effect on oscillatory Cl- currents elicited by ACh. However, the calcium-dependent neurotransmitter

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Journal ofPhysioloyy (1989), 416, pp. 601-621 601
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EFFECTS OF DEFOLLICULATION ON MEMBRANE CURRENT
RESPONSES OF XENOPUS OOCYTES
BY R. MILEDI AND R. M. WOODWARD
From the Laboratory ofCellular andMolecularNeurobiology, Department of
P'sychobiology, UlniversityofCalifornia, Irvine, CA 92717, USA
(Received 12September 1988)
SUMMARY
1. Catecholamines, adenosine, gonadotrophins, vasoactive intestinal peptide
(VIP) and E-series prostaglandins all elicit K+ currents in follicle-enclosed Xenopus
oocytes. Evidence suggests that cyclic nucleotides act as intracellular messengers in
the activation ofthisK+ conductance. Muscarinic agonists and somedivalent cations
(e.g. Co2+, Mn2+, Ni2+ and Cd2+) elicit slow oscillatory Cl- currents, which are
activated through hydrolysis of inositol phospholipids and mobilization of intra-
cellular calcium by inositol phosphates.
2. We investigated whether these membrane current responses were generated in
the oocyte itselfor in enveloping follicular cells which are coupled to the oocyte by
gap junctions. Oocytes were defolliculated, either enzymatically using collagenase,
or by manual dissection combined with rolling over poly-L-lysine-coated slides.
Removal of follicular cells was checked using scanning electron microscopy.
AIemnbrane current responses ofdefolliculated oocytes were compared with responses
seen in follicle-enclosed oocytes taken from the same ovary.
3. The K+ responses evoked by all the various hormones/neurotransmitters were
either drastically reduced (> 90%) or abolished by defolliculation. K+ currents
generated by the adenylate cyclase activator forskolin and by intraoocyte injection
of adenosine 3',5'-cyclic monophosphate (cyclic AMP). or guanosine 3',5'-cyclic
monophosphate were similarly reduced in defolliculated oocytes. In contrast,
oscillatory Cl- currents to acetylcholine and divalent cations were selectively
preserved through defolliculation.
4. Injection ofcyclic AMP (1-20 pmol) into defolliculated oocytes had little or no
effect on oscillatory Cl- currents elicited by ACh. However, the calcium-dependent
transient Cl- current, activated by depolarization of the oocyte membrane, was
consistently potentiated (100-900%) by injections ofcyclic AMP (1-10 pmol).
5. These experiments suggest that cyclic nucleotide-activated K+ currents arise
essentially in follicular cells and are monitored within the oocyte through electrical
coupling by gap junctions. Oscillatory Cl- responses evoked by ACh and divalent
cations are produced largely or wholly in the oocyte itself.
INTRODUCTION
Oocytes of the frog Xenopus laevis are now used widely in studies of mammalian
neurotransmitter/hormone receptors and ion channels, which can be 'induced' in the
602 R. MILEDI A-.D R. 31. WOODI'4RD)
oocyte membrane by microinjection of foreign messenger RNAs (for examples see,
(Gundersen, Miledi & Parker. 1983, 1984a, b; Sumikawa, Parker & Mliledi, 1984;
Fukuda, Kubo, Akiba, Maeda, Mishina & Numa, 1987; Masu, Nakayama, Tamaki,
Harada, Kuno & Nakanishi, 1987; Takahashi, Neher & Sakmann, 1987; Julius,
MaeDermott, Axel & Jessell, 1988; Meyerhof, Morley, Schwarz & Richter, 1988).
Examining receptor systems and channels existing in native oocytes is therefore
important, firstly in terms of ovarian physiology, but also for predicting and
interpreting the behaviour offoreign receptors when expressed in oocytes.
