F is h... .. .... ... ... .... ... . ... 76

TELEOSTS WITH EMPHASI SON.I~'TERFLOUNDER (Ps'udop1eurone c t ,s~ )

by

ClJama sJos ep h Nagler, B.Sc., H.Sc.

AthesissUblllttedtotheSchool of Gradua t eStudies inpar tia l fulfillmentof therequire me nts for th e

degree of Doct o rof Philosophy

Dep a r t llen t of Bi ology Memo r i al Uni v e r sit yof Newfo und land

June1991

St. John' s Newfou ndland

I i ABSTRACT

Theovarianuptakeofthe homol og o us serum proteins vitellogenln (VGI and ve ry highdensitylipoproteinII (VHDL II ) (form e rlypeak,.prot ein) werestud i ed as potent i alyolk precu rs o rsinvolv ed invite l logenesisin winterfloundl:![

(Pseudop l !!!urom!ctes~).ThellIil j o r yolkpr e c u rso r appea r s to be VG based on thequanti tyof yolk proteinthi1t recog n iz es the VG andVHDLIIant i s erabyWe s ter nblo t t ing.

The rateof upta keof VGby the ova r yisabo ut; three times greaterthan VHDLII. rneeen eiIaec VG is processe dinto a 280,000relativemolecularmass(MrJ yolk protein (lipovite llin) thatcontribu t es to the major fractio n (82\) of ovaria n pr o t e in . Ac culIlulationof VHDL II occurs in an unp rocessedform.and contr ibutes to a fract i onofova r i an proteinre pr e s e nting 12 \ of the total. Phosv it inand a low Hrphos phoprote in were apparentbut insdallamounts.

In~ovarian incubationsdoneduringtheprespawn ing to early vitellogenic pha s e s of the re prod uc t i ve cyclein vfntier-flounde r showedthatpitu i t a r yex tra c t stimulates est radio l-17P (Ell prod uct iononly during the vite lloge'ic phase, whil e indu ced testosterone (TI produc ti o n was greatest shortly bef o respa wn i ng. Theseobs ervat ionswere reflected in the seas o nal patternof ser um Lev eLs of El and T in femal ewint e r flound e r. To investi ga te theeffe ctof soc key e sal mon carbohyd r ate-po o r (ConA 1) andca rbohyd r ate -

rich (conAII) pi tu ita ry pr otei nfra ct i ons on Ez pr od uct i on, ova ria nfol licl es with (inta ct) or without the sur fa c e epithe lium-t hecalcellla yer (defol lic u l a t ed) from ra inbow tro ut (Onc o rhynchu s~) were inc ubatedjnti.tt2.

It was demon s t r ate d tha t ConAI inthe pre se nceof Tis ca pa bl eof sig nif icantl y increa sing Ez pr od uc t i o nin defoll icul a ted ovari anfolli cleswhileunder similar condi tions co n A 11 (con t a in ing the maturati onal gona dotrop in ) wa s not. Purifi e d sa l mo n i d and pleuronect id growthhormones (GHs ) we rete sted for theirabi lit y to inc r ea se ei t her Ezand T prod uc tionduri ng.in~ovar ian inc uba tions inboth rainbowtrout and winter flounder res pectivel y , but wer efound to be inactive .

Gr owt h hormoneswere isola t ed from the pituitaries of soc keyesal mon (On co rhynchus ~) and Americanplaice (Hippogl ossoi des clate s s ol de s j. A bioassaybasedon the inc r ea s e Clf se r um triiodo thyronine in ri!linb owtrout was developedto fo ll ow GHbiologicalactivityduringpituitary fra ct ionati on. The isolationof a pituitaryprote infrom sock eye sal mo n that wasac t i ve in the bioassaywas conf irmed asmonome ricGHby an amino-terminal (N-T) amino acid sequence. In plai ce GH variantswere isolated from two Mr reg ions withinthe pituitary , 42, 000 and<33 ,0 00 , that were acti ve in thebioa ssay and had ident ical N-Tami no acid sequences .The 42,000 Mr form predominates in the plaice pitu itarymakingup 93\ of the total.

iv ACKNOWLEDGEMENTS

I wo u l d like toex p r e s s my sincerestgr a t it ud e to Dr. DavidR. Idl er for his su pe r v is i o n andco n s t a nt support th r o u g h ou t my program of study. Hiske e nint e r e s t and insi g ht were greatly appreciatedduringthis research . 1 alsothankthe other members of my thesis advisory commi~:tee, Dr.Jo h nH. Green and Dr. Sailen Mcokerjea, fo r the i r as s i s t a nc e .

I amindebted to Mr.ShinnJiaHwa ng and Dr. 'ling P.So for theirtechnicalexpertise, comput e r know-how and helpfu l discussions. It was always an educationalexpe r i e nc e when working withthese individuals.

I greatfullyacknowledgethe MSRL custod iansHr.·sMi ke Dyer,Wa y ne Morrissey,JimYoungand Randy Cahill tor thei r lateni g ht assistance inthe coldroom.special thankstothe lat e Miss Beryl Truscott,Mr.John M. Walsh, Mr. surceh Bel kode, th eMs Rt diving unit and the re s t of the MSRI, staff.

The conti nualsuppor t of my family and wifeFaye were the great estsourceof encouragement during myst ud Ies ,

Title page.•.... •..•...•. ...•... ... ..•· 1o.!:;;<:ract. " .... ... ... ... . ... ... . ... ... ii Ac ltnowledqeme nt s. ... ... . ... ..•.... . iv Tableof Contents.. ... ... ...•.... •• •.•.•... List ofTables. .. .. ... ... ... .. . ...• . . •...• ... ix Lis t ot Figures.... • •...•... ....•... ... ... Listot Abbreviations.... . . . ....•. .•.. .. ....• • .•.. • .•. xvii

CHAPTER 11 GENER1r.L INTRODUCTION•••••••• ••• ••• ••••• • •• •••

CHAPTER2: OOCYTE YOLK FORKATION IN THE WINTER FLOUNDER••••• ••••••••• • ••• •••• •••• ••• • •••••• •

Introduction .. ...•..•••. ..•.. •.•... . . . .. ... .. . Materialsan4 methods....•... ... .. ... •.. •... Fish ... .. •...•. .•... •. ..•... .... .. •... . •.

Isol at i on of serumviteUoge ni nandpe a k A prote i n . . 10 Radio labell ingofvitellogen i nandpe a k Apr ote in.. . 14 Radioi mmu noa ssaysfor vitel logen in and pea kA pro te in . ... .. ... .. .. ... . ... . . . . ... ... .... 17 Sa l t solUbleovaria nextract immunoreactiv itywith vitel logcni nand pe a k Aprot einanti s era ... ... 17 Denat ur i ngelec tropho r esisof oocy t e prote i ns.... .. . 19

vi West e rn blotting and Ima uno a aaay anal ysisof

oocyte proteins. .... ... . ... 21 Experiments usingradioiodina ted vi te ll og en in and peakAprote in.. ... .. ... ... .. 22 li:xperiments using tr i t i u m labelled vitelloge n in and pe a k A pr otei n.... .... . ... .... ... .. . .... 23

R esults... .... ... ... .... ...

2~ Bioche mica l ana lys esof vitel loge ni nand pe ak1\

protein... .... ... ... ... .... ... .... ... .. ... .

²⁵

Ultracen trifugationanalysisof femalewinter flou nd e r plasma... .. ... ...•... .. ...• ... .. 28 Rad i ol abe l l l ng of vitelloge ni nand peak.A protei n... )) Seasona l serum pr of iles of vitellogenin and

peak Aprote in... ... ... .. ... JJ Stabi li tyofradiol a belle d vitelloge ninand

pe a kApr oteininjectedinY.iY2in theblood.. . ... J5 Immun o r e act i vi t y of thesal t sol ubleovarian

ext ractwithvitellogen i n andpeak A protein antisera. ...•... ...•... . . . ...•.... . 42 Rel atio nshipof oocyte proteinsto vitellogenin and peakApro tein... .... ... . ... 42 Ovaria nuptakeof radiolabelled vite ll og eninand peak Aprote i n... . . . ... ... ... ... .... ·14 Discussion. . ... .• ...•. ... ...• ... 60

vii CnPT£ RJ: PI TOIT~ RYREGULAT IONor VITELLOGENESIS IN

WI NTERFLOUNDER1JrlDRAINBOWTRODT... ... ... 72

Intro4uetion. ... .. ... . .. .... .. .. .... . . ... .. ... .. . 7 :.1

"ata r ial eand••tbods.. ... . .. . ... .... 76

F is h... .. .... ... ... .... ... . ... 76

InillL2inc uba tions of ovarianti s sue s... ... ... 77

Hormoneprepar8tio n ~I.. . ......... .................... 82

steroid radi oimmunoassays... .. ... ... ... ... 84

Dou bl e equilibriumdialysis . .. . . ... ... .... ... 67

Experiments on hy p o phy s ect o my and ov ari an uptake of vi telloqenin andveryhigh de ns i t y lip oprote inII ••••• ••••••••••••••• •••••••• •• •• • •••• 87 statistical anal )·ses ... ... ... .. 89

Relllulta. ... ... .... ... . .. ... ... ... . .. 89

Seasonalqo nados olllati c indicesand serum level s of estra diol-I7,Oandtestostero neinwinter flounder... ... .•••...••.••. . ... •••..• ..• ..•.... .. 89

Ovaria nestradi ol -I7" andte st osterone re spo ns e s topitu itary ext r a c tinrlYJldudngthe pres pawningtovit ell og e nl cpha s e... . .. .... .. . ... 91 Ovarianaroma t a s eregu lat i on inrainbo,", trout by sock e ye salmonpituilary proteintr a c t i ons

in

titJ:g.. ... . ... ... . . .. .. .. .... .. . ... ....

