Developmental Changes in Egg Yolk Proteins of Walleye Pollock

Bull. Fish. Sci. Hokkaido UnIV. 53(3),95-105,2002

Developmental Changes in Egg Yolk Proteins of Walleye Pollock, Theragra chalcogramma, and a Comparative Study of Immunoreactivity of Other North Pacific Teleosts and Invertebrate Eggs

2 Kevin M. BAILEY!), Nazila MERATI1J, Michael HELSER ),

3 4 Naoshi HTRMIATSU ) and Akihiko HARA ) (Received 3 Seprernber 2002 Accepred 10 October 2002)

Abstract

Changes In egg proteins during embryonic development in walleye pollock, Theragra chalcogramma, were shown by SDS-PAGE. Western blotting with a polyclonal antibody developed against proteins from hydrated eggs showed major reactive bands in serum of vltelJogenic females at 175,76 and 66 kDa. Vitellogenic ovaries had major reactive bands at 97 kDa, and extruded, hydrated eggs had bands at 94 kDa. Fertilized, late-stage developIng eggs and yolked larvae had major bands at 66 kDa. These results suggested proteolytie cleavage ofvitellogenin and other egg proteins upon uptake by oocytes and fmiher digestion of egg proteins dUrIng development. lmmunoblots run to test cross-reaclivity potential between anti-pollock egg yolk protein antibodies and various protellls of Invenebrate and other teleost species demonstrated Ihat antigenic slmilanties exist between most teleosls and walleye pollock egg proteins, but not between pollock and invertebrate eggs. Subsequent Western blotting shov"ed that several major iJT)munoreacrive egg proteins are shared in dIstantly related fish faInilies. II is thought that egg-yolk proteins are antigenlcally conserved among teleosts.

Key words: Egg development, Egg protein" Vitellogenin, Walleye pollock, Theragra chalcogramma, Cross­ reactivity. Immunorecognioon

Introduction pattems have shown that several new protein bands appear in oocytes during vitellogenesis and because these During the egg and early larval stages of marine fishes, proteins are smaller than vitellogenin, it has been sug­ maternally supplied yolk provides the components gested that vitellogenin is the precursor for the smaller required for energy production, biosynthesis and mainte­ egg proteins observed (Wallace and Selman, 1985). nance. Vitellogenin, a lipoglycophosphoprotein (Ber­ Greely et a!. (1986) also observed changes in the protein gink and Wallace, 1974; Christmann et al., 1977; Ng component of teleosts during oocyte maturation and and Idler, 1983; Mommsen and Walsh, 1988; Specker suggested that these changes result from the alteration of and Sullivan, 1994), is the precursor of the major yolk existing proteins. In marine fishes, information for the proteins of fishes and many other oviparous animals. mechanisms of yolk breakdown and the fate of yolk Vitellogenin is synthesized in the liver of females under products is limited to a few species (Matsubara et a!., the influence of estrogen, secreted into the blood, and 1999; Carnevali et aI., 1999; Harting and Kunkel, 1999). then incorporated into egg yolk proteins. They are Aside from the initial processing of vitellogenin, the proteolytically cleaved into lipovitellin, phosvitin and occurrence of additional cleavage of yolk protei ns along proteins called YGP40 or j3f-component in amphib­ with final oocyte maturation has been discovered in ians, birds and fishes (Wallace, 1978; Matsubara and some particular pelagic egg-laying marine li.sh (Wallace Sawano, /995; Yamamura et aI., 1995; Hiramatsu and and Begovac, 1985; Wallace and Selman, 1985; Greely Hara, J996). Previous studies using electrophoretic et aI., 1986; Carnevali et aI., 1993; Matsubara and

I) National Marine Fisheries SerVIce, Alaska Fisheries Science Center, 7600 Sand Point Way NE, BIN C15700, Seattle, WA 981lS­ 0070, USA

2) Depanment of Food Science, Cornell University, Ithaca, NY 14850, USA

3) Department of Zoology. North Carolina State University, Raleigh, NC 27695-7617, USA

'j Laboratory of Comparative PhYSiology, Graduate School of Fisheries Sciences, Hokkaido University. 3-1- J, Minato, Hakodate. Hokkaido 041-8611, Japan (~ ~J1'ij:Jt*$*~I%*i£~4$liJTy'Cff~ljf!ttl~~~~mD

.- 95 ­ Bull. Fish. Sci. Hokkaido Univ. 53(3), 2002.

