Primary Structure and Biological Activity of a Third Gonadotropin-Releasing Hormone from Lamprey Brain*

Transcription

1 /93/1~23-,,2.5~03.00/0 Endocrinology Copyright B 1993 by The Endocrine Society Primary Structure and Biological Activity of a Third Gonadotropin-Releasing Hormone from Lamprey Brain* STACIA A. SOWER, YUEH-CHIN CHIANG, SANDOR LOVAS, AND J. MICHAEL CONLON Department of Biochemistry and Molecular Biology (S.A.S., Y.-CC.), University of New Hampshire, Durham, New Hampshire, 03824; and Regulatory Peptide Center (XL., J.M.C., Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, Nebraska ABSTRACT Previous studies have led to the identification of two molecular forms of GnRH (GnRH-I and II) in the brain of the sea lamprey, Petromyzon marinas. From analysis of these two forms, the primary structure of GnRH-I and the amino acid composition of GnRH-II were determined. We have now isolated a third molecular form of GnRH (GnRH-III) from the brain of this species that is different from GnRH- I and -II. The primary structure of GnRH-III is pglu-his-trp-ser- His-Asp-Trp-Lys-Pro-Gly-NH2. A synthetic decapeptide with this amino acid sequence was chromatographically identical to natural GnRH-III. A MAJOR hypothalamic peptide is GnRH, whose action in the various reproductive processes of mammals, birds, and to a lesser extent in some amphibians, reptiles, teleosts, and agnathans has been well defined. Research during the past several years, has now established that there is considerable diversity in the structure of GnRH and related molecular forms. The primary structure of GnRH has been determined in mammal (1,2); salmon (3); two forms in catfish (catfish-i and chicken GnRH-II) (4); two forms in chicken 1 (5-7) and II (8); one form in ratfish (chicken GnRH-II) (9); two forms in alligator (chicken-l and II) (10); two forms in dogfish (dogfish GnRH and chicken GnRH-II) (11) and lamprey (W. Lampreys are one of only two extant representatives of the vertebrate class, the Agnathans. Agnathans are of particular importance in understanding the evolution of GnRH since they represent the oldest lineage of vertebrates that diverged over 550 million years ago. Lamprey GnRH-I differs in five amino acids compared with mammalian GnRH, catfish GnRH-I, and chicken GnRH-I and in four amino acids compared with salmon GnRH, dogfish GnRH, and chicken GnRH-II. Certain regions of the molecule have been highly conserved including the NH,-terminal, pglu -His* and Ser and the COOH-terminal cu-amidated dipeptide. These regions and the length of the molecule have remained unchanged during 500 million years of evolution. The conservation of the NH,- and COOH-termini suggests these regions are of functional significance for conformation, receptor Received September 18, Address all correspondence and request for reprints to: Dr. Stacia A. Sower, Department of Biochemistry and Molecular Biology, Spaulding Life Science Building, University of New Hampshire, Durham, New Hampshire * fhis work was supported by National Science Foundation Grants DCB and DCB (to S.A.S.) and the Great Lakes Fisherv Commission (to S.A.S.). Intraperitoneal injection of synthetic lamprey GnRH-III (0.1 pg/g) produced a significant (P < 0.05) elevation of plasma progesterone (31% over basal values) in female lampreys that was comparable to that produced by the same dose of lamprey GnRH-I (36% over basal). The elevation in plasma estradiol produced by lamprey GnRH-III (244% over basal) was significantly (P < 0.05) less than the elevation produced by GnRH-I (322% over basal). We propose based on the biological activity of lamprey GnRH-III in these studies and the occurrence of this peptide during metamorphosis in lampreys, that both lamprey GnRH-I and -III are neurohormones involved in reproduction in lampreys. (hdoc~inobgy 132: , 1993) binding, resistance to enzymatic degradation, and in receptor-mediated events required for gonadotropin release (5, 6). This structural information combined with later immunocytochemical (13-l 6) and