GENERAL
AND
COMPARATIVE
Isolation
37, 508-520 (1979)
ENDOCRINOLOGY
and Characterization of Luteinizing Hormone and Follicle-Stimulating Hormone from Pituitary Glands of the Turkey (Meleagris gallopavo) W. H. BURKE,*
P. LIGHT,? H. PAPKOFF,$ AND A. BONA GALLot
*Departmettt of Animal Science, University of Minnesota, St. Paul, Minnesota 55108; *Departmen: of Zoology, Utdversity of California. Berkeley, California 94720; and *Hormone Research Laborator?: and Reproductive Endocrinology Center, University of California. San Francisco. California 94143
Accepted January 9. 1979 Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were purified from the pituitary glands of the turkey (Meleagris gallopavo). These hormones were characterized biochemically and biologically and compared with chicken gonadotropins prepared in several independent laboratories. Amino acid and carbohydrate analyses demonstrated homology between turkey and other species of gonadotropin. The turkey LH purified here had significantly higher carbohydrate content than a previous preparation of turkey LH. Immunological studies further confirmed that the turkey FSH and LH were distinct from one another and that each was homologous to the respective gonadotropin from other vertebrates: the immunopotencies of the turkey hormones were similar to those from chickens. A variety of bioassays and radioreceptor assays (RRAs) confirmed the biological activity of the two turkey gonadotropins and revealed that the turkey LH was distinct from that of the chicken. As expected, the two types of turkey hormones were approximately equipotent in total gonadotropin bioassays (frog spermiation and $*P uptake by chick testes), and only the turkey LH was active in the rat Leydig cell assay and in RRA for LH in mammals. However, the turkey LH was also highly potent in several assays considered to be relatively FSH specific, including the Anolis lizard assay and several RRA systems using mammalian, turtle and avian gonadal receptors with ‘ZSI-labeled human FSH as tracer. Turkey and chicken FSH are similar in the RRAs, but the turkey LH was consistently more potent than either avian FSH in competing for FSH-binding sites. Chicken LH had relatively low activity by comparison. It is suggested that the evolution of the structure of active sites in turkey LH has involved convergence on those of the FSH molecule.
Several laboratories have reported on the separation of gonadotropins from the pituitaries of two species of galliform birds, the domestic fowl (Stockell-Hartree and Cunningham, 1969) and turkey (Farmer et al., 1975, Wentworth, 1971) and one gonadotropin has been partially purified from an anseriform bird, the duck (Licht et al., 1977d). Limited chemical data for these hormones suggest significant interspecific differences among avian hormones. For example, the luteinizing hormone (LH)
from the turkey appeared unique among all vertebrates examined in its low carbohydrate content, and the duck gonadotropin exhibited a different spectrum of activities than those of the other avian species (Licht et al., 1977d); unfortunately, folliclestimulating hormones (FSH) from turkey and duck have not been prepared in sufficiently purified form to allow meaningful comparisons. Such information on the extent of divergence in hormones among closely related species, especially within a 508
0016-6480/79/040508-13$01.00/O Copyright All rights
@ 1979 by Academic Press, Inc. of reproduction in any form reserved.
GONADOTROPINS
FROM
single order, are basic to evaluating the importance of variations in structure and function of hormones among higher taxa. At present, it is difficult to generalize about the nature of avian gonadotropins. In an attempt to gain more detailed information on the avian gonadotropins, we undertook further studies to purify and characterize the two gonadotropins from the turkey, Meleagris gallopavo. We also took this opportunity to compare the turkey hormones with those derived from the related galliform, the chicken Gaffus donzesticus. For the latter purpose, preparations of chicken hormones from three independent laboratories were compared. MATERIALS AND METHODS Ftiactionation of Turkey Gonadotropins Isolations of gonadotropins were performed with two batches of turkey glands. The first batch, 203 g welt wt, representing about 9000 glands, was obtained primarily from sexually mature females, with about IO& of the glands from a mixture of mature and 21- to 22Jweek old immature males. The second batch of glalnds. weighing 554 g and representing about 25,000 birds, was primarily from 20- to 21-week-old males, buq included a small portion of immature females and sexually mature animals of both sexes. All glands were collected at poultry processing plants. The heads were obtained from birds within 20 mill of death, opened quickly, and the pituitary glands ha&ested on dry ice. They were stored at -20” until used for extraction, within about 10 weeks of collection. The fractionation procedures were similar to those summarized by Licht et al. (1977d) which have been suocessfully used for the purification of hormones from a wide variety of species. The glands were homogenized in a Waring Blender with cold water, usijlg about 2.5 ml of water per g of fresh tissue, then diluted to a final concentration of about 30 g of tissue pen 100 ml H,O, and the pH was then adjusted to 9.5 with Ca(OH),. After centrifugation, the supernatant wad, adjusted to 0.15 M (NH&SO, and the pH was adjusted to 4 by the addition of metaphosphoric acid. The material was then centrifuged and the supematan{, containing the glycoprotein hormones, was collected. The precipitate, containing growth hormone ancf prolactin, was dialyzed, lyophilyzed, and used in stu lies to be reported elsewhere. The supernatant wa 1 brought to 0.8 saturation with ammonium sulfate and1 adjusted to pH 6.5 with NaOH. The resulting
TURKEY
PITUITARIES
509
precipitate was collected by centrifugation. dissolved in a small volume of 0.2 M K,H PO,, and heated to 55-60” for 2 min to reduce activity of proteases and carbohydrases (Papkoff ef al., 1965). It was then dialyzed against water for several days and lyophilized. The glycoprotein was extracted from the lyophilized material with cold 10% NH,Ac pH 5.140% ethanol (Stockell-Hat-tree, 1966) and further purified by ion-exchange and gel filtration chromatography as follows: Material from the first batch of glands was first applied to a DEAE-cellulose column equilibrated with 0.03 M NH,HCO,, pH 9 (Licht and Papkoff, 1974a). The unabsorbed fraction was then run on a sulfoethyl-Sephadex C-50 (SE-CSO) column equilibrated with the same buffer. as described by Papkoff et al. (1965). The material that absorbed to this SE-C50 column was eluted with 1 M NH,HCO, and twice chromatographed on a 2.5 x 95-cm Sephadex G-100 column to yield the final LH fraction (designated B25B). FSH from this batch of glands was not purified. Several additional ion-exchange columns were introduced in processing the second, larger batch of glands. After the DEAE-cellulose column, the unabsorbed fraction (containing almost all of the gonadotropic activity) was chromatographed on Amberlite CG-50 (Licht and Papkoff, 1974a). The unabsorbed (pH 5.1) eluate (containing all FSH and half the LH) was chromatographed on SE-CSO: the unabsorbed fraction from this column contained the FSH and the absorbed fraction the LH. The remainder of the LH which was initially absorbed on the Amberlite column was also further purified by SE-CSO. The FSH fraction was further purified on a column of CMSephadex. The gel was equilibrated with pH 5.4. 0.1 M sodium acetate and the sample applied in the same buffer. The hormone passed through the column unretarded and was then purified by gel filtration on G-100 (designated B150A). The two LH factions were also subjected to G-100 (material unabsorbed on Amberlite was designated Bl IOB and absorbed LH, B 1I IB).
