422 M.N. SKOBLINA AND I. HUHTANIEMI

© 1997 WILEY-LISS, INC.

Involvement of Chloride Channels in ProgesteroneProduction During Meiotic Maturation ofFollicle-Enclosed Oocytes of Rana temporariaand Xenopus laevis

M.N. SKOBLINA1* AND I. HUHTANIEMI2

1Institute of Developmental Biology, Russian Academy of Sciences,117808 Moscow, Russia

2Department of Physiology, University of Turku, 20520 Turku, Finland

ABSTRACT The chloride channel blockers SITS (4-acetamido-4´-isothiocyanostibene-2,2´-disulphonic acid) and DIDS (4,4´-diisothiocyanostilbene-2,2´-disulphonic acid) markedly suppressedprogesterone production in the Rana temporaria and Xenopus laevis follicle-enclosed oocytes andoocyte maturation stimulated by the homologous pituitary suspension and hCG, respectively. In-hibition was dose-dependent and decreased with the increase of the hormone concentration. SITSdid not affect progesterone production in the R. temporaria follicle-enclosed oocytes stimulated bydbcAMP. Substitution of sodium chloride for equimolar concentrations of sodium gluconate,methanesulfonate, glutamate, or formate significantly potentiated the gonadotropin-stimulatedprogesterone production and oocyte maturation in the both species. Possible involvement of chlo-ride channels in progesterone production by the gonadotropin-stimulated amphibian follicle-enclosed oocytes is discussed. J. Exp. Zool. 278:422–428, 1997. © 1997 Wiley-Liss, Inc.

The study of action of the gonadotropic hor-mones on maturation of the amphibian follicle-enclosed oocytes (FEO) (Skoblina et al., ’95a,b) ledus to search for the role of interactions of follicleand medium in FEO maturation especially at lowgonadotropin concentrations. It was shown thatincreased NaHCO3 concentration decreases theminimal concentration of gonadotropic hormonesthat stimulated oocyte maturation in sturgeon(Goncharov, ’78) and Xenopus laevis (Skoblina, un-published results). Ca2+-dependent (Supplission etal., ’91) and cAMP-dependent (Arellano andMiledi, ’93, ’94) chloride channels have been de-scribed in the amphibian FEOs. It was proposedthat chloride channels or chloride ions are in-volved in luteinizing hormone (LH)-stimulated tes-tosterone production in the rat Leydig cells (Choiand Cooke, ’90) and progesterone production inthe chicken granulosa cells (Morley et al., ’91).Nothing is known about their involvement in theregulation of progesterone production in the fol-licle-enclosed oocytes of low vertebrates. We stud-ied the influence of chloride channel blockers andchloride-deficient media on the progesterone pro-duction in FEOs of R. temporaria and X. laevisand oocyte maturation stimulated by the homolo-gous pituitary suspension (PS) and hCG, respec-

tively. Progesterone, a physiological inducer of am-phibian oocyte maturation (Masui and Clarke, ’79),was assayed by radioimmunoassay (RIA).

MATERIALS AND METHODSExperiments were carried out on FEOs of 20 R.

temporaria and 21 X. laevis females. R. temporariafemales were collected during late autumn andhoused in a cold room in darkness at 4°C. X. laevisfemales were bred in the animal rooms of the In-stitute of Developmental Biology, Russian Acad-emy of Science. One or two months before theexperiments, X. laevis females were injected withhuman chorionic gonadotropia (hCG) (Profasi,Italy) (350–500 IU/female) and 3 to 5 days beforethe experiments with PMSG (Sigma, St. Louis,MO; 100–350 IU/female). They were anesthetizedby immersion into 0.2% ethyl m-aminobenzoate(MS-222; Sigma) and operated. R. temporaria fe-males were killed by decapitation. The ovarieswere removed, immersed in Ringer solution, di-vided with forceps into fragments containing oneto ten FEOs each, and carefully mixed. Individual

*Correspondence to: M.N. Skoblina, Institute of Developmental Bi-ology, 26 Vavilov Street, 117808 Moscow, Russia. E-mail: SKOB-LINA@IBRRAN.MSK.SU.

