BIOLOGY OF REPRODUCTION 67, 837–846 (2002)
DOI 10.1095/biolreprod.102.003889
Ionic Currents Activated via Purinergic Receptors in the Cumulus Cell-Enclosed
Mouse Oocyte1
Rogelio O. Arellano,2,3 Ataulfo Martı́nez-Torres,4 and Edith Garay3
Centro de Neurobiologı́a,3 Universidad Nacional Autónoma de México, Querétaro 76230, México
Department of Neurobiology and Behavior,4 University of California, Irvine, California 92697
ABSTRACT
that form the follicle; an important network of communication is also provided by gap junctions between granulosa
cells and the oocyte [4, 5]. Communication via gap junction
channels between the oocyte and its closely associated
granulosa, a subgroup of cells named cumulus cells, is essential for the development of the gamete, as was shown
in early studies [6], and more recently by experiments in
which two of the more important follicular gap junction
proteins were knocked out [7, 8].
The variety of substances that might be involved in folliculogenesis is extensive and includes compounds in several categories such as peptides, neurotransmitters, and steroids. Functional membrane receptors for several of these
substances have been shown to exist in either granulosa
cells or in oocytes of mammals. However, particularly for
receptors to neurotransmitters, their molecular characteristics and the physiological consequences of their activation
have not been completely elucidated (e.g., [9, 10]). In other
species, membrane receptors have also been demonstrated
in distinct cellular components of the follicle. For example,
a bewildering number of functional membrane receptors
have been investigated in the membrane of the Xenopus
oocyte and in the follicular cells that surround it [11–14].
So far, stimulation of membrane receptors that respond to
acetylcholine, adenosine, or angiotensin II, and the consequent activation of ionic currents in Xenopus oocytes and
follicles have been implicated in the modulation of several
important physiological processes, such as oocyte maturation [11, 12, 15, 16]. Nonetheless, comparable studies of
mammalian oocytes and follicles have been lacking. Here,
we study the electrophysiological characteristics of cumulus cell-enclosed oocytes (CEOs) in the mouse and describe
ionic current responses elicited by either membrane depolarization or by activation of purinergic receptors. This
method may allow further studies of the intercellular electrical and chemical communication in CEOs and may help
to elucidate the mechanisms and molecular structures involved in these processes, which are important for gamete
development [17].
There is previous evidence that granulosa and luteal cells
of different species are endowed with purinergic receptors
[18–21], and it has been proposed that purines and their
derivatives might have important effects on the development of the oocyte [22–24]. Although an increase in intracellular calcium produced by purinergic agents has been
shown in various studies (e.g., [20, 21]), there is no information on the membrane electrical events that might be
produced. More importantly, it is not known whether purinergic receptors are present in cumulus cells or whether
their stimulation alters the characteristics of the oocyte
through gap junction communication, as has been shown in
Xenopus follicles [14, 25, 26]. Therefore, this study was
performed to investigate these questions using the two-electrode voltage clamp method in CEOs.
Several chemical signals synthesized in the ovary, including
neurotransmitters, have been proposed to serve as regulators of
folliculogenesis, however, their mechanisms of action have not
been completely elucidated. Here, electrophysiological and molecular biology techniques were used to study responses generated via purinergic stimulation in cultured mouse cumulus
cell-enclosed oocytes (CEOs). Application of extracellular ATP
elicited depolarizing responses in CEOs. Using the voltage
clamp technique by impaling oocytes with two microelectrodes,
we determined that these responses were mainly due to activation of two distinct ionic currents. The first corresponded to
the opening of Ca21-dependent Cl2 channels (ICl(Ca)) and the second to the opening of Ca21-independent channels that are permeable to Na1 (Ic1). The potency order for different nucleotides
(50 mM) was UTP . ATP . 2meS-ATP . ADP, and a,bme-ATP
and adenosine were found to be inactive. Suramin (100 mM)
blocked the response elicited by ATP or UTP. In addition, voltage
dependent K1 currents activated by depolarization of CEOs were
characterized. All CEO ionic currents recorded from the oocyte
were completely inhibited by octanol (1 mM), a gap junction
blocker. Thus, purinergic responses and K1 currents originate
mainly in the membrane of cumulus cells. Transcripts of the purinergic receptor P2Y2 subtype were amplified by polymerase
chain reaction from the cDNA of granulosa cells or cumulus
cells. This study shows that P2Y2 receptors are expressed in
CEOs, and that their stimulation opens at least two different
types of ion channels. Both the ion channels and the receptors
seemed to be located in the cumulus cells, which transmit their
corresponding electrical signals to the oocyte via gap junction
channels.
cumulus cells, follicular development, granulosa cells, neurotransmitters, signal transduction
INTRODUCTION
The growth, development, and maturation of the oocyte
in mammals is dependent on multiple factors. In addition
to the control exerted by the neuroendocrine system via
hormones, it is known that chemical signals that originate
in the follicle are essential, beginning with the early stages
of oocyte growth and development [1–3]. Moreover, signaling via diverse paracrine and autocrine substances are
not the only mechanisms of interaction between the cells
1
This work was supported by grants from CONACyT México (3713-PN,
32364-N) to R.O.A., and from The Grass Foundation to E.G.
2
Correspondence: Rogelio O. Arellano, Centro de Neurobiologı́a UNAM,
Domicilio Conocido Km. 15 Carretera QRO-SLP, Juriquilla Querétaro, CP
76230, México. FAX: 52442 238 1062;
e-mail: [email protected]
Received: 24 January 2002.
First decision: 5 March 2002.
Accepted: 4 April 2002.
Q 2002 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
837
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ARELLANO ET AL.
FIG. 1. Phase contrast of CEOs maintained in culture and depolarization elicited by ATP. A) CEOs maintained in culture
for 48 h. B) For electrophysiological recording, the CEO area was standardized
by removing excess cumulus cells. C) Vm
response to ATP (100 mM) recorded with a
microelectrode inserted into the oocyte. In
this and subsequent records the solid bars
at bottom or top indicate the time of drug
application. OO, Oocyte; ZP, zona pellucida; CC, cumulus cells.
