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BOR - Papers in Press, published online ahead of print November 12, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.021428
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BIOLOGY OF REPRODUCTION 70, 737–743 (2004)
DOI: 10.1095/biolreprod.103.021428
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Hormonal Regulation of Vasa-Like Messenger RNA Expression in the Ovary of the Marine Teleost Sparus aurata

M. Cardinali2, G. Gioacchini2, S. Candiani3, M. Pestarino3, G. Yoshizaki4, and O. Carnevali1,2

Department of Marine Science,2 Polytechnic University of the Marches, 60131, Ancona, Italy Department of Experimental,3 Environmental and Applied Biology, University of Genova, 16132 Genova, Italy Department of Aquatic Biosciences,4 Tokyo University of Fisheries, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The vasa gene is an important maternal regulator of primordial germ cell (PGC) development in both vertebrate and invertebrate models. It is also expressed in the mature gonads, but its role in these tissues is still unclear. In oviparous species, oogenesis is a complex process under hormonal control: estrogens, gonadotropins, and other hormones operate at different stages of oogenesis, regulating meiosis, vitellogenesis, follicle maturation, and egg release. The aim of this work is the determination of a regulative role of hormones controlling oocyte maturation on vasa mRNA expression in the sea bream ovary through a molecular biology approach. By in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR), reaction (the vasa mRNA in the sea bream ovary was found to be expressed at higher levels in the advanced stages of oocyte maturation. After in vivo hormonal treatment, the effect on ovarian vasa mRNA expression was studied through semiquantitative RT-PCR. The quantification of vasa-like mRNA expression in sea bream ovary demonstrates that estradiol (E2), growth hormone (GH), and the combination of gonadotropin-releasing hormone (GnRH) with GH are able to induce an increase in vasa mRNA expression. In contrast, the treatments with GnRH alone or E2 plus GH significantly decreased vasa mRNA expression. These data suggest a regulative interplay between the vasa gene expression and the endocrine system that controls the oogenesis in the ovary of the sea bream.

gametogenesis, gene regulation, mechanisms of hormone action, oocyte development, ovary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Germ cells are highly specialized cell types that transmit genetic information between generations. The commitment of primordial germ cells (PGCs) during the first phases of development is made by specialized cytoplasm within the egg, the germ plasm, which is asymmetrically localized after fertilization [1, 2]. This cytoplasm contains molecules that are indispensable for PGC commitment: in Drosophila the transplantation of the germ plasm is sufficient to direct the development of ectopic germ cells at the site of injection [3]. The molecular composition of the germ plasm has been thoroughly studied in Drosophila [4], where eight genes were identified as necessary maternal factors for PGC formation. Among these genes, the vasa gene codes for a DEAD box family protein, a putative ATP-dependent RNA helicase [5]. In the fruit fly, the enzymatic activity of vasa protein shares the function of a germ-line-specific translational regulator, acting as the eukariotic Initiation Factor 4A (eIF4A) [6]. Moreover, vasa protein may also play an indirect role in gene transcription, regulating the expression of transcription factors [7]. In addition to Drosophila melanogaster [8], the vasa gene was cloned and sequenced in several other species of invertebrates and vertebrates: nematode, Caenorhabditis elegans [9], toad, Xenopus laevis [10], mouse, Mus musculus [11], and recently in several teleost species, including zebrafish, Danio rerio [12, 13], tilapia, Oreochromis niloticus [14], and rainbow trout, Oncorhynchus mykiss [15]. The vasa gene is also expressed in the mature gonads, and, despite extensive progress in comprehending the function of the vasa gene in PGC determination, much remains to be studied about its function in these tissues. In fact, little information about the involvement of the vasa gene in gonadal function is available to date. In Drosophila, null-vasa mutants fail to develop mature oocytes, even if some early oocytes are present, indicating that, in mature gonads, the vasa gene is involved in oocyte maturation [16]. This was the initial evidence, later confirmed by other studies in invertebrates [17] that coupled gamete maturation and the vasa function, suggesting an interplay between oogenesis and the activity of the vasa protein in Drosophila [18]. Recently, vasa mRNA expression has been related to gametogenesis in tilapia as well [14].

