The response of hypophysectomized male killifish (Fundulus heteroclitus) to thyrotropin preparations and to the bovine heterothyrotropic factor

GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

10,

1-7

(1968)

The Response of Hypophysectomized (Fundulus heterocl;tus) to Thyrotropin and

to the

GRACE Department

Bovine

Heterothyrotropic

E. PICKFORD1 of Biology, Xeaz

The

AND

July

GRANT” I’ale

Ulriversity

20, 1967

thyroid glands of hypophysectomizcd male Fundulus heteroclitus were by chronic administration of bovine heterothyrotropic factor (HTF), bovine thyroid stimulating hormone (TSH) and reference standard TSHdoses of 1 pg/gm, 1 mU (0.07 pg)/gm, and 0.1 mU/gm, respectively. Thyroid was measured in four ways with similar results: thyroid cell height, Ii3’ in the thyroid area on day in the thyroid area on day 2 (probe), Ilzl uptake A relatively low-order counter), and serum butanol-extractable 113’ (BEY). to HTF and purified TSH appears to be related to the absence of some pituitary factor that is present in the reference standard, but studies of the response and the hematocrit showed that it is not related to somatotropin an erythrocyte-stimulating factor. A slight stimulation of the regressed testes from treatment with HTF and standard TSH, but not with purified TSH.

mammalian hypophysis contains glycoproteins, in addition to that have a powerful thyroideffect on the thyroid of the

undertaken to ascertain if the heterothyrotropic factor (HTF) could act directly on the inactive thyroids of hypophysectomized Fmd~lus heteroclitus. Unpublished experiments of Fontaine (personal communicationj3 have shown this to be true of the hypophysectomized eel.

one or more thyrotropin, stimulat,ing

Factor

F. BLAKE

The Bingham Laboratory, Haven, Connecticut 06620

Rccc,ived The stimulated purified USP at function uptake 4 (well response unknown growth nor to resulted

Male Killifish Preparations

trout’, but little or no effect on the thyroid of the mouse (Fontaine, 1963). This heterothyrotropic activity, measured by a teleostean bioassay, is also present in the pit’uitary of other vertebrates (Fontaine and Le Belle, 1965). The t.eleost’ean assay is based on thyroid stimulation of starving trout, (Fontaine and Fontaine, 1956). Such fish are in a state of physiological hypopit’uitarism and consequent hypothyroidism, but the possibility of indirect stimulation through the action of a hypothalamic thyrotropin-releasing factor is not excluded. The experiments reported in the present investigation were

M.4TERIALS

’ Y. A. Fontaine, 1

Press

Inc.

METHODS

Male F. heteroclitus, captured in the vicinity of Clinton, Connecticut in July 1965, were maintained in recirculating sea water at ca. 20°C on an S-hour day, and fed daily with Aronson’s mixture supplemented twice weekly with frozen brine shrimp. The fish were hypophysectomized in mid August and the experiment was started on October 11, 9.5 weeks after hypophysectomy. Experience has shown that, under the conditions of these experiments, a period of about 8 weeks is required for all target organs of the adenohypophysis to pass into complete regression (Pickford and Atz, 1957). The fish were grouped in separate tanks in recirculating sea water (ca. 3.0% salinity) and

1 Supported by a grant from the National Science Foundation (NSF-GB 132). ‘Address after September 1, 1967: Fish-Pesticide Research Laboratory, Columbia, Missouri 65201. @ 1968 by Scademic

AND

September

9, 1966.

