GENERAL AND COMPARATIVE ENDOCRINOLOGY 36, 48-58 (1978)

Cytological Changes in Prolactin, ACTH, and Growth Hormone Cells of the Pituitary Gland of Fungitim

pungitius L. in Response to Increased Environmental Salinities

MICHAEL BENJAMIN

Department of Anatomy, University College, Cardiff CFl lXL, United Kingdom

Accepted May 19, 1978

Adult, nine-spined sticklebacks, Pungitius pungitius, were gradually tmnsferred from fresh water to dilute and full strength seawater for periods of 10 hr, 3, 6, 9, and 21 days. Light microscopy studies of the pituitary gland revealed unusual and marked changes in the prolactin, ACTH, and GH cells of the adenohypophysis. In at least some animals from all experimental groups there were intercellular cysts among the prolactin cells. These became so large in the 21-day group as virtually to obliterate the RPD. In view of the enormous size of some cysts and the great diminution in prolactin cell numbers, they were presumed to be signs of decreased secretory activity. Transfer of animals from fresh water to dilute and full strength seawater was generally associated with decreased nuclear diameters in prolactin, ACTH, and GH cells. The GH cells were markedly degranulated in all animals from the 21-day seawater group. Evidently the euryhaline members of the Gasterosteidae show widely differing pituitary responses to altered ambient salinities.

In many teleosts the pituitary is impor- tant in osmo(iono)-regulation (Ball, 1969a, b; Ball and Baker, 1969; Olivereau and Ball, 1970; Schreibman et al., 1973). Cytological changes in adenohypophyseal cells (par- ticularly prolactin and ACTH cells) in fish collected from, or transferred to, different ambient salinities, have been described by numerous authors (e.g., Abraham, 1971- Mugil cephalus; Holtzman and Schreibman, 1971-Xiphophorus maculatus; Leather- land, 1972-Carassius auratus; Naga- hama et al., 1973-Gillichthys mirabilis and Platichthys stellatus; Leatherland et al., 1974-various Tilapia species). However, within the family Gasterosteidae, attention has been restricted entirely to Gasterosteus. In the migratory form of Gasterosteus (form trachurus), several adenohypophyseal cells respond markedly to changes in ambient salinity (Leatherland, 197Oa, b; Honma et al., 1976). In a briefly reported study of the freshwater form

(leiurus), Oreg and Chadwick (1975) described pronounced changes in the prolactin cells in fish adapted to seawater for 3 weeks. However, Benjamin (1974) and Benjamin and Ireland (1974) could find no obvious changes in the prolactin and ACTH cells in the freshwater form adapted to 70% seawater for shorter periods.

Although all the Gasterosteidae are euryhaline, there are marked differences in their salinity tolerance and habitat. They may be confined to fresh water or to seawa- ter or live in both (Wheeler, 1969). Nelson (1968) reported a decrease in salinity toler- ance with increasing utilization of fresh- water habitats in the following order: Spinachia, Apeltes, Gaderosteus, Pun- gitius, and Culaea. The present paper deals with the response of three adenohypoph- yseal cells (prolactin, ACTH, and growth- hormone-secreting cells) in the pituitary of the nine-spined (also called ten-spined) stickleback, Pungitius pungitius, to in-

48 0016.6480/78/0361-0048$01.00/O Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any fom reserved.

SALINITY EFFECTS ON PUNGITIUS PITUITARY 49

creased environmental salinities. This stickleback is locally distributed compared with G. aculeatus (Wheeler, 1969) and in- deed has been reported absent from Wales (Bagenal, 1973). Although generally found in fresh water, Pmgitim does live in salt waters (Forbes, 1897; Heuts, 1943; Rawson and Moore, 1944, Dartnall, 1973).

MATERIALS AND METHODS

Adult Pungitius puagitius (body length, 30-45 mm) of both sexes were collected by hand-netting from weed-choked reens at Marshfield near Cardiff in January 1976. Examination of the caudal peduncle and lateral bony plates showed the population con- tamed Pungitius pungitius pungitius and Pmgitius pmgitius luevis (Wootton, 1976). Water samples were taken with each collection and titrated against a standard silver nitrate solution (Tait, 1972). The salin- ity of these samples never exceeded 0.29 g/lner.

