Changes in Specific Gonadotropin Binding Sites in the Liver during Metamorphosis in the Bullfrog, Rana catesbeiana(Endocrinology)

7 Pages·1991·4.2 MB·English

by  Kaoru Kubokawa

ZOOLOGICAL SCIENCE 8: 759-765 (1991) \ 1991 Zoological Societ) ofJapan
Changes in Specific Gonadotropin Binding Sites in the Liver
during Metamorphosis in the Bullfrog,
Rana catesbeiana
Kaoru Kubokawa
DepartmentofBiology, SchoolofEducation, and Advanced Research Centerfor
Human Sciences. Waseda University, Xishi-W'ascda, Tokyo 169-50. Japan
—
ABSTRACT The author studied bindings of radioiodinated bullfrog follicle-stimulating hormone
(fFSH) and luteinizing hormone (fLH) to a crude plasma membrane traction ofthe liveroftadpoles of
various stages and adults ofthe bullfrog. Ranacatesbeiana. Slight specific bindingsofthe labeled II SII
and fLH were detected in tadpoles between Stage V and XX. Specific bindings o\ the radioiodinated
hormones increased significantly from Stage XXL which is just after "climax" of metamorphosis.
Furthermore, accumulation of3'-5'cyclicadenosine monophosphate wasstimulated by both fFSH and
fLH in small blocks of the liver derived from individuals after the climax of metamorphosis but not in
those derived from individuals before the climax. These resultsstrongly suggest that, in bullfrogs after
the climax of metamorphosis, gonadotropins act on the liver through specific membrane receptors
coupled with the adenylate cyclase system, and stimulate some biochemical process in the liver.
tissue isequal to or higherthan the total numberof
INTRODUCTION
the sites in the ovaries. However, none of these
Challenging the general belief that distribution studies has revealed the final physiological action
of gonadotropin receptors in the body of verte- of gonadotropin in these organs mediated by the
brates is restricted to the gonads, Kubokawa and extragonadal gonadotropin receptors.
Ishii [1] demonstrated that in adult amphibians the Since amphibians show drastic metamorphosis,
liver as well as the gonad has specific receptors for it is important and interesting to know the time ol
gonadotropins, and also that bullfrog follicle- occurrence of the hepatic gonadotropin receptors
stimulating hormone (fFSH) and luteinizing hor- through the life of the bullfrog. The present
mone (fLH) can stimulate accumulation of 3',5'- author sutdied change in gonadotropin bindings to
cyclic adenosine monophosphate (cAMP) in the the liver, if any. during the metamorphosis of the
liver in adult bullfrogs. They showed that hor- bullfrog.
mone-binding propertiesofthese hepatic gonadot-
ropin receptors are almost identical to those ol MATERIALS AND METHODS
idal gonadotropin receptors. Thus, they de-
monstrated that specific bindings ofgonadotropins
Materials
to the hepatic receptor are not nonspecific uptake
of glycoproteins, and should play a specific phy- Bullfrog {Rana caleshetana) tadpoles were
siological role, although it is unknown. Beside our purchased from a commercial dealer. The) had
study, extragonadal gonadotropin receptors was been kept under a constant temperature (25
shown in the pig by Ziecik et al. (2). The) and light condition (121 121)) lor several days to
estimated that in pregnancy the total number of Several months until they reached appropriate
rific gonadotropin binding sites in the uterine stages, and then used for experiments which Were
performed between fcbniar\ and \1a\.
1
Accepted April 5. 1991 Stanes of the tadpoles at the time <-l experiments
Received March 1. 1991 were V. \ I. and \l\ XXV of fayloi and Kollms
C
760 K. KUBOKAWA
[3]. Adult male bullfrogs of over several months concentration of 10mg of the original fresh tissue
after metamorphosis were also purchased and in 100ju\, and used as the crude plasma membrane
used. preparation for binding experiments. The tissue
concentration in the membrane preparation
Hormones and chemicals ranged between 10mg and 20mg per 100ju\
Bullfrog FSH and LH used were FF1341B and among samples of different metamorphic stages.
