D EVELO PM EN T AL BIO LO G Y 181, 47 –63 (1997) ART IC LE N O . D B968441 D istinct Stress-Inducible and D evelopm entally Regulated Heat Shock Transcription Factors in Xenop us Oocytes Sandra Gordon,1 Steve Bharadw aj, Alex Hnatov, Adnan Ali, and N ick Ovsenek 2 D epart m en t of A n at om y an d C ell Biology , C ollege of M ed icin e, U n iv ersit y of Sask at ch ew an , 107 W iggin s R oad , Sask at oon , Sask at ch ew an , C an ad a S7N 5E5 The presence of a m aternal pool of heat shock factor (HSF) in Xenop us oocytes has been suggested by tw o lines of evidence from previous studies. First, heat shock response elem ent (HSE)-binding activity is induced in heat-shocked eggs and em bryos prior to expression of zygotic HSF. Second, expression from m icroinjected heat shock protein prom oters in oocytes is induced upon heat shock. To date, how ever, endogenous oocyte HSF m olecules have not been detected, nor has induction of HSE-binding activity been directly dem onstrated. Here w e report the detection of distinct stress-inducible and developm entally regulated HSE-binding activities of endogenous oocyte factors. Exposure of defolliculated oocytes to heat, cadm ium , and arsenite resulted in the form ation of an HSE-specific com plex detectable by gel m obility shift assay. Induction of HSE-binding activity by each of these stressors corresponded to increased expression from a m icroinjected hsp70 prom oter. The stress-inducible HSE-binding com plex w as recognized by antiserum against m am m alian HSF1, but not by HSF2 antiserum , suggesting that a Xenop us hom ologue of HSF1 is the m ajor com ponent of this activity. The HSE-binding activity of HSF1 w as induced by stress treatm ents of stage I through VI oocytes, an indication that it is responsive to stress throughout oogenesis. D uring recovery from heat shock, the HSF1 –HSE com plex rapidly declined to control levels, but w as induced for prolonged periods in oocytes exposed to continuous stress, a pattern unlike the transient activation previously observed in fertilized eggs or em bryos. The kinetics of HSF1 activation in oocytes suggests that a key protein(s) regulating attenuation of the stress response is present at exceedingly low levels or is som ehow m odified during preem bryonic developm ent. We also detected an unusual constitutive HSE-binding com plex in unstressed stage I and II oocytes, but not in later stage oocytes, eggs, developing em bryos, or A6 cells. This constitutive com plex w as unaffected by heat or chem ical treatm ents and w as not recognized by either HSF1 or HSF2 antiserum . Appearance of the constitutive HSEbinding activity during oogenesis corresponded closely w ith peak levels of hsp70 m RN A detected by N orthern blot analysis of RN A from staged oocytes. We suggest that the constitutive HSE-binding activity in early oocytes is form ed by a unique developm entally regulated heat shock factor that m ay play a role in the expression of heat shock proteins during early stages of oogenesis. q 1997 Academ ic Press IN TROD U CTION Eu k aryot ic cells respon d t o st ress by rapidly in creasin g t h e syn t h esis of a set of h eat sh ock prot ein s (H SPs) (review ed by M orim ot o et al., 1990). G en es en codin g t h e H SP fam ily of prot ein s are su bject t o com plex regu lat ory m ech an ism s, as illu st rat ed by H SP expression in respon se t o a 1 2 T h e fi rst t w o au t h ors con t ribu t ed equ ally t o t h is m an u script . T o w h om correspon den ce sh ou ld be addressed. w ide variet y of st ress st im u li, as w ell as expression in t h e absen ce of st ress du rin g developm en t an d differen t iat ion , ch an ges in t h e cell cycle, an d exposu re t o grow t h fact ors (review ed by M orim ot o et al., 1990). In m ost eu k aryot es, t h e k ey bioch em ical st ep facilit at in g st ress-in du ced t ran script ion of H SPs in volves post t ran slat ion al act ivat ion of a con st it u t ively expressed t ran script ion act ivat or, t h e h eat sh ock fact or (H SF), w h ich bin ds t o h igh ly con served h eat sh ock respon se elem en t s (H SEs) presen t in t h e u pst ream regu lat ory region of all h eat sh ock gen es (Lis an d Wu , 1993; M orim ot o, 1993). U pon exposu re t o st ress, H SF is con vert ed 0012-1606/ 97 $25.00 C opyrigh t q 1997 by Academ ic Press All righ t s of reprodu ct ion in an y form reserved. AID DB 8441 / 6x15$$$$81 47 12-09-96 18:37:34 dbas 48 G ord on et al. from an in act ive m on om er t o a h om ot rim er w it h h igh -affi n it y H SE bin din g act ivit y (Wu et al., 1987; M osser et al., 1988; West w ood et al., 1991; West w ood an d Wu , 1993). Alt h ou gh H SF is regu lat ed prim arily at t h e level of oligom erizat ion an d D N A bin din g, fu rt h er post t ran slat ion al m odifi cat ion s m ay be requ ired for fu ll act ivat ion of t h e st ress respon se (Larson et al., 1988). In vert ebrat es, t h e H SF fam ily of prot ein s in clu des H SF1, H SF2, an d H SF3 (Wiederrech t et al., 1988; Sorger an d Pelh am , 1988; C los et al., 1990; Rabin dran et al., 1991; Sarge et al., 1991; Sch u et z et al., 1991; N ak ai an d M orim ot o, 1993). H SF1 m ediat es t h e rapid in du ct ion of H SP gen e expression ch aract erist ic of t h e classic st ress respon se (Rabin dran et al., 1993; Sarge et al., 1993; Baler et al., 1993). In con t rast , H SF2 is n ot act ivat ed by st ress, bu t appears t o acqu ire H SE-bin din g abilit y in cert ain cells du rin g differen t iat ion an d developm en t (Sist on en et al., 1992; M u rph y et al., 1994; Sarge et al., 1994; Sist on en et al., 1994). H SF3 sh ares som e of t h e propert ies of H SF1, bu t it s act ivit y h as on ly been described in a rest rict ed su bset of avian cells (N ak ai et al., 1995). Sequ en ce com parison s of H SF prot ein s from differen t species reveal several com m on feat u res in clu din g a con served h elix –t u rn –h elix class D N A-bin din g dom ain (H arrison et al., 1994, Vu ist er et al., 1994), an adjacen t am in o-t erm in al t rim erizat ion dom ain con t ain in g leu cin e zipper m ot ifs (Sorger an d N elson , 1989; Pet eran dl an d N elson , 1992), a carboxy-t erm in al leu cin e zipper w h ich appears t o regu lat e oligom erizat ion (Lis an d Wu , 1993; N ak ai an d M orim ot o, 1993; Rabin dran et al., 1993; Z ou et al., 1994), an d t ran script ion act ivat ion dom ain s n ear t h e C -t erm in u s (Sorger, 1990; G reen et al., 1995; Z ou et al., 1995; Sh i et al., 1995). T h e m ech an ism by w h ich cells det ect variou s st ress st im u li an d gen erat e t h e sign al(s) m odu lat in g H SF act ivit y h as becom e on e of t h e cen t ral qu est ion s of t h e st ress respon se. H SP gen e expression appears t o be st rict ly regu lat ed t o en su re t h at it is proport ion al t o t h e severit y of st ress an d sw it ch ed off u pon resu m pt ion of n orm al en viron m en t al an d ph ysiological con dit ion s. At t en u at ion of t h e t ran script ion al respon se du rin g prolon ged exposu re of cells t o m ild st ress is associat ed w it h con version of t h e H SF t rim er t o in act ive m on om ers (C los et al., 1990; Rabin dran et al., 1991; N ak ai an d M orim ot o, 1993). It h as been proposed t h at t h e oligom erizat ion an d D N A-bin din g st at e of H SF is regu lat ed by t h e in t racellu lar level of free H SP70 fam ily prot ein s (C raig an d G ross, 1991; M orim ot o, 1993). Alt h ou gh experim en t s aim ed at t est in g t h e au t oregu lat ory effect s of H SP70 in v iv o su pport an act ive role for m odu lat in g t h e H SE-bin din g act ivit y of H SF (M osser et al., 1993; Rabin dran et al., 1994), a m ech an ism in volvin g free in t racellu lar H SPs as a cellu lar st ress sen sor h as n ot been clearly est ablish ed. A con siderable degree of u n cert ain t y h as su rrou n ded earlier st u dies of t h e st ress respon se in X en opu s oocyt es. In fact , t h e oocyt e w as at on e t im e t h ou gh t t o represen t an except ion t o t h e u n iversalit y of t h e h eat sh ock respon se becau se in it ial experim en t s su ggest ed t h at H SP m RN A w as n ot in du ced u pon h eat sh ock an d t h at H SP syn t h esis w as con t rolled prim arily at t h e level of t ran slat ion (Bien z an d G u rdon , 1982; Bien z, 1984a,b). Alt h ou gh su bsequ en t experim en t s con fi rm ed t h at t ran script ion of H SP gen es in h eat -sh ock ed oocyt es is exceedin gly low , it w as sh ow n t h at follicle cells su rrou n din g t h e oocyt e con t ribu t ed t o t h e expression of H SPs fi rst t h ou gh t t o represen t t ran slat ion al u pregu lat ion of preexist in g m RN A (H orrell et al., 1987; Kin g an d D avis, 1987). In con sist en t resu lt s h ave also been obt ain ed u pon in ject ion of report er con st ru ct s u n der t h e con t rol of H SP prom ot ers. Wh ile in som e experim en t s H SP70 prom ot ers direct ed con st it u t ive expression in oocyt es (Bien z, 1984a; Bien z, 1986; H orrell et al., 1987), ot h er sh ow ed in du cible t ran script ion in respon se t o h eat st ress or coin ject ion