94

viii Th eeffectof grow th ho rmon e on ovarian

estr a d iol- 17P andtestoste ro neprOduction

.inillnlin rainbowtr out and winter flounder.• .•... 102

Thyroxinestatus as acriterion fo r suc cessful hYVOph ysect o llly•.•... .•... ....•.•. .... ...•.. 10 5 Effe ctof hypophysectomy on ova rianupt a ke of (U1IJ-v itell og e n in and (UlIJ-v e r y high dens ity lipop r otein II ••.. . ••... .. •... .. •.• . ... ... ... 105

Di s cu s sio n•....•... .... ... ...•..•...•... ... 11 1 CHAPTER 4: PITUITARY GROWTHHORMONEISOLATION••••• •••• ••1:l0 Int ro duc ti on•..•. • •... .... .• •.. ..•. .•.... •..•...• ...120

Materialsand metbods.•...•...•. ••. .. •....• ... 123

Fish ••.•••.•• • ••.•••.• ...•• .•...•. ...• . ...•.. 123

Pitu ita ry proteinfractionation.•.•• . ... ....•.... 123

DeveLopseent; of growth bormone bio assa y.•.•. ..•. . . 126

statl~ t i cal analyses...•....••... .•... ... . 128

Results..•... •...•..•.• •.... •....• •••..•.•.•..•.. ... 129

Soc ke ye salmongr owt hhc reone isol at i o n•... .• .... ... 129

Amer i canpl ai c egr o wth hormo neisolati on.•... ....•.. 136

Discussion•.. . .•. ... .. . ...•.. ..•..• . .. . .. .• • 149

CHAPT ER5:GENERAL CONCLUSION••••• •••••• ••••• ••••••••••, 157

REFERE NCES••• ••••••••• • ••••••••••••• • •••••••• ••••••••••• 161

LISTOF TABLES

Table 1:Amino acid composition (\:) of female winter flounder vitellogeninandpeakA.protein.. .. . .. 31 Table 2: Ultracentrifugationanalysisof femalewinter

flounder plasma... ... ... 32 Table J:comparison of theovarianupt a k e of t ritiated

{lHl vitellogeninand peak A protein

preparations la b e lle dinyJ..yQ andin

ill.r,Q. . ...

58 Table 4: The propo rti on of injectedradioactivity due

to[lH]v i te llog e ni n and [3H]peakAprotein present in saltsolubleand insoluble

r ract Ionsof ovarianextracts. ... ... .. ... 59 Table 5:Ova rian uptake of (131I) v i t e llo g e n i n over 48

hoursby hypophysectomized (hypex) and

sham-operated female winterflounder .•.•..•••.•loa Table 6:Ova rian uptakeof (UlI]veryhi g h density

lipoproteinII over 48ho ur s by

hypophysectomized (hyp ex) and sham-operated femalewinter flounder..•..• ... ...••. • . .•.. . 109 Tab le7: The effectof diffe rent soc ke ye salmon

carbohydrate- poor<25,000 M_rpituitary prote ins, isolatedby preparat ive polyacryl am':'degel electrophoresisonserum triiodothyronineeleva tionin rainbowtrout 24hou r s post injectio n.. .... . ... . ... 134

LIST OF FIGURES

Figure1: Calibra tionlinesfor sodiulldodecyl sUlfatepolyac ry l amidegel

elec t rop horesis... ... . .... .... ... .... . .. . 13 Figure2: Sta nda r d curve sdevelop ed from

vltellog e nl nand pe a k A protein

rad io i mmunoa s s ays.. ... .. ... ... 16 Figure 3: Gel fi l t ra tionchr oma t og r a phyof3 m1 of

femal ewinterflounderse r a .. ... .. ... . 26 Figure4: Ge l filt ration chromZltOl']raphy of 3.5ml

ofse rum from an estrogeniz ed fe male

winte r flo under.. ... ... ... 21 Figure 5: Nati v epolyacrylamidegel el ectrophores is

analysis... .. ... .... .... . ... ... 29 Fi g u re 6: Sodiumdodecyl su lfa t epolya crylamide gel

el ect ro pho res is analys i s... . . . ... 30 Figu r e 7: Seasona l seru m profileof vite ll og e ni n and

peakA protein in femalewinter flou nder (19 8 6-87 ) . ... ... . . .... ... 34 Figure 8: Gel filtrat ionchromatog r ap hy of female

winterflounde r plas ma from fish injected 48hea rlier with(131I ]vitellogeni n.. ... 36 Fig u re 9: Gel filt ra tio nch roma tograph yof female

wInt e r floun d e rplas ma from fish inj e c t ed 48h earlie rwith[lJ1I) pea kAprote in... ... n

Figure 10: Gelfiltration chromatographyof female wi nt e r flounder plasma from fIshinjected 2we e k s earlier witheH)propionyl

-·vitellogenin... . . .. ... 38 Figure 11: Gel filtrationchromatogra ph yoffe mal e

winter flounder plasma fromfish injected 2 weeks eer-LLer with[3H]vite1 1ogenin. . .. .... 39 Figure 12: Gel filtration chromatog raphy of fe mal e

winter flounderplasma fromfish injected 2 weeks earlierwith [3H]propionyl -peak A

protein... .. .. ... .... .... ... .. . .. .. . ...

40 Figure 13:Gel filtrationchromatographyof female

winter flounder plasmafrom fish inj e cted 2 weeks ear lierwith[lH]p eakA pr ot ein . . .. . . 41 Figure 14: Gel filtration chromatographyof ovarian

extract withaliquots measured in the peak A proteinand vi te llogenin

radioimmunoassays.. ...•. .. . •... . .. 43 Figure 15:Sodium dodecyl sulfatepolyacrylamidegel

electrophoresis of oocytepr otei ns... . .... . .. 45 Figure16: Westernblotofwinte rflounder oocyte

proteinsprobed with the vite ll ogenin antibody...•.. .. ....•...•. •• ..• •. . .• •... 46 Figure17 : Western blot of winterflounder oocyte

proteins probed withthe peakAprotein antibody.... ... .••••.. ..• .•.• ••... .•..••• 47

xii Figure 18:Ti me course plotof [13' I] v ite ll oge n i n and

(1l1I] p e a k Apr o t e i n ovarian uptake....•. .. . •• 48 Figure19:Gel fil t ra tio nchromatogra p hy of sal t

soluble ovarianext ract fr o m female wint e r flounderinjec ted2 weeks earlierwi th [3H) p r op i o nyl-v ite ll o g e n in .••...•.... ••...• 49 Figure20:Gel fil trationchromatogr a phyof salt

solubleovarianextract fro m femalewinter ::l ou nd e r inj e cted 2 weeksea r li e r with

(3H]v it e ll ogenin. . . .. . .• 51

Figure 21:Gel filtration chromatography of salt solubleovarian extract fromfemale winter flou nd e r inj e c t ed 2 weeks earlier with [3H] p r o p i o ny l-p e a k Aprot e i n... ... . .. 52 Figure 22:Ge l fil t rationchr o ma t og r a ph y of salt

solubleovarian extract mo nitoredfor protein and alkali-labileprotein

phospho rus. ... ... ..•... . 53 Fi g u r e 23: Ge l filtrationchr oma tographyof salt

sol u ble ov ar i a n extract on UI trogelAcA 54 fr om a femal ewinter flounder injected2 weeks earlie r with [3H]propionyl

-v i te l log e n in . .... .. . .. .... . .. . . ... ... .. 55

xiii figur e 24: Gel filtration chromatography of salt

sol ubleovarianextract monitoredfor proteinand alk.ali-labile protein

phosphorus. •.... •...• •....•.••.. .••••.•. ..• 56 figure 25: The effect of bovineserumalbuminaddition

to incubationme d i a on estradiol -l7j3 production by winterflounderovarian fragmentsin~.... ....... 79 Fig ure 26: The effect of bovine serum albuminaddition

toincubati on med i a on testosterone productionby winterflounderovarian fragment s inili1;:Q... .. • ...•. .. 80 Figure27: Seasona lgo n ados o matic indicesof female

winter fl ounder.••....••...•. ••• .•••••.•. 90 Fi g u r e28: Seasonal serumconcentrationsof

estradiol-17/3in femalewinter flounder... 92 Fi gur e29: Seasonal serumconcentrations of

testosteronein female winter flounder... 93 Fi g u r e30: Product ionof estradiol-17,Oby winter

flounder ovarian fragmentsin~during the prespawningto earlyvi t e ll oge n i c phasesof the reproductivecycl e... .... .... .. 95 Figure31: Productionof testosteroneby wjOlter

flounderovarian fragments.inilll.:2.during the prespawni ngtoea r l yvi t e lloge nlc phasesof the reproductivecycle..••...••... • 96

x Lv Figure 32: Productionof est radiol -17/3 bywinte r

flounde r ov a rian fragments.inY.it..r2

incuba tedwi t h te sto stero ne. . ... .. .. ....•. ... 97 Figure33: Product i onofes t radiol-17 ,8by is ol ated

intact ova rian follic les fromrainbow trout incub ate dinti.t..r2 withsockeye salmon pi t uita r ypro teinfr act I on s . Figu re34: Produc tionof estradiol-17/3by isolat ed

intact ova r i a n folliclesfrom rainbo wtrout incubatedinY.ti..J;:Qwith testosterone... ... .100 Figure35: Pr oduc t ion of estrad iol -17,Bby isolated

defolliculatedovarianfolliclesfrom rainbowtrout inc ub atedin ill.r.2with testosterone.... ...•. .. . .•....•... ...• . . .. •..101 Figur e36: Pr od uc t i onof estradiol-17{j by isolated

de f oll i cul a t c d ovarian follicl e s from ra i nbow tro ut .\nc uba ted.iniliJ;:Qwith testosterone andsoc keye salmonpi t uit ary protei n frac tions..•...•...• •...•..•. ...•..10) Figure 37: Serum thyr o x inele v el s during Nov embe r in

male winte r flou nde r sampledafter

hyp oph y s e ctomy or shamop erat ion 106 Figu re 38: Ser um thyr o x ineLev e Isdu r i ngJanuary in

femal ewinte r floundersampled after

hypophy s e ctomy or sham ope ration... .... .•.107

F'igure 39:The effect of sockeye eet aon cr ude pi tuitllry extracton serumt ri i odothyronine lev e l s in rainbowtrou t•...•.. ...••.... •• ... . 130 Figure 40: The ef f e ct of sockeye salmo n carbohydra te