The regulation of gonadal functions falls under the hypothalamic-pituitary-gonadal axis. In particular, oogenesis is controlled mainly by the pituitary gonadotropins through the local action of estradiol-17ß. Oogenesis in teleost fish, as in other nonmammalian vertebrates, occurs in two phases: vitellogenesis and maturation. In the first phase, oocyte meiosis is stopped at the first meiotic prophase, and vitellogenin (VTG) uptake from the blood leads to a dramatic increase in oocyte volume. In the second phase, meiosis restarts, and oocyte maturation takes place. The roles of several different hormones in gonadal maturation have been well documented. During the first phase, the maturation of the oocyte is strictly correlated to vitellogenin synthesis, and vitellogenesis is regulated mainly by FSH and estradiol-17ß [19], but other hormones are also involved in regulating the synthesis of this protein. In fact, there is clear-cut evidence for the action of growth hormone (GH) on VTG synthesis induction [20, 21]. In addition, during the second phase, GnRH promotes germinal vesicle breakdown (GVB) [22].

Since the role of vasa in mature gonads is still unclear and its possible involvement in gamete maturation must still be elucidated, this study was undertaken to explore a possible correlation between vasa gene expression and the hormones involved in oocyte maturation, such as GnRH, estradiol (E2), and GH.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Treatment and Sampling

Adult female sea bream were obtained from a commercial fish farm (La Rosa, Orbetello, Italy) during the prespawning period. All the animals used for this experiment were matched for size and age, and they have been kept under the same conditions: 37 psu of salinity, temperature of 18°C, 14L:10D. Groups of five animals were treated as follows: E2 (3.5 µg/animal; Sigma, St. Louis, MO), GH (sbGH, 50 µg/animal; GroPep, Adelaide, Australia), GnRH (aGnRH, 20 µg/animal; Sigma), a combination of GH + GnRH (50 µg/animal GH and 20 µg/animal GnRH; Sigma), and E2 + GH (3.5 µg/animal E2 and 50 µg/animal GH). Two different vehicles were used for the injection of hormones: physiological saline solution (113 mM NaCl, 1 mM KCl, 2 mM CaCl2, 5 mM Hepes) for GH, GnRH, and GnRH + GH and cocoa butter for E2 [23]. In order to check possible differences due to these different vehicles, two control groups were established: one injected with saline and the other with cocoa butter.

After 3 days of treatment, the animals were killed, and the entire ovary, brain, liver, and muscle were rapidly removed in ribonuclease (RNase)-free conditions and stored at -80°C for RNA extraction.

Oocytes at different maturation stages were dissected from five different prespawning female sea bream. Oocytes were separated under micrometric microscopy in F.O. solution (NaCl 113 mM, KCl 1 mM, CaCl2 2 mM, Hepes 5 mM, pH 7.5) in five stages: stage I, oogonia (<0.025 mm); stage II, previtellogenic oocytes (0.025–0.1 mm); stage III, early vitellogenic oocytes (0.15–0.3 mm); stage IV, midvitellogenic oocytes (0.3–0.4 mm); and stage V, late vitellogenic oocytes (>=0.5 mm). After their separation, total RNA was immediately extracted, without any freezing step.

The animal studies were approved by the animal care and treatment committee of the University of Genova.

RNA Extraction

For each tissue, 100 mg were used for total RNA extraction with Trizol Reagent, following manufacturer's protocol (Invitrogen Life Technologies, Milan, Italy). To avoid possible genomic DNA contamination, RNA samples were incubated with 1 unit RNase-free DNase (MBI Fermentas, St. Leon-Rot, Germany) in 10 mM Tris-HCl pH 7.5, 2.5 mM MgCl2, and 0.1 mM CaCl2. After DNase treatment, the samples were reextracted with phenol:chloroform:isoamilic alcohol (25:24:1) and then precipitated with ethanol [24]. The integrity of the RNA was assessed by denaturing agarose gel electrophoresis, followed by ethidium bromide staining, and the quantity was determined by UV reading at 260 nm.

