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BOR - Papers in Press, published online ahead of print April 27, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.039313
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BIOLOGY OF REPRODUCTION 73, 358–365 (2005)
DOI: 10.1095/biolreprod.104.039313
© 2005 by the Society for the Study of Reproduction, Inc.

Serotonin and Its Antidepressant-Sensitive Transport in Mouse Cumulus-Oocyte Complexes and Early Embryos1

Pascal Amireault , and François Dubé 2 

Département d'obstétrique-gynécologie, Université de Montréal and Centre de recherche, Centre hospitalier de l'Université de Montréal (CHUM)-Hôpital Saint-Luc, Montréal, Québec H2X 1P1, Canada


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Serotonin (5-hydroxytryptamine [5-HT]), is a neurohormone found in various nonneural tissues, including the gonads of many invertebrates, in which it regulates spawning and oocyte meiotic maturation. The possibility that a local serotonergic network might also exist in the female gonads of vertebrate species, including mammals, remains poorly documented. To clarify this possibility, we investigated mouse cumulus cells, oocytes, and embryos for three key serotonergic components, namely, 5-HT itself; the rate-limiting enzyme for its production, tryptophan hydroxylase 1 (TPH1); and the 5-HT-specific transporter (SLC6A4) required for modulating its cellular effects. Using a combination of reverse transcription-polymerase chain reaction analysis and immunofluorescence confocal microscopy, we showed that mouse cumulus cells, oocytes, and embryos contain 5-HT and SLC6A4, while only cumulus cells possess the 5-HT-producing enzyme TPH1 and may thus be the local source of 5-HT observed in their neighboring cells. With a semiquantitative assay in single cells, we demonstrated that 5-HT can actively be taken up by isolated oocytes when it is supplied exogenously in vitro. This 5-HT transport in isolated oocytes is driven by a classical serotonin transporter, expressed up to the blastocyst stage, that is sensitive to the antidepressants fluoxetine and fluvoxamine, which belong to the selective serotonin reuptake inhibitor family. All together, our results show that 5-HT may be produced locally by cumulus cells and that it can be actively taken up by mammalian oocytes and embryos as part of a likely larger serotonergic network possibly regulating various developmental processes much earlier than previously thought.

cumulus cells, embryo, follicle, oocyte development, serotonin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Serotonin (5-hydroxytryptamine [5-HT]) is known to regulate spawning and to induce oocyte meiotic maturation in a number of invertebrates, such as mollusks and nemerteans [1–3]. By analogy with these invertebrate species and based on several independent observations, it has long been suspected that 5-HT may have a role as local regulator of various early developmental events in mammalian species. First, 5-HT itself can be detected in female rodent genital tracts [4] and in human follicular fluid [5]. A 5-HT effect on reproductive tissues is illustrated by its stimulation of steroidogenesis in human [6], rat [7], and hamster [8] granulosa cells, this action being possibly mediated through HTR7 as identified in cultured human granulosa-lutein cells [9]. The oocyte itself could be a target for 5-HT, which elicits typical intracellular Ca2+ oscillations, similar to those seen after fertilization, but sensitive to HTR2A antagonists, in isolated hamster metaphase II (MII) oocytes [10]. Moreover, it has been shown that the sensitivity of hamster oocytes to produce Ca2+ spikes on 5-HT addition differs according to the oocyte maturation stage [11]. Also, recently, the Htr1d mRNA was detected in mouse MII oocytes and preimplantation embryos and was suggested to regulate cleavage divisions of embryos cultured in vitro [12]. Other studies have reported the possible involvement of serotonin in oocyte maturation and early embryogenesis [13, 14], but they lacked clear demonstrations of endogenous serotonergic components in these cells. More recently, however, a direct suggestion was made that an unidentified receptor, possibly related to HTR7 and sensitive to the serotonin antagonist ritanserin, could be involved in maintaining prophase I arrest in both Xenopus and mouse oocytes [15]. The potential involvement of 5-HT, not only in meiotic maturation but also in several other aspects of follicular growth or early embryogenesis, is further suggested by our recent demonstration of HTR7 or HTR2A in mouse cumulus cells, oocytes, and early embryos (unpublished results). All together, these recent reports point to the possibility that a complete and functional serotonergic network might regulate several, though as yet not fully characterized, processes of ovarian and oocyte physiology.

