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G E N E R A L AND C O MP AR A T I V E E N D O C R I NO L O G Y
57, 3 5 2 - 3 5 9 (1985)
Environmental Control of Prolactin Synthesis in the Teleost Fish
Oreochromis (Formerly Sarotherodon) mossambicus
S. E . W e n d e l a a r B o n g a . G. F l i k , C. W. G. M. L o w i k , a n d G. J. J. M. v a n E y s
Department o f Zoology, University o f Nijmegen, 6525 ED N ijm egen . The Netherlands
A c c e p t e d M ay 22, 1984
In the cichlid fish Oreochromis mossambicus prolactin cell activity is inversely related
to the o s m o la rity and the C a : ~ c o n c e n tr a t io n o f the am bien t water. Prolactin cell activity
was e s t i m a t e d , at the end o f a 3-week e x p e rim e n ta l period, by d e te rm in a tio n o f the rate of
prolactin s y n th e s is during incubation o f the rostral parts o f the pituitary gland, in the p re s ­
ence o f 13HJlysine. Since the s e c r e t o r y activity o f isolated prolactin cells is k n o w n to be
inversely related to the o s m o la rity o f the incubation m ediu m , the possibility was investi­
gated that the effects o f c h a n g e s in ionic c o m p o s itio n o f the ambient w a t e r on prolactin
secretion in vivo are m ediated by c h a n g e s in o sm o la rity o f the blood plasma. N o s u p p o rt
was found for this h y p o th e s is . In fish e x p o s e d to high w a t e r osmolarities prolactin cell
activity was re d u c e d , while plasm a o sm o la rity increased. In c o n t r a s t , at high C a : ~ c o n ­
c e n t r a ti o n s o f the water, w h e n prolactin secretion was inhibited to a similar exten t, plasma
o sm o la rity was significantly re d u c e d . Although direct effects o f plasm a osm olarity on p ro ­
lactin cells c a n n o t be e x c l u d e d completely, it is unlikely that plasma osm olarity is the
p re d o m in a n t factor in the control o f prolactin cell activity in situ. The physiological signif­
icance o f the c a p a c i ty o f isolated prolactin cells to re s p o n d to c h a n g e s in osm olarity o f the
ambient m e d i u m — a c a p a c i ty s h are d with s o m e o t h e r e n d o c r in e cell t y p e s — is therefore
unclear. V 1985 Academic Press. Inc.
In g e n e ra l th e r e is c o n s e n s u s a b o u t the
o s m o la r ity o f the w a t e r as an i m p o r t a n t a m ­
bient f a c to r c o n tro llin g p ro la c tin s e c r e ti o n
in fish ( E n s o r a n d Ball, 1972; D u b o u r g ct
cil., 1980; W e n d e l a a r B o n g a a n d Van d e r
M eij, 1981). F o r s p e c i e s s u c h as s t i c k l e ­
b a c k s a n d th e c i c h lid O reochrom is m osscimhicus ( f u r t h e r called tilapia), the w a t e r
c o n c e n t r a t i o n s o f C a 2+ a n d M g 2+ an d the
pH o f the w a t e r a re i m p o r t a n t a d d itio n a l
f a c t o r s ( W e n d e l a a r B o n g a , 1978; O g a s a w a ra a n d Y a m a d a , 1979; W e n d e l a a r B o n g a
a n d Van d e r Meij, 1980, 1981; W e n d e l a a r
B on ga cl ciL, 1983, 1984). H o w e v e r , s p e ­
cies-specific d iff e re n c e s h a v e b e e n r e p o r t e d
f o r t h e r e s p o n s e s o f p r o l a c t i n c e l l s to
c h a n g e s in e x t e r n a l C a :+ levels ( D u b o u r g
ct a i , 1983).
W ith r e s p e c t to th e i n t e r n a l c o n t r o l o f
p ro lactin s e c r e t i o n , tw o m e c h a n i s m s h av e
been p r o p o s e d : neural c o n tro l a n d re g u la ­
tion by blood f a c t o r s a c tin g d ire c tly on the
p ro la c tin cells, in p a r tic u la r p l a s m a o s m o 352
0016-6480/85 $1.50
Copyright « 1985 by Academic Press. Inc.
