GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

Control

25, 166- 188 ( 1975)

of Prolactin Secretion in Teleosts, with Special Reference to Gillichthys mirabilis and Tilapia mossambical

YOSHITAKA HOWARD Department University

NAGAHAMA,

RICHARD

S. NISHIOKA,

A. BERN, AND ROBERT L. GUNTHER of Zoology of Californiu,

and Cancer Berkeley,

Reseurch Laboratory, California 94720

Accepted October 22, 1974 Two euryhaline teleosts, Gillichthys mirabilis (seawater) and Tilapia mossambica (freshwater), were used to study the control of prolactin secretion. Cytological changes of prolactin cells, plasma sodium measurements, and densitometry of disk electrophoretograms of pituitary extracts and incubation media provided the data for the study. When seawater Gillichthys are transferred to a hypotonic environment, both morphological and physiological data indicate that prolactin secretion is activated dramatically, confirming the physiological role of prolactin in adaptation to hyposmotic conditions. Prolactin cells are cytologically activated by pituitary transplantation as well as by injections of reserpine and 6-hydroxydopamine with a significant elevation of plasma sodium concentration. In contrast, the cells were inactivated by the injection of L-DOPA. When seawater fish bearing an autografted pituitary were transferred to fresh water, a significant decrease in prolactin cell granules was observed. In addition, the injection of estradiol-17P caused cytological activation of both prolactin cells and ACTH cells. Pituitary glands of Tilapia were incubated with 3H-leucine for 5-6 hr, and the radioactive prolactin present in the pituitary gland and that released in the incubation medium were measured. Prolactin release is directly stimulated by low osmotic pressure of the incubation medium. Addition of dopamine caused a significant decrease in the amount of radioactive prolactin released into the hyposmotic medium. The results indicate that at least four mechanisms may be involved in the control of prolactin secretion in teleosts: (1) inhibitory control from the hypothalamus presumably mediated by aminergic fibers, (2) direct stimulation due to decreasing plasma osmotic pressure, (3) negative feedback by either prolactin itself or increased plasma sodium level, and (4) stimulation by circulating estrogen.

The multiple actions of prolactin among the vertebrates have been summarized recently by Nicoll and Bern (1971). In at least some euryhaline fishes, there seems no doubt that prolactin is the most important hormone regulating hydromineral metabolism in freshwater environment (see reviews by Ball, 1969; Ensor and Ball,

1972; Lam, 1972; Utida et al., 1972; Johnson, 1973). Detailed cytological changes of the prolactin cells in some euryhaline teleost fishes have been described in relation to environmental salinity (Ball and Baker, 1969; Olivereau, 1969; Sage and Bern, 1971; Schreibman et al., 1973; Holmes and Ball, 1974). Recently, disk electrophoresis has been used to demonstrate various pituitary hormones, and the use of electrophoresis coupled with a densitometric procedure has

’ Some of the material contained in this paper was presented at the VII International Symposium on Comparative Endocrinology, Tsavo, Kenya, in July 1974. 166 Copyright 0 1975 by Academic Press, Inc. All tights of reproduction in any form reserved.

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been validated (Nicoll et al., 1969; Yanai and Nagasawa, 1969) for prolactin determinations. This technique has been used in Poecilia latipinna (Ball and Ingleton, 1973; Ingleton et al., 1973), Anguilla anguifla (Ingleton et a/., 1973), and Tilapia mossambica (Clarke, 1973; Zambrano et al., 1974) to demonstrate the amount of prolactin present in the pituitary or secreted into incubation or culture media. Pituitary transplantation and the use of various drugs such as reserpine and 6hydroxydopamine (6-HODA) indicate that the hypothalamus exerts an inhibitory effect upon the prolactin cells in some fishes (for reviews, see Olivereau, 1969; Ball and Baker, 1969; Ball et al., 1972; Peter, 1973; Holmes and Ball, 1974). There is another mechanism which controls prolactin secretion in some teieosts. Sage ( 1968) found in Xiphophorus that prolactin cells cultured for 2 days degranulate more rapidly in a dilute medium than in a normal medium. More recently, Ingleton et al. (1973) and Zambrano et al. (1974) demonstrated in Poecilia latipinna and Tilapia mossambica, respectively, that incubated prolactin cells are directly responsive to changes in the osmotic pressure of the surrounding medium. Gillichthys mirabiiis, a euryhaline seawater gobiid fish, is an excellent subject for the study of possible aminergic influences on prolactin secretion because of the direct innervation of the prolactin cells by type B fibers from the hypothalamus (Zambrano et al., 1972; Nagahama et al., 1974). Moreover, the prolactin cells of Gillichthys exhibit dramatic responses to changes in external salinity (Nagahama et al., 1973). In the present study, therefore, this fish was used to correlate changes in the cytology of the prolactin cells with those in pituitary prolactin content as determined by disk gel electrophoresis, in response to external salinity and to treatment with various drugs affecting cat-

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echolamine

levels. In addition, Tifapia a euryhaline freshwater cichlid fish, was used to investigate in vitro secretion by the pituitary exposed to various incubation media; a single Tilapia pituitary incubated for 5-6 hr secretes enough prolactin to be determined by disk electrophoresis, providing an excellent model system. mossambica,

MATERIALS

AND

METHODS

Animals. Gillichrhys mirabilis, ranging from 20 to 40 g in body weight, were obtained commercially from south San Francisco Bay. The fish were acclimated for at least 1 week in lo-gallon aquaria containing natural seawater that was continuously aerated and filtered. During the experiments, fish were kept at approximately 14°C under artificial light (12-hr light/l 2-hr dark) and were fed live brine shrimp. The acclimated fish were transferred to aquaria containing water of various salinities (see Results). Tilapia mossumbica, ranging from 9 to 15 cm in body length, were taken from a freshwater pond at the University of California, Berkeley. Most fish providing pituitaries for short-term incubation were taken directly from the pond. However, two groups of about 30 fish were acclimated in the laboratory at 26 ? 1°C in freshwater and 33% seawater aquaria for about 4 months under 12-hr photoperiod (light: 0800-2000). The fish were fed five times per week with tropical fish food (Tetramin; Kordon Corp., Hayward, CA). Aquarium water was continuously aerated and filtered. Injections. Two milligrams of 6-HODA (Regis Co., Chicago, IL) were dissolved in 1 ml 0.1% ascorbic acid, adjusted to pH 5.5 with hydrochloric acid. The solution was prepared immediately before use and kept on ice. Half the Gillichthys. including both seawater- and 5% seawater-adapted fish, were given three intracisternal injections, 24 hr apart, of 100 pg 6-HODA/fish, while the remaining fish were injected with the ascorbic acid vehicle alone to serve as controls. Eight daily intraperitoneal injections of either reserpine (100 pglfish) or L-DOPA (100 pglfish) were given to both seawater- and 5% seawateradapted fish. Controls were injected in the same manner with an equal volume of the carrier solution alone. Fifty milligrams of estradiol- 17p were mixed in 50 ml saline after grinding with a small amount of gum arabic. Seawater Gillichthys were given intraperitoneal injections of estradiol- 17p for 19 days at a daily dose of 100 pg/fish. Controls were injected with the vehicle alone. In vitro incubation. Tilupiu were killed by decapita-