Xenopus ovarian follicles, manually 'plucked' from the ovary, consist ofan oocyte
surrounded by a vitelline envelope, follicular cells (follicle cells) and thecal tissues, all
ofwhich are enveloped within the inner ovarian epithelium (Dumont & Brummett,
1978). A variety of hormones and neurotransmitters elicit slow membrane current
responses in follicle-enclosed oocytes (stages V and VI; Dumont, 1972). In broad
terms, these reponses can be divided into two major types ofmembrane current:
Acetyleholine (ACh) acting on muscarinic receptors (Kusano, Miledi & Stinnakre,
1977, 1982) and divalent cations such as Cd21. Ni2 and Co2+ (Miledi. Parker &
Woodward, 1988) evoke oscillatory Cl- currents. The different agonists appear to
activate a common mechanism involving hydrolysis of inositol phospholipids,
mobilization of intracellular Ca2± and activation of Ca2+-sensitive Cl- channels
(Miledi & Parker, 1984; Oron, Dascal, Nadler & Lupu, 1985; Parker & Miledi, 1986).
These responses can be largely preserved in oocytes denuded of envelopinig tissues
(i.e. defolliculated oocytes), indicating that the currents and intracellular messenger
system are located in the oocyte itself (e.g. Kusano et al. 1977, 1982).
Catecholamines (Kusano et al. 1982), adenosine (Lotan, Dascal, Cohen & Lass,
1982), gonadotrophins (Woodward & Miledi, 1987a), vasoactive intestinal peptide
(VIP) (Woodward & Miledi, 1987b) and prostaglandins (see accompanying paper;
Miledi & Woodward, 1989) all elicit slow K+ currents which desensitize upon
extended exposure. These agonists bind to different receptors, which can be
distinguished either pharmacologically or by comparing changes in the relative
sensitivities of follicles taken from different frogs. All the responses seem to be
generated primarily through stimulated synthesis of adenosine 3',5'-cyclic mono-
phosphate (cyclic AMP) and activation ofa K+ gating mechanism sensitive to cyclic
nucleotides (Lotan, Dascal, Oron, Cohen & Lass, 1985; Van Renterghem, Penit-Soria
& Stinnakre, 1985; Stinnakre & Van Renterghem, 1986; Smith, Brooker & Brooker,
1987; Woodward & Miledi, 1987a, b; Miledi & Woodward, 1989). Defolliculating
oocytes with collagenase was reported to reduce or abolish responses to,f-adrenergic
agonists and dopamine (Kusano et al. 1982; Van Renterghem et al. 1985). However,
these experiments were largely qualitative and concerns were raised overthe possible
non-specific effects of proteases which contaminate collagenase preparations. In
direct contrast, K+ responses to adenosine were reported to be only 'rather reduced'
in completely denuded oocytes (Lotan et al. 1982), which was subsequently
interpreted as indicating that purinergic receptors and cyclic nucleotide-activated
K+ channels were located in the oocyte memnbrane itself (e.g. Dascal, Lotan, Gillo,
Lester & Lass, 1985; Lotan etal. 1985; but for further discussion see Dascal, 1987).
The location within follicles of cyclic nucleotide-activated K+ responses therefore
l)EFOLLIClULATION ON XENOPl.!S OOCYTES 603
remained unclear. This uncertainty arises because follicular cells are electrically
coupled to oocytes by gap junctions, which raises the possibility that while voltage
clamping an enclosed oocyte, currents arising in the follicular layer are recorded
together with those generated in the oocyte itself (Browne, Wiley & Dumont, 1979;
Browne & Werner, 1984; van den Hoef, Dictus, Hage & Bluemink, 1984; authors'
unpublished results). Alternatively, K+ responses could arise entirely in the
oocyte, or it is even possible that receptors and K+ channels are located in
different compartments (e.g. receptors in the follicular cells and K+ channels in the
oocyte membrane), with cyclic nucleotides acting as diffusible intercellular
messengers.
We subsequently found that cyclic nucleotide-mediated K+ responses were also
evoked by mammalian gonadotrophins (Woodward & Miledi, 1987a). This again
implicated a role for the follicular cells in this type of response, because
gonadotrophini receptors were considered to be located in the follicular layer (e.g.
Masui. 1967; Mulner & Ozon, 1981; Kwon & Schuetz, 1986). In this paper we return
to the problem oflocalizing membrane currents, comparing effects ofenzymatic and
manual defolliculation on the different electrical responses.
