Gen Phvsiol B i o p l n s (19%) 1 5 , H71 387 3 7 1

The Ionic Basis of Membrane Potential Changes from Before Fertilization Through the First Cleavage in the Egg of the Frog Rana cameranoi

S, E R D O G \ N G L O G O G L U and 1 O Z G U N E N

(I'ukaiova Unniersitij School of Medicine Department of Physiology 01310 Ada n a Tin Lei/

A b s t r a c t . Expenments weie peifoiined to identify the ionic basis of membiane potential changes m the Rana nimnanoi egg fiom pnoi to feitih/ation t luough the fiist clea\age The membiane potential was monitoied contmuoush duiing this penod Ten pel cent Ringei was used as the lecoidmg solution m the (ontiol gioup The effects of N a + oi C a J f conductances weie obsci\ed b\ alteimg < \ te ina l coiuentiatioiis oi these ions K+ and Cl~ conductances weie tested b\ adding channel blockeis of these ions (TEA and SITS íespec tnely) to the extiacellulai medium

Tin lestmg i)otential of the unfeitihzeel egg is mainl> affected In Iv + (on due tance Chlonde conductance is u sponsible foi the depenalization phase of the feitih/ation potential cnoked b\ spenn e n t n and K + conductance is íesponsible loi the íepolaiiľation phase oi tins potential We suggest that N a + penneabiliťs does not d n e c t h coutiibute to the feitih/ation potential howevoi feitih/ation po­tential peak is signihi anth leduced upon a lediut ion of e\tiac ellulai sodium The1

leitili/ation potential is not signihe anth influenced In extiacellulai C a 2 + and eggs íeitili/ed m < ak íum-íiee solutions maintain then noiinal d( \olopment these lesults suggest that extiacellulai C a 2 + does not signihe a n t h couti ibute to the elcetncal and mechanical blocks that pie\ent pol>speim\ The membiane potential of the feitilized egg does not altei significantly until the fiist clea\age Potassium conduc­tance contnbutes to hvpeipolan/ation geneiated upon the hist clea\age wheieas sodium is the basic ion íesponsible foi the phase which follows peak In peipolan/a-tion and which pla\s a íole m the íetuín of the1 post-e lea\ age membiane potential to a steach le\el CT~ conductance which is n n p o i t a n t as the ionic basis oi the fei­tih/ation potential does not significantly influence any paiametei of the cleavage c\cle

C oiiespoiulencc to Seiel bido{,an M D C ukuioxa Uni\cisit\ School of \h flic ine D c p . u t i i u n t oi Ph\siolo},\ 0 1 » 0 - \ . c k i i d / i u i k t \ Iel (90) Í22-Í-5S (>S 17 l a x (90) 522-33X6") 72 Ľ-nidil seido^an " p a i m i k c r ru cdu 1i

372 Lido^an c t al

K e y w o r d s : Membiane potential Fei t ih/at ion — Clea\age Fiog egg

In troduc t ion

It is essential ioi the \ iabiht \ oi the embivos of main animals rind e\en some plants that onh one spermatozoon fertilize the egg Block to poh speinn is assuiecl m most cases In two 01 thiec plienomc na The fust block is the iast tiansient c lee t in al block which is the sudden potential change m the oocyte plasma membiane when the c gg is feitili7cd by a speimato/oon (feitilization potential FP) (C haibonneau et al 1983 lafie et al 1985 lafie and Schhchtei 1985 laife 198G Klme et al 1985 Webb and Nucitelh 1985a b Klme et al 1986 Biawlcn 1991) In most species FP is the c le< t iua l depolan/at ion of the ooc i te plasma membiane and it p ie \cn t s poh s])cim\ until a permanent and a ielati \ol\ slow mechanic al l)loc k is established ( l a f fee ta l 1985, lafie and Se hhchtei 1985 Klme et al 1985 lafie 1986) F P is well documented in \ anous species oi animals such as staihsh Uic>chis and amphibia and in plants like Ftuoul alynt but its ionic basis is different ioi diffeient species ( lafie 1986 B iawk) 1991)

Dining c a ih clea\agc of fiog eggs electiical h\ perpolaiization of the fertilized egg plasma nienibiane is followed b\ a le tu in of the membiane potential to a stable \a lue (Webb and NIK itelli 1985a b) Woodwaid (1968) and de Laat and Bluemmk (1974) stated that this In peipenalization might develop due1 to the addition of a new nienibiane with a high potassium penneabili t \ (Cioss and Elinson 19 SO) In another stuch pciioimed b\ nicasuiing extiae edlulai ion euncrits dunng elevnage of \titoj)W> eggs it was íepoited that K + might be íesponsible ioi membiane hy­pe lpolai i /a t iou and N a + , Ca 2 + and CT~ togethei foi the le tu in of the membiane potential to a stable \a lue (Klme et al 1983) Membiane potential changes ha \e been documented iioni ieitilization t lnough the hist elevuage stage especially in amphibia such as Ac nupiit, hit t/s and Runa pipicns but the ionic basis oi these po­tential changes has not been evaluated en tneh In continuous íecendings me hiding this pc nod

The ami of this stuch was to deteimine membiane potential changes and then ionic basis fiom piioi to feitilization t lnough the hist chvnage stage in the1 Rami

(aiiKianui fiog egg b\ lecoichng membiane potentials continuoush dunng this eail\ developmental pcnod

Mater ia l s and M e t h o d s

Selected flogs of Rana cameranoi species weie kept until use at +4°C, in plastic

boxes which weie filled with small amounts of stock solution iWcbb and Xuc itelli

1985a)

Membrane Potential from Fertilization 373

Obtaining eggs and spermatozoa: insemination procedure

The study was s ta i ted in November, and tcimiiiated in June. The pituitary glands

were removed from the female frogs, homogenated, and injected intraperitoneally

into the sexually mature female frogs (Perkins et al. 1981). The number of pituitary

glands injected to one individual was adjusted according to the season (Rugh 1962).

