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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 12 4750-4757
Copyright © 2000 by The Endocrine Society

Original Studies

D1-Receptor, DARPP-32, and PP-1 in the Primate Corpus Luteum and Luteinized Granulosa Cells: Evidence for Phosphorylation of DARPP-32 by Dopamine and Human Chorionic Gonadotropin

Artur Mayerhofer, Stephanie Fritz, Robert Grünert, Sheryl L. Sanders, Diane M. Duffy, Sergio R. Ojeda and Richard L. Stouffer

Anatomisches Institut (A.M., S.F., R.G.), Universität München, D-80802 München, Germany; and Oregon Regional Primate Research Center-Oregon Health Sciences University, Divisions of Reproductive Sciences (S.L.S., D.M.D., R.L.S.) and Neuroscience (A.M., S.R.O.), Beaverton, Oregon 97006

Address correspondence and requests for reprints to: Artur Mayerhofer, M.D., Professor of Molecular Anatomy, Anatomisches Institut, Universität München, Biedersteiner Str. 29, D-80802 München, Germany. E-mail: Mayerhofer{at}lrz.tum.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The multifunctional phosphoprotein "dopamine and cAMP-related phosphoprotein, Mr 32,000" (DARPP-32), which is able to act as an intracellular third messenger, was previously found to be present in human luteinized granulosa cells (GCs) and human ovary. DARPP-32 phosphorylation in GCs was increased by dopamine (DA) acting via a DA-1 receptors (D1-R). In the present study, we examined whether the major endocrine signaling molecule for GCs, LH/human CG (hCG), could also affect DARPP-32 phosphorylation. Immunoprecipitation studies showed that hCG, as well as DA, increased phosphorylation of DARPP-32 at threonine residues within 10 min, indicating that the signal transduction pathways of a hormone and a neurotransmitter involve DARPP-32 in GCs. Phosphorylated DARPP-32 is known to inhibit a cellular phosphatase (PP-1), which was also found to be expressed by GCs. Using RT-PCR and sequence analyses we showed that DARPP-32, PP-1, and D1-R genes were not restricted to cultured luteinized GCs, but were expressed in vivo, in the corpus luteum (CL) of the rhesus monkey throughout its entire life span. Whereas hCG increased steroid production in monkey luteinized GCs and in isolated luteal cells, DA failed to affect basal or hCG-stimulated progesterone production. This indicates that, unlike the LH/hCG receptor, the D1-R is not directly linked to steroid production. Although the precise role of D1-R in the CL remains to be shown, the presence of D1-R, DARPP-32, and its target PP-1 in this endocrine tissue, as well as the phosphorylation of DARPP-32 by a gonadotropin and by DA in luteinized GCs, indicate that the signal transduction pathways of the neurotransmitter DA and the gonadotropin hCG/LH involve DARPP-32. The PP-1 inhibitor DARPP-32 may, thus, be a third messenger used by both DA and hCG/LH to exert common regulatory influences on the cells of the CL.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MAMMALIAN OVARY and testis are regulated by gonadotropic hormones and are also subjected to regulatory influences by locally produced factors. These include neurotransmitters (1, 2, 3, 4). Among those, catecholamines are likely to play a special part in this "neuroendocrinotrophic stimulatory complex" (for a review see Ref. 4). The catecholamines found in the gonads may be derived from sympathetic innervation (5) or from the adrenal via the blood stream. In human and nonhuman primate species another potential source of gonadal catecholamines exists, namely neuron-like cells (6, 7, 8). Catecholamines act via receptors ({alpha}- and ß-adrenergic types) present on steroidogenic cells to affect steroid production. In addition, catecholamines are able to induce gene expression, because activation of ß- adrenergic ovarian receptors initiates FSH receptor expression and follicular growth in the neonatal rat ovary (9). That major regulatory actions of neurotransmitters are exerted also in vivo is indicated by the consequences of neonatal ovarian denervation, which included abnormal ovarian development and function (10). A role of catecholamines in vivo is also implied by the hyperactivation of the ovarian catecholaminergic system (11) associated with polycystic ovaries in rats.

