Presence of corticotropin activity in the pituitary gland of chondrichthyean fish

CENERlL

.4ND

Presence

COMPARATIVE

ESDOCRISOLOGY

of Corticotropin

267-275

9,

Activity

(1%‘)

in the

of Chondrichthyean ROGER Col,umbia,

Missouri

and

Friday Friday AND

Department

Harbor,

grant AM-06259 Arthritis and Public Health

CAROLYN of Zoology,

from the Metabolic Service.

Fish’

Laboratories, Washington

February

National Diseases, 267

University

of

Washington.

DEROOS University

Corticoid hormones chemically identical to those produced by the mammalian adrenal cortex have been demonstrat.ed, either in vivo or in vitro, as secretory products of the adrenocortical (interrenal) tissue from representatives of the major vertebrate groups (Holmes et al., 1963; Bern and Nandi, 1964). In contrast, relatively little attention has been directed to the control of the adrenocortical tissue in nonmammalian vertebrates. Present information is sufficient. to conclude that corticotropin (ACTH) activity is present in the pituitary glands of teleost fish and terrestrial vertebrates (Knobil and Sandler, 1963; deRoos and C. deRoos, 1964a ; Gist and deRoos, 1966) ; however, the degree and type of pitiutary control of the adrenocortical tissue is largely unknown. The lack of information concerning a possible pituitary control of adrenocortical function is particularly evident when considering the phylogenetically more primitive fish, the chondrichthyeans and cyclostomes. Cyclostomes remain virtually uninvestigated, although the unpublished observations of both Strahan (see Chester Jones et al., 1962a, discussion) and Nowell and Phillips (see Chester Jones et al., 196213) suggest that both lamprey and hagfish pituitaries contain ACTH activity. ‘Aided by Institute of United States

Glan

DEROOS

Harbor

Received

Pituitary

of

Missouri

1, 1967

There is no direct evidence for AC, activity in the chondriehthyean pituit and the ind.irect. evidence is contradictory. Dittus (1941) reported that hypophysectomy of elasmobranch species, prlmariiy of Torpedo, resulted in atrophic changes in the adrenocortical tissue whie ached a maximum degeneration within days. However, Dodd (1961) stated that total hypophysectomy of the dogfish (presumably Scyliorhinus caniculus) was without visible effect on the adrenocortical tissue even after one year. The present communication represents part of a continuing effort to docum.entf the phylogenetic existence of the hormones necessary for a possible pituitary-adrenocortical axis in vertebrates, The results described herein demonstrate ACTFI aetivity in pituitary glands obtained from three chondrichthyean fish. Portions of this report, have appeared in abstract, form (deRoos and C. deRoos, 1964b). MATERISLS

AK-D

METBODS

BNIM.4LS

The assay for ACTH activity was performed on pituitary glands from three chondrichthyrarr species: Lwo elasmobranches, the spiny dogfish shark (SqzLabus acalzO&xs) and the longnose skstc (Raja rhino,), and one hoiocephaian, the rat%&: (Hydrolagus colliei). The fish were rolleeted frcn! the waters near the Friday Harbor Laboxtories, San Juan Island, Washington, hy means of XL

268

DEROOS

AND

otter trawl operated from the research vessel “Hydah.” The fish were kept in large outdoor holding pens for 1-12 days before sacrifice for pituitary collection. The 75 dogfish, 16 skates, and 60 ratfish, were of mixed sexes and breeding conditions ; no’ obviously pregnant dogfish were used. Additional dogfish were maintained in the laboratory as described below. The 280 White Leghorn cockerels” used as the assay animals were obtained as l-day-old chicks and raised in a finishing cage on commercial feed to 3.238 days of age. PITUITARY

NOMENCLATURE

Descriptions of the morphology of the elasmobranch pituitary and discussions of the problems of nomenclature have been reviewed by Pickford and Atz (1957) and Dodd (1963). The recent work by Sathyanesan (1965) described the microscopic anatomy of the pituitary of a holocephalan, the ratfish, and included a review of the literature. In the present study the nomenclature used by Gorbman and Bern (1962) is employed. The following terms are used for the lobes or regions of the elasmobranch pituitary : the pars distalis divisibIe histologically and by gross morphology into the rostra1 and proximal pars distalis, the neurointermediate lobe, the ventral lobe, and the saccus vasculosus. The same pituitary lobes are present in the holocephalan with the exception of the ventral lobe which has not been found (Bern and Nandi, 1964; Sathyanesan, 1965). However, the Rachendachhypophyse, a glandular structure located under the pharyngeal mueosa, and which may be comparable to the elasmobranch ventral lobe (Sathyanesan, 1965), was collected for assay. PITUITMLY