-poor and carbohydrate-rich fr ac t i o ns on serumtriiodothYronine level s in rai nbow trout. ...•... .•... . • •.••. ••• ••. •••...••..•. 132 Fi g ur e 41:The eff ect of sockeye salmon carbo hy dra te

-poo r fractions on serumtr i iodothyronine le v e l s in rainbow trou t... .• .. . .•••. . ..•• ...• 133 Figure 42:Sodium dodecyleujr et.e polyac r ylam idegel

electrophoresisof sockeye salmon growth hormone... . .•.• ....••....•... •. .. . . • • •..• 135 Fi g ure43: The eff ect ofdif f er e ntdos e s of sockeye

salmongrowth hormone on sec-un triiodothyro nine levelsin ra i nbow trout. Ins e t isthelog-line ar dose-response

relaH.mshipbetween diff erent doses.•. ••••.. ~37 Figure 44: The effectof plaice crudepi t uit ary

extractand carb o hyd r ate- poor fr a c t i o ns on serumtr iiod o t hyr on i nele v el s in

rainbowtrout...••...• •.. ..••.. .• • ...••• •.•.. 138 Figure45: Gel filtrationchroma tographyof plaice

carbohydra te-poor peak C on Ultr ogel AcA 54monitored for absor b ance... . .... . ~3 9

Figure 46:Re-chromatographyof aliquo tsof plaice F II and F III frac tionsonUltroge l !leA 54.. 141 Fi gur e 47:Theeffect of pl a ice FI, F IIand FIl l

fractionson serulII t ri i odo thyron i ne levels in rainbowtr o ut•.. •• ..• ...••... •••...•.. . . . • 142 Figure 48: Theeffect of pla i c e FIII fr act i ons on

se rumtriiodot hyron i n e levelsin ra inbow trout... •.... . . .••..• ..••. •.•... ••.. .•...• • 143 Figure 49:Theeffect of pl a i c e F II RfS0.22and 0.28,

andFIl lRj 0. 2 2proteinson serum

triiodothyroninelevels in rainbowtrout... 144 Figure 50: Densitometricscans of plaiceF II Rj0.22,

R_f 0.28 and F III R_f 0.22 onnative

polyacry lamidegel electrophoresis... .•...• 145 Figure 51:Sodium dodecyl sulfatepolyacrylamide ge l

el ectrop horesisof plaicegrowt h hormo nes unde r non-reducingor reducingcondit ions.... 147 Fi gur e 52:Amino - terminal amin o acid seque ncesof the

plaiceFII an dF III rela tive mo b il i t y 0.22proteins...•.. . •.•• ., . • .•... . .. ... •. 148

LIST OF ABDREV:I:Jl.TION S

Ab, anti body AcA 54,Ul. tr ogel AcA 54

ALP P , alkali-lab ile prote i nphos phorus B/Bo' 50!!;bi ndi n g /O\:binding BSA, bovine seru m albumin C-T , carbo x yl-termina l ConA, Con canava linASepharo se ConA I,ca rboh y d rate-poorfra ct ion ConA II, carb oh yd ra te-rich fraction cpm, count s per minute

dpm, degrada tions pe r mi nute E2,es trad iol-17 P G,gravity GH, gr owth hormone GSI, gonadosornatic ind e x GTH, gonadot r opin 3H, t ri tium 1311 , 13'i Odine Ip , intr ape ri t on e a l Iv , int r avenous 2- ME, 2-me r c apt o e th ano l

mGTH, mat u r atio n al-typ e gonadotropin Mr, re l at iv emolecul armass

HSRL, Marine Sciences Res earch Lab orato ry

xvii

N-T, ev rncterminal

PAGE, jfo l y acr y lam i de gel el e ct r o p ho r e s is PE, pituitary ext ract

Pk A,peakAprotein Rf• relativemobil ity RIA ,radioimmunoassay 5-300,Sephacry l5-300 50S, sod iumdodecyl sulfate SE, standar derror

SSOE, sa l t sol u bl e ovarian extract T, testosterone,

T3, triiodothyronine T4, thyr ox i ne

TCA, trichloroace t i c acid Vo. void volume VG. vitell oqeni n

VHDL, veryhi ghdensityli p opr o t e ill

xvlLi

GENERALINTRODUCTZ ON

A major componen t of oocyt e gro\rith in all ovip a ro us vert ebra t e sderives fromtheac c umula t i on ofbloo d plas ma

jLpoproteina 1ai ddownas yolk in the ovar y (Wallace1985 ) . Thi s yol k rep r e sentsthe solenutritionalreserve ofthe even t ual offsp rinq . The proces s of yol.k pr o t ein fonat ion and it s ho rmona lregu l at i on ca n be br o a dly tieraed vi tellogenesis.The mos twellstudied yolk pre curs orprote i n is vitelloqenin^{(VG )} (Wa l lace^{1985 ).}Vitellogenin.

sy nthe s i z e din the li ver and released into the blood,enter s theooc y t e via rece ptor - me d i at e d endocytosisand is pro teolyti cally cl e a v ed toform the yolk proteins lipovitell i n, phosvit inand sma l l e r units ca l l ed phosvettes.

In aephIjaf ans themaj ority ofyolkformedis at t r i but ed to uptakeofVG(Wa lla c e§..t..sU.1972). However, in birdsserum veryrev density lipopr ot e i n is activelyacquired by the ovaryand cont ributesmore totheyolkthan VG(Wa llac e 1985) . with re s pect tofishVGi s establishedas a yol k pre cur sor (we i ga nd~98 2, Ng and Idler 1983, wallace§.t.g l. 198 7,Tylert t Ai. 1988a,b ).

There aredifferencesO'll1'longst marine te l e o st s andother ovipar ous ver t e brate s withrespect to yol kformationand co mpos i t i on. Theyol k proteinslipovitellin andphosvitin

arepresent In a solu b leform inmo stma r i ne te l eo s t oocytes.Thisis in con t ra st to othe regglayi ng vertebrate s where the yo l kis insoluble dueto its fOrJIati on in to cry sta lline structureste rmed yol k platelet s (Wa llacen ill.

1966, Wallace andSel ma n198 1 ,wallace 19 85). Some mad n(!

teleost s mayhav elit t leor nophosviti n withi nthe yo l k (Jare dand Wa ll a c e 1968) and teleosts ingene r al ha v e be en regar d edas havi ngat ypical yolkprotein patte rns when comp a redwith ot he rovipar o u svert e bra tes(Wal lace 1985). Fin ally , in tel e ost s plasma lipi ds, car o teno i d s, glyco p ro t e insan dvit a min - bi nding proteinsalsogainacc e s s totheovaryinaddit i on toVG(Wei g and 1982, MOllmscnand Walsh1988 ) . Rel ative toVG the spe c ific contribu ti o nsof these other extraovari ancompo nentsto teleo s t yolkprotein areno t known.

Thepitui ta ry gland regu la t es re produc ti on in teleosts (Idle rand Ng1983) .Go nadotropins are the pi tu itary hormonestha t regulate reproduc eIveprocesses andinrceerc

!ishthe irbiological actio n s can becate goriZedas vitel logen lcand matu rationa l. Vitellog eni cacti v it iesarc invol v edwith yo lkfor matio nwhichent ai lsextr aova rian lipoprote in synt hesis and it sova rianuptake. Matura ti on al activities deal principallywith the regulationof ovarIn n sex stero id hormo nesbefore spawningwhichre-in it iate meio s isvit h in theooc yte s andreg ulat e ovul a tio n.

Initia lly fish were thoug httoha ve onegonadotro pin

jGTH) whichw~s responsible forbo t h vitellogenicand maturationalevents. To date thereisnocons e nsus as tothe number of definitive GTHsthat re g u l a t e thesere prod uc t i v e processes. However , Id le r!itli. (1975a.b)proposedthe existence of a second pituita r yGTH besides the"cl a s s i ca l"

GT Hknownin fishand showed using chum salmon (Oncorhync hus 1s;~) pituitarieshow it couldbe separ a tedonConcanaval in Aaf! in ity media due to its lowcarbohydrate conte nt. Thi s ledto the recognitio n of two groups of GTHsinfis h termed ConAI (carbohydra te-poor) and Can AII (carbo hyd rate- rich). Subsequently, Ng and Idl e r (1979) det-.ermined that America n pla i ce (Hippoqlo sso ldesplates!io ides) andwinter flounder (Pseud o pleuronectes~) pituitaryCon AI fa c il itatedthe uptakeofra d i o l a b e lled leuc ineand phosphorusinto the develop ing ov a r y of winter flounder, whileConA II was primarily involved ... i th mat urat ional activities.Lat er, a purif i e d protein(R_t 0.7 pr ot e i n ) fr om chumsalmo nCon A I was sho wnto stimul a teov aria nupta ke of rad iolabel ledVG in land loc ked Atl a nt i c salmon (~~

Q1!A.rn..!!..!£hg) (I dl. eran d So 1987). The work of Id le r and colleaguessuggest that Con A I typ e GTH is vi tellogenic while ConAII typ e GT Hcontrolsmatura tiona l events, al though the Con A II GTH is thou gh tto regulateest rogen synthesiswhich induceshepaticVGprod u ction (Ngand Idler 19 8 3,MommsenandWalsh 1988 ).Mo r e recentlyS....ansonllll.