marked with fin clips for individual identification. At the time of hypophysectomy and again at the start of the experiment, measurcmcnts were made of weight and standard length. In addition, at, the start of the experiment, the interocular width, which averaged 9.9 mm across the outermost points of the eyes, was measured to the nearest 0.1 mm. At this time, no fish had increased significantly in length since the date of hypophysrctomy, and none showed any significant tracr of nuptial coloration. These indications, together with an overall pallor, provide adequate evidence of the absence of a pituitary remnant. The dosago for the TSH preparations was selrrted on the basis of previous studies in which, under conditions similar to those of the present experiments, injections of 0.04 mU TSH/gm body wvright wrrc barely effertive, whereas 0.4 mU TSH/gm resulted in strong thyroidal stimulation measured by mean cell height (Pickford, 1954). This dosage is of the samr order (units per hod? wright per injection) as that required for starving trout (Fontaine and Fontaine. 1956) (total don 8.6 m11/20-30 gm fish in four injections) and h~po~)h.vsectomiaed ~1s CI,eloup and Fontaine. 1956) (total dosr 8.6 mlTr/50-SO gm fish in three injections). Intraprritom~al injections were ndministerecl thrictl w~kly for a month. On the day of injrction 12, the weight, length, and interocular width were again recorded. Subsequent to this, I’“’ was administered and hormonal injections were rontinued according t,o thr following sc~hrdule for days before autopsy. Dag 5: Final wrighing and measuring, hormone injection 12. Day 4: Injection of a tracer dose of carrier-free 113’ (0.5 pc/gm of body weight in 0.01 ml solvent, measured to the nearest 0.0005 ml). Day 3: Hormonal injection 13. Day 2: Thyroid area counted i,z Z~~LW with a collimated scintillation probe (6 minutes under anesthesia.). Day 1: hormonal injection 14. Day 0 : Autopsies. At autopsy, blood was taken from the cut end of the tail. Heparinizrd, microhematocrit tubes were used for the hematocrit ; nonheparinized microhrmatocrit tubes, graduated at 10 pl intervals. were employed for the serum. The weights of the tests and of the liver mere recorded to give indices of gonad stimulation and possible depletion of liver reserves. The thyroid region was fixed in spa water Bouin’s fiuid for subsequent tracchr counts and histological study. The fixing

fluid was changrd after (IPPS .soluble radioiodidrs Pitltitccry

24 holu-r; to rcmovc rsrxtractcd by thr mrdium.

Preparalions

As customary in our investigations, all prrparations were administered in proportion to the weight of the fish, in a standard volume of solvent (0.01 ml/gm body weight to the nrarrst 0.0005 ml). These precautions are ncscrasary because tho wright of the fish varies (in the prrscnt rxperiment from 2.30 to 6.05 gm) and brcausr a volume per gm weight in escess of that employed is highly stressful to the firh (Rlichrr rt nl., 1966). Thr solvent was a buffrred gelatinr solution (100 ml of 0.5 M NaCl, 19.5 ml of 0.2 M NaH2POI, 30.5 ml of 0.2 M Na?HPO,. 0.5 gm gr’latine, adjusted to 500 ml with H?O; IIH 7.0). This solution was suggested to us by Dr. Y. ‘1. Fontaine to prevent, tlrnat,uration of preparations at low concentrations. Stock solutions wer(’ I)rrparcd in awl\-:mcr and frozc,n in dail)- injpciion cltlnta; (Iilutions werfl made on the tl:~y of injection. Ctrntrols received solvcxnt only. Bovine hrterothyrotropic factor (HTF 7-51-C, T. A. Fontaine), prcparcd as des;caribed by T. A. Fontaine (1964) and Y. A. Fontaine and Burzawa-Ckard (1966) was used. The activit,p, assayed on starving trout, was 8.2 TSH-USP units/my; on micr 0.07 TSH-ITSP units/mg. This preparation \vvl~s adminiskrctl at thrrcs dosapr: levels: 1 pg/gm, 0.1 &gm, and 0.01 pg/gm. Purified bovine thyrotropin (TSH S-l&i\, Y. A. Fontaine) was also usrd. prepared as dcscribed by Condliffe in Ratrs and Condliffc (1966). The activity of this prcpnration, assayed on mice, was cakulatrd to bc 14.2 TSH-USP units/mg. It 1va.s administc~red at two dosagcx levels: 1 mU/gm and 0.05 I~U, gm. Kefrrrnce standard USP-TSH, administered at a single dose>, was used : 0.1 mU/gm. Thr activity of I his prrparation i;: arc.?I)tcTd :ts 0.074 units/me.