Progesterone (Sigma, P-0130) was dissolved in vegetable oil at 10 mg/ml by

gently heating the test tube, and the stock solution was stored at room temperature.

Progesterone was injected into the female's thigh muscle. Pi tui tary homogenates

and progesterone were injected consecutively The amount of the injected proges­

terone was also adjusted according to the season (Jaffe et al. 1985). The injected

frogs were kept at 18 °C for 36 h, or at 25 °C foi 24-36 h, and mature eggs were

then obtained by squeezing from the cloaca. To obtain mature eggs over 2-3 days,

female frogs were kept at +4°C.

Mature eggs appiox. 1.75 mm in diameter, surrounded with vitelline membrane

and the jelly envelope, with a pigmented animal moiety and a greyish-white vegetal

moiety were included in the study (Rugh 1951).

Sperm suspensions were obtained by maceiating frog testes m 10% Ringei,

2 5 h after a male was injected with 300 IV human choiionic gonadotropin (hCG,

Sigma CG-2) intraperitoneally Foi the activation of sperms, the suspensions were

kept at loom temperature for 5-10 min. To remove debris, sperm .suspensions were

then nlteicd through a fine nylon mesh. Sperm was examined foi motility and

morphology under a microscope at 1000 x magnification.

Five to ten niatuie eggs weie placed m a div plastic Petri dish: they weie

immobilized by the natural sticking of their jelly to the plastic. For fertilization,

the eggs were covered with a few drops of a sperm suspension (approx. 5 x 10''

sperm/ml) . Aftei 1 min. the recoiding solution was added to the dish, and the eggs

were impaled with a micioelectrode. Fertilization occurred approx. 5 min after the

insemination Nonnally, more than 90' / of the inseminated eggs were fertilized.

Xoimal fertilization was scored by a shift m membrane potential towaids positive

values (about ó min J, íotation (about 30 mm), noiinal first cleavage (about 2 .5

h). and embryo formation (Jaffe and Schlichter 1985) Records obtained from eggs

that showed membrane depolarization'and rotation but did not cleave normally or

did not develop until the embryo formation stage, were excluded from the study

Experiments were perfonneel at 21-25 lJC.

Control and e.ipcivinen.tal groups

Contiol records weie cairieel out in standard 10% Ringer solution which contained

(in mniol/1): NaCl. 11.1: KC1. 0.19, CaClo. 0.11, MgS(X,, 0 08; KaOH, 0 4: HEPES.

0.25; pH 7.8 (.laife and Schlichter 1985) Solutions to be tested for the effects of

ions were1 piepareel by modifying the Ringer solution, and nvords performed in

374 Ei doga n et al

these modified solutions seived as experimental gioups Feitilization, hist cleavage and e a i h embivonal development were uoimal in all these1 solutions

Ten minol/1 te^iaethylammonium clilonde (TEA., Sigma) and 1 mmol/1 4-acetamido-4'-isothioeyanatostilbene-2,2'-disulphonic acid (SITS, Sigma) weie used to block K + and Cl~ channels. íespectively The composition of the solutions in the expeinnental gioups were as follows [Na+]o •; and [Na+]o i solutions weie piepaic>d bv leducing N a + to 0 5 oi 0 1 of that found in the standaid solution [Na+]o solution winch was fiee of N a + TEA and SITS solutions were piepaied bv adding bloekeis of K + and CT~ channels íespectiveh [Ca2 +]o r, solution was piepaied bv leducmg C a 2 + to 0 5 of that found in the standaid solution, [Ca 2 + ]o solution, which was fiee of C a 2 + All solutions weie adjusted to be isoosmolai with 10% Rmgei, bv substituting choline CI foi NaCl and MgCh foi C a C h To avoid C a 2 + contamination Ca 2 + -fiee solution contained 5 mmol/1 EGTA (ethylene gheol bis( J-ammoethyl ether )-N N N' N'-tetiaacetic acid Sigma)

Electiophipiioloqital measinemenť,

Micioelectiodes were noimally pulled (Haiwaid Micioelectiode Puller) fiom 1 2 min OD thin glass capillanes (Intiacel Ltd) and filled with 3 mol/1 KC1 Then had tip lesistanees of 10 20 MSi and tip potentials of 2 5 m\ These micioelectiodes were used to measuie membiane potential with lespect to the solution bath, which was giounded t lnough an agai budge that served as the lefeience elec t iode Undei a stcreonncioscope (Olympus YMZ). the tip of the eleetiode was genth piessed on the egg s animal moiety bv the aid of a hydiauhe miciomanipulatoi (Nanslnge MO-203) The eleetiode was mseited into the egg by t iansienth m< leasing the1

negative capacitance oi the pieamplifiei (Nihon Kohden MEZ-7200) to i>ioduce an oscillating cuiient Electncal lecoidmgs were made continuously until the end oi the fiist cleavage Membiane potentials were monitoied on a stoiage1 oscillo­scope (Nihon Kohden \ C-10) tlnough an ami)hfiei (Nihon Kohden AVB-10) and íecoided on a chait íecoidei (Palmei Bioscience) The following paiameters were measuied dunng electneal leeoidings

Resting membiane1 potential oi the unfertilized m a t n i e egg {RAW) feitili­zation tune (F,) peak fertilization potential {FPp), duiat ion oi the fertilization potential (FPj) membiane potential of the fertilized egg (MPf), the peak of mem­biane by peipolanzation obseived dunng the hist cleavage (CPľ), tune fiom the onset oi mernbiane In perpolanzation upon the hist cleavage, to the l e t u i n of the potential to a steady value1 (cleavage1 cycle C\ m this cycle phase1 1 is the time to the1 peak hy peipolanzation and phase 2 is the time fiom peak hy perpolanzation to the generation oi a steady nienibiane potential), membiane potential aftei the fiist cleavage (MP,,r)

Membiane Potent ia l fiom i e i t ihzat ion 3 7 5

Statistical analysis

All aveiages weie expiessed as mean i S E M Significance of differences between means was deteimmed using one way Anova test and Student s ŕ-test foi paned data The ei i tcnon foi significance was p < 0 05

