GENERAL AND COMPARATIVE ENDOCRINOLOGY 36, 306-316 (1978)

Prolactin and Pituitary Control of Calcium Regulation in the Killifish, Fund/us heteroclitus

PETER K. T. PANG,* MARTIN P. SCHREIBMAN,? FERNANDO BALBONTIN,~ AND ROSEMARY K. PANG*

*Department of Pharmacology and Therapeutics, School of Medicine, Texas Tech University Health Sciences Centers, Lubbock, Texas 79409; ‘tDepartment of Biology, Brooklyn College of the City University of

New York, Brooklyn, New York 11210; and $Department of Oceanology, University of Chile at Valparaiso, Casilla 13-D, Viiia del Mar, Chile

Accepted June 1, 1978

Hypophysectomized killifish (Fundulus heteroclitus) adapted to calcium-deficient seawa- ter showed significant decreases in plasma calcium levels 8 days after the operation. At Day 16, plasma calcium levels were no longer significantly different from those of operated control fish. Plasma sodium and potassium levels were unaffected at all time periods. Injections of whole carp (Cyprinus carpio) pituitary homogenate corrected the hypocalcemia seen in the hypophysectomized killifish. When the carp glands were divided into “anterior,” “middle,” and “posterior” portions and tested in the hypocalcemic fish, the “middle” portion had the greatest corrective effect. Histological studies demonstrated that this portion probably contains the largest number of prolactin cells. In all cases, plasma sodium levels were elevated. In one experiment, a prolactin cell-free “posterior” portion had no hypercal- cemic or hypematremic effect. Injections of prolactin produced a log-dose-related hypercal- cemia and hypernatremia in the hypophysectomized killifish. The highest dose of prolactin used (10 pg/g of body wt per injection) had a reduced hypercalcemic effect. Based on all these data, we suggest that prolactin may be the pituitary hypercalcemic principle in the killifish.

Fishes maintain their plasma calcium levels quite precisely, even in low-calcium environments. It is very likely that they have hypercalcemic regulatory mecha- nisms. However, the identity of the hyper- calcemic hormones in fishes has not yet been clearly established. Although most tetrapods possess parathyroid glands which serve to raise serum calcium levels, these glands are absent in fishes. Hypophysec- tomy has been shown to produce hypocal- cemia and hyponatremia in fishes (Fon- taine, 1956; Chan and Chester Jones, 1968; Chan et al., 1968). In killifish (Fundulus heteroclitus) adapted to calcium-deficient seawater, hypophysectomy resulted in hypocalcemia and tetanic seizures, while the levels of other serum electrolytes were unaffected (Pang et al., 1971). This study indicates that killifish pituitary glands may

contain a specific hypercalcemic factor. In a subsequent investigation, prolactin was implicated as a possibly hypercalcemic hormone in the killifish pituitary gland (Pang et al., 1973b).

In the present study, the hypercalcemic function of the killifish pituitary gland was investigated more extensively. The effect of administering mammalian prolactin or homogenates of different parts of carp (Cyprinus carpio) pituitary glands was tested in hypophysectomized killifish that had been maintained in calcium-deficient seawater for at least 3 weeks.

MATERIALS AND METHODS Male killifish, Fundulus heteroclitus, were pur-

chased from a commercial supplier in Queens, New York. The fish were kept at 15” in calcium-deficient seawater prepared from calcium-deficient Instant Ocean sea salts (Aquarium Systems, Inc.) for at least

306 0016~6480/78/0362-0306$01.00/O Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

PITUITARY REGULATION OF FISH CALCIUM 307

3 weeks before use in experiments. This artificial sea- water contains 2 to 3 mg of calcium/ml. The animals were fed a low-calcium Aronson food mixture (Pang et al., 1971). The fish were anesthetized with Finquel (Ayerst) and hypophysectomized or mock operated by an opercular approach. The fish were not fed for 3 days after the operation. In experiments which in- volved injections of substances the hormonal prepa- rations were injected intraperitoneally with a 23-gauge needle. Daily injections started 3 days after the opera- tion and continued for 5 days. The animals were killed 2 hr after the final injection. Blood was col- lected from the cut end of the tail with heparinized he- matocrit tubes. Plasma calcium was determined with the Yanagisawa (1955) method. Plasma sodium and potassium were analyzed with an Instrumentation Laboratory flame photometer and chloride and with a Buchler-Cotlove chioridometer. Three main groups of experiments were conducted. They will each be described below.