168

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tion. Pituitaries were removed and preincubated in isosmotic medium (Na+, 145 meq/liter: 300 mOsm) for 1 hr. Incubations were done in small vials (0.9-cm i.d., 4.5 cm in length) containing 0.2 ml medium. For the study of release, the pituitary glands were incubated in isosmotic medium containing 2 &i/4,53H-t-leucine (specific activity 60 Ci/mmole; Schwartz BioResearch Inc., Orangeburg, NY) for 1 hr. After rinsing with isosmotic medium, the glands were incubated in various media for 5-6 hr. For the study of synthesis, the glands were continuously incubated in various media for 5-6 hr. Incubation media were derived from that previously described for freshwater Tilupia (Colombo et al., 1972) by altering the concentration of sodium chloride. The hyposmotic medium was 250 mOsm (Na+, 126 meq/liter); the hyperosmotic medium was 350 mOsm (Nat, 170 meq/liter). In one experiment, mannitol was added to the medium containing 126 meqlliter Na+ to bring the osmotic pressure to about 350 mOsm, the same as that of the hyperosmotic medium. Dopamine hydrochloride (Sigma, St. Louis, MO) was added to hyposmotic media (0.1 fig or 1 pg10.2 ml). Before use the incubation media were gassed with 95% 0,/j% CO,. During the incubation period, each vial was capped with a small balloon inflated with 95% 0,/S% CO, and incubated in a Dubnoff shaker at 26°C. Gel electrophoresis. Polyacrylamide gels were prepared by using the solutions outlined by Davis (1964). Seven percent acrylamide gels were made in 3-mm (i.d.) glass tubes. The glass tubes were sealed on one end with parahlm, placed in a holder, and filled with gel to a height of about 5.5 cm (1.0 ml). After polymerization, a l-cm column of large-pore spacer gel was placed on top of the running gel. Pituitaries were homogenized in large-pore gel (0.2 ml), and the incubation medium was mixed with largepore gel without sucrose solution. Tissue homogenates or media were layered on top of the spacer gel. After filling the tubes with Tris-glycine buffer, the gel tubes were allowed to polymerize before connecting them with the electrophoresis apparatus. Both chambers were filled with buffer (6 g Trizma Base, 28.8 g glycinelliter), a few drops of bromphenol blue tracking dye were placed in the upper chamber, and the current was regulated at 1.5-2 mA/gel tube throughout the run. The power supply used was either a Heathkit lP-32 or Buchler 3-l 155. A watercooled chamber was used to keep gels at a constant temperature of about 6°C. Running gels at cold temperature gave much better band definition. The run was allowed to continue until the tracking dye had reached an etched mark on the glass tube. At the termination of the run the gels were removed, the tracking dye front was marked with India ink, and the gels were placed in aniline blue-black stain overnight, destained the following day, and stored in 7% acetic

ET AL.

acid. Photographs and optical density readings were taken within a few days after destaining. For measuring radioactivity of the prolactin band, the appropriate slice from the gels was removed and put in 0.5 ml 30% H,O, in a scintillation vial and warmed at 50-55°C overnight. The dissolved gel slices were mixed with 15 ml of scintillation fluid (8 g BBOT. 160 g naphthalene, 1200 ml toluene, and 800 ml 2-ethoxyethanol). Radioactivity was counted in a liquid scintillation counter (Packard Model 3330). Blood collecrion. At the end of the transfer experiments, each Gillichthys was measured, and after careful wiping of the caudal region, the tail was cut off at the level of the anal fin. Blood was collected from the exposed caudal artery into sodium-free heparinized capillary tubes. After centrifugation the plasma was analyzed for sodium in a Perkin-Elmer atomic absorption spectrophotometer. Cytology. The pituitary glands from Gillichrhys and Tilapia were fixed for light and electron microscopy as described previously (Nagahama et al.. 1973). Granule numbers and sizes of prolactin cells were recorded only on cells sectioned through the nucleus. Thirty prolactin cells from at least three animals were assessed by measuring the major diameter of each cell. Statistical evaluation. All data were analyzed for significance using the Student t test.

RESULTS Gillichthys

mirabilis

Prolactin Cells and Their Neurosecretory Innervation

The

cytology of prolactin cells of has been described in detail (Zambrano, 1970b; Nagahama et al., 1973). In Gillichthys, the prolactin cells present in the rostra1 pars distalis (RPD) are the only cell type to show marked cytological changes in relation to external salinity (Nagahama et al., 1973). The prolactin cells of seawater Gillichthys have relatively large numbers of dense, membrane-bound, round to oval secretory granules with an average diameter of 140 nm (90-230 nm). The nuclei are irregular and lack a prominent nucleolus. Cell organelles such as rough endoplasmic reticulum (ER), Golgi apparatus and mitochondria are poorly developed. Two Gillichthys

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169

types of neurosecretory axons innervate the RPD of Gillichthys. Type B fibers containing large dense-cored or granulated vesicles (LGV), 90-100 nm in diameter, make direct contact with prolactin cells. On the other hand, type A fibers containing granules measuring 150- 160 nm in diameter, are found only in the proximity of ACTH cells. Identijication of the Prolactin by Gel Electrophoresis

Band

A preliminary experiment was designed to detect the prolactin band after electrophoresis. The prolactin cells in Gillichthys form a distinct homogeneous mass at the rostra1 end of the gland, the RPD (Nagahama et al., 1973). With a dissecting microscope the mass of prolactin cells can easily be distinguished from the rest of the gland. Pituitaries of five seawater Gillichthys were divided into RPD, proximal pars distalis (PPD), and pars intermedia (PI). No prominent band was found in the RPD, but one prominent band with a mobility of 0.46 was found in the PPD, suggestive of growth hormone. The second experiment was done using 5% seawater-adapted fish in which more prolactin can be expected when compared with seawater fish. A homogenate of 10 RPD from 5% seawater-adapted fish was used per column. One prominent band with a mobility of 0.12 was found in the RPD (Fig. 1). Bioassay of this band yellowing activity revealed Gillichthys (Sage, 1970). The optical density of the stained band was proportional to the number of Gillichthys RPD used. Changes in Prolactin Cells, Pituitary Prolactin Content, and Plasma Sodium Concentration in Transfer Experiments a. Seawater to fresh water. A previous paper described the detailed changes of prolactin cells and plasma sodium concentrations after transfer of seawater

FIG. 1. Polyacrylamide gel electrophoresis mirabilis. itary regions of seawater Gillichlhys

of pitu-

Gillichthys directly to fresh water (Nagahama et al., 1973). In the present study,

changes of pituitary prolactin content were studied during similar transfer experiments. Forty-five seawater Gillichthys were separated into three groups: one group was sacrificed as control, while the others were transferred directly to fresh water and killed 10 and 24 hr later. Ten hours after the transfer, pituitary prolactin content was reduced to about 60% of seawater level and 24 hr later, the content was still lower than the seawater level (Fig. 2). b. Seawater to fresh water to seawater.

