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Prolactin and freshwater osmoregulation of juvenile chum Oncorhynchus keta and sockeye O. nerka salmon Neuman, H. R. 1974

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tl  PROLACTIN AND FRESHWATER OSMOREGULATION OF JUVENILE CHUM (ONCORHYNCHUS KETA) AND SOCKEYE (0. NERKA) SALMON  by H. Ross Neuman B.Sc.(Hons), University of Victoria, 1971  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Zoology We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA January, 1974  In  presenting  an  advanced  the I  Library  shall  scholarly  by h i s this  written  thesis  degree  f u r t h e r agree  for  of  this  at  it  purposes  for  may  financial  is  British  by  for  gain  Columbia  shall  the  that  not  requirements  Columbia,  I  agree  r e f e r e n c e and copying  t h e Head o f  understood  of  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  of  for extensive  be g r a n t e d  It  fulfilment of  available  permission.  Department  Date  freely  permission  representatives. thesis  partial  the U n i v e r s i t y  make  that  in  of  or  that  study.  this  thesis  my D e p a r t m e n t  copying  for  or  publication  be a l l o w e d w i t h o u t  my  ABSTRACT The possible role of prolactin in the freshwater osmoregulation of laboratory reared juvenile chum (Oncorhynchus keta) and sockeye (0_. nerka) salmon was investigated.  Pituitary cytology indicated that prolactin  cells of both species develop gradually during freshwater residence and downstream migration.  During this time the prolactin cells increase  slightly in size; the number of prolactin cell follicles also increases. Simultaneously, the intensity of cytoplasmic staining with erythrosin increases.  Alternate day injections of 5 or 15 ug/g body wt prolactin (ovine)  did not affect survival of chum fry in deionized water.  Thirty micrograms  per gram slightly increased survival while 60 ug/g decreased survival in deionized water.  Prolactin injections prolonged, to a small extent, the  survival of sockeye smolts in deionized water.  Sockeye fry suffered only  slight mortality after transfer from fresh water to deionized water. A prolactin dose of 5 ug/g did not alter this survival; however, doses of 15 ug/g or higher resulted in 40 to 70% mortality after 10 days i n deionized water.  Alternate day injections of 10 ug/g prolactin had no effect on  plasma sodium concentrations of chum fry, sockeye fry, o^ sockeye smolts after transfer from sea water to either fresh water or deionized water. It is concluded, from histological and physiological evidence, that prolactin does not play an obvious role in the freshwater osmoregulation of juvenile chum and sockeye salmon.  The possible role of prolactin in the  spawning migration of adults i s discussed.  iii TABLE OF CONTENTS Page LIST OF TABLES  v  LIST OF FIGURES  vi  ACKNOWLEDGEMENTS  >.vii  INTRODUCTION  .  1  MATERIALS AND METHODS  4  Fish  4  Experimental Temperatures and Age and Weight of Fish  4  Note on Hypophysectomy  4  Injections  ••••  6  Histological Study  6  Survival Study  .  Plasma Sodium Study  1 7  RESULTS  '  Histological Study  10 10  Prolactin cells of juvenile chum salmon......  10  Prolactin cells of juvenile chum salmon during seaward migration  11  Prolactin cells of juvenile sockeye salmon  .. 12  Prolactin cells of sockeye smolts salmon during seaward migration  12  Survival Study Plasma Sodium Study  13 . ...  15  iv Table of Contents (cont'd)  Page  DISCUSSION  27  Prolactin cells  27  Physiological effects of prolactin  29  Possible role(s) of prolactin i n adult migratory salmon  36  SUMMARY REFERENCES  .  39 41  V  LIST OF TABLES  TABLE I  Page Experimental temperatures and age and weight of fish  II  .  Electrolyte content of fresh and deionized water  5 8  vi LIST OF FIGURES FIGURE  Page  1.  Pituitary gland of a 1-month-old chum salmon  17  2.  Pituitary gland of a 6-month-old chum salmon  17  3.  Pituitary gland of a juvenile chum salmon during seaward migration  4.  17  Pituitary gland of a sockeye fry at the time of yolk absorption  .. 18  5.  Pituitary gland of a 15-month-old sockeye salmon  18  6.  Pituitary gland of a sockeye smolt during seaward migration..  18  7.  Prolactin and survival of chum fry i n deionized water  19  8.  Prolactin and median survival times of chum fry in deionized water  20  9.  Prolactin and survival of sockeye fry in deionized water  10.  Prolactin and survival of sockeye smolts in deionized water.. 22  11.  Prolactin and plasma sodium of chum fry after transfer to fresh water  12.  23  Prolactin and plasma sodium of sockeye fry after transfer to fresh water  13.  .-  24  Prolactin and plasma sodium of sockeye smolts after transfer to fresh water  14.  21  25  Prolactin and plasma sodium of chum fry, sockeye fry, and sockeye smolts after transfer to deionized water  26  vii  ACKNOWLEDGEMENTS  I would like to express my sincere appreciation to my supervisor, Dr. W. S. Hoar, for suggesting the problem and for his guidance and valuable criticism throughout the course of this study. I also wish to thank my committee members, Dr. A. M. Perks, Dr. T. G. Northcote, and Dr. P. Ford, for c r i t i c a l l y reading the manuscript. Thanks also to my lab companions, Mr. K. Khoo, Mr. K. Chan, Mr. N. Stacey, Mr. W. Marshall, and Mr. J.G. Godin for the many pleasant gatherings around a beaker of tea. Mr. K. Khoo was particularly helpful with some histological procedures and Mr. N. Stacey's constructive criticism of the manuscript is much appreciated. Dr. N. R. Liley kindly supplied laboratory space in which many of the experiments were conducted. This investigation was supported by a National Research Council Grant in aid of Research to Dr. W. S. Hoar and by N.R.C. Postgraduate Scholarships to the author.  The prolactin used in this study was donated  by Dr. R. W. Bates of the National Institutes of Health.  INTRODUCTION Prolactin is believed to play an important role in the hydromineral regulation of many teleosts.  Since Burden (1956) demonstrated the ina-  b i l i t y of hypophysectomized Fundulus heteroclitus to survive in fresh water, this phenomenon has been reported in a variety of teleost species (Schreibman and Kallman, 1966a, 1969).  On the other hand, some species  such as the goldfish, Carassius auratus (Lahlou and Sawyer, 1969) and the eel, Anguilla anguilla (Callmand, et_ al_., 1951) do not require their pituitary gland to survive in fresh water.  Studies on replacement therapy  have shown that prolactin alone promotes freshwater survival in hypophysectomized F_. heteroclitus (Burden, 1956; Pickford and Phillips, 1959; Pickford, et_ al_. , 1965) and Xiphophorus maculatus (Schreibman and Kallman, 1966a); other hypophyseal hormones or hormones of other endocrine glands are ineffective.  