MATERIALS AND METHODS
Reagents
L-15 medium, a-minimal essential medium (a-MEM), and their supplements (insulin, transferrin, penicillin, streptomycin, and fetal bovine
serum [FBS]) were all obtained from Gibco BRL (Gaithersburg, MD).
Follicle-stimulating hormone was from Calbiochem (La Jolla, CA). BAPTA-AM was purchased from Molecular Probes (Eugene, OR). ATP,
uridine-59-triphosphase (UTP), ADP, adenosine, a,b-methyleneadenosine
59-triphosphate [a,bme-ATP]), 2-methylthioadenosine 59-triphosphate
(2meS-ATP), suramin, 1-octanol, tetraethylammonium (TEA1) chloride,
Hepes, NMDG1, dimethyl sulfoxide (DMSO), and all other salts were
from Sigma Chemical Company (St. Louis, MO).
CEO Culture
Female C57Bl mice, 20–60 days of age and at random stages of the
estrous cycle, were killed by cervical dislocation, and the ovaries were
dissected through dorsolateral incisions. The ovaries were transferred to a
Petri dish containing L-15 medium supplemented with 5% FBS and were
cleaned of adjacent tissues under a microscope using fine forceps in order
to visualize the antral follicles. The antral follicles (8–15 per each ovary)
were isolated and transferred to a-MEM, in which each follicle was
opened using forceps, and the CEO was carefully removed from the rest
of the follicle cellular mass. Finally, using a pipette, individual CEOs were
placed on glass coverslips coated with poly-D-lysine (Fig. 1A). CEOs were
maintained in culture (378C, 5% CO2) in 200–250 ml of a-MEM supplemented with 1 mM sodium pyruvate, 10 mg/ml of bovine insulin, 10 mg/
ml of human transferrin, 100 ng/ml of porcine FSH, 100 U/ml of penicillin, 100 mg/ml of streptomycin, and 10% FBS [27].
Electrophysiological Techniques
After 1–5 days in culture, CEOs were studied electrically using both
intracellular recording of the membrane potential (Vm) and the two-microelectrode voltage clamp technique [28] in order to record the current (Im)
flux through the membrane of the entire CEO complex. Recording electrodes, made of borosilicate glass, had resistances of 10–15 MV when
used in membrane potential studies, or 2–3 MV when used for the voltage
clamp technique. Both types of electrodes were normally filled with 3 M
KCl, but some experiments were performed with electrodes that were filled
with 2 M K-acetate. With both recording techniques, an Axoclamp 2B
(Axon Instruments, Foster City, CA) amplifier was used, the bath was
grounded through silver electrodes using saline agar bridges, and the monitored signals were digitized and recorded using the Axon digidata 1200
converter for subsequent analysis.
At least 30 min before recording, the CEO area was standardized by
removing excess cumulus cells with a fine needle (Fig. 1B). This procedure permitted most of the CEO to be voltage clamped using parameters
that allowed a stable holding membrane potential to be reached in 1–1.5
msec during stepping protocols (see Figs. 5A and 8A) at the maximum
amplifier gain and with the microelectrode straight capacity fully compensated.
A CEO was placed in a chamber mounted on an inverted microscope
and continuously superfused at ;0.5 ml/min with Krebs solution (KS)
containing 150 mM NaCl, 6 mM KCl, 1.5 mM CaCl2, 2 mM MgCl2, 5
mM Hepes, and 4 mM glucose (adjusted to pH 7.4). For Vm recording,
oocytes (74–80 mm in diameter) were impaled with one electrode, whereas
the second microelectrode was used to impale different cumulus cells in
order to explore the isopotentiality of the preparation during responses
elicited by the agonists, or during current pulses injected via the electrode
located within the oocyte itself.
For the voltage clamp technique, the two electrodes were inserted into
the oocyte and were separated by a distance of 60–70 mm. The electrodes
were introduced at an angle greater than 808, and the bath fluid level in
the chamber was kept as low as possible (350–500 mm). Insertion of the
electrodes produced a sudden depolarization for several seconds, but in
most cases the impaled oocytes recovered their membrane potential to
steady and control values within 1–3 min. Considering the initial amplitude of purinergic and K1 currents of the CEOs described here, the coupling rate among cumulus cells and oocytes was not greatly affected by
electrode impaling even after several hours of recording. The common
procedure was to voltage clamp a CEO at a holding potential (Vh ) and
then record the ion currents that resulted when different reagents were
superfused, or when the membrane was hyperpolarized or depolarized by
steps of various intensities, or both. For instance, voltage dependent currents were elicited holding a CEO at 260 mV and then the potential was
briefly displaced in the 2100 to 160 mV range in steps of 110 mV (see
Fig. 5). To obtain information on ions that carried the currents generated
using this protocol, we measured the reversal potential (Vrev) of the tail
currents recorded on returning the Vh to different levels following a large
(150 or 160 mV) depolarization step. Similarly, current/voltage (I/V) relationships were built at different times (Fig. 6A) during purinergic stimulation in CEOs that were bathed in external solutions of various ionic
compositions, the Vh was varied from 140 to 2100 mV in steps (100–
150 msec) of 120 mV. The currents recorded during the response were
subtracted from the control current (before application of the reagent), and
the I/V relations were plotted as shown in Figures 6 and 7.
Agonists such as ATP, UTP, or ADP, as well as other reagents such as
1-octanol, suramin, or TEA1 were added to the KS from concentrated
stock solutions. In order to chelate any increase in intracellular calcium,
in some experiments CEOs were incubated for 5–10 h in a-MEM containing 10 mM 1,2-bis(2-aminophenoxy)-ethane-N,N,N9,N9,-tetra-acetic
acid tetrakis (acetoxymethyl ester) (BAPTA-AM) in 0.1% DMSO. Krebs
solutions containing different concentrations of K1 were prepared by substituting the corresponding amount of Na1 in the KS, and solutions with
different chloride concentrations were prepared by mixing the necessary
proportion of KS with a solution that had 11 mM Cl2, containing 75 mM
Na2SO4, 6 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 75 mM sucrose, 4
mM glucose, and 5 mM Hepes (adjusted to pH 7.4). External solutions
containing different concentrations of cations were prepared by mixing KS
with a solution in which all Na1 was substituted with N-methyl-D-glucamine (KS-NMDG1).