In order to investigate the changes of plasma sodium concentration and prolactin cells during the transfer from a hypotonic to a hypertonic environment, 33 seawater Gillichthys were transferred directly to fresh water and five fish were killed after 7 days in fresh water. At least five animals were killed 0, 3, 6, 24, and 72 hr after retransfer to seawater. In addition, six seawater fish were killed at the same time.

170

NAGAHAMA

0 30

ET

meq/liter. During the period from 3 to 72 hr after retransfer to seawater after 7 days in fresh water, the plasma sodium concentrations gradually increased, rising to 148 meq/liter 72 hr after transfer. In animals kept for 7 days in fresh water, the prolactin cells contained fewer secretory granules. The rough ER and Golgi apparatus were well developed. Seventy-two hours after retransfer to seawater, the prolactin cells had relatively large numbers of secretory granules in the peripheral cytoplasm (Fig. 4). The cells still contained well-developed Golgi apparatuses with many profiles suggestive of new formation of secretory granules, and rough ER. For the study of pituitary prolactin content during this transfer experiment, 60 seawater Gillichthys were separated into four groups (seawater control, 3 and 5 days in fresh water, and 3 days in fresh water followed by 2 days in seawater). The pituitary prolactin content of animals 3 and

r

: -

i

FIG. 2. Effect of transfer from seawater to fresh water on pituitary prolactin content ( 15 pooled pituitaries in each group) in Gillichthys.

The results of plasma sodium determination are shown in Fig. 3. Seven days after transfer to fresh water the plasma sodium level showed a dramatic fall to 104

7

160

-

150

-

AL.

140.

tz E +-

130-

P z 6 a

120-

110-

100-

11 5

5

7daysFW

7doysFW

SW (3hfSl

FIG.

7 ‘do’

SW 16 hrs)

dw

I24

SW hrs)

SW 172 hrs)

3. Effect of transfer from fresh water to seawater on plasma sodium concentration

in

Gillichfhys.

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FIG. 4. Prolactin cells of Gillichthys in seawater for 2 days after 3 days in fresh water. Relatively large numbers of secretory granules as well as well-developed rough ER and Golgi apparatus (G). Compare : with prolactin cells of Gillichtlzys 5 days after transfer from seawater to fresh water (Nagahama et al.. 1973, p. 160). x 12,000.

5 days after transfer to fresh water was depressed to 47 and 40% of seawater levels, respectively. In contrast, the prolactin content of animals 3 days in fresh water followed by 2 days in sea water was elevated to 130% over seawater levels (Fig. 5). c. Seawater to 5% seawater. The cytoprolactin logical changes of Gifkhthys cells during the period from 3 hr to 3 days after transfer to 5% seawater are similar to those seen after direct transfer to fresh water: rapid disappearance of secretory granules and well-developed cell organelles such as rough ER, Golgi apparatus, and mitochondria. However, during the interval from 10 to 21 days after transfer to 5% seawater, the prolactin cells appeared differently from the cells after direct transfer to fresh water. The cells contained numerous larger secretory granules as well as well-developed cell organelles (Fig. 6).

0.3c

0.2c z. C E $ 0 ” 8

O.IC

0.c

S V

lJ 3dOYS FW

5dOYS 3ovys F.W

A_.

FW

followed

2 doye

by SW

FIG. 5. Effect of transfer from fresh water to seawater on pituitary prolactin content (15 pooled pituitaries in each group) in Gillichthys.

172

NAGAHAMA

ET AL.

FIG. 6. Prolactin cells of Gillichthys 3 weeks after transfer from seawater to 5% seawater. Large number of secretory granules and prominent rough ER. Compare with prolactin cells of seawater Gillichthys (Nagahama et al., 1973, p. 156). ~13,000.

Plasma sodium concentrations are shown in Fig. 7. During the first 3 days after transfer, the plasma sodium values were not significantly different from those after transfer to fresh water. However, 10

II

S.W. 3.

I

I

I

Ihr 3hrs

days after transfer to 5% seawater, the plasma sodium concentration was again elevated to 149 meq/liter, which is in contrast with 110 meq/liter after transfer to fresh water.

c

6 hrs

12 hrs

rt

24 hrs.

”

4

3 days

I

5days

IOdoys

FIG. 7. Plasma sodium concentration in Gillichthys transferred directly from seawater to fresh water or 5% seawater.

CONTROL

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In order to study the effect of transfer from seawater to 5% seawater on pituitary prolactin content in Gillichfhys, 60 seawater fish were separated into four groups. Two groups of 15 fish served as seawater controls, whereas the other two groups were transferred to 5% seawater and killed after 3 days. By 3 days after transfer, pituitary prolactin content had fallen significantly to about one-half the seawater level (Fig. 8). Other seawater Gillichthys were separated into four groups: one served as control, while the others were transferred to 5% seawater and killed after 1, 6, or 21 days (Fig. 9). One day after transfer, pituitary prolactin content was reduced to about 42% of seawater level. Even 6 days after transfer, the content remained lower than normal seawater level. However, by 21 days after transfer, the prolactin content had recovered to almost normal seawater level. Changes in Prolactin Cells after Pituitary Autotransplantation a. One to 4 weeks in seawater. Nagahama et al. (1974) demonstrated that

FIG. 8. Effect of transfer from seawater to 5% seawater on pituitary prolactin content ( 15 pooled pituitaries in each of four groups) in Gillichthys.

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IN TELEOSTS 0.3OL

f

). 0.20 -

k D 0 .s 0” O.lO-

0.0 5% S.W.

5% SW.

5% s w

FIG. 9. Effect of transfer from seawater to 5% seawater on pituitary prolactin content (15 pooled pituitaries in each group) in Gillichthys.

the prolactin cells of the autografted pituitary exhibited cytological signs of active synthesis and release of secretion. In the present study, the number of secretory granules and cell size at various times after autotransplantation were recorded (Fig. 10). One to 2 weeks after autotransplantation, the number of secretory granules was reduced to about 9- 10% of that seen in normal seawater fish (P < 0.01). However, by 4 weeks after autotransplantation, the granule number had recovered to within 70% of normal seawater fish (P < 0.05). During these periods, the size

and diameter of auFIG. 10. Granule tografted prolactin cells in Gillichthys in seawater.