Received 7 November 1996; Revision accepted 26 February 1997

JEZ 805THE JOURNAL OF EXPERIMENTAL ZOOLOGY 278:422–428 (1997)

IONIC CHANNELS IN AMPHIBIAN OOCYTE MATURATION 423

FEOs were isolated with forceps for every experi-mental variant. Progesterone production in R.temporaria FEOs was stimulated by the homolo-gous PS (0.00125–0.01 pit/ml) and in X. laevisFEOs by hCG (5–40 IU/ml). These concentrations,except the highest, induce complete or near 100%oocyte maturation without overdosage of hor-mones (Skoblina and Kondratieva, ’92).

Two chloride channel blockers were used: SITS(4-acetamido-4´-isothiocyanostibene-2,2´-disul-phonic acid) and DIDS (4,4´-diisothiocyanostil-bene-2,2´-disulphonic acid) at 0.01, 0.1, 1 and 10µM. FEOs of both species were incubated in theamphibian Ringer solution or in a Ringer solu-tion with an inhibitor for 1 hour. Some FEOs werethen left in either of these solutions and the restof them were treated by the hormones with orwithout SITS or DIDS until the end of the ex-periment. The effect of SITS on the progesteroneproduction in R. temporaria FEOs stimulated bydbcAMP (Sigma; 2 and 3 mM) was also studied.Besides the Ringer solution, we used a saline withchloride salts substituted for equimolar concen-trations of the corresponding gluconates or withsodium chloride substituted for sodium meth-anesulfonate, glutamate, or formate. All solutionshad pH 7.8 and osmolarity 217 to 228 mOsM.

Progesterone content was assayed in the FEOsof R. temporaria and in the incubation medium ofthe FEOs of X. laevis. Good correlation of theprogesterone content between the FEOs and themedium was shown earlier (Schuetz and Lessman,’82; Fortune, ’83; Patino and Purkiss, ’93). In theR. temporaria FEOs, whose maturation was in-duced by low PS concentrations, progesterone pro-duction sharply increased 18 h after the beginningof PS treatment and remained at the high levelthereafter. In our conditions, the highest concen-tration of progesterone in the medium was at-tained 6 h after the beginning of hCG treatment(Skoblina et al., ’95b). Samples for progesteroneRIA in R. temporaria were prepared as follows:progesterone was extracted 20–24 h after the be-ginning of experiment. FEOs from all experimen-tal variants (three repetitions, n = 20 in eachrepetition) were placed in 5-ml glass test tubes. TheRinger solution transferred with the FEOs was re-moved by filter paper. Steroids were extracted asdescribed by Lessman and Schuetz (’82). One mil-liliter of methanol (Merck, Darmstadt, Germany,analytical grade) was added to each tube. Extrac-tion was carried out in a shaker (100 oscillations/min) at the room temperature for 15 min. Metha-nol was carefully transferred into another set of

tubes and evaporated under nitrogen flow. Samplesfor progesterone RIA in X. laevis were preparedas follows: progesterone was extracted within 6hours after the beginning of the hormonal treat-ment. The medium in the well was carefully mixedand 1 ml of it was removed and extracted by 2 mlof freshly distilled diethyl ether. All samples werestored at –20°C until RIA. The efficiency of steroidextraction was higher than 95% (Skoblina et al.,’95a). The progesterone content was measured asdescribed elsewhere (Vourento et al., ’89).

The rate of oocyte maturation (two repetitions,n = 30) was determined within 48 hours in R.temporaria (16–17°C) and within 24 hours in X.laevis (20–22°C). The FEOs were boiled and cutunder a dissection microscope. The absence of ger-minal vesicle served a criterion of FEO matura-tion. In the X. laevis FEOs, the presence of a whitespot at the animal pole was an additional crite-rion. The percentage of maturation determinedusing these two criteria was similar in all cases.

