Purification of thyrotropin from the pituitaries of two turtles: The green sea turtle and the snapping turtle

GENERAL

AND

Purification

COMPARATIVE

of Thyrotropin from the Pituitaries of Two Turtles: Green Sea Turtle and the Snapping Turtle DUNCAN

Depurtment

45, 39-48 (1981)

ENDOCRINOLOGY

S. MACKENZIE,’

PAUL

LICHT

AND HAROLD

of Zoology, University of California, Berkeley, Culiforniu Luhorutory, University of Caiiforniu. Sun Fruncisco,

The

PAPKOFF*

94720; und *Hormone Cuiiforniu 94143

Research

Accepted January 21, 1981 The purification of thyrotropin from the pituitaries of two species of turtles was followed using a bioassay based on the uptake of radioiodide by the thyroid of the baby slider turtle, Pseudemys scriptu. Conventional chromatographic techniques which had previously resulted in the separation of pituitary gonadotropins were used initially, yielding a potent thyrotropin (TSH) from the snapping turtle (Chelydru serpentinu) that was relatively free of gonadotropic activity. Several pituitary fractions high in thyrotropic activity were obtained from the sea turtle (Cheloniu mydus), but all had significant gonadotropin contamination (primarily as luteinizing hormone, LH). Successful removal of this LH contamination was achieved by subjecting these glycoprotein preparations to countercurrent distribution, a technique which destroyed the gonadotropic activity while leaving the thyrotropic activity intact. Both species of turtle TSH resembled mammalian TSH in fractionation behavior. Additionally, the amino acid composition of the sea turtle TSH was strikingly similar to that of bovine TSH. Antisera raised against snapping turtle TSH, when used in conjunction with radiolabeled sea turtle TSH, allowed the development of a specific turtle thyrotropin radioimmunoassay able to detect blood levels of TSH in turtles treated with goitrogens. This represents the first successful purification of TSH from a nonmammalian tetrapod.

The extensive recent research into the nature of pituitary glycoprotein hormones from nonmammalian vertebrates has centered almost exclusively on gonadotropins (see reviews in Licht et al., 1977b; Fontaine and Burzawa-GCrard, 1977) with little work on thyrotropin (TSH). Fontaine (1969) reported on the purification of TSH from two teleost fish, and MacKenzie et al. (1978) reported on the separation of TSH from an amphibian, although this preparation contained significant gonadotropic contamination. Nonmammalian thyrotropins have presented the same purification problems as those encountered with mammalian hormones (Fontaine, 1969; Pierce, 1974). The relatively small amounts of this molecule in the pituitary necessitate the processing of enormous numbers of glands to insure adequate recovery of the purified product, while the structural similarity

between thyrotropin and the gonadotropins (primarily luteinizing hormone, LH) makes their separation difficult. In addition, great caution must be exercised in employing bioassays for thyrotropins because the heterothyrotropic activity of the gonadotropins in various TSH bioassays can often give misleading results (Fontaine, 1969; Grau and Stetson, 1977; MacKenzie et al., 1978; MacKenzie, 1979). The fractionation of a relatively large number of pituitary glands from two species of turtles, the green sea turtle Chelonia mydas and the snapping turtle Chelydra serpentina, provided an opportunity to examine the properties of thyrotropins in reptiles. Descriptions of the isolation of two distinct gonadotropins from each of these chelonian species have been published elsewhere (Licht et al., 1976; Papkoff et al., 1976; Licht et al., 1977b). This report describes the separation of the thyrotropin from the gonadotropins, as well as studies

l Present address: Department of Zoology, University of Alberta, Edmonton, Alberta T6G 2E9, Canada. 39

0016-6480/81/090039-10$01.00/O Copyright All rights

@ 1981 by Academic Press, Inc. of reproduction in any form reserved.

