Studies on several marsupial anterior pituitary hormones

GENERAL AND COMPARATIVE ENDOCRINOLOGY 43, 336-345 (1981)

Studies on Several Marsupial Anterior Pituitary Hormones

SUSANWALKERFARMER," PAULLICHT,? ANTONELLABONAGALLO,~ ROSALIE MERCADO-SIMMEN,* FRANCESE.DELISLE,* AND HAROLDPAPKOFF*+

*Hormone Research Laboratory, University of California, San Francisco, California 94143, iDepartment of Zoology, University of California, Berkeley, California 94720, and $Reproductive Endocrinology Center,

University of California, San Francisco, California 94143

Accepted July 14, 1980

The anterior pituitary hormones LH, GH, and PRL were purified from several marsupial species, Eastern grey kangaroo (Macropus giganteus), Western grey kangaroo (Macropus fuliginosus), tammar wallaby (Macropus eugenii), and brush possum (Trichosurus vulpecula). LH was isolated only from the kangaroos, whereas GH and PRL were isolated from all four species. The purified marsupial hormones were identified on the basis of bioassay, radioreceptor assay, and radioimmunoassay. They were found to be less potent biologically than the respective eutherian hormones in eutherian assays. Chemical characterization, including NH,-terminal analysis, amino acid composition, disc gel electrophoresis, and molecular weight determination, revealed basic similarities to eutherian hormones. A homologous, double-antibody radioimmunoassay for Eastern grey kangaroo PRL was developed. This assay was specific for marsupial PRLs; there was very low or no cross-reaction with other kangaroo hormones or eutherian PRLs. In general, the marsupial hormones were found to resemble their eutherian counterparts, but there were differences in biological and immunological potencies, emphasizing the need for purified marsupial hormone standards in studies of marsupial endocrine systems.

Marsupial reproductive physiology is characterized by several unusual aspects. In the kangaroo, for example, the female gives birth to embryonic young, supplies milk which differs in composition and volume for a neonate in the pouch and for an older offspring “at-foot,” and can be si- multaneously carrying a blastocyst in embryonic diapause (Dawson, 1979). Studies with marsupials have shown that pituitary hormones may be involved in the regulation of these reproductive processes (Tyndale- Biscoe et al., 1974). However, very little information is available on the nature of marsupial pituitary hormones. Farmer and Papkoff (1974) isolated partially purified preparations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), growth hormone (GH), prolactin (PRL), and ad- renocorticotropin (ACTH) from pitutiaries of the red kangaroo (Macropus rufa). The limited quantity of materials obtained precluded any characterization beyond iden-

t&cation of the hormonal principles. In a second study (Bona Gallo et al., 1978), we isolated LH and FSH from a glycoprotein extract of tammar wallaby (M. eugenii) pituitaries. Sufficient material was obtained to determine the amino acid composition of these hormones and to evaluate their biological and immunological activities.

This report describes the continuation of our studies on marsupial pituitary hormones. These studies have been extended to include additional species, Eastern grey (EG) kangaroo, M. giganteus; Western grey (WG) kangaroo, M. fuliginosus; and brush possum, Trichosurus vulpecula; as well as fractionation of whole pituitaries from tammar wallabies, M. eugenii. Rela- tively small pools of pituitary tissue were collected and limited yields of hormones obtained. Purifed preparations of kangaroo LH, GH, and PRL were prepared in sufficient quantities for chemical and biological characterization, as well as development of

336 00166480/81/030336-10$01.00/O Copyright @ 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

MARSUPIAL HORMONES 337

a homologous Eastern grey kangaroo PRL radioimmunoassay (RIA). GH and PRL were also prepared from tammar wallaby and brush possum pituitaries.