The fish were maintained in the laboratory in lo- line plastic tanks at 16 * lo in dechlorinated tap water for at least 1 week before experiments. They were fed daily on “Tetramin” or live Tubifex. Twenty-five fish were transferred to 33% seawater, and tive were killed after 10 hr. The remainder were transferred to 50% seawater and then to 66% sea water (after 24 hr) and finally to full strength seawater (after 48 hr). Groups of five of these fish were killed, 3, 6, 9 and 21 days from the beginning of the experi- ment. Five fish were maintained in tap water throughout and served as controls.

The brains with pituitaries attached were dissected out and fixed in Bouin-Hollande for 24 hr. Six- micrometer paraffin wax sections were stained with alcian blue-PAS-orange G (Kerr, 1965). Nuclear diameters were measured on prints at a magnification of x 815 by taking the average of the major and minor axes of 20 nuclei per cell type per gland. Prolactin cell nuclei in a standard area were counted to assess cell size; the fewer the nuclei, the larger the cells. Sig- nificant differences were assessed by Student’s t test.

RESULTS General Morphology of the Pituitary

Gland in Freshwater Animals

The general structure of the pituitary in adult Pmgitim pungitius is shown dia- grammatically in Fig. 1. It is connected to the brain by two short stalks that form a hollow cylinder in three dimensions, Four regions of the pituitary could be easily dis-

FIG. 1. Diagrammatic representation of the pituitary gland of Pungitius (median sag&al section) to show the distribution of the adenohypophyseai cell types. Rostra1 pars distahs (RPD): (0) ACTH ceils; (A) prolactin cells. Proximal pars distahs (PPD): (0) growth hormone cells; (@I basophils, gonadotropbs, and thyrotrophs. Pars intermedia (PI); (A) PAS- positive cell: ( x ) PAS-negative cell.

tinguished; the neurohypophysis (IV), the rostral pa,rs distalis (RPD), the proximal pars distalis (PPD), and the pars i~terrn~di~ (PI). The prolactin cells of the RPD were unusually small. They were closely packed and not arranged in follicles (though excep- tions were noted). Their nuclei were rouml or oval, though occasionally deeply invagj- nated (Fig. 2). The staining reaction of the cytoplasm. varied. Although the prolactin cells of one animal stained strongly with orange G, in all others they stained poorly (Table 1). The ACTH cells of the RFD we& arranged in a narrow band dorsal to’ ,the prolactin cells. With alcian blue-PA3+- orange G staining they were chromophobic cells with round or oval nuclei? slightly smaller than those of neighbouring pralactm cells (Figs. 2 and 5). Intermingled am~n/g the prolactin cells were numerous stellate or “nongranulated cells.” The whole RPD had a well-developed blood supply, with blood vessels running ‘beneath, the cormec- tive tissue capsule and penetrating between endocrine cells.

In the PPD, intensely acidophilic growth hormone cells (GH cells) were locate@ mainly dorsally and the ventral portions were occupied by large, coarsely grand-

50 MICHAEL BENJAMIN

FIG. 2. Pituitary gland of an animal maintained in fresh water throughout the experiment. Both the prolac- tin (PRL) and GH cells of this animal are strongly acidophilic, and the ACTH cells are conspicuous yet chromophobic. The ventral region of the PPD is occupied predominantly by large, coarsely granulated basophils (arrows). CT = connective tissue capsule. Alcian blue-PAS-orange G. Blue-green filter.

lated, basophilic cells (Fig. 2). The GH cells were round or pear-shaped and larger than the prolactin cells, though with smaller and eccentrically located nuclei (Figs. 2 and 6). Within the PPD it was impossible to distin- guish gonadotrophs from thyrotrophs using the alcian blue-PAS-orange G staining method. Basophilic cells typical of the PPD were often found among the prolactin cells of the RPD.

The PI contained two cell types. One was PAS- and orange-G-positive; the other was chromophobic. Both the PPD and PI had a well-developed blood supply.