All rights of reproduction in anv form reserved.
larity. T h e r e is a m p le e v i d e n c e for the p r e s ­
e n c e o f h y p o t h a l a m i c c o n tro l (W ig h am ct
ul., 1975; P e te r a n d M c K e o w n , 1975; Ball,
1981). T h e o s m o l a r i t y o f b l o o d p l a s m a
could be a f a c t o r in m e d ia tin g the effe cts o f
the o s m o l a r i t y o f the w a t e r on p ro la c tin s e ­
c r e t i o n ( S a g e , 1968; N a g a h a m a ct cil.,
1975), since p ro la c tin cells in vitro r e s p o n d
to o s m o l a r i t y o f t h e i n c u b a t i o n m e d i u m
( S a g e , 1968; N a g a h a m a ct cil., 1975;
W i g h a m ct cil., 1977; G r a u ct cil., 1981).
H o w e v e r it s h o u ld be s t r e s s e d that the e v ­
id en ce s u p p o r t in g this h y p o t h e s i s is indi­
r e c t a n d b a s e d on th e s t u d y o f p r o l a c t i n
cells that w e re d e p r iv e d o f th e ir h y p o t h a ­
lamic c o n n e c t i o n s . T h e q u e s tio n r e m a in s to
be a n s w e r e d w h e t h e r p ro la c tin cells in situ
can r e s p o n d a u t o n o m o u s l y to p l a s m a o s ­
m o la rity if the h y p o t h a la m i c c o n tro l m ec h a n is m s are intact.
In o u r s tu d ie s on the c o n tro l o f p ro la c tin
s e c r e tio n in tilapia w e m a d e sev e ra l o b s e r v a to n s th at w e r e c o n s i s t e n t with the c o n ­
C O N T R O L O F P R O L A C T I N S E C R E T I O N IN FI SH
ce p t o f d ire ct c o n tr o l o f p ro la c tin cell a c ­
tivity by p l a s m a o s m o la rity . But the re s u lts
o f som e e x p e rim e n ts raised d o u b t about
this c o n c e p t . If s t i c k l e b a c k s o r tilapia w e re
e x p o s e d to s e a w a t e r with highly r e d u c e d
c o n c e n t r a t i o n s o f b o th C a 2+ a n d M g :+ (but
not in l o w - C a 2+ s e a w a t e r ; in this r e s p e c t
this w o r k has o fte n b e e n m i s q u o t e d ) , p r o ­
lactin cells w e re a c tiv a te d e v e n th o u g h
plasm a osm olarity increased (W endelaar
B o n g a, 1978; W e n d e l a a r B o n g a a n d Van d e r
M eij, 1980). In a n o t h e r e x p e r i m e n t , fish
w ere e x p o s e d to fre s h w a t e r with high C a 2+
o r M g :+ levels. T h e re su ltin g d e c r e a s e in
p ro la c tin cell a c tiv ity w a s a c c o m p a n i e d by
a d r o p in ste a d o f a rise in p l a s m a o s m o la r i ty
( W e n d e l a a r B o n g a a n d Van d e r Meij, 1980).
In the e x p e r i m e n t s d e s c r i b e d in the p r e s e n t
paper, p l a s m a o s m o l a r i t y w as m o d ifie d by
e x p o s i n g t i l a p i a to f r e s h w a t e r w i t h d i f ­
ferent o s m o la r i tie s o r differen t c a lc iu m c o n ­
c e n t r a t i o n s . It w a s i n v e s t i g a t e d w h e t h e r
th e re is a c o n s i s t e n t r e l a t io n s h ip b e t w e e n
p l a s m a o s m o l a r i t y a n d p r o l a c t i n cell a c ­
tivity. W hile in p r e v i o u s e x p e r i m e n t s p r o ­
lactin cell a c tiv ity w a s e s t i m a t e d by m o r ­
p h o m e t r y at light a n d e l e c tr o n m i c r o s c o p e
levels, in this s tu d y p ro la c tin cell activ ity
w as m ainly a s s e s s e d by d e t e r m i n a t i o n o f
the rate o f p ro la c tin s y n t h e s i s o f the p itu ­
itary g land s.
MATERIALS AND METHODS
F r e s h w a t e r male tilapia {O. mossambicus; this name
has r e c e n t l y b e e n p r o p o s e d for Sarotherodon m o s­
sambicus; T re w a v a s , 1981) o f about 20 g were used.
T h e s e tlsh w ere acc lim a ted e ith e r to fresh w a t e r s u p ­
plem en ted with NaCl o r to artificial fresh w a t e r (for
com p o sitio n see W e n d e la a r Bonga and Van d e r Meij,
1980) with different C a 2 + c o n c e n t r a t i o n s . T h e o s m o ­
larity o f the N a C l - e n r ic h e d fresh w a t e r varied from 10.