Reverse Transcription-Polymerase Chain Reaction
and Sequencing
Amplification of P2Y2 transcripts from total RNA purified from granulosa cells and cumulus cells was made using reverse transcription-polymerase chain reaction (RT-PCR). For this, using the guanidine isothiocyanate method [29], total RNA was isolated from granulosa or cumulus
cells collected from CEO preparations similar to those used for electrophysiological recording. First-strand cDNA was synthesized using reversetranscriptase, 1 mg of RNA, oligo(dT), and random hexamers as primers
in a total volume of 50 ml. The reverse-transcribed DNA (1 ml) was used
in a PCR using two pairs of oligonucleotides. The first were degenerate
oligonucleotide primers based on the sequence of transmembrane domains
III and VII of chick P2Y1 and murine P2Y2 with a forward sequence of
59-GCAGCATCCTC/GTTCCTCACC/GT-39, and a reverse sequence of
59-CCCA/T/GGCCAGGAAGTAGAGT/C/GAC/TC/GGG-39 [30]. The
second pair was designed specifically for the P2Y2 receptor subtype; the
forward sequence was 59-TGCTGGTGCTGGCCTGCCAGGCAC-39, the
reverse sequence was 59-GCCCTGCCAGGAAGTAGAGTACCG-39. The
PCR amplification conditions were 948C for 60 sec, 558C for 45 sec, and
ION CURRENTS IN CUMULUS CELL-ENCLOSED MOUSE OOCYTE
839
FIG. 2. Isopotential response to ATP in
CEOs. Depolarization caused in one CEO
by ATP (50 mM). The Vm was recorded simultaneously by one microelectrode inserted into the oocyte (Vm2) and another
inserted sequentially into a different cumulus cell (Vm1) around the oocyte (A–F; approximate position of the cumulus cell
electrode is indicated by the corresponding letters on the CEO image). Responses
generated by ATP were identical in both
cell types. The arrow in E indicates the
sudden depolarization produced by the
cumulus cell electrode coming out; note
that this was also detected by the oocyte
electrode.
728C for 60 sec for 30 cycles, followed by 5 min at 728C. If necessary,
PCR products were identified by Southern blot using a homologous probe,
or they were gel-isolated using a QIAquick PCR purification kit (Qiagen
Inc., Valencia, CA) and subcloned using the p-GEM-T vector system (Promega Co., Madison, WI). Sequences of the recombinant fragments were
determined on both strands by the dideoxy chain termination method.
RESULTS
Electrical Responses Elicited by ATP
and Isopotentiality of CEOs
CEOs (n 5 73) used in this study were 205 6 36 mm
in diameter (all data are given as means 6 SEM; Fig. 1B).
In KS, CEOs had a mean resting potential (Vm) of 253.6
6 8.4 mV when measured with a microelectrode that was
inserted either into the oocyte (n 5 59) (Fig. 1C) or into
one of the surrounding cumulus cells (n 5 46). The mean
CEO input resistance (Ro) measured under voltage clamp
(see below) was 2.5 6 0.38 MV. The Vm and Ro maintained
their regular control values for several hours (4–6 h) in
preparations superfused (0.5 ml/min) continuously with KS
at room temperature (23–268C). In these conditions, ATP
(5–100 mM) added to KS always produced a multiphasic
membrane depolarization ranging from 20 to 40 mV (23
CEOs from 9 mice; Fig. 1C). This depolarization was dosedependent (data not shown) and, in most cases, the initial
Vm recovered completely within 60–90 sec after washing
with KS. The ATP depolarization was regularly composed
of at least three phases: 1) an early spike-like response that
frequently presented the larger amplitude, 2) a slowly developing and inactivating smooth component, and 3) small
oscillations that superimposed on the previous components.
The relative proportion of the different phases varied greatly between CEOs from different donors, but were more
consistent among different CEOs dissected from the same
mouse. Such variability caused the shape of the overall purinergic responses to differ (compare Fig. 1C and Fig. 2).
It is well known that an oocyte and its surrounding cumulus cells are electrically coupled through gap junction
channels [4, 5, 31]. It is expected that if this coupling is
strong enough, a CEO would behave as an isopotential cellular aggregate, and would be amenable to recording using
the two-electrode voltage clamp technique, as has been
shown for other cell aggregates such as cardiomyocytes,
acinar cells, and Xenopus follicles [14, 32–36].
In order to test this possibility, we simultaneously monitored the Vm from the oocyte (Vm2) with one microelectrode and from one of the cumulus cells (Vm1) with a second microelectrode, usually by choosing a cumulus cell located at the edge of the cumulus (Fig. 2). During simultaneous Vm1 and Vm2 recordings, application of ATP (50–100
mM) elicited identical membrane depolarization changes in
cumulus cells and in the oocyte. This protocol was repeated
in seven CEOs in which four to eight different cumulus
cells surrounding each oocyte were recorded, and in each,
the same result was obtained. Although Vm1 recordings
from cumulus cells were less stable, they were rarely lost
during the short period needed to test a drug. When this
occurred, and Vm1 was lost (arrow in Fig. 2E), a fast, simultaneous depolarization was detected by Vm2. This fast
depolarization always recovered within a few seconds,
probably due to resealing of the cumulus cell membrane.
A similar set of experiments with multiple impaling of
oocytes and different cumulus cells was made to examine
the Vm changes produced when current pulses (2 sec) were
injected into the oocyte. The changes in membrane potential elicited in four to eight cumulus cells of a CEO (seven
CEOs in total) were plotted against the membrane potential
changes recorded in the oocyte itself. The ensuing relationship was well-fitted by a straight line with a slope of 0.93
6 0.4 (r 5 0.98) (data not shown). These results clearly
indicate that CEOs behave as a strongly electrically coupled
cellular aggregate. Therefore, the two-electrode voltage
clamp technique was used to study the characteristics of the
membrane currents underlying the depolarization response
evoked by ATP.
