Effect of hypothalamic extracts on prolactin secretion in the goldfish, Carassius auratus L

Camp. Biochem. Physiol., 1974, Vol. 47A, pp. 419 to 426. Pergamon Press. Printed in Great Britain

EFFECT OF HYPOTHALAMIC EXTRACTS ON PROLACTIN SECRETION IN THE GOLDFISH, CARASSIUS AURATUS L. JOHN

F. LEATHERLAND

and

D. M. ENSOR

lDepartment of Zoology, University of Guelph, Guelph, Ontario, Canada; and *Department of Zoology, University of Liverpool, England

(Received 13 April 1973) Abstract-l.

A single-injection of an acid extract of hypothalamus from distilled water or 30% sea water-acclimated donors (DdWand D,, respectively) was administered intracranially to recipients acclimated to either distilled (Rdw) or 30% sea water (R,,). fish was significantly lower than in R,, 2. The plasma Naf level in R,,D, D, fish at 12 and 24 hr after the injection; the level in R&l, fish at 24 hr was significantly lower than in similarly treated groups at 1, 4 and 12 hr. 3. The data are interpreted as indicating a temporary fall in plasma prolactin levels as a result of hypothalamic prolactin release inhibiting factor (RIF) in the extract. 4. The plasma Na+ levels in 30% sea water-acclimated recipients (R#,) administered hypothalamic extracts of D, and D,, fish did not differ significantly at any of the times investigated. 5. There were no significant differences in the K+ data from the R,, or R, groups.

INTRODUCTION THE NATURE of hypothalamic regulation of adenohypophysial function in teleost fishes has been the subject of a number of investigations in recent years (Ball & Olivereau, 1965 ; Ball et al., 1965 ; Knowles & Vollrath, 1966; Knowles et al., 1967; Olivereau & Ball, 1966; Olivereau, 1969a, b, 1971a, b; Leatherland, 1970, 1971, 1972; Peter, 1970, 1971, 1972; Zambrano, 1970a, b, 1971; Abraham, 1971; Ball et al., 1972; Chidambaram et al., 1972; Leatherland & Ensor, 1973). Hypothalamic regulation of prolactin cell activity has received particular attention. In PoeciZiu formosa (Ball et al., 1965 ; Olivereau & Ball, 1966), P. Zutipimu (Ball & Olivereau, 1965), AnguiZZu unguilla (Olivereau, 1971 a, b), IctuZurus melus (Chidambaram et al., 1972) and Gusterosteus uculeutus form truchurus (Leatherland, 1970, 1971) prolactin secretion appears to be under inhibitory hypothalamic control. However, in P. Zutipinnu (Ball & Olivereau, 1965) and Carussius uurutus (Leatherland & Ensor, 1973) transplanted prolactin cells appeared less active in fish in hyperosmotic ambient media than in similar fish in fresh or distilled water which suggests that in these species the prolactin cells are more independent of 419

JOHNF. LEATHRRLAHD ANDD. M. ENSOR

420

hypothalamic control. However, the close proximity of neurosecretory synapses and prolactin cells (Leatherland, 1972) and the presence of a circadian rhythmicity in levels of circulating prolactin in both 30% sea water- and fresh water-acclimated goldfish (Leatherland & McKeown, 1973) are suggestive of some form of hypothalamic intervention. The purpose of this investigation was to determine whether extracts of hypothalamic material affect the secretory activity of prolactin cells. Since prolactin is known to be at least in part responsible for the maintenance of plasma Naf in goldfish (Lahlou & Sawyer, 1969; Lahlou & Giordan, 1970) for the purpose of this investigation the plasma Na+ concentration was used as an index of circulating prolactin levels.