Mean ± S.E.M. were calculated for different ex-perimental groups. The data were compared us-ing Student’s t-test.

RESULTSThe influence of SITS and DIDS onprogesterone production in FEOs of

R. temporaria stimulated by PS or cAMPand in FEOs of X. laevis stimulated by hCGSince the initial progesterone level and its pro-

duction stimulated in FEOs of different femalesby the same PS or hCG concentration can varysignificantly, the results presented in each figurewere obtained for FEOs of one female. The influ-ence of the chloride channel blockers was studiedon the FEOs of 11 R. temporaria and six X. laevisfemales. The both inhibitors (1 and 10 µM) sig-nificantly decreased the progesterone productionstimulated by PS (0.00125–0.005 pit/ml) or hCG (5–20 IU/ml) in FEOs of all females (Figs. 1A, 2A, 3A,and 4A). They did not affect the progesterone pro-duction stimulated by the highest gonadotropin con-centration in both species (data not provided). SITSand DIDS (0.1 µM) significantly decreased theprogesterone production in FEOs of eight R. tem-poraria females at all PS concentrations, unlike inFEOs of three other females. The inhibitory effectsof SITS and DIDS at 0.1 µM on the hCG-stimu-lated progesterone production in X. laevis FEOswere insignificant (data not provided). The influ-ence of SITS and DIDS at 0.01 µM was studied onthe FEOs of two R. temporaria females only: proges-terone production was significantly decreased (Fig.

424 M.N. SKOBLINA AND I. HUHTANIEMI

1A). SITS (1 and 10 µM) did not affect the proges-terone production stimulated in FEOs of three fe-males by dbcAMP (2 and 3 mM; Fig. 5).

The influence of SITS and DIDSon oocyte maturation

Some oocytes of six (out of 11) R. temporariafemales matured spontaneously. We will not con-sider here the influence of the chloride channelblockers on oocyte maturation in these females.SITS and DIDS (10 µM) inhibited oocyte matura-tion almost completely in five other females stimu-lated by all PS concentrations. The inhibitors at1 µM markedly suppressed oocyte maturation ofall females, stimulated by one or two PS concen-trations only and at 0.1 µM oocyte maturation ofthree females stimulated by the lowest PS con-centration (Figs. 1B, 2B). The oocytes of both fe-

males treated with the inhibitors at 0.01 µM ma-tured in all PS concentrations.

None of the oocytes of 11 X. laevis females usedin experiments did mature spontaneously. Bothinhibitors at 10 µM significantly suppressed oo-cyte maturation of all females stimulated by allhCG concentrations, at 1 µM suppressed oocytematuration stimulated only by lower hormone con-centrations (Figs. 3B, 4B), and at 0.1 µM did notaffect oocyte maturation (data not provided).

The influence of chloride ions substitutionon progesterone production in FEOs ofR. temporaria and X. laevis stimulated

by PS or hCG, respectively

The influence of chloride ions substitution onthe progesterone production was studied in FEOsof 12 females of R. temporaria and six females ofX. laevis. The absence of extracellular chloride ions

Fig. 1. The influence of SITS on the progesterone produc-tion (A) and maturation (B) of the follicle-enclosed oocytes ofRana temporaria stimulated by pituitary suspension (PS).Each point is the mean ± S.E.M. of three measurements. Theasterisks indicate significant differences from the precedingtime-point of the same treatment (P < 0.05).

Fig. 2. The influence of DIDS on the progesterone pro-duction (A) and maturation (B) of the follicle-enclosed oo-cytes of Rana temporaria stimulated by pituitary suspension(PS). For details see Figure 1.

IONIC CHANNELS IN AMPHIBIAN OOCYTE MATURATION 425

(it was substituted for equimolar concentrations ofgluconates) or their decreased concentration (sodiumchloride was substituted for sodium glutamate,methanesulfonate or formate) significantly in-creased the progesterone production in FEOsstimulated by lower PS or hCG concentrations(Figs. 6A, 7A). In chloride-deficient media, theprogesterone production by unstimulated FEOsincreased. In some cases the increase was signifi-cant (Fig. 6A). Progesterone production stimulatedby the highest PS or hCG concentration in mostcases decreased insignificantly (data not provided).

