GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

(1992)

88,29-39

Induction by P-Estradiol of Vitellogenin in Striped Bass (Morone saxatilis): Characterization and Quantification in Plasma and Mucus’ MITSUYO

KISHIDA,

THOMAS

R. ANDERSON,*

AND JENNIFER

L. SPECKER

Department of Zoology, Biological Sciences Center, University of Rhode Island, Kingston, Rhode Island 02881; and *Berkeley Antibody Company, 4131 Lakeside Drive, Richmond, California 94806-1965 Accepted February 15, 1992 Striped bass (Morone saxatilis) were implanted with p-estradiol to induce the production of vitellogenin, the egg yolk precursor produced by the liver. Electrophoretic analysis revealed that g-estradiol caused marked production of a plasma protein of apparent molecular mass 170 kDa. Size exclusion chromatography suggested that the estradiol-induced protein circulated as a dimer. This protein was purified from the plasma of estradiol-treated fish by DEAE-agarose column chromatography and used to induce antibodies in rabbits and goats. Western blots revealed that the antiserum bound to the putative vitellogenin in plasma from estradiol-treated fish and adult females, but not with any proteins in male plasma. Western blot of ovarian extract revealed several smaller immunoreactive protein bands and supported the identity of the purified protein as vitellogenin. A competitive ELISA was developed with sensitivity in a range from 8 to 1000 &ml. Plasma concentrations of adult females during their spawning migration ranged from 100 to 600 pg/ml. Western blot of mucus extract revealed the presence of a 170-kDa protein in vitellogenic female fish along with several minor bands ranging from 50 to 110 kDa. Positive immunoreactivity was present in the surface mucus of all females and in none of the males collected during a spawning migration in the Hudson River. B lwz Academic press, IX

Vitellogenin is the precursor molecule for the primary egg yolk proteins of all egglaying animals. In teleost fishes, as in other nonmammalian vertebrates, its synthesis by the liver is regulated by estradiol (recently reviewed by Byrne et al., 1989; Selman and Wallace, 1989). Detection of vitellogenin or other sex-specific proteins in the serum and surface mucus has been suggested as a means for determining sex of salmonid fish (Le Bail and Breton, 1981; Gordon et al., 1984). This approach would be particularly useful for teleosts such as striped bass (Morone sax&is), in which management of both wild stocks and captive broodstock is complicated by their lack of sexual dimorphism.

The striped bass is a temperate bass in the Family Moronidae, which includes the white bass (M. chrysops) and white perch (M. americana) (Smith, 1985). The striped bass is among the most important commercial and sport fishes of the United States. Hybrid striped bass (M. saxutilis x M. chrysops) are also of growing economic importance (Hodson er al., 1987). Despite historic and current enthusiasm for these fish, much remains to be known about their reproductive biology. Recent analyses of oocyte development and measurement of levels of gonadal steroids indicate that in maturing and mature females, vitellogenesis occurs throughout much of the year (Berlinsky and Specker, 1991). The objectives of this study included purifying vitellogenin from striped bass, generating antibodies against the molecule, developing a specific enzyme-linked immunosorbent assay (ELISA) for measuring

r The U.S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged. 29

0016~6480192

$4.00

Copyright 0 1992 by Academic Press, Inc. Au rights of reproduction in any form reserved.

30

KISHIDA,

ANDERSON,

striped bass vitellogenin, and applying the ELBA to detect vitellogenin in plasma and mucus from striped bass. The results indicate that striped bass vitellogenin production is stimulated by estradiol and that vitellogenin-like immunoreactivity is present in the plasma, ovary, and the surface mucus of vitellogenic fish. These results have been previously reported in abstract (Kishida et al., 1990). MATERIAL

AND METHODS

Sample collection. Striped bass were caught by haul seine from the Hudson River, New York, in May 1991. The females (n = 14) weighed 5.7 t 1.8 kg (mean rf: SE) and had a fork-length of 73 + 8 cm. Their paired ovaries were 551 * 352 g, constituting 9.5 2 4.1% of their body weight. The males (n = 3) had a body weight of 1.5 2 0.7 kg and a fork-length of 49 of:5 cm. Paired testes weighed from 8 to 172 g (73 a 87 g), accounting for 3.9 * 3.4% of body weight. Blood was collected from the caudal vasculature through heparinized 20gauge, l-in. needles into lo-ml syringes in the presence of 500 kallikrein units aprotinin (Sigma)/ml blood. Plasma was separated from the cellular components by centrifugation and stored at - 80” until used. The fish were then killed by a blow to the head. Gonads were dissected from body, weighed, and stored at - 80” until used. Using standard procedures described in Berlinsky and Specker (1991), about 1 g of gonadal tissue was fixed prior to freezing for histological examination. Mucus was collected by scraping the body surface with a stainless steel spatula into a microcentrifuge tube containing aprotinin (500 kallikrein units/ml mucus) and stored at -80” until used. Induction of vitellogenesis. Several striped bass (2-3 kg) were held at the Narragansett Bay Campus of the University of Rhode Island under natural photoperiod in a 2-m circular tank with flow-through Narragansett Bay water (26 ppt, varying between 5 and 20” seasonally). They were implanted with silastic tubes sealed at one end only, each containing 20 mg of p-estradiol (Sigma). These lish were also injected two or three times intraperitoneally at roughly weekly intervals with 2 mg estradiol dissolved in a small amount of ethanol and diluted with peanut oil. The fish were anesthetized (0.02% 2-phenoxyethanol) and bled 2 or 3 days after the injection. Ovarian tissue from one female was taken for analysis by electrophoresis and Western blot. Purification of vitellogenin. Vitellogenin was purified by ion-exchange chromatography using a DEAEagarose (Bio-Rad) column equilibrated with 0.025 M Tris-HCl, pH 7.5, 0.07 M NaCl, 0.1% aprotinin. A