MATERIALS AND METHODS Source and maintenance of animals Three hundred goldfish (Carassius auratus L.) were obtained from a local supplier in early August 1970. Two groups of 150 fish were maintained in the laboratory in large constantly aerated and filtered aquaria which contained either glass distilled water or 30% sea water at lO-11°C. Both groups were subjected to a “natural” (16 hr light) photoperiod. The fish were fed to excess three times a week with a commercial gold&h pellet food and the water was changed 1 hr after feeding; they were starved for 1 week prior to removing the donor hypothalami and subsequently for the remaining experiment period. After 1 month 100 fish were removed from each group to serve as hypothalamic donors. They were weighed and the hypothalamus removed and placed on dry ice. Blood samples were taken from the first 6 fish in each group for plasma Naf and K+ analysis (see Table 1). TABLE ~-BODY WRIGHTANDPLASMANa+ ANDK+ CONCRHTRATIONS IN DISTILLEDWATRRAND 30% SEA WATER-ACCLIMATED DONORFISH

Donor

Mean body weight (g k S.E.M.)

Mean plasma concentration (m-equiv/l. f S.E.M.) Naf

K+

Ambient concentration (m-equiv/l.) Naf

Kf

Distilled water-acclimated

3.03 f 0.08

115~07+4*37

7.90 + 1.57

0.04

0.01

30% sea water-accli-

3.02 f 0.07

166.80 + 5.36

5.30 f 0.34

180.20

6.10

mated * Samples of plasma taken from the first six fish in each group.

Two groups of 24 “recipient” fish were taken from each of the ambient salinites and placed in smaller experimental aquaria which had interconnected circulating water supplies. The fish were retained in the experimental aquaria for 10 days before receiving the hypothalamic extracts.

EFFECT OF HYPOTHALAMIC

Preparation

of hypothalamic

EXTRACTS ON PROLACTIN

SECRETION IN GOLDFISH

421

extracts

The hypothalamus was removed from each of the “donor” fish between 09.00 and 11 .OO hours. The brain was exposed by removing the cranium and the cerebral hemispheres and optic lobes were dissected free. An incision was then made just anterior to the optic chiasma and at the anterior margin of the cerebellum so that the hypothalamic region could be removed from the skull. Care was taken to ensure that the pituitary gland was not attached to the hypothalamus; it was then rapidly frozen on dry ice and stored overnight at -60°C. The two pools of hypothalami (from distilled water- and sea water-acclimated donors) were separately homogenized with 5 ml of ice-cold 0.1 N NC1 and centrifuged for 30 min at 15,000 g. The supematant was retained and the tissue was extracted a second time with 5 ml of O-1 N HCl. The pooled supernatants were freeze dried. The procedure was modified from that used in mammals by Grosvenor et al. (1965), Grosvenor (1965) and Grosvenor et al. (1967). The extracts were dissolved in sufficient 0.9% NaCl (solvent) so that each “recipient” was given the equivalent of 1.5 hypothalami in 0.02 ml of solvent. Experimental procedure The distilled water recipients (I&,) and 30% sea water (I&J acclimated recipients were injected intracranially with the hypothalamic extracts of donors acclimated to either distilled water (Dad or 30% sea water (0,) (see Table 2). The injections were performed between 11.00 and 12.00 hours when plasma prolactin levels were at their lowest (Leatherland & McKeown, 1973). TABLE

~-NOMENCLATURE

USED FOR GROUPS OF EXPERIMENTAL ANIMALS

Donors (D) Recipients (R)

Distilled water-acclimated

30% sea water-acclimated

Distilled water-acclimated 30% sea water-acclimated Samples of six fish from each group were killed 1, 4, 12 and 24 hr after the injection. Plasma samples were taken and the Na+ and K+ concentrations measured by the procedure described earlier (Leatherland & Ensor, 1973). Statistics Naf and Kf concentrations in distilled water- and 30% sea water-acclimated recipients were separately subjected to one-way analysis of variance. Individual means were compared by least significance difference (1.s.d.) (Steel & Torrie, 1960). RESULTS

The mean plasma Na+ concentrations of the sixteen groups of recipient fish are shown in Fig. 1. The plasma Na+ in R,,D,, fish was significantly lower than that of the RdwDsw animals in the groups killed 12 hr (P
JOHN F. LEATHERLAND ANDD. M. ENSOR

422

recipients of hypothalami from distilled water and 30% sea water acclimated donors

distilled water recipients 120

110 1

s

100

F L

E ZE.