physiological studies (17-24), provide evidence for the regulatory influence of the hypothalamus on the pituitary-gonadal axis which implies that certain aspects of the GnRH molecule have been conserved throughout vertebrate evolution. However, there are many questions concerning hypothalamic regulation that remain unresolved. Chromatographic and immunological studies of various vertebrate brain extracts have indicated two or more GnRHlike peptides in representative species of agnathans, teleosts, amphibians, birds, and mammals (11, 25-29). HPLC followed by RIA with specific antisera raised against the known GnRHs have been used to separate and identify the GnRHs. We had determined that there was a second form of GnRH in lamprey brain but only the amino acid composition and not the sequence was known (12). This form was more hydrophobic and differed from lamprey GnRH I by three residues (Ile, Phe, and His instead of Glu, Lys, and Tyr). In later studies, using the lamprey GnRH antibody in RIA of fractions after chromatography of sea lamprey brain extract on HPLC, we detected a third form of immunoreactive (ir)gnrh which we now call lamprey GnRH-III. This third form was detected in brain samples of lampreys undergoing different stages of metamorphosis coinciding with the acceleration of gonad maturation and appeared to represent a higher portion of GnRH than in adult brain (25). The structural identification and biological activity of GnRHs in the oldest lineage of vertebrates is important especially in understanding the evolution of these peptides in vertebrates. The present paper describes the isolation, structural determination, and biological activity of the third form of GnRH in lamprey brain. 1125

2 1126 PRIMARY STRUCTURE OF THIRD LAMPREY GnRH lhi<l * Isolation of lamprey GnRH-III Materials and Methods Whole brains from 2150 mature male and female sea lampreys (Petronlyzotl nlarirlus) were collected in June and July 1990 at Hammond Bay Biological Station (Millersburg, MI). The lampreys were captured as they migrated from Lake Huron into the Cheboygan river for spawning and transported to the Biological Station. Brains were rapidly removed and immediately frozen on dry ice and stored at -80 C until extracted. Frozen brains were weighed (180 g) and boiled for 10 min in 2 M acetic acid (1 liter) and homogenized using a I olytron blender. After centrifugation (18,000 X g for 60 min at 4 C), the supernatant (950 ml), containing 0.1% (by vol) trifluoroacetic acid, was passed through 10 tandemly connected Sep-Pak C-18 cartridges (Water Associates, Milford, MA) at a flow rate of 2 ml/min. Bound material was eluted from the Sep-Paks with 80% (vol/vol) acetonitrile/water containing 0.1% trifluoroacetic acid as previously described (31). The volume of the effluent from the cartridges was reduced to 10 ml (Savant Speed-Vat, Farmingdale, NY) and this material was chromatographed on a 2.5 X 100~cm volume of Sephadex G-25 (Sigma, St. Louis, MO) equilibrated with 2 M acetic acid at a flow rate of 2 ml/min. Fractions (10 ml) were collected and the concentration of GnRH-like ir was determined by RIA at appropriate dilution. The fractions containing maximum ir (see Fig. 1) were pooled and pumped at a flow rate of 1.5 ml/min onto a 1 x lo-cm Aquapore Cx column (Applied Biosystems, Foster, CA) equilibrated with 0.1% trifluoroacetic acid. The concentration of acetonitrile in the eluting solvent was raised to 21% (vol/vol) over 10 min, held at this concentration for 30 min, and raised to 50% (vol/vol) for another 60 min using linear gradients, Absorbance was measured at 214 nm and 2 ml fractions were collected. Fractions containing GnRH-like ir were rechromatographed on a 0.46 X 25.cm Vydac 219TP54 phenyl column (Separations Group, Hesperia, CA) equilibrated with 0.1% trifluoroacetic acid at a flow rate of 1.5 ml/min and individual peaks were collected by hand. The concentration of acetonitrile was raised to 35% (vol/vol) over 60 min using a linear gradient. A very late peak (probably ir-lamprey-gnrh-ii) was