40

MACKENZIE,

LICHT,

on the biological and immunological characterization of turtle TSH. MATERIALS

AND METHODS

Chemicul techniques. Thyrotropin was purified from the same pools of pituitaries previously employed for the isolation of gonadotropins from the snapping turtle (Papkoff et ul., 1976) and the sea turtle (Licht et al., 1976). The methods of extraction, fractionation, and chromatography have been described in detail elsewhere (Licht rt ul., 1977b; Farmer and Papkoff, 1979); a summary of the chromatographic behavior of each turtle thyrotropin appears under Results. Amino acid analyses were performed by the method of Spackman et al. (1958) in a Beckman amino acid analyzer (Model 120B). The countercurrent distribution technique is that described by Papkoff and Samy (1967). In the present studies, solvents and salts were removed from desired fractions by passage over a Sephadex G-25 column equilibrated with 0.04 M NH,OH. Fractions were then lyophilized. Thyrotropin hioussay. The primary assay used to determine the thyrotropic activity of the turtle pituitary fractions was the baby slider turtle (Pseudemys scriptu) thyroid radioiodine uptake bioassay (described in MacKenzie et ul., 1978). Except for initial studies with the snapping turtle fractions, the “thyroxine-blocked” protocol was used, i.e., endogenous thyrotropin secretion in the assay turtles was reduced by injection of 0.5 pg Na-L-thyroxine (T,) in 50 ~1 phosphate-buffered saline (PBS) for 2 days prior to bioassay. This treatment was continued during the four daily injections of test substance by suspending pituitary fractions in PBS containing 10 pg/ml T,. All assays included saline controls, as well as a standard curve of two doses of NIH TSH-B4 (2.12 USP U/mg). The sensitivity of this assay was 0.2 mlU of thyrotropin per animal, and the index of precision (A) ranged from 0.05 to 0.2. Bioassay potencies were calculated with a computer program for parallel line assays based on Finney (1964). Gonudotropin tr5su?‘. Gonadotropic activity of the snapping turtle preparations was determined by bioassay as described in Papkoff et ul. (1976). Gonadotropic activity of the sea turtle fractions was quantified by one of two methods: (a) nonspecific gonadotropin radioreceptor assay utilizing Cheloniu ovary homogenates as receptor and radioiodinated human FSH as radioligand (Licht and Midgley, 1976), or (b) highly specific Chrloniu follicle-stimulating hormone (FSH). and LH-P subunit radioimmunoassays (Licht et al., 1977a). Illlrnrrrtologi~crl techniques. Immunization of rabbits with thyrotropin preparations was by the technique of Vaitukaitus et ul. (1971). and radioimmunological techniques were the same as those used for turtle gonadotropins and reported in Licht et ul. (1977a). Rab-

AND

PAPKOFF

bits were initially immunized with 50 pg of either highly purified Chelydru or Cheloniu hormone, and boosted every 4-6 weeks thereafter with lo- 15 pg of the same preparation. Antiserum to sea turtle LH-/3 was that described in Licht et ul. (1977a). All fractions and bloods were tested in RIAs at several twofold dilutions, and RIA potencies were calculated using a computer program for parallel line assays. For immunoabsorption and neutralization studies, 25 pg of hormone was incubated with 1 ml of undiluted antiserum at 37” for 1 hr, then at 4” overnight. Goat antirabbit gamma-globulin serum was then added to precipitate the antibody-hormone complex. and the resulting supernatant after centrifugation was diluted in PBS and tested for biological activity. For controls, hormones were incubated with normal rabbit serum (NRS) instead of antiserum. Biological testing. Tests of the sea turtle hormones in the homologous species were made possible by the acquisition of several dozen recently hatched C. mydus weighing between 20 and 200 g which were maintained and tested at 30”. Both Cheloniu TSH and the NIH bovine TSH were tested for their ability to stimulate in vivo T, release in these animals. Blood samples were taken from animals 15 min to 8 hr after intracardiac TSH injection, and different pretreatments of 1, 2. or 3 daily injections of hormone were tested. Injected doses of the two thyrotropins ranged from 50 to 450 pg/lOO g body wt in the different protocols. At the same time, thyroids weighing 7, 8, 37, and 162 mg were removed from juvenile C. mydus, diced, and tested for their in vitro thyroxine release response to the thyrotropins. The procedures for the in ,titro thyroxine release bioassay and for the radioimmunoassay of thyroxine were as described in MacKenzie rf al. (1978). NIH bovine TSH and both turtle TSHs were tested in vitro at doses ranging from 0.25 to 25 pg hormone/mg tissue in 250 ~1 incubation medium. Juvenile C. m)ldus and adult P. scriptu were also treated with 500 pg/lOO g body wt per day of goitrogen methimazole (Tapazole, Lilly) for 2-5 weeks. At the end of this time, blood samples were taken and thyroids were removed for histological examination.