AND SPECKER

plasma sample from an estradiol-treated fish or a mature male fish was diluted three times with the starting buffer and applied to a 1 x 27 cm column. and the column washed with 80 ml of the starting buffer. The elution was performed by a gradient of 0.07 to 0.5 M NaCl which was produced in a mixing chamber of 150 ml. The flow rate was 36 ml/hr and the eluate was collected by 2.0 ml/tube. Optical density of the eluate was measured at 280 nm. An estradiol-induced peak, designated as Fraction A, was concentrated by ultrafiltration using a YM-10 membrane (Amicon), and analyzed further using size exclusion chromatography and polyacrylamide gel electrophoresis. The concentrated material was applied to a Bio-Gel A-1.5m (Bio-Rad) column (2.5 X 48 cm) equilibrated with 0.025 M TrisHCl, pH 7.5, 0.07 M NaCl, 0.1% aprotinin. The flow rate was 30 ml/hr. The eluate was collected by 2.5 ml/tube and monitored at 280 nm. The column was calibrated by gel filtration standards (Bio-Rad) including bovine thyroglobulin (670 kDa), bovine y-globulin (158 kDa), chicken ovalbumin (44 kDa), horse myoglobin (17 kDa), and vitamin B-12 (1.35 kDa). SDS-polyacrylamide PAGE). Plasma,

gel

electrophoresis

(SDS-

ovarian extract, and mucus extract from fish were analyzed using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970), followed by Western blot. Ovarian and mucus extracts were prepared as described below (under ELISA). Molecular mass markers (Bio-Rad) included myosin (200 kDa), Escherichia coli p-galactocidase (116 kDa), rabbit muscle phosphorylase b (97 kDa), bovine serum albumin (66 kDa), and hen egg white ovalbumin (43 kDa). Coomassie brilliant blue R 250 was used to stain the proteins. Immunization. Polyclonal antibodies against vitellogenin were induced in rabbits and goats. Initial injections were made proximal to the axillary and inguinal lymph nodes, and subsequent boosts were made intramuscularly such that the antigen would drain through the sensitized lymph nodes. Immunizations were performed using minced pieces of electrophoresis gels containing a vitellogenin band or with native vitellogenin isolated using DEAE ion-exchange chromatography, as described above. Each animal was initially injected with approximately 0.5 mg of immunogen emulsified in Freund’s complete adjuvant. Subsequent immunizations were made at 4-week intervals, and consisted of approximately 0.25 mg of material emulsified in Freund’s incomplete adjuvant. Serum samples were drawn 10 days after each immunization. Antibody purification. Antibodies were initially isolated from crude antisera by Protein G chromatography using conventional methodologies (Bjorck and Kronwall, 1984). Very briefly, antisera were diluted with an equal volume of 0.1 M sodium acetate, pH 5, and applied to a Protein G column. The column was washed with acetate buffer until the optical density of the flow-through was reduced to background. Anti-

ESTRADIOL

INDUCTION

OF

VITELLOGENIN

bodies were then eluted from the column with 0.1 M glycine-HCl, pH 2.8, and rapidly brought to neutrality with 1 M sodium carbonate. This antibody preparation was then applied to an affinity resin prepared using a “total protein” preparation harvested from male striped bass plasma adhered to this column. Antibodies which flowed through this column were used for Western blot and ELISA. Western

blot of plasma,

mucus,

and gonad

samples.

Proteins separated on SDS-PAGE were transferred to a nitrocellulose membrane in 25 mM Tris, pH 8.3, 192 rnM glycine, 20% (v/v) methanol, at a constant voltage (100 V) for 1 hr. The protocol in Bio-Rad Immun-Blot Assay Kit instruction manual was followed for immunostaining. After blocking in 3% gelatin in 20 mM Trisbuffered saline and washing, the membrane was incubated with the diluted antiserum or purified antibody. After washing, the antigen-antibody complex was visualized with goat anti-rabbit colloidal gold conjugates (Bio-Rad) or goat anti-rabbit IgG alkaline phosphatase conjugate or rabbit anti-goat IgG alkaline phosphatase conjugate (Bio-Rad) followed by the incubation with a substrate solution (alkaline phosphate conjugate substrate kit, Bio-Rad). Prestained SDS-PAGE standards (high range) (Bio-Rad) including myosin (205 kDa), P-galactosidase (116.5 kDa), bovine serum alubumin (77 kDa), and ovalbumin (46.5 kDa) were employed as molecular mass markers. ELBA for striped bass vitellogenin. A competitive ELISA for quantitative measurement of vitellogenin was developed based on the study by Kaneko et al. (1988) with some modifications. Fraction A from a DEAE column was employed as a standard and the protein concentration was determined by Bio-Rad Protein Assay and by the Hartree (1972) method using bovine serum albumin as a standard. The wells of the microtiter plates (Flow Laboratories) were coated overnight at 4” with 20 ng striped bass vitellogenin in 200 ~1 0.1 M sodium carbonate buffer, pH 9.6. The plate was covered with paralilm, incubated for 1 hr at room temperature, and then kept at 4” overnight in a moist chamber. Serial dilutions of striped bass vitellogenin (8-1000 &ml) in 0.1 M phosphate buffer saline, pH 7.4, containing 0.05% Tween 20 (PBST) were placed in polypropylene tubes and incubated with the purified antibody derived from a goat (2.5 &ml in 0.1 M PBST) for 1 hr at room temperature, and then at 4 overnight. On the second day, after washing the plate three times with 0.05 M PBST, the antigen-antibody mixture (200 ~1) was added to each well and incubated overnight at 4”. On the third day, after the plate was washed, HRP conjugated-rabbit anti-goat IgG (1: 17000) (Sigma) was added to the well and incubated as before. The next day, after the final series of washes, 200 pl of the substrate solution (50 ml 0.1 M citrate buffer, pH 4.5, containing 10 pl H,O, and 20 mg OPD) was added to each well and incubated in the dark at room temperature for 25 min. The reaction was

