Table Of ContentZOOLOGICAL SCIENCE 8: 879-884 (1991) © 1991 Zoological Society ofJipan
Fat Body Involvement in the Plasma Vitellogenin Changes
During Recovery Phase in Rana esculenta
Oliana Carnevali and Gilberto Mosconi
Department ofBiology {MCA) University ofCamerino, via F. Camerini
2, 62032 Camerino MC, Italy
—
ABSTRACT The role of fat body in the reproductive physiology of Rana esculenta was investigated
during recovery phase. Changes in the ovarian weight, plasma vitellogenin (VTG) and estradiol-17,3
levelswere analyzedinintactfrogs, inthosewithoutfatbodies(FBD)andinmalesandfemalesinjected
with estradiol-17^. The ovarian weight decreased by day 28 in intact females but not in FBD animals.
At the same time plasma VTG titers sharply decreased on day 21 in intact animals, but no significant
changes were found in FBD females. Male frogs never synthetize VTG; they do so, however, after
injectionofestradiol-17,?. FBDmalesshowedhigherVTGtitersthanintactmales. The resultsobtained
in vitroshowed asharpdecreaseofvitellogenin added inthe mediumofcultured fat body. Thus, thein
vivoandin vitroresultsallowustosuggestthatfatbodiesmetabolizeplasmavitellogenin,atleastduring
recover) phase; therefore ovarian growth may depend on an interplay among hepatic vitellogenin
synthesis, fat body metabolic activity and ovarian uptake.
been demonstrated to be correlated with the plas-
INTRODUCTION
maestradiol-17/3(E2) profile. These authorsfound
A peculiarity, at least so it seems, of the repro- that fat body does contain vitellogenin, at least
ductive physiology of amphibians is the insertion during the pre-spawning and spawning periods,
of the fat body (FB) in the hypothalamo- but does not synthesize it.
hypophysial-gonadal axis [1]. The importance of In ordertoclarify the involvement offat body in
the fat body in the regulation of testicular activity the regulation of Rana esculenta plasma vitel-
in Ranaesculentahasbeenwidelydemonstratedby logenin, this paper describes: i) the effects of FB
Chieffi et al. [2J. These authors found that the fat removal during the recovery phase on ovarian
bodies are the main deposit for lipids in the body; weight and in the plasma levels of VTG and E2 in
the lipids, synthesized by the liver are released in female frogs; ii) the variations of VTG and E:
the plasma and subsequently incorporated in the plasma titers in estrogenized intact and fat body
fat body and testis; the annual changes in these deprived males and females; iii) whether female
parameters correlate with gametogenetic activity fat body can release VTG using an in vitro culture
The relationship between fat body and ovarian system.
cycle have been ascertained by Pierantoni etal. [3]
in the same species. Fat body removal impairs the MATERIALS AND METHODS
ovarian growth, during the recoveryperiod, result-
ing in a lack of lipid uptake by the gonad and in a
reduction in the protein synthesis. Recently Var- Animals
nale et al. [4] investigated the annual variations of Adult male (15-20g) and female (20-25 g)frogS
\TG in the FB. liver, plasma and ovaries of adult were collected in the mountain pond (CoIfioritO,
female. Ranaesculenta frogs, these variations have Umbria) in November and FB removal was im-
mediately performed. The experiments began Oil
Accepted June 20, 1991 the 1st November: the animals wen maintained
Received April 20, 1991 in tanks with deep water al 18 C Oi temperature
.
O. Carnevali and G. Mosconi
and fed on bovine liver and fly larvae. which uses bovine serum albumin (BSA) as stan-
dard following Lowry [7].