For histological studies, several carp brain- pituitary complexes were fixed in Helly’s solution and embedded in paraffin. Five-micrometer serial sections were stained with Masson’s trichrome stain or the lead-hematoxylin-Pas method (Solcia ei a/., 1969).

The three main groups of experiments were as fol- lows.

(a) Hypophysectomized or mock-operated killifish were killed 4, 8, or 16 days after the operation.

(b) In this group of experiments, different regions of the carp pituitary gland were tested on hypo- physectomized killifish adapted to calcium-defi- cient seawater. One hundred fifty sexually mature male and female carp (Cyprinus car@) were pur- chased from a New York City distributor. These fish weighed between I and 1.5 kg each. The fish were used as soon as they arrived at the laboratory. The gonads of the fish occupied the major portion of their body cavity and contained either ripe (yolky) eggs or free-flowing sperm. Gametes could be expressed eas- ily by gentle pressure on the fish abdomen.

A total of three experiments were performed. The design of the first two was the same, but that of the third was modified. In the first two experiments, the carp were decapitated, the cranium opened, and the brain with the attached pituitary gland removed. Under a dissecting microscope. the pituitary gland was divided with a surgical blade into three portions (“anterior.” “middle,,” and “posterior”). Each region was immediately frozen on dry ice in separate con- tainers and then stored at -55”. In addition, several brain-pituitary complexes were fixed for histological examination to correlate the regional distribution of cell types.

The results of the: first two experiments revealed that the posterior portion of the pituitary gland con- tained some prolactin-like activity. Therefore, in the third experiment, a smaller portion of the posterior

part was used to avoid contamination with prolactin cells. To compensate for the smaller size of this por- tion, the number of glands per gram of body weight per injection was doubled in this experiment.

In each experiment, the sectioned glands were fro- zen for no longer than 2 months. The day before the injections started, the portions of the glands were thawed and homogenized in 0.6% saline with a Teflon pestle homogenizer. The volume of the homogenate was adjusted so that 0.01 ml of the solution contained 4% of a carp gland portion or whole giand. In the third experiment, the posterior portion homogenate contained 8% of a carp gland portion. The homoge- nates were divided into aliquots sufficient for each day’s injection and were frozen immediately. Just prior to the injection each day, tubes containing the various portions or the whole gland were thawed. In this way, the homogenates were only thawed once. In each experiment, one hypophysectomized and one mock-operated group of fish were injected with 0.6% saline, 0.01 ml/g of body wt per injection. Four hypo- physectomized groups received 0.01 ml/g of body wt per injection of “anterior,” “middle,” or “poste- rior” portions or whole carp pituitary glands, respec- tively. In the third experiment, the smaller posterior portion, which was relatively free of prolactin cells, was used. For each injection of “posterior”’ portion, 0.01 ml of the homogenate contained twice the num- ber of gland portions.

(c) In this third main experiment, five groups of fish were hypophysectomized and one was mock oper- ated. One group of hypophysectomized fish and the mock-operated fish were injected with 0.6% saline. The other four groups of hypophysectomized fish were injected with various doses of ovine prolactin (NIH-P-S-8 ovine). The doses of hormone were 0.3, 1.0, 3.0, and IO &g of body wt per injection. The hormone was dissolved in 0.6% saline and aliquots for daily injection were stored frozen in individual vials. They were thawed just prior to use.

RESULTS

In the experiment which tested the ef- fects of hypophysectomy at different times after the operation, serum calcium levels were significantly lower in hypophysec- tomized fish compared to the mock- operated controls at Days 4 and 8. How- ever, the effect of the removal of the pitui- tary gland on plasma calcium levels became minimal by Day 16. At all three times, there was a detectable, but statistically indignifi- cant, decrease in hematocrit, No changes were seen in plasma sodium, potassium,

308 PANG ET AL.

or chloride levels. The data are given in Table 1. The percentage changes in plasma calcium and sodium levels of hypophysectomized fish compared with those of mock-operated controls are plotted against the number of days after the opera- tion in Fig. 1. This shows that the decrease in plasma calcium levels is greatest at Day 8 and is minimal at Day 16. At the latter time, plasma calcium levels are near normal. Changes in plasma sodium levels are not obvious at any of the three sampling dates.