Further, i t should be noted that adrenocorticotropic  hormone (ACTH) as well as prolactin, prolongs survival of Gambusia sp. i n fresh water (Chambolle, 1967) as well as the marine form (trachurus) of the threespine stickleback (Gasterosteus aculeatus) in deionized water (Lam and Leatherland, 1970). Death of hypophysectomized fish in fresh water i s associated with a decline in plasma electrolytes.  The effects of prolactin on plasma elec-  trolytes have now been recorded in many species (see reviews by Ball, 1969; Lam, 1972, and Ensor and Ball, 1972).  Hypophysectomy causes a f a l l in  plasma osmolality which is restored by prolactin in F_. heteroclitus (Pickford, et a l . , 1966) and partially prevented in Tilapia mossambica (Dharmamba, 1970).  Prolactin also reduces the decline in plasma sodium  and chloride in early-winter G_. aculeatus (Lam, 1968) and hypophysectomized  2 goldfish (Lahlou and Sawyer, 1969). Thus, while prolactin is requisite to freshwater survival in some fishes and not in others i t does appear to be universal in its ability to maintain a normal electrolyte balance. Since salmon experience considerable changes in environmental ion concentrations during diadromous migrations, the role of prolactin in hydro-mineral regulation of these species is of interest.  Activity of  prolactin cells showed only slight changes in sockeye salmon during their anadromous migration (Cook and van Overbeeke, 1969; McKeown and van Overbeeke, 1969), while circulating levels of prolactin f i r s t increased as the fish approached the river and then dropped after the fish had entered fresh water (McKeown and van Overbeeke, 1972).  Three investigators  have examined the prolactin cells of juvenile salmonids in fresh water (Olivereau, 1969; Nagahama and Yamamoto, 1970; McKeown and Leatherland, 1973); however, presently there is no information on the effects of prolactin on freshwater osmoregulation of salmonids. The purpose of this investigation was to determine whether prolactin plays a role in the freshwater osmotic and ionic regulation of juvenile salmon and, i f so, whether changes in prolactin secretion and/or effects of exogenous prolactin are evident at the time of seaward migration. Pacific salmon can be grouped into two broad categories according to the age at which juveniles migrate downstream.  Chum and pink salmon enter  sea water shortly after emerging from the gravel while sockeye and coho remain in fresh water until the spring of their second year.  Chinook  salmon are somewhat intermediate, spending a few months to a year in fresh water before migrating.  Juvenile chum (Oncorhynchus keta) and  sockeye (0_. rierka) salmon were chosen for this study as representatives  3 of these two migratory patterns.  Since chum migrate to the sea as fry  and possess the capacity to osmoregulate in sea water at that time (Houston, 1961) they may be considered "seawater fish".  Sockeye fry,  on the other hand, almost always remain in fresh water an additional year and undergo a parr-smolt transformation prior to seaward migration. It was reasoned that i f changes in the secretion or effects of prolactin occur in conjunction with seaward migration, this would be evident in a comparison of pituitary histology and physiological responses to prolactin of downstream migrants (chum fry and sockeye smolts) with freshwater residents (sockeye fry).  4  MATERIALS AND METHODS Fish Fertilized chum (Oncorhynchus keta) and sockeye (0. nerka) eggs were collected in the f a l l of 1971 and 1972 from the spawning channel at Weaver Creek, British Columbia, Canada.  Eggs were incubated and  hatched at the University of British Columbia in "Heath trays" (Heath Aircraft Inc., Auburn, Wash., USA).  Shortly before the yolk was ab-  sorbed, alevins were moved to holding tanks. Temperatures of fresh water (Vancouver dechlorinated) varied seasonally between 5 and 16°C; the  photoperiod was of natural day length.  Feeding was initiated with  frozen brine shrimp and changed to Oregon moist pellets (R.V. Moore Co., La Conner, Wash., USA) when the fish were feeding well.  A feeding  schedule of three to four times daily for young fish was gradually reduced to once per day after two or three months. Experimental Temperatures arid Age arid Weight of Fish Experimental temperatures were allowed to vary seasonally.  Body  weights of fish used in different experiments also varied since the fish were growing rapidly. are  Temperatures and age and weight of fish  shown in Table I.  Note on hypophysectomy Due to the location of the pituitary gland hypophysectomy of juveni l e salmon is not feasible.  The pituitary i s situated above the attach-  ment of the g i l l arches to the roof of the pharynx.  Attempted removal  of the gland would result in rupture of the dorsal aorta.  Hypophysectomy  Table I Experimental temperatures and age and weight of fish  chum fry Survival Study  wt(g) rO,  Plasma Sodium Study  4.0-5.0  sockeye fry 3.0-4.0  sockeye smolts 7.0-8.0  temp CQ  12±1  9±1  12+1  age(mo)  3-4  3-4  15-16  wt(g) temp (°C) age(mo)  2.0-3.0  2.0-3.0  7.0-8.0  14+1  14+1  10+1  3-4  3-4  15-16  6  of rainbow trout (Salmo gairdneri) (Donaldson and McBride, 1967).  has been successfully carried out  The position of the pituitary relative  to the g i l l arches appears to be different in this species or in fish of this size (250 g as compared to a 2-3 g sockeye fry). Injections Before injection, fish were anaesthetized in a 0.004% solution of MS222 (tricaine methane-sulfonate). using an 0.05  Injections were intraperitoneal,  ml Hamilton syringe with a 30 guage needle.  volume was either 0.02  The injected  ml or 0.03 ml dependong on the size of the fish.  Ovine prolactin (NIH-P-S10; 25.6  IU/mg) was used in this study; the sol-  vent was buffered (pH 7.5) 0.7% NaCl. Histological Study Pituitaries were prepared for light microscopy at various times commencing when 50% of the fish had absorbed the yolk, until the age of six months for chum and 15 months for sockeye.  Field samples of migrat-  ing juveniles of both species were also collected.  Fish were killed by  decapitation and the head fixed in sublimated Bouin Hollande for 24 to 48 hr.  After fixation the heads were trimmed and where necessary, de-  calcified in 5% formic acid in 5% formalin: this solution was changed daily for two weeks. Tissue was embedded in "Tissueprep" and sagittal sections cut at 5 U.  Slides were stained with Cleveland-Wolfe trichrome  (Cleveland and Wolfe, 1932).  The tinctorial differentiation of various  cell types in the adenohypophysis of juveniles was usually not possible. Staining procedures were, therefore, determined on adult pituitaries and directly applied to juvenile pituitaries.  7  Survival Study Groups of 20 fish were removed from holding tanks and placed separately in 35 l i t e r tanks of flowing dechlorinated fresh water. Fish were held under these conditions for 48 hr.  Following this  period, one group was injected with solvent and a second group was uninjected.  The remaining groups were injected with different doses  of prolactin. the experiment.  