‘I’m-Day Uptake. Thr radioactivity in the thyroid area was dr,tclminrtl 4S hours after injection of I’“’ (24 hours after hormonal or solvent injection). The instrument employed was a collimated scintillation probe (Baird-Atomic, Model 812s. 2.5-cm lead Phield and 0.65~cm collimation bore). Anesthetized fish held on a form-fitting Lucite carriage wpre positioned with a mechaniral ;;tage about 1 mm vertically below and horizontally (:dong thr medial asis of the lower jaw) alignrd with the collimation bore so that the wholr thyroid region (thr point rcntcrcd in thr peak of radioactivity) wad clsl)osed to the srintill:ltion cryst:kl through the collim:l ior. firplicnt 13

TsH

AA-D

HTF

OS

HTE’OPHYSECTOMIZEU

counts with this geometric arrangement gave acceptable reproducibility. Counts from the body (taken above the pelvic fins on the inverted fish) were substracted from counts from the thyroid area; the results are expressed as cpm/gm hod) weight. Four-Day c.pt&. The percrnt uptake of a tracer dose of P’ in the thyroid arra was measured 4 days after injection of radioiodine and 24 hours after the last injection of solvent or hormone. Uptake was measured in a well scintillation counter (Baird-Atomic, Model SlOA) and is expressed as percent uptake per gm body weight. Bdanol-Extractable P’ (BEPI). Blood samples taken in graduated microhematocrit tubes were allowed t,o rlot and the separated sera were subjected to Mougey and Mason’s (1962) modification of the Taurog and Chaikoff (1948) butanolextraction procedure. The extracts were counted for estimations of the relative amounts of mdiothyroxine and radiotriiodothyronine. This fraction of serum radioiodine is expressed as cpm/lO ~1 of serum. The procedures were scaled down for the small serum volumes that were available (lo-40 ~1 per fish). Thyroid Cell Height. After a few weeks, when the Bouin-fixed thyroids were no longer significantly radioactive, the glands were washed in repeated changes of 70% ethanol containing LiCOI to remove picrate, decalcified in 70% ethanol containing 3% HNO,, dehydrated through 80, 95, and 100% ethanol, cleared in methyl salicylate, embedded in paraffin, and sectioned at 7 p. Blocks of five sections each were taken from the ribbon at intervals of 20 sections, and mounted alternately on two slides. Each slide thus ~amplcd the ribbon at intervals of 50 sections (350~). Slide 1 was stained with hematoxylin and eosin, slide 2 with modified Hcidenhain’s Azan. Histological procedures for evaluating thyroid activityin teleost fishes vary greatly (Pic~kford and Atz. 1957). Our method, which has proved reliable over the years, is a compromise between t,he more elaborate and time-consuming studies recommended by many authors, and sul)erficial ratings that do not permit statistical treatment. The height of a single representative crll is measured in each of 100 follicles. The following precautions are imperative : every follicle must be studied on each selected section to avoid discrimination in fav-or of “good-looking” follicles, successive sections must be at least 200 p apart (the length of the largest follicles) and the cell t,hat is selected for measurement must represent the mean epithrlial cell height of the follicle (the lowest and highest cells may be averaged if desired, but usually visual inspection suffices).

The tive”

Azan method and “resting”

Fmdulus permits follicles.

3 differentiation

of “ac-

RESULTS

The data are summarized in Table 1. All fish, irrcspectire of treatment, showed a small decrease in weight. There was no significant increase in length except in the two groups receiving the higher doses of HTF. This small length increment of the order of O.lc/c, however, merely reflects a partial restoration of the length decrement resulting from hypophysectomy. The mean pcrccntage length decrease for all 34 fish from the day of hypophysect,omy to the start of the experiment, was -0.5 Jc 0.12 tSE 1. Clearly, a mean increase of 0.1s did not, reflect true growt’h. The tcst’es were slightly, but significantly, stimulated by HTF at the two higher doses and by TSH-USP, but not by purified TSH 8-18-A. The liver showed no significant changes in weight. The hematocrit remained uniformly low as in the controls; pancytopcnia is characteristic of hypophyscctomized F. heteroclitus (Slither, 19611. The interocular widths showed no significant changes, although the possibility that cxophthalmos-producing substance ( EPS) was prcsclnt at a low level in standard TSH-USP was not excluded. Two of the recipients showed increases of 2.2 and 355c/, at the termination of the experiment (24 hours after hormonal injection 12j, but the mean for the group was only 0.8%.