MATERIALS AND METHODS

Pituitary Tissue and Fractionation Procedures

Pituitaries were obtained in Australia (kangaroo and wallaby) and New Zealand (brush possum) within 30 min of death by Dr. C. H. Tyndale-Biscoe and col- leagues (CSIRO, Division of Wildlife Research, Can- berra). They were frozen immediately, stored in dry ice, and later lyophilized for shipment to San Fran- cisco. Glands were taken from mature animals of both sexes, and as either whole pituitaries or separated anterior pituitaries.

Pituitaries were fractionated by methodology similar to tbat outlined by Licht et al. (1977). Details presented in that report will not be repeated here. In brief, pituitaries were homogenized in a Waring Blendor and extracted at pH 9.5, 4”. The supernatant was adjusted to 0.15 M (NH&SO, and to pH 4 with HPO,. The supernatant contained the glycoprotein hormones and was heat treated (60” for 2 min) to destroy proteolytic enzymes and extracted with NH,Ac-EtOH to obtain a glycoprotein concentrate. Subsequent separation and purification of LH and FSH was achieved by ion- exchange chromatography employing Sephadex (SE) C-50, pH 9. LH was adsorbed and eluted with 1 M NH,HC03. Where sufficient material was available (kangaroo pituitaries) additional purification was achieved by chromatography on DEAE-cellulose and Sephadex G-100.

The precipitate resulting from addition of HPO, to the initial alkaline pituitary extract contained GH and PRL. This fraction was extracted with pH 5.1 phosphate buffer adjusted to 12% saturation with (NH&SO+ The supernatant contained the GH and was chromatographad on Amberlite CG-50 equilibrated with the same buffer. GH eluted with pH 6 buffer and was further purified on DEAE and Sephadex G-100 (for details see Farmer et al., 1976a).

The insoluble residue remaining after extraction with pH 5.1 buffer and the residue from the initial alkaline extraction of the pituitaries were combined and extracted with 40% &OH, pH 9.5 for PRL (Ellis et al., 1969). The soluble, PRL-containing extract was pre- cipit.ated by addition of 3 vol ice-cold ethanol, dialyzed, and lyaphilized. Additional purification was achieved by ion-exchange chromatography on DEAE-cellulose equilibrated with 0.03 M NH,HCO,. PRL was adsorbed on this column and eluted with 0.2 M NH,HCQ, (kangaroo) or 1 M NH,HCOB (wallaby). Final purification was achieved on Sephadex G-100 equilibrated with 0.05 M NH,HCO,.

Chemical Characterization

Where sufficient material was obtained, the following determinations were made on the marsupial hormones: amino acid composition (Spackman et al., 1958); NH,-terminal amino acid analysis by the dansyl procedure (Gray, 1967; Woods and Wang, 1967); molecular weight determination by SDS-polyacrylamide disc gel electrophoresis (Weber and Osborn, 1969); and gel electrophoresis at pH 8.3 in 7.5% gels stained with Coomassie blue (Ornstein, 1964).

Assays

The following bioassays were performed for the respective marsupial hormones: the rat tibia assay for GH (Greenspan et al., 1949); the pigeon crop sac assay for PRL (Nicoll, 1967); and the rat Leydig cell assay for LH (Moyle and Ramachandran, 1973).

Additional measurements of gonadotropin activities were obtained from radiureceptor assays employing gonadal homogenates (receptors) prepared from a eutherian (porcine ovary) or a marsupial (iMacropus testis} with either 1251-labeled hFSH (LER-1801-S) or hCG (CR-119) to quantify FSH and LH binding activities, respectively.

Radioimmunoassays employed for the identification of protein hormones were: for GH, both a monkey anti-rat GH serum (Hayashida, 1970) and a monkey anti-snapping turtle GH serum (Hayashidaet ai., 1975) with iodinated rat GH (NIAMDD); for FSH, a rabbit anti-ovine FSH serum (obtained from Dr. Gary Hodgen, as employed by Licht and Bona Gsllo, 1978) and iodinated hFSH (LER-1801-3); and.forLH, a, rabbit anti-ovine LH serum (No. 15, obtained from Dr. G. D. Niswender, Niswender et al., 1968) and “251- ovine LH (Papkoff, G3-222B).