Chemical Analysis Carbohydrate compositions were determined using colormetric techniques described previously (Farmer et al., 1975). Amino-terminal amino acids were determined by the dansyl chloride technique (Gray, 1%7; Woods and Wang, 1967). Amino acid analyses were performed on unoxidized preparations. They were hydrolyzed in constant boiling HCI in sealed evacuated tubes at 105” for 20 hr before being analyzed in the Beckman Model 119C amino acid analyzer according to the method of Spackman et al. (1958). Spectroscopic analyses to determine tyrosine and and tryptophan content were performed by the method of Beavan and Holiday (1952).
510 Gonadotropin
BURKE
Assays
Monitoring of gonadotropins during fractionation and characterization of final products were accomplished in a variety of radioimmunoassays (RIAs), radioreceptor assays (RRAs), and bioassays. RZA. Avian LH was measured by a homologous RIA system for the turkey hormone. An antiserum was raised in a rabbit against a previously purified preparation of turkey LH (Farmer er n[., 1975) using the method of Vaitukaitis et al. (1972) and iZ51-labeled turkey LH was used as a tracer. Initially, an LH (W28BG) prepared by Farmer et al. (1975) was used as the tracer and standard but after purification of the first batch of glands in this study, the newly prepared LH (B25B) was used for these purposes. Preliminary validation of this assay was accomplished by demonstrating that the tracer could be displaced specifically by LH, from both turkey and chicken (see Results); data for serum samples were consistent with previous physiological data obtained with a described turkey LH-RIA (Wentworth et al., 1976); dose-response curves with turkey pituitary extracts and sera were parallel with the standard curve; and the expected rise in serum LH followed castration, injection of LH-RH, and long-day stimulation. Assays for pituitary fractions consisted of incubation for 1 day at room temperature in a total volume of 250 ~1 (see Licht et al., 1977~). All samples were tested in duplicate at multiple dilutions. FSH was monitored by several heterologous RIAs based on mammalian FSH. Preliminary studies employed the heterologous system (anti-ovine FSH serum with ‘*51-labeled rat FSH) described and characterized by Follett (1976). We subsequently developed a similar RIA with an independently derived anti-ovine FSH serum with ‘251-labeled human FSH as tracer (Licht and Bona Gallo, 1979). This latter assay proved to be highly FSH specific with wide phylogenetic cross-reactivity in tests with purified amphibian, reptilian, avian, and mammalian gonadotropins. Finally, the turkey FSH (Bl50A) prepared here was labeled with radioiodine and used as a tracer with this last anti-ovine FSH serum. RRA. Radioreceptor assays were based on previously described studies from this laboratory (Licht and Midgley, 1976a,b; Licht et al., 1977a,d). Basically, receptor preparations consisted of homogenates of gonads from various species (porcine ovary, sea turtle ovary and chicken testis) with ‘Z51-labeled human FSH and ‘*Y-labeled human chorionic gonadotropin (hCG) as tracers to produce tests for FSH and LH binding sites, respectively. The hCG was used only with the mammalian receptor since specific binding could not be obtained with the reptilian and avian tissues (see also Licht et al., 1977d). Ovine FSH (NIH-FSH-Sll) and LH (G3-222B = 2.75 X NIHLH-Sl) served as standards in these assays.
ET AL.
Eonssays. Five different bioassay systems were employed to monitor fractionation of gonadotropins or to compare final purified preparations of turkey and chicken gonadotropins; all assays have been used previously for such studies. The assays based on s2Puptake by l-day-old cockerel testis (according to the method of Kamiyoshi et al., 1972) and spermiation in the frog Hyla regilla (Licht, 1973) were used as total gonadotropin assays. Limited tests were performed with two mammalian bioassays considered to be hormone specific-the in vitro assay for LH based on androgen production by rat Leydig cells (Ramachandran and Sairam, 1975) and the in viva assay for FSH based on augmentation of ovarian weight in rats (Steelman and Pohley, 1953). Finally the stimulation of testis weight and androgen production by the lizard Anolis carolinensis (Licht and Pearson, 1969; see Licht et al., 1977b) was initially chosen as an FSH assay since turkey LH was previously found to be relatively inactive (Farmer er al., 1975). The later tests were performed with both hypophysectomized and intact lizards (Licht and Pearson, 1%9). Several attempts were made to measure thyrotropin activity using the uptake of 32P by chick thyroids (Licht and Papkoff, 1974a) and lZsI uptake by baby turtle thyroids (MacKenzie et al.. 1978) as well as stimulation of in viva thyroxine production in baby quail and turkeys. Bovine thyrotropin (NIH-TSH-B4) was used as a standard.
Chicken
Gonadotropins
Chicken (Callus domesticus) gonadotropins prepared in three independent laboratories were employed for comparative purposes in these studies. One batch of chicken FSH and LH was derived from studies in our laboratory (Licht et al., 1977d). The LH (W306B) was considered highly purified, but the FSH (W315C) was in a somewhat cruder stage. Another batch of chicken LH (IRC-2) and FSH (CPI-Seph 3) considered to be highly purified was supplied by Dr. B. K. Follett (see Scanes and Follett, 1972: Jenkins et al., 1978) and a third purified chicken FSH preparation (AGCHD-11113A) was supplied by Dr. S. Ishii (see Furuya and Ishii, 1974). Since these were available in only limited quantities, attention was focused on some of the more sensitive RIA and RRA procedures. Some of these results have been reported previously (Licht and Bona Gallo, 1978). Limited comparisons were also performed with the turkey LH (W28BG) reported previously (Farmer et al., 1975).