Whereas norepinephrine (NE) seems to be the major catecholamine responsible for these changes, an involvement of dopamine (DA) is less clear. DA, like NE, is present in the ovary (12, 13) (e.g. in antral fluid of preovulatory human follicles; Ref. 14). DA does not seem to affect follicular cells, which lack D1-R directly, but rather it may serve as a precursor for NE synthesis in oocytes. Oocytes lack the rate-limiting enzyme of catecholamine biosynthesis tyrosine-hydroxylase, but possess a functional DA transporter and the enzyme DA-ß-hydroxylase (7). However, DA may act directly on luteal cells, because luteinized human granulosa cells (GCs) express a DA receptor of the D1 type, which is linked to stimulation of cAMP production (15). Activation of D1-R did not significantly alter progesterone production by human luteinized GCs. Instead, DARPP-32, a target protein for D1-R, present in nerve cells, was identified in human GCs. DARPP-32 was phosphorylated and, thus, activated by a DA and a D1-R agonist. DARPP-32 is a potent inhibitor of PP-1 and can affect intracellular signal transduction pathways (16, 17, 18, 19). Importantly, transduction pathways of different signaling molecules, including neuropeptides and catecholamines can converge onto DARPP-32 in neurons (15, 18). Thus, different neurotransmitters can affect the phosphorylation state of DARPP-32, emphasizing the importance of DARPP-32 as a common "third messenger." Whether such a mechanism exists also in the ovary (e.g. in luteinized GCs) is not known.

Because LH/human CG (hCG) is the major signaling molecule for luteinizing GCs and the corpus luteum (CL) in primates we examined, whether DARPP-32 serves as a "third messenger" for a gonadotropin, and whether this hormone can affect DARPP-32 phosphorylation in human GCs. We also examined whether a target of DARPP-32, PP-1, is present. Because luteinized GCs are models for the CL, we subsequently studied whether D1-R, DARPP-32, and PP-1 are present in vivo in the CL of a primate species, the rhesus monkey, and tested the possibility of an effect of DA on steroid production in this tissue.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissues

The care and housing of rhesus macaces (Macaca mulatta) at the Oregon Regional Primate Research Center (ORPRC) was described previously (20). Animal protocols and experiments were approved by the ORPRC Animal Care and Use Committee, and studies were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Ovaries were collected from rhesus macaces undergoing ovariectomy (see below) for other purposes or were obtained at necropsy from the tissue distribution program at the ORPRC. In total, ovaries from 25 monkeys, ranging from 1.5–12 yr of age, were examined. On collection, all tissues were rapidly frozen on dry ice (for extraction of RNA) or immersed in fixative for immunohistochemistry (see below). Individual CLs were obtained as described previously (20). Adult female monkeys with regular menstrual cycles were bled daily by saphenous venipuncture beginning on day 8 after the onset of menses. Serum concentrations of estradiol and progesterone were measured by RIA in the Endocrine Services Core Laboratory at the ORPRC (21). The day of the precipitous fall in circulating estradiol levels after the midcycle peak was designated day 1 of the luteal phase (21). CLs were surgically removed from anesthetized monkeys for RNA extraction on different days of the luteal cycle and for cell culture on day 6 of the luteal phase, as described previously (20). For subsequent RT-PCR studies CL samples were grouped in the following way: samples from days 3–5 were assigned to the early phase, from days 6–9 to the mid phase, from days 10–12 to the mid-late phase, and from days 13–15 to the late phase.

