GENERAL
AND
COMPARATIVE
Isolation
ENDOCRINOLOGY
81, 391-402 (1991)
and Characterization
of Japanese
Eel Prolactins
REIKO SUZUKI, AKIKAZU YASUDA,* JUN KONDO,~ HIROSHI KAWAUCHI,* AND TETSUYA HIRANO Ocean Research Institute, Universty of Tokyo, Nakano, Tokyo 164; *Laboratory of Molecular Endocrinology, School of Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-01, Japan; fMitsubishi Kasei Co., Ltd., Research Center, Yokohama, Kanagawa 227, Japan Accepted April 6, 1990 A highly purified prolactin (PRL) was isolated from the pituitary of the Japanese eel (Anguilla japonica) by extraction with acid-acetone, gel filtration on Sephadex G-75, and reversed-phase HPLC on TSK-gel ODS 120T and on TSK-gel TMS 250. Eel PRL is comprised of two variants (ePRL I and II), which were separated by HPLC on an ODS column. The two PRLs were also secreted by organ-cultured pituitaries in a defined medium. After being dialyzed against distilled water and lyophilized, the medium was dissolved in 0.01 M ammonium acetate (pH 9.0), and then the insduble fraction was subjected to HPLC on an ODS column to isolate the secreted PRLs. The ePRL I and II were equipotent in retaining plasma Na in the hypophysectomized killiiish, Fund&s heteroclitus, transferred from seawater to fresh water. The putative PRL-producing cells in the rostral pars distalis of the eel pituitary were specifically stained with the antiserum against the mixture of ePRL I and II. Both PRLs had a molecular weight of 22 kDa in SDS-PAGE, an isoelectric point of 10.1 by gel electrofocusing, and an N-terminal residue of valine. Amino acid compositions and the partial amino acid sequences of ePRL I and II show that they are highly homologous with a limited number of substitutions, and that they are more closely related to those of teleostean PRLs than to those of mammalian PRLs. o IWI Academic PRSS, h.
and homologous radioimmunoassays are available to measure their plasma and pituitary concentrations (Hirano et al., 1985). The effects of homologous PRL on hydromineral balance as well as the changes in plasma PRL concentrations following exposure to various salinities suggest that PRL is involved also in freshwater adaptation in salmonid fishes, even though it is not essential for their survival in fresh water (Hirano et al., 1985; Prunet et al., 1985; Hasegawa et al., 1986; Ogasawara et al., 1989). The migratory pattern of the eel is opposite to that of salmon; eels grow in fresh water and, as they mature, go downstrean to spawn. The actions of PRL in eel osmoregulation may be different from those in salmonids. However, eels have received less attention than salmonids, although there are a few histological studies indicat-
Prolactin (PRL) is an important freshwater-adapting hormone for several euryhaline teleosts such as the killifish, tilapia, and molly, in which hypophysectomy without PRL therapy impairs survival in fresh water (see Clarke and Bern, 1980; Hirano, 1986). In contrast, the eel and salmonids survive in fresh water after hypophysectomy (Fontaine et al., 1949; Nishioka et al., 1987). While studies on the role of PRL in teleost osmoregulation have been extended to examine the direct effects of PRL on ionand water-transporting epithelia, the differences in the mode of PRL action between the species dependent on PRL for their survival in fresh water and those not so dependent have not been elucidated. In salmonids, chum, chinook, and Atlantic salmon PRL have been isolated (Idler et al., 1978; Kawauchi et al., 1983; Prunet and Houdebine, 1984; Andersen et al., 1989) 391
00164480191 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
392
SUZUKI
ing that PRL cells are more active in fresh water than in seawater (Olivereau and Ball, 1970; Naito et al., 1983). We report here the isolation of two variants of eel PRL and their physicochemical and biological characteristics, including partial amino acid sequences. MATERIALS
AND METHODS
Materials. Pituitary glands were collected from immature, cultured Japanese eel (Anguillu juponicu), weighing 150-200 g. Four thousand pituitaries were frozen on dry ice, and stored for 1-2 months at - 80”. Nine hundred pituitaries were cultured in a defined medium following the protocol of Kishida et al. (1987), under which condition the pituitaries were viable to continue to synthesize and secrete growth hormone for several weeks. Twenty pituitaries were cultured in a plastic dish (diameter 59 mm, depth 15 mm) in 8 ml of Medium 199 with Earle’s salts supplemented with 100 U/ml streptomycin, and 0.25 p&ml Fungizone 100 U/ml penicillin, (M. A. Bioproducts, Walkersville, MD) in a gas phase of 95% 0, and 5% CO, at 22” for 4-6 weeks. The pH of the medium was adjusted to 7.3-7.4 by adding sodium bicarbonate and the osmotic pressure was adjusted to 290-300 mOsm. The medium was changed once a week and stored at -20”. Isolation. The frozen pituitaries (5 g) were extracted with acid-acetone as described for salmon PRL (Kawauchi et al., 1983). The extract (100 mg) was subjected to gel filtration on a Sephadex G-75 column (2 x 90 cm) equilibrated with 0.1 N acetic acid. Pooled fractions were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970). The fraction containing the protein with a molecular weight of 22 kDa was dissolved in 0.1% trifluoroacetic acid (TFA) and subjected to highperformance liquid chromatography (HPLC) using a reversed-phase column (0.46 x 25 cm, TSK-gel ODS 120T and TMS 250) at a column temperature of 40” and a flow rate of 1 mUmin. Linear gradient elution was performed with acetonitrile containing 0.1% TFA. The eluate was monitored by measuring absorbance at 220 nm. Each peak was collected in one tube and the main fraction was analyzed by Western blotting after SDSPAGE following the protocol of Miyajima et ul. (1988) using an antiserum against chum salmon PRL (Kawauchi et al. 1983). PRL secreted into the culture medium was purified after dialyzing the pooled medium against distilled water. The dialyzate was lyophilized and then dissolved in 0.01 M ammonium acetate (pH 9.0). The insoluble fraction was dissolved in 0.1% TFA containing 1 M urea and subjected to HPLC on an ODS column (0.46
ET
AL.
X 25 cm) under the same elution conditions described above. Chemical and physicochemicul churucterizution. Molecular weight was estimated by SDS-PAGE. Molecular weight markers (14,300-71,500, BDH Chemicals) were employed as the reference standards. The isoelectric point was estimated by gel isoelectric focusing in 5% polyacrylamide gel at 2% ampholine (pH 3.5-10) and staining with 0.06% Coomassie brilliant blue G-250 in 3.2% sulfosalicylic acid, 5% trichloroacetic acid, and 50% ethanol. An isoelectric focusing p1 calibration kit (Pharmacia) was used for determination of the pH gradient profile. For amino acid analysis, the proteins were hydrolyzed for 18-22 hr at 110” with constantly boiling HCl containing 0.6% phenol (Muramoto et al., 1987). Amino acid analyses were carried out by HPLC on a TSK-gel ODS 80TM (0.46 x 25 cm) with a precolumn labeling method using 6 3:
4
0
50
100
Fraction Number FIG. 1. Elution profile of an acid-acetone extract of Japanese eel (Anguilla juponica) pituitary glands on a Sephadex G-75 column (2 x 90 cm) equilibrated with 0.1 N acetic acid. Flow rate, 15 ml/hr; fraction size, 2.5 ml. VJV, of peak 3 was 1.54.
ISOLATION
393
OF EEL PROLACTIN
phenylisothiocyanate (Bidlingmeyer et al., 1984). Phenylthiocarbamyl amino acids were developed using a linear gradient of 0.14 M sodium acetate (pH 5.4) and acetonitrile. Half-cystine content was estimated after performic acid oxidation of the protein. Nterminal analysis was performed by the dansyl method (Gray, 1%7). Intact PRL was digested with lysyl endopeptidase (E/S = l/60, by weight) in 0.1 M ammonium bicarbonate containing 4 M urea, pH 8.0, at 37 for 4 hr. The resulting fragments were subjected to HPLC on a TSK-gel ODS 120T column using a linear gradient with isopropanol in 0.1% TFA. Elution was monitored at 210 nm and individual peaks were collected in separate tubes. Partial sequences of some fragments were determined using gas-phase sequencers (Applied Biosystem 470A and 477A-120A).