165, 320. and 670 to 980 ( ± 1%) mosmol/liter. T h e fish
were a d a p t e d to these solutions in several steps, as
described earlier ( W e n d e l a a r Bonga and Van d e r Meij,
1981). Transfer to w a t e r with different C a 2~ c o n c e n ­
trations was p e rfo rm ed in the following way: groups
o f tlsh w ere tra nsfe rred from t a p w a t e r ( C a 2^, 0.8 mM)
to artifical fresh w a t e r with the s a m e C a 2~ c o n c e n t r a ­
tion. After 2 d a y s three groups o f f i s h w ere transferred
to w a t e r containing 0.2 m M C a 2~, and 2 d a y s later for
two o f these g ro u p s transfer followed to either 0.1 or
353
0.05 myVI C a 2*: three o t h e r groups o f fish were t r a n s ­
ferred directly to w ater with 2.5. 5.0. 7.5, or 10.2 mA7
C a 2+ ( ± 2 % ) .
After an e x p o s u r e period o f 21 d a y s the fish were
lightly anesthetiz ed in MS-222. Blood was collected
from the caudal blood vessels and the pituitary glands
w ere dissected. M eth o d s for d e te rm in a tio n o f plasma
osmolarity. for plasma total calcium, o r for light mi­
cro sc o p y have been described earlier (W endelaar
Bonga and Van d er Meij. 1980). The glands were used
either for light m icro sco py or for amino acid incor­
poration studies.
Pulse incubations. T h e rostral pars distalis was s e p ­
a ra te d from freshly d isse c te d pituitary glands and
t r a n s f e r r e d to D u l b e c c o ' s m odified E a g l e ' s m e d i u m
(M D M ). as modified by Van Eys el al. (1983). T h e final
osm olarity and C a 2~ c o n c e n tr a t io n s were 310 m osmol/
liter and 1.25 m M, respectively, similar to the o s m o ­
lality and free Ca c o n c e n tr a tio n o f the blood plasma
o f S. mossambicus. T h e prolactin lobes were placed
in 100 fj.1 MDM and p re incubated for I hr 30 min in a
metabolic s h a k e r at 24°. S u b s e q u e n tly they were in­
c u b a t e d in 100 p .I MDM containing 15 ( j l C i [3H]lysine
( N e w England Nuclear, sp act 90 Ci/mmol) for 3 hr at
24°. A f te r w a r d s the lobes w e re h o m o g e n iz e d in 500 p.1
0 . 1 M acetic acid in an all-glass homogenizer. T he hom o g en a te was centrifuged at lO.OOOg for 5 min in a
B e c k m a n microfuge and the s u p e r n a t a n t was stored at
- 2 0 ° for later h i g h - p r e s s u r e liquid c h r o m a t o g r a p h y
( H P L C ) or freeze dried for later gel ele ctro p ho resis.
H PLC analysis. The 500-p.l sam ples were analyzed
with a Sp e c tra Physics SP 8000 high-pressure liquid
ch ro m ato g rap h (Spectra Physics, E indhoven, The
N e th e r la n d s ) e q u ip p e d with a stainless-steel column
p a c k e d with S p h e ris o rb 10 O D S ( C h r o m p a c k BV, Middelburg. The N e th erla nd s). T h e linear gradient c o n ­
sisted o f a 0.5 M formic a c i d - 0 . 14 M pyridine mixture
(p H 3.0) a n d l - p r o p a n o l . T h e flo w r a t e o v e r the
co lu m n was 2 ml/min, and 0.5-min fractions were col­
lec te d with a L K B R e d i r a c 2112 fr a c t io n c o llec tor.
F o u r milliliters A q u a L u m a ( B a k e r C h e m i c a l s ) was
a d d e d a n d th e f r a c t i o n s w e r e c o u n t e d in a Ph ilip s
liquid scintillation a n a l y z e r (Model PW 4540).
Proteins were se p a ra te d by sodium dodecyl sulfate
( S D S ) - g e l e le ctro p h o res is after L a em m li, with 15%
a c r y l a m i d e , as d e s c r i b e d r e c e n t l y (Van E y s et al.,
1983). T h e c e l s w e r e s i l v e r s t a i n e d a f t e r M o r i s s e v
(1981) and s c a n n e d with a Bio-Rad Model 1650 d e n ­
sitometer.
Statistics. T h e results were tested for statistical sig­
nificance by S t u d e n t ' s t test, at the 5% level.