of den at u red prot ein in du cers (Voellm y an d Ru n gger, 1982; An an t h an et al., 1986; M iffl in an d C oh en , 1994). Wolffe an d colleagu es h ave recen t ly accou n t ed for variabilit y of expression from H SP70 prom ot ers in t h e oocyt e by dem on st rat in g t h at appropriat e regu lat ion is depen den t u pon effi cien t assem bly of in ject ed t em plat e D N A in t o ch rom at in (Lan dsberger an d Wolffe, 1995; Lan dsberger et al., 1995). T h u s, t h e X en opu s oocyt e appears t o be com pet en t for t h e st ress respon se at t h e t ran script ion al level, even t h ou gh in creased H SP gen e t ran script ion is n ot apparen t from t h e gen om ic D N A in a sin gle n u cleu s (H orrell et al., 1987). D espit e t h e con t roversy arisin g from earlier st u dies, t h e X en opu s oocyt e h as em erged as a con ven ien t m odel syst em in w h ich t o st u dy t h e act ivit ies of in ject ed H SF m olecu les. T h is h as been illu st rat ed by expression of clon ed D rosoph ila H SF in oocyt es sh ow in g part ial su ppression of H SE-bin din g at t h e n orm al grow t h t em perat u re of X en opu s (C los et al., 1990) an d t h e u se of oocyt es by Voellm y an d colleagu es for m u t agen ic an alyses of h u m an H SF1 (Baler et al., 1993; Z ou et al., 1994, 1995). A k ey assu m pt ion of t h ese experim en t s, h ow ever, is t h at en dogen ou s oocyt e H SF does n ot con t ribu t e su bst an t ially t o t h e act ivit y of in ject ed fact ors. Wh ile t h is is con sist en t w it h t h e failu re t o det ect H SF w it h an t ih u m an H SF1 an t ibodies in oocyt e ext ract s, or H SE-bin din g act ivit y in gel sh ift assays (Z ou et al., 1994; St u m p et al., 1995; Lan dsberger an d Wolffe, 1995), it is n ot com pat ible w it h st ron g eviden ce in su pport of a m at ern al pool of H SF in clu din g H SE-bin din g act ivit y in u n fert ilized eggs an d em bryos before expression from t h e em bryon ic gen om e at lat e blast u la (O vsen ek an d H eik k ila, 1990; Karn et al., 1992) an d t ran script ion al u pregu lat ion from m icroin ject ed H SP70 prom ot ers in t h e absen ce of exogen ou s H SF (Lan dsberger et al., 1995). U n t il n ow , h ow ever, t h e D N A-bin din g act ivit y of en dogen ou s oocyt e H SF h as n ot yet been exam in ed direct ly. Fu rt h erm ore, very lit t le is k n ow n abou t t h e early bioch em ical st eps of t h e st ress respon se du rin g vert ebrat e oogen esis. In t h is st u dy, w e h ave in vest igat ed t h e st ress respon se of t h e X en opu s oocyt e, an d report t h e det ect ion of t w o dist in ct D N A-bin din g act ivit ies of en dogen ou s H SF m olecu les. An en dogen ou s oocyt e H SE-bin din g act ivit y w as in du ced by h eat , cadm iu m , an d arsen it e at all st ages of oogen esis. T h is C opyrigh t q 1997 by Academ ic Press. All righ t s of reprodu ct ion in an y form reserved. AID DB 8441 / 6x15$$$$81 12-09-96 18:37:34 dbas 49 H SF A ct iv it ies in X en opu s O ocy t es st ress-in du cible act ivit y w as su persh ift ed by an t ibodies again st H SF1, bu t n ot by H SF2 an t ibodies. T h e D N A-bin din g act ivit y of oocyt e H SF1 persist ed t h rou gh ou t t h e du rat ion of prolon ged st ress t reat m en t s, su ggest in g t h at fact ors regu lat in g at t en u at ion of H SE-bin din g abilit y are lim it in g or m odifi ed in t h e oocyt e. In addit ion , w e report a dist in ct developm en t ally regu lat ed H SE-bin din g act ivit y presen t in st age I an d II oocyt es, bu t absen t in lat er st age oocyt es, em bryos, an d som at ic cells. T h is act ivit y w as n ot recogn ized in v it ro by eit h er H SF1 or H SF2 an t iseru m an d w as u n affect ed by st ress t reat m en t s of early oocyt es. N ort h ern blot experim en t s sh ow ed t h at h sp70 m RN A levels are also m axim al in early st age oocyt es. T ak en t oget h er, t h ese dat a provide eviden ce of a u n iqu e, developm en t ally regu lat ed H SF w h ich m ay play a role in expression of h eat sh ock prot ein gen es in previt ellogen ic oocyt es. MATERIALS AN D METHOD S O ocy t es X en opu s laev is frogs w ere pu rch ased from Xen opu s I (An n Arbou r, M I). O vary port ion s w ere su rgically obt ain ed from adu lt fem ale frogs an d follicle cells w ere rem oved from oocyt es by t reat m en t in calciu m -free O R2 bu ffer (82.5 m M N aC l, 2.5 m M KC l, 1 m M M gC l 2 , 1 m M N aH 2 PO 4 , 5 m M H epes, pH 7.8, 10 m g/ lit er st rept om ycin su lfat e, 10 m g/ lit er ben zyl pen icillin ) con t ain in g 2 m g/ m l collagen ase (t ype II, Sigm a) for 2 –3 h r at 187C . O ocyt es w ere w ash ed ext en sively an d allow ed t o recover overn igh t at 187C in O R2 (as above /1 m M C aC l 2 ; Wallace et al., 1973). Effi cien cy of follicle cell rem oval w as m on it ored by fl u orescen ce m icroscopy aft er st ain in g oocyt es in 1 mg/ m l H oecsh t dye (H orrell et al., 1987). In som e experim en t s, oocyt es w ere separat ed in t o pools represen t in g each of t h e in dividu al st ages accordin g t o t h e crit eria described by D u m on t (1972). C on t rol oocyt es w ere m ain t ain ed at 187C in O R2, ch em ically st ressed oocyt es w ere in cu bat ed in O R2 su pplem en t ed w it h sodiu m arsen it e or cadm iu m ch loride, an d h eat sh ock ed oocyt es w ere in cu bat ed in O R2 preequ ilibrat ed t o in dicat ed t em perat u res. In all experim en t s, at least 20 oocyt es w ere u sed for each sam ple. Follow in g st ress t reat m en t s, oocyt es w ere qu ick ly w ash ed in O R2 an d collect ed for prot ein ext ract s or for C AT expression an alysis. from X en opu s t est es an d rat ovary m at erial w ere m ade essen t ially as described above. G el Mob ilit y Shift A ssay s D N A m obilit y sh ift assays w ere perform ed as previou sly described (O vsen ek an d H eik k ila, 1990). D N A-bin din g react ion s w it h eit h er em bryon ic or st age V an d VI oocyt e sam ples con t ain ed 10 ml ext ract (on e em bryo equ ivalen t is approxim at ely 20 mg solu ble prot ein ). For experim en t s u sin g early st age oocyt es, prot ein levels w ere qu an t ifi ed w it h a Bio-Rad k it (Bio-Rad, H ercu les, C A), equ ivalen cy w as ch eck ed by C oom assie st ain in g of SD S–polyacrylam ide gels, an d ext ract volu m es w ere adju st ed so t h at equ al prot ein (20 mg) w as added t o each bin din g react ion . H SE oligon u cleot ide probes u sed in ou r assays w ere as described in O vsen ek et al. (1990). Bin din g react ion s w ere perform ed in t h e presen ce of 1 mg poly(dI-dC ), 10 m M T ris (pH 7.8), 50 m M N aC l, 1 m M ED T A, 0.5 m M dit h iot h reit ol, an d 5% glycerol, in a fi n al volu m e of 20 ml. React ion s w ere in cu bat ed on ice for 20 m in an d im m ediat ely loaded on t o 5% n on den at u rin g polyacrylam ide gels con t ain in g 6.7 m M T ris –C l (pH 7.5), 1 m M ED T A, 3.3 m M sodiu m acet at e. G els w ere elect roph oresed for 2.5 h r at 150 V, dried, an d exposed overn igh t t o X-ray fi lm (Kodak X-om at 5). For su persh ift an alysis, polyclon al an t iseru m again st eit h er H SF1 or H SF2 (k in dly provided by D r. Kevin Sarge, D epart m en t Bioch em ist ry, C h an dler M edical C en t er, U n iversit y of Ken t u ck y; Sarge et al., 1993) w as in cu bat ed w it h ext ract s for 15 m in on ice prior t o addit ion of ext ract s in t o bin din g react ion s. N ort hern Blot A naly sis O ocyt es at differen t st ages of oogen esis w ere collect ed an d t ot al RN A w as isolat ed aft er h om ogen izat ion in T RIzol reagen t (G ibco-BRL, Bu rlin gt on , O n t ario, C an ada). Sam ples con t ain in g t ot al RN A (10 mg) w ere elect roph oresed on 1.2% form aldeh yde agarose gels an d t ran sferred t o H ybon d-N / m em bran e (Am ersh am , O ak ville, O n t ario, C an ada; Sam brook et al., 1989). H ybridizat ion react ion s w ere perform ed u sin g 32 P-labeled X en opu s h sp70B gen e probe (Bien z, 1984a). Aft er post h ybridizat ion w ash es, blot s w ere exposed t o X-ray fi lm at 0807C . Equ al loadin g of t h e RN A gels w as det erm in ed by spect roph ot om et ry an d by visu alizat ion of t h e RN A aft er st ain in g w it h et h idiu m brom ide. Au t oradiograph s w ere scan n ed on a U M AX Vist a-S6E scan n er an d relat ive m essage levels w ere det erm in ed u sin g t h e N IH Im age Version 1.59.1 an alysis program . M essage sizes w ere det erm in ed u sin g a st an dard RN A ladder (G ibco BRL). Prot ein Ext ract s For prot ein ext ract s, oocyt es w ere h om ogen ized in Bu ffer C (50 m M T ris –C l, pH 7.9, 20% glycerol, 50 m M KC l, 0.1 m M ED T A, 2 m M dit h iot h reit ol, 10 mg/ m l aprot in in , an d 10 mg/ m l leu pept in (D ign am et al., 1983)) in a D ou n ce h om ogen izer w it h a t igh t fi t t in g pest le. H om ogen at es w ere t ran sferred t o eppen dorf t u bes an d spu n for 5 m in at 15,000g (47C ). T h e resu lt an t su pern at an t s w ere rem oved t o a fresh t u be, im m ediat ely frozen in liqu id n it rogen , an d st ored at 0807C . O ocyt es (st age V an d VI) w ere h om ogen ized in 10 ml bu ffer C per oocyt e, w h ich