259 in Atlantic salmon (Salmo salar L.) occurs by zonagenesis preceding vitellogenesis in vivo and in vitro

T Celius and B T Walther Department of Molecular Biology, University of Bergen, HIB, N-5020 Bergen, Norway (Requests for offprints should be addressed to T Celius)

Abstract

Fish oogenesis represents pleiotropic cytodifferentiative injected with high E2 concentrations (0·4 mg or programs including hepatic synthesis of the molecular 1·1 mg/kg bw). After E2 treatment, hepatocytes isolated components for both the eggshell and the oocytic energy from male fish synthesized zr-proteins and vitellogenin deposits. Both hepatic processes are directly controlled by in vitro. However, zr-proteins were secreted into the plasma levels of estradiol (E2), and injected E2 induces both medium two days before vitellogenin, as measured by biogenetic processes in prepubertal fish of both sexes. This ELISA. The data indicate a preferential induction of work compares the temporal pattern of E2-induced bio- zr-proteins compared with vitellogenin, both with regard synthesis of zona radiata proteins (zr-proteins) and vitello- to E2 sensitivity and response time to E2 treatment. These genin in Atlantic salmon (Salmo salar L.) in vivo and in vitro. findings suggest an obligate sequence in salmon oogenesis. We monitored the presence of plasma zr-proteins and During sexual maturation low E2 levels at first induce only vitellogenin, using homologous polyclonal antiserum to zonagenesis, while increasing levels of E2 subsequently zr-proteins and a monoclonal antibody to vitellogenin. induce both zonagenesis and vitellogenesis. In nature, the Zr-proteins were induced by all E2 concentrations interval between zonagenesis and vitellogenesis may, (0·001–1·1 mg/kg body weight (bw)) within one week of therefore, be considerable. The data suggest new control exposure while vitellogenin was not induced until two mechanisms in fish oogenesis. weeks post-injection and then only in plasma from fish Journal of Endocrinology (1998) 158, 259–266

Introduction genin in teleosts is interrelated because both processes are controlled by estradiol-17â (E2) (Wallace 1985, Hyllner Sexual maturation in female fish has been extensively et al. 1991, Murata et al. 1991, Oppen-Berntsen et al. reviewed (Guraya 1986). The rapid growth of the oocyte 1992a,b, 1994). E2 synthesis is, in turn, regulated by prior to in most oviparous vertebrates is mainly gonadotropins (GtHs) secreted by the pituitary (Xiong et due to the incorporation of the protein precursor al. 1994). Pituitary secretion is triggered by seasonal signals vitellogenin (Wallace 1985). Vitellogenin is synthesized by such as light and temperature, and complex neuro-sensory the liver (Chen 1983, Bidwell & Carlson 1995) and pathways ending in the hypothalamus, which transmit transported to the where it is sequestered to serve as environmental cues to initiate pituitary secretion an energy reserve for the developing embryo. Another (Sherwood et al. 1994). feature of oocyte development is the formation of an In Atlantic salmon (Salmo salar L.) zona radiata is extracellular eggshell, or vitelline envelope (Dumont & composed of three protein monomers named á, â and ã Brummet 1985). Zona radiata constitutes the major part of zr-proteins (Oppen-Berntsen et al. 1994). Zr-proteins are the fish eggshell, with the outer zona pellucida as the minor synthesized in extra-ovarian tissues in response to E2 part (Lønning 1972, Oppen-Berntsen et al. 1992b). During in both rainbow trout (Hyllner et al. 1991) and cod the eggshell provides protection (Oppen-Berntsen et al. 1992b). Oppen-Berntsen et al. for the embryo. It is shed when hatching is induced by (1992a) reported that hepatocytes from E2-treated fish developmental and environmental signals (DiMichele & synthesized zr-proteins when further treated with E2 Powers 1984, Yamagami 1988, Helvik & Walther 1992). in vitro. Olsen & Walther (1993) reported that plasma The teleostean eggshell consists of two to four protein levels of zr-proteins correlated with activity of pituitary monomers (zona radiata (zr)-proteins) (Begovac & Wallace gonadotropic cells and pituitary contents of GtH I. Salmon 1989, Oppen-Berntsen et al. 1990, Brivio et al. 1991, plasma levels of zr-proteins are broadly correlated to Hyllner et al. 1991, Iuchi et al. 1991, Masuda et al. 1991, plasma levels of both E2 and GtH I (but not GtH II) during Hyllner et al. 1994). Synthesis of zr-proteins and vitello- sexual maturation (Oppen-Berntsen et al. 1994).