Monkey luteal cells, monkey GCs, and human GCs

Luteal tissue was dissociated in Ham’s F10 medium (Life Technologies , Grand Island, NY) and 1% BSA (Sigma, St. Louis, MO) containing 0.16% collagenase (type IV; Worthington Biochemical Corp., Freehold, NJ) and 0.02% deoxyribonuclease I (Sigma), as described previously (22). Luteal cells were incubated in Ham’s F10 medium and 0.1% BSA in a shaking water bath at 37 C for 3 h under 95% O2-5% CO2. Cells were incubated at a density of 12,500 cells per 0.25 mL, and incubations were performed in triplicate with or without the addition of DA-HCl (10 nmo/L or 10 µ mol/L; Sigma) alone or in combination with hCG (NIH, Bethesda, MD; CR-123; 1 ng/mL or 100 ng/mL; equivalent to ~0.013 and 1.3 IU/mL). To prevent breakdown of DA, 10 µg/mL ascorbic acid were added and all media contained 0.5 mmol/L isobutylmethylxanthine (Sigma). Media samples were stored at -20 C until RIA for progesterone in the Endocrine Services Core Laboratory, ORPRC (23, 24). For all assays, the intra- and interassay coefficients of variation were less than 10% and less than 15%, respectively. Data were normalized to 50,000 cells/mL.

Monkey GCs were retrieved by follicle aspiration after administration of human gonadotropins to promote development of multiple preovulatory follicles (25). Rhesus monkeys were treated for 7 days with a GnRH antagonist (Antide; Are-Serono Group, AAT Inc., Randolph, MA; 1 mg/kg body weight), which continued during sequential treatment with recombinant human FSH (rhFSH) (Gonal-F; Ares-Serono; 30 IU, im BID) for 6 days, followed by rhFSH + rhLH (30 IU each, im BID) for 1–3 days. When ultrasonography revealed follicles 4 mm or more in diameter, rhCG (1000 IU, im; Ares Serono) was given the next day to induce periovulatory events, including follicular luteinization. Follicles were aspirated from anesthetized monkeys 27 h after hCG injection (25). After removal of the oocytes for in vitro fertilization procedures, the follicular aspirates from each animal were pooled to obtain the luteinizing GCs. An enriched fraction of GCs was obtained by Percoll gradient centrifugation of the pooled aspirates (26). Four preparations of cells from different animals were used. In every case, cells were plated at 100,000 per well, cultured for 24 h in DMEM:F12 containing 10% calf serum, and then used for incubation experiments lasting 6 h. DA-HCl and hCG (Sigma) were used at 10 µmol/L or 10 IU/mL, respectively. Medium progesterone concentrations in quadruplicate wells were measured by RIA, and results were expressed as progesterone accumulation per cell number.

Human GC culture was performed as described in detail previously (15, 27). Follicular fluid containing GCs was derived from in vitro fertilization patients. The experimental procedure and the use of the cells were approved by the local ethics committee, and the women gave written consent to the use of these cells. Cells were cultured and used for experiment and immunoprecipitation as described previously (15)