RESULTS
Prolactin S y n t h e s i s in Vitro
A fte r in c u b a tio n o f the rostral p ars d is ­
talis o f the p itu ita ry g la n d s (p ro la ctin lobes)
354
W E N D E L A A R B O N G A ET A L .
in the p r e s e n c e o f [3H ]lysine, h o m o g e n a t e s
o f th e lobes an d the in c u b a tio n m e d i a w e re
a n a ly z e d by H P L C . A n a ly s is o f lobes o f
fish from n o rm a l fre sh w a t e r (10 m o s m o l /
liter; 0.8 mA</ C a 2+ ) r e v e a ls only o n e m a jo r
labeled and th e re fo re n ew ly s y n th e s iz e d
p r o d u c t that e lu te s a f te r 65 min. T h e s a m e
p r o d u c t is re le a s e d in the i n c u b a tio n m e ­
dium d u rin g in c u b a tio n (Fig. 1). O n ly t r a c e s
o f th i s p r o d u c t w e r e o c c a s i o n a l l y f o u n d
a fte r in c u b a tio n o f the p r o x im a l p a rs distalis, while it w a s a b s e n t a f te r in c u b a tio n o f
the p a rs in te r m e d ia . S D S e l e c t r o p h o r e s i s o f
this p e a k r e s u lte d in tw o b a n d s w ith M r o f
20.5K and 2 1 .5 K , w h ic h are typical fo r tila p ia p r o l a c t i n u n d e r o u r e l e c t r o p h o r e t i c
c o n d i ti o n s ( W e n d e l a a r B o n g a et al., 1984).
H P L C p e a k s eluting in the first 10 min are
13H ] 1ysine a n d small p r o d u c t s r e p r e s e n t i n g
c o n t a m i n a t i o n s o f the [3H ]Iysine p r e p a r a ­
tion (Van E y s et al., 1984). U l t r a s t r u c t u r a l
e x a m in a tio n s h o w e d th at the p ro la c tin
lobes did not c o n ta in a n y g r o w th h o r m o n e
cells o r o t h e r ty p e s o f e n d o c r i n e cells, e x ­
c e p t for s o m e A C T H cells. M in u te a m o u n t s
o f new ly s y n t h e s i z e d A C T H — eluting a f te r
40 min u n d e r the c h r o m a t o g r a p h i c c o n d i ­
tions u s e d — co u ld be o b s e r v e d (Fig. 1).
T h e total a m o u n t o f labeled p ro la c tin r e ­
c o v e re d a fte r in c u b a tio n fro m lobes and
m e d i a r e f l e c t s th e h o r m o n e - s y n t h e s i z i n g
c a p a c i t y o f the p ro la c tin lobes at the m o ­
m e n t o f d is s e c tio n a n d is th e r e f o r e u sed as
a p a r a m e t e r for p ro la c tin cell activity.
E ffec ts o f E x ter n a l O s m o l a r i t y a n d C a 2 1
on P ro la c tin S y n t h e s i s
As s h o w n in Fig. 2, e x p o s u r e o f fish for
3 w e e k s to fre sh w a t e r e n r ic h e d with N aC l
leads to a m a r k e d r e d u c tio n o f p ro la c tin cell
a c t i v i t y . In a m e d i u m t h a t is i s o - o s m o t i c
with the blood p l a s m a (330 m o sm o l/lite r),
the ra te o f p ro la c tin s y n t h e s is is only 20 %
o f that o f fish from n o rm a l fre sh w ater. At
h ig h e r o s m o la r itie s the rate o f p ro la c tin
s y n t h e s i s is s lig h tly b u t n o t s i g n i f i c a n t l y
hieher.
c
E x p o s u r e to w a t e r e n r i c h e d with C a C l 2
in d u c e s a r e d u c tio n that is e v e n m o re p r o ­
n o u n c e d if e x p r e s s e d on a m o la r basis. For
a 50% r e d u c tio n o f p ro la c tin s y n t h e s i s , as
o b t a i n e d a f te r e x p o s u r e to 2.5 m M C a C l 2
(17 m o sm o l/lite r), a c o n c e n t r a t i o n o f 80 m M
N a C l (165 m o sm o l/lite r) is re q u ir e d (Fig. 2).
O f the fish e x p o s e d to the h igh est N a C l
c o n c e n t r a t i o n (980 m o sm o l/liter) a b o u t 50%
died in the c o u r s e o f the e x p e r i m e n t . T h e
fish o f the o t h e r g r o u p s did not s h o w e n ­
h a n c e d mortality.
In b o t h e x p e r i m e n t a l s e r i e s , p r o l a c t i n
cell s iz e s h o w e d a s i m i l a r d e c r e a s e at
hig her o s m o la r itie s o r C a 2+ c o n c e n t r a t i o n s
as p ro la c tin s y n th e s i s (Fig. 2).