Prolactin cells. Within IO hr of transfer of the fish to one-third seawater, the nuclei were significantly smaller than in animals kept in fresh water (Fig. 4) and the cyto- plasm stained more heavily with orange G. In two animals there were small cysts among the prolactin cells, near blood ves- sels. Both the cells and their nuclei con- tinued to decrease in size throughout the experiment (Figs. 3 and 4). In all sub- sequent experimental groups at least some animals had cysts in the prolactin zone of the RPD (Table 1). Possible stages in their

formation are shown in Figs. 7-10. A few cysts became so large as almost to obliter- ate the whole RPD (Fig. 10). Such cysts had diameters of 200 pm and were bounded in parts by only a thin, connective tissue membrane (Fig. 10). Fragments of alcian blue- and PAS-positive material were fre- quently found within the cysts, but the lat- ter contained neither healthy nor degenerat- ing cells (Fig. 9). In serial sections, small blood vessels could be traced through the middle of even the largest cysts. Although these larger cysts were usually lined by a PAS-positive membrane (Figs. 8-lo), this was not always true of smaller ones (Fig. 7). The heavy cytoplasmic staining of prolactin cells in animals killed 10 hr after transfer was maintained for 6 days, but in animals from the 9- and 21-day seawater groups, most cells were poorly stained (Table 1).

ACTH cells. ACTH cell nuclei decreased in size throughout the experiment (Fig. 5) and were inconspicuous 21 days after the initial transfer (Fig. 10). With alcian blue- PAS-orange G staining, they were chromophobic in all experimental and con- trol groups.

TABL

E I

SUMM

ARY

or= T

HE

INCI

DENC

E OF

CY

ST

FORM

ATIO

N IN

TH

E RP

D AN

D OF

TH

E ST

AINI

NG

REAC

XION

S OF

PR

OLAC

TIN

AND

GH

CELL

S FR

OM

ANIM

ALS

TRAN

SFER

RED

ro SE

AWA~

ER~

Expe

rimen

tal

grou

pb

Prola

ctin

cells

FW

Contr

ol Po

orly

gran

ulated

(4)

; no

cy

sts

Heav

ily

gran

ulated

(I)

; no

cy

sts

IO-hr

SW

Po

orly

gran

ulated

(I)

; ve

ry sm

all

cysts

(1)

He

avily

gr

anula

ted

(4);

very

small

cy

sts

(1)

3-day

SW

Po

orly

gran

ulated

(1)

; no

cy

sts

Mode

ratel

y gr

anula

ted

(1);

large

cy

sts

(1)

Heav

ily.gr

anula

ted

(3);

iarge

cy

sts

(3)

&day

SW

He

avily

gr

anula

ted

(5);

small

cy

sts

(i)

9-day

SW

Po

orly

gran

ulated

(3)

; sm

all

cyst

(I);

large

cy

st (I)

Mode

ratel

y gr

anula

ted

(I);

no

cysts

He

avily

gr

anula

ted

(1);

no

cysts

-

21-d

ay

SW

Poor

ly gia

iimat

ed

(5);

small

cy

sts

(4);

large

cy

st (I)

--~-

U Fig

ures

in

pare

nthes

es

indica

te

the

numb

er

of an

imals

. b

FW

= fre

shwa

ter,

SW

= se

awate

r.

Grow

th ho

rmon

e ce

lls

Heav

ily

gran

ulated

(5)

Heav

ily

gran

ulated

(5)

Mode

ratel

y gr

anula

ted

(1)

Heav

ily

gran

ulated

(4)

Heav

ily

gran

ulated

(5)

Poor

ly gr

amda

ted

(3)

Mode

ratel

y gr

anula

ted

(1)

Heav

ily

gran

ulated

(1)

Poor

ly gr

anula

ted

(5)

WI

52 MICHAEL BENJAMIN

FW SW SW SW SW SW loh sd 6d 9d md

FIG. 3. Changes in the number of prolactin cell nuclei per unit area in animals adapted to seawater or dilute seawater. Mean values ? SEM. Probabilities are comparisons with FW controls; asterisks (*) indi- cate p < 0.05.