The influence of chloride ionssubstitution on oocyte maturation

The effect of chloride ions substitution on thegonadotropin-stimulated oocyte maturation wasstudied on FEOs of 12 R. temporaria and 10 X.laevis females. The absence of chloride ions ortheir lowered concentration increased the rate ofoocyte maturation in both species. The effect of

chloride-deficient media was significant in all X.laevis females, especially at low hCG concentra-tion (Fig. 7B). The high rate of R. temporaria oo-cyte maturation in the control made this effectinsignificant in the most cases. The rate of “spon-taneous” oocyte maturation in all R. temporariafemales was significantly higher after substitu-tion than in the Ringer solution (Fig. 6B).

DISCUSSIONIt was proposed that chloride channels can play

the role of a second messenger at low gonadotro-pin concentrations (Choi and Cooke, ’90). But itwas shown that the chloride channels blockers(SITS and DIDS) reduced the accumulation of ex-tracellular cAMP in the chicken granulosa cells(Morley et al., ’91) and that only the cAMP-dependent chloride conductance is activated at theresting potential in the Leydig cells (Noulin andJoffre, ’93). Nevertheless, in amphibians, as well

Fig. 3. The influence of SITS on the progesterone produc-tion (A) and maturation (B) of the follicle-enclosed oocytes ofXenopus laevis stimulated by hCG. For details see Figure 1.

Fig. 4. The influence of DIDS on the progesterone pro-duction (A) and maturation (B) of the follicle-enclosed oo-cytes of Xenopus laevis stimulated by hCG. For details seeFigure 1.

426 M.N. SKOBLINA AND I. HUHTANIEMI

as in mammals and birds, chloride channel blockersinhibited gonadotropin-stimulated steroidogen-esis, especially at low PS or hCG concentration.The increased hormone concentration was fol-lowed by decreased inhibitory influence of SITSand DIDS on the progesterone production byFEOs. We did not observe any effect of SITS andDIDS on testosterone or estradiol production bythe R. temporaria FEOs (Skoblina et al., ’95b), sothey seem not to enhance the progesterone me-tabolism. Note that SITS and DIDS can affectsome other pathways of chloride transfer (Russeland Brodwick, ’79). The data about the regula-tion of chloride transfer in amphibian FEOs arerather contradictory. In some cases, the perme-ability of FEOs for chloride ions is connected withchloride channels (Schlichter, ’89; Katayama andWiddicombe, ’90; Chao and Katayama, ’91), whilein others, with Na+-K+-Cl–-cotransfer (Chipperfieldand Fry, ’82; Bartel et al., ’89; Keicher and Meech,’94). In our preliminary experiments, bumetanideat 100 µM did not inhibit the progesterone pro-duction in X. laevis FEOs stimulated by all hCGconcentration, although at 10 µM it strongly inhib-ited chloride reuptake by Rana oocytes (Keicher andMeech, ’94). SITS and DIDS can also affect theintracellular pH, chloride activity (Vaugham-Jones, ’79; Kaila et al., ’92), and calcium level(Kimura et al., ’88; Romero and Ortiz, ’88). In ourpreliminary experiments, SITS did not affect theintracellular pH of R. temporaria and X. laevisfollicle cells (Nikiforova et al., unpublished re-sults). The inhibition by DIDS of Ca2+-ATPase

(Kimura et al., ’88; Romero and Ortiz, ’88) couldincrease rather than decrease the intracellularCa2+ (and steroid) level.

