Fish Physiology and Biochemistry vol. 12 no. 4 pp 337-345 (1993) Kugler Publications, Amsterdam/New York
Chromatographic and immunological identification of gonadotropinreleasing hormone in five marine teleosts Koichi Okuzawa,l Masafumi Amano, 2 Katsumi Aida, 2 Yoshihisa Hasegawa, 3 Hideki Tanaka and Hirohiko Kagawal INational Research Institute of Aquaculture, Nansei, Watarai, Mie 516-01, Japan; 2 Department of Fisheries, Faculty of Agriculture, University of Tokyo, Bunkyo, Tokyo 113, Japan; 3 School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034, Japan Accepted: July 23, 1993 Keywords: GnRH, salmon GnRH, chicken GnRH-II, HPLC, RIA, bluefin tuna, red seabream, black seabream, red spotted grouper, Japanese flounder
Abstract Brain extracts from bluefin tuna, Thunnus thynnus, red seabream, Pagrusmajor, black seabream, Acanthopagrus schlegeli, red spotted grouper, Epinephelus akaaraand Japanese flounder, Paralichthysolivaceus, were analyzed by high performance liquid chromatography (HPLC) and specific radioimmunoassays. Immunoreactive material co-eluting from HPLC with salmon gonadotropin-releasing hormone (GnRH) and chicken GnRH-II, respectively, was found in all five species. In addition, a GnRH immunoreactive fraction showing the same HPLC retention time as lamprey GnRH-I was detected in the brain extracts of all species examined when using an unspecific radioimmunoassay which detects several GnRH forms, including lamprey GnRH-I. In the Japanese flounder brain extract, a fourth GnRH immunoreactive fraction was detected with the unspecific radioimmunoassay which did not co-elute with any of the six synthetic GnRH standards used in the present study.
Introduction Eight different forms of gonadotropin-releasing hormone (GnRH), mammalian GnRH (Burgus et al. 1972; Matsuo et al. 1971), chicken GnRH-I (King and Millar 1982a, b; Miyamoto et al. 1982, 1983), chicken GnRH-II (Miyamoto et al. 1984), salmon GnRH (Sherwood et al. 1983), lamprey GnRH-I (Sherwood et al. 1986), catfish GnRH (Ngamvongchon et al. 1992a; Bogerd et al. 1992), dogfish GnRH (Lovejoy et al. 1992), and lamprey GnRH-III (Sower et al. 1993) have been identified in vertebrate brain extracts, which constitute a
family of structurally related decapeptides. In teleosts, three of the eight known GnRH molecules, namely salmon GnRH, chicken GnRHII and catfish GnRH have been purified and their amino acid sequences have been determined. Salmon GnRH was first purified and characterized from chum salmon Oncorhynchus keta (Sherwood et al. 1983), and later from codfish Gadus morhua morhua brain extracts (Wu et al. 1986). In the Thai catfish Clarias macrocephalus (Ngamvongchon et al. 1992a) and in the African catfish Clariasgariepinus (Bogerd et al. 1992), both catfish GnRH and chicken GnRH-II were identified. Recently, the
Correspondenceto: Koichi Okuzawa, National Research Institute of Aquaculture, Inland Station, Tamaki, Waterai, Mie 519-04, Japan
338 Bluefin tuna s
M CIL CII
3
V
.
A. sGnRH Ab (Lot No.2)
I
(3 'L mmm
t3
3-
B. cGnRH-I Ab (Lot R-E)
r
(: .)
0 4-
3-
C. cGnRH-II Ab (Lot R-II)
,
I n
1
10
20
30
40
50
Fractions Fig. 1. Reverse-phase HPLC of bluefin tuna Thunnus thynnus brain extract followed by salmon GnRH RIA (A), chicken GnRH-II RIA (B) and "GnRHs" RIA (C). Arrows indicate the elution positions of synthetic GnRH standards: M, mammalian GnRH; CI, chicken GnRH-I; L, lamprey GnRH-I; CII, chicken GnRH-II; S, salmon GnRH. The amount of ir-GnRH in the 1-ml HPLC fractions is represented by black bars. The dotted line represents the percentage of acetonitrile in the mobile phase.