Ionic Currents Elicited by ATP
CEO voltage clamp was performed by inserting two microelectrodes into the oocyte. After the Vm was stable, the
CEO was clamped at a Vh of 260 mV. The membrane
potential was monitored continuously, and voltage steps (10
mV for 1–2 sec) were applied in order to monitor the CEO
membrane conductance (Fig. 3). Under voltage clamp, superfusion of CEOs with KS containing ATP (5–1000 mM)
elicited mainly inward ionic currents composed of several
phases. In general, these current components corresponded
in time-course and pharmacological characteristics with the
three components described above for the depolarization
responses. All three were associated with an increase in
membrane conductance and included 1) an early and fast,
rising-inward current that inactivated in seconds (;10 sec);
2) a slowly developing smooth current that inactivated in
several tens of seconds; and 3) oscillatory currents with
variable amplitudes that were usually superimposed on
components 1 and 2. The amplitude of the oscillatory currents was the most variable among different CEOs from
distinct donors, and sometimes this component was even
absent.
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ARELLANO ET AL.
these experiments, CEOs were superfused with solutions
containing increasing concentrations of ATP or UTP in KS
(0.1–1000 mM range) at 15-min intervals. Peak currents
were then normalized against the largest response obtained
with a supramaximal concentration of UTP (1 mM), averaged, and fit to the Hill equation (Fig. 4B). The half-maximal effective concentration for UTP was of 23 6 2 mM,
whereas that for ATP was of 59 6 3 mM. The maximal
current responses in this study usually corresponded to that
of the first component of the current. Further studies will
be necessary in order to determine a more precise efficacy
of different nucleotides on the distinct components of the
purinergic response.
Overall, these results support the idea that a P2Y receptor is involved in the generation of the ATP currents and,
given the sensitivity to UTP, these results also suggest that
the receptor subtype involved has strong similarities to the
P2Y2, P2Y4, or the P2Y6 subtypes.
K1 Conductance and Effective Membrane
Capacity in CEOs
FIG. 3. Purinergic current response of CEOs. Membrane current response elicited by ATP (50 mM), recordings obtained using the two-electrode voltage clamp technique. Traces show both the total CEO membrane current (Im) and Vh measured in the oocyte. CEOs were superfused
in KS and held at 260 mV; in this and subsequent records, periodic depolarizing steps of 10 mV (1–2 sec) were applied to monitor membrane
conductance. The two main current components are indicated with numbers.
Pharmacological Characteristics of Purinergic Receptors
The current response elicited by ATP was mimicked by
other purinergic substances and UTP. A series of agonists
were tested at 50 mM in CEOs (n 5 8) held at 260 mV.
The maximal current amplitude obtained with each agonist
was normalized with respect to the UTP current, which
elicited the largest responses. As shown in Figure 4A, the
descending order of potency was UTP . ATP . 2meSATP 5 ADP, whereas ADO and a,bme-ATP were ineffective. This suggested that the receptor mediating the CEO
current responses was related to P2-type purinergic receptors. In accord with this, the ATP and UTP (50 mM) currents were both blocked completely by 100 mM suramin,
an antagonist of P2-type receptors (n 5 6 for each agonist;
(Fig. 4, A and C).
Dose-response curves were constructed for the currents
elicited by ATP and UTP in CEOs (n 5 7) (Fig. 4B). For
FIG. 4. Pharmacology of CEO purinergic
responses. A) The columns show means of
the peak Im generated by different nucleotides (50 mM) in eight CEOs (four donors)
with or without suramin. B) Dose-response
relationships for Im activation in CEOs elicited by the agonists ATP (v) or UTP (V).
The peak Im elicited by each agonist concentration was normalized with respect to
the maximal response obtained with 1
mM UTP. Curves are the fit to Hill equation. C) The middle trace shows an example of the effect of suramin on the purinergic response generated by ATP (50
mM). The ATP responses obtained before
and after (top and bottom traces) application of the antagonist are also shown. All
CEOs were held at 260 mV.
Before studying the ionic basis of the CEO purinergic
responses, we carried out a brief study of voltage dependent
outward currents that were activated by membrane depolarization, and also of the capacitance currents associated
with voltage steps. As noted below, these results confirmed
that CEO currents recorded under voltage clamp were efficiently controlled.
As shown in Figure 5A, when Vh was made more positive than about 210 mV, a steady outward current began
to be generated. This outward current increased slowly after
a large capacitance current component, which was provoked by the onset of the voltage step, reached a peak
amplitude in about 100 msec at 210 mV, and remained
fairly steady for several hundred milliseconds. The rising
phase of the outward current was voltage dependent; thus,
for steps to 160 mV, the maximum peak amplitude was
reached in less than 25 msec. Leak currents activated were
subtracted (Fig. 5A, middle and bottom traces) using a p/4
protocol of prestimulation, and both total peak currents and
leak-subtracted currents were plotted as in Figure 5B,
showing that after the threshold at about 210 mV, the outward current amplitude increased almost linearly with Vh.
From such I/V relations, it is estimated that the CEO input
resistance (Ro) at rest was 2.5 6 0.38 MV, whereas during
the outward current it decreased to 660 6 180 KV (16
CEOs, 7 donors).