IN

STRIPED

31

BASS

stopped by adding 20 ~1 of 6 N HCI per well. The intensity of color developed was then quantified at 490 nm with an automated ELISA reader (MR 300 MicroELISA Reader, Dynatech Laboratories, Inc.). Application ian extract.

of ELISA

to plasma,

mucus,

and ovar-

The plasma samples were diluted (1:200 to 1:13800) with 0.1 M PBST and measured in the assay. The mucus extract was prepared by suspending thawed mucus in an equal volume of 0.1 M PBST, vortexing vigorously, and removing debris by centrifugation at 3000 rpm for 10 min. The mucus extract was diluted (1:20) with 0.1 M PBST and measured in the assay. Protein concentrations in mucus extract were determined by the Bio-Rad Protein Assay. The ovary was sonicated in the same volume of 0.9% NaCl and centrifuged at 3000 rpm for 10 min. The middle layer was diluted with 0.1 M PBST and measured in the assay. The recoveries of vitellogenin from plasma and from mucus extract were examined by adding known amounts of vitellogenin (50 and 100 ng) to 1 ml of 0.1 M PBST containing 50 ul of plasma or mucus extract from male fish.

RESULTS

An electrophoretic profile of plasma samples drawn from estradiol-treated fish and from immature fish and male fish is shown in Fig. 1. A major change caused by estradiol treatment was an induction of a protein with an apparent molecular mass of approx1

2

3

4

5

6

FIG. 1. Electrophoretic profiles (SDS-PAGE, 7%) of striped bass plasma (5 pl) from a mature male before any treatment (lane 1), after the implant and the first injection of estradiol (lane 2), after the second injection (lane 3), and from an untreated mature male (lane 4), and untreated immature striped bass (lanes 5 and 6). The numbers on the right indicate molecular mass markers (kDa).

32

KISHIDA,

ANDERSON,

imately 170 kDa which was not present before estradiol treatment. This protein was not found in any immature striped bass of either sex or in mature male fish. A similar protein was seen in the plasma of mature wild female striped bass (not shown). A protein of 4.5 kDa molecular mass also appeared after estradiol treatment, and amounts of proteins of 32, 36, and 38 kDa decreased. It was presumed that a 170-kDa protein was the primary component of striped bass vitellogenin, due to its presence only in mature females, and its massive induction by estradiol. In the absence of a protease inhibitor, aprotinin, a protein with a molecular mass of about 100 kDa, appeared along with the 170 kDa. Vitellogenin was purified using DEAEagarose ion-exchange chromatography with an increasing salt concentration. Figure 2

AND

SPECKER

shows the elution profile using plasma from an estradiol-treated fish (upper panel) and plasma from a male fish (lower panel). Clearly, a peak eluting from fractions 7.5 to 85, Fraction A. was found in the plasma from an estradiol-treated fish but was completely absent in untreated male plasma. This fraction was pooled and analyzed for purity by SDS-PAGE. The inset in Fig. 2 shows the purity of Fraction A and the presence of the protein band at 170 kDa. Fraction A subsequently served as an immunogen, and as the standard in the quantitative ELISA. The molecular weight of native vitellogenin was estimated by passing Fraction A over a Bio-Gel A-1Sm column which had been calibrated with proteins of known molecular weight. Figure 3 shows the molecular weight of native striped bass vitelloge-

lLk-2~ “A”

E2 NaCl

6

I.0

20

30

40

50

60

70

80

90

50

60

70

80

90

NaCl Gradient

0.5

0

10

20

30

40

Tube Number FIG. 2. Purification of vitellogenin from plasma (0.5 ml) of estradiol-treated striped bass (upper panel) by ion-exchange chromatography using a DEAE-agarose column. Elutiun profile of pfasma (0.3 ml) from male striped bass is shown in lower panel. The gradient from 0.07 to 0.5 M NaCl.began at fraction 42 as indicated by the arrow. Insert shows an electrophoretic profile (SDS&GE, 7%) of Fraction A from DEAE agarose chromatography. The nnmbers indicate molecular mass markers NW.

