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Dopamine, prolactin control, and osmoregulation in eels
GENERAL
AND
COMPARATIVE
Dopamine,
ENDOCRINOLOGY
Prolactin
26, 550-561 (1975)
Control,
and Osmoregulation
in Eels’
MADELEINE OLIVEREAU Laboratoire de Physiologic, 195, rue Saint-Jacques,
Institut 75005
Oceanographique, Paris, France
Accepted April 6. 1975 In freshwater eels, administration of t-Dopa or 2-Br-or-ergocryptine induced a granule storage in pituitary prolactin cells. These cells appear less active, as shown by a significant decrease in nuclear diameter and atrophy of the endoplasmic reticulum; blood sodium level and skin mucus content (assayed by N-acetyl-neuraminic acid determination in the skin), both under pro&tin control in freshwater, are reduced. These two drugs act through a dopaminergic mechanism in mammals and some fish. Conversely, pituitary transplantation or 6-hydroxy-dopamine treatment stimulates prolactin cells which show an intense degranulation. In seawater eels, no clear effect was detected after a similar treatment with L-Dopa; prolactin cells remained sparsely granulated, blood sodium and skin mucus content were not affected. It appears, therefore, that both the release and the synthesis of prolactin have already decreased after 1 mo in seawater and that dopamine has no further effect. In eels, prolactin secretion is regulated by external and blood osmolality; pituitary activity is also controlled by prolactin circulating levels through a direct short feedback. Hypothalamus control, from the nucleus lateralis tuberis (NLT), seems to involve a dopaminergic mechanism. Recent evidence has shown that dopamine may be the prolactin-inhibiting factor (PIF) in the rat; this hypothesis may apply to freshwater eels, but in seawater, the regulation of proiactin synthesis and release may well be different or more complex. The role of a prolactin releasing factor (PRF) simultaneously modulating these processes must be taken into consideration.
Among the pituitary hormones involved in osmoregulation, prolactin plays the main role in freshwater; generally, pituitary prolactin concentration is much higher in freshwater than in seawater, and prolactin secretion seems to increase with decreasing external salinity, in several teleosts, as recently reviewed by Fontaine and Olivereau (1975). However this may not be true for some salmonids: in Oncorhynchus nerka, pituitary prolactin content in river fish was lower than that of seawater fish, and serum prolactin concentration was reduced in freshwater, indicating an enhanced utilization of the hormone as the fish enters the river (McKeown and Van Overbeeke, 1972).
In salmon smolts, the plasma prolactin level was also much higher in one-third seawater or in pure seawater than it was in freshwater (Leatherland and McKeown, 1974). Prolactin secretion is affected by variations in the blood sodium level, an effect which is also evident in vitro with Xiphophorus pituitary (Sage, 1968). However, when eels are adapted to seawater, blood sodium concentration is about the same as in freshwater [155-l 65 meqllitre (Olivereau and Lemoine, 1973a)], the main difference being the greater sodium turnover in seawater than in freshwater (Maetz et al., 1967). It seems unlikely that sodium turnover may directly affect prolactin cell activity. In teleost fish, prolactin secretion is localized in the rostra1 pars distalis, an area where innervation seems rather complex,
’ A report based on this paper was given at the Seventh Symposium on Comparative Endocrinology. Tsavo National Park, Kenya in July 1974. 550 Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any fom reserved.
DOPAMINE,
PROLACTIN
CONTROL,
especially in the roach (Big, et al., 1974) and the mullet (Abraham, 1971, 1974); in Tilupia, several subtypes of type B fibres, probably aminergic, predominate in the rostra1 lobe (Zambrano, 1972), but no obvious changes were evident in fish adapted to seawater (Nishioka et al., 1972). In Anguilfu, the rostra1 lobe is in contact with the anterior part of the neurohypophysis which rarely contains peptidergic neurosecretory fibers; many other fibres, often PAS positive, are present (Olivereau, 1967) and most of them come from the NLT. In electron microscope studies, the nerve fibre tracts do not make direct contact with the endocrine cells, but are separated from them by extravascular spaces. Most of the fibres in these tracts contain membrane-bound vesicles with an electron-dense core about 700 A in diameter; these vesicles closely resemble B type vesicles; the new fibres with membrane-bound vesicles and an electron dense content about 1400 A in diameter are probably type A fibres, as seen in the neurosecretory tract (Knowles and Vollrath, 1966). It was interesting therefore to study the possibility of an aminergic control of prolactin secretion in eels. Several drugs were used: L-Dopa, an immediate precursor of dopamine, which increases dopamine content of the hypothalamus; 6-hydroxydopamine, which destroys dopaminergic nerve terminals (type B fibres) and reduces brain norepinephrine and dopamine; the ergocryptine, 2-Br-a-ergocryptine. which prevents prolactin release and was recently shown to directly stimulate dopamine receptors, this being a long-term effect (Corrodi et al., 1973); it slows dopamine turnover in rat tuberoinfundibular neurons (Hokfelt and Fuxe, 1972). To evaluate the effect of these drugs, several criteria were selected: (1) the histological structure of the prolactin cells, mainly the density of granules and the development of the endoplasmic reticu-
AND
OSMOREGULATION
551
lum; (2) the blood sodium level, essentially dependent upon prolactin secretion in freshwater; (3) the amount of sialic acid (N-acetyl-neuraminic acid or NANA) in the skin, also correlated to prolactin secretion in freshwater (Olivereau and Lemoine, 197 la,b); NANA is a major component of fish mucus as in eels (Lemoine and Olivereau, 197 1). MATERIAL
AND METHODS
Male silver eels (Anguilla anguillu L.) (50-75 g) were kept in freshwater at 20 2 I”. with a photoperiod of 12 hr (light from 8 to 20). Thirty freshwater eels were injected in the body cavity with t.-Dopa ( 15 mg/kg/day for 7-20 days, or 30 mg/kg, twice a day, for 8 days): 20 others were injected with 2-Br-aergocryptine methanesulfonate or CB 154 Sandoz ( IO eels with 0.5 mg/animal/day and IO with I mg/day for up to 14 days, after dissolution in tartaric acid). The animals were killed l-3 hr after the last injection of L-Dopa or CB 154. Twenty-six eels received one, two, or three injections of 6-hydroxydopamine (6.OH-DA) of l-4 mg/day (Fremberg and Olivereau, 1973) in the body cavity and were killed l-9 days after the last injection. Seventeen eels were injected intracranially once or twice with 100 pg of 6-OH-DA and sacrificed 3 hr to 9 days later; 6-OH-DA was dissolved in a I% ascorbic acid solution to prevent its oxidation. Control eels were, respectively, injected with the same volume of solvent: NaCl 6 per 1000. tartaric acid (5 mglml), or ascorbic acid I%, for the same length of time, in the body cavity or intracranially. Sixteen eels were gradually transferred into seawater (in 36 hr) and kept for 3 wk in natural pure seawater (20”, 12 hr light). Eight animals were injected with L-Dopa, twice a day for 8 days (I4 injections, 30 mg/kg), and 8 with NaCl 6 per 1000: control and injected eels were killed after I mo in seawater. After MS222 anaesthesia, blood was collected foi Nat. K+, Cat, and Cll determinations, a piece of skin was removed as previously described (Lemoine and Olivereau, 1971) and the NANA content estimated using the thiobarbituric acid technique of Aminoff. Pituitaries still attached to the brain were quickly fixed in Bouin-Hollande sublimated, cut at 4 pm, and stained mainly with Herlant’s tetrachrome and Cleveland-Wolfe, to reveal the granular content of prolactin cells and with methyl green-pyronine for ribonucleic acid detection in the endoplasmic reticulum. Nuclear diameter (mean of two perpendicular axes of the ellipse) was measured in 50 cells per pituitary.