174 of prolactin changes.

NAGAHAMA

cells did not show significant

b. Transfer of autografted fish into fresh water for 3 days. When Gillichthys

bearing an autografted pituitary were transferred directly to fresh water, exocytosis of the secretory granules from the prolactin cells was frequently observed (Nagahama et al., 1974). In the present study, more detailed observations were carried out and the results are shown in Fig. 11. The number of granules in prolactin cells after transfer of autografted fish to fresh water for 3 days decreased to approximately 25% of the number seen in grafts from fish maintained in seawater for 4 weeks (P < 0.01). Again, there were no significant changes in the size of prolactin cells. Eflects of Various Drugs on Prolactin Cells and Plasma Sodium Concentration of Seawater and 5% Seawater Gillichthys a. Seawater fish. In seawater Gillichthys treated with vehicle alone, the

prolactin cells had relatively large numbers of secretory granules and poorly developed cell organelles typical of seawater Gillichthys (Nagahama et al., 1973). Pro-

ET AL.

lactin cells of seawater fish given three daily injections of 6-HODA contained larger numbers of secretory granules (Fig. 12). Rough ER and Golgi apparatus were well developed. In seawater fish which received eight daily injections of reserpine, many mitotic figures were found in the RPD; these cells possessed the ultrastructural features of prolactin cells (Fig. 13). Many prolactin cells exhibited cytological features indicating high activity. Prolactin cells treated with L-DOPA exhibited a slight increase in granule number. After both 6-HODA and reserpine treatments, the plasma sodium levels of seawater fish showed a significant elevation (P < 0.05) (Fig. 14). b. 5% Seawater jish. In 5% seawateradapted fish treated with vehicle alone, the prolactin cells contained numerous larger secretory granules as well as welldeveloped cell organelles. After three injections of 6-HODA, the prolactin cells contained fewer secretory granules and prominent rough ER. In fishes given eight daily injections of reserpine, a few mitotic figures were found in the RPD, although their frequencies were markedly lower than in seawater fishes which received reserpine. The cytoplasmic granulation of the prolactin cells in the L-DOPA-injected 5% seawater Gillichthys appeared to exhibit a stronger stainability than in the vehicle-injected 5% seawater fish. No alterations of plasma sodium concentrations were found in 5% seawater fish after 6-HODA, reserpine, or L-DOPA. Changes in Prolactin Cells after Injection of Estradiol-I 7/3

In order to study the effect of estrogen the prolactin cells, 16 seawater Giflichthys were injected with estradiol17/3. After the treatment, the prolactin cells exhibited marked activation. The cells contained relatively large numbers of secretory granules and well-developed rough ER, Golgi apparatus, and mion

4 weeks S.W Auto-T

3 dayso:W,Auto-T 4 weeks in S W

FIG. 11. Granule number and diameter of autografted prolactin cells in Gillichthys in fresh water for 3 days after 4 weeks in seawater.

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FIG,. 12. Prolactin cells of seawater Gillichthys after 6-HODA injection. Large number of secretory granules and Krell-developed rough ER and Golgi apparatus (G). x 15,000. after reserpine treatment. X 15.000. FIG . 13. Mitosis of prolactin cells of seawater Gillichthys

176

NAGAHAMA

Vehicle

FIG. 14. Effect adapted Gillichthys.

of 6-HODA

6-HODA

and reserpine

6 r iW Reserplne

on plasma

tochondria (Fig. 15). A few mitotic figures were found. The ACTH cells were also activated by the treatment with estradiol17p, as shown by increase in cell size and well-developed organelles (Fig. 16). Both prolactin cells and ACTH cells treated with vehicle alone showed no cytological changes compared with those of intact seawater controls. There was no significant difference in plasma sodium concentration between estradiol- 17p- and vehicle-treated groups. Tilapia mossambica Prolactin Cells and Their Neurosecretory Innervation The cytology of the prolactin cells of Tilapia mossambica has been described in detail (Dharmamba and Nishioka, 1968; Bern et al., 1974). The prolactin cells of freshwater Tilapia include numerous membrane-bound secretory granules, from 150 to 3 10 nm in diameter. The nuclei are round in shape and have a prominent nucleolus. Well-developed rough ER and Golgi apparatus occur (Fig. 17). In contrast, the prolactin cells from 33% seawater-adapted fish showed ultrastructural features of low secretory activity, as indicated by fewer small secretory

ET AL.

6 YASW Vehicle

sodium

6 5%SW B-HOOP,

concentration

6 S%SW Aeserpine

in seawater-

and 5% seawater-

granules, poorly developed cell organelles, dilated perinuclear cisternae of the rough ER, and small Golgi apparatuses and mitochondria (Fig. 18). Processes of the neurohypophysis are clearly separated from the prolactin cells and ACTH cells by a distinct basement membrane. Only rarely were axons seen beyond the basal lamina-stellate cell process layer and therefore only rarely made direct membrane-to-membrane contact with prolactin and ACTH cells. Three kinds of neurosecretory axons are found in the neurohypophysis innervating the dorsal areas of the rostral lobe (types A, B, and C) (Bern et al., 1974). Identification of the Prolactin by Gel Electrophoresis

Band

Pituitaries of five freshwater Tilapia were divided into three parts (RPD, PPD, and PI). After electrophoresis, a prominent band with a mobility of 0.40 was found in the RPD. Bioassay of this band revealed Gillichthys-yellowing activity. Clarke (1973) has also demonstrated a positive reaction for sodium-retaining activity of this band. The second major band with a mobility of 0.38 was found in the proximal pars distalis and is suggestive of growth hormone.

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FIGi. 15. Prolactin cells of seawater Gillichrhys after estradiol- 17p treatment. Well-developed rough ER and Golgi apparatus (G). X 15,000. FIG;. 16. ACTH cells of seawater Gillichthys after estradiol-17P treatment. Well-developed rough ER. x lS,( 100.

NAGAHAMA

FIG i. 17. Prolactin ER. t 3olgi apparatus FK ;. 18. Prolactin those of freshwater

ET

AL.

cells of freshwater Tilapiu. 1 arge number of sec5’etory granules and well-developed r ough (G), and mitochondria CM). x 15.000. cells of 33% seawater-adapted Tihpiu. Smaller size and fewer secretory granules than fish. Poorly developed rough ER. X 15,000.

