for other ovarian sources for NE in two strains of rats and extend the study to human beings.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Ovaries from adult and prepubertal Wistar and Sprague Dawley rats, derived from stocks maintained at the University of Chile, were used. The ovaries of Wistar rats are reported to contain neuron-like cells, whereas Sprague Dawley rats lack these cells (16). The ovaries derived from adult Wistar and Sprague Dawley rats were used either to quantify NE, incorporate and release NE, RT-PCR procedures, and/or fixed for immunohistochemistry (n = 30 for each rat strain), whereas the ovaries from prepubertal Wistar and Sprague Dawley rats were used to isolate granulosa cells (n = 12 for each rat strain). Some of the Wistar and Sprague Dawley rats underwent sympathetic ovarian nerve denervation (SONX) 11–12 d before the experiments. This procedure was intended to eliminate the extrinsic contribution of NE via the sympathetic innervation of the ovary. For the procedure, rats were anesthetized with xylazine (10 mg/kg) and ketamine (90 mg/kg), and the ovaries were denervated under dorsal approximation as described (14, 17). All the animals were euthanized by decapitation. The ovaries were rapidly removed, cleaned of adherent tissue, and immediately used for release and incorporation experiments. Other ovaries of the same age were frozen at –80 C for total RNA isolation and NE quantification or embedded in Zamboni’s fixative for immunohistochemistry. In addition, Bouin’s fixed and paraffin-embedded ovaries from adult Sprague Dawley rats were used for the preparation of sections and immunohistochemistry. These samples had been used in previous studies (18). All animal procedures were previously approved by the Institutional Ethic Committee of the Faculty of Chemistry and Pharmaceutical Sciences of the Universidad de Chile, and the experiments were conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals as promulgated by the Society for the Study of Reproduction.

Rat granulosa cells
Rat granulosa cells were purified as previously described (14, 19). To eliminate the contribution of luteal cells present in the ovary of adult rats, the ovaries of prepubertal (25 d old) Sprague Dawley rats were used as recommended (19). The ovaries were punched with a needle and squeezed out gently to obtain the granulosa cells. Cells were suspended in 1 ml Krebs-Ringer buffer (KRB) and centrifuged at 3000 x g for 5 min, and the pellet of cell was suspended in 1 ml KRB. Cells were counted, and aliquots of 50,000 cells were used for the NE release and incorporation experiments. RNA was isolated from several batches of 100,000 cells each.

Human granulosa cells
Human granulosa cells were obtained from follicular aspirates of women undergoing in vitro fertilization, as described (20, 21). They were separated by centrifugation at 560 x g for 3 min and subsequently washed in serum-free 1:1 DMEM/Ham’s F-12 medium (Sigma-Aldrich, Deisenhofen, Germany). Washed cells were suspended in culture medium supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), and 10% fetal calf serum as previously described (22). The Ethics Committee of the Ludwig-Maximilians University of Munich had approved the use of the cells for scientific experiments, and written consent of the patients was obtained. Cells from two to four patients were pooled for all experiments.

Uptake and release of NE
The procedure used has been previously described (23, 24, 25). In brief, the ovaries were preincubated for 20 min in KRB (pH 7.4), gassed with 95%O2-5%CO2, and then incubated for 30 min at 37 C with 2 µCi 3HNE (New England Nuclear Life Science Products, Boston, MA). After washing the tissue in KRB (six washes of 10 min each) to remove nonincorporated radioactivity, the ovaries were transferred to a thermoregulated superfusion chamber and perifused at a flow rate of 1.5 ml/min for 10 min with KRB plus 10 mM tetraethylammonium, a potassium channel blocker that enhances the release of NE from nerve terminals in response to electrical stimulation (26). One-minute fractions were collected. After 3 min the gland was subjected to a train of monophasic electrical pulses (80 V, 10 Hz, 10 msec/pulse for 1 min), delivered through a parallel set of platinum electrodes and generated by a Grass S-4 stimulator (Grass Instruments, Quincy, MA). One-minute fractions were collected for 5 min more. In each experiment, ovaries from SONX and control rats were simultaneously stimulated in parallel superfusion chambers. At the end of the experiment, the ovaries were homogenized in 0.4 N perchloric acid, and 3Hcatecholamine remaining in the tissue was determined by scintillation counting [Tri-Carb Liquid Scintillation Analyzer 1600TR (Packard Instruments, Meriden, CT); 72.5% efficiency for 3H] to calculate the radioactivity remaining in the tissue after the experiment. To calculate the amount of NE released, the radioactivity present in each of the 1-min samples was determined in the same way. The release, which represents 3HNE overflows from ovaries structures, was then expressed as fractional release, i.e. as a percentage of the total radioactivity present in the tissue (24, 25).

