| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Studies |
Institute for Hormone and Fertility Research, University of Hamburg, 22529 Hamburg, Germany
Address correspondence and requests for reprints to: Amal K. Mukhopadhyay, Institute for Hormone and Fertility Research, University of Hamburg, Grandweg 64, 22529 Hamburg, Germany. E-mail: amal{at}ihf.de
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Apart from its well-known effects within the nervous system, 5-HT seems also to have hormonal function in nonneuronal cells in many peripheral tissues. For example, 5-HT increases proliferation of vascular endothelial cells (6) and stimulates collagenase production (7) in rat and human myometrial cells. In testis, 5-HT has been demonstrated (8) to induce the secretion of corticotropin-releasing factor by Leydig cells. A stimulatory effect of 5-HT on adrenal steroid production has also been reported for human (9) and rat (10) adrenals. In the ovaries of the teleost fish Medaka, 5-HT seems to regulate both steroidogenesis and oocyte maturation (11). In isolated bovine luteal cells, 5-HT has been reported (12, 13) to stimulate progesterone production.
In human granulosa cells cultured in vitro, a stimulation of progesterone production was observed following exposure of the cells to 5-HT (14). 5-HT has been found in human follicular fluid (15).
Pharmacological analyses and molecular cloning have revealed the existence of at least fourteen 5-HT receptor subtypes (5, 16, 17) in diverse tissues. All 5-HT receptors belong to the family of seven transmembrane domains receptors that are coupled to different intracellular effectors via guanine nucleotide binding proteins (G-proteins), the only exception being the 5-ht3 receptor, which is a transmitter-gated Na+/K+ channel of pentameric subunit structure. At least five subtypes of 5-ht1 have been identified (5-ht1a, 5-ht1b, 5-ht1d, 5-ht1e and 5-ht1f), the two last being only partially characterized, and these receptors are coupled to Gi-proteins, leading to an inhibition of adenylate cyclase, when activated. The three subtypes of 5-ht2 receptor subfamily (5-ht2a, 5-ht2b and 5-ht2c) are all coupled to Gq-proteins linked to phospholipase C-ß activation. Other 5-HT receptor classes, namely 5-ht4, 5-ht6 and 5-ht7, are linked to a Gs-protein-mediated stimulation of adenylate cyclase. The effector system of 5-ht5 receptors (5-ht5a and 5-ht5b) remains to be molecularly elucidated, although there it has been reported (18) that a recombinant 5-ht5A expressed in HEK-293, is negatively coupled to adenylate cyclase.
Although a possible physiological relevance of this neurotransmitter in human ovary (see above) has been suggested, neither the mechanism of action of 5-HT nor the type of 5-HT receptor on granulosa cells has yet been identified. In a preliminary study, Tanaka et al. (19) suggested that 5-ht2 receptors may be involved in 5-HT-induced steroidogenesis in rat ovarian follicles. On the other hand, Terranova et al. (20) showed that in hamster preovulatory ovarian follicles estradiol production in vitro was stimulated by 5-HT via 5-ht1 receptors. However, these studies have been hampered by the fact that only a limited number of specific 5-HT receptor antagonists was available at that time.