ESTRADIOL

INDUCTION

OF

VITELLOGENIN

.c‘

IN

STRIPED

33

BASS

205 -

-

117-

-117

77-

-77

47 -

3. Molecular weight determination of native striped bass vitellogenin by size exclusion chromatography. Fraction A was concentrated by ultrafiltration (YM-10) to 4 ml and applied to a Bio-Gel A-l .Sm column. The arrows indicate elution positions of the markers (kDa) and vitellogenin (Vg).

205

-47

FIG.

nin as estimated by the interpolation between standards to be approximately 300 kDa. Given the inherent inaccuracies of this method of molecular weight determination, these results suggest a possible native structure of a dimer of the 170-kDa subunit. The second peak, eluting at around 2 kDa, appeared in some but not all runs and is thought to contain fragments derived from vitellogenin by nonspecific cleavage due to the prolonged handling of the sample, since a minor band at 100 kDa appeared in the vitellogenin fraction after chromatography. Specificity of the antibodies was confumed by Western blot, as shown in Fig. 4. The antibody identified a protein of molecular mass 170 kDa. The antibody did not cross-react with any proteins in the plasma from male or immature fish. In the ELISA for vitellogenin, serially diluted plasma from female striped bass showed parallelism with the standard curve (Fraction A) (95% confidence intervals about slopes were overlapping) (Fig. 5), indicating that the ELBA could be used to quantify vitellogenin in plasma. Plasma samples from male fish failed to generate any displacement in the assay. Serially diluted ovarian extract also showed cross-reactivity in ELISA, although parallelism was not retained (Fig. 5). SDS-PAGE of ovarian extract showed several protein bands with the major bands

FIG. 4. Western blot of Fraction A (lane l), plasma from estradiol-treated striped bass (0.05 ~1, lane 2) using the rabbit antiserum to striped bass vitellogenin (1:20000) and colloidal gold, mucus extract from female fish (20 ~1, lane 3), and from male fish (20 pl, lane 4) using the purified rabbit antibody (5 tq@nl) and goat anti-rabbit alkaline phosphatase conjugate, resolved by SDS-PAGE (7%). The numbers on the sides indicate molecular mass markers (kDa). The arrows indicate vitellogenin.

at the molecular weights of 113, 98, 69, 44 kDa, and at the dye front, instead of a vitellogenin band (Fig. 6). The presence of these proteins was unaffected by the addi-

100 . ..g p 50 2

. “..

vg

.. .

..

\ 0

FIG. 5. ELISA for striped bass vitellogenin showing serial dilution of the standard (Vg, closed circles on a solid line) as compared with serial dilution of ovarian extract (closed circles on a broken line), and serial dilution of plasma from a female striped bass (open circles). No displacement occurred with plasma from a male fish (open triangles). Samples of plasma and ovarian extract were diluted in 0.1 it4 PBST and used in the ELISA at ranges shown in top of scale. In each case, standards and unknowns were used in a total volume of 200 pl in the assay. Each point represents the mean of duplicates.

34

KISHIDA,

ANDERSON,

AND

SPECKER

Mucus Extract (ul’ml: ln -m (\j s m CD x /___ T-,---r .--,

116 97-

66-

43 -

FIG. 6. SDS-polyacrylamide gel (7%) electrophoresis of the ovarian extract (0.25 ~1) stained by Coomassie brilliant blue (lane l), and followed by Western blot using the antiserum to striped bass vitellogenin (1:2000) (lane 2). The numbers indicate molecular weight standards (kDa). The antibody binds proteins at 113,98, 88, and 69 kDa and at the dye front, but not the 44-kDa protein.

tion of the proteaseinhibitor to the buffer for extraction. Western blot of the gel revealed that most of the bandsobservedby CBB staining were immunoreactive to the antibody, except the band at 44 kDa. An additional band at 88 kDa was recognized more clearly in Western blot, although it was very faintly stainedby CBB. The presenceof immunoreactivity in the ovarian extract is taken as confirmation of the identity of the purified compound as vitellogenin. ELISA was similarly performed using surfacemucus taken from male and female fish (Fig. 7). A serial dilution of mucus extract from maturefemale fish showeda displacement curve, the slope of which was not parallel to that of the standardcurve.

FIG. 7. ELBA showing detection of immunoreactive vitellogenin in the mucus of striped bass. Mucus samples were weighed, diluted in equal volumes of 0.1 M PBST, vigorously mixed, and centrifuged at 3OtN rpm for 10 min. The supematant was used in the assay at ranges shown in top of scale. Serial dilution of the mucus extract from a female striped bass (open squares) shows a displacement curve, whereas mucus extract from a male fish (open circles) shows no crossreactivity. Each point represents the mean of duplicates.