(19 87 a , b ) and Suz uki !it.u . (1988a,b)haveisolate d mUlt iple

glycoproteinGTHs from coho (Oncor h ync hus~ )andchu m sal monrespectively,butthese preparat i o n s have bi o l og ica l activities such as inducing gon ad a l ste r oidproductio n that classifythem as maturational-typeGTHs (rnGTII) .

Vitellogeninproducedby the liveris under th e control of ovari a n estrogens, principallyestrad i o l -17/3_{(E l}) , in fish(Ng andIdler 1983,Momms e nand Wal s h198 8 ).Thesite of El biosynthesisin the salmonidovary isthe qr-anul cen cellla y e r withintheovar-Lanfollicle (Kagawa t lill. 1982 ) . Thisfollows fromthe "t wo-c e ll"model first proposedIn mammals byFalck (1959) whe r ebytestosterone (T) synthe sized in the outer th eca lcell layer is aromat i z ed via the aromatase enzyme in theinnergranulosa callla y e r to El • In salmonids ovarianEl synthesis is control ledby the pituitaryandmGTH(Le. ConAII) is considered the hormonal mediato r dist i nct fromvitellogenicG'I'lItLe .Co n1\

I) (Idler and Campbe l l 1980, NgI Illl, 19 80). 1\ number of experimenta lstudies bo t hinY.iY.,Q(Billardtl sU.1978, Bretoni lU.19 8 5) and.loYil.I:,Q (Kagawa !l.t!tl. 1982, zotrer- g!;.Al. 1982 , YoungI I ai,1983b, Van Oer Kraak and Donaldson 1986,Kanamor i ll.ill. 1988 )ha ve demonstrated thatmGTII can inc reaseovaria n Elpraductio n. In contrastaccumulated evidencefrom studieson natura l blood levelsoC mGTHandF.2 in fe mal eaaLacnLds du ring therep r oductive cycledo not indicateanint erdependence of one with the ot he r(Billard g!;.

.su.

197 8 , Scot tan dSumpte r 198 3, Sumptert l~, 198 4 ),

2 increaseprogressively to the i rhighest levels in fema le rainbo w trout(Qn.&Q.rhynchus~) (s cott and Sumpter1983, Scott~U. 1980) plasma levels of l'IGTH remain very low

«0 .5 ng/ml ) andunc ha nge d (Sumpterand Scott 1989). Inthe aneso salmon (Oncorhynchus~ J mGTHwa s showntobe inactive inenhancingE2 production by ovarian gr a nulo s a cellpreparations incub a t edinillmwithexogenousT as aromatizable substrate (Kagawa

n

i\l.1982. Youngitl i.

1983a).The inabil i t y of mGTH to affect granulosa cel l aromatizatlonpersiststhroughout the reproducti v ecycle of thi sspecies (Kana morl !tl.1!l. 1988). Clearlydiscrepa ncies remainconcerningth erol e of rnGTH in regUlating E2 productioninthe salmonidovary.

Another ho r mone of pituita r yorigi n,growth hormone (GH), has recently been found to infl uencethepro duc t i on of E2by the ovary.StudiesshowedthatGH couldincrease theleve l of ovarian E2 production , both.invivoand .in Y.it.!:2,in killif ish (Fundu lus het e r oclitus) andbyra i nbow tr out ovarianfragments .in~(Singh~li.19 8 8). In goldfish ( ~aura tus) GH appears topotentia tethe effec t of mGTHinstimUlating both E2 and T productio n byov arian toj Llc Iesin illI:2 while by itself i tis inactive (Van ner Kraakfi.ill. 1990). These studiesin dicate that GH could have effectson the synt hesis of ovarian sexsteroids. This is particula r ly inter estingsin ceGH is foundin the ConA I

pituitaryfr a c t ion (Komourdjian andIdler 19 7 9, Idlert lej, 1.989). Pr ev i o u s studies in male teleosts have documented steroidogen iccapabilitiesof theCon A II fraction (Le.

containing mGTH) and have also reported some androgen stimulatingcapacity in theCon AI fraction (Ng andIdle r 1979, Van Der Kraak and Peter 1987). Itis possible that compone ntsof the Con At fraction may be involved J.n the synthesis of ovarian sex steroids particularly when mG'rHis unavailableor unable to regulateprocesses att ributable to the pituitarygland.

Theobjectivesof this study therefore are to: 1) explore the uptake and processing ofVG and another homologous serum protein. as potentia 1 yolk precursors in the winter flounder, 2) examine the pituitary regulation and possible involvement of GH, inovarian Ez synthesis of winter flounder and ra i nbow trout, and 3) isolatethesalmonid and pleuronectidGHs to be used for objective

'2.

OOCYTEYOLJtFORKATIO N INTHE WINTERFLOUNDER

INTRODUCTION

St udies have been undertakento ascertainif serum proteins othe r than VGbec omeassoc iatedwiththe ovary of wtnter flounder. Bhat (19 A6) and So and Id l er (19 87 ) ha v e prov Lde devidence that other ser umproteinsbesid esVG are incor poratedintowinter flou nderovaries . All thecr ude ser um prot e in tra ct i o nsseparated ba s e d onmol e cul a r mass thatare normallypresent inthese r a of adult fe mal e s were shown tobecc e e associ a t ed wi ththe ovaryto some ext en t (So and Idlerunpubli shed ).Of particularint e r e s t isahigh re l at i ve mol ecul a r .ass (Hr) serumpr ote in(1,170,000) found inbothsexes.Th i s protein ha s been termedpeak A protein (Pk A) inrefe r e nc e toit be i ngthe firstserull.

prote inelu ti ngonsepha c ry l 5-300 (S-300) gel fil t r a tion medi a. Bycompa rison winterflounderVG ha sa native"r of 514 , 0 00 . Peak Apr o t e in in termsof its biochemical cha r a c t eristi c s isJI'IorelikeVGth an any othe r ser um pr ote i n both beingglycolipophosp hop r o t eins . However , na tive and dena turing polya c r yl a mide gel electropho resis (PAGE) , enzymaticdegr adatio nand immunocross- reaction st ud i e s indi c a t eth a t thetwoproteinsarest ru c t u r ally disti nct

fr omone another (Bhat1986).

Froma compar-a'tLv e viewpoint PkAappears/I1ikel y ca ndidate to contribu t e to ovaria nyolkprotein along with VG due to their simil a r biochemicalmakeupa I though a number ofqu e st ionsrema i n . Wha t are theseaso na l serum

concentrat ionsof these two prote ins inr-epr-oduc t.Lve l y active female s? In other female teleostsde s c ribe dto date VGlevels ar etyp i c all y elevatedduring the vitellogenic period (So!!!....t.1!l. 1985, Scot t andSumpt er1983).

vite l logeninsareusuallycharacterized asve r yhi gh density lipop r o t"i ns (VHDL)(Ba b Ln and Vernier1989, Schje ideand Schjeide 1981 )butthe lipoproteindensityof bothwinter flounderVG andPk A are not known. The identificationof theeverrtucI end product(s) of PkA in thedeveloping oocyte wouldaf Low a compariso nwithyolk proteins derived fromvn.

In summary, the specific contributio nbyvoand PkA towards winter flounder yolkboth intermsof qualitative and quan titativecharac teris t ics is unknown.

To da teno othe r studies onfish have compared th e up t a ke of VGandan y othe r homologousserumprotein knowntobe inc o rpo r a t e dbythe ovary during th e reproductivecycle.The present studysought tocompar e VG and Pk A interms of th ei r season a lserumlevels byradio i mmunoassay (RIA) , plasma li po protein class, uptakeby the ova r y, and qual itativeand quantitativere latio nsh ipto yo lkproteins in the wi nter flounder .

Fish

Mature winter flounder (Ps eud o p l e u r o ne c t e s ~!i Wa l ba um) were captured throughout the years 1986-1990 by SCUBA divers usingha ndnets from conception Bay, Newfoundland. Fish were brought to theMa r i ne Sciences Research Laboratory (MSRL) wherethe y weremaintainedin tanks(10-15 fish/2401 tank) with ambient seawater and natural photoperiod. Fishhe l dfr o m June toOctoberwere fed chopped caplin ( ~ ~ )to satiationthreetimes per week.Since feeding normally ceases inthis species during winter and early spring (Fletcher and King19 78 ) no food was providedat this time of year. Experimentsre po r t e d he r e i n were conducted from October to January duringthe rnid -vitellogen icphase of the female reproductive cycle which is characterized by rapid gonadgr owt h as a resul t of yolkformationwi t h in the developingoocytes. Female winter flo undertypically weighed 425-750 9 with average gonadosornatic indices (GSI) (ova ry weight/bodyweight-ovary weight x100 ) that varied from10 to 18. sampling and experiments were always conducted approximately2 weeks after the fish were obtained.