Using cultured human granulosa-lutein cells (hGLC) aspirated for oocyte retrieval procedure from women undergoing in vitro fertilization protocol, we have carried out a detailed investigation to demonstrate the presence of serotonergic receptors on hGLC. Based on pharmacological and molecular characterization, we report here that human granulosa cells express a 5-ht7 receptor involved in the regulation of steroidogenesis via adenylate cyclase activation. Furthermore, we show that that this receptor is involved in a heterologous down-regulation of gonadotropin receptor-coupled cAMP signaling system in hGLC, pointing to a possible implication in the corpus luteum insufficiency.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The sources of various chemicals used were:
3-isobutyl-1-methylxanthine (IBMX), forskolin, dimethyl sulfoxide,
isoproterenol, propanolol, 5-methoxytryptamine, dithiothreitol,
phenylmethylsulfonylfluoride, and guanosine 5'-[ß,
-imido]
triphosphate [Gpp(NH)p], serotonin creatine sulfate, DMEM/nutrient
mixture F12 Ham (DMEM-Ham’s F-12 medium), penicillin, and streptomycin
were from Sigma (Deisenhofen, Germany);
(+/-)-2-methylserotonin maleate, 
-methyl-5-hydroxytryptamine
maleate, histamine dihydrochloride, (-)-pindolol, butaclamol
hydrochloride, loxapine succinate, amoxapine,
5-carboxy-amidotryptamine maleate,1-(3-chlorophenyl) piperazine
dihydrochloride (m-CPP), mianserin, ritanserin, clozapine,
serotonin HCl, pindolol, ketanserin, and
8-hydroxy-2-(di-N-propylamino)-tetralin were from Research
Biochemical Incorporated (Köln, Germany); BSA was from
Merck & Co., Inc.(Darmstadt, Germany); FBS was obtained
from Serva (Heidelberg, Germany); plastic 6- and 24-well plates were
from Nunc GmbH (Wiesbaden, Germany);
5-[1,2-3H(N)]-serotonin creatine sulfate was
from NEN Life Science Products Du Pont (Boston, MA);
Isothiocyanate of guanidium solution (RNA clean) was from AGS
(Heidelberg, Germany); SuperscriptTM II Rnase H-
reverse transcriptase, Taq DNA polymerase, and 100-bp DNA
ladder and RNA ladder were from Life Technologies, Inc./BRL (Eggenstein, Germany); primers were from MWG-Biotech
GmbH (Ebersberg, Germany); [-32P] dCTP,
Kodak Biomax MS and Kodak X-OMAT, and Hybond-N membrane were from
Amersham Pharmacia Biotech (Freiburg, Germany); highly
purified human CG (hCG) and Klenow enzyme was from B
rhinger Mannheim
Biochemica (Mannheim, Germany); Percoll, Sephadex G25, and NAP-5 column
was from Pharmacia Biotech (Freiburg, Germany); QIAEX DNA
Gel Extraction Kit and QIAPREP spin plasmid kit were from
QIAGEN (Hilden, Germany); ABI PRISM Dye Terminator Cycle
Sequencing Ready Reaction Kit was from Perkin-Elmer Corp.
(Weiterstadt, Germany); Taq polymerase was from Genecraft
(Münster, Germany).
Methods
hGLC.The hGLC were obtained from follicular fluid collected after the aspiration of hyperstimulated preovulatory follicles from women undergoing oocyte retrieval for in vitro fertilization. The female patients seeking treatment for infertlity at a private infertility clinic, Gemeinschaftspraxis, received GnRH antagonist treatment to block endogenous gonadotropin release. Ovarian stimulation was initiated by human menopausal gonadotropin/FSH (150 IU/day), and follicular growth was routinely monitored by ultrasonography. Once the largest follicle reached the diameter of approximately 19 mm, the patient received 10,000 IU hCG, and 36 h later follicular puncture was performed to retrieve the oocytes. The granulosa cells were transferred to our lab for culturing. All steps of cell purification were performed at room temperature, under sterile conditions. After collecting the cells by centrifugation at 600 x g for 10 min, follicular fluid was removed. The cells were resuspended in DMEM-Ham’s F-12 medium, containing 0.01% BSA and centrifuged at 1000 x g for 25 min on a 40% Percoll cushion. The cells were collected by aspiration and washed in DMEM-Ham’s F-12 medium. After counting with an hemocytometer, cells were plated into 24-well or 6-well plates, at a density of 2 x 105 cells/mL, in DMEM-Ham’s F-12 medium supplemented with 10% heat-inactivated FCS, 50 U/mL penicillin, and 50 µg/mL streptomycin and cultured in a humidified 95% air-5% CO2. After 1 day of culture, medium was replaced by DMEM-Ham’s F-12 medium containing 0.1% of BSA, 50 U/mL penicillin, and 50 µg/mL streptomycin. Cells were cultured for 3 days in this medium, following which experimental incubations were initiated.
Freshly prepared cells were found to be contaminated by mostly erythrocytes and other blood cells, which could be easily washed off after 1 day of culture because these cells did not adhere to the culture plates. Also, staining for 3ß-hydroxysteroid dehydrogenase revealed over 95% positively stained cells, suggesting that we had almost pure GLCs in culture.
For collection of human granulosa cells, we have received the approval from the local Medical Practitioner’s Ethical Committee according to the declarations of Helsinki.