R e s u l t s

Fertilization of the Rana canieianoi fiog egg was accompanied by a sudden and lapid depolanzation of the nienibiane The duiat ion of this potential change was appiox 15 mm The membiane potential of the feitihzed egg which did not change

F i g u i e 1. ( ontinuous ieioiHm„s of the lneinbiane potential fiom impalement t l n o u g h fe lli l i/dtion to I lie 1st cleavage m ňet/ioeggs 1 In 1 0 ' / Ringer B In N . i + lief solution ( In solution with 1 1 V D In solution with SI 1 S

376 1 i c l o ^ m e t a l

T a b l e 1. hlectiical p iopei t ies of Rana cairn ninoi egg riming le itilization m clifleient solutions (mean ± S I )

Membiaiii pote ntial (m\ ) lime (nun) Solutions

RMľ Fľľ Ml'f I, ľľ,

10'/ Ringei -2(> 35 ± 1 2b h 25 ± 0 79 -22 55 ± 0 M> (> 30 ± 0 55 15 OS ± 105 ( \ = ,s n " = 20)

N a J r - 2 9 5 ± 1 19 2 2 ± 1 2 1 ' - 2 0 55 ± 1 OS 5 5(> ± 0 2S I t OS ± i) S 7 { \ = 5 „ = 20)

v l o i ( \ = 3 n = 21) Na+ - 1 . ( \ = 2 ci = 10)

( i(-;+, - 2 S 57 ± 1 92 9 02 ± 1 25 -22 S5 ± 1 1 I 10 ± 0 1 19 20 ± 1 25 ( \ =2 n = 21) ( a ' + - 2 7 SI ± 1 3 5 7 79 ± 1 2J - 2 1 10 ± 1 05 5 55 ± 0 75 17 52 ± 1 35

\ a + j - 2 9 SI ± 0 s i 1 (>7 ± 0 (KS - 2 5 71 ± 0 99 5 49 ± 0 15 12 51 ± 0 79

Nd+ - 12 (>9 ± 1 9(>'' 1 0 ± 0 93" - 2 7 SI ± L 5* 5 25 ± 0 57 13 I 3 ± 0 09

(\ = 3 n = 19)

SITS ( \ = 3 n =

n \ (\ = i n =

- 2 5 71 ± 1 53 = 21)

-20 is ± 0 so = 21)

- f 0 5 ± 1 0 1 '

12 7(i ± 0 «)(.

- 2 0 9 ± 1 2 3

- 1 5 55 ± 0 SI

5 10 ± 0 5S

1 5 1 ± 0 5

7 10 ± 1

17 05 ± 1

. 12

. 12

\ u m b e i ol hogs ** Numbe i oi eggs " Significant \s the eoiitiol g ioup /) < 0 05 Significant \s othei \ a + g i ( i u p s /i < 0 05

signihc a n t h unt i l the h i s t eleavage s h o w e d a slow n e g a t i v e shift al t in o n s e t of

t h e e leavage T h i s p o t e n t i a l e h a n g e m the1 h y p e i p o l a i i / m g d n e e t i o n l a s t e d a p p i o x

25 m m a n d was followed by a l e t u m of the m< mbiane p o t e n t i a l towaiel the level

of t h e unfer t i l ized state (1 lg 1 1)

Ri •ttini/ mi iiihinni pott nfin! oj tin nnjt i tih~<d iI/I/ mid Itit/h ntion potintml

\\ h e n t h e t i a c i n g of each egg was e x a m i n e d individual ly t h e í e s t m g p o t e n t i a l of

the unfer t i l ized egg (RMP) was m o i e negative in [ N a + ] n s o l u t i o n but general ly less

n e g a t i v e in T E \ s o l u t i o n (Fig 1 1 B C ) The 1 m e a n RMP va lues m t h e c o n t i o l

N a , | a n d T E A g i o u p s w e r e 1 - 2 6 5 > ± 1 2 6 i n \ - 1 2 6 9 ± 1 9 0 m \ a n d - 2 0 4 S ± 0 SO

m \ l e s p e e t n e h the moie negative value in t h e \ a , | g i o u p a n d the less negative

value in t h e T E A g i o u p were b o t h s tat i s t ica l ly significant (Tabic 1 1 F igs 2 5)

T h e m e a n fer t i l izat ion time did n o t diffei significantly m modif ied s o l u t i o n s

c o m p a i e d t o t h a t m 10'/ R i n g e i ( T a b l e 1)

Membrane Potential fiom Fertilization 377

> E

c tu o Q. 0) C <u

10

o -

-10 -

-20

-30 -

- 4 0 - . ..

-50 -

-60

i • -

1 F _ l

E

'Y* 'V 1 v 1 \ # • . .

j v * I

• *

•

•

1 1

Control

Na0+

- - i \ T

\A \ \

i

i /

, / T*

I i i

20 40 60 BO 100 120 140 160

Time (min)

F i g u r e 2 . 1 lectncal piopei t ies of Rana < anuunioi egg horn feitilization until the hist cleavage contiol and NajJ" gioups (mean ± S L) (*) Significant vs the contiol g ioup (/< < 0 05)

Blocking of the K-1- eoncluc tanc e me leased the peak of the fertilization potential

(moie positive FPt) vvheicvis blocking oi the C I - conductance1 i educed it (moie

negative FPV) (Figs I f D , 1 5 Table 1) Reducing oi extiacellulai N a + i educed

the FP,, level m a concentiation-dependent manner, and lesulted m significantly

less positive mean FPp values (Fig 2 Table 1) Reducing of extiac ellulai C a 2 + did

not change the amplitude oi the peak significantly (Table 1 Fig 3)

Blocking of the1 K+ conductance piolonged the duiation of the feitilization

potential (FPi) whereas blocking of the Cl~ conductance shoitened it maikedly

(Fig \C D) FP, /d id not change1 significantly m solutions erf different N a + cone en-

tiations and m Ca2 +-f iee gioups The mean FP,i values m SITS and Ca^t, gioups

differed significantly eonipaied to that m the contiol gioup (Table 1 Fig 4)

\ s also observed with the1 membiane1 potential of unfertilized e^g stable mem­

biane potential following feitilization (MPt ) was ahvav s moie negative1 m N a j solu­

tions and less negative m TEA solutions compaie d to those m the contiol t iacings

(Fig ID.C) The mean MP f values m all gioups were significantly less negative

3 7 8 Eidogan et al

0) C (0

n E a>

20

10

> E

J -10 c d) * - < O

a - 2 0 -

-30 ; .