Biological activi@. Sodium-retaining activity of the eel PRL was examined using intact tilapia and hypophysectomized Fundulus. The assay for intact tilapia, Oreochrumis niloticus, was carried out following the slightly modified protocol of Clarke (1973). Tilapia (4-5 g) were acclimated to 50% seawater (23”) for more than 2 weeks before use under natural photoperiod in Tokyo. They were anesthetized with 0.03% 2phenoxyethanol and given intraperitoneal injections of 1.5 pl of saline (0.9% NaCl), homogenate of eel rostra1 pars distalis (RPD), chum salmon PRL, or ovine PRL (NIAMDD o-PRL-11) between 1800 and 1900 hr daily for 5 days. Eel RPD was homogenized in a small volume of 0.1 N acetic acid and diluted with saline to prescribed doses which were determined by a homologous RIA for eel PRL (Suzuki and Hirano, 1991). The
I II
3
14 0
40 Time
(min)
, ,I L 0
40 Time
(min
)
FIG. 2. High-performance liquid chromatography (HPLC) patterns of peak 3 in Fig. 1. The sample was dissolved in 0.1% trifluoroacetic acid (TFA). The column temperature was maintained at 40”. Flow rate was I ml/min. Elution was performed by a linear gradient of acetonitrile containing 0.1% TFA. Five micrograms of proteins in main peaks were subjected to SDS-PAGE and stained with Coomassie blue. (A) Elution pattern of fraction on a TSK-gel ODS 120T column (0.46 x 25 cm, 5 Km particle size). Fractions I and II were designated as ePRL I and ePRL II. (B) Repeated chromatography of fraction I on a TSK-gel TMS 250 column (0.46 x 25 cm, 10 pm particle size).
394
SUZUKI
preparation contained only 3% eel growth hormone (GH) which was detected by a specific RlA for eel GH (Kishida and Hirano, 1988). Hormones were dissolved in a small volume of 0.1 N acetic acid and diluted with saline. The fish were sacrificed 16 hr after the last injection, and the blood was collected from the caudal artery. Plasma was separated by centrifugation at 10,OtM rpm for 10 min. Sodium levels were determined by atomic absorption spectrophotometry (Hitachi 18050). Male kill&h, Fund&s hereroclitus, weighing from 3 to 5 g, were kept in seawater at 22”. Fundulus assay was carried out following the protocol of Grau et al. (1984). Hypophysectomy was performed as described by Grau and Stetson (1977). Following hypophysectomy, the fish were held for 1 week in seawater. Hypophysectomized fish were injected intraperitoneally with 25 pl saline, homogenate of eel RPD, or hormones between 1800 and 2000 hr. After the injections, fish were maintained in seawater for 1 hr and then transferred to freshwater aquaria (Na, 0.4 mM; Ca, 0.35-0.45 n&f) for 22-24 hr until blood sampling. The statistical analysis used was the Duncan’s multiple range test. Immunocytochemistry. The eel PRL antiserum was raised in a young rabbit. Emulsion (100 pl) containing 25 pg eel PRL (mixture of ePRL I and II) and 0.5 mg killed and dried Mycobacterium butyricum (Difco) was injected into each lymph node of the right and left hind limbs. Emulsion (2 pl) containing 50 pg eel PRL and 1 mg killed and dried M. butyricum was subcutaneously injected in the back of the rabbit three times at intervals of 3 weeks after the first inguinal injection. Eel PRL was dissolved in a small volume of 0.1 N acetic acid and diluted with saline. The emulsion was made by mixing equal volumes of a saline solution of eel PRL and of Freund’s complete adjuvant. The rabbit was completely bled from the carotid artery 2 weeks after the last injection and the serum was stored at - 40”. A pituitary of the eel was fixed in Bouin’s fluid and embedded in paraplast; serial sagittal sections were cut (4 pm thick). Immunocytochemical staining of PRL producing cells was carried out using the peroxidase-antiperoxidase method (Naito et al., 1983). and an adjacent section was also stained with the eel GH antiserum (Kishida et al., 1987).
RESULTS
Isolation of eel PRL. When an acidacetone extract of eel pituitaries and the culture medium in which eel pituitaries were incubated were subjected to SDSPAGE and then to Western blotting, the band corresponding to a molecular weight
ET AL.
of 22 kDa reacted with the antiserum against salmon PRL. The elution pattern of an acid-acetone extract of pituitaries on Sephadex G-75 is shown in Fig. 1. Peak 3 containing the protein of molecular weight
#’I’ #’ ,I’#’ I: a’,’ #’ I’ ,#’ /,’ , 1,.8’
L 0
I 40
Time (min) FIG. 3. Analytical HPLC of PRL fractions on a TSK-gel ODS 120T column (0.46 x 25 cm, particle size 5 pm). (A) The insoluble fraction of culture medium in 0.01 M ammonium acetate (pH 9.0). The fraction was dissolved in 0.1% TFA containing 1 M urea. (B) The acid-acetone extract of eel pituitaries (see legend to Fig. 2A).
ISOLATION
395
OF EEL PROLACTIN
B \
5.0
4.8 9 2 5 Ip8
s"
ePRL I,11 (22,000)
4.2
(14,300)
6
/-
4.0 5
J
0
0.2
0.4
0.6
0.8
,,‘-,, (28,600)
4.4
7
,
(42,900)
\
4.6
1.0
0
0.2
0.4
Mobility
0.6
0.8
1.0
Mobility
FIG. 4. (A) Estimation of the isoelectric point of ePRL I and II by gel isoelectric focusing. The pH gradient profiile was determined using the pI calibration kit (Pharrnacia; open circles) and chum salmon PRL of p1 10.3 (open triangle). (B) Estimation of the molecular weight by SDS-PAGE. Open circles indicate molecular weight markers (BDH Chemicals).
AMINO
ACID
COMPOSITIONS
Eel PRL”
Asp Thr Ser Glu FYO
GlY Ala CYS Val Met Ile Leu ‘W Phe Trp His LYS Arg Total N-aa a Value b Taken ‘Taken d Taken
TABLE 1 PRLs COMPARED
OF EEL
Salmon PRLb
Carp’ PRL
I
II
I
II
19.5 9.0 30.6 15.9 10.3
19.3 8.6 31.4 15.2 10.3 10.9 8.7 4.1 3.0 3.3 5.2 31.2 2.5 5.5
20 8 28 12 12 9 6 4 4 9 9 26 2 6
21
11.0 8.5 4.3 3.9 3.3 4.7 27.3 2.7 4.8
1.0
WITH THOSE OF OTHER
1.0
6 28 12 12 9 6 4 4 8
10 27 2 6
1
1
6.2 8.9 12.2
6.4 9.6 11.6
6 13 12
6 12 I3
184.1 Val
187.8
187
187
Val
Be
Be
was calculated on the basis of the molecular weight of 22 kDa. from Yasuda et al. (1986). from Yasuda et ak(l987). from Yamaguchi et al. (1988).
24 8 27 14
10 5 9 4 8 5 3 31
1 7
1 7
11 11 186 Val
PRLs
Tilapia PRLd 20K
24K
16 12
16 9 29 18 12 6
24 22 8 4 12 4 7 4
11 4 7 7
10
10
25 2 4 I 4
26 2 5
11
11
7
9
1 7
177
188
Val
Val
396
SUZUKI ET AL.
of 22 kDa was pooled and applied to an ODS column. The elution profile of peak 3 on an ODS column is shown in Fig. 2A. Two main peaks (I and II) cross-reacted with the anti-salmon PRL in Western blotting. When stained with Coomassie blue after SDS-PAGE, peak I was contaminated with the earlier-eluting 17 kDa component, whereas peak II contained only the 22 kDa protein (Fig. 2A). When a fraction of Peak I was subjected to a TMS column with a steep gradient, the 22 kDa protein was eluted earlier than the 17 kDa protein. Peak I yielded 350 p,g and peak II yielded 1.27 mg of 22 kDa proteins. They were designated as ePRL I and ePRL II, respectively. The proteins were also isolated from the culture medium of the eel pituitary. When the insoluble fraction in 0.01 M ammonium acetate (pH 9.0) was subjected to HPLC, the retention times of the two main peaks were the same as those of PRL I and II from eel pituitary extracts (Fig. 3). Physicochemical characterization. Both PRLs had an identical isoelectric point of 10.1 (Fig. 4A). The amino acid compositions of ePRL I and II were estimated on the basis of the molecular weight of 22 kDa as determined by SDS-PAGE (Fig. 4B). As shown in Table 1, they are highly similar to each other, each having four cysteine and one tryptophan residues, as is the case for other teleostean PRLs. Valine was identified as the sole amino-terminal residue of both ePRL I and II. Figure 5 shows the elution patterns in HPLC of the fragments resulting from digestion with lysyl endopeptidase of ePRL I (130 pg) and II (250 pg). The peptide maps indicate that they are highly homologous. There was a replacement between ePRL I and II in LE 6 (BeNal), in accord with the difference in their amino acid compositions. Sequences of LE 1, 2, and 3 of ePRL II, which were identical to those of ePRL I, were determined (Fig. 6). The sequences of eel PRLs showed stronger resemblances with those
h-
LE1.2.5
,’
,-
LE’i2’5
(min) Time FIG. 5. HPLC patterns of the digests pg) (A) and ePRL II (250 pg) (B) with tidase on a TSK-gel ODS 120T column 5 pm particle size) by linear gradient containing 0.1% TFA.