In the carp pituitary gland-replacement studies, hypophysectomized killifish were hypocalcemic compared with mock- operated and injected controls. In the first two experiments, homogenate containing whole carp pituitary glands was effective in correcting plasma calcium to near normal levels. The three different portions of the carp pituitary gland were also able to in- crease plasma calcium levels. However, of the three parts, the “middle” portion was the most effective. Administration of this part raised plasma calcium levels above normal values. In both experiments, hypophysectomy did not affect plasma sodium or potassium levels. Injections of pituitary gland homogenates tended to in- crease plasma sodium levels. The max- imum effect was seen with the “middle” portion of the gland. The data, summa- rized in Table 2, show that the rise in plasma sodium levels seems to correlate well with the increase in plasma calcium levels.

In the third carp pituitary gland homoge- nate injection experiment, hypophysec- tomy again produced a significant hypocal- cemia. Once again, the “middle” portion of the gland produced a greater increase in plasma calcium levels than did the “an- terior” portion of the gland. However, this time, the “posterior” portion of the gland, which was presumably free of pro- lactin cells, had no corrective effect on plasma calcium levels. In fact, the plasma calcium levels were lower than those of the

PITUITARY REGULATION OF FISH CALCIUM 309

Time in days

FIG. 1. Effects of hypophysectomy on male killifish adapted to calcium-deficient seawater.

hypophysectomized fish injected with saline. These results are given in Table 3.

The gross morphology and, to a limited extent, the cytology of the carp pituitary gland has been described previously (Bil- lard et al., 1971; Charipper, 1937; Pickford and Atz, 1957; Schruggs, 1951). Two major lobes can be recognized easily upon gross examination of the egg-shaped pituitary gland. The larger, rounded end of the gland is attached to the floor of the brain by a narrow stalk. This region comprises the rostral and caudal portions of the pars dis- talis. The smaller and more tapered region is the pars intermedia. The hypophysis is suspended in an anteroventral direction. Histological examination of mid-sagittal sec- tions reveals that the neurohypophysis en- ters one end as a broad band and tapers as it extends to the pars intermedia. Smaller lat- eral branches arborize all regions (Fig, 2.).

The pars distalis is clearly separable from the pars intermedia on the basis of its loca- tion, the cell types that populate it, and, in

some portions, the presence of connective tissue septa and basement membranes. The boundaries between the rostra1 and caudal pars distalis are less bbvious but these re- gions can nevertheless be delineated by differential distribution of cell types. Fuchsinophilic cells are the predominant cell type of the rostra1 pars distalis and the “cap” that they form on the anterodorsal side of the pituitary gland extends from the entry of the neurohypophyseal stalk to the point, of juncture with the pars intermedia (Fig. 2). Based on their location, morphol- ogy, and tinctorial properties, it ‘is pre- sumed that these cells synthesize prolactin (Schreibman and Holtzman, 1975). In the sexually mature animals used for the pres- ent study, the prolactin cell layer; may be wider near the hypophyseal stalk and taper gradually towards the pars inter-media, or it may be fairly uniform from one end to the other. This invariability is produced by ex- tensive hyperplasia of the gonadotropes of the caudai pars distalis at the prespawn-

TABL

E 2

EFFE

CTS

OF

CARP

PI

TUIT

ARY

HOM

OGEN

ATES

ON

HYPO

CALC

EMIC

KIL

LIFI

SH~

Plas

ma

Ca”+

Pl

asm

a Na

+ Pl

asm

a K+

Gr

oup

(mm

(m

eq/lit

er)

(meq

iliter

)

Expe

rimen

t 1

Oper

ated

co

ntro

ls +

salin

e 2.

73 -

+ 0.

12 (

7)

Hypo

phys

ecto

mize

d +

salin

e 2.