complementary DNA (cDNA) encoding the precursor for salmon GnRH was characterized in an African cichlid Haplochromis burtoni (Bond et al. 1991), in Atlantic salmon Salmo salar(Klungland et al. 1992), and in the masu salmon Oncorhynchus masou (Suzuki et al. 1992), confirming the presence of salmon GnRH in these species. In addition to these studies, a combination of high performance liquid chromatography (HPLC)
and radioimmunoassay (RIA) techniques has been applied to study GnRH peptides in teleosts, revealing that at least two members of the GnRH peptide family are present in the brain of several species. Both salmon GnRH and chicken GnRH-II are common in many teleost fish such as the rainbow trout Oncorhynchus mykiss (Sherwood et al. 1984; Okuzawa et al. 1990), the masu salmon (Amano et al. 1992a), the goldfish Carassiusauratus(Yu et al. 1988; Kobayashi et al. 1992), the common carp Cyprinus carpio (Amano et al. 1992b), the molly Poecilia latipinna (Coe et al. 1992), the herring Clupeaharenguspallasi(Sherwood 1986), the milkfish Chanos chanos (Sherwood et al. 1984), and the mullet Mugil cephalus (Sherwood et al. 1984). However, mammalian GnRH has been identified in the eel Anguilla anguilla (King et al. 1990). Moreover, the possible presence of novel GnRH forms has been suggested in the hake Merluccius capensis (King and Millar 1985), in tilapia Tilapia sparrmanii (King and Millar 1985), and in the wrasse Coris julis (Powell et al. 1986). In marine teleosts, such as perciform fish (the largest group of teleosts), only a limited number of studies (see above) has been carried out; so that detailed information on the nature of the GnRH peptides is missing. The purpose of the present study therefore was to collect data on the identity of the GnRH peptides in a number of marine fish species. To this end, brain extracts were subjected to HPLC and four different RIA systems were then used to detect GnRH immunoreactive material in the HPLC fractions. Three of these RIAs were specific for salmon GnRH, chicken GnRH-II and mammalian GnRH, respectively, whereas the fourth one was an unspecific system and detected several different GnRH forms.
Materials and methods Fish brains Specimen of underyearling bluefin tuna, and of adult red seabream, black seabream, and red spotted grouper were purchased from local fishermen in the Gokasho bay, Mie prefecture. The fish
339 Black seabream
Grouper s
M CILCH
3-
4-
T
I C
0
o
A. sGnRH Ab
2.
s
M CILCII
A_
3
(Lot No.2)
I L 1C
A. sGnRH Ab (Lot No.2)
I rC
CD 1 U,
L U
=
...........
..-
.-.-. .... ....................................
'
'
~`
'~ '
'
'
3
_
Z'
o
o
4.
.t
B. cGnRH-II Ab (Lot R-E)
c
S 3
B. cGnRH-II Ab (Lot R-E)
± 2-
a
C
O 1 L
0 5
40
6
-40
5'
4
C'E .2 Ct 3
C. cGnRH-II Ab
.
30
-.......................
S
20
"E
O
10 0
C.
Chromatographic and immunological identification of gonadotropinreleasing hormone in five marine teleosts Koichi Okuzawa,l Masafumi Amano, 2 Katsumi Aida, 2 Yoshihisa Hasegawa, 3 Hideki Tanaka and Hirohiko Kagawal INational Research Institute of Aquaculture, Nansei, Watarai, Mie 516-01, Japan; 2 Department of Fisheries, Faculty of Agriculture, University of Tokyo, Bunkyo, Tokyo 113, Japan; 3 School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034, Japan Accepted: July 23, 1993 Keywords: GnRH, salmon GnRH, chicken GnRH-II, HPLC, RIA, bluefin tuna, red seabream, black seabream, red spotted grouper, Japanese flounder
Abstract Brain extracts from bluefin tuna, Thunnus thynnus, red seabream, Pagrusmajor, black seabream, Acanthopagrus schlegeli, red spotted grouper, Epinephelus akaaraand Japanese flounder, Paralichthysolivaceus, were analyzed by high performance liquid chromatography (HPLC) and specific radioimmunoassays. Immunoreactive material co-eluting from HPLC with salmon gonadotropin-releasing hormone (GnRH) and chicken GnRH-II, respectively, was found in all five species. In addition, a GnRH immunoreactive fraction showing the same HPLC retention time as lamprey GnRH-I was detected in the brain extracts of all species examined when using an unspecific radioimmunoassay which detects several GnRH forms, including lamprey GnRH-I. In the Japanese flounder brain extract, a fourth GnRH immunoreactive fraction was detected with the unspecific radioimmunoassay which did not co-elute with any of the six synthetic GnRH standards used in the present study.