HPLC elution time Imin)
F ig . I . H ig h -p e rfo rm a n c e liquid r a d i o c h r o m a t o g r a m
o f newly s y n th es ize d proteins in prolactin lobes (ro s­
tral pars distalis) o f tilapia pituitary glands. Two lobes
of fre sh w a te r tilapia w ere in c u b a te d for 3 hr in m edium
c o n t a i n i n g f H ] l y s i n e . S u b s e q u e n t l y , e x t r a c t s o f the
lobes (left) and the m edium (right) w e r e subm itted to
H P L C . and the radioactivity o f the elution s a m p le s was
d e te rm in e d . Peaks eluting within 10 min re p r e s e n t u n ­
incorporated | 3H ]1ysine and c o n ta m i n a t i o n ; the peak
e l u t i n g a f t e r 65 min w a s i d e n t i f i e d as p r o l a c t i n by
S D S - g e l e le c t r o p h o r e s i s ; d o tte d line is elution g r a ­
dient as pe rc en tag e o f s e c o n d a r y solvent ( 1-propanol).
P l a s m a O s m o l a r i t y a n d Total Ca
rnr
An in c r e a s e in the N aC l c o n c e n t r a t i o n o f
the w a t e r leads to a small but c o n s i s t e n t
in c r e a s e in p l a s m a o s m o la r ity (Fig. 3). T h e
v a lu e s for fish fro m w a t e r with an o s m o ­
larity o f 980 m o s m o l/lite r differed signifi­
ca n tly from the c o n tr o l v a lu e (P < 0.05).
P la s m a total C a did not c h a n g e . E x p o s u r e
o f fish to high C a 2+ c o n c e n t r a t i o n s leads to
a slight d r o p in p l a s m a o s m o la r ity , s ta tis ti­
C O N T R O L O F P R O L A C T I N S E C R E T I O N IN FI SH
incorporation
cell volume
incorporation-
355
cell volume
Fig. 2. Effect o f w a t e r o s m o la rity (left; NaCl in fresh water) or C a 2 ' c o n c e n tr a t io n (right; C aC L in
lresh water) on prolactin cell volum e and rate o f prolactin synthesis. After e x p o s u r e o f the fish for 3
w e e k s the pituitary glands w ere r e m o v e d and either p ro c e s s e d for m icro scop y to d e te rm in e prolactin
cell volume ( s q u a re s ; n = 6) o r incubated in the p re s e n c e o f | ; H|lysine; the a m o u n t o f newly s y n ­
thesized prolactin r e c o v e r e d from prolactin lobe h o m o g e n a t e s and incubation media was estimated
by d e te rm in a tio n o f the radioactivity o f the H P L C fractions eluting b e tw e e n 60 and 70 min (dots; n
= 4; e x p r e s s e d as the p e rc e n ta g e radioactivity found for fish from normal fresh water, 10 mosmol/
liter; 0.8 m M C a 2~).
cally significant at 5 m M C a 2+ a n d higher.
If the C a 2 + c o n c e n t r a t i o n is r e d u c e d b e lo w
norm al fre s h w a te r levels, p la sm a o s m o ­
larity also d e c r e a s e s . P l a s m a total C a levels
s h o w a c o m p l i c a t e d p a t t e r n . At a m b i e n t
C a 2 + levels up fro m 7.5 m M it is signifi­
c a n t l y i n c r e a s e d {P < 0 . 0 5 ) , w h e r e a s at
c o n c e n t r a t i o n s b e lo w the c o n tro l level ( 0.8
m M C a 2+ ) p l a s m a C a first i n c r e a s e s and
then d r o p s rap id ly (Fig. 3).
DISCUSSION
H P L C a n a ly s is o f p ro la c tin lobe h o m o g ­
e n a t e s a n d in c u b a tio n m e d ia s h o w e d o n e
m a jo r p e a k th at y ield ed tw o p e a k s on S D S
gel, with M r v a lu e s o f 2 0 .5 K a n d 2 1 .5 K ,
th at are typical for p r o la c tin u n d e r o u r e l e c ­
tr o p h o r e t ic c o n d i t i o n s a n d th a t a p p e a r e d to
be specific for the p ro la c tin lobe a n d not for
the o t h e r p a rts o f the h y p o p h y s i s . F o r tilapia p ro la c tin slightly lo w e r (C la rk e , 1973;
F a r m e r et a l . , 1977) o r h ig h e r ( S p e c k e r et
cil. , 1984) v a lu e s h a v e b e e n r e p o r t e d a fte r
S D S e l e c t r o p h o r e s i s . We c o n s i d e r s u c h dif­
fe re n c e s ty pical for the S D S p r o c e d u r e ,
w h ic h in v o lv e s d e n a t u r a t i o n an d binding o f
an a n io n ic d e t e r g e n t , t r e a t m e n t s that m ay
a f f e c t e l e c t r o p h o r e t i c m o b ility . We c o u l d
m o d ify the mobility o f the 21.5-D a b a n d to
a value c o r r e s p o n d i n g to 24 Da by p r o ­
longing the d e n a t u r a t i o n t r e a t m e n t from 1
(as usual) to 3 min. A p p a re n tly , S D S - g e l
e l e c t r o p h o r e s i s is not the m e t h o d o f c h o ic e
for d e te rm in a tio n o f prolactin m o lecu lar
w eight in tilapia.