RESULTS
Chromatographic
Fractionation
Fractionation of the first batch of glands yielded one major LH fraction (B25B),
GONADOTROPINS
FROM TURKEY
whereas in the second batch of glands, t& purification of LH on the Amberlite CG-50 column resulted in two potent LH preparations (BllOB and BlllB). As indicated under Materials and Methods, the mtajority of FSH activity was lost from the first batch of glands. The second batch yij:lded a single FSH fraction (B150A). The y&d of the final LH fractions from the two batches of glands were equivalent to approximately 75 and 122 mg/kg wet wt, resdectively, (the yields of the two LH fractions obtained with the second batch of gl mds were about equal). The yield of FSH ( 4 150A), from the second batch of glands, was about 25 mg/kg. Immunological Characterization Gonadotropins
of chicken LH were also about equipotent to one another, but only about half as active as the turkey LHs in this homologous turkey RIA. In constrast, in a homologous chicken LHRIA, our turkey LH (B25B) was only half as active as the chicken LH (Jenkins et al., 1978). Neither turkey nor chicken FSH had appreciable immunoactivity in the LHRIA, being equivalent to about 3% of the purified LHs. These values, indicating low LH contamination, are consistent with those reported by Jenkins et al., (1978) for the same preparations. Results in the various heterologous FSH-RIA systems (with anti-ovine FSH sera) gave similar results whether human or turkey FSH were used as tracers; results for the assay with 1251-labeled human FSH are shown in Table 1. In these assays, turkey and chicken FSH showed a high degree of cross-reactivity with parallelism between inhibition slopes of avian and mammalian hormones. The avian FSH preparations were only slightly less active than those of mammals (cf. Licht and Bona Gallo, 1978) and there was little difference between the
of
TABLE POTENCIES
511
al., 1975). The two preparations
The activity of the final turkey fractions and various chicken gonadotropins available for comparison in the two types of RlAs are summarized in Table 1. The three turkey LH preparations obtained from the ttio batches of glands were essentially uipotent to one another and to the one 128BG) previously purified (Farmer et IMMUNOLOGICAL
PITUITARIES
1
OF TURKEY
AND
CHICKEN
GONADOTROPINS
-
Preparation identification
RIA (X turkey LH)”
Mammalian FSH-RIA (X ovine FSH)b
B86 B25B BllOB BlllB B150A W136E W306B IRC-2 w315c CPlSeph3 AGCHD-I 1113A
0.0094 1.0 0.9 0.7 0.035 0.005 0.49 0.40 0.01 Not Tested 0.03
0.0003 nil’ nil nil 0.30 0.009 0.001 Not tested 0.10 0.08 0.67
Turkey
Species
Hormone
Turkey
Pituitary extract LH
Cl-f’ r15 >15 15.4 ( 4.0-59.0) 12.4 ( 5.0-13.0) 15.3 ( 7.0-34.0) 14.2 ( 5.0-44.0) 24.1 (12.0-49.0)
0 Based on surgically hypophysectomized lizards in spring; 2+2 assay design. b Mean potency with 95% confidence interval shown in parentheses. c Based on “physiologically” hypophysectomized lizards in fall; 2+2 assay design.
key LH and FSH were especially pronounced; i.e., the LH was up to twice as potent in competing for FSH binding sites on the avian gonad. Limited supplies of the turkey LH prepared earlier (W28BG) did not permit direct comparisons in these RRAs. but previous data (Licht and Midgley , 3976b) demonstrate that it had relatively low activity in the FSH-RRA, being comparable to the
dent between the two species of LH in the FSH-RRA systems. All avian FSH preparations were more potent than chicken LH in the three FSH-RRA systems, especially in the chicken receptor. These results suggest some specificity of these FSHbinding sites. However, the three turkey LH preparations were consistently equal to or more active than all avian FSHs in all FSH-RRAs. Differences between the turTABLE POTENCY
OF TURKEY
AND CHICKEN RADIORECEFI-OR
5 GONADOTROPINS ASSAYS
IN SEVERAL
hFSH-RRA” Species
Hormone
Turkey
LH
Chicken
FSH LH FSH
ID B25B BlllB BllOB BISOA W306B IRC-2 w315c CPI-Seph 3 AGCHD-11113A
LH-RRAb
Chicken S
Chelonia 0
Pig P
14.83 25.87 16.72 10.70 0.08 NT’ 2.08 NT 16.53
8.00
0.32 0.98 0.21 0.39 0.06 NT 0.16 NT 0.24
9.80 3.23 2.43 0.20 0.08 0.50 1.10 2.50
pig0 0.018 0.027 0.009 llett, B. K. (1976). Plasma follicle-stimulating hormone during photoperiodically induced sexual maturation in male Japanese quail. J. Endocrinol. 60, 117-126. Furuya, T., and Ishii, S. (1974). Separation of chicken adenohypophysial gonadotropins. Endocrinol. Japan
21, 329-334.
Gbdden, P. M. M., and Scanes, C. G. (1975). Studies on the purification and properties of avian gonadotropins. Gen. Comp. Endocrinol. 27, .538542.
Gray, W. R. (1967). Sequential degradation plus dansylation. In “Methods in Enzymology” (C. W. Hirs, ed.), Vol. 11, pp. 469-475. Academic Press, New York. Isihii, S., and Adachi, T. (1977). Binding of avian testicular homogenate with rat follicle-stimulating hormone and inhibition of binding by hypophyseal extracts of lower vertebrates. Gen. Camp. Endocrinol.
31, 287-294.