,
of ePRL I(130 lysyl endopep (0.46 x 25 cm, of isopropanol
of other teleostean PRLs than with higher vertebrate PRLs. Biological properties. When tested in tilapia, the eel RPD homogenate as well as salmon PRL were inactive, although ovine PRL exhibited significant sodium-retaining activity (Fig. 7A). When hypophysectomized Fund&s were injected with eel RPD homogenate or ovine PRL and transferred from seawater to fresh water, both the RPD homogenate and ovine PRL caused a significant increase in plasma sodium after 24 hr (Fig. 7B). The PRL content in the RPD homogenate was estimated using a homologous RIA raised against the fractions containing both ePRL I and II. In the next ex-
ISOLATION
rat ovine sea turtle bullfrog tilapia 20K tilapia 24K carp chum salmon I eel
OF
EEL
397
PROLACTIN
20 30 40 50 10 KAINDPP’S;SSEADPEDR LPVCS=G=GDCQTPliPELFDR& VH~.tiYIBTtYODMFIEFDK QYVQDREFIA TPVCPNGPGDCQYSERBLFD@$ VHVBHYIBNGLSEHFIEFBK RYAPGKGFTTklALNS#W&PIPEDK LPlCPSGSVGCIJ&XENLFDiU VKf&HYIT&la SISEHFIIEFRERYAQGRGFLTKAINGI?&ZSSbT2PEDR QPlCPNGGTNCQIPTSALFDBh VKi&HYIlW SSEHFDEFDERDTDGRRFLAKSGIS@CJSS&NIPEP
. I.__
=====C&T.S&$$PTDX S$f,flYPTP S%QVPNDX ~S~~~PKDK ~~~~AS.~GHDK
FIG. 6. Alignment of the partial amino acid sequences of ePRL I and II with the corresponding N-termini of PRLs from rat (Gubbins et al., 1980), sheep (Li et al., 1970), sea turtle (Yasuda et al., 199Oa), bullfrog (Yasuda et al., 199Ob), tilapia (Yamaguchi er al., 1988). carp (Yasuda et al., 1987), and chum salmon (Yasuda et al., 1986).
periment, the sodium-retaining effect of ePRL I and II was examined in a Fundulus assay. As shown in Figure 8A, ePRL I was almost equipotent with ePRL II and ovine PRL in retaining plasma sodium, although ePRL II was significantly (P < 0.01) less potent than ovine PRL at the dose of 1 l&g. All PRL preparations also increased plasma calcium levels in hypophysectomized Fundulus transferred to fresh water (Fig. 8B).
Zmmunocytochemistry. Immunocytochemical staining of the eel pituitary with the antiserum against eel PRL showed strong cross-reactivity only with the follicular cells of the RPD, and none with the GH cells identified by an anti-eel GH (Fig. 9). DISCUSSION
Amino
acid sequences of both eel PRLs
130120-
Saline +
110 6 1 I 1 , 100 L 0.5 2.5 5 0.2 1 2.5 5 PRL (,ug/g BW) PRL Ggfg BW) FIG. 7. Sodium-retaining activity of eel PRL. (A) Tilapia assay. Eel RPD homogenate (A), salmon PRL (O), or ovine PRL (0) were injected into tilapia acclimated to 50% seawater daily for 5 consecutive days. The fish were sacrificed 16 hr after the last injection for blood sampling. Vertical bars indicate standard errors of the means (N = 5-12 per treatment). *Signiticantly (P < 0.01) different from saline-injected group (m) by the Duncan’s multiple range test. (B) Fundulus assay. Hypophysectomized Fundulus were injected with eel RPD homogenate (A) or ovine PRL (0) and transferred from seawater to fresh water for 22-24 hr until blood sampling. Vertical bars indicate standard errors of the means (N = 6-9 per treatment). *Significantly (P < 0.01) different from saline-injected group (m) by the Duncan’s multiple range test. The doses used are indicated per gram of body weight (BW). 12oL
I 0.05
398
SUZUKI ET AL.
A 180-
-
o’“&’
160-
0 Intact FW
E z”
140-
iz ia
120HX FW
+ (Saline) 1OOL
B 2.8
-
6 Q 0
2.4
-
ii s a
2.0
-
7 0’”
9
+ 1.6 -
HX I
0.2 PRL$.@g
I
AW,
FIG. 8. Effects of ePRL I (A), ePRL II (v), and ovine PRL (0) on plasma sodium (A) and calcium (B) concentrations in hypophysectomized Fundulus. The assay protocol was the same as in Fig. 7B. Vertical bars indicate standard errors of the means (N = 7-10). All PRLs restore plasma sodium concentration significantly (*P < 0.01 by the Duncan’s multiple range test) from that found in saline-injected group (m) to that of intact fish (0) in fresh water (FW) 22-24 hr after transfer from seawater (SW). The doses used are indicated per gram of body weight (BW).
are highly similar to those of the other teleostean PRLs. The sequences of eel PRLs, like salmon, carp, and tilapia PRLs, differ from mammalian PRLs in that one disulfide loop is absent from the N-terminal region. They show 60-80% sequence identity with other teleost PRLs and 20-30% with mammalian PRLs. It is to be noted that sequence comparison with other vertebrate GHs revealed that eel GH is closer to avian and mammalian GHs with 55% identity than to salmon GH with 48% identity (Yamaguchi et al., 1987).
PRL cells of the eel pituitary have been identified by immunocytochemistry using the antiserum against chum salmon PRL (Naito et al., 1983); PRL cells are arranged in follicles and form the bulk of the RPD, whereas GH cells are found exclusively in the proximal pars distalis (Kishida et al., 1987). Immunocytochemistry of the eel pituitary showed that only the follicular cells were immunoreactive with anti-eel PRL raised in the present study. It has been well established that hypophysectomy results in a significant decrease in plasma sodium levels in some fish in fresh water and that PRL treatment restores the plasma sodium level to normal. PRL treatment of fish in seawater causes an elevation of plasma sodium levels. The sodium-retaining bioassay using intact tilapia acclimated to seawater (Clarke, 1973) and the assay using hypophysectomized Fundulus are specific for PRLs. In the present study, when assayed using hypophysectomized Fundulus, both ePRL I and II maintained plasma sodium. Although they seemed to be equipotent with each other and possibly with ovine PRL, no definite potency was calculated as only two doses of each hormone were tested. However, the chinook and chum salmon PRL were clearly more potent than ovine PRL in the assay of hypophysectomized Fund&us (Grau et al., 1984; Hasegawa et al., 1986). On the other hand, both eel RPD homogenate and chum salmon PRL lacked sodiumretaining activity in the assay using tilapia acclimated to 50% seawater (Fig. 7A) and in an assay using hypophysectomized tilapia treated similarly to Fund&s (data not shown), although ovine PRL had a significant effect. The present findings are in agreement with the earlier observation by Grau et al. (1984), who reported that the coho salmon RPD homogenate lacked consistent activity in sodium-retaining assay in tilapia. Thus there seems to be species difference between various PRLs and the test animals. Hypercalcemia along with hyper-
ISOLATION
OF EEL PROLACTIN
399
FIG. 9. (A) Sagittal section of an eel pituitary stained with an anti-eel PRL rabbit serum at a dilution of 1:8000. (B) Adjacent section of an eel pituitary stained with anti-eel GH rabbit serum at a dilution of 1:8000. Both sections were also stained with Mayer’s hematoxylin. X70.