Adenylate cyclase assay and cAMP RIA.Preparation of cell membrane was essentially carried out as described (21), with minor modifications. In brief, the cells (106 cells per mL) were resuspended in a solution containing 50 mM Tris-HCl (pH 7.4), containing 50 mM NaCl, 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonylfluoride and homogenized with a polytron homogenizer. Homogenates were centrifuged at 150 x g for 10 min to remove unbroken cells and debris. The supernatant fractions were centrifuged at 30,000 x g for 30 min. Resulting membrane pellets were suspended in ice-cold 50 mM Tris-HCl (pH 7.4) containing 50 mM NaCl, 1 mM dithiothreitol, and 5 mM EDTA. This suspension was recentrifuged at 30,000 x g for 30 min. Membranes were resuspended in 50 mM Tris-HCl (pH 7.4) containing 50 mM NaCl, snap-frozen in dry ice alcohol bath, and stored at -80 C until binding assays are performed. Protein concentrations were determined according to Bradford (22).
Adenylate cyclase assays and cAMP RIAs were performed as described previously (21). Adenylate cyclase assays were performed for 20 min at 37 C using 5 µg membrane proteins. In case of cAMP assays performed with intact cells, incubation of cells was terminated by adding ice-cold ethanol to a final concentration of 80% ethanol.
Progesterone RIA.Progesterone concentrations in the media of cultured hGLCs were measured by RIA, as described previously (23).
Statistical analysis.Student’s t test was performed for statistical analysis, using the software package provided by GraphPad Software, Inc. (San Diego, CA). All experiments were repeated at least three times, and results are represented as means ± SEM.
RT-PCR.RT: After 3 days of culture in 6-well plates, hGLCs were washed, scraped off, and collected by centrifugation at 1800 x g. Total RNA was isolated using RNA clean solution, according to the instruction of the manufacturer. An aliquot of 5 µg RNA, heated for 10 min at 70 C and quenched on ice, was subjected to first-strand complementary DNA synthesis by using 200 U Superscript II Rnase H- reverse transcriptase for 1 h at 42 C in a volume of 20 µl, as described by the supplier. After completion of first-strand synthesis, the reaction was diluted to 100 µl with sterile distilled water. Two microliters each were used for following PCR assays.
RT-PCR.PCR: For amplification of specific cDNA sequences,
the following pairs of primers (see Fig. 1
) designed according to the published
h5-ht7 receptor cDNA sequence (24, 25) were used:
|
The last set of primers was specifically used to identify splice variants by RT-PCR, as reported previously (24). Amplifications were performed in a total volume of 25 µl of 20 mM Tris HCl (pH 8.4) containing 50 mM KCl, 1.5–2.5 mM MgCl2, 200 µM dNTP each, 100 nM primer each, and 0.5 U Taq polymerase, with the following cycles: 95 C for 2 min; touch down: Tm + 14C -Tm + 4C for 1 min (2C increments), 72 C for 1 min and 95 C for 1 min for two cycles; Tm + 4C for 1 min, 72 C for 1 min and 95 C for 1 min for 20 cycles and 72 C for 10 min. Fragments to be sequenced were obtained with PCR and sometimes with nested-PCR. For amplification, the lowest melting temperature from the two primers was taken as Tm for the reaction.
Sequencing.A quantity of 400-ng PCR fragments was used for each reaction of sequencing. Sequencing was performed at least three times for both strands, with the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit, as prescribed by the manufacturer. Sequences were performed by the Laboratory Services of the University of Hamburg, with the PE Applied Biosystems DNA sequencing equipment (model 377A).
Northern blot hybridization.Random priming labeling: After
agarose gel electrophoresis, the PCR-amplified cDNA fragments were
isolated and purified using QIAEX DNA Gel Extraction Kit. An aliquot of
100 ng h5-ht7 receptor RT-PCR fragment (nt 867-1393)
obtained with primers e and b or glyceraldehyde-3-phosphate
dehydrogenase RT-PCR fragment (nt 361–558), according to the published
sequence (26), was labeled by random priming with
[-32P]dCTP (3000 Ci/mmol) using 8 U Klenow enzyme in
20 µl 1x Klenow buffer for a 1-h incubation at 37 C. Specific
activities of 109 cpm/µg were obtained. Labeled cDNAs
were purified from unincorporated [-32P]dCTP by NAP-5
column chromatography.