A
FL421B of Takada and Ishii [4], respectively.
gonadotropin rich glycoprotein fraction was pre- Binding experiments
pared from bullfrog pituitaries according to Taka- One hundred microliters of the plasma mem-
da and Ishii [4] and used as competitor for obtain- brane preparation were mixed with 50^1 of 125I-
ing nonspecific binding. Carrier-free Na[125I] was fFSHor 125I-fLH (ca. 10,000cpm) and50ju\ofthe
purchased from Amersham Japan Corporation, bullfrog pituitary glycoprotein solution containing
Tokyo. An assay kit for radioimmunoassay of 5jug of the glycoprotein or the same volume of
cAMP was purchased from Yamasa Shouyu Cor- buffer alone. The radioligands and the pituitary
mM
poration, Chiba. As an inhibitor of phosphodies- glycoprotein were dissolved in 40 Tris-HCl,
terase, l-methyl-3-isobutylxanthine (MIX) of Sig- pH7.4, containing 50mM MgS04 and 0.1 % BSA
ma Chemical Co. Ltd. was used. (incubation buffer). The mixture was incubated
for2.5 hr at20°C when 125I-fLHwasused, andfor
Iodination 17hr at 15°C when 125I-fFSH was used. Mem-
Bullfrog FSH and LH were radioiodinated by brane-bound hormone was separated from free
the lactoperoxidase (LPO) method [5]. Briefly, hormone by centrifugation at 10,000Xg for 3 min
2.5jug ofbullfrog FSH or LH was reactedwith0.5 after addition of 1 ml ofchilled incubation buffer.
jug LPO and 0.2mQ ofNa[125I] for 1 min at room The pellet was washed twice in an aliquot of the
temperature. Free and bound iodide were sepa- buffer, andthencountedforradioactivity. Specific
rated by a 1.5x20cm column of Sephadex G-75. bindingwas determined by subtracting nonspecific
SpecificradioactivitiesofradioiodinatedfFSH and binding(bindingofradioligandunderthepresence
fLH were estimated by matching data ofcompeti- of the pituitary glycoprotein) from the total bind-
tion and saturation experiments as described by ing (binding of the radioligand under the absence
Kubokawa and Ishii [5]. Specific radioactivities of of the pituitary glycoprotein). All the determina-
125I-fFSH and -fLH were about 40juOJjug. tions were done in duplicate.
Plasma membranepreparation Experiments on effects offFSH and fLH on the
cAMP
Tadpoles of different stages and adult males concentration in the liver
were anesthetized in ice water and decapitated. Liversweredissectedoutfrom tadpolesofStage
Livers were dissected out immediately, washed in XVIII, XIX, XX, XXI, XXIV and adults (3-6
chilled amphibian saline, and put into a plastic months old). Each fresh liver was minced finely
tube individually numbered. They were frozen by into 1-2mm cubes and washed several times by
—
immersing in liquid nitrogen and stored at 80° decanting and adding chilled amphibian Krebs-
until use. Ringer bicarbonate (aKRB). The tissue cubes
Each frozen liver was thawed, weighed and were dispersed in aKRB as to 1 ml of the disper-
homogenized for 1 min with micro homogenizer sioncontainsabout80mgofthetissue. Analiquot
(Muromachi Co. Ltd., Tokyo) in 5 volumes of of250ju\ ofthe cube dispersion was transferred to
chilled 40mM Tris-HCl buffer, pH7.4 containing each well of a 24-well microplate. One hundred
50mMMgS04 (receptorbuffer). The homogenate microliters of fLH or fFSH solution in aKRB
was centrifuged at 10,000xg for 20min at 4°C. containing 1 jug of hormone and 150jA of aKRB
The pellet was washed by suspending in 20ml of containing 10mM MIX were added to each well.
buffer and centrifuging again. The washed pellet Then, the microplates were incubated for 2hr at
was resuspended in the receptor buffer at the 25°C with shaking under 95% 2 and 5% C02.