Hormone preparations employed as standards in these assays included bovine GH (1.5 IUimg), ovine PRL (30 IU/mg), and porcine PRL (30 IU/mg) prepared by Dr. C. H. Li; ovine LH (2.8 x NIH-I&-$1), ovine FSH (25 x NIH-FSH-Sl), and canine PRL (Papkoff, 1976) prepared by Dr. H. Papkooff, and rat GH and PRL from the NIAMDD.

Kangaroo PRL Radioimmunoassay

A young male albino New Zealand rabbit was used for production of antiserum against highly purified Eastern grey kangaroo PRL. Five injections of 300, 300, 300, 150, and 100 pg each of kPRL were given in complete Freund’s adjuvant at 15 to 30-day mtervals. Each injection was divided into three equal ‘portions, two given SC and one ip. The presence of antibodies was verified by RIA. A booster of 50 gg kPRL was given in saline, and a week later the animal,was bled completely by cardiac puncture. Merthialate (l:lO,OOO) was added to the serum as a preservative.

The double-antibody radioimmunoassay procedure

338 FARMER ET AL.

employed has been previously described (Farmer and Papkoff, 1979). Eastern grey kangaroo PRL was iodinated by the chloramine-T method (Greenwood et al., 1963) employing 4 pg chloramine-T’/pg protein and 1 mCi lZ51/5 pg protein. Specific activity of the iodinated kPRL ranged from 60 to 118 /.Xi/~g (N = 6). The assay buffer employed was 0.01 M sodium phosphate, pH 7.4, containing 0.14 M NaCl, 0.5% BSA, and 0.01 M EDTA. Serial dilutions of standard and unknown preparations were added in 0.1 ml assay buffer. The kPRL antiserum was added in a volume of 0.1 ml at a final concentration of 18000, which resulted in -30% total binding in the absence of unlabeled hormone. Iodinated kPRL was added in a concentration of lO,OOO-15,000 cpm/50 ~1. Assays were performed under both equilibrium (addition of all of the above solutions on Day 1) and disequilibrium conditions. In the latter case, iodinated hormone was added after a 24hr 4” incubation of test preparations and antiserum. Second antibody, sheep anti-rabbit y-globulin, was a gift of Dr. Ted Hayashida. Each unknown was assayed in duplicate at six dilutions and the results are ex- pressed as percent B/B,. Results were computer analyzed using the program “Analysis of Radioim- munoassay,” 1975 edition, by V. B. Vader and D. Rodbard, NICHD.

RIAs of intermediate gonadotropin fractions indicated that each of these three marsupial species contained a separate LH and FSH which could be isolated from one another, and which fractionated in essentially the same manner as the respective eutherian hormones. Purification of the LH activity from the two kangaroos resulted in fractions which were relatively free of FSH, GH, and PRL activities, and these were subjected to more extensive analysis.

RESULTS

Gonadotropins

Limited yields of materials from the brush possum pituitaries precluded purification of gonadotropins from this species, and difficulties encountered with FSH from the two kangaroos prevented its final purification and characterization. How- ever, bioassays, radioreceptor assays, and

The yield (Table l), amino acid composition (Table 2), and biological and immunological properties of the two species of kangaroo LH were similar to one another and closely resembled those of the LH previously isolated from wallaby pituitaries (Bona Gallo et al., 1978). Attention is fo- cused here on several interesting properties of these Macropus LH preparations. When tested in a eutherian (ovine) LH RIA, the kangaroo LHs were, like wallaby LH, essentially equipotent to highly purified ovine LH. Repeated potency estimates in this RIA for kangaroo LHs ranged from 0.8 to 1.5 x ovine LH (G3-222B) and dose-response curves were parallel. FSH contamination of the kangaroo LHs appeared low; when tested in the mammalian FSH RIA their activities were on!y 0.6% (Western grey) and 3.8% (Eastern grey) of ovine FSH. For reference, wallaby FSH was 31%