cDNA Cloning

The partial sea bream vasa-like cDNA (sbvasa cDNA) was cloned and sequenced with 3' RACE. A total amount of 5 µg of RNA was used for cDNA synthesis, employing 0.5 µg oligo d(T) + adapter primer, 5'- GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTTT-3', in a buffer containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 0.5 mM each dNTP and 200 units of Superscript II RT (Invitrogen Life Technologies, Milan, Italy), with incubation at 42°C for 50 min. The cDNA obtained was amplified by polymerase chain reaction (PCR) using a degenerate forward primer previously used on O. mykiss [15], vasa For 5'-ATGGCNTGYGCNCARACNG-3', and a vasa Rev primer specific for the adapter region of the oligo d(T) used for cDNA synthesis (see previous discussion), 5'-GACTCGAGTCGACATCG-3'.

For qualitative and quantitative analyses, a short fragment of sbvasa was isolated by PCR and cloned. The vasa For 2, 5'-GTGTGCGTCCAGTAGTAGTGT-3', generated on the homologous sequence of sea bream, was used as the forward primer, while the vasa Rev 2, 5'-CCACCTGGGTTGAACCCTGTG-3', used as the reverse primer, was generated on the conserved regions of vasa-like cDNAs available in the GenBank (888 base pairs [bp] long; NCBI, Bethesda, MD).

PCR reactions were carried out with 0.1 µl of synthesized cDNA, 1x PCR buffer (10 mM Tris-HCl pH 8.8, 50 mM KCl, 1% Triton X-100), 1.5 mM MgCl2, 0.2 µM of each primer, 250 µM of each nucleotide, and 1 unit of Taq Dynazyme (Finzyme, Espoo, Finland). The thermocycle profile (PCR Express; Hybaid, Ashford, UK) for vasa For and vasa Rev primers was denaturation phase at 94°C for 5 min, 30 cycles of 94°C for 30 sec, 53°C for 1 min and 72°C for 1 min and 30 sec, followed by a phase at 72°C for 8 min.

The PCR product was purified using the MinElute PCR Purification Kit for PCR product purification (Qiagen, Milan, Italy) and then cloned into the pGEM T easy vector, using the pGEM T easy vector system (Promega, Milan, Italy) in accordance with the manufacturer's protocol. The plasmid was transformed into DH5{alpha} host cells by the TransformAid kit (MBI Fermentas). Since several isoformos of vasa-like mRNA have been reported for other species [25], we analyzed several positive clones to determine if that was also the case in the sea bream. All the cloned inserts were sequenced by an ABI model 310 DNA sequencer (Perkin-Elmer, Oak Brook, IL).

Probe Synthesis

For Northern blot and in situ hybridization analysis, digoxigenin (DIG)-labeled RNA probes were prepared by in vitro transcription using the homologous sbvasa short fragment cloned in pGEM T easy vector (Promega) as a template. For the antisense and sense probe, SP6 and T7 RNA polymerase (Roche, Basel, Switzerland) were used with the DIG-RNA labeling kit (Roche) following the manufacturer's protocol. For in situ hybridizations, the probe was purified after labeling by precipitation with 4 M LiCl and 100% ethanol.

For Southern blot analysis a DIG-labeled DNA probe was made using the same homologous fragment as the template. The labeling was performed using the DIG DNA labeling kit (Roche), based on random primed reaction, following the manufacturer's instructions.

Northern Blot Analysis

For qualitative analysis on vasa-like mRNA expression in sea bream tissues, total RNA extracted from gonads, brain, liver, and muscle was used for Northern blot. An amount of 20 µg of total RNA per sample was diluted in a sample buffer (50% formamide, 1x MOPS, 2 M formaldehyde), denatured at 65°C for 10 min, and then loaded in a denaturing gel. After running, the blotting was made under a rapid protocol, using a light alkaline buffer [26], on a nylon membrane (Schleicher & Schuel, Dassel, Germany). The membrane was then hybridized overnight by the homologous RNA probe at 65°C in a standard hybridization buffer (5x saline-sodium citrate [SSC], 0.1% N-lauroylsarcosine, 0.02% SDS, 2% blocking reagent, 50% formamide). After the hybridization, the blot was first washed two times in a solution containing 0.1% SDS and 2x SSC for 5 min at room temperature, then twice in 0.1% SDS and 0.1x SSC at 65°C for 15 min. After these washings, a chemiluminescent detection procedure was performed with the DIG Luminescent detection kit (Roche) according the manufacturer's protocol. The signal was then evidenced by autoradiography using the Kodak BioMax Light-1 film (Sigma).

