1 Dipartimento di Scienze Morfologiche e Biochimiche Comparate, Università degli Studi di Camerino, 62032 Camerino (MC) Italy; 2 Istituto di Scienze del Mare, Università di Ancona, 60131 Ancona, Italy; and 3 Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4
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ABSTRACT |
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Experiments were carried out to study in
vitro the effects of 17-estradiol (E2), homologous
pituitary homogenate (HPH), and recombinant red sea bream growth
hormone (sbGH) on vitellogenin (VTG) secretion from cultured sea bream
liver fragments. Basal secretion of VTG was found to be significantly
higher in the prespawning period, compared with sea bream liver in the
spawning and postspawning periods. Similarly, the sea bream liver
obtained during the prespawning period responded more significantly to
treatments with E2, HPH, or sbGH compared with sea bream
liver during spawning. In the postspawning period, treatments with
E2, HPH, or sbGH were without significant effect on VTG
secretion level in sea bream liver. The level of E2
receptors was also analyzed by Western blot analysis. The result
demonstrates a significantly higher level of E2 receptors in the sea bream liver at the prespawning stage compared with those at
the spawning and postspawning stages. The findings support the
hypothesis that homologous upregulation of estrogen receptors plays an
important role in the estrogen-sensitive control of VTG synthesis in
the sea bream liver.
vitellogenin induction; hormones; estradiol receptor
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INTRODUCTION |
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THE GILTHEAD SEA
BREAM, Sparus aurata, is an economically
important species because of high demand and the high quality of its
meat. Recent studies have provided information on reproductive endocrinology of sea bream (3), and there is evidence for
the presence of three forms of gonadotropin-releasing hormones
(GnRH) in the brain of this species (4, 11). GnRH in turn
stimulates gonadotropin production, which is the main regulator of
gonadal development, steroidogenesis, and ovulation. In female fish,
the accumulation of yolk proteins, i.e., vitellogenesis, is crucial for
oocyte development and spawning. As in a number of oviparous species,
production of vitellogenin (VTG) in the liver is controlled by a number
of gonadal and pituitary hormones (6, 12, 14). S. aurata is a true hermaphrodite and functions as a male during the
first 2 years of life. In the third year, there is testicular degeneration and ovary development, resulting in transformation of the
fish to functional females. Sea bream is classified as a "group
synchronous" spawner (15) with short and regular
spawning cycles. Because spawning may be prolonged over months,
vitellogenesis, which is part of yolk protein formation, is continuous
throughout the breeding season (1). However, apart from
the work by Mosconi et al. (7), little information is
available on hormonal control of hepatic VTG synthesis in sea bream
maintained under intensive culture conditions. Recent studies
characterized the S. aurata VTG molecule and developed an
enzyme-linked immunosorbent assay (ELISA) method for its measurement
(7). Changes in plasma VTG were shown to be correlated
with those of 17-estradiol (E2) in both prespawning and
spawning female sea bream. In the postspawning period, however, VTG was
found to be undetectable when the circulating level of E2
was the same as that in the spawning period. This may, therefore, raise
questions about the role of estradiol in triggering and
terminating VTG synthesis in sea bream. There is also evidence that
estrogen-induced vitellogenesis is influenced by growth hormone (GH) in
silver eel (9) and by other pituitary hormones in certain
oviparous species (10).
In the present study, sea bream liver was cultured in the presence of E2, homologous pituitary homogenate (HPH), and recombinant red sea bream growth hormone (sbGH), followed by measurement of VTG in the culture media (CM). We also measured the estradiol receptor level in the same hepatic tissue as that used in incubation experiments by Western blot analysis.
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MATERIALS AND METHODS |
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Animals.
Gilthead sea bream (S. aurata) living in the Mediterranean
area naturally spawn from November until February. In the present study, adult females (between 1.5 and 2 kg body wt) were obtained from
a commercial fish farm (La Rosa, Orbetello, west coast of Italy;
42°28' N, 11°12' E). The animals were rapidly anesthetized in a
tank containing water plus 100 mg/l of MS222 (Sigma), and blood samples
were collected from the caudal vein with a syringe. The blood samples
were placed in heparinized tubes containing 1 mM phenylmethylsulfonyl
fluoride (PMSF) and centrifuged (1,500 g for 15 min at
4°C), and plasma samples were stored at 20°C. The liver was
removed and processed for in vitro incubation and Western blot analysis.