natremia were observed in response to ePRL I, ePRL II and ovine PRL in hypophysectomized Fundulus. This is in accord with the report by Pang et al. (1973) on a specific hypercalcemic action of PRL in Fundulus held in calhypophysectomized cium-deficient seawater. The elution profiles of the two forms of eel PRL isolated from culture medium were identical to those of the two PRLs isolated from the pituitary extract, suggesting that the two PRLs are released into the circula-
tion. Two molecular forms of PRL are found in tilapia with 70% homology (Specker et al., 1985; Yamaguchi et al., 1988). Two forms of PRLs with greater homology (more than 97%) have also been found in salmon (Yasuda et al., 1986) and carp (Yasuda et al., 1987). Although a single PRL with trace amounts of variants has been found in mammals (Lewis, 1984; Sinha and Gilligan, 1984) two PRLs are equally dominant in these fishes as well as in the eel (see Figs. 2A and 3). The signifi-
400
SUZUKI
cance of two variants of great homology and overlapping biological activity remains to be elucidated. A previous attempt was made to isolate eel PRL. Ingleton and Stribley (1977) reported that the putative PRL band was separated by PAGE of pituitary extract of the European eel (A. anguillu), and the band was shown immunocytochemically to originate from PRL cells. However, its molecular weight was not determined. Furthermore, Knight et al. (1978) have judged the bioactivity of the European PRL band in the PAGE gel by the pigeon crop sac assay in which teleost PRL is devoid of specific lactogenic activity (Nicoll and Bern, 1968; Farmer et al., 1977) and by xanthophore dispersion test in the goby in which specificity for PRL was doubtful (Farmer et al., 1975). The European eel PRL separated by PAGE is different from the PRLs obtained in the present study. Ingleton and Stribley (1977) identified PRL as a band with Rf0.46 in PAGE, pH 9.5, whereas Japanese eel PRLs obtained in this study were not detectable with PAGE, pH 9.5, since their isoelectric point was 10.1. Another attempt to purify eel PRL secreted from the organcultured pituitaries failed to obtain PRL but resulted in isolation of GH (Kishida et al., 1987). In the present study, 400 l.r,geel PRL per 1000 pituitaries was isolated from the organ-culture medium of the eel pituitaries, although 12.7 mg of secreted GH per 1000 pituitaries was isolated from the medium (Kishida et al., 1987). However, GH content in the pituitary of freshwater eel was only 3-4 times greater than PRL content, indicating that greater amounts of GH than PRL are secreted from organ-cultured pituitaries (unpublished data). Secreted PRLs have been successfully isolated from the organ-cultured pituitaries of mouse and hamster (Kohmoto, 1975; Shoer et al., 1978; Colosi et al., 1981) and of tilapia (Specker et al., 1985), in which PRL secretion is primarily under inhibitory control by the hypothalamus. In the eel, the inhibitory con-
ET AL.
trol by the hypothalamus seems to be weaker on PRL secretion than on GH secretion (Suzuki et al., 1990). ACKNOWLEDGMENTS We express our gratitude to Professor A. Urano, Ocean Research Institute, University of Tokyo, for his continuous interest and encouragement, to Dr. K. Yamaguchi, Tokyo Laboratory, Kyowa Hakko Kogyo, for help in sequencing eel PRL, and to Dr. K. Ikuta, Ocean Research Institute, University of Tokyo, for assisting with the raising of antibody. We are also grateful to Professor H. A. Bern, University of California at Berkeley, for his encourgement and also for critical reading of the manuscript. This study was supported by a fellowship of the Japan Society for the Promotion of Science for Japanese Junior Scientists to R.S. and aided in part by grants-in-aid for scientific research from the Ministry of Education to T.H., H.K. and R.S. and also from the Fisheries Agency to T.H. and H.K.
REFERENCES Andersen, Id., Skibeli, V., and Gautvik, K. M. (1989). Purification and characterization of Atlantic salmon prolactin. Gen. Camp. Endocrinol. 73, 354-360. Bidlingmeyer, B. A., Cohen, S. A., and Tarvin, T. L. (1984). Rapid analysis of amino acids using precolumn derivatization. J. Chromarogr. 336, 93104. Clarke, W. C. (1973). Sodium-retaining bioassay of prolactin in the intact teleost Tilupiu mossambica acclimated to sea water. Gen. Comp. Endocrinol. 21,498-512. Clarke, W. C., and Bern, H. A. (1980). Comparative endocrinology of prolactin. In “Hormonal Proteins and Peptides” (C. H. Li, Ed.), Vol. 8, pp. 105-197. Academic Press, New York. Colosi, P., Markoff, E., Levy, A., Ogren, L., Shine, N., and Talamantes, F. (1981). Isolation and partial characterization of secreted hamster pituitary prolactin. Endocrinology 108, 850-854. Farmer, S. W., Clarke, W. C., Papkoff, H., Nishioka, R. S., Bern, H. A., and Li, C. H. (1975). Studies on the purification and properties of teleost prolactin. Life Sci. 16, 149-159. Farmer, S. W., Papkoff, H., Bewley, T. A., Hayashida, T., Nishioka, R. S., Bern, H. A., and Li, C. H. (1977). Isolation and properties of teleost prolactin. Gen. Comp. Endocrinol. 31, 60-71. Fontaine, M., Callamand, O., and Olivereau, M. (1949). Hypophyse et euryhalinite chez I’Anguille. C. R. Acad. Sci. 228, 513-514. Grau, E. G., Prunet, P., Gross, T., Nishioka, R. S.,
ISOLATION
OF
and Bern, H. A. (1984). Bioassay for salmon prolactin using hypophysectomized Fund&us heteroc&us.
Gen.
Comp.
Endocrinol.
53, 78-85.
Grau, E. G., and Stetson, M. H. (1977). Pituitary autotransplants in Fund&s heteroclitus: Effect on thyroid function. Cert. Comp. Endocrinol. 32, 427-431. Gray, W. R. (1967). Sequential degradation plus dansylation. In “Methods in Enzymology” (C. H. W. Him, Ed.), Vol. 11, pp. 46-75. Academic Press, New York. Gubbins, E. J., Maurer, R. A., Lagrimini, M., Erwin, C. R., and Donelson, J. E. (1980). Structure of the rat prolactin gene. J. Biol. Chem. 255, 865% 8662. Hasegawa, S., Hirano, T., and Kawauchi, H. (1986). Sodium-retaining activity of chum salmon prolactin in some euryhaline teleosts. Gen. Comp. Endocrinol. 63, 309-317. Hirano, T. (1986). The spectrum of prolactin action in teleosts. In “Comparative Endocrinology: Developments and Directions” (C. L. Ralph, Ed.), pp. 53-74. A. R. Liss, New York. Hirano, T., Prunet, P., Kawauchi, H., Takahashi, A., Ogasawara, T., Kubota, L., Nishioka, R. S., Bern, H. A., Takada, K., and Ishii, S. (1985). Development and validation of a salmon prolactin radioimmunoassay. Cert. Comp. Endocrinol. 59, 266-276. Idler, D. R., Shamsuzzaman, K. M., and Burton, M. P. (1978). Isolation of prolactin from salmon pituitary. Cert. Comp. Endocrinol. 35, 409-418. Ingleton, P. M., and Stribley, M. F. (1977). Immunofluorescent identification of the cells of origin of eel (Anguilla anguilla) pituitary hormones separated by polyacrylamide gel electrophoresis. Gen. Comp. Endocrinol. 31, 37-44. Kawauchi, H., Abe, K., Takahashi, A., Hirano, T., Hasegawa, S., Naito, N., and Nakai, Y. (1983). Isolation and properties of chum salmon prolactin. Cert. Comp. Endocrinol. 49, 446-458. Kishida, M., and Hirano, T. (1988). Development of radioimmunoassay for eel growth hormone. Nippon Suisan Gakkaishi 54, 1321-1327. Kishida, M., Hirano, T., Kubota, J., Hasegawa, S., Kawauchi, H., Yamaguchi, K., and Shirahata, K. (1987). Isolation of two forms of growth hormone secreted from eel pituitaries in vitro. Gen. Comp. Endocrinol.
65, 47W88.
Knight, P. J., Chadwick, A., and Ball, J. N. (1978). Biological tests on eel “prolactin” separated by gel electrophoresis. Gen. Comp. Endocrinol. 36, 30-32. Kohmoto, K. (1975). Mouse prolactin obtained by pituitary organ culture in a serum-free medium. Endocrinol.
Laemmli,
Japon.
22, 465-469.
U. K. (1970). Cleavage of structural pro-
EEL
401
PROLACTIN
teins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. Lewis, U. J. (1984). Variants of growth hormone and prolactin and their posttranslational moditications. Annu. Rev. Physiol. 46, 33-42. Li, C. H.. Dixon, J. S., Lo, T.-B., Schmidt, K. D., and Pankov, Y. A. (1970). Studies on pituitary lactogenic hormone. XXX. The primary structure of the sheep hormone. Arch. Biochem. Biophys. 141, 705-737.