Growth hormone cells. The nuclei were much smaller 21 days after initial transfer to one-third seawater and slightly smaller in all other experimental groups (Fig. 6). After 9 days the GH cells of several animals were poorly granulated (Table l), and by 21 days they were virtually chromophobic and

--I F-l FIG. 4 PRL CELLS

difficult to identify in all animals (Fig. 10). Cysts .were not found among ACTH cells - 4l

or in the PPD.

DISCUSSION

Numerous cytological studies of teleost pituitaries have shown that prolactin cells in many species respond markedly to al- tered environmental salinities (Dharmamba and Nishioka, 1968; Olivereau, 1969; Ball and Baker, 1969; Schreibman et ul., 1973). The cells are usually more active in animals adapted to fresh water than to seawater or dilute seawater. Inactive prolactin cells have shrunken nuclei, inconspicuous nuc- leoli, and poorly granulated cytoplasm. In- deed such is the morphology of prolactin cells from the 21-day seawater group of Pungitius. However, what is most unusual in the current work are the cysts that form between prolactin cells.

Abraham et al. (1977) found enlarged in- tercellular spaces between prolactin cells in Aphcmius dispar, but these were very small compared with those in Pungitius and as-

FIG. 5. ACTH CELLS

FIG. 6. GH CELLS

. . . . . . . . . . . . . . . . , , , , ,

. . . . . . . . . . .

. . . . . . ““’

. . . . . . . , . , ,

. . . . . . . . . . .

. . . . . . ““’

. . . . . .

. . . . . .

:W s2Y

FIGS. 4-6. Changes in the nuclear diameter of prolactin (PRL) (Fig. 4) ACTH (Fig. S), and GH cells (Fig. 6) in animals adapted to seawater or dilute sea- water. Mean values + SEM. Probabilities are com- parisons with FW controls. Asterisk (*), p < 0.05; double asterisks (**), J’ < 0.01.

sociated with increased cell activity. Honma and Yoshie (1974) studied seasonal changes in the prolactin cells of the ayu, Plecoglossus altivelis , and noted several “lacunae” between the mass of prolactin cells in one post-spawning fish. In Pungitzks,

SALINITY EFFECTS ON PUNGITIUS PITUITARY 53

FIG. 7. Pars distalis of a stickleback killed 3 days after initial transfer to dilute seawater~ Note the early signs of cyst formation among the well granulated prolactin cells (arrows). These cysts are not lined by a connective tissue membrane such as that surrounding the whole gland (CT). The ACTH cells are conspicuous, and the GH cells are heavily granulated. Alcian blue-PAS-orange G. Blue-green filter.

FIG. 8. Pars distalis of a stickleback killed 3 days after initial transfer to dilute seawater, showing a shgbtly later stage of cyst formation than that in Fig. 7. The cyst is lined by a distinct membrane (M) and is adjacent to a small blood vessel (BV). Alcian blue-PAS-orange G. Blue-green filter.

54 MICHAEL BENJAMIN

FIG. 9. Pituitary gland of a stickleback killed 9 days after initial transfer to dilute seawater. Note the large cyst dominating the RPD. The cyst contains only fragments of PAS- and alcian blue-positive material (arrows). The GH cells of this animal are still heavily granulated yet slightly smaller than in FW controls (Fig. 2). The remaining prolactin cells (PRL) are poorly granulated. The ACTH cell nuclei are less distinct and smaller than in FW controls, BV = blood vessel. Alcian blue-PAS-orange G. Blue-green filter.

FIG. 10. Pituitary gland of a stickleback killed 21 days after initial transfer to dilute seawater. Note the enormous cyst almost obliterating the RPD. In places the cyst is maintained only by the connective tissue capsule surrounding the whole gland (CT). The remaining prolactin cells (PRL) are small and indistinct. The GH cells are also small and almost completely degranulated; the ACTH cells are similarly poorly defined. Alcian blue-PAS-orange G. Blue-green filter.

SALINITY EFFECTS ON PUNGITIUS PITUITARY 55

the cysts are assumed to be signs of inactiv- ity, simply because the larger ones must develop at the expense of prolactin cells. It is interesting that the cysts in Pz4agifius can almost obliterate the prolactin zone, be- cause we do not understand the role of pro- lactin in seawater fish. Perhaps in Pungitius at least, there is no need at all for prolactin in seawater-adapted fish.