PREPARATION

AND

ASSAY

The fish were killed by pithing and the pituitary exposed under a dissecting microscope. The lobes of the pituitary were removed, and from 4 to II equivalent lobes placed in fresh acetone. The lobes were dehydrated with a minimum of 16 changes of acetone over a 3-day period, dried under vacuum, placed in sealed vials, and returned to the University of Missouri for ACTH assay. The samples were stored at 15°C. The acetone-dried samples were extracted on the day prior to assay. The samples were placed in 0.1 N HCI, homogenized, and allowed to stand ’ Intraflock-Single Comb White Leghorn chickens made available through the courtesy of the Department of Poultry Husbandry, University of Missouri.

DEROOS

for 30 minutes at room temperature. Following centrifugation for 10 minutes in a Beckman Spinco microfuge, the supernatant fluid was transferred and diluted with either distilled water to 0.01 N HCl or with an equal volume of 0.1 N NaOH to obtain a neutral extract of small volume. The final volumes of the extracts used for assay ranged from 1.5’ to 4.0 ml. The prepared samples were stored at -26°C. The pituitary extracts were assayed for ACTH activity by measuring their ability to increase corticoid production (corticosterone and aldosterone) by chicken adrenal tissue in vitro. The methods of incubation, extraction, paper chromatography in the toluene-75% methanol system (Bush, 1953), and quantitative estimation calculated from the absorption spectra of the eluted corticoids in ultraviolet light were essentially as described previously (deRoos, 1961). Pairs of adrenals from four to eight birds were used in each experiment based on the anticipated corticoid production under the various treatments. The tissue was incubated for 30 minutes (preincubation), after which time the medium was changed and the preincubation medium discarded. Incubation proceeded for 3 hours with a change of the medium after 1% hours. On the day of assay the pituitary extract was thawed and added to the incubation medium at the level of 0.2 ml of extract/100 mg of incubated adrenal tissue. The extract was added to the medium following the preincubation period and renewed at the second change of the medium. ROSTRAL

PARS

DISTALIS

REMOVAL

Sixteen fasting dogfish of mixed sexes, equally divided into four experiments, were maintained in circular concrete aquaria. The animals were kept in the aquaria for one week prior to use. The dogfish were anesthetized with 106 ppm tricaine methanesulfonate (MS-222, Sandoz) in sea water. The pars distalis and neurointermediate lobe of the pituitary were exposed essentially following the procedure described for the spotted dogfish (S. calziculus) by Dodd et al. (1960). The forward projecting, tongue-like pars distalis displays a prominently tapered constriction approximately two-thirds along its distal length; this divides the pars distalis into the proximal and rostra1 regions (Pickford and Atz, 1957; Gans and Parsons, 1964). The pars distalis was transected at the center of the constriction and the rostra1 pars distalis removed by suction. The dogfish were autopsied on the eleventh day after the operation and the completeness of the operation was determined visually. The neurointermediate lobe was removed, acetone-dried, and

< KOXDRICHTHYEAN

ACT13

vasculosus did not increase corticoid production over control levels (Tabie 1) ; ACTH activity was found in both the pars distalis and the neurointermediate lobe. The effects of acid extracts of the dogfish pars distalis on corticoid production by chicken adrenal tissue are presented in Table 2. Extracts of the entire pars distalis increased corticosterone and aidosterone production over the levels of these two corticoids secreted by control adrenal tissue. 1n subsequent experiments, the acetone-dried samples of the pars distalis were

assayed for ACTH actix-ity as described above. Control samples of the rostra1 pars distalis and neurointermediate lobe were obtained from intact animals. RESULTS DOGFISH

The initial assays of each of the lobes of the dogfish pituitary employed a variable number of lobes and different extract volumes. These experiments were designed to establish the presence and location of ACTH activity within the pituitary, and TABLE EFFECTR

OF DOGFISH OS

(Squalus CORTICOID

None 11 9 5 5 5

Ventral Saceus

1

acanthias) VENTRAZ LOBE PRODUCTION BY CHICKES

ASD

Lobes extracted/ assay

Lobe Assayed

vasculosus

a Corticoid product,ion standard error is given.

presented

SMTUS

4.0 4.0 4.0 1.5 1.5

7 1 1 2 1 2

per 100 mg of incubated

adrenal

production

&)a

-

Corticcsterone

Aldosterone

0.2

0.3

Ik 0.05 0. 1 0 1 0.2 0.1 0.1

tissue.