Gonadotropin Receptors in Tadpole Liver 761
After the incubation, liver blocks were collected Stage \1\. although it was detectable throughout
from each well and used for determination of the metamoiphic stages studied (Fig. 2). From
cAMP as follows. The liver blocks were homoge- Stage XXI, the specific binding started to increase
nized in one milliliter oia 6rr trichloro acetic acid and exceeded the nonspecific binding level from
solution, and cAMP in the tissue was extracted.
The bomogenate was centrifuged at 3.000 rpm for
5 min. and the resulting supernatant was transfer-
red to a glass tube. Five volumes of water satu-
rated with ethyl etherwas added to each tube, and
the tube was vigorously shaken for removing lipid. 6000
The aqueous layer was separated, and cAMP in
the aqueous layer was determined by radioimmu-
noassay using the kit according to the maker's
instruction.
The protein concentration in the tissue blocks
was determined by the Follin-Lowry method after eft
solubilizing the tissue blocks in 2 N NaOH. 2000-
Bovine serum albumin was used as standard.
^nh
RESULTS
UUVI UIX UXIVUXV UXVI UXVIIUXIXUXXI UXXIIUXXIIIUXXIVUXXVUR
Change in the liver weight during metamorphosis
1000
The liver weight varied among stages (the mean
ranging between 0.15 and 0.35 g) (Fig. 1). It also S20001
varied widely among individual tadpoles in most C
stages. Accordingly the variation in the mean
among stages was not statistically significant (P> Fig. n2.onspMeeciafnics(pdecoiwfnicwar(dupcwoalrudmncso)lbuimnndsi)ngsan(depmmepaenr
0.05). 10mg ofwet tissue) ofradioiodinated bullfrog FSH
to a plasma membrane fraction of the liver ol
Bindings offFSHto livermembranepreparation of tadpolesofvariousstagesand adultsofthe bullfrog.
tadpoles at different stages Each vertical bar shows the standard error of the
Specific bindingoffFSH wasextremely low unitl tmheeant.wo Mhoertiazmoontraplhiacxess.tag"eAs"ariendiincdaitcesat"eaddublett"w.een
200
> 100
-
o-1
I VI XIV XV XVI XVIIXVIII XIX XX XXI XXIIXXIII XXIV XXV
St age
Fig. 1. Changes in liver weight of bullfrogs during metamorphosis. The arrow at Stage XX indicates the dill
metamorphosis. Each point andvertical barindieate the mean and standard errorof the mean. rCSpettivel) Fhe-
number of individuals used at each stage are indicated upside of the horizontal axis
762 K. KUBOKAWA
Stage XXIV. The specific binding level at Stage were, as a whole, similar to those of fFSH. The
XXVwasabout ahalfofthatintheadult. Over-all specificbindingwaslowinearlystages, andstarted
difference in specific binding of fFSH to tadpole to increase from Stage XXI (Fig. 3). However, in
liver among metamorphic stages was statistically fLH bindings, nonspecific levels were higher than
highly significant (P<0.01 by one-way layout those in fFSH bindings, and the rate ofthechange
analysis of variance after log-transformation of in the specific binding of fLH with stage was less
data). Kendall's rank correlation coefficient (r) marked than that of fFSH. By the final stage of
between metamorphic stages and mean specific metamorphosis, the specific binding of fLH had
bindingsafterStageXIXwas0.905 andstatistically also reached a level about a halfofthe adult level.
highly significant (P<0.01). Nonspecific binding Variation of the specific binding of fLH among
of fFSH also showed statistically significant varia- stagesoftadpoleswasstatisticallyhighlysignificant
tion among stages, but Kendall's rank correlation (P<0.01) when tested by the Kruscal-Wallis test.
coefficient (r=0.047) between stages and mean Kendall's rank correlation coefficient (r=0.905)
nonspecific bindings after Stage XIX was not sta- between metamorphic stages after Stage XIX and
tistically significant (P>0.05). mean specific bindings was also highly significant
(P<0.01). Variation of the nonspecific binding
Bindings offLH to liver of tadpoles at different among stages of tadpoles was also statistically
stages highly significant (P<0.01), when tested by the
Changes in specific binding of fLH with stage analysis of variance. However, Kendall's rank
=
correlation coefficient (r 0.52) between meta-
morphic stages and nonspecific bindings after
Stage XIX was not significant (P>0.05).