Miyajima, K., Yasuda, A., Swanson, P., Kawauchi, H., Cook, H., Kaneko, T., Peter, R. E., Suzuki, R., Hasegawa, S., and Hirano, T. (1988). Isolation and characterization of carp prolactin. Gen. Comp.
Endocrinol.
10,407-417.
Muramoto, K., Sunahara, S., and Kamiya, H. (1987). Measurement of tryptophan in peptides by acidhydrolysis in the presence of phenol and its application to the amino acid sequence of a sea anemone toxin. Agric. Biol. Chem. 51, 1607-1616. Naito, N., Takahashi, A., Nakai, Y., Kawauchi, H., and Hirano, T. (1983). Immunocytochemical identification of the prolactin-secreting cells in the teleast pituitary with an antiserum to chum salmon prolactin. Gen. Comp. Endocrinol. 50, 282-291. Nicoll, C. S., and Bern, H. A. (1968). Further analysis of the occurrence of pigeon crop sac-stimulating activity (prolactin) in the vertebrate adenohypophysis. Gen. Comp. Endocrinol. 11, 5-20. Nishioka, R. S., Richman, N. H., III, Young, G., Prunet, P., and Bern, H. A. (1987). Hypophysectomy of coho salmon (Oncorhynchus kisutch) and survival in fresh water and seawater. Aquaculture 65, 343-352. Ogasawara, T., Hirano, T., Akiyama, T., Arai, S., and Tagawa, M. (1989). Changes in plasma prolactin and growth hormone concentrations during freshwater adaptation of juvenile chum salmon (Oncorhynchus keta) reared in seawater for a prolonged period. Fish Physiol. Biochem. 7, 309-313. Olivereau, M., and Ball, J. N. (1970). Pituitary influences on osmoregulation in teleosts. In “Hormones and the Environment.” Mem. Sot. Endocrinol.
18, 57-85.
Pang, P. K. T., Schreibman, M. P., and Griffith, R. W. (1973). Pituitary regulation of serum calcium levels in the killifish, Fundulus heteroclitus L. Gen. Comp. Endocrinol. 21, 536-542. Prunet, P., Boeuf, G., and Houdebine, L.-M. (1985). Plasma and pituitary prolactin levels in rainbow trout during adaptation to different salinities. .Z. Exp. Zool. 235, 187-l%. Prunet, P., and Houdebine, L.-M. (1984). Purification and biological characterization of chinook salmon prolactin. Gen. Comp. Endocrinol. 53, 49-57. Shoer, L. F., Shine, N.R., and Tammantes, F. (1978).
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Isolation and partial characterization of secreted mouse pituitary prolactin. Biochim. Biophys. Acta 537, 336-347. Sinha, Y. N., and Gilligan, T. A. (1984). A cleaved form of prolactin in the mouse pituitary gland: Identification and comparison of in vitro synthesis and release in strains with high and low incidences of mammary tumors. Endocrinology 114, 20462053. Specker, J. L., King, D. S., Nishioka, R. S., Shirahata, K., Yamaguchi, K., and Bern, H. A. (1985). Isolation and partial characterization of a pair of prolactins released in vitro by the pituitary of a cichlid fish, Oreochromis mossambicus. Proc. Natl. Acad. Sci. USA 82, 7490-7494. Suzuki, R., and Hirano, T. (1991). Development of a homologous radioimmunoassay for eel prolactin. Gen. Comp. Endocrinol., in press. Suzuki, R., Kishida, M., and Hirano, T. (1990). Growth hormone secretion during long-term incubation of the pituitary of the Japanese eel, Anguilla japonica. Fish Physiol. Biochem., 8, lS9165. Yamaguchi, K., Specker, J. L., King, D. S., Yokoo, Y., Nishioka, R. S., Hirano, T., and Bern, H. A.
(1988). Complete amino acid sequences of a pair of fish (tilapia) prolactins, tPRLtT7 and tPRL1ss. J. Biol. Chem. 263, 9113-9121. Yamaguchi, K., Yasuda, A., Kishida, M., Hirano, T., Sano, H., and Kawauchi, H. (1987). Primary structure of eel (Anguilla japonica) growth hormone. Gen. Comp. Endocrinol. 66,447-453. Yasuda, A., Itoh, H., and Kawauchi, H. (1986). Primary structure of chum salmon prolactins: Occurrence of highly conserved regions. Arch. Biothem. Biophys. 244, 528-541. Yasuda, A., Miyazima, K., Kawauchi, H., Peter, R. E., Lin, H.-R., Yamaguchi, K., and Sano, H. (1987). Primary structure of common carp prolactins. Gen. Comp. Endocrinol. 66, 280-290. Yasuda, A., Takehara, H., Papkoff, H., and Kawauchi, H. (1990). The complete amino acid sequence of prolactin from the sea turtle (Chelonia mydas). Gen. Comp. Endocrinol., 80, 363-371. Yasuda, A., Yamaguchi, K., Kobayashi, T., Yamamoto, K., Kikuyama, S., and Kawauchi, H. (1991). The complete amino acid sequence of prolactin from the bullfrog, Rana catesbeiana. Gen. Comp. Endocrinol., in press.
AND
COMPARATIVE
Isolation
ENDOCRINOLOGY
81, 391-402 (1991)
and Characterization
of Japanese
Eel Prolactins
REIKO SUZUKI, AKIKAZU YASUDA,* JUN KONDO,~ HIROSHI KAWAUCHI,* AND TETSUYA HIRANO Ocean Research Institute, Universty of Tokyo, Nakano, Tokyo 164; *Laboratory of Molecular Endocrinology, School of Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-01, Japan; fMitsubishi Kasei Co., Ltd., Research Center, Yokohama, Kanagawa 227, Japan Accepted April 6, 1990 A highly purified prolactin (PRL) was isolated from the pituitary of the Japanese eel (Anguilla japonica) by extraction with acid-acetone, gel filtration on Sephadex G-75, and reversed-phase HPLC on TSK-gel ODS 120T and on TSK-gel TMS 250. Eel PRL is comprised of two variants (ePRL I and II), which were separated by HPLC on an ODS column. The two PRLs were also secreted by organ-cultured pituitaries in a defined medium. After being dialyzed against distilled water and lyophilized, the medium was dissolved in 0.01 M ammonium acetate (pH 9.0), and then the insduble fraction was subjected to HPLC on an ODS column to isolate the secreted PRLs. The ePRL I and II were equipotent in retaining plasma Na in the hypophysectomized killiiish, Fund&s heteroclitus, transferred from seawater to fresh water. The putative PRL-producing cells in the rostral pars distalis of the eel pituitary were specifically stained with the antiserum against the mixture of ePRL I and II. Both PRLs had a molecular weight of 22 kDa in SDS-PAGE, an isoelectric point of 10.1 by gel electrofocusing, and an N-terminal residue of valine. Amino acid compositions and the partial amino acid sequences of ePRL I and II show that they are highly homologous with a limited number of substitutions, and that they are more closely related to those of teleostean PRLs than to those of mammalian PRLs. o IWI Academic PRSS, h.