The data are summarized in Tab!e 2. Within each group there was considerable individual variation ; some fish responded more than others. There is, however, close agreement between the four methods of evaluating thyroid activity: t,he clistribution of means within columns are similar for corresponding injection groups between columns (Table 21. The values for 2-day uptake, 4-day uptake, and BEI’“’ were correlated to respective values of thyroid ccl1 height on an individual basis, irrespec-

TABLE

1

RESPONSE OF HYPOPHYSECTOMIZED KILE F. h~terocIitus OF A HF,TEROTHYROTROPIC FACTOR .IND THYROTROPINS: Keight70chnnge mean

N

Solvent

5

HTF

3

HTF 0.1 e/w HTF 0.01

5

Length SE

-cl.73 *0.2.5 0. l?’ -tn.“‘?d_ n.ow

i3.6

i-o.42 -0.75 +o 21'

-8.”

5

+2.4 - 7.8

4

Testis

GSI’J

Liver

IISIc

mean

* SE

mean

i SE

0.295

Hematocrit mean zk SE

( ) .393”

kO.48

+0.05,5 0.471" +0.034 0.265

5.97 fO.Si 5.X-1

+0.:310

fO.XX 4.X

+(I.:{1 5x2 in, 41

-5.; k3.0

-0. 16

-0 46fP

+0.4x

f0

+o.::o 072

s4

-0.04

0.01 -2.0

k4.i :;1 .:;

0.344

-7.6 --f”.L’

-0

* 8. 'I 25,s

kO.041 0 34!l

kO.35 -0.15 iO.li

Interoculnr width change % mean

25.7 *::.9 25.9 + ‘) ,9I -r, 1.5.9

d.66 +0.55 5.14

kO.044

-0.20

k1.i 5

change % f SE

mean

-8.1 +2 3 --‘2.0 &I.6 -4.1

5

e/w

TSH 8-18-9 1 mU/gm TSH 8-18-A 0.05 mU/gm TSH-TISP 0.1 mU/gm

f

TO CHRONIC ADMINISTRATION XONTHYROID.IL EFFECTS

0.5

k4.7

2R.S rt4.6

-2. i

5.87

:20 .6

0.x

+0.46

kTJ.4

a Significantly different from the controls (p = < 0.05). b GSI = gonosomatic index (testes weight X lOO/hody weight). c HSI = hepatosomatic index (liver weight X lOO/body weight 1. tive

of hormonal

treatment.

The

resulting

radioisotope

Pearsonian correlat,ion coefficients ( rr,, + SE) for the 2-day uptake, 4-day uptake, and BEI131 correlated to cell height were respectively 0.524 f 0.124 (p < 0.005)) 0.467 & 0.134 (p < 0.005), and 0.570 * 0.121 Ip < 0.0005). Moreover, the three TABLE THTROID.IL

RESPONSE

OF HYPOPHYSECTOMIZED

Thyroid

receiving

Solvent HTP 1 PdW HTF 0.1 /.Lg/gm HTF 0.01 h&m TSH-S-18-9 1 mU/gm TSH 8-18-A 0.05 mC/gni TSH USP 0 1 mlT/gm

n-

5 5

cell height

mean’;

SE

F’. heteroclifus

F.KWR

.\ND

1131 uptake

day

(probe) cpm/gm mean f SE

3 .7:; +0.36 -6.41 rto.30

67.9 f16.5 669 I<& +“lS. 1 102.0

5

4.16

5

+0.27 :; i!)

+16." 70.1

10.16

+ 1” ‘i -A(

5

5 4

gave

activity

indices

2

MILE

OF .L HETEROTHYROTROPIC

Group

methods

that indicated comparable degrees of variability (21%32.5%) attributable to or determined by their association with cell height. Also, there was (expectedly) almost a perfect correlation between 2-day and 4day uptakes jr,,, t SE = 0.978 XL-0.007,

5. l? +o.:i1 3.46 k 0.3s d O!P

+0.11

u Significantly greater than the controls (p 7 0.05). * Only forlr fish.