To be functional, a local serotonergic network requires that 5-HT reaches target cells through neural innervation or vascular irrigation or, more directly, that it be produced locally by surrounding cells. While serotonin has been reported in the ovaries, oviducts, uterus, and even oocytes and preimplantation embryos [16] of some rodents, its origin, although tentatively ascribed to mast cells [4], has remained obscure. The capacity of producing 5-HT by given cells or tissues is conferred by the enzyme tryptophan hydroxylase (TPH), a member of the tetrahydropterin-dependent amino acid hydroxylase family, that catalyzes the conversion of tryptophan into 5-hydroxytryptophan, the rate-limiting step in 5-HT synthesis [17]. Tryptophan hydroxylase 1 (TPH1) has long been considered as the single isoform of TPH until the recent cloning of a second isoform, TPH2 [18]. These authors also showed that TPH1 is the isoform expressed in peripheral tissues, while TPH2 is expressed solely in the brain [18]. Tph1 cDNAs of various mammalian species, including humans, rats, and mice, have been cloned and sequenced [19–21]. Interestingly, Tph1 mRNA, along with 5-HT, has recently been detected in epithelial cells of the mouse mammary gland, supporting a central regulative role for this enzyme in local 5-HT production within a peripheral autonomous serotonergic network [22]. However, the activity or presence of TPH has never been reported in any ovarian tissues that would have substantiated their capacity to produce serotonin locally.

Besides 5-HT itself and its receptors, local serotonergic regulation also requires an active serotonin uptake mechanism to ensure removal of 5-HT from the cellular environment of 5-HT-sensitive cells when it is no longer needed. This function of 5-HT reuptake, in vertebrate species, is normally achieved by a conserved serotonin transporter (SLC6A4, also known as SERT) that is a member of Na+- and Cl–-dependent transporters comprising also noradrenaline and dopamine transporters. Slc6a4 cDNA encodes a protein of 630 amino acids that contains 12 potential transmembrane domains and potential N-glycosylation and phosphorylation sites permitting regulated trafficking to the membrane [23]. In the central nervous system, SLC6A4 is expressed in presynaptic neurons, where it removes 5-HT after its vesicular release, but it is also found in the gastrointestinal tract [24], platelets [25], lymphocytes [26], and the placenta [27]. In addition to its role in removal of extracellular 5-HT, SLC6A4 has also been shown to be essential, in platelets, for a newly discovered signaling process called "serotonylation" and requiring intracellular accumulation of 5-HT [28].

SLC6A4 is the target of selective serotonin reuptake inhibitors (SSRIs), which include an array of widely used antidepressants for human health care, such as fluoxetine (Prozac) and fluvoxamine (Luvox) [29]. SSRIs have a high affinity for SLC6A4 and are used to treat psychiatric disorders associated with the serotonin system, including anxiety, obsessive-compulsive symptoms, and premenstrual dysphoric disorders [30].

The aim of the present work was to clarify scattered earlier reports of serotonergic components in various regions of female reproductive organs in diverse mammalian species. To do so, we focused on a more restricted cellular system, the mouse cumulus-oocyte complex, and on the characterization of three serotonergic effectors: 5-HT itself, TPH1, and SLC6A4. Here, we show that all three serotonergic effectors are present in mouse cumulus cells, oocytes, or embryos and are components of a local serotonergic network exhibiting potential 5-HT-producing capacity and active 5-HT uptake. Our study thus supports the view that a local serotonergic network could serve several functions pertaining to cell communications required for follicular growth, oocyte meiotic maturation, or early embryogenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Oocyte and Embryo Collection

Fully grown germinal vesicle (GV)-stage oocytes, ovulated MII-arrested oocytes, and preimplantation embryos at various stages were obtained from 3- to 4-wk-old female B6C3F1 mice (Charles River) after standard gonadotropin injection. For GV-stage oocytes, the mice were primed with 5 IU of equine gonadotropin (eCG, Sigma), and cumulus-enclosed, fully grown oocytes were collected 46–48 h later by puncturing of antral follicles with a 30-gauge needle, under a dissecting microscope, in M2 medium containing 100 µM 3-isobutyl 1-methylxanthine (IBMX). When needed, the cumulus cells were removed by repeated pipetting with a small-bore pipette. For MII-arrested oocytes, the mice were primed with 5 IU of eCG, followed (44–48 h later) by a 5-IU human chorionic gonadotropin (pregnyl-hCG; Organon Canada) injection, and cumulus-oocyte complexes (COC-MII) were collected from the oviduct 18–20 h later in M2 medium. When needed, the cumulus cells were dispersed in M2 medium containing 10 mg/ml bovine testis hyaluronidase (Sigma), and the oocytes were washed and collected in M2 medium. For embryos, female mice were submitted to the gonadotropin protocol and were allowed to mate with a male the night after the second injection. Embryos were collected by flushing the oviducts or uterus with M2 medium via a 30-gauge needle mounted on a syringe. The timing of embryo collection was as follows: one-cell, 19 h post-hCG; two-cell, 43 h; four-cell, 50 h; eight-cell, 67 h; morula, 74 h; blastocysts, 91 h. All animal experiments were approved by the Institutional Animal Use and Care Committee and were consistent with the Canadian Council on Animal Care guidelines.