and homologous radioimmunoassays are available to measure their plasma and pituitary concentrations (Hirano et al., 1985). The effects of homologous PRL on hydromineral balance as well as the changes in plasma PRL concentrations following exposure to various salinities suggest that PRL is involved also in freshwater adaptation in salmonid fishes, even though it is not essential for their survival in fresh water (Hirano et al., 1985; Prunet et al., 1985; Hasegawa et al., 1986; Ogasawara et al., 1989). The migratory pattern of the eel is opposite to that of salmon; eels grow in fresh water and, as they mature, go downstrean to spawn. The actions of PRL in eel osmoregulation may be different from those in salmonids. However, eels have received less attention than salmonids, although there are a few histological studies indicat-
Prolactin (PRL) is an important freshwater-adapting hormone for several euryhaline teleosts such as the killifish, tilapia, and molly, in which hypophysectomy without PRL therapy impairs survival in fresh water (see Clarke and Bern, 1980; Hirano, 1986). In contrast, the eel and salmonids survive in fresh water after hypophysectomy (Fontaine et al., 1949; Nishioka et al., 1987). While studies on the role of PRL in teleost osmoregulation have been extended to examine the direct effects of PRL on ionand water-transporting epithelia, the differences in the mode of PRL action between the species dependent on PRL for their survival in fresh water and those not so dependent have not been elucidated. In salmonids, chum, chinook, and Atlantic salmon PRL have been isolated (Idler et al., 1978; Kawauchi et al., 1983; Prunet and Houdebine, 1984; Andersen et al., 1989) 391
00164480191 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
392
SUZUKI
ing that PRL cells are more active in fresh water than in seawater (Olivereau and Ball, 1970; Naito et al., 1983). We report here the isolation of two variants of eel PRL and their physicochemical and biological characteristics, including partial amino acid sequences. MATERIALS
AND METHODS
Materials. Pituitary glands were collected from immature, cultured Japanese eel (Anguillu juponicu), weighing 150-200 g. Four thousand pituitaries were frozen on dry ice, and stored for 1-2 months at - 80”. Nine hundred pituitaries were cultured in a defined medium following the protocol of Kishida et al. (1987), under which condition the pituitaries were viable to continue to synthesize and secrete growth hormone for several weeks. Twenty pituitaries were cultured in a plastic dish (diameter 59 mm, depth 15 mm) in 8 ml of Medium 199 with Earle’s salts supplemented with 100 U/ml streptomycin, and 0.25 p&ml Fungizone 100 U/ml penicillin, (M. A. Bioproducts, Walkersville, MD) in a gas phase of 95% 0, and 5% CO, at 22” for 4-6 weeks. The pH of the medium was adjusted to 7.3-7.4 by adding sodium bicarbonate and the osmotic pressure was adjusted to 290-300 mOsm. The medium was changed once a week and stored at -20”. Isolation. The frozen pituitaries (5 g) were extracted with acid-acetone as described for salmon PRL (Kawauchi et al., 1983). The extract (100 mg) was subjected to gel filtration on a Sephadex G-75 column (2 x 90 cm) equilibrated with 0.1 N acetic acid. Pooled fractions were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970). The fraction containing the protein with a molecular weight of 22 kDa was dissolved in 0.1% trifluoroacetic acid (TFA) and subjected to highperformance liquid chromatography (HPLC) using a reversed-phase column (0.46 x 25 cm, TSK-gel ODS 120T and TMS 250) at a column temperature of 40” and a flow rate of 1 mUmin. Linear gradient elution was performed with acetonitrile containing 0.1% TFA. The eluate was monitored by measuring absorbance at 220 nm. Each peak was collected in one tube and the main fraction was analyzed by Western blotting after SDSPAGE following the protocol of Miyajima et ul. (1988) using an antiserum against chum salmon PRL (Kawauchi et al. 1983). PRL secreted into the culture medium was purified after dialyzing the pooled medium against distilled water. The dialyzate was lyophilized and then dissolved in 0.01 M ammonium acetate (pH 9.0). The insoluble fraction was dissolved in 0.1% TFA containing 1 M urea and subjected to HPLC on an ODS column (0.46
ET
AL.
X 25 cm) under the same elution conditions described above. Chemical and physicochemicul churucterizution. Molecular weight was estimated by SDS-PAGE. Molecular weight markers (14,300-71,500, BDH Chemicals) were employed as the reference standards. The isoelectric point was estimated by gel isoelectric focusing in 5% polyacrylamide gel at 2% ampholine (pH 3.5-10) and staining with 0.06% Coomassie brilliant blue G-250 in 3.2% sulfosalicylic acid, 5% trichloroacetic acid, and 50% ethanol. An isoelectric focusing p1 calibration kit (Pharmacia) was used for determination of the pH gradient profile. For amino acid analysis, the proteins were hydrolyzed for 18-22 hr at 110” with constantly boiling HCl containing 0.6% phenol (Muramoto et al., 1987). Amino acid analyses were carried out by HPLC on a TSK-gel ODS 80TM (0.46 x 25 cm) with a precolumn labeling method using 6 3:
4
0
50
100
Fraction Number FIG. 1. Elution profile of an acid-acetone extract of Japanese eel (Anguilla juponica) pituitary glands on a Sephadex G-75 column (2 x 90 cm) equilibrated with 0.1 N acetic acid. Flow rate, 15 ml/hr; fraction size, 2.5 ml. VJV, of peak 3 was 1.54.
ISOLATION
393
OF EEL PROLACTIN
phenylisothiocyanate (Bidlingmeyer et al., 1984). Phenylthiocarbamyl amino acids were developed using a linear gradient of 0.14 M sodium acetate (pH 5.4) and acetonitrile. Half-cystine content was estimated after performic acid oxidation of the protein. Nterminal analysis was performed by the dansyl method (Gray, 1%7). Intact PRL was digested with lysyl endopeptidase (E/S = l/60, by weight) in 0.1 M ammonium bicarbonate containing 4 M urea, pH 8.0, at 37 for 4 hr. The resulting fragments were subjected to HPLC on a TSK-gel ODS 120T column using a linear gradient with isopropanol in 0.1% TFA. Elution was monitored at 210 nm and individual peaks were collected in separate tubes. Partial sequences of some fragments were determined using gas-phase sequencers (Applied Biosystem 470A and 477A-120A).
Biological activi@. Sodium-retaining activity of the eel PRL was examined using intact tilapia and hypophysectomized Fundulus. The assay for intact tilapia, Oreochrumis niloticus, was carried out following the slightly modified protocol of Clarke (1973). Tilapia (4-5 g) were acclimated to 50% seawater (23”) for more than 2 weeks before use under natural photoperiod in Tokyo. They were anesthetized with 0.03% 2phenoxyethanol and given intraperitoneal injections of 1.5 pl of saline (0.9% NaCl), homogenate of eel rostra1 pars distalis (RPD), chum salmon PRL, or ovine PRL (NIAMDD o-PRL-11) between 1800 and 1900 hr daily for 5 days. Eel RPD was homogenized in a small volume of 0.1 N acetic acid and diluted with saline to prescribed doses which were determined by a homologous RIA for eel PRL (Suzuki and Hirano, 1991). The
I II
3
14 0
40 Time
(min)
, ,I L 0
40 Time
(min
)
FIG. 2. High-performance liquid chromatography (HPLC) patterns of peak 3 in Fig. 1. The sample was dissolved in 0.1% trifluoroacetic acid (TFA). The column temperature was maintained at 40”. Flow rate was I ml/min. Elution was performed by a linear gradient of acetonitrile containing 0.1% TFA. Five micrograms of proteins in main peaks were subjected to SDS-PAGE and stained with Coomassie blue. (A) Elution pattern of fraction on a TSK-gel ODS 120T column (0.46 x 25 cm, 5 Km particle size). Fractions I and II were designated as ePRL I and ePRL II. (B) Repeated chromatography of fraction I on a TSK-gel TMS 250 column (0.46 x 25 cm, 10 pm particle size).
394
SUZUKI
preparation contained only 3% eel growth hormone (GH) which was detected by a specific RlA for eel GH (Kishida and Hirano, 1988). Hormones were dissolved in a small volume of 0.1 N acetic acid and diluted with saline. The fish were sacrificed 16 hr after the last injection, and the blood was collected from the caudal artery. Plasma was separated by centrifugation at 10,OtM rpm for 10 min. Sodium levels were determined by atomic absorption spectrophotometry (Hitachi 18050). Male kill&h, Fund&s hereroclitus, weighing from 3 to 5 g, were kept in seawater at 22”. Fundulus assay was carried out following the protocol of Grau et al. (1984). Hypophysectomy was performed as described by Grau and Stetson (1977). Following hypophysectomy, the fish were held for 1 week in seawater. Hypophysectomized fish were injected intraperitoneally with 25 pl saline, homogenate of eel RPD, or hormones between 1800 and 2000 hr. After the injections, fish were maintained in seawater for 1 hr and then transferred to freshwater aquaria (Na, 0.4 mM; Ca, 0.35-0.45 n&f) for 22-24 hr until blood sampling. The statistical analysis used was the Duncan’s multiple range test. Immunocytochemistry. The eel PRL antiserum was raised in a young rabbit. Emulsion (100 pl) containing 25 pg eel PRL (mixture of ePRL I and II) and 0.5 mg killed and dried Mycobacterium butyricum (Difco) was injected into each lymph node of the right and left hind limbs. Emulsion (2 pl) containing 50 pg eel PRL and 1 mg killed and dried M. butyricum was subcutaneously injected in the back of the rabbit three times at intervals of 3 weeks after the first inguinal injection. Eel PRL was dissolved in a small volume of 0.1 N acetic acid and diluted with saline. The emulsion was made by mixing equal volumes of a saline solution of eel PRL and of Freund’s complete adjuvant. The rabbit was completely bled from the carotid artery 2 weeks after the last injection and the serum was stored at - 40”. A pituitary of the eel was fixed in Bouin’s fluid and embedded in paraplast; serial sagittal sections were cut (4 pm thick). Immunocytochemical staining of PRL producing cells was carried out using the peroxidase-antiperoxidase method (Naito et al., 1983). and an adjacent section was also stained with the eel GH antiserum (Kishida et al., 1987).