564.6"

+ 131.3 x5.x * 22 :; i5R * -. -I +3’?.2

TO CHRONIC

ADMINISTRATION

THYROTROPINS 2

wt

1’31 uptake

day

(well) 70 dose mean f SE

0.90 io.30 8.31 +2.9s 2.42 kO.5!J O.Y4 +0.24 9.50zk2.60 1.50 f0.58

11 .9w k4.94

4

BEP day 4 cpm/lO pl serum mean f SE

5.9

Lk2.1 299 !F +x7.0 7 .!I +o.s 7.6

+n.t 1’28 ‘3 k41.3 6.5"

IL-l.6 18%. 3’1 f82.5

TSH

AKD

HTF

ON

p < 0.0005). The degree of correlation between the 4-day uptake and the BEI131 was also high (rsJ + SE = 0.872 -+ 0.043, p < 0.0005) * HTF was effective at the highest dose (1 pg/gm) and the response was approximately equivalent to that elicited by TSHUSP at 0.1 mC/gm. The two lower doses of HTF had no effect, although a nonsignificant trend was evident at the intermediate dose that suggested an incipient stimulation. Purified TSH 8-18-A was active at, t,he higher but not at’ the lower dose; this was disappointing since we had selected a range in which a log dose-response had been anticipated (see Materials and Methods). The evidence suggests that 1 pg HTF (7-51-C) is approximately equivalent to 1 mU (0.07 pg) of purified TSH (8-18-A) and t’o 0.1 mU (1.35 ,ag) of TSH-USP. DISCUSSION

The results reported in this inr-est,igat,ion indicate that HTF acts directly in stimulating the regressed thyroids (and testes) of hypophysectomized F. heteroclitus. Comparison of the methods used in this study for rating thyroid activity introduces an interesting point for consideration that concerns t,he several effects on thyroid funct,ion attributed to the thyroid-stimulating hormone. Mean thyroid cell height is generally thought to be an index that reflects thyroidal activity in terms of a single functional unit,, although Eales (1964) has suggested that it is a strictly morphological characteristic controlled solely by TSH that does not necessarily reflect the rate of iodine metabolism in salmonids. The radioisotope methods, on the other hand, give indices that reflect total t,hyroid activity and, under chronic treatment, include the factor of follicular proliferation. In our experiments, correlation coefficients between the radioisotope methods were high in comparison with correlation coefficients between radioisotope methods and cell height. The mean thyroid cell height provides a reliable assay for TSH in goldfish (Ortman and Billig, 1966), but simultaneous

5

Fundulus

HYPOPHYSECTOMIZED

studies of teleostean t’hyroid function by cell height and radioiodine methods are not numerous and have given equivocal results (Matty, 1960; Swift, 1960; and Eales, 1963, 1964). These experiments were concerned with thyroid function in intact fish, under seasonal or experimental conditions and, with the exception of Fontaine et al. (1953) and Chavin (1956) little attention has been given to the hypophysect.omized recipient. Even then, the number of injections was limited so that follicle proliferation resulting from chronic stimulation was obviated. In our experiments no attempt was made to estimate the total number of thyroid follicles since this was not a foreseen purpose of the investigation, but the striking difference between the magnitude of the response measured by radioiodine assays and the relatively low, but more uniform increase in thyroid cell height appears to involve this parameter. The response to the reference standard TSH-USP was of the expected order at 0.1 mU/gm, and corresponded approximately wit’h the minimum effective doses that had been established for the trout and for the eel (see Materials and Methods). On the other hand, hypophysectomized F. heteroclitus were relatively less responsive to HTF and to purified TSH than the trout (Table 3). The specific activity of purified TABLE 3 ACTIVITY OF PURIFIED TSH (8-lg-AQ AND HTF (7-51-c) IX TERMS OF STANDARD BOVINE TSH-USP (U/MG)

RELATIVE

Recipient Sample 8-18-A

7-51-c

Mice ca.

Starved (Salmo

15”

0.07

trout* gairdnwi)

ea. 15 8.2

Hypophysectomized F. hetmoclitus

l-2 0.1

a 14.2, see Materials b Assayed

and Methods. by Y. A. Fontaine.