00 -

c 0.

04 (

7)*

Hypo

phys

ecto

mize

d +

whole

gla

nd

2.72

f

0.11

(6)

**

Hypo

phys

ecto

mize

d +

ante

rior

part

of g

land

2.48

f 0.0

6 (8

)**

Hypo

phys

ecto

mize

d +

midd

le pa

rt of

glan

d 2.8

6 +

0.09

(8)**

Hy

poph

ysec

tom

ized

+ po

sterio

r pa

rt of

glan

d 2.5

6 -t

0.07

(8)**

Expe

rimen

t 2

Oper

ated

co

ntro

ls +

salin

e 2.6

0 I

0.09

(6)

171.

5 t

6.1

(6)

2.12

+ 0.3

1 (6)

Hy

poph

ysec

tom

ized

+ sa

line

1.79

c 0.0

4 (8

)* 17

3.9

-+ 4.5

(8)

2.1

5 2

0.22

(6)

Hypo

phys

ecto

mize

d +

whole

gla

nd

2.56

? 0.0

7 (5

)**

183.

6 t

1.7

(5)*

1.90

t 0.2

1 (4)

Hy

poph

ysec

tom

ized

+ an

terio

r pa

rt of

glan

d 2.3

8 2

0.05

(9)**

17

9.4

t 6.6

(9)

2.0

3 t

0.42

(8)

Hypo

phys

ecto

mize

d +

midd

le pa

rt of

glan

d 2.7

6 zt

0.04

(7)**

20

2.6

+ 4.8

(7)

*” 2.0

3 r

0.22

(6)

Hypo

phys

ecto

mize

d +

poste

rior

part

of g

land

2.44

+ 0.0

4 (6

)**

185.

6 t

2.7

(6)*

2.15

-c 0.4

6 (6)

” Da

ta a

re g

iven

as m

ean

c SE

with

nu

mbe

r of

ani

mal

s ind

icate

d wi

thin

pa

rent

hese

s.

* Si

gnific

antly

di

ffere

nt

from

th

e op

erat

ed

cont

rols.

P

< 0.

01 (

Stud

ent’s

t

test

). **

Sig

nific

antly

di

ffere

nt

from

th

e hy

poph

ysec

tom

ized

+ sa

line

grou

p.

P <

0.01

(St

uden

t’s

t te

st)

PITUITARY REGULATION OF FISH CALCIUM 311

TABLE 3 EFFECTS OF CARP PITUITARY HOMOGENATES ON HY~OCALCEMIC KILLIFISH”

Plasma Ca’+ Plasma Na+ Piasma K+ Group mm fmm mw

Operated controls + saline 2.26 t 0.07 (9) 177.2 i 2.1 (10) 3.22 15 0.32 (IO) Hypophysectomized + saline 1.93 s 0.09 (9)* 168.4 -t_ 4.6 (10) 3.48 F 1.16 (10) Hypophysectomized + anterior part

of gland 2.26 t 0.10 (9)*” 176.5 t_ 1.6 (10) 4.08 i- 0.32 (10) Hypophysectomized + middle part

of gland 2.49 + 0.10 (6)** 184.6 + 2.5 (8)** 3.71 ” 0.56 (8) Hypophysectomized + prolactin

cell-free posterior part of gland 1.68 i- 0.14 (8)* 171.1 r 1.2 (8) 3.90 t 0.75 (8)

n Data are given as mean + SE with number of animals indicated within parentheses. * Significantly different from the operated controls. P < 0.02 (Student’s f test).

** Significantly different from the hypophysectomized + saline group. P < 0.05 (Student’s t test).

ing period. The neurohypophysis pervades the prolactin cell region and appears as small islands of tissue. Lead-hemotoxylin- positive cells, presumably the cortico- tropes, are scattered along the border of the neurohypophyseai tissue.