Effect of cocaine on ovarian incorporation and release of 3HNE
To elucidate the effect of cocaine on the NE incorporation by the ovary, rat ovaries were halved. One half was incubated with KRB and the other with KRB plus 10 µM cocaine (Sigma Chemical Co., St. Louis, MO) for 10 min at 37 C. Later, the ovaries were incubated with 2 µCi/ml 3HNE (l-[7,8-3H]-NE, 40 Ci/mmol; Amersham Biosciences, Buckinghamshire, UK) for 30 min at 37 C. Each ovary was washed six times with KRB for 10 min to eliminate 3HNE excess. Ovarian halves treated initially with cocaine were also washed with 10 µm cocaine the first two times. Finally, the halved ovaries were homogenized in 0.4 N perchloric acid, and the remaining 3Hcatecholamine in the tissue was determined by scintillation counting (Packard Liquid Scintillation Analyzer 1600TR; 72.5% efficiency for 3H) to calculate the radioactivity incorporated by the tissue.

Incorporation of NE in isolated granulosa cells
To analyze the releasing capacity of NE in granulosa cells, they were isolated from the Sprague Dawley and Wistar rats’ ovaries as previously explained. Granulosa suspension was incubated in 1 ml KRB, containing 0.25 µCi/ml of 3HNE (l-[7,8-3H]-NE) for 30 min at 37 C. Granulosa cell suspension was centrifuged, and the cellular pellet was washed five times by suspension with 1 ml KRB during 10 min, followed by 5 min centrifugation (3000 x g) each time to eliminate nonincorporated 3HNE. To generate a depolarizing stimulus, granulosa cell suspension was incubated with KRB supplemented with 80 mM potassium for 10 min, and then it was centrifuged for 5 min at 3000 x g and supernatant saved for radioactivity counting. A post-stimulus fraction was obtained by suspending the cells in KRB during 10 min and then centrifuging them at 3000 x g for 5 min. The final pellet was homogenized in 0.4 N perchloric acid, and the remaining 3Hcatecholamine in the tissue and that of the lavages was determined by scintillation counting (Packard Liquid Scintillation Analyzer 1600TR; 72.5% efficiency for 3H).

The effect of cocaine and calcium dependence in the 3HNE uptake and release in rat granulosa cells
To evaluate the effect of cocaine in the 3HNE granulosa incorporation, the protocol used was the same as the one explained before, except that 10 µM cocaine was present during incorporation of 3HNE and during the first two lavages. To study the dependence of the release of NE on extracellular calcium, granulosa cells were incubated in KRB with 3HNE, washed, and stimulated with corresponding KRB solutions lacking calcium and containing 0.1 mM EGTA.

NE determination by HPLC
To quantify the concentration of NE in the ovaries, they were homogenized in 1:4 Dulbecco’s PBS (pH 7.35) and centrifuged for 15 min (13,000 x g) at 4 C. Supernatant was acidified with 0.25 N perchloric acid in a relation of 1:4, then centrifuged at 13,000 x g for 3 min, and stored at –80 C. For quantification the supernatant was filtered (sterile filter 0.22 µm), and 20 µl of the solution was injected into a Waters HPLC system (P600; Waters Corp., Milford, MA) with a C18 reverse-phase column (MF 6213 ODS; BASi, West Lafayette, IN) coupled to an electrochemical detector (464; Waters Corp.). The mobile phase contained 100 mM NaH2PO4, 1.29 mM octyl-sulfate, 0.02% EDTA, and 0.5% acetonitrile (pH 2.5), with a flow rate of 1 ml/min. The potential of the amperometric detector was set at 0.65 V, and the sensitivity was 1 nÅ. Under these experimental conditions, retention time was 4 min for NE. A standard curve between 25 and 400 pg was measured parallel to NE’s sample quantification.