Chloride-deficient media exert different effectson the gonadotropin-induced steroidogenesis invarious systems: they stimulate steroidogenesisin mammals (Choi and Cooke, ’90) and amphib-ians (our results) and inhibit it in birds (Morleyet al., ’91). Enhanced steroidogenesis was ob-served at low hormone concentrations and alsoin unstimulated amphibian FEOs. It is knownthat some anions substituted for chloride stimu-late chloride transfer, while others inhibit it(Russel and Brodwick, ’79). Since all chloride sub-stitutes used in our experiments enhanced theprogesterone production in FEOs of the both spe-cies, the mechanism of their action appears to bethe same. The stimulating influence of chloride-

Fig. 5. The influence of SITS on the progesterone pro-duction of the follicle-enclosed oocytes of Rana temporariastimulated by dibutyrylcyclic AMP (dbcAMP). For detailssee Figure 1.

Fig. 6. The influence of substitution of chloride ions forequimolar concentrations of corresponding gluconate on theprogesterone production (A) and maturation (B) of the fol-licle-enclosed oocytes of Rana temporaria stimulated by pitu-itary suspension (PS). For details see Figure 1.

IONIC CHANNELS IN AMPHIBIAN OOCYTE MATURATION 427

still more elevated concentration inhibited it(Kleis-SanFrancisco and Schuetz, ’86). It is pos-sible that the different effects of chloride-deficientmedia on steroidogenesis in birds (Morley et al.,’91) and mammals (Choi and Cooke, ’90) are con-nected with different intracellular Ca2+ levels intheir cells, especially because LH concentrations(0.01–0.1 ng/ml), whose effect was enhanced inchloride-deficient medium in mammals, were notused in birds. At LH concentration higher than 1ng/ml, a decreased progesterone production wasalso observed in mammals. Insignificant decreaseof progesterone production at the highest PS orhCG concentrations was also observed in our ex-periments. But one cannot also exclude differenteffects of chloride-deficient media on the cAMPlevel in steroid-producing cells of different species.Measurements of cAMP and intracellular calciumin the amphibian follicle cells will help to solvethis problem.

Since progesterone is a physiological inducer ofamphibian oocyte maturation (Masui and Clarke,’79), changes in its production should affect therate of oocyte maturation. The inhibition of proges-terone production by the chloride channel blockersdid reduce the rate of oocyte maturation, whilestimulation of the progesterone production afterchloride ions substitution increased it. However,oocyte maturation proved to be less sensitive tothe chloride channel blockers and more sensitiveto chloride ion substitution. We propose that eventhe lowest PS concentration used stimulates pro-duction of more progesterone than is necessaryfor the induction of oocyte maturation. Hence, itspartial reduction is not accompanied by the inhi-bition of oocyte maturation. The higher sensitiv-ity of oocyte maturation to chloride-deficientmedia can be explained by their stimulating ef-fect on Ca2+ level in the oocyte, in addition to itsaction on progesterone production. It is knownthat oocyte maturation can be stimulated by in-creased or decreased concentration of extracellu-lar Ca2+ (Cicirelli and Smith, ’87).

To answer the question whether the influenceof chloride channel blockers and chloride-deficientmedia is connected with the intracellular Ca2+

level, Ca2+ should be assayed in the follicle cellsof the FEOs stimulated with gonadotropins in dif-ferent conditions. It can not also be excluded thatSITS, DIDS and chloride-deficient media affectedcAMP level differently in different systems. Wecould not find any published data on chloride mea-surements in the steroidogenic cells and we aregoing to carry out these studies further.

Fig. 7. The influence of substitution of sodium chloridefor equimolar concentration of sodium methanesulfonate onthe progesterone production (A) and maturation (B) of thefollicle-enclosed oocytes of Xenopus laevis stimulated by hCG.For details see Figure 1.

deficient media can be explained by an increasedintracellular Ca2+ level. Two facts favor this sug-gestion: (1) substitution of chloride ions forisothionate prevents the agonist-stimulated Ca2+

ion efflux from the rat neonatal pituitary (Acs etal., ’93). If the intracellular Ca2+ level of amphib-ian follicle cells is also elevated in the chloride-deficient media, one could expect an increasedprogesterone production in the amphibianFEOs, since it is calcium-dependent (Kleis-SanFrancisco and Schuetz, ’86, ’87); and (2) addi-tion of Ca2+ ions to gluconate solution leads tosignificant differences in the rate of spontane-ous maturation of FEOs. If the rate of oocytematuration in gluconate solution is low, addi-tion of Ca2+ ions increases it; if it is high, de-creases it (Skoblina et al., ’96).

