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Original Studies |
Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada V6H 3V5
Address correspondence and requests for reprints to: Dr. Peter C. K. Leung, Department of Obstetrics and Gynecology, University of British Columbia, Room 2H30-4490 Oak Street, Vancouver, British Columbia, Canada, V6H 3V5. E-mail: peleung{at}interchange.ubc.ca
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Purinergic receptors have been classified as P1 receptors and P2 receptors. Pharmacologically, the P1 receptors have a high affinity for extracellular adenosine and AMP (Adenosine > AMP > ADP > ATP), whereas P2 receptors have a high affinity for ATP and ADP (ATP > ADP > AMP > adenosine) (1, 3, 4). Six subtypes of P2 purinergic receptors (P2X, P2Y, P2D, P2T, P2Z, and P2U) have been identified in pharmacological and molecular cloning studies (5).
Functionally, a P2U purinoceptor (P2UR) has been detected in human granulosa-luteal cells (hGLCs) using microspectrofluorimetry (6). Autonomic nerves have been shown to innervate the ovary and may be involved in regulating steroidogenesis (7, 8, 9). It is tempting to speculate that the coreleased ATP from autonomic nerve endings in the ovary may play a role in regulating ovarian function. ATP has been shown to increase the production of progesterone and estradiol in hGLCs (6). These findings provide further evidence that ATP is able to regulate ovarian function through binding to ATP receptors.
Although P2UR has been identified pharmacologically in human ovary (6, 10), its expression and regulation at the messenger RNA (mRNA) levels have not as yet been characterized. To understand further the potential role of ATP and the receptor of this neurotransmitter in the ovary, the present study was designed to detect the expression of the P2UR in hGLCs and to examine the regulation and signaling of this receptor in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Prostaglandin F2
, GnRH, human CG, estradiol, progesterone,
8-bromo-adenosine-3',5'-cyclic monophosphate (8-bromo-cAMP), forskolin,
ATP, and uridine triphosphate (UTP) were obtained from
Sigma (St. Louis, MO). DMEM (phenol-red free) and
penicillin-streptomycin, were obtained from Life Technologies, Inc. (Burlington, Ontario, Canada). Fura-2 AM was purchased
from Molecular Probes, Inc. (Eugene, OR).
hGLCs culture and treatments
hGLCs were collected from patients undergoing an In
Vitro Fertilization-Embryo Transfer program. The use of hGLCs was
approved by the University of British Columbia Clinical Screening
Committee for Research and Other Studies Involving Human Subjects.
Granulosa cells were separated from red blood cells in follicular
aspirates by centrifugation through Ficoll Paque, washed in phenol-red
free DMEM and suspended in DMEM containing 100 U penicillin G
sodium/mL, 100 µg streptomycin/mL, and 10% FBS. The cells were
plated at a density of approximately 200,000 cells/dish in 35-mm
culture dishes. The dishes were incubated at 37 C under a
water-saturated atmosphere of 5% CO2 in air for
2 days. To examine the regulation of the P2UR mRNA, hGLCs were
incubated in serum-free medium for 24 h before treatment with
estradiol (10-7 M),
progesterone (10-7 M),
prostaglandin F2 (10-7
M), GnRH, (10-7
M), hCG (5 IU/mL), or ATP (10
µM) for 24 h. For dose-response
experiments, hGLCs were treated with different concentrations of hCG
(0.1, 1, 5, 10 IU/mL) for 24 h. For time-course analysis, hGLCs
were treated with 5 IU/mL hCG for 0, 3, 6, 12, 24, or 48 hours. To
further delineate the underlying mechanism, by which the expression of
P2UR mRNA was regulated, cells were treated with 8-bromo-cAMP (1
mM) or forskolin (10 µM)
for 24 h before the determination of P2UR mRNA levels.
