Comp. Biochem. Physiol. Vol. 93B, No. I, pp. 57-60, 1989 0305-0491/89 $3.00 + 0.00 Printed in Great Britain © 1989 Pergamon Press plc

EXPERIMENTAL INDUCTION OF VITELLOGENIN SYNTHESIS IN EEL (ANGUILLA ANGUILLA) ADAPTED

TO SEA-WATER OR FRESHWATER

INGRID PETERSEN* and BODIL KORSGAARDt

*Institute of Biochemistry and tlnstitute of Biology, Odense University, Campusvej 55, DK 5230, Odense M, Denmark (Tel: 09 15 8600)

(Received 20 July 1988)

Abstract--l. One week after treatment with a single dose of estradiol, the vitellogenin (Vg) concentration was significantly higher in the freshwater group than in the sea-water group.

2. DNA concentration in the liver was increased in the freshwater group compared to the control, while there was no effect on the sea-water group.

3. RNA concentration in the liver was significantly increased in both groups compared to the controls. 4. Treatment with estradiol did not affect the osmotic concentration when compared to the control

group, but a significant decrease is seen in the freshwater control group compared to the sea-water control group.

5. Treatment with estradiol decreased the concentration of chloride in plasma in both sea-water and freshwater groups compared to the controls.

6. Gel filtration of plasma from estradiol-treated animals shows that the elution profile contains a peak, which coincides with a Vg peak.

7. One week after treatment with estradiol the concentration of estradiol in plasma in the treated sea-water group was twice as high as in the treated freshwater group.

INTRODUCTION

In sexually mature oviparous vertebrates a precursor protein for egg-yolk, vitellogenin (Vg), is synthesized in the liver. The process is thought to be primarily regulated by ovarian estrogen as high titers of estro- gen are detected in circulation either before or during the onset of vitellogenesis (Idler and Campbell, 1980; van Bohemen and Lambert, 1981; Sundararaj et al., 1982; Ueda et al., 1984). The hepatocytes synthesize a polypeptide, which after various post-translational modifications is secreted into the blood and transported to the developing oocytes, where it is taken up in the form of the yolk proteins phosvitin and lipovitellin (Jared and Wallace, 1968). The syn- thesis of Vg can be induced by exogenous estradiol and this induction of female-specific protein synthesis does not only take place in females but also in male and immature animals.

Vitellogenesis has been studied in several teleosts and elasmobranchs, particularly in trout and salmon (Crim and Idler, 1978; Hara and Hirai, 1978; van Bohemen et al., 1981; Ueda et al., 1984). A few papers concerning vitellogenesis in eel have been published (Yamamoto et al., 1974; Olivereau and Olivereau, 1979; Hara et al., 1980). Vitellogenesis in eels in nature is presumed to take place during the marine migration towards the Sargasso sea. It has been shown that it is possible by treatment with gonado- tropin to induce female eels to a striptive condition in the laboratory (Boetius and Boetius, 1980). Con- sidering the sparse knowledge of the reproductive processes of the eel we decided, as a first approach, to study the hormonal induction of Vg synthesis. The present paper describes changes in biochemical

components related to the synthesis of Vg induced by a single injection of estradiol-17fl in eels adapted to sea-water and freshwater, respectively.

57

MATERIALS AND METHODS

Fish

Immature silver eels [Anguilla anguilla (L)] weighing between 125 and 350 g were a gift from Bogense eel farm. The eels were kept in fresh water at the farm. One group (20 fish) was kept in a temperature-regulated (21-23°C) aquarium with circulated and aerated sea-water (salinity 20%0), and one group (20 fish) was kept at the same tempera- ture but in circulated and aerated freshwater. The eels were not fed during the experimental period.