10 IsolationoflIIerum vitellog'eDinanc3peak Apr ote i n

To increase the seru m VG t i trefi shwere injected in tra p e ri t o ne a lly (ip ) wi t hp-e s t r a dio l - J - b e n zo a t e (Sigma) su s pe n d ed in peanut oil (10 lIl9/kg) 1 llIon t h beforeblood collection(S o

n

Al.1985).SerumVG degradat i onwas preven tedby injectingal lfish via caudal vesselswith 2 ml of aprotinin (1 5-3 0trypsin inhibitor units/mI l (Sigma) 15 minutespriorto blood samplin g (Wallaceand Selma n 1982) . Blood was sampledby ne edl eandsyringe fromthecauda l vascul a tureofuna ne sthe ti z e d fish, usually in themorning, andallowedto clot in sterile pla s tic tubes

e'

2006, Falcon) overnight at 4°C. Serum was sepa ra t e d by cent rifugationat 13,OOOxgravity (e) the followingday and then used immediately or frozen at-60OCfr.lr upto ) years . The bloodserulll proteinsof reeeIe winte r flounder we re eeparaeedbased on their",by low pre s surege lfiltrati o n cenuen chromatography on5-300 (2 .6 x 90ce ) (Pharmac ia) using VG buffer (pH 8. 0 ) (So~ill. 19 8 5 ) contain ing 0.4Ill) ap rotinin/ m1ascolumn eluent. Column chr o ma t og r a p h y was con du ctedat SClcin a cold room at a flow rate of 15 ml/hour using as c e nd ing elution. The columnwasca li bra t e dwith th yrogl o bulin (669 , 0 00), ferriti n (440,000) and al dolase (1S8 ,00 0 )M, sta nda rdssup plied byPharmacia. The op tical densityat 280 nm ofthe eluatefractionswere moni tored in a spect rophotometer (Ultrospec II, LJ<B) using a1 em quartz

cuvette. To isolate VG the peak tubes were concentratedand re-chromatog raphedan 8-300tore mov e any conta minationfrom adjacent peaks. Both the VG and PkAfra c tionswerefurt he r pu r i f i e dbyaffinity chromatography on Concanavalin A sepne rcse (ConA) (Pharmacia) collect i ng the adsorbed fractionfollowingelutionwi t h a-methyl -D -glucaside (Idler andHwang 197 8 ). Only the peaktu b e s were utilizedwhe n recoveringse r umproteins.PurifiedPk A andVG were aliquoted, storedat-60°C andnever re- fr o ze naf t erward.

Serumproteinconcent ration wa s determinedby trich loroaceticacid (TeA) pr e c i p i t at i o n followedby the Lowry me t h o d (LowryII U.1951) using bov i ne gamma globulin (BloRad) as standard.

Na t i ve (ana lytical) PAGE of serum pr ote i ns isolatedon s- 300 was done using themethod of Davi s (1964). Briefly, 10 cm gels(5%acrylamide: 0.2% N,N'-methyl e nebisacrylamide, pH 8.9) were cast in5x125mm glasstubesand elect rophoresed using a disconti nuous bu f f e r system(S mMtris, 38 mM glycine , pH8.3) inaBi oRad Model15 0electrophoresiscell.

Electrophoresis was conductedat a cons tant 2 milliamperes/gel (300volts ) at room tempe r ature wit h tapwater coolingunt ilthebr-cmcphencI bluetracker dye reached 1cm fromthe bott omofthe ge l (us uall y abo ut 3 hours). Fol lowingelectrophoresisgelswere stainedwi th 0.1\ CoomassieR-250and destainedby diffusi onin7%acetic acid.

12 Denaturi ng sodiu mdod!;!c yl sul fate (50s) PAGE ofVGand Pk Awere performed on uni form 10\ slollbgels (33.5\acrylamlde: 0.3\N,N'-meth ylene b l sa cryla mide. pH9.1,conta ini ng 10\

80s). 1.5x 12 0x 140IIUII! . witha1emH stackIn ggel (30\

acryl a midQ: 0.44 \;N,N'-methylenebi sacryl a l lid e . pH6.8, conta i ning 10\ 80s) followIngDreyfu s s~ll. (198 4) . SaJnp l e s were mi xed wit h the samevolumeof sample bu ffer

(0.5HTr i s-He l, pH6.8, 10\ SOS,20 \ gly c e r ol) cont ai n i ng 10\ 2-mercapto e th anol (2-ME) andplac ed in boili ng water for 5minu tesbefo r eapp licat i o n ontothegels.A Hoeffer Mode l SE40 0was used at roomte mpera ture run at constan t 10 0 vol ts fo r 16hours (o runtilthebr omo phe nol bl ue tra c ker dyehad runoff the gel) vith atypi c alrese rv o irbu f fer (25 DIM Tris ,pH 8.3, 192mMgl ycine, O.H. 50s).Gels we re fixe d for 1hour in 50\ metha nol -lOtacet ic acid,stai n e d for 2 hoursin0.3\ COOllltls s i e BlueR-2 50in 50\ metha nol-1St aceticacid and de-sta inedbydiff usion in 10\me t ha nol-lot ac eti c aci d . Rel at i ve ecj. e cu jexmas s eswereestimated frolll proteinstandards (UfWandHMW, Pharmacial run onadjacen t lane s of thegels . Censitomet ricsc ans (Hod el 165 0, Bi oRad) were us e d to calculatetherelativ emobility(R,) of stand a r d sandun k now ns, and a semi-logar ithm i cplot of st a n d ard l s log MrvsR, (Fig. 1) wa s us ed to det e r mine the M, ofthe unknownsba s e d on their R_f•

Proteinphospho r usdeterminat i o ns ofserumpr otein s were made usingthe alka li-l abileprot e in phosphoru s (ALPP )

400 300 •

200

A

.2~

:30 100

~0 0o o~

Eo 50

.~

'" ~

1.0 .8 .6 .4 .'1

10'---~--~--~---'----'

o

RelativeMobility

Figu r e1: Calibration line s for sodiumdcdecyj,sulfat e poly a c r y l ami de gel elect r ophoresis . repres ented by semi-logarithmicplots of prot e in standard 's lo g rela tivemolecu l a rmassve rsus the irrela t i v e mobilit y . A..HMWPhar macia , 8'"LMWPharmaci a.

14 protocoloutlinedinNagler

n

l i. (1987).

The aminoacid compos i t i on ofPk A and VC vere determine d by phenyl-thiocyanatederi vitizatio n (Plc o-T ag .Waters) and quantificationby revers e-ph a s e (C-18) highpressure liquid ch r omatog r a p h y (Spect ra-Ph y sics ) .

For ultracentri fugati onanalysisblood""asobtained fro ,.

IS femalesand pooled. Plasmawasis olated by placingblood in hepa r i n iz e d glasstubes (vecu eemer4783, Becton - Di ck inson) , kept at 4°Cove r nig ht and cent rifugedas above the fol lowi ng day. Five mlali q uo tsof plasmawer e used to isola t e the various lipopr otei nclassesbased on the same densi ty inte rva l s us ed to separ a te huma n plasma lipoproteins : verylowdensitylipo prot e in,d<l.006i low density lipoprotein ,d"'l.006-1.06 3 ; high dens ity lipoprotei n,d- l. 063-1. 21, andVHDL, d>1.21. Preparative ul tra c e nt r i f uga ti on wa s co nd uc t e d in po1yc arbonate tubes witha BeckmanLS-50 (50Ti rotor) at HO,OOOxg (lO"C) followingth e Ille t h o d of Ewingt lAl. (1965). Lipo p r o tei ns re c ov ere d werefr oz e n at -60"C until used.Prote incontent was dete rmi n e d by theTCA-Lowrymethoddes cribedabove using bov i ne gamma globulinas standa rd.

Radi olabellingot vitellageninand pe ak A protein

Vitelloge n in andPk A were ra d i ol a b ell e dwi t h l3Iiodine CUll ) (NENDuPo nt)in Yll.r.2us ing lodogen (P i e r c e ) following

tine protocol described by Son li. (19 8 5). This involved reacting 101-19 of either VG or Pk A (in 25 1J1 of 0.5 M phosphate bUffer, pH7.4) ina 6 x 50 mm borosilicateglass tube with 10

""1)

of lodogen and L 5 mCl of131tfa r 12 minutes .Thereaction was terminate d by adding 500~lof 50 roMphosphate buffer (pH7.4) for a furthe r 12 minu tesbefore applying themixt ur e to a disposab leSe phadexG-25Hcolumn

(PO-IO, Pharmacia) to separate la b ell e d proteinfrom free isotope. The firstradioactive fraction el u t ed contai ning the la be l led protein was subsequentlycleaned-upbeforeus e on a disposableplastic co l u mn (0.8x 18 em, Kontes) packed with5- 30 0. Theco l umn was previous lyrinsedwith1ml 5%

bovine serum albumin(BSA) (Sigma) and onlythe pea ktube fromthe first radioactive fractionelutedin eachcase was recovered. Iodinated VG and Pk A were usedin RIA and for sho rtterm ovarianupt a ke studiesinYiY2 .

vitellogeninand Pk A were alsoradiola belled wit h tr itium (3H). for longer term ovarianuptakestudiesby both .inriY.2and..in~te c hn i q ue s. Labellingof VGin~ involvedip injectionoffe male s with ,8-estradiol -3 -benzoate one day prior toipin jec t i onof a [3H] ami noacid hydrolysa te (TRK 440, Amersham) carrie r -freeat 0.5mei/kg fi s h.After two weeks fish werebloodsampledandthe r-eeuLt.Lnqserumappl iedon 5-300as previo us lydescribed for the isola tionofVG(pg. 10 ) . The protocol wa s similar for 3Hla be ll i ng of Pk Ainyj"yQ, except that the ,8-estradiol-3 -

16 benzoate in ject ions we re omi t t e d and fi sh were bloo dsample d after four weeks. For bothproteinsthe fract i on cont ai ni ng the [3H]VG or [3M]Pk Afro m a number ofchromatograph i c separationswerepoo l e d .conce nt r a t e d by ul traf il tration (YMIO, Amic on) and fr o z en at -60°Cuntil required.

