18. R. E. Nyholm and G. A. Currie, Br. J. Cancer37, 337 (1978).

19. D. W. Hedley and G. A. Currie, ibid., p. 747.20. J. S. Haskill, J. W. Proctor, Y. Yamamura, J.

Natl. Cancer Inst. 54, 387 (1975).21. J. L. Boak and T. C. Agurunobi, in The Macro-

phage and Cancer, K. James, W. McBride, A.Stuart, Eds. (James McBride and Stuart, Edin-burgh, 1977), p. 418; J. Rhodes and N. Plow-man, in preparation.

22. R. Evans, Transplantation 14, 468 (1972); R. S.Kerbel and H. F. Pross, Int. J. Cancer 18, 432(1976).

23. J. Rhodes, Nature (London) 257, 597 (1975); R.S. Bar, C. R. Kahn, H. S. Koren, ibid. 265, 632(1977); R. H. Muschel, N. Rosen, 0. M. Rosen,

be regulated by gonadotropins.

The functional relation between theamphibian oocyte and the single layer offollicle cells that surrounds it is not wellunderstood. Follicle cells have been im-plicated in a number of gonadotropin-regulated processes involved with am-phibian oocyte growth and development.These include steroidogenesis (1), initia-tion of yolk protein (vitellogenin) uptake(2), and increases in amino acid uptakeand protein synthesis in the oocyte (3).Ultrastructural examination of the ovaryof Xenopus has shown that the folliclecells possess numerous macrovilli whichproject through the vitelline envelopeand contact the oocyte surface, formingjuhctional complexes of an unidentified

B. R. Bloom, J. Immunol. 119, 1813 (1977).24. 0. Oelz, E. R. Foresch, H. F. Bunzli, R. E.

Humbel, W. J. Ritschard, in Handbook ofPhys-iology, vol. 1, Endocrinology, D. F. Steiner andN. Frienkel, Eds. (Williams & Wilkins, Balti-more, 1972), p. 685.

25. T. Hyodo, K. Kegyesi, C. R. Kahn, J. P.McLean, H. G. Friesen, J. Clin. Endocrinol.Metab. 44, 1175 (1977); J. E. DeLarco and G. J.Todaro, Nature (London) 272, 356 (1978).

26. We thank N. M. Bleehen, N. Plowman, D.Rapp, D. K. C. Cooper, and F. Howlett and hisstaff for cooperation. Supported by the CancerResearch Campaign of Great Britain.

17 August 1978

nature with the oocyte membrane (4).Recently we have observed, with the aidof lanthanum tracers, that there aresmall gap junctions between the folliclecell macrovilli and the oocyte in Xen-opus (Fig. IA). Gap junctions have alsobeen identified in the mammalian ovarybetween granulosa cells and oocytes (5).In this report we present evidence thatthe junctional complexes between theamphibian oocyte and its follicle cells aregap junctions and that these junctionalcomplexes may be hormonally regulat-ed.Gap junctions, through their structural

modifications of the membranes of ad-jointed cells allow for cell to cell commu-

Fig. 1. (A) A small gap junction between a follicle cell macrovil4is (MA) and an oocyte (0)demonstrated by means of a lanthanum tracer. (B) Phase-contrast and (C) fluorescent micro-scope images of the same field of follicle cells from an oocyte injected with 6-carboxyfluoresceinfrom an hCG-stimulated animal. (D) Two oocytes enclosed in a common follicle (FW, follicularwall) but having individual follicle cell layers (arrows); BV, blood vessels in theca.

182 0036-8075/79/0112-0182$00.50/0 Copyright K 1979 AAAS

nication by the passage of ions and ofmolecules of low molecular weight (6).To determine whether the junctionalcomplexes observed between the am-phibian oocyte and its surrounding fol-licle cells are gap junctions, we isolatedcomplete follicles (4) from Xenopus ova-ries and injected approximately 50 nl ofthe fluorescent dye, 6-carboxyfluores-cein through the follicular wall into theoocytes. Fluorescein and its derivativespass freely through gap junctions, but donot readily permeate nonjunctionalmembranes (6). The injected dye was al-lowed to diffuse through the ooplasm for1 to 2 hours. Smaller injection volumesor shorter diffusion times result only inlower levels of fluorescence in respond-ing oocyte-follicle cell complexes. Twohours after injection the oocytes in theirfollicles were transferred to a solution of1 mM phenylarsine oxide in a double-strength salt solution [OR 2 (7)]. Phenyl-arsine oxide, a sulfhydryl agent, stiffensthe theca, the follicle cell layer, and theacellular vitelline envelope, while the hy-pertonic solution causes the oocyte toshrink slightly and separate from thetheca and the follicle cell layer. This sep-aration allows the two layers to be easilyremoved, one after the other, withwatchmaker's forceps. Very often the vi-telline envelope and the follicle cell layerare removed as a single unit. For ourpurposes this technique proved to be su-perior to the standard means for remov-ing follicle cells which involves placingdissected oocytes in Ca2+-Mg2+-free orEDTA-containing media (8, 9).