RT-PCR analyses and Western blotting

Total RNA was prepared from samples by a cesium chloride ultracentrifugation method (28), by acid phenol-extraction method, or by a commercial kit (RNeasy; QIAGEN, Hilden, Germany), as described (29, 15). We used 100–500 ng total RNA for RT using a 18-mer polydeoxythymidine primer and Moloney’s murine leukemia virus reverse transcriptase (7, 15, 29). In addition, a commercial human complementary DNA (cDNA), reverse transcribed from pooled adult ovarian messenger RNA (mRNA), was used (Invitrogen, DeSchelp, The Netherlands), as described before (15). Amplifications of D1-R and DARPP-32 were performed as described (15), with minor modifications, including a nested PCR step for D1-R in some cases. The nucleotide primers for the first PCR of D1-R were 19-mers (sense primer: 5'-CTGAAGACTCTGTCAGTGA-3', complementary to nt 811–829 of the monkey D1-R mRNA; antisense primer: 5'ACTCACCGTCTCTATGGCA-3', complementary to nt 1068–1086 of the monkey D1-R mRNA; GenBank accession number AF077862). The "nested" oligonucleotide primers were 20-mers (sense: 5'-TGTGTTTGTGTGCTGTTGGC-3' (nt 836–855); antisense: 5'-GCA AAG TCT GTA GCA TCC TA-3'; (nt 1033–1052), yielding a 216 bp fragment. For DARPP-32 PCR, two oligonucleotide primer sets were used as described previously (15) yielding a 289 bp fragment. For PP-1, the oligonucleotide primers were selected according to the published human sequence (GenBank accession number NM 006732 and U48707.1; Ref. 30). They were 17-mers (sense: 5'-ACA TCT CAA GTC CAC TT-3' (nt 182–198) and antisense: 5'-GGAATCCAGTGGTGGTA-3', nt 503–487). All PCR reactions were performed as described (15) after testing the quality of the RT reactions by PCR amplification of tubulin. PCR amplification consisted of 35 cycles of denaturing (94 C, 15 sec), annealing (55 C, 1 min), and extension (72 C, 2 min). The PCR reaction products were separated on 2% agarose gels and visualized with ethidium bromide. In some cases, PCR products were subcloned into the pGEMT vector (Promega Corp., Mannheim, Germany) before sequencing; in another case, PCR fragments were directly sequenced using one of the PCR primers, as described (15).

Immunoprecipitation and Western Blot studies were performed as described (15, 31), with minor changes. Namely, we now used a commercial DARPP-32 antiserum (Chemicon, Hofheim, Germany) and protein A-Sepharose for immunoprecipitation. The DARPP-32 antiserum recognizes in rat brain homogenate a single protein of about 30–32 KDa, which most likely represents DARPP-32 (see Fig. 1AGo). Cells were treated on the second or fourth day after isolation for 10 min with/without hCG (10 IU/mL; Sigma, Deisenhofen, Germany) or hCG and DA (10 µ mol/L; Sigma). After electrophoretic separation and blotting onto nitrocellulose, blots were developed using a monoclonal antibody against phosphothreonine (Sigma; 1:100). In rat brain homogenate this antibody reacts as expected with numerous proteins, including a doublet band at about 30–32 KDa, likely to represent DARPP-32 (see Fig. 1AGo). For analyses of changes, integrated optical densities of the bands were determined using an edited version of the program NIH Image, as described previously (15).



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  		Figure 1. DARPP-32 is phosphorylated by DA and by hCG in human luteinized GCs. A, Immunoblot characterization of DARPP-32 antiserum and phospho-threonine antibody using rat brain homogenate: the DARPP-32 antiserum recognizes a single protein band of about 30–32 KDa, whereas the monoclonal antibody against phosphothreonine (a-P-Thr) reacts with numerous proteins, including a doublet band at about 30–32 KDa, likely to represent DARPP-32. B, Increased phosphorylation of DARPP-32 in human GC by DA: GCs were incubated with 10 µ mol/L DA for 10 min, and DARPP-32 was subsequently immunoprecipitated and threonine phosphorylation was detected using a monoclonal antiphosphothreonine antibody. Note that phosphorylated DARPP-32 migrates as a doublet. The optical density of both bands were analyzed, as reported (15 ). Co, Control, no treatment. C, Phosphorylation of DARPP-32 in human GCs by 10 IU/mL hCG (representing the maximal concentration for progesterone production) for 10 min. Results shown are representative of five independent experiments (see Results); optical densities of experiment shown (in arbitrary units) are 27 (Co) and 50 (hCG).

 
Western Blot analyses were performed as described (15) using cultured human GC protein. PP-1 antibody was obtained from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany) and used at a 1:500 dilution.