RESULTS

Chromatographic Purification Chelydru. Thyrotropic activity in pituitary extracts chromatographed with LH activity over Amberlite CG-50 at pH 6.0 and sulfoethyl- Sephadex (SE) C-50 equilibrated to 1 M NH,HCO, during the initial purification of snapping turtle hormones. There was a clear separation of these two activities, however, on DEAE -Sephadex with the LH passing through unadsorbed

TURTLE

ACTIVITIES

TABLE 1 (Chdydra

OF SNAPPING

TURTLE

THYROTROPIN

41

THYROTROPINS

AND

serpentinn)

GONADOTROPIN

HORMONES

IN

BIOASSAYS

Potency Fraction (ID)

TSH bioassay”

LH (T28B,37C) TSH (T36GC) FSH (T48C)

0.05 1.0 -

LH bioassay*

FSH RRA’

2.0

0.05

co.04

0.05

1.0

” Pseudemys radioiodine uptake, potency x NIH TSH-B4 (2.12 USP U/mg). b Xenopus ovulation bioassay, potency x NIH LH-S18 (I U/mg). (’ Chelonicr FSH radioreceptor assay, potency x NIH FSH-S9 (1 U/mg).

and the TSH remaining adsorbed. The adsorbed TSH was eluted with 1 M NH,HCO, and designated fraction T36B. This fraction was further purified on G-100 to give a product designated T36GC, which was used as the primary snapping turtle TSH in these studies. In Table 1, this TSH is compared to the highly purified FSH (which separated from LH and TSH on CG-50) and LH which were also produced from the same batch of glands; the characterization of these two Chelydra gonadotropins is given in Licht et al. (1977b). Chelonia. The fractionation of the Chelonia pituitaries differed from that of Chelydra in that thyrotropic and gonadotropic activities were not as easily separated. Thyrotropic activity of the initial extract split on DEAE-Sephadex, and was found in both the unadsorbed (LH) fraction and the adsorbed (FSH) fraction. SubTABLE ACTIVITY

OF MAJOR

SEA

TURTLE

FRACTIONS

sequent purification of these two fractions on sulfoethyl-Sephadex C-50 and Amberlite CG-50 yielded an FSH (TC73BRB) free of TSH and an LH (TC75BRB) with low thyrotropic contamination (Table 2) but did not allow the removal of gonadotropic contamination from the various thyrotropin fractions. A single fraction eluted from SE C-50 with 1 M NH,HCO, (TC76D) represented both the most potent TSH and LH (Table 2); this is comparable to the Chelonia LH (TC48B) described previously (Licht et al., 1977a, b) and represents the largest LH fraction obtained. Although the TSH activity always tended to occur in this SE C-50 fraction during purification, we were unable to prepare any fractions using these chromatographic techniques which were potent thyrotropins, but which lacked significant gonadotropic contamination. Other chromatographic techniques (e.g., 2

IN THYROTROPIN

AND

GONADOTROPIN

BIOASSAYS

RRAb

LH-P RIA’

Potency Fraction (ID) Extract LH/TSH (TC76D) LH (TC75BRB) FSH (TC73BRB) TSH (TC88B) ” Pseudemys

’ Cheloniu LH-S 18). (’ Chelonicr

TSH bioassay” 0.018 0.9

0.06 co.05 1.0

Gonadotropin 1.0 1.0 -

1.0 1.0 -

0.07

0.06

radioiodine uptake bioassay, potency x TCSSB (TC88B = 5 x NIH TSH-B4). homologous radioreceptor assay, potency x TC76D (TC76D = 4.5 x NIH TSH-B4, homologous LH-P radioimmunoassay,

potency x TC76D.

1 x NIH

42

MACKENZIE,

LICHT,

AND

PAPKOFF

carboxymethyl-cellulose, carboxymethylSephadex) also were ineffective at separating TSH and gonadotropin.