552
MADELEINE
OLIVEREAU
DOPAMINE,
PROLACTIN
CONTROL,
RESULTS I” Freshwater (a) Control eels injected with various solvents. Prolactin cells organized in fol-
licles were more or less granulated; the apical zone rarely contained many granules (Fig. I); the cells surrounded a small lumen with little or no secretion (either PAS positive, mucopolysaccharidic, or stainable with the orange G), the origin of which is not clearly established. Immunofluorescence technique can detect prolactin in the follicular cells of the rostral pars distalis of Oncorhynchus nerka, mainly in the granulated cytoplasm, but the apex and the content of the lumen do not react with labeled anti-ovine prolactin (McKeown and van Overbeeke, 1969). The nucleus lay either centrally or in the basal third of the cell, and RNA was detected in most of the cells. None of the injected solvents caused a significant modification in these cells. (h) L-Dopa. As previously reported (Olivereau and Lemoine, 1973b), L-Dopa administration induces a retention of cytoplasmic granules which accumulate throughout the cell (Fig. 2); the nucleus is adjacent to the basal part of the cell where the endoplasmic reticulum is normally detected, but which, after L-Dopa, is no
AND
OSMOREGULATION
553
longer apparent. A short treatment with 15 mg/kg/day does not clearly modify the blood sodium level and the cell nuclear diameter (Figs. 7 and 8); however, plasma sodium is slightly but significantly reduced after 10 days or more. With a dose of 30 mg/kg, blood sodium level, prolactin cell nuclear diameter and mucus content of the skin are significantly decreased (P < 0.001). (c) 2-Br-a-ergocryptine. The effects of treatment with CB 154 are quite similar to those obtained after L-Dopa administration: granule retention, nuclear diameter reduction, decrease of blood sodium level, and NANA skin content (Figs. 7 and 8), atrophy of the endoplasmic reticulum which may be still visible in a few cells, in which case the nucleus in not so close to the follicle basal membrane as after L-Dopa treatment. In two animals, the lumen is enlarged (Fig. 3); more often, the maximal cell height is reduced (Olivereau and Lemoine, 1973~). (d) 6-Hydroxy-dopamine. Injected into the body cavity, 6-OH-DA stimulates prolactin cell activity (Olivereau, 1973); this effect is more evident after two intracranial injections: most of the prolactin cells are completely degranulated (Fig. 4) with an enlarged nucleus, a hypertrophied nucleolus, a strongly developed endoplasmic
FIG. 1. Control eel in freshwater. The apical area is less granulated than the basal portion of the prolactin cells organized in follicles with a small lumen. FIG. 2. Freshwater eel treated with L-dopa (0.75 mg/day) for 20 days. Prolactin cells are heavily granulated with basal nuclei often smaller than those of control eels. FIG. 3. Freshwater eel treated with CB 154 (ergocryptine) (1 mg/day) for 12 days. Prolactin cells are well granulated, but often smaller than in control eels. The nuclear diameter is reduced, the follicle lumen may be slightly enlarged. Some TSH cells (light grey) are visible on the bottom of the picture. FIG. 4. Freshwater eel treated with Ghydroxydopamine (100 &day) and killed 9 days after the second injection. The prolactin cells are stimulated and often degranulated; the nucleus is frequently apical, the enlarged endoplasmic reticulum being basal. FIG. 5. Control eel after 1 mo in seawater. The pro&tin cells contain only a few erythrosinophilic granules and are smaller than in freshwater eels. FIG. 6. Eel adapted to seawater for 1 mo and treated for 8 days with L-dopa (4 mg/day). No accumulation of granules can be detected in the prolactin cells which appear similar to those of control animals (Fig. 5). Figs. 1-6, ~925. Herlant’s tetrachrome.
554
MADELEINE
fw dose “‘g/day
L.oopa 0.76
4
OLIVEFCEAU
fw CB 154 0.6
c
c
fw
fw graft
6-OH-DA 0.1
FIG. 7. Variations of the nuclear diameter of prolactin cells in eels after treatment with 6-OH-DA, or pituitary autotransplantation in freshwater (F.W.) or seawater (SW.). The arrows in the development of the endoplasmic reticulum (e.r.) and of the amount of granules.
reticulum (Fig. 7). This picture resembles the one often observed in autotransplanted pituitaries. 2. Seawater (a) Control eels injected with vent. Compared with freshwater
the sol-
animals, the rostra1 pars distalis was reduced in eels adapted to seawater for 1 mo. The cells are almost devoid of erythrosinophilic granulations (Fig. 5), the nucleus is much
L-dopa, indicate
CB 154, changes
smaller, the amount of RNA greatly reduced; the lumen is sometimes increased. This cytological picture is not affected by the solvent. Blood sodium (154.8 * 2.16 meq/liter) is higher than in freshwater eels (143.1 ? 1.4 meq/litre) in this experiment, but skin NANA values are similar (Fig. 8). (b) L-Dopa. No clear effect from L-Dopa treatment could be detected, neither on the histological structure of prom Sodium m
FIG. 8. Simultaneous changes in blood sodium levels of plasma neuraminic acid) of the skin of eels injected with the vehicle (V) hypophysectomized (H) eels or after pituitary autotransplantation.