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Sumpter et al. (1984) reported that significant increases (St Louis, MO, USA). Other chemicals for Western in plasma levels of GtH, vitellogenin, E2, and testosterone blotting and ELISA analysis were from Bio-Rad occurred at least one year before ovulation in trout. Laboratories (Hercules, CA, USA). Diethyl ether and 3 Formation of yolk vesicles in oocytes (termed endogenous heptane were from Merck (Darmstadt, Germany). [ H]E2, vitellogenesis) began a full year before ovulation, while for determination of recovery in E2 extraction, was ovarian uptake of liver-derived vitellogenin (exogenous obtained from Amersham International (Amersham, vitellogenesis) started three months later, or about ten Bucks, UK), and soybean oil was from Idun (Oslo, months before ovulation. Hyllner & Haux (1992) reported Norway). Monoclonal antibody against vitellogenin that the amount of vitelline envelope proteins in the (KB-1) was from Biosense (Bergen, Norway). plasma of maturing rainbow trout varied during the year, AutoDELFIA Estradiol kit was from Wallac Oy in a manner that was similar to vitellogenin. In Atlantic (Gaithersburg, MD, USA). Dulbecco’s modified Eagle’s salmon, Lindhom (1997) observed parallel changes in medium (DMEM), fetal bovine serum (FBS), glutamine plasma levels of zr-proteins and vitellogenin during a and antibiotic/antimycotic solution (AAS) were ob- 21-month period before ovulation. Both data sets were tained from GIBCO BRL (Life Technologies Ltd, interpreted in terms of a similar hormonal regulation of Paisley, Renfrewshire, UK). Secondary antibodies for liver synthesis of vitelline envelope proteins and vitello- Western blotting and ELISA analysis were purchased genin. However, Hyllner et al. (1994) later reported that from Bio-Rad Laboratories. Primaria plates for cell zr-proteins were detected in plasma nearly a year before cultures were from Falcon, Becton Dickinson Labware, vitellogenin, and that the initial formation of the eggshell Oxnard, CA, USA. Other chemicals were of the highest started before the active uptake of vitellogenin. They also commercial purity. stressed that the low pre-vitellogenic plasma levels of E2 in females are of physiological significance. These obser- vations in rainbow trout appear to differ from the findings and treatments of Lindhom (1997) in Atlantic salmon, where monthly Juvenile Atlantic salmon were obtained from Salar Arlvik sampling of plasma did not distinguish the chronology of (Arlvik, Norway), belonging to the Mowi salmon strain appearance of zr-proteins and vitellogenin during sexual (Bergen, Norway). A total of 36 individuals (an even mix maturation. The reason for this seeming discrepancy is not of both sexes and weighing 89&12 g) was used for the known. experiment. Fish were kept in continuously running sea Selective uptake of vitellogenin by trout oocytes is water at a constant temperature of 8·5 )C(&0·5 )C) at the thought to occur by receptor-mediated endocytosis in a Industrial Laboratory (ILAB), HIB, Bergen. The exper- process involving specific cell surface receptors (Tyler et al. iment was performed in January; six groups of six fish each 1988, 1990). Tyler et al. (1991) reported that in rainbow were injected once with a single intraperitoneal dose of trout, receptor-mediated uptake of vitellogenin is devel- various amounts of E2 (0·001, 0·01, 0·1, 0·4, and 1·1 opmentally regulated and that oocytes must reach a certain mg/kg body weight (bw)) and subsequently maintained in size before they are able to sequester vitellogenin. How- separate tanks for two weeks. E2 was dispersed in soybean ever, Lancaster & Tyler (1994) demonstrated that vitello- oil and sonicated for 10 min (50% duty cycle and an output genin receptors are expressed at the oocyte surface in of 60 W) with an Ultrasonic Homogenizer (Cole Palmer follicles as small as 0·3 mm, although they were not shown Instrument Co, Chicago, IL, USA) just prior to injection. to be active. Any presence of vitellogenin in the oocyte The experiment also contained a control group of fish would, therefore, seem to suggest both its synthesis by the injected with soybean oil only. The sex of all fish was liver and its uptake by the oocyte. determined at the end of the experiment. In order to resolve the apparent discrepancies in the literature, we investigated the E2 induction of salmon zr-proteins and vitellogenin, in vivo and in vitro. Specifi- Collagenase perfusion of liver cally, we compared the inducibility of biosynthesis of Hepatocytes were isolated from prepubertal Atlantic zr-proteins and vitellogenin, both in terms of E2 concen- salmon (male, ]300 g) by a two-step perfusion method of tration, and in terms of the lag time needed for E2 to Berry and Friend (1969) as modified for fish by Andersson induce biosynthesis of zr-proteins and of vitellogenin. et al. (1983). The liver was perfused using a peristaltic pump at a flow rate of 22 ml/min. First, the liver was perfused in situ for 10 min with a -free solution Materials and Methods containing: NaCl (7·14 g/l); KCl (0·36 g/l); MgSO4 (0·15 g/l); Na HPO (1·6 g/l); NaH PO (0·4 g/l); Chemicals 2 4 2 4 NaHCO3 (0·31 g/l) and EGTA (20 mg/l) at ambient Estradiol-17â (E2), 4-chloro-1-naphthol tablets, O- temperature, until all blood was washed out. The phenylenediamine dihydrochloride, EGTA, HEPES, whitened liver was then perfused in situ for about 10 min BSA, and collagenase were purchased from Sigma in the same buffer but with calcium (0·22 g/l) instead of