t ypically yielded a fi n al prot ein con cen t rat ion of 2.0 m g/ m l. Early st age oocyt es w ere h om ogen ized in t h e follow in g volu m es in order t o obt ain approxim at ely equ ivalen t fi n al prot ein con cen t rat ion (2.0 m g/ m l) for each sam ple: st ages III an d IV, 4 ml/ oocyt e; st ages I an d II, 1 ml/ oocyt e. Prot ein ext ract s O ocy t e Inject ions and C A T A ssay s Plasm id con st ru ct s u sed for m icroin ject ion experim en t s w ere t h e h u m an C M V-C AT an d t h e X en opu s H sp70-C AT clon es (k in dly provided by D r. Alan Wolffe, N IC H H D , N at ion al In st it u t es of H ealt h , Bet h esda, M D ) previou sly described in Lan dsberger et al. (1995). Follow in g defollicu lat ion , oocyt es w ere in cu bat ed for several h ou rs at 187C , aft er w h ich h ealt h y oocyt es w ere select ed an d in ject ed in t o n u clei w it h 20 n l of a solu t ion con t ain in g 100 n g/ ml (2 n g) of eit h er C M V-C AT or H sp70-C AT plasm id u sin g a N arash ige IM 300 m icroin ject or. Aft er in cu bat ion overn igh t at 187C , h ealt h y oocyt es w ere select ed an d st ressed for 2 h r by h eat sh ock at 337C or by in cu bat ion for 2 h r in 50 m M cadm iu m or 5 m M arsen it e at 187C . Follow in g t h ese t reat m en t s, oocyt es w ere in cu - C opyrigh t q 1997 by Academ ic Press. All righ t s of reprodu ct ion in an y form reserved. AID DB 8441 / 6x15$$$$82 12-09-96 18:37:34 dbas 50 G ord on et al. bat ed for an addit ion al 2 h r at 187C , w ash ed in O R2, an d assayed for C AT act ivit y. As a con t rol for oocyt e in ject ion s, D N A w as recovered from at least 5 in dividu als ou t of t h e pool of in ject ed oocyt es, an d t h e equ ivalen cy of in ject ed plasm id D N A w as con fi rm ed by Sou t h ern blot t in g. C AT assays w ere perform ed u sin g 1 oocyt e equ ivalen t of w h ole cell ext ract from u n in ject ed or in ject ed oocyt es as previou sly described (O vsen ek et al., 1990). A pool of at least 20 oocyt es w ere u sed for each an alysis. T h e acet ylat ed produ ct s w ere separat ed by t h in -layer ch rom at ograph y an d visu alized by au t oradiograph y. C AT act ivit y w as qu an t ifi ed by scin t illat ion cou n t in g an d expressed as percen t age con version of ch loram ph en icol t o acet ylat ed form s. RESU LTS St ress Ind uct ion of an End ogenous HSE-Bind ing A ct iv it y in St age V I O ocy t es We in vest igat ed t h e D N A-bin din g act ivit ies of en dogen ou s H SF m olecu les in X en opu s oocyt es. Resu lt s from earlier w ork su ggest ed t h at en dogen ou s H SF m olecu les do n ot con t ribu t e t o t h e H SE-bin din g act ivit y observed in gel sh ift assays perform ed w it h ext ract s from oocyt es in ject ed w it h clon ed H SF m olecu les (C los et al., 1990; Baler et al., 1993; Z ou et al., 1994, 1995). It h as been est ablish ed, h ow ever, t h at t h e oocyt e is capable of u pregu lat in g t ran script ion from m icroin ject ed D N A con st ru ct s con t ain in g H SP70 prom ot ers (Lan dsberger et al., 1995). Sin ce t h is expression w as presu m ably at t ribu t able t o in du ct ion of en dogen ou s H SF m olecu les, w e reason ed it w ou ld be possible t o det ect oocyt e H SE-bin din g act ivit y in h eat -sh ock ed oocyt es. In order t o t est for H SF act ivat ion , a gel m obilit y sh ift assay w as perform ed w it h oocyt e ext ract s an d an H SE oligon u cleot ide probe (Fig. 1A). Wh ile H SE-bin din g act ivit y w as n ot det ect ed in oocyt es in cu bat ed at con t rol t em perat u res (Fig. 1A, lan e 1), a prom in en t H SE–prot ein com plex w as in du ced aft er t reat m en t of oocyt es bet w een 30 an d 367C (Fig. 1A, lan es 4 –9). In du ct ion w as rapid, as det ect able levels of com plex w ere observed im m ediat ely aft er 30 m in of h eat sh ock (Fig. 1A, lan es 4, 6, an d 8). T h e resu lt s of t h is experim en t su ggest t h at t h e X en opu s oocyt e is com pet en t t o act ivat e t rim erizat ion of an en dogen ou s H SF, t h e prin cipal st ep in t h e st ress respon se pat h w ay. H eat -in du cible bin din g of en dogen ou s oocyt e H SF is en t irely con sist en t w it h previou sly observed t ran script ion al u pregu lat ion from in ject ed H SP70 prom ot ers (Lan dsberger et al., 1995; also see Fig. 2B). Absen ce of sign ifi can t H SE-bin din g act ivit y in con t rols (Fig. 1A, lan e 1) su ggest s t h at pu t at ive oocyt e H SF1 m olecu les are presen t as lat en t n on -D N A-bin din g m on om ers, an d m ay be n egat ively regu lat ed at t h e level of oligom erizat ion u n der n on st ress con dit ion s. Sin ce earlier st u dies h ave sh ow n t h at h eat sh ock , h eavy m et als, an d arsen it e t reat m en t s brin g abou t act ivat ion of t h e st ress respon se an d accu m u lat ion of H SP m RN A in developin g em bryos (H eik k ila et al., 1987), w e w ere prom pt ed t o exam in e t h e effect s of m et al an d arsen it e on H SE-bin din g act ivit y in t h e oocyt e (Fig. 1B an d 1C ). H SEbin din g act ivit y w as in du ced in oocyt es aft er a 1-h r exposu re t o eit h er cadm iu m ch loride (Fig. 1B, lan e 9) or sodiu m arsen it e (Fig. 1C , lan es 6 –8). T h ese resu lt s in dicat e t h at pu t at ive H SF1 m olecu les are act ivat ed at t h e level of D N A bin din g by at least t w o differen t form s of ch em ical st ress. M oreover, t h e oocyt e appears t o be com pet en t for t h e recept ion an d t ran sdu ct ion of disparat e st ress sign als. We con fi rm ed t h at t h e H SE-bin din g act ivit y observed in ou r experim en t s w as n ot at t ribu t able t o con t am in at in g follicle cells. Bat ch es of oocyt es u sed in t h ese experim en t s w ere det erm in ed t o be free of som at ic cells aft er st ain in g w it h H oesch t dye an d exam in at ion by fl u orescen ce m icroscopy (n ot sh ow n ). In addit ion , w e direct ly com pared t h e levels of in du cible H SE-bin din g act ivit y in en zym at ically defollicu lat ed oocyt es an d m an u ally separat ed oocyt es con t ain in g several t h ou san d follicle cells (Fig. 1D ). In creased levels of in du cible H SE-bin din g act ivit y w ere n ot observed u pon deliberat e in clu sion of follicle cells in st ress experim en t s (Fig. 1D , com pare lan es 2 an d 3 t o lan es 5 an d 6). T h u s, it appears as t h ou gh an y pot en t ial con t ribu t ion from follicle cell H SF act ivit y is below t h e level of det ect ion in t h ese assays. In order t o det erm in e t h e com posit ion of t h e st ress-in du cible H SE-bin din g act ivit y (s1) in oocyt es, gel su per-sh ift experim en t s w ere perform ed u sin g an t i-H SF1 an d an t i-H SF2 an t ibodies. For t h is experim en t , ext ract from h eat -sh ock ed st age VI oocyt es w as prein cu bat ed w it h polyclon al an t iseru m raised again st eit h er m ou se H SF1 or H SF2 prot ein s (Sarge et al., 1983). T h ese an t isera h ave previou sly been sh ow n t o exclu sively recogn ize t h eir respect ive an t igen s in gel m obilit y sh ift assays (Sarge et al., 1993). Specifi c recogn it ion by eit h er an t iseru m sh ou ld resu lt in n eu t ralizat ion or decreased m obilit y of t h e H SE–H SF com plex (s1). Addit ion of H SF1 an t iseru m t o pre-st ressed oocyt e ext ract resu lt ed in t h e loss of h eat sh ock -in du ced H SE-bin din g act ivit y (Fig. 2A, com pare lan es 1 –4). C on versely, addit ion of t h e polyclon al an t iseru m again st H SF2 did n ot affect t h e form at ion of t h e st ress-in du cible com plex (lan es 5 –7). T h e h eat -in du cible H SE-bin din g act ivit y w as u n affect ed even at very h igh H SF2 an t ibody con cen t rat ion (1:10 dilu t ion ; dat a n ot sh ow n ), w h ereas com plet e n eu t ralizat ion of bin din g w as observed at a relat ively low H SF1 an t ibody con cen t rat ion (1:200 dilu t ion ; see lan e 3). N eit h er an t iseru m affect ed t h e n on specifi c com plex. We also det erm in ed t h at t h e fact or con t ribu t in g t o t h e st ress-in du ced H SE-bin din g act ivit y observed in t h ese experim en t s is relat ed t o t h e previou sly clon ed XH SF1 (St u m p et al., 1995). In cu bat ion of h eat st ressed st age VI ext ract s w it h a polyclon al rabbit an t iseru m m ade again st in v it ro-syn t h esized XH SF1 (M ercier, O vsen ek , an d West w ood, u n pu blish ed resu lt s) also resu lt ed in k n ock ou t of t h e st ress-in du cible com plex (dat a n ot sh ow n ). We con clu de from t h is experim en t t h at t h e st ress-in du ced H SE-bin din g act ivit y in st age VI oocyt es is form ed by a X en opu s h om ologu e of m am m alian H SF1. T h e presen t an alysis, h ow ever, does n ot allow u s t o con clu de t h at t h e C opyrigh t q 1997 by Academ ic Press. All righ t s of reprodu ct ion in an y form reserved. AID DB 8441 / 6x15$$$$82 12-09-96 18:37:34 dbas 51 H SF A ct iv it ies in X en opu s O ocy t es A Temp. (°C) 18 Time (h) 27 30 33 36 39 0.5 1.0 0.5 1.0 0.5 1.0 0.5 1.0 0.5 1.0 s1 ns 1 2 3 4 5 6 7 8 9 10 11 D Stress Follicle C _ _ HS _ Ars _ – + HS + Ars + 1 2 3 4 5 6 [AsO2] (µM) C H 1 10 (mM) 100 1 10 100 s1 s1 ns ns 1 