RESULTS
Isolation of eel PRL. When an acidacetone extract of eel pituitaries and the culture medium in which eel pituitaries were incubated were subjected to SDSPAGE and then to Western blotting, the band corresponding to a molecular weight
ET AL.
of 22 kDa reacted with the antiserum against salmon PRL. The elution pattern of an acid-acetone extract of pituitaries on Sephadex G-75 is shown in Fig. 1. Peak 3 containing the protein of molecular weight
#’I’ #’ ,I’#’ I: a’,’ #’ I’ ,#’ /,’ , 1,.8’
L 0
I 40
Time (min) FIG. 3. Analytical HPLC of PRL fractions on a TSK-gel ODS 120T column (0.46 x 25 cm, particle size 5 pm). (A) The insoluble fraction of culture medium in 0.01 M ammonium acetate (pH 9.0). The fraction was dissolved in 0.1% TFA containing 1 M urea. (B) The acid-acetone extract of eel pituitaries (see legend to Fig. 2A).
ISOLATION
395
OF EEL PROLACTIN
B \
5.0
4.8 9 2 5 Ip8
s"
ePRL I,11 (22,000)
4.2
(14,300)
6
/-
4.0 5
J
0
0.2
0.4
0.6
0.8
,,‘-,, (28,600)
4.4
7
,
(42,900)
\
4.6
1.0
0
0.2
0.4
Mobility
0.6
0.8
1.0
Mobility
FIG. 4. (A) Estimation of the isoelectric point of ePRL I and II by gel isoelectric focusing. The pH gradient profiile was determined using the pI calibration kit (Pharrnacia; open circles) and chum salmon PRL of p1 10.3 (open triangle). (B) Estimation of the molecular weight by SDS-PAGE. Open circles indicate molecular weight markers (BDH Chemicals).
AMINO
ACID
COMPOSITIONS
Eel PRL”
Asp Thr Ser Glu FYO
GlY Ala CYS Val Met Ile Leu ‘W Phe Trp His LYS Arg Total N-aa a Value b Taken ‘Taken d Taken
TABLE 1 PRLs COMPARED
OF EEL
Salmon PRLb
Carp’ PRL
I
II
I
II
19.5 9.0 30.6 15.9 10.3
19.3 8.6 31.4 15.2 10.3 10.9 8.7 4.1 3.0 3.3 5.2 31.2 2.5 5.5
20 8 28 12 12 9 6 4 4 9 9 26 2 6
21
11.0 8.5 4.3 3.9 3.3 4.7 27.3 2.7 4.8
1.0
WITH THOSE OF OTHER
1.0
6 28 12 12 9 6 4 4 8
10 27 2 6
1
1
6.2 8.9 12.2
6.4 9.6 11.6
6 13 12
6 12 I3
184.1 Val
187.8
187
187
Val
Be
Be
was calculated on the basis of the molecular weight of 22 kDa. from Yasuda et al. (1986). from Yasuda et ak(l987). from Yamaguchi et al. (1988).
24 8 27 14
10 5 9 4 8 5 3 31
1 7
1 7
11 11 186 Val
PRLs
Tilapia PRLd 20K
24K
16 12
16 9 29 18 12 6
24 22 8 4 12 4 7 4
11 4 7 7
10
10
25 2 4 I 4
26 2 5
11
11
7
9
1 7
177
188
Val
Val
396
SUZUKI ET AL.
of 22 kDa was pooled and applied to an ODS column. The elution profile of peak 3 on an ODS column is shown in Fig. 2A. Two main peaks (I and II) cross-reacted with the anti-salmon PRL in Western blotting. When stained with Coomassie blue after SDS-PAGE, peak I was contaminated with the earlier-eluting 17 kDa component, whereas peak II contained only the 22 kDa protein (Fig. 2A). When a fraction of Peak I was subjected to a TMS column with a steep gradient, the 22 kDa protein was eluted earlier than the 17 kDa protein. Peak I yielded 350 p,g and peak II yielded 1.27 mg of 22 kDa proteins. They were designated as ePRL I and ePRL II, respectively. The proteins were also isolated from the culture medium of the eel pituitary. When the insoluble fraction in 0.01 M ammonium acetate (pH 9.0) was subjected to HPLC, the retention times of the two main peaks were the same as those of PRL I and II from eel pituitary extracts (Fig. 3). Physicochemical characterization. Both PRLs had an identical isoelectric point of 10.1 (Fig. 4A). The amino acid compositions of ePRL I and II were estimated on the basis of the molecular weight of 22 kDa as determined by SDS-PAGE (Fig. 4B). As shown in Table 1, they are highly similar to each other, each having four cysteine and one tryptophan residues, as is the case for other teleostean PRLs. Valine was identified as the sole amino-terminal residue of both ePRL I and II. Figure 5 shows the elution patterns in HPLC of the fragments resulting from digestion with lysyl endopeptidase of ePRL I (130 pg) and II (250 pg). The peptide maps indicate that they are highly homologous. There was a replacement between ePRL I and II in LE 6 (BeNal), in accord with the difference in their amino acid compositions. Sequences of LE 1, 2, and 3 of ePRL II, which were identical to those of ePRL I, were determined (Fig. 6). The sequences of eel PRLs showed stronger resemblances with those
h-
LE1.2.5
,’
,-
LE’i2’5
(min) Time FIG. 5. HPLC patterns of the digests pg) (A) and ePRL II (250 pg) (B) with tidase on a TSK-gel ODS 120T column 5 pm particle size) by linear gradient containing 0.1% TFA.
,
of ePRL I(130 lysyl endopep (0.46 x 25 cm, of isopropanol
of other teleostean PRLs than with higher vertebrate PRLs. Biological properties. When tested in tilapia, the eel RPD homogenate as well as salmon PRL were inactive, although ovine PRL exhibited significant sodium-retaining activity (Fig. 7A). When hypophysectomized Fund&s were injected with eel RPD homogenate or ovine PRL and transferred from seawater to fresh water, both the RPD homogenate and ovine PRL caused a significant increase in plasma sodium after 24 hr (Fig. 7B). The PRL content in the RPD homogenate was estimated using a homologous RIA raised against the fractions containing both ePRL I and II. In the next ex-
ISOLATION
rat ovine sea turtle bullfrog tilapia 20K tilapia 24K carp chum salmon I eel
OF
EEL
397
PROLACTIN
20 30 40 50 10 KAINDPP’S;SSEADPEDR LPVCS=G=GDCQTPliPELFDR& VH~.tiYIBTtYODMFIEFDK QYVQDREFIA TPVCPNGPGDCQYSERBLFD@$ VHVBHYIBNGLSEHFIEFBK RYAPGKGFTTklALNS#W&PIPEDK LPlCPSGSVGCIJ&XENLFDiU VKf&HYIT&la SISEHFIIEFRERYAQGRGFLTKAINGI?&ZSSbT2PEDR QPlCPNGGTNCQIPTSALFDBh VKi&HYIlW SSEHFDEFDERDTDGRRFLAKSGIS@CJSS&NIPEP
. I.__
=====C&T.S&$$PTDX S$f,flYPTP S%QVPNDX ~S~~~PKDK ~~~~AS.~GHDK
FIG. 6. Alignment of the partial amino acid sequences of ePRL I and II with the corresponding N-termini of PRLs from rat (Gubbins et al., 1980), sheep (Li et al., 1970), sea turtle (Yasuda et al., 199Oa), bullfrog (Yasuda et al., 199Ob), tilapia (Yamaguchi er al., 1988). carp (Yasuda et al., 1987), and chum salmon (Yasuda et al., 1986).
periment, the sodium-retaining effect of ePRL I and II was examined in a Fundulus assay. As shown in Figure 8A, ePRL I was almost equipotent with ePRL II and ovine PRL in retaining plasma sodium, although ePRL II was significantly (P < 0.01) less potent than ovine PRL at the dose of 1 l&g. All PRL preparations also increased plasma calcium levels in hypophysectomized Fundulus transferred to fresh water (Fig. 8B).