90

56 Ool

80 B

++I ;%

18

EF i P

15

T

Irl-i

30% sea water recipients

FIG. 1. Effect of hypothalamic extracts from distilled water- and 30% sea wateracclimated donors on the plasma Na + levels in distilled water- and 30% sea water-acclimated recipient C. au~utus.

The plasma Na+ concentration in the R,,D, group killed at 24 hr cantly (PC 0.01) smaller than in similar fish at the three other times. Na+ levels in the RdwDm groups at 12 and 24 hr were significantly in similar fish at 1 and 4 hr (PC 0.01 except for comparison of 4 and

was signifiThe plasma larger than 24 hr when

P< O-05). In the groups of recipients acclimated to 30% sea water there were no significant differences between the plasma Na+ concentrations of the R&, and R,Dm fish at any time. The plasma Na + level in the RswDdw fish killed 12 hr after the injection of hypothalamic extracts was significantly (P
EFFECT

OF

HYPOTHALAMIC

EXTRACTS

ON

PROLACTIN

SECRETION

IN

GOLDFISH

423

recipients of hypothalami from distilled water and 30% sea water 51 acclimated donors

r

distilled water recipient

30%

S

sea water recipients &I

T

time after injection(h) FIG. 2. Effect of hypothalamic extracts from distilled water- and 30% sea wateracclimated donor on the plasma K+ levels in distilled water- and 30% sea wateracclimated recipient C. auratus.

DISCUSSION

The inhibitory hypothalamic regulation of prolactin cell activity has been clearly demonstrated in a number of teleosts (see review by Ball et al., 1972). However, in the goldfish the evidence of hypothalamic involvement is conflicting. Autotransplanted prolactin cells appear to respond to the ambient salinity in the same manner as in situ cells (Leatherland & Ensor, 1973) which suggests that they are relatively independent of hypothalamic control and yet there is a clear circadian rhythmicity in the concentration of plasma prolactin which may be associated with hypothalamic involvement (Leatherland & McKeown, 1973). In addition, the close proximity of B-type neurosecretory synapses to the prolactin cells may indicate a functional relationship between these elements (Leatherland, 1972). If the hypothesis of a prolactin release-inhibiting factor (RIF) that is found in other teleosts is valid in C. auratus the situation represented diagramwill be higher matically in Fig. 3 may occur. The concentration of prolactin-RIF in distilled water-acclimated compared with 30% sea water-acclimated fish since in the former group the prolactin cells are markedly more active than in the 30% sea water-acclimated animals (Leatherland, 1972) thus indicating that the RIF in the distilled water-acclimated animals is stored within the hypothalamus while that in the 30% sea water-acclimated fish is released in order to decrease prolactin

424

JOHNF. LEATHERLAND ANDD. M. ENSOR distilled water acclimated 0.04 mEq/l

30% sea water acclimated 160.2 mEq/l

166.6

plasma mEq/l Na*

FIG. 3. Diagrammatic representation of the situation assuming the presence of prolactin-RIF in the hypothalamus. The closely cross-hatched hypothalamus and prolactin zones represent accumulations of secretory product. The black arrows represent a blocked release and the white arrows a release of material.

cell activity. Hence, injection of hypothalamic extracts from D,, fish to R, fish would be expected to depress plasma Na + levels by inhibiting prolactin secretion (synthesis or release). This effect was found in R,,D,, fish which were killed 12 and 24 hr after receiving the injection of hypothalamic material (Fig. 1); the response was confirmed by subsequent experiments (present authors, unpublished data). Conversely, extracts of hypothalami from 30% sea wateracclimated donors caused an apparent increase in plasma Na+ levels in R, fish which were killed 12 and 24 hr after the injection compared with similarly treated fish killed 1 and 4 hr after injection. The reason for this is not known but the Naf levels in the 1 and 4 hr R,,D,, and R&l,, groups seem to be artificially lower than in uninjected fish (115.1 m-equiv/l. in donor fish (Table 1) compared with 100~3-103~0 m-equiv./l. in recipient fish) possibly due to Na+ loss resulting from the stress of the injection procedure. The extracts used in the investigation were acid extracts of the whole hypothalamic region and probably contained release factors (RF) and RIF for a number of adenohypophysial hormones as well as octapeptide hormones from the hypothalamic neurosecretory centres. Lahlou & Giordan (1970) showed that hormones other than prolactin are involved in osmotic homeostasis in C. aurutus and these may also affect plasma electrolytes. However, Lahlou & Sawyer (1969) found that ACTH was ineffectual in maintaining plasma Na+ in hypophysectomized goldfish and in prolonged treatments of tap water-acclimated intact goldfish with metapirone, cortisol, thyroxine and thiourea the plasma Na+ and K+ levels were