2 3 4 5 6 7 8 FIG. 1. St ress-in du ct ion of H SE-bin din g act ivit y in st age VI oocyt es. (A) G el m obilit y sh ift assays w ere perform ed w it h prot ein ext ract s prepared from st age VI oocyt es in cu bat ed at con t rol t em perat u re (187C , lan e 1) or differen t h eat sh ock t em perat u res for eit h er 30 m in (lan es 2, 4, 6, 8, an d 10) or 60 m in (lan es 3, 5, 7, 9, an d 11). Ext ract equ ivalen t t o on e oocyt e (20 mg) w as u sed in each bin din g react ion . T im e an d t em perat u re of h eat t reat m en t s are in dicat ed above each lan e. T h e h eat -in du cible H SE-bin din g com plex is in dicat ed by an arrow labeled s1 (sh ift -1). T h e relat ive in t en sit y of t h e n on -specifi c com plex (n s), t ypically fou n d in em bryon ic an d oocyt e ext ract s, is an in dicat ion of cell viabilit y. T reat m en t s at 397C w ere let h al t o oocyt es, as in dicat ed by disappearan ce of t h e n s com plex associat ed w it h oocyt e deat h . (B) H SE-bin din g act ivit y w as m easu red by gel m obilit y sh ift assay in st age VI oocyt es exposed t o differen t con cen t rat ion s of cadm iu m ch loride (C dC l 2 ) for 1 h r (lan es 4 –9). C adm iu m con cen t rat ion s are in dicat ed above each lan e. Exposu re t o 100 m M C dC l 2 in du ced form at ion of t h e H SE-bin din g com plex (lan e 9). Ext ract s prepared from u n t reat ed (187C , lan e 2) an d h eat -sh ock ed oocyt es (337C for 1 h r, lan e 3) w ere in clu ded as con t rols. Lan e labeled P (lan e 1) con t ain ed probe alon e. (C ) H SE-bin din g act ivit y w as det erm in ed by gel m obilit y sh ift assay in st age VI oocyt es exposed t o in creasin g con cen t rat ion s of sodiu m arsen it e (N aAsO 2 ) for 1 h r (lan es 3 –8). Ext ract s from u n t reat ed con t rols (C , lan e 1) an d h eat -sh ock ed oocyt es (H , lan e 2) w ere in clu ded as con t rols. (D ) H eat -in du cible H SE-bin din g act ivit ies w ere com pared in en zym at ically defollicu lat ed oocyt es (lan es 1 –3) an d m an u ally separat ed oocyt es con t ain in g t h ou san ds of follicle cells as det erm in ed by H oesch t st ain in g (lan es 4 –6). Bin din g react ion s con t ain ed con t rol oocyt es at st age VI (lan es 1 an d 4), oocyt es h eat sh ock ed at 337C for 1 h r (lan es 2 an d 5), or oocyt es t reat ed w it h 10 m M arsen it e for 2 h r (lan es 3 an d 6). C opyrigh t q 1997 by Academ ic Press. All righ t s of reprodu ct ion in an y form reserved. AID DB 8441 / 6x15$$8441 12-09-96 18:37:34 dbas 52 G ord on et al. A Anti-HSF2 No Ab 1:500 1:200 1:100 1:500 1:200 1:100 Anti-HSF1 1 2 3 4 5 6 7 s1 ns B CMV-CAT HSP70-CAT C H Ars Cd C H Ars Cd 0.1 0.1 0.1 0.1 0.1 89.2 84.3 83.7 FIG. 2. An t ibody recogn it ion of t h e fact or form in g t h e st ress-in du cible H SF–H SE com plex, an d st ress regu lat ion of it s t ran sact ivat ion abilit y. (A) Aliqu ot s of ext ract con t ain in g h eat -sh ock ed (337C , 1 h r) st age VI oocyt e (20 mg) w ere in cu bat ed eit h er alon e (n o an t ibody, lan e 1) or w it h a 1:500, 1:250, or 1:100 dilu t ion of t h e H SF1 an t iseru m (lan es 2 –4) or H SF2 an t iseru m (lan es 5 –7) prior t o gel m obilit y sh ift an alysis. T h e specifi c H SE–H SF1 com plex is in dicat ed by an arrow (labeled s1), an d t h e n on specifi c com plex is in dicat ed as n s. (B) C AT assays w ere perform ed on st age VI oocyt es in ject ed w it h C M V-C AT or h sp70-C AT plasm id D N As an d su bject ed t o st ress. M icroin ject ion s w ere perform ed as described u n der M at erials an d M et h ods. O ocyt e t reat m en t s w ere as follow s: con t rol, 187C ; h eat sh ock , 337C for 1 h r; C d, 50 m M C d for 1 h r; Ars, 5 m M arsen it e for 1 h r, as in dicat ed above t h e pan el. Percen t age con version of ch loram ph en icol t o acet ylat ed form s is in dicat ed below . H SF1 prot ein presen t in oocyt es is iden t ical t o XH SF1, an d so t h e possibilit y rem ain s t h at oocyt e H SF1 is en coded by a differen t gen e. We n ext exam in ed t h e relat ion sh ip bet w een in du ct ion of t h e H SE-bin din g abilit y of H SF1 an d t ran script ion al regu lat ion of t h e X en opu s h sp70 prom ot er m icroin ject ed in t o oocyt es. Solu t ion s of plasm id D N As con t ain in g t h e bact erial C AT report er gen e lin k ed t o eit h er t h e h sp70 prom ot er or t h e C M V prom ot er w ere m icroin ject ed in t o oocyt e n u clei, an d oocyt es w ere in cu bat ed overn igh t t o allow for ch rom at in assem bly (Lan dsberger an d Wolffe, 1995). T h e n ext day, oocyt es w ere su bject ed t o t h e sam e st ress t reat m en t s sh ow n t o resu lt in act ivat ion of H SF1 an d form at ion of t h e s1 com plex (337C , 100 m M C d an d 5 m M arsen it e; see Fig. 1). C AT act ivit y w as u n det ect able in oocyt es in ject ed w it h C M V prom ot er con st ru ct s (Fig. 2B, lan es 3 –6) an d u n st ressed oo- C opyrigh t q 1997 by Academ ic Press. All righ t s of reprodu ct ion in an y form reserved. AID DB 8441 / 6x15$$$$82 12-09-96 18:37:34 dbas 53 H SF A ct iv it ies in X en opu s O ocy t es oocyte stage: V VI C I H Cd C II H Cd C III H Cd C IV H Cd C H Cd C H Cd 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 s1 s2 ns FIG. 3. D evelopm en t al pat t ern of st ress-in du cible H SE-bin din g act ivit y in developin g oocyt es. O ocyt es w ere separat ed in t o pools represen t in g each in dividu al st age (I t o VI (D u m on t , 1972), st ages in dicat ed above each lan e). St aged oocyt es w ere in cu bat ed u n der con t rol con dit ion s (in dicat ed as C ; lan es 1, 4, 7, 10, 13, an d 16), h eat sh ock ed at 337C for 1 h r (in dicat ed as H ; lan es 2, 5, 8, 11, 14, an d 17), or exposed t o 50 m M C dC l 2 for 1 h r (in dicat ed as C d; lan es 3, 6, 9, 12, 15, an d 18). G el m obilit y sh ift an alysis w as perform ed essen t ially as in Fig. 1. Each bin din g react ion con t ain ed prot ein equ ivalen t t o on e st age IV oocyt e (20 mg; see M at erials an d M et h ods). An H SE-bin din g com plex (arrow labeled s1) w as in du ced aft er h eat sh ock or cadm iu m t reat m en t s at each st age of oogen esis. A secon d fast er m igrat in g com plex (see arrow labeled s2) is det ect able in ext ract s of con t rol an d st ressed st age 1 an d II oocyt es, bu t n ot in lat er st age oocyt es. cyt es in ject ed w it h H sp70-C AT (lan e 7). In con t rast , t reat m en t of H sp70-C AT -in ject ed oocyt es w it h h eat , cadm iu m , an d arsen it e resu lt ed in very h igh levels of C AT act ivit y (Fig. 2B, lan es 8 –10). T h ese experim en t s clearly est ablish a fu n ct ion al relat ion sh ip bet w een form at ion of t h e s1 com plex in oocyt es an d t ran script ion from an exogen ou s h sp70 prom ot er. It is clear, t h erefore, t h at a variet y of st ress t reat m en t s in du ce bot h t h e H SE-bin din g abilit y an d t h e t ran sact ivat ion pot en t ial of oocyt e H SF1. T he St ress Resp onse in Early St age of O ocy t es Very lit t le is k n ow n abou t t h e st ress respon se du rin g oogen esis, part icu larly at early st ages. T h e m ajorit y of st u dies exam in in g t h e fu n dam en t al m olecu lar even t s of t h e h eat sh ock respon se in preem bryon ic developm en t h ave focu sed on lat er st age (st age VI) X en opu s oocyt es as a t est syst em . We t h erefore w ish ed t o exam in e w h et h er oocyt es at earlier st ages w ere capable of respon din g t o st ress at t h e level of H SF act ivat ion . In order t o t est t h is, in dividu al oocyt es w ere separat ed in t o pools represen t in g each of t h e st ages of oogen esis as est ablish ed by D u m on t (1972). O ocyt es at each st age w ere exposed t o h eat sh ock or cadm iu m t reat m en t s, an d t h e resu lt s of t h e st an dard H SE-bin din g assay are sh ow n in Fig. 3. In creased H SE-bin din g act ivit y w as det ect ed aft er h eat (Fig. 3, lan es 2, 5, 8, 11, 14, an d 17) an d cadm iu m st ress (lan es 3, 6, 9, 12, 15, an d 18) in oocyt es at st age I t h rou gh t o st age VI of developm en t (see arrow labeled S1). T h e resu lt s of t h is assay sh ow t h at a st ress-in du cible H SE-bin din g fact or, presu m ably H SF1, is presen t in early oocyt es an d is respon sive t o h eat an d ch em ical st ress at all st ages of oogen esis. In order t o con fi rm t h ese resu lt s, w e repeat ed t h is experim en t several t im es (dat a n ot sh ow n ). T h e apparen t declin e in t h e in t en sit y of st ress-in du cible H SF1 –H SE com plex (s1) at st age V an d VI (lan es 14, 15, 17, an d 18) w as n ot observed in m ost experim en t s an d t h erefore is n ot lik ely t o refl ect real differen ces in t h e levels of in du ct ion of H SF1 bet w een early an d lat e st age oocyt es. Id ent ificat ion of a C onst it ut iv e, D ev elop m ent ally Regulat ed HSE-Bind ing A ct iv it y in Early St age O ocy t es In ou r in it ial experim en t s u sin g early st age oocyt es, an u n u su al H SE-bin din g act ivit y w as observed in u n st ressed con t rols (Fig. 3, lan es 1 –6; see arrow labeled s2). T h is com plex w as elect roph oret ically dist in ct from