Similar to the responses elicited by ATP, the outward
ION CURRENTS IN CUMULUS CELL-ENCLOSED MOUSE OOCYTE
FIG. 5. Voltage dependent K1 currents of the CEO. A) A series of voltage
steps applied to a CEO from 160 to 2100 mV (Vh 260 mV), evoked leak
and outward ionic currents that did not inactivate (top traces, V). Long
transient capacitance current components were usually associated with
the on and off voltage steps. A similar series of voltage steps were applied
to a different CEO in KS (middle traces, m) or in KS containing 20 mM
TEA1 (bottom traces), where most of the voltage dependent outward currents were blocked, in these series the leak currents were subtracted using
a p/4 prestimulation protocol. B) Current-voltage (I/V) relationships for
both total Im (V) and the outward current after leak subtraction (m).
current was generated in most CEOs tested, and its amplitude of 178 6 58 nA at 160 mV (55 CEOs, 23 mice) was
independent of the time in culture. Two classic blockers of
K1 channels were effective in inhibiting the CEO outward
currents: TEA1 (20 mM) rapidly (;40 sec) blocked 91%
6 3.7% of the outward currents (15 CEOs, 6 donors) (bottom traces in Fig. 5A), whereas Ba21 (10 mM) blocked
about 47% after 15 min of continuous application (not
shown). The tail current Vrev in KS was 276.3 6 2.6 mV
(12 CEOs, 5 donors), and a similar Vrev value was found
for CEOs that were superfused in KS with 50% Cl2 (substituted with SO422), in KS in which all Na1 was substituted
with NMDG1, or in KS without divalent cations (see below; three CEOs in each solution). Overall, this suggested
that outward currents are carried mainly by K1 ions, and
this notion was confirmed by experiments in which the concentration of K1 in the KS was decreased to 3 mM, or
increased to 20 mM by substituting Na1. This shifted the
841
tail current Vrev to about 292 and 250 mV, respectively (7
CEOs, 3 donors); the relation between Vrev and the logarithm of the extracellular K1 concentration fit well to a line
with a slope of 256 mV (r 5 0.96).
Further analysis of the voltage dependent K1 current indicated that 1) it was not dependent on extracellular or intracellular Ca21, and 2) K1 currents were completely inhibited by 0.5–1.5 mM octanol (4 CEOs, 2 donors; see Fig.
8A), a drug that closes gap junction channels [37]. In all
cases, the inhibitory effect of octanol on the voltage dependent K1 currents was reversible after washing with KS
for 4–6 min.
The amplitude of the voltage dependent K1 currents provide a way to assess the electrical coupling between the
cells in a given CEO, because its amplitude is proportional
to the number of cells and their coupling. This was also
studied in more detail by analyzing the capacitance current
component that occurs in the on and off voltage steps [38,
39]. For this, the integral of the capacitance currents (Q)
was estimated from steps in the 220 to 120 mV range,
and the slope of the Q/V relation was calculated by linear
regression, giving an effective CEO membrane capacity of
6.4 6 0.35 nF. Assuming a typical value of 1 mF/cm2 for
the specific capacitance of the membrane, we calculated
that CEOs were coupled to about 2500 cumulus cells. Also,
from the peak capacitance current at the onset of the small
voltage steps [32], we estimated that the series resistance
(Rs) that developed between the CEO and the ground electrode had values in the 70–110 KV range.
Ionic Basis of the Purinergic Current Response in CEOs
Experiments were subsequently aimed at determining
the ionic basis of the two main components of the purinergic activated currents. From I/V relations constructed
during the purinergic response it was clear that the Vrev for
component 1 was always more negative than that of component 2 (Fig. 6A). In fact, the Vrev of the current became
progressively more positive as component 1 faded. In KS
the Vrev of current components 1 and 2, measured at their
peak, were 229 6 6 mV and 17 6 4 mV, respectively (28
CEOs, 8 donors), and no difference was found between the
Vrev of currents elicited by ATP or UTP. This difference
was maintained in responses elicited by ATP in KS con-
FIG. 6. I/V relationship of the purinergic response. A) I/V relationship for the current elicited by 100 mM ATP in KS. A series of voltage steps from
140 to 2100 mV (Vh 260 mV) was applied at control conditions and at different times during the purinergic response as indicated by symbols in the
trace and that corresponded to component 1 (V) and component 2 (□, n, v). Each data point represents the mean of three CEOs from the same ovary
(SEM was within the symbol size). Similar results were obtained from 28 CEOs from 8 donors. B) Currents elicited by ATP in a CEO held at different
Vh as indicated in each trace. At 240 and 260 mV only inward currents were generated, whereas at 210 and 220 mV, currents were composed of
early outward currents (component 1) and slow inward currents (component 2). At these potentials, during wash in KS, an outward current increased
slowly, but this was not further characterized.
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ARELLANO ET AL.
FIG. 7. Ionic basis for purinergic current components 1 and 2 and dependency on intracellular Ca21. A) I/V relationships for component 1 of the
current elicited by ATP (100 mM) in solutions containing three different concentrations of Cl2 (replaced by SO422), 170 mM (v), 85 mM (□), and 10
mM (n). B) I/V relationships for component 2 elicited by 100 mM ATP in solutions containing three different concentrations of Na1 (replaced by
NMDG1), 150 mM (v), 75 mM (n), and 37.5 mM (□). Data in A and B are means of three CEOs in each condition. C) Response elicited by ATP in
the same CEO in KS (top trace, numbers indicate current components) or in KS-NMDG1 (middle trace). Component 2 was eliminated in KS-NMDG1.
The bottom trace shows the response to ATP in one CEO (same mouse) incubated in medium containing BAPTA-AM. In this case, component 2 was
not affected, whereas component 1 disappeared. All CEOs were held at 260 mV.
taining 20 mM TEA1 to block the voltage dependent K1
channels (5 CEOs, 3 donors). Further demonstration that
components 1 and 2 had distinct Vrev was obtained by recording ATP currents (Fig. 6B) or UTP currents while holding the potential between 210 mV and 220 mV, in which
component 1 became outward, while component 2 remained inward (6 CEOs, 3 donors). This strongly suggested
that the ionic basis of the main components of the purinergic responses involved at least two different pathways.
Current-voltage relations were constructed for purinergic
responses in CEOs bathed in solutions with low Cl2 concentration (Fig. 7A). Only the Vrev values for component 1
shifted notably in solutions containing 50% and 6% normal
Cl2 to 122 6 8 mV and 156 6 3 mV, respectively, and
when plotted against the external Cl2 concentration, the relation fit well with the Nernst equation prediction for Cl2
ions with a slope of 60 mV (r 5 0.98). Thus, these results
indicate that component 1 is a current that flows through
ionic channels that are permeable mainly to that anion.