Islhii, S., and Furuya, T. (1975). Effects of purified chicken gonadotropins on the chick testis. Gen. Comp. Endocrinol. 25, l-8. I&ii, S., Tsutsui, K.. and Adachii, T. (1978). Effects of gonadotropins on elements of testes of birds. In “Comparative Endocrinology” (P. J. Gaillard and H. H. Boer eds.) pp. 73-76. El SevieriNorthHolland Biomedical Press. Jenkins, N., Sumpter, J. P.. and Follett, B. K. (1978). The effects of vertebrate gonadotropins on androgen release in virro from testicular cells of Japanese quail and a comparison with their radioimmunoassay activities. Gen. Comp. Endocrinol. 35, 309-32 1. Kamiyoshi, M., Tanaka, K., and Tanabe, Y. (1972). A 6-hour bioassay for pituitary gonadotropins based on radioactive phosphorus uptake by chick testes. Endocrinology 91, 385-388. Licht, P. (1973). induction of spermiation in anurans by mammalian pituitary gonadotropins and their subunits. Gen. Comp. Endocrinol. 20, 522-529. Licht, P., and Bona Gallo. A. (1978). Immunochemical relatedness among pituitary follicle-stimulating hormones of tetrapod vertebrates. Gen. Comp. Endocrinol.
36, 575-584.
Licht, P., Bona Gallo, A., and Daniels, E. L. (1977a). III vitro binding of radioiodinated sea turtle
TURKEY
519
PITUITARIES
(Chelonia mydas) follicle-stimulating reptilian gonadal tissues. Gen. Comp.
hormone to Endocrinol.
33, 266-230.
Licht, P., Bona Gallo, A., Hat-tree, A. S., and Shownkeen, R. C. (1977b). Physiological actions of human follicle-stimulating hormone and its P-subunit in reptiles. J. Endocrinol. 74, 441-447. Licht, P., Farmer, S. W., and Papkoff, H. (1976). Further studies on the chemical nature of reptilian pituitary gonadotropins: FSH and LH in the American alligator and green sea turtle. Biot. RPprod.
14, 222-232.
Licht, P., MacKenzie, D. S., Papkoff, H., and Farmer, S. W. (1977~). Immunological studies with the gonadotropins and their subunits from the green sea turtle Chelonia mydas. Gen. Camp. Endocrinol.
33, 226-230.
Licht, P., and Midgley, A. R., Jr. (1976a). In vifro binding of radioiodinated human folliclestimulating hormone to reptilian and avian gonads: Radioligand studies with mammalian hormones. Biol. Reprod. 15, 195-205. Licht, P., and Midgley, A. R., Jr. (1976b). Competition for the in vitro binding of radioiodinated human follicle-stimulating hormone in reptilian, avian and mammalian gonads by nonmammalian gonadotropins. Gen. Camp. Endocrinol. 30, 364-371. Licht, P., and Papkoff, H. (1974a). Separation of two distinct gonadotropins from the pituitary gland of the bullfrog Rana catesbeiana. Endocrinology 94, 1587-1594. Licht, P., and Papkoff, H. (1974b). Phylogenetic survey of the neuroaminidase sensitivity of reptilian gonadotropin. Gen. Comp. Endocrinol. 23, 415420.
Licht, P., and Papkoff, H. (1978). Species specificity as a key to structural evolution in pituitary glycoprotein hormones. In “Comparative Endocrinology” (P. J. Gaillard and H. H. Boer eds.) pp. 405-408. El Sevier/North-Holland Biomedical Press. Licht, P., and Pearson, A. K. (1%9). Effects of mammalian gonadotropin (FSH and LH) on the testes of the lizard Anolis curolinensis. Gen. Comp. Endocrinol.
13, 367-381.
Licht, P., Papkoff, H., Farmer, S. W., Muller, C. H., Tsui, H. W., and Crews, D. (1977d). Evolution in gonadotropin structure and function Rec. Prog. Horm. Res. 33, 169-248. MacKenzie, D., Licht, P., and Papkoff, H. (1978). Thyrotropin from amphibian (Rana catesbeiana) pituitaries and evidence for heterothyrotropic activity of bullfrog luteinizing hormone in reptiles. Gen Camp.
Endocrinol.
36, 566-574.
Papkoff. H., Gospodarowicz, D., Candiotti, A., and Li, C. H. (1%5). Preparation of ovine interstitial cell-stimulating hormone in high yield. Arch. Biochem.
Biophys.
111, 431-438.
520
BURKE
Ramachandran, J., and Sairam, M. R. (1975). The effects of interstitial cell-stimulating hormone, its subunits and recombinations on isolated rat Leydig cells. Arch. Biochem. Biophys. 167, 297300. Sairam, M. R., and Papkoff, H. (1974). In “Handbook of Physiology” (E. Knobil and W. H. Sawyer, eds.), Vol. IV, Pt. 2, Sec. 7, pp. 111-131. American Physiological Society, Washington, D. C. Scanes, C. G., and Follett, B. K. (1972). Fractionation and assay of chicken pituitary hormones. &it. Poultry Sci. 13, 603-610. Spackman, D. H., Stein, W. H., and Moore, S. (1958). Automatic recording apparatus for use in chromatography of amino acids. Anal. Chem. 30, 1190-1206. Stockell-Hartree, A. (1966). Separation and partial purification of the protein hormones from human pituitary glands. Biochem. J. 100, 754-761. Stockell-Hartree, A., and Cunningham, F. J. (1969). Purification of chicken pituitary follicle-
ET AL. stimulating hormone and luteinizing hormone. J. 43, 609-616. Steelman, S. L., and Pohley, F. M. (1953). Assay of the follicle-stimulating hormone based on the augmentation with human chorionic gonadotropin Endocrinology 53, 604-616. Vaitukaitis, J., Robbins, J. B., Nieschlag, E., and Ross, G. T. (1972). A method for producing specific antisera with small doses of immunogen. J. Endocrinol.
Clin.
Endocrinol.
Metabol.
33, 988-991.
Wentworth, B. C. (1971). Isolation and purification of follicle stimulating hormone and luteinizing hormone from turkey pituitary glands. Biol. Reprod. 5, 107-108. Wentworth, B. C., Burke, W. H., and Birrenkott, G. P. (1976). A radioimmunoassay for turkey luteinizing hormone. Gen. Comp. Endocrinol. 29, 119-127. Woods, K. R., and Wang, K. T. (1967). Separation of dansyl-amino acids by polyamide layer chromatography. Biochim. Biophys. Acta 133, 369-370.