IIow these cysts originate is unknown. Several prolactin cells may die and disin- tegrate or portions of the connective tissue capsule may invaginate the RPD and be- come pinched off. However, not all the small cysts are lined by a membrane. It seems likely that the subsequent develop- ment of cysts must involve considerable cell death in view of the enormous size of some cysts and the drastic reduction in pro- lactin cell numbers. Thus it is puzzling that there were no signs of degenerating cells. Blood vessels passing through even the largest cysts were not destroyed along with the prolactin cells presumably because they supplied regions that remained intact.

The staining reaction of prolactin cells in freshwater Pungitius varied. Whereas in most animals the cells were chromophobic, in one animal they were heavily granulated. Jose and Sathyanesan (1977) reported a similar variability in the prolactin cells of Lubeo rohita . Chromophobic prolactin cells in freshwater animals were also de- scribed by Baker et al. (1974) in Heterop- neustes fossilis. In nine-spined sticklebacks transferred to seawater, the immediate pro- lactin cell response was for an increase in cytoplasmic staining. It was only after longer periods in seawater (2 I days) that the cytoplasm was once more degranulated. These findings can be explained if seawater adaptation differentially affects hormone synthesis and release, as has been suggested for Poecilia lutipinna (Ball and Ingleton, 1973), Anguillu anguilla (Aler, 1971; Qlivereau and Lemoine, 1972), and Puecilia reticulata (Ethridge and Benjamin, 1977).

Evidently the behaviour of prolactin ceils to altered ambient salinities is very different in the three members of the Gasterosteidae now investigated. In the migratory stickleback, G. aculeatus form trachwus, the prolactin cells are stimulated when this fish migrates from the sea into fresh water (Leatherland, 197Oa; Honma et al., 1976). According to Benjamin and Ireland (1974), the freshwater stickleback, G. aculeatus form leiurus, shows no pronounced changes in its prolactin cells when fish are transferred to 70% seawater for 1 week. Nevertheless it is possible that long-term adaptation to seawater does inactivate t prolactin cells as Benjamin (1973) and Oreg and Chadwick (197.5) suggest. In neither form of the three-spined stickleback have cysts been described in the RPD. It remains of interest to investigate other Gasteros- teidae, i.e., Culaea inconstans, Apeltes quadrucus, and Spinuchia spinachia.

The role of ACTH in osmo(iono)- regulation in teleosts is unclear. There have been many conflicting reports about changes in ACTH cells with changes in am= bient salinity. In Pungitius it seems the ceils may be less active in animals transfused from fresh water to seawater. In G. aculeatus form trachurus the response of ACTH cells to salinity transfers varies with the season (Leatherland, 197Oa). In fish transferred from fresh water to ‘seawater in spring, the response was similar to that of P. pungitius. In fish transferred from sea- water to fresh water in winter, the cells showed no response (Leatherland, 197Oa). The ACTH cells in the freshwater stickleback, G. aculeatus form /eiwus, did not resend markedly to changes in en- vironmental salinity (Benjamin and Ireland, 1974). Nevertheless their interrenal cells were markedly hypertrophied. ACTH cells in Anguilla are activated when f&h enter the sea from fresh water (Olivereau and 1970), and in Mugil cephalus Q~ey h trophy in transfer from seawater to fresh water (Abraham, 1971).

56 MICHAEL BENJAMIN

On the evidence of nuclear size and cyto- plasmic stainability, it is tentatively con- cluded that high salinities decrease the ac- tivity of GH cells in Pungifiu,s. Such a pro- nounced change is unusual; in many tele- osts the GH cells are singularly um-espon- sive to most experimental procedures. Their identification as growth-hormone- secreting cells largely depends on the cir- cumstantial evidence that other pituitary hormones have been allocated to other sites (Holmes and Ball, 1974). However, evi- dence is beginning to accumulate to suggest that in some teleosts, growth hormone is implicated in osmo(iono)-regulation. Pickford et aZ. (1965) showed that growth hormone promotes the survival of hy- pophysectomized Fund&s heteroclitus in fresh water. Whether this was a specific effect of growth hormone or was caused solely by prolactin impurities in the prepa- ration unfortunately is not clear. According to Olivereau and Ball (1970), the GH cells of eels are less active after transfer of ani- mals from fresh water to seawater. Likewise work in vitro has shown that growth hormone release from eel pituitaries is stimulated by a low sodium medium (Ingleton et aZ., 1973).