Either

the mean

IO.07 0.1 0 .2 i: 1 0.1 0 ::

or the mean

k

transected with the aid of a miel=oseope at the center of the constriction which morphologically divides the rostral pars distalis and the proximal pars distalis. The two regions were assayed separately. The addition of extracts of the rostra1 pars

TABLE DOGFISH (Squalus CORTICOID PRODUCTION OF

Ex~~~.ic-ns

V~scu~osus

TISSUIZ

ADREX~L

Corticoid

to determine the number of lobes and the extract volume required to obtain a significant submaximal stimulation of corticosterone production by the chicken adrenal tissue in vitro. Extracts obtained from the ventral lobe and the saccus EFFECTS ON

9iS!l

AC!TIVITY

2

acunttias) PARS DISTALIS BY CHICKEN ADRENAL

EXTRACTS TISSUE

-Lobe assayed

Lobes extracted/ ZLS%y

Corticoid

None Pars distalis Rostra1 Proximal

pars distalis pars distalis

a Corticoid prodllction standard error is given. b Significant increase 5’)/. level.

presented

11 9

3 0 3.0

i 1 1

5 5 5 5

4.0 2.0 1.5 1.5

2 1 4 4

per 100 mg of incubated

in corticosterone

production

(,a)”

Corticosterone

or aldosterone

0.2

2.8 0.2 adrenal

production

2 0.05 6.4 4.4

over

0.3

0.S 2.4 i O.&Y +_ 0.06

tissue.

Either control

-

Aldosternne

1.0 0.3 the mean (none)

when

j: 0.07 i.3 jL.I 9 11.5 ii .5 -L 0 21’ + ().(I:: or the mean tested

t

at the

270

DEROOS

AND

TABLE EFFECTS

DEROOS

3

OF DOGFISH (Xqualus aeanthias) NEUROINTERMEDIATE ON CORTICOID PRODUCTION BY C~IICFXN ADRENAL Corticoid

Lobes

Extract volume (ml)

extracted/assay

Number

of assays

Corticosterone

None

-

7

0.2

11 9 5 5

4.0 5.0 4.0 1.5

1 1 3

1.0

1

fk 0.05 0.3 0.3 * 0.206 1.4

LOBE TISSUE production

EXTRACTS

!/Lp). Aldosterorie

0.3

0.7

f 0.07 1.1 1.0 f 0.156 0.7

a Corticoid production presented per 100 mg of incubated adrenal tissue. Either the mean or the mean f standard error is given. 6 Significant increase in corticosterone or aldosterone production over control (none) when tested at the 5%

level.

distalis to the medium resulted in a significant increase in the production of both corticosterone

and

aldosterone;

extracts

of

the proximal pars distalis failed to increase corticoid production over that of the control experiments. Extracts

from

the neurointermediate

lobe

neurointermediate lobe, and the neurointermediate lobe from dogfish sacrificed 10 days after removal of the rostra1 pars distalis were compared (Table 4). The assays of the normal rostra1 pars distalis and neurointermediate lobe served as procedural controls and confirmed the previous results presented in Tables 2 and 3. Extracts of the neurointermediate lobe from