Experimental design
Five days after fat body removal (day 0), ex- Measurement of VTG concentrations by ELISA
perimental groups were composed of intact frogs Purified VTG was used to raise antibodies by a
in captivity, intact frogs injected with 100ju\ of E2 protocol developed in our laboratory [8] and the
(7^g/ml, Sigma), fat bodiesdeprivedfrogs (FBD) polyclonal antibody raised in the rabbit was used
and FBD frogstreatedwith 100fAofE2(7/ig/ml). to determine VTG titerin the mediumbyenzyme-
Ten animals, for each experimental group, were linked-immuno-sorbent-assay (ELISA)
weighed andkilledatweeklyintervalsuntilday28. Buffer and reagents for ELISA (i) Coating
Theovarieswerethenremovedandweighedtothe buffer: phosphate buffered saline (PBS) 0.15 M
nearest milligram and blood samples were taken pH7.2 plus 0.02% sodium azide. (ii) Washing
by capillary inserted in the "conus arteriosus"; buffer: PBSplus0.05% Tween20. (iii) Rabbitanti
after centrifugation at 2500g the supernatants VTG antiserum, (iv) Dilution buffer per conju-
were frozen at —80°C until assayed. gate: PBS plus 0.05 Tween20, 0.1% BSA and 5%
pig serum, (v) Conjugate: alkaline phosphatase-
Culture Media and Preparation of Fat Body Cul- conjugated goat anti rabbit immunoglobulin anti-
tures serum (Sigma), (vi) Substrate buffer: diethanola-
Ten adult female frogs were anesthetized in ice; mine buffer 1 M, pH9.8, containing 0.5 mM
the abdomen was cleaned with 5% chlorhexadine MgCl2. (vii) Substrate: p-nitrophenylphosphate 1
gluconate in 70% methanol and was then opened mg/ml in substrate buffer.
taking care not to cut the abdominal vein. Fat
bodies were removed into sterile petri dishes con- Assayprocedure
taining Eagle-Earle's salts (Gibco Bio-Cult Glas- Flat bottomed microtiter plates (Greiner,
gow Scotland) prepared as describedbyWang and medium affinity; SIAL, Rome, Italy) were coated
Knowland [5] and containing a final concentration overnight with 100//l of culture media from in-
of 20mM HEPES, 2 mM glutamine, 50units/ml cubations. Standard curve was carried out with
penicillin and 50/zg/ml streptomycin. All solu- gradual dilution (10-1280ng) of purified VTG.
tions and glassware were sterilized before use by Plates were washed 4 times, post-coated overnight
filtrationorautoclaving. ExplantedFBwerechop- with 10jug/m\ BSA in PBS, washed with PBS and
ped with scissors and rinsed thoroughly and distilled water and dried under vacuum in a desic-
drained on autoclaved 3MM paper. Sixty mg cator. Rabbit anti VTG antiserum was diluted
(about 6-7 pieces) were then weighted and placed 1 :1000 in buffer containing 5% pig serum to
in each well of a Falcon culture dish, and covered reduce non specific binding.
with 1 ml of culture medium. Plateswere washed after 1 h and incubatedwith
M
Culture were incubated at room temperature substrate. The reaction was stopped with 3
(20°C) in air on a rocking table (4 cycles/min). NaOH, 25^/1/well after 30-60min.
The medium was daily changed foruptofive days. The optical density was read at a wavelength of
After incubation for two days in culture medium 405 nm. All steps were performed at room
alone, estradiol-17/? (2xlO"6M), was added to temperature. Post coating was done with 150/^1/
the wells. Finally 60mg/well incubated for 24hr well, antigen (VTG), samples, rabbit anti-VTG
with lOmg/ml of VTG. antiserum, conjugate and substrate were added in
volumes of 100^1/well.
Purification of VTG A reliable calibration curve enables the antigen
VTG was purified from the plasma of estroge- titer (VTG) to be measured in all cultured media
nized males by MgCl2/EDTA precipitation and in the plasma.
according to Wiley etal. [6]. The concentration of
purified VTG was determined by a procedure
1
Fat Body and Vitellogenin in Rami esculenta 881
sequently. radioimmunological analysis (RIA) of
Estradiol-17^ assay estradiol-17,i (E:) was carried out as previously
Plasma samples, were extracted with ether; sub- described by Polzonetti-Magni ct til. [8]. A sensi-
I
. _L
.