h eat -in du cible H SF act ivit y, m igrat in g bet w een h eat -in du cible an d n on specifi c ban ds. In order t o clearly est ablish t h e st age-depen den cy of t h is apparen t ly con st it u t ive com plex, an d t o ru le C opyrigh t q 1997 by Academ ic Press. All righ t s of reprodu ct ion in an y form reserved. AID DB 8441 / 6x15$$$$82 12-09-96 18:37:34 dbas 54 G ord on et al. ou t t h e possibilit y t h at it s form at ion in con t rol oocyt es (Fig. 3, lan es 1 an d 4) w as a con sequ en ce of in advert en t st ress, st aged oocyt es w ere isolat ed from t h e ovary of a differen t fem ale an d a gel m obilit y sh ift assay w as perform ed as above (Fig. 4A). An H SE-bin din g com plex (s2) w as det ect ed in u n st ressed st age I an d II oocyt es (Fig. 4A, lan es 1 an d 2), bu t n ot in lat er st age oocyt es from st ages III t o VI (Fig. 4A, lan es 3 –6). We also t est ed for t h e presen ce of t h is act ivit y in X en opu s em bryos at variou s developm en t al st ages an d in som at ic cells (Fig. 4B). T h e con st it u t ive H SE-bin din g act ivit y w as n ot det ect ed in ext ract s from u n fert ilized eggs, cleavage or gast ru la st age em bryos, or in A6 cells (Fig. 4B, lan es 2 –5). T h erefore, act ivit y of t h e s2 com plex appears t o be rest rict ed t o early oogen esis. We n ext perform ed com pet it ion experim en t s t o det erm in e t h e sequ en ce specifi cit y of bot h H SE–prot ein in t eract ion s s1 an d s2 (Fig. 4C ). T h e fast er-m igrat in g con st it u t ive H SE-bin din g com plex (s2) presen t in ext ract s from u n st ressed st age II oocyt es w as dim in ish ed by addit ion of H SE oligon u cleot ide com pet it or t o D N A-bin din g react ion s (Fig. 4C , lan e 2), bu t n ot by excess am ou n t s of oligon u cleot ides con t ain in g AP-2, C EBP, or G AL4 bin din g sit es (lan es 3 –5). T h ese resu lt s clearly est ablish t h e con st it u t ive com plex (S2) as a sequ en ce-specifi c H SE-bin din g act ivit y. It w as also im port an t t o det erm in e w h et h er st ress t reat m en t s w ou ld brin g abou t an elevat ion in t h e levels of t h e con st it u t ive com plex (Fig. 4C , lan es 6 –10). T reat m en t s of st age II oocyt es w it h 50 m M cadm iu m resu lt ed in form at ion of t h e m ore slow ly m igrat in g H SE–H SF1 com plex (s1), bu t h ad n o sign ifi can t effect on form at ion of t h e s2 com plex (com pare lan es 1 –5 t o lan es 6 –10). In n u m erou s experim en t s u sin g a variet y of st ress regim es, t h e con st it u t ive H SE-bin din g act ivit y of st age I an d II oocyt es w as n ot sign ifi can t ly affect ed by con dit ion s k n ow n t o elicit act ivat ion of H SF1 (dat a n ot sh ow n ; also see Fig. 3). In addit ion , bot h h eat -in du cible an d con st it u t ive com plexes w ere fou n d t o be sequ en ce specifi c, as sh ow n by com pet it ion w it h u n labeled H SE oligon u cleot ide (lan e 7). O ligon u cleot ides con t ain in g k n ow n bin din g sit es for est ablish ed t ran script ion fact ors (AP-2, C EBP, an d G AL4) h ad n o com pet it ive effect on form at ion of eit h er com plex (lan es 8 –10). O n t h e basis of it s beh avior in st age I an d II oocyt es, w e specu lat ed t h at t h e con st it u t ive H SE-bin din g com plex (s2) w as form ed by a X en opu s h om ologu e of m am m alian H SF2. Previou s experim en t s w it h m ou se an d h u m an cells h ave sh ow n t h at H SF2 is act ive in cells u n dergoin g differen t iat ion an d developm en t , exh ibit s con st it u t ive H SE-bin din g act ivit y in t h e m ale germ lin e an d em bryon al carcin om a cells, an d is n ot in du ced by st ress at t h e level of D N Abin din g act ivit y (M ezger et al., 1989; Sist on en et al., 1992; M u rph y et al., 1994; Sarge et al., 1994; Sist on en et al., 1994; G oodson et al., 1995). T h e con st it u t ive com plex observed in ou r experim en t s appeared t o sh are several feat u res in com m on w it h H SF2, in clu din g it s presen ce in cells u n dergoin g grow t h an d differen t iat ion (st age I an d II oocyt es), affi n it y for t h e H SE in t h e absen ce of st ress, an d it s lack of in du ct ion u pon st ress t reat m en t s. In order t o ext en d t h is com parison , w e exam in ed t h e possibilit y t h at a sim ilar act ivit y w as presen t in t est es isolat ed from adu lt X en opu s m ales. T h is t issu e w as ch osen becau se t h e D N A-bin din g act ivit y of H SF2 is k n ow n t o be part icu larly st ron g in m am m alian t est es (M u rph y et al., 1994; Fioren za et al., 1995). Resu lt s of com parat ive gel sh ift an alyses sh ow t h at con st it u t ive H SE-bin din g act ivit y form ed in st age II oocyt es closely co-m igrat es w it h an act ivit y presen t in prot ein ext ract s m ade from t est es exposed t o con t rol an d h eat sh ock con dit ion s (Fig. 5A, com pare lan e 1 t o lan es 3 an d 4). We also t est ed for t h e presen ce of a con st it u t ive H SE-bin din g act ivit y in m am m alian ovary t issu e. In t erest in gly, a sim ilar act ivit y w as also det ect ed in rat ovary t issu e (lan e 2), im plyin g t h e possible in volvem en t of a relat ed fact or in m am m alian oogen esis. All of t h ese act ivit ies w ere fou n d t o com igrat e w it h an H SF2 ban d in con t rols u sin g m u rin g t est es (dat a n ot sh ow n ). Alt h ou gh t h is com igrat ion st ren gt h en ed t h e correlat ion bet w een t h e s2 com plex an d a pu t at ive X en opu s H SF2 h om ologu e, it w as n ot su ffi cien t t o posit ively iden t ify t h e H SE-bin din g fact or as H SF2. In order t o m ore clearly det erm in e t h e com posit ion of t h e con st it u t ive H SEbin din g act ivit y, w e perform ed gel su per-sh ift experim en t s w it h t h e sam e an t i-H SF1 an d an t i-H SF2 an t ibodies u sed in Fig. 2. For t h is experim en t , ext ract s from u n st ressed an d h eat -sh ock ed st age I oocyt es w ere prein cu bat ed w it h t h e polyclon al an t isera again st m ou se H SF1 or H SF2 prot ein s (Sarge et al., 1983). First , n eit h er an t iseru m specifi cally recogn ized t h e con st it u t ive (s2) com plex in early oocyt es (Fig. 5B, com pare lan es 2, 3, an d 4). O n t h e ot h er h an d, H SF1 an t iseru m n eu t ralized t h e st ress-in du cible H SE-bin din g act ivit y (s1) in h eat -sh ock ed st age 1 oocyt es (a su persh ift ed com plex is apparen t in lan e 6), bu t h ad n o effect on t h e s2 com plex in t h e sam e ext ract (Fig. 5B, com pare lan es 5 an d 6). T h ese resu lt s con fi rm t h e iden t it y of t h e st ress-in du cible fact or as H SF1 an d su ggest t h at t h e con st it u t ive com plex is form ed by a dist in ct fact or. H SF2 an t iseru m did n ot recogn ize eit h er com plex (com pare lan es 4 an d 7). H SF2 an t iseru m did, h ow ever, recogn ize t h e H SE-bin din g com plex in rat ovary t issu e, an d con t rol experim en t s con fi rm t h at t h e an t iseru m u sed in t h ese experim en t s su persh ift ed t h e H SE– H SF2 com plex presen t in ext ract s of m u rin e t est es (dat a n ot sh ow n ). T h e sim plest in t erpret at ion of t h is dat a is t h at t h e prot ein com prisin g t h e con st it u t ive com plex (s2) in early oocyt es is a u n iqu e H SE-bin din g prot ein u n relat ed t o eit h er H SF1 or H SF2, alt h ou gh it is possible t h at t h e polyclon al an t ibodies presen t in t h e an t iseru m direct ed again st m ou se H SF2 do n ot su ffi cien t ly recogn ize a pu t at ive X en opu s H SF2 h om ologu e. In cu bat ion of st age I an d II oocyt e ext ract s w it h very h igh an t i-H SF2 an t iseru m con cen t rat ion s (1:10 dilu t ion ) did n ot affect t h e s2 com plex (dat a n ot sh ow n ). We w ere in t erest ed in exam in in g a possible relat ion sh ip bet w een t h e developm en t al pat t ern of t h e con st it u t ive H SEbin din g act ivit y (s2) an d t h e syn t h esis of H SP m RN A du rin g oogen esis. N ort h ern blot an alysis w as perform ed u sin g t h e C opyrigh t q 1997 by Academ ic Press. All righ t s of reprodu ct ion in an y form reserved. AID DB 8441 / 6x15$$$$83 12-09-96 18:37:34 dbas 55 H SF A ct iv it ies in X en opu s O ocy t es A oocyte stage I II III IV V VI B s2 stI E CL G A6 2 3 4 5 6 s2 ns ns 1 2 3 4 5 6 C 1 Stage II oocytes 50 mM Cd HSE CEBP AP-2 GAL4 - HSE CEBP AP-2 GAL4 Competitor: - Control 1 2 3 4 5 6 7 8 9 10 s1 s2 ns FIG. 4. A con st it u t ive, n on in du cible, an d developm en t ally regu lat ed H SE-bin din g act ivit y in st age I an d II oocyt es. (A) H SE-bin din g act ivit y w as exam in ed by gel m obilit y sh ift an alysis u sin g ext ract s m ade from st aged oocyt es u n der con t rol con dit ion s (187C ). A con st it u t ive H SE-bin din g com plex (arrow labeled s2) w as det ect ed in bin din g react ion s w it h st age I an d II oocyt es, bu t n ot w it h ext ract s from oocyt es at st ages III t o VI. T h e n on specifi c com plex is in dicat ed by an arrow labeled n s. (B) H SE-bin din g act ivit y in ext ract from u n st ressed st age 1 oocyt es (lan e 2) w as com pared t o act ivit y in u n st ressed ext ract s from