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a Laboratoire de Biologie de la Reproduction des Poissons, Unité Associée INRA, Université Bordeaux I, 33405 Talence, France
b Department of Biology and Biochemistry, Brunel University, Uxbridge, Middlesex, UB8 3PH, United Kingdom
c Laboratoire d'Histologie-Embryologie, Université Bordeaux II, 33076 Bordeaux, France
d Laboratoire d'Endocrinologie Moleculaire de la Reproduction, UPRES-A 6026, CNRS, Université de Rennes I, 35042 Rennes, France
e Department of Molecular Genetics, Biocenter and University of Vienna, A-1030, Vienna, Austria
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The uptake of VTG into growing oocytes occurs by receptor-mediated endocytosis [2–6, 10]. Receptors for VTG (VTGR) have been identified at the oocyte cell surface in several vertebrate groups, including birds [11–13], amphibians [5, 14], fish [6, 15–19], and some invertebrates [20–23]. Studies on VTGR protein in the rainbow trout have shown that it is present in oocytes throughout the major part of ovarian development and that it can be detected even before the onset of vitellogenesis (defined as the period when there is a rapid uptake of VTG into oocytes) [24].
Molecular characterization of the cDNA of the gene encoding the VTGR in chickens, Xenopus, and rainbow trout demonstrated that the VTGR gene is a member of the low-density lipoprotein (LDL) receptor (LDLR) gene superfamily [25–28]. The deduced amino acid sequence of VTGR cDNA revealed that this protein typically contains 1) a ligand binding domain at the N terminus with eight cysteine-rich repeats, 2) an epidermal growth factor precursor homology domain, 3) a transmembrane domain, and 4) a cytoplasmic region containing the signal (FDNPVY) involved in receptor internalization via coated pits [29, 30].
In the rainbow trout ovary, the cDNA encoding for a second lipoprotein receptor has been partially characterized from the ovary, which differs only in sequence from the VTGR, by an additional 105 base pairs (bp) (this constitutes the so-called O-linked sugar domain) [27]. This receptor has been referred to as the somatic lipoprotein receptor, and it is expressed both in the ovary and in somatic tissues. The precise function of the somatic lipoprotein receptor is not known, but it is thought to mediate the uptake of lipoproteins other than VTG, for example, very low-density lipoprotein (VLDL) [27].
Preliminary studies have been conducted on the expression of VTGR mRNA in rainbow trout [27, 28], but nothing is known about when expression of the VTGR gene is initiated or the control of its expression. Furthermore, nothing is known about the expression of VTGR mRNA in relation to the synthesis of VTG and/or uptake of VTG into the ovary.
Given the importance of the vitellogenin receptor in the mediation of oocyte growth in all oviparous animals, this study aimed to quantify the dynamics of expression and localization of VTGR mRNA throughout ovarian development in rainbow trout, using both Northern blotting and in situ hybridization. In parallel, light and electron microscopy and immunocytochemistry were employed on histological sections of ovaries to determine the timing of the uptake of VTG into oocytes in relation to the timing of the synthesis of VTGR mRNA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female rainbow trout at different stages of ovarian development were obtained from a commercial trout farm in Hampshire (UK). All fish had been maintained in outdoor tanks/earthen ponds under conditions of natural photoperiod and temperature. Fish were anesthetized in 2-phenoxyethanol (Sigma, London, UK), and blood samples were collected from the caudal sinus using heparinized syringes. Blood was centrifuged at 3000 x g for 10 min, and the plasma was collected and frozen at -20°C until required. Different tissues (ovary, ovulated eggs, intestine, heart, and muscle) were rapidly removed in ribonuclease (RNase)-free conditions, and parts of each tissue were processed for both Northern and in situ analysis. Testicular tissue was also collected from four male fish (2 sexually immature and 2 sexually mature). Fish of various ages from a number of different populations were sampled in order to get ovaries at all the required stages of development. Body and ovary weights were measured to the nearest gram to determine the gonadosomatic index (GSI = [gonad weight/body weight] x 100).
Tissue Fixation and Slide Preparation
Immediately after sampling the fish, pieces of tissue of approximately 1 cm3 were cut off and fixed in Bouin's fluid for 6 h. Tissue samples were then extensively washed and subsequently stored in 70% ethanol at 4°C before being embedded into paraffin wax. Paraffin-embedded tissues were then sectioned at 5 µm on a rotary microtome, and the sections were lifted onto baked glass slides pre-coated with a 2% solution of TESPA (3-aminopropyl triethoxysilane; Sigma, