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