Proteinswer e labelledin illx.2bymodi fic a t ions tothe method of Ashergt!ti.. (198 3 ).Thismethod,used for rad io labelling locust VG, isbase d on che mical aod LrtcatIon of lysi n e residuesby reactionwith [3H]-sllC cin i mid y l propionate. Pr oteinswereultrafil t e red (YM5. Ami con) aga ins t 0.1Mbora te buff er , pH8.5. guc cLnImfdy I propionate, N-[propionate- 2, 3-3Hj (56.8Ci/mmoll, was purchased from NEN DuPont . Modificat i o ns toAshe r t l21. (1983) includ ed 1) increas ing the amount ofprotein rea cted (18~g), 2) decreas ing there a c ti on time when itwas found that1 hour at roomtemperaturegavea better recov e r yth a n 16hour sat4°C, 3) st op p i n g the reac t i o n wi th 250III of boratebUffer, and4) apply ingthe rea c t i on mixturedirectly onto a dis posableplast iccolumn (0 .8 x18 cm, Ko nt e s ) packed withS-300 at5°C,pre vious ly rins e dwi th 1 ml 5\ BSA and using bor a t e bufferaseluent. Pe ak tube s of the first radioact iv efract ioneluted containingeither [3U) p ro p iony l- VG or [3H)propionyl-PkAwere lyophili zedand recons t i t uted with0.9% salinefo r injection.

Polyc lona l anti bodIeswe re ra i s ed fo r 6 Jlon t h s inmale New Ze aland Whit e ra b b i ts ag ai n stpuri fi ed PkA andVG following theprotoco l of Id l eran d Nq (1979).The Pk A ant i bod y(Ab) ob tainedwas dilu t e d at 1:60 0 , 0 0 0while tho se of VG1:1 50,0 00togiv esal binding of radioiodinated antige n , respect ively. The RIApr o t oc o l, usi ng a double-Ab method, was thesameasthat described forsal mo n VGbySo

g .1!l. (1985).The log-logitweighted regre s sionline s for

Pk A and ve, re specti vely ,had rls- 0.993 and 0.995, slopes of-1.936and -1.5 0 6, interceptsof 0.82 6 an d1.87 6 and50\

binding/olbind ing (8/80) of 2.67 and 17.6 nq,Theuseful rang e of thestandardcurve for Pk A was 0.13-65.4 n9 and fo rVG0.85-2 19 n9 (Fi g. 2).Vitellogeni n ha d01cros s- rea ction inthe PkARIA and Pk A ha d 01 cr oss -re ac tion in the VG RI A. Bloodsamples we r ecollectedmont hl y during 1986-1987 fro m femalewint e r flounde r to provide seas ona l serum pro fi lesfor PkAandVG.

Saltsolubleovarianextract illllllunoreacti vitywith vitelloqen i nand peak A protein anthera

Ovarian folliclesare small «0.5mm) and difficult to isolateindiVidually in any number, ther efore pieces of ovary frolll2female winterflou nderwe r edis sectedfree of

~

o⁵⁰

10

0.1 1.0 10

ngprotein

100 1000

Figure2: Standard curvesdeveloped from r-a dIoimmunoa aaaya, representedas %b ind i ng/ O% binding (B/ Bal versu s log dosefor winter floundervitellogen i n (9) and peak A protein (,).

,.

the ovar-ia nwalland ut il i z edto ob tainsal t sojub j.e ova rian extrac ts (SSOE). Teleost yolkprotei nsaresolu bl e in0.5 M Ha C! (Wa l l ace d,AJ..19 6 6, Plac k£tp.!. 1971). Porti o ns of

ovary (3)0II<)l were homogenized (powersettinq3, Polytron,

Bclnkmann) andext ractedwith4volumuofVGbuffer. After 24 hours at 4°Cthesalt solub l e portionofthe ovarian ext r ac t W;JSseparated fromfollic le celland vitelline envelopedebrisbycentrif ugatio nat 19,000x Gfor 1 hour. Sa lt solubleextrac tswer ethen placed indialytic bags (6 , 0 0 0-8 , 0 00 Mrcu t -o ff, SpectraPor) , preci p i tate d by dialys isag a inst 5mMCa C12• andseparated bycen t r ifu ga ti on at 19,000x G for1hour.The pre cip ita t ed fract i onwa s re- disso lve dinVGbu!fer andchr omatog r a ph e don 5-3 00. Fo llowing chromatogra ph yev e ry fift heluatefract i on fr om acrossth e profilewas measured for i.Munor e activ i tyinboth theVGandPk IIRI lls.

Denaturi ngele ctrop hores isof oocyte prote ins

Oocyte pr oteins fo rdenatur ing 50S PAGEwereobtained fromfr eshl yremo vedovaries of2 femal e....inter flounder.

Foll ic l e s (0 .2 ·0 . 3mm) ....er e dissected fromst r o ma l tissues wit h fi ne forceps underast ere os c o p i c micros cope and transferredwitha gla s s Pa ste u r pipette. They ....ereri ns ed twi c e inmodified (158 mMHaCI) FOsol utio n(a physiolog ical fish oocytebUf fer; Wall aceandSelman1978)and collected

20 each timeby ge nt l ecentri fug at ion. Batc h esof10 or 20 follicle s....ere pla cedin 500 #1 polyp r opy l ene mic rote s t tubes (BioRad )wit ha min i mum of Fa solu t i o n , lyo phil ized, and stored de sic cate dat-20°C until used. Lyoph ilized follicl es wer e groundupwith a small glass rod, takenup in samp lebuf f e r (62.5mMTris- He l , pH6.8, 2% 50S, 12.5%

gl yc ero l, 40 mMdi th l o t h rei toland0.002% b romophe noj, blue) and plac ed inboi l ing water for 5minu te s be fo r e cent r ifug ingtoremov e follicula r deb ris . Total protein conte nt of oocy teprotein s (insamplebufferwititiout;

br omop he nol blue) was es t i ma ted using the Pierce Coomassie prot ein assayrea ge nt and eSAstandard.Sampleswere applied to^5-20%lineargrad i e nt slabgels (30% acrylamide:o.8%

N,N'-me t hylen e bi s a cryla mi d e, pH 8.8.contai ning 10 \ SOS) (Hames 1981 ), 1.5 x 120 X140mm3, overlaidwitha 1 cm3\

stackinggel (acr y lamiderat ioas above, pH6.8, containi ng 10%5DS ).Elec t r ophor e s i s cond itions , st ai ningand de- stai ni ngwe r eiden tical toth e pro t oc ol outlinedfor serum proteinsde s c r i bed earlier Cpg. 12). TheM~Swe r e estimated fromprotei nst andards (505-7and SOs-6H,Sigma) run on adja centlan e s ofthe gels. De ns ito me t ric scanswere used to calc u l atethe R_fof standard s and unknowns allowing the lowe stM~standi!.r d (14,200)an Rf valueof 1.0. Similar to 50S PAGE anal ysis for serumproteins asemi- logarithm icplot of standa r dIs logM~vsR,was used to determine the Mrof the unkn owns basedon the ir RI.

weste rnblotting and immuno ass ay anal y s i s ofoo c y t e prote ins

Oocytepr ote ins sep ara t e d by50S PAGEabove ....ere el ectrcph ore t l cally t ra nsfe r r e d ontonylon membran e s (Zeta- Pro b e , Bi oR a d ) inaHc effer- Mod elTE 52 equipped withahe a t exchange r. Thegel was inc u b a t ed in 200 rnlof t ran sfer buffe r (25 mMTri s ,pH8.3, 19 2:rnMglycine) for 10 minutes and th en plac ed inth e transfercasse t t e sandwiched against thenyl on memb rane bet ·...eensh eets of bl otterpaper (3MM, Wha tm an ) and foam pads, and cl ampedtog e ther.Tran s f er conditio ns we r etypi cal l y0.6 ampe r esat 2°Cfo r 10 hours to remo veall theprotein from thegel.

Followingtra ns fe r the membraneswereanal y z ed using an enz yme-immunoa s say emp l oy i ng a primaryAb(eit h e r Pk Aor VG) and a se cond ary al ka line phosphatase con j u ga t ed Ab (Pr o t ob lot, prcmaqa ] forirnmunodetectionand subsequent colourreact io n. Mod if icationstoth e protocolsuppliedby Promega included bl ockingthe nylonmembranewith10% BSA in Tris-buf fer edsalinewithTween 20 for 12 ho ursat 50°C, inC"u b a ti ng wi ththe primaryAb in 1%BSA+Tr i s-bu f f e red sa linewit hTwee n20 for 2 hou rsand simil a rly with the seco ndary Abfo r 1hour.