Sawano, 1995; Thorsen et aI., ]996). The secondary net-pens and fed a diet of herring and squid until processing of yolk proteins is thought to be an important spawning commenced. Eggs were stripped, fe11ilized event giving rise a pool of free amino acids that causes with milt, and then reared according to techniques in oocyte hydration for the acquisition of buoyancy. Bailey and Stehr (1986). These free amino acids are also responsible for the rapid embryonic growth after fertilization. In teleosts, bio­ Eggs and invertebrate collections chemical infOlmation on yolk degradation during em­ Ovaries and egg samples from fish and invertebrates bryogenesis is limited to a few species (Olin and Yon were coUected in 1986-1987 from Puget Sound and Der Decken, 1990; Hartling and Kunkel, 1999: Mat­ stored at - 80T. Samples tested for potential cross­ subara et aI., 1999), and the mechanisms of yolk hreak­ reactions are summarized in Table I. Approximately down and the fate of yolk products remains to be 0.16 g of each sample was weighed and homogenized in verifIed. a glass tissue grinder in 500 j.ll Tris-buffered saline (TBS) Walleye pollock is thought to have an annual spawn­ (20 mM Tris, 500 mM NaCl, pH 7.5). The ing cycle and its oocyte maturation has been classifted as homogenant was centrifuged for 3 min at I 1,750x g at partially synchronous (Hinckley, 1986). Walleye pol­ room temperature. The supematant was drawn off and lock currently supports a large commercial fishery in the frozen at - 80'C until needed. Protein stocks were North Pacific Ocean. It is the sUbject of an intensive diluted 1 : 100 in TBS and absorbance was read at 280 study to detennine abiotic and biotic factors critical to nm to measure protein concentration (Shimadzu UY­ the recruitment of fish stocks. In this study, we used an 120-2). Final protein concentration used in the assay antibody probe developed against egg yolk proteins of was l5j.lg/100j.l1. One hundred microliters of protein walleye pollock (Theragra chalcogramma) to show that extract was applied to 0.45 j.lm nitrocellulose membrane the sma]] proteins in egg yolk are derived from vite!­ in a microfiltration apparatus and allowed to gravity logenin. Using SDS-PAGE and Western blots, we filter at 4'C. When filtering was complete and the traced the major changes in egg proteins from sera of membrane was dry, the membrane was removed from mature females through oocyte development and yolk the filtering apparatus and blocked iu Blotto/Tween (5% absorption by the larvae. We also compared pollock nonfat milk, 0.05% Tween-20, 0.01% Antifoam A, and egg protein components with eggs of other teleosts and phosphate-buffered saline, pH 7.4). Immunoblotting invertebrate species. Immunoblots were initially used procedures followed Theilacker (t986) except th at 10% to quantify the amount of immunological specificity normal goat serum was added to the blocking solution between walleye pollock and various other marine before and during second antibody incubation. The species. SDS-PAGE and Western blotting techniques primary antibody used in the assay was an unabsorbed were utilized to determine which specific egg proteins polyclonal rabbit anti-pollock egg yolk protein rgG were immunoreactive with our anti-pollock egg yolk fraction (see below for antibody production in details). protein antibody. The titre used in tllese experiments was I: 5,000. The conjugate antibody used was a I: 3,000 dilution of Materials and Methods alkaline phosphatase-labelled goat-rabbit IgG (Bio-Rad Lab.). Proteins were visualized using BCIP (p-nitro Fish and biochemicals blue tetrazolium chloride) and NBT (5-bromo-4-<:hloro­ Adult pollock were caught in the Tacoma Narrows 3 indyl phosphate p-toluidine salt). vicinity of Puget Sound, Washington (U.S.A) by hook A series of positive and negative controls were run and line in autumn and winter months, and transported along with the rest of the samples to monitor the inten­ to the Manchester Aquaculture Facility of the Northwest sity of the color reaction and to calibrate the scale or Fisheries Science Center (U.S. National Marine Fish­ cross-reactivity. The maximum amount of cross­ eries Service). Blood and ovaries of some of the fish reaction was observed as an intense purple-black color were collected immediately after capture. Blood was present in the homogenate of one pollock yolk-&ac collected by excising the tail. Blood samples were larvae. To this color we assigned a grade of ++-+, and transported to the laboratory on ice and allowed to clot scaled this as a very strong reaction. For strong reac­ at approximately 4°C overnight. The resulting clot was tions exhibiting a purple lavender color, a grade of ++ removed and serum was separated by centrifugation at was assigned. Weak reactions displaying a light laven­ room temperature for 10 min. Ovaries were collected der were graded as +. Samples that showed no reac­ from approximately 40 em fish and were frozen on dry tion were graded as -. Positive controb consisted of ice for about 6 hr, after which time ovaries and blood pollock egg and yolk-sac larvae extracts and female sera were stored at - 80'C. Other fish were held in serum. Negative controls included normal pooled goat

96 BAlLEY et al.: Egg yolk proteins of walleye pollock

Table I. Samples used In cros~-reactivity study

Sample Common Name Scientific Name Family

IA Pacific cod egg Gadus macrocephalus Gadidae IB Pacific tomcod ovary Microgadus proximus Gadidae IC Sand sole egg Pseltichthys melanostictus Pleuroncctidae lD Pacific tomcod egg Microgadus proximus Gadidae IE Pacific hake ovary Merluccius productus GadIdae IF Sableflsh egg A rlOplopoma fimbria Anoplopomalidae IG Pacific halibut egg Hippoglossus stenolepis Pleuronectjdae IH Pacific herring egg Clupea harengus pallasi Clupeidae I I Rockfish embryo Sebastes sp. Scorpaenidae

2A Padded sculpin egg A rtedius fenestralis Cottidae 2B Kelp greenling egg Hexagrammos decagrammus Hexagrammidae 2C Angelfish egg Cichlidae 2D Cabezon egg Scolpaeniclhys marrnoratus COludae 2E Whitespotted greenling egg Hexagrammos stelleri Hexagrammidae 2F Decorared warbonnet egg Chirolophis decoratus Stichaeidae 2G Lingcod egg Ophiodion elongatus Hexagram midae 2\-1 Pollock tlesh (female) Theragra chalcogramma Gadidae 21 Pacific spmy lumpsucker egg Eumicrotremus orbis Cyclopteridae

3A Grunt sculpin egg Rhamphocottus richardsoni Cottidae 38 Coho salmon egg Oncorhynchus kisurch Salmonidae 3C Chinook salmon egg Oncorhynchus tshawytscha Salmonidae

4A Frilled dogwinkle egg Nucella lamellosa Muricidae 4B Rosy tritonla egg Tritonia diomedea Tntonildae