In Situ Hybridization

Using the homologous sbvasa RNA as a probe, in situ hybridization was performed using ovaries from prespawning sea bream. Ovarian samples were fixed in 4% paraformaldehyde in 20 mM, pH 7.4, phosphate-buffered saline (260 mM NaCl, 10 mM Na2HPO4, 10 mM NaH2PO4) at 4°C overnight.

Tissue samples were then extensively washed and subsequently embedded in Tissue Freezing Medium (Electron Microscopy Science, Fort Washington, PA) at -25°C, after which they were cut in transverse sections (10 µm thick) using a Frigocut 2800E cryostat (Leica, Wetzlar, Germany). The frozen sections were mounted on glass slides coated with 10% poly-L-lysine-hydrobromide (Sigma) and used for the experimental procedures.

Frozen sections were treated with 0.2 N hydrochloric acid for 15 min at room temperature, equilibrated in 20 mM PBS, pH 7.4, and then partially digested with proteinase K (20 µg/ml; Sigma) for 7 min at room temperature. Sections were hybridized overnight at 55°C with a DIG-labeled probe (100 ng/ml) in hybridization buffer containing 50% formamide, 100 µg/ml heparin, 5x SSC, 5 mM EDTA, pH 8.0, 1x Denhardt solution, 1 mg/ml total yeast RNA. After hybridization, the coverslips were floated off in 50% formamide, 2x SSC, on a rotary shaker at 55°C. Subsequently, the slides were treated with RNase A (10 µg/ml) for 20 min at 37°C and then washed in 2x SSC (2 x 15 min) at 55°C, 0.2x SSC (2 x 15 min) at room temperature, after which they were incubated 1 h in blocking solution containing 10% normal sheep serum and 2 mg/ml BSA. After incubation overnight at 4°C with anti-DIG alkaline phosphatase conjugated antibody (1:5000; Roche), the sections were washed in PBS, and the reaction was developed with nitrotetrazolium blue chloride (2.5 µl/ml)/5-bromo-4-chloro-3-indolyl phosphate (3.5 µl/ml) in alkaline phosphatase buffer containing 0.05M MgCl2, 0.1 M NaCl, 0.1 M Tris-HCl, pH 9.6. After fixing and washing, the sections were mounted in DABCO (Sigma) and observed by a Leica DMRB microscope.

Semiquantitative RT-PCR

For quantitative analysis of sbvasa mRNA expression in the ovary of hormonally treated animals and in separate oocytes, a semiquantitative RT-PCR method, coupled with Southern blot analysis, was optimized on the basis of that previously described [27, 28]. The EF-1A cDNA was used as an internal standard to normalize the yield of the vasa PCR product. For sbvasa mRNA expression, the primers vasa For 2 and vasa Rev 2 were used as described previously, while for EF-1A mRNA expression, a forward primer deduced from sea bream EF-1A sequence (NCBI GenBank accession no. AF184170) was employed, with EF-1A 5'-TGCTGCCATTGTAAACT-3' as the forward primer and the vasa Rev 1 (see previous discussion) as the reverse primer. This cDNA is considered to be a housekeeping gene, and it is widely used as an internal standard in quantitative evaluations of small changes in the relative amounts of specific mRNA, with the results proving reasonably accurate [29–31]. The PCR reaction for EF-1A was performed with 0.1 µl of synthesized cDNA, 1x PCR buffer, 1.5 mM MgCl2, 10 µM of each primer, 250 µM of each nucleotide, 0.2 units of Taq Dynazyme (Finzyme). After a denaturation phase of 5 min at 94°C, the amplification consists of cycles of 20 sec at 94°C, 30 sec at 58°C, 20 sec at 72°C, and a single final phase at 72°C for 5 min. Preliminary experiments were conducted in order to determine the optimal number of PCR cycles and the amount of cDNA template needed to ensure that the reactions were in the exponential phase. The products obtained were separated in a 1.5% agarose gel and then subjected to Southern blot hybridization using homologous DNA probes. As for the Northern blot analysis, the hybridization was followed by a chemiluminescent detection, and the signal was evidenced by autoradiography. The films were scanned by a laser scanner (Sharp Electronics, Milan, Italy) and then subjected to densitometric analysis by ImageQuant software version 1.2 (Molecular Dynamics, Amersham Biosciences, Sunnyvale, CA).