NINA
and of the amount of NANA (N-acetyl or L-dopa, CB 154, and in intact (I) or
DOPAMINE,
PROLACTIN
CONTROL,
lactin cells (Fig. 6) nor on blood sodium level (153.9 * 1.60 meqllitre) (Fig. 8); the NANA skin content reduction is not significant (A. M. Lemoine, unpublished results). DISCUSSION
1. Control of Prolactin in Freshwater
Secretion
Several factors may affect prolactin secretion. They were recently reviewed for Gillichthys mirabilis and Tilapia mossambica (Nagahama et al., 1975). (a) External control. In various species, it is mainly exerted through the blood sodium level or serum osmolality, and in vitro prolactin release is directly stimulated by a hyposmotic medium (Sage, 1968; Nagahama et al., 1975). However, in eels which have been gradually transferred to seawater (avoiding stress), after 1 mo (or less) plasma sodium is approximately similar to the level in freshwater, but the prolactin cell structure is very different. Prolactin cell stimulation still occurs in grafted pituitaries in Poecilia transferred from one-third seawater to freshwater (Ball and Olivereau, 1965). (6) Blood prolactin level. In the eel, injection of ovine prolactin for more than 20 days induces degranulation and atrophy of prolactin cells and their nuclei; this result suggests that an increase of circulating prolactin may produce a reduction of prolactin cell activity (unpublished data cited in Ball and Olivereau, 1964). Equally suggestive is the fact that when putuitary glands are transplanted into intact eels, after 10-l 5 days, prolactin cells of the transplant remain granulated without evidence of stimulation (Olivereau et al., 1971). No effect is detectable in the in situ pituitary after 2 wk. The prolactin secreted by the in situ gland seems able to prevent, or at least greatly reduce prolactin release by the disconnected gland which would normally be under hypothalamic inhibition
AND
OSMOREGULATION
555
(Olivereau and Dimovska, 1969). Recently, Nagahama’s findings (1975) on intact Gillichthys receiving pituitary homotransplants indicate that the prolactin cells of the in situ glands become cytologically inactive within 40-50 days, prolactin cells in homotransplants contain more granules than in autotransplants (Nagahama et al., 1974) a result which agrees with our previous observations on the eel. In mammals, the pituitary also appears as a possible site of prolactin feedback involved in its autoregulation (Spies and Clegg, 1971) (see also no. 2 discussion). (c) Hypothalamic control. An inhibitory hypothalamic control of prolactin secretion was first suspected in Poecilia latipinna after pituitary autotransplantation (Ball et al., 1965; Olivereau and Ball, 1966) and observed in several other species at various salinities (Ball et al., 1972). In Anguilla, an autotransplanted pituitary contains prolactin cells which are always stimulated as compared with in situ glands (Olivereau and Dimovska, 1969). In the goldfish, there is a dual control correlated to the NLT: lesions in the NLT pars lateralis causes a significant increase in serum prolactin without effect on pituitary prolactin levels; large lesions of the anterior-medial thalamus and anterior hypothalamus dorsal to the NLT cause a decrease in serum prolactin, but have no significant effect on pituitary prolactin levels (Peter and McKeown, 1973). As the NLT area does not contain neurosecretory fibres (Type A), it seems that the inhibitory control may be mostly mediated through aminergic fibres (B type). Several monoaminergic bundles have been described, and numerous aminergic fibres supply the NLT in the eel (L’Hermite and Lefranc, 1972). Treatment with L-Dopa which increases the amount of dopamine in the brain and/or the pituitary, and treatment with CB 154 which stimulates dopamine receptors, both inhibit prolactin release in
556
MADELEINE
eels. The granule release is reduced in freshwater in correlation with a decrease of plasma Na+ and NANA skin content. An inhibition of prolactin secretion seems evident and agrees with results obtained on Poecilia (McKeown, 1972) or Fund&us (Kramer et al., 1973) treated with CB 154 and on Gillichthys injected with L-Dopa (Nagahama et al., 1975). Inhibition of prolactin release in mammals with L-Dopa and CB 154 is well documented and the literature was recently reviewed (Olivereau and Lemoine, 1973b,c). However, during the last 2 yr, more data were obtained about the mechanism of action of these drugs. L-Dopa (or dopamine) was supposed to act at the hypothalamic level to increase its PIF concentration (Lu and Meites, 1972) and its release in vivo (Kamberi et al., 1970) and in vitro (Quijada et al., 1974), as well as to reduce prolactin discharge when injected into the third ventricle, but not in the pituitary (Kamberi et al., 1971). More recently, it was shown that L-Dopa directly acts on prolactin release even after lesions of the median eminence (Donoso et al., 1973) or pituitary transplantation (Donoso et al., 1974); dopamine acts in vitro to prevent prolactin release by incubated pituitaries (MacCann et al., 197 1; McLeod and Lehmeyer, 1974), or by dissociated rat prolactin cells (Hymer et al., 1974). An interesting hypothesis has been proposed with dopamine as the physiological inhibitor of prolactin secretion, acting directly on the pituitary to stimulate specific receptors (McLeod and Lehmeyer, 1974); dopamine might be the PIF (Shaar et al., 1973; Clemens, Shaar, Smalstig, and Matsumoto, 1974). Indeed, a correlation between PIF activity and the catecholamine content of one porcine hypothalamic extract was found (Takahara et al., 1974), but such a correlation was lacking in another preparation. Moreover, by adding glucose to the extract to prevent its rapid oxidation, Takahara et al. (1974)
OLIVEREAU
were able to inhibit prolactin release by infusing this preparation or dopamine in the rat hypophyseal portal vessel, a confirmation of a direct action on the pituitary gland. CB 154 also acts directly on the pituitary, whether ectopic or in vitro (literature in Olivereau and Lemoine, 1973~; Hoshino, 1973; Clemens et al., 1974; Marko and Niederer, unpublished data cited in Del Pozo et al., 1974). Since CB 154 stimulates dopamine receptors, the hypothesis of dopamine as a PIF is still valid and agrees with our findings on eels. However, it is difficult to ascertain if prolactin synthesis is simultaneously depressed by these substances. Conversely, 6-OH-DA administration induces a destruction of dopaminergic nerve terminals in mammals and an increased release of prolactin (Olivereau, 1973) and MSH (Fremberg and Olivereau, 1973) in eels. In Gillichthys and Tilapia, ultrastructural studies also indicate a strong stimulation of prolactin cells in freshwater, but this effect could not be demonstrated in seawater (Zambrano et al., 1974). In freshwater eels, 6-OH-DA clearly seems to increase prolactin secretion. However, after reserpine administration, no apparent effect was detected in male eels (Olivereau, 1971), but a brief stimulation can be observed in females (unpublished data); this stimulation seems more evident in the roach (Bsge et al., 1974) and Gillichthys (Zambrano et al., 1972). 2” Control of Prolactin in Seawater
Secretion
The role of dopamine in osmoregulation in seawater is not as evident as in freshwater. Prolactin release not only appears reduced, but its synthesis also seems inhibited after some weeks of adaptation to seawater. L-Dopa has no further effect on either prolactin cell structure, blood sodium level, or NANA skin content.
DOPAMINE,
PROLACTIN
CONTROL,
However, it does increase the fluorescence of the rostra1 lobe in the dogfish (Wilson and Dodd, 1974). 6-OH-DA has no effect on prolactin cells in seawater Tilupia (Zambrano et al., 1974); some modification of the rostra1 lobe innervation may occur during adaptation to seawater as suggested for Mugil kept at various salinities (Abraham, 197 l), although a thorough study has not yet been done, but no effect was observed in Tilapia (Nishioka et al., 1972).