METHODS
Oocytes at stages V or VI (Dumont, 1972) were either plucked as ovarian follicles or were
dissected directly from the ovaries oflab-reared adult Xenopus laevis. Frogs were purchased from
Xenopus Ltd. (Surrey, UK), Xenopus I (Ann Arbor, MI, USA) and Nasco (Fort Atkinson, WI,
USA) and killed by decapitation. Defolliculation of oocytes was carried out enzymatically with
collageniase or manually.
Collagenase. Groups often to twenty follicles wereplaced in vials containing collagenase (Sigma
type I or type VII, 0-5-2-0 mg ml-') in 1-2 ml frog Ringer solution (in mM): NaCl. 115; KCI. 2;
CaCl2 1-8; HEPES. 5: pH 70. and gently spun (5 r.p.m.) on a Pelco 1050 rotator at room
temperaturefor 1-2 h. Vialswerethen brisklyshaken byhandwhich usually loosened and removed
innlerovarian epithelia, seen as 'ghosts' floating in the medium (see also Kusano etal. 1982; Miledi
& Parker. 1984). In some cases epithelia were not shed, and were either removed later with
watchmakersforceps, orwere left envelopinig the oocytes during subsequent experiments. Treated
follicles were repeatedly washed and then returned to sterile Barth's medium (in mm): NaCl. 88;
KCl. 1; NaHCO3. 24; MgS04. 0-82;Ca(NO3)2. 033;CaCl2.0-41; HEPES, 5; pH to74. usually with
nyastatin (50 U ml-') and gentamycin (0-1 mg ml-') for storage at 16 °C.
Manual defolliculation. Preparations ofcollagenase used to defolliculate oocytes (Sigma type I
and even the higher purity Sigma type VII) were contaminated with proteases. This concern
prompted us to develop a manual defolliculation procedure which effectively removed follicular
cells without the use of enzymatic or chemical treatments which might disrupt and inactivate
electrical responses located intheoocyte membrane itself.
Maanual defolliculation was done in a glass Petri dish in Barth's medium, first freeing oocytes
from the ovary by peeling away inner ovarian epithelia with sharp watchmakers forceps (as
opposed to 'plucking' the oocytes as follicles). During this dissection it was important to ensure
that theepithelial covering, which often consisted oftwo distinct layers, was completely removed.
These layers appear to be (1) the epithelium itself, which was usually removed together with
associated thecal blood vessels and (2) loose bundles and tangles ofcollagen overlyingthe follicular
cells (e.g. Plate 1A and B). Complete removal of epithelial/thecal tissues only occurred when
dissection produced a characteristic 'unzipping effect', where the oocyte begins to extrude itself
through a rip in the enclosing epithelium (e.g. Plate IC and D). Only oocytes whose epithelial
coverings had been completely removed were used further. These oocytes will be referred to either
as ERoocytes (epithelium removed, see Kusanoetal. 1982), oras 'epithelium-free' oocytes.
604 R. MILEDI AND R. M. VI'OOD W'ARD
Fig. 1. For legend see facing page.
D)EFOLLI(CU1'LATIO)N ON XENLOTIUS 0O0(YTES 66(0)55
(Oocytes free(d from the ovary in this manner were still enveloped by large niumbers offollicular
cells (see text). We had nioticed in early experiments (University College London) that epithelium-
free oocvtes often be(anmestuck to p)lasti( Peti'i (lishes and ap)p)eared to leave a residue offollicular
cells when mnoved. To exp)loit this p)roperty. we took a l)oly-I.-lysine-treate(l glass sli(le illmnlelse(l
in saline an(l silni)ly iolle(l epithelitum-free oocytes over the lenigth of thle slide. LUsing a clissectinlg
mlicroscope ( x 50) atll( olbliq1ue transmnitted liglht to rod(luce a (lark hiel( effect. it was clear that
rolling cauised follicular cells to l)e (lislo(lged fiomil the vitellinie envelope a(li stick. somiletimies as
Irared(ldisontinuious sheets. to the surface oftheslidle (e.g. Fig. 1). The rollinig procedure was (lolle
in Barth's medieinu, frog Ringer soluitioni or zero (Ca2+ Ringer solutioni (in mni): NaCl. 115 RCl.L2
MgCl2. 5-10: EGTA (ethy\leeglyc(lol-bis-(/i-amllinoethy-lethler) N.N'-tetraacetic acid) I: HEI)ES. 5:
pH 70. ()ocytes were rolled till they nio loniger shedl follictilar cells and(I the vitellille elVelope
apIpearedi to l)e f'ree ofoverlying tissue. All oocvtes uise(l in thissttudy werestill surrou(liledl b thleir-
protectix'e vitellilleenvelopes. Remnoval ofthe vitellille layerrequireseither (1)extend(le(d treatmenit
with collagenase in comnbination with proteases (e.g. pronase) (A. R. Limbrick & R. Miledi.