also were found to contain ACTH activity (Table 3) ; however, the increases in corticosterone production by neurointermediate TABLE 4 lobe extracts were less than those obtained EFFECTS OF EXTRACTS OF DOGFISH with extracts of the entire pars distalis or (Squalus acanthius) ROSTRAL PARS DISTALIS, the rostra1 pars distalis (Table 2, see also NEUROINTERMEDIATE LOBE, AND Table 4). The greater amount of ACTH NEUROINTERMEDIATIZ LOBE SFTER REMOVAL activity found in the pars distalis as OF THE ROSTR~L PARS DISTALIS ON CORTICOID PRODUCTION BY CHICKEN ADRENAL TISSUE compared to that of the neurointermediate lobe was not due to differences in the Corticoid production (Fg)b N”i?ber amounts of tissue extracted. The following Corticosterone ~SXLYS Aldosterone average wet weights of the relevant lobes 7 0.2 IO.05 0.3 IO.07 of the pituitary were recorded from 11 dog- None 3 4.3 I% 1.66” 1.0 + 0.5oc fish: neurointermediate 28.5 mg, total pars Normal rostral pars distalis 11.7 mg, rostra1 pars distalis 4.1 distalis mg, and proximal pars distalis 7.6 mg. Normal neu3 1.9 f 0.93” 1.2 k 0.36c Weights of acetone-dried tissues from seven rointermeanimals revealed similar ratios : neurointerdiate mediate 5.2 mg, total pars distalis 1.4 mg, Experimental 4 2.1 i: 0.746 1.1 + O.OW rostra1 pars distalis 0.5 mg, and proximal neurointerpars distalis 0.9 mg. mediate Following the localization of ACTH a Four lobes extracted per assay; exkact volume, activity in the rostra1 pars distalis and the 1.5 ml. neurointermediate lobe, additional experiBCorticoid production presented per 100 mg of ments were performed to determine the incubated adrenal tissue. The mean i standard effect of the removal of the rostra1 pars error is given. distalis on the amount of ACTH activity c Significant increase in corticosterone or aldosin the neurointermediate lobe. Extracts of terone production over control (none) when tested the normal rostra1 pars distalis, the normal at the 5y0 level.

CHONDRICHTHYEAN

dogfish in which the rost.ral pars distalis had been removed did not show any change in ACTH activity ; the increase in corticosterone and aldosterone production over control levels was the same as that ohtained with extracts of the normal neurointermediate lobe.

271

ACTH ACTIVITY

in amounts comparable to that of the dogfish; no acti.vity was associated with t,he ventral lobe. RATFISH

The effects of acid extracts of the lobes of t’he raffish pituitary on cortieoid production by chicken adrenal tissue Zn SKATE vitro are presented in Table 5. The initial Two assays were performed on extracts experiment using an extract obtained from whole pituitaries indicated the presence of of the pars distalis, neurointermediate ACTH activity. lobe, and ventral lobe of the skate pituiSubsequent. experiments tary. The saccus vasculosus was not using ext’racts from the separate lobes in cortieoici proinvestigated. These experiments were de- resulted in an increase signed to provide confirmatory data on duction over control levels when an the existence and location of ACTH extract of either the pars distalis or ncuroactivity in the elasmobranch pituitary. We intermediate lobe was added to the mewere particularly desirous of confirming dium. Extracts of the Rachendachhypothe absence of ACTH activity in the dog- physe and saccus vasculosus did not infish ventral lobe. The ventral lobe of the crease corticoid production over that of the dogfish lies in a deep depression in t,he control experiments. As was true of the dogchondocranium, and is heavily invested by fish, the ratfish pars distalis was eonsidcrfibrous connect,ive tissue which cannot be ably smaller than the neurointermediat8e be excluded in dissection. The large amount lobe as shown by the following average of comlective tissue included in the ventral wet. weights of the two lobes from seven lobe samples made the tissues difficult to animals: pars distalis 4.0 mg, and neurohomogenize and extract. In. contrast, the intermediate lobe 9.0 mg. However, the skate ventral lobe, located superficially on pars distalis extract’s increased corticosthe floor of the chondocranium, is without terone production to a greater amount than a heavy connective tissue investment and did the neurointermediat’e lobe extra& was easily extracted. The results of the assays confirmed the The results of the present study provide presence of ACTH activity in the pars distalis and the neurointermediate lobe and definitive evidence for the presence of TABLE

5

&FECTS OF RATFISH (Hyarolagus co&?) PIT~JITART ox CORTICOID PRODUCTION BY CHICKEN ADnENlr,

Esu~.4c~rs l'issm

Corticoid Lobe

assayed=

None Whole pituitary Pars distalis Ne\lrointermediate Rachendachhppophyse Saceus vasculosus

Extract volume (ml)

3.0 1.5 2.0 1.5 1.5 1 .?I

Number ssssys

7 1 3 3 1 3 a

production

(i&F

-

of Corticosterone

Aldosterme

0.2

f 0.05 4.2 5.1 IL 0.59c 1.0 F 0.2@ 0.7 0.2 AZ 0.05 0.3 t 0.09

a Four pituitaries or lobes extracted per assay. b Corticoid production presented per 100 mg of incubated adrenal standard error is given. c Significant, increase in corticosterone or aldosterone production 570 level.

