Changes in Plasma and Pituitary Prolactin Levels in Toad (Bufo japonicus) Larvae during Metamorphosis(Endocrinology)

5 Pages·1991·1.7 MB·English

by  Kazuhiko Niinuma

ZOOLOGICAL SCIENCE 8: 97-101 (1991) 1991 ZoologicalSocietyofJapan
Changes in Plasma and Pituitary Prolactin Levels in Toad
(Bufojaponicus) Larvae during Metamorphosis
Kazuhiko Niinuma, Kazutoshi Yamamoto
and Sakae Kikuyama1
Department ofBiology, School ofEducation, Waseda University,
Shinjuku-ku, Tokyo 169-50, Japan
—
ABSTRACT Bothplasma andpituitaryprolactin (PRL) concentrationsinBufojaponicustadpolesat
variousdevelopmentalstagesweredeterminedbyahomologousradioimmunoassay. PlasmaPRLlevels
continued to rise gradually as metamorphosisproceeded, the values at late climaxbeingabout 3 times
higher than those at the premetamorphic stage. PRL concentrations and the amount of PRL in the
pituitaryglandalsoincreasedduringthepreclimaxperiodandreachedamaximumatmid-climaxwitha
slightdeclineattheendofmetamorphosis. PRLsynthesis,asmeasuredbyincorporationof 3H]leucine
[
into pituitary PRL in vitro, was relatively low duringpremetamorphosis, continuedto rise throughout
the prometamorphic period, reached amaximum atclimax anddeclined at the endofmetamorphosis.
Taken together, these data indicate that the function ofthe pituitary gland in toad larvae in terms of
PRL secretion is gradually enhanced as metamorphosis progresses. The present resultsobtained with
toad tadpoles and those obtained previously with Rana catesbeiana larvae are discussed, taking the
differences in metamorphic pattern between the two species into consideration.
homologous radioimmunoassay for toad (Bufo
INTRODUCTION
japonicus) PRL has been developed [7]. It is
The discovery that mammalian prolactins therefore of interest to see whether the plasma
(PRLs) exert anti-metamorphic [1] and growth- PRL levels in toad tadpoles, which are compara-
promoting [2, 3] effects on amphibian larvae tively small and undergo complete metamorphosis
prompted a hypothesis that PRL levels are high rapidly, exhibitchangessimilartothoseinbullfrog
during premetamorphosis and prometamorphosis larvae, which metamorphose rather slowly with a
and begin to decline once metamorphosis has long growth phase. Changes in the PRL contents
started, so thattadpolesgrowduringthepreclimax and PRL synthesis in the pituitary gland during
period and metamorphose rapidly once the anim- metamorphosis were also studied.
alshave reached climax [4]. However, withregard
to bullfrog (Rana catesbeiana) larvae, it has been MATERIALS AND METHODS
found that plasma PRL values obtained by homo-
logous radioimmunoassay do not fit this hypoth-
esis. Immunoassayable PRL levels are relatively Animals
low during the preclimax period [5] and begin to Eggs ofB. japonicus collected in the suburbs of
rise at mid-climax when the tissues have under- Tokyo were hatched in our laboratory at 23°C.
gone considerable transformation through the Tadpoles were fed on boiled spinach. The stages
effect of thyroid hormone [5, 6]. Up to now, of metamorphosis were classified according to
plasma PRL levels in anuran larvae have been Limbaugh and Volpe [8].
determined only for R. catesbeiana. Recently, a
Plasma andpituitary samples
Blood was taken from tadpoles by insertion ofa
Accepted September 1, 1990
ReceivedJuly 11, 1990 heparinized capillary into the heart. Each sample
1 To whom all correspondence should be addressed. (about 200-400/d) was collected from 20-30
98 K. Niinuma, K. Yamamoto and S. Kikuyama
animals at the same developmental stage. After metabolic shaking incubator gassed with 95% 2-
collection, bloodsampleswere centrifugedandthe 5% C02. After incubation, the glands together
plasmawasstoredat —70°Cuntiluse. The anteri- with the medium were homogenized. An aliquot
or pituitary gland was quickly dissected out under of the homogenate was used for protein deter-
adissectingmicroscope. Eachsampleconsistingof mination. Tothehomogenate, anequalvolumeof
10 pituitaries taken from animals at the same 5% polyacrylamide sample gel (pH8.6) was
developmental stage was homogenized with a added. Themixturewashomogenizedandlayered
Teflon homogenizer in 500/A distilled water and onthetopofpolyacrylamidegels. Electrophoresis
stored at —70°C until assay. was performed according to the procedure de-
scribed elsewhere [12]. After electrophoresis, the
PRL radioimmunoassay gel was stained with amido black 10B in 7% acetic
PRL for antiserum production, radioiodination acid for identification ofthe PRL band. The band
and use as a reference standard was purified from was dissected out, placed in a screw-capped vial
H
anterior pituitary glands of adult toads [9]. Anti- containing 200//l 2 2, and heated in an oven
serum against the toad PRLwas raised in afemale at 80°C. After solubilization, scintillation fluid
rabbitbythe multiple-siteinjectiontechnique [10]. (Aquasol 2, NEN) was added to the vial and
Radioiodination oftoad PRLwith Na125I (carrier- radioactivity was measured in a liquid scintillation
free;The RadiochemicalCentre,Amersham, Eng- counter.
land) was carriedout atroom temperature accord-
ing to the modified lactoperoxidase method [11]. Statistical analysis
The specific radioactivity of the radioiodinated Statistical analysis was performed using Stu-