Total RNA isolation and RT-PCR
Total RNA was prepared from the cultured hGLCs by the phenol-chloroform method of Chromczynski and Sacchi (11). Briefly, hGLCs were lysed in solution D [4 M guanidine thiocynate, 25 mM sodium citrate (pH 7.0), 0.5% N-lauroyl sarcosine, and 0.1 M ß-mercaptoethanol], followed by acid-phenol extraction. The RNA concentration was determined based on absorbance at 260 nm, and the mRNA integrity was checked by separation in 1% agarose denaturing gel. One microgram of total RNA obtained from hGLCs was reverse transcribed into complementary DNA (cDNA) using the First Strand cDNA Synthesis Kit (Pharmacia Biotech, Morgan, Canada). One set of oligonucleotide primers (5-CCTGGAATGCGTCCACCACATAT-3 and 5-GACGTGGAATGGCAGGAAG CAGA-3) based on the published human P2U receptor sequence (12) was designed for PCR to amplify the P2UR from hGLCs. PCR reactions were performed in the presence of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 400 µM dNTPs, 0.25 U Taq DNA polymerase, 2 µM primers, and 1 µl cDNA template per 25 µl reaction. Amplification was carried out for 33 cycles with a condition of denaturation at 94 C for 60 sec, annealing at 64 C for 35 sec, and extension at 72 C for 90 sec, and a final extension at 72 C for 15 min. The same amount of cDNA of each sample was used for amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Primers for GAPDH (5'-ATGTTCGTCATGGGTGTGAACCA-3' and 5'-TGGCAGGTTTTTCTAGACGGCAG-3') were designed based on published sequence (13). Amplification was carried out for 18 cycles with a condition of denaturation at 94 C for 60 sec, annealing at 55 C for 35 sec, and extension at 72 C for 90 sec, and a final extension at 72 C for 15 min.
Cloning and sequencing of RT-PCR product
Ten microliters of PCR products of P2UR were fractionated in a 1% agarose gel stained with ethidium bromide. The expected PCR products (599 bp) were isolated from the gel, cloned using the TA cloning kit (Invitrogen, San Diego, CA) and sequenced by the dideoxy chain termination method using a T7 DNA polymerase sequencing kit (Pharmacia Biotech, Morgan, Canada). The sequence of the cDNA was sent to GenBank at NCBI (National Center for Biotechnology Information) through the internet (www.ncbi.nlm.nih.gov) to compare the identity with published human P2UR. This cDNA was then used as the template for making probes for Northern and Southern blot analyses.
Northern blot analysis
Approximately 15 µg total RNA was separated by electrophoresis in a 1% agarose-formaldehyde gel and transferred onto a charged nylon membrane (Hybond-N; Amersham Pharmacia Biotech, Oakville, Canada). The Northern blots were incubated in 5x SSPE for 30 min. They were then transferred to a prehybridization solution of 5x SSPE containing 50% deionized formamide, 5x Denhardt’s, 1% SDS, water and heat-denatured salmon sperm DNA (final concentration, 0.2 mg/mL). The blots were prehybridized at 42 C for 3 h. The radiolabeled P2UR probe was then added to the prehybridization solution. The probe was radiolabeled by the random primer method of Feinberg and Vogelstein (14) and heat-denatured before being added to the prehybridization solution. The blots were incubated in the presence of the radiolabeled probe at 42 C for 16 h, then washed twice with 2x SSPE at room temperature (5 min/wash), twice with 2x SSPE containing 1% SDS at 55 C (30 min/wash), twice with 0.2x SSPE at room temperature (30 min/wash), and finally exposed to Kodak Omat x-ray film.
Southern blot analysis
After sequencing, the cloned cDNA of the P2U receptor was used as the template to make digoxigenin (DIG)-labeled probe using a DIG DNA Labeling Kit following the protocol provided by the manufacturer (Roche Molecular Biochemicals, Laval, Canada). The PCR products in 1% agarose gel were transferred to Hybond-N nylon membranes (Amersham Pharmacia Biotech Inc.) and hybridized with DIG-labeled P2U receptor cDNA. The membranes were processed as per the manufacturer’s protocol. Finally, the membranes were exposed for 10 min at room temperature to x-ray film. The autoradiograms were scanned with a laser densitometer (Model 620, Video Densitometer; Bio-Rad Laboratories, Inc., Richmond, CA), and P2UR mRNA levels were standardized against GAPDH.