unp)ublished results) or (2) maniuial remioval. usually after incubation in hypertonic salinie (e.g.
Methifessel. WVitzemiann, 'I'akahashi. MIixshina. Nuimiai & Sakmiainin. 1986). Only after- removal ofthe
v-itelline envelope is the surface of the oocvte itself rev-ealed, with its characteristic covering of
microvilli (PIlate 1E ain( F) (cf. Dtuimzonit & lBruimimilett. 1978).
The efficacy of' different def'olliculation procedures. in terms of' removing follicular cells, was
m(lonitored usinig scanning electroni microscop)y (SEM). Samples of' (lifferenit types of oocvte
(follicles. ER oocytes. rolled oocytes and collagenase-treated oocvtes which had shed epithelia)
were all prepared in the same way. Oocytes were fixed in glutaraldehyde (25%/, in 50 mM-
phosphate buffer. pH 74) for 3 h. washed in buffer, dehydrated through a graded acetone series
(20 min intervals) and critical-point dried in CO2 (Ladd 28000 critical-point dryer) Oocytes were
mounted on stubs, sputter-coated with gold to a thickiness of ca 6nm (l'elco 9500 coater) and
examined with a Hitachi 8-500 microscope at 10 kV (cf. Dumont & Brummett, 1978). Oftell these
preparations had small dimpling or cratering effects (e.g. Plates 1A and E. 2C and E) and
occasionally somie crackinig in oocvtes (e.g. I"late 2A, top)) neither ofwhich siginificaiitlv obscured
the vitelline surIface.
Electrophysiology was done at room temperature (22-25 °C) in a bath contilluously perfused
with f'rog Riliger solutioln usinigfollicles/oocytesoverthe first 4daysfollowing reliloval froim fi'ogs
(for further (letails see Kusanio et al. 1982:; Mliledi, 1982). Membrane current responises of
dlefolliculated oocvtes 'were comiipared with responses recorded in control follicles which had been
store(l for the same p)eriod. Electrical recordinig from manually defolliculated oocytes was usually
(lonie within 1-2 h ofrolling anid ofteni within 2-10 mill. The large majority of' rolled oocytes ha(d
good restitig potentials (-40 to -90 nV) and high input resistance (0(5-22 il) ); (lamaged
oocvtes were (liscar(lel. Intraoocyte inijections of' cyclic nucleotides were ma(le by pneumatic
pressure ejection fromil micropipettes (Miledi & Parker, 1984), usinig 1-10 miMi-cyclic AMP or cyclic
Ml,113. in 10nmnm-HElI'ES. pH adjusted to 7(0 with KOH. The pressure regulator was usualil set at
200 kPa anid the lenigth ofthe pulse (50- 2000 ms) was selectedl to eject droplets of' 100-250 pl.
Prostaglandins P(GE1 anicl PUE2E were purehased f'rom eayman Chemical (Anin Arbor. MI.
USA) orCalbiochem. Forskolin (Coleusforskohlii) andporcinefollicle stimulating hormone (pFSH)
wereobtainedfrom Calbiochem; all otherdrugsandreagentswerefrom Sigma.