RNA preparation, reverse transcription (RT), and PCR (RT-PCR) from rat tissues
RT-PCR was used to determine whether mRNAs encoding Net (SLC6A2), Dat (SLC6A3), and Vmat2 (SLC18A2) are present in the rat ovary, granulosa cells, and/or celiac ganglion. The celiac ganglion was dissected as described earlier (27). Total RNA was extracted as recommended (28). A total of 5 µg RNA of each sample was subjected to RT at 42 C for 60 min, using 1.6 mM deoxynucleotide triphosphate, 10 mM dithiothreitol, 176 nM random hexamers (Invitrogen Corp., Carlsbad, CA), 25 U RNaseOUT (Invitrogen), 125 U reverse transcriptase SuperScript II (Invitrogen), and first-strand buffer in a 30-µl volume. The reaction was finished by heating the samples at 75 C for 10 min.

Dilutions of the RT reaction were subjected to PCR amplification using 1 U DNA Taq polymerase (Promega Corp., Madison, WI), 1 mM deoxynucleotide triphosphate, and 0.5 µM each gene-specific primer in a total volume of 30 µl. The PCR consisted of 32 cycles (denaturation at 94 C for 60 sec, annealing at 60 C for 60 sec, and extension at 72 C for 60 sec) using a DNA thermal cycler (MJ Research, Watertown, MA). The primers for Net and 18s (the latter used as constitutively expressed gene for normalization purposes) were those previously reported by other authors: Net (29); and those for 18s were obtained from Ambion (Wiesbaden, Germany). Amplification of 18s RNA was performed in a different tube to avoid interference with the amplification of the mRNAs of interest. The other primer sequences are detailed in Table 1Go. Reaction tubes lacking RT product input were used as PCR controls. The RT-PCR products were separated on 2.0% agarose gels, stained with ethidium bromide, and photographed digitally. The identities of all PCR products were verified by direct sequencing, using one of the specific primers.


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  		TABLE 1. Primer information for rat Dat (SLC6A3) and Vmat2 (SLC18A2)

 
RT-PCR for human granulosa cells
Total RNA from several batches of cultured granulosa cells (3 and 4 d after isolation) was prepared using the RNEasy kit (QIAGEN, Hilden, Germany). As previously described (20), total RNA (200–500 ng) was subjected to RT, using random primers (pdN6) and Superscript-RT II (Life Technologies, Karlsruhe, Germany). Commercial human brain and ovary cDNAs (BD Clontech, Inc., Heidelberg, Germany) were used as well.

PCR amplifications (Table 2Go) consisted of 34 cycles of denaturing (at 94 C for 60 sec), annealing (at 60 C for 60 sec), and extension (at 72 C for 60 sec). Reaction tubes lacking RT product input were used as PCR controls. In some cases, control reactions, in which cDNA was replaced by RNA, were also performed. The PCR products were separated on 2.0% agarose gels and visualized with ethidium bromide. The identities of all PCR products were verified by direct sequencing, using one of the specific primers.


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  		TABLE 2. Primer information for human SNAP25, DAT (SLC6A3), NET (SLC6A2), and VMAT2 (SLC18A2)

 
Immunocytochemistry of human granulosa cells
DAT protein in cultured human granulosa cells was examined by immunocytochemistry using a commercially available polyclonal antiserum (rabbit anti-DAT, 1:200–1:500; Alpha Diagnostics International Inc., San Antonio, TX), as described (30). This antiserum recognizes rat and mouse DAT but cross-reacts with human DAT because their sequences posses a 72% homology. Cells were cultured for this purpose on glass coverslips, fixed (4% paraformaldehyde in 10 mM PBS), and used after rinsing in 10 mM PBS (pH 7.4). Incubations with the antiserum were performed overnight in a humidified chamber at 4 C. After washing steps, a fluorescein isothiocyanate-labeled second goat antirabbit antiserum was used. For control purposes, the first antiserum was omitted, and incubations with normal rabbit serum were performed instead. In a similar way, sections were incubated with rabbit anti-NE transporter (NET) antiserum (ab41559, 1:500–1:1000; BIOZOL Diagnostica Vertrieb GmbH, Eching, Germany) was used. Sections were examined with a Zeiss Axiovert microscope (Zeiss, Oberkochen, Germany) equipped with a fluorescein filter set.