The absence of granule storage in seawater may be due either to the suppression of prolactin synthesis, or to a reduced synthesis still1 taking place and a simultaneous release. The second possibility seems less likely as L-Dopa does not appear to promote cytoplasmic granule accumulation; more probably, prolactin synthesis is depressed after 1 mo in seawater. This conclusion would agree with results obtained on Mugil (Blanc-Livni and Abraham, 1970), Tilapia (Clarke, 1973) and Poecilia (Ball and Ingleton, 1973) which indicate that pituitary prolactin content, determined by various biological tests, is reduced in seawater, but other tests or radioimmunoassays gave divergent results on prolactin in seawater fishes. The xanthophore dispersion test for prolactin assay used on Gillichthys (Sage and Bern, 1972) when applied to Mugil cephalus, showed a similar yellowing activity in the pituitary of fish whether kept in seawater or adapted to freshwater from 30 min to 6 wk; the prolactin content has no correlation with the blood Na level in seawater (Sage, 1973). In Carassius auratus, plasma prolactin levels were similar in tap water and in 30% seawater (length of adaptation not indicated (Leatherland and McKeown, 1973)). However, with an heterologous raresults must be indioimmunoassay, terpreted with some caution. In Oncorhynchus nerka directly transferred to seawater, prolactin accumulates in the pituitary, but is reduced in the serum
AND
OSMOREGULATION
557
after lo-20 days, suggesting that its utilization is increased in seawater (McKeown, 1970). More recently, in the same fish kept for 15 days in full seawater after a gradual adaptation over 14 days, plasma prolactin is about three times higher than in freshwater salmon; according to Leatherland and McKeown (1974) “this suggests that, at least in this species, it is the rate of usage of the hormone by the target organs that dictates the plasma concentration.” As prolactin cells are less active in hyperosmotic media, one may wonder about the physiological significance of this high level of prolactin that is neither utilized, excreted nor inactivated after 2 wk; ovine prolactin injected into cichlid fish disappears from the serum within 3-7 hr (Bliim, 1968) and the half-life of human prolactin is only 15 min (Miyai et al., 1974). The high serum concentration in experimental seawater goldfish or salmon might be responsible for the reduced prolactin cell activity through a short feedback system. However other target organs must be studied before definite conclusions can be drawn about the physiological significance of the prolactin level in seawater animals. At least, in Anguilla, such a high level does not seem likely according to the normal plasma sodium concentration (155-l 65 meq/litre) which does not change after hypophysectomy but is greatly increased after ovine prolactin injection (up to 200 meq/litre in 10 days) (Olivereau and Lemoine, 1973a) with a simultaneous augmentation of K+, Ca”+, and Cl-, in intact or hypophysectomized animals. To explain these discrepancies, it may be supposed that freshwater and seawater prolactin are not similar compounds or have different biological activities or are present in multiple forms as it has been proposed for some mammalian hormones (Nicoll et al., 1974). Also, radioimmunoassays may detect immunoreactive components without biological activity and
558
MADELEINE
may give an overestimation of prolactin in certain cases. The most likely reason for the depression of prolactin cell activity is the high osmotic pressure of seawater and the increased blood osmolality, but the role of dopamine is not clear. In conclusion, a dopaminergic control of prolactin secretion must be seriously considered: in freshwater, dopamine may interfere to modulate the release, and perhaps the synthesis of prolactin, and seems to act as a PIF, or at least regulate PIF secretion. Its effect can be associated with those of serum osmolality and feedback to the pituitary from blood prolactin level after prolactin injection or prolactin secretion by a pituitary transplanted in an intact fish. In seawater, the role of dopamine is not apparent and prolactin regulation may depend more on serum osmolality; however, as prolactin cells are poorly granulated in control seawater eels, any effect of dopamine on the amount of granules would be easier to demonstrate if some synthesis were still taking place. Actually, the regulation of prolactin secretion may be more complex, involving the participation of a PRF: there is increasing evidence that a PRF might be present in the hypothalamus of the rat as well as in the goldfish (Peter and McKeown, 1973). The possible participation of the thyrotrophin releasing factor which stimulates prolactin release in several mammals, must also be considered (Hill-Samli and MacLeod, 1974).
We are indebted to Sandoz Laboratories (Basel) for the gift of CB 154 used in this experiment, to A. M. Lemoine for permission to quote her unpublished data on the effect of L-Dopa on NANA skin concentration in seawater, to J. Olivereau and S. Warrot for
technical
assistance
and
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(CNRS)
OLIVEREAU
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DOPAMINE,
PROLACTIN
CONTROL,
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OSMOREGLJLATION
traventricular injection of catecholamines on prolactin release. Endocrinafogy 88, IO 1 Z- 1020. Knowles. F., and Vollrath, L. (1966). Neurosecretory innervation of the pituitary of the eels Anguilfa and Conger. Il. The structure and innervation of the pars distalis at different stages of the lifecycle. Phil. Trans. Ray. Sot. London Ser. B 250, 329-342.
Kramer, K. L., Schreibman, M. P., and Pang, P. K. T. (1973). The effect of 2-Br-a-ergocryptinemethane-sulfonate on freshwater survival and pituitary cytology in the teleost, Fundulus hetrroclitus.
Amer.
Zool.
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1779.
Leatherland, J. F., and McKeown, B. A. (1973). Circadian rhythm in the plasma levels of prolactin in goldfish, Carassius aurafus L. J. fnterdiscipf. Cycle Res. 4, 137- 143. Leatherland, J. F., and McKeown, B. A. (1974). Effect of ambient salinity on prolactin and growth hormone secretion and on hydromineral regulation in kokanee salmon smolts (Oncorhynchus nerka).
J. Camp.
Physial.
89, 215-226.
Lemoine. A. M., and Olivereau, M. (197 I). Presence d’acide N-acetylneuraminique dans la peau d’Anguiffa anguilla L. IntCret de son dosage dans I’Ctude de I’osmoregulation. Z. I/ergl. Phgsiol. 73, 22-33.
L’Hermite, A., and Lefranc, G. (1972). Recherches sur les voies monoaminergiques de I’enctphale d’Anguilla phol. Exp.
vulgaris.
Arch.
61, 139-l
52.
Anat.
Microsc.
Mar-
Lu, K. U., and Meites, J. (1972). Effects of L-dopa on serum prolactin and PIF in intact and hypophysectomized pituitary-grafted rats. Endocrinology 91,824~828.
McCann, S. M., Kabra, P. S., Donoso, A. O., Bishop, W., Schneider, H. P. G., Fawcett, C. P., and Krulich, L. (1971). The role of monoamines in the control of gonadotropin and prolactin secretion. In “Brain-Endocrine Interaction. Median Eminence: Structure and Function” (International Symposium, Munich 19711, pp. 224-235. Karger, Basel. McKeown, B. A. ( 1970). Immunological identification and quantification of pituitary hormones in the sockeye salmon (Oncorhynchus nerka. Walbaum). Ph. D. Thesis, Simon Fraser Univ.. pp. I-83. McKeown, B. A. (I 972). Effect of 2-Br-cY-ergocryptine on freshwater survival in the teleosts, Xiphaphorus hefferii and Poe&a Iatipinna. Experientia
28, 675.
McKeown, B. A., and Van Overbeeke, A. P. ( 1969). lmmunohistochemical localization of ACTH and prolactin in the pituitary gland of adult migratory
560
MADELEINE
sockeye salmon (Oncorhynchus nerka). J. Fish. Res. Bd. Can. 26, 1837-1846. McKeown, B. A., and Van Overbeeke, A. P. (1972). Prolactin and growth hormone concentrations in the serum and pituitary gland of adult migratory sockeye salmon (Oncorhynchus nerka). J. Fish. Res. Bd. Can.
29, 303-309.
MacLeod, R. M., and Lehmeyer, J. E. (1974). Studies on the mechanism of the dopamine-mediated inhibition of prolactin secretion. Endocrinology 94, 1077-1085. Maetz, J., Mayer, N., and Chattier-Baraduc, M. M. (1967). La balance min&ale du sodium chez Anguih anguih en eau de mer, en eau deuce et au tours de transfert d’un milieu g I’autre: effets de l’hypophysectomie et de la prolactine. Gen.
Camp.
Endocrinol.
8, 177- 188.
Miyai, K., Onishi, T., Hosokawa, M., Ishibashi, K., and Kumahara, Y. (1974). Inhibition of thyrotropin and prolactin secretions in primary hypothyroidism by 2-Br-cY-ergocryptine. J. C[in. Endocrinol.
Metab.
39, 391-394.
Nagahama, Y., Nishioka, R. S., and Bern, H. A. (1974). Structure and function of the transplanted pituitary in the seawater goby, Gillichthys mirabilis. I. The rostra1 pars distalis. Gen.
Comp.
Endocrinof.
22, 2 I-34.
Nagahama, Y., Nishioka, R. S., Bern, H. A., and Gunther, R. (1975). Control of prolactin secretion in teleosts, with special reference to Gillichrhys mirabilis and Tilapio mossambica. Gen. Comp. Endocrinol. (in press). Nicoll, C. S., Asawaroengchai, H., and Vodian, M. A. (1974). Multiple forms of pituitary hormones. Gen. Comp. Endocrinol. (in press). Nishioka, R. S., Bern, H. A., and Nagahama, Y. (1972). Innervation of the pituitary of the teleost, Tilapia
mossambica.
Amer.
2001.
12, 678.