4C Purple sea urch 10 egg Strongylecentrotus purpuratus Strongy1ecentrotidae 4D Sand dollar egg Dendraster excentricus Dendrasteridae 4E Lion nudibranch egg Melibe leonina Tethyidae 4F Starfish egg Pisaster ochraceous Asteriidae 4G Orange peel nudibranch egg Tochuina letraqueta Tntonlldae 4H Pacific sea-lemon nudlbranch egg A nisodoris nobifis Discodorididae 41 Hermissenda nudibranch egg Hermissenda crassicornis Face1Jnidae

SA Northern shrimp egg Pandalus borealis PandalIdae 5B Spot prawn egg Pandalus platyceros Pandalidae SC Gamrnand roe Cyphocoris challengeri LysIanassidae 5D Pacific Lyre crab Cancer oregonensis Cancridae 5E Eyed crab roe Hyas lyratus Majidae

6A Diatom Phaeodactylum tricomutum CymbelIaceae 6B Dinoftaggelate A mphidium corleri Dinophyceae 6C Green algae Plalymonas suecia Chlamydomonadaccae

7A 1/ '0 pollock egg Theragra chalcogramma Gadidae 7B 1/100 pollock egg Theragra chalcogramma Gadidae 7C 1/1000 pollock egg Theragra c!wlcogramma Gadidae 7D 1 yolk-sac larvae Theragra chalcogramma Gadidae 7E l/IO yolk-sac larvae Theragra chalcogramma Gadidae 7F Female pollock blood serum Theragra chalcogramma Gadidae 7G Male pollock blood serum Therogra chalcogramma Gadidae 7H Protein extraction buAer 7 I Normal goat serum pooled

- 97 ­ Bull fish Sci. Hokkaido Uoiv. 53(3), 2002. serum, TBS extraction buffer, and male pollock serum. 11,750 X g: the supernatant was collected and stored All negative controls remained clear. at - 80T. Protein concentrations of sera, ovalY, egg, and larval homogenates were determined by absorbance Antibody preparations at 280 nm. Egg yolk proteins were extracted as follows. Hydrat­ Prior to electrophoresis, the homogenates were diluted ed, ovulated and unfertilized eggs were extruded from a to the same concentrations in sample buffer and heated ri pe female, fmzen over dly ice and transferred to liquid to 95'C for 4 min. Sample wells were loaded with 20 nitrogen. Five grams of eggs were thawed, rinsed with ,.ug/5,.ul protein. The SDS-PAGE gradient gels (10­ 0.9% NaCI and homogenized using a ground-glass tissue 20%) were run at constant voltage until the dye front grinder over ice. The egg homogenate was centrifuged migrated to the bottom. Coomassie Brilliant Blue at 8,160xg for 4min. The supernatant solution R-250 stain or silver stain was used for protein visualiza­ contained some floating panicles that were col1ected and tion. Molecular weights of egg proteins were deter­ filtered through a 0.20,.um filter membrane (Plack et aI., mined by plotting the molecular weight of protein 1971; Hara and Hirai, 1978) The clear solution was standards (Bio-Rad Lab.) versus the distance they mi­ dripped into 30 ml chil1ed, distilled water and allowed grated from the interface of tbe stacking and separating to flocculate. The precipitate, collected by centrifuga­ gel. tion. was dissolved in 1 ml 0.02 M Tris-HCl buffer pH 80 containing 0.2% NaCl, dripped into chilled, deion­ Western blotting ized distilled water and allowed to stand at 4'C for 2 hI'. For Western blots, 3.5 ,.ug/5,.u1 protein per well was The supernatant was decanted, and rhe precipitate was loaded for each sample on SDS-PAGE gels which were dissolved in Tris-HC! buffer (above mentioned) and constructed and run as described above. Duplicate gels precipitated into distilled water again. The flocculate were silver stained. After running the gels, they were was centrifuged at 1,200 X gat 4'C for IS min, the super­ equilibrated in Tris-glycine buffer (25 mM Tris, 192 roM narant was discarded, and precipitate was dissolved in glycine, 20% v/v methanol, pH 8.3) for 30 min. The 2.S ml Tris-HCl buffer. A stock solution for rabbit proteins were transferred to nitrocellulose paper at 50 V injection was diluted to 2.3 mg protein/ml, mixed with for the first hour and at 100 V for the second hour. The an equal volume of Freund's complete adjuvant, and nitrocellulose paper with the transfe!Ted protei ns was then it was frozen. A New Zealand male rabbit was blocked with BLOTTO block solution (5% nonfat dry injected four times at 10 day intervals. Blood was milk, 0.05% Tween-20 in TBS) overnight. Following collected about 6 weeks after the initial injecrion. two 10 min washes in TTBS (005% Tween-20 in TBS), Blood was allowed to stand for I hr at room tempera­ the nitrocellulose paper was incubated with the anti­ ture, it was then stjned once, allowed to stand overnight pollock egg yolk antibody (diluted I : 1,000 in BLOTTO at 4'C, and then centri fuged ar 8, I60 X g at 4'C for 10 block solution) for 2 hr. The egg protein developmenr mm. The IgG fraction was salted out from the blood blots used the absorbed antibody, and the cross­ sera by using a 40% solution of saturated ammonium reacrivity blots used the unabsorbed antibody. The sulfate; the precipitate was dissolved in 0.01 M phos­ membrane was washed in BLOTTO wash solution (5% phate buffer at pH 8.0 and was purified by passing the nonfat dly milk, 0.3% Tween-20%, in TBS), three times solution through a DEAE column using DE52 (What­ for 10 min each, and in TTBS two times for 10 min each. man). A faint reactivity of anti-egg protein antisera The reactive proteins were rhen probed by incubation in with male sera detected on dot-blots and Western blots the second antibody solution (alkaline phosphatase­ was removed by affinity purification. Male blood sera labelled goat anti-rabbit IgG) diluted I: 3,000 in were atrached to a column of Sepharose 4B (Pham1acia) BLOTTO block solution. Excess antibody was activated wirh cyanogen bromide, and the IgG solution removed by washing in BLOTTO wash solution four was passed through it. The resulting absorbed IgG times, TTBS two times and TBS two times, all for 10 fraction showed no cross-reactivity to male sera. min each. The reactive proteins wcre then visualized by color development as described above. Electrophoresis