Immunohistochemistry for DAT and NET in rat and human ovaries
Rat ovary sections from Wistar and Sprague Dawley rats and human ovarian sections (from the tissue collection of the Anatomical Institute, Munich, Germany) (21, 30, 31, 32) were subjected to immunohistochemistry. The same specific DAT and NET antisera used for human granulosa cells were used for these tissues, according to the procedure previously reported (21, 30, 31). In addition, vesicular monoamine transporter 2 (VMAT2) rabbit antiserum was used (Phoenix Pharmaceuticals, Inc., Mountain View, CA; 1:1.000–1:2:000). In brief, sections of 6 µm were deparaffinized, and endogenous peroxidase activity was blocked. After that, those sections were submitted to antigen retrieval by heating in citrate buffer [1.8 mM citric acid, and 8.2 mM sodium citrate (pH 6.0)] at 90 C for 30 min, plus 20 min cooling in the same solution. Sections were incubated overnight at 4 C with the antisera, followed by a second antibody (antirabbit biotinylated {gamma}-globulin, 1:250; Vector Laboratories, Burlingame, CA), and avidin-biotin complex peroxidase (Vector Laboratories). The binding was visualized with 3'3-diaminobenzidine HCl (Vector Laboratories). As a control for DAT specificity, some sections were incubated with anti-DAT, which had been preabsorbed with DAT peptide, provided by Alpha Diagnostics International. Other controls consisted of replacing the primary antiserum by buffer or incubation with nonimmune serum. Sections were observed with a Zeiss Axiovert microscope.

Statistical analysis
The results were expressed as mean ± SE. Comparisons among several groups were made using the Student’s t test, considering P < 0.05 as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ovarian NE uptake studies and effects of cocaine
Surgical denervation of the ovary by SONX 11- 12 d before the NE uptake and release experiments was performed to eliminate the most important source of extraovarian NE (sympathetic innervation via SON). As shown in Fig. 1Go, surgical denervation of the ovary from Wistar or Sprague Dawley rats decreased the amount of 3HNE released upon electrical depolarization. The effect of denervation on endogenous ovarian NE levels is shown in Table 3Go. In the ovary of Wistar rats, it decreased by approximately 80% (108 ± 33 to 14 ± 5.5 ng/mg tissue; P < 0.01) as consequence of denervation. A similar reduction was found in Sprague Dawley rats (data not shown) (14). However, the amount of 3HNE released upon electrical depolarization decreased only by 48% (15.3 ± 1.6 to 8.0 ± 1.0 5% fractional release; P < 0.01) for Wistar and 59% (10.3 ± 1.6 vs. 4.3 ± 1.5) in Sprague Dawley rats.


Figure 1
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  		FIG. 1. Induced release of 3HNE from the ovary of Wistar and Sprague Dawley rats. Ovaries from control and denervated (SONX) animals were incubated with 3HNE and stimulated by transmural electrical pulses (80 V, 2 msec, 10 Hz) as described in Materials and Methods. The rectangle represents the time of the stimulus, and each bar corresponds to the dpm of 3HNE released by ovary per minute. Results are means ± SEM from four to six individual experiments for each condition in each rat strain.

 

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  		TABLE 3. Changes in the NE concentration, 3HNE release, 3HNE incorporation, and the effect of cocaine in ovaries of sham and denervated Wistar rats (SONX)

 
The capacity for incorporating NE after blocking the NET and DAT catecholamine transporters with cocaine, a well-known inhibitor of NE and DA neuronal uptake (33, 34), was then compared. A decrease of 85% was found in the NE incorporation capacity, an effect that had the same magnitude for control and SONX ovaries (Table 3Go). In the ovary of Sprague Dawley rats, cocaine produced a similar effect but in a lower magnitude (56% for control and 58% for denervated ovaries). Thus, we conclude that a cocaine-sensitive uptake mechanism exists, and although in the Wistar and Sprague Dawley rat ovaries the sympathetic innervation is very important as a source of NE, there must be other intraovarian cellular structures that can take up and incorporate NE to maintain the steady-state levels of the neurotransmitter.