On the other hand, mucus extract from male fish did not show any significant cross-reactivity in the assay. Western blot of mucus extract from female fish showed the intensely stainedband at the same position of vitellogenin along with severalminor bands ranging from around SOto 110 kDa. Mucus extract from male fwh did not show any cross-reactivityat the position of vitellogenin, althougha faint band was observedat 206kDa, which also appearedto be presentfaintly in the female. The identity of the 206kDa protein is unknown. Theseresults suggestthat immunoreactivevitellogenin was presentin the mucus. The recovery of vitellogenin added to plasma and mucus was 113and lOO%,respectively. The intra- and interassayvariation was 13% (n = 8) and 14% (n = 7), respectively, at around 50% of binding. In light of the recovery of vitellogenin from mucus extract at the fried amount used in the assay,we report the mucusextract con-

ESTRADIOL

INDUCTION

OF VITELLOGENIN

centrations as quantitative, although because of the lack of parallelism of serially diluted mucus with the standard curve, these may be only relative. The assay was sensitive enough to measure vitellogenin levels in plasma and mucus. The limit of detection in the assay was 1.6 ng/ well (91.4 2 4.1% of binding, 12 = 10). Therefore, the detection limit for plasma or mucus level was 160 &ml, since plasma and mucus extract had to be diluted at least 20 times (10 PYwell). Recoveries of vitellogenin from plasma or mucus extract became unreliable when plasma and mucus were used at higher concentrations. Vitellogenin concentrations were measured in plasma and in mucus extract from wild fish caught in the Hudson River during the spawning migration (Fig. 8). The females which had not spawned (13/14) had plasma vitellogenin concentrations ranging from 114 to 558 pg/ml(306 + 135 l&ml) and positive immunoreactivity in their surface mucus indicating the presence of 48 to 433 ng vitellogenin/mg protein (176 + 101 ng/mg protein). The oocytes in these females were roughly 1 mm and were probably nearing 3500 0 3000 x 8 a

L

500

F 400 ??I 5 $I 300 2 2

200

. .

0 .

I 100

l .

l

.

. . 0

L

IIll “8

8 -cdm*m(D

Plasma

8

8

5:

8

Vg &g/ml)

FIG. 8. Relationship between concentrations of vitellogenin in plasma and mucus extract from female striped bass caught during a spawning migration in Hudson River. Closed circles represent fish which had not spawned. The open circle represents a fish which had recently spawned.

IN STRIPED BASS

35

germinal vesicle breakdown. One female had recently spawned, as indicated histologically by the presence of many convoluted empty follicles, and had vitellogenin concentrations of 660 &ml in plasma and 3213 ng/mg protein in mucus extract. DISCUSSION Vitellogenin, a phospholipoglycoprotein, is produced by the liver and serves as a precursor to the egg yolk proteins of oviparous vertebrates. The synthesis of vitellogenin is controlled by ovarian estrogens. It has been well established that like in other oviparous vertebrates, vitellogenesis in fish can be induced in both males and females by exogenous estrogens (Byrne et al., 1989; Selman and Wallace, 1989). The studies described here demonstrate that administration of estradiol induces the synthesis of vitellogenin in both male and female striped bass. Vitellogenin was present naturally only in mature females. Using specific antiserum, positive immunoreactivity was detected in the ovary, indicating uptake and processing of the yolk precursor by the oocytes. We confirmed for the first time the presence of vitellogenin in the surface mucus of a teleost fish, by detecting vitellogenin on female striped bass during their spawning migration. The molecular weight of striped bass vitellogenin was estimated to be 170 and 300 kDa by SDS-PAGE and size exclusion chromatography, respectively, suggesting the possibility of a dimeric form of a native molecule. The molecular weight of the subunit is similar to those of turbot, Scophthalmus maximus (185 kDa, Silversand and Haux, 1989), catfish, Zctalurus punctatus (150 kDa, Bradley and Grizzle, 1989), and tilapia, Oreochromis aweus (180 and 130 kDa, Ding et al., 1989). Tao et al. (1991) have recently reported that striped bass vitellogenin may circulate as a homotetramer of 640 kDa. Our experiments show that a single step

36

KISHIDA.

ANDERSON,

of ion-exchange chromatography on a DEAE-agarose column is sufficient to isolate striped bass vitellogenin from plasma. The elution of vitellogenin with the highest salt concentration from an anion-exchange column seems to be a common characteristics of vitellogenin probably due to a highly charged surface of the molecule (de Vlaming et al., 1980; Norberg and Haux, 1985; Silversand and Haux, 1989; Tyler and Sumpter, 1990). Initially the plasma was subjected to size exclusion chromatography prior to DEAE-agarose chromatography. Although this procedure reduced the amount of protein eluting before the vitellogenin fraction, it did not improve the purity of vitellogenin but instead seemed to facilitate proteolysis. There are a number of techniques developed for the measurement of vitellogenin in the blood of fish. Plasma calcium and protein-bound phosphate have been used as indirect indices of vitellogenin (Baily, 1957; Craik, 1978; Whitehead et al., 1978; Elliott et al., 1979; Bjornsson and Haux, 1985). Immunoassays have been also developed, including rocket immunoelectrophoresis (Maitre et al., 1985), radial immunodifusion (Hara, 1978), immunoagglutination (Le Bail and Breton 1981), and radioimmunoassays (So et al., 1985; Sumpter, 1985; Norberg and Haux, 1988; Benfey et al., 1989; Tyler and Sumpter, 1990). Homologous radioimmunoassays seem to be most useful in terms of specificity, sensitivity, and accuracy. However, because of the instability of labeled vitellogenin another step was required to purify the intact labeled vitellogenin (Sumpter, 1985), or an egg yolk protein was used as standard and label (Idler et al., 1979). On the other hand, ELISA employed in this study does not require labeling vitellogenin and still possesses advantages of radioimmunoassay. Recently, ELISA has also been developed for sole, Mea vulgaris, vitellogenin (Rodriguez et al., 1989). The Western blot analysis clearly showed the specificity of the antiserum for