After a week of acclimation the eels received 1 mg estradiol-17-fl in 0.1 ml peanut oil/250 g fish. The eels were killed a week after the injection. Before injection and killing the eels were anaesthetized for about 1 rain in a 2%0 solution of phenoxyethanol. Blood samples were taken from the caudal vein into heparinized vials and the plasma obtained was frozen at -20°C until use. Livers were immediately removed and frozen in liquid nitrogen. The livers were then kept in a -80°C freezer until use.

Quantitative determination of Vg

The amount of Vg in plasma and in the fractions from gel filtration was determined by the amount of alkali-labile phosphorus as described by Emmersen and Petersen (1976), and is expressed as/~g orthophosphate/ml plasma or eluate. Plasma Vg was also quantitated by measuring the concen- tration of Ca 2+ by atomic absorption spectrophotometry, and is expressed as mg/1.

Estradiol in plasma

Estradiol concentration of plasma was determined by direct radioimmunoassay using a kit from Biogenesis Ltd,

58 INGRID PETERSEN and BODIL KORSGAARD

Bournemouth. Sensitivity of the method is approximately 10 pg.

Gel filtration

Gel filtration was performed as described by Emmersen and Petersen (1976).

Protein #1 liter

This was determined as described by Nicholls et al. (1976).

DNA and RNA in liter

The sampling and determination of total liver DNA and RNA was carried out as described by Emmersen and Emmersen (1976).

Osmolarity

The osmotarity of plasma was determined on a Knauer automatic osmometer using the freezing point depression method, and is expressed as mosmol/kg plasma.

Chloride

Chloride content of plasma was determined by titration on a radiometer chloride automatic titrator, and is expressed as meq/ml plasma.

Statistics

Results were expressed as arithmetic means with standard errors of the mean (SEM). Statistical significance of differences between means was estimated by Student's t-test.

RESULTS

Table 1 shows that treatment with estradiol in- creases the level of alkali-labile phosphorus in both treated groups, with the most marked increase in the freshwater group compared to the group in sea-water. When Ca 2+ is used as an indicator for Vg a marked increase is seen in both treated groups, but in this case the most marked increase is seen in the sea-water group. Estradiol has a small effect on the hepato- somatic index (HSI) as the HSI of the treated fresh- water group is increased while there is no effect on the sea-water group. The R N A content of the liver is significantly increased in both estradiol-treated groups compared to the respective control groups. D N A in the liver is unaffected in the sea-water group, but slightly increased in the freshwater group com- pared to the controls. The protein concentration of the liver is increased significantly in the treated sea-water group, while no change is seen in the freshwater group.

Figure 1 shows the elution profiles of (A) plasma from a control group and (B) plasma from a treated group. The elution profile shows that the plasma can

. . . . . . . . . ] 0 A

50 L/#~ A ! 4 \ / / ~ 2

oooooo ooooooo I 0

T % ~ • ~ ' ,ug/ml

,\

10 20 30 40

F r o c t i o n n u m b e r

Fig. 1. Gel filtration performed at 4 C on a 1.5 x 90cm column containing Ultrogel ACA34. The eluant used was 0.1 M Tris, 0.1M KC1 pH 7.0. The flow rate was 8 ml/hr and fractions were collected every 30 min. The transmittance of the effluent is indicated by a solid line, and fractions analysed for alkali-labile protein phosphorus by O . . . . . ©. (A) 0.5ml plasma from an immature control animal (B) 0.5ml plasma from an animal treated with I mg

estradiol/250 g.

be separated into six main fractions of which four are corresponding. The protein fraction in the treated animal with a peak in fraction number 19 is due to Vg as the protein peak and the peak of alkali-labile protein phosphorous coincide. No alkali-labile protein phosphorus could be detected in the first very large fraction. Prolonged centrifugation before gel filtration, in order to remove aggregated material, did not decrease this peak. The prolonged centrifugation did, however, result in some floating material, sug- gesting the presence of lipid, which was removed before the plasma was gel filtrated. No attempt was made to identify the u.v.-absorbing material in the first major peak.