Identification of DAT, NET, and VMAT2 in rat granulosa cells
The effects of cocaine, a blocker for NET and DAT, suggested the presence and involvement of these membrane transporters in the ovary. Thus, immunohistochemical studies attempted to localize NET and DAT in the ovary of Wistar and Sprague Dawley rats (Fig. 2Go). Immunoreactive NET, DAT, and VMAT2 proteins were mainly located in granulosa cells and the oocyte in Wistar rats. Signals were found in luteal cells as well, albeit with a lower intensity. DAT and VMAT2 immunoreactivity was found in the ovary of Sprague Dawley rats, but immunoreactivity for NET was not. Signals were absent in negative controls, indicating the specificity of anti-NET, anti-DAT, and anti-VMAT2. However, Fig. 2Go shows that Dat and Net mRNAs are present in the ovary of both strains of rats, in the isolated granulosa cells, and, as expected, in the celiac ganglion. Furthermore, mRNA for the vesicular transporter (Vmat2) was also detected in both the ovary and isolated granulosa cells.


Figure 2
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  		FIG. 2. Expression of NET and DAT in the ovary (Ov) of Wistar (A) and Sprague Dawley (B) rats. A, On the left, DAT and NET and VMAT2 immunoreactive cells in a tissue slice from the ovary of Wistar rats. There is an intense mark in granulosa cells (GC) and the oocyte (DAT) of the follicle that disappears when the specific DAT antiserum is preabsorbed with excess of specific peptide (Co) (middle). The additional control (Co) on the right side of VMAT2 immunostaining is a consecutive section of the one stained for VMAT2, in which the antiserum was replaced by the buffer. Other controls, such as incubation with nonimmune serum, are not shown. Right, Ethidium bromide-stained agarose gels depict results of RT-PCR experiments for Net, Dat, and Vmat2. Co represents the PCR negative control without RT product. Cloning and sequencing of RT-PCR products confirmed their identity. B, On the left, DAT and VMAT2 immunoreactive granulosa cells in ovary of Sprague Dawley rats. Control (Co) is a consecutive section of the one stained for VMAT2 (specific antiserum was omitted). Right, Ethidium bromide-stained agarose gels depict results of RT-PCR experiments for Net, Dat, and Vmat2. Cloning and sequencing of RT-PCR products confirmed their identity. CGgl, Celiac ganglion.

 
Uptake and release studies in rat granulosa: uptake is blocked by cocaine, and the release of NE is calcium dependent
Because of the presence of NET and DAT (catecholamine membrane transporters), the vesicular transporter VMAT2 and the known presence of the exocytotic protein synaptosome-associated protein of 25 kDa (SNAP25) (18, 35) in granulosa cells, in the present experiment, it was tested whether they are able to incorporate and release 3HNE. Figure 3Go shows the percentage of 3HNE fractional release from granulosa cells, which were isolated from Wistar and Sprague Dawley prepubertal rats. High potassium stimulation (80 mM) evoked the release of a substantial quantity of 3HNE, followed by a post-stimulation release, higher in magnitude than the basal release, indicating that granulosa cells can take up NE and release it in a controlled way.


Figure 3
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  		FIG. 3. Induced release of 3HNE from Wistar (A) and Sprague Dawley (B) rat granulosa cells. Granulosa cells were incubated with 3HNE and stimulated with KCl-80 mM as described in Materials and Methods. The asterisks represent the significant increase of 3HNE release in response to the stimulus, and results are expressed in percent (%) fractional release. Results are means ± SEM from four individual experiments in each rat strain. *, P < 0.05; ***, P < 0.001.