Statistics

One-way ANOVA and Fisher post hoc tests were used to evaluate the progesterone data, and the t test was used to analyze increases in optical densities.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Phosphorylation of DARPP-32 by hCG in human luteinized GCs and presence of PP-1 in human GCs and ovary

Using immunoprecipitation and Western blotting, we found that not only DA (Fig. 1BGo), as expected (15), but also hCG can phosphorylate DARPP-32 in human GCs at threonine residues (Fig. 1CGo). The increases induced by hCG were statistically significant (P < 0.05; t test) and ranged in five independent experiments from 1.3- to 6.1-fold (mean, 2.95) over basal phosphorylation levels. Concomitant treatment of cells with DA and hCG did not further increase the levels of DARPP-32 phosphorylation over the already elevated levels by either treatment (three independent experiments; results not shown).

RT-PCR of human GC and ovary, as well as Western blotting (human GC) indicated that PP-1 is expressed in the human ovary and in luteinized human GCs (Fig. 2AGo). Two clones were sequenced, and the results indicated that they were identical to the known PP-1 sequence. Treatment of GCs with hCG (10 IU/mL) for 24 h did not alter the levels of PP-1 (Fig. 2BGo).



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  		Figure 2. PP-1 in the human ovary and luteinized GCs. A, Ethidium bromide-stained agarose gel showing the result of a RT-PCR experiment; PP-1 cDNA (324 nt) was detected in samples of human GCs (day 2 in culture) and in the human ovary (Ov). B, Western blot detection of PP-1 in cultured human GCs on day 2 in culture. Levels of PP-1 were not altered after incubation with 10 IU/mL hCG for 24 h in two experiments.

 
Presence of D1-R, DARPP-32, and PP-1 in the monkey CL

Subjecting total RNA from the monkey ovaries (data not shown), CL, and GCs to RT, followed by PCR amplification with primers directed against the monkey D1-R sequence, yielded cDNA fragments that were 100% homologous to the previously described rhesus monkey D1-R (four CL cDNAs, two ovarian cDNAs, and one monkey GC cDNA were sequenced; see Ref. 32). A nested labeled oligonucleotide for Southern blot detection was further used in some cases to confirm identity of the cDNAs instead of sequencing (data not shown).

To examine D1-R gene expression in the CL throughout the menstrual cycle, samples from the early (days 3–5), from the mid (days 6–9), mid-late (days 10–12), and from the late (days 13–15) phases of the life cycle of the CL were used for RT-PCR. The results obtained (Fig. 3AGo) indicate that D1-R mRNA is present in all phases in the CL.



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  		Figure 3. Detection of D1-R (A), DARPP-32 (B), and PP-1 (C) mRNAs in monkey CL samples and human GCs: ethidium bromide-stained agarose gels showing RT-PCR products, which correspond to respective cDNAs in samples from early (E), mid (M), mid to late (M/L) and late (L) luteal phases of the menstrual cycle. Identity of cDNAs was verified after sequencing. The results indicate that D1-R, DARPP-32, and PP-1 mRNAs are present in all phases in the CL (D1-R: n = 3 samples from E; n = 4 from M; n = 5 from M/L; n = 5 from L; DARPP-32 n = 2 samples from E; n = 2 from M; n = 3 from M/L; n = 6 from L; PP-1: n = 2 from E, n = 2 from M, n = 3 from M/L, n = 2 from L). Note that fewer mRNA samples from the CL were used for the DARPP-32 and PP-1 experiments compared with the experiments for D1-R. Co, Control, no template for PCR.

 
In addition, both DARPP-32 and PP-1 mRNA were identified in monkey ovary (data not shown), GC, and CL samples (Fig. 3Go, B–C). DARPP-32 mRNA was present in the CL throughout its life span (Fig. 3BGo). PP-1 is, likewise, expressed in the monkey CL throughout is entire life cycle (Fig. 3CGo).