Olivereau, M. (1967). Observations sur I’hypophyse de I’Anguille femelle, en particulier lors de la maturation sexuelle. Z. Zellforsch. Mikrosk. Anat. 80, 286-306. Olivereau, M. (1971). Action de la r&erpine chez I’Anguille. I. Cellules ?I prolactine de l’hypophyse du m%e. Z. Zeflforsch. Mikrosk. Anat. 121, 232-243. Olivereau, M. (1973). Dopaminergic control of prolactin secretion in eels. VI International Symposium on Neurosecretion, London 1973, p. 60. Olivereau, M., and Ball, J. N. (1966). Histological study of functional ectopic pituitary transplants in a teleost fish (Poecilia formosa). Proc. Roy. Sot.
Ser. B 164, 106-129.
Olivereau, M., and Dimovska, A. (1969). Prolactinsecreting cells in the autotransplanted pituitary of the eel. Gen. Comp. Endocrinol. 13, 523-524. Olivereau, M., and Lemoine, A. M. (197 la). Action
OLIVEREAU
de la prolactine chez 1’Anguille intacte et hypophysectomisCe. VII. Effet sur la teneur en acide sialique (N-ac&yl-neuraminique) de la peau. Z. Vergl.
Physiol.
73, 34-43.
Olivereau, M., and Lemoine. A. M. (1971b). Teneur en acide N-acCtyl-neuraminique de la peau chez I’Anguille apr&s autotransplantation de I’hypophyse. Z. Vergl. Physiol. 73, 44-52. Olivereau, M., and Lemoine, A. M. (1973a). Action de la prolactine chez I’Anguille intacte et hypophysectomiske. VIII. Effets sur les tlectrolytes plasmatiques en eau de mer. J. Comp. Physiol. 86, 65-75. Olivereau, M., and Le’hoine, A. M. (1973b). Action de la L-dopa sur la s&cr&tion de prolactine chez I’Anguille. C. R. Acad. Sci. Ser. D 276, 1325-1327. Olivereau, M., and Lemoine, A. M. (1973~). Effet de la 2-Br-a-ergocryptine (CB 154) sur la s&r&ion de prolactine chez I’Anguille. C. R. Acad. Sci. Ser. D 276, 1883-1886. Olivereau, M., Lemoine, A. M., and Dimovska, A. (197 1). Don&es sur le contrble de la fonction prolactinique chez I’Anguille. Ann. Endocrinol. 32, 271. Peter, R. E., and McKeown, B. A. (1973). Control of prolactin secretion in the goldfish, Carussius auratus. VI International Symposium on Neurosecretion, London 1973, p. 25. Quijada, M., Illner, P., Krulich, L., and McCann, S. M. (1974). The effect of catecholamines on hormone release from anterior pituitaries and ventral incubated in vitro. Ncuroenhypothalami docrinology 13, I5 l- 163. Sage, M. (1968). Responses to osmotic stimuli of Xiphophorus prolactin cells in organ culture. Gen.
Comp.
Endocrinol
10, 70-74.
Sage, M. (1973). The relationship between the pituitary content of prolactin and blood sodium levels in mullet (Mugil cephnlusf transferred from sea water to fresh water. Contrib. Marine Sci. 17, 163-I
67.
Sage, M., and Bern, H. A. (I 972). Assay of prolactin in vertebrate pituitaries by its dispersion of xanthophore pigment in the teleost Gillichthys mirabilis. J. Exp. Zool. 180, 169- 174. Shaar, C. J., Smalstig, E. B., and Clemens, J. A. (1973). The effect of catecholamines, apomorphine and monoamine oxidase on rat anterior pituitary prolactin release in tzitro. Pharmacologist
15, 256.
Spies, H. G., and Clegg, M. T. (197 1). Pituitary as a possible site of prolactin feedback in autoregulation. Neuroendocrinology 8, 205-2 12. Takahara, J., Arimura, A., and Schally, A. V. (1974). Suppression of prolactin release by a purified
DOPAMINE,
PROLACTIN
CONTROL,
AND OSMOREGULATION
561
porcine PIF preparation and catecholamines infused into a rat hypophysial portal vessel. En-
hydroxydopamine on hypothalamic control of prolactin and ACTH secretion in the teleost fish.
docrinology
Tilapia
95, 467465.
Wilson, J. F., and Dodd, J. M. (1973). Distribution of monoamines in the diencephalon and pituitary of the dogfish, Scyliorhinus canicula L. Z. Zellforsch.
Mikrosk.
A nat.
137, 45 1-469.
Zambrano, D. (1972). Innervation of the teleost pituitary. Get]. Comp. Etzdocrinol. Suppl. 3, 22-3 I. Zambrano, D., Clarke, W. C., Hawkins, E. F., Sage, M., and Bern, H. A. (1974). Influence of 6-
tnossambica.
Nrurorndocritzology
13,
284-298. Zambrano, D., Nishioka, R. S., and Bern, H. A. ( 1972). The innervation of the pituitary gland of teleost fishes. Its origin, nature and significance. In “Brain-Endocrine Interaction. Median Eminence: Structure and Function” (International Symposium. Munich 197 1). pp. 50-66. Karger. Base].
AND
COMPARATIVE
Dopamine,
ENDOCRINOLOGY
Prolactin
26, 550-561 (1975)
Control,
and Osmoregulation
in Eels’
MADELEINE OLIVEREAU Laboratoire de Physiologic, 195, rue Saint-Jacques,
Institut 75005
Oceanographique, Paris, France
Accepted April 6. 1975 In freshwater eels, administration of t-Dopa or 2-Br-or-ergocryptine induced a granule storage in pituitary prolactin cells. These cells appear less active, as shown by a significant decrease in nuclear diameter and atrophy of the endoplasmic reticulum; blood sodium level and skin mucus content (assayed by N-acetyl-neuraminic acid determination in the skin), both under pro&tin control in freshwater, are reduced. These two drugs act through a dopaminergic mechanism in mammals and some fish. Conversely, pituitary transplantation or 6-hydroxy-dopamine treatment stimulates prolactin cells which show an intense degranulation. In seawater eels, no clear effect was detected after a similar treatment with L-Dopa; prolactin cells remained sparsely granulated, blood sodium and skin mucus content were not affected. It appears, therefore, that both the release and the synthesis of prolactin have already decreased after 1 mo in seawater and that dopamine has no further effect. In eels, prolactin secretion is regulated by external and blood osmolality; pituitary activity is also controlled by prolactin circulating levels through a direct short feedback. Hypothalamus control, from the nucleus lateralis tuberis (NLT), seems to involve a dopaminergic mechanism. Recent evidence has shown that dopamine may be the prolactin-inhibiting factor (PIF) in the rat; this hypothesis may apply to freshwater eels, but in seawater, the regulation of proiactin synthesis and release may well be different or more complex. The role of a prolactin releasing factor (PRF) simultaneously modulating these processes must be taken into consideration.
Among the pituitary hormones involved in osmoregulation, prolactin plays the main role in freshwater; generally, pituitary prolactin concentration is much higher in freshwater than in seawater, and prolactin secretion seems to increase with decreasing external salinity, in several teleosts, as recently reviewed by Fontaine and Olivereau (1975). However this may not be true for some salmonids: in Oncorhynchus nerka, pituitary prolactin content in river fish was lower than that of seawater fish, and serum prolactin concentration was reduced in freshwater, indicating an enhanced utilization of the hormone as the fish enters the river (McKeown and Van Overbeeke, 1972).
