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Biology of Reproduction 67, 837-846 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Ionic Currents Activated via Purinergic Receptors in the Cumulus Cell-Enclosed Mouse Oocyte1

Rogelio O. Arellano2,,a, Ataulfo Martínez-Torresb, and Edith Garaya

a Centro de Neurobiología, Universidad Nacional Autónoma de México, Querétaro 76230, México b Department of Neurobiology and Behavior, University of California, Irvine, California 92697


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 Ca2+-dependent Cl- channels (ICl(Ca)) and the second to the opening of Ca2+-independent channels that are permeable to Na+ (Ic+). The potency order for different nucleotides (50 µM) was UTP > ATP > 2meS-ATP > ADP, and {alpha},ßme-ATP and adenosine were found to be inactive. Suramin (100 µM) blocked the response elicited by ATP or UTP. In addition, voltage dependent K+ 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 K+ 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
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents

L-15 medium, {alpha}-minimal essential medium ({alpha}-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-5'-triphosphase (UTP), ADP, adenosine, {alpha},ß-methyleneadenosine 5'-triphosphate [{alpha},ßme-ATP]), 2-methylthioadenosine 5'-triphosphate (2meS-ATP), suramin, 1-octanol, tetraethylammonium (TEA+) chloride, Hepes, NMDG+, 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 {alpha}-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 (37°C, 5% CO2) in 200–250 µl of {alpha}-MEM supplemented with 1 mM sodium pyruvate, 10 µg/ml of bovine insulin, 10 µg/ml of human transferrin, 100 ng/ml of porcine FSH, 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 10% FBS [27].



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  		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 µM) 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

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 M{Omega} when used in membrane potential studies, or 2–3 M{Omega} 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.



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  		FIG. 5. Voltage dependent K+ currents of the CEO. A) A series of voltage steps applied to a CEO from +60 to -100 mV (Vh -60 mV), evoked leak and outward ionic currents that did not inactivate (top traces, {circ}). 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, {block}) or in KS containing 20 mM TEA+ (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 ({circ}) and the outward current after leak subtraction ({block})



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  		FIG. 8. Effect of octanol on CEO ionic currents. A) A series of voltage steps from +40 to -100 mV (-20 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 µM ATP. The top trace illustrates the control current, then the same CEO was superfused with solution containing octanol and ATP was reapplied (bottom trace)

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 µm 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 µm. The electrodes were introduced at an angle greater than 80°, and the bath fluid level in the chamber was kept as low as possible (350–500 µm). 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 K+ 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 -60 mV and then the potential was briefly displaced in the -100 to +60 mV range in steps of +10 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 (+50 or +60 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 +40 to -100 mV in steps (100–150 msec) of +20 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.



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  		FIG. 6. I/V relationship of the purinergic response. A) I/V relationship for the current elicited by 100 µM ATP in KS. A series of voltage steps from +40 to -100 mV (Vh -60 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 ({circ}) and component 2 ({square}, {bigtriangleup}, ). 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 -40 and -60 mV only inward currents were generated, whereas at -10 and -20 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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  		FIG. 7. Ionic basis for purinergic current components 1 and 2 and dependency on intracellular Ca2+. A) I/V relationships for component 1 of the current elicited by ATP (100 µM) in solutions containing three different concentrations of Cl- (replaced by SO42-), 170 mM (), 85 mM ({square}), and 10 mM ({bigtriangleup}). B) I/V relationships for component 2 elicited by 100 µM ATP in solutions containing three different concentrations of Na+ (replaced by NMDG+), 150 mM (), 75 mM ({bigtriangleup}), and 37.5 mM ({square}). 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-NMDG+ (middle trace). Component 2 was eliminated in KS-NMDG+. 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 -60 mV

Agonists such as ATP, UTP, or ADP, as well as other reagents such as 1-octanol, suramin, or TEA+ 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 {alpha}-MEM containing 10 µM 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N',-tetra-acetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM) in 0.1% DMSO. Krebs solutions containing different concentrations of K+ were prepared by substituting the corresponding amount of Na+ 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 Cl-, 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 Na+ was substituted with N-methyl-D-glucamine (KS-NMDG+).