When the intact hypophysis is examined under the dissecting microscope, the pars

intermedia is clearly distinguished and can be easily separated by surgical means from the remainder of the gland. The rostra1 and caudal pars distalis, by comparison, cannot be delineated because of the lack of gross structural landmarks. As a result, both the “anterior” and the “middle” portions of the gland contained rostra1 and caudal

FIG. 2. Sagittal section through a carp pituitary gland. The neurohypophyseal stalk (N) enters and passes through the pars distalis (right) and terminates in the pars intermedia (left). The broken line marks the bound- ary of these two regions. Prolactin cells form a cap on the pars distalis (light-staining cells outlined by the fetter P on black stars). Cuts that were made through the gland to separate the various portions are marked by lines A and B. Area to the right of line B is the “anterior” portion. “Middle” portion is between the tines and “posterior” portion is to the left of line A. Masson’s stain. 39 x

312 PANG ETAL.

pars distalis. It follows, therefore, that prolactin-producing cells were also present in both portions. However, the results clearly indicate that the “anterior” portion contained considerably fewer prolactin cells than did the “middle” portion.

In the ovine prolactin-injection experi- ment, hypophysectomy again elicited hypo- calcemia. The hormone produced a dose- related hypercalcemic response up to 3 ,uglg of body wt. The highest dose admin- istered (10 pg/g) had a reduced effect. These data are given in Table 4 and Fig. 3. A linear relationship was observed up to a dose of 3 pg/g. The changes in plasma sodium levels are different from those of plasma calcium levels. There is a progres- sive increase in plasma sodium with the increase in doses, all the way up to the dose of 10 ,ug/g. Moreover, the percentage in- crease in plasma calcium is mugh higher than that of plasma sodium.

DISCUSSION

The participation of the pituitary gland in the hypercalcemic control of bony fishes has been suggested repeatedly. Administra- tion of pituitary gland extract elicits a hypercalcemia that is related to ovarian maturation (Fontaine et al., 1964). This would explain the hypercalcemia observed in female fish. However, testicular matura- tion does not affect the plasma calcium levels of male killifish (Balbontin et al., 1978) and in our experiments, only male killifish were used. Data from this and other previous studies show that hypophysec- tomy in male killifish produces hypocal- cemia (Pang et al., 1971). It is, therefore, possible that there may be a hypercalcemic hormone in the killifish pituitary gland and that this hormone is not related to gonadal maturation.

There have been other studies on eels to show that hypophysectomy elicited hypocalcemia (Fontaine, 1956; Chan and Chester Jones, 1968; Chan et al., 1968). However, in those investigations, there are

PITUITARY REGULATION OF FISH CALCIUM 313

+ 30

-Ca++

,,-,,Nd+

.

_ ____ *-e--c-- -_--- 1‘

C 0.3 1 3 .lO

Prolactin in pg/gm

FIG. 3. Effects of ovine prolactin injections on hypophysectomized, hypocalcemic male killifish adapted to calcium-deficient seawater.

changes in other electrolytes in addition to calcium. It would, therefore, be difficult to conclude that the pituitary gland has a spe- cific effect on calcium regulation, espe- cially since this endocrine gland is known to affect osmoregulation in fishes. However, in our present studies as well as those pre- viously reported, hypophysectomy pro- duces a distinct decrease in plasma calcium while plasma sodium, potassium, and chloride are not affected (Pang et al., 1971). This strongly indicates that the killifish pitu- itary gland has a specific hypercalcemic ac- tion. All these killifish experiments were conducted with fish adapted to artificial calcium-deficient seawater. In addition, the hypocalcemic effect of hypophysectomy was separable from the hyponatremic ef- fect under some circumstances in another species of killifish, Fundulus diaphanus, which was adapted to soft fresh water (Pang et al., 1973a). The physiological implication

of the hypercalcemic action of the k~iIi~~h pituitary gland was demonstrated: by exper- iments which showed that hypoealcemia could only be seen in fish adapted to calcium-deficient environments (Pang et cd., 1971).

In our time-sequence experiment, the hypocalcemia observed after hypbphysec- tomy increased from Day 4 to Day’8 bnt &he response disappeared by Day 16. This indi- cates that there may be some compensatory mechanism. One possibility is the’presence of another hypercalcemiti controlling sys- tem. For example, the hypercalcemic vita- min D may ,be important in fish calcium regulation, although such a function of this vitamin and its metabolite remains to be demonstrated. Another explanatiqi ,for the ev.entual return of plasma calcium letiels to near normal after hypophysectomy may depend upon the inhibition of some hy- pocalcemic mechanism by the hypocal-

314 PANG ETAL.

cemia produced by hypophysectomy. All these hypotheses remain to be proved.