.
_
.
O.CK
days
days
5000
4000
3000
2 2000 -
-
Fir,. i. a) Ovarian weight (gonadosomatk index. (rSli. b) plasma vitellogenin and c) estradiol 17>9titers in the intaci
i iS) and estrogemzed fgggg ) female frogs, in those deprived ol their f;it bodies ( ^r) and those without fat
bodies and injected with estradiol- 1,3( | Vertical lines indicate SD
882 O. Carnevali and G. Mosconi
tivity of 7 pg (intraassay variation, 4.5%; interas-
say variation 7.5%) was observed. Steroid anti- Statistical analysis
serum was provided by G. Bolelli (Physiopatholo- Results were analyzed using™a statistical soft-
gy of Reproduction Service, University of Bolo- ware package, Stat View 512+ (Brain Power
gna, Italy); tritium labelled E2 were purchased Inc. Calabasas, CA, USA). Since ovary weight
from Amersham International (Buckinghamshire, was highly correlated with body weight, somatic
England) and authentic steroids were obtained index (GSI=ovary wt/body wt) was employed for
from Sigma. analyses and presentation of data. A probable
level of0.05 was taken to indicate a significance in
comparison of means.
days
5000
4000
3000
2000
1000-
days
Fig. 2. a)Plasmavitellogeninandb)plasmaestradiol-17/?titersinestrogenizedintact( )andfatbodies-deprived
( ) male frogs. Vertical lines indicate SD.
Fat Body and Vitellogenin in Runa esculenta ^}
RESULTS DISCUSSION
Following a summer post-reproductive refrac-
/;; Vivo experiments toriness, frog ovary enters in the recovery phase.
Females: The ovary weight (GSI) does not During this period the ovarian recrudescence is
display significant changes by day 21 in neither mainly due to hepatic vitellogenin synthesis and its
intact nor FBD frogs. In estrogenized females, the uptake in the growingoocytes [8]. The increase of
GSI significantly (P<0.01) increased up to 0.25 ovarian weight dependsalsoon the lipid accumula-
within a week, remained high until day 21 and tion, commonly transported from FB to the ovary
sharplydecreased at day28in intact animals, while [9-10].
no significant changes were found in FBD ones The present datasuggest that FB are involved in
(Fig. hi). autumn recovery phase but do not impair the
Plasma VTG titers in intact and FBD female ovarianweight aspreviouslyfound by Pierantoniet
frogs were found to be in the range of0.8-1.1 mg/ al. [3]. Although the ovarian weight changes,
ml on day (i.e. 5 days after fat body removal). found in these experiments, could arise from the
While in FBD frogs plasma VTG levels remained stress of confinement, they are mainly consistent
high during all the experimental time, a significant with ovarian decrease in intact females. Also the
decrease of VTG titers (P<0.01) was found on highest E2 plasma levels found at day 21, in
days 21 and 28 in intact frogs (Fig. lb). A signi- females as well as in estrogenized males, may be
ficant increase of plasma VTG levels (P<0.05), due to the captivity stress acting on the hypothala-
was found in both intact and FBD animals after mo-hypophysial-gonadal axis; in fact androgen and
seven days of E2 injection until day 28 (Fig. lb). estrogen plasmachanges, even afterashortcaptiv-
With respect to the plasma E2 trend, a very ity have been observed in this anuran [11].