u n fert ilized egg (lan e 3), cleavage st age em bryos at st age 6 (lan e 4), gast ru la st age em bryos at st age 10 (lan e 5), an d A6 cells (lan e 6). Each bin din g react ion con t ain ed 20 mg of prot ein ext ract , except for lan e 1 (P) w h ich con t ain ed H SE oligon u cleot ide probe alon e. (C ) Prot ein ext ract s w ere prepared from oocyt es at st age II t h at w ere eit h er u n st ressed (C on t rol; lan es 1 –5) or in cu bat ed for 2 h r in cadm iu m (50 m M C d, lan es 6 –10) an d u sed in a com pet it ion experim en t t o det erm in e t h e sequ en ce specifi cit y of st ress-in du cible (com plex labeled s1) an d con st it u t ive (com plex labeled s2) H SE-bin din g act ivit ies. Ext ract equ ivalen t t o approxim at ely fi ve st age II oocyt es (20 mg) w as u sed in each bin din g react ion . C om pet it or D N As w ere added in t o bin din g react ion s at a 50-fold m olar excess relat ive t o labeled H SE probe. Form at ion of bot h con st it u t ive (s2) an d st ress-in du cible (s1) com plexes w as dim in ish ed by H SE com pet it or (lan es 2 an d 7), bu t n ot by oligon u cleot ides con t ain in g C EBP (lan es 3 an d 8), AP-2 (lan es 4 an d 9), or G AL4 (lan es 5 an d 10) bin din g sit es. C opyrigh t q 1997 by Academ ic Press. All righ t s of reprodu ct ion in an y form reserved. AID DB 8441 / 6x15$$8441 12-09-96 18:37:34 dbas 56 G ord on et al. B Anti-HSF1 Anti-HSF2 oocyte stage I heat shock – – + + + – – + – – control + – 2 3 s1 s2 A I-II Rat ov. 1 2 X. testes C HS ns s2 ns 3 4 1 4 5 6 7 FIG. 5. T h e developm en t ally regu lat ed H SE-bin din g act ivit y of early st age oocyt es is u n relat ed t o H SF2. (A) C om parison of con st it u t ive H SE-bin din g act ivit ies in early oocyt es, t est es, an d rat ovary t issu e. M igrat ion of t h e con st it u t ive com plex (s2) w as com pared in ext ract s from st age I an d II oocyt es (lan e 1), rat ovary (lan e 2), u n st ressed X en opu s t est es (lan e 3), or h eat -sh ock ed t est es (lan e 4). (B) An t ibody recogn it ion of t h e con st it u t ive H SE-bin din g act ivit y (s2) in st age I oocyt es. Ext ract con t ain in g u n st ressed (lan es 2 –4) or h eat -sh ock ed (337C , 1 h r) st age I oocyt es (lan es 5 –7) w as in cu bat ed eit h er alon e (n o an t ibody; lan es 2 an d 5), or w it h a 1:250 dilu t ion of t h e H SF1 an t iseru m (lan es 3 an d 6) or H SF2 an t iseru m (lan es 6 –8) prior t o gel m obilit y sh ift an alysis. 20 mg of ext ract w as u sed in each bin din g react ion . Lan e 1 (P) con t ain ed probe alon e. X en opu s h sp70B gen e probe (Bien z, 1984a) an d t ot al RN A isolat ed from oocyt es at each st age of oogen esis (Fig. 6). H igh levels of H SP70 m RN A w as det ect ed at st age I an d II (Fig. 6, lan es 1 an d 2). Levels declin ed t h rou gh ou t lat er oogen esis (lan es 3 –6). We n ot e t h at t h e t ot al RN A con t en t of oocyt es is k n ow n t o in crease approxim at ely fi vefold du rin g developm en t from st age II t o VI (D oleck i an d Sm it h , 1979), an d so t h is experim en t sh ou ld n ot be seen as a m easu rem en t of absolu t e ch an ges in H SP70 m RN A accu m u lat ion in a sin gle oocyt e. Regardless, a clear correlat ion is apparen t bet w een t h e early appearan ce of h sp70 m RN A an d t h e presen ce of t h e s2 com plex du rin g oogen esis. We su ggest t h at t h e con st it u t ive H SE-bin din g act ivit y plays a role in t h e expression of H SPs du rin g early st ages of oocyt e developm en t . A ct iv at ion of HSF1 d uring C ont inuous St ress It h as been post u lat ed t h at t h e D N A-bin din g an d oligom eric st at e of H SF1 is regu lat ed by t h e in t racellu lar level of H SP70 fam ily prot ein s (M orim ot o, 1993). In oocyt es, h ow ever, in creased H SP70 syn t h esis is n ot in du ced by h eat sh ock (H orrell et al., 1987). T h u s, t h e oocyt e represen t s a u n iqu e syst em in w h ich st ress-in du ced act ivat ion of H SF can be assessed w it h ou t con cu rren t elevat ion of in t racellu lar H SP con cen t rat ion s. T h erefore, w e exam in ed t h e k in et ics of H SF1 act ivat ion in oocyt es du rin g prolon ged st ress. St age VI oocyt es w ere con t in u ou sly exposed t o h eat sh ock an d ext ract s prepared at variou s t im e poin t s w ere u sed in t h e H SE-bin din g assay presen t ed in Fig. 7A. T h is experim en t sh ow s t h at t h e st ress-in du cible H SE-bin din g com plex rem ain ed act ive for as lon g as 9 h r of h eat sh ock at 337C (Fig. 7A, lan es 3 –12). H SE-bin din g act ivit y of H SF1 persist ed u n t il oocyt e deat h (Fig. 7A, lan e 13). A sim ilar pat t ern w as observed in several differen t experim en t s perform ed at differen t h eat sh ock t em perat u res, w it h H SF1 rem ain in g act ive t h rou gh ou t t h e du rat ion of oocyt e viabilit y (dat a n ot sh ow n ). T h is ext en ded pat t ern con t rast s w it h t h e t ran sien t u pregu lat ion an d deact ivat ion of H SE-bin din g act ivit y (w it h in 2 h r) observed u n der sim ilar h eat sh ock con dit ion s in X en opu s em bryos (O vsen ek an d H eik k ila, 1990; Karn et al., 1992). T o ext en d t h is an alysis, w e also t est ed w h et h er t h e H SEbin din g abilit y of H SF1 rem ain ed act ive du rin g prolon ged C opyrigh t q 1997 by Academ ic Press. All righ t s of reprodu ct ion in an y form reserved. AID DB 8441 / 6x15$$$$83 12-09-96 18:37:34 dbas 57 H SF A ct iv it ies in X en opu s O ocy t es oocyte stage: l ll lll lV V Vl 1 2 3 4 5 6 28S Hsp70 18S 18S Hsp70 mRNA Levels 50 40 30 20 10 0 I II III IV Oocyte Stage V VI FIG. 6. N ort h ern blot exam in at ion of h sp70 m RN A expressed du rin g X en opu s oogen esis. T ot al RN A isolat ed from oocyt es at differen t st ages w as elect roph oresed on a 1.2% agarose form aldeh yde gel an d t h en t ran sferred t o H ybon d-N / m em bran e. T h e blot s w ere h ybridized again st t h e 32 P-labeled X en opu s h sp70B D N A probe. All lan es con t ain 10 mg of t ot al RN A from each oocyt e st age as in dicat ed above. An arrow in dicat es t h e posit ion of t h e 2.7-k b h sp70 t ran script s. Ribosom al RN A ban ds are sh ow n on t h e left . As a con t rol, levels of 18S rRN A w ere det ect ed w it h a radiolabeled rRN A oligon u cleot ide probe an d are sh ow n below t h e pan el. Relat ive levels of H SP70 m RN A w ere det erm in ed by den sit om et ry an d are sh ow n at t h e bot t om of t h e pan el. exposu re of oocyt es t o arsen it e an d cadm iu m (Figs. 7B an d 7C ). H SE-bin din g act ivit y rem ain ed act ive for as lon g as 24 h r in oocyt es exposed t o 500 mM arsen it e (Fig. 7B, lan es 3 – 10) an d also in oocyt es exposed t o 5 m M cadm iu m (Fig. 7C , lan es 7 –11). St ress-in du cible H SE-bin din g act ivit y w as presen t in oocyt es u p u n t il oocyt e deat h at 36 h r, as det ect ed by dim in ish m en t of t h e n on specifi c ban d (n ot sh ow n ). Low er con cen t rat ion s of arsen it e an d cadm iu m w ere u sed for t h ese t im e cou rse experim en t s t h an in previou s experim en t s exam in in g acu t e act ivat ion of H SE-bin din g abilit y (Figs. 1B an d 1C ) becau se h igh con cen t rat ion s resu lt ed in oocyt e deat h aft er on ly a few h ou rs of t reat m en t (dat a n ot sh ow n ). T h e sam e pat t ern of H SF act ivat ion w as det ect ed w it h ch em ical st ress as w it h h eat sh ock ; H SF rem ain ed act ive for t h e du rat ion of st ress t reat m en t s u p u n t il oocyt e deat h , t h e t im e of w h ich w as depen den t u pon t h e severit y of t reat m en t (dat a n ot sh ow n ). T h ese resu lt s su ggest t h at alt h ou gh t rim erizat ion of en dogen ou s oocyt e H SF1 m olecu les is apparen t ly in du ced by st ress, t h e cellu lar m ech an ism s regu lat in g con version of H SF1 t o t h e m on om eric st at e are absen t or in act ivat ed in t h e oocyt e. Sin ce H SP70 fam ily prot ein s appear t o fu n ct ion in t h e disassem bly of H SF t rim ers an d at t en u at ion of H SE-bin din g act ivit y (M osser et al., 1993; Rabin dran et al., 1994; Baler et al., 1996), w e specu lat e t h at t h e ext en ded pat t ern of H SF1 act ivat ion in oocyt es is a con sequ en ce of low d e n ov o H SP prot ein syn t h esis (H orrell et al., 1987). We n ext t est ed t h e at t en u at ion of H SF1 act ivit y in oocyt es du rin g recovery from an acu t e h eat sh ock (Fig. 8). As expect ed, a h igh level of H SE-bin din g com plex w as in du ced aft er 1 h r of h eat sh ock (Fig. 8, lan e 2). H SE-bin din g act ivit y ret u rn ed t o con t rol levels w it h in 5 m in aft er resu m pt ion of n orm al t em perat u res (Fig. 8, lan es 3 –6). In sim ilar recovery experim en t s, H SF1 act ivit y rem ain ed h igh in ext ract s m ade from oocyt es follow in g brief exposu res t o arsen it e or cadm iu m (dat a n ot sh ow n ). T h is w as probably du e t o t h e persist en t effect s of arsen it e or cadm iu m absorbed in t o oocyt es an d n ot rem oved even aft er ext en sive w ash es. T ak en t oget h er, t h e resu lt s of experim en t s exam in in g t h e k in et ics of H SF1 act ivat ion (Figs. 7 