PRL was about 40-50pCi/jug. A 30% specific dent's t-test.
binding of the added radioligand was obtained in
the absence of any unlabeled PRL when the anti-
RESULTS
serum was used at a final dilution of 1:20,000.
Details of the radioimmunoassay have been de- AsindicatedinFigure 1, plasmaPRLlevelsrose
scribed elsewhere [7]. It had been confirmed that graduallyasmetamorphosisprogressed. The aver-
the neurointermediate lobe homogenate and age value at the end of metamorphosis (stage 46)
mammalian PRL and growth hormone did not was about 3 times higherthan that at thepremeta-
cross-reactwiththeantiserum, andthattheplasma
of hypophysectomized toads showed the least de-
gree of cross-reaction. Sensitivity of the radioim-
munoassay averaged 0.12ng per 100/A of assay
E
buffer. The interassay coefficient ofvariation was tn
9.2% and the intraassay coefficient of variation
Q.
8.0%.
Determination of PRL synthesis in the pituitary
gland a.
PRL synthesis in the pituitary glands from tad- c
poles at various developmental stages was mea-
sured in vitro. Whole pituitary glands from 50 36 38 40 42 44 46
tadpolesatthe same developmentalstagewereput
into a vial containing 150fA of67% Eagle's MEM Stages
in which the leucine concentration was reduced to Fig. 1. PlasmaPRLlevelsintoadtadpoles. Eachpoint
10% and supplemented with 1 //Ci 3H]leucine and vertical line represent the mean of 15 deter-
[ minations and standard error of the mean, respec-
(NEN; spec, act., 145 Ci/mmol). The pituitary tively. * Significantly different from preceding
glands were incubated at 25°C in a Dubnoff- value at 5% level.
Prolactin in Bufo Larvae 99
morphic stage (stage 36). tration but not total amount of pituitary PRL
BoththetotalamountandconcentrationofPRL declined significantly (Fig. 2).
in the pituitary gland were low at stage 36, but Pituitary glands of stage 46 animals were incu-
increased throughout the prometamorphic stages bated in the presence of 3H]leucine. The incor-
[
and reached a maximum at mid-climax (stage 44). poration of the isotope into PRL increased over a
After completion of metamorphosis, the concen- period of 24hr (Fig. 3). PRL synthesis in the
pituitary glands of tadpoles at various stages was
500
. measured by monitoring the incorporation of
3H]leucine into PRLfor20hr. PRLsynthesiswas
[
I 400 found to be enhanced during prometamorphosis
o and remained high during mid-climax. At the end
a. 300 of metamorphosis, PRL synthesis declined con-
u> siderably (Fig. 4).
15
2 200
cc o
Q. 100
5
c 10
c
36 38 40 42 44 46 fl)
o
Stages a
Fig. 2. Changes in PRL levels in the anterior pituitary 5
of toad tadpoles at various developmental stages. 8o*
Eachpointandverticallinerepresentthemeanof13
determinations and standard error of the mean,
respectively. * Significantly different from preced- aE
ing value at 5% level.
°
36 38 40 42 44 46
Stages
Fig. 4. PRL synthesis in the pituitary glands of toad
tadpoles during metamorphosis. Whole pituitary
glands from tadpoles at various developmental
stages were incubated with [3H]leucine for 20hr at
25°C. Radioactivityofelectrophoreticallyseparated
PRL from the medium and pituitary glands repre-
sents PRLsynthesized in the pituitaryglandsduring
the incubation period. Each point and vertical line
represent the mean of 6 determinations and stan-
darderrorofthemean,respectively. * Significantly
different from preceding value at 5% level.
Incubation Time (hr)
Fig. 3. Timecourseofincorporationof 3H]leucineinto DISCUSSION
[
prolactin in the pituitary glands of toad larvae.
Whole pituitary glands were incubated with The present experiment revealed that plasma
[3H]leucine for 24hr at 25°C. PRL in the medium PRL levels in toad tadpoles are initially low, and
watranosdphmpoiertaeussiiutsra,reyda.gnldaEnatdhcsehwrpaaosdiinostaecrpteaiprvraiettsyeednotfbsytthdheiesPcmRegeLalnbeolaefncd2- otfhebnulrlifsreoagstmaedptoalmeosr,pthhoesipslapsrmoacePedRsL. lIenvetlheisclaoswe
determinations. and remains rather constant until the animals
100 K. Niinuma, K. Yamamoto and S. Kikuyama
reach theclimaxstage [5]. Duringtheearlyclimax PRLlevelsbetweenthe twospecies atlate climax.
stages, elevation ofthe PRLlevel is not so conspi- Inconclusion, PRLsynthesis andreleaseintoad
cuous. Then the level rises markedly during mid- tadpoles are enhanced as metamorphosis progres-
climax and reaches a maximum at late climax [5, ses. However, their pituitary function in terms of
6]. Although the PRL levels in toad tadpoles PRL secretion seems to be lower than that of
continue to rise throughout the larval period, they bullfrog tadpoles or adult toads.
are about 1/10-1/20 of the levels in bullfrog
tadpoles [5] at comparable stages. Endogenous ACKNOWLEDGMENTS
PRL is known to have antimetamorphic and
growth-promoting activities [5, 13, 14]. Accord- This work was supported by a research grant from
ingly, thedifferenceinPRLlevelsbetweenthetwo Waseda University and Grants-in-AidfromtheMinistry
ofEducation and Science Culture ofJapan to S.K.
species may reflect the difference in the larval size
aswellasthedurationofthelarvalperiodbetween
the two species. Toad tadpoles complete meta- REFERENCES
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