Life~Sciences Vol. 20, pp . 1227-1232, 1977 . Priated in the~II .S .A .

Perganon Press

A TELECBT (TILAPIA biGÔSAMBICA) GO~IVADOTROPIN THAT EgTI2:ING HORMONE Susan Wallcar Farmgirl and Harald Papkoffl'

2

lHormane Research Laboratory and 2Reproductive Endocrinology Center University of Calüornia 3sn Frsncieco, California 94143

(Beceived is final fora February 25, 1977) A purified gonadotropin preparation was obtained from pituitaries of a teleost fleh (Tilspia mossambica). This gonadatropin was found to resemble LH in t it be ved identically to mammalian and non-mammalian LHs in several chromatographic systems, and stimulated testerone production in isolated rat Leydig cells. In this assay, specific for LH, the Tilapia ganadatrapin was lass pote~ than mammalian LHa but significantly more active than avian, reptilian or amphibian LHe . The Tilapia gonadotropin was found tobe a glycopratein ; preliminary amigo acid campoeitian data show resemblancee to both mammalian and non-mammaliaa LHs . Reports from several laboratories have confirmed the existence of two gonadotropins, resembling mammalian luteioi$iag hormone (LH) and follicle-etimalsting hormone (FSH) in the pituitaries of birds, reptiles, and amphibians (1-7). Studies on fish gonadotropina, however, are ambiguous . Fractionation studies from several laboratories po~iat to the presence of s single gonadotropin (8-11) . However, other fractionation studies (12-14) end many histochemical and cytological studies on the fish pituitary (15-17) indicate the presence of two chemically distinct gonadotropine in fish . Thus, the question of whether fish have one or two gonadotropine remains an open and interesting question . We previously fractionated pituitaries from a euryhaline teleost, Tile is mossambica*, and prepared highly purified pralsctin and growth ormona 1 amination of side fractions from these ezperiments hoe led to the purification of a ganadotropin which behaves chramatographically identically to ell of the mammalian end non-mammalian LHs ezamined to date in our laboratory, and which is able to stimulate testosterone production in vitro in rat Leydig cells, a specific property of LH (21, 22). MATERLAI9 AND METHOiD3 A total of 20, 001 pituitaries (63 g wet weight) from mature Tila mossambica collected in Wahiawa Reservoir, Oahu, Hawaii, were removed immediately, fro$en, and kept at -20'C u~il fractionation. The initial puri flcation procedures have been described previously (19, 20). The alkaline *This species hoe recently been renamed arotherodan mossambicu s . 1227

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extract was chromatographed on columns of Amberlite CG-50 and DEAEcellulose. The lyophilized DEAF uaadeorbed fraction was dissolved is 0 . 15M (NI~)2S04 and adjusted to pH 3. 5 with freshly prepared 0. 2 M HP03 . The material that remained in solution following removal of the pH 3, 5 precipitate was dialyzed, lyophilized and then applied to a Sephadea G-100 column (2, 5 x 92 cm) equilibrated with 0 . 05 M NH4HC03. Bioassay revealed the presence of significant LH activity in an area frown a Ye/Vo of 1 . 5 to 2. 0, just proceeding a major peak with a Ve/Vo of 2.4 . The biologically active fraction was obtained is a yield of 4 mg . Characterization of this material included bioassay in an in vitro rat Leydig cell aeeay (21) and an Amphibian ovulation assay (23) ; amino acid analysis by the method of Spackman et al . (24) in an automatic amino acid analyzer (Beckman Model 120B) ; aac~carbohydrate analyses by micro calorimetric analyses ae previously described (4). Ia the rat Leydig cell assay, ovine LH (25) and bullfrog LH (3) were included as standards . itESULTS AND DLSCUSSIOTi During fractionation, the T~ila i~s gonadotropin was absorbed on Amberlite CG-50 and eluted with pH 6 buffer, and was unabsorbed on DEAEcellulose equilibrated to 0. 03 M NH4HC03, pH 9. 0 . Hoth mammalian and non-mammalian LHe have been shown to fractionate identically. Conversely, FSHs from both mammalian and non-mammalian species are unabsorbed on Amberlite and absorbed on DEAE under these conditions (26) . Although no clear peak was obtained on gel filtration, the Tila is gonadatropin emerged at a Ye/Vo of apprcocimately 1 .8, similar to t observed for a wide variety of LHs . The yield, 4 mg, ie equivalent to 63 mg/kilo pituitaries . The Tilapia goaadatropin was assayed for biological activity is an in vitro rat Le ig cell aeeay. Previous studies (21, 22) have shown this to be a highly sensitive and specific assay for marnrmslian LH, which can be measured at a dose range of 1-10 ng . We have also tested non-mammalian gonadotropias in this aeeay and found that they produce a similar response in terms of the dose-response elope and the maximal production of testosterone, but at considerably higher doses, approximately 1-10 ~g (3 ; Farmer_et al . , unpublished data). Figure 1 shows the results obtained with sheep, ~, and bullfrog LHe in the rat Leydig cell aeeay . The bullfrog LH data 3) is very similar to that obtained with other avian and reptilian LHs (Farmer gooadatropin, which had a potency of et al . , unpublished data). The Tila 0 . 009 z highly purified ovine LH . 002 z NIH LH, was significantly more active than any of the ocher non- :mmmalian LHe (17 x bullfrog LH, Fig. 1) . However, despite this potent LH bioactivity, the Tilapia gonadotrapin was not active is stimulating ovulation is the Xen s frog . Ali other LHs tested have been active in this assay, which routinely used for the purification of non-mammalian LHs (26) . Another preparation was obtained from the Tilapia pituitaries which fractionated identically to mammalian and nanmammalian FSHs . This material was inactive in the Leydig cell aeeay, se were purified Tilapia growth hormone (19) and prelactin (18, 20). The amino acid composition aä the Tilapia gonsdotropin ie presented is Table 1, with data fraan sheep and sea turtle LHs included for coanparison.