Injections were continued on alternate days throughout Immediately following the second injection, fish were  transferred to tanks of flowing deionized water.  Fish were not fed  for 48 hr before or subsequent to transfer into deionized water.  The  day of transfer from fresh water to deionized water was designated Day 0.  Each morning, for a period of 10 days, the dead fish in each group  were removed and the number recorded.  The experiments with sockeye fry  were duplicated, thus, each group contained a total of 40 fish. Water was deionized by passing i t consecutively through two Barnstead demineralizer cartridges (Table II).  The data were analyzed  statistically using the Kolmogorov-Smirnov two-sample test (Siegel, 1956) . Plasma Sodium Study Groups of approximately 50 seawater acclimated fish were placed separately in three 35 l i t e r tanks of flowing sea water.  Fish had been  adapted to seawater (ca 25°/oo) for at.least three weeks before starting the experiments. After 48 hr under these conditions, one group was i n jected with solvent, one group with 10 ug/g body wt prolactin, while the third group was uninjected.  Injections were continued on alternate  days throughout the experiment,  Twentyr-four hours after the third  8  Table II  Electrolyte content of fresh and deionized water at 10°C measured as resistance in megaohms.  Resistance + SD (megaohm)  Dechlorinated fresh water  0.092 +_ 0.007  Deionized dechlorinated water  0.421 + 0.158  standard deviation  9 injection, fish were transferred to tanks of flowing dechlorinated fresh water.  Blood was collected from six fish in each group at  intervals of 0, 2, 4, 12, 24, 48, and 72 hr.  The sample at 0 hr was  taken immediately before transfer to fresh water.  Fish were not fed  for 48 hr before transfer to fresh water or subsequently.  Blood was  collected after wiping the t a i l region of the fish dry and severing the t a i l with a clean razor blade.  The cut end was blotted lightly  with facial tissue to remove any water or tissue fluids; blood was collected i n heparinized microhematocrit capillary tubes as i t flowed from the cut end of the caudal artery.  The tubes were sealed with  plastic clay and centrifuged for 5 min; 2 u l aliquots of plasma were added to 5 ml of glass-distilled water in plastic vials. samples were prepared for each fish.  Duplicate  Sodium concentrations were deter-  mined by flame photometry using a Techtron AA120 atomic absorption spectrophotometer (Varian Techtron Pty. Ltd.).  Data were analysed  statistically by t-test (Sokal and Rohlf, 1969).  10 RESULTS Histological Study The pituitary gland of salmon consists of four regions: rostral pars distalis, proximal pars distalis, pars intermedia, and neurohypophysis. Prolactin cells (eta cells) and corticotrophs compose the rostral pars distalis which occupies the anterior portion of the gland (van Overbeeke and McBride, 1967; Nagahama and Yamamoto, 1970).  The prolactin cells are  easily distinguished by their cellular arrangement.  They are columnar and  arranged in f o l l i c l e s each with a central lumen which often contains material stained with aniline blue.  The nucleus of the eta cells is situated  peripherally on the side opposite the follicular lumen.  The cytoplasm  stains with erythrosin particularly in the nuclear region. Prolactin cells of juvenile chum salmon In chum fry at the time of yolk sac absorption  (age 0 fry) and at one  month of age (1-month-old fry) the pituitary gland is dorsoventrally flattened, in close contact with the floor of the brain, and measures 100 to 150 u in the minor axis and 400 to 500 u in the major axis.  The neurohypo-  physis exhibits very l i t t l e interdigitation with the adenohypophysis which consists of a narrow band of cells attached to the periphery of the neurohypophysis.  Vascularization of the adenohypophysis is slight.  The cells  of the adenohypophysis display similar staining with the Cleveland-Wolfe technique; however, the eta cells may be distinguished by their f o l l i c u l a r arrangement (Fig. 1). The pituitary of each fish contains one or two f o l licles with lumina which are generally larger than in older juveniles and rarely contain aniline blue stained material.  Cytoplasm i n the region of  11 the eta cell nucleus stains weakly with erythrosin; the distal  cytoplasm  is not stained by the dye. In 2-,4-, and 6-month-old fry the pituitary gland increases considerably in volume.  It reaches approximately 400 u and 800 u for minor and  major axes by the age of six months.  Interdigitation of the neurohypophy-  sis with the adenohypophysis also increases and is extensive in 6-monthold fry. The gland is well vascularized at this time and capillaries are frequently observed near the prolactin f o l l i c l e s . The prolactin cells show a gradual development during the interval between 1-month-old fry and 6-month-old fry. The stage of development at the age of six months will be described.  The diameter of the f o l l i c l e s or the  number of cells in each f o l l i c l e has not changed noticeably from the 1-monthrold fry. However, the prolactin cells have increased in height, resulting in a decrease in the diameters of the lumina (Fig. 2).  The number of  f o l l i c l e s has also increased gradually over the period of observation; four or five f o l l i c l e s per median sagittal section are present in 6-monthold fry. Material stained with aniline blue is recognized in many of the f o l l i c u l a r lumina.  The intensity of cytoplasmic staining with erythrosin has  also increased and the distal cytoplasm is stained. Prolactin cells of juvenile chum salmon during seaward migration. The morphology of the pituitary and prolactin cells of chum salmon sampled during the downstream migration closely resemble that of fry sampled from laboratory stock during their f i r s t month (Fig. 1 and 3).  This might  be expected since chum fry normally migrate to the sea shortly after emerging from the gravel,  The neurohypophysis is only slightly interdigitated  with the adenohypophysis which is poorly vascularized. The gland contains  12 two or three f o l l i c l e s ; stained material is usually not found in the lumina. The cytoplasm of the prolactin cells is stained lightly with erythrosin. Prolactin cells of juvenile sockeye salmon. Development of prolactin cells in juvenile sockeye i s comparable to that in similar age chum but appears to proceed more slowly.  In age 0 and  1- and 2-month-old sockeye, the pituitary gland measures 100 to 125 u in the minor axis and 300 to 400 u in the major axis.  Vascularization is poor and  a l l c e l l types display similar staining properties. one f o l l i c l e in each pituitary. stained material.  