Follicle cell layers were examinedfrom stage IV, V, and VI oocytes (10).Oocytes were taken from unstimulatedanimals and from animals that had beenstimulated with 1000 I.U. of human cho-rionic gonadotropin (hCG) (11) 24 hourspreviously. Ten healthy females werestimulated with hCG. Nearly all folliclecell layers removed from the fluorescein-injected oocytes of the ten stimulated fe-males showed fluorescence, indicative ofthe passage of a dye through a gap junc-tion (Fig. 1, B and C). Fluorescence wasobserved in the follicle cell layers from,oocytes of all stages examined. Normal-ly not all of the follicle cells from thesame oocyte fluoresced with the same in-tensity, ,although occasionally all wereequally fluorescent. Neither uninjectedoocytes enclosed in their intact folliclesnor dissected oocytes onclosed only infollicle cells and exposed directly in fluo-rescein-containing medium cause fluo-rescence in follicle cells.

In some Xenopus ovaries, folliclescontaining two oocytes are present. Al-though these oocytes are enclosed within

SCIENCE, VOL. 203, 12 JANUARY 1979

Oocyte-Follicle Cell Gap Junctions in Xenopus laevis and theEffects of Gonadotropin on Their Permeability

Abstract. Junctions between Xenopus laevis oocytes and follicle cells have beenidentified as gap junctions by the passage of microinjectedfluorescent dye from oo-cytes tofollicle cells. The opening or assembly ofthesejunctions, or both, appears to

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a common theca, each oocyte has itsown follicle cell layer (Fig. ID). To showthat the fluorescence observed in folliclecells is due to direct passage of the dyefrom the oocyte to the follicle cells andnot due to leakage from the oocyte intothe medium, we conducted ten experi-ments in which only one oocyte of thedouble follicle pair was injected withdye. Only the follicle cells from the in-jected oocytes showed fluorescence,leading to the conclusion that the dyewas reaching the follicle cells directlyfrom the oocytes and not from the intra-follicular space.

Unstimulated animals that did not re-ceive hCG prior to dye injection hadvery low or no fluorescence in their fol-licle cells. This suggests that there werefew, if any, functional gap junctions be-tween these occytes and follicle cells. Theonly unstimulated animals that showedfluorescence in the follicle cells after in-jection of fluorescein into the oocyteswere 5 out of 13 females that had beenrecently acquired from the supply house(South African Snake Farm, Fish Hoek).Follicle cells from oocytes from tenunstimulated animals that had beenmaintained in the laboratory for longperiods of time did not fluoresce afterinjection of the dye into the oocyte.To examine further the effects of hCG

on intercellular communication betweenthe follicle cells and the oocyte, weanesthetized seven recently acquired un-stimulated animals by hypothermia andremoved a portion of the ovary. Afterthe incision was closed the animals wereinjected with 1000 I.U. of hCG. Oocytesfrom the excised ovary were injectedwith fluorescein and the follicle cellswere examined for fluorescence. Twen-ty-four hours later, partial ovariectomieswere again performed on the now hCG-stimulated animals, the o6cytes injectedwith fluorescein, and the level of fluores-cence in the follicle cells noted. In allseven animals the follicle cells shpwedlittle or no fluorescence. Twenty.fourbours after hCG stimulation, however,joth the number of follicle cells that fluo-

resced and the intensity of their fluores-cence was greatly increased.Apparentty hCG, which seems to elicit

effects similar to endogenous pituitarygonadotropins in Xenopus, stimulatesthe opening or assembly of gap junctionsbetween the oocyte and its follicle cells.Variations in the levels of fluorescence inadjacent follicle cells suggest that the ef-fect of hCG may be at the level of indi-vidual cells rather than affecting all cellsof the follicular layer simultaneously.Such variations also imply that the fol-12 JANUARY 1979

licle cells may not be completely inter-connected by functional gap junctions asare mammalian granulosa cells (5).