Northern blot hybridization.Northern blot: Aliquots of 10–20 µg total RNA isolated from hGLC were denatured for 10 min in 2.2 M formaldehyde containing 50% (v/v) deionized formamide, 50 mM MOPS (pH 7.0), and 1 mM Na2 EDTA at 60 C and quenched on ice. Samples were fractionated by agarose gel electrophoresis on 1.2% agarose gels containing 4.0% (v/v) formaldehyde, 50 mM MOPS (pH 7.0), 1 mM Na2 EDTA, and 1 µg/mL ethidium bromide. Fractionated RNAs were transferred onto nylon membrane (Hybond-N membrane) and ultraviolet cross-linked. To confirm equal amounts of RNA loaded into gel, blots were hybridized with labeled glyceraldehyde-3-phosphate dehydrogenase RT-PCR fragment.
Hybridization of blots was performed, and blots were washed with 1x SSC/5% SDS at RT and at 65 C for 10 min each and with 0.1 SSC/0.5% SDS at 65 C for 20 min. Membranes were exposed to Kodak Biomax MS.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
At the outset, we examined whether 5-HT was able to stimulate progesterone production by cultured hGLCs. For this purpose, after 3 days of hGLC culture, medium was replaced by incubation medium, which was identical to culture medium but contained 0.25 mM IBMX in addition. Following incubation of the cells for 3 h in the absence or presence of 10-5 M 5-HT, the amount of progesterone produced was found to be 39.86 ± 0.66 and 70.98 ± 2.03 ng/105 cells·3 h, respectively, clearly confirming earlier observations by Bodis et al. (17).
Effect of 5-HT on cAMP formation in hGLCs
To investigate whether 5-HT-mediated increase in progesterone
production by hGLCs is related to elevated intracellular cAMP levels,
cells were incubated with various concentrations of 5-HT in the
presence or absence of 0.25 mM phosphodiesterase inhibitor,
IBMX, for 60 min, and cAMP formed was measured. Fig. 2 clearly shows that 5-HT was able to
stimulate cAMP formation in these cells in a dose-dependent manner,
with a maximum stimulation being achieved at a concentration of
10-5 M 5-HT. The presence of IBMX
increased the sensitivity of the response 4- to 5-fold, by
shifting the dose-response curve to the left, without
affecting the level of maximum response.
|
we have presented the
time course of cAMP formation in the cells, following
stimulation with 10-5 M
5-HT, in the presence of 0.25 mM IBMX. For the
sake of comparison, we have also included data obtained when the cells
were incubated with 10 ng/mL hCG. It is evident from the data shown in
Fig. 3 that there is a rapid increase in the level of cAMP in response
to 5-HT, until a maximum is reached by 45 min; thereafter, the level
declines somewhat, with a plateau being maintained until 180 min. With
hCG there was also a rapid increase during the first 45 min,
but a slower increase still continued until 180 min.
|
, dose-response curves of
cAMP formation in hGLCs in response to an incubation with various
concentrations of 5-HT, 5-CT, and 5-MT are shown. Results
have been presented as percentage of maximum cAMP response obtained
with 10-5 M 5-HT. It is evident that
although all three compounds were able to stimulate cAMP formation in a
dose-dependent manner, 5-CT has been the most potent stimulator.
|
show clearly that whereas a robust
stimulation of cAMP formation was observed for 5-HT-treated cells over
untreated cells, none of the ß-adrenergic antagonists used had any
effect on the response of the cells to 5-HT.
|
presents the results of an
experiment where hGLCs were incubated with three different
concentrations of a series of 5-HT agonists (-methyl 5-HT, 2-methyl
5-HT, and m-CPP), in addition to ß-adrenergic agonists (isoproterenol
and epinephrine). Also, dopamine and histamine were examined. None of
these substances had any effect on cAMP formation by hGLCs. However,
the addition of both hCG and 5-HT resulted in a marked stimulation of
cAMP formation.
|
), loxapine, and amoxapine (Fig. 7) produced a dose-dependent inhibition
of 5-HT-stimulated cAMP formation in hGLCs. However, none of
these compounds had any inhibitory effect on hCG-stimulated cAMP
formation in these cells (data not shown). This last result
demonstrates that inhibition of cAMP formation by these pharmacological
agents was not due to unspecific toxic effects. It may be noted that
the absolute levels of cAMP measured varied somewhat among different
batches of cell preparations. However, the qualitative aspects of the
results obtained in different experiments were always reproducible.