TSH was not higher than that of the reference standard, and the activity of HTF was ten times lower. In this respect, the response of F. heteroclitus to HTF more nearly resembled the low response of the mouse. But F. heteroclitus also responded much less (relative to the standard) to

6

PICKFOHI)

purifietl bovine TSH (8-18-A) than the mouse. Therefore, the effect, of HTF was probably not due to contaminating residual t,races of TSH. If bovine TSH 8-18-A is taken as the standard, HTF was more active on Fundtdus (0.1/l = 0.1 unit/mg) than on mice (0.07/15 = 0.005 units/mg) . This indicates a true heterothyrotropic effect not mediated through the pituit,ary. The low relative activity of purified bovine TSH, as well as bovine HTF, suggests that there was somet,hing else in the reference standard that had a hct,erothyrotropic activity to which Fundulus was more responsive t’han the trout. This factor appears to have been deficient, in both of the derived fractions. The nature of this “missing” hypophysial factor remains unresolved. Our experiments appear to exclude certain possibilities. Now of the preparations contained significant growth-promoting activity and participation of somatotropin may be tentatively excluded. The adrenals were not studied. but the absence of an erythropoietic effect presumably reflects the absence of ACTH. The absence of exophthalmos-stimulating activity was unexpect,ecl ; EPS may have been present at a low level in TSH-USP since two of the recipients showed a small increase in interocular width at the termination of the experiment. Some gonadotropic activity was present both in the standard TSH-USP and in thcl heterothyrotropic preparation 7-51-C, but it was not detected in the purified bovine TSH 8-18-A. Stimulation of the testes could be explained by contamination of the preparations with a residual trace of luteinizing hormone (LH) . The regressed test’es of hypophysectomized F. heteroclitus can 1~ stimulated by repeated injections of ovine or bovine LH in doses as low as O.Ol0.02 pg/gm wt.. (Pickford and Atz, 1957; Pickford et al., unpuhlished).4 Thus, to elicit a minimal significant stimulation of the testes, 0.1 mU TSH-PSP ( 1.35 ;~g) -I G. E. Pickford, G. Bara. B. Lofts and J. FV. Atz : unpublished investigations on the, responscx of h?l)ol)h?sectomized F. heteroclit~s to bovine LH and GH. separately or in combination, 1966.

.ISI)

GK,\ST

.~hould contain at least the cquivaient of 0.01 pg LH-NIH. Similarly, even the intermediate dose of bovine HTF (0.1 pg), which caused significant tcrtis stimulation, should, on this hypothesis, contain at least t’hc cquivalcnt of 0.01 pg LH-NIH-S-l. The question of a lack of testis st’imulation by purified bovine TSH 8-18-A is more puzzling since this preparation was reported to contain considerable LH activity. Dr. T. A. Fontaine has stated (personal communication)” that it was assayed by the Parlow method and cont’aincrl 0.7 X LHNIH equivalent. Thus, at the 1 mI’ dose (0.07 pg) , each fish would have received the equivalent of 0.05 pg of LH-NIH, more than sufficient for significant testis stimulation. Our injection solutions were prepared in a gelatine solution for stahilization (SW Materials and Methods), but the possibility of inactivation, hoth of TSH and LH activit’y, is not entirely excluded. Y. A. Fontaine and Burzawa-Gdrard ( 1966) showed that HTF preparations possess FSH-like properties when tested by standard mammalian assays. However, the regressed testes of hypophysectomizcd F. heterorlks do not respond to mammaIian (porcine1 FSH, except at high nonphysiological doses (5 pg/gm, Pickford and Atz, 1957). At t’his dose, the known contamination by LH sufficed to explain the response. ACKNOWLEDGMEKTS This investigation could not have been undertaken without the rlosr rollahorxtion of Dr. YVC,S Alain Fontaine. Dr Fontaine provided the bovine HTF and TSH preparations. and recommended suitable injection dosrs. In addition, he read the manuscript and made many helpful suggestions. H e was invited to he a co-author, but refused on the grounds that t,he work was ours. We are indebted to Mrs. Ethel Hafter 3tz for her microscopical examination of the thyroid sections. REFERENCES R. W., ASD CONDLIFFE, P. G. (1966). Thr physiology and chemistry of thyroid stimulating hormone. ILL “Thp Pituitary Gland,” (G. W. Harris and B. T. Donovan, cds.), Vol. I, pp. 374410, Univ. of California Press, Berkeley, California.