Introduction Eight different forms of gonadotropin-releasing hormone (GnRH), mammalian GnRH (Burgus et al. 1972; Matsuo et al. 1971), chicken GnRH-I (King and Millar 1982a, b; Miyamoto et al. 1982, 1983), chicken GnRH-II (Miyamoto et al. 1984), salmon GnRH (Sherwood et al. 1983), lamprey GnRH-I (Sherwood et al. 1986), catfish GnRH (Ngamvongchon et al. 1992a; Bogerd et al. 1992), dogfish GnRH (Lovejoy et al. 1992), and lamprey GnRH-III (Sower et al. 1993) have been identified in vertebrate brain extracts, which constitute a
family of structurally related decapeptides. In teleosts, three of the eight known GnRH molecules, namely salmon GnRH, chicken GnRHII and catfish GnRH have been purified and their amino acid sequences have been determined. Salmon GnRH was first purified and characterized from chum salmon Oncorhynchus keta (Sherwood et al. 1983), and later from codfish Gadus morhua morhua brain extracts (Wu et al. 1986). In the Thai catfish Clarias macrocephalus (Ngamvongchon et al. 1992a) and in the African catfish Clariasgariepinus (Bogerd et al. 1992), both catfish GnRH and chicken GnRH-II were identified. Recently, the
Correspondenceto: Koichi Okuzawa, National Research Institute of Aquaculture, Inland Station, Tamaki, Waterai, Mie 519-04, Japan
338 Bluefin tuna s
M CIL CII
3
V
.
A. sGnRH Ab (Lot No.2)
I
(3 'L mmm
t3
3-
B. cGnRH-I Ab (Lot R-E)
r
(: .)
0 4-
3-
C. cGnRH-II Ab (Lot R-II)
,
I n
1
10
20
30
40
50
Fractions Fig. 1. Reverse-phase HPLC of bluefin tuna Thunnus thynnus brain extract followed by salmon GnRH RIA (A), chicken GnRH-II RIA (B) and "GnRHs" RIA (C). Arrows indicate the elution positions of synthetic GnRH standards: M, mammalian GnRH; CI, chicken GnRH-I; L, lamprey GnRH-I; CII, chicken GnRH-II; S, salmon GnRH. The amount of ir-GnRH in the 1-ml HPLC fractions is represented by black bars. The dotted line represents the percentage of acetonitrile in the mobile phase.
complementary DNA (cDNA) encoding the precursor for salmon GnRH was characterized in an African cichlid Haplochromis burtoni (Bond et al. 1991), in Atlantic salmon Salmo salar(Klungland et al. 1992), and in the masu salmon Oncorhynchus masou (Suzuki et al. 1992), confirming the presence of salmon GnRH in these species. In addition to these studies, a combination of high performance liquid chromatography (HPLC)
and radioimmunoassay (RIA) techniques has been applied to study GnRH peptides in teleosts, revealing that at least two members of the GnRH peptide family are present in the brain of several species. Both salmon GnRH and chicken GnRH-II are common in many teleost fish such as the rainbow trout Oncorhynchus mykiss (Sherwood et al. 1984; Okuzawa et al. 1990), the masu salmon (Amano et al. 1992a), the goldfish Carassiusauratus(Yu et al. 1988; Kobayashi et al. 1992), the common carp Cyprinus carpio (Amano et al. 1992b), the molly Poecilia latipinna (Coe et al. 1992), the herring Clupeaharenguspallasi(Sherwood 1986), the milkfish Chanos chanos (Sherwood et al. 1984), and the mullet Mugil cephalus (Sherwood et al. 1984). However, mammalian GnRH has been identified in the eel Anguilla anguilla (King et al. 1990). Moreover, the possible presence of novel GnRH forms has been suggested in the hake Merluccius capensis (King and Millar 1985), in tilapia Tilapia sparrmanii (King and Millar 1985), and in the wrasse Coris julis (Powell et al. 1986). In marine teleosts, such as perciform fish (the largest group of teleosts), only a limited number of studies (see above) has been carried out; so that detailed information on the nature of the GnRH peptides is missing. The purpose of the present study therefore was to collect data on the identity of the GnRH peptides in a number of marine fish species. To this end, brain extracts were subjected to HPLC and four different RIA systems were then used to detect GnRH immunoreactive material in the HPLC fractions. Three of these RIAs were specific for salmon GnRH, chicken GnRH-II and mammalian GnRH, respectively, whereas the fourth one was an unspecific system and detected several different GnRH forms.
Materials and methods Fish brains Specimen of underyearling bluefin tuna, and of adult red seabream, black seabream, and red spotted grouper were purchased from local fishermen in the Gokasho bay, Mie prefecture. The fish
339 Black seabream
Grouper s
M CILCH
3-
4-
T
I C
0
o
A. sGnRH Ab
2.
s
M CILCII
A_
3
(Lot No.2)
I L 1C
A. sGnRH Ab (Lot No.2)
I rC
CD 1 U,
L U
=
...........
..-
.-.-. .... ....................................
'
'
~`
'~ '
'
'
3
_
Z'
o
o
4.
.t
B. cGnRH-II Ab (Lot R-E)
c
S 3
B. cGnRH-II Ab (Lot R-E)
± 2-
a
C
O 1 L
0 5
40
6
-40
5'
4
C'E .2 Ct 3
C. cGnRH-II Ab
.
30
-.......................
S
20
"E
O
10 0
C.