In view of the considerable variation shown by different species in the impor- tance of prolactin in the electrolyte economy of fish (Holmes and Ball, 1974), it is to be expected that the role of growth hormone in osmo(iono)-regulation will also vary. Indeed one of the earliest studies (Smith, 1956) showed growth hormone im- proved the survival of brown trout in sea- water. More recently Komourdjian et af. (1976) and Clarke et al. (1977) reported that growth hormone promotes the adaptation of two salmonids (S. salar and 0. nerka, respectively) to seawater. Nagahama et al. (1977) have postulated an osmoregulatory role for growth hormone in young coho salmon and found that GH cells in yearling fish acclimated to seawater were more numerous and more granulated than in

freshwater fish. They presumed that growth hormone secretion was greater in seawater.

In the migratory stickleback, G. aculeatus form trachurus, GH cells re- spond to natural salinity changes and are most active in seawater fish caught just be- fore the spring migration (Leatherland, 197Ob). The differing involvements of growth hormone in osmo(iono)-regulation in salmon, eels, and migratory sticklebacks should be considered in the light of their natural migrations. The pattern of GH cell response in nonmigratory fish may not be comparable, as the cells cannot pre-adapt to salinity changes. Where growth hormone is involved in osmo(iono)-regulation in such fish, perhaps the GH cells are more active in the natural environment (at least in fully adapted, rather than adapting fish); other- wise the young fish may not grow properly. Whether young Pungitius maintained in- definitely in seawater can grow normally is an important question that is currently being investigated. It is interesting that young coho salmon prematurely transferred to seawater were stunted, yet apparently had more numerous and heavily granulated GH cells (Clarke and Nagahama, 1977).

Finally, it must be admitted~that with any pituitary cell, an altered morphology ac- companying a change in the ambient salin- ity of the fish can only suggest an osmo(iono)-regulatory function. A cell could respond to salinity changes without being osmo(iono)-regulatory.

ACKNOWLEDGMENTS I would like to thank Mr. P. F. Hire for his photo-

graphic assistance.

REFERENCES Abraham, M. (1971). The ultrastructure of the ceil

types and of the neurosecretory innervation in the pituitary of Mugil W&/W L. from fresh water, the sea, and a hypersaline lagoon. 1. The rostra1 pars distalis. Gerz. CWZ~. Emfocrind. 17, 334- 350.

Abraham, M., Dinari-Lavie, V., and Lotan, R. (1977). The pituitary of Aphunius &qxzr (Rfippeh) from hypersahne marshes and freshwater. II. Uhra-

SALINITY EFFECTS GN PUNGITIUS PITUITARY Y-l

structure of the rostra1 pars distalis. Ceil Tissue Res. 179> 317-330.

4ler, G. M, (1971). The study of prolactin in the pitui- tary gland of the Atlantic eel (Anguillu unguilhz) and the Atlantic salmon (S&no salar) by im- munofluorescence technique. Acta Zool. (Stock- holm] 52, 145-l%.

Bagenal, T. B. (1973). “Identification of British Fishes,” p. 160. Hulton, Amersham.

Baker, B. I., Keshavanath, P., and Sundararaj, B. I. (1974). The cell-types in the adenohypophysis of the Indian catfish ZZeteropneustes fossilis (BIoch). Cell Tissue Res. 148, 347-358.

Ball, J. N. (1969a). Prolactin (fish prolactin or paralac- tin) and growth hormone. Zn “Fish Physiology” (W. S. Hoar and D. J. Randall, eds.), Vol. 2, pp. 207-240. Academic Press, New York and Lon- don.

Ball, J. N. (1969b). Prolactin and osmoregulation in teleost fishes: A review. Gen. Camp. Endocrinol. Suppl. 2, 10-2.5.