It was shown earlier that the increased extra-cellular calcium concentration stimulated proges-terone production by the amphibian FEOs, but

428 M.N. SKOBLINA AND I. HUHTANIEMI

ACKNOWLEDGMENTSThe skillful technical assistance of Ms. Olga T.

Kondratieva, Ms. Galina P. Nikiforova, Ms. AiliMakitao, and Ms. Tarja Laiho is gratefully ac-knowledged. This study was supported by grantsfrom The Academy of Finland and Russian Foun-dation for Basic Research (96-04-49109).

LITERATURE CITEDArellano, R.O., and R. Miledi (1993) Novel Cl– currents

elicited by follicle-stimulating hormone and acetylcho-line in follicle-enclosed Xenopus oocytes. J. Gen. Physiol.,102:833–857.

Arellano, R.O., and R. Miledi (1994) Osmo-dependent Cl– cur-rents activated by cyclic AMP in follicle-enclosed Xenopusoocytes. Proc. R. Soc. Lond., 258:229–235.

Acs, Z., L. Zsom, Z. Mergl, and G.B. Makara (1993) Signifi-cance of chloride channel activation in the gammaamino-butyric acid induced growth hormone secretion in theneonatal rat pituitary. Life Sci., 52:1733–1739.

Bartel, D., S. Lepke, G. Layh-Schmitt, B. Legrum, and H.Passow (1989) Anion transport in oocytes of Xenopus laevisinduced by expression of mouse erythroid band 3 protein-encoding cRNA and of a cRNA derivative obtained by site-directed mutagenesis at the stilbene disulfonate binding site.EMBO J., 8:3601–3609.

Chao, C., and Y. Katayama (1991) Regulation of endogenouschloride conductance in Xenopus oocytes. Biochem. Biophys.Res. Commun., 180:1377–1382.

Chipperfield, A.R., and D.J. Fry (1982) Potassium and chlo-ride influxes in immature amphibian oocytes: Evidence fora diuretic-sensitive cotransport. J. Physiol., 322:24P.

Choi, M.S.K., and B.A. Cooke (1990) Evidence of two inde-pendent pathways in the stimulation of steroidogenesis byluteinizing hormone involving chloride channels and cyclicAMP. FEBS Lett., 261:402–404.

Cicirelli, M.F., and L.D. Smith (1987) Do calcium andcalmodulin trigger maturation in amphibian oocytes? Dev.Biol., 121:48–57.

Fortune, J.E. (1983) Steroid production by Xenopus ovarianfollicles at different developmental stages. Dev. Biol.,99:502–509.

Goncharov, B.F. (1978) The influence of the composition ofculture medium on the capacity of follicle-enclosed oocytesof sturgeons to mature after treatment with gonadotropichormones. Problems of early fish ontogenesis. (in Russian)Naukova dumka, Kiev, pp. 77–78.

Kaila, K., B. Rydqvist, M. Pasternack, and J. Voipio (1992)Inward current caused by sodium-dependent uptake ofGABA in the crayfish stretch receptor neurone. J. Physiol.,453:627–645.

Katayama, Y., and J.H. Widdicombe (1990) Halide transportby Xenopus oocytes. J. Cell Biol., 111:64a.

Keicher, E., and R. Meech (1994) Endogenous Na+-K+ (orNH4

+)-2Cl– cotransport in Rana oocytes: Anomalous effectof external NH4

+ on pHi. J. Physiol., 475:45–57.Kimura, S., B.C. Robison, and N. Kraus-Friedmann (1988)

Inhibitor of anion transport, DIDS, releases Ca2+ from he-patic microsomes. Biochem. Biophys. Res. Commun.,151:396–401.