Statistical Analysis

The mRNA quantification data are expressed as mean ± SD. The data were analyzed by ANOVA, followed by Student t-test to test the difference between experimental groups; P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning of sbvasa cDNA

The PCR amplification made by vasa For 1 and vasa Rev 1 primers gave a product of 1586 base pair (bp), which includes approximately 75% of the full-length sequence (NCBI GenBank accession no. AF520608). The sbvasa cDNA showed a high homology with other teleost vasa-like cDNA sequences: 85% with O. niloticus, 80% with O. latipes, and 79% with O. mykiss. In addition, the deduced amino acid sequence showed marked homology with vasa proteins of several organisms, including O. mykiss (84%) and O. niloticus (88%). Moreover, this sequence presents all the consensus sequences typical of DEAD box family protein, ATP-dependent helicases [32], including the ATP-A motif (AHTGSGKT), the ATP-B (DEAD), and the PTRELI motif, which shows a substitution of the last amino acid, as previously found in X. laevis [10] (Fig. 1).



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  		FIG. 1. Nucleotide and deduced amino acid sequences of sbvasa cDNA. The boxes indicate the consensus sequences typical of the DEAD box family ATP-dependent RNA helicases, including the ATP-A motif (AHTGSGKT), the ATP-B (DEAD), and the PTRELI motif, which shows a substitution of the last amino acid.

Northern Blot Analysis

The qualitative analysis of sbvasa mRNA expression in the sea bream tissues showed a tissue specific presence of a single vasa mRNA of approximately 2100 nucleotides only in the gonads but not in other tissues, such as brain, liver, or muscle (Fig. 2).



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  		FIG. 2. Qualitative analysis of sbvasa mRNA expression in the ovary (1), in the brain (2), in the liver (3), and in the muscle (4). Total RNA from tissues was loaded in a denaturing 1% agarose gel (A)—arrows indicate ribosomal RNAs—and then transferred onto a nylon membrane by Northern blot. After homologous hybridization, the specific sbvasa expression was observed only in the ovary (B).

In Situ Hybridization

The sea bream ovary showed a great number of ovarian follicles at different stages of growth, varying in size from 70 to 500 µm (Fig. 3). The oocyte's nucleus was centrally located in the cytoplasm. The vasa RNA was localized in midvitellogenic (Fig. 3a) and vitellogenic (Fig. 3b) oocytes; a particularly strong signal was visible in the perinuclear cytoplasm of midvitellogenic oocytes and among the yolk granules in vitellogenic oocytes. No vasa RNA expression was evident in oogonia and previtellogenic oocytes. The absence of sbvasa mRNA in the previtellogenic oocytes could be related to the low levels of transcripts. Finally, no detectable signal was observed using the sense probe.



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  		FIG. 3. Vasa mRNA expression in ovary of S. aurata. Signals are visible in the cytoplasm of the oocytes at the midvitellogenic (MV) (a) and vitellogenic (V) (b) stages; the expression is only cytoplasmic, at first perinuclear (arrowheads) in MV oocytes and subsequently scattered among the yolk (arrowheads) in V oocytes. Oogonia (asterisks) and previtellogenic (PV) oocytes are negative. Bar = 100 µm.