Ball, J. N., and Baker, B. I. (1969). The pituitary gland: anatomy and histophysiology. Zrr “Fish Physiology” (W. S. Hoar and D. J. Randall, eds.), Vol. 2, pp. l-110. Academic Press, New York and London.

Ball, J. N., and Ingleton, P. M. (1973). Adaptive var- iations in prolactin secretion in relation to external salinity in the teleost Poecilia latipirma. Gen. Comp. Endocrmol. 20, 312-325.

Benjamin, M. (1973). “Studies on the Pituitary Gland and Saccus Vasculosus of Teleost Fishes.” Ph.D. thesis, University of Wales.

Benjamin, M. (1974). Seasonal changes in the prolactin cell of the pituitary gland of the freshwater stickleback, Gasterosteus aculeatus form leiurus. Cell Tissue Res. 152, 93-102.

Benjamin, M., and Ireland, M. P. (1974). The ACTH- interrenal axis in the freshwater stickleback, Gas- terosteus aculeatus form leiurus. Cell Tissue Res. 155, 10.5-l 1.5.

Clarke, W. C., Farmer, S. W., and Hartwell, K. M. (1977). Effect of teleost pituitary growth hormone on growth of Tilapia mossambica and on growth and seawater adaptation of sockeye salmon (on- corhynchus nerkcz). Gen. Comp. Endocrinoi. 33, 174- 178.

Clarke W. C., and Nagahama, Y. (1977). Effect of premature transfer to sea water on growth and morphology of the pituitary, thyroid, pancreas, and interrenal in juvenile coho salmon (C&z- corhynchus kisutch). Canad. J. 2001. 55, 1620- 1630.

Dartnall, H. J. G. (1973). Parasites of the nine-spined stickleback Pungitius pzmgitius (L). J. Fish Biol. 5, 505-509.

Dharmamba, M., and Nishioka, R. S. (1968). Re-

sponse of “prolactin-secreting” cells of Tilapia mossumbica to environmental salinity. Gen. Camp. Endocrinol. 10, 409-420.

Ethridge, L., and Benjamin, M. (1977). The time- sequence of response of the prolactin cells of the freshwater teleost, Poecilia reticuluta, to WI al- tered environmental salinity. Cell Tissue Res. 182, 371-382.

Forbes, H. G. (1897). Habitat of Gasterosteus purz- gitius, Linn. Bull. Liverpool h4us. 1, 24.

Heuts, M. J. (1943). La regulation osmotique chez 1’Epinochette (Pygostezzs pungitius L.). 50~. Roy. Zool. BeZg. 74, 99-105.

Holmes, R. L., and Ball, J. N. (1974). “The Pituitary Gland. A Comparative Accoum.” University Press, Cambridge.

Holtzman, S., and Schreibman, M. P. (1971). His- tophysiological responses of the prolactin cell to changes in the environmental salinity of the freshwater teleost, Xiphophorus mmzhtus. Amer. Zool. 11, 653-654.

Honma, Y., Teshigawara, H., and Chiba, A. (1976). Changes in the cells of the adenobypophysis as- sociated with the diadromous migration of the threespine stickleback, Gasterosteus aczdeatus L. Arch. Histol. Japan. 39, l-14.

Honma, Y., and Yoshie, S. (1974). Studies on the endocrine glands of a salmonoid fish, the ayu, Plecoglossus altiveiis Temminck et Scblegel. LX. Seasonal changes in the cells of the adenohypophysis, especially the prolactin prodnc- ing cells. Arch. Histol. Japan. 36, 237-250.

Ingleton, P. M., Baker, B. L, and Ball, J. N. (1973). Secretio~n of prolactin and growth hormone by teleost pituitaries in vitro. 1. Effect of sodium concentration and osmotic pressure during short- term incubations. J. Comp. Physiol. 87, 3 1’7.-?28.

Jose, T. M., and Sathyanesan, A. G. (1977). Pituitary cytology of the Indian carp Lubeo rohita @am.). Anat. Anz. 142, 410-423.

Komourdjian, M. P., Saunders, R. L., and Fenwick, J. C. (1976). The effect of porcine somatotropin on growth, and survival in seawater of Atlantic sal- mon (Salmo salar) Parr. Canad. J. Zoo/. 54, %I.- 53s.