which eluted before the LH-a subunit on the G-100 column (fraction “N” in elution profile in Licht et at. (1977a)). This technique was used again in this study with the Separation from Gonadotropin TC76D preparation to selectively dissociate by Immunoabsorption its LH contamination into subunits, thereby allowing the separation of TSH. In an attempt to demonstrate that a sea Table 4 shows that when the TSH/LH turtle TSH could be produced which did not contain significant LH activity, the TC76D fraction is treated in this manner, the LH-P fraction was incubated with anti-Chelonia activity appears in the upper phase of the LH-P antiserum and the supernatant, rep- countercurrent system, whereas the thyroresenting the hormone which did not bind tropic activity, along with 10% of the LH-P to the antiserum, was tested for activity in activity and the LH-c~ subunit, is found both thyrotropin and gonadotropin assays. primarily in the lower phase. Consequently, The results, shown in Table 3, demonstrate various Chefonia fractions produced during that the LH-P antiserum is able to remove the present chromatographic purification or block 99% of the gonadotropic activity which had high ratios of thyrotropic to while the thyrotropic activity of the fraction gonadotropic (primarily LH) activity were remains unchanged. The opposite results pooled and subjected to countercurrent were obtained in neutralization studies distribution. The resulting lower counterusing an antiserum against turtle TSH (see current phase yielded a potent thyrotropin (TCSSB) with low LH contamination (Table below). 4) after chromatography on G-100 to reSeparation from Gonadotropin by move the subunits; 3.5 mg of this material Countercurrent Distribution was produced, and this is the fraction used Previous work with sea turtle fractions as a sea turtle TSH for subsequent studies. demonstrated that thyrotropic activity could be separated from LH subunits dur- Chemical Characterization ing their preparation by countercurrent The extremely small quantities of TSH distribution (Licht et al., 1977a). The LH obtained in these studies severely limited subunits resulting from this treatment could the chemical characterization which could be reassociated after Sephadex G-100 gel be performed. Results obtained during filtration to give full gonadotropic activity, purification are, however, suggestive and free of thyrotropic contamination (Table 4). informative. In general, the TSHs deThe thyrotropic contamination, in turn, scribed here behaved in a manner similar to could be concentrated in a separate fraction bovine TSH during fractionation and chroTABLE

3

ACTIVITY OF Chelonicr TSH/LH AFTER ABSORPTION WITH NORMAL RABBIT SERUM (NRS), ANTISERUM (A/S)TO Chelonia LH-p SUBUNIT.• R ANTISERUM TO Chelydra TSH Potency Hormone TSH/LH

(TC76D)

Treatment

TSH bioassay”

NRS LH-P A/S TSH AIS

1.0 1.1 -co.04

Gonadotropin

bioassay*

1.0 0.01 0.9

o Pseudemys thyroid radioiodine uptake bioassay, potency x untreated TC76D. * Xenopus ovulation bioassay for TSH A/S, Thamnophis testis testosterone production bioassay for LH-/3 A/S (Licht et al., 1977b). All potencies x untreated TC76D. NRS-treated hormone was determined in both assays.

TURTLE

TABLE USE OF COUNTERCURRENT ACTIVITIES

43

THYROTROPINS

4

DISTRIBUTION THROUGH FORMATION

TO SEPARATE TSH AND OF LH SUBUNITS

LH

Potency” Hormone

TSH bioassay

Intact TSH/LH (TC76D) Subunit studies LH-cx LH-P LH-a + LH+ (recombinant) Countercurrent distribution CCD upper (LH-P) CCD lower (LH-cx + TSH) Final TSH (TC88B)

Gonadotropin

RRA

LH-P RIA

1.0

1.0

1.0

0.1 0.01 0.05

0.01 0.03 1.4

0.008 4.0 -

0.5 2.7 1.1

0.06

1.0 0.1 0.07

(( Assays as described in Table 2.

matography (Pierce et al., 1971; Pierce, 1974). For this reason, one can infer similar chemical properties. It is likely that the two turtle TSHs are glycoprotein in nature. Furthermore, their elution profiles on Sephadex G-100 columns (VJV, = 1.72) suggest a similar molecular weight, i.e., approximately 30,000 daltons. Limited disc gel electrophoresis experiments showed the AMINO

ACID

Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine’ Valine Methionine’ Isoleucine Leucine Tyrosine Phenylalanine r( Calculated DCalculated (’ Values for forms of these

COMPOSITION

microheterogeneity typical of the glycoprotein hormones (a diffuse and poorly stained band) and a mobility (R, = 0.150.30) similar to many LH preparations (Licht et al., 1976). Sufficient sea turtle TSH was available to determine its amino acid content. These results are shown in Table 5 and are compared to the composition of the present sea

TABLE 5 LH AND TSH COMPARED

OF SEA TURTLE

Sea turtle TSH” (TC88B) 17.9 4.5 8.0 19.2 20.4 12.6 16.6 15.2 11.4 12.7 11.4 11.4 5.3 9.5 11.1 17.6 9.7

Bovine TSHO (Pierce) 19 6 15 20 11 15 14 8 13 22 11 9 8 6 16 9

TO BOVINE

TSH

Sea turtle LH” (TC75BRB) 13.5 5.4 6.8 16.5 18.2 15.3 16.9 24.8 13.3 16.5 11.3 11.2 3.1 7.8 10.4 12.0 12.1

on the basis of 215 residues per mole. from the structures of TSH-a and TSH-P (Pierce er al., 1971). half-cystine and methionine are low in sea turtle preparations because performic acid-oxidized hormones were not available for analysis.