There is generally only  Lumina are large and usually.devoid of  Until the age of two months, the cells do not resemble  normal prolactin cells as they are not elongated and contain l i t t l e cytoplasm (Fig. 4). The cytoplasm in the region of the nucleus has a weak affinity for erythrosin; the distal cytoplasm is unstained. Pituitary glands from freshwater fish sampled at 4, 6, 8, 11, 13 and 15 months showed gradual eta cell development. month-old sockeye will be described.  The prolactin cells of 15-  At the age of 15 months the pituitary  gland has increased in size to measure 300 u and 600 u for minor and major axes respectively.  The neurohypophysis is well interdigitated with the  adenohypophysis which is richly vascularized.  The prolactin cells have i n -  creased in height resulting in a decrease in lumen size (Fig. 5). Though the f o l l i c l e s have increased in number to three to five per median sagittal section they remain about the same size as those of younger fish.  Aniline  blue stained material is present in many of the lumina. The erythrosinophilic stainability of the cytoplasm is also enhanced. Prolactin cells of sockeye smolts during seaward migration. Pituitary and prolactin cell morphology of sockeye smolts sampled in  13 May during the peak of their seaward migration resembles that of similar age fish reared in the laboratory (Fig. .5 and 6). Sockeye smolts migrate downstream in the spring of their second year. to 14 months old at this time.  Laboratory fish were 13  The minor and major axes of the pituitary  gland measure 250 to 300 u and 500 to 000 V respectively i n migrating smolts.  Interdigitation of the neurohypophysis with the adenohypophysis  is extensive and the adenohypophysis is richly vascularized.  The prolactin  cells have increased in height in comparison with those of fry. sity of cytoplasmic staining with erythrosin has increased.  The inten-  The gland con-  tains three to five f o l l i c l e s per median sagittal section and material stained with aniline blue is present in many of the lumina. Survival Study The data on survival of chum fry in deionized water are presented in Fig. 7.  Chum fry cannot tolerate deionized water and i t appears that pro-  lactin injections have no effect on survival; in both control and experimental groups 100% mortality occurs within 6 to 8 days.  There was no differ-  ence in mortality between solvent-injected and uninjected fish or between these control groups and prolactin injected fish (Kolmogorov-Smirnow twosample test). A more sensitive method for analysing time-percent effect curves has been developed by Litchfield (1949).  For each prolactin dose, time was  plotted against cumulative percent mortality on logarithmic-probability paper and a straight line fitted by eye.  Median survival times or times to  50% mortality were then read directly from the graph.  The slope of each  prolactin line was found to differ from the slope of the solvent line  14 (p .05); the slopes of the prolactin lines did not differ significantly <  from one another.  This slope difference indicates that the course of  mortality has been altered by prolactin treatment.  A logarithmic plot of  prolactin dose against median survival time gives a straight line for doses of 5 to 30 ug/g body wt.  (Fig. 8).  This indicates that prolactin has a  small dose effect and prolongs survival of chum fry in deionized water. comparison of median survival times (Litchfield, 1949) 15 Ug/g  showed that 5 and  prolactin did not alter survival; the effect of 30 ug/g  showed border-line significance Cp <.05).  A  on survival  Sixty micrograms per gram re-  duced survival of chum fry in deionized water (p <.05)  (Fig. 8).  Since 100% mortality was not reached during the 10 day observation period, the time-percent effect curves for sockeye fry and smolts are truncated (incomplete).  Truncated curves can be solved by Litchfield's  (1949) method; however, due to the less accurate nature of such a curve at least 50% of the individuals must react.  Since this was  often not the case,  the method of Litchfield (1949) cannot be applied to the data on sockeye fry and smolts.  The solution to these curves, however, appears obvious  (Fig. 9 and 10) and the less sensitive Kolmogorov-Smimow method (Siegel, 1956)  is sufficient, Sockeye fry survive well in deionized water; only 7.5 to 15% mortality  had occurred in control groups after 10 days (Fig. 9).  A prolactin dose of  5 yg/g did not significantly affect this survival but doses of 15, 30, 60 ug/g  and  increased mortality in deionized water (P <.01). A dose effect  appears to be present; however, 15 ug/g resulted in greater mortality than 30 Ug/g.  There was no significant difference in mortality between solvent-  injected or uninjected fish or between these control groups and 5 yg/g prolactin.  15 Sockeye smolts do not survive in deionized water (Fig. 10). There was no difference in mortality between solvent-injected and uninjected fish.  However, prolactin injections promote survival in deionized water  (P <.01).  Significance was not found between the 5 and 15  ug/g doses  or between the 30 and 60 ug/g doses. However, mortality i s different between the low prolactin doses (5 and 15 ug/g) and the high prolactin doses (30 and 60  ug/g) (P <.01).  Thus, prolactin has a beneficial effect on  smolt survival in deionized water, albeit a small effect with doses less than 60 ug/g.  A dose effect is present; however, adjacent doses do not  differ in their effect. Plasma Sodium Study The data on plasma sodium in chum fry are presented in Fig. 11.  Both  solvent-injected and uninjected controls showed a rapid decline in plasma sodium after transfer from sea water to fresh water. levels had dropped about 25%.  At the end of 24 hr,  After this time the decline was less drastic  though i t continued throughout the 72 hr experimental period. parent that 10  It is ap-  ug/g body wt prolactin had no effect on plasma sodium of  chum fry. Differences between plasma sodium levels of solvent-injected and uninjected controls were not significant; therefore, only solvent-injected controls were used for sockeye fry and smolts. Plasma sodium values in sockeye fry are presented in Fig. 12.  Sockeye  fry showed an instantaneous regulation of plasma sodium after transfer from sea water to fresh water; the slight drop in levels was not significant. Prolactin had no effect on plasma sodium. Fig. 13 shows plasma sodium values in sockeye smolts.  Plasma sodium •  in solvent-injected fish declined steadily and had dropped about 30% by  16 72 hr.  Prolactin had no effect on plasma sodium; plasma concentrations  in solvent-injected and prolactin-injected fish did not differ. To determine whether prolactin has an effect in conditions more hyposmotic than fresh water, experiments were repeated in deionized water. Seawater^adapted fish were injected, on alternate days, with either solvent or 10  ug/g body wt prolactin.  Twenty-four hours after the third injection,  fish were transferred to flowing deionized water.  Plasma sodium was esti-  mated before transfer and after 72 hr in deionized water.  