an d 8) su ggest t h at t h e cellu lar m ech an ism (s) requ ired for disassem bly of H SF1 t rim ers is presen t in t h e oocyt e, bu t at levels in su ffi cien t for at t en u at ion of t h e respon se du rin g con t in u ou s st ress. D ISCU SSION T h ese experim en t s represen t t h e fi rst direct dem on st rat ion of a m at ern al pool of dist in ct st ress-in du cible an d con st it u t ive H SE-bin din g act ivit ies in X en opu s oocyt es. We h ave est ablish ed t h at an en dogen ou s H SF is in du cible by h eat sh ock an d ch em ical st ress an d is readily det ect able by gel m obilit y sh ift assay. G el su persh ift experim en t s u sin g an t isera again st m am m alian H SF1 an d H SF2 su ggest t h at t h e st ress-in du cible H SE-bin din g act ivit y det ect ed t h rou gh ou t oogen esis is form ed by a X en opu s h om ologu e of H SF1. H SF1 appears t o be presen t in u n st ressed oocyt es in a lat en t form t h at is con vert ed t o t h e H SE-bin din g con form at ion u pon h eat an d ch em ical st ress at all st ages of oogen esis (Fig. C opyrigh t q 1997 by Academ ic Press. All righ t s of reprodu ct ion in an y form reserved. AID DB 8441 / 6x15$$$$83 12-09-96 18:37:34 dbas 58 G ord on et al. A Time (h) at 33°C 0 0.1 0.5 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 11 12 13 s1 ns FIG. 7. T im e cou rse of H SE-bin din g act ivit y in oocyt es con t in u ou sly exposed t o st ress. (A) St age VI oocyt es w ere m ain t ain ed at a h eat sh ock t em perat u re of 337C , an d prot ein ext ract s w ere obt ain ed at t h e t im es in dicat ed above each lan e. A h eat -in du cible H SE-bin din g com plex (arrow labeled s1) w as det ect ed aft er 30 m in of exposu re (lan e 3), an d persist ed t h rou gh ou t t h e t im e cou rse of t h e experim en t (lan es 3 –12) u p u n t il oocyt e deat h (lan e 13). D im in ish m en t of t h e n s com plex at 10 h r is in dicat ive of oocyt e deat h . (B) T im e cou rse of H SE-bin din g act ivit y in oocyt es in cu bat ed in 500 mM sodiu m arsen it e. Lan es 1 an d 2 con t ain ed a con t rol ext ract (187C ) t ak en at T im e 0 an d at 24 h r, respect ively. T im e of arsen it e t reat m en t is in dicat ed above each lan e (lan es 3 –10). T h e in du cible com plex is in dicat ed by an arrow labeled s1. (C ) T im e cou rse of H SE-bin din g act ivit y in oocyt es in cu bat ed in 5 m M cadm iu m ch loride. C on t rols w ere t ak en at t h e on set of t reat m en t (T im e 0; lan e 1) an d aft er 12 (lan e 2) an d 24 h r (lan e 3). T h e in du cible com plex (arrow labeled s1) w as presen t u p u n t il oocyt e deat h aft er 24 h r. T h e n on specifi c ban d is in dicat ed by an arrow labeled n s. 2). Experim en t s w it h st aged oocyt es provide eviden ce for a con st it u t ive H SE-bin din g act ivit y t h at is elect roph oret ically dist in ct from t h e H SF1 –H SE com plex an d does n ot appear t o be affect ed by st ress. T h is act ivit y appears t o be developm en t ally regu lat ed, as ju dged by it s presen ce in st age I an d II oocyt es, bu t n ot in lat er st age oocyt es, eggs, em bryos, or som at ic cells (Figs. 3 an d 4). Alt h ou gh t h e beh aviou r of t h e fact or form in g t h is com plex is sim ilar t o t h at of m am m alian H SF2 (Fig. 5A), gel su persh ift experim en t s su ggest it m ay be a u n iqu e H SE-bin din g fact or u n relat ed t o H SF1 or H SF2 (Fig. 5B). A close correlat ion w as observed bet w een t h e developm en t al pat t ern of H SP70 m RN A accu m u lat ion (Fig. 6) an d form at ion of t h e con st it u t ive com plex (Figs. 3 an d 4), im plyin g a role for t h is fact or in t h e n orm al expression of H SP gen es du rin g oogen esis. St ress-Ind ucib le HSE Bind ing in Xenop us O ocy t es Experim en t s w it h st age VI oocyt es sh ow t h at st ress-in du cible H SE-bin din g abilit y of en dogen ou s oocyt e H SF m olecu les is det ect able u sin g a st an dard gel m obilit y sh ift assay (Figs. 1 an d 2). T o ou r k n ow ledge t h e on ly previou s dem on st rat ion of in du ced H SE-bin din g du rin g preem bryon ic vert ebrat e developm en t w as in m at u re ovu lat ed m ou se oocyt es (M ezger et al., 1994). In du ct ion of h eat sh ock fact ors en dogen ou s t o t h e oocyt e is en t irely con sist en t w it h earlier report s of t ran script ion al u pregu lat ion from in ject ed H SP70 prom ot ers in X en opu s oocyt es (Lan dsberger et al., 1995). Alt h ou gh w e are u n able t o explain t h e failu re t o det ect H SE- bin din g act ivit y in earlier st u dies (C los et al., 1990; Baler et al., 1993; Z ou et al., 1994, 1995), t h is w as lik ely du e t o t h e low level of m at ern al fact ors relat ive t o t h e robu st act ivit y of exogen ou sly expressed H SF1 m olecu les. H ere w e h ave est ablish ed t h e in v iv o cellu lar st ress con dit ion s for reprodu cible act ivat ion of H SE bin din g u pon acu t e exposu res t o h eat sh ock (337C ), cadm iu m (50 m M ), an d arsen it e (1 m M ). T h ese dat a provide direct eviden ce t h at t h e frog oocyt e h as a vigorou s respon se t o h eat sh ock an d diverse form s of ch em ical st ress as m easu red by t h e act ivat ion of H SF1 D N A bin din g abilit y. An t ibody recogn it ion experim en t s (Figs. 2A an d 5B) sh ow t h at t h e fact or bin din g t o H SE form in g t h e s1 com plex in st ressed oocyt es is relat ed t o m am m alian H SF1. T h e st ressin du cible propert ies of oocyt e H SF1 det ect ed in t h ese experim en t s, bot h in D N A-bin din g abilit y an d t ran sact ivat ion pot en t ial (Figs. 1 an d 2), closely resem ble t h at of vert ebrat e H SF1 (Baler et al., 1993; Sarge et al., 1993). Fu rt h erm ore, an t ibody recogn it ion experim en t s u sin g an t iseru m direct ed again st XH SF1 su ggest t h e fact or form in g t h e h eat -in du cible com plex is relat ed t o XH SF1 (dat a n ot sh ow n ), for w h ich a cD N A clon e h as recen t ly been isolat ed (St u m p et al., 1995). H ow ever, ou r an alysis falls sh ort of dem on st rat in g a direct relat ion sh ip bet w een t h e XH SF1 cD N A an d t h e gen e en codin g oocyt e H SF1. Sin ce XH SF1 cD N A w as isolat ed from an d A6 cell library (St u m p et al., 1995) it rem ain s possible t h at oocyt e H SF1 det ect ed in t h is st u dy is en coded by a differen t gen e. O u r experim en t s su ggest t h at H SF1 is n orm ally presen t C opyrigh t q 1997 by Academ ic Press. All righ t s of reprodu ct ion in an y form reserved. AID DB 8441 / 6x15$$$$83 12-09-96 18:37:34 dbas 59 H SF A ct iv it ies in X en opu s O ocy t es B Time (h) in 500µM AsO2 C1 C2 1 2 4 6 8 10 12 24 3 4 5 6 7 8 9 10 s1 ns 1 C 2 Control Time (h) Time (h) in 5 mM Cd 0 12 24 1 2 3 4 6 8 12 18 24 1 2 3 4 5 6 7 8 9 10 11 12 s1 ns FIG. 7— C on t in u ed in an in act ive n on -D N A-bin din g st at e in t h e oocyt e an d is n egat ively regu lat ed at t h e level of oligom erizat ion u n der n on st ress con dit ion s (Figs. 1 an d 3). As w it h m ost cell t ypes, con version of H SF1 t o an act ive D N A-bin din g t rim er appears t o be an early st ep in t h e st ress in du ct ion pat h w ay in oocyt es. Lan dsberger an d Wolffe (1995) report ed t h at overexpression of XH SF1 in oocyt es leads t o in appropriat e oligom erizat ion an d act ivat ion of H SP70 t ran script ion u n der n on st ress con dit ion s. T h is im proper regu lat ion cou ld be du e t o t it rat ion of regu lat ory m olecu les t h at n orm ally fu n ct ion C opyrigh t q 1997 by Academ ic Press. All righ t s of reprodu ct ion in an y form reserved. AID DB 8441 / 6x15$$$$83 12-09-96 18:37:34 dbas 60 G ord on et al. Recovery (min) C HS 5 15 30 60 s1 script ion of H SP m RN A in early oocyt es an d in t h e absen ce of sign ifi can t H SP m RN A syn t h esis in st age VI oocyt e, deserves fu rt h er an alysis. A D ev elop m ent ally Regulat ed HSF A ct iv it y in Early St age O ocy t es ns 1 2 3 4 5 6 FIG. 8. T im e cou rse of H SE-bin din g act ivit y in oocyt es recoverin g from a 1-h r 337C h eat sh ock . Lan e 1 con t ain s an u n t reat ed con t rol ext ract (C ; lan e 1), an d lan e 2 con t ain s ext ract obt ain ed from oocyt es im m ediat ely aft er h eat t reat m en t (H S; lan e 2). O ocyt es w ere allow ed t o recover at 187C for t h e t im e in dicat ed above each lan e (lan es 3 –6). St ress-in du cible an d n on specifi c com plexes are in dicat ed w it h arrow s (s1 an d n s, respect ively). t o m ain t ain H SF in an in act ive m on om eric st at e. In ligh t of t h is, t h e abilit y t o det ect D N A-bin din g act ivit ies of en dogen ou s oocyt e H SF m olecu les great ly en h an ces t h e pot en t ial of t h e oocyt e as a m odel syst em t o det erm in e t h e fu n dam en t al bioch em ical even t s an d sign al t ran sdu ct ion pat h w ays of t h e st ress respon se, especially sin ce problem s associat ed w it h t h e regu lat ion of exogen ou s H SF can be avoided. It is also n ot ew ort h y t h at H SE-bin din g act ivit y is readily det ect able in ext ract from a sin gle oocyt e, w h ereas approxim at ely 100 m at u re ovu lat ed m ou se oocyt es are