Sequence analyses of DARPP-32 (three cDNAs from the CL) indicated that the monkey DARPP-32 cDNA sequence is 98% homologous to human DARPP-32 and 87% homologous to bovine DARPP-32 (GenBank accession number M27444; see Ref. 17) at the nucleotide level (Fig. 4Go). Sequence analyses of PP-1 (n = 3 cDNAs from the CL) showed that the monkey form is 97.5% homologous to human PP-1 (alignment human vs. monkey; Fig. 5Go). The sequences were submitted to GenBank, accession numbers AF233349 (DARPP-32 human), AF233348 (DARPP-32 monkey), and AF233347 (PP-1 monkey).



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  		Figure 4. Alignment of bovine, human, and monkey DARPP-32 nucleotide sequences and deduced amino acid sequences. Nucleotide and amino acid differences between primate and bovine sequences are indicated by highlighted letters. The positions of the PCR primers are underlined. Alignments are based on published sequence information [GenBank accession numbers: bovine DARPP-32, M27444 (17 ); human DARPP-32, AF233349; monkey DARPP-32, AF233348; protein sequences: bovine DARPP-32, AAA57248; human DARPP-32, AAB30129].

 


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  		Figure 5. Alignment of human and monkey PP-1 sequences. Nucleotide and deducted amino acid differences are highlighted. The positions of the PCR primers are underlined. Alignments are based on published sequence information [GenBank accession numbers: human PP-1, U48707.1 and NM 006741 (30 ); monkey PP-1, AF233347; human PP-1, NP 006732].

 
Progesterone release is not stimulated by DA in monkey GCs and CL cells

To examine whether DA-dependent activation of D1-R could be involved in the regulation of ovarian progesterone synthesis, isolated CL cells from the active mid phase (day 6 after ovulation) CL and luteinized GCs derived from preovulatory follicles were treated with DA in vitro, and the changes in progesterone output were measured by RIA. In neither case did DA affect progesterone secretion on its own or altered the progesterone response to hCG (Fig. 6Go).



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  		Figure 6. Lack of a stimulatory effect of DA on progesterone production by monkey luteal cells and luteinized GCs. A, Luteal cells from day 6 of the luteal phase were incubated for 3 h with or without hCG (0.013 IU/mL or 1.3 IU/mL) in the absence or presence of 10 nmol/L or 1 µ mol/L DA. The concentration of 1.3 IU/mL of this preparation of hCG represents a maximal stimulatory concentrations for progesterone production (P < 0.05). DA did not alter this response (means + SEM of 3 wells/group). B, Luteinized GCs were isolated from preovulatory follicles and incubated for 6 h in the presence of hCG (10 IU/mL), DA (10 µ mol/mL), or both. The experiments were conducted 2 days after plating (100,000 cells per plate, n = 4 plates/group). Each bar represents means + SEM of 4 wells/group. The results shown are representative of four similar experiments. Note that for these experiments, as in a previous study (15 ), a commercial hCG preparation was used to obtain comparable results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study and a previous report (15) reveal that human luteinized GCs, cells known to express functional receptors for the hormone LH/hCG, also possess D1-R, as well as DARPP-32 and PP-1. The hormone hCG and the neurotransmitter DA can both increase phosphorylation of DARPP-32 in these cells. Thus, the signal transduction pathway of a hormone and of a neurotransmitter involve phosphorylation of DARPP-32. Although presently it is not known whether DA and LH/hCG pathways could converge in vivo, the presence of D1-R, DARPP-32, and PP-1 mRNAs in the cells of a primate CL supports this hypothesis. Our results indicate that the proteins required for an interaction of DA and gonadotropins are present in the primate CL and, in addition, show that steroidogenesis is not a target for DA in the CL.