- 4 0

- 5 0

- 6 0

• Control

r Ca02+

— I —

20 — I —

40 60 BO 100 120 140

Time (min)

— i 160

F i g u r e 3 . Flectiieal piopei t ies of Rana caineianoi egg liom íei t ihzal ion until the ínst eleavage contiol and < a("j+ gioups (mean ± S L) (*) Significant vs Ihi contiol g ioup (l>< 0 05)

eompaied to tlie mean RMP leveds (p < 0 05 Table- 1) As also determined ioi

the mean RMP value the mean MP j levels differed significantly m N a,"j" and TEA

gioups eompaied to that m the contiol gioup (Table 1 Figs 2.5)

C'/e a c a e / e - c / s s o e iat<d iiKiiibiaiK pottntial (hangts

L pon cleavage1, fiist the nieiiibiane giadualh hvpeipolan/ed (phase 1) and tins

hy perpolanzation was followed by a leeoverv of the membiane1 potential to a new

steady value (phase- 2) (Fig 14) This post-cleavage membiane potential (MP,,, )

was moie negative m all gioups eompaied to the nienibiane potential following

fertilization (MPS) (Fig 1 4 B C D)

The1 peak potential of hy perpolanzation upon the hist cleavage1 (CP,,) was

always less negative in TEA solutions (Fig 1C), whereas it did not maikedlv differ

m the other gioups The mean CP,, value1 of —45 12 ± 2 23 niY foi the- contiol

gioup was significantly less negative1 m TEA gioup ( - 2 S Gi ± 1 86 m Y p < 0 05,

Table 2 Fig 5)

The new nienibiane potential geneiated following hy peipolanzation (MP,,, )

Membiane Potential from Feitilization 379

10 n

> E

c d) *-» o a a> c TO

d)

-10

-20 -

-30 -

-40 -

-50 -

- 6 0

• Control

T SITS

20 — I — 40 60 80 100

Time (min)

120 140 —i 160

F i g u i e 4 Llcctucal piopei t ies of Rana < amnanoi egg fiom feitilization until the fust elrav tge contiol end S í l S gioups (mean ± S I ) (*) Significant vs the contiol g ioup (p < 0 05)

cliff t ic el signific anth m \ a ( { Na,f and TEA gioups eompaied to that m the contiol

gioup (íespectivery - 4 4 79 ± 4 4G ni \ - 4 3 25 ± 3 3 nA - I S 44 ± 1 03 in \ and

- 3 2 24 ± 1 28 m \ Table 2 Figs 2 5)

The cleavage cycle (C ) ic the tunc fiom the onset of membiane In pei

polaii /ation upon cleavage to the i c tum oi the potential to a steady value was

2G 42 ± 1 73 linn in the contiol gioup The duiation of this cycle was signific an th

shoiter m TEA. and Ca 2 + gioups the mean ± S E values being 17 36 ± 1 29 mm

and 17 3 S ± 195 mm uspectively (p < 0 05 Tables 2 3) The cluiations of the

cleavage cycle phases an also shown in Table 3

D i s c u s s i o n

R< stun/ mtnibiant pott ntial of unjt itdut d tgq

In the piesent study the ic stmg potential of the unfcitilizcd Rana taint itinoi e g„

bathe el m 1 0 / Ringc i was —26 35± 1 26 m \ Snmlai value s have be c n íepoifeel loi

3 8 0 Eidogan et al

E d) O Q. O Ľ

E tu

• Control

• TEA

— i — 40

— I — 60 BO 100 120 140 160

Time (min)

F i g u r e 5. E l e c t i u a l piopeit ies of Rana t aineianoi egg fiom Ic itilization until the- hist cle-avage m Hie contiol and 4 4 A gioups (mean ± S Ľ) (*) Sigmhe ant \s the- contiol g ioup (/-< 0 05)

Rana pipuns Rana t scitlt nta . nd Rana tt nipoiai iti eggs bathc-d m solutions of ionic content snnilai to 10'/ Ringer (Ciossand Elmson 1980 Schlichtei and Elmsoii 1981, laffe et al 1985 Jaffe and Schlichtei 1985) Foi Rana pipit ns and Xtiiojuis /c/cc/s eggs bathe d m solutions of different e (imposition the1 ic poitecl lost nig pot cut nd was appiov - 5 5 niY and - 1 9 niY lospoetivch (Webb and Nue itelli 1985a)

In solutions oi ledueed Na ' (Na, |- and N a ( | j gioups) the lestmg potential was insignificantly moie negative whereas In perpolanzation observed m Na+-fiee solution (Na("| R l o u p ) was signific a n t h different fiom the contiol and other N a +

gioups (Table 1) Significant livpeipolan/atiou detected m Na f - f iee solution was considered to be- clue to Na * efflux tlnough the membiane1 and tins conclusion was suppoited bv insignificantly moie negative1 lestmg potential values m oocytes bathed in low external sodium (Table 1)

As also lepoi ted foi A'c n o p u s lat ois c-ggs (Pcre-s and Mancinellí 1985) c ale min and chlonde ions did not signihc anth afiec t the íesting potential m Rana cainaanoi e'ggs (Table 1)

Membrane Potent ia l fiom Feitil ization 3 8 1

T a b l e 2 . solutions

Solutions

Elect i (mean

107c Ringer

^ o + . Na +

SI I s

I L \

(N

(\ (A

(A

(A

( \

ical p ioper t ies ± S E )