EFFECTOF HYPOTHALAMIC EXTRACTSON PROLACTINSECRETIONIN GOLDFISH

425

not significantly changed (Leatherland & Ensor, unpublished data). Thus prolactin is considered to be the predominant pituitary hormone involved in plasma Na+ maintenance. The hypothalamic extracts were administered between 11.00 and 12.00 hours and the 1 and 24 hr recipients were killed during the same period of the day; plasma prolactin levels in untreated tap water- and 30% sea water-acclimated goldfish were found to be minimal at this time (Leatherland & McKeown, 1973). The 4 and 12 hr recipient fish were killed at 04.00 and 24.00 hours when plasma prolactin levels in untreated fish were higher than at the time of injection (Leatherland & McKeown, 1973). There is a possibility therefore that the plasma Na+ responses to the hypothalamic extracts were modified by the inherent prolactin circadian rhythm. Since comparisons at any one time were made between two groups of recipients (R,,D,, and RawDsw and R,,D,,) and since plasma prolactin levels in both tap water-acclimated and 30% sea water-acclimated untreated fish exhibit a circadian rhythmicity of approximately equal mode and intensity the differencs between the recipient groups were attributed to differences in the quantity of RIF. Acknowledgements--We are indebted to Professor J. G. Phillips for his support during the period of this research. The work was supported by grants in aid of research from S.R.C. and N.R.C. (J. F. L.). The paper is No. 061 in the migration series. REFERENCES ABRAHAMM. (1971) The ultrastructure of the cell types and of the neurosecretory innervation in the pituitary of Mugil cephalus L. from fresh water, the sea, and a hypersaline lagoon-I. The rostral pars distalis. Gen. & Compar. Endocr. 17, 334-350. BALL J. N., BAKERB. I., OLIVEREAU M. & PETERR. E. (1972) Investigations on hypothalamic control of adenohypophysial functions in teleost fishes. Gen. & Compar. Endocr. Suppl. 3, 11-21. BALL J. N. & OLIVEREAUM. (1965) Pituitary autotransplants and fresh-water adaptation in the teleost Poecilia latipinna. Am. Zoologist 5, 167A. BALL J. N., OLWEREAUM., SLICHERA. M. & KALLMANK. D. (1965) Functional capacity of ectopic pituitary transplants in the teleost Poecilia formosa, with a comparative discussion on the transplanted pituitary. Phil. Trans. R. Sot. B 249, 69-99. CHIDAMBARAM S., MEYER R. K. & HAULERA. D. (1972) Effects of hypophysectomy, pituitary autografts, prolactin, temperature and salinity of the medium on survival and natremia in the bullhead, Ictalurus melas. Camp. Biochem. Physiol. 43A, 443-458. GROSVENOR C. E. (1965) Effect of nursing and stress upon prolactin-inhibiting activity of the rat hypothalamus. Endocrinology 77, 1037-1042. GROWENORE. C., MCCANN S. M. & NALLARR. (1965) Inhibition of nursing-induced and stress-induced fall in pituitary prolactin concentration in lactating rats by injection of acid extracts of bovine hypothalamus. Endocrinology 76, 883-889. GROSVENOR C. E., MENA F., DHARIWALA. P. S. & MCCANN S. M. (1967) Reduction of milk secretion by prolactin-inhibiting factor: further evidence that exteroceptive stimuli can release pituitary prolactin in rats. Endocrinology 81, 1021-1028. KNOWLES F. & VOLLRATHL. (1966) Neurosecretory innervation of the pituitary of the eels Anguillu and Conger-II. The structure and innervation of the pars distalis at different stages of the life cycle. Phil. Trans. R. Sot. B 250, 329-342.