Preparation of samples for sodium dodecyl sulfate Results (SDS)-polyacrylamide gel electrophoresis (PAGE) was perfOlmed as follows. Ovaries, eggs and larvae were Egg protein development homogenized in sample buffer (0.16 g/0.5 mI TBS) with A composite gel of sera. ovarian oocytes, hydrated a ground glass rissue grinder on ice. The homogenate fertilized eggs, yolk-sac and post-yolk-sac feeding larvae was centrifuged at room temperature for 3 min at IS shown in Fig. \. This gel was used ro calculate

98 ­ BAILEY el al.: Egg yolk proteins of walleye pollock

LANE

1 2 3 4 5 6 7 8 9 10 11 200 116 97 97 66 66 45 45

31

21.5

14

Fig. J. SDS-PAGE of serum, ovarian and egg proteins of walleye pollock. Gradient gel, 10-20% stained with Coomassie Blue. Lane: 1, low molecular weight marker; 2, pre-vitelJogenic ovary extract; 3, pre-vitellogenic female serum; 4, virelJogenic ovary extract; 5, vltellogenic female serum; 6, crude purified unfertilized egg exrract; 7, early fertilized egg extract; 8, late fertilIzed egg extract: 9, yolk-sac larva extract; 10, post-yolk-sac larVa extract; and II, high molecular weight marker. molecular weights for comparison with Western blots completed spawning in May had no reactive proteins in and their duplicate silver-stained gels. Bands were their ovaries, but weak immunoreactions in the May sera assigned to either major or minor categories based on sample were observed that corresponded ro the major their initial staining intensity, The molecular weights bands found in the blots of sera from preceding months. of the main bands of yolk protein were observed to be These weak bands did not correspond closely to those of 97 kDa for vitellogenic ovary extract and 94 kDa for the egg extracts, indicating that in walleye pollock, either hydrated feltilized eggs (Fig. 1, lanes 4 and 6) egg yolk proteins are not being resorbed directly or that Western blots indicated that the polyclonal antibodies resorption had already taken place. Sera from male developed against crude, purified pollock egg proteins walleye pollock collected in March and May did not were immunoreactive with egg yolk protein and its react with the antibody (Fig. 2, lanes 12 and 13). derivatives in sera and ovary extracts (Fig. 2). Ovaries Fertilized walleye pollock eggs up to age 4 days had from female walleye pollock collected in December that major reactive bands of 94, 76, 58 and 25 kDa and were in the early stages of egg development did not react another just under 14 kDa (referred to as 14 kDa-l; 14 with the egg yolk protein antibodies (Fig. 2, lane I), kDa was the smallest molecular weight marker used) indicating that the individuals were previtellogenic. (Fig. 3). Minor bands were found at 96 and 66 kDa. However, sera collected during this month exhibited Late-stage eggs and yolked larvae had major bands of three minor bands which reacted ,vith the antibodies at 94,66 and 25 kDa, minor bands of 76 and 14 kDa-l. 175, 76 and 66 kDa (Fig. 2, lane 7). As oocytes matur­ As well as another minor band which migrated slightly ed from Febntary to April, ovaries reacted to the anti­ beyond 14 kDa-l. First-feeding pollock larvae which bodies revealing major proteins of 97,58,25 and 14. had resorbed their yolk had no reactive yolk proteins During these months blood sera samples showed major (Fig. 3, lane 7). bands of 175, 76 and 66 kDa in addition to the minor proteins observed in December. Individuals that had

~ 99­ Bull. Fish. Sci. Kokkaido Univ. 53(3). 2002.

LANE

1 2 3 4 5 6 7 8 9 10 11 12 13 --Q 175 o 97 ~ 76 G 66 ~ ~ 58 ~ < 25 Su ~ o~ ~ 14

Fig. 2. Western blotting of ovarian and serum protems of walleye pollock. SDS-PAGE linear gradient 10-20%; proteins transferred to nitrocellulose paper for IInmunodetection with rabbit antibody developed against crudely purified egg proteins. Lane: I. pre-vitellogenic ovary extract; 2, developing ovary extract; J, vltelJogenic ovary extract; 4, spawning ovary extract; 5, spent ovary extract; 6, pre-vitellogenic female serum; 7. pre-vitellogenic female serum; 8, developing female serum; 9. virellogenic female serum: 10. spawning female serum: II, spent female serum; 12, mawre male serum; and IJ, spent male serUlll.