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 reverse-transcriptase, 1 µg of RNA, oligo(dT), and random hexamers as primers in a total volume of 50 µl. The reverse-transcribed DNA (1 µl) 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 5'-GCAGCATCCTC/GTTCCTCACC/GT-3', and a reverse sequence of 5'-CCCA/T/GGCCAGGAAGTAGAGT/C/GAC/TC/GGG-3' [30]. The second pair was designed specifically for the P2Y2 receptor subtype; the forward sequence was 5'-TGCTGGTGCTGGCCTGCCAGGCAC-3', the reverse sequence was 5'-GCCCTGCCAGGAAGTAGAGTACCG-3'. The PCR amplification conditions were 94°C for 60 sec, 55°C for 45 sec, and 72°C for 60 sec for 30 cycles, followed by 5 min at 72°C. 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
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Electrical Responses Elicited by ATP and Isopotentiality of CEOs

CEOs (n = 73) used in this study were 205 ± 36 µm in diameter (all data are given as means ± SEM; Fig. 1B). In KS, CEOs had a mean resting potential (Vm) of -53.6 ± 8.4 mV when measured with a microelectrode that was inserted either into the oocyte (n = 59) (Fig. 1C) or into one of the surrounding cumulus cells (n = 46). The mean CEO input resistance (Ro) measured under voltage clamp (see below) was 2.5 ± 0.38 M{Omega}. 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 (23–26°C). In these conditions, ATP (5–100 µM) 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 dose-dependent (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).



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  		FIG. 2. Isopotential response to ATP in CEOs. Depolarization caused in one CEO by ATP (50 µM). 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

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 µM) 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 ± 0.4 (r = 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 -60 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 µM) 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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  		FIG. 3. Purinergic current response of CEOs. Membrane current response elicited by ATP (50 µM), 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 -60 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 µM in CEOs (n = 8) held at -60 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 > 2meS-ATP = ADP, whereas ADO and {alpha},ßme-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 µM) currents were both blocked completely by 100 µM suramin, an antagonist of P2-type receptors (n = 6 for each agonist; (Fig. 4, A and C).



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  		FIG. 4. Pharmacology of CEO purinergic responses. A) The columns show means of the peak Im generated by different nucleotides (50 µM) in eight CEOs (four donors) with or without suramin. B) Dose-response relationships for Im activation in CEOs elicited by the agonists ATP () or UTP ({circ}). 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 µM). The ATP responses obtained before and after (top and bottom traces) application of the antagonist are also shown. All CEOs were held at -60 mV

Dose-response curves were constructed for the currents elicited by ATP and UTP in CEOs (n = 7) (Fig. 4B). For these experiments, CEOs were superfused with solutions containing increasing concentrations of ATP or UTP in KS (0.1–1000 µM 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 ± 2 µM, whereas that for ATP was of 59 ± 3 µM. 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.

K+ Conductance and Effective Membrane Capacity in CEOs

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 -10 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 -10 mV, and remained fairly steady for several hundred milliseconds. The rising phase of the outward current was voltage dependent; thus, for steps to +60 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 -10 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 ± 0.38 M{Omega}, whereas during the outward current it decreased to 660 ± 180 K{Omega} (16 CEOs, 7 donors).

Similar to the responses elicited by ATP, the outward current was generated in most CEOs tested, and its amplitude of 178 ± 58 nA at +60 mV (55 CEOs, 23 mice) was independent of the time in culture. Two classic blockers of K+ channels were effective in inhibiting the CEO outward currents: TEA+ (20 mM) rapidly (~40 sec) blocked 91% ± 3.7% of the outward currents (15 CEOs, 6 donors) (bottom traces in Fig. 5A), whereas Ba2+ (10 mM) blocked about 47% after 15 min of continuous application (not shown). The tail current Vrev in KS was -76.3 ± 2.6 mV (12 CEOs, 5 donors), and a similar Vrev value was found for CEOs that were superfused in KS with 50% Cl- (substituted with SO4-2), in KS in which all Na+ was substituted with NMDG+, or in KS without divalent cations (see below; three CEOs in each solution). Overall, this suggested that outward currents are carried mainly by K+ ions, and this notion was confirmed by experiments in which the concentration of K+ in the KS was decreased to 3 mM, or increased to 20 mM by substituting Na+. This shifted the tail current Vrev to about -92 and -50 mV, respectively (7 CEOs, 3 donors); the relation between Vrev and the logarithm of the extracellular K+ concentration fit well to a line with a slope of -56 mV (r = 0.96).

Further analysis of the voltage dependent K+ current indicated that 1) it was not dependent on extracellular or intracellular Ca2+, and 2) K+ 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 K+ currents was reversible after washing with KS for 4–6 min.

The amplitude of the voltage dependent K+ 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 -20 to +20 mV range, and the slope of the Q/V relation was calculated by linear regression, giving an effective CEO membrane capacity of 6.4 ± 0.35 nF. Assuming a typical value of 1 µF/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 K{Omega} 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 -29 ± 6 mV and +7 ± 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 containing 20 mM TEA+ to block the voltage dependent K+ 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 -10 mV and -20 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 Cl- concentration (Fig. 7A). Only the Vrev values for component 1 shifted notably in solutions containing 50% and 6% normal Cl- to +22 ± 8 mV and +56 ± 3 mV, respectively, and when plotted against the external Cl- concentration, the relation fit well with the Nernst equation prediction for Cl- ions with a slope of 60 mV (r = 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 (Na+, Ca2+, and K+), we performed experiments in which all Na+ in KS was substituted with NMDG+, 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-NMDG+ was -26 ± 4 mV (6 CEOs, 3 mice). Moreover, the voltage dependent K+ current was not altered in the KS-NMDG+ (not shown), strongly suggesting that inhibition of component 2 was not due to an unspecific effect of NMDG+. As shown in Figure 7B, in KS containing 50% or 25% Na+, the amplitude of component 2 decreased, and Vrev shifted to +2 ± 3 mV and -5 ± 2 mV, suggesting that Na+ is a main carrier of current (7 CEOs for each concentration of NMDG+, 4 donors). However, these experiments did not rule out the possible participation of other cations in this component and, therefore, we named it Ic+ 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 Cl-, whereas the second had a high permeability for Na+.