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In Vitro Incubation of Tilapia Pituitary a. Comparison of prolactin synthesis by freshwater- and 33% seawater-adapted

Tilapia. In order to compare the synthetic activity of the prolactin cells of freshwater and 33% seawater Ti/apia, three pituitaries from each group were incubated in isosmotic medium with 3H-leucine for 5 hr. The results are shown in Fig. 19. About 4 times more 3H-leucine was incorporated into the prolactin cells of freshwater fish than into those of 33% seawater fish (P < 0.01). b. Effect of various media on release of newly synthesized prolactin in vitro. To de-

termine whether low sodium or low osmotic pressure directly influences release of prolactin from the pituitary, pituitaries were incubated in three different media (Fig. 20). About 10 times more newly synthesized prolactin was released into hyposmotic medium (122 meq/liter Na+, 240 mOsm) than into hyperosmotic media, prepared by addition of NaCl alone (170 meqlliter Na+, 355 mOsm) or by addition of mannitol to low Na+ medium (122 meq/liter Na+, 355 mOsm) (P < 0.01). There was no significant difference in the amount of newly synthesized prolactin released between the latter two groups.

FW

13

b s w

19. Comparison of prolactin freshwater- and 33% seawater-adapted itary incubated with 3H-leucine in vitro. FIG.

synthesis by Ti!u/k pitu-

Osmollc pressure mOsm

355

240

355

FIG. 20. Effect of osmotic pressure on release of newly synthesized prolactin by Ti/q& pituitary in

vitro.

Epon-embedded 1-vm sections were made to compare the cytological difference between pituitaries incubated in the three different media for 6 hr. In the RPD incubated in media with high sodium and high osmotic pressure or low sodium and high osmotic pressure, the prolactin cells contained many fine secretory granules. In electron micrographs, these prolactin cells had numerous electron-dense secretory granules and well-developed cell organelles (Fig. 21). No exocytoses were observed. These cytological features of prolactin cells are similar to those of normal freshwater Tilapia. On the other hand, in the pituitaries incubated in the medium with low sodium and low osmotic pressure, the prolactin cells in the dorsal half of the RPD near the neurohypophysial processes were degranulated. In electron micrographs, the cells contained few secretory granules (Fig. 22). The rough ER occupied most of the cytoplasm. Golgi apparatuses were also prominent, and frequent exocytoses were seen. The ACTH cells did not show any cytological differences. c. Effects of dopamine on the release of newly synthesized prolactin by Tilapia pituitary in vitro. In order to study the ef-

180

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ET AL.

cells of Tilapin incubated for 6 hr in hyperosmotic media. FIG . 21. Prolactin les. X 12,000. FIG (. 22. Prolactin cells of Tilnpia incubated for 6 hr in hyposmotic medium. granu les, well-developed rough ER and many exocytoses (arrows). x 12,000.

Large Small

numbers

of seer ‘etory

number

of seer ‘etory

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181

than that of Tilapia prolactin reported by Clarke (1973), probably owing to our use of lower running temperature. In addition, the possible Gillichthys prolactin has a considerably lower mobility (0.12), which corresponds closely to the value reported for prolactin from Poecilia latipinna (0.16) (Ball and Ingleton, 1973). These Rfs obtained from fish prolactins are considerably lower than those of tetrapod prolactins (Nicoll and Nichols, 1971). -

Adaptive Alterations of Prolactin Secretion in Relation to External Salinity

FIG. 23. In vitro effect of dopamine on the release of newly synthesized prolactin by Tilapia pituitary.

fects of dopamine on the release of prolactin, a total of 15 pituitaries was incubated in hyposmotic media with or without dopamine for 5 hr. The addition of dopamine to hyposmotic media (0.1 or 1.0 pg dopamine/0.2 ml) caused about 30% decrease in the amount of labeled prolactin released into the incubation medium (P < 0.05) (Fig. 23). DISCUSSION Gel Electrophoresis of Fish Prolactin

The present study has clearly demonstrated that in Gillichthys there is a good correlation between morphological data and pituitary prolactin content measured by disk electrophoresis coupled with a densitometric procedure. The first applications of polyacrylamide gel electrophoresis to teleostean prolactin determination were those of Knight et al. (1970) and Chadwick (1970), who demonstrated a Rf of 0.46 for eel (Anguilla anguilla) prolactin and a Rf of 0.80 for flounder (Pleuronecks Jesus) prolactin, respectively. Recently, Clarke (1973) reported Rf’s for Tilapia (0.47) and Cichlasoma (0.42) prolactin. In the present study, however, the Rf of Tilapia prolactin was slightly lower

Many morphological studies have demonstrated that the prolactin cells in various teleosts respond to environmental salinity and usually are more active in fresh water than in seawater (see Olivereau, 1969; Ball and Baker, 1969; Sage and Bern, 1971; Schreibman et al., 1973; Nagahama, 1973; Holmes and Ball, 1974). In a previous paper (Nagahama et al., 1973), we reported that within 3 hr after transfer of Gillichthys from seawater to fresh water, the prolactin cells exhibited definite functional activation. This is also true after transfer of Gillichthys to 5% seawater. This dramatic functional activation of Gillichthys prolactin cells during the early stages of adaptation to lower salinities is similar to those reported from other transfer experiments using euryhaline and stenohaline freshwater fish (Fund&u heteroclitus, Ball and Pickford, 1964; Poecilia latipinna, Ball, 1969; Oryzias latipes, Nagahama and Yamamoto, 1971; Xiphophorus maculatus, Holtzman and Schreibman, 197 1). The pituitary prolactin content of Gillichthys dropped to 60% of the seawater level 6 hr after transfer to fresh water. Ball and Ingleton (1973) measured a 75% decrease in the prolactin content of Poecilia latipinna pituitaries 18 hr after transfer from 33% seawater to fresh water. Clarke (1973) also reported in Tilapia mossambica that pituitary prolactin concentration was reduced signifi-

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cantly 6 hr after transfer from seawater to fresh water. Pituitary prolactin levels in Tilapia gvahami were reduced by more than 50% 1 day after transfer from Lake Magadi water (osmolality 650 mOsm/liter) to fresh water (Clarke, 1973). When taken together, these cytological and physiological data indicate that the rate of prolactin release is higher than the synthetic rate during the first stage of transfer from a hypertonic environment to a hypotonic environment. The plasma sodium concentration of Gillichthys showed a sharp decline for the first 3 hr after transfer to a hypotonic environment. However, 6- 24 hr after the transfer, there was a slight overall increase in plasma sodium concentration when compared to the initial drop, which suggests that the circulating levels of prolactin were elevated by this time. Most Gillichthys cannot survive in fresh water for more than 10 days, but can live in 5% seawater indefinitely (Nagahama, unpublished data). These differences in survival are of great interest when evaluated in terms of the present morphological and physiological data. Ten days after transfer to fresh water, the plasma sodium concentration was about 108 meq/liter and the prolactin cells contained few secretory granules. In contrast, 10 days after transfer to 5% seawater, the plasma sodium concentration was about 150 meqlliter and the prolactin cells contained relatively large numbers of secretory granules. Moreover, the pituitary prolactin content recovered to the normal seawater level 3 weeks after transfer to 5% seawater. Thus, in Gillichthys the quantity of secreted prolactin seems to be sufficient to allow survival in 5% seawater. However, this pattern is different from that of the euryhaline freshwater fish. Nagahama and Yamamoto (197 1) demonstrated that 5 days after transfer of seawater Oryzias latipes to fresh water, the prolactin cells began to recover and 10 days later, the cells showed about the