44

MACKENZIE,

LICHT,

AND

PAPKOFF

turtle LH and a bovine TSH. The striking these antisera; this lack of specificity made similarity to bovine TSH is evident. While them unsuitable for use in further studies. TSH is known to be similar in amino acid Boosting these rabbits with TC88B incontent to LH (Pierce et al., 1971), the creased apparent titers, but did not improve content of certain amino acids readily dis- the specificity. tinguishes the two. Thus, TSHs possess a The most specific system was a heterolohigher content of lysine, methionine, and gous one in which anti-Chelydru TSH antityrosine-characteristics exhibited by sea serum was used with radioiodinated Cheturtle TSH when compared to sea turtle LH hiu TSH as radioligand. Both rabbits (Table 5). LHs, on the other hand, have an immunized with Chelydru TSH produced unusually high content of proline which is sera which showed maximum titers 2 weeks always higher than that of TSH, as seen in after the third boosting. The specificity of the present case. these antisera decreased with repeated boosting, however, and for this reason, an Biological Testing earlier bleeding taken from one rabbit 1 Both in vivo and in vitro T, release bioas- week after the first boost was used to desays were performed with a total of 20 velop the heterologous RIA. This antiserum juvenile C. mydus. In both cases, doses of bound 30% of the radioiodinated Cheloniu 50-450 pg/lOO g body wt of turtle TSH TSH at final antiserum dilutions of 1:2500 were unable to stimulate any significant to 1:5000. A representative radioimmunoacute thyroxine release by the thyroids of assay using this system is shown in Fig. these animals at intervals of 15 min to 8 hr 1. The sensitivity for Cheloniu TSH is after treatment. Doses of bovine thyrotroapproximately 1 &tube, and for Chelydru pin in the same range and with the same TSH approximately 10 rig/tube. In both protocols also had no effect. species, the assay is relatively specific for thyrotropin. Cross-reactivities of purified Immunological Studies turtle LH and FSH are less than 6 and l%, We were unable to successfully iodinate respectively, that of the homologous the Chelydru TSH, so antisera produced thyrotropins; these values are consistent against both sea turtle and snapping turtle with the estimates of TSH bioactivity in thyrotropins were studied with radioiodithese gonadotropins. The assay system nated Cheloniu TSH (TC88B) as radioligand (label). Sensitivity of the radioimmunoassays (RIA) developed was defined as the amount of unlabeled TSH required to displace 20% of this label from the appropriately diluted antiserum; specificity was determined by testing various turtle gonadotropins for their ability to displace label from the antiserum. Although all three rabbits immunized with Cheloniu TSH produced measurable titers of antisrum after 4-6 weeks, the speciNG IJNLABELLED HORMONE ficity of these antisera was poor. In particuFIG. 1. Representative radioimmunoassay showing lar, purified Chelonia LH which was low in ability of unlabeled turtle thyrotropins and gonadotroTSH bioactivity (TC75BRB, Table 2) pins to displace radioiodinated Cheloniu mydus (C.m.) cross-reacted significantly (30-70% as po- TSH from anti-ChefydrLt serpentinu (C.S.) TSH antitent as sea turtle TSH) in all systems using serum. Cm

FSH

,,,,,

IO

5

IO

50

100

/I,,

500

1000

TURTLE

45

THYROTROPINS

does not cross-react with purified thyrotropins and gonadotropins from avian or mammalian species. Results of TSH determinations by RIA of plasma are shown in Table 6. The assay was not sensitive enough to detect normal levels of thyrotropin in adult or juvenile sea turtles. It was possible, however, to detect immunoreactive TSH in both sea turtles and slider turtles treated for 2-4 weeks with a goitrogen in which there was histological evidence that a goiter had been produced. In addition, further evidence for the specificity of the RIA came from tests in which bloods from nesting, wild-caught female sea turtles were tested for TSH with this RIA, and for LH with a highly specific sea turtle LH-P RIA (Licht et al., 1979). An ovulating animal with high circulating LH levels (shown in Table 6) showed low and unchanging levels in the TSH RIA. Antiserum generated against the Chelydra TSH could also be used to neutralize the thyrotropic activity of the TSH/LH fraction (Table 3). After treatment of this fraction with the antiserum, the thyrotropic activity was reduced by more than 96%, while the gonadotropic activity remained unchanged.