Effects offLH andfFSH on the concentration of
cAMP
in liver
Changes in the cAMP concentration in liver in
,3000
response to gonadotropin treatments were ex-
pressed in terms ofthe ratio ofthe cAMP concen-
-2000" tration in hormone treated tissue to that in corres-
ponding untreated tissue (Figs. 4, 5). Data of
Stages XVIII to XX were combined, since only a
1000 single determinationwasperformedin SageXVIII
nn^h^hMl and XIX, and also gonadotropin bindings did not
change so much until Stage XX. In individuals at
V VI IX XIV XV XVI XVII XIX XX XXI XXII XXIV XXV R Stage XVIII to XX, the cAMP concentration in
the liverwasnotinfluencedbybothfLHandfFSH
T treatments, the ratio to the control being about 1.
-o 1000 yrj In contrast, the cAMP concentration was in-
TT creased by both of the treatments in the liver of
individuals at and afterStage XXL The difference
in the response from the youngest Stage (Stage
Fig. 3. Mean specific (upward columns) and mean XVIII-XX) group was significant in Sage XXI and
nonspecific (downward columns) bindings (cpm of the adult stage in the FSH treatment (Fig. 4), and
10mgofwettissue)ofradioiodinatedbullfrogLHto also in Sage XXI, XXIV and the adult stage in the
aplasma membrane fraction ofthe liveroftadpoles LH treatment (Fig. 5). The concentration of
of various stages and adults of the bullfrog. Each cAMPin the untreated control tissuesvariedwith-
vertical bar shows the standard error of the mean.
Metamorphic stages are indicated between the two in a small range (the mean and standard deviation
horizontal axes. "A" indicates "adult". being 0.062 and 0.014 pmoles per mg protein,
Gonadotropin Receptors in Tadpole Livet 763
respectively).
: 3T
DISCISSION
In the present study, significant amounts of
specific gonadotropin bindings were demonstrated
> in the liverofbullfrog larvae ofthe late stagesafter
Stage XXI. Slight specific bindings of i'FSH and
fLH to the liver were observed in larvae o\ the
younger stages before Stage XXI. However, the
XVI[I4I1-XX X(2X)I X(X5I)V Rd(u3l)t specific bindings to the liver of the younger stage
Fig. 4. Effect of bullfrog FSH in vitro on the cyclic larvae are too small and hence considered to be
AMP concentration in the liveroftadpoles ofdiffe- physiologically insignificant. Similar low levels of
rent stages and adults of the bullfrog. Changes in specific bindings of gonadotropins have been de-
the cAMP concentration are expressed in terms of tected in a number of non-gonadal and non-
tsrtheapegetemiteniauomnnbseorifs.rianTtdhiioecsatvteeodrticiconanltprbaoalrr.enatthTethsheeestnouupnmdobeferreatchohef thuerptalteic[7]t,isrsauiesnbionwvtarroiuotu[scX]vaerntdebbrualtlefsrog(in[9)r)a.t T[h6|e.
column shows the standard error ofthe mean. increase in the specific bindings of fFSH and fLH
* Difference from the youngest stagegroupissigni- at Stage XXI is considered to indicate appearance
ficant at P<0.05. of specific gonadotropin receptors in the hepatic
* Difference from the youngest stage group is tissue. This conclusion is supported also by the
significant at P<0.01. cAMP experiment. The hepatic tissue started to
respond to gonadotropins by increasing the cAMP
concentration from Stage XXI.
: 2.5 Stage XX, the stage just before gonadotropin
co receptors appear in the liver, has been referred to
o 2 the onset of metamorphic climax. A number of
o
physiological changes occur in the bullfrog larva
- 1.54 around this stage. For example, Kistler el al. [10]
reported that a steep increase in the free amino
acid concentration took place in the liver around
this stage. An increase in secretion of glucocorti-
coids toward the climax was reported by Jaffe [ 1 1 1.