The expression of D1-R in endocrine gonadal cells, namely in cells of the monkey CL, is in keeping with previous findings in human luteinized GCs (15) and with results showing the binding of a D1-R antagonist to cells derived from pregnant mare’s serum gonadotropin-treated immature rat ovaries (33). In another steroidogenic tissue, the rat adrenal zona glomerulosa, D1-R mRNA was, likewise, demonstrated using in situ hybridization techniques (34). Clearly, the expression of D1-R mRNA alone cannot be construed as evidence for the presence of functional receptors. However, functionality of D1-R in human GCs was shown by the ability of DA and a DA agonist to increase cAMP levels and change cell morphology (15), although steroid production was not altered by these ligands. Additional studies examining steroid production were conducted with monkey GCs and luteal cells. The results obtained are comparable with those obtained with human GCs and do not provide evidence for an involvement of DA/D1-R in progesterone or estradiol (data not shown) production. In contrast, Mori et al. (33) demonstrated a stimulatory effect of D1-R agonists on progesterone and cAMP production by cultured rat ovarian cells. Bodis et al. (35) reported a modulatory effect of DA on estradiol synthesis by human granulosa-luteal cells; however, this effect was abolished by the ß-receptor antagonist propranolol. Thus, it is likely that the effects on progesterone and estradiol secretion are not a consequence of a direct action of DA, but instead are due to its conversion to NE, which can activate ovarian adrenergic receptors (36). Indeed, evidence for the intraovarian conversion of DA into NE has been shown in the bovine CL (37) and monkey oocytes (7).

The present study also demonstrates that the monkey CL, like human luteinized GCs, contains the typical targets of D1-R, namely DARPP-32 and PP-1. The partial cDNA sequences isolated indicated a close similarity to the previously reported human DARPP-32 (15) and PP-1. Importantly, our RT-PCR results indicate that both DARPP-32 and PP-1 are present in the young, active CL, as well as the old, regressing CL.

Only on phosphorylation at threonine 34 can DARPP-32 function as a potent inhibitor of PP-1 (18, 38). We previously found that DA is able to induce DARPP-32 phosphorylation in human GCs (15). In nerve cells, several other first messengers besides DA regulate the phosphorylation state of DARPP-32. These include neuropeptides and neurotransmitters (e.g. cholecystokinin, vasoactive intestinal peptide, NE) and nitric oxide (39, 40). Therefore, DARPP-32 serves as a "third messenger." It has been suggested that it acts as a multifunctional integrator for the cellular effects of several "first messengers," at least in the systems examined so far. That phosphorylation of DARPP-32 by other factors besides DA can also occur in the ovary is supported in the present study. Thus, the first messenger hCG is able to phosphorylate DARPP-32 in human GCs in the same way as does DA (Ref. 15 and the present study). Most likely the effects of hCG and DA are mediated by a similar signal transduction pathway, involving cAMP, which is strongly increased by hCG (15).

In an individual GC endowed with both D1-R and receptors for LH/hCG one may speculate that concomitant stimulation with DA and hCG could further increase the phosphorylation levels of DARPP-32. We did not study individual cells, but rather homogenates of thousands of cells, and did not find an additive effect of hCG and DA. Most likely, this is due to the fact that all GCs are endowed with the LH/hCG receptor, but that the D1-R is restricted to certain GCs. This is supported by immunocytochemical staining of D1-R (15). Moreover, strong increases in cAMP after hCG treatment contrasted with only moderate increases induced by DA treatment (see Ref. 15 for details). Despite this assumed heterogeneous expression of D1-R and LH/hCG receptors, the fact that either signaling molecule can increase DARPP-32 phosphorylation in GC indicates that the signal transduction pathway of a gonadotropin and the signaling pathway of a neurotransmitter in human GC involve DARPP-32. The mere presence of D1-R, DARPP-32 and LH/hCG-receptors in the CL suggest a similar situation in vivo.