Hormones. HPH was prepared by homogenizing homologous total female pituitary glands in CM. E2 and sbGH were purchased from Sigma (St. Louis, MO) and GroPrep (Adelaide, Australia), respectively.
Liver incubation.
Sixty milligrams of liver (~6-7 pieces) were weighed, placed in
a Falcon culture dish well, and covered with 1 ml of medium 199 (Sigma)
with Hanks' salts and L-glutamine and without
NaHCO3, and the salinity was adjusted with NaCl and
NaHCO3. The medium, enriched with oxygen, was supplemented
with 1% (vol/vol) Ultraser (IBF Biotechnics), 100 U/ml penicillin, and
100 mg/ml streptomycin. The pH was adjusted to 7.4. Liver fragments
were incubated at 18°C on a rocking table (4 cycles/min) for up to 8 days. E2 (106 M), HPH (1/20
eq · gland
1 · ml
1), and/or
sbGH (1 µg/ml) were added to each well. In this study, five
wells were prepared for each treatment. The media containing appropriate hormones were oxygen enriched and changed daily. The control wells contained CM plus vehicle only. At the end of incubation, tissue was examined under light microscopy to be sure of its
preservation. The liver incubations were performed during prespawning,
spawning, and postspawning periods.
ELISA for VTG in CM. The VTG concentration in the CM was assayed with an ELISA method previously validated and described by Mosconi et al. (7). The sensitivity (amount of VTG that gave 90% of binding) was ~8 ng/ml, with an intra-assay variation of 4.8% (n = 16) and an interassay variation of 7.2% (n = 12) around 50% of binding. VTG content was normalized against the tissue total protein content of each sample. The results are expressed as accumulated levels of VTG.
E2 assay in plasma. Plasma E2 levels were analyzed by an enzyme immunoassay (EIA) method (estradiol EIA kit, Cayman) with a sensitivity of 8 pg/ml.
In vivo experiments. Adult female sea bream (n = 6) were injected during the postspawning period with 5 mg/kg body wt of E2 dissolved in peanut oil (5 mg/ml); after 5 days, both E2-treated and control animals (6 females injected with vehicle only) were rapidly anesthetized as described in Animals, and the liver was removed and processed for Western blot analysis.
Western blot analysis for estradiol receptor evaluation.
Total protein extraction was carried out with RIPA buffer (25 mM Tris
pH 7.5, 0.1% SDS, 1% Triton X-100) to homogenize the liver; then 10 µl/ml of PMSF (10 mg/ml) was added before centrifugation (30 min at
4°C at 12,000 rpm); the supernatant was quantified with a Hitachi
U2000 spectrophotometer (Tokyo, Japan), and the bicinchoninic acid
(BCA) method for protein quantification (Pierce, Rockford, IL) was
applied. For the protein gels, 10 µg of total protein was loaded per
lane of gel. The protein samples were resolved on a 10% resolving gel
and a 3.75% stacking gel. Samples were electrophoresed at constant
voltage (150 V) until the bromophenol blue dye front reached the bottom
of the gel. Gels used for Western blot transfer were run with Amersham
molecular weight markers (rainbow markers) and used for transfer
immediately without staining. Western blotting proteins were
transferred to polyvinylidene difluoride (PVDF) plus membrane (Micron
Separations) in a Bio-Rad Mini-Protean 2 cell apparatus at 100 V
for 1 h. The ECL Western blotting system (Amersham Life Sciences)
was used for detection purposes. The primary antibody used was
polyclonal antibody raised against human estrogen receptor (ER)- in
rabbit (Sigma) at a dilution of 1:1,000 for a minimum time of 1 h
overnight. Horseradish peroxidase-labeled secondary antibody
(conjugated anti-rabbit) was used at a dilution of 1:3,000 for 1 h. Blots were exposed on Kodak autoradiography-sensitive film for as
little as 40 s, and a clear signal was detected. The dilution of
the second antibody was optimized to give a clear background.
Statistical analysis. The results were analyzed by ANOVA with Stat View 512+ (Brain Power) statistical software package. A probability level of 0.05 was taken to indicate a statistical difference between means. Results are expressed as means ± SD (n = 5).
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RESULTS |
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VTG secretion from cultured sea bream liver fragments in vitro.
Experiments were carried out to study the effects of E2,
HPH, and recombinant sbGH on VTG secretion on cultured sea bream fragments in vitro. Cultured sea bream liver was found to produce and
spontaneously release VTG in the CM in a time-related manner. Treatment
with sbGH resulted in a dose-related increase in secretion of VTG
compared with controls (Fig.