TABLE 1 YIELDS OBTAINED OF PURIFIED MARSUPIAL PITUITARY HORMONES

Species

Eastern grey kangaroo (M. giganteus)

Western grey kangaroo (M. fuliginosus)

Tammar wallaby (M. eugenii)

Brush possum (T. vu/pecula)

Dry weight of pituitaries (g)

5.76

5.04

1.20

0.98

Hormones

LH GH PRL LH GH PRL GH PRL

GH PRL

Yield Identification Ox) No.

3.0 EG 9B 8.9 EG 7C

10.8 EG 13BR 3.4 WG 9B 4.8 WG 7C 3.7 WG 13C 1.8 WL 7B 0.9 WL 9c

1.4 BP 7B 1.9 BP 6C


TABLE 2 AMINO ACID COMPOSITION OF LUTEINIZING

HORMONE FROM SHEEP,” KANGAROO,~ AND WALLABY’

Eastern grey Western grey Tammar Sheep kangaroo kangaroo wallaby

Lys I2 14.0 12.2 12.5 His 6 6.1 2.6 5.6 Arg 11 10.1 10.0 9.1 Asp 11 13.1 11.6 10.0 Thr 16 15.7 15.6 15.8 Ser 14 17.6 17.6 16.6 Glu 14 17.3 16.6 14.2 Pro 27 22.6 21.7 25.1 Gly 11 13.0 12.2 15.5 Ala I5 19.2 20.0 18.1 cys 22 14.6 13.4 19.7 val 13 11.5 11.2 11.6 Met 7 6.6 6.1 5.6 Be 7 6.4 6.9 6.7 Leu 14 15.6 16.3 14.1 TY~ 7 6.7 12.4 7.2 Phe 8 10.5 11.2 9.6

a Data taken from Papkoff et al. (1973). * Amino acid analysis: 20-hr hydrolysis; half-cystine and

methiomne content calculated from performic acid-oxidized samples, uncorrected for hydrolytic destruction, calculated on the basis of 215 residues/mole.

c Data taken from Bona Gal10 et cl. (1978).

as potent as ovine FSH in this assay (Bona Gallo et al., 1978). Despite the above indication of immunological identity with ovine LH, the biological and binding activities of the kangaroo LH preparations in eutherian assays were only a small fraction of the potency of ovine LH. In the rat Leydig cell bioassay measuring testosterone production, Eastern grey kangaroo LH had only 3.2% (2.8-3.8%, 95% confidence limits) and Western grey kangaroo LH had only 2.9% (2.3-3.6%) the activity of ovine LH; these values were similar to those previously obtained for wallaby LH. These low biological potencies in a eutherian bioassay were consistent with results from LH binding assays employing porcine ovaries: both kangaroo LH preparations were ap- proximately 6.5% as potent as ovine LH. However, when tested in a binding assay employing kangaroo testes, the kangaroo LHs were 33% as potent as ovine LH. These binding activities are also similar to those obtained for wallaby LH in the same assays (cf. Bona Gallo ef aE., 1978).

An indication of the molecular weight of the kangaroo LHs was obtained from ;lu- tion profiles on Sephadex,G-100. The VJV, ratio for both kangaroo LHs, 1.9, was iden- tical to that of ovine LH, which corre- sponds to a molecular weight of 28,000- 30,000 (Sairam and Papkoff, 1974). Amino- terminal analyses of kangaroo LIIs revealed the presence of phenylalanine and alanine for both, with additional trace amounts of tyrosine and aspartic acid in WG kangaroo LH. Analysis of kangaroo LH by disc gel electrophoresis showed a single diffuse band at an Rf of approxi- mately 0.1, similar to the pattern observed with a variety of eutherian and nonmammalian LHs in our laboratory.