Given that inward currents with a Vrev close to 0 mV, or
that more positive currents such as those for component 2,
can be driven by a mixture of cations (Na1, Ca21, and K1),
we performed experiments in which all Na1 in KS was
substituted with NMDG1, a large, nonpermeable cation. As
shown in the middle trace of Figure 7C, this abolished component 2, which had been elicited by ATP (or UTP), whereas component 1 was unaffected. The Vrev of the later component measured in CEO bathed in KS-NMDG1 was 226
6 4 mV (6 CEOs, 3 mice). Moreover, the voltage dependent K1 current was not altered in the KS-NMDG1 (not
shown), strongly suggesting that inhibition of component 2
was not due to an unspecific effect of NMDG1. As shown
in Figure 7B, in KS containing 50% or 25% Na1, the amplitude of component 2 decreased, and Vrev shifted to 12
6 3 mV and 25 6 2 mV, suggesting that Na1 is a main
carrier of current (7 CEOs for each concentration of
NMDG1, 4 donors). However, these experiments did not
rule out the possible participation of other cations in this
component and, therefore, we named it Ic1 until we obtain
further experimental evidence.
Thus, all these results indicated that the current responses elicited via stimulation of P2Y receptors involved ions
moving through at least two different channels that mediate
the two major components of the response. One of the
channels was permeable mainly to Cl2, whereas the second
had a high permeability for Na1.
Purinergic Activated Currents and Ca21
Several subtypes of purinergic P2Y receptors generate
an increase in intracellular calcium either via activation of
G proteins and enzymes that synthesize intracellular messengers such as IP3, or by influx of calcium from the extracellular medium passing through ionic channels [40].
However, large current responses, similar to controls,
were still generated in CEOs bathed in KS with no calcium
added (9 CEOs, 5 donors). A similar result was obtained
in four CEOs (two mice) superfused with medium containing high Ca21 (20 mM, not shown). In both cases, the Vrev
of the purinergic currents behaved like those described in
KS (i.e., component 1 always had a Vrev more negative than
that for Ic1). These results indicated that activation of purinergic currents were not dependent on extracellular calcium.
In contrast, Figure 7C shows an example in which current responses elicited by ATP were recorded in a control
CEO (top trace) and in one CEO loaded with BAPTA-AM
(bottom trace) from the same donor. Notice that in the latter
condition, the first component carried by Cl2 was completely abolished, while component Ic1 was still fully activated
(13 CEOs, 5 donors). These results strongly suggested that
component 1 was dependent on an intracellular Ca21 increase, whereas Ic1 was independent, and further suggested
that the membrane mechanisms elicited via the P2Y receptor were also diverse. Based on its Ca21 dependence and
its permeability to chloride, component 1 will be named
ICl(Ca).
Effects of Cellular Uncoupling on CEO Ionic Currents
As suggested by the measurements of both Vm and Im,
CEO cellular components maintain a strong electrical coupling. There is ample evidence that the molecular basis of
this electrical communication is the existence of gap junction channels between the granulosa cells, which comprise
ION CURRENTS IN CUMULUS CELL-ENCLOSED MOUSE OOCYTE
FIG. 8. Effect of octanol on CEO ionic currents. A) A series of voltage
steps from 140 to 2100 mV (220 mV steps) was applied to a CEO in
KS (top superimposed traces) and after 10 min in KS supplemented with
1 mM octanol (bottom traces). B) Effect of 1 mM octanol (10 min) on the
ionic response generated by 100 mM ATP. The top trace illustrates the
control current, then the same CEO was superfused with solution containing octanol and ATP was reapplied (bottom trace).
the cumulus, and between those cells and the oocyte. To
investigate whether electrical coupling via gap junctions is
absolutely necessary in order to generate purinergic and K1
currents, recordings were made in CEOs exposed to octanol. Figure 8A shows an example of the inhibitory effect
produced by octanol (1 mM, 0.1% DMSO) on the voltage
dependent K1 currents and the capacitance transients generated by voltage steps in the 140 to 2100 mV range. The
octanol-induced inhibition was associated with an increase
in the input resistance of the CEO from a control value of
2.1 6 0.4 MV to 32 6 7 MV (9 CEOs, 4 donors). These
data supported the idea that octanol uncoupled the cells in
the CEO, an effect that was reversible when the CEO was
washed for about 3 min with KS. Furthermore, application
of ATP or UTP (100 mM) during the octanol-elicited uncoupled condition did not produce electrical responses in
the CEO (7 CEOs, 2 donors), whereas the same CEO in
KS generated ICl(Ca) and Ic1 currents of 87 6 36 nA and
69 6 13 nA, respectively (Fig. 8B). Thus, it is clear that
the oocyte did not contain all the necessary elements to
generate purinergic responses of the CEO.
Amplification of P2Y2 Receptor Subtype Transcripts
in Cumulus and Granulosa Cells
Some pharmacological characteristics of the purinergic
receptor involved in the CEO responses, such as the sensitivity to UTP and that ICl(Ca) was activated through a
Ca21-dependent mechanism, implicated P2Y2, P2Y4, or
P2Y6 as the receptor subtype involved. Therefore, to determine whether a receptor of any of these subtypes might
be expressed in the CEO, we first examined the possibility
that transcripts for P2Y receptors were expressed in cumulus and granulosa cells.
Degenerated oligonucleotide primers based on the sequences encoding the highly conserved transmembrane domains III and VII of the P2Y receptors were used to carry
out RT-PCR to amplify the corresponding sequences from
cDNA made from the RNA extracted from granulosa cells
grown in culture, and from cumulus cells obtained from
843
FIG. 9. RT-PCR analysis of P2Y2 receptor RNA in granulosa and cumulus
cells. A) Ethidium bromide-stained gel of RT-PCR amplified products using
P2Y2 specific primers and cDNA from granulosa cell total RNA. Line M
contains DNA markers (in base pairs), line 1 is the product of amplification, and line 2 is the negative control (without reverse transcriptase).