HATES,

‘Y.

A. Fontaine,

September

9, 1966.

TSH

ATTD

HTF

ON

W. (1956). Thyroid distribution and function in the goldfish, Carassius awatus L. J. Exptl. Zool. 133, 259-279. EALES, J. G. (1963). A comparative study of thyroid function in migrant juvenile salmon. Canad. J. Zool. 41, 811-524. EALES, J. G. (1964). The influence of temperature on thyroid histology and radioiodine metabolism of yearling steelhcad trout, Salmo gairdneri. Canad. J. Zool. 42, 829-841. FONTAINE, M., AND FONTAINE, Y. A. (1956). DBtermination du pouvoir thyrkotrope de l’hypophysr et du milieu intkrieur de tkleostkens par mesurr de la fixation de 13’1 par la thyroide de la truitr arc-en-ciel (Salmo gairdneti Rich.). J. de Physiol. 48, SSl-892. FONTAIKE. M., J,ELOUP, J., AND OLIVEREAIJ, M. (1953). fitude histologiquc et biochimique de la glandr thyroide de l’anguille hypophysectomisde. Compt. Rend. Sot. Biol. 147, 255-257. FONTAINE. T. A. (1963). Sum des facteurs & activite h4terothyrbotrope presents dans les hypophyses de mammiferes. Definition et purification Gen. Comp. Endocrinol. 3, 701 (Abstract). FONTAINE, Y. A. (1964). Caracteres biochimiqucs d’un facteur purifie B partir d’hypophyses de mammiferes et doue d’une activite hkterothyrkotrope (HTF). Sixth Internat. Congress of Biochemistry Abstracts. CHAVIN,

FONTAINE,

E.

(1966).

ActivitC hCtCrothyr&otrope et hormones tropcs des hypophyses de mammiferes. Rend Acad. Sci. 263, 400-403. FONTAINE, Y. A., AND LE BELLE, N. (1965). paraison des activites thyreotropes et thyreotropes des hypophyses de different brCs. Gcrr. Camp. E,ctlocrinol. 5, (Abstract).

T.

A.,

.~ND BURZAwA-GbRARD,

gonadoCompt.

7

Fund&s

HYPOPHYSECTOMIZED

J. AND FONTAINE, M. (1956). &ude du dosage de l’hormone thyreotropes par la mesure de la fixation du radio-iode par la thyroid& de l’anguille hypophysectomishe. Arch. Sci. Physiol. 10, 201-214.

LELOUP,

MATTY,

Zool.

A. J. (1960). Thyroid Sot. 2, 17-27.

cycles

in fish. Symp.

E. H.. .~ND MESON, J. W. (1962). Mcasurement of butanol-extractable iodide in the rhesus monkey. J. Lab. Clin. Med. 59, 672-680.

MOUGEY,

R., AND BILLIQ, R. D. (1966). A reexamination of the goldfish microhistometric assay met,hod for thyrotropin. Gen. Comp. Endocrinol. 6, 362370.

ORTMAN,

G. E. (1954). The physectomizcd male killifish ment with small doses of crinology 55, 274-257.

response of hypoto prolonged treatthyrotropin. Endo-

PICKFORD,

PICKFORD,

Physiology New l.ork

G.

E.,

.~ND

ATZ,

of the Pituitary Zool. Sot., New

J.

W. (1957). “The Gland of Fishes,” York, 613 pp.

A. M. (1961). Endocrinological and hematological studies in Fundulus heteroclitlts (Linn.). Bull. Bingham Oceanographic Collection, 17, (3), 55 pp.

SLICHER,

A. M., PICKFORD, G. E., AND PANG, K. T. P. (1966). Effects of “training” and of volume and composition of the injection fluid on stressinduced lrukopenia in the mummichog. Plogr. Fish-Cult. 28, 216219.

SLICHER,

D. R. (1960). Cyclic activity of the thyroid gland of fish in relation to environmental changes. Synzp. Zool. Sot. 2, 17-27.

SWIFT,

Comhe&overte67fk679

A., AND CHAIBOFF, nature of the circulating Biol. Chem. 176, 639-656.

TAUROG,

I.

thyroid

1,.

(1948).

hormone.

The J.