= 8 n = 17)

= 5 n = 14) = 5 n = 19) = 2 o = 12)

= 2 n = 19) = 3, n = 14)

= 2 n = 22)

= 4 ;i = 16)

of Ran a camertnioi

CPP

( m \ )

- 4 5 12 ± 2 23

- 5 2 29 ± 4 18 - 1 5 11 ± 2 8 1 - 5 4 67 ± 5 01

- 5 1 11 ± 4 56 - 3 7 79 ± 3 63

- 5 5 05 ± 3 23

- 2 8 63 ± 1 86°

egg upon t he fiist

C, (mm)

26 42 ± 1 73

19 56 ± 3 24 19 56 ± 1 88 19 00 ± 1 30

21 08 ± 1 85 17 38 ± 1 95"

21 13 ± 1 53

17 36 ± 1 29"

cleavage in different

MPpc

(mV)

- 3 2 24 ± 1 28

- 4 4 79 ± 1 46" - 3 6 47 ± 2 f 3 - 4 3 25 ± 3 3 '

- 2 9 53 ± 1 39 - 2 5 29 ± 1 17

- 3 8 11 ± 2 55

- 1 8 44 ± 1 03"

Significant vs the contiol gioup p < 0 05

T a b l e 3 . Duia t ions ol phases of the hist cleavage m rliffeient solutions (mean ± S 1 )

107 Rmger

\ a ( !

Na(T

c 4+

SI I s

11 \

(ll =

(n =

(n =

(n =

(,,=

(» =

in =

('• =

IT)

11) 19) 12)

19) 14)

22)

16)

Phase 1 (nun)

15 21 ± 2 15

11 52 ± 1 17 12 18 ± 1 1 11 06 ± 0 96

s 25 ± 0 9 2 " 8 29 ± 0 78 '

9 f 2 ± 0 97

7 50 ± 0 76"

Phase 2 (mm)

13 18 ± 0 92

8 2 3 ± 2 2 3 7 19 ± 1 48 7 51 ± 1 2 5

12 13 ± 1 19 9 OS ± 1 65

12 51 ± 0 98

9 16 ± 0 80

1 ot al (mm j

20 12 ± 1 7 5

19 56 =c 3 2 1 19 36 ± 1 8 \ 19 00 ± 1 30

21 08 ± 1 85 17 38 ± 1 95

21 13 ± 1 33

17 36 ± 1 2 9 '

Significant vs the contiol gioup p < 0 05

In solution containing K + channel blocker the nienibiane potential dec leased

significantly eompaied to the contiol gioup (Table 1) It has been lepoited foi

Rana pipit ns eggs that potassium ions aie piesent in higher cone entiations in the

382 I l d o g a n e t al

ICF (appiox 121 mmol/1) than in the- e ell extcnoi (appiox 0 19 mmol/1) and they tend to diffuse- out of the e ell along this c one entiation giaehent (laffe and Schlichtei 1985) We suggest that the1 sigmhe ant depolanzation observed in the FE \ gioup is due to doe H a seel K + efflux due to the blocking of K""" e hauliers

In suininaiv the ic stmg potent nil oi the unfertilized Rana taint rnnoi egg is stiongly infiiKiKed by the1 chemical and electncal giadients of K+ (E/K = —150 m \ ) (Jaffe and Sehliehter 1985) and it also differs significantly in sodium-fiee solutions

Fc ; titration turn (Ft )

The1 mean feitilization time was 6 3 0 ± 0 53 nun m the contiol gioup and it did not diffei sigmfieanth in any of the modified solutions (Table 1) Snnilai findings have been lepoited foi vanous fiog c-ggs inseminated by snnilai sperm c one entiat ions (Chaibonne-au et al 1983 Jaffe et al 1985 Jaffe and Schlichter 1985 Webb and Xin itelli 1985a,b)

Tht ptak ft itilization potential (FP,,)

Fertilization is accompanied bv nienibiane depolanzation In 10'/ Ringer the1 peak of this potential change1 was 6 25±0 79 niY (Table1 1) Foi Rana pipit ns e ggs bathed in the same solution it has been lepoited to be 13 nA ( Jaffe and Se hhc lite i 1985) different values have been nieasuic-cl foi Rana pipit ns and Xtnopus eggs ( — 5 1 and ± 3 0 n i \ lespee tiveh ) bathed in solutions of different composition (Webb and Nue itelli 1985a)

Jaffe and Schlichtei (1985) has lepoited that CI™ channel blockeis (SITS and DIDS) had no effect on the- feitilization oi activation of Rana pipit ns eggs but in another study peifoiined with Xtnopus eggs DIDS was found to deciease C I -

conductance which men asc el upon feitilization (Webb and Nue itelli 19S2) When CT~ channels were- blocked by SITS m oui expeinnents the1 fertilization potential peak (FP,,) was significantly moie negative (—4 05 ± 1 04 m\ Table 1 Fig 4) eompaied to the contiol gioup nicont ias t blocking of K + channels bv TEA caused this peak to become signific anth moie positive (12 7(> ± I) 96 m\ Table1 1 I lg 5) In the TEA. gioup FP,, was yen e lose to the1 Cl~ e epuhbiium pote ntial (E( /) w Inch foi Rana pipit ns egg is ±18 in\ (Jaffe and Sehliehter 19S5) m the SITS gioup on the other hand FP,, shifted tern aids the calculated E^ of —150 niY ( Jaffe and Schlichter 1985) but this shift was not as piomment as that seen in the TEA gioup These lesults suppoit the c one lusions that Iv+ and C 1™ c onelue tam e s w hie h aie both piesent m higher c one entiations m the ooplasm than m the egg exte-noi (laffe and Schlichter 1985) coutiibute to the fc itilization potential and that the depolanzation is clue to a laigei efflux of chlonde ions t lnough the Rana ctuiKiaiwi