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ao ao

g

0

av

~oM

i

a~

HORw10f~E

Fig. 1 Stimnlstion aE testwterone producèion in isolated nt testis Leydig ceIIs by sheep, ~ia and bullfrog t-T~~ . Nate diffsre~ concentralian rsage employed for the n'+a^'.rnalian (ßg ) and nOn- mwmrnallwn (i=g) I,Il", Since the Tilspia ganadolropin may not be ad ultimate purity this data i~ regarded se prelinninary, but came comparisons with ganadatropiw of other species are of interest . The Tilspis gonadatropin campo~ition iw similar in many respects to s variety od nasazalalian (Z7) and non-mammalian (28) LHs, with the ezception of the low half-cp~tine content, and the higher aspartic and glatamic acid contents . In the laäer respect, the Tit~s s ganadatropin resembles more the mammalian FSHs (27) and Ssh ganadatropins (9-11) . Haavaver, the glycine and alanine caote~ of the Tilspia ganadotropin is more like the LHs and different from that of other fish g~otropins, Tilapia gonadotropin was found to pwsess carbohydrate, a uaiwrsal characteristic of LHs and FSHs . The content, 5. 91~ hs:ose, 5, OS~G hazwamine, and O. T0~(r sialic acid, is not significatnly different from other maannoalian and nm-mammalian ganadotropins (28),

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TABLE 1 Amino acid campositiona of Tilapia ganadatropin compared with sheep and sea turtle LH Amino acid

Tilapia

Sheepb

Sea turtle

c

Lye 9. 4 12 14 Hie 6. 1 6 5 Arg 8. 2 11 8 Asp 21 . 5 11 17 Thr 14 .4 16 20 Ser 16 . 6 14 14 22 . 9 14 15 Glu Pro 17 . 2 27 20 Gly 15 . 2 11 14 15 Ala 15 . 8 14 22 21 1/2 Cys 7 .6 Val 14 . 6 13 12 Met 6. 2 7 4 IIe 11 .4 7 8 Leu 14 . 5 14 10 Tyr 6. 9 7 10 Phe 7. 1 8 9 a Amino acid analysis : 20 hr hydrxlyeis, not corrected for hydrolytic destruction, calculated on the basis a~f 215 residues per male ; cystine and methionine values were obtained with performic acid-mddi~ed preparations . b Calculated from structural analysis, 215 residues (29) . c Data taken from Licht et al . (4). In conclusion, we have prepared a Tilapia ganadotropin which ie very similar to a variety of mammalian and non-mammalian LHs in its fractionation behavior and in its significant biological activity in a eepcific mammal ian LH bioassay . In amino acid composition, the Tilapia goaadotrapin was found to primarily resemble LHe, but also possess anew FSH-like features similar to other fish gonadotropins . Our data neither support nor refute the hypothesis that fish have only ~e ganadotropin . However, if there are two gonadotropine in telexet pituitaries, haanxlogous to LH and FSH, it seems likely that the material we have isolated represents teleost LH . Fish pituitary fractionation has been hindered in the past because fish pituitary hormones have little or no activity in moat non-fish bioassays (9). Thus, the finding of Tilapia gonadotropin activity in the rat Leydig cell sassy ie a very interesting and important observation, especially since the fish LH was much more potent than highly purified avian, reptilian or amphibian LHe . ACKNOWLEDGEMENT The collection of Tilapia pituitaries was made possible by a grant to Dr . Howard A. Bern from the Graduate Division of the University of Calif. ,

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Berkeley, and was accomplished by a team under the leadership of R S. Nishioka, consisting of Lauren Bern, Peggy Braadley, Karen T. Mills, Daniel Swanson and L. J. Wiley. To them, to the etudes of the Aiea HIgh School Biology Club and their advisor, Ms . Iris Shinseki, and Mr, William S. Devick of the Hawaiian Department of Land and Natural Resources, Division aä Fish and Game, we express our appreciation . We thank Professor Choh Hso Li for his support of these studies and for critically reading the manuscript . The technical assistance of Alan Suyama and Kenway Hoey ie appreciated. We thank Dr . Paul Licht for per forming the Xenopue assay. This work was supported in part by s grant from the National Science Foundation, BMS 75-16138 and a Rockefeller Foundation Center Grant . REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 .