In salmon smolts, the plasma prolactin level was also much higher in one-third seawater or in pure seawater than it was in freshwater (Leatherland and McKeown, 1974). Prolactin secretion is affected by variations in the blood sodium level, an effect which is also evident in vitro with Xiphophorus pituitary (Sage, 1968). However, when eels are adapted to seawater, blood sodium concentration is about the same as in freshwater [155-l 65 meqllitre (Olivereau and Lemoine, 1973a)], the main difference being the greater sodium turnover in seawater than in freshwater (Maetz et al., 1967). It seems unlikely that sodium turnover may directly affect prolactin cell activity. In teleost fish, prolactin secretion is localized in the rostra1 pars distalis, an area where innervation seems rather complex,
’ A report based on this paper was given at the Seventh Symposium on Comparative Endocrinology. Tsavo National Park, Kenya in July 1974. 550 Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any fom reserved.
DOPAMINE,
PROLACTIN
CONTROL,
especially in the roach (Big, et al., 1974) and the mullet (Abraham, 1971, 1974); in Tilupia, several subtypes of type B fibres, probably aminergic, predominate in the rostra1 lobe (Zambrano, 1972), but no obvious changes were evident in fish adapted to seawater (Nishioka et al., 1972). In Anguilfu, the rostra1 lobe is in contact with the anterior part of the neurohypophysis which rarely contains peptidergic neurosecretory fibers; many other fibres, often PAS positive, are present (Olivereau, 1967) and most of them come from the NLT. In electron microscope studies, the nerve fibre tracts do not make direct contact with the endocrine cells, but are separated from them by extravascular spaces. Most of the fibres in these tracts contain membrane-bound vesicles with an electron-dense core about 700 A in diameter; these vesicles closely resemble B type vesicles; the new fibres with membrane-bound vesicles and an electron dense content about 1400 A in diameter are probably type A fibres, as seen in the neurosecretory tract (Knowles and Vollrath, 1966). It was interesting therefore to study the possibility of an aminergic control of prolactin secretion in eels. Several drugs were used: L-Dopa, an immediate precursor of dopamine, which increases dopamine content of the hypothalamus; 6-hydroxydopamine, which destroys dopaminergic nerve terminals (type B fibres) and reduces brain norepinephrine and dopamine; the ergocryptine, 2-Br-a-ergocryptine. which prevents prolactin release and was recently shown to directly stimulate dopamine receptors, this being a long-term effect (Corrodi et al., 1973); it slows dopamine turnover in rat tuberoinfundibular neurons (Hokfelt and Fuxe, 1972). To evaluate the effect of these drugs, several criteria were selected: (1) the histological structure of the prolactin cells, mainly the density of granules and the development of the endoplasmic reticu-
AND
OSMOREGULATION
551
lum; (2) the blood sodium level, essentially dependent upon prolactin secretion in freshwater; (3) the amount of sialic acid (N-acetyl-neuraminic acid or NANA) in the skin, also correlated to prolactin secretion in freshwater (Olivereau and Lemoine, 197 la,b); NANA is a major component of fish mucus as in eels (Lemoine and Olivereau, 197 1). MATERIAL
AND METHODS
Male silver eels (Anguilla anguillu L.) (50-75 g) were kept in freshwater at 20 2 I”. with a photoperiod of 12 hr (light from 8 to 20). Thirty freshwater eels were injected in the body cavity with t.-Dopa ( 15 mg/kg/day for 7-20 days, or 30 mg/kg, twice a day, for 8 days): 20 others were injected with 2-Br-aergocryptine methanesulfonate or CB 154 Sandoz ( IO eels with 0.5 mg/animal/day and IO with I mg/day for up to 14 days, after dissolution in tartaric acid). The animals were killed l-3 hr after the last injection of L-Dopa or CB 154. Twenty-six eels received one, two, or three injections of 6-hydroxydopamine (6.OH-DA) of l-4 mg/day (Fremberg and Olivereau, 1973) in the body cavity and were killed l-9 days after the last injection. Seventeen eels were injected intracranially once or twice with 100 pg of 6-OH-DA and sacrificed 3 hr to 9 days later; 6-OH-DA was dissolved in a I% ascorbic acid solution to prevent its oxidation. Control eels were, respectively, injected with the same volume of solvent: NaCl 6 per 1000. tartaric acid (5 mglml), or ascorbic acid I%, for the same length of time, in the body cavity or intracranially. Sixteen eels were gradually transferred into seawater (in 36 hr) and kept for 3 wk in natural pure seawater (20”, 12 hr light). Eight animals were injected with L-Dopa, twice a day for 8 days (I4 injections, 30 mg/kg), and 8 with NaCl 6 per 1000: control and injected eels were killed after I mo in seawater. After MS222 anaesthesia, blood was collected foi Nat. K+, Cat, and Cll determinations, a piece of skin was removed as previously described (Lemoine and Olivereau, 1971) and the NANA content estimated using the thiobarbituric acid technique of Aminoff. Pituitaries still attached to the brain were quickly fixed in Bouin-Hollande sublimated, cut at 4 pm, and stained mainly with Herlant’s tetrachrome and Cleveland-Wolfe, to reveal the granular content of prolactin cells and with methyl green-pyronine for ribonucleic acid detection in the endoplasmic reticulum. Nuclear diameter (mean of two perpendicular axes of the ellipse) was measured in 50 cells per pituitary.
552
MADELEINE
OLIVEREAU
DOPAMINE,
PROLACTIN
CONTROL,
RESULTS I” Freshwater (a) Control eels injected with various solvents. Prolactin cells organized in fol-
licles were more or less granulated; the apical zone rarely contained many granules (Fig. I); the cells surrounded a small lumen with little or no secretion (either PAS positive, mucopolysaccharidic, or stainable with the orange G), the origin of which is not clearly established. Immunofluorescence technique can detect prolactin in the follicular cells of the rostral pars distalis of Oncorhynchus nerka, mainly in the granulated cytoplasm, but the apex and the content of the lumen do not react with labeled anti-ovine prolactin (McKeown and van Overbeeke, 1969). The nucleus lay either centrally or in the basal third of the cell, and RNA was detected in most of the cells. None of the injected solvents caused a significant modification in these cells. (h) L-Dopa. As previously reported (Olivereau and Lemoine, 1973b), L-Dopa administration induces a retention of cytoplasmic granules which accumulate throughout the cell (Fig. 2); the nucleus is adjacent to the basal part of the cell where the endoplasmic reticulum is normally detected, but which, after L-Dopa, is no
AND
OSMOREGULATION
553
longer apparent. A short treatment with 15 mg/kg/day does not clearly modify the blood sodium level and the cell nuclear diameter (Figs. 7 and 8); however, plasma sodium is slightly but significantly reduced after 10 days or more. With a dose of 30 mg/kg, blood sodium level, prolactin cell nuclear diameter and mucus content of the skin are significantly decreased (P < 0.001). (c) 2-Br-a-ergocryptine. The effects of treatment with CB 154 are quite similar to those obtained after L-Dopa administration: granule retention, nuclear diameter reduction, decrease of blood sodium level, and NANA skin content (Figs. 7 and 8), atrophy of the endoplasmic reticulum which may be still visible in a few cells, in which case the nucleus in not so close to the follicle basal membrane as after L-Dopa treatment. In two animals, the lumen is enlarged (Fig. 3); more often, the maximal cell height is reduced (Olivereau and Lemoine, 1973~). (d) 6-Hydroxy-dopamine. Injected into the body cavity, 6-OH-DA stimulates prolactin cell activity (Olivereau, 1973); this effect is more evident after two intracranial injections: most of the prolactin cells are completely degranulated (Fig. 4) with an enlarged nucleus, a hypertrophied nucleolus, a strongly developed endoplasmic
FIG. 1. Control eel in freshwater. The apical area is less granulated than the basal portion of the prolactin cells organized in follicles with a small lumen. FIG. 2. Freshwater eel treated with L-dopa (0.75 mg/day) for 20 days. Prolactin cells are heavily granulated with basal nuclei often smaller than those of control eels. FIG. 3. Freshwater eel treated with CB 154 (ergocryptine) (1 mg/day) for 12 days. Prolactin cells are well granulated, but often smaller than in control eels. The nuclear diameter is reduced, the follicle lumen may be slightly enlarged. Some TSH cells (light grey) are visible on the bottom of the picture. FIG. 4. Freshwater eel treated with Ghydroxydopamine (100 &day) and killed 9 days after the second injection. The prolactin cells are stimulated and often degranulated; the nucleus is frequently apical, the enlarged endoplasmic reticulum being basal. FIG. 5. Control eel after 1 mo in seawater. The pro&tin cells contain only a few erythrosinophilic granules and are smaller than in freshwater eels. FIG. 6. Eel adapted to seawater for 1 mo and treated for 8 days with L-dopa (4 mg/day). No accumulation of granules can be detected in the prolactin cells which appear similar to those of control animals (Fig. 5). Figs. 1-6, ~925. Herlant’s tetrachrome.