Amino acid composition data for kangaroo LH are compared with previously published data for sheep and wallaby.LH in Table 2. In general, the analyses of the four LH preparations were very similar to one another, with the exception of the low his- tidine and high tyrosine content of Western grey kangaroo LH. The relatively low. val-, ues of half-cystine in both kangaroo LIIs probably reflect the usual difficulty in ob- taining accurate analyses of this amino acid. The analyses indicated the presence of carbohydrate in the kangaroo ~ LI-Is, but amounts of individual sugars were not determined due to the small amount of mate- riais available.

Growth Hormone and Prolactin

Purified GH and PRL prep&-ations were obtained from the four marsupial species studied (Table 1). The -grey kangaroo preparations of GH and PRL were ‘characterized chemically, immunochemically, and biologically. More limited characterization studies were performed with waIlaby and brush possum hormones, which were obtained in lower yields. GH and PRL fractions previously obtained from red kangaroo pituitaries (Farmer and Papkoff, 1974) were further purified by isoelectric precipitation and were used in some of the

340 FARMER ET AL.

characterization studies for comparative purposes.

Preparations of GH from Eastern and Western grey kangaroos were both found to have phenylalanine as the principal amino-terminal residue, with trace amounts of proline and leucine also identified. Molecular weights determined by SDS gel electrophoresis were 23,200 and 22,800, respectively. On disc gel electrophoresis at pH 8.3, these GHs showed a major band at Rf 0.32, with several closely associated additional bands at higher Rf values. Similar patterns were observed for the other marsupial GHs isolated (Fig. l), with some additional diffuse staining seen with wallaby and brush possum GHs. Amino acid analyses of the two grey kangaroo GHs are presented in Table 3. Their compositions closely resemble one another as well as that of ovine GH.

The grey kangaroo GH preparations were tested for biological activity in the rat tibia assay. Both significantly stimulated the growth of tibia1 cartilage, but the dose- response slopes obtained with these GHs were not parallel to that of the bGH standard (Table 4).

Two radioimmunoassays, employing an-

FIG. 1. Disc gel electrophoresis patterns of purified marsupial growth hormone preparations. The gels were 7.5%, run at pH 8.3 and stained with Coomassie blue.

tisera to rat and snapping turtle GHs, were used to identify GH fractions during puriti- cation. Inhibition curves obtained with the purified marsupial GHs were parallel to one another in the rat assay, but not to the rat GH standard (Fig. 2A). In the snapping turtle GH RIA, inhibition curves obtained with marsupial GHs were parallel to one another and to the rat GH standard (Fig. 2B). In both assays, marsupial PRL preparations showed less than 2% cross-reaction relative to their respective species of GH (data not shown).

Eastern and Western grey kangaroo PRLs were tested for biological activity in the pigeon crop sac assay. Both had signiti- cant activity but the dose-response slopes were not parallel to that of the oPRL standard’(Table 5). Chemical characterization included molecular weight determination by SDS gel electrophoresis. Values of 25,800 and 26,000 were determined for M. giganteus and M. fuliginosus PRL, respectively, and both had leucine as their amino-terminal residue. Disc gel electrophoresis at pH 8.3 revealed a major stained band at Rf 0.80 with closely associated, faster running bands for all marsupial PRL preparations (Fig. 3). Western grey kangaroo and brush possum PRLs also showed faint bands, indicating some GH contamination, and some additional diffuse staining. The amino acid compositions of the grey kangaroo PRLs are very similar to one another (Table 3); the composition of ovine PRL is included for comparison.