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EGTA, and collagenase (80 mg/150 ml). All glassware and analysis, and a polyclonal antiserum that recognized all instruments were autoclaved before use. Solutions were three zr-proteins was raised in rabbit. The specificity of the sterilized by filtration using a 0·22 µm millipore filter polyclonal antiserum was checked by Western blotting, (Millipore AS, Oslo, Norway). and no cross-reactivity with purified vitellogenin or other plasma proteins was seen (Oppen-Berntsen et al. 1992a, Lindhom 1997). Preparation and culture of hepatocytes After perfusion the liver was dispersed in a plate together SDS-PAGE and Western blotting with calcium-containing buffer supplemented with BSA (0·1 g/100 ml). The suspension was filtered through a Proteins were separated by SDS-PAGE as described by 150 µm nylon monofilament filter and centrifuged at 50 g Laemmli (1970) using 4% stacking and 9% separation gels. for 2 min. Cells were washed with medium three times, Proteins were electrophoretically transferred onto a nitro- and finally resuspended in medium. Viability of the cellulose membrane at 100 V, 400 mA for 1 h as described hepatocytes used for experiments was always over 90%, as by Towbin et al. (1979). Salmon zr-proteins were probed determined by Trypan blue exclusion. After measuring with homologous antiserum (1:2000) prepared in rabbit, cell yield, cells were plated on 24-well multiwell Primaria and salmon vitellogenin was probed with monoclonal plates (]0·5#106 cells per well) in DMEM medium antibody (1:500). Incubation with secondary antibodies (without phenol red) containing 0·5% (v/v) FBS, and was performed with goat anti-rabbit IgG horseradish 15 mmol/l HEPES, glutamine (0·3 g/l medium) and AAS peroxidase conjugate (1:3000) and goat anti-mouse IgG (1000 units/l penicillin, 1 µg/ml streptomycin, and horseradish peroxidase (1:3000). Blots were developed 0·75 µg/ml amphotericin). Cells were kept at a constant with 4-chloro-1-naphthol (30 mg) dissolved in 10 ml temperature of 10·0 )C(&0·5 )C) in ambient atmos- methanol, and mixed with 50 ml TBS containing 0·03% phere. After plating, cells were cultured for two days in H2O2. DMEM medium. Then the medium was removed and replaced by new medium containing different concen- ELISA analysis trations of E2 (0·1, 1·0, 10·0 nmol/l), changed daily for five days. Cells were plated and treated in triplicate. Collected Enzyme-linked immunosorbent assay (ELISA) was based culture media were stored at "20 )C until analyzed. on the method of Oppen-Berntsen et al. (1994), except that non-specific protein binding to the wells was blocked using 2% BSA (200 µl). The wells were incubated with Collection of plasma and extraction of E 2 100 µl polyclonal antiserum against zr-proteins, diluted Blood was collected from the caudal vein by heparinized 1:1000 in 1% BSA, or 100 µl monoclonal antibody against syringes, both from treated and control fish. Blood was vitellogenin at a concentration of 1:1000 in 1% BSA. centrifuged at 5000 r.p.m. for 5 min, and the collected Secondary antibodies were goat anti-rabbit IgG horse- plasma was stored at "45 )C. E2 was extracted using a radish peroxidase conjungate (1:3000) and goat anti-mouse modified method of Methven et al. (1992) with an average IgG horseradish peroxidase (1:3000). Absorbance was recovery of 84%. E2 was extracted from aliquots of plasma measured at 492 nm. (150–200 µl) by adding 4 ml of a mixture of diethyl ether:heptane (4:1), followed by vigorous vortex mixing for 1 min. Test tubes containing the mixture were placed Results in liquid nitrogen until the bottom water phase was frozen. The top organic phase was decanted to a new test tube and Figure 1A documents the induction of zr-proteins at all E2 the extraction repeated. The resulting diethyl ether: concentrations. At 0·001 mg/kg only the â-monomer of heptane mixtures, containing the extracted E2, were the zr-proteins was induced. This monomer was also seen evaporated to dryness on a heater (45 )C) under nitrogen in plasma from control fish, but to a much lesser extent. In gas flow. The dry sample was dissolved in PBS buffer contrast with the induction of zr-proteins, vitellogenin was (containing 0·50 mol/l NaH2PO4, 0·10 mol/l Na2HPO4, not detected at any E2 concentrations after one week of 1·01 mol/l NaCl, 2% BSA, pH 7·0) and frozen at "20 )C exposure (data not shown). At the end of the experiment until analysis. Concentrations of E2 were measured by (14 days), fish were killed and plasma collected. Plasma AutoDELFIA Estradiol kit. samples were analyzed by Western botting and ELISA analysis. Figure 1B presents a Western blot showing that the three monomers of zr-proteins are induced at all Antiserum preparation and specificity concentrations of E2 given. Western blot analysis probed Antiserum to zr-proteins from Atlantic salmon was pre- with monoclonal antibody to vitellogenin (Fig. 2) reveals pared by Rong (1992) as follows: zr-proteins collected induction of vitellogenin, but only at the two highest doses from plasma were purified by preparative SDS-PAGE of E2 (1·1 and 0·4 mg/kg bw).