Purinergic Activated Currents and Ca2+

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 Ca2+ (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 Ic+). 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 Cl- was completely abolished, while component Ic+ was still fully activated (13 CEOs, 5 donors). These results strongly suggested that component 1 was dependent on an intracellular Ca2+ increase, whereas Ic+ was independent, and further suggested that the membrane mechanisms elicited via the P2Y receptor were also diverse. Based on its Ca2+ 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 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 K+ 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 K+ currents and the capacitance transients generated by voltage steps in the +40 to -100 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 ± 0.4 M{Omega} to 32 ± 7 M{Omega} (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 µM) 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 Ic+ currents of 87 ± 36 nA and 69 ± 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 Ca2+-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 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).



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  		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


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
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 Ca2+-dependent and driven by chloride ions (ICl(Ca)), and the other carried by cations and not dependent on intracellular Ca2+ (Ic+). 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 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 µm and cumulus cells of 10 µm 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 + Rs; see for example [32]), and when Rm >> Rs, Vm is close to Vh. Thus, at rest, when the CEO input resistance was about 2.5 M{Omega}, the estimated Rs of 100 K{Omega} will cause Vm to differ from the holding potential by about 4%; and at the peak of large currents, such as the voltage dependent K+ current in which the input resistance decreased to 600 K{Omega}, 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 TEA+, which effectively blocked the K+ 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 (-20 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 Ic+. 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 K+ and Cl- 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 experimental 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 Ca2+ 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 Ca2+ 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 Ic+, 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 Ic+ channels are activated via the associated pathway of the phospholipase C cascade (i.e., synthesis of diacylglycerol), or alternatively, via another membrane mechanism activated in parallel such as stimulation of phospholipase D or A (see for example [44]).

Electrical Characteristics of Mammalian Granulosa Cells and Oocytes

Electrical activity of granulosa cells and oocytes from mammals has been reported in several studies. For example, Ca2+, K+, and Cl- channels have been shown in granulosa cells from different species. A delayed rectifier K+ channel has been described in avian and porcine granulosa cells [45–47]. Their properties seem to be similar to those of the voltage dependent K+ current studied here (e.g., they all have a threshold around -20 to -10 mV, they do not present inactivation, and are blocked by extracellular TEA+). The strong inhibition of K+ currents by octanol indicates that the channels involved are located in the cumulus cell membrane. Thus, K+ 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 (-15 to -38 mV) [48, 49].

In contrast to what has been shown for porcine granulosa cells stimulated by LH [50], the delayed rectifier K+ channel in mouse cumulus cells was not affected by extracellular or intracellular changes in Ca2+, and stimulation of P2Y2 receptors by ATP or UTP had little or no effect on this K+ current. However, further experiments after minimizing the purinergic responses, ICl(Ca) and Ic+, may allow the detection of small effects of purinergic substances on the voltage dependent K+ 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 Ic+.

Calcium-dependent Cl- channels have also been described in avian granulosa cells [51], but there is no previous information about their possible activation by the Ca2+ 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, K+ channels, and Ca2+-dependent Cl- 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 Ic+, 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 Ca2+ 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 Ca2+ and Cl- ions [52, 53]. Also, it has been shown that intracellular Ca2+ has a central role in the regulation of important processes in the oocyte [54]. Thus, ATP and UTP acting via P2Y2 purinergic receptors might be important paracrine signals in the physiological regulation of CEO growth and maturation in mice.


   ACKNOWLEDGMENTS
 
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 Díaz and Dr. Dorothy D. Pless for critical comments and help with the manuscript, and to Lic. María del Pilar Galárza Barrios, to M.V.Z. Martin Garcia Servin, Ing. Alberto Lara, and Mr. Horacio Ramírez Leyva for technical assistance.


   FOOTNOTES
 
First decision: 5 March 2002.

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. Back

2 Correspondence: Rogelio O. Arellano, Centro de Neurobiología UNAM, Domicilio Conocido Km. 15 Carretera QRO-SLP, Juriquilla Querétaro, CP 76230, México. FAX: 52442 238 1062; arellano{at}calli.cnb.unam.mx Back

Accepted: April 4, 2002.

Received: January 24, 2002.


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