What is the pituitary hypercalcemic prin- ciple(s)? We have suggested previously that prolactin may be such a hormone (Pang et al., 1973b). The hypercalcemic effects of prolactin have been suggested for mammals (Mahajan et al., 1974) and amphibians and birds (Baksi et al., 1978). In our present studies, ovine prolactin injections yielded a dose-related response in increasing plasma calcium levels. However, the highest dose of prolactin (10 pg/g per injection) produced a lesser effect. It has been reported that high doses of hormones often produce less or opposite effects. An example of such a pharmacological phenomenon can be seen with the neurohypophyseal hormone, meso- tocin, which produces diuresis in bull- frogs at low doses but is less diuretic or, even at times, antidiuretic at high doses (Pang, ‘1977; Pang and Sawyer, 1978). Why high doses of prolactin produce less of a hypercalcemic effect is not understood at present.

The carp pituitary gland-replacement experiments further substantiate the hypothesis that prolactin may be the hyper- calcemic hormone in the fish pituitary gland. From the histological examination of the carp hypophysis, we observed that the prolactin cells are not restricted to the an- terior part of the gland. In fact, the prolac- tin cells spread to the “middle” part of the gland as well. We estimated that because of the sheer bulk of the “middle” portion of the carp pituitary parts used in our experi- ment, this portion probably contained most of the prolactin cells. The results indicate that this portion of the gland exhibits the highest hypercalcemic activity in our hy- pocalcemic fish in all three experiments. The histological data also indicate that the posterior portion that we separated out from the carp pituitary gland may contain some prolactin cells. This would explain the hypercalcemic effect of this portion in the first two experiments. To show that the

hypercalcemic effect is related to prolactin cells, a smaller piece of the posterior por- tion was collected from the carp pituitary glands in our third experiment. To compen- sate for the smaller size of this prolactin cell-free posterior portion, the number of portions per gram of body weight per injec- tion was doubled. This preparation had no hypercalcemic effect and, in fact, the plasma calcium levels of fish receiving in- jections of this portion were decreased. This finding, along with our other data, lends strong support to the participation of prolactin cells in hypercalcemic regulation.

Further evidence for the specific hyper- calcemic action of prolactin comes from the analysis of plasma sodium data. In the carp pituitary gland-replacement experiment, the hypercalcemic effect was accompanied by hypernatremia. The absence of hyper- calcemia in fish injected with the prolactin cell-free posterior portion was also accom- panied by the absence of hypernatremia. In the ovine prolactin injection experiment, a dose-related hypernatremia was seen in the hormone-treated fish. In teleosts adapted to sea water, prolactin injections have been shown to produce dose-related hyper- natremia (Ball and Ensor, 1967; Clarke et al., 1971). The simultaneous appearance of hypernatremia and hypercalcemia in carp pituitary gland and ovine prolactin-treated fish strongly indicates that prolactin may be the hypercalcemic hormone in some fishes.

It has been suggested that the hypercal- cemic effect of prolactin may not be specific and may only reflect a change in osmoregu- lation which is known to be greatly affected by prolactin. In our hypophysectomized fish, the only changes we observed in all our experiments were on calcium, and not on sodium levels. Furthermore, when the fish were injected with the highest dose of ovine prolactin, the effect on calcium was minimal but the effect on plasma sodium was the greatest. In addition, the percent- age of decrease in calcium is always much greater than the percentage of increase in

PITUITARY REGULATION OF FISH CALCIUM 315

plasma sodium. All these data suggest that the effects of prolactin on these two electro- lytes are separable. In other words, prolac- tin has a specific effect on killifish calcium regulation.