Quantification of P2UR mRNA
To compare the expression and regulation of P2UR mRNA, semiquantitative PCR was performed. For validation, various cycles of (27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38) PCR were performed to amplify P2UR mRNA. GAPDH was used in the present study to normalize the PCR product of P2UR mRNA. For GAPDH, 15–27 cycles of PCR were performed for validation. Southern blot analysis was carried out using equal amounts of amplified PCR products (10 µg), and the results were quantified using a laser densitometer.
Microspectrofluorimetry
hGLCs were seeded onto 25-mm circular glass cover slips (5000 cells/slip) and incubated for 3 days at 37 C in humidified air with 5% CO2 before microfluorimetric experiments. Cytosolic calcium concentrations were measured using the dual-excitation single-emission fluorimetric technique, as described previously (15). Briefly, the cells were incubated with 5–10 µM fura-2 AM acetoxymethyl ester (Molecular Probes, Inc.) for 30 min at 37 C in humidified air with 5% CO2. The cover slip was mounted onto the perifusion chamber and equilibrated for 10 min with balanced salt buffer [137 mM NaCl, 5.36 mM KCl, 1.26 mM CaCl2, 0.81 mM CaCl2·7H2O, 0.34 mM Na2HPO4·7H2O, 0.44 mM KH2PO4, 4.17 mM NaHCO3, 10 mM HEPES, 2.02 mM glucose (pH7.4)] in humidified air with 5% CO2. The fura-2 ratio measurements were performed using the Attoflour Digital Fluorescence Microscopy System (Atto Instruments, Rockville, MD). The perifusion chamber was connected to a multiunit six-channel perifusion system with a flow rate of 1–2 mL/min. Fura-2-loaded cells were observed through a x40 fluorescent objective lens and were illuminated alternatively with light at 340 nm and 380 nm. Emitted light was filtered using a 510-nm long-pass filter and detected using a low light-sensitive camera. Measurements of cytosolic calcium were performed at 1- to 2-sec intervals. All records were corrected for background fluorescence (determined from cell-free region of cover slip). Changes in the fluorescence ratio recorded at 340 and 380 nm correspond to changes in cytosolic calcium. To confirm the presence of functional P2UR in hGLCs, cells were treated with 100 µM ATP or UTP (Sigma). For dose-response experiments, cells were treated with various concentrations of ATP or UTP (1, 10, and 100 µM) before cytosolic calcium determinations.
Data analysis
Relative P2UR mRNA levels were expressed as the ratio of P2UR to GAPDH. For each patient, the data are represented as the percentage change relative to the control. Data of the same treatment groups are represented as means ± SE. Statistical analysis was performed by one-way ANOVA, followed by Tukey test. Differences were considered significant at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expression of P2U receptor mRNA in hGLCs was examined by
RT-PCR using one set of primers designed on the basis of the published
human P2UR expressed in airway epithelium. The positions and sequences
of primers are shown in Fig. 1A
. An
expected 599-bp DNA fragment was observed in ethidium bromide-stained
gel from hGLCs isolated from three different patients (Fig. 1B). No
product was obtained from the negative control (without first strain
cDNA template in PCR reaction). The PCR products from hGLCs were
subcloned and sequenced. Sequence analysis revealed that the cloned
cDNA is identical to nucleotide position 436-1034 of the published
human P2U receptor (12). This cDNA was then used as a template for
making probes for Northern and Southern blot analyses. Using Northern
blot analysis, two P2UR transcripts of 2.0 kb and 4.6 kb were detected
in hGLCs, as shown in Fig. 1C.