tissue. over

Either control

Q 3 I 0.07 1.2 1.4 i O.Ii~ 1.3 i 0 :.4c 07 0.2 t 0 02 0.4 * 0.21 1__-the mean (none)

when

or the mean teswd

2

at thr

272

DFROOS

AiiD

ACTH activity within the pituitary glands of chondrichthyean fish. Mellinger (1962) suggested on the basis of pituitary cytology that the “Q cells” present in the proximal pars distalis of the spotted dogfish were corticotrophs; however, the present results establish ACTH activity in the rostra1 pars distalis, a region of the pituitary reported to contain only basophils (Dodd et al., 1960; Mellinger, 1962). The bioassay procedures employed in this study were essentially the same as the methods used in earlier experiments to determine the in vitro steroidogenic response of chicken adrenal tissue to graded doses of added mammalian ACTH and to acid extracts of chicken and alligator (AZligator mississipienlsis) adenohypophyses (deRoos and C. deltoos, 1964a ; Gist and deRoos, 1966). A comparison of these results provides the opportunity to draw tentative conclusions concerning the phylogenetic distribution of pituitary ACTH activity and the biological specificity of the various ACTH principles. The steriodogenic response of chicken adrenal tissue to extracts from the chondrichthyean pars distalis and neurointermediate lobe was qualitatively the same as the response obtained using mammalian, chicken, or alligator pituitary preparations. These data, together with the report of Carstensen et al. (1961) that both mammalian ACTH and saline preparations of American bullfrog (Rana mtesbeiana) pituitaries increased corticoid production by bullfrog adrenal tissue in vitro, suggest that species specificity is absent in the biological activity of ACTH preparations obtained from different pituitaries. Although teleost and cyclostome pituitary preparations have not been assayed for their effects on adrenocortical steroidogenesis, the results of other bioassay methods for ACTH activity do not contradict this conclusion. The administration of teleost pituitary extracts maintained adrenal weight and resulted in ascorbic-acid depletion in hypophysectomized rats (Rinfret and Hane, 1955; Woodhead, 1960). Van Overbeeke and Ahsan (1966) demonstrated that hypophysectomy of the cyprinid, Couesius

CEROOS

plumbeus, resulted in adrenocortical tissue atrophy which could be reversed by the administration of pituitary extracts from Pacific salmon (Oncorhynchus tshawytscha and 0. kisutch). The unpublished observations of both Strahan (see Chester Jones et al., 1962a, discussion) and Nowell and Phillips (see Chester Jones et al., 1962b) indicate that cyclostome pituitary preparations bioassayed in mice increased adrenal weight and resulted in adrenal ascorbic acid depletion. Although the present study was not designed to provide precise information concerning the amount of ACTH activity within the chondrichthyean pituitary, a gross comparison can be made between the responses observed with the various pituitary preparations. Extracts of the rostra1 pars distalis from four dogfish, or the entire pars distalis from four ratfish, resulted in a stimulation of corticosterone production that was 5O-100% of the increase obtained with an equal number of chicken or alligator adenohypophyses. The response obtained with extracts from four neurointermediate lobes from the dogfish or ratfish was 10-25s of the increase obtained with an equal number of chicken or alligator adenohypophyses. These approximate values are presented only to emphasize that the ACTH activity was demonstrable with extracts from but a few pituitaries, and that the amount of ACTH activity extracted from chondrichthyean pituitaries was comparable to that of other vertebrates studied. The demonstration of ACTH activity in the chondrichthyean pituitary and the information which has established that chondrichthyean adrenocortical tissue (including the three species used in this study) secretes cortisol and/or corticosterone and aldosterone (Bern et al., 1962; Holmes et al., 1963), documents the existence of the requisite hormones for a possible pituitary-adrenocortical axis. However, any conclusions regarding the physiological role of the ACTH activity would seem premature. The evidence that the pituitary, via ACTH, is involved in the control of the adrenocortical tissue is based largely on the studies of Dittus (1941). Dittus