A homologous double-antibody radioimmunoassay was developed using EG kangaroo PRL as antigen and iodinated tracer. Typical competitive binding curves were obtained with unlabeled marsupial pituitary PRL preparations in this assay when results were plotted as percent B/B,, vs log of hormone concentration (Fig. 4). The slope of the inhibition curve obtained with kangaroo PRL was -60 + 5, 50% inhibition of binding was achieved with 4.5 +- 1.5 ng per tube under disequilibrium condi-


TABLE 3 AMINO ACID COMPOSITION OF GROWTH HORMONE AND PROLACTIN PREPARATIONS FROM

SHEEP, EASTERN GREY KANGAROO, AND WESTERN GREY KANGAROOS

Growth hormone Prolactin

EG WG EG WG Sheep kangaroo kangaroo kangaroo kangaroo Sheep

LYS 11 10.2 9.7 8.4 9.9 9 His 3 3.8 4.0 1.3 5.1 8 Arg 13 11.6 10.8 9.4 9.2 11 Asp 16 17.3 11.0 23.7 21.5 22 Thr 12 9.6 10.0 5.5 6.4 9 Ser 13 16.8 16.6 16.8 14.8 15 Glu 24 29.8 29.0 30.9 31.4 22 Pro 6 5.5 8.2 10.0 12.9 II GUY 10 10.0 10.5 10.0 14.1 11 Ala 15 14.0 14.0 11.5 11.3 9 CYS 4 5.0 4.5 5.5 6.8 6 Val 6 9.4 8.9 9.1 8.3 10 Met 4 3.5 3.0 4.2 4.0 7 Be 7 5.2 6.0 9.4 8.1 11 Leu 27 22.6 24.0 25.2 19.6 23 Tyr 6 5.1 5.4 5.5 5.1 7 F’he 13 10.6 10.1 5.8 7.0 6 Tw 1 1.3 1.7 1.8 ND 2

0 Amino acid analysis: 20-hr hydrolysis; not corrected for hydrolytic destruction; calculated on the basis of 191 residues/mole for GH (ovine structure) and 199 residues for PRL (ovine structure); values for half-cystine and methionine were obtained with performic acid-oxidized preparations; tryptophan values were determined by spectrophotometric analysis. ND, not determined. Data for sheep GH taken from Li et al. (1973); for sheep PRL from Li et al. (1970) and Li (1976).

tions (see Materials and Methods), and the sensitivity of the assay was 0.8 + 0.07 ng [data averaged from six assays).

Assay specificity was assessed by testing various marsupial and eutherian preparations (Fig. 4). No cross-reaction was observed with Eastern grey kangaroo gonadotropin fractions. The cross-reaction ob-

TABLE 4 ASSAY OF BOVINE AND KANGAROO GH

BY THE TIBIA TEST

Species of GH

Saline Bovine

Eastern kangaroo

Western kangaroo

Dose Tibia width t/x4 (pm ? SEM)

- 164.7 2 8.2 20 254.8 f 12.3 60 282.7 f 5.0 30 237.9 t 2.5 90 250.7 k 4.8 30 245.2 i 7.7 90 253.9 i 5.8

served with Eastern grey kangaroo GH (2.4%) was undoubtedly due to a small PRL contamination of this preparation. Inhibi- tion curves obtained with other marsupial PRL preparations and a pituitary extract from an American marsupial (Did&&is vir- ginianus) were all parallel to the standard (EG kangaroo PRL). Highly purified eutherian PRLs showed a variety of degrees of cross-reaction. A parallel inhibition curve of low potency was observed with porcine PRL, significant but nonparallel inhibition with rat PRL, a minimal cross-reaction with dog PRL, and no inhibition with sheep PRL (Fig. 4).