554
MADELEINE
fw dose “‘g/day
L.oopa 0.76
4
OLIVEFCEAU
fw CB 154 0.6
c
c
fw
fw graft
6-OH-DA 0.1
FIG. 7. Variations of the nuclear diameter of prolactin cells in eels after treatment with 6-OH-DA, or pituitary autotransplantation in freshwater (F.W.) or seawater (SW.). The arrows in the development of the endoplasmic reticulum (e.r.) and of the amount of granules.
reticulum (Fig. 7). This picture resembles the one often observed in autotransplanted pituitaries. 2. Seawater (a) Control eels injected with vent. Compared with freshwater
the sol-
animals, the rostra1 pars distalis was reduced in eels adapted to seawater for 1 mo. The cells are almost devoid of erythrosinophilic granulations (Fig. 5), the nucleus is much
L-dopa, indicate
CB 154, changes
smaller, the amount of RNA greatly reduced; the lumen is sometimes increased. This cytological picture is not affected by the solvent. Blood sodium (154.8 * 2.16 meq/liter) is higher than in freshwater eels (143.1 ? 1.4 meq/litre) in this experiment, but skin NANA values are similar (Fig. 8). (b) L-Dopa. No clear effect from L-Dopa treatment could be detected, neither on the histological structure of prom Sodium m
FIG. 8. Simultaneous changes in blood sodium levels of plasma neuraminic acid) of the skin of eels injected with the vehicle (V) hypophysectomized (H) eels or after pituitary autotransplantation.
NINA
and of the amount of NANA (N-acetyl or L-dopa, CB 154, and in intact (I) or
DOPAMINE,
PROLACTIN
CONTROL,
lactin cells (Fig. 6) nor on blood sodium level (153.9 * 1.60 meqllitre) (Fig. 8); the NANA skin content reduction is not significant (A. M. Lemoine, unpublished results). DISCUSSION
1. Control of Prolactin in Freshwater
Secretion
Several factors may affect prolactin secretion. They were recently reviewed for Gillichthys mirabilis and Tilapia mossambica (Nagahama et al., 1975). (a) External control. In various species, it is mainly exerted through the blood sodium level or serum osmolality, and in vitro prolactin release is directly stimulated by a hyposmotic medium (Sage, 1968; Nagahama et al., 1975). However, in eels which have been gradually transferred to seawater (avoiding stress), after 1 mo (or less) plasma sodium is approximately similar to the level in freshwater, but the prolactin cell structure is very different. Prolactin cell stimulation still occurs in grafted pituitaries in Poecilia transferred from one-third seawater to freshwater (Ball and Olivereau, 1965). (6) Blood prolactin level. In the eel, injection of ovine prolactin for more than 20 days induces degranulation and atrophy of prolactin cells and their nuclei; this result suggests that an increase of circulating prolactin may produce a reduction of prolactin cell activity (unpublished data cited in Ball and Olivereau, 1964). Equally suggestive is the fact that when putuitary glands are transplanted into intact eels, after 10-l 5 days, prolactin cells of the transplant remain granulated without evidence of stimulation (Olivereau et al., 1971). No effect is detectable in the in situ pituitary after 2 wk. The prolactin secreted by the in situ gland seems able to prevent, or at least greatly reduce prolactin release by the disconnected gland which would normally be under hypothalamic inhibition
AND
OSMOREGULATION
555
(Olivereau and Dimovska, 1969). Recently, Nagahama’s findings (1975) on intact Gillichthys receiving pituitary homotransplants indicate that the prolactin cells of the in situ glands become cytologically inactive within 40-50 days, prolactin cells in homotransplants contain more granules than in autotransplants (Nagahama et al., 1974) a result which agrees with our previous observations on the eel. In mammals, the pituitary also appears as a possible site of prolactin feedback involved in its autoregulation (Spies and Clegg, 1971) (see also no. 2 discussion). (c) Hypothalamic control. An inhibitory hypothalamic control of prolactin secretion was first suspected in Poecilia latipinna after pituitary autotransplantation (Ball et al., 1965; Olivereau and Ball, 1966) and observed in several other species at various salinities (Ball et al., 1972). In Anguilla, an autotransplanted pituitary contains prolactin cells which are always stimulated as compared with in situ glands (Olivereau and Dimovska, 1969). In the goldfish, there is a dual control correlated to the NLT: lesions in the NLT pars lateralis causes a significant increase in serum prolactin without effect on pituitary prolactin levels; large lesions of the anterior-medial thalamus and anterior hypothalamus dorsal to the NLT cause a decrease in serum prolactin, but have no significant effect on pituitary prolactin levels (Peter and McKeown, 1973). As the NLT area does not contain neurosecretory fibres (Type A), it seems that the inhibitory control may be mostly mediated through aminergic fibres (B type). Several monoaminergic bundles have been described, and numerous aminergic fibres supply the NLT in the eel (L’Hermite and Lefranc, 1972). Treatment with L-Dopa which increases the amount of dopamine in the brain and/or the pituitary, and treatment with CB 154 which stimulates dopamine receptors, both inhibit prolactin release in
556
MADELEINE
eels. The granule release is reduced in freshwater in correlation with a decrease of plasma Na+ and NANA skin content. An inhibition of prolactin secretion seems evident and agrees with results obtained on Poecilia (McKeown, 1972) or Fund&us (Kramer et al., 1973) treated with CB 154 and on Gillichthys injected with L-Dopa (Nagahama et al., 1975). Inhibition of prolactin release in mammals with L-Dopa and CB 154 is well documented and the literature was recently reviewed (Olivereau and Lemoine, 1973b,c). However, during the last 2 yr, more data were obtained about the mechanism of action of these drugs. L-Dopa (or dopamine) was supposed to act at the hypothalamic level to increase its PIF concentration (Lu and Meites, 1972) and its release in vivo (Kamberi et al., 1970) and in vitro (Quijada et al., 1974), as well as to reduce prolactin discharge when injected into the third ventricle, but not in the pituitary (Kamberi et al., 1971). More recently, it was shown that L-Dopa directly acts on prolactin release even after lesions of the median eminence (Donoso et al., 1973) or pituitary transplantation (Donoso et al., 1974); dopamine acts in vitro to prevent prolactin release by incubated pituitaries (MacCann et al., 197 1; McLeod and Lehmeyer, 1974), or by dissociated rat prolactin cells (Hymer et al., 1974). An interesting hypothesis has been proposed with dopamine as the physiological inhibitor of prolactin secretion, acting directly on the pituitary to stimulate specific receptors (McLeod and Lehmeyer, 1974); dopamine might be the PIF (Shaar et al., 1973; Clemens, Shaar, Smalstig, and Matsumoto, 1974). Indeed, a correlation between PIF activity and the catecholamine content of one porcine hypothalamic extract was found (Takahara et al., 1974), but such a correlation was lacking in another preparation. Moreover, by adding glucose to the extract to prevent its rapid oxidation, Takahara et al. (1974)
OLIVEREAU