There are, most likely, other hypercal- cemic endocrine systems in fishes. Estro- gen is hypercalcemic in female fish. In addi- tion, the renal enzyme for the formation of the vitamin D active metabolite (1,25-di- hydroxyvitamin DJ is present in fishes (I-Ienry and Norman, 1975; Baksi et ~2.) 1978). Although this active vitamin D hor- mone has not been demonstrated to affect calcium regulation, it has been related to phosphate metabolism in fish (Lopez et al., 1977). In birds, it has been reported that prolactin stimulates the formation of the ac- tive vitamin D, metabolite (Spanos et af., 1976a,b). It will be extremely interesting if this relationship can also be established for fishes. In a limited study on the bullfrog, prolactin elicited hypercalcemia without a significant effect on renal metabolism of vitamin D, (Bakis et al., 1978). However, further studies on lower vertebrates are needed.

We believe that our present studies strongly support the hypercalcemic func- tion of prolactin in the killifish. It is difficult to ascertain the physiological significance of this hormone in calcium regulation at this stage. It is, nevertheless, tempting to con- sider this hormone as an important hyper- calcemic mechanism in the normal physiol- ogy of killifish because in the freshwater environment both sodium and calcium are deficient and a hormone which regulates these two electrolytes is of great advantage to the animal.

ACKNOWLEDGMENT This investigation was supported by NSF Grant

No. TCM 76-82239 to P. K. T. Pang.

REFERENCES Baksi. S. N., Kenny, A. D., Galli-Gallardo, S. M., and

Pang, P. K. T. (1978). Vitamin D metabolism in bullfrogs and Japanese quail: Effects of estradiol

and prolactin. Cen. Comp. Endocriilol. 35, 258-262.

Balbontin, F., Espinosa, X., and Pang, P. K. T. (1978). Gonadal maturation and serum calcium levels in two teleosts, the hake and killifish. Camp. Biockem. Pkysioi.. in press.

Ball, J. N., and Ensor, D. M. (1967). Sp&cific action of prolactin on plasma sodium levels in hypophysec- tomized Poecilia lcrtipinna (Teleosteii). Grn. Comp. Endocrinol. 8, 432-440.

Billard. R., Breton, B., and Dubois, M. P. (197 If. Immunocytologie et histochemie des cell&es gonadotrophs et thyeotropes hypophysaires chez la carpe Cyprinus carpio. C. R. N. Acad. Sci. (Paris) 272, 981-983.

Chan, D. K. O., and Chester Jones, I. (1968). Regula- tion and distribution of plasma calcium and itior- ganic phosphate in the European eel [Anguiila anguilla L.). J. Endocrirzol. 32, 109-117.

Chan, D. K. O., Chester Jones, I., and Mosley, W. (1968). Pituitary and adrenocortical factors in the control of the water and electrolyte composition of the freshwater European eel (An&!/a unguilla L.). J. Endocrinol. 42, 91-98.

Charipper, H. A. (1937). The morphology of the hypophysis in lower vertebrates, particularly fish and amphibia, with some notes on the cytology of the pituitary of Cnrassius uuratus (the .go~dfish> and Necturus maculosus (the mudpuppy). Cold Spring Harbor Symp. Quant. Biol. 5, 151-164.

Clarke, W. C. (1971). Bioassay for prolactin using intact Tilapia mossambicn acclimated to sea

water. Amer. Zool. 11, 647. Fontaine, M. (1956). The hormonal control of water

and salt-electrolyte metabolism in fish. Mem. Sot. Endocrinol. S, 69-81.

Fontaine, M.. Bertrand, E., Lopez, E., and Cal- lamand, 0. (1964). Sur la maturation des organes gCnitaux de l’anguille femelle (AnguiIla angzdle L.) et l’tmission spontanee des oeufs en aquarium. C. R. H. Seances Acad. Sci. fParis) 259, 29Ol-2910.

Henry, H., and Norman, A. (1975). Presence of 25- hydroxyvitamin D-l-hydroxylase in species of alI vertebrate classes. Comp. Biockem. Physioi. 50& 43 l-434.

Lopez, E., Peignoux-Deville, J., Lallier, F., Colston. K. W., and Maclntyre, T. (1977). Response of bone metabolism in the eel. (Anguilla unguilla) to injections of 1,25-dihydroxyvitamin R,. C&if Tisslte Aes. 22 (Suppl.), 19-23.

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