LANE Immunoblotting and immunospecijicity studies 1 2 3 4 5 6 7 Anti-pollock egg proteins cross-reacted most intensely with proteins from members of the Gadidae (Table 2, 94 Fig. 4). Strong reactions were also noted with proteins of Pacific halibut (Hippoglossus steno!epis) , rockfish 58 (Sebastes spP.), sablefish (Anop!opoma fimbn'a), and sand sole (Psettichthys melanostictus) eggs or yolked embryos. VelY weak or no reactions occurred with members of the Salmonidae, Hexagrammidae, Conidae and Stichaedidae. A weak reaction was noted with female pollock flesh tested using unabsorbed antibody. For a complete summary, see Table 2 and Fig. 4. 25 Gel electrophoresis and iml1lunoblots of fertilized 24-hour old walleye pollock eggs and eggs of selected marine fish species revealed that they possess several bands in common. The molecular weights of egg 14 proteins observed in SDS-PAGE gels (Fig.5) were

Fig. 3. Western blotting of egg, yolk-sae larva and post­ yolk-sac larva proteins of walleye pollock. SDS­ PAGE linear gradient 10-20%; proteins transferred to nitrocellulose paper for immunodeteclion With rabbit antibody developed against crudely purified egg pro­ tein. Lanes: I, crudely purified unfeniJized egg extract; 2, unfertilized egg extract; 3. I-day-old fertil­ ized egg; 4, 4-day-old fertilized egg; 5, 8-day-old fertil­ ized egg; 6, yolk-sac-larva: and 7, post-yolk-sac larva.

-100­ BAILEY et al.: Egg yolk proteins of walleye pollock

Table 2. Results of dot-blots testing potential cross­ LANE reactivity berween selected invertebrate and fish eggs. ReactIons are scored by comparison w positive control. In this case, we used one ABCDEFGHI walleye pollack yolk-sac larvae as the guide and assigned grades based on the intensity of 1 •• reacti vi ty. 2 Cross­ Sample Type reactivity 3 Pacific cod egg Teleost + -'--'­, , Pacific tolDcod ovary Teleost +++ 4 Sand sole cgg TeleoSI ++ Pacific lomcod egg Teleost +++ 5 Pacific hake ovary Teleost ++ 6 Sablefish egg Teleost ++ Paeific halibut egg Teleost +++ 7 Pacific herri ng egg Teleost + Fig. 4. Immunoblot of teleost and invertebrate eggs to Sebastes sp. embryo Teleost ++ determine cross-reactivity potential. Samples were Padded sculpin egg Teleost + exposed to a I: 5,000 dilution of rabbit anti-pollock egg yolk protein antibody. Secondary antibody used Kelp greenling egg Teleost + was it I: 3.000 dilution or goat anti-rabbit alkaline Angelfish egg Teleost + phosphatase. See Table I for immunoblot legend. CabelOn egg Teleost ++ Whitespotted greenling egg Teleost + LANE Decorated warbonnet egg Teleost + Lingcod egg Teleost + 1 2 3 4 5 6 7 8 9 10 11 12 Pollock flesh (female) Teleost + 200 Pacific spiny lumpsucker egg Teleost 116 Grunt sculpin egg Teleost 97 97 66 Coho salmon roe Teleost + 66 Chinook salmon roe Teleost + 45 45 Pollock yolk-sac larvae Teleost (+++) Sea snaiI egg Invertebrate 31 Gianr orange lludibranch egg Invertebrate

Sea urch JU egg Invertebrate 21.5 Sand dollar egg 1uvertebrate 14 /VJelibe leonina Invertebrate Starfish egg Invertebrate Fig. 5. SDS-PAGE of eomparative egg proteins. Gradi­ ent gel 10-20%, stained with Commassie Blue. Lancs: Orange peel nudibranch egg Invertebrate I, high molecular weight marker: 2, Pacific cod egg; 3, Sea lemon nudibranch egg Invertebrate Pacific tomcod egg; 4, Pacific halibut egg; 5, Pacific nudibranch egg lnvertebrate hake ovary; 6, sablefish egg; 7, Sebastes sp. embryo; 8, Pacific herring egg; 9, Grunt sculpin egg; 10, coho Pink shrimp egg ] nvertebrate salmon egg; ll, 24-h fertilized walleye pollock egg; Spot prawn egg Invertebrate and 12, low molecular weight marker. Gammarid roe Invertebrate Pygmy rock crab roe lnvertebrate blots of egg proteins from Pacific cod (Gadus macroce­ Pacific lyre crab roe Invertebrate phalus), tomcod (Microgadus proximus), sablefish, Diatom Algae Pacific halibut, Pacific hake (Merluccius productus), Dinoflagellate Algae Pacific herring (Clupea harengus), coho salmon (Oncor­ Green algae Alg:a~ hynchus kisutch), and embryo proteins of rockfish (Sebastes spp.) also possessed the approximately 94 kDa similar to those seen in the Western blots (Fig. 6). band. Grunt sculpin (Rhamphocottus richardsoni) egg Western blots of walleye pollock egg proteins (Fig. 6, extract reacted with the walleye pollock egg yolk anti­ lane 10) showed peptide bands of 94 kDa, several bands bodies only in the approximately 20 kDa band (Fig. 6, between 94 and 66 kDa, 25 kDa, and 14 kDa. Western lane 8).

- 101­ Bull. Fish. Sci. Hokkaido Univ. 53(3), 2002.