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Figure 1 Western blots of plasma from Atlantic salmon injected with various concentrations of E2. Blot A: plasma sampled after one week of exposure. Lane 1, 1·1 mg/kg bw; lane 2, 0·4 mg/kg bw; lane 3, 0·1 mg/kg bw; lane 4, 0·01 mg/kg bw; lane 5, 0·001 mg/kg bw; lane 6, control. Blot B: plasma sampled after two weeks of exposure. Lane 7, 1·1 mg/kg bw; lane 8, 0·4 mg/kg bw; lane 9, 0·1 mg/kg bw; lane 10, 0·01 mg/kg bw; lane 11, 0·001 mg/kg bw; lane 12, control. Plasma was diluted 1:100 and 10 ìlwas applied to each lane. Western blots were probed with polyclonal antiserum to salmon zr-proteins (á, â, ã). Each lane shows representative plasma from one fish from an experimental group consisting of six fish, all of which were analyzed with the same results. The sexes of all fishes were characterized after the experiment, and each group contained at least two individuals of male or female, except for the control group which contained females only. Results are therefore representative for both sexes. F=female, M=male.

Figure 2 Western blot of plasma contents of vitellogenin from Atlantic salmon injected with various concentrations of E2, analyzed after two weeks of exposure. Lane 1, 1·1 mg/kg bw; lane Figure 3 ELISA analysis of zr-proteins from Atlantic salmon plasma 2, 0·4 mg/kg bw; lane 3, 0·1 mg/kg bw; lane 4, 0·01 mg/kg bw; after two weeks of exposure to various concentrations (mg/kg bw) lane 5, 0·001 mg/kg bw; lane 6, control (soybean oil only). of E2. Control, fish treated with soybean oil. Absorbance was Western blot was probed with monoclonal antibody to salmon measured at 492 nm using a polyclonal antiserum to zr-proteins. vitellogenin (vtg). Each lane shows representative plasma (dilution Each experimental group consisted of six fish. The sexes of all 1:100 with 10 ìl applied to each lane) from one fish from an fishes were characterized after the experiment, and all groups experimental group consisting of six fish, all of which were contained at least two individuals of male or female, except for analyzed. The sexes of all fishes were characterized after the the control group which contained females only. Results experiment, and all groups contained at least two individuals of (means&S.E.M.) are therefore representative for both sexes. To male or female, except for the control group which consisted of calculate P values, Microsoft Excel 5·0 was used. *P<0·01, females only. Results are therefore representative for both sexes. **P<0·001 compared with control (t-test). F=female, M=male.