Fig. 1. Removal offollicular cellsby rolling oocytes overpoly-L-lysine-treated slides. An
oocyte with itsepithelial covering manually removedwasplacedona clean poly-i.-lysine-
coatedslide,freshly immersedinBarth'smedium. Theoocytewasviewedunderdarkfield
illumination, at x50 A,oocyteandslidepriortorolling.B,afterrollingtheoocytealittle
wayalongthesurfaceoftheslide. Atthisearlystagein theproceduretheoocytehasshed
ca2000follicularcells, roughly200/) ofitstotalfollicularlayer (oocytediameterca I mm;
see text). The black and white fields on the oocvte are animal and vegetal poles. In this
figure the l)lane offocus was kept on the slide and effects of'rolling on1 the oocyte surf:ace
are not illustrated. Failure toseeshedding offollicular cellswas usually a consequence of
incomiiplete removal ofepithelial coverings. Oocytes were rolled equatorially and pole-to-
pole until they no longer shed follicular cells: up and down a 75 mm slidie was usuallv
sufficient.
606 66 R. MIILEDI AND R. Mll. WlOODWlA4RD
RESULTS
Oocyte histology and the effects ofdefolliculation
Scanning electron microscopy confirmed that oocytes (stages V and VI), freed
from their enclosing epithelia simply by dissection, invariably had large numbers of
follicular cells still covering their vitelline envelopes (e.g. Plate 1 G-1). The follicular
layer consisted of a discontinuous monolayer of irregular 'ovoid' cells, flattened
against the outer surface of the vitelline envelope. Follicular cells typically had
diameters of 15-25 pum and each cell extended numerous processes. making contacts
with their immediate neighbours and the underlying envelope (I'late 1I) (cf. Dumont
& Brummett, 1978). Depending on the ovary from which oocvtes were dissected, and
variations between individual oocytes, 2-60% of follicular cells were grossly
damaged following removal of epithelia and subsequent handling of oocytes (e.g.
Plate 1G and H). We found no clear difference in the density of follicular cells
covering animal and vegetal poles. The number of follicular cells per oocyte was
therefore estimated by assuming a spherical oocyte and counting cells in 100-200,um
squares ofundamaged follicular layer. Forty-three counts, taken from eleven oocytes
(five frogs) gave a density of32+7 follicular cells per 10000,um2 ofvitelline surface
(mean+s.D.). Therefore, as a rough approximation, oocytes with diameters of 1-0
and 1P2 mm are surrounded by respectively 10000 and 14000 follicular cells.
Removal of follicular cells by collagenase treatments and the manual defollicu-
lation procedure were then compared. For whole mounted oocytes it was possible,
using tilt, to scan 60-70% ofthe vitelline surface, which was improved to > 95% by
slicing fixed oocytes in two and mounting as hemispheres.
Our standard collagenase treatments (1-2 h at 05-20 mg m-1'. Sigma Type I)
typically removed large numbers of follicular cells though efficacy varied greatly,
particularly when comparingoocytestaken from differentfrogs, andwhen comparing
the effectiveness of different batches of collagenase. The degree to which follicular
cells were removed also showed some dependence on the length of incubation and
concentration of collagenase used. For the least effective treatments 30-70% of
follicular cells remained on the vitelline envelope (Plate 2A andB), ofwhich ca 20%
were obviously damaged, consisting of residual cell debris (e.g. Plate 2D). In
contrast, the best treatments generally left < 5% of follicular tissue, at least 50%
of which was debris (Plate 2E and F). Furthermore, even follicular cells which still
looked as if they remained intact often had lost their characteristic flattened ovoid
shape, and appeared to have become partly dislodged from the vitelline surface (e.g.
compare Plates 11 with 2B and D). Amongst these remnants it remains difficult to
estimate the number of follicular cells which might be electrically coupled to the
oo(eyte. In summary, our collagenase treatments typically removed or destroyed at
least 40%, and in the best cases > 99% of enveloping follicular cells (comparing
sixty-five oocytes, eight treatments, six frogs). In general, the longer treatments
using higher concentrations of collagenase were more effective. However, we stress
that even within the same treatment there could be conspicuous variations between
oocytes in the number offollicular cells remaining. Forexample, we examined twelve
oocytes which had been defolliculated using 2 mg ml-' Sigma Type I collagenase for
LDEFOLLICLTLATI)NO ON XNENOPlS OOCYTE'S 607
2 h. Ten oocytes were essentially free of follicular cells, but in two cases ca 40% of
the follicular layer still remained attached to the vitelline envelope.