AND
COMPARATIVE
Isolation
37, 508-520 (1979)
ENDOCRINOLOGY
and Characterization of Luteinizing Hormone and Follicle-Stimulating Hormone from Pituitary Glands of the Turkey (Meleagris gallopavo) W. H. BURKE,*
P. LIGHT,? H. PAPKOFF,$ AND A. BONA GALLot
*Departmettt of Animal Science, University of Minnesota, St. Paul, Minnesota 55108; *Departmen: of Zoology, Utdversity of California. Berkeley, California 94720; and *Hormone Research Laborator?: and Reproductive Endocrinology Center, University of California. San Francisco. California 94143
Accepted January 9. 1979 Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were purified from the pituitary glands of the turkey (Meleagris gallopavo). These hormones were characterized biochemically and biologically and compared with chicken gonadotropins prepared in several independent laboratories. Amino acid and carbohydrate analyses demonstrated homology between turkey and other species of gonadotropin. The turkey LH purified here had significantly higher carbohydrate content than a previous preparation of turkey LH. Immunological studies further confirmed that the turkey FSH and LH were distinct from one another and that each was homologous to the respective gonadotropin from other vertebrates: the immunopotencies of the turkey hormones were similar to those from chickens. A variety of bioassays and radioreceptor assays (RRAs) confirmed the biological activity of the two turkey gonadotropins and revealed that the turkey LH was distinct from that of the chicken. As expected, the two types of turkey hormones were approximately equipotent in total gonadotropin bioassays (frog spermiation and $*P uptake by chick testes), and only the turkey LH was active in the rat Leydig cell assay and in RRA for LH in mammals. However, the turkey LH was also highly potent in several assays considered to be relatively FSH specific, including the Anolis lizard assay and several RRA systems using mammalian, turtle and avian gonadal receptors with ‘ZSI-labeled human FSH as tracer. Turkey and chicken FSH are similar in the RRAs, but the turkey LH was consistently more potent than either avian FSH in competing for FSH-binding sites. Chicken LH had relatively low activity by comparison. It is suggested that the evolution of the structure of active sites in turkey LH has involved convergence on those of the FSH molecule.
Several laboratories have reported on the separation of gonadotropins from the pituitaries of two species of galliform birds, the domestic fowl (Stockell-Hartree and Cunningham, 1969) and turkey (Farmer et al., 1975, Wentworth, 1971) and one gonadotropin has been partially purified from an anseriform bird, the duck (Licht et al., 1977d). Limited chemical data for these hormones suggest significant interspecific differences among avian hormones. For example, the luteinizing hormone (LH)
from the turkey appeared unique among all vertebrates examined in its low carbohydrate content, and the duck gonadotropin exhibited a different spectrum of activities than those of the other avian species (Licht et al., 1977d); unfortunately, folliclestimulating hormones (FSH) from turkey and duck have not been prepared in sufficiently purified form to allow meaningful comparisons. Such information on the extent of divergence in hormones among closely related species, especially within a 508
0016-6480/79/040508-13$01.00/O Copyright All rights
@ 1979 by Academic Press, Inc. of reproduction in any form reserved.
GONADOTROPINS
FROM
single order, are basic to evaluating the importance of variations in structure and function of hormones among higher taxa. At present, it is difficult to generalize about the nature of avian gonadotropins. In an attempt to gain more detailed information on the avian gonadotropins, we undertook further studies to purify and characterize the two gonadotropins from the turkey, Meleagris gallopavo. We also took this opportunity to compare the turkey hormones with those derived from the related galliform, the chicken Gaffus donzesticus. For the latter purpose, preparations of chicken hormones from three independent laboratories were compared. MATERIALS AND METHODS Ftiactionation of Turkey Gonadotropins Isolations of gonadotropins were performed with two batches of turkey glands. The first batch, 203 g welt wt, representing about 9000 glands, was obtained primarily from sexually mature females, with about IO& of the glands from a mixture of mature and 21- to 22Jweek old immature males. The second batch of glalnds. weighing 554 g and representing about 25,000 birds, was primarily from 20- to 21-week-old males, buq included a small portion of immature females and sexually mature animals of both sexes. All glands were collected at poultry processing plants. The heads were obtained from birds within 20 mill of death, opened quickly, and the pituitary glands ha&ested on dry ice. They were stored at -20” until used for extraction, within about 10 weeks of collection. The fractionation procedures were similar to those summarized by Licht et al. (1977d) which have been suocessfully used for the purification of hormones from a wide variety of species. The glands were homogenized in a Waring Blender with cold water, usijlg about 2.5 ml of water per g of fresh tissue, then diluted to a final concentration of about 30 g of tissue pen 100 ml H,O, and the pH was then adjusted to 9.5 with Ca(OH),. After centrifugation, the supernatant wad, adjusted to 0.15 M (NH&SO, and the pH was adjusted to 4 by the addition of metaphosphoric acid. The material was then centrifuged and the supematan{, containing the glycoprotein hormones, was collected. The precipitate, containing growth hormone ancf prolactin, was dialyzed, lyophilyzed, and used in stu lies to be reported elsewhere. The supernatant wa 1 brought to 0.8 saturation with ammonium sulfate and1 adjusted to pH 6.5 with NaOH. The resulting
TURKEY
PITUITARIES
509
precipitate was collected by centrifugation. dissolved in a small volume of 0.2 M K,H PO,, and heated to 55-60” for 2 min to reduce activity of proteases and carbohydrases (Papkoff ef al., 1965). It was then dialyzed against water for several days and lyophilized. The glycoprotein was extracted from the lyophilized material with cold 10% NH,Ac pH 5.140% ethanol (Stockell-Hat-tree, 1966) and further purified by ion-exchange and gel filtration chromatography as follows: Material from the first batch of glands was first applied to a DEAE-cellulose column equilibrated with 0.03 M NH,HCO,, pH 9 (Licht and Papkoff, 1974a). The unabsorbed fraction was then run on a sulfoethyl-Sephadex C-50 (SE-CSO) column equilibrated with the same buffer. as described by Papkoff et al. (1965). The material that absorbed to this SE-C50 column was eluted with 1 M NH,HCO, and twice chromatographed on a 2.5 x 95-cm Sephadex G-100 column to yield the final LH fraction (designated B25B). FSH from this batch of glands was not purified. Several additional ion-exchange columns were introduced in processing the second, larger batch of glands. After the DEAE-cellulose column, the unabsorbed fraction (containing almost all of the gonadotropic activity) was chromatographed on Amberlite CG-50 (Licht and Papkoff, 1974a). The unabsorbed (pH 5.1) eluate (containing all FSH and half the LH) was chromatographed on SE-CSO: the unabsorbed fraction from this column contained the FSH and the absorbed fraction the LH. The remainder of the LH which was initially absorbed on the Amberlite column was also further purified by SE-CSO. The FSH fraction was further purified on a column of CMSephadex. The gel was equilibrated with pH 5.4. 0.1 M sodium acetate and the sample applied in the same buffer. The hormone passed through the column unretarded and was then purified by gel filtration on G-100 (designated B150A). The two LH factions were also subjected to G-100 (material unabsorbed on Amberlite was designated Bl IOB and absorbed LH, B 1I IB).