It i s apparent  from Fig. 14 that prolactin has no effect on plasma sodium in chum fry, sockeye fry, or sockeye smolts transferred from sea water to deionized water.  17  Figure 1  Sagittal section of the pituitary gland of a 1-month old chum salmon (Cleveland-Wolfe trichrome).  The gland  contains two prolactin cell f o l l i c l e s (P).  Figure 2  Sagittal section of the pituitary gland of a 6-monthold chum salmon (Cleveland-Wolfe  trichrome).  Note the  increase in the number of prolactin cell f o l l i c l e s (P) and the decrease in lumen size compared to 1-month-old fish (Fig.  Figure 3  1).  Sagittal section of the pituitary gland of a juvenile chum salmon during seaward migration trichrome).  (Cleveland-Wolfe  The gland closely resembles that of 1-month-  old fish (Fig. 1) .  17a  18  Figure 4  Sagittal section of the pituitary gland of a sockeye fry at the time of yolk absorption (age 0) trichrome).  (Cleveland-Wolfe  Note the single prolactin cell f o l l i c l e (P)  with a large lumen.  Figure 5  Sagittal section of the pituitary gland of a 15-month-old sockeye salmon (Cleveland-Wolfe trichrome).  The prolactin  cells have increased in height resulting in a decrease in lumen size compared to age 0 sockeye (Fig. 4).  Figure 6  Sagittal section of the pituitary gland of a sockeye smolt during seaward migration (Cleveland-Wolfe  trichrome).  18a  19  Figure 7  Prolactin and survival of chum fry in deionized water. Abscissa represents the days after transfer from fresh water to deionized water.  20  Figure 8  Prolactin and median survival times of chum fry in deionized water.  Median survival time for solvent-  injected fish i s 3.4 (3.0-3.8) days. are logarithmic. dence limits.  Both scales  Vertical bars enclose 95% confi-  20a  U)  u _o O Q_  OO>C0 N <0 u) *f  (S/DQ) a L U j i  pAjAjng  uojpayy  21  Figure 9  Prolactin and survival of sockeye fry in deionized water.  Duplicate experiments were not significantly  different and the results have been pooled.  Abscissa  represents the days after transfer from fresh water to deionized water.  21a  i  i  i  i  i  i  i  1  1  1  1  r  1  60//g/g  y*  l5//g/g  •  • 30//g/g  m  P Uninjected p — o — o  •/ J 0  I  2  3  4  5  / /*  — • — • Solvent ^ 5//g/g  J  I  6  7 Days  8  I  I  I  9  10 II  I  12  L  13 14  22  Figure 10  Prolactin and survival of sockeye smolts in deionized water.  Abscissa represents the days after transfer  from fresh water to deionized water.  22a  100 i  0  1  1  1  1  1  1  1  i  1  1  1  I 2  3  4  5  6  7  8  9  10  II  12 13 14  1  Days  r  23  Figure 1 1  Prolactin ( 1 0 ug/g) and plasma sodium of chum fry after transfer from sea water to fresh water. Abscissa represents hours after transfer. point represents the mean of six fish. bars enclose 9 5 % confidence limits.  Each  Vertical  i  r  • Prolactin o Solvent * Control  _JL_  72  CO  24  Figure 12  Prolactin (10 yg/g) and plasma sodium of sockeye fry after transfer from sea water to fresh water. represents hours after transfer. the mean of six fish. fidence limits,  Abscissa  Each point represents  Vertical bars enclose 95% con-  25  Figure 13  Prolactin (10 ug/g) and plasma sodium of sockeye smolts after transfer from sea water to fresh water. Abscissa represents hours after transfer. point represents the mean of six fish. bars enclose 95% confidence limits.  Each  Vertical  26  Figure 14  Prolactin (10 ug/g) and plasma sodium of chum fry, sockeye fry, and sockeye smolts after transfer from sea water to deionized water. done on six fish.,  Each determination was  Vertical bars show the upper 95%  confidence limit. SW  = sea water  DW  = deionized water  I  250  i i i i  I Solvent Prolactin  C- 200 CT  LU  E E  JL  150  O  CO  a E  CO  JL  100  X  -X.  CL  50  Ohr 72 hr (SW) (DW) CHUM FRY  0 hr 72 hr (SW) (DW) SOCKEYE FRY  0 hr 72 hr (SW) (DW) SOCKEYE SMOLTS CTi  27 DISCUSSION Prolactin cells In the present study the prolactin cells of chum fry and sockeye fry and smolts were similar to those of chum fry described by Nagahama and Yamamoto (1970).  Only slight changes occurred in the eta cells of  both species during the period of observation (to the age of six months in chum and 15 months in sockeye).  Also, cytological changes concomitant  with seaward migration were not observed.  It appears, rather, that the  prolactin cells of juvenile salmon develop gradually during freshwater residence arid seaward migration.  The cells increase slightly in size  during the juvenile freshwater stage of both species; the number of prolactin cell f o l l i c l e s also increases.  Simultaneously, the intensity of  cytoplasmic staining with erythrosin increases.  These cytological features  are undeveloped compared to the adult where a large increase in both the size and number of prolactin cells as well as intensified cytoplasmic staining with acidic dyes is evident (van Overbeeke and McBride, 1967; Nagahama and Yamamoto, 1970). Nagahama and Yamamoto (1970) examined the histology and fine structure of the prolactin cells of chum salmon at various stages of the life-cycle from fry in fresh water to sexually mature fish on the spawning grounds. They observed a progressive development of the prolactin cells throughout the life-cycle.  Functionally active features were not observed until the  sexually maturing fish reached the coastal area.  The cells of fish at this  stage differed considerably from those of immature adults in the north Pacific,  The rostral area of the pituitary had increased greatly in volume  due to an increase in number and size of the prolactin cells.  The cytoplasm  28  of the eta cells was stained strongly with azocarmine G.i'Ultrastructural features, such as a well-developed rough-surfaced endoplasmic reticulum arranged in concentric whorls, numerous electron-dense granules in the cytoplasm, and a well-developed Golgi apparatus, indicated increased eta cell  activity. In the Atlantic salmon (Salmo salar) the eta cells also appear to  develop as the fish matures (Olivereau, 1969).  Only a small increase i n  cell height was observed i n the smolt when compared to the parr.  In the  sexually maturing adult, having spent several years in the ocean, the prolactin cells were further developed and mitoses were observed (Olivereau, 1969) . Observations using light and electron microscopes have shown that the prolactin cells of most teleosts are more active in fresh water than in. sea water (Ball and Pickford, 1964; Olivereau and Ball, 1964; Dharmamba and Nishioka, 1968; Nagahama and Yamamoto, 1971; Nagahama et_ al_. , 1973; Ball and Ingleton, 1973).  After a transfer of fish from sea water to fresh  water the size of the rostral lobe increases due to an increase i n the size and number of prolactin cells.  The intensity of cytoplasmic staining with  acidic dyes i s also stronger in the eta celiEs of freshwater fish.  