 
Figure 4Go depicts the incorporation rate of 3HNE in granulosa cells under the presence of cocaine (10 µM). In whole tissue of the Wistar rats, cocaine decreased the 3HNE incorporation in granulosa cells (Fig. 4AGo, left), indicating that NE enters the cells through a cocaine-dependent transporter. In addition, cocaine strongly decreases the subsequent fractional release of 3HNE in granulosa cells (Fig. 4AGo, middle), suggesting that an important fraction of 3HNE was incorporated to granulosa cells by DAT/NET and that this way of uptake is a prerequisite for subsequent NE release. Next, it was examined whether calcium was also involved in the release of NE, by removing extracellular calcium during high-potassium induced NE release. It was discovered that the induced release from granulosa cells was clearly dependent on extracellular calcium (Fig. 4AGo, right), as it could be expected for a neuronal release mechanism. The same effect was found in the granulosa cells of the ovary of Sprague Dawley rats (Fig. 4BGo, right).


Figure 4
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  		FIG. 4. Effect of 10 µM cocaine on incorporation (left) and release (middle) of 3HNE from rat granulosa cells and the effect of the absence of extracellular calcium on KCl-80 mM induced release (right). Results from Wistar (A) and Sprague Dawley (B) rats are expressed either as percentage (%) of incorporation in relation to total 3HNE in the incubation medium or as percent (%) fractional release calculated as net release (minus basal release). Results are means ± SEM from four individual experiments in each rat strain. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
DAT, NET, VMAT2, and SNAP25 in human granulosa cells
Finally, the entire ovary, isolated luteinized human granulosa cells, and a commercial cDNA from brain where screening by RT-PCR to look for the presence of NET and DAT mRNAs to examine whether the results obtained in rat granulosa cells, could also be of relevance for the human ovary (Fig. 5AGo). DAT and NET were already found in the brain, and using identical PCR conditions, NET mRNA was detected in the entire ovary, but it was present only in traces in human granulosa cells. On the other hand, DAT mRNA was strongly present in both ovary and granulosa cells. To further examination of DAT, immunohistochemistry and immunofluorescence methods were used, and revealed that DAT is present in human granulosa cells in vivo (large antral follicle) and isolated human granulosa cells as well (Fig. 5BGo). RT-PCR studies revealed that human granulosa cells, besides expressing SNAP25 (18), also express VMAT2 (Fig. 5CGo). Thus, these results correspond to the ones found in rats, and support the existence of a similar uptake, storage, and secretory machinery in both rat and human granulosa cells.


Figure 5
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  		FIG. 5. Expression of DAT, NET, SNAP25, and VMAT2 in human ovary and cultured human granulosa cells. A and C, Ethidium bromide-stained agarose gels depict results of RT-PCR experiments for NET and DAT and VMAT2 in human brain, ovary, and granulosa cells (hGC). B, DAT immunoreactivity in cultured granulosa cells (top) and in granulosa cells (arrows) of a large antral follicle of the human ovary (bottom). There is a distinct mark in granulosa cells that disappears when the first antibody is preabsorbed with excess of specific peptide (CO for human ovary) or when the specific antiserum was replaced by the buffer (CO in granulosa cells). co(-), Control reaction shown without cDNA input.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A large body of evidence supports a role for the sympathetic nervous system in the regulation of steroid production and follicular maturation. Changes in the sympathetic outflow of the ovary could participate in the control of follicular development, and, thus, changes in the sympathetic input (by stress or steroidal hormones) could modify ovary function (14, 15). Recent studies in ovaries have shown expression of genes by granulosa cells that are normally associated with nerve cells (36, 37). In addition, differentiation of granulosa cells into tumors, expressing neuronal markers, has likewise been observed (38). These results imply that there could be similarities in the behavior of ovarian and neuronal cells.

On the other hand, granulosa cells (through membrane receptors to DA and NE) are under the control of catecholamines, which are mainly derived from extrinsic sympathetic nerves or released from intrinsic sources, present in at least some species (16, 39), and could travel through the blood stream to the ovary from the adrenal. However, neither sympathetic innervation nor neuron-like cells or blood vessels are found in direct contact with granulosa cells and do not penetrate the basal lamina, which separates the granulosa and theca cell compartments. This implies that, first, catecholamines must diffuse through this structural barrier to reach granulosa cells. Although in small follicles this may be a strong possibility, the diffusion distance increases in multilayered growing follicles.