In Vitro Culture of COCs, Oocytes, and Embryosfor 5-HT Detection

MII-arrested oocytes, COC-MII, four-cell embryos, and blastocysts were collected as described previously and cultured in small Petri dishes containing a drop of medium, under paraffin oil, equilibrated previously in a humidified chamber at 37°C and 5% CO2. The cells were incubated for 20 min in M16 medium alone, in M16 medium containing 500 nM 5-HT, or in M16 medium containing 500 nM 5-HT and 1 µM fluvoxamine. For the fluvoxamine treatments the cells were preincubated for 15 min in fluvoxamine alone before being transferred to medium containing also 5-HT. After incubation, the cells were immediately transferred to a paraformaldehyde solution and submitted to the indirect immunofluorescence protocol. Each experiment was performed at least three times with at least 10 cells/condition.

mRNA Isolation and Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Collected cells were incubated in acidic Tyrode solution (Sigma) to remove the zona pellucida of GV-oocytes, MII oocytes, and one-cell embryos. The cells were kept in a minimum of M2 medium at –80°C until mRNA isolation. mRNAs of 10 oocytes or 10 embryos or cumulus cells from 30 to 50 COC-MII were isolated according to the microscale protocol with the Dynabeads mRNA direct kit (Dynal). The mRNAs were reverse transcribed using Superscript II enzyme (Invitrogen) in a 20-µl reaction at 42°C, for 45 min, to construct a cDNA library immobilized on beads, following the manufacturer's specifications. The first PCR run (50 µl) was performed on cDNA beads in suspension. The PCR program, of 26 cycles with a hot start, consisted of denaturation of 90 sec at 95°C, primer annealing of 90 sec at 65°C, and primer extension of 90 sec at 72°C (last primer extension of 15 min). The second PCR run was performed with one-tenth of a microliter of products from the first amplification and the same PCR program (28 cycles). For Slc6a4, two pairs of primers were used in a nested PCR strategy to produce amplicons of 559 bp (forward 5'-CCAACTACTTCGCCCAGGACAACATCAC-3' and reverse 5'-TCTTCGTTCCTCATCTCAGCCATGTAGCC-3') and 533 bp (forward 5'-TCGGACACGTCTTCGTTCCTCATCTCAG-3' and reverse 5'-GACCAACATCACCTGGACACTCCATTCCAC-3'). For Tph1, two pairs of primers in a nested PCR strategy produced amplicons of 563 bp (forward 5'-GACATCGGATCAGAAGACTCCC-3' and reverse 5'-CGTGAATTCAATCTTGGGAATGG-3') and 389 bp (forward 5'-CACCATGATTGAAGACAACAAGG-3' and reverse 5'-CATACAACAGCACTCTGTTGGCG-3'). Primers for Actb as positive controls yielded 540-bp (forward 5'-GTGGGCCGCTCTAGGCACCAA-3' and reverse 5'-CTCTTTGATGTCACGCACGATTTC-3') and 277-bp (forward 5'-TGTGATGGTGGGAATGGGTCAGAAGGAC-3' and reverse 5'-TACGTACATGGCTGGGGTGTTGAAGG-3') amplicons. Twenty-five microliters of each reaction were loaded on agarose gel stained with ethidium bromide. Each amplification was executed at least three times yielding similar results. All PCR products obtained were cloned in pCRII (Invitrogen) and sequenced on both strands by the Université Laval (Ste-Foy, Canada) sequencing service to confirm the sequence.

Experimental Conditions of the 5-HT Uptake Study

MII-arrested oocytes were cultured in M16 medium containing 500 nM 5-HT creatinine sulfate (5-HT; Sigma) for 0, 2, 5, 10, or 20 min. In other experiments, MII-arrested oocytes were cultured for 10 min in M16 medium containing 0–1000 nM 5-HT. MII-arrested oocytes were also cultured for 10 min in M16 medium containing 500 nM 5-HT and 0–1000 nM fluoxetine hydrochloride (fluoxetine; Sigma) after 15-min preincubation with fluoxetine alone. Controls with different concentrations of fluoxetine alone and without 5-HT or fluoxetine were also included. Next, MII-arrested oocytes were cultured for 10 min in M16 medium containing 500 nM 5-HT and 0–1000 nM fluvoxamine (Sigma) after 15-min preincubation with fluvoxamine alone. Controls with different concentrations of fluvoxamine alone and without 5-HT or fluvoxamine were also included. Controls for the reversibility of fluoxetine and fluvoxamine inhibitions were performed by washing MII-arrested oocytes three times (10 min each) in M16 medium, after a 15-min preincubation in 1 µM of each inhibitor alone, and finally incubating them after for 10 min in M16 medium containing 500 nM 5-HT.

In all experiments, the oocytes were transferred to a paraformaldehyde solution and submitted to the indirect immunofluorescence protocol for 5-HT detection. Each of these experiments included at least 10 oocytes/ condition, pooled from three to six mice, and was performed at least three times for a minimum total of 30 oocytes/condition.