Sbvasa mRNA Expression

The products obtained at different cycles of amplification were analyzed by Southern blot hybridization and densitometry in order to verify the threshold number of cycles: the optimum conditions were found at 28 cycles for vasa and 26 for EF-1A, within the exponential phase of the amplification. The PCR product obtained for EF-1A, employed to normalize the signal of vasa mRNA expression, was 550 bp long, while the vasa product was 888 bp. No significant differences were found between the two control groups of animals for vasa-like mRNA expression; consequently, in the quantification experiments, the control group is a mean of all the animals injected with saline solution or cocoa butter. Vasa-like mRNA expression under hormonal treatment, expressed as arbitrary units compared to the control group (1.04 ± 0.11 a.u.), showed a stimulatory effect of E2 (1.73 ± 0.07 a.u., P < 0.01) on vasa-like mRNA expression in the ovary. GH treatment also increased vasa mRNA content in the ovary (1.60 ± 0.04 a.u., P < 0.01), while treatment with GnRH significantly decreased vasa mRNA expression (0.71 ± 0.03 a.u., P < 0.05). Simultaneous treatment by GH and GnRH showed a counteracting effect of GnRH on the stimulatory effect that GH alone had on the expression of this mRNA (1.39 ± 0.06 a.u., P < 0.01), while E2 plus GH completely inhibited vasa mRNA expression (Fig. 4).



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  		FIG. 4. A) Representative Southern blot of the amplification of sbvasa and EF-1A mRNA by RT-PCR in sea bream oogonia (I), previtellogenic oocytes (II), early vitellogenic oocytes (III), midvitellogenic oocytes (IV), and late vitellogenic oocytes (V). B) Relative concentration of sbvasa mRNA in sea bream oocytes, as determined by semiquantitative RT-PCR, followed by Southern blot and densitometric analysis. The sbvasa mRNA concentration has been normalized using the EF-1A as the internal standard and is expressed as the mean result for five separate experiments. All the differences between all samples are statistically significant (P < 0.01).

In the oocytes at different maturation stages, sbvasa mRNA expression showed a progressive increase, together with oocyte maturation. In fact, in oogonia, sbvasa mRNA is not present; its expression starts at the previtellogenic stage (0.58 ± 0.03 a.u.), increasing in early and midvitellogenic oocytes (0.94 ± 0.04 a.u. and 1.22 ± 0.06 a.u., respectively) and reaching its maximum in late vitellogenic oocytes (1.65 ± 0.06 a.u.; Fig. 5).



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  		FIG. 5. A) Representative Southern blot of the amplification of sbvasa and EF-1A mRNA by RT-PCR in the ovary of control sea bream (1), treated with E2 (2), GH (3), GnRH (4), E2 + GH (5), and GnRH + GH (6). B) Relative tissue concentration of sbvasa mRNA in the ovary of sea bream, as determined by semiquantitative RT-PCR, followed by Southern blot and densitometric analysis. The sbvasa mRNA concentration has been normalized using the EF-1A as the internal standard and is expressed as the mean figure for five animals in each experimental group, compared to vehicle-treated control group, by the Student t-test. Values with different letters indicate statistical significance (P < 0.01).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present paper, the vasa-like gene was cloned and characterized for the first time in a marine teleost, the gilthead sea bream Sparus aurata. The sbvasa cDNA partial sequence cloned showed appreciable similarity when aligned with the freshwater teleosts vasa-like sequence; in addition the deduced AA sequence contains all the consensus sequences for the vasa-like proteins. Taken as a whole, these data confirmed that the cDNA cloned and characterized is homologous to the vasa mRNA in S. aurata.

The mRNA for the vasa-like gene was detected by Northern blotting in adult fish only in gonadal tissue, suggesting that it may be involved in oocyte maturation. This hypothesis is supported by the data of vasa-like mRNA expression in oocytes at different stages of maturation, as detected by both in situ hybridization and semiquantitative RT-PCR in oocytes of different size. The increasing expression of vasa mRNA observed as oocytes progress through the various stages of vitellogenesis in the sea bream suggests a potential regulatory role for vasa in oocyte maturation in this species, as has been suggested for Drosophila [19] and tilapia [14].