|
|
5-HT was able to stimulate adenylate cyclase activity in the
membrane preparation from hGLCs in the presence of 100 µM
of a nonhydrolysable GTP analog, Gpp(NH)p. The enzyme assay was carried
out either with constant amount (10 µg) of protein added over a
varying duration of incubation (Fig. 8A)
or with varying amounts of membrane protein added for a period of 60
min (Fig. 8B). The addition of Gpp(NH)p increased the enzyme activity
considerably over basal activity, but the highest activation
resulted when both 5-HT and Gpp(NH)p were added together. The cyclase
activity increased linearly with increasing duration of incubation
(Fig. 8A). The enzyme activity was also dependent on the membrane
protein added (Fig. 8B).
|
Sequencing of 5-HT receptor transcript from hGLCs
Sequencing data of RT-PCR products amplified from hGLC RNA using sets of primers covering all the h5-ht7 transcript correlated totally with the sequence published (23). A silent substitution (Gly-654-Ala) was also found in some patients. Silent substitution (Ala-1233-Gly) and other polymorphisms (Pro-279-Leu, Th-92-Lys) have already been reported (27) for the h5-ht7 receptor cDNA.
Northern blot analysis of 5-HT receptor from hGLCs
Transcripts of expected size (2.8 kb) coding the
h5-ht7 receptor were detected by Northern blot
(Fig. 9) in hGLC RNA, the expression of
which decreased during culture from 20–70 h. Neither the addition of
5-CT nor hCG had any effect on h5-ht7 receptor
mRNA level in the cultured cells. In a separate experiment where period
of culture was extended to 140 h, Northern blotting revealed a
drastic reduction of h5-ht7 transcription, even
below the limit of detection (data not shown).
|
Using primers n and b, as used by Heidmann et
al. (24), the presence of three splice variants
of the h5-ht7 gene could be demonstrated by
RT-PCR in human granulosa cells, as shown in Fig. 10. Specificity of these amplified
fragments were confirmed by Southern blot (data not shown).
|
Having demonstrated that h5-ht7 receptors are present on hGLCs, it was of interest to examine whether there was any interaction between h5-ht7 and hCG receptors, because both of these receptors are coupled to adenylate cyclase activation and progesterone release in luteal cells. Also to be noted, the luteal cells in the corpus luteum of early pregnancy are prevented from demise by the action hCG secreted by feto-placental unit.
For this purpose, we have preincubated the cells for 3 h,
with or without 5-HT (10-6 M),
following which the cells were washed. Fresh medium containing 100
ng/mL hCG was added, and the incubation was continued for various
durations. It is clear from the results presented in Fig. 11 that hCG-stimulated cAMP response of
the cells preincubated with 5-HT was markedly lower at all time points,
compared with the cells that have not seen 5-HT.
|
show that the desensitizing effect of 5-HT on hCG-stimulated cAMP
response was clearly dose-dependent, with maximum effect being seen at
a 5-HT/5-MT concentration of 10-6 M.
Similar results were obtained with 5-CT (data not shown).
|
presents the results of
preincubating the cells with 10-6 M
5-HT together with various concentrations of clozapine. Preincubation
with 5-HT alone reduced the amount of cAMP formed in response to
subsequent stimulation with hCG by about 70%. This reduction could be
completely reversed in a dose-dependent manner by the inclusion of
clozapine during the preincubation period. Clozapine added alone during
the preincubation period had no effect on cAMP formation during the
subsequent stimulation by hCG.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Incubation of hGLCs with 5-HT for different periods of time resulted in a time-dependent increase in the amount of cAMP formed. We observed a rapid increase in cAMP formation in hGLCs, which reached a maximum after about 45 min and thereafter decreased. In comparison, hCG-stimulated cAMP formation kept increasing throughout the incubation period. One explanation for this difference may be due to relatively less stability of 5-HT in culture medium. In addition, using membrane preparation from hGLCs, it could be shown that adenylate cyclase activity was stimulated by 5-HT in the presence of a stable GTP analogue, Gpp(NH)p. Enzyme activity increased linearly with increasing amounts of hGLC membrane protein added and with an increasing duration of incubation. The pEC50 determined for adenylate cyclase activity with respect to stimulation by 5-CT, 5-HT, and 5-MT was found to agree very well with the data reported for recombinant h5-ht7b receptor expressed in transfected HEK 293 cells by Jasper et al. (28).