Interestingly, the role of DARPP-32 may extend beyond the inhibition of a phosphatase to the inhibition of a kinase. Thus, Bibb et al. (19) reported that after phosphorylation of DARPP-32 at threonine residue 75 by cyclin-dependent kinase 5 (Cdk-5), DARPP-32 inhibits protein kinase A in neurons. The existence of this mechanism in human GC is unlikely, because we did not detect immunoreactive Cdk-5 in human GCs (Mayerhofer, A., unpublished data). However, phosphorylation of DARPP-32 at other sites than threonine 34, such as serine 137, may occur in GCs. This was indicated by the migration pattern of phospho-DARPP-32, resulting in a double band (see also Ref. 15). Phosphorylation of DARPP-32 at serine 137 decreases dephosphosphorylation of threonine 34 and may reinforce the inhibitory action of DARPP-32 on PP-1 (38).

The consequences of phosphorylation of DARPP-32 in ovarian cells are presently not known. Indeed, even in neurons only few of the targets of PP-1 have been identified. Among those known are proteins responsible for the regulation of the membrane potential, including Na+/-K+-ATPase and ion channels (such as Na+channels and Ca2+channels; Refs. 16, 18 and 41). A similar target may exist in human luteinized GCs and monkey CL cells, which possess Na+/-K+-ATPase and voltage-activated Na+ and K+ channels (42, 43). Additional studies need to be conducted to determine whether these or other proteins are targets for the DARPP-32/PP-1 system and, thus, for DA and LH/hCG.

In this context, it is also important to mention that human and monkey preovulatory GC and luteal cells contain progesterone receptors (PRs; see Refs. 44 and 45). Moreover, during the luteal phase of the menstrual cycle the ratio of the two isoforms (A/B) of this receptor change (46). Whereas the end points of the activation of PR in the CL of primate species are unknown (47), a role of progesterone/PR in the process of ovulation and luteinization appears essential, based on studies in mice lacking the PR gene (48) and monkeys (49). A potential relationship between ovarian PR and ovarian DA/D1-R may exist, based on the previous findings that DA binding to D1-R can activate progesterone-dependent pathways (50, 51). Thus DA, in the absence of progesterone, increased transcriptional activity of PR and caused translocation of the PR from the cytoplasm to the nucleus (50). PR activation by DA in the brain mimicked the progesterone-induced sexual behavior in rats (52), indicating that cross-talk between these different classes of receptors occurs also in vivo. That DARPP-32 is involved in this, signal transduction pathway and receptor cross-talk has recently been shown in the brains of mice lacking DARPP-32 (53). The presence of D1-R in the primate ovary raises the possibility of a direct D1-R-mediated role of DA on a progesterone-dependent pathway in this organ, as well.

DA may reach its ovarian targets after delivery from innervation, neuron-like cells, and the blood stream. In fact, levels of DA in the follicular fluid of preovulatory follicles are sufficiently elevated to activate D1-R (see Ref. 14). Whether these sources are important for the activation of luteal cell D1-R remains to be shown. Despite this uncertainty and the lack of effect of DA on luteal cell steroidogenesis, the mere presence of D1-R, and moreover the expression of the third messenger DARPP-32 and PP-1 in the luteal compartment of the ovary, suggest that this catecholamine and DARPP-32 are involved in the regulation of processes underlying luteal function. The observation that LH/hCG can phosphorylate DARPP-32 identifies a novel part of the signaling pathway of this gonadotropin in GC and the CL. Moreover, these results show how a gonadotropin and a neurotransmitters may interact in ovarian cells and, thus, offer a novel perspective on how hormonal and local signals may be integrated to control ovarian function.


   Acknowledgments
 
We thank Prof. F. Berg and Dr. U. Berg (Munich, Germany) for providing hGCs. We are grateful to the ORPRC ART Core laboratory and Dr. Ted Molskness for providing monkey GCs and to the ORPRC Endocrine Services Laboratory and Dr. David Hess for performing RIAs. The expert technical assistance of Andrea Thalhammer, Marlies Rauchfuß, and Barbara Zschiesche are gratefully acknowledged. Human gonadotropins and GnRH antagonist were donated by Ares Advanced Technology Inc., a member of the Ares-Serono Group.

Received March 24, 2000.

Revised August 16, 2000.

Accepted September 5, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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