Chemical Analysis Carbohydrate compositions were determined using colormetric techniques described previously (Farmer et al., 1975). Amino-terminal amino acids were determined by the dansyl chloride technique (Gray, 1%7; Woods and Wang, 1967). Amino acid analyses were performed on unoxidized preparations. They were hydrolyzed in constant boiling HCI in sealed evacuated tubes at 105” for 20 hr before being analyzed in the Beckman Model 119C amino acid analyzer according to the method of Spackman et al. (1958). Spectroscopic analyses to determine tyrosine and and tryptophan content were performed by the method of Beavan and Holiday (1952).
510 Gonadotropin
BURKE
Assays
Monitoring of gonadotropins during fractionation and characterization of final products were accomplished in a variety of radioimmunoassays (RIAs), radioreceptor assays (RRAs), and bioassays. RZA. Avian LH was measured by a homologous RIA system for the turkey hormone. An antiserum was raised in a rabbit against a previously purified preparation of turkey LH (Farmer er n[., 1975) using the method of Vaitukaitis et al. (1972) and iZ51-labeled turkey LH was used as a tracer. Initially, an LH (W28BG) prepared by Farmer et al. (1975) was used as the tracer and standard but after purification of the first batch of glands in this study, the newly prepared LH (B25B) was used for these purposes. Preliminary validation of this assay was accomplished by demonstrating that the tracer could be displaced specifically by LH, from both turkey and chicken (see Results); data for serum samples were consistent with previous physiological data obtained with a described turkey LH-RIA (Wentworth et al., 1976); dose-response curves with turkey pituitary extracts and sera were parallel with the standard curve; and the expected rise in serum LH followed castration, injection of LH-RH, and long-day stimulation. Assays for pituitary fractions consisted of incubation for 1 day at room temperature in a total volume of 250 ~1 (see Licht et al., 1977~). All samples were tested in duplicate at multiple dilutions. FSH was monitored by several heterologous RIAs based on mammalian FSH. Preliminary studies employed the heterologous system (anti-ovine FSH serum with ‘*51-labeled rat FSH) described and characterized by Follett (1976). We subsequently developed a similar RIA with an independently derived anti-ovine FSH serum with ‘251-labeled human FSH as tracer (Licht and Bona Gallo, 1979). This latter assay proved to be highly FSH specific with wide phylogenetic cross-reactivity in tests with purified amphibian, reptilian, avian, and mammalian gonadotropins. Finally, the turkey FSH (Bl50A) prepared here was labeled with radioiodine and used as a tracer with this last anti-ovine FSH serum. RRA. Radioreceptor assays were based on previously described studies from this laboratory (Licht and Midgley, 1976a,b; Licht et al., 1977a,d). Basically, receptor preparations consisted of homogenates of gonads from various species (porcine ovary, sea turtle ovary and chicken testis) with ‘Z51-labeled human FSH and ‘*Y-labeled human chorionic gonadotropin (hCG) as tracers to produce tests for FSH and LH binding sites, respectively. The hCG was used only with the mammalian receptor since specific binding could not be obtained with the reptilian and avian tissues (see also Licht et al., 1977d). Ovine FSH (NIH-FSH-Sll) and LH (G3-222B = 2.75 X NIHLH-Sl) served as standards in these assays.
ET AL.
Eonssays. Five different bioassay systems were employed to monitor fractionation of gonadotropins or to compare final purified preparations of turkey and chicken gonadotropins; all assays have been used previously for such studies. The assays based on s2Puptake by l-day-old cockerel testis (according to the method of Kamiyoshi et al., 1972) and spermiation in the frog Hyla regilla (Licht, 1973) were used as total gonadotropin assays. Limited tests were performed with two mammalian bioassays considered to be hormone specific-the in vitro assay for LH based on androgen production by rat Leydig cells (Ramachandran and Sairam, 1975) and the in viva assay for FSH based on augmentation of ovarian weight in rats (Steelman and Pohley, 1953). Finally the stimulation of testis weight and androgen production by the lizard Anolis carolinensis (Licht and Pearson, 1969; see Licht et al., 1977b) was initially chosen as an FSH assay since turkey LH was previously found to be relatively inactive (Farmer er al., 1975). The later tests were performed with both hypophysectomized and intact lizards (Licht and Pearson, 1%9). Several attempts were made to measure thyrotropin activity using the uptake of 32P by chick thyroids (Licht and Papkoff, 1974a) and lZsI uptake by baby turtle thyroids (MacKenzie et al.. 1978) as well as stimulation of in viva thyroxine production in baby quail and turkeys. Bovine thyrotropin (NIH-TSH-B4) was used as a standard.
Chicken
Gonadotropins
Chicken (Callus domesticus) gonadotropins prepared in three independent laboratories were employed for comparative purposes in these studies. One batch of chicken FSH and LH was derived from studies in our laboratory (Licht et al., 1977d). The LH (W306B) was considered highly purified, but the FSH (W315C) was in a somewhat cruder stage. Another batch of chicken LH (IRC-2) and FSH (CPI-Seph 3) considered to be highly purified was supplied by Dr. B. K. Follett (see Scanes and Follett, 1972: Jenkins et al., 1978) and a third purified chicken FSH preparation (AGCHD-11113A) was supplied by Dr. S. Ishii (see Furuya and Ishii, 1974). Since these were available in only limited quantities, attention was focused on some of the more sensitive RIA and RRA procedures. Some of these results have been reported previously (Licht and Bona Gallo, 1978). Limited comparisons were also performed with the turkey LH (W28BG) reported previously (Farmer et al., 1975).