Cross-reaction of kangaroo PRL was tested in two homologous eutherian P RIAs. One employed a rabbit anti-o,vine PRL serum and the other a guinea pig anti- porcine PRL serum: In both, kangaroo (Eastern grey or red) PRLs showed parallel

342 FARMER ET AL.

RAT GH RIP. . Rot GH statild SNAf?lNG * Eastern Grey Kmwm TURTLE

100 - o WedemGwK~o GH RIA -co 0 Tmlmrwllbby A Brush Possum

-80

FIG. 2. Competitive binding curves for purified preparations of rat and marsupial growth hormones in two double-antibody radioimmunoassays employing ‘251-labeled rat GH as tracer. In panel A a monkey anti-rat GH serum was used and in panel B a monkey anti-snapping turtle Gh serum was used. Each point represents the mean of duplicate determinations.

inhibition curves but with very low potency relative to the eutherian standards, ~1% (data not shown).

Rigorous assessment of either purity or cross-contamination of marsupial GHs and PRLs with other pituitary hormones was not possible in this study due to limited yields of hormone preparations. Disc gel electrophoretic analysis indicated that WG kangaroo and brush possum PRLs contained small amounts of GH as well as other unidentified stainable material. However, only leucine was identified at the amino terminus of the grey kangaroo PRLs, and all of the marsupial PRLs showed less than 2% cross-reaction relative to their respective GHs in both of the GH RIAs. Disc gel electrophoretic analysis of the marsupial

TABLE 5 ASSAM OF OVINE AND KANGAROO PRL BY THE

PIGEON CROP SAC ASSAY

Dose Mucosal dry weight Species of PRL (WLg) (mg ? SEM)

Saline - 9.0 k 0.9

Ovine 2 17.2 T 0.6

10 28.4 -+ 3.0

Eastern kangaroo 4 16.7 2 1.4

20 21.0 +- 2.1

Western kangaroo 4 19.2 -t 2.6 20 20.6 f 2.5

GHs indicated a faint PRL band in the grey kangaroo preparations and traces of leucine were found at the amino terminals of these GHs. Nevertheless, in the kangaroo PRL RIA both EG and WG kangaroo GHs had only 2% cross-reaction relative to the EG PRL standard. However, the WG kangaroo GH had 14% activity relative to WG kangaroo PRL. Similarly, although brush possum GH had a low cross-reaction in the kangaroo PRL RIA relative to the EG kangaroo PRL standard (0.2%), it had 7%

Aed Earna w&tern Tamrn7DOf ElFush KOR+pRFi Grey Grey Wotlu@ Possum

Ita KW@QO

FIG. 3. Disc gel electrophoresis patterns of purified marsupial prolactin preparations. The gels were 7.5%, run at pH 8.3 and stained with Coomassie blue.

343 MARSUPIAL HORMONES

80

100

ng Hormone FIG. 4. Competitive binding curves for purified preparations of eutherian and marsupial hormones,

and a crude pituitary extract (PE) from the opossum in a homologous double-antibody radioimmunoassay employing Eastern grey kangaroo PRL as antigen and tracer. Each point represents the mean of duplicate determinations.

cross-reaction relative to brush possum PRL. These results may be due to the relatively low cross-reaction of WG kangaroo and brush possum PRLs in this assay (Fig. 4).

DISCUSSION

This report extends our studies on the purification and characterization of marsupial pituitary hormones. The purification methodology employed was similar to that used previously for a variety of eutherian and nonmammalian pituitary hormones (Licht et al., 1977). In all cases, kangaroo hormones behaved identically to their eutherian counterparts in the series of purification procedures employed. Close chemical homologies with eutherian hormones were also indicated by data on amino acid’ composition, NH,-terminal residues, molecular weights, and disc gel electrophqresis. Similar electrophoretic patterns have been reported for GHs and PRLs from a diversity of species (Nicoll and Nichols, 1971; Nicoll and Licht, 1971; Farmer& aE., 1974; Farmer et al., 1976a, b). The amino acid composition of the marsupial hormones reflects many features

found for a variety of eutherian species. Noteworthy is the high content of prolille and half-cystine in marsupial and eutherian LHs, and the high content of glutamic acid and leucine in the GHs. The GH compositions are also characterized by four half- cystines and a single tryptophan residue5 while PRL compositions are ‘characterized by high values, of aspartic acid, gfutamic acid, and leucine, six half-cystine residues, and two tryptophan residues.