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To determine the condition under which the amplification of P2UR
was in the logarithmic phase, various cycles of PCR were performed.
Equal aliquots (1 µl) of cDNA were amplified by different PCR cycles.
Ten microliters of the PCR products were fractionated, transferred onto
a charged nylon membrane, detected by DIG-labeled cDNA, and finally
subjected to radioautography. The results were quantified using a laser
densitometer. A linear relationship between PCR products and
amplification cycles was observed in both P2UR (Fig. 2) and GAPDH (data not shown).
Thirty-three cycles for P2UR and 18 cycles for GAPDH were used for
quantification in subsequent regulation studies.
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To examine the regulation of P2UR mRNA, hGLCs were treated with
hCG (5 IU/mL), estradiol (10-7 M),
progesterone (10-7 M), ATP(10
µM), prostaglandin F2 (10-7
M), or GnRH (10-7 M),
respectively. As shown in Fig. 3, no
significant change of P2UR mRNA levels were observed in the groups
treated with estradiol, progesterone, ATP, prostaglandin F2, or
GnRH. In contrast, about a 30% decrease of P2UR mRNA
(P < 0.05) was noted in the hCG-treated group. To
further examine the effect of hCG on P2UR expression, hGLCs were
treated with increasing concentrations of hCG for 24 h. As shown
in Fig. 4, hCG down-regulated the level
of P2UR mRNA in a dose-dependent manner. Furthermore, a time-course
analysis revealed that hCG down-regulated P2UR mRNA in a time-dependent
manner (Fig. 5). It is well established
that hCG activates adenylate cyclase and increases the production of
cAMP in ovarian cells. To examine the possible mechanism by which P2UR
mRNA is regulated by hCG, hGLCs were treated with 8-bromo-cAMP and
forskolin, an activator of adenylate cyclase. As shown in Fig. 6, both cAMP and forskolin significantly
down-regulated the expression of P2UR mRNA.
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The P2UR expressed in hGLCs was tested functionally and
pharmacologically using microspectrofluorimetry in single cell studies.
As shown in Fig. 7, hGLCs responded
equally well to 100 µM ATP and UTP, indicating the
expression of a functional P2UR in these cells at the level of calcium
signaling. The cytosolic calcium mobilization was characterized by a
spike and a marked increase in cytosolic calcium, followed by numerous
oscillations with decreasing amplitudes. To examine further the
dose-response relationship, hGLCs were treated with increasing
concentrations of ATP or UTP (1–100 µM). It has been
demonstrated that submicromolar concentrations of ATP were incapable of
mobilizing cytosolic calcium (10). As shown in Fig. 8, both ATP and UTP were able to induce
cytosolic mobilization in micromolar levels with maximal responses
reached when treated with 10 µM of ATP or UTP, and no
difference was noted between cells treated with 10 µM and
100 µM.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adrenergic and cholinergic nerves have been shown to innervate the ovary and may be involved in the regulation of steroidogenesis (24, 25, 26). In human granulosa cells, epinephrine and norepinephrine have been shown to stimulate progesterone secretion via interaction with the ß-adrenergic receptor (27). In other reproductive tissues, extracellular ATP has been shown to activate contraction in the intact myometrium (28). Amnion cells isolated from the human placenta express ATP receptors that are coupled to inositol phospholipid breakdown and Ca2+ mobilization (29). ATP can trigger the acrosome reaction in human sperm in vitro (30). Following binding to P2-purinergic receptors, ATP can increase the secretion of testosterone in rat Leydig cell (31).
The human P2UR gene has been mapped to chromosome 11q 13.5–14.1 (32). The human P2UR cDNA was cloned and sequenced from airway epithelium (12). The P2UR in the human ovary has not as yet been characterized. The present study demonstrates for the first time the expression of P2UR in human ovarian cells. Northern blot analysis revealed that two species of mRNA, 2.0 kb and 4.6 kb, were expressed in hGLCs. Interestingly, human uterine cervical cells express at least four distinct transcripts [2.0, 2.2, 3.0, and 4.6 kb (33)], whereas human nasal and proximal-tubule epithelia and liver express only a single 2.1 kb mRNA (12). The expression of P2UR in hGLCs supports the hypothesis that extracellular ATP might play a role in the regulation of ovarian function.