Plasma was also investigated by ELISA analysis. both proteins. Plasma sampled one week after injection Figures 3 and 4 present ELISA measurements of zr- was also analyzed by ELISA, yielding the same result proteins and vitellogenin respectively, after two weeks of except that the plasma contents of zr-proteins and exposure. Results show a dose-dependent induction of vitellogenin were much lower (data not shown).

Journal of Endocrinology (1998) 158, 259–266

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Figure 4 ELISA analysis of vitellogenin from Atlantic salmon plasma after two weeks of exposure to increasing concentrations (mg/kg bw) of E2. Control, fish treated with soybean oil. Absorbance was measured at 492 nm using a monoclonal antiserum to vitellogenin. Each experimental group consisted of six Figure 5 Plasma content (ng/ml) of E in salmon treated with fish. The sexes of all fishes were characterized after the 2 various concentrations (mg/kg bw) of E2. Control, fish treated with experiment, and each group contained at least two individuals of soybean oil. Each experimental group consisted of plasma male or female, except for the control group which contained extracted from five fish. The sexes of all fishes were characterized females only. Results (means&S.E.M.) are therefore representative after the experiment, and each group contained at least two for both sexes. To calculate P values, Microsoft Excel 5·0 was individuals of male or female, except for the control group which used. *P<0·01 compared with control (t-test). contained females only. Results (means&S.E.M.) are therefore representative for both sexes.

Measurements of plasma concentrations of E2 show that the levels of plasma E2 at the end of the experiment (Lindhom 1997) depending on how rapid the plasma E2 reflected the amounts injected and the consequent induc- concentration increases during sexual maturation. tion of zr-proteins and vitellogenin (Fig. 5). In vitro studies Data on zonagenesis compared with vitellogenesis are demonstrated that both zr-proteins and vitellogenin at present limited. In our studies primary cultures of were synthesized and secreted during E2 treatment of hepatocytes from male salmon showed differential induc- primary hepatocyte cultures from male Atlantic salmon ibilities for zr-proteins and for vitellogenin. Secretion of (Fig. 6A,B). ELISA analysis reveals induction of zr- both zr-proteins and vitellogenin was observed in culture proteins after 24-h treatment of cells, while vitellogenin media after E2 treatment. Zr-proteins were more respon- was detected in the medium after 72 h of cell exposure. sive than vitellogenin to induction by E2, both in terms of However, we were not able to detect vitellogenin in these E2 concentration and in terms of the lag time before samples by Western blotting. zr-proteins were detected in the culture medium. Zr- proteins were present in the medium within 24 h of E2 treatment, while no vitellogenin was detected at this time Discussion using ELISA (Fig. 6B). Only after induction for 72 h was vitellogenin detected. Secretion of zr-proteins and vitello- The present study reports different inducibilities of zr- genin into culture media may mimic secretion of the proteins and vitellogenin in Atlantic salmon. This finding proteins into the blood stream in vivo. was observed both by in vivo and in vitro experiments. E2 The sequential appearance of zr-proteins and vitello- induced zr-proteins more rapidly than vitellogenin. Also, genin was also observed in vivo. We detected zr-proteins in at lower levels of E2 only zr-proteins were induced, and plasma from juvenile salmon for all E2 concentrations not vitellogenin. Similar results were also obtained when given, within one week of exposure. Vitellogenin was primary hepatocyte cultures from male salmon were detected only in plasma from fish injected with the two treated with increasing concentrations of E2. This work highest amounts of E2, and then only those sampled two suggests an ordered sequence of initiation of zonagenesis weeks after treatment. The lack of detectable vitellogenin before initiation of vitellogenesis during salmon sexual in our studies could possibly be due to insufficient maturation. However, the pattern of in vivo accumulation sensitivity of the methods used. The absolute sensitivity of of zr-proteins and vitellogenin in various fish species our ELISA assay was not determined. Sensitive radio- may be distinct (Hyllner et al. 1994) or concerted immunoassays have permitted measurements of very low