5-HT-stimulated cAMP formation was not affected by
ß-adrenergic receptor antagonists like propanolol or pindolol,
confirming that 5-HT is acting in these cells via its own receptor and
not by cross-activation of ß-adrenergic receptors. Isoproterenol and
epinephrine produced no stimulation of cAMP formation (Table 1
). Both
dopamine and histamine were ineffective, thus precluding
that 5-HT might be acting through either dopaminergic or histaminergic
receptors. Not surprisingly, -methyl-5-HT, 2-methyl-5-HT, and m-CPP,
even at concentrations up to 10-5
M, were fully ineffective (results not
shown).
Antagonists for 5-ht6 and 5-ht7 receptors like mianserine, loxapine, and amoxapine were nearly equipotent in inhibiting 5-ht-stimulated cAMP formation, but ketanserine was found to be approximately 10-fold less potent. These results point to the fact that hGLC cells may have either 5-ht6 or 5-ht7 class of receptors.
Sequence analysis revealed complete identity of the 5-HT receptor from hGLC with the cloned human 5-ht7 receptor (25). Our preliminary RT-PCR analysis using a set of primers (see Materials and Methods) corresponding to h5-ht6 receptors among others did not yield any corresponding fragment, revealing an absence of this receptor in hGLCs. Northern blot analysis confirmed the presence of a 2.8-kb expected size of 5-ht7 transcript in hGLCs. An increase in the duration of in vitro culture of hGLCs seems to lead to a decreased level of the receptor transcript. During in vitro culture, granulosa cells have been reported (30) to undergo differentiation from early to late luteal cells. It will be of interest to determine whether changes in the 5-ht7 receptor transcript level during the culture would correlate with this phenomenon of in vitro differentiation, which requires further investigations.
Based on Northern blotting, PCR, and in situ hybridization histochemistry, localization of the 5-ht7 receptor was demonstrated in some specific areas of the central nervous system, particularly in rat brainstem, hypothalamus, hippocampus, and thalamus (29, 31). Minor expression was detected in cortex, striatum, olfactory bulb, olfactory tubercle, amygdala, superior colliculus, cerebellum, and the raphe nuclei (29, 31, 32). This was generally in agreement with the binding data obtained in the guinea pig (33). In the periphery, 5-ht7 receptors have been localized by RT-PCR and Northern blot in spleen (31), liver (31, 32, 34), stomach, descending colon and ileum (31) of rat, coronary artery, descending colon and ileum in human (25), and intestine and heart in mouse (35). The 5-ht7 receptor mRNA was not detected in the rat bladder, testis, adrenal gland, or kidney (32). There is so far no report documenting the expression of 5-ht7 receptor in ovarian cells of any mammalian species. In the rat ovary, a transcript of r5-ht7 receptor could not be detected by Northern blotting analysis (29). This could be a species-specific phenomenon. Alternatively, it could be due to the use of whole ovary rather than the granulosa cells for preparing RNA used for Northern blotting. This may reduce the relative abundance of 5-ht7 transcript making it difficult to detect by Northern analysis.
The 5-ht7 receptor has been cloned in a variety of species, including xenopus (36), guinea pig (37), mouse (35), rat (31, 32, 34), and human (25). In fact, a fragment of the 5-htdro1 cDNA cloned from Drosophila melanogaster (38) cDNA library, and used as a probe, permitted molecular identification of the human 5-ht7 receptor, the homology amounting to 57% between these two species. The amino acids sequence of this receptor has a low homology with other 5-HT receptors (39–53%), which indicates a direct lineage between 5- htdro1 and 5-ht7 receptor. The percentage of interspecies homology for mammalian 5-ht7 receptors reach 95%, which explains the relatively high resemblance in pharmacological profile among different species studied. This conservation during the evolution suggests an important role for this receptor. Gene of human 5-ht7 receptor (h5-ht7) has been localized in the 10q23.3-q24.4 chromosomal region (27, 39). Genes of rat 5-ht7 (r5-ht7) and h5-ht7 receptors contain at least two introns: the first one is localized after the third transmembrane domain (25, 31, 33), and the second one is in the terminal position of the open reading frame (33).