S. W. FARMER, H. PAPKOFF, and P, LICHT, Baal . Raprod . 12, 415-422 (1975) . H, PAPKOFF, S. W. FARMER and P, LICHT, Endocrinology 98, 767-777 (1976) . H. PAPKOFF, S. W. FARMER and P. LIGHT , Life Sci. 18, 245250 (1976) . P, LICHT, 3. W. FARMER and H. PAPKOFF, Biel . Rsprod . 14, 222-232 (1976) . A, S~. HARTREE and F . J. CUNHINGHAM, J, Endocrinol . 43, 609616 (1969) . C, G. SCANES and B, K, FOLLETT, Br . Point, Sci. 13, 603-610 (1972 ). 5. ISHI and T. FURUYA, Gen. Comp . Endocrinol . 25, 1-8 (1975) . E. M. DONALDSON, F, YAMAZAKI, H, M. DYE and W, W. PHILLEO, Gen. Comp . Endocrinol . 18, 469-481 (1972), E, BUR,ZAWA-GERARD and Y. A, FONTAINE, Gen. Camp . Endocrinol. Suppl. 3, 715-728 (1972), E, BURZAWA- GERARD, B. F GONCHAROV and Y. A. FONTAINE, Gen. Comp . Endociinol . 27, 296-304 (1975) . B. BRETON, B. JALABERT and P. REINAUD, Ann. Biol . Anim. Biochem, Biophys . 16, 25-36 (1976) . D. R» IDLER, L. S-$AZAR and S. J. HWANG, Endocr . Rae. Common, 2, 215-235 (1975), D. R. IDLÉR, S. J. HWANG and L. S. BAZAR, Endocr. Res . Commun . 2, 237-249 (1975) . S . G. HADER and Y. GLUM, Gen. Caanp, Endocrinal . _29, 251 (Abstr. ) (1976) . R. REINBOTH, Amer, Zoal, 12, 307-324 (1972) . M. P, SCHR .ELBMAN, J. F. LEATHERLAND and B. A, McKEOWN, Amor . Zoal, 13, 719-?42 (1973). M. OLIVEREAÛ, Gen. Comp. Endocrind. 28, 82-95 (1976) . S, W. FARMER, W. C. CLARKE, H, PAPKOFF, R. S, NiSHIOKA, H. A, BERN and C . H. LI, Life Sci. 16, 149-159 (1975) . S, W. FARMER, H. PAPKOFF, T. H r~YASHLDA, T, A, BEWLEY, H. A, BERN, and C. H. LI, Gen. Canzp. Endocrinal, 30, 91-100 (1976) .

123 2 20 . 21. 22. 23. 24. 25. 26. 27. 28. 29.

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5. W. FARMER, H. PAPKOFF, T. A BEWLEY, T. HAYASHiDA, R S. NISHIOKA, H. A. Bern, and C. H. LI, Gen . Ccanp. Endocrinal. 31, 60-71 (1977) . J. RAMACHANDRAN and M. R SAIRAM, Arch. Hiochem . Biophye . 167, 294-300 (1975). M . L. DUFAU, R. POCK, A. NENBAUER, and K. J. CATT, J. Clin. Endocrlnal . Metab . 42, 958-969 (1976) . P. LICHT and H. PAPKOFF, Gen . Camp. Endocrinol. 22, 218-237 (1974 ). D. H. SPACKMAN, W. H. STEIN, and S. MOORE, Anal. Chem . _30, 1190-1206 (1958) . H. PAPKOFF, D. GC6PODAROWICZ, A. CANDIOTTI, and C. H. Li, Arch. Biochem . Biophye . 111, 431-438 (1965). . W. FARMER, C. H. MÜLLER, H. W. P. LICHT, H. PAPKOFF, S TSUI, and D. CREWS, R.ec . Prog. Hor. Res . , in press, (1977). M. R. SAIRAM and H. PAPKOFF , in Handbook auf Physiology, Endocrinology IY, Part 2, pp 111-131, eds . E. Knobil and W. H. Sawyer, Amer. Phyeial . Soc . , Baltimore (1974). H. PAPKOFF, S. W. FARMER and P. LICHT, 5th Intl. Cangr . End ocrinal . , Hamburg, July, 1976 . Ezcerpta Medics Intl. Coagr . Ser. , in press (1977). H. PAPKOFF, M. R SAIRAM, S. W. FARMER and C. H. LI, Rec . Prog. Hor . Ries . 29, 563-588 (1973).

A teleost (Tilapia mossambica) gonadotropin that resembles luteinizing hormone.

Life~Sciences Vol. 20, pp . 1227-1232, 1977 . Priated in the~II .S .A . Perganon Press A TELECBT (TILAPIA biGÔSAMBICA) GO~IVADOTROPIN THAT EgTI2:ING...
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