DISCUSSION

In this report, we have described the purification and biochemical characterization of thyrotropin from the pituitaries of two turtles. Both the Chelonia and Chelydra hormones described here behaved in a similar manner to mammalian thyrotropins during chromatographic purification (Pierce et al., 1971; Pierce, 1974) except that separation of thyrotropin from gonadotropin during chromatography on DEAE-cellulose was complete in Chelydra, but only partial in Chelonia. Due to the relatively small amounts of material available at this stage, extensive rechromatography to remove the gonadotropin contamination from the Chelonia preparation was impractical. This was achieved, however, in two simple steps through countercurrent distribution, a technique which had been used successfully in the past to separate bovine TSH from LH (Pierce et al., 1971), and which had previously enabled us to prepare the first Chelonia LH free of thyrotropic contamination (Licht et al., 1977a). In the latter case the LH subunits resulting from countercurrent distribution and gel filtration could be reassociated to give full LH activ-

TABLE 6 TESTS OFTURTLE PLASMA IN HOMOLOGOUS Chelonia LH-p RADIOIMMUNOASSAY AND HETEROLOGOUSTURTLE TSH RADIOIMMUNOASSAY Sample Adult Chelonicr pool Juvenile Chelonia pool Chelonio (1 month Tapazole) Chelonia (2 weeks Tapazole) Psedetnys (saline injected) Psedetnys (2 weeks Tapazole) Chelonin

TSH RIA” (rig/ml) ‘c7.5 c7.5 40.0 49.0 c4.0

10.0

LH-P RIA” b-&ml) co.5

co.4 co.4 -

-

#21’

Day 0 Day 1, AM Day 1, PM

12.0 approx 8.0 approx 8.0

2.1 13.6 20.4

” TC88B used as standard. Approximate potencies are given for bloods nonparallel to the standard or at the lower limit of assay sensitivity. All other bloods ran parallel to the standard. b TC76D used as standard. ’ Adult ovulating female (see Licht et al., 1979).

46

MACKENZIE,

LICHT,

AND

PAPKOFF

ity devoid of thyrotropic activity. The only laboratory which do not respond to purified significant thyrotropic contamination of the bovine thyrotropins at any dose. The pituChelonia LH subunit preparations was itary-thyroid axis in these animals does found in the LH-a! subunit, and the thyroappear to be active at this stage, as evidenced by the effectiveness of methimazole tropic activity of the recombinant, although low (less than 5% that of the starting mate- in producing goiter and high levels of imrial), was fully accounted for by the thyro- munoreactive TSH, and it is possible that tropic activity of the LH-a! fraction used for some other aspect of thyroid function, such recombination. In the present study, the as iodine uptake, will provide a more sensibulk of any LH-(x contamination which ac- tive indication of thyroid stimulation than companied the intact thyrotropin in the does T4 release. Although our data reprelower (aqueous) phase of our countercursent tests on a relatively small number of rent system was removed by gel filtration juvenile green sea turtles, these preliminary on Sephadex G-100, resulting in gonado- results do indicate that there may be funtropin-free Chelonia TSH. damental differences in the physiology of In addition to behaving during purificathe thyroid system between families of turtion like a mammalian (bovine) TSH, the tles. Pseudemys (Chrysemys) scripta, the amino acid content of Chelonia TSH is also species used for bioassay, responds very similar to that of mammalian thyrotropins, sensitively to both bovine and Chelonia and distinct from Chelonia LH and FSH thyrotropins under the same conditions, (Table 5, Licht et al., 1976). It seems likely and at much lower doses than those used in as well on the basis of solubility consid- C. mydas in this study (see MacKenzie et erations that both thyrotropins are glyco- al. (1978) for T, release response in proteins. Thus, on the basis of this prelimiPseudemys to the same NIH bovine TSH). nary biochemical characterization, we can Since much of the knowledge of turtle conclude that there are three distinct hor- thyroid physiology is based on experiments monal entities (TSH, LH, and FSH) pres- in Pseudemys and Chrysemys (Lynn, 1970), ent in these turtles which correspond to it will be particularly interesting in the futheir mammalian counterparts. The simiture to see how applicable these data are to larities in the amino acid compositions of other distant chelonian groups. the Chelonia glycoprotein hormones and The antisera generated in this study may those of mammalian origin are again indi- prove to be an important tool in furthering cative of the high degree of chemical con- this knowledge of turtle thyroid physiology. servation that has been maintained during The differences in purification between evolution (Licht et al., 1977b; Papkoff et the Chelonia and Chelydra thyrotropins al., 1977). seemed to be reflected in the characteristics Proof that these thyrotropins are active in of the antisera raised against these preparastimulating the thyroids of homologous tions. If the easily separated Chelydra TSH species is still not available. Although the was used to immunize rabbits, the resulting Chelonia thyrotropin was tested at high antisera were relatively specific for turtle doses in juvenile C. mydas, there was no thyrotropin when tested with various chelonian and nonchelonian thyrotropins stimulation whatsoever of T, release either and gonadotropins. In contrast, antisera in vivo or in vitro. It appears that this lack of response is due to the insensitivity of from rabbits immunized with the Chelonia juvenile sea turtles to exogenously adminthyrotropin produced by the countercurrent istered thyrotropin rather than to a basic technique cross-reacted significantly with inactivity of the TSH preparation; they rep- homologous gonadotropins. This may indiresent the only vertebrates tested in this cate that this preparation still possesses a