RESULTS
Chromatographic
Fractionation
Fractionation of the first batch of glands yielded one major LH fraction (B25B),
GONADOTROPINS
FROM TURKEY
whereas in the second batch of glands, t& purification of LH on the Amberlite CG-50 column resulted in two potent LH preparations (BllOB and BlllB). As indicated under Materials and Methods, the mtajority of FSH activity was lost from the first batch of glands. The second batch yij:lded a single FSH fraction (B150A). The y&d of the final LH fractions from the two batches of glands were equivalent to approximately 75 and 122 mg/kg wet wt, resdectively, (the yields of the two LH fractions obtained with the second batch of gl mds were about equal). The yield of FSH ( 4 150A), from the second batch of glands, was about 25 mg/kg. Immunological Characterization Gonadotropins
of chicken LH were also about equipotent to one another, but only about half as active as the turkey LHs in this homologous turkey RIA. In constrast, in a homologous chicken LHRIA, our turkey LH (B25B) was only half as active as the chicken LH (Jenkins et al., 1978). Neither turkey nor chicken FSH had appreciable immunoactivity in the LHRIA, being equivalent to about 3% of the purified LHs. These values, indicating low LH contamination, are consistent with those reported by Jenkins et al., (1978) for the same preparations. Results in the various heterologous FSH-RIA systems (with anti-ovine FSH sera) gave similar results whether human or turkey FSH were used as tracers; results for the assay with 1251-labeled human FSH are shown in Table 1. In these assays, turkey and chicken FSH showed a high degree of cross-reactivity with parallelism between inhibition slopes of avian and mammalian hormones. The avian FSH preparations were only slightly less active than those of mammals (cf. Licht and Bona Gallo, 1978) and there was little difference between the
of
TABLE POTENCIES
511
al., 1975). The two preparations
The activity of the final turkey fractions and various chicken gonadotropins available for comparison in the two types of RlAs are summarized in Table 1. The three turkey LH preparations obtained from the ttio batches of glands were essentially uipotent to one another and to the one 128BG) previously purified (Farmer et IMMUNOLOGICAL
PITUITARIES
1
OF TURKEY
AND
CHICKEN
GONADOTROPINS
-
Preparation identification
RIA (X turkey LH)”
Mammalian FSH-RIA (X ovine FSH)b
B86 B25B BllOB BlllB B150A W136E W306B IRC-2 w315c CPlSeph3 AGCHD-I 1113A
0.0094 1.0 0.9 0.7 0.035 0.005 0.49 0.40 0.01 Not Tested 0.03
0.0003 nil’ nil nil 0.30 0.009 0.001 Not tested 0.10 0.08 0.67
Turkey
Species
Hormone
Turkey
Pituitary extract LH
Cl-f’ r15 >15 15.4 ( 4.0-59.0) 12.4 ( 5.0-13.0) 15.3 ( 7.0-34.0) 14.2 ( 5.0-44.0) 24.1 (12.0-49.0)
0 Based on surgically hypophysectomized lizards in spring; 2+2 assay design. b Mean potency with 95% confidence interval shown in parentheses. c Based on “physiologically” hypophysectomized lizards in fall; 2+2 assay design.
key LH and FSH were especially pronounced; i.e., the LH was up to twice as potent in competing for FSH binding sites on the avian gonad. Limited supplies of the turkey LH prepared earlier (W28BG) did not permit direct comparisons in these RRAs. but previous data (Licht and Midgley , 3976b) demonstrate that it had relatively low activity in the FSH-RRA, being comparable to the
dent between the two species of LH in the FSH-RRA systems. All avian FSH preparations were more potent than chicken LH in the three FSH-RRA systems, especially in the chicken receptor. These results suggest some specificity of these FSHbinding sites. However, the three turkey LH preparations were consistently equal to or more active than all avian FSHs in all FSH-RRAs. Differences between the turTABLE POTENCY
OF TURKEY
AND CHICKEN RADIORECEFI-OR
5 GONADOTROPINS ASSAYS
IN SEVERAL
hFSH-RRA” Species
Hormone
Turkey
LH
Chicken
FSH LH FSH
ID B25B BlllB BllOB BISOA W306B IRC-2 w315c CPI-Seph 3 AGCHD-11113A
LH-RRAb
Chicken S
Chelonia 0
Pig P
14.83 25.87 16.72 10.70 0.08 NT’ 2.08 NT 16.53
8.00
0.32 0.98 0.21 0.39 0.06 NT 0.16 NT 0.24
9.80 3.23 2.43 0.20 0.08 0.50 1.10 2.50
pig0 0.018 0.027 0.009 llett, B. K. (1976). Plasma follicle-stimulating hormone during photoperiodically induced sexual maturation in male Japanese quail. J. Endocrinol. 60, 117-126. Furuya, T., and Ishii, S. (1974). Separation of chicken adenohypophysial gonadotropins. Endocrinol. Japan
21, 329-334.
Gbdden, P. M. M., and Scanes, C. G. (1975). Studies on the purification and properties of avian gonadotropins. Gen. Comp. Endocrinol. 27, .538542.
Gray, W. R. (1967). Sequential degradation plus dansylation. In “Methods in Enzymology” (C. W. Hirs, ed.), Vol. 11, pp. 469-475. Academic Press, New York. Isihii, S., and Adachi, T. (1977). Binding of avian testicular homogenate with rat follicle-stimulating hormone and inhibition of binding by hypophyseal extracts of lower vertebrates. Gen. Camp. Endocrinol.
31, 287-294.