Yamamoto and Kikuyama [12] revealed that the
XVIII-XX XXI XXIV Rdult
(4) (2) (5) (3) concentration of prolactin in plasma increased
Fig. 5. Effect ofbullfrogLHin vitroon thecyclicAMP rapidly from Stage XXII and reached the max-
concentration in the liver of tadpoles of different imum at Stage XXIV. However, it is difficult to
stages and adults of the bullfrog. Changes in the correlate a certain one among these phenomena to
cmeAaMnPofcornatcieonstrtoatcioonntroalr.e eTxhperensusmebdeirnotferrempsetoiftitohnes the appearance of gonadotropin receptors in the
is indicated in parentheses under the stage number liver.
The vertical barat the topofeach column shows the Janssens and Mahei [13] found in the adult
standard error of the mean. axolotl that glucagon and adrenalin increased the
Difference from the youngest stage group issigni- cAMP concentration in the liver. Janssens and
ficant at P<0.Q5. Cirigg [14| reported in the adult Xcnopus that
Difference from the youngest stage group is
significant at P<0.01. adrenalin stimulated glycogenosis in the liver in
vitro. The results bv the present author in conjunc-
tion with these previous results suggest a possibil-
ity that gonadotropin mimics or swicrgisticalls
764 K. KUBOKAWA
intensify the effect ofglucagon or adrenalin in the bullfrog seem to have some physiological function
liver in bullfrogs after the metamorphic climax. as well as those of the adult bullfrog. Further
This possibility should be tested by examining in biochemical study to find the final hepaticfunction
vitro effects of frog gonadotropins and cyclic nuc- that is mediated by the gonadotropin receptors is
leotide on the carbohydrate metabolism in liver of needed.
bullfrogs after the climax of the metamorphosis.
It will be interesting to compare the change in ACKNOWLEDGMENTS
the hepatic gonadotrpin receptors with changes in
The author is grateful to Professor Susumu Ishii,
hepaticreceptorsforthe otherpituitaryhormones. Department of Biology, Waseda University for his re-
White and Nicoll [15] failed to detect significant viewing the manuscript and criticisms and suggestions
specificbindingsofprolactinin the liverofbullfrog provided duringthe course ofthisstudy. Thisstudywas
tadpoles at any stage, while they demonstrated supported by a grant from Waseda University.
high specific bindings in other organs such as tail,
gill and kidney. Carr et al. [16] detected specific REFERENCES
bindings of prolactin in the liver as well as in the
tail and kidney. However, the level ofthe binding 1 Kubokawa, K. and Ishii, S. (1987) Native gonadot-
ropin receptors in amphibian liver. Gen. Comp.
was lowest in the liver. They further showed that Endrocrinol., 68: 260-270.
thenumberofprolactinbindingsitesinthetadpole 2 Ziecik, A. J., Stanchev, P. D. and Tilton, J. E.
liver increased during early metamorphic stages (1986) Evidence for the presence of luteinizing
anddecreased attheclimaxofmetamorphosis. No hormone/human chorionic gonadotropin-binding
study hasbeen reportedforreceptorsforthe other sites in the porcine uterus. Endocrinology, 119:
1159-1163.
pituitary hormones in the tadpole liver.
3 Taylor, A. C. andKollros,J. J. (1946) Stagesinthe
As far as hepatic gonadotropin receptors appear normal development ofRanapipiens larvae. Anat.
at Stage XXI and if these receptors play some Rec, 94: 7-23.
physiologicalrole, synthesisandsecretionofgona- 4 Takada, K. and Ishii, S. (1984) Purification of
dotropins should have started before this stage. bullfrog gonadotropins: Presence ofnew subspecies
An immunocytochemical study showed that FSH ofluteinizing hormone with high isoelectric points.
and LH became detectable from Stage V and X, Zool. Sci., 1: 617-629.
5 Kubokawa, K. and Ishii, S. (1984) Adaptation of
respectively [17]. This study also revealed with testicular follicle-stimulating hormone receptors to
radioimmunoassay that the content of FSH had a ambient temperatures in vertebrates: Equilibrium
peak at Stage XVIII and that of LH at around analysis. Gen. Comp. Endocrinol., 54: 277-282.