egg membiane

M e m b i a n e Potential fiom feiti l ization 383

In eggs bathed in low [Na+Jn 01 in sodiuni-tiee solution the1 fertilization poten­tial peak was found to dec lease significantly, Na + -fiee solutions did not niteifeie with the development oi the depolaiizing fertilization potential (Table 1 Fig IB) Sodium channels which open when the1 membiane potential is biought to positive-values have been lepoited in eggs of Rana and Xenopus (Baud et al 1982 Schlichter 1983) m low external sodium as m pond water sodium cuiient t lnough these chan­nels will be outwaid and thcrefoie it will have a hvpeipenalizing effect causing the potential change to become less positive1 When the lesults lepoited in om vvoik aie eompaied with those of othei studies, it may be suggested that sodium does not diiectly coutiibute1 to the development oi the feitilization potential but X a + efflux t lnough the N a + channels which aie opened by the Cl~-mduced depenalization will pi event the pe>ak feitilization potential fiom i caching moie positive values in eggs bathed in low external sodium

We could observe no significant effect of extiacellulai calcium on the feitiliza­tion potential (Table 1 Fig 3)

Duiation of tht ft- itilization pott ntial (FP,i)

Fhe duiation of the feitilization potential was 15 08 ± 1 (13 mm snnilai ie-sults have bee n lepoited foi Ranu pipit ns and A'c nop u s lut ľ is eggs (Jaffe and Schlichtei 19S5 Webb and Nueitelh 1985a b) Blocking of C I " channels by SITS ledueed this value significantly (7 46 ± 1 12 nun Table 1 Fig 4) vvheicvis blocking of Iv + channels bv T E \ piolonged tins duiat ion insignificantly (17 05 ± 1 4 2 mm Tabic 1 Fig 5) These findings suggest that when Cl~ conductance íesponsible fiom the1 depenalization phase of the feitilization potential is limited the1 c -f tec t of K + conductance- becomes moie pionnne nt causing the feitilization potential peak to become signific a n t h i educed (Table 1 Fig 4) and the nienibiane to become polan/ed again moie easily and hence in a shoitci time On the contiaiv when K + channels aie blocked the moie piomiiient effect of C I - conductance causes the feitilization potential peak to meiease and the ic polanzing fences to become weakei m this c ondition the nic nibiane will be polaiized foi a longer time1 e ausing an insignificant piolongation of the- duiation of the1 feitilization potential The effects of blocking of K + and Cl~ channels on the duiation of the1 fertilization potential have1 been observed cle-aily m the tiacings n c ended fiom each Runa egg (Fig IC D)

In e-ggs bathed m low e xtc-inal calcium the1 duiation of the feitilization poten­tial was sigmhe antly piolonged whcieas it did not change signihc a n t h in ealcium-fice solutions (Table 1) Moie over Ca~ + conductance did not seem to plav a sig­nificant idle in othei paiameteis of the- ie itilization potential and low extiacellulai < ale mm oi C.r'+-fiee solutions did not niteifeie with the moiphologn ally noimal development of fertilized eggs t lnough the neuial fold stage1 These lesults suggest that extiacellulai e ale mm plays no significant íole in the generation of the fast

384 Eielogan e 1 al

tiansieiit d e n t i n a l block and the1 lelativelv slow pennanent mechanical block nec-essaiv to pic'vent polyspeimy Tins conclusion is paitly suppoited by the lesults of Busa et al (1985) who have lepoited that mtiacellulai calcium stoics can be important in piodueing the pennanent mechanical block In the light of all these le­sults we conclude that the significantly long-lasting fertilization potential observed in the Ca5"t gioup can be explained as an effect of a leakage conductance of othei ions which may have developed d u n n g the- experiments

The insignificant effee t of N a + eonduc tance on the duiat ion of the feitilization potential (Table 1) suppoits oui findings eonfiimmg that X a + does not diiectly coutiibute1 to the fertilization potential

In summaiy Cl~ conductance is íesponsible foi the depolanzation phase vvheicvis Iv+ eonduc trine e is íesponsible foi the- lepolan/ation phase1 of the1 fer­tilization potential m the egg of the fiog Rana tanmunoi

Mtinbiant poltntial of tilt ftitihztd tgtj (MP/)

In all gioups, the membiane1 potential of the feitih/ed egg was significantly less negative than the lestmg potential Snnilai lesults were- lepoited foi Rana pipit ns and Xtnopus lat vis eggs and the less negative1 MP f values have1 been explained as an effect of leakage c u n e n t s which may develop d u n n g mcasuieunonts with an mtiacellulai eleetiode (Webb and Nucitelh 1985a, Peios et al 1986)

The membiane potential ol the1 fertilized egg was —22 5 5 ± 0 86 in\ m the- con­tiol gioup In solution with a K + blocker this value was significantly less negative ( — 15 35 ± 0 84 niY Table 1 Fig 5) leading to the conclusion that KH is one-of the ions li-spousiblo foi the lepolanzation phase of the feitilization potential i c-foi membiane lepolan/ation

In eggs bathed m Xa+-fiee solution MPj was significantly moie negative (—27 81 ± 1 50 niY) eompaied to the1 contiol gioup (Table 1 Fig 2) tins ie-sult can be explained as an effect of sodium efflux developing m this condition Although sodiuni-fiee solution pioeluc ed such a hy peipolaiizing effect the mean MP/ values of the- Na,"!^, Naf"!, and Na,! gioups did not show íelativo and significant hypcipolaiľ/ations when eompaied to each othei (Table 1), moieovcr Na^-t iee so­lutions did not niteifeie with the development of the feitilization potential and these solutions did not significantly affect the duiation of this potential (Table 1 Fig 1 4 B) These findings suggest that Na f eouduetance does not couti ibute to the geneiation oi the ieitilization potential and the meinbiaiic- potential following fertilization

Oui lesults also show that t hloiide oi c ale mm e onduc fane es do not sigmhe a n t h affect the nienibiane potential of the fertilized egg (Table 1)

Tin {>tal, pott ntml upon tin fast tltauatjt (CP,,)