not investigated because of the very low level of ir (Fig. 2). Lamprey GnRH-I and III were purified to apparent homogeneity by chromatography on a 1 x 25.cm Vydac 218TP510 (C,,) column under the same conditions used for the phenyl column. RIA GnRH-like ir was measured by RIA as previously described (23) using antiserum (1467) raised against lamprey GnRH-I at a dilution of 1:25,000, synthetic lamprey GnRH-I (Penmsula Laboratories, Belmont, N ; 1200 lx 800.t TIME (MINUTES) FIG. I. Initial purification of GnRH on Sephadex G-25 gel permeation column. Fractions with K., between 0.33 and 0.56 (considered maximum ir) were pooled and subjected to further purification by reversedphase HPLC. CA) as standard and [ 251]lamprey GnRHI as tracer. The antibody bmding ranged between 42-60%. Antiserum 1467 demonstrated less than 0.03%, 0.02%, and 0.01% cross-reactivity with chicken GnRN-II, mammal CnRH, and chicken GnRH-I, respectively. Pyroglutamyl aminopeptidase digestion Lamprey GnRH I (-2 nmol) and lamprey GnRH 111 (-4 nmol) were separately incubated for 12 h at 37 C with 2.5 rg pyroglutamyl aminopeptidase from calf liver (excision grade free from contaminating proteases; Calbiochem, San Diego, CA) in 0.1 M sodium phosphate buffer, ph 8.0, containing 5% (vol/vol) glycerol, 10 mm EDTA, and 5 mm dithiothreitol (300 ~1). The reaction mixture was chromatographed on a (0.46 x 25.cm) Vydac 218TP54 column. Structural analysis Amino acid compositions were determined by precolumn derivatization with phenylisothiocyanate using an Applied Biosystems model 420A derivatizer, followed by separation of the phenylthiocarbamyl amino acids by reversed phase HI LC (32). Hydrolysis in 5.7 M hydrochloric acid (24 h at 110 C) of approximately 500 pmol peptide was performed. Tryptophan and cysteine residues were not determined. The primary structures of lamprey GnRH-I and III were determined by automated Edman degradation of approximately 500 pmol peptide using an Applied Biosystems model 471A sequenator modified for on-line detection of phenylthiohydantoin amino acids under gradient elution conditions, The detection limit was 0.5 pmol. Synthesis of lamprey GnRH-III The peptide was synthesized (0.25 mmol scale) using an Applied Biosystems model 430A peptide synthesizer on a 4-(1,l dimethoxyphenyl-fmoc-aminomethyl)phenoxy resin. All amino acids, except pyroglutamic acid, were N-cr-Fmoc protected and were coupled as their I- hyroxybenzotriazole active esters. The reactive side-chain groups were protected as follows: His, N-im-trityl; Asp,@-t-Butyl ester, Lys, N-c-t- Boc; Ser, t-butyl ether. The peptide was cleaved from the resin by stirring with trifluoroacetic acid/l,2-ethanedithiol/anisole (95:1.6:3.4, by vol) for 90 min at room temperature. The yield of crude product was 280 mg. The synthetic peptide was purified to apparent homogeneity by chromatography on a (1 X 50-cm) Vydac C-18 (15-20 p) column equilibrated with 50 rnm ammonium acetate, ph. 4.5, at a flow rate of 5 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 33% (vol/vol) over 100 min. The identity of synthetic lamprey GnRH 111 was confirmed by amino acid analysis and by low resolution fast atom bombardment mass spectrometry. Biological activity Forty adult female sea lampreys (10 lampreys per treatment group) were tested with one injection of lamprey GnRH-I (0.1 kg/g) or one of two doses (0.2 or 0.1 rg/g) of synthetic lamprey GnRH-III. Thirty minutes before injection, peptides were dissolved in 0.6% NaCl in distilled water and injected ip relative to body weight. The animals were anesthetized with ethyl m-amino benozate methanesulfonate during blood collection. Blood samples were collected in heparinized syringes by cardiac puncture. Plasma was drawn off after centrifugation and stored frozen at -20 C until assayed for estradiol and progesterone. Plasma estradiol and progesterone were measured by RIA, as previously described (17, 33). Data for hormone concentrations were analyzed by the Student-Newman-Kuel test after a preliminary analysis of variance. In all tests, the level of significance for differing groups was P < Results Isolation of lampwy GnRH-I and III The initial extract of lamprey brain contained GnRH-like immunoreactivity equivalent to 280 pmol/g wet tissue weight. The immunoreactivity in serial dilutions of the extract