AND

SPECKER

the vitellogenin band. No other proteins in plasma from female or male fish crossreacted with the antiserum. Similar results were obtained when the purified antibodies from a goat were used. The results from ELISA also show the specificity of the assay, in which the serial dilution of the plasma from male fish did not show any cross-reactivity, while the plasma from mature female fish showed significant crossreactivity. Ovarian extract cross-reacted in the assay, suggesting the incorporation of vitellogenin by the oocytes and adding further support to the identity of 17%kDa protein as vitellogenin. However, when the ovarian extract was analyzed by SDS-PAGE, several protein bands at smaller molecular weights (113, 98, 88, 69, and 44 kDa) than that of vitellogenin were observed instead of vitellogenin. Similarly smaller proteins were found in ovaries of Fundulus heteroclirus (Wallace and Selman, 1985). The protein bands found in striped bass ovarian extract were revealed to be immunoreactive (except the 44-kDa protein) to the striped bass vitellogenin antiserum by Western blot. Therefore, this antiserum recognizes the egg yolk proteins derived from vitellogenin, which probably accounts for the displacement of vitellogenin by the ovarian extract in the ELISA and also the lack of parallelism. The absence of the 170-kDa protein in the ovarian extract suggests rapid proteolysis of the vitellogenin molecule during uptake by the oocyte. Our results show that vitellogenin is not the only protein induced by estradiol treatment. An additional protein at 45 kDa appears in the plasma of estradiol-treated striped bass. The possibility that this protein is the same as the 44-kDa protein seen in the ovarian extract merits further attention. It has been reported that in Orysias latipes a spawning female-specific substance distinct from vitelbgenin is also produced in the liver under estrogen& control. This substance serves as a precursor of the

ESTRADIOL

INDUCTION

OF

VITELLOGENIN

major glycoprotein component of the inner layer of the ovarian egg envelope (chorion) and has an apparent molecular mass of 49 kDa (Hamazaki et al., 1987, 1989). In striped bass, this 44-kDa protein did not show cross-reactivity with the antibody against striped bass vitellogenin. Similarly sized chorion proteins (47, 54, and 74 kDa) have been shown to constitute the eggshell of the cod, Gadus morhua (OppenBernsten et al., 1990). Further research is required to identify the 44-kDa protein as a component of the chorion in striped bass. The serial dilution of the mucus extract taken from mature female fish showed a displacement curve, whereas the mucus extract from male fish did not show any significant cross-reactivity. Western blot of mucus extract indicated that an intact molecule of the vitellogenin monomer is present in the mucus extract from female fish but not from male fish. Cross-reactivity was also found with several bands ranging from 50 to 110 kDa, which might have been derived from vitellogenin by nonspecific cleavage, considering that vitellogenin is highly susceptible to proteolysis. This finding might explain why the displacement curve of mucus extract from vitellogenic female fish was not parallel to the standard curve. Although vitellogenin was not found in mucus extract from male fish by Western blot, a protein band at 206 kDa showed very faint cross-reactivity with the antibody. This band also appeared to be present with less intensity in the mucus extract from female fish. The identity of this protein is not clear; however, because none of the serial dilutions of mucus extract from male fish assayed showed any displacement in the assay, the cross-reactivity of this protein may not be significant in a competitive assay such as employed in this study, Because vitellogenin is thought to be evolutionally related to apolipoprotein B-100, the protein component of low-density lipoprotein (Byrne et al., 1989), it might be possible that the antibody to striped bass vitelloge-

IN

STRIPED

BASS

37

nin cross-reacts with this apolipoprotein in noncompetitive conditions. It has been reported that the molecular weight of apoBlike apolipoprotein in rainbow trout is 240 kDa (Babin, 1987). Vitellogenin was detected both in plasma and in mucus extract from wild female fish caught during the spawning migration up the Hudson River. This is the first confirmation of the presence of vitellogenin in the surface mucus of any fish to our knowledge. The presence of a sex specific protein, which was presumed to be vitellogenin, in the mucus from female coho salmon, has been reported (Gordon et al., 1984). In striped bass, no significant relationship was found between concentrations in plasma and in mucus extract among the females in the late stages of vitellogenesis, even though the amount of mucus extract was normalized by protein content. The relatively high concentration of vitellogenin in the surface mucus of the recently spawned female suggests that hepatic synthesis might continue beyond ovulation and that the skin serves as an excretory pathway. Another possibility is that vitellogenin in surface mucus has a pheromonal function, conveying information to other individuals. Whether vitellogenin moves from plasma to mucus by diffusion or is transported by a regulated mechanism is unknown. Vitellogenin has been suggested as a candidate pheromone used by female garter snakes to elicit male courtship behavior (Garstka and Crews, 1981). Although recent studies indicated that methyl ketones present in the skin serve as sex pheromones (Mason et al., 1989, 1990), the significance of the presence of vitellogenin in the snake skin remains to be found. In fish, many serum proteins are known to occur in skin mucus (Smith and Ramos, 1976, 1980); however, little is known about the translocation of these proteins to the surface or their functions. The significance of the presence of vitellogenin in mucus of striped bass is un-