In the contiol gioup the peak value of nienibiane In peipolanzation upon the fust

Membrane Potential from Fertilization 3 8 5

cleavage was —45 12 ± 2 23 mV Upon blocking of K+ channels, CPV changed to a significantly less negative value (—28 63 ± 1 86 mV Table 2, Fig 5), whereas Na+ , Cl~ and Ca2+ conductances did not affect it significantly (Table 2) It has been suggested by Woodward (1968), and de Laat and Bluemink (1974) that membrane hyperpolanzation obseived dunng the first cleavage is due to the addition of a new membiane with different electncal piopeities, and a high potassium peimeabihty The increased K+ conductance upon cleavage will cause the membrane potential to become much closer to Ejs (which is approx —150 mV), leading to membrane hypeipolarization Therefore, blocking of K+ channels by TEA will cause this peak potential to become significantly less negative, as observed in the present study

Cleavage cycle (Cc)

In the contiol gioup, the cleavage cycle lasted foi 26 42 ± 1 73 mm Calcmm-fiee solutions and blocking of K+ channels by TEA shoitened the duration of this cycle eompaied to the contiol gioup (Tables 2,3, Figs 3,5) We suggest that the significantly less negative peak potential measuied in the TEA gioup upon the fiist cleavage may lead to a shoitening of the cleavage cycle and hence to the letuin of the post-cleavage potential to a steady value in a much shoiter time This suggestion is suppoited by oui finding of a significant and stiong conelation between the peak potential upon cleavage and the cleavage cycle, when Iv+ channels aie blocked by TEA (?, = 16, 7 = —0 678 p < 0 01) In oui expenments, phase 1 of the cleavage cycle (the time fiom the onset of membiane hypeipolaiization to the peak value of hypeipolaiization) was most stiongh affected when K+ conductance was limited by TEA As shown m Table 3 the duiation of phase 1 was significantly shoitei in the TEA gioup wheieas the shoitening m phase 2 was not significant eompaied to the contiol group

In eggs bathed m low external Ca2+ oi in Ca2+-fiee solutions we weie unable to detect íeasonable and significant differences in the mean peak hypeipolaiization values (i e íelatively moie negative peak hypeipolaiization with the reduction of extiacellulai Ca2 +) as eompaied to the contiol gioup (Table 2), so, the effect of Ca2+ conductance on significant shoitening m phase 1 seen m these gioups (Table 3), could not be explained by oui piesent knowledge

Membrane potential of the cleaved egg (MPpc)

Reducing of extiacellulai Na+ shifted the membiane potential of the cleaved egg towards moie negative values the diffeiences in Nar}5 and Naj gioups being signif­icant eompaied to the contiol gioup (Table 2) in addition phase 2 of the cleavage cycle was shoitei in all Na+ gioups as eompaied to each of the othei gioups (Ta­ble 3), these findings suggest that sodium is the basic ion coiitnbuting to phase 2 of cleavage (i e to the phase which follows peak hypeipolaiization, and which plays a íole m the letuin of the post cleavage membiane potential to a steady level)

386 F i d o 0 a n et al

Klme et al (1983) have lepoitenl that C a 2 + and Cl~ conductances m addition to that of N a + , may also be involved m the generation of phase 2 howevei we could demolish ate no significant effect of CT~ conductance on any paiainc-tei of the eleavage cycle1 (Tables 2 3 Fig 4), upon changing extiacellulai calcium com­position, we could not explain the c onti ibutiou of C a 2 + conductance to phase 2 by o m findings i elated to cleavage

In all gioups the1 nienibiane potential oi the cleaved egg (the potential winch follows the hvpeipolanzmg cleavage potential) was signific a n t h moie negative as eonipaied to the nienibiane potential of the feitilized egg Snnilai lesults have been lepoited ioi Rana pipit ns and A'c nopus lut vis eggs (Webb and Nue itelli 1985a) We suggest that the addition of a new membiane with a high potassium permeability (Woodwaid 1968 de Laat and Bluemink 1974) may cause the nienibiane potential oi the cleaved egg to become moie negative1, tins suggestion is suppoited bv oui finding that this potential was signihc a n t h less negative when a K"1" c liannel bloeke i was added to the extiacellulai medium (Table 2)

The oveiall companson of oui lesults with those lepoited in the- h t e i a t u i e has led us to conclude that the íesponse to feitilization with a lapid membiane depo­lanzation is an aetivre phenomenon evoked bv the sperm entiy into the Rana t am tiunoi egg. whereas membiane hypeipolaiization observed dunng the fiist e ioavage appeals to be a passive pioccss which develops slow h and lasts longer and which is piodueed by the addition of a new membiane with diffeient electiual p iopei t ies

A c k n o w l e d g e m e n t s . This vvoik was s u p p o i t e d bv giant I I 95 E 39 fiom Gukuiova University Research Council

References

Baud C K a d o R T Maichei K (1982) Sodium channels induced bv d e p o l a n z a t i o n ol t h e Xen op u s ta t ins oocvte P i o r Na ti \cad Sci I S \ 7 9 , 5188-3192

Biawlcy S H (1991) T h e last block against polvspcimv m ľutoiil alt/a e is an e lec tora l block Develop Biol 144, 91—106

Busa W B P e i g u s o n L 1 loscph S K W i l l i a m s o n ) R Nue c ite 111 R (1985) Valua­tion ol fiog (Xenopus latins) eggs bv inositol I nphosphate I ( 'ha iac tc i i za t ion of C a " + lelease fiom nitiae ellulai stoics I Cell Biol 1 0 1 , 677 682

C h a i b o n n e a u M Moieau M Piclieial B G u e i u e i P \ ilam I P (1985) \ o l t a g e noise changes d u n n g monospc i inir and p o h spe i line Ieitilization ol matnie eggs of the a n u i a n Rana teinpoiai ta Develop Glow 111 Difle i 2 5 . 185 191