LANE 175 kDa could be a monomeric form of pollock vitel­ logenin. 1 2 3 4 5 6 7 8 9 10 Female sera and maturing unhydrated oocyles both had an immunostaining band indicating a 97 kDa 94 protein. This band was much more intense in the 66 oocyte homogenate blo!. Traces of the 97 kDa protei n found in the sera suggest that it could result from processing of the 175 kDa serum peptide. The other major immunostaining proteins in the pollock oocytes were 58 and 25 kDa and th us much smaller than those found in sera. Wallace and Selman (1985) found major 25 proteins in Fundulus oocytes at 122, 103,75,45,42,26 and 20 kDa. De Vlaming et a1. (1980) found proteins at 105, 110 and 19-25 kDa originating from lipovitellin and phosvitin-derived proteins of 7.6 and 14.5 kDa. 14 Greeley et al. (1986) found that the major yolk protein of homogenates of premature marine pelagic fish eggs is Fig. 6. Western blotting of various egg. ovary and yolk­ 90-108 kDa and stated that, generally, lipovitellin is sacs of teleost species. SDS-PAGE linear gradient 10­ 100-130 kDa and phosvitin is 33-40 kDa. 20%; proteins transferred to nitrocellulose paper and Matsubara et al. (1999) provided a multiple vitel­ incubated with rabbit anti-pollock egg yolk protein amibody (I: 5,000). The secondary antibody used logenin model in barfin flounder ( Verasper moseri.), and was a I: 3,000 dilution of goat anti-rabbit alkaline two lipovitellins were identified electrophoretically and phosphatase. The reaCtion was visualized using an immunologically in postvitellogenic oocyres. Each NET/BCIP conjugate reaction. Lanes: I. Pacific cod appeared to be composed of distinct heavy chains (107 egg; 2, Pacific tomt:od egg; 3. Pacific halibut egg; 4, Pacific hake ovary; 5. sablellsh egg; 6. Sebastes sp. kDa and 94 kDa) and light chains (30 kDa and 28 kDa) embryo; 7, Pacific herring egg; 8, Grunt sculpin egg; when analyzed by SDS-PAGE. As to jJ'-component 9, coho salmon egg; 10. 24-h fertilized walleye pollock and phosvitin in the vitellogenic oocyres of barfin egg. flounder, rheir molecular weights on SDS-PAGE were estimated as 17 kDa and 38 kDa, respectively (Matsubar­ Discussion a and Sawano, 1995). Although the proteins from walleye pollock oocytes ,,·ere generally smaller than Our results showed that there was egg yolk-related these, our results are similar, with the 97 kDa protein protein in the serum of matu ring female walleye pollock probably representing a heavy chain of lipovitellin. because of its immunoreactivity with antibody devel­ Greeley et al. (1986) found that hydrated eggs of oped against pollock egg yolk proteins. As shown by mari ne pelagic fishes had a major yolk protein of 90 SDS-PAGE, followed by Western blotting, the serum kDa. Theilacker et al. (t 986) reported seven reacti ve egg-related proteins are comprised of a major monomer­ bands to antisera developed against Northern anchovy ic prorein with a molecular weight of 175 kDa and (Engraulis mordax) egg extract corresponding Lo pro­ minor proteins with 97, 76 and 66 kDa. Conversely. teins at 91.2,80.7,72.9,30.3,28.2,18.2 and 16.7 kDa. Hamazaki et a1. (1987) and Babin (1987) found that the Matsubara et al. (1999) found that one lipovitellin heavy antibody developed against purified vitellogenin reacted chain decreased in size from 94 kDa to 92 kDa, while with the smaller proteins found in egg yolk. The value another heavy chain showed degradation into free of 175 kDa is somewhat smaller than that of 200 kDa amino acids duting oocyte maturation in barfin floun­ found for rhe major serum vitellogenin in the killifish der. Li povitelJin light chains ,vere also processed into Fundulus heteroclitus (Selman and Wallace, 1983) and smaller peptides (22 kDa and 15 kDa) along with the for tbe medaka Oryzias latipes (Hamazaki et aI., 1987), oocyte maturation. Similar proreolytic c1eavege of and larger than tbat of the 140-147 kDa protein found lipovitellin associated with the final oocyte maturation, in the sera of goldfish Carassius auratus (de Vlaming et and followed by embryogenesis was observed in another aI., 1980). It is identical in weight to the protein found flounder, Pleuronectes ameri.canus (Hartling and Kun­ in the sera of Oncorhynchus mykiss, formerly Salmo kel, 1999). Carnevali et al. (1999) suggested that cathe­ gairdneri (Babin, 1987). Considering orher investiga­ psin D and L seemed to be involved in the proteolysis tions about subunit slnlCture of fish vitellogenins (Speck­ of vitellogenin and yolk proteins in red sea bream er and Sullivan, 1994), the band witb molecular mass of (Sparus aurota). In our study, we obtained similar