Further-  more, the endoplasmic reticulum and Golgi apparatus become well-developed and the number of electron-dense granules and the frequency of granule release increases. It i s interesting to note that Nagahama and Yamamoto (1970) observed a slight increase in the number and size of prolactin cells as well as an increase in staining intensity in chum fry approximately one month after  29  entering the sea when compared to freshwater fry. They assume that these changes are the result of a chronological development of the prolactin cells accompanied by growth of the fish.  Recently McKeown and Leatherland  (1973) studied the fine structure of the prolactin cells of sockeye smolts collected during the downstream migration.  There was no change i n prolactin  cell activity, asjjudged by fine structural characteristics, after a transfer of smolts from fresh water to sea water. Hormone release from prolactin cells by the extrusion of secretory granules through the cell membrane has been observed in a number of teleosts (Weiss, 1965; Leatherland, 1970; Nagahama and Yamamoto, 1971) including the adult migratory sockeye salmon (Cook and Van Overbeeke, 1969).  McKeown and  Leatherland (1973) did not observe this phenomenon in the sockeye smolt. They suggest that granules may be released i n a form not visible in the electron microscope.  On the other hand, the observations of McKeown and  Leatherland may indicate that prolactin release does not occur in sockeye smolts. Thus, on the basis of histological and fine structural evidence i t appears that the prolactin cells of juvenile freshwater salmon are functionally inactive.  The presence of secretory granules in the cytoplasm indi-  cates that hormone synthesis is occurring but there is no evidence of hormone secretion (McKeown and Leatherland, 1973).  The cells probably do not  become active until the fish is reaching sexual maturity. Physiological effects of prolactin Many euryhaline fish do not survive in fresh water after hypophysectomy; prolactin prolongs survival i n a l l species that have been tested (Pickford  30  and Phillips, 1959; Ball and Olivereau, 1964; Schreibman and Kallman, 1964, 1966a; Chambolle, 1965; Pickford et a l . , 1965; Dharmamba et a l . , 1967; Utida et d . , 1971; Chidambaram ert al_. , 1972).  If chum and pink  (0_. gorbuscha) fry are retained i n fresh water beyond the normal time of seaward migration they often experience osmoregulatory difficulties and die (Black, 1951; Baggerman, 1959; Houston, 1961).  As hypophysectomy was  not feasible in the present study (see Materials and Methods), i t was decided to use deionized water as a "dilute fresh water" to intensify osmoregulatory difficulties the fish may encounter i n fresh water. Under the present experimental conditions, chum fry cannot live in deionized water (Fig. 7). Prolactin injections of 5 or 15 ug/g body wt do not alter survival; however, 30 ug/g has a barely significant effect in prolonging survival.  On the other hand, 60 yg/g significantly reduced  survival of chum fry i n deionized water (Fig. 8). This adverse effect of prolactin i s similar to that observed with doses above 15 yg/g i n sockeye fry which will be discussed later. Sockeye smolts also die in deionized water; however, prolactin has some effect in prolonging survival.  This effect is small compared to other  species and doses of 30 to 60 yg/g were necessary to reduce mortality below 50% (Fig. 10). This response may be pharmacological since doses in the range of 5 to 10 yg/g are effective in other species (Schreibman and Kallman, 1966a; Dharmamba et a l . , 1967; Lam and Leatherland, 1969; Utida et^ al_., 1971; Chidambaram et a l . , 1972). It i s apparent that sockeye fry are better hyposmotic regulators than either chum fry or sockeye smolts; they suffered only slight mortality i n  31  deionized water.  A prolactin dose of 5 yg/g had no effect on survival,  however, doses of 15 yg/g or higher had an adverse effect resulting in 40 to 70% mortality after 10 days in deionized water (Fig. 9). It i s d i f f i cult to reconcile this detrimental effect of prolactin in both chum and sockeye fry.  It i s possible that high doses of prolactin may alter mem-  brane permeabilities.  High prolactin doses have been shown to affect water  and sodium permeabilities in the kidney of the flounder (R. Foster, personal communication).  Also, high doses of prolactin may be toxic or impuri-  ties i n the hormone preparation may reach toxic levels i n high doses.  This  possibility was tested by injecting a group of 20 sockeye fry i n fresh water with ,60 yg/g prolactin on alternate days for a period of 10 days. No mortality occurred.  Death as a result of high doses of prolactin occurs  only in deionized water. Sockeye fry differ from smolts and chum fry either i n tissue tolerance to reduced ion levels or i n the mechanisms of osmoregulation or the e f f i ciency of these mechanisms. ACTH and adrenocorticosteroids play an important role in hydromineral regulation of both seawater and freshwater fishes. (Chester Jones et^al_., 1969) . High doses of prolactin may interfere pharma"-'cologically with adrenal steroid actions or secretion and may upset hormonal balances causing increased osmoregulatory failure of chum and sockeye fry in deionized water.  Prolactin injections led to inactivation of the ACTH cells  in Hippocampus (Boisseau, 1967 cited i n Ball and Baker, 1969).  On the other  hand, prolactin may mimic ACTH and stimulate the interrenal in Gambusia (Chambolle, 1967).  32  The effects of prolactin on survival of chum fry and sockeye fry and smolts in deionized water i s confusing and at this stage i t is d i f f i cult to obtain a clear understanding of the role of prolactin in these species.  Chum fry and sockeye smolts cannot survive in deionized water.  This might be expected since these fish are seaward migrants and can be considered to be physiologically primed for osmoregulation in sea water. Sockeye fry, on the other hand, are freshwater residents and their better hyposmotic capacity is reflected in their ability to withstand deionized water.  Prolactin has some effect in prolonging survival of sockeye smolts  in deionized water while there is l i t t l e more than a suggestion of an effect in chum fry.  This difference may be due to an age dependent sensi-  t i v i t y of osmoregulatory tissues which will be discussed later.  Sixty  micrograms per gram prolactin had an adverse effect on survival of chum fry in deionized water as was found for the higher doses in sockeye fry. may,  This  also, be due to age differences in tissue response. Juvenile salmon experience hyponatremia following transfer from sea  water to fresh water.  At a dose of 10  ug/g, within the range found effec-  tive in other species, prolactin injections did not affect plasma sodium concentrations on chum fry, sockeye fry, and sockeye smolts after a transfer from sea water to either fresh water or deionized water (Tig. 11, 12, and 14).  Fish were injected on alternate days; blood was  24 to 48 hr after the time of injections.  