External fa c to rs controlling prolactin se­
cretion. O s m o l a r i t y a n d C a 2+ c o n c e n t r a ­
tion are p ro b a b ly im p o r ta n t e n v i r o n m e n t a l
f a c to r s in the c o n tro l o f p ro la c tin s e c r e tio n
in f r e s h w a t e r tila p ia . I n c r e a s e d le v e ls o f
N a C l a n d C a C l 2 o f t h e w a t e r le a d to a
m a r k e d d e c r e a s e o f p ro la c tin cell activity.
On a m o la r basis, C a C l 2 is m u c h m o r e e f­
fective than N a C l. O b v io u sly , C a a n d not
Cl ions are the re le v a n t factor, since th e re
is no c le a r c o r r e la tio n b e t w e e n the ch lo rid e
c o n c e n t r a t i o n and the rate o f p ro la c tin s y n ­
th esis if the d a t a on N a C l an d C a C l 2 e x ­
p o s u r e are c o m p a r e d . A lth o u g h we a s s u m e
that the inhibitory effect o f the N a C I-c o n t a i n i n g w a t e r o n p r o l a c t i n s y n t h e s i s is
c a u s e d by the o s m o la r i ty o f the w a te r, a d ­
ditional effects o f e i t h e r N a + o r C l " c a n n o t
be ruled out co m p lete ly .
W E N D E L A A R BONGA ET AL.
356
Ca ImMI
mosmol/1
mosmol ' \
plasma osmolarity
plasma Ca
plasma osmolarity
plasma Ca
^
U5
350
Ca (mM>
U5
350
¿0
L0
300
300
35
35
30
250
30
250
25
25
200
200
L I______I_____ I_____ L
0
J_____ ______ I--------L
500
J_____ I_____ L. J
J -
1000
ambient osmolarity (mosmol I I
0
10
ambient Ca‘ * ImM )
3. Effect o f w a t e r o s m o la rity (NaCl in fresh water) o r ambient C a 2* (CaCL in fresh water) on
plasm a o sm o la rity (dots: // = 8) and plasm a total Ca (s q u a re s ; n = 8). Fish w ere acclim ated for 3
weeks.
F ig .
*
T h e m a r k e d r e d u c t i o n o f p ro la c tin s y n ­
thesis s h o w n by p r o la c tin cells o f fish e x ­
p o s e d to high C a 2+ levels c o n f ir m s e a r lie r
re su lts o b ta in e d by u l t r a s tr u c tu r a l m o r ­
p h o m e t r y ( W e n d e l a a r B o n g a and Van d e r
M e ij, 1980, 1981). To a l a r g e e x t e n t t h e
s a m e c o n c l u s i o n c an be d r a w n for th e ef­
fects o f a m b ie n t o sm o la rity . We have
s h o w n b e fo re th at an i n c r e a s e in the o s ­
m olarity o f fre sh w a t e r leads to a r e d u c t io n
o f p r o l a c t i n cell a c t i v i t y a s r e f l e c t e d by
m o r p h o m e t r i c a l a n a l y s i s in t i l a p i a ( W e n ­
d e l a a r B o n g a a n d Van d e r M e i j , 1980,
1981). H o w e v e r , in t h e s e e a r l i e r e x p e r i ­
m e n ts p ro la c tin cells s h o w e d signs o f high
activity in fish a d a p t e d to w a t e r with an o s ­
m o larity h ig h er th an th a t o f the blood
p la s m a , if the C a 2+ a n d M g 2+ levels w e re
k e p t low . T h i s w a s n o t o b s e r v e d in th e
p r e s e n t study. T h e r e a s o n for this d i s c r e p ­
a n c y m ay be c o n n e c t e d with the s tra in s o f
tilapia used.