CHOKDRICHTHYEAN

reported that hypophysectomy of species of TorjX?do was followed by atrophic changes in the adrenoeortical tissue and that these changes occurred within 8-9 days ; mammalian ACTH administration resulted in morphological indices of adrenocortical stimulat,ion in both hypophysectomized and intact animals. These results are in contrast to the recent report by Dodd (1961) that total hypophysectomy of the spotted dogfish was without visible effect on the adrenocort.ical tissue, even after more than one year. The two studies which investigated the effects of mammalian ACTH on corticoid production by chondrichthyean adrenocortieal tissue in vitro are equally contradictory. Macchi and Rizzo (1962) suggested that ACTH increased the production of materials presumed to be corticoids by skate (Raj, erinacea) adrenocortical tissue; however, Bern et ~1. (1962) failed to obtain evidence that, ACTR increased corticoid production by the adrenocortical tissue of three chondrichthyean species, including the skate (Raju &inn). The finding of ACTH activity segregated within the chondrichthyean pituitary was anticipated from the existing literature. Gonadotrophin function has been shown to reside in the ventral lobe of the spotted dogfish (Dodd et nt., 1960)) and there is evidence that thyroid stimulating hormone (TSE) activity also is present in this lobe (Mellinger, 1960; Dent and Dodd, 1962). A factor which will induce the return to water (“‘water-drive”) of the terrestrial eft stage of the spotted newt, Notophthalmus (~ie~i~t~lz~~) viridescens, and which is associated wit.h the prolactin principle in tetrapods, appears to be present in the rostra1 lobe (pars distalis) of the skate, Raja erinacen, and the spiny dogfish (Grant, 1962; Grant and Waterman, 1962). The elasmobranch neurointermediate lobe contains both melanophore stimulating hormone (h4SH) (Pickford and Atz, 1957; Fingerman, 1963; Mellinger, 1963) and neurohypophyseal principles (Sawyer, 1965). Although the known distribution of hormonal activities wit.hin the pituitary does not support, the suggestion that the ventral lobe may be the homologue of the

ACTH

ACTIVITY

2’7:3

pars distalis of higher vertebrates (Dodd et al., L960; Dodd, 1963)) it does emphasize the segregation of the hormonal a&ivities within the distinct and largely separate lobes, a situation without parallel in other vertebrates and of considerable experimental potential. The presence of ACTH activity within both the rostra1 pars distalis and % neurointermediate lobe was unexpected. The proximai pars distalis, interposed between the rostra1 pars distalis and the neurointermediate lobe, was without detectable ACTH activity and thus the poesibility that the results were due to contamination is excluded. In addition, the fact that the proximal pars distalis was without activity indicates that the ACTH activity is well removed from the points of dissection in the separation of this region. Studies on both birds and mamma?shave shown that the posterior lobe of the pituitary of these speciescontains ACTH act,ivity that is not due to MSH and which originates wit,hin the pars distalis (MialheVoIoss: 1958; Itoh, 1962; Smelik et- & 1962). The experiments in which we removed the rostra1 pars distalis from dogfish and subsequently assayed the neurointermediate lobe for ACTH activity were designed to test the hypothesis that neurointermediate ‘lobe ACTH activity came from the rostra1 pars distalis. The observation that the amount of ACT ext’racted from the dogfish neurointermediate lobe remained unchanged 10 days after removal of the rostra1 pars distalis indicates that the activity originates within the neurointermediate lobe. While our results do not allow us to exclude th.e possibility that ACTH is produced in two distinct and spatial’ly separate regions of the chondrichthyeaa pituitary, we favor an alternative explanat.ion. The published information that suggests a single location for the known hormonal act’ivities of the chondrichthyean pituitary and the following indirect evidence suggest to us that the ACTH activity of the nemointermediate lobe is probably due to t,he MSH principle. As noted above, the elasmobranch neuroint’erlncdiat,e lobe