significant increase (P<0.01) was found on day 21 As previously demonstrated in this species by
in intact females, while a much lesser increase of Polzonetti-Magni et al. [8], ovarian growth paral-
plasma E2 content was found in FBD frogs (Fig. lels the plasma vitellogenin trend. The plasma
lc). vitellogenin decrease, observed on days 21 and 28
Males: Male frogs do not synthesize VTG; in intact frogs, but not in FBD animals, suggests
plasma VTG was found in both intact (0.3 mg/ml) that fat body affects the ovarian growth by acting
and FBD males (0.5 mg/ml) after a week of E2 on plasma VTG concentration. The same results,
injection: thereafter, VTG peaked on day 14 (P< obtained in male and female estrogenized frogs,
0.01). and this increase was significantly greater (P allow us to suppose that the VTG decrease in
<0.01) in FBD males than in intact ones (Fig. 2a). intact animals may depends on FB metabolic acti-
With respect to plasma E2 levels a significant peak vity in sequestering plasma vitellogenin. This
was found at day 21 in both intact and FBD hypothesis is also supported by the results
estrogenized males (Fig. 2b). obtainedin vitro, showingthat, although fat bodies
do not release VTG in the in vitro culture system
In vitro" experiments. nor in presence of E2, they induce a significant
Female frog fat body did not release VTG when decrease ofthe VTG added in the medium. The in
cultured for 5 days with medium alone, nor in the vivo and in vitro results reported here, indicate
presence ofE In ordertoestablish whetherVTG that fat body plays a role in the frog gonadal
2.
is utilized in some way by FB. 10/yg/ml of VTG function. Such a role, in ouropinion, is particular-
were added to FB culture. After 24 h incubation. ly consistent with the hypothesis that plasma vitel-
VTG concentration decreased (P<0.01) to around logenin is metabolized bj the fat body and. in
5//g/ml. As control. VTG was added in the consequence, the ovarian growth depends Oil an
medium without FB and no changes of VTG interplay among hepatic vitellogenin synthesis, fat
concentration were observed (data not shown). body metabolie aeti\it\ and ovarian uptake. More
detailed studies are needed to investigate hou IB
1 ,
884 O. Carnevali and G. Mosconi
utilize this glycolipophosphoprotein in view of vitellogenin content of liver, fat body, plasma and
their involvement in the lipids storage as ascer- ovary in the frog {Rana esculenta). Gen. Comp.
Endocrinol., 69: 328-334.
taiTnheed ilnotwheirs fprolgasbmyaMiEl2onleeveetlsal.f[o1u2n].d in FBD 5 Wviatnegllho,geLn.inJ.inacnudltKunreoswlofanmda,leJ.an(1d9f75e)malSyenftrhoegsilsivoefr
females, with respect to intact animals, agree with regulated byestradioltreatmentin vitro. Proc. Nat.
the results obtained by Chieffi et al. [1], showing Acad. Sci. U.S.A., 72: 3172-3175.
FB involvement in the frog gonadal steroidogene- 6 Wiley, H. S., Opresco, L. and Wallace, R. A.
sis; in addition, the plasma levels of this hormone (1979) New methods for the purification of verte-
brate vitellogenin. Anal. Biochem., 97: 48-53.
in intact female frogs, reported here, confirm a
multihormonal control of VTG synthesis, as sug- 7 LRaonwdrayl,l,O.R.H.J,. R(o1s9e51n)broPurgotheiNn. mJ.e,aFsaurrremAe.ntL.waintdh
gessted by Carnevali and Polzonetti [13], since the follenphenolreagent.J. Biol. Chem.,193:256-275.
peak values of this hormone are concomitant with 8 Polzonetti-Magni,A. M.,Curini,R.,Carnevali,O.
the lowest plasma VTG levels. Novara, C, Zerani, M. and Gobbetti, A. (1990)
Ovarian development and sex steroid hormones
during the reproductive cycle of Rana esculenta
ACKNOWLEDGMENTS complex. Zool. Sci., 7: 265-271.
9 Iela, L., Milone, M., Caliendo, M. F., Rastogi, R.
This work was supported by grants to A. M. Pol- K. and Chieffi, G. (1979) Role of lipids in the
zonetti-MagnifromMURST(40%-60%)andCNR. We physiology of the testis of Rana esculenta: annual
are grateful to Prof. A.M. Polzonetti-Magniforreading changesinthe lipidandproteincontentoftheliver,
this manuscript. fat body, testis and plasma. Boll. Zool., 46: 11-16.
10 Milone, M., Iela, L., Esposito, V., Rastogi, R. K.
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