13,  sampled from  It is possible that an effect of  prolactin would have been observed i f fish had been injected daily or i f sodium had been estimated at shorter intervals after injections.  However,  results on other species indicate that this is not a source of error.  Lam  33  (1968) found that a single injection of prolactin significantly reduced the f a l l in plasma sodium and chloride concentrations in freshwater Gasterosteus aculeatus for a period of at least 48 hr. Similarly, a single injection of prolactin elevates plasma sodium in hypophysectomized Ictalurus me las in fresh water for seven days (Chidambaram et al_., 1972) and prolongs freshwater survival of hypophysectomized Oryzias latipes for at least 20 days (Utida'et'al_., 1971).  It appears, therefore, that the  effect of prolactin injections is long lasting and that alternate day i n jections are sufficient.  Also, i t i s possible that an effect of prolactin  on plasma sodium would be apparent with higher doses as there was a slight effect on smolt survival in deionized water with doses above 30  yg/g.  Higher doses were not tested due both to a lack of time and the large amounts of hormone that would be required. These results are at variance with observations on other euryhaline species.  The green molly (Pbecilia latipinna) shows a transient drop in  plasma sodium after transfer from dilute sea water to fresh water.  Levels  return to normal after three days in fresh water (Ball and Ensor, 1965; Ball and Ingleton, 1973).  Hypophysectomized mollies maintain nearly normal sodium  levels in dilute sea water, but on transfer to fresh water they loose sodium about twice as fast as intact controls and are unable to return levels to normal.  Ovine prolactin largely prevents sodium loss in hypophysectomized  P_. latipinna in fresh water (Ball and Ensor, 1965); prolactin is specific in this effect (Ball and Ensor, 1967). In freshwater Furidulus heteroclitus, hypophysectomy results in a drop in serum osmotic pressure, sodium, and chloride (Burden, 1956; Pickford  34  et a l . , 1966).  These changes are opposed by exogenous prolactin  (Pickford  et_ al_., 1966).  Prolactin also reduces the f a l l in plasma sodium and  chloride  concentrations in "physiologically hypophysectomized" early-winter Gasterosteus aculeatus in fresh water (Lam,  1968).  Similar results in other species (e.g. Carassius auratus, Ti1apia mossambica, Oryzias latipes, Ictalurus melas) have shown that prolactin plays an important role in freshwater teleosts by maintaining normal plasma osmol a l i t y and electrolyte concentrations (Lalhou and Sawyer, 1969; 1970;  Utida et_ al_., 1971;  Dharmamba,  Chidambaram e_tal_. , 1972). Prolactin exerts i t s  effects via a number of pathways, particularly at the g i l l s and kidney.  The  major actions of prolactin appear to be an ability to decrease electrolyte efflux at the g i l l s , reduce the passive influx of water, and stimulate production of volumous amounts of dilute urine.  A f u l l consideration of the  mechanisms of action of prolactin is beyond the scope of this discussion  and  the reader i s referred to recent reviews by Ball, 1969;  1972;  and Lam,  Ensor and Ball,  1972.  The results of this study, based on both prolactin c e l l cytology and physiological effects of ovine prolactin, indicate that prolactin does not play an obvious physiological role in the freshwater osmoregulation of juveni l e Pacific salmon,  Since prolactin has been shown to play an important  osmoregulatory role in many other freshwater teleosts one might expect similar effects in juvenile salmon residing in fresh water.  However, other  osmoregulatory studies using prolactin have been conducted on mature fish. It is possible that target tissues in immature fish (such as the present study) are insensitive to prolactin.  In mammals, the response of the gonads,  35  the accessory reproductive organs, and the brain to exogenous hormones depend not only upon the hormone administered, the length of treatment, and the dosage employed but also upon the age of the animal (Barraclough, 1967).  It appears that a c r i t i c a l stage of differentiation exists for  target tissues which must be attained before the tissues are sensitive to hormonal control (Barraclough, 1967).  Osmoregulatory tissues in salmon  may not be sensitive to prolactin until sexual maturity is reached and the adult is preparing to return to fresh water.  On the other hand, the slight  effect of prolactin on prolonging survival of sockeye smolts in deionized water may indicate that osmoregulatory mechanisms in smolts are starting to become sensitive to prolactin before the hormone is secreted and plays a physiological role. In concluding that prolactin does not play an.osmoregulatory role in juvenile salmon I must emphasize the fact that the fish were not hypophysectomized. out.  Therefore, the presence of endogenous prolactin cannot be ruled  Exogenous hormones may produce spurious effects by upsetting  the  balance of naturally occurring hormones or by initiating immunologic reactions, thereby, negating the effect of injected hormone.  It is also possible  that prolactin action in salmon is species specific; mammalian prolactins be ineffective. This is unlikely, however, since mammalian prolactins are effective in other teleosts and bovine prolactin has been shown to affect water flux in the g i l l s of a salmonid (Salmo gairdrierii)  (Ogawa, 1973).  Furthermore, i t is possible that a threshold concentration exists for prolactin action and that this level was surpassed by endogenous secretion. If such a situation exists in juvenile salmon, injection of exogenous  may  36  hormone would not have an observable effect.  However, the effect of pro-  lactin on the survival of sockeye smolts in deionized water appears to be dose-dependent.  Also, a dose-response relationship exists between prolac-  tin and plasma sodium in hypophysectomized Oryzias latipes 1971).  (Utida et a l . ,  Tilapia mossambica (Dharmamba, 1970), and Poecilia latipinna  (Ball  and Ensor, 1967) . This effect iii P_. latipinna has been developed into a bioassay for fish "prolactin" (Ensor and Ball, 1968).  On the other hand,  Leatherland and Lam (1969) did not find an obvious dose-effect of prolactin on survival of Gasterosteus aculeatus in deionized water. The difficulties inherent in studying prolactin physiology in.fish that cannot be hypophysectomized might be overcome by pharmacologically blocking prolactin secretion. Ergocornine methanesulfonate has repeatedly been used to inhibit prolactin secretion in mammals (Nagasawa and Meites, 1970; Wuttke ejt al_., 1971) and has recently been found effective in three teleost species (McKeown, 1972; Olivereau and Lemoine, 1973).  I proposed  to use ergocornine to test the hypothesis that prolactin secretion does not occur in juvenile chum and sockeye salmon in fresh water..  