B) Top figure illustrates the ethidium bromide-stained gel of RT-PCR amplified products in similar reaction conditions as those in A from cumulus
cell total RNA. Line 1 shows the amplified product, and line 2 is the
control without reverse transcriptase. The bottom figure illustrates the
Southern blot of the same top gel hybridized with a specific fragment for
P2Y2 subtype receptors as probe.
CEO preparations similar to those used for electrophysiological studies.
A band of about 500 base pairs containing the presumed
sequences of P2Y receptors was amplified in both cellular
preparations and excised from the gel. After elution and
subcloning the PCR fragments into the pGEM-T vector, 25
independent clones were subjected to sequence analysis.
Analysis showed that the products encoded a P2Y2 receptor
subtype that was identical to that reported previously for
the P2Y2 receptor in mice [41]. To verify this result, P2Y2
specific primers were used in PCR analysis, and a product
was obtained with the expected size of 445 base pairs in
both granulosa and cumulus cells (Fig. 9, A and B).
DISCUSSION
Voltage Clamp Analysis of CEOs
We have shown that activation of a purinergic receptor
of the P2Y2 subtype in the cumulus cell-enclosed oocyte
of mice elicits two types of currents; one Ca21-dependent
and driven by chloride ions (ICl(Ca)), and the other carried
by cations and not dependent on intracellular Ca21 (Ic1).
Our results suggest that the receptors, the activated membrane mechanisms, and probably the ionic channels, are
located in the cumulus cells, and that the ensuing membrane currents are transmitted to the oocyte via gap junction
intercellular communication.
The current responses and the general electrical characteristics of the CEO were monitored using the two-electrode voltage clamp technique, as has been used in other
cellular aggregates. This method takes advantage of the fact
that some cells, like those composing the CEO, are strongly
electrically coupled via gap junction channels. The electrical coupling makes the spherical aggregates behave as an
isopotential complex, and allows a current generated at one
point to spread over the entire aggregate to control its membrane potential in a manner comparable to a voltage clamp
in giant spherical cells [42]. In this respect, several specific
characteristics of the mouse CEO must be considered before any conclusion can be drawn from the studies presented here.
The first important consideration is our demonstration
that the cumulus cells and the oocyte of a CEO maintained
in culture were strongly electrically coupled, and in control
conditions, had an isopotential behavior. It is also important
844
ARELLANO ET AL.
that this isopotentiality was maintained during and after the
Vm changes produced by applying purinergic agonists, suggesting that receptor activation does not strongly affect the
electrical coupling between the cells. A second important
consideration is the possibility of a spatial inhomogeneity
that might occur in these aggregates; for example, when the
current source was a point located in the oocyte, or due to
the existence of a finite Rs (see below). This and specific
characteristics of the aggregate, such as an hypothetical
space constant shorter than the diameter of the CEO, might
give rise to areas with distinct Vm. In order to reduce this
possibility, an important step in the preparation of the CEO
for voltage clamp recording was the appropriate delimitation of their area. In normal culture conditions the CEO
tended to extend on the slide by hundreds of micrometers,
which affects the efficacy of the clamp. This did not permit
the voltage to be clamped with the maximal gain available,
and it produced unstable recordings by eliciting oscillations
at the on and off voltage steps. Thus, all CEOs in our experiments were restricted to approximately three times the
diameter of their enclosed oocytes. Under these conditions,
current pulses injected into the oocyte produced similar potential changes in the cumulus cells located at different regions around the oocyte, thus suggesting that a point current source within the oocyte produced isopotential changes
in the entire complex. From the effective membrane capacity, we calculated that, on average, 2500 cumulus cells were
coupled to the oocyte. Estimates, considering an oocyte of
80 mm and cumulus cells of 10 mm in diameter, indicate
that in general, CEOs were composed of a similar number
of cumulus cells. This information indicates that the recording technique used reflects closely the total membrane
area of the CEO.
A last important consideration is the efficacy of the
clamp in the voltage range we used. It is known that Rs
prevents a proper measurement of the potential directly
across the cell membranes in such a way that Vm deviates
from Vh by the ratio of membrane resistance (Rm) to total
input resistance (i.e., Rm 1 Rs; see for example [32]), and
when Rm k Rs, Vm is close to Vh. Thus, at rest, when the
CEO input resistance was about 2.5 MV, the estimated Rs
of 100 KV will cause Vm to differ from the holding potential by about 4%; and at the peak of large currents, such as
the voltage dependent K1 current in which the input resistance decreased to 600 KV, the Vm might have differed by
about 17%. Taking this into account, the Vrev values of the
currents described here may have an error factor, but in
general, the results of the ion substitution studies will not
be distorted. This is supported by a similar behavior of Vrev
during the purinergic response in the presence of TEA1,
which effectively blocked the K1 currents, thus increasing
Rm at positive potentials, and decreasing the error factor
introduced by Rs. It is also supported by the biphasic membrane current detected at Vh (220 mV) near the Vrev for
ICl(Ca), in which this current developed as an early and transient outward component followed by an inward current
corresponding with Ic1. It is expected that at these potentials, the ATP-elicited Rm decrease would produce smaller
currents that have less effect on the relation between Rm
and the total input resistance. Finally, the Vrev values obtained from ion substitution studies for K1 and Cl2 currents
were close to those predicted by the Nernst equation.
Thus, it seems clear that a more detailed description of
the electrical parameters of the CEO during a two-electrode
voltage clamp will help to clarify specific aspects of the
behavior of the cellular complex under different experi-
mental conditions. This, together with additional studies to
confirm the electrical characteristics of isolated cumulus
cells, will certainly help to establish the relatively easy
whole-CEO voltage clamp method as a practical way to
investigate the physiological and pharmacological effects
on intercellular communication that may be produced by
substances involved in the development of the follicle.
Studies on their mechanisms of action also become amenable, thus helping to define the identity of participating
membrane proteins.