Indirect Immunofluorescence Confocal Microscopy for 5-HT and SLC6A4 Detection

Oocytes and embryos were collected and treated as described previously, then fixed in fresh Dulbecco phosphate-buffered saline (D-PBS) paraformaldehyde 4% for 30 min at room temperature. They were washed three times for 5 min in D-PBS before a 1-h blocking step in D-PBS/milk 5%/Triton 0.5%/normal goat serum (NGS) 5%. They were next incubated overnight at 4°C with a primary antibody raised against 5-HT (1/25 000; Incstar) in D-PBS/milk 1%/Triton 0.1%/NGS 1%. After three washes in D-PBS, they were incubated for 1 h at room temperature in a Cy-3-conjugated goat anti-rabbit antibody (1/2000; Jackson Immunoresearch) and washed three times in D-PBS. Finally, MII-arrested oocytes, COC-MII, and four-cell embryos were mounted with Fluoromount (Electron Microscopy Sciences), and GV-stage oocytes and blastocysts were mounted with 50% glycerol in D-PBS. Images were collected with a Zeiss Axiovert 100M microscope coupled with the LSM510 system. Competition controls were included for each cell type and culture condition tested. In these controls, the first antibody (1/25 000) was incubated at room temperature, with gentle shaking, in D-PBS/milk 1%/Triton 0.1%/NGS 1% with 5-HT-BSA (10 µg/µL) for 4 h before overnight incubation with the cells. Controls without the first antibody were also included for each cell type tested.

For the 5-HT uptake study, fluorescence quantification of individual oocytes was undertaken with the LSM510 program. A circle was drawn around each oocyte, and the mean intensity of each pixel inside this circle served as a measure of 5-HT incorporation. These data were then analyzed statistically.

The procedures for the immunodetection of SLC6A4 were identical to those deployed for the detection of 5-HT, but with a different first antibody. A rabbit anti-rat antibody directed against amino acids 579–599 (Oncogene) was used at a 1/5000 dilution. Controls without the first antibody were also included for each cell type tested.

Western Blotting

For each detection of TPH or SLC6A4, approximately 300 oocytes and their corresponding surrounding cumulus cells were lysed in 30 µl of 0.5% SDS and kept at –80°C until electrophoresis. The frozen samples were diluted in 4x sample buffer, loaded on 7.5% SDS-polyacrylamide gel, run at 200 V for 45 min, and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% milk and 0.1% Tween 20 in D-PBS for 1 h at room temperature before overnight incubation at 4°C in fresh blocking solution containing the appropriate diluted first antibody. The membrane was washed three times in 0.1% Tween 20 in D-PBS and incubated in fresh blocking solution containing the appropriate diluted second antibody. Finally, the membrane was washed several times in 0.1% Tween 20 in D-PBS before the detection protocol, using the enhanced chemiluminescence plus assay kit (Amersham). The same SLC6A4 (1/20 000) antibody as for indirect immunofluorescence detection was employed for Western blotting with a goat anti-rabbit-horseradish peroxidase (HRP) secondary antibody (1/20 000; Bio-Rad). TPH1 was detected with a sheep anti-rabbit antibody (Chemicon) at 1/300 dilution and a donkey anti-sheep-HRP secondary antibody (1/20 000; Bio-Rad).

Hamster Oocyte Collection

Hamsters were primed with 25 IU of eCG, followed by a 25-IU hCG injection 52 h later, and cumulus-enclosed oocytes were collected from the oviduct 18 h later in M2 medium. The cumulus cells were dispersed in M2 medium containing 10 mg/ml bovine testis hyaluronidase, and the oocytes were washed and collected in M2 medium. The oocytes were then submitted to the indirect immunofluorescence confocal microscopy protocol for 5-HT detection.

Statistical Analysis

Data were compared statistically by Kruskal-Wallis one-way ANOVA. Groups were then compared by the Dunn method. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Using a specific anti-5-HT antibody, followed by indirect immunofluorescence observations of serial optical sections under confocal microscopy, we detected 5-HT in COCs, isolated mature MII oocytes, and GV-stage oocytes (Fig. 1, A–A' D–D', and G–G', shows phase contrast and corresponding immunofluorescence images). Interestingly, oocyte labeling with the anti-5-HT antibody was enhanced considerably when these cells were preincubated in medium containing 500 nM 5-HT for 20 min, further confirming the antibody's specificity for 5-HT (Fig. 1, B, E, and H). Similarly, 5-HT immunoreactivity was observed in cumulus cells, either isolated (not shown) or in intact COCs (Fig. 1, A–A'), and was also enhanced by incubation in the presence of 5-HT (Fig. 1B). No cell labeling was found when the anti-5-HT antibody had been previously depleted by preincubation with a BSA-5-HT complex (Fig. 1, C, F, and I) or when only a secondary antibody was used (data not shown).