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same cytological features as are seen in freshwater fish. In Poecilia latipinncr by 72 hr after transfer from 33% seawater to fresh water, the prolactin cells had attained the typical freshwater morphology and the plasma sodium concentration also increased to the freshwater-adapted level (Ball. 1969), although the pituitary prolactin content was still only 50% that of the 33% seawater controls (Ball and Ingleton, 1973). In Tilapia mossambica after 1 week in fresh water, the pituitary prolactin level recovered to that of seawater fish (Clarke, 1973). Thus, in euryhaline freshwater fish transferred from a hypertonic environment to fresh water, synthesis and release of prolactin appear to come into balance within a few days. When Gillichthys after 7 days in fresh water are transferred to seawater, release and synthesis become dissociated. Seventy-two hours after transfer, the prolactin cells had larger numbers of secretory granules than the fish in seawater or in fresh water for 7 days, and their synthetic organelles were still more developed than in prolactin cells from seawater fish. This suggests that, although the release of hormone had stopped, synthesis of the hormone was still occurring, which may explain the elevated pituitary prolactin content of fish 2 days after transfer from fresh water to seawater. In P. latipinna, however, 72 hr after transfer from fresh water to seawater, synthesis and release were reduced to the level seen in 33% seawateradapted fish (Ball and Ingleton, 1973). Moreover, Holtzman and Schreibman (1972) found in Xiphophorus that the prolactin cells 72 hr after transfer from fresh water to 33% seawater were slightly smaller than in freshwater fish, and most of them lose their staining intensity. Thus, when Gillichthys is transferred from one environmental salinity to another, the processes of synthesis and release of pituitary prolactin exhibit quick changes in response to surrounding medium. However, the

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pituitary prolactin level allows no conclusions about the rates of synthesis and release. Reliable data on prolactin concentrations in fish plasma are needed (cf. McKeown and van Overbeeke, 1972). In addition, there is little information on how Gillichthys prolactin acts on various target organs in osmoregulation, although data on urinary bladder function are forthcoming (Doneen and Nagahama, 1973; Doneen and Bern, 1974). Direct Control of Prolactin due to Osmotic Changes

Secretion

The dramatic changes in plasma sodium concentration prior to cytological alterations of the prolactin cells after transfer of Gillichthys to a hyposmotic environment and after reverse transfer suggest that the secretion of prolactin is directly influenced by the alterations of plasma ion concentration. Sage ( 1968) reported that in Xiphophorus the prolactin cells in organ culture respond directly to osmotic stimuli. In the present study, in fact, when Gillichthys bearing an autografted pituitary were transferred directly to fresh water, the secretory granules of the grafted prolactin cells were decreased by 25% from the initial number, which may suggest that change in environmental salinity has a direct stimulating effect on the pituitary to increase prolactin release, although there is always the possibility of some residual control of the grafted pituitary by the hypothalamus. Immediately after transfer to fresh water, the fish loses plasma sodium ions and the plasma osmolality is also decreased. The increased prolactin secretion, then, could be due to the low Na+ concentration in particular or to low osmotic pressure in general. In Tilapia approximately 10 times more newly synthesized prolactin was released into the hyposmotic medium than into the hyperosmotic media, whether the latter was achieved by increasing Na+ alone or by addition of mannitol to a low Na+ medium. In a previous paper (Naga-

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et al., 1974), we concluded that Gillichthys prolactin cells may be directly

controlled by plasma Na+ concentration based on the results of transfer of seawater Gillichthys bearing an autograft into fresh water. When taken together, however, the cytological and physiological data from both Gillichthys and Tilapia indicate that the prolactin cells in those fishes respond directly to changes in osmotic pressure rather than to changes in sodium concentration, as has already been demonstrated in Poecilia latipinna and Angailla anguilla (Ingleton et al., 1973). Zambrano et al. (1974) also reported in Tilapia that pituitaries incubated in hyposmotic medium showed a reduction in prolactin content and released a greater amount of bioassayable prolactin into the medium. In the present study, we did not examine whether the hyposmotic medium can stimulate uptake of labeled leucine during short-term incubation. However, electron micrographs of the prolactin cells incubated in the hyposmotic medium revealed a greater activation of synthetic cellular organelles, which suggests that this medium stimulates synthesis as well. Zambrano et al. (1974) found by electronmicroscope autoradiography that incorporation of leucine was stimulated by hyposmotic medium. However, in P. latipinna, the overall rate of synthesis by freshwater pituitaries for the 12-hr incubation period was not significantly affected, although a difference in prolactin synthesis was indicated by cytological examination (Ingleton et al., 1973). Inhibitory Control of Prolactin by the Hypothalamus

Secretion

There is another factor controlling prolactin secretion in teleost fishes. Histological studies of pituitary transplants in Poecilia formosa (Ball et al., 1965; Olivereau and Ball, 1966), Anguilla anguilla (Olivereau and Dimovska, 1969), and Gasterosteus aculeatus (Leatherland,

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1970) have shown that the prolactin cells remain active when removed from hypothalamic connections. Moreover, Chidambaram et al. (1972) found that the catfish tctalurus melas with an autotransplanted pituitary maintains about normal plasma Na+ levels in fresh water, whereas hypophysectomized catfish show marked reduction of plasma Na+ and usually die within a few days. However, Leatherland and Ensor (1973) found no differences in the appearance of prolactin cells between goldfish with autotransplanted pituitaries and sham-operated control animals. With the appearance of the prolactin cells, the effects measured in the eel by Olivereau (197 1b) and Olivereau and Lemoine (197 1) indicate that less than normal amounts of prolactin are secreted by the transplanted pituitary, which may suggest the presence of a prolactin-releasing factor (PRF). However, evidence for a PRF in the eel is contradictory (Olivereau, 197la; Olivereau and Dimovska, 1969). The grafted prolactin cells of seawater Gillichthys exhibited obvious activation with a significant elevation of plasma sodium concentration, strongly indicative of the presence of a prolactin-inhibiting factor from the hypothalamus. Recently, Leatherland and Ensor (1974) provided evidence for a factor inhibiting prolactin cells in the goldfish hypothalamus, judging from the decrease in plasma sodium after treatment with a crude hypothalamic extract. Peter and McKeown ( 1974) provided more convincing evidence for the hypothalamic control of prolactin secretion in Carassius auratus, demonstrating by the heterologous radioimmunoassay that lesions in the pars lateralis of the nucleus lateralis tuberis caused a significant increase in serum prolactin, but had no effect on pituitary prolactin levels. They concluded that this region of the hypothalamus is the source of a factor that inhibits prolactin release from the pituitary. Thus, from the cumulative evidence, it seems certain that