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KNOWLES F., VOLLRATHL. & NISHIOKA, R. S. (1967) Dual neurosecretory innervation of the adenohypophysis of Hippocampus, the sea horse. Nature, Lond. 214, 309. LAHLOU B. & GIORDANA. (1970) Le controle hormonal des &changes et de la balance de l’eau chez le teleosteen d’eau deuce Carassius auratus, intact et hypophysectomisee. Gen. U Compar. Endocr. 14, 491-509. LAHLOU B. & SAWYER W. H. (1969) Electrolyte balance in hypophysectomized goldfish, Carassius auratus L. Gen. & Compar. Endocr. 12, 370-377. LEATHERLANDJ. F. (1970) Histological investigation of pituitary homotransplants in the marine form (trachurus) of the threespine stickleback, Gasterosteus aculeatus L. Z. Zellforsch. mikrosk. Anat. 104, 337-344. LEATHERLANDJ. F. (1971) Effect of pituitary homotransplants on peripheral target organs in intact threespine sticklebacks, Gasterosteus aculeatus L. form trachurus. Can. J. Zool. 48, 1341-I 344. LEATHERLANDJ. F. (1972) Histophysiology and innervation of the pituitary gland of the goldfish, Carassius auratus L. : a light and electron microscope investigation. Can. J. Zool. 50, 835-844. LEATHERLANDJ. F. & ENSOR D. M. (1973) Activity of autotransplanted pituitary glands in goldfish Carassius auratus L. maintained in different ambient salinities. Can. J. Zool. 51, 225-235. LEATHERLANDJ. F. & MCKEO~N B. A. (1973) Circadian rhythm in the plasma levels of prolactin in the goldfish, Carassius auratus L. J. Interdisc. Cycle Res. (In press.) OLIVEREAUM. (1969a) ActivitC de la pars intermedia de l’hypophyse autotransplantee chez l’anguille. Z. Zellforsch. mikrosk. Anat. 98, 74-87. OLIVEREAU M. (1969b) Cytologie de l’hypophyse autotransplantee chez l’anguille, comparaison avec celle de Poecilia. Neuroendocrinologie 927, 2.51-260. OLIVEREAUM. (197la) Structure histologique de quelques glandes endocrines de l’anguille apres autotransplantation de l’hypophyse. Acta x001. 52, 69-83. OLIVEREAU M. (197lb) Structure histologique du rein et electrolytes plasmatiques chez l’anguille apres autotransplantation de l’hypophyse. Z. vergl. Physiol. 71, 350-364. OLIVEREAUM. & BALL J. N. (1966) Histological study of functional ectopic pituitary transplants in a teleost fish (Poecilia formosa). Proc. R. Sot. B 164,106-129. PETER R. E. (1970) Hypothalamic control of thyroid gland activity and gonadal activity in the goldfish, Carassius auratus. Gen. &Y Compar. Endocr. 14, 334-356. PETER R. E. (1971) Feedback effects of thyroxine on the hypothalamus and pituitary of goldfish, Carassius auratus. J. Endocr. 51,31-39. PETER R. E. (1972) Feedback effects of thyroxine in goldfish Carassius auratus with an autotransplanted pituitary. Neuroendocrinology 10,273-282. STEEL R. G. D. & TORRIE J. H. (1960) P rinciples and Procedures of Statistics. McGraw-Hill, New York. ZAMBRANOD. (197Oa) The nucleus lateralis tuberis system of the gobiid fish Gillichthys mirabilis-I. Ultrastructure and histochemical characterization of the nucleus. Z. Zellforsch. mikrosk. Anat. 110,9-26. ZAMBRANOD. (1970b) The nucleus lateralis tuberis system of the gobiid fish Gillichthys mirabilis-II. Innervation of the pituitary. Z. Zellforsch. mikrosk. Anat. 110,496-516. ZAMBRANOD. (1971) The nucleus lateralis tuberis system of the gobiid fish Gillichthys mirabilis-III. Functional modification of the neurons and gonadotropic cells. C-.+X & Compar. Endocr. 17, 164-182. Key

Word IndetiHypothalarnus;

prolactin;

Carassius auratus;

plasma sodium.