Oocytes which had been dissected out offollicles and then rolled on poly-L-lysine-
treated slides were examined using the same procedures. When compared with
collagenase treatment, we found that manual defolliculation was consistently a more
effective and reliable means of removing follicular cells. In twenty-four of the
twenty-six rolled oocytes examined (five frogs), there were simply no detectable
follicular cells, not even damaged ones, the vitelline envelope clean except for a light
scattering of small debris (Plate 2G-I). In two oocytes a few isolated groupings of
follicular cells remained, many of which were obviously damaged. We invariably
found less than 100 potentially intact follicular cells per rolled oocyte, constituting
< 1% ofthe original follicular layer.
Effects ofcollagenase treatment on memnbrane currentt responses
In all experiments membrane current responses were recorded while voltage
clamping follicle-enclosed or defolliculated oocytes at -660 mV. This potential is
away from the oocyte's reversal potentials for Na' (ca +50 mV), K+ (ca - 100 mV)
and Cl- (ca -20 mV) (see Kusano et al. 1982). The responsiveness offollicle-enclosed
oocytes was established by exposing five to twelve untreated follicles to a series of
different agonists. The mean membrane current produced by each agonist was then
compared to the mean response seen in a similar number of collagenase treated
oocytes. Results from three treatments (three frogs) are given as examples in
Table 1.
Following treatment with collagenase, the K+ currents elicited by catecholamines
(noradrenaline and dopamine), purinergic agonists (adenosine and ATP), gonado-
trophins, VIP and prostaglandins were all either substantially reduced (e.g. Table
1A; Fig. 2B) or effectively abolished (e.g. Table 1B; Fig. 20). In this study we have
examined more than seventy-five oocytes, taken from twelve frogs, and in all cases
collagenase-treated follicles responded with < 10% of the mean K' currents seen
in follicle-enclosed oocytes taken from the same frog. Sensitivity to collagenase
treatment was approximately the same for responses to all the various neuro-
transmitters/hormones which evoked K+ currents. In particular, gonadotrophin
responses were not obviously more sensitive to defolliculation. However, the overall
level ofreduction did vary somewhat between oocytes from different frogs (compare
Table 1A (day 1) andB). Ifepitheliaremained covering oocytes then reduction ofthe
K+ responses was still substantial, but tended to be less pronounced than ifepithelia
were shed (e.g. Table 1A and B). It was also clear that K+ currents produced by
forskolin, which acts intracellularly on the catalytic subunit of adenylate cyclase,
were reduced or abolished in parallel with receptor-mediated responses (Table
lA-C). Residual K+ currents seen in treated follicles were lost overa subsequent 1-2
days storage in Barth's medium (e.g. Table lA) and K+ responses which had been
abolished by treatment did not then reappear overthe following 1-3 days.
Effects ofmanual defolliculation on membrane current responses
Mlerely dissecting away inner ovarian epithelia typically caused significant
reductions in K+ responses. This effect was quite variable and epithelium-free
608 R. MILEDI AND R. M. WOODWARD
oocytes in some cases responded with up to 70% ofthe mean current seen in control
follicle-enclosed oocytes (e.g. Table 2A andB). In contrast, 'epithelium-free' oocytes
which had been rolled upon poly-L-lysine-treated slides (to remove remaining
follicular cells) consistently gave no detectable K± response to noradrenaline,
TABLE 1. Effect ofdefolliculationi using collagenase oIn embrane current responses
A pFSH (5 Itg m1-) NA (1I0,Im) AD (100,uM) AT1P (100jim)
Day1 Con 210+64 (5) 283+ 12 (5) 201+64 (5) 70+59 (4)
CT 15+9 (6) 18+10 (6) 13+7 (6) 3+3 (6)
Day 3 CoIn 163+63 (5) 198+ 108 (4) 203±+75 (4) 138+71 (3)
CT (4) (4) (4) (4)
B PGE2 (2gM) V'IP (60 nM) AD (100(,um) FOR (1 pii)
Day 2 Con 213+72 (11) 202+125 (11) 485+234 (11) 482+225 (11)
(I11)(1)(1 -- l)
CT
C FOR (10,UM) ACh (100,CCM)
Day I CoIn 231+33 (7) 1341+482 (6)
CT 4+4 (6) 1285+311 (6)
All membrane currentsare innA, given asthe mean+S.D.; number offollicles per oocytestested
given in parentheses; (-) denotes a mean response of < 1 nA; clamp potential -60 mV. A C are
experiments on oocytes taken from three different frogs. Day 1-3 indicates the time following
collagenase treatment at which oocvtes and controls were tested, day 1 being the saame day as
treatment. Con, control follicle-enclosed oocytes; CT, collagenase-treated oocytes; pFSH, porcine
follicle stimulating hormone; NA, noradrenaline; AD, adenosine; FOR. forskolin; PGE2,
prostaglandin E2; VIP, vasoactive intestinal peptide. ACh responses (part C) are inward currents
measured at the initial peak oscillations.