Although chemical differences between homologous marsupial and eutherian hormones were n.ot major, biological and binding assays provided evidence of i&- portant structural divergence between marsupial and eutherian hormones in regard #to “active sites” on the hormones. A relatively high degree of species specificity was observed in all of these assays. ‘Marsupial hormones, LH, GH, and PRL, were of ldw potency relative to euiheriar! standards in eutherian assays, but both kaqg&roo and wallaby LHs exhibited consider8bl.y greatq activity relative to eatherian ‘LB when tested in a m’arsupial receptor assay. Further data OI$ the biologicd activities .of kangaroo GH arid PRL in homblogous or at

344 FARMER ET AL.

least related marsupial species are required before the importance of species specificity can be evaluated for these hormones. In general, these biological data indicate that differences in biochemical characteristics existing between marsupial and eutherian hormones can be important for determining biological activity. These findings also em- phasize the importance of having purified marsupial hormones for use as standards in studies of marsupial endocrine systems.

An antiserum was raised against kangaroo PRL, which was found to be suitable for radioimmunoassay. This RIA was highly specific for PRL relative to other kangaroo hormones (LH, FSH, and GH); it was also relatively species specific. In fact, there was evidence of reduced immunochemical relatedness of even other marsupial PRL preparations, including those from other Macropus species. The PRLs of WC kangaroo and wallaby were only about 20% as potent as EC kangaroo PRL, and both of these were considerably more active than a more distantly related marsupial, the brush possum (Fig. 4). The dis- parity in immunoreactivity of PRLs from the two grey kangaroos adds further sup- port to the distinctive species status of these two Macropus, a situation which has only recently been recognized (cf. Poole and Catting, 1974).

The kangaroo PRL RIA proved very useful for identifying PRL-enriched fractions from other marsupial species in which the characteristic disc gel electrophoretic band was faint and there was insufficient material for bioassay. However, serum samples from wallabys in various reproductive states, including lactating females, showed no cross-reaction in this assay, although purified kangaroo PRL added to wallaby serum was readily measured. This suggests that the PRL levels in all of the sera were below the detectable level of the assay (-10 rig/ml) but further studies should be conducted on this problem.

It was of interest that the only eutherian PRL which showed a parallel inhibition

curve relative to kPRL in the kPRL RIA was of porcine origin. Porcine PRL, but not ovine or bovine, showed a line of identity with red kangaroo PRL in agar diffusion experiments using ovine PRL antiserum (Farmer and Papkoff, 1974) and porcine PRL antiserum (unpublished). Thus, a closer immunochemical identity is seen between kangaroo PRL and porcine PRL than with other eutherian PRLs. However, EG and red kangaroo PRLs also showed a weak but parallel cross-reaction in both ovine and porcine PRL RIAs.

ACKNOWLEDGMENTS

We thank Dr. C. H. Tyndale-Biscoe and his col- leagues of the CSIRO, Division of Wildlife Research, Canberra, Australia, for the collection of pituitaries; Dr. Ted Hayashida and Mrs. Eleanor Rowley for second antibody; Dr. Seina Kaplan for performing the protein iodinations; Mr. J. D. Nelson, Mr. Kenway Hoey, and Ms. Kathy Hines for expert technical as- sistance; and Dr. Choh Hao Li for critical reading of the manuscript. This work was supported in part by a Lalor Foundation Fellowship to S.W.F. and a grant from the National Science Foundation, PCM 78-12470 (H.P. and P.L.).

REFERENCES

BonaGallo, A., Licht, P., Farmer, S. W., Papkoff, H., and Hawkins, J. (1978). Fractionation and biological actions of pituitary gonadotropins from a marsupial, the wallaby (Macropus eugenii). Biol. Reprod. 19, 680-687.

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Studies on several marsupial anterior pituitary hormones