1A). The dose-related effect of sbGH became apparent after 6 days of culture in vitro. In a similar experiment, we also tested the effect of various
concentrations of E2 on cultured sea bream liver. Treatment
with E2 also resulted in a dose-related increase in VTG
secretion in vitro, and the steroid action became apparent after 7 days
of treatment (Fig. 1B). In these experiments, the most
effective doses were found to be 106 M and 1 µg/ml for
E2 and sbGH, respectively.
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Plasma E2, VTG, and liver ER levels at different
reproductive stages in sea bream.
The levels of plasma E2 show seasonal changes, with the
highest levels found during prespawning (2,188 ± 158 pg/ml),
compared with levels at spawning (1,010 ± 95 pg/ml) and
postspawning (1,100 ± 90 pg/ml) periods (Table
1). The highest VTG plasma levels were
found during prespawning (1.5 ± 0.16 mg/ml), significantly decreasing during spawning (0.7 ± 0.08 mg/ml) and reaching a
minimum (0.01 ± 0.001 mg/ml) at the postspawning period (Table
1).
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DISCUSSION |
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The present study provides information on VTG secretion level in sea bream liver in response to in vitro treatments with E2, HPH, and sbGH; moreover, it gives novel information on in vivo ER level in the sea bream liver at different stages of reproduction, demonstrating a positive correlation between E2 receptor level and liver responsiveness to hormonal treatments. The findings provide an insight into the mechanism by which estradiol may effect hepatic VTG synthesis in sea bream liver and its release. Our results demonstrate for the first time in fish that E2 is not the only hormone that exerts direct stimulatory action on hepatic VTG secretion. In this context, treatments with HPH and sbGH stimulated VTG synthesis in in vitro cultured sea bream liver. The involvement of pituitary hormones such as GH and prolactin in induction of VTG synthesis was previously demonstrated in Anguilla japonica (5) and A. anguilla (9). In female silver eel, GH treatment was shown to stimulate VTG synthesis in cultured hepatocytes (9). The multihormonal stimulation of GH and/or prolactin as well as E2 was found to be essential for the active synthesis of VTG in A. japonica (5). In S. aurata, which is a marine teleost with a reproductive strategy different from that of eels, the liver responds directly to GH stimulation and produces VTG in concentrations higher than those in eels. There appears to be a specific liver sensitivity to estrogenic compounds (estradiol) and pituitary hormones (GH), depending on the reproductive phase. In this context, although estradiol was found to be more potent than sbGH in the prespawning period, pituitary hormones induced more significant stimulatory effect on VTG secretion than did estradiol during spawning period. The present results clearly demonstrate that basal VTG production has seasonal changes and that the responsiveness to E2 and pituitary hormones of sea bream liver varies during the reproductive cycle. The stimulatory action of pituitary hormones, as well as estradiol, on liver VTG production, was also previously demonstrated in other oviparous vertebrates (10). GH was shown to act as a coregulator of VTG synthesis in fish (12), and there is evidence for the involvement of GH via production of insulin-like growth factor I (IGF-I) activating second messenger system (2, 16).
In this study, a positive correlation between E2 receptor
level and liver responsiveness to E2 was clearly observed.
During prespawning, when E2 receptor levels are highest,
the liver produces the highest levels of VTG in response to
E2. These data are supported by the in vivo results showing
the band of ~66 kDa, corresponding to human ER-, suggesting
positive response to E2 treatment.
In this study, our findings suggest that estradiol upregulates ERs that may, in part, be responsible for changes in seasonally dependent sensitivity to circulating estrogen level in sea bream.
In summary, the present results demonstrate a direct action of GH and estradiol on sea bream liver VTG secretion in vitro. Furthermore, the findings demonstrate a seasonal variation in hormone-induced VTG secretion response correlated with the E2 receptor level in sea bream liver.
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ACKNOWLEDGEMENTS |
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This research was supported by the Ministero per le Politiche Agricole e Forestali (MIPAF) of Italy.
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. M. Polzonetti-Magni, Dipartimento di Scienze Morfologiche e Biochimiche Comparate, via Camerini 2, 62032 Camerino (MC) Italy (E-mail: alberta.polzonetti{at}unicam.it).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00411.2001
Received 22 August 2001; accepted in final form 16 April 2002.
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