Here, novel evidence for granulosa cells of ovarian follicles, including large follicles, is shown. These follicles will become a cellular compartment that is able to regulate intrafollicular catecholamine homeostasis in an unexpected way. We show that granulosa cells take up NE, store it, and, upon depolarization, release it. The presence of these mechanisms in the ovary of Wistar and Sprague Dawley rats and the prerequisites for this mechanism in human ovary strongly suggest that these cells serve as unexplored catecholamine-storing cells within the ovarian follicle, ensuring that NE and/or DA is present in the granulosa cell compartment, which lacks direct contact to catecholamine sources, such as innervation. This perspective would be in accordance with a role of NE and DA as important factors of the follicular microenvironment.

Many studies have demonstrated that after ovarian denervation (surgical cutting of the sympathetic nerves arriving to the ovary), there is a residual intraovarian NE independent of the rat’s strain used in the study (3, 40). This implies the presence of another intraovarian catecholamine storage compartment besides nerve fibers, which degenerate after the denervation procedure. Intraovarian neuron-like cell bodies have been described only in the ovary of Wistar but not in Sprague Dawley rats (41) and could be used for such storage of NE. As we previously demonstrated in the Sprague Dawley rat (14) and in the present study for the Wistar rat, we found a similar decrease in endogenous NE after surgical denervation, suggesting that the remaining NE is still present in another cellular compartment present in both ovaries.

Although granulosa cells are not able to perform de novo synthesis of catecholamines because they do not express tyrosine hydroxylase, they were found to possess DAT and NET. Interestingly, granulosa cells from both strains of examined rats express DAT and NET. However, the amount of mRNA and protein for NET seem to be low, especially in Sprague Dawley, in which it was not possible to detect NET by immunohistochemistry. Nevertheless, the use of cocaine, although clearly indicating functionality of DAT and/or NET, does not allow to judge the degree of participation of DAT vs. NET because both transporters can be affected by cocaine (42). In addition to intraovarian neuronal cells (16) only present in Wistar rats, granulosa cells of the two strains may also differ in relation to NET. Yet, they do not differ in relation to DAT because it is a prerequisite for catecholamine uptake, and functional catecholamine uptake, possibly suggesting that this mechanism may be of greater importance.

There are some subtle differences between the capacities for releasing NE between both strains. Sprague Dawley rats, which do not posses intraovarian neurons, have a higher capacity to release NE under electrical depolarization, and sympathetic denervation is more effective to decrease the NE. It remains to be established whether these differences reflect a special contribution of intraovarian neurons or granulosa cells. Nevertheless, it is suggestive that the capacity for releasing NE from granulosa cells is higher in the cells of Sprague Dawley rats than those of Wistar rats. It is proposed that in the ovary of Sprague Dawley rats, NE comes preferentially from extrinsic sympathetic nerves and granulosa cells, whereas in Wistar they are derived from three different compartments, which are: sympathetic nerves, intrinsic neurons, and granulosa cells. Granulosa and extrinsic nerves are minor components compared with the innervation of Sprague Dawley rats. Regarding intraovarian nerves, it is suggested that the differences in intraovarian neurons between Wistar and Sprague Dawley ovaries may be a result of the actions of different intraovarian regulators, which may include estrogen, on target cells. Supporting this assumption, it was found that tyrosine hydroxylase-immunoreactive cells (presumably intraovarian neurons) also become detectable in denervated (guanethidine treated) Sprague Dawley rat ovaries after the treatment with estradiol (2). This may be related to the fact that the gene for nerve growth factor NGFb, present in the rat ovary, the gene for low-affinity NGFb receptor, which provides trophic support to the sympathetic ovarian nerves (9), and presumably intraovarian neuron-like cells, contain estrogen-responsive elements (43).

The transmembrane transporters DAT and NET mediate Na+-dependent reaccumulation of released catecholamines into the presynaptic terminals of neurons (44) in the nervous system. Functionality of ovarian DAT and NET was proved by the ability of cocaine to block NE uptake in isolated rat granulosa cells. NE is the largest catecholamine detected in the rat ovary (45) and that was the reason it was chosen to perform the experimental uptake and release studies using radioactive NE. Although NET has a preference for NE, and DAT for DA (42), both catecholamines can be taken up by the transporters. Because both transporters are inhibited by cocaine, a specific involvement of NET or DAT in the uptake of NE or DA in rat granulosa cells cannot be concluded for the reasons mentioned previously. However, it is possible to speculate that due to the strong expression of DAT, in contrast to NET, in human granulosa cells, the human ovary, besides containing NE, also contains a high concentration of DA (46). Thus, in a DAT-mediated uptake, granulosa cells are likely to uptake not only DA, but also NE, and could make these cells a storage compartment for these catecholamines.