Four isoforms of r5-ht7 and h5-ht7 receptors have been described, differing in the intracytoplasmic C-terminal region (24). In the rat, three isoforms have been named as r5-ht7a, r5-ht7b and r5-ht7c. The two isoforms r5-ht7a (448 amino acids) and r5-ht7b (435 amino acids) differ from each other due to splicing at two donor sites arranged in tandem. An insertion of five bases (GTAAG) in the carboxyl-terminal region introduceds a stop codon, resulting in a variant truncated by 13 amino acids (r5-ht7b). The third isoform, r5-ht7c (470 aa), arises from the maintenance of an exon in C-terminal position. Also in human, three isoforms have been found corresponding to h5-ht7a (445 aa) and h5-ht7b (432 aa) by homology to rat 5-ht7 (24, 28). On the other hand, a third isoform in human, named h5-ht7d (479 aa), lacking any homology to the r5-ht7c isoform, arises from the inclusion of an exon at C-terminal position. RT-PCR analysis performed on hGLC RNA with sets of specific primers for h5-ht7 splice variants revealed the presence of the three splice variants h5-ht7a, h5-ht7b and h5-ht7d in hGLC. The rat homologue of all three splice variants seem to code for receptor proteins having similar binding and activation capacity (40). Thus, in an analogous manner, it may be contemplated that all of these three isoforms may contribute to the action of 5-HT in hGLCs. The presence of a pseudogen having 88% homology to the 5-ht7 receptor has been recently detected (41) in the cerebellum, cerebral cortex, medulla and putamen. This was however, not detected in hGLCs by Northern blotting.
Finally, we observed that a prior exposure of the cells to 5-HT leads to a marked decrease in the ability of these cells to respond to a stimulation with hCG. This desensitizing effect of serotonin on hCG/LH receptors on hGLC could be prevented by a 5-ht7 receptor antagonist, clozapine. Because the cells used by us could be considered to be similar to the cells of corpus luteum of early pregnancy, which depend on hCG for survival, it may not be difficult to contemplate that an increase in 5-HT concentration at the time of the formation of corpus luteum of early pregnancy, could possibly result in a failure of the luteal cells to respond to hCG effectively. This may, thus, lead to a corpus luteum insufficiency that could be prevented by administration of the antagonist clozapine. Whether this may happen in vivo under certain stressful conditions or not, of course, remains to be a matter of conjecture. There is, however, a need for a detailed evaluation, especially as we do not completely understand the causative factor(s) involved in the corpus luteum inefficiency as a cause for infertility. Also, additional studies will be necessary to understand the molecular mechanisms involved in 5-ht7 receptor-mediated desensitization of hCG/LH receptor-coupled cAMP response of the hGLC. It will be important to understand if this desensitization results from a down-regulation of LH-receptor protein or uncoupling of adenylate cyclase signaling from the receptor activation or both.
In conclusion, we have been able to demonstrate the presence of a 5-HT receptor positively coupled to adenylate cyclase activation in hGLC and have characterized this receptor by pharmacological and molecular analyses to be a h5-HT7 receptor. Furthermore, we have shown that the interaction between this receptor and the gonadotropin receptor may have potential clinical relevance.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received June 23, 1999.
Revised October 4, 1999.
Accepted November 19, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  P. Amireault and F. Dube Intracellular cAMP and Calcium Signaling by Serotonin in Mouse Cumulus-Oocyte Complexes Mol. Pharmacol., December 1, 2005; 68(6): 1678 - 1687. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Amireault and F. Dube Serotonin and Its Antidepressant-Sensitive Transport in Mouse Cumulus-Oocyte Complexes and Early Embryos Biol Reprod, August 1, 2005; 73(2): 358 - 365. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
and reverse primer 
) used
to amplify the h5-ht7 receptor cDNA.
) or presence
(
) of 0.25 mM IBMX. cAMP formed was measured by RIA.
Data represent mean ± SEM from triplicate
determinations.
),
with 10-5 M 5-HT (•), or with 10 ng/mL hCG
(
)
alone, or with GppNHp plus 10-7 M 5-HT (
)
for 3 h, washed, and reincubated with 100 ng/mL hCG for 30 min.
The amount of cAMP formed was measured. Each data point represents
mean ± SEM from triplicate determinations.