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  		FIG. 1. Serotonin in mouse cumulus-oocyte complexes (COCs) and oocytes. Indirect immunofluorescence confocal microscopy (1 µm middle optical section), using an anti-5-HT antibody, of a COC (A, A', and B), an isolated MII oocyte (D, D', and E) and a germinal vesicle (GV)-stage oocyte (G, G', and H). Panels A, D, and G show phase contrast images of the same cells seen in immunofluorescence in panels A', D', and G'. Similarly prepared COC and oocytes that had been previously incubated with 500 nM 5-HT for 20 min reveal enhanced 5-HT-specific fluorescence (B, E, and H). Panels C, F, and I show, respectively, a COC, an isolated MII oocyte, and a GV-stage oocyte prepared using an anti-5-HT antibody previously depleted by preincubation with a BSA-5-HT complex. Bars = 10 µm

These observations that 5-HT is normally present within mammalian cumulus cells and oocytes and that it can accumulate within them, when 5-HT is supplied exogenously, suggest possible local production of serotonin and an active 5-HT uptake mechanism. We, therefore, examined whether the key enzyme TPH1 [14], found only in 5-HT-synthesizing cells, and the classical mammalian serotonin transporter, known to drive 5-HT uptake in other cells, could be expressed in oocytes, embryos, or cumulus cells. Figure 2 shows the amplified bands seen after nested RT-PCR analysis, using oligonucleotides specific for Slc6a4 or Tph1 with mRNA extracted from ovaries, cumulus cells, oocytes, and embryos at various stages. As depicted, bands of expected sizes (verified by cloning and sequencing) revealed Tph1 mRNA in total ovaries and isolated cumulus cells but not in isolated oocytes or embryos at any stage, whereas Slc6a4 mRNA was detected in all cell types examined, including embryos up to the blastocyst stage (Fig. 2). No other contaminating bands were found by RT-PCR analysis.



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  		FIG. 2. Expressions of serotonin transporter and tryptophan hydroxylase mRNAs in cumulus cells, oocytes, and embryos. Reverse transcription-polymerase chain reaction-amplified bands for Slc6a4, Tph1, and Actb control obtained with mRNA extracted, respectively, from (lanes 1–11) total ovaries (1); isolated cumulus cells (2); germinal vesicle (GV) stage oocyte (3); MII oocytes (4); one-cell (5), two-cell (6), four-cell (7), and eight-cell embryos (8); morulas (9); blastocysts (10); and a representative negative control sample without cDNA (11)

Western blotting with antibodies specific for either TPH1 or SLC6A4 was then performed and confirmed the presence of SLC6A4 in isolated oocytes and cumulus cells, whereas TPH1 was detected only in cumulus cells, with both proteins at expected size ranges comigrating with their counterparts from brain samples (Fig. 3, A and B). An immunofluorescence study with the same anti-SLC6A4 antibody also confirmed positive SLC6A4 immunoreactivity in COCs, in isolated MII oocytes, as well as in four-cell embryos and blastocysts (Fig. 4). While anti-SLC6A4 labeling was spread over the surface of cumulus cells and MII oocytes in a punctate pattern, it appeared more localized in patches in four-cell stage embryos, with strong labeling of polar bodies, and also in blastocysts in which it was restricted to distinct spots of the trophectoderm (Fig. 4, A'– D').



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  		FIG. 3. Expressions of serotonin transporter and tryptophan hydroxylase proteins in cumulus cells and MII oocytes. A) Western blotting using an anti-SLC6A4 antibody against samples from brain (1), isolated oocytes (2), and cumulus cells (3) showing a common major band at an apparent molecular weight of 57 kDa. B) Western blotting using an anti-TPH antibody against samples as in A, displaying a common major band (52 kDa) in brain (1) and cumulus cells (3) but not in isolated oocytes (2). An additional band (127 kDa) of unknown nature is also detected in cumulus cells (3). Similar results were obtained in three separate experiments



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  		FIG. 4. Expression of serotonin transporter protein in cumulus-oocyte complexes (COCs), isolated MII oocytes, and early embryos. Immunofluorescence of cells prepared with an anti-SLC6A4 antibody and observed by confocal microscopy. Phase contrast (A–D) and corresponding fluorescence (A'–D') images of a mouse COC (A–A'), isolated MII oocyte (B– B'), a four-cell stage embryo (C–C'), and a 4-day-old blastocyst (D–D'). Bars = 10 µm

Based on the observed increase in immunolabeling by the anti-5-HT antibody when cells were incubated with exogenous 5-HT (Fig. 1) and the demonstration that both SLC6A4 mRNA (Fig. 2) and protein (Fig. 3) were present in oocytes and embryos, we designed an experimental approach to more directly characterize the activity of 5-HT transport into isolated MII oocytes. This was performed by incubating MII oocytes in the presence of externally added 5-HT and assessing its internal accumulation by a semiquantitative assay, with fluorescence intensity being measured in our immunological detection of cellular 5-HT by confocal microscopy (Fig. 5; see Materials and Methods). Figure 5A illustrates the linear uptake of exogenous 5-HT (500 nM) by isolated MII oocytes over a 20-min incubation. When MII oocytes were incubated with varying concentrations of 5-HT over a 10-min period, a typical saturation curve resulted with a Km for this SLC6A4 estimated to be 280 nM (Fig. 5B). 5-HT uptake by oocytes was also found to be abolished by low temperature (4°C), further suggesting its enzymatic nature (Fig. 5C). The latter experiments thus establish the specificity and sensitivity of our assay of 5-HT uptake by oocytes.