Islhii, S., and Furuya, T. (1975). Effects of purified chicken gonadotropins on the chick testis. Gen. Comp. Endocrinol. 25, l-8. I&ii, S., Tsutsui, K.. and Adachii, T. (1978). Effects of gonadotropins on elements of testes of birds. In “Comparative Endocrinology” (P. J. Gaillard and H. H. Boer eds.) pp. 73-76. El SevieriNorthHolland Biomedical Press. Jenkins, N., Sumpter, J. P.. and Follett, B. K. (1978). The effects of vertebrate gonadotropins on androgen release in virro from testicular cells of Japanese quail and a comparison with their radioimmunoassay activities. Gen. Comp. Endocrinol. 35, 309-32 1. Kamiyoshi, M., Tanaka, K., and Tanabe, Y. (1972). A 6-hour bioassay for pituitary gonadotropins based on radioactive phosphorus uptake by chick testes. Endocrinology 91, 385-388. Licht, P. (1973). induction of spermiation in anurans by mammalian pituitary gonadotropins and their subunits. Gen. Comp. Endocrinol. 20, 522-529. Licht, P., and Bona Gallo. A. (1978). Immunochemical relatedness among pituitary follicle-stimulating hormones of tetrapod vertebrates. Gen. Comp. Endocrinol.
36, 575-584.
Licht, P., Bona Gallo, A., and Daniels, E. L. (1977a). III vitro binding of radioiodinated sea turtle
TURKEY
519
PITUITARIES
(Chelonia mydas) follicle-stimulating reptilian gonadal tissues. Gen. Comp.
hormone to Endocrinol.
33, 266-230.
Licht, P., Bona Gallo, A., Hat-tree, A. S., and Shownkeen, R. C. (1977b). Physiological actions of human follicle-stimulating hormone and its P-subunit in reptiles. J. Endocrinol. 74, 441-447. Licht, P., Farmer, S. W., and Papkoff, H. (1976). Further studies on the chemical nature of reptilian pituitary gonadotropins: FSH and LH in the American alligator and green sea turtle. Biot. RPprod.
14, 222-232.
Licht, P., MacKenzie, D. S., Papkoff, H., and Farmer, S. W. (1977~). Immunological studies with the gonadotropins and their subunits from the green sea turtle Chelonia mydas. Gen. Camp. Endocrinol.
33, 226-230.
Licht, P., and Midgley, A. R., Jr. (1976a). In vifro binding of radioiodinated human folliclestimulating hormone to reptilian and avian gonads: Radioligand studies with mammalian hormones. Biol. Reprod. 15, 195-205. Licht, P., and Midgley, A. R., Jr. (1976b). Competition for the in vitro binding of radioiodinated human follicle-stimulating hormone in reptilian, avian and mammalian gonads by nonmammalian gonadotropins. Gen. Camp. Endocrinol. 30, 364-371. Licht, P., and Papkoff, H. (1974a). Separation of two distinct gonadotropins from the pituitary gland of the bullfrog Rana catesbeiana. Endocrinology 94, 1587-1594. Licht, P., and Papkoff, H. (1974b). Phylogenetic survey of the neuroaminidase sensitivity of reptilian gonadotropin. Gen. Comp. Endocrinol. 23, 415420.
Licht, P., and Papkoff, H. (1978). Species specificity as a key to structural evolution in pituitary glycoprotein hormones. In “Comparative Endocrinology” (P. J. Gaillard and H. H. Boer eds.) pp. 405-408. El Sevier/North-Holland Biomedical Press. Licht, P., and Pearson, A. K. (1%9). Effects of mammalian gonadotropin (FSH and LH) on the testes of the lizard Anolis curolinensis. Gen. Comp. Endocrinol.
13, 367-381.
Licht, P., Papkoff, H., Farmer, S. W., Muller, C. H., Tsui, H. W., and Crews, D. (1977d). Evolution in gonadotropin structure and function Rec. Prog. Horm. Res. 33, 169-248. MacKenzie, D., Licht, P., and Papkoff, H. (1978). Thyrotropin from amphibian (Rana catesbeiana) pituitaries and evidence for heterothyrotropic activity of bullfrog luteinizing hormone in reptiles. Gen Camp.
Endocrinol.
36, 566-574.
Papkoff. H., Gospodarowicz, D., Candiotti, A., and Li, C. H. (1%5). Preparation of ovine interstitial cell-stimulating hormone in high yield. Arch. Biochem.
Biophys.
111, 431-438.
520
BURKE
Ramachandran, J., and Sairam, M. R. (1975). The effects of interstitial cell-stimulating hormone, its subunits and recombinations on isolated rat Leydig cells. Arch. Biochem. Biophys. 167, 297300. Sairam, M. R., and Papkoff, H. (1974). In “Handbook of Physiology” (E. Knobil and W. H. Sawyer, eds.), Vol. IV, Pt. 2, Sec. 7, pp. 111-131. American Physiological Society, Washington, D. C. Scanes, C. G., and Follett, B. K. (1972). Fractionation and assay of chicken pituitary hormones. &it. Poultry Sci. 13, 603-610. Spackman, D. H., Stein, W. H., and Moore, S. (1958). Automatic recording apparatus for use in chromatography of amino acids. Anal. Chem. 30, 1190-1206. Stockell-Hartree, A. (1966). Separation and partial purification of the protein hormones from human pituitary glands. Biochem. J. 100, 754-761. Stockell-Hartree, A., and Cunningham, F. J. (1969). Purification of chicken pituitary follicle-
ET AL. stimulating hormone and luteinizing hormone. J. 43, 609-616. Steelman, S. L., and Pohley, F. M. (1953). Assay of the follicle-stimulating hormone based on the augmentation with human chorionic gonadotropin Endocrinology 53, 604-616. Vaitukaitis, J., Robbins, J. B., Nieschlag, E., and Ross, G. T. (1972). A method for producing specific antisera with small doses of immunogen. J. Endocrinol.
Clin.
Endocrinol.
Metabol.
33, 988-991.
Wentworth, B. C. (1971). Isolation and purification of follicle stimulating hormone and luteinizing hormone from turkey pituitary glands. Biol. Reprod. 5, 107-108. Wentworth, B. C., Burke, W. H., and Birrenkott, G. P. (1976). A radioimmunoassay for turkey luteinizing hormone. Gen. Comp. Endocrinol. 29, 119-127. Woods, K. R., and Wang, K. T. (1967). Separation of dansyl-amino acids by polyamide layer chromatography. Biochim. Biophys. Acta 133, 369-370.