3 PRIMARY STRUCTURE OF THIRD LAMPREY GnRH 1127 diminished in parallel with the synthetic lamprey GnRH-I standard in RIA. The immunoreactivity in the extract, after partial purification on Sep-Pak cartridges, was eluted from a Sephadex G-25 gel permeation column as three incompletely resolved peaks (Fig. 1). Maximum ir was detected at the elution volume of synthetic lamprey GnRH-I and the fractions denoted by bar with K,, between 0.33 and 0.56 were pooled and subjected to further purification by reversed-phase HPLC C, column. Maximum ir was detected for lamprey GnRH III in fractions collected between min and lamprey GnRH I in fractions collected between min (Fig. 2). Fractions from each peak were then separately eluted on a phenyl column. Data are combined and shown on one graph (Fig. 3). Maximum ir was detected for lamprey GnRH III in fractions collected between min and lamprey GnRH I in fractions collected between min. As shown in the final reverse-phase Clx HPLC chromatogram (Fig. 4), lamprey GnRH-I was well separated from lamprey GnRH- III. Structure of lamprey GnRH-I and-iii Lamprey GnRH-I and-111 were subjected to automated Edman degradation but no amino acid phenylthiohydantoins were identified thereby indicating that the peptides did not contain free a-amino groups. After incubation of lamprey GnRH I with pyroglutamyl aminopeptidase, it was possible to identify without ambiguity amino acid phenylthiohydan I TIME (~vinutes) FIG. 2. Further purification of GnRH on reverse-phase Cx column. Maximum ir was detected for lamprey GnRH III in fractions collected between min and lamprey GnRH I in fractions collected between min. Late peak was considered to be ir-gnrh II which was not further investigated because of low abundance. Absorbance at 214 nm (uwr graph). I N \ is t i? IO TIME (MINUTES) FIG. 3. Fractions from each peak from Cs column were then separately eluted on a phenyl column. Data are combined and shown on one graph. Maximum immunoreactivity was detected for lamprey GnRH III in fractions collected between min and lamprey GnRH I in fractions collected between min. Absorbance at 214 nm (upper graph). toins during eight cycles of operation of the sequencer and the derivative of glycine was detected in trace concentration during cycle 9 (Table 1). Similarly, after incubation of lamprey GnRH III with pyroglutamyl aminopeptidase, it was possible to identify without ambiguity amino acid residues during nine cycles of operation (Table 1). The primary structures of lamprey GnRH-I and -111, including the presence of a NH>-terminal pyroglutamyl groups and a COOH-terminal glycine residue, were confirmed by the results of amino acid analysis (found residues/m01 peptide: GnRH I Glx 2.0, Ser 1.0, Gly 1.1, His 1.0, Pro 1.1, Tyr 0.8, Leu 1.0, Lys 1.0; GnRH-III Asx 0.8, Glx 1.0, Ser 1.0, Gly 1.0, His 1.8, Pro 1.1, Lys 1.0). The final yields of pure peptides, based on the amino acid analysis data, were 8.2 nmol (GnRH-I) and 10.5 nmol (GnRH-III). Under the elution conditions shown in Fig. 5, a synthetic replicate of the COOH-terminally cu-amidated form of lamprey GnRH-III co-eluted with endogenous lamprey GnRH-III from a Vydac Cls column as a single sharp peak. Biological activity of lamprey GnRH III One injection of lamprey GnRH-III (0.2 or 0.1 pg/g lamprey) significantly stimulated plasma estradiol compared to