Relatively few studies have focused on the regulation of P2UR mRNA in
response to hormone treatments. For example, retinoids have been shown
to regulate the expression of P2UR mRNA in human uterine cervical cells
(33). In the present study, hGLCs were treated with estradiol,
progesterone, PGF2, GnRH, ATP, and hCG. The result shows that only
hCG attenuated the expression of P2UR mRNA in these cells, suggesting
that LH/hCG may play a role in regulating the expression of P2UR in the
human ovary. It is well established that activation of the LH/CG
receptor activates adenylate cyclase and PKA (34). To further elaborate
the mechanism by which hCG regulates the expression of P2UR mRNA, hGLCs
were treated with exogenous 8-bromo-cAMP and forskolin, an activator of
adenylate cyclase. Our results show that both 8-bromo-cAMP and
forskolin markedly down-regulated the expression of P2UR mRNA levels,
supporting the notion that hCG down-regulation of the expression of
P2UR mRNA may be mediated by adenylate cyclase and cAMP. Recently, we
and others have shown that hCG can alter the mRNA levels of GnRH
receptor and PGF2 receptor (35, 36, 37) in hGLCs. There seems to be a
complex interaction of GnRH and PGF2 on
steroid hormone production in hGLCs (38). In the present study, hCG,
but not GnRH or PGF2, has been demonstrated to down-regulate the
expression of P2UR in dose- and time-dependent manners. The
physiological significance of the hCG effect on P2UR remains to be
determined. It has been reported that ATP may act as a trigger for
apoptosis or programmed cell death (39) and that ATP at a concentration
of 2.0 mM causes cell necrosis and death in the ovary (40).
It is conceivable that hCG is capable of minimizing the detrimental
effect of ATP, at least in part, by down-regulation of P2UR expression
in hGLCs.
ATP has been shown to induce cytosolic calcium oscillations in hGLCs
(5, 10, 15). Pharmacologically, the order of agonist potency for P2UR
is ATP = UTP > ATP
S >> 2MeSATP (6, 41). It also has been
demonstrated that the cytosolic calcium oscillations evoked by ATP are
initiated by the release of calcium from cytosolic stores and
maintained by extracellular calcium influx (10). In the present study,
we confirmed the presence of a functional P2UR in hGLCs. Furthermore,
our results clearly indicate that, like ATP, UTP is also capable of
evoking cytosolic calcium mobilization in a dose-dependent manner.
These data provide further evidence that the P2UR expressed in hGLCs is
functional following receptor activation by the ligand, at the level of
signal transduction.
In summary, our results demonstrate for the first time the expression of P2UR in the human ovary at the mRNA level. We have determined that the level of P2UR mRNA is down-regulated by hCG, presumably via a cAMP-mediated mechanism. The P2UR expressed in hGLCs is functional, in terms of calcium signaling. Taken together, these findings further support a role for ATP and the P2UR in the regulation of human ovarian function.
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Acknowledgments |
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Footnotes |
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2 Studentship recipients of the British Columbia Research Institute
for Children’s and Women’s Health.
Received August 2, 1999.
Revised November 30, 1999.
Accepted December 23, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor expression in cultured human
granulosa-luteal cells. Endocrinology. 138:191–195.-receptor mRNA in human
granulosa-luteal cells by human chorionic gonadotropin and
prostaglandin F2. Endocrine. 8:261–267.[CrossRef][Medline]
and
gonadotropin-releasing hormone on progesterone and estradiol production
in human granulosa-luteal cells. Biol Reprod. 57:1346–1353.[Abstract]
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