-102­ BAILEY et al.: Egg yolk proteins or walleye pollock resu Its with the major reactive bands in hydrated unier­ amphibians and birds by hybridization to cON A probes tilized and fertilized eggs of 94 and 25 kDa and other indicate a high degree of conservation (James et al., 1982; minor bands of 76,66 and ]4 kDa. Based on strong Nardelli et aL, 1987), similarities in antigenic determi­ immunostaining and the molecular mass, the 25 kOa nants between fish and invel1ebrates have been lost. band could be a ,B'-component or smaller molecule of Algal extracts were tested against poJlock egg-yolk Iipovitellin fragments (a light chain of lipovitellin). protein antibodies because they serve as common foods The decreased intensity of this band in late-stage em­ for invel1ebrare species tesred (Crook and Sunderland, bryos indicates that this component was being rapidly 1984), and no cross-react.ion was noted. catabolized The shifting distribution of polypeptide [mmunoblotting of egg proteins showed that matured molecular weight towards smaller polypeptides from oocytes of other NOt1h Pacific fishes exhibited simi lar hydrated eggs through late-stage embryos indicates rhar banding patteOls as hydrated walleye pollock eggs. vitellogenin-derived polypep6des are being utilized to Different developmental stages i.e., Pacific hake ovary meet anabolic and maintenance requirements of devel­ with maturing oocytes, possessed the same banding oping embryos. Our results generally support the con­ pattern as 24-hour waJJeye pollock egg. The occurrence cept rhat proteolytic cleavage of native vitellogenin of immunological cross-reactivity between walleye pol­ occurs during receptor mediated endocytosis into yolk lock egg protein and distantly related teleost families as globules of the developing oocytes (Wallace, 1985; demonstrated by Western blots reflects the highly conser­ Busson-Mabillot, 1984). Trace presence of the major 76 vative nature of teleost egg-yolk protein. kDa egg protein in sera could have resulted from proteolytic processing that results in smaller polype­ Acknowledgements ptides as a result of lysosomal activity (Perona er aI., 1988; Waiiace and Selman, 1985) and hydration We would like to thank R.A. WaJlace and P. Babin (Greeley et al., 1986). Further proteolytic activity for their helpful comments. C. Mills, M. Orellana and through the iertilized egg and yolk-sac larval stages that P. McMahon provided samples for the cross-reactivity results in a shift from large to smaller polypeptides is a studies. We also thank WW. Dickhoff for facilities function of molecular cleavage at specific stages in and suppon. This article is contribution FOCI-OI16 to development. The products of this activity yield quite the Fisheries-Oceanography Coordinated Investigations. special functions in rlle development process (Indrasith et a!., 1987), whereas others may act as energy reserves (Zhu et al, 1986). References Utter and Ridgway (1967) reported that antisera Babin, P.l (1987) Apolipoproteills and the association or developed against serum vitellins of several teleost egg yolk protein; WIth plasma high density lipo­ species cross-reacted at the family level, and in some proteins aner ovulation and rolllCular atresia in cases, the antibodies reacted outside of the family group. the ralnbow trout (Salmo gairdneri). 1. Biol Utter and Ridgway (1967) also showed thar rhe amount Chan.. 262, 4290-4296. Bailey, K. and Stehr. c.L. (1986) Laboratory sludies on of specificity between the serum vitell ins decreased as the the early lire history or the walleye pollock, families tested became more genetically distant. Theragra chalcogramma (Pallas). 1. Exp..Mar. Theilacker et al. (1986) rep0l1ed tilar antisera raised Bial Eco!., 99, 233-246. against anchovy egg proteins cross-reacted among mem­ Bcrgink, E.W. and Wallace, R.W. (1974) Precursor­ bers of the clupeidae. Covens et a1. (1987) observed product relationship between amphibian vilel­ 10genin and the yolk proteins, lipovHcllin and high levels of immunorecognition when subunits of phosvitin. 1. Biol. Chem., 249, 2897-2903. stickleback (Gasterosteus acu!eatus) and seabass Bussoll-Mabillot, S. (1984) Endosomcs transfer yolk pro­ (Dicentrarchus labrax) vitellogenin were tested against teins to lysosome., in the vilellogenic oocyte or the estradiol-induced vitellogenic plasma of fish of different trout. Biol Cell, 51, 53-66. orders. From our results and those of others (Covens et Carnevah, 0., Cadetia. R., Cambi, A., Vita, A. and Bromage, N. (1999) Yolk rormation and degra­ aI., 1987; Hara et aI., 1983; Utter and Ridgway, 1967), it dation during oocyte maturation in seabream seems that teleost. egg protei ns are conserved. There Sparus aurata: involvement of two lysosomal may be some changes in configuration, amino acid protei nase. Bio!. Reprod., 60, 140-146. composition and molecular weight of the egg proteins, Carncvali, 0., Mosconi, G., Roncaratl, A .. Belvedere, P., but the critical determinants have remained intact. Limatola, E. and Polzonetr.i-Maglll, A.M. (1993) Yolk protein changes dunng oocyte growth in Invel1ebrate eggs tested showed no specificity to anti­ European sea bass Dicenlrarchlls labrax L 1. pollock egg yolk proteins. Although studies of vitel­ Appl. Icl1lhyol.. 9, 175-184. logenin coding sequences in genomic DNA of insects, Christmann, J.L, Grayson, MJ. and Huang, R.C.C (1977)

-103­ Bull. Fish. Sci. Hokkaido Univ. 53(3), 2002.