Kleis-SanFrancisco, S., and A.W. Schuetz (1986) Calciumeffects on progesterone accumulation and oocyte matu-

ration in cultured follicles of Rana pipiens. J. Exp. Zool.,240:265–273.

Kleis-SanFrancisco, S., and A.W. Schuetz (1987) Sources ofcalcium and the involvement of calmodulin during steroido-genesis and oocyte maturation in follicles of Rana pipiens.J. Exp. Zool., 244:133–143.

Lessman, C.A., and A.W. Schuetz (1982) Insulin induction ofmeiosis of Rana pipiens oocytes: Relation to endogenousprogesterone. Gamete Res., 6:95–106.

Masui, Y., and H.J. Clarke (1979) Oocyte maturation. Int.Rev. Cytol., 57:185–282.

Morley, P., J.L. Schwartz, J.F. Whitfield, and B.K. Tsang(1991) Role of chloride ions in progesterone production bychicken granulosa cells. Mol. Cell Endocrinol., 82:107–115.

Noulin, J.-F., and M. Joffre (1993) Cyclic AMP- and calcium-activated chloride currents in Leydig cells isolated frommature rat testis. Archiv. Intern. Physiol. Bioch. Biophys.,101:35–41.

Patino, R., and R.T. Parkiss (1993) Inhibitory effects of n-alkanols on hormonal induction of maturation in follicle-enclosed Xenopus oocytes: Implications for gap junctionaltransport of maturation-inducing steroid. Gen. Comp.Endocrinol., 91:189–198.

Romero, P.J., and C.E. Ortiz (1988) Electrogenic behavior ofthe human red cell Ca2+ pump revealed by disulfonicstilbens. J. Membr. Biol., 101:237–246.

Russell, J.M., and M.S. Brodwick (1979) Properties of chlo-ride transport in barnacle muscle fibers. J. Gen. Physiol.,73:343–365.

Schlichter, L.C. (1989) Ion channels in Rana pipiens oocytes.Changes during maturation and fertilization. In: Mecha-nism of Egg Activation. R. Nuccitelly, G.N. Cherr, W.H.Clark, eds. Plenum Press, New York, pp. 89–131.

Schuetz, A.W., and C.A. Lessman (1982) Evidence for folliclewall involvement in ovulation and progesterone productionby frog (Rana pipiens) follicles in vitro. Differentiation,22:79–84.

Skoblina, M.N., and O.T. Kondratieva (1992) Follicle cell nu-clei do not control amphibian oocyte maturation inducedby gonadotropic hormones in vitro. Russian J. Dev. Biol.,23:289–292.

Skoblina, M.N., T. Matikainen, and I. Huhtaniemi (1995a)Involvement of cAMP in inhibition of maturation of follicle-enclosed oocytes by actinomycin D in Xenopus laevis andRana temporaria. J. Exp. Zool., 273:142–148.

Skoblina, M.N., I. Huhtaniemi, and T. Matikainen (1995b)Nuclei of the follicle cells regulate steroidogenesis stimu-lated by the pituitary suspension in the common frog fol-licles in vitro. Russian J. Dev. Biol., 26:446–452.

Skoblina, M.N., O.T. Kondratieva, G.P. Nikiforova, and I.Huhtaniemi (1996) The influence of a chloride channelblocker and chloride-deficient media on spontaneous matu-ration of Rana temporaria and Xenopus laevis oocytes. Rus-sian J. Dev. Biol., 27:379–383.

Supplisson, S., R.T. Kado, and C. Bergman (1991) A possibleNa/Ca exchange in the follicle cells of Xenopus oocyte. Dev.Biol., 145:231–240.

Vaugham-Jones, R.D. (1979) Regulation of chloride in quies-cent sheep-heart Purkinje fibers studied using intracellu-lar chloride and pH-sensitive micro-electrodes. J. Physiol.,295:111–137.

Vuorento, T., A. Lahti, O. Hovatta, and I. Huhtaniemi (1989)Daily measurements of salivary progesterone reveal a highrate of anovulation in healthy students. Scand. J. Clin. Lab.Invest., 49:395–401.