Stage XXIII. Thus, synthesis and secretion of 6 Steinberger, A., Thanki, K. H. and Siegal, B.
gonadotropins precedes the appearance ofhepatic (1974) Cellular localization of FSH receptors, In
"Hormone Binding and Target Cell Activation in
gonadotropin receptors. Unfortunately, change in theTestis". Eds. byM. L. Dufau andA. R. Means,
plasma gonadotropin levels in bullfrog tadpoles is Plenum Press, New York, pp. 179-191.
notclear. Plasmaconcentrationsofgonadotropins 7 Adachi, T. and Ishii, S. (1977) Binding of rat
in bullfrog tadpoles of Stage XXIII to XXV were follicle-stimulating hormone to the turtle ovary in
low, rangingbetween0.3 and0.8ng/ml inLH and vitro and inhibition ofthe binding by gonadotropin
preparations of various verbebrates. Gen. Comp.
between 1.0 and 2.2ng/ml in FSH, and it was
Endocrinol., 33: 1-7.
difficult to detect the change clearly (personal 8 Schlaghecke, R. (1983) Binding of 125J-hCG to
communications with Dr. S. Tanaka, Gunma Uni- rainbow trout (Salmon gairdneri) testis in vitro.
versity, Maebashi). Furthermore, no ontogenic Gen. Comp. Endocrinol., 49: 261-269.
study of gonadal gonadotropin receptors was re- 9 Takada, K., Kubokawa, K. and Ishii, S. (1986)
ported in amphibians, although sex differentiation Specific gonadotropin binding sites in the bullfrog
ofthe gonads has been reported to start in the late testis. Gen. Comp. Endocrinol., 61: 302-312.
10 Kistler, A., Miyauchi, H. and Frieden, E. (1980)
metamorphic stage in Rana nigromaculata [18]. Changes in amino acid metabolism and protein
In conclusion, the hepatic gonadotropin recep- synthesis during spontaneous metamorphosis in the
tors of the late metamorphic stage larvae of the bullfrog Rana catesbeiana tadpole liver. Int. J.
Gonadotropin Receptors in Tadpole Liver 765
Bioehem.. 12: 395-400. and metamorphosis. Science. New York 204: 851-
11 Jaffe, R. C. (1981) Plasma concentration of corti- 853.
COSterone during Rana catesbeiana tadpole meta- 16 Carr. F. F.. Jacobs. P. J. and Jaffe. R. C. (1981)
morphosis. Gen. Comp. Endrocrinol., 44: 314-318. Changes in specific prolactin binding in Rana cates-
12 Yamamoto, K. and Kikuyama, S. (1982) Radioim- beiana tadpole tissues during metamorphosis and
munoassay of prolactin in plasma of bullfrog tad- following prolactin and thyroid hormone treatment.
poles. Endocrinol. Japon, 29: 159-67. Mol. Cell Endocrinol., 23: 65-76.
13 Janssens. P. A. and Maher. F. (1986) Glucagon and 17 Tanaka, S.. Sakai. M.. Park. M. K. and kurosumi.
insulin regulate in vitro hepatic glycogenolysisin the K. (1991) Differential appearance ofthe subunitsof
axolotl ambystoma mexicanum via changes in tissue glycoprotein hormones (FH. FSH and TSH) in the
cyclic AMP concentration. Gen. Comp. Endocri- pituitary o\ bullfrog (Rana catesbeiana) larvae dur-
nol.. 61: 64-70. ing metamorphosis. Gen. Comp. Endocrinol., in
14 Janssens. P. A. and Grigg. J. A. (1984) Adrenergic press.
regulation of ghcogenolvsis in liver of Xenopus 18 Tanimura, A. and Iwasawa, H. (1988) Ultrastructu-
laevis in vitro. Comp. Bioehem. Physiol.. 77: 403- ral observations on the origin and differentiation of
406. somaticcellsduringgonadaldevelopmentinthefrog
15 White. B. A. and Nicoll. C. S. (1979) Prolactin Rana nigromaculate. Develop. Growth and Differ..
receptors in Rana catesbeiana during development 30: 681-691.