What is the possible role of a granulosa cell storage compartment for catecholamines? An immediate consequence is a reduced amount of catecholamines in the surrounding extracellular space or the follicular fluid of larger follicles. This may have consequences such as lower concentrations of catecholamines that may reduce the ability of signaling molecules to activate their receptors. Recently, four of the five dopaminergic receptors described in the central nervous system (47) have been described in human granulosa cells and two of them in rat granulosa cells. Granulosa cells also support typical receptors for NE, specifically β- and {alpha}-adrenergic receptors (10). Therefore, it is possible that uptake mechanisms compete with the actions performed by catecholamines, after binding to their receptors. This situation is comparable to the one in the synaptic cleft. In monkeys functional DAT has also been found in another cell type contained in the follicle, specifically the oocyte (11). Furthermore, oocytes possess a catecholamine-metabolizing enzyme (DA β-hydroxylase). Thus, it can synthesize NE from its precursor DA. Uptake of catecholamines may be just part of a more complex process supporting intrafollicular catecholamine homeostasis because experimental evidence for storage and regulated release was found, as well as the presence of typical molecules involved in these tasks in neurons, specifically VMAT2 and SNAP25. The presence of the VMAT2 in both rat and human granulosa cells was reported. In neurons, VMATs, which are present in two isoforms (VMAT1/VMAT2), package monoamines into synaptic and secretory vesicles by exchange of protons (42). A similar role and evidence of a vesicular storage compartment must now be assumed in case of ovarian granulosa cells, as well.

The concept that granulosa cells are targets for neurotransmitters is not a new one. However, the idea that these cells, which are present in a compartment of the ovary and lack direct innervation, can take up, store, and release neurotransmitter, including catecholamines, is only an emerging insight. From a pathological perspective, it has been previously postulated that the development and maintenance of polycystic ovary are associated with an activation of sympathetic nerves (12). Adult rats exposed to chronic stress develop a polycystic condition similar in morphological aspect to the human PCOS, suggesting a higher sympathetic input and role in the derangement of follicular development characteristically seen in human beings with the pathology (15). From this point of view, it has recently been demonstrated that women with PCOS have a higher sympathetic tone (13). Bearing this in mind, granulosa cells as a cellular compartment in general with the ability of storing and releasing NE could serve as a reserve to maintain a higher availability of catecholamines in the follicles. Experiments are currently in progress to examine this hypothesis.

In summary, this study shows that ovarian nonneuronal, endocrine granulosa cells can take up NE, presumably derived from several potential sources, and can then serve as an intrafollicular catecholamine-storing compartment. Exocytotic NE release from granulosa cells can occur in a regulated manner, as described for neuronal cells. These results provide novel insights into the neuronal nature of granulosa cells and suggest a more complex involvement of catecholamines in normal ovarian functions as they are currently recognized. It remains to be shown whether and how these mechanisms are involved in ovarian disorders.


   Acknowledgments
 
We thank N. Singer for final review of the text.


   Footnotes
 
This work was supported by Fondecyt Grant 1050765 (to H.E.L.), Conicyt fellowship (to M.G.), and by DFG MA1080/17-1 (to A.M.).

Disclosure Statement: The authors have nothing to declare.

First Published Online June 19, 2008

Abbreviations: DA, Dopamine; DAT, dopamine transporter; KRB, Krebs-Ringer buffer; NE, norepinephrine; NET, norepinephrine transporter; NGF, nerve growth factor; PCOS, polycystic ovary syndrome; RT, reverse transcription; SNAP25, synaptosome-associated protein of 25 kDa; SON, superior ovarian nerve; SONX, sympathetic ovarian nerve denervation; VIP, vasoactive intestinal peptide; VMAT2, vesicular monoamine transporter 2.

Received November 30, 2007.

Accepted for publication June 9, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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