Comparat.ive study of hen yolk phOSVItin and and oocyte assembly. pp. 347-406, Hoar, W.S. and plasma vitellogenin. Biochemistry, 16, 3250­ Randall, DJ. (eds), Fish Physiology, Vol. XIA, 3256. Academic Press, New York: Covens, /III .. Covens, L., Ollevier, F. and De Loof, A. Nardelll, D., Gerber-Huber, S., van hel Schip, r.D., Gruber, (1987) A comparative study of some properties M., Geert, A.B. and Whali, W. (1987) Verte­ of vitellogenin (Vg) and yolk proteins in a num­ brate and nemarode genes coding for yolk proteins ber of freshwater and marine teleost fishes. are derived from a common ancestor. Biochemis­ Comp. Biochem. Physiol., 88B, 75-80. Ir)), 26, 6397-6402. Crook, N.E. and Sunderland. KDA. (1984) Detection of Ng, TB. and Idler, D.R. (1983) Yolk formation and aphid remains in predatory insects and spiders by differentialion in teleost fishes. pp. 373-404, Hoar, ELISA. Ann. Appl Biol, lOS, 413-422. W.S, Randall, D.J. and Donaldson, E.M (eds), Greeley, M.S., Calder, D.R. and Wallace. R.A. (1986) Fish Physiology, Vol. IX, Academic Press. New Change.s in teleost yolk proteins during oocyte York. maturation: correlation of yolk proteolysis with Olin, T. and Von Del' Decken, A. (1990) Yolk protcms m oocyt.e hydrat.ion. Comp. Biochern. Physiol., 84B, salmon (Sa/mo salar) oocytes. eyed eggs, and 1-9. aJevins differing in viability. Can. J. Zool.. 68. Hamaz.aki, TS., luchi, 1. and Yamagami, K. (1987) 895-900. Purification and idemification of vitellogenm and Perona, R., Bes, J.e. and Vallejo, e.G. (1988) Degrada­ its immunohistochemical detection In growing tion of yolk in the brine shrimp Anemia. Bio­ oocytes of the teleost, Oryzias lalipes. J. Exp. chelTIlcal and morphologIcal studIes on r.he Zool, 242, 333-341. involvement. of the lysosomal system. Biol of Hara. A. and Hirai, H. (1978) Comparative studies on the Cell. 63, 361-366. immunochemical propertles of female-specific Plack, P.A .. Pritchard, DJ. and Fraser, N.W. (1911) Egg serum protein and egg yolk proteins in rainbow proteins in cod serum. Naturai occurrence and trout (Salmo gairdnel'i). Compo Biochem. mduction by injections of ocstradiol 3-benwate. PhysioL. 59B, 339-343. Biochem. J., 121, 847-856. Hara, A., Takano, K. and Hlfai, H. (1983) lmmuno­ Selman. K. and Wallace, R.A (1983) Oogenesis in Fun­ chemical identificmion of female-specific serum dulus heteroc!itus. Vitellogenesis. J. Exp. protein, vitellogenin. in the me{]aka, Olyzias Zool, 226,441-457. lalipes (Teleosts). Compo Biochem. Physiol., 76A, Specker, lL. and Sullrvan, e. V. (1994) Vltellogenesis in 135-141. fishes: status and perspectives. pp.304-315, Hartling, R.e. and Kunkel, J.G. (1999) Developmental Davey, K.G., Peter, R E. and Tobe, S.S. (e{]s), fate of the yolk protein lipovltcllin in embryos and Perspeclives in Comparative Endocrinology, larvae of winter flounder, Ple1.lronectes amer­ National Reserch Council. Ottawa. ieanus. 1. Exp. Zool, 284. 686-696. Thcilackcr, G.H.. Kimball, A.S. and Trimmer, 1.S. (1986) Hinckley, S. (1986) Spawning dynamics and frecundity of Use of an ELlSPOT immunoassay to det.ect eu­ walleye pollock (Theragra chalcogramma) in the phausiid predation on larval anchovy. Mar. Bering Sea. M.S. thesis, Univ. Washington, Seattlc, Ecol Prog. Ser., 30. 127-131. 104 p. Thorsen, A, Kjesbu. 0., Fyhn, H.J. and Solemdal, P. Hiramatsu, N. and Hara. A. (1996) Relat.ionship between (1996) Physiological mechanisms of byoyancy in vitellogcnin and its related egg yolk proteins In eggs from brackish war.er cod. J. Fish BioI., 48. Sakhalin taimen (Hucho penyi). Compo Bio­ 457-477. chern. Physiol., USA. 243-251. Utter, F.M. and Ridgway, G.J. (1967) Cross-reactive lndrasnh, L.S.. Furusawa, T .. Shikata, M. and Yamashita, prapenies of anlisera prepared in rabbits by stImu­ O. (1987) Limited degradation of vitellin and latious with teleost vitellins. Fish. Bull U.s., 66, egg-specific protein in Bombyx cggs during em­ 203-207 bryogenesis. Insect Biochem., 17, 539-545. de Vlaming, V.L, Wiley, H.s., Delahunty, G. and Wallace, James, T.e., Boud, Y.M., Maack, e.A, Applebaum, S.W. R.A. (1980) Goldfish (Carassius auraws) vitel­ and Tala, J.R. (1982) Evolutionary converva­ logenin: inductlon, isolation, properties and rela­ tion of vitellogenin genes. DNA, I. 345-351. tionship to yolk proteins. Compo Biochem. Mat.subara, T and Sawano. K. (1995) Proteolytic cleav­ Physiol, 67B, 613-623. age of vitellogenin and yolk proteins duriug vit.el­ Wallace, R.A. (1978) Oocyte growth in nonmammalian logenin uptake and oocyte maturation in barfiu vertebrates. pp.469-502, .Jones, R.E. (ed). The flounder (Verasper moseri). J Exp. Zool, 272, Vertebrate Ovary, Plenum Press, New York. 34-45. Wallace, R.A (1985) VitclJogenes1s and oocyte growt.h In Matsubara, T. Ohkubo, N., Andoh, T., Sullivan. e.V. and non-mammlian venebrales. pp. t27-177, Browder. Hara, A. (1999) Two forms of vitelJogenin, L. (ed), Developmental Biology, Vol. 1,. Pergamon yielding [\>'0 distinct lipovirellins, play differeut Press, New York. roles during oocyte maturatiou and early develop­ Wallace, R.A. and Begovac, P.e. (1985) Phosvitins in ment of badin flounder, Verasper moseri, a Fundulus oocytes a nd eggs. Prelimlllary mariue teleost that spawns pelagic eggs. Dev. chromatographIC and electrophoretic analyses Bioi., 213, 18-32. together wnh biological cousiderations. 1. BioI. Mommsen, T.P. and Walsh, P.L. (1988) VItellogenesis Chern., 260, 11268-11274.

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Wallace. R A and Selman, K (1985) Major prorein C-terminal cysteine-rich domain oC"llellogeninlT. changes dUring "itellogencSlS and mdturaLion of Biochim. Biophys. Ana, 1244. 384-394. Fundulus oocyte;. De vel. Bioi., 110. 492-498. Zhll. 1., lndra<'lth. L.S. Jnd Yamashita. O. (1986) Charac­ Yamamura. J, l.. Adachi, T., Aok i. N" Nakajima. 1-1., lerizatlon of vltellm. egg-speufic proLeln and 30 Nakamur'l. R. and MaLsuda. T (1995) kDa proLem trom Bomb)'x eggs, and theil' f

- 105­