274

DEROOS

AND

contains an MSH principle and the available evidence indicates that the elasmobranch principle is similar to the MSH principles of other vertebrates. In our studies related to the present report (deRoos and C. deRoos, unpublished) we observed permanent blanching of dogfish following removal of the neurointermediate lobe; this did not occur in animals in which only the pars distalis or rostra1 pars distalis was removed. Administration of aqueous homogenates of the neurointermediate lobe resulted in a prompt darkening of animals which were blanched. Importantly, the administration of mammalian ACTH generally darkened intact dogfish or dogfish in which either the pars distalis or rostra1 pars distallis was removed, an observation previously reported by Dittus (1937, 1939) and which suggests a similarity in the chemical structure of the MSH and ACTH principles. In mammals, the entire amino acid sequence of #(r-MSH is repeated in the ACTH molecule. Not only does ACTH possess inherent MSH activity, but a-MSH has been shown to possess inherent ACTH activity both in vivo and in vitro (Steelman and Guillemin, 1959; Hofman and Yajima, 1961; Lebovitz et al., 1966). Thus, it seems reasonable to suggest that the consistent ACTH activity present in extracts of the neurointermediate lobe may reflect the inherent ACTH activity of the chondrichthyean MSH principle, at least with regard to its ability to promote an increase in corticoid production. ACKNOWLEDGMENTS We are grateful for the interest and cooperation extended to us, by the staff of the Friday Harbor Laboratories, University of Washington. We wish to extend particular thanks to the Director, Dr. Robert L. Fernald and to the Captain of the “Hydah,” Mr. Cleave C. Vandersluys. The skilled technical assistance of Mr. David Eden and Mr. Max W. Stutz was of immeasurable help. REFERENCES A., AND NANDI, J. (1964). Endocrinology of poikilothermic vertebrates. 1n “The Hormones” (G. Pincus, K. V. Thimann, and E. B.

BERN,

H.

DEROOS Astwood, eds.), Vol. 4, pp. 1991~298. Academic Press, New York. BERN, H. A., DEROOS, C. C., AND BIGLIERI, E. G. (1962). Aldosterone and other corticosteroids from chondrichthyean interrenal glands. Gen. Comp. Endocrinol. 2, 490-494. BUSH, I. E. (1953). Species differences in adrenocortical secretion. J. .EndocrinoZ. 9, 95-100. CARSTENSEN, H., BURGERS, A. C. J., AND LI, C. H. (1961). Demonstration of aldosterone and corticosterone as the principal steroids formed in incubates of adrenals of the American bullfrog (Rana catesbeiana) and stimulation of their production by mammalian adrenocorticotropin. Gen. Camp. Endocrinol. 1, 37-50. CHESTER JONES, I., PHILLIPS, J. G., AND BELLAMY, D. (1962a). The adrenal cortex throughout the vertebrates. Brit. Med. Bull. 18, 110~114. CHESTER JONES, I., PHILLIPS, J. G., AND BELLAMY, D. (1962b). Studies on water and electrolytes in cyclostomes and teleosts with special reference to Myzine glutinosa L. (the hagfish) and Anguilln anguillu L. (the Atlantic eel). Gen. C,omp. Endocrinol. Suppl. 1, 3&47. DENT, J. N. AND DODD, J. M. (1962). Some effects of mammalian thyroid stimulating hormone, elasmobranch pituitary gland extracts and temperature on thyroidal activity in newly hatched dogfish (Scyliorhinus caniculus). J. Endocrinol. 22, 395-402. DEROOS, R. (1961). The corticoids of the avian adrenal gland. Gen. Comp. Endocrinol. 1, 4% 512. DEROOS, R., AND DEROOS, C. C. (1964a). Effects of mammalian corticotropin and chicken adenohypophysial extracts on steroidogenesis by chicken adrenal tissue in vitro. Gen. Comp. Endocrinol. 4, 6@&607. nnRoos, R., AND DEROOS, C. C. (196413). Demonstration of corticotropin activity in the pituitary gland of chondrichthyean fishes. Am. Zoologist 4, 393 (Abstract). DITYPUS, P. (1937). Experimentelle Untersuchungen am Interrenalorgan der Selachier. 1. Atemfrequenz und Melanophoren bei interrenopriven und mit corticotropem Hormon behandelten Selachiern. Publ. Staz. Zool. Napoli 1~6, 402435. DITTUS, P. (1939). Das Verhalten der Melanophoren hypophysektomierter Selachier und Amphibien nach Zufuhr von kortikotropem Hormon. Biol. Zentr. 59, 627-652. DITTUS, P. (1941). Histologie und Cytologie des Interrenalorgans der Selachier. Ein Beitrag zur Kenntnis der Wirkungsweise des kortikotropen Hormons und des Verhaltnisses von Kern au Plasma. Z. Wiss. Zool. Abt. A. 154, 401124.

CHONDRICHTHYEAN DODD,

J. M. (1961). Adenohypophyscal

of fishes. Abst?-acts Pac$c Sci. Congress,

Symposium Honolulzc,

ACTH

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10th

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