Individual follicles were exposed to a series of agonists in the sequence and concentrations
indicated. Treatment A failed to loosen epithelia, which were left surrouniding oocytes during
electrophysiology. In B and C, inner ovarian epithelia were shed as 'ghosts'. Treatment A was
1-0 mgml-' collagenase (Sigma Type VII) for 1 h; B, 05 mg ml-' for 1 h (Sigma Type I)
C, 1-0 mgml-' for 1 h (Sigma Type I).
dopamine, adenosine, gonadotrophins, VIP or prostaglandins (e.g. Table 2A and B;
Fig. 3). This was equally true for oocytes with low or high levels ofresponsiveness to
these agonists. As with collagenase treatment, K+ currents elicited by the different
hormones/neurotransmitters appeared to be similarly sensitive to manual defollicu-
lation, and currents generated by forskolin were reduced or abolished in parallel
with the receptor-mediated responses (Table 2A and B). Again, K' responses
abolished by manual defolliculation did not start to reappear over a subsequent 1-2
days storage in Barth's medium.
Follicles taken from some frogs had good levels ofresponsiveness both to agonists
which elicit K+ currents and to agonists (e.g. ACh andl Co2+) which produce
oscillatory Cl- responses. In these follicles we found that enzymatic or manual
defolliculation selectively reduced or abolished K+ currents, while in the same
defolliculated oocytes Cl- responses remained substantially intact (e.g. Table 1C;
Table 2B; Fig. 3A and B). The oscillatory Cl- currents evoked by ACh and divalent
cations were consistently well preserved in oocytes with high or medium levels of
responsiveness to these agents. But for oocytes with low sensitivity, e.g. which gave
ca 10-50 nA ofoscillatory Cl- current, the response was often significantly reduced,
I)EFOLLIJCl LATON ON XEVOPU!S OOCYTES 609
and in some cases removed (ef. Kusano etal. 1977). This wasparticularly conspicuous
for small ACh ressponses consisting ofa single transient Cl- current occurring at short
latency (e.g. Fig. 3D), which wvere invariably abolished by manual defolliculation
(sixteen oocvtes. four frogs). WVe were also unable to preserve the small (15-25 nA)
K' cuirrents elicited by ACh in follicle-enclosed oocytes from some frogs (ef. Dascal
& Cohein. 1987).
A
100 nA (A)
50 nA (Band C)
4 min
PGE2 (2pM) VIP (60nM) Adenosine (100pM) Forskolin (1 pM)
B
a I I I IIpa a
PGE2 (2pM) VIP (60nM) Adenosine (100pM) Forskolin (1pM)
C
A
PGE2 (2pM) VIP (60 nM) Adenosine (100pM) Forskolin (1 pM)
Fig. 2. Effect of defolliculation ith collagenase oIn membrane currenits elicited by
prostaglandin (PGE2). vasoactive initestinal peptide (VIP). adenosinie andforskolin.
E2 A.
cutrrenits elicited in a follicle-enclosed oocvte. B and C, examples of responses in two
collagenase-treated oocytes fiom the same frog. B, the only treated oocyte, of eleven
tested. where small residual K+ currents were detected. C, one ofthe ten treated oocytes
where responses were abolished. In this and all following figures the clamp potential was
-60 mV and membrane conductance monitored by periodic 6-10s steps to -50 mV.