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  		FIG. 5. Serotonin transport in mouse MII oocytes. A) 5-HT uptake over time as measured by the fluorescence intensity (fluorescence arbitrary units [FAU]) of oocytes prepared with an anti-5-HT antibody. Mean fluorescence intensities of 30–50 oocytes (± SEM). The upper panels show a typical oocyte prepared as in Figure 1 and seen by immunofluorescence microscopy for the corresponding time points. B) 5-HT uptake at different external concentrations (0–1000 nM) over a 10-min incubation period. C) 5-HT uptake inhibition at low temperature (4°C). D) Effects of fluoxetine and (E) fluvoxamine on 5-HT transport by oocytes with 10-min incubation in the presence of the indicated drug concentrations (0–1000 nM). F) Lack of effects of 1 µM fluoxetine or fluvoxamine added alone on basal 5-HT levels and reversibility of their effects on the transport of added 5-HT after washing of cells (w). Letters a and b indicate means that are significantly different or not from one another (P < 0.05). Scale information is the same as provided for Figure 1, D–D'

To further verify that this 5-HT uptake occurs through a classical serotonin transporter, we tested the effects of SSRIs comprising several different antidepressants that can reduce or abolish SLC6A4 activity. One such widely used SSRI, fluoxetine (Prozac), was found to inhibit 5-HT uptake by MII oocytes in a dose-dependent manner with a calculated IC50 of 355 nM (Fig. 5D), confirming the involvement of a classical serotonin transporter. Similarly, another SSRI, fluvoxamine (Luvox), was even more potent in suppressing 5-HT uptake with a calculated IC50 of 6 nM (Fig. 5E). These inhibitions were reversible upon washing of the cells (Fig. 5F), and active SSRI concentrations were in the usual range of specificities reported in other cells [26]. Control oocytes incubated in fluoxetine or fluvoxamine alone without added 5-HT displayed fluorescence levels not statistically different from those of untreated oocytes (Fig. 5F). We also detected endogenous 5-HT in four-cell stage embryos and 4-day-old blastocysts (Fig. 6, A–A' and D–D', shows phase contrast and corresponding immunofluorescence images). Embryo labeling with the anti-5-HT antibody was also enhanced when the embryos were preincubated in medium containing 500 nM 5-HT for 20 min (Fig. 6, B and E). No cell labeling was found when the anti-5-HT antibody had been depleted previously with a BSA-5-HT complex or when only a secondary antibody was used (data not shown). We also confirmed fluvoxamine-sensitive 5-HT uptake in four-cell stage embryos as well as in blastocysts (Fig. 6, C and F). Moreover, we performed some preliminary experiments demonstrating that incubating MII oocytes in the presence of fluvoxamine at 1 µM did not result in reduced fertilization on insemination and also that embryos progressed normally, as compared to controls, from two-cell to the blastocyst stage when cultured in vitro in the continuous presence of fluvoxamine (data not shown). These experiments suggest that 5-HT transport is not required for either fertilization or preimplantation development and also confirm that SSRIs have no toxic effects in these cells.



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  		FIG. 6. Serotonin in mouse embryos. Indirect immunofluorescence confocal microscopy (1 µm middle optical section), using an anti-5-HT antibody, of a four-cell stage embryo (A–A', B, and C) and a blastocyst (D– D', E, and F). The same untreated four-cell embryo (A–A') or blastocyst (D–D') are seen in phase contrast with their corresponding immunofluorescence image. Similarly prepared embryos that had been previously incubated with 500 nM 5-HT for 20 min reveal enhanced 5-HT-specific fluorescence (B and E) that can be inhibited by a 15 min preincubation in 1 µM fluvoxamine (C and F). Bars = 10 µm

To determine the extent to which our observations could apply to other mammalian species besides the mouse, we observed the presence of 5-HT in hamster oocytes that can also actively take up external 5-HT (Fig. 7), indicating the probable universality of such a serotonergic network in mammalian COCs. All together, our results suggest that, on the one hand, the presence of the 5-HT-synthesizing enzyme TPH1 in cumulus cells makes them the best candidates for the origin of 5-HT, seen also in neighboring oocytes. On the other hand, the absence of TPH1 in oocytes and embryos renders SLC6A4 the likely candidate for transporting 5-HT into these cells.