The gonadal functions are strictly correlated with hormonal levels. In particular, the gametogenic process is under the control of the hypothalamic-pituitary-gonadal axes; the hypothalamic factor, GnRH, stimulates the production of gonadotropins in the pituitary; and the latter stimulates the production of sex steroids in the gonads. One of the most important sex steroid hormones for oocyte maturation is estradiol-17ß, which also induces the hepatic synthesis of vitellogenin during oocyte maturation [33].

To elucidate the effects of the typical signals involved in gonadal function on the vasa gene expression, mature females were injected, during the prespawning period, with hormones strictly correlated to gamete maturation. In addition, one more group was treated with GH since this growth factor has been found to be involved in the vitellogenic process [20, 21]. In the present work, the hormonal control of vasa-like gene expression was demonstrated for the first time. In teleosts, the vasa mRNA and not its protein is part of the germ plasm [34]. In the sea bream, this mRNA is localized differently during the different phases of oocyte maturation. It has recently been demonstrated that a specific nucleotide sequence of the 3'-end of vasa mRNA is important for its correct positioning within the oocyte [35]. The observation that vasa-like mRNA is first expressed during midvitellogenesis and is localized to the perinuclear area at that time, while vasa-like mRNA fills up the cytoplasm in late vitellogenesis, appears to indicate that the accumulation of this maternal factor occurs during active vitellogenesis, when E2 levels in the blood are highest [23]. This idea is supported by the observed stimulatory effects of vasa-like expression induced by both E2 and GH treatment; in fact, both these hormones are correlated with vitellogenin synthesis. The proliferation of oogonial cells that may be induced by E2 or GH treatment does not influence the sbvasa expression since these cells, in sea bream, do not express the vasa gene, as demonstrated by RT-PCR on separate oocytes.

Totally unexpected was the inhibitory effect of the GnRH treatment; however, similar negative effects were found using GnRH analogue treatment, which, instead of acting as a positive regulator, may have negative interaction in the gonads [36] as well as playing a role in the resumption of oocyte meiosis in sea bream. Moreover, GnRH also appears to have a negative effect on the release of the insulin-like growth factors (IGFs) in the liver [37]. A negative effect of GnRH on IGF expression and/or release may explain, in part, the suppressive effect of GnRH on GH-enhanced vasa-like mRNA expression in these studies. In order to further investigate this aspect, a bidimensional study on GnRH dose-response effect on GH stimulatory action on vasa gene expression should be made in the future. This hypothesis is supported by the results obtained through the simultaneous administration of GnRH plus GH. In this group of fish, the expression of vasa is higher than in the control but lower compared to that found with the GH treatment, suggesting a counteracting effect of GnRH on GH action.

The most surprising result, however, was the total inhibition of vasa gene transcription when GH and E2 were administered simultaneously. A similar unconventional effect was previously observed in transgenic mice (MT-hGHRH), in which high GH, combined with E2 treatment, inhibited the GH receptor and the GH binding protein, both of which are involved in IGF induction [38]. It is well known that GH and E2 regulate the interplay between body growth and gonadal function in a manner that depends on their circulating concentrations with GH playing a major role in regulating the energy allocation needed for increasing body mass while E2 plays a major role in regulating reproductive function. We speculate that the high levels of GH and E2 simultaneously circulating in the blood may cause a downregulation of vasa gene transcription. These data open questions on the mechanisms regulating the control of the maternal messenger transcription responsible for gonadal ontogenesis. More in-depth studies are necessary to elucidate the negative effects of two signals that, in contrast, have a positive effect on vasa gene transcription when administered alone.


   FOOTNOTES
 
1 Correspondence: Oliana Carnevali, Department of Marine Science, Polytechnic University of the Marches, Via Brecce Bianche, 60131 Ancona, Italy. FAX: 39 071 220 4650; carnevali{at}univpm.it Back

Received: 24 July 2003.

First decision: 18 August 2003.

Accepted: 4 November 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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