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teleost prolactin cells are regulated by an inhibiting factor from the hypothalamus. There is evidence indicating that the inhibitory control over teleost prolactin cells may be mediated by neurosecretory fibers from the hypothalamus which make direct contact with the adenohypophysial cells or end upon a basement membrane adjacent to these cells (Zambrano, 1972; Peter, 1973). In Gillichthys, type B axons containing LGV, which possibly originate in the nucleus lateralis tuberis, traverse the basement membrane, and make direct contact with the prolactin cells of the rostra1 lobe, suggesting that catecholamine-containing axons may play an important role in the regulation of their secretory activity (Zambrano et al., 1972; Nagahama et al., 1974). In the present study, the prolactin cells became extremely active after reserpine treatment, which stimulates prolactin secretion in mammals, probably by reducing catecholamines. At the same time, the plasma sodium concentration was elevated to a significant level. Similar activation of prolactin cells after reserpine was reported in Heteropneustes fossilis (Sundararaj and Nayyar, 1972), Mugil platanus (Zambrano, 1972), and Leuciscus rutilus (Bige ef al., 1974). However, in the eel, Olivereau ( 197 lc) could find no evidence, using several criteria, for the release of prolactin following reserpine treatment. After 6-HODA treatment, type B fibers exhibit various stages of degenerative changes in Gillichthys mirabilis (Zambrano et al., 1972), Gasterosteus aculeatus (Foll&ius, 1972), and Tilapia mossambica (Zambrano et al., 1973). The injection of 6-HODA caused a definite functional activation of prolactin cells in seawater fish, with evidence of an elevation of plasma sodium concentration. However, neither the prolactin cells nor plasma sodium concentration showed any clear alterations after 6-HODA treatment of 5% seawater fish, which suggests that the prolactin cells of 5% seawater fish

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were already fully activated. In the present study, after intraperitoneal injections of L-DOPA, an immediate precursor of dopamine, the prolactin cells of Gillichthys were more granulated and appeared less active than the cells of fish injected with the vehicle alone. This result is similar to the report by Olivereau and Lemoine (1973), who found in Anguillu anguilla that L-DOPA injections promoted granule retention and significant nuclear atrophy, the nucleus migrating to the cell periphery where the ER was no longer discernible. Our results from pituitary transplantation, and from 6-HODA and L-DOPA treatments, taken together, support the probable transport of a prolactin-inhibiting factor from the hypothalamus by type B fibers containing catecholamines. In mammals, considerable evidence supports the view that hypothalamic catecholamines play a role in the regulation of prolactin secretion (see Meites and Clemens, 1972). However, it is not certain whether this control is direct or indirect. On the one hand, it has been clearly demonstrated that catecholamines can directly inhibit the secretion of prolactin in vitro (MacLeod, 1969; Birge et al., 1970; Quijada ef al., 1974; MacLeod and Lehmeyer, 1974). On the other hand, hypothalamic catecholamines may be acting as neurotransmitters which release PIF from other neurons, because perfusion of the pituitary with catecholamine does not inhibit release whereas intraventricular infusion causes marked inhibition of prolactin secretion (Kamberi et al., 1971). In our experiments on Tilupiu, we demonstrated that dopamine may have a direct inhibiting action on the pituitary gland. However, it is not absolutely certain that this action occurs directly on the prolactin cells because some neurohypophysial elements are invariably included with the explanted pituitary. Dopamine could cause PIF release from the severed neurohypophysial axonal endings. In addition, it remains to be deter-

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mined which catecholamine is actually involved in control of prolactin secretion in Tilupiu. Further studies on Tilupiu pituitary in progress in our laboratory should provide more detailed information of the control by catecholamines of prolactin secretion. Although Zambrano (1970a) described fluorescent cells in the NLT in Gillichthys, Swanson (personal communication) has been unable to confirm this claim. He finds no fluorescence indicative of monoamines in the area of the RPD or in the NLT of Gillichrhys, even after treatment with drugs to raise the concentration of catecholamines, results which are consistent with those of Honma and Honma (1970) and Ekengren (1973). A nonfluorescent amine, such as octopamine (Zimmerman, personal communication), could be the active agent in this innervatory system, and this is being investigated. Other Possible Mechanisms Proluctin Secretion

Controlling

Nagahama ef al. (1974) demonstrated that in Gillichthys in situ prolactin cells became inactive 40-50 days after homotransplantation. In contrast, grafted prolactin cells exhibited activation. These results seem to indicate that the high circulating levels of prolactin secreted by grafted prolactin cells may act directly or indirectly on the prolactin cells (a “short” feedback mechanism). The elevated plasma sodium resulting from the pituitary transplant could also act to control prolactin secretion. Prolactin cells of seawater Gillichrhys were activated by treatment with estradiol-17p in agreement with results in mammals, where estrogen stimulates pituitary prolactin release by direct action on the pituitary and via the hypothalamus by reducing PIF content (see Meites and Clemens, 1972). In the present study, however, plasma sodium concentration showed no significant elevation after estrogen treatment. Inasmuch as ACTH cells are also cytologically activated by the

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FIG. 24. Possible. factors controlling prolactin secretion in teleosts, based on studies of Gillichthys and

Tilapia.

estrogen treatment, it is possible that the absence of an elevation of plasma sodium concentration after estrogen may be due to antagonism of the increased prolactin by increased cortisol; such antagonism is demonstrable in the hormonal control of the teleost urinary bladder (Johnson, 1973 ; Doneen and Bern, 1974). Figure 24 summarizes the possible factors which may control the secretion of prolactin in teleosts. This diagram is slightly modified from the previous one (Nagahama et al., 1974) by inclusion of information from the present study of both Gillichthys and Tilapia. At least four basic mechanisms seem to be involved; further clarification will require hypothalamic manipulations, as well as determination of plasma prolactin levels. ACKNOWLEDGMENTS We thank Dr. Kaoru Kohmoto and Dr. Tetsuya Hirano for much helpful discussion, Mr. John Underhill for photographic assistance, Ms. Ann Mos for microtechnical assistance, and Ms. Emily Reid for preparation of the graphs. This study was aided by NIH Grant CA-05388 and NSF Grant GB 35239X.