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  		FIG. 7. Presence of serotonin in hamster oocytes. Immunofluorescence of isolated MII hamster oocytes prepared with an anti-serotonin (5-hydroxytryptamine [5-HT]) antibody, as described, and examined by confocal microscopy. A and B) Same hamster oocyte seen in phase contrast (A) and in fluorescence (B) without any further treatment. C) Hamster oocyte similarly prepared after incubation in the presence of 5-HT added externally at 500 nM for 20 min. Bars = 10 µm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our work establishes unequivocally, for the first time, the presence of TPH1, SLC6A4, and early 5-HT transport within mammalian cumulus cells, oocytes, or early embryos, which are likely part of a regulatory local serotonergic network. In addition, we confirmed the presence of 5-HT in oocytes and early embryos [16] and demonstrated that cumulus cells also contain 5-HT. It was thought, until now, that mast cells were the only source of 5-HT in the ovary [4], but our demonstration of TPH1 and 5-HT in cumulus cells suggests that these cells are also a likely ovarian source of 5-HT. Indeed, similar assumptions of local 5-HT synthesis by peripheral cells, based on the presence of both 5-HT and TPH mRNA and/or protein, were proposed for gut enterochromaffin cells [31], embryonic stem cells [32], and epithelial cells of the mammary gland [22]. Further studies, such as in vitro activity assays, would more firmly establish this assumption for cumulus cells, but the presence of both TPH1 mRNA and protein makes them the most plausible source for, at least, the 5-HT detected in follicular fluids [5] and oocytes [this work, 16], even though additional indirect sources cannot be excluded at this time. Also, an intrafollicular source of 5-HT could allow a bidirectional communication between ovarian steroids and a follicular serotonergic network. In this respect, it is noteworthy that in macaque and guinea pig dorsal raphae, TPH mRNA and protein expressions are stimulated by estrogen [33]. If a similar mechanism was active in cumulus cells, their production of 5-HT could be regulated by local estrogen levels. Conversely, since 5-HT stimulates estradiol and progesterone secretions by rat [7] and hamster [8] follicles and by cultured porcine [34] and human [5, 6, 9] granulosa cells, cumulus cells could provide 5-HT-driven autocrine or paracrine regulation of local steroidogenesis. A potential link between serotonergic regulation and local steroidogenesis, to promote folliculogenesis, is further suggested by the observation that total ovarian 5-HT levels fluctuate in phase with the estrous cycle in rats [35]. Interestingly, an unsuspected autonomous peripheral serotonergic network has recently been uncovered in the mammary gland, with Tph1 mRNA expression influenced by prolactin, leading to coordinated local production by epithelial cells of 5-HT, which, in turn, exerts autocrine/paracrine functions in the mammary gland development [22]. Likewise, TPH1 expression in follicles might thus be involved in the regulated production of local 5-HT with potential yet uncovered effects on follicular development.

In addition to cumulus cells potentially producing 5-HT in a coordinated fashion, the functional SLC6A4 expressed in oocytes and cumulus cells would permit the reuptake of 5-HT released in the extracellular space and would contribute to further regulate the magnitude and temporal and spatial distribution of 5-HT produced locally and, hence, its possible interaction with membrane-located receptors such as the HTR1D [12] and HTR7 expressed in oocytes or the HTR7 and HTR2A expressed by cumulus cells (unpublished results). Alternatively, 5-HT uptake by SLC6A4 could allow its interaction with 5-HT receptors located to intracellular structures of oocytes, embryos, or cumulus cells, as shown for the HTR7 and HTR2A in some regions of the rat brain [36, 37]. Along this line, the possibility that intracellular 5-HT might affect early embryonic development was proposed long ago [13, 14], but this hypothesis was lacking clear experimental evidence or any known supportive mechanism. In light of our work, this early proposal gains a renewed interest and also that of the recent discovery of a new receptor-independent signaling process set by intracellular 5-HT and involving an active role for SLC6A4-driven 5-HT uptake [28]. Indeed, this signaling mechanism called serotonylation, which consists of the transamidation of 5-HT to GTPases rendering them constitutively active, was shown to regulate alpha-granule exocytosis from platelets and to require prior SLC6A4-induced intracellular accumulation of 5-HT [28]. Whether this signaling mechanism is of widespread occurrence remains to be seen, but it should predictably be predominant in peripheral tissues exhibiting a local serotonergic network, including SLC6A4-driven 5-HT transport, such as shown here for the mammalian follicle. From previous studies, SLC6A4 was believed to appear no earlier than around Day 10 of rodent embryonic development [38, 39], and its involvement at much earlier, even prefertilization, stages was thus not investigated until now. This implies that SSRIs, through their action on SLC6A4, could impact on reproductive processes at much earlier stages than expected and underscores that antidepressants of the SSRI family, as well as other serotonergic drugs of wide use in human health care, deserve new evaluations for their potential effects on reproductive functions at early stages.