Kerr, T. (1965). Histology of the distal lobe of the pituitary of Xenopus laevis Daudin. Gen. Camp. Endocrinol. 5, 232-240.

Leatherland, J. F. (197Oa). Seasonal variation in the structure and ultrastructure of the pituitary of the marine form (TracZzuru.r) of the threespine stickleback, Gasterosteus aculeatus L. 1. Rostra1 pars distalis. z. Zeh!forsch. 104, 301-317.

Leatherland, J. F. (19’7Ob). Seasonal variation m the strncture and ultrastructure of the pituitary gland in the marine form (Trachurus) of the threespine stickleback, Gasterosteus aczdeatzzs L. 11. Prox-

58 MICHAEL BENJAMIN

imal pars distalis and neuro-intermediate lobe. 2. Zelljorsch. 104, 3 18-336.

Leatherland, J. F. (1972). Histophysiology and inner- vation of the pituitary gland of the goldfish, Curassius aurutus L.: A light and electron micro- scope investigation. Canad. .I. Zool. SO, 835-844.

Leatherland, J. F., Ball, J. N., and Hyder, M. (1974). Structure and fine structure of the hypophyseal pars distalis in indigenous African species of the genus Tilapia. Cell Tissue Res. 149, 245- 266.

Nagahama, Y., Clarke, W. C., and Hoar, W. S. (1977). Influence of salinity on ultrastructure of the se- cretory cells of the adenohypophyseal pars dis- talis in yearling coho salmon (~~co~~~x/zus kisutch). Cunud. J. Zool. 55, 183-198.

Nagahama, Y., Nishioka, R. S., and Bern, H. A. (1973). Responses of prolactin cells of two euryhaline marine fishes, Gillichthys mirabilis and Platichthys stellatus , to environmental salinity. .Z. Zelvorsch. 136, 153-167.

Nelson, J. S. (1968). Salinity tolerance of brook sticklebacks, Culaea inconstans, freshwater ninespine sticklebacks, Pungitius pungitius, and freshwater fourspine sticklebacks, Apeltes quad- races. Canad. J. Zool. 46, 663-667.

Olivereau, M. (1969). Functional cytology of prolactin-secreting cells. Gen. Camp. Endocrinol. Suppl. 2, 32-41.

Olivereau, M., and Ball, J. N. (1970). Pituitary influ- ences on osmoregulation in teleosts. Mem. Sot. Endocrinol. 18, 57-82.

Olivereau, M., and Lemoine, A. M. (1972). Effets des variations de la salinite externe sur la tenuer en acid Ar-acetylneuraminique (ANAN) de la peau chez 1’Anguille. Modifications simultanees des cel- lules a prolactine de l’hypophyse. J. Camp. Physiol. 79, 41 l-422.

Oreg, F. B., and Chadwick, A. (1975). Cytological changes in the pituitary gland of the stickleback, Gasterosteus aculeatus, associated with hormone secretion. J. Endocrinol. 64, 1OP.

Pickford, G. E., Robertson, E. E., and Sawyer, W. H. (1965). Hypophysectomy, replacement therapy, and the tolerance of the euryhaline killifish, Fun- dulus heteroclitus, to hypotonic media. Gen. Camp. Endocrinol. 5, 160-180.

Rawson, D. S., and Moore, J. E. (1944). The saline lakes of Saskatchewan. Cunud. J. Res. 22(D), 141-201.

Schreibman, M. P., Leatherland, J. F), and McKeown, B. A. (1973). Functional morphology of the teleost pituitary gland. Amer. Zool. 13, 719-742.

Smith, D. C. W. (1956). The role of the endocrine organs in the salinity tolerance of trout. Mem. Sot. Endocrinol. 5, 83-101.

Tait, R. V. (1972). “Elements of Marine Ecology,” p. 13. Butterworths, London.

Wheeler, A. (1969). “The Fishes of the British Isles and North-West Europe,” pp. 506-511. Macmil- lan, London.

Wootton, R. J. (1976). “The Biology of the Sticklebacks,” p. 267. Academic Press, London.