P ro lactin cell a c tiv ity in tilapia in vivo is
s t i m u l a t e d n o t o n l y b y lo w w a t e r o s m o ­
larity o r C a 2+ levels, but also by low w a t e r
pH ( W e n d e l a a r B o n g a et al., 1984). R e d u c ­
tions in w a t e r o s m o la r ity , C a 2+ level, o r pH
have th re e e ffe cts in c o m m o n : the b ra n c h ia l
influx o f w a t e r a n d the p a s s iv e o u tflo w o f
ions are initially i n c r e a s e d , p l a s m a o s m o ­
larity and ion c o n c e n t r a t i o n s a re d e c r e a s e d ,
and p ro la c tin cells b e c o m e a c t iv a t e d ( W e n ­
mm
d e l a a r B o n g a et al., 1984). S ince p ro la c tin
r e d u c e s gill p e rm e a b ility to w a t e r a n d ions
and i n c r e a s e s p l a s m a e le c t r o l y te levels in
fish (C la rk e and B e rn , 1980), s tim u la tio n o f
p ro la c tin s e c r e tio n s e e m s an a d e q u a t e r e ­
s p o n s e to c o u n t e r a c t the effects o f r e d u c e d
w a t e r o s m o la rity , C a 2+ c o n c e n t r a t i o n s , o r
pH.
Internal f a c t o r s controlling p r o la c ti n s e ­
cretion. If fish p ro la c tin cells a re in c u b a te d
in c u ltu r e m ed ia, th eir s e c r e t o r y activ ity is
s tim u la te d w h e n the o s m o l a r i t y o f the in c u ­
b a t i o n m e d i u m is r e d u c e d ( N a g a h a m a et
al., 1975: W ig h a m et al., 1975, 1977: B e n ­
j a m i n and B aker, 1976, 1978: G r a u et al.,
1981; o u r u n p u b lis h e d o b s e r v a t i o n s ) . T his
r e s p o n s e has led to the su g g e s tio n that the
s tim u la tio n o f p ro la c tin cells as o b s e r v e d
a f te r tr a n s f e r o f fish fro m s e a w a t e r to fresh
w a t e r is m e d ia te d by a r e d u c ti o n o f p l a s m a
o sm o la rity . O u r p r e s e n t d a t a d o not s u p p o r t
th is s u g g e s t i o n , s i n c e t h e r e d u c t i o n o f
p la s m a o s m o la r it y in fish e x p o s e d to high
w a t e r C a 2+ levels w as a c c o m p a n i e d by a
r e d u c tio n o f p ro la c tin s y n th e s is . T h u s it is
unlikely that p ro la c tin s y n th e s is in tilapia is
p r e d o m i n a n t l y c o n tro lle d by d ire c t o r in­
dire ct effects o f p la s m a o s m o la r it y on th e s e
cells. B r e w e r an d M c K e o w n (1980) c a m e to
the s a m e c o n c l u s i o n fo r c o h o s a l m o n , a f te r
th e y fo u n d th at th e s tim u la tio n o f p ro la c tin
s e c r e tio n follow ing t r a n s f e r o f the fish from
357
C O N T R O L O F P R O L A C T I N S E C R E T I O N IN F I SH
s e a w a t e r to fresh w a t e r w a s not a c c o m p a ­
nied by n o ta b le c h a n g e s in p l a s m a o s m o larity.
A p p a r e n tly , tilapia p ro la c tin cells s h o w a
c o n s i s t e n t r e s p o n s e to o s m o l a r i t y o f th e
s u r r o u n d i n g fluid only in the a b s e n c e o f in­
tact h y p o t h a l a m i c c o n tr o l m e c h a n i s m s . A
sim ilar c o n c l u s i o n ca n be d r a w n fo r p r o ­
lactin cells o f E u r o p e a n eels a n d rats that
are also s tim u la te d in vitro a f t e r r e d u c tio n
o f the o s m o l a r i t y o f the in c u b a tio n m e d iu m
(B e n ja m in a n d B a k e r, 1976, 1978; L a B e lla
et a l., 1975). In c o n t r a s t , if in E u r o p e a n eels
p l a s m a o s m o l a r i t y is r e d u c e d by e x p o s i n g
the fish to d e io n iz e d w a te r, the p ro lac tin
cells are u n a ff e c te d ( O liv e r e a u et al., 1980,
1981). Similarly, p l a s m a p ro la c tin levels in
rats are not n o tic e a b ly in flu e n c e d by e x ­
perim entally induced in c re a se s or d e ­
c r e a s e s in p l a s m a o s m o l a r i t y ( M a t t h e i j ,
1977a,b). T h u s , t h e p h y s i o l o g i c a l s ig n if i­
c a n c e o f the r e s p o n s e o f p ro la c tin cells in
vitro to c h a n g e s in o s m o l a r i t y s e e m s u n ­
clear. T his is the m o r e so sin ce su ch a r e ­
s p o n s e is not typical for p ro la c tin cells. It
has also b een d e s c r ib e d for in c u b a te d
A C T H a n d g r o w th h o r m o n e cells o f E u r o ­
p e a n eels a n d the molly Poecilia latipinna
(B e n ja m in a n d B ak e r, 1978; B a tte n et al.,
1983) a n d b o v i n e a d r e n a l m e d u l l a r y c e lls
( H a m p t o n a n d H o lz , 1983).