If gonadotropin stimulation is respon-sible for the opening or assembling offunctional gap junctions between theoocyte and follicle cells, the low levels offluorescence found in follicle cells from asmall number of unstimulated dye-inject-ed oocytes is not surprising. The consid-erable variation observed in the rate ofvitellogenin uptake in unstimulated (con-trol) animals in vitro (2) suggests thatnatural hormonal levels are difficult tocontrol under long-term laboratory con-ditions. This is supported by the obser-vation that the unstimulated laboratory-maintained animals whose oocyte-fol-licle cell junctions either were not pres-ent or did not pass dye had a poor ovula-tory response to hCG, whereas all newlyacquired animals that showed some pas-sage of dye prior to hCG stimulation andenhanced fluorescence after hCG stimu-lation had excellent ovulatory responses.It is possible that higher levels of endog-enous gonadotropins may have beenpresent in those animals which showedthe presence of functional gap junctionswithout exogenous hormonal stimula-tion.The functional significance of the gap

junctions between the amphibian oocyteand its surrounding follicle cells is stillunclear. In response to hCG stimulationthe follicle cells secrete two clearly dif-fusible steroid hormones, one similar toprogesterone that leads to oocyte matu-ration (9), and estrogen, which inducesthe synthesis and secretion of the yolkprecursor protein vitealogenin by theliver (12). It has also been postulated thatthe follicle cells may be the source of arelatively nondiffusible factor which in-fluences endocytotic uptake of vitelloge-nin by the oocyte in response to gonado-tropic stimulation (2). Since it appearsthat follicle cells must be present on thesurface of the oocyte for initiation of thein vitro uptake of vitellogenin, it is pos-sible that this "initiator factor" is trans-mitted from the follicle cells to theoocyte directly by means of the gap junc-tions. In the mammalian ovary, the oo-cyte-granulosa cell gap junctions havebeen suggested as a means of maintain-ing meiotic arrest in the oocyte by trans-mitting an inhibitor from the follicle cells(13). There are as yet no data to supportthe theory of a similar function in am-phibians.

It is significant not only that the gapjunctions between the amphibian ooeyteand its follicle cells may serve to trans-mit a hormonal stimulus, but also that

the means of communication, a per-meable gap junction, is hormonally regu-lated. It has been proposed that an im-portant function of gap junctions is to al-low the synchronized movement of smallregulatory molecules between function-ally related cells during growth and dif-ferentiation (14). More recently it wasshown that rat granulosa cells pass hor-monal stimuli by means of a second mes-senger through gap junctions formed inculture with mouse myocardial cells (15).Thus, in hormonally regulated devel-oping systems such as the amphibianoocyte, gap junctions may allow the pas-sage of secondary messages from the fol-licle cells to the oocyte in response tohormonal stimuli. Many aspects ofXen-opus oocyte growth and differentiation.such as vitellogenin and amino acid up-take, protein synthesis, and maturationare under the control of hormones andcan be induced in vivo and in vitro withhCG. Thus the ovary of Xenopus pro-vides an excellent system in whichthe transmission of hormonal stimulithrough naturally occurring junctionscan be studied with respect to growthand differentiation.

CAROLE L. BROWNEH. STEVEN WILEY

University ofTennessee-Oak RidgeGraduate School ofBiomedicalSciences, Biology Division,Oak Ridge National Laboratory,Oak Ridge, Tennessee 37830

JAMES N. DUMONTBiology Division,Oak Ridge National Laboratory

References and Notes1. M. R. Redshaw,Am. Zool. 12, 289 (1972); A. W.

Schuetz, Adv. Reprod. Physiol. 4, 100 (1969).2. H. S. Wiley and J. N. Dumont, Biol. Reprod., in

press.3. R. L. Hallberg and D. C. Smith, Dev. Biol. 18,

303 (1976).4. J. N. Dumont and A. R. Brummett, J. MorphoL

155, 73 (1978).5. D. F. Albertini and E. J. Anderson, J. Cell Biol.

63, 234 (1974).6. Y. Kanno and W. R. Loewenstein, Nature

(London) 212, 629 (1966).7. R. A. Wallace, D. W. Jared, J. N. Dumont, M.

W. Sega, J. Exp. Zool. 184, 321 (1973).8. R. A. Wallace, T. Ho, D. W. Salter, D. W. Jar-

ed, Exp. Cell Res. 82, 287 (1973).9. Y. Masui, J. Exp. Zool. 166, 365 (1967).

10. J. N. Dumont, J. Morphol. 136, 156 (1972).11. R. A. Wallace and D. W. Jared, Can. J. Bio-

chem. 46, 953 (1968).12. __, Dev. Biol. 19, 498 (1969).13. E. Anderson and D. F. Albertini, J. Cell Biol.

71, 680 (1976).14. W. R. Loewenstein, Dev. Biol. Suppl. 2, 157

(1968).15. T.S. Lawrence, W. H. Beers, N. B. Gilula, Na-

ture (London) 272, 501 (1978).16. C.L.B. was supported by subcontract 3322 from

the Biology Division of Oak Ridge NationalLaboratory to the University of Tennessee and-H.S.W. by grant 6M-07431-Cell Biology fromNational Institute of General Medical Sciences.The research was sponsored by the Division ofBiomedical and Environmental Research, U.S.Department of Energy, under contract W-7405-eng-26 with the Union Carbide Corporation. Wethank B. Rose for the 6-carboxyfluorescein.

30 May 1978; revised 18 July 1978

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