Table 2 shows that there is no effect of treatment with estradiol on the osmolarity when compared to the respective controls in the freshwater as well as in sea-water. When however the sea-water and freshwater groups are compared the osmolarity of the latter is significantly lower. The chloride

Table I. Changes in the hepatosomatic index HSI, DNA, RNA and protein concentration in liver and vitellogenin and Ca: ' in plasma of eel in sea-water and freshwater treated with estradiol-17-fl*

HSI DNA RNA Vitellogenin Ca -~ * Protein N (g × 100/g B.W.) (mg/g liver) (mg/g liver) (#g P/ml) (rag:l) (mg/g liver)

Sea-water control (8) 0.945 + 0.068 5.37 + 0.71 E-treated group (10) 1.059 ± 0.045 7.56 -+ 0.85

Freshwater control (10) 0.853 -+ 0.045 4.14 _+ 0.49 E-treated group (10) 0.942 4- 0.036~ 6.97 _+ 0.72:~

Data expressed as mean ± SEM. N number of individuals. *1 mg estradiol 17-,6 injected once during a period of 1 wk. tP < 0.02 compared to the control. :~P < 0.01 compared to the control. §P < 0.001 compared to the control.

5.90+0.19 0.001 4-0.001 134.3_+ 1.8 171.2+4.4 8.92_+0.42§ 15.4+_4.2" 485.6_+50.5§ 212.7+6.8§

5.02_+0.25 0.50+0.15 150.7+50.0 164.4_+.3.1 8.26-+0.63§ 28.9_+7.9§ 376.1+56.2§ 162.0±16.4

Vitellogenin synthesis in eels 59

Table 2. Changes in osmolarity and CI- concentration in eels treated with estradiol-17-/~ in sea-water and freshwater

Osmol. CI- N (mosmol/kg) (meq/ml)

Sea-water control (8) 332 + 7.9 135 + 3.0 E-treated group (10) 331 + 3.6 125.7 +_ 1.4"

Freshwater control (10) 290 __+ 7.3 I 11.6 + 1.1 E-treated group (10) 289.4 + 3.9t 101.2 + 1.7t~

Data expressed as mean + SEM. N = number of individuals. *P < 0.02 compared to the control. t P < 0.001 compared to the control. :~P < 0.001 compared to the E-treated group in sea-water.

concentration in plasma is significantly decreased in both treated groups compared to the controls, and here also there is a significant difference in the level between the sea-water and freshwater group. Table 3 shows that the concentration of estradiol measured in the sea-water group is twice as high as in the fresh- water group. The concentration in the sea-water and freshwater control groups, respectively, is very low compared to the treated groups.

DISCUSSION

In the present paper it has been shown that treatment with a single dose of 1 mg estradiol/250 g animal induces the synthesis of Vg in eels adapted to sea-water and freshwater, respectively. In the Japanese eel Anguilla japonica the appearance of Vg was demonstrated after injection with an extract prepared from salmon pituitary (Hara et al., 1980). The response is significantly higher in the freshwater group compared to the sea-water group when Vg is quantitated as alkali-labile phosphorus. When Ca 2÷ is used as an indicator of Vg, the response in the treated group is less pronounced. Alkali-labile phos- phorus thus is a much more sensitive indicator for Vg than Ca 2+, which is in accordance with observations on Carrasius carrasius (Tinsley, 1985). The difference in response in Ca 2+ compared to the response in alkali-labile phosphorus could be due to osmo- regulatory processes although changes in Ca 2+ in serum in the eel Anguilla japonica were shown to be of a transient nature, reaching a peak value on the 4th day and declining to the control value after 7 days (Chan et al., 1978). In the European eel kept in freshwater, plasma calcium levels were considerably increased by estradiol injection over a period of 15-78days (Olivereau and Olivereau, 1978). The observation in the present paper, that the concen- tration of CI- was reduced in the estradiol-treated freshwater and sea-water groups compared to controls, is in accordance with the observation by

Table 3. Concentration of estradiol in plasma

Estradiol (mg/ml) N

Sea-water control 0.65 + 0.22 (5) E-treated group 167.5 _+ 21.2 (10)

Freshwater control 0.50 + 0.33 (5) E-treated group 78.2 + 8.00 (5)

Data expressed as mean +_ SEM. N = number of individuals.