C'ioss N L Flinson R P (1980) \ fast block to polvspc nnv m h o g me chate d In e hanges m the me m b i a n e pote ntial Develop Biol 75 , 187 198

de Laat S W Bluemink 1 G (1971) New membiane foi mat ion d u n n g cytokinesis m noiinal and e v tochalasin B-tieated eggs of Xt nopus laevis J Cell Biol 6 0 , 5 2 9 — 540

Jaffe L A (1986) Electiical regulation of speim-egg fusion \nnu Rev Phvsiol 4 8 , 191—200

M e m b i a n e P o t e n t i a l h o r n 1 c í t i l i z i t ion 3 8 7

I ifľe L A S c h l i c h t e i t C ( 1 9 8 5 ) f e r t i l i z a t i o n i n d u c e d ionic c o n d u c t a n c e s in e g g s of

t h e h o g Rant, Pipitns 1 Phv siol 3 5 8 . 299 — 319

laffe L \ h a d o R 1 M u n c v I (19S5) P i o p i g a t m g p o t a s s i u m a n d e hlonclc e o n d u c

t a n c e s d u n n g i c t i v a t i o n end I e i t i l i z a t i o n eif the e g r t ol t h e h o g liana Pipit ns I

P h y s i o l ( l o n d o n ) 3 6 8 . 227 212

Klme D R o b i n s o n K R \ u c i t e l h R ( 1 9 8 3 ) I o n c u i i c n l a n d m e m b i a n e d o m a i n s m t h e

c l e a v i n g Y e n o / N c s t g g J Cel l Biol 9 7 . 1 7 5 3 — 1 7 6 1

K l i n e D laffe L A T u c k r i R P ( 1 9 8 5 ) I e i t i l i z a t i o n p o t e n t i a l end p o l v s p e i m y p i c

vc nl i o n m the e g g of the Nt met tain ( eiebratiilus latteus 1 L \ p e i / o o l 2 3 6 ,

1 5 - 52

Klme D Jaffe 1 \ K a d o R I ( 1 9 8 6 ) \ c íle m i n u l i v a t o d s o d i u m e oudi ie t me e c o u ­

t i i b u t e s t o t he f c i t i h z i t i o n p o t e n t i a l m t l i e egg of t h e Neinciltan Wuiiu (tnbiat

iilui lacttus D e v e l o p Biol 1 1 7 , 181 193

P e n s V M a n r m c l l i L ( 1 9 8 5 ) S o d i u m c o n d u e t a n c e a n d t h e act iv i t i o n p o t e n t i d ill Xcno

pus latins eggs P f l u g c i s Vich 4 0 5 , 2 9 — 3 6

P e r e s A B e i n a i d n n G \ e g i i m C ( 1 9 8 6 ) Mc n i b i me p o t c n t i d me a s u i e n i e n t s of u life l

t i h z e d mi l f e i t i l i z e d Xtnopus tin i is eggs ue i f fected bv el l i n a g e i nisi d bv the

e l e e t i o d e F x p ( ell P e s 1 6 2 . 159 168

P c i knii, K W T i a n k s R 1 W h i t t i n R I I ( 1 9 8 1 ) R e p t i l e s end A m p h i b i m s C a i e a n d

C u l t u i e ( a i o l m a Biologic il S u p p l y C o i n p a n v U S A

R u g h R ( 1 9 5 1 ) T h e f iog I t s R c p i o d u c t i o n a n d De \c l o p m e nt M c G i a w Hill B o o k C o n i

panv USA R u h h R ( 1 9 6 2 ) F x p e l line n t a l l i n b i y o l o g y le c lmic[ue s m d P i o c e d u i i s l l m d I dit ion

Binge ss P u b l i s h i n g C o m p a n y

Schl ichtc i I ( ( 1 9 8 3 ) S p o n t a n i o u s i c t i o n p o t i nt i ds pioelue c cl by \ a + mi l ( 1 e h m n e l s

m m l i m i n g Rana pipient, ooc v t e s D e v e l o p Biol 9 8 , 1 7 — 5 9

S c h l i c h t e i I ( Ľ l i n s o n R P ( 1 9 8 1 ) Elee t n c a l ic s p o u s e s of l m m a t u i e a n d m a l i n e Rana

pipiens oocv tc s t o s p e n n a n d o t h e i act iv i t i n g s t i m u l i D e v e l o p Biol 8 3 , 33 If

W e b b D I \ u c i t e l h R ( 1 9 8 2 ) I n t i a c e l l u ' a i |>lf change s ae e o m p mv m g t hi act iv i t i o n D1

d e v e l o p m e n t m f iog i „ g s l o m p a n s o n ol p H n m ioi li i t i o d i s m i l P \ M R m e a

s u i e m e n t s In I n t i a c i l lul u pf 1 ft s Me i s u i e m i n t s R e g u l i t i o n s m d J t i l l / i t ion in

( i l lu la i 1 u n c t i o n s ( I els R r\uc itell i m d D De u m i ) p p 29 5 321 W m R I iss

[lie \e\y 5 o i k

W e b b D I \ u c iti Hi R ( 1 9 S 5 i ) y. c o m p a i a t i v e s t u d y of the m e i u b i a n e f i o m befoie

f ( i t i l i7" i t io i i t l n o u g h clc a v age in t w o h o g s Rana pi pun s and \t n op u s lat m s ( o m p

B i o c h e m P h y s i o l 8 2 A . 3 5 — 1 2

W<l>l> D I N u i i t i l h R ( 1 9 8 5 b ) f e i t i l i / it ion pote nt i d a n d eh i t i n il p i o p e i t n s of t h e

Xtnopus lat ns i „ g D i v . l o p Biol 1 0 7 , 395 106

W o o d w a i d D 1 ( 1 9 6 8 ) I li 11 lie a l s igns ol new mi m b i a i i e p i o d u i t i o n d u n n g i It i v a g e o f

Rana pipitns e g g s 1 G i n P h y s i o l 5 2 , 509 551

I m a l v e i s i o n u c o p i e d O c t o b c i 21 1996