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Figure 6 ELISA analysis of zr-proteins and vitellogenin in medium from hepatocytes treated with increasing concentrations of E2. Doses of E2 given were 0·1, 1·0 and 10·0 nmol/l, and medium was changed every day for five days. Induction of zr-proteins (A) and induction of vitellogenin (B) are shown, both in terms of E2 concentration, and in terms of time of exposure.

levels of vitellogenin. Examining the earliest stages of multiple EREs than is necessary for activating the zr- vitellogenesis with this technique, Copeland et al. (1986) protein genes. One of the alternative explanations is that concluded that the sensitivity of the assay employed to two different estrogen receptors are responsible for acti- measure vitellogenin determines the conclusion reached vation of such genes. However, in pilot studies we tested about its absence or presence in plasma. the induction of zr-proteins and vitellogenin with E2 in The basis for a time- and concentration-dependent concentrations from 1–10 mg/kg bw, and found that both mechanism of E2 induction of different oogenetic par- were equally induced after two weeks exposure, as ameters is not known. Sathya et al. (1997) reported that measured by Western blot and ELISA. many estrogen-responsive genes possess multiple estrogen- Previous work on the teleostean eggshells has revealed responsive elements (EREs) that act synergistically to that formation of the eggshell starts from the outside. It is activate gene expression. They demonstrated that the most peculiar that fish eggshells are composed of a thin synergism among EREs depends on the number of EREs, outer layer (zona pellucida) and a thicker inner part, the their spacing, and the distance of the EREs from the zona radiata (Guraya 1986). Ovarian synthesis of zona promoter. The differential induction of zr-proteins and pellucida occurs before the formation of the zona radiata vitellogenin seen in our study may potentially be explained takes place by deposition of bloodborne hepatic compo- by the vitellogenin gene requiring more E2 to activate nents on the oocytic aspect of the zona pellucida (Wourms

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1976, Dumont & Brummet 1985, Guraya 1986, Copeland PA, Sumpter JP, Walker TK & Croft M 1986 Vitellogen Matsuyama et al. 1991). It remains a mystery how the zona levels in male and female rainbow trout (Salmo gairdneri Richardson) at various stages of the reproductive cycle. Comparative Biochemical radiata becomes specifically deposited next to the oocytes, Physiology 83B 487–493. what causes soluble plasma proteins to become insoluble DiMicheleL&PowersDA1984Therelationship between oxygen macromolecular aggregates, and why zonagenesis should consumption rate and hatching in Fundulus heteroclitus. Physiology occur before vitellogenesis. and Zoology 57 46–51. Based on immunological methods this work suggests an Dumont JN & Brummet AR 1985 envelopes in vertebrates. In Developmental Biology: A Comprehensive Synthesis, vol 1, pp 235–288. ordered sequence of initiation of zonagenesis and of Ed RW Browder. New York: Plenum Press. vitellogenesis during teleostean sexual maturation. Since Guraya S.S 1986 The cell and molecular biology of fish oogenesis. In synthesis and deposition of zonagenetic and vitellogenetic Monographs in Developmental Biology, vol 18, pp 1–262. Ed HW components take place in different cells, in vitro obser- Sauer. Basel: Karger. vations on zr-protein synthesis do not readily translate into Helvik JV & Walther BT 1992 Photo-regulation of the hatching process of halibut (Hippoglossus hippoglossus) . Journal of the plasma zr-protein contents observed in vivo.Inour Experimental Zoology 263 204–209. experiments there is, nevertheless, a general correspon- Hyllner SJ & Haux C 1992 Immunochemical detection of the major dence between in vivo and in vitro observations. This would vitelline envelope proteins in the plasma and oocytes of the female seem to strengthen the interpretation that the differential rainbow trout, Oncorhynchus mykiss. Journal of Endocrinology 135 and sequential induction of zonagenesis and vitellogenesis 303–309. 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Journal of Endocrinology (1998) 158, 259–266

Downloaded from Bioscientifica.com at 09/25/2021 02:59:45AM via free access