38

KISHIDA,

ANDERSON,

known. Some cichlid fish are known to feed the young on mucus (Jalabert and Zohar, 1982). Hence, it is intriguing to suggest that vitellogenin in mucus in some fish might serve as a source of nutrition for the young. The measurement of vitellogenin concentrations in blood will add to our efforts to understand the reproductive biology of striped bass. The noninvasive assay for vitellogenin in surface mucus may be a useful technique for aquaculturists to detect maturing females and for biologists concerned with managing wild populations. ACKNOWLEDGMENTS The authors thank Dr. Craig Sullivan (North Carolina State University) for his generosity in providing male striped bass plasma; Krishna Balakrishnan, Jorge Garcia, Liyue Huang, Janis Peterson, Philip Veillette, and Ming-Hsien Wu for assistance; John F. O’Brien (Rhode Island Fish and Wildlife) and Andy Kahnle (New York Environmental Conservation) for coordinating collections of the striped bass; Kenneth Davignon for graphics; and Dr. Anne Richards (National Marine Fisheries Service) for supporting the study. This research was sponsored by the Department of Commerce through an Emergency Striped Bass Project Grant (NASSEA-D-0040 to J.L.S.), a Small Business Innovative Research Award (89-l-l 16 to T.R.A.), and NOAA Office of Sea Grant (Grant NA89AA-D-SG082 to J.L.S.). The U.S. Government is authorized to produce and distribute reprints for governmental purposes notwithstanding any copyright notation that may appear hereon.

AND

SPECKER

some properties of streptococcal protein G, a novel IgG-binding reagent. .I. Zmmunol. 133, 969974. Bjornsson, B. I-., and Haux, C. (1985). Distribution of calcium. magnesium and inorganic phosphate in plasma of estradiol-178 treated rainbow trout. J. Comp.

Physiol.

B 155, 347-352.

Bradley. J. T., and Grizzle, J. M. (1989). Vitellogenin induction by estradiol in channel catfish, Ictalurus punctatus.

Gen.

Comp.

Endocrinol.

73, 28-39.

Byrne, B. M., Gruber, M., and AB, G. (1989). The evolution of egg yolk proteins. Prop. Biophys. Mol.

Biol.

53, 33-69.

Craik, J. C. A. (1978). Kinetic studies of vitellogenin metabolism in the elasmobranch Scyliorhinus canicula L. Comp. Biochem. Physiol. A 61, 355361.

de Vlaming, V. L., Wiley, H. S., Delahunty, G., and Wallace, R. A. (1980). Goldfish (Carassius auratus) vitellogenin: Induction, isolation, properties and relationship to yolk proteins. Comp. Biothem.

Physiol.

B 67, 613423.

Ding, J. L.. Hee, P. L., and Lam, T. J. (1989). Two forms of vitellogenin in the plasma and gonads of male Oreochromis uureus. Camp. Eiochem. Physiol. 93B, 363-370. Elliott, J. A. K., Bromage, N. R., and Whitehead, C. (1979). Effects of estrddiot-178 on serum calcium and vitellogenin levels in rainbow trout. J. Endocrinol. 83, 54P-55P. Garstka, W. R., and Crews, D. (1981). Female sex pheromone in the skin and circulation of a garter snake. Science 214, 681-682. Gordon, M. R., Owen, T. G., Ternan, T. A., and Hildebrand, L. D. (1984). Measurement of a sexspecific protein in skin mucus of premature coho salmon (Oncorhynchus kisutch). Aquaculture 43, 333-339.

REFERENCES Babin, P. J. (1987). Plasma lipoprotein and apolipoprotein distribution as a function of density in the rainbow trout (S&no gaira’neri). Biochem. J. 246, 425-429. Bailey, R. E. (1957). The effect of estradiol on serum calcium, phosphorus, and protein of goldfish. J. Exp.

2001.

136, 455-469.

Benfey, T. J., Donaldson, E. M., and Owen, T. G. (1989). An homologous radioimmunoassay for coho salmon (Oncorhynchus kisutch) vitellogenin, with general applicability to other pacific s~almonids. Gen. Comp. Endocrinol. 75, 78-82. Berlinsky, D. L., and Specker, J. L. (1991). Changes in gonadal hormones during oocyte development in the striped bass, Morone saxatilis. Fish Physiol. Biochem. 9, 51-62. Bjorck, L., and Kronwall, G. (1984). Purification and

Hamazaki, T. S., Iuchi, I., and Yamagami, K. (1987). Production of a “spawning female-specific substance” in hepatic cells and its accumulation in the ascites of the estrogen-treated adult fish, Oryzias latipes.

J. Exp.

Zool.

242, 325-332.

Hamazaki, T. S., Nagahama, Y ., luchi, I., and Yamagami, K. (1989). A glycoprotein from the liver constitutes the inner layer of the egg envelope (zona pellucida interna) of the fish, Oryzias latipes. Dev. Biol. 133, 101-110. Hara. A. (1978). Studies on female specific serum proteins (vitellogenin) and egg yolk proteins in teleosts: Immunochemical, physicochemicai and structural studies. Mem. Fat. Fish. Hokkaido 34, l-59.

Hartree, E. F. (1972). Determination of protein: A modification of the Lowry method that gives a linear photometric response. Anal. Biochem. 48, 422-427.

ESTRADIOL

INDUCTION

OF

VITELLOGENIN

Hodson, R. G., Smith, T., McVey, J., Harrell, R., and Davis, N. (1987). “Hybrid Striped Bass Culture: Status and Perspective,” 105 pp. North Carolina Sea Grant College Program, NC. Idler, D. R., Hwang, S. J., and Crim, L. W. (1979). Quantification of vitellogenin in Atlantic salmon (Salmo s&r) plasma by radioimmunoassay. J. Fish.