TURTLE

47

THYROTROPINS

low, but important contamination with LH-a subunit which results in antisera containing a significant number of antibodies directed against this subunit. Since there is now evidence that this LY subunit is very similar in most vertebrate pituitary glycoprotein hormones studied (Fontaine and Burzawa-Gerard, 1977), these antibodies could be the cause of the nonspecificity of the antiserum. This a! subunit contamination appears to be one major disadvantage of using countercurrent distribution to purify thyrotropin from small amounts of pituitary material. Alternatively, the presence of a common a! subunit within each glycoprotein hormone may be the basis for this cross-reactivity. Antisera generated against Chelydra TSH are sufficiently specific to allow selective neutralization of the thyrotropic activity in various Chefonia glycoprotein fractions, just as an antiserum generated against Chelonia LH-P subunit is able to differentially neutralize the LH activity in these fractions. In addition, radioiodinated Chefonia TSH binds to the anti-Chelydra TSH antiserum in such a way as to give a specific RIA for turtle TSH. Sensitivity of the RIA is sacrificed, however, by employing this heterologous system to gain specificity. This heterologous turtle TSH RIA is only I- 10% as sensitive as the nonspecific homologous Chelonia TSH RIA or the homologous RIAs developed for Chelonia gonadotropins (Licht et al., 1977a), and, as a result, it is unable to detect thyrotropin in bloods from normal adult and juvenile C. mydas. This RIA appears to be most useful for evaluating the thyrotropic contamination of pituitary preparations, or measuring elevated TSH levels in serum, such as those in animals treated with giotrogenic drugs (Table 6). On the basis of the work presented in this report, it seems reasonable to conclude that the turtle pituitary contains three separate and distinct glycoprotein hormones: TSH, LH, and FSH. Conventional column chro-

matography can be used to produce purified gonadotropins, and in the case of Chelydra, a purified thyrotropin. In Chelonia, the major contaminant of thyrotropin preparations during chromatography (LH) can be selectively removed by countercurrent distribution to give purified TSH. The LH so removed is split into subunits which can subsequently be reassociated to give an LH without thyrotropic activity. In addition, antisera can be generated in rabbits against both the Chelydra TSH and the p subunit of Chelonia LH which can selectively neutralize either the thyrotropic or LH activity of turtle pituitary fractions, and which can be used to develop specific radioimmunoassays for turtle TSH and LH. The two species of turtle TSH shows a high degree of immunoc hemical relatedness, and the biochemical evidence that we have presented indicates that, as with gonadotropins, the turtle and mammalian thyrotropins appear to be structurally homologous. ACKNOWLEDGMENTS The authors wish Cayman Turtle Farm, Bill Rainey for their ously provided goat work was supported Grant PCM-78-12470 koff.

to thank Dr. Jim Wood of the Dr. Antonella Bona-Gallo, and assistance. Dr. J. Garcia generanti-rabbit gamma-globulin. This by National Science Foundation to Paul Licht and Harold Pap-

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