The action of 5-HT on any early reproductive processes should involve at least one of its receptors expressed in these cells. Indeed, we recently found that mouse oocytes express the HTR7 and that cumulus cells express two 5-HT receptors, the HTR7 and HTR2A subtypes (unpublished results). We also demonstrated that 5-HT could regulate the cAMP and intracellular calcium levels of cumulus cells, thus confirming the expression of a complete and autonomous serotonergic network in mouse COCs (unpublished results). The present work suggests that an autocrine or paracrine cell communication system based on 5-HT could be involved in several key reproductive processes driven by these cells, including follicular growth and steroidogenesis, ovulation, fertilization, and early embryonic development. In a preliminary study, we performed some in vitro fertilization experiments that showed no effect of 5-HT or fluvoxamine on this process (data not shown). Since such in vitro studies can only partially approximate physiological conditions, we cannot exclude an in vivo serotonergic regulation of fertilization, but an intraovarian role seems more likely. Such an intraovarian role is suggested for other animals, such as the teleost Fundulus heteroclitus, in which 5-HT inhibits the 17{alpha},20ß-dihydroxy-4-pregnen-3-one (17,20ßP)-induced resumption of oocyte meiosis of both denuded and follicle-enclosed oocytes [40]. Even though these pharmacological studies did not identify the 5-HT receptor subtype involved, they demonstrated clearly that 5-HT increased the cAMP level of follicles and that it blocked the cAMP decrease observed after 17,20ßP treatment [41].

Along this line, a recent work proposes that an unidentified receptor, sharing common properties with the HTR7 and sensitive to the serotonin antagonist ritanserin, could be involved in maintaining prophase I arrest in both Xenopus and mouse oocytes [15]. On the other hand, the Gi-coupled HTR1D [12] and Gs-coupled HTR7 (unpublished results) were reported in mouse oocytes, and these receptors are, respectively, negatively or positively coupled to adenylate cyclase and thus directly regulate intracellular cAMP levels through binding of their ligand. It has been known for a long time that the reinitiation of meiotic maturation in prophase I-arrested oocytes, as triggered either by an LH surge in mice or by progesterone in frogs, is linked to a decrease of intraoocyte cAMP [42], while maintenance of oocytes in prophase I requires constant Gs protein activity and high cAMP content in both frog and mouse oocytes [43, 44]. This constant Gs protein activity has recently been shown to be maintained by the orphan GPR3 receptor since oocytes from Gpr3 knockout mice resume meiosis within antral follicles [45]. Nevertheless, it remains possible that oocyte HTR1D and HTR7, through control of cAMP levels, could be involved in the fine-tuning of the meiotic maturation process or, alternatively, be involved in later steps of oocyte and embryo development. As for a possible role of 5-HT in early embryogenesis, we performed experiments showing that 5-HT or fluvoxamine had no effect on the percentage of embryos reaching the blastocyst stage when cultured in vitro (data not shown). However, 5-HT was recently shown to lower the mean cell number of blastocysts in mouse embryos cultured in vitro, thus suggesting a negative effect of 5-HT on early cleavage divisions [16]. However, further experiments are required to assess the role, if any, of 5-HT in mouse oocyte meiotic maturation as well as in other early developmental processes. To do so, it would be required to further characterize the coordinated activity of all the serotonergic components reported here in the in vivo context. The rapidly expanding panel of genetically modified mice might prove useful to assess the potential roles, in reproductive tissues, of the various serotonergic components uncovered by our work.

Interestingly, besides 5-HT, other locally regulated biogenic amines could be involved in these same processes since it was demonstrated that a dopamine transporter and the enzyme dopamine hydroxylase, both present in monkey oocytes, were responsible, respectively, for dopamine uptake and its subsequent conversion to norepinephrine, within oocytes, for later stimulation of ß-adrenergic receptors of surrounding granulosa cells [46]. In conclusion, our present work confirms the existence of a local serotonergic network in mammalian COCs and, thus, provides a stronger basis for extending earlier observations of the effects of 5-HT on early processes such as follicular growth and steroidogenesis and possibly other early developmental events.


   ACKNOWLEDGMENTS
 
We thank B.G. Allen and the Institut de cardiologie de Montréal for providing access to their confocal microscope and L.R. Villeneuve for technical assistance with it. M. Sainte-Marie is acknowledged for help with collecting oocytes and embryos. Thanks are also due to W. Eckberg, F. Pothier, P. Ribeiro, and R. Sullivan for critical reading of early versions of this manuscript. The editorial work of O.M. Da Silva is appreciated.


   FOOTNOTES
 
1 Supported by scholarships from the Fonds de recherche en santé du Qué bec (FRSQ) and Fonds pour la formation de chercheurs et l'aide à la recherche (FCAR) and the Université de Montréal to P.A. and by a Natural Sciences and Engineering Research Council (NSERC-Canada) grant to F.D. Back

2 Correspondence. FAX: 514 412 7314; francois.dube{at}umontreal.ca Back

Received: 3 January 2005.

First decision: 18 January 2005.

Accepted: 18 April 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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