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Leuciscus ncrillrs. 1. The rostra1 pars distalis and its innervation. Actn Zoo/. 55, 25-45. Ball, J. N. (1969). Prolactin and osmoregulation in teleost fishes: a review. Gen. Comp. Endocrinol. Suppl. 2, 10-25. Ball. J. N., and Baker, B. I. (1969). The pituitary gland: anatomy and histophysiology. In “Fish Physiology” (W. S. Hoar and D. J. Randall, eds.). Vol. 2, pp. l- 110. Academic Press, New York. Ball, J. N., Baker, B. I., Olivereau, M., and Peter, R. E. (1972). Investigations on hypothalamic control of adenohypophysial functions in teleost fishes. Gen. Comp. Endocrinol. Suppl. 3, 1 l-21. Ball, J. N., and Ingleton, P. M. (1973). Adaptive variations in prolactin secretion in relation to external salinity in the teleost Poecilia Iutipinnu. Gen. Comp. Endocrinol. 20, 3 12-325. Ball, J. N., Olivereau, M., Slither, A. M., and Kallman, K. D. (1965). Functional capacity of ectopic pituitary transplants in the teleost Poeciliu formosu, with a comparative discussion on the transplanted pituitary. Phi/. Trans. Roy. Sot.

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Ball, J. N., and Pickford, G. E. (1964). Pituitary cytology and freshwater adaptation in the killifish, Fundulus heteroclitus. Anat. Rec. 148, 358. Bern, H. A., Nishioka, R. S., and Nagahama, Y. (1974). The relationship between nerve fibers and adenohypophysial cell types in the cichlid teleost Tilapia mossumbica. In “Manfred Gabe Memorial Volume” (L. Arvy, ed.) (in press). Birge, C. A., Jacobs, L. S., Hammer, C. T., and Daughaday, W. H. (1970). Catecholamine inhibition of prolactin secretion by isolated rat adenohypophyses. Endocrinology 86, 120-l 30. Chadwick, A. (1970). Pigeon crop sac-stimulating activity in the pituitary of the flounder (Pleuronectes Jesus).

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Chidambaram, S., Meyer, R. K., and Hasler, A. D. (1972). Effects of hypophysectomy, pituitary autografts, prolactin, temperature and salinity of the medium on survival and natremia in the bullhead, Ictalurus melus. Comp. Biochem. Physiol. A 43, 443-457.

Clarke, W. C. (1973). Disc-electrophoretic identification of prolactin in the cichlid teleosts Tilapiu and Cichlasoma and densitometric measurement of its concentration in Tilapiu pituitaries during salinity transfer experiments. Can. J. Zoo/. 51, 687-695. Colombo, L., Bern, H. A., Pieprzyk, J., and Johnson, D. W. (1972). Corticosteroidogenesis in vitro by the head kidney of Tilapia mossambica (Cichlidae, Teleostei). Endocrinology 91, 450-462. Davis, B. J. (1964). Disc electrophoresis. II. Method

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and application to human serum proteins. Ann. N. Y. Acad. Sci. 121,404-427. Dharmamba, M., and Nishioka, R.’ S. (1968). Response of “prolactin-secreting” cells of Tilapia mossambica to environmental salinity. Gen. Comp. Endocrinol. 10, 409-420. Doneen, B. A., and Bern, H. A. (1974). In vitro effects of prolactin and cortisol on water permeability of the urinary bladder of the teleost Gillichthys mirabilis. J. Exp. 2001. 187, 173-179. Doneen, B. A., and Nagahama, Y. (1973). Roles of proiactin and cortisol in osmoregulatory functions of the urinary bladder of the euryhaline teleost, Gillichthys mirabilis. Amer. 2001. 13, 1278. Ekengren, B. (1973). The nucleus preopticus and the nucleus lateralis tuberis in the roach, Leuciscus rutilus. Z. Zellforsch. Mikrosk. Anat. 140, 369-388. Ensor, D. M., and Ball, J. N. (1972). Prolactin and osmoregulation in fishes. Fed. Proc. Fed. Amer. Sot. Exp. Biol. 31, 161.5-1623. Follenius, E. (1972). Cytologie fine de la digentrescence des fibres aminergiques intrahypophysaires chez le Poisson telbosteen Gasterosteus aculeatus apres traitement par la 6-hydroxydopamine. 2. Zellforsch. Mikrosk. Anat. 128, 69-82. Holmes, R. L., and Ball, J. N. (1974). “The Pituitary Gland: A Comparative Account,” pp. l-397. Cambridge University Press, London. Holtzman, S., and Schreibman, M. P. (1971). Histophysiological responses of the prolactin cell to changes in the environmental salinity of the freshwater teleost Xiphophorus maculatus. Amer. Zoo/. 11, 653-654. Holtzman, S., and Schreibman, M. P. (1972). Morphological changes in the “prolactin” cell of the freshwater teleost, Xiphophorus helierii, in salt water. J. Exp. Zool. 180, 187-196. Honma, S., and Honma, Y. (1970). Histochemical demonstration of monoamines in the hypothalamus of the lamprey and ice-goby. Bull. Jap. Sot. Sci. Fish. 36, 125-134. Ingleton, P. M., Baker, B. I., and Ball, J. N. (1973). Secretion of prolactin and growth hormone by teleost pituitaries in vitro. 1. Effect of sodium concentration and osmotic pressure during shortterm incubations. J. Comp. Physiol. 87, 3 17-328. Johnson, D. W. (1973). Endocrine control of hydromineral balance in teleosts. Amer. Zool. 13, 799-818. Kamberi, I. A., Mical, R. S., and Porter, J. C. (1971). Effect of anterior pituitary perfusion and intraventricular injection of catecholamines on prolactin release. Endocrinology 88, 1012-1020. Knight, P. J., Ingleton, P. M., Ball, J. N., and Han-

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Olivereau, M., and Dimovska, A. (1969). Prolactinsecreting cells in the autotransplanted pituitary of the eel. Cert. Comp. Endocrinol. 13, 523-524. Olivereau, M., and Lemoine, A. M. (1971). Teneur en acide N-acetyl-neuraminique de la peau chez 1’Anguille apres autotransplantation de l’hypophyse. Z. Vergl. Physiol. 73, 44-52. Olivereau, M., and Lemoine, A. M. (1973). Action de la L-Dopa sur la secretion de prolactine chez 1’AnguiIle. C. R. Acad. Sci. Paris 276, 132% 1327. Peter, R. E. (1973). Neuroendocrinology of teleosts. Amer.

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