22 Ezperi me n t s us i n grlldi o iodin a te dvite l lOCjllni nand pea k A prot e i n

Toesta b lish thest ability of injected [1l1IJPkAand [13lI)VGinthebloodup to48 hours postinjectionsix fe llla le winterflounderwereanesthetizedwith tr i c aine meth a n esul fonat e (125 mg/l;CrescentResearchChemicals, Inc. ) and3 each injected intra ve nousl y(IVI. viacaudal vessels, witheithe r (1l1IJPk Aor [lStI ] VG at 0.5JLCi/ kg fi sh. Aft er48 hours2ml of blood was withd r awn fromeac h fishand dilut ed4: 1 with PMSFsolutio n (a VG stabilizi n g solution;de vla ming

n u.

19 80) on icc.The bloo dwa s centrifuge d and 1 ml plasma aliquots frn!ll fishof each group pooled and chro matographedon 5-30 0asabove . Eluat e fract ionswe r e mo n ito r edforabs orban ce at 280Rilland specificactiv ityin a qa mmlloscint illa t i on count er (Packa rd 56 50)•

Toassess short-tera (48 hour) ova rian uptake 6females were ana esthe tized and in j e ct ed, asabove, witheither radio la bell e d [tl1IJPkAor [131 I JVCat 10 JlCi /kgfish. Two fishper groupwere killed byde ca pi t at ionat 12, 24, and48 hourspost injectionandthe ovary dis s e c t ed fr eeof the ovarianwall. Thespeci fi c acti vityin2. 5g of ovar y was de t e rmine dina gammasc intil latio n counter asabove.

Experim ents using'trit iumlabel ledvitBllcqeninand peakA protein

Two experimentswere undertakento examine longerterm (2 week) ovarianuptakebybothinyj.yQand1niliJ,:Q[3H]

lab e l led VG and Pk A. Inthe firs t exper iment 2 female winter flounde rwere anaesthetized and injected as above with either 13.6 X 10" degradationsper minut e (dpm) [lHJpr opionyl -VGor 7.4 x 106dpmr' Hj prop i o ny l-Pk A/kg fish.

Th e fi sh, killed aft e r 2weeks were weighed ,blood sampled and plasmaprepared as ab ov e. Ovarie swere removed, weighed and placed on ic e.

Plasma aliquots(3 ml) from eachfi s h were chr o ma t og r a p h edon5-300 with VG buffer, modifiedby addi t i on of thepro te o l y t icen zyme in h i b i t ors pepstatinA and leupeptin (si g ma ) each at 0.4~9/ml , ascolumneluent . Sal t soluble ovarian extractswere preparedfrom2 9 portionsofova r y asdescribedpreviously (pg. 17 )but using modif ied VGbuffer (s e e above) andomitting the dialysis step inorder to obtain allthe salt solubleovari a n pro teins.Aliquots (4 mIl of th e SSOE, alw aysusedfresh, wereehromatographedon S-300orUl tr oge l AcA 54 (AcA 54) (2.6 x 84em) (I BF Biotechnics) using modifiedVGbuffe r as columneluent. The AcA54 columnwas use d at a flow rateof 12 ml/hour with upward elutionand calibratedusing

24 ovalbum in (43,000) and carbonican hydrase(29,000) (Sig ma), andribonucleaseA (13 ,700) Mr sta nd ard s (phar-maci a j,

Elua te fractions were moni t oredfor absorbance at 280 nm.

To de t ermin e radiolabel content1 ml el uatealiquo tswere added to 14 ml of scinti l la tion fluid (Re ady -S afa, Beckman or Aquas ol-2 , NENDu Po n t ) in gla s s count i ng via lsand counted ina PackardTr i·Ca r b JODe liquid scintillation counte r adjusted for quenching. Pl a sma aliquots (100JlI, n= 5 ) were combined with 0.5 ml 0.5 M tissuesolubil izer (Protosolor Solvab le, NEN DuPont), digested at es-c overnightand neutralized with 50 pI acetic acid before addingscintillation fluid. similarly , ovarian fragments

(1 8 0-2 0 0 mg, n=5) dissected fr e e ofthe ovaria nwall were digeste d in 1 ttllti s s ue solubiliz e rand prepared for scintil lationcounting as above .

Thepr o t e i n content of eluat e fractionswa s measured by the TCA-Lowrymethod us i ng the Pi e r c e BSA standard.

An ALPP determinatio nwas madeon el uatefractions by the followin gprocedure. Aliquots(200111) were incub ated for 15 mi n utes at 10 0 °C in2 M NaOH,neutralizedwith HCL (Wallace and Jared 1968) and precipita ted with 2.5 volumes of cold 15 %TCA for 10 minutesat room temp e r at u r e.This was followedby centrifugat ion at /I,OOOX Gfo r 10 minutes and removal of 1 ml of the supernata ntwhichwas combi nedwith 1 ml each ofth e colorimet r ic reagen tsammonium mol yb d a t e and ammon iumvunadat;e,The colo urinte nsi tyof the complexwas

lIeasuredspectrophotollletrical l yagainsta standardcurve derived froIDKH2PO.l.asin Nllg l er

n

Al. (1987) .

Th esecondex p e riDen tus ed4 fernal e s , 2 eacn inj ec te d

\litheither [3HJVGor(JHJPkAat 9.2 ^l( 10"and 5.0x10"

dpm/kg fish , respectively . PlaslU an dovarysample s were obta in ed andpre pa red forcol u mn ch r ollAt o g rllphy or scint i llat i o ncou n ting asabove.In add ition ,aliquots of the SSOE(0.5mI, n-5) andthe ins o l ublepell et fraction (di vid e din to two 0.5ml aliquots l fromtheovarian extractionproc edurewe re alsodi gested and prepared for sci nt i lla t ioncounting.

RESULTS

BiochemicalaDal y se. of vite l l oqe ni n &:14peakApro t ein

Poo ledsera from 5fe.alewi nt er flounder, taken du ring thevi tellogenicphase ofthe rep roducti v ecyc le,wa s pr eparedto provide a re presentati v e assessment of the serum pr oteinpro f ileon 5-30 0. Th isse ra was se pa r a t ed into4 fracti ons la be lled A, B,C, and0(F i g.3). Thesefr a c ti ons hadMrs of 1,170,000, 500,000, 270,000 ,and 150,000, respectively.The serumof an es trog e niz e d female on 5-300 demonstra tes the ma s s iveproduction of VG, due toEl, whi ch el ut e s intheregionof peak B (Fig. 4). I t isnot e dth a t est r og e ntr eatment redu ces the amou n ts of otherserum

26

2.0

A

I

^1.5

~

^1.0

0

[!,l

0

0 0.5

Va 0

100 12 0 14 0 160 240

tube no. (1.5 ml/tube)

Figure 3: Gelfiltrat ionchromatography of 3 ml of reecte winter floundersera on sephacryl5-300 and monitored for absorbance. Void volume (Vo, 1.5 X106 H_r) indicatadwith an arrow and pe aks la bell e d A-D.

2.5

2.0

I

1.5

0 1.0

eoN 0 0

0.5

0 100

Vo

I

120 140 160 180 200 220 240

tubeno.(1.5ml/tube)

Fi gure 4: Gelfil t ration chromatographyof3.5 ml of se rum froman estrog e niz edfemal. e ",interflounder as in Fig . J.

28 proteinspar ticularlyPk A.NativePAGE analysis revealed that th epeakA fractio ncontained onepro tein (Pk 1\) oflow R_f, 0.06(Fig.5, lane a).Followingestrogentreatment two proteins at prev Lous l y low levelsinthe serum (Fig. 5,lane b)appear as two distinctbandswith Rls of 0.30and 0.J5on nati vePAGEandareattributab le to VG. OngOSPAGE Pk A is separatedinto threesUbunits,sharpbandsat 210,000and 70,000 Mr' anda broad diffuse<14,000 Mr band (Fig. 6,lane b).Vitellogeni n givesa major band at18 0, 00 0 M_rplUS some minor compo n ent s (Fig. 6, lane c). comparisons ofthe amino acid composi ti onsfor Pk A and VG showthattheyare not great lydifferent the onlyaminoacids differingby 50\or more bet ween the twopro t e i n s bei ng his t i d i ne , isoleucine and!s cysti ne(Ta b l e1).Me thionine wasno tde t e r mi nedIn eithercase.

Ul t rac ent ri f uqa tio n analys is of femalewint er Uounde r plasma

u~tracentrifugationof female plasma indicated4 density cj.aases of li pop r ot e i n (Table2).TheVHDL fraction representing27.5\of the total pla s maprot e in contained both Pk A andVG. The concent r atio nof Pk A (1470~g/ml)wa s about twi ce that for VG (737 J.j9/ml) in theVHDLfraction. A smal ~amoun t of VG was also found inthe hi gh density lipo prot e in fr act ion.

a b

Figure 5: Native polyacrylamide gel electrophoresis analysis of winter flounder peak A protein, lane ,a.; serum proteins ta k e n from peak B (Fig. 3) , lane12; and winter flounder vi t e l l og e n i n , lane g. 25 ~g protein applied to each gel.

30

a b d

~

330 220 94 ⁶⁷

~^~

- - ^{.. ... 67 ... 60}

43~

~

"'36

30~

20.1

~

... 18.5

14.4

~

Figure 6: Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of LMW standards (Pharmacia), lane,5li winter flounder peak A protein, lane Q; winter flounder vitellogenin, lane~; and HMW standards (Pharmacia), laneg. M_r (xl, 000) as calculated in Materials and Methods indicated by arrowheads.

Table 1:Amino acidcomposition(%) of female winter flounder vitellogeni nandpeak Aprot e i n .