requ ired for det ect ion (M ezger et al., 1994). Experim en t s sh ow in g in du ct ion of H SF1 act ivit y by h eat an d ch em ical st ress in early st age oocyt es (Fig. 3) represen t t h e fi rst dem on st rat ion t h at oocyt es at all st ages of developm en t are capable of a rigorou s st ress respon se at t h e level of H SF t rim erizat ion . It is diffi cu lt t o com m en t on t h e biological sign ifi can ce of H SF1 in du ct ion du rin g oogen esis, sin ce H SPs are n ot expressed in respon se t o st ress in st age VI oocyt es (H orrell et al., 1987) an d sin ce it is n ot k n ow n w h et h er H SP syn t h esis is u pregu lat ed by st ress in early st age oocyt es at eit h er t h e t ran script ion al or t ran slat ion al levels. In addit ion , very lit t le is k n ow n abou t t h e early bioch em ical even t s of t h e st ress respon se in t h e fem ale germ lin e as previou s st u dies in X en opu s h ave focu sed prim arily on t h e act ivit ies of exogen ou s prot ein s in st age VI oocyt es (Baler et al., 1993; Z ou et al., 1994, 1995; Lan dsberger an d Wolffe, 1995; St u m p et al., 1995). It is clear, t h erefore, t h at t h e pot en t ial role of st ress-in du cible H SF, bot h in t h e t ran - In t h e cou rse of exam in in g st ress-in du cible H SF act ivit ies in early st age oocyt es, w e also observed an u n u su al con st it u t ive H SE-bin din g act ivit y in u n st ressed con t rols. T h e con st it u t ive H SE-bin din g com plex (s2) w as det ect ed in u n st ressed st age I an d II oocyt es, bu t n ot in lat er st age oocyt es, eggs, em bryos, or A6 cells (Figs. 3 an d 4). C om pet it ion experim en t s est ablish ed t h e sequ en ce specifi cit y of bot h con st it u t ive an d st ress-in du cible H SE–prot ein in t eract ion s (Fig. 4C ). T h e con st it u t ive act ivit y also w as fou n d t o com igrat e w it h an H SE–prot ein com plex presen t in frog t est es as w ell as an abu n dan t act ivit y in rat ovary t issu e (Fig. 4A). In it ially w e com pared t h is dat a w it h t h at t h e D N A-bin din g act ivit y of H SF2 in m am m alian t issu es w h ere H SF2 is rest rict ed t o cells u n dergoin g differen t iat ion an d developm en t , exh ibit s con st it u t ive H SE-bin din g act ivit y in t est es, an d is n ot sign ifi can t ly in du ced by st ress (Sist on en et al., 1992; M u rph y et al., 1994; Sarge et al., 1994; Sist on en et al., 1994; Fioren za et al., 1995). T h e con st it u t ive com plex presen t in oocyt es beh aved in a rem ark ably sim ilar fash ion : it com igrat ed w it h an act ivit y presen t in t est es, exh ibit ed con st it u t ive H SE bin din g, an d did n ot appear t o be st ress-in du cible. O n t h e st ren gt h of t h is com parison , w e origin ally specu lat ed t h at t h e con st it u t ive com plex observed in st age I an d II oocyt es is form ed by a X en opu s h om ologu e of H SF2. H ow ever, an t ibody recogn it ion experim en t s u sin g an t isera direct ed again st m ou se H SF1 an d H SF2 in con ju n ct ion w it h gel m obilit y sh ift assays (Fig. 5B) provide st ron g eviden ce t h at t h is fact or is u n relat ed t o eit h er H SF1 or H SF2. We propose t h at t h e s2 com plex is form ed by a n ovel developm en t ally regu lat ed H SE-bin din g fact or, bu t cau t ion t h at t h e presen t an alysis can n ot ru le ou t t h e possibilit y t h at t h is fact or is relat ed t o H SF2 or n egat e t h e presen ce of a gen e en codin g an H SF2 h om ologu e in X en opu s. In order t o posit ively iden t ify oocyt e H SFs, w e are presen t ly at t em pt in g t o isolat e gen es en codin g H SF fam ily m em bers from a X en opu s ovary cD N A library. C learly t h ou gh , t h e im plicat ion of t h e presen t st u dy is t h at addit ion al H SF fam ily m em bers are expressed in oocyt es an d adu lt t issu es of X en opu s. T h e det ect ion of a con st it u t ive H SE-bin din g act ivit y in rat ovary an d st age I an d II oocyt es su ggest s t h at H SP gen es m ay be regu lat ed du rin g vert ebrat e oogen esis by a developm en t ally regu lat ed H SF. A com parison bet w een ou r dat a sh ow in g h igh levels of con st it u t ive H SE-bin din g in early developin g oocyt es, w it h t h e act ivit y of H SF2 in cells of t h e t est es (Sarge et al., 1994), gives rise t o t h e in t rigu in g possibilit y t h at H SF fam ily m em bers m ay play relat ed roles in bot h m ale an d fem ale germ lin es. As t o t h e pot en t ial fu n ct ion of t h e con st it u t ively act ive H SF, w e fi n d it in t erest in g t h at it s H SE-bin din g act ivit y coin cides w it h t h e pres- C opyrigh t q 1997 by Academ ic Press. All righ t s of reprodu ct ion in an y form reserved. AID DB 8441 / 6x15$$$$83 12-09-96 18:37:34 dbas 61 H SF A ct iv it ies in X en opu s O ocy t es en ce of H SP70 m RN A in st age I an d II oocyt es (Fig. 6). Fu rt h erm ore, t h is act ivit y coin cides w it h t h e m ost act ive period of m RN A syn t h esis in oocyt es; m ost of t h e poly(A)/ m RN A con t en t of oocyt es is syn t h esized by st age II an d t u rn s over very slow ly t h rou gh ou t t h e rem ain der of oogen esis (Perlm an an d Rosbash , 1978; D oleck i an d Sm it h , 1979). T h erefore, it is lik ely t h at t h e con st it u t ively act ive H SF is a posit ive regu lat or of H SP expression du rin g very early st ages of oogen esis w h en t h e dem an d for cellu lar ch aperon es is very h igh , an d it w ill be in t erest in g t o det erm in e of t h e m ech an ism s con t rollin g t h e st age specifi cit y of t h is fact or. A t t enuat ion of Ind ucib le HSF A ct iv it y in O ocy t es An alysis of t h e k in et ics of H SF1 act ivat ion in oocyt es sh ow ed a rapid resu m pt ion t o con t rol H SE-bin din g levels u pon ret u rn t o n orm al t em perat u res (Fig. 8), bu t a t em porally ext en ded act ivat ion profi le u pon con t in u ou s exposu re of oocyt es t o h eat , cadm iu m , an d arsen it e (Fig. 7). T h is prolon ged pat t ern of act ivat ion is fu n dam en t ally differen t t h an t h e t ran sien t in du ct ion of H SE bin din g previou sly report ed in em bryos su bject ed t o iden t ical con dit ion s (O vsen ek an d H eik k ila, 1990; Karn et al., 1992). T h e rapid declin e of H SEbin din g levels du rin g recovery in dicat es t h at a m ech an ism for at t en u at ion of H SF1 act ivit y by t rim er disassem bly is presen t in t h e oocyt e. It appears, h ow ever, t h at t h e cellu lar m ach in ery perform in g t h is t ask in oocyt es is u n able t o overcom e t h e effect s of con t in u ou s st ress. In t erest in gly, H SF1 is t ran sien t ly act ivat ed in em bryos u n der sim ilar con t in u ou s st ress regim es, even prior t o t h e on set of H SP m RN A syn t h esis an d acqu isit ion of t h erm ot oleran ce at t h e m idblast u la st age (H eik k ila et al., 1985; O vsen ek an d H eik k ila, 1990). T h e sim plest in t erpret at ion of t h e differen ce bet w een oocyt es an d em bryos is t h at a cellu lar fact or(s) regu lat in g at t en u at ion of t h e st ress respon se an d disassem bly of H SF1 t rim ers is eit h er lim it ed or post ran slat ion ally m odifi ed in t h e oocyt e. We propose t h at t h e delayed at t en u at ion of H SF1 act ivit y in st ressed oocyt es is lin k ed t o t h e low level of in du ced H SP syn t h esis (H orrell et al., 1987). In t h is regard, t h e idea t h at H SF1 oligom erizat ion an d acqu isit ion of D N A bin din g act ivit y is regu lat ed by in t racellu lar levels of H SP70 fam ily prot ein s h as been t h e su bject of con siderable specu lat ion (M orim ot o, 1993, an d referen ces t h erein ). In ligh t of t h is, in effi cien t at t en u at ion of H SF1 act ivit y in t h e absen ce of H SP syn t h esis in t h e oocyt e w ou ld be con sist en t w it h a pot en t ial role for H SP70 in t h e disassem bly of H SF t rim ers an d at t en u at ion of t h e st ress respon se (M osser et al., 1993; Rabin dran et al., 1995). Fu t u re experim en t s w ill be aim ed at det erm in in g t h e pot en t ial role of H SPs on t h e act ivit ies of en dogen ou s H SF1 in X en opu s oocyt es. ACKN OWLED GMEN TS T h e au t h ors t h an k Bren da Bart n ik for su pplyin g rat ovary t issu e, Kevin Sarge for H SF1 an d H SF2 an t isera, Alan Wolffe for C AT report er con st ru ct s, an d Jeff G u t t m an n for assist an ce w it h fi gu res. T h is w ork w as su pport ed by gran t s from t h e M edical Research Fou n dat ion of Sask at ch ew an an d t h e M edical Research C ou n cil of C an ada t o N .O . A.A. w as su pport ed by a post doct oral fellow sh ip from t h e H ealt h Scien ces U t ilizat ion Research C ou n cil of Sask at ch ew an , an d A.H . w as su pport ed by an N SERC (C an ada) su m m er research sch olarsh ip. REFEREN CES An an t h an , J., G olberg, A. L., an d Voellm y, R. (1986). Abn orm al prot ein s serve as eu k aryot ic st ress sign als an d t rigger t h e act ivat ion of h eat sh ock gen es. Scien ce 232, 522 –525. Baler, R., Welch , W. J., an d Voellm y, R. (1992). 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