Outward current isdenoted byupwarddeflection; drugsappliedasindicatedbybarswith
perfusiondeadtimesof15-25 s. RecordsselectedfromtheexperimentinTable 1B.
Effect.s ofdefolliculation on currents elicited by intracellular injection ofcyclic
ucleotides
n
The receptor-mediated and forskolin-activated K+ currents were essentially
abolished in defolliculated oocytes. Butaspreviously discussed, even ifreceptors and
adenylate cyclase were located in follicular cells. it remained possible that the cyclic
nucleotide-activated K+ channels themselves were nevertheless located in oocytes.
241 I'H 416
610 R. JIILED)I AND R. 31. WOODIVARD
We therefore compared the K' currents produced by direct intraoocyte injections of
cyclic nucleotides in follicle-enclosed and defolliculated oocytes.
Pressure injecting cyclic AMP or cyclic GMP into follicle-enclosed oocytes usually
elicits substantial K+ currents (cf. Dascal et al. 1985; Dascal, Lotan & Lass, 1987),
TABLE 2. Effect ofmanual defolliculation on membrane current responses
A V'IP (30 InM) FOR (1 ,uM) pFSH (25l,g mn-1)
Con 550+145 (5) 4159+137 (5) 411+347 (5)
ER 45+37 (6) 63+50 (6) 109+68 (5)
Rolled (5) (5) 1+2 (5)
B NA (100()M) FOR (1 ,UM) Al) (100,M) COCI2 (1 mM)
CoIn 450+ 137 (6) 230+57 (6) 319+131 (5) 371+ 161 (6)
ER 42+23 (4) 123+23 (4) 115±32 (4) 315+ 100 (4)
Rolled (5) (5) (5) 493+65 (7)
Dataarepresented asinTable 1;Con, control untreatedfollicles;ER,follicleswithinnerovarian
epithelia removed manually; rolled. ER oocytes which were rolled on polV-L-lysine-treated slides.
All oocytes tested on the day ofdefolliculation.
though sensitivity to cyclic nucleotides does vary between oocytes from different
frogs. For example, at a clamp potential of -60 mV, highly responsive follicles
elicited outward currents > 600 nA to injections ofca 2 pmol cyclic AMP, whereas
follicles oflow sensitivity generated < 20 nA to injections of20 pmol. Testing more
than forty oocytes (ten frogs) with high or low sensitivity, we invariably found that
the K+ currents produced by intraoocyte injection of cyclic nucleotides were either
greatly reduced (< 10% of controls) or abolished by defolliculation. Similar effects
were seen with collagenase treatment (e.g. Table 3A and B) and manual
defolliculation (Fig. 4). As with the responses to neurotransmitters/hormones,
manual defolliculation was again more consistent in abolishing responses to cyclic
nucleotides. K+ currents evoked by either cyclic AMP or cyclic GMP were equally
sensitive to defolliculation, and again, whereas responses to intraoocyte cyclic AMP
were abolished, in the same rolled oocytes oscillatory Cl- currents elicited by ACh
were selectively preserved (e.g. Table 30).
Effects ofcyclic nucleotides on membrane currents arising in the oocyte itself
Having found that cyclic nucleotide-activated K+ currents were effectively
abolished by defolliculation, we considered it worthwhile to then look for any
modulatory effects of cyclic nucleotides on membrane current responses actually
preserved in defolliculated oocytes. This was particularly important in the context
offinding an electrophysiological assay for foreign (induced) receptors which might
be expected to couple to adenylate cyclase. We therefore checked to see ifoscillatory
Cl- responses to ACh, or some ofthe native voltage-gated currents, were themselves
modulated by intraoocyte injections ofcyclic AMP orcyclic GMP.
Cyclic nucleotides onresponses toACh. To testforeffects ofcyclic nucleotides on the
oscillatory Cl- currents produced by ACh we used defolliculated oocytes with high
sensitivity to ACh. In these oocytes, repeated exposures to ACh (50-500 nM) elicited
reproducible membrane currents, whereas oocytes with low sensitivity required