Amino Acid Vitellogenin Pk Aprotein

aspartic acid 10.5 7.3

glutamic acid 15.0 10. 8

serine 8.3 9••

glyci ne 8.0 7.4

hi s t i d i ne D.s 2.4

arginine _7.' 4••

th reo n i ne '.8 10.6

al anine 4.9 4.2

proline 7.6 9.8

tyrosine 3.7 3.1

valine a.7 7.7

isoleucine 2.2 4 . '

leucine

5.'

^6.7

phenylalani ne 2.8 4. 1

lysine 9.0

6.'

'~cystine 2.0 1.0

32

Table 2: Ul t racen t r i fug a tionan a lys isof fem alewinter floun de r plasma.

Li po prote in Densit y Pr o tein Vite llogenin PEak A pr otein Fractions (9/ 11I1) (mg/_l) (# 9'/111) (/-19/ 11I1)

'VLO L <1.00 6 0. 01

LOL 1.006 - 1.063 2.91

HDL

VHDL

1.0 63 - 1.2 1 26.6

>1.21 11.2

25

73 7 1470

l VLDL= very 10'01densi tyli popr ote i n, LOL- 10'11'dens i ty lipoprote in, HOL- highdensity lipo proteIn , VHDL"" ver y hIghdensitylipo prote i n.

Rac!iolabelling ofvitellogeninand peak Aprotein

Both VG and PkA were successfully radiolabelled wi t h UlI, lH and ('H)-succ1nimi dyl propionate . The speci f ic acti vitiesonaverage fo r (1l 1II VG, eHlp r opion yl-VGan d [ 3H] V G were 16. 8and0.82~Ci/ug, and 15nCi / mg respective ly.

similarly, specific act ivitiesfor

r'31

1]Pk A, [3Hlpropionyl - Pk A and[3 H]PkA were 13.6 and0.66/Jcijug,and 4 nCi/mg respe c ti vel y .

Seasonalse r ulII profilesof vi telloqe llin and peak A protein

TheRIAs developed for Pk AandVG al lowed the det e rminationof seasonalserum profiles of these two protei ns1n femalewinter flounder (Fig. 7).BothPk Aand VG followthe same general pattern overthe ann ual reproductivecycle with PkAconcentrationsus u a lly about twicethose of VG.Dut"ing the summer following the spawning per iod (May-June ) the le vel s aretypica l lylow but increase dr a ma tical lyby la t e Aug ustandSeptemb e r,drop inOctobe r and increaseagain torea chthehi ghestleve l s in early December.Levels declineover the wint er and in tothespring with the onsetof spawning.

34

J J

o

N

o

J F ... A

(~ ~/'\J~I ^{')1\, (0)1/ (.)} _(.~ ^/t'(') _. ^{( .) (.) (.)(')}

A M J O N D J F M A

Month

Figure7: Seasonal serum profileof vitellogenin(A) and peak A protein(B) infemale win t er flounder

(1986-87).Each point representsth e mean standarderror(S E ) (nu mb er of fish); •'" pooled sera from5 fi sh .

stabilityof ra4i oll1belled vi t el lo ge n in and peakApr otein injected!nYi.Y.Qin the blood

Theintegrity ofra d i o l ab e l l e d prot einsinth eblo od , followingiv injection. were determi n ed by the elut ion position of fractionscontaining radiolabel when pla s ma r rcm trea ted fish were chromatograp hedon 5-300. In fishsampled 48hoursafter[B1IJVGin j e c t ion most of the radioactivity was found infr act ionselutingat the approximate M_rregion ofVG(50 0 ,0 00), althoughthere wereseveral obviouspeaks of radi oactivitysepa ratedon 5-300 (Fig. 8).However, in (llIIJPklI.injec ted fis h after 48 hoursall the radioactivity measured was found in fractionsassociatedwith the first proteinpeakon5-300 (Fig. 9). wi t h [3H] pr o p i ony l -VG and [3HJVG inj ect e dfis h after2weeks a singlepeak of radioact ivitywas measured amongst fractionselutingat the appr oxima teMrregionof VG(5 0 0,0 00 ) (1"igs. 10 and 11).A single peak of radioactivity wa s ap parentfor

r

3H] prop i ony l- Pk A and [3H] Pk Ainjec t e d fish aftertwo weekstha t was found in fractionselutingwiththe first proteinpea k (Figs. 12and 13) . Thecle a r a n c e from thebl ood of all radiolabelled VG preparations is rapid, as re fl e ctedbythe amount of radi olabel present in the pla s ma , in comparison with similarlyradiolabelledPk A pr eparationsaft er 48 ho ur s or2weeks . The clearanceofra d i o l ab e lle d vc s in all cases is at least 20 times that of comparablePk A

J6

1.5 500

T

^1.0 400³⁰⁰

i

§

.0

.

0 200

Z

coN 0.5 E

c [S

0 100

Vo

o

0

80 100 120 14 0 160 180 200 220

tubeno.(1.5ml/tu be )

Figure 8: Gel filtration chromatographyof 3 ml of female winterflounderplasma on Sephacryl5-300from

fishinjected 48 hours earlier wi t h

[1311]v i t ellog e ni nat 0.5 /lei/k.g and monitared for absorbanceand ra di o a cti v i t y (coun tsper minute [cpm} l l voidvolume (Va, 1.5 x 10 6 Mr) indi cated with anarrow.

3.0 5.0

2.5 4.0

j

T

^2.0 3.0 .0

.

§

1.5 ^~

~

0 2.0

eo _~

N 1.0

C> 0

C>

1.0

~

0.5

0

140 200 220

tube no.(1.5ml/t ube)

Figure9: Gel filtra tionchr omatogra ph yof 3 mlof female winterflounde r plasma from fish inj ected 48 hoursearl ier with (131 I ]peak A pro te i n at 0.5 pe l/kg, as inFig. a,

3.

1.0 2.5

2.0

j

I

^0.75

~

^1.5

~

^0;

a 0.5

E

ro 1.0 ^<,

N

"b

a0 0.25

Va 0.5 3

0 0

1

100 240

Fi g u r e10 : Gel filtration chromatographyat Jml ofrenaie winter flounder plasmafrom fish injected 2 weeksearlier with13.6x 106 degradations per minu te(dprn)[3U] prop i o nyl -v i t ellog eni n, asin Fig. B.

2.0 200 175

1.5 150

I

¹²⁵

1

~ ^{1.0 100}

i

~

₇₅

..

~

00 0.5 _Vo 50

i

I

2.

0 0

'00 120 1'40 160 220 240

tube no. (1.5 ml/tube)

Figure 11: Gel filtrati onchromatographyof3mlof female winter flou nde r plasma from fis h injected 2 weeksearlierwith9.2 x105 de g rad a tio ns per minute(dpm)

r

3Hlvi te ll og e nin,as in Fig .8.

30 1.0

T

^0.75

o

~

0.5

~o o 0.25

Va

1

12 0 140 16 0 18 0 200 220

40 35

i

25

g .

20

-.

E 15

~

_o

10 -- 40

tubeno.(1.5ml/tube)

Figur<> 12: Ge l fil t r ationchr oma tog r a phyofJml of fe male

winter flounderpl a sma fr o m fish injected2 weeksea r lier with 7.4X 106degradations per minute (dpm) [l Hjpro p i onyl - pe ak A protein, asin Fig . 8.

2.0 2.0

A

I

1.5 1.5

I _i

1.0 1.0

..

~ ~

8

0.5 0.5

Vo ^~

I

^.g-E

O 0

100 120 2<0

tube no. (i.sm1/tube)

Fi g u r e 13: Gel filt r a ti on chromatographyof 3 ml of female winter flounderplasma from fishinj e c t e d 2 weeks earlier with 5.0x lOSdegradatio ns per minute(dpm) ['H]peakA protein, asinFig.8.

"

ra d i olabel s when the differentspecific activitiesar eta ken intoaccount (F i qs . 8- 13 ). Infish sampl e d two weeks afte r

r

3H) VG and [~] PkAin j e c t i onthe injectedradiolabel calculatedto bere lDai ni n g inthe plasmawas2and 52\, respectively,of thetotal injected.

ImJlIunoreactivityof thesalt sol ub l e ov arian ex t rac t with vitell og eninandpeak~proteinan tis e r a

Followingdi a l y s i s SSOE could be separated in totwoma j o r protein pea ks by5-300chromatog ra p hy,the firstnear the void vol ume(Vo)ofthe columnand the second at 280,000 M,

(Fig. 14). The best immunoreactivityfor Pk A occurredon thetr a il i ng edge of the firstproteinpeak and to aless e r extent in theseco ndpeak.However, fo r VGthe lIla jor siteof illlmunoreactionoccurred on the second peakand in a conc e n tra t i ondependent fa shionrelative to the absorbance.

Thereliassomeminor imlDunore ac ti v ityals oon the firstpe ak and the tra il i ng ed ge ofth e second.

Re l a t i ons h i p ofooc y t e proteins to vitelloqen inan d peak ~ pr otein

Oocyte prote ins from vitellogen i c ova ria n fo llicles sep a r a t e don5DSPAGE produ c e d5 major subuni ts; 101,400,

J.O

J.' 2.'

J.O

I

^2.01.5 ^{2. '}2.0

ⁱ ..

~

~

~

^{1.0 1.5}

^ll:

"

^'.0

o

0.' 0.'

0 0

80 100 '20 280

tubeno.(1.5ml/tubo)

Figure 14:Gel fil trationchroma tographyof 2 ml of ovar ian extract on sephacryl5-300andmonitoredfor absorbance. An aliquot of every sixthel uate fraction was measured inthe peakA proteinand viteUogeni nradioimmunoassays as indicated .