Zmmunocytochemistry. Immunocytochemical staining of the eel pituitary with the antiserum against eel PRL showed strong cross-reactivity only with the follicular cells of the RPD, and none with the GH cells identified by an anti-eel GH (Fig. 9). DISCUSSION
Amino
acid sequences of both eel PRLs
130120-
Saline +
110 6 1 I 1 , 100 L 0.5 2.5 5 0.2 1 2.5 5 PRL (,ug/g BW) PRL Ggfg BW) FIG. 7. Sodium-retaining activity of eel PRL. (A) Tilapia assay. Eel RPD homogenate (A), salmon PRL (O), or ovine PRL (0) were injected into tilapia acclimated to 50% seawater daily for 5 consecutive days. The fish were sacrificed 16 hr after the last injection for blood sampling. Vertical bars indicate standard errors of the means (N = 5-12 per treatment). *Signiticantly (P < 0.01) different from saline-injected group (m) by the Duncan’s multiple range test. (B) Fundulus assay. Hypophysectomized Fundulus were injected with eel RPD homogenate (A) or ovine PRL (0) and transferred from seawater to fresh water for 22-24 hr until blood sampling. Vertical bars indicate standard errors of the means (N = 6-9 per treatment). *Significantly (P < 0.01) different from saline-injected group (m) by the Duncan’s multiple range test. The doses used are indicated per gram of body weight (BW). 12oL
I 0.05
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SUZUKI ET AL.
A 180-
-
o’“&’
160-
0 Intact FW
E z”
140-
iz ia
120HX FW
+ (Saline) 1OOL
B 2.8
-
6 Q 0
2.4
-
ii s a
2.0
-
7 0’”
9
+ 1.6 -
HX I
0.2 PRL$.@g
I
AW,
FIG. 8. Effects of ePRL I (A), ePRL II (v), and ovine PRL (0) on plasma sodium (A) and calcium (B) concentrations in hypophysectomized Fundulus. The assay protocol was the same as in Fig. 7B. Vertical bars indicate standard errors of the means (N = 7-10). All PRLs restore plasma sodium concentration significantly (*P < 0.01 by the Duncan’s multiple range test) from that found in saline-injected group (m) to that of intact fish (0) in fresh water (FW) 22-24 hr after transfer from seawater (SW). The doses used are indicated per gram of body weight (BW).
are highly similar to those of the other teleostean PRLs. The sequences of eel PRLs, like salmon, carp, and tilapia PRLs, differ from mammalian PRLs in that one disulfide loop is absent from the N-terminal region. They show 60-80% sequence identity with other teleost PRLs and 20-30% with mammalian PRLs. It is to be noted that sequence comparison with other vertebrate GHs revealed that eel GH is closer to avian and mammalian GHs with 55% identity than to salmon GH with 48% identity (Yamaguchi et al., 1987).
PRL cells of the eel pituitary have been identified by immunocytochemistry using the antiserum against chum salmon PRL (Naito et al., 1983); PRL cells are arranged in follicles and form the bulk of the RPD, whereas GH cells are found exclusively in the proximal pars distalis (Kishida et al., 1987). Immunocytochemistry of the eel pituitary showed that only the follicular cells were immunoreactive with anti-eel PRL raised in the present study. It has been well established that hypophysectomy results in a significant decrease in plasma sodium levels in some fish in fresh water and that PRL treatment restores the plasma sodium level to normal. PRL treatment of fish in seawater causes an elevation of plasma sodium levels. The sodium-retaining bioassay using intact tilapia acclimated to seawater (Clarke, 1973) and the assay using hypophysectomized Fundulus are specific for PRLs. In the present study, when assayed using hypophysectomized Fundulus, both ePRL I and II maintained plasma sodium. Although they seemed to be equipotent with each other and possibly with ovine PRL, no definite potency was calculated as only two doses of each hormone were tested. However, the chinook and chum salmon PRL were clearly more potent than ovine PRL in the assay of hypophysectomized Fund&us (Grau et al., 1984; Hasegawa et al., 1986). On the other hand, both eel RPD homogenate and chum salmon PRL lacked sodiumretaining activity in the assay using tilapia acclimated to 50% seawater (Fig. 7A) and in an assay using hypophysectomized tilapia treated similarly to Fund&s (data not shown), although ovine PRL had a significant effect. The present findings are in agreement with the earlier observation by Grau et al. (1984), who reported that the coho salmon RPD homogenate lacked consistent activity in sodium-retaining assay in tilapia. Thus there seems to be species difference between various PRLs and the test animals. Hypercalcemia along with hyper-
ISOLATION
OF EEL PROLACTIN
399
FIG. 9. (A) Sagittal section of an eel pituitary stained with an anti-eel PRL rabbit serum at a dilution of 1:8000. (B) Adjacent section of an eel pituitary stained with anti-eel GH rabbit serum at a dilution of 1:8000. Both sections were also stained with Mayer’s hematoxylin. X70.
natremia were observed in response to ePRL I, ePRL II and ovine PRL in hypophysectomized Fundulus. This is in accord with the report by Pang et al. (1973) on a specific hypercalcemic action of PRL in Fundulus held in calhypophysectomized cium-deficient seawater. The elution profiles of the two forms of eel PRL isolated from culture medium were identical to those of the two PRLs isolated from the pituitary extract, suggesting that the two PRLs are released into the circula-
tion. Two molecular forms of PRL are found in tilapia with 70% homology (Specker et al., 1985; Yamaguchi et al., 1988). Two forms of PRLs with greater homology (more than 97%) have also been found in salmon (Yasuda et al., 1986) and carp (Yasuda et al., 1987). Although a single PRL with trace amounts of variants has been found in mammals (Lewis, 1984; Sinha and Gilligan, 1984) two PRLs are equally dominant in these fishes as well as in the eel (see Figs. 2A and 3). The signifi-
400
SUZUKI
cance of two variants of great homology and overlapping biological activity remains to be elucidated. A previous attempt was made to isolate eel PRL. Ingleton and Stribley (1977) reported that the putative PRL band was separated by PAGE of pituitary extract of the European eel (A. anguillu), and the band was shown immunocytochemically to originate from PRL cells. However, its molecular weight was not determined. Furthermore, Knight et al. (1978) have judged the bioactivity of the European PRL band in the PAGE gel by the pigeon crop sac assay in which teleost PRL is devoid of specific lactogenic activity (Nicoll and Bern, 1968; Farmer et al., 1977) and by xanthophore dispersion test in the goby in which specificity for PRL was doubtful (Farmer et al., 1975). The European eel PRL separated by PAGE is different from the PRLs obtained in the present study. Ingleton and Stribley (1977) identified PRL as a band with Rf0.46 in PAGE, pH 9.5, whereas Japanese eel PRLs obtained in this study were not detectable with PAGE, pH 9.5, since their isoelectric point was 10.1. Another attempt to purify eel PRL secreted from the organcultured pituitaries failed to obtain PRL but resulted in isolation of GH (Kishida et al., 1987). In the present study, 400 l.r,geel PRL per 1000 pituitaries was isolated from the organ-culture medium of the eel pituitaries, although 12.7 mg of secreted GH per 1000 pituitaries was isolated from the medium (Kishida et al., 1987). However, GH content in the pituitary of freshwater eel was only 3-4 times greater than PRL content, indicating that greater amounts of GH than PRL are secreted from organ-cultured pituitaries (unpublished data). Secreted PRLs have been successfully isolated from the organ-cultured pituitaries of mouse and hamster (Kohmoto, 1975; Shoer et al., 1978; Colosi et al., 1981) and of tilapia (Specker et al., 1985), in which PRL secretion is primarily under inhibitory control by the hypothalamus. In the eel, the inhibitory con-
ET AL.
trol by the hypothalamus seems to be weaker on PRL secretion than on GH secretion (Suzuki et al., 1990). ACKNOWLEDGMENTS We express our gratitude to Professor A. Urano, Ocean Research Institute, University of Tokyo, for his continuous interest and encouragement, to Dr. K. Yamaguchi, Tokyo Laboratory, Kyowa Hakko Kogyo, for help in sequencing eel PRL, and to Dr. K. Ikuta, Ocean Research Institute, University of Tokyo, for assisting with the raising of antibody. We are also grateful to Professor H. A. Bern, University of California at Berkeley, for his encourgement and also for critical reading of the manuscript. This study was supported by a fellowship of the Japan Society for the Promotion of Science for Japanese Junior Scientists to R.S. and aided in part by grants-in-aid for scientific research from the Ministry of Education to T.H., H.K. and R.S. and also from the Fisheries Agency to T.H. and H.K.
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