Olivereau and Olivereau (1979), who found that plasma ion levels were significantly reduced by estradiol treatment of freshwater eels. However the significance of this effect of estradiol on plasma ion levels remains to be investigated further, especially because water salinity does not seem to induce ovarian development (Querat et al., 1986). In the Japanese eel serum osmolarity increased by 15-20% and serum chloride by 60-65%, 3--4 hr after transfer to sea-water, but the increase levelled off during the following week (Chan et al., 1978).

During the present relatively short-lasting experi- ment only a slight effect was seen on the HSI by estradiol treatment in the freshwater group, while there was no effect on the sea-water group. This index, however, was significantly increased by estradiol treatment in the 15-78 day experiment per- formed by Olivereau and Olivereau (1979). However, the concentration of liver RNA was increased signifi- cantly in the present work and thus depicts the enhanced metabolic activity of the liver in relation to the estradiol-induced synthesis of Vg, as is ob- served in many teleosts (Sundararaj and Nath, 1981; Korsgaard et al., 1983; Ueda et al., 1984; Haux and Nordberg, 1985). Measurement of the concentration of estradiol in plasma (Table 3) shows that the estradiol concentration in the sea-water group was twice the concentration measured in the freshwater group. This is in agreement with the observations of Querat et al. (1986) who found that 2 months after the transfer of eels to sea-water, the plasma level of estradiol was largely increased in sea-water eels compared to freshwater eels. They suggest that the increase in salinity may involve an increase in the volume of distribution of steroids. The higher con- centration of estradioi in the sea-water group may also be the result of a slower clearance of estradiol in the sea-water group compared to the freshwater group.

The difference in vitellogenic response in the sea- water and freshwater group could therefore be the result of a difference in the uptake of estradiol or a difference in the clearance of estradiol in the two groups. The higher response in the freshwater group compared to the sea-water group when Vg is quanti- tated as alkali-labile phosphorus indicates that the concentration of estradiol has been higher in the freshwater group, as it has been shown that the vitellogenic response is dose-dependent in trout (van Bohemen et al., 1981) and flounder (Korsgaard et al., 1983).

Furthermore it has been shown that peak levels of estradiol preceded maximal serum Vg levels and that Vg synthesis continues despite the return of circulat- ing estrogen to basal levels. (Whitehead et al., 1983; Korsgaard et al., 1983; Scott and Sumpter, 1983). The length of time between the peak level of estradiol and the maximal Vg may be different for the eels adapted to sea-water and the eels adapted to freshwater.

The increase in liver DNA in the freshwater group was unexpected. Reports on experimental induction of Vg synthesis in flounder (Emmersen and Emmersen, 1976) and rainbow trout (Haux and Norberg, 1985) show that the liver DNA is unaffected by experimental induction of Vg synthesis. In con- trast to this there was a three-fold increase in liver

60 INGRID PETERSEN and BOD1L KORSGAARD

D N A by endogenous estradiol in vitellogenic female flounders (Emmersen and Emmersen, 1976).

The increased liver D N A in the freshwater group corresponds with the increased HSI suggesting hyperplacia and hyper t rophy. Expressing the D N A content on the basis o f mg protein (not shown) did not change the increase in D N A seen in the fresh- water group.

Consider ing the aquacul tura l interest in eel pro- duct ion, fur ther studies on the progress of the vitello- genie response in freshwater as well as sea-water are impor tant . The different metabol ic aspects of Vg synthesis reported in this work will be fur ther eluci- dated in t ime-course and dose response studies in our laboratories.

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