Purinergic Responses in CEOs
Previous evidence suggests that purines and their metabolism might be important in different aspects of folliculogenesis, particularly during oocyte maturation. Also, purinergic receptors have been detected using different approaches in granulosa cells from various species; for example, by measuring Ca21 increases in granulosa cells of
humans, pigs, and chickens [18, 19], and by recording
membrane currents elicited by several purinergic agonists
in frog follicles [14, 15, 25, 26]. Here we showed that the
cumulus cell-enclosed mouse oocyte responds electrically
to different purinergic agonists, primarily to ATP and UTP.
This response was probably elicited via stimulation of a
P2Y2 receptor subtype, as defined by the pharmacological
profile of the response and the presence in both granulosa
and cumulus cells of transcripts coding for the P2Y2 receptor. The depolarization produced by stimulation of the P2Y2
receptors affects the entire CEO, indicating that physiological actions elicited via this pathway affect both the cumulus cells and the oocyte. The effects on the oocyte might
include not only direct Vm changes, but may also include
diffusion to the oocyte of second messengers produced by
the cumulus cells in a manner similar to that shown for the
Xenopus follicle [16]. For example, evidence exists that in
sheep, CEO stimulation of LH receptors, which are located
only in the membrane of cumulus cells, produces an increase in the intracellular calcium concentration in these
cells and provokes a similar increase in the companion oocyte, an increase that is dependent on the electrical coupling
of the complex [43]. In the CEOs of mice, the ICl(Ca) response generated by UTP or ATP was dependent on an
increase in intracellular calcium, and it may be that, similar
to what happens in other cellular systems, activation of the
P2Y2 receptor in the CEO leads to an increase in intracellular Ca21 and activates the chloride channels involved. The
precise mechanism of this calcium increase remains to be
elucidated, however, it seems likely that it occurs through
IP3 synthesis because the P2Y2 receptor subtype couples
efficiently to phospholipase C in several cell types, both in
native form and in those in which the cloned receptors have
been transfected [40]. It is also possible that not all the
response elements are located in cumulus cells, and that
second messengers diffuse from the cumulus cells via gap
junctions, and then activate ionic channels in the oocyte
membrane, in this way generating at least part of the response described.
The second phase of the purinergic response, the Ic1, was
clearly calcium-independent, but its pharmacological characteristics strongly suggested that the P2Y2 receptor was
also responsible for activation of this component. Thus, it
might be that Ic1 channels are activated via the associated
pathway of the phospholipase C cascade (i.e., synthesis of
diacylglycerol), or alternatively, via another membrane
ION CURRENTS IN CUMULUS CELL-ENCLOSED MOUSE OOCYTE
845
mechanism activated in parallel such as stimulation of
phospholipase D or A (see for example [44]).
might be important paracrine signals in the physiological
regulation of CEO growth and maturation in mice.
Electrical Characteristics of Mammalian Granulosa Cells
and Oocytes
ACKNOWLEDGMENTS
Electrical activity of granulosa cells and oocytes from
mammals has been reported in several studies. For example, Ca21, K1, and Cl2 channels have been shown in granulosa cells from different species. A delayed rectifier K1
channel has been described in avian and porcine granulosa
cells [45–47]. Their properties seem to be similar to those
of the voltage dependent K1 current studied here (e.g., they
all have a threshold around 220 to 210 mV, they do not
present inactivation, and are blocked by extracellular
TEA1). The strong inhibition of K1 currents by octanol
indicates that the channels involved are located in the cumulus cell membrane. Thus, K1 channels in these cells
seem to play a central role in also controlling the membrane
potential of the oocyte. This is supported by the observation
that denuded oocytes have a lower Vm (215 to 238 mV)
[48, 49].
In contrast to what has been shown for porcine granulosa
cells stimulated by LH [50], the delayed rectifier K1 channel in mouse cumulus cells was not affected by extracellular or intracellular changes in Ca21, and stimulation of
P2Y2 receptors by ATP or UTP had little or no effect on
this K1 current. However, further experiments after minimizing the purinergic responses, ICl(Ca) and Ic1, may allow
the detection of small effects of purinergic substances on
the voltage dependent K1 current. Even so, it is already
clear that the depolarizing responses produced by purinergic agents in the mouse CEO were mainly due to the activation of ICl(Ca) and Ic1.
Calcium-dependent Cl2 channels have also been described in avian granulosa cells [51], but there is no previous information about their possible activation by the
Ca21 released via stimulation of membrane receptors. However, neither the presence of cationic channels nor their activation via membrane receptors had been shown previously
in granulosa cells, and it will be interesting to know whether such channels are present in cumulus cells from other
species.
It seems clear that similar molecular components are located in the membrane of granulosa cells from different
species. We show here that macromolecules such as P2Y2
receptors, K1 channels, and Ca21-dependent Cl2 channels
are present in the cumulus cells that surround the oocytes
of mice, and their expression in other species suggests conserved roles in the physiology of ovarian systems. Activation of the purinergic receptors in the mouse CEO leads to
activation of ICl(Ca) and Ic1, but the physiological significance of these effects for cumulus cells and the oocyte remains to be elucidated. It is possible that depolarization
regulates the secretion of substances such as steroids that
might act as paracrine or autocrine regulators. However, it
is also expected that cellular processes activated by purinergic stimulation, and consequent release of intracellular
Ca21 might have important effects on the metabolism of
both the cumulus cell and oocyte compartments. It is important to mention that steroidogenesis, a principal functional role of granulosa cells, is affected by changes in the
levels of both intracellular Ca21 and Cl2 ions [52, 53]. Also,
it has been shown that intracellular Ca21 has a central role
in the regulation of important processes in the oocyte [54].
Thus, ATP and UTP acting via P2Y2 purinergic receptors
We are deeply indebted to Professor Ricardo Miledi F.R.S. for the
many discussions that originated the background of important aspects of
this study and for his constant advice and encouragement. We are grateful
to Dr. Mauricio Dı́az and Dr. Dorothy D. Pless for critical comments and
help with the manuscript, and to Lic. Marı́a del Pilar Galárza Barrios, to
M.V.Z. Martin Garcia Servin, Ing. Alberto Lara, and Mr. Horacio Ramı́rez
Leyva for technical assistance.
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