4 PRIMARY STRUCTURE OF THIRD LAMPREY GnRH Endo * 199:x VIII I?! * No :i IGnRH Ill IGnRH I TIME (MINUTES) FIG. 4. As shown in the final reverse-phase C,, HPLC chromatogram (Fig. 2), lamprey GnRH-I was well separated from lamprey GnRH-III at absorbance of 214 nm. Peak fractions were used for structural characterization. TABLE 1. Automated Edman degradation of lamprey GnRH-I and III after digestion with pyroglutamyl aminopeptidase CnRH-I GnRH-III Cycle no. Amino Yield Amino Yield acid (pmol) acid (pmol) 1 His 114 His Tyr 252 Trp Ser 44 Ser 76 4 Leu 198 His Glu 128 Asp Trp 116 Trp 130 I LYS 131 LYS Pro 28 Pro 47 9 GUY Trace Gly 6 controls (Table 2). The elevation in plasma estradiol produced by lamprey GnRH-III (244% over basal levels) was significantly (P < 0.05) less than the elevation produced by GnRH- I (322% over basal). Lamprey GnRH-III at 0.2 or 0.1 &g and lamprey GnRH I at 0.1 pg/g were equally effective in stimulating plasma progesterone compared to controls. IA I I I I TIME (MINUTES) FIG. 5. Coelution of lamprey GnRH III (1 nmol) with a synthetic replicate of the COOH-terminally a-amidated form of lamprey GnRH- III (1 nmol) from a Vydac C,, column as a single sharp peak. Absorbance at 214 nm. TABLE 2. In viva biological activity of lamprey GnRH-I or III in adult female sea lamprey Treatment Estradiol Mean + SE (ng ml)f rogesterone Control 0.64 f Lamprey GnRH-I (0.1) " " Lamprey GnRH-III (0.1) " 0.71 f 0.06" Lamprey GnRH-III (0.2) 1.79 f " Plasma e&radio1 or progesterone (rig/ml) of female lampreys injected with saline (control) or lamprey GnRH-I at 0.1 rg/g lamprey or lamprey GnRH-III at 0.2 or 0.1 *g/g. Mean f SE (n = 10). Denotes significance at P < Discussion The decapeptide reported here is the second peptide from the lamprey brain to be fully characterized structurally. Lam-

5 PRIMARY STRUCTURE OF THIRD LAMPREY GnRH 1129 prey GnRH-III is a member of the GnRH family as structur- the same species is presently under study. The presence of ally defined by the amino and carboxyl termini of pglu - irgnrh in extrahypothalamic brain regions is well docu- His and Pro9Gly NH2, the conservation of Ser4 and its mented which is suggestive of multiple functions such as length of 10 amino acids (Fig. 6). It is the first peptide of this neurotransmitters and/or neuromodulators (37, 38). Differfamily to have an Asp in the sixth position compared to Glu ent forms of GnRH within the hypothalamic region have in lamprey GnRH-I and Gly in mamma1 GnRH, salmon suggested that gonadotropin secretion is probably regulated GnRH, catfish GnRH-I, dogfish GnRH, and chicken GnRH- by a dual mechanism (39). More recent evidence in the I and -11. Lamprey GnRH-III is more closely related to the chicken indicates that while both chicken GnRHs induce other members of the GnRH family compared to lamprey gonadotropin secretion; chicken GnRH-II is 10 times more GnRH-I. Lamprey GnRH-III has 80% identity with chicken potent than chicken GnRH-I (39). GnRH-I in lamprey ap- GnRH-II and dogfish GnRH; 70% identity with catfish pears to be more restricted in its distribution in the brain GnRH I, lamprey GnRH I, and salmon GnRH; and 60% compared to GnRH in mamma1 brains (16, 40) suggesting a identity with mammal GnRH and chicken GnRH-I. more restricted function. The present study demonstrates that at least three distinct GnRHs exist in one of the two most primitive vertebrates. Chromatographic and immunological studies of various vertebrate brain extracts have indicated two or more GnRH-like peptides in representative species of agnathans, teleosts, amphibians, birds, and mammals (12, 26-29). In lampreys, we had previously determined that there was a second form of GnRH (lamprey GnRH II) in lamprey brain but only the amino acid composition and not the sequence was known (12). This form was more hydrophobic and differed from lamprey GnRH-I by three residues (Ile, Phe, and His instead of Glu, Lys, and Tyr). The present study has shown that lamprey GnRH-II is also distinctly different from lamprey GnRH-III (Ile, Phe, and Leu instead of Asp, Lys, and Trp). In hagfish, the only other living representative of the oldest class of vertebrates, the presence of irgnrh was reported in Heptatretus hexamtrema (34) and Eptatretus stouti (35) brain; whereas in other studies GnRHs were not detected in hagfish brain (13, 15, 36). More recently, we demonstrated two irgnrh forms in hagfish brain using a lamprey GnRH