were able to inhibit prolactin release by infusing this preparation or dopamine in the rat hypophyseal portal vessel, a confirmation of a direct action on the pituitary gland. CB 154 also acts directly on the pituitary, whether ectopic or in vitro (literature in Olivereau and Lemoine, 1973~; Hoshino, 1973; Clemens et al., 1974; Marko and Niederer, unpublished data cited in Del Pozo et al., 1974). Since CB 154 stimulates dopamine receptors, the hypothesis of dopamine as a PIF is still valid and agrees with our findings on eels. However, it is difficult to ascertain if prolactin synthesis is simultaneously depressed by these substances. Conversely, 6-OH-DA administration induces a destruction of dopaminergic nerve terminals in mammals and an increased release of prolactin (Olivereau, 1973) and MSH (Fremberg and Olivereau, 1973) in eels. In Gillichthys and Tilapia, ultrastructural studies also indicate a strong stimulation of prolactin cells in freshwater, but this effect could not be demonstrated in seawater (Zambrano et al., 1974). In freshwater eels, 6-OH-DA clearly seems to increase prolactin secretion. However, after reserpine administration, no apparent effect was detected in male eels (Olivereau, 1971), but a brief stimulation can be observed in females (unpublished data); this stimulation seems more evident in the roach (Bsge et al., 1974) and Gillichthys (Zambrano et al., 1972). 2” Control of Prolactin in Seawater
Secretion
The role of dopamine in osmoregulation in seawater is not as evident as in freshwater. Prolactin release not only appears reduced, but its synthesis also seems inhibited after some weeks of adaptation to seawater. L-Dopa has no further effect on either prolactin cell structure, blood sodium level, or NANA skin content.
DOPAMINE,
PROLACTIN
CONTROL,
However, it does increase the fluorescence of the rostra1 lobe in the dogfish (Wilson and Dodd, 1974). 6-OH-DA has no effect on prolactin cells in seawater Tilupia (Zambrano et al., 1974); some modification of the rostra1 lobe innervation may occur during adaptation to seawater as suggested for Mugil kept at various salinities (Abraham, 197 l), although a thorough study has not yet been done, but no effect was observed in Tilapia (Nishioka et al., 1972).
The absence of granule storage in seawater may be due either to the suppression of prolactin synthesis, or to a reduced synthesis still1 taking place and a simultaneous release. The second possibility seems less likely as L-Dopa does not appear to promote cytoplasmic granule accumulation; more probably, prolactin synthesis is depressed after 1 mo in seawater. This conclusion would agree with results obtained on Mugil (Blanc-Livni and Abraham, 1970), Tilapia (Clarke, 1973) and Poecilia (Ball and Ingleton, 1973) which indicate that pituitary prolactin content, determined by various biological tests, is reduced in seawater, but other tests or radioimmunoassays gave divergent results on prolactin in seawater fishes. The xanthophore dispersion test for prolactin assay used on Gillichthys (Sage and Bern, 1972) when applied to Mugil cephalus, showed a similar yellowing activity in the pituitary of fish whether kept in seawater or adapted to freshwater from 30 min to 6 wk; the prolactin content has no correlation with the blood Na level in seawater (Sage, 1973). In Carassius auratus, plasma prolactin levels were similar in tap water and in 30% seawater (length of adaptation not indicated (Leatherland and McKeown, 1973)). However, with an heterologous raresults must be indioimmunoassay, terpreted with some caution. In Oncorhynchus nerka directly transferred to seawater, prolactin accumulates in the pituitary, but is reduced in the serum
AND
OSMOREGULATION
557
after lo-20 days, suggesting that its utilization is increased in seawater (McKeown, 1970). More recently, in the same fish kept for 15 days in full seawater after a gradual adaptation over 14 days, plasma prolactin is about three times higher than in freshwater salmon; according to Leatherland and McKeown (1974) “this suggests that, at least in this species, it is the rate of usage of the hormone by the target organs that dictates the plasma concentration.” As prolactin cells are less active in hyperosmotic media, one may wonder about the physiological significance of this high level of prolactin that is neither utilized, excreted nor inactivated after 2 wk; ovine prolactin injected into cichlid fish disappears from the serum within 3-7 hr (Bliim, 1968) and the half-life of human prolactin is only 15 min (Miyai et al., 1974). The high serum concentration in experimental seawater goldfish or salmon might be responsible for the reduced prolactin cell activity through a short feedback system. However other target organs must be studied before definite conclusions can be drawn about the physiological significance of the prolactin level in seawater animals. At least, in Anguilla, such a high level does not seem likely according to the normal plasma sodium concentration (155-l 65 meq/litre) which does not change after hypophysectomy but is greatly increased after ovine prolactin injection (up to 200 meq/litre in 10 days) (Olivereau and Lemoine, 1973a) with a simultaneous augmentation of K+, Ca”+, and Cl-, in intact or hypophysectomized animals. To explain these discrepancies, it may be supposed that freshwater and seawater prolactin are not similar compounds or have different biological activities or are present in multiple forms as it has been proposed for some mammalian hormones (Nicoll et al., 1974). Also, radioimmunoassays may detect immunoreactive components without biological activity and
558
MADELEINE
may give an overestimation of prolactin in certain cases. The most likely reason for the depression of prolactin cell activity is the high osmotic pressure of seawater and the increased blood osmolality, but the role of dopamine is not clear. In conclusion, a dopaminergic control of prolactin secretion must be seriously considered: in freshwater, dopamine may interfere to modulate the release, and perhaps the synthesis of prolactin, and seems to act as a PIF, or at least regulate PIF secretion. Its effect can be associated with those of serum osmolality and feedback to the pituitary from blood prolactin level after prolactin injection or prolactin secretion by a pituitary transplanted in an intact fish. In seawater, the role of dopamine is not apparent and prolactin regulation may depend more on serum osmolality; however, as prolactin cells are poorly granulated in control seawater eels, any effect of dopamine on the amount of granules would be easier to demonstrate if some synthesis were still taking place. Actually, the regulation of prolactin secretion may be more complex, involving the participation of a PRF: there is increasing evidence that a PRF might be present in the hypothalamus of the rat as well as in the goldfish (Peter and McKeown, 1973). The possible participation of the thyrotrophin releasing factor which stimulates prolactin release in several mammals, must also be considered (Hill-Samli and MacLeod, 1974).
We are indebted to Sandoz Laboratories (Basel) for the gift of CB 154 used in this experiment, to A. M. Lemoine for permission to quote her unpublished data on the effect of L-Dopa on NANA skin concentration in seawater, to J. Olivereau and S. Warrot for
technical
assistance
and
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