We h a v e s u g g e s te d th at p l a s m a c a lciu m
in stead o f p l a s m a o s m o l a r i t y m a y influen ce
p r o l a c t i n s e c r e t i o n in fish ( W e n d e l a a r
B o n g a a n d G r e v e n , 1978). H o w e v e r , th e
p r e s e n t d a t a s h o w that th e r e is no c o n s i s ­
tent r e la tio n s h ip b e t w e e n p l a s m a c a lc iu m
levels a n d p ro la c tin s y n t h e s i s in tilapia. A l­
th ou gh the p r e s e n c e o f c a lc iu m ions m a y
be a p r e r e q u is ite for basal p ro la c tin re le a s e
( G r a u et al., 1981; M a c D o n a l d a n d M cK e o w n , 1983), as it is fo r re le a s e in m a n y
gland cells, it is u nlikely th at the p ro la c tin
cells in tilapia a re p r e d o m i n a n t l y c o n t r o l l e d
by c h a n g e s in p l a s m a c a lc iu m levels.
In the p r e s e n t e x p e r i m e n t s a c o m p l i c a t e d
re la tio n s h ip w a s fo u n d b e t w e e n the c a lc iu m
c o n c e n t r a t i o n s o f the w a te r and blood
p la sm a . A lth o u g h in general the p la s m a c a l­
ciu m levels are c o r r e la t e d positively to the
e x te r n a l c a lc iu m c o n c e n t r a t i o n , a re d u c tio n
in the ca lciu m c o n c e n t r a t i o n o f the w a t e r
from 0.8 to 0.2 m/VZ leads to an in c re a se in
p l a s m a c alciu m . T h e m ost simple e x p l a n a ­
tion is that p la s m a c a lciu m levels a re d e ­
te r m in e d by both the c alciu m c o n c e n t r a t i o n
o f the w a t e r and the rate o f s e c r e tio n o f a
h y p e r c a lc e m ic h o r m o n e , for w h ich p r o ­
l a c t i n is a lik e ly c a n d i d a t e ( P a n g , 1981;
W e n d e l a a r B o n g a a n d Flik, 1982).
We suggest that p ro lactin s y n th e s is and
re lea se a re mainly c o n tro lle d by h y p o t h a ­
lamic tr a c ts , and not by d ire c t effects o f
p l a s m a f a c t o r s on t h e p r o l a c t i n c e l l s .
W h e r e a s i n h i b i t o r y c o n t r o l by d o p a m i ­
nergic fibers has b e e n well e s ta b lis h e d for
teleo st p ro la c tin cells, th e re is g ro w in g e v ­
id e n c e that th e re a re in ad dition s tim u la to r y
tr a c ts that m a y be s e r o to n e r g ic (Ball, 1981).
T h e p ro b le m r e m a in s to be so lved as to how
in fo rm a tio n a b o u t the ionic c o m p o s i t i o n o f
the w a t e r is m e d i a te d to the cen tral n e r v o u s
s y s t e m . M o re a tte n ti o n sh o u ld be paid to
th e s e n s o r y i n n e r v a t i o n o f th e b r a n c h i a l
a re a , since M a y e r-G o s ta n and H ira n o
( 1976) s h o w e d that in eels t r a n s e c t i o n o f the
vagal an d g l o s s o p h a r y n g e a l n e r v e s , w hich
in n e r v a te the b ra n c h ia l a re a , has p r o ­
n o u n c e d e f f e c t s on o s m o r e g u l a t i o n . T h e
a u t h o r s s u g g e s te d that efferent n e rv e fibers
m ay send inform ation from branchial re ­
c e p t o r s to the brain that m ay reg u la te the
re le a se o f h o r m o n e s .
ACKNOWLEDGMENTS
T h e a u t h o r s are indebted to Mr. A. C o e n e n , Mr.
G. H. G o y v a e r t s , and Mr. P. Cruijsen for technical a s ­
sistance.
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*
*
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