antibody (Sower, S. A., and King, J. A., unpublished; 30). One of the ir forms elutes with the new lamprey GnRH-III and the other form with chicken GnRH-II. However, further studies are required to verify the existence of a GnRH-like molecule in hagfish brain. The functional significance of multiple forms of GnRH within the brain and in extrahypothalamic locations within Lamprey-III pglu-his-trp-ser-his-asp-trp- Lys- Pro-Gly-NH2 Lamprey-I pglu-his-b-ser-leu-glu-trp -Lys- Pro-Gly-NH;? Dogfish pglu-his-trp-ser-his-m- Trp- &- Pro-Gly-NI 12 Catfish I pglu-his-trp-ser-his-(&- Leu- Asn- Pro-Gly-NH2 Salmon pglu-his-trp-ser-tyr-gly- Trp- &- Pro-Gly-NH2 Chicken-II pglu-his-trp-ser-his-c&- Trp- m- Pro-Gly-NH2 Chicken-I pglu-his-trp-ser- l&-g&- Leu- Gin- Pro-Gly-NH2 Mammal pglu-his-trp-ser-tvr-gly- Leu- Arg- Pro-Gly-NH2 FIG. 6. Primary structures of the eight known vertebrate GnRH molecules. Based on the known sequences, a phylogenetic tree is presented in Fig. 7 (developed by Dr. Russell Doolittle, University of San Diego, San Diego, CA). We suggest that there are three sets of GnRH molecules with nine single-base changes in genetic codons not including position 8. Mammal GnRH and chicken GnRH I can be converted from each other by single-base changes. The conversion of lamprey GnRH-III to chicken GnRH-II would require a minimum of two base changes. Lamprey GnRH I is far removed from the other peptides by a minimum of five to seven base changes and is considered the outlier and closer to the ancestral molecule. We would predict that there are at least two other GnRH forms because of at least one nonconnected residues in position 5 between Leu and His. In addition, we would predict that one of the unidentified GnRH forms would have a His in the eighth position. Miyamoto et al. (39) and Lovejoy et al. (9) have suggested that chicken GnRH-II has occurred at a much earlier stage of evolution compared to the other forms (except lamprey GnRH-I). Chicken GnRH-II was structurally characterized in the ratfish (class Chondrichthyes) which diverged from the line of evolution about 400 million years ago (9). This information combined with the structure of lamprey GnRH-111 and dogfish GnRH suggest that these three molecules are more closely related to the ancestral molecule(s). We would thus predict that Leu5 evolved first followed by a conversion to Tyr then to His or that His and Leu5 evolved separately. The sixth position is considered critical for function of the molecule. Conformation energy analysis of mammal GnRH suggests that the termini of the molecule are proximal (41) and that the GnRH interacts with the receptor in the folded conformation due to the 6-70 turn (42). Interestingly, both lamprey GnRH I and III are the only molecules to have substitutions in the sixth position, Glu and Asph, respec- tively. Glu appears to have evolved first with a single base change to Asp followed by a single base change to Cly. We have tested the biological activity of Gly lamprey GnRH in lampreys and determined that the substitution of Gly for Glu totally inhibited the biological activity (Sower, S. A., J. G. Abrams, and M. Goodman, unpublished). These data would suggest that the receptor requirements for GnRH are different in the lampreys from those in other vertebrates. Trp occurs in lamprey CnRH I and 111, chicken GnRl1 II, dogfish CnRH, and salmon GnRH; whereas Leu is in catfish GnRH-I, chicken GnRH-I, and mammal. The appearance of Trp in lamprey (GnRH-I and III), dogfish, and ratfish (chicken GnRH-II), which represent the two oldest lineages