Res.

Can.

36, 574-578.

Jalabert, B., and Zohar, Y. (1982). Reproductive physiology in cichlid fishes, with particular reference to Tilapia and Sarotherodon. In “The Biology and Culture of Tilapias” (R. S. V. Pullin and R. H. Lowe-McConnell, Eds.), pp. 129-140. ICLARM, Manila. Kaneko, T., Fraser, R. A., Labedz, T., Harvey S., Lafeber, F. P. J. G., and Pang, P. K. T. (1988). Characterization of antisera raised against hypocalcin (teleocalcin) purified from corpuscles of stannius of rainbow trout, Salmo gairdneri. Gen. Comp.

Endocrinol.

69, 238-245.

Kishida, M., Wu, M.-H., Balakrishinan, K., Anderson, T. R., and Specker, J. L. (1990). Purification of vitellogenin from striped bass plasma and development of an enzyme-linked immunosorbent assay (ELISA). Proc. Western Regional Conf. Camp. Endocrinol., Abstr. 24. Le Bail, P. Y., and Breton, B. (1981). Rapid determination of the sex of puberal salmonid fish by a technique of immunoagglutination. Aquaculture 22, 367-375. Laemmli, II. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. Maitre, J. L., Le Guellec, C., Derrien, S., Tenniswood, M., and Valotaire, Y. (1985). Measurement of vitellogenin from rainbow trout by rocket immunoelectrophoresis, application to the kinetic analysis of estrogen stimulation in the male. Can. J. Biochem.

Cell Biol.

63, 982-987.

Mason, R. T., Fales, H. M., Jones, T. H., Pannell, L. K., Chinn, J. W., and Crews, D. (1989). Sex pheromones in snakes. Science 245, 290-293. Mason, R. T., Jones, T. H., Fales, H. M., Pannell, L. K., and Crews, D. (1990). Characterization, synthesis, and behavioral responses to sex attractiveness pheromones of red-sided garter snakes (Thamnophis

sirtalis

parietalis).

J. Chem.

Ecol.

16, 2353-2369. Norberg, B., and Haux, C. (1985). Induction, isolation and a characterization of the lipid content of plasma vitellogenin from two Salmo species: Rainbow trout (Salmo gairdnert) and sea trout (Salmo trutta). Comp. Biochem. Physiol. B 81, 869-876.

IN

STRIPED

BASS

39

Norberg, B., and Haux, C. (1988). An homologous radioimmunoassay for brown trout (Salmo trutta) vitellogenin. Fish Physiol. Biochem. 5, 5p-68. Oppen-Bemsten, D. O., Helvik, J. V., and Walther, B. T. (1998). The major structural proteins of cod (Gadus morhua) eggshells and protein crosslinking during teleost egg hardening. Dev. Biol. 137, 258-265. Rodriguez, J. N., Kah, O., Geffard, M., and Le Menn, F. (1989). Enzyme-linked immunosorbent assay (ELISA) for sole (Solea vulgaris) vitellogenin. Comp. Biochem. Physiol. B 92, 741-746. Selman, K., and Wallace R. A. (1989). Cellular aspects of oocyte growth in teleosts. 2001. Sci. 6, 211-231. Silversand, C., and Haux, C. (1989). Isolation of turbot (Scophthalmus maximus) vitellogenin by high-performance anion-exchange chromatography. .I. Chromatogr. 478, 387-397. Smith, C. L. (1985). “The Inland Fishes of New York State.” The New York State Department of Environmental Conservation, NY. Smith, A. C., and Ramos, F. (1976). Occult haemoglobin in fish skin mucus as an indicator of early stress. J. Fish Biol. 9, 537-541. Smith, A. C., and Ramos, F. (1980). Automated chemical analysis in fish health assessment. J. Fish Biol. 17, 445-450. So, Y. P., Idler, D. R., and Hwang, S. J. (1985). Plasma vitellogenin in landlocked Atlantic salmon (Salmo salar Ouananiche): Isolation, homologous radioimmunoassay and immunological crossreactivity with vitellogenin from other teleosts. Comp.

Biochem.

Physiol.

B 81, 63-71.

Sumpter, J. P. (1985). The purification, radioimmunoassay and plasma levels of vitellogenin from the rainbow trout, Salmo gairdneri. In “Current Trends in Comparative Endocrinology” (B. Lofts and W.N. Holmes, Eds.), pp. 355-357. Hong Kong Univ. Press, Hong Kong. Tao, Y., Hodson, R. G., Woods III, L. C., Bennett, R. O., and Sullivan, C. V. (1991). Vitellogenin and vitellogenesis in striped bass (Morone suxatilis).

Amer.

Zool.

31, 41A.

Tyler, C. R., and Sumpter, J. P. (1990). The development of a radioimmunoassay for carp, Cyprinus carpio, vitellogenin. Fish Physiol. Biochem. 8, 129-140. Wallace, R. A., and Selman, K. (1985). Major protein changes during vitellogenesis and maturation of Fundulus oocytes. Dev. Biol. 110, 492498. Whitehead, C., Bromage, N. R., and Fostier, J. R. M. (1978). Seasonal changes in reproductive function of the rainbow trout. J. Fish. Biol. 12, 601-608.