Unfortunately,  due to delays in obtaining the correct ergocornine derivative and unsatisfactory results in preliminary experiments with goldfish, the experiments were not conducted. Possible role(s) of prolactin in adult migratory salmon Although prolactin does not play an obvious role in juvenile salmon in fresh water i t may be involved in one or more ways with the anadromous migration of adults. Prolactin may function in the adaptation of migratory adults to fresh water.  This is apparently the role of prolactin in the marine threespine  37 stickleback, Gasterosteus aculeatus (Lam, 1972).  As mentioned earlier, i t  appears that the prolactin cells of salmon are activated when the fish i s reaching sexual maturity.  This increased activity is maintained throughout  the anadromous migration (Nagahama and Yamamoto, 1970).  Similarly, serum  prolactin increased in seawater sockeye as they approached the river and then declined by 27% after they had entered fresh water (McKeown and van Overbeeke, 1972).  Since salmon prolactin i s known to have an osmoregulatory  capacity (Donaldson ejtal_,, 1968) i t seems likely that i t plays a role i n the adaptation of adults to the freshwater condition. Alternatively, prolactin may function in the control of migratory behavior.  Prolactin has been shown to induce the return of the terrestrial  stage (red eft) of the salamander, Notophthalamus (Diemictylus) viridescens, to fresh water (Chadwick, 1941; Grant and Grant, 1958).  This migration,  which involves a readaptation to aquatic l i f e , is from a dehydrating environment (land) to a hydrating environment (fresh water) and imposes problems i n hydromineral regulation similar to those encountered by salmon migrating from sea water to fresh water.  Prolactin may induce the spawning  migration of salmon as i t does the "water-drive" of urodele efts. Finally, prolactin may be involved i n pre-migratory fattening and growth of adult salmon.  Prolactin has been implicated in the regulation of growth  in a l l vertebrate classes (Bern and Nicoll, 1968; Meier, 1972).  In two  species of sparrows, prolactin causes an increase in body weight and fat stores during the intermigratory period (Meier and Farner, 1964; Meier and Davis, 1967).  This fattening response i s equivalent to that found during  the migratory period and is possibly related to preparation for migration.  38  Fattening responses to prolactin have also been found in three teleost species (Lee and Meier, 1967; Mehrle and Fleming, 1970; Joseph and Meier, 1971).  Since migratory salmon do not feed after entering fresh water,  they must rely on stored energy reserves during this phase of the migration.  Prolactin may induce fattening and energy storage in salmon prior  to the onset of anadromous migration.  39  SUMMARY  The possible role of prolactin in the freshwater osmoregulation of laboratory reared juvenile chum (Oncorhynchus keta) and sockeye (0_. nerka) salmon was  Pituitary histology was  investigated.  examined at intervals from the time of yolk  absorption to the age of six months in chum and 15 months in sockeye. Field samples of downstream migrants of both species were also examined.  Pituitary cytology indicated that the prolactin cells of both species develop gradually during freshwater residence and downstream migration. During this time the prolactin cells increase slightly in size; the number of prolactin cell f o l l i c l e s also increases.  Simultaneously,  the intensity of cytoplasmic staining with erythrosin increases.  The effect of prolactin on survival in deionized water was examined by timed mortality tests.  Chum fry suffered a high mortality after transfer from fresh water to deionized water; alternate day injections of 5 or 15 ug/g body wt prolactin (ovine) did not affect survival.  Thirty micrograms per gram  slightly increased survival while 60 yg/g decreased survival in deionized water.  40 Sockeye smolts also suffered high mortality in deionized water; however, prolactin prolonged survival to a slightly small extent.  Sockeye fry suffered only slight mortality after transfer to deionized water.  A prolactin dose of 5 g/g did not alter this sur-  vival; however, doses of 15 g/g or higher resulted i n 40 to 70% mortality after 10 days i n deionized water.  The effect of prolactin on plasma sodium was examined by estimating sodium concentrations by flame photometry at intervals after transfer of fish from sea water to either fresh or deionized water.  Alternate day injections of 10 g/g prolactin had no effect on plasma sodium concentrations of chum fry, sockeye fry, or sockeye smolts after transfer from sea water to either fresh water or deionized water.  It i s concluded from histological and physiological evidence, that prolactin does not play an obvious role in the freshwater osmoregulation of juvenile chum and sockeye salmon.  The possible role of pro-  lactin in the spawning migration of adults is discussed.  41 REFERENCES  Baggerman, B. 1959. The role of external factors and hormones in migration of sticklebacks and juvenile salmon. In_ Comparative endocrinology (Edited by A. Gorbman), pp. 24-37, Wiley and Sons. New York. Ball, J. N. 1969. Prolactin (fish prolactin or paralactin) and growth hormone. In_Fish physiology (Edited by W. S. Hoar and D. J. Randall) Vol. II. pp. 207-240. Academic Press. New York. Ball, J. N. and B. I. Baker. 1969. The pituitary gland; anatomy and histophysiology. In_ Fish physiology (Edited by W. S. Hoar and D. J. Randall). Vol. II. pp. 1-110. Academic Press. New York. Ball, J. N. and D. N. Ensor. 1965. Effect of prolactin on plasma sodium in the teleost, Poecilia latipinna. J. Endocrin. 32_. 269-270. Ball, J. N. and D. M. Ensor. 1967. Specific action of prolactin on plasma sodium levels in hypophysectomized Poecilia latipinna (Teleostei) . Gen. Comp. Endocrin. 8^. 432-440. Ball, J. N. and P. M. Ingleton. 1973. Adaptive variations in prolactin secretion in relation to external salinity in the teleost Poecilia latipinna. Gen. Comp. Endocrin. 20: 312-325. Ball, J. N. and M. Olivereau. 1964. Role de l a prolactine dans la survie en eau douce de Poecilia latipinna hypophysectomise et arguments en faveur de la synthese par les cellules erythrosinophile^s eta de l'hypophyse des Teleosteens. C. R. Acad. Sci. Paris'. 259. 1443-1446. Ball, J. N. and G. E. Pickford. 1964. Pituitary cytology and freshwater adaptation in the k i l l i f i s h , Fundulus heteroclitus. Anat. Record. 148. 358. Barraclough, C. A. 1967. Modifications in reproductive function after exposure to hormones during the prenatal and early postnatal period. Tn Neuroendocrinology (Edited by L. Martini and W. F. Ganong) Vol. II. pp. 61-99. Academic Press. New York. Bern, H. A. and C. S. Nicoll. 1968. The comparative endocrinology of prolactin. Recent Prog. Hormone Res. 24_. 681-720. Black, V. S. 1951. Changes in body chloride, density, and water content of chum (Oncorhynchus keta) and coho (0_. kisutch) salmon fry when transferred from fresh water to sea water. J. Fish. Res. Bd. 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