6 1130 PRIMARY STRUCTURE OF THIRD LAMPREY GnRH Endo. 199:i Vol 132. No :J ;- L 7* +jf?%- ;:;;;g :;,;;;;, Y,+H FIG. 7. Based on the known sequences, a phylogenetic tree is proposed (devel- L,zW QHWSHGLNPG CATFISH oped by Dr. Russell Doolittle using his QHWSYGWLPG SALMON own system). One arroul signifies one Y,*W base change. Lamprey GnRH-I is con- I --+Yr-- D,- G I QHWSHGWYPG CHICKEN II sidered to be the outlier. L5 a E,-+D Y,- H - QHWSHGWLPG DOGFISH of vertebrates, provides further evidence that these molecules evolved first or from an ancestral molecule with Trp in the seventh position. The most variable position is position eight. Each of the GnRH peptides except lamprey GnRH-I and III has a different amino acid. Both lamprey GnRH-I and III have Lys, suggesting Lys* may have occurred in the ancestral molecule or that given the substitutions in the sixth position of Glu and Asp, Lys may reflect changes in receptor molecules that are different in lampreys compared to other vertebrates. Ovulatory and steroidogenic responses to lamprey GnRH- I have been well documented in adult female sea lampreys (19, 22). As in previous studies, plasma estradiol and progesterone were measured in the lamprey as potential indicators of pituitary responsiveness to GnRH. Gonadotropin(s) (GTH) have not yet been isolated from the lamprey pituitary glands, so that RIA of secreted gonadotropins is not possible at this time. We have shown direct pituitary responsiveness of lamprey pituitaries cultured irz vitro with lamprey GnRH; elevated immunoreactive GTH measured with a heterologous assay (salmon GTH and antibody) (Sower, S. A., and P. Swanson, unpublished) and elevation of estradiol in pituitary ovary cocultures with lamprey GnRH (Sower, S. A., unpublished, cited in 20). The steroid hormones estradiol and progesterone measured in this study are associated with reproductive activity (19, 43, 44) and are present in the ovary (45). In the present study, lamprey GnRH-III was shown to be biologically active as determined by increased levels of plasma steroids. Administration of lamprey GnRH-III significantly stimulated plasma estradiol compared to controls. Lamprey GnRH-Ill was slightly less effective at 0.1 fig/g compared to lamprey GnRH-I in stimulating plasma estradiol. Lamprey GnRH-I or -III were equally effective in stimulating plasma progesterone compared to controls. As stated earlier, ir-lamprey GnRH-III was detected in brain samples of lampreys undergoing metamorphosis (25). In these studies, there was an increase of brain GnRH during metamorphosis which coincided with the acceleration of gonadal maturation. Further studies would be required to determine the difference in potency and function of lamprey GnRH molecules in sea lampreys. We would speculate at this time based on the biological activity of lamprey GnRH-III in these studies and the occur- rence of this peptide during metamorphosis in lampreys, that both lamprey GnRH-I and -III are neurohormones involved in reproduction in lampreys. Acknowledgments We thank Thomas Bolduc and Mary Jane James for their help during sampling of lamprey brains and Dr. Andrew I. Laudano for his ad& We thank Dr. Russell F. Doolittle for his critical review of this manuscript. We thank Drs. J. A. King and R. I. Millar for lamprey GnRH antisera. References 1. Matsuo H, Baba Y, Nair RMG, Arimura A, Schally AV 1971 Structure of the porcine LH- and FSH-releasing hormone. I. The proposed amino acid sequence. 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J Biol Chem 257: Miyamoto K, Hasegawa Y, Minegishi T, Nomura M, Takahashi Y, Igarashi M, Kangawa K, Matsuo H 1982 Isolation and characterization of chicken hypothalamic luteinizing hormone-releasing hormone. Biochem Biophys Res Commun 107: Miyamoto K, Hasegawa-Y, Igarashi M, Chino N, Sakakibara S, Kannawa K, Matsuo H 1983 Evidence that chicken hvpothalamic lute&zing hormone-releasing hormone is [Gln ]LHdH. Life Sci 32: Lovejoy DA, Sherwood NM, Fischer WH, Jackson BC, Rivier JE, Lee T 1991 Primary structure of gonadotropin-releasing hormone from the brain of a holocephalan (Ratfish: Hydrolagus colliei) Gen Comp Endocrinol82: Lovejoy DA, Fischer WH, Parker DB, McRory JE, Park M, Lance V, Swanson P, Rivier KE Sherwood NM 1991 Primary structure of two forms of gonadotropin-releasing hormone from brains of the American alligator (Alligntor mississippiensis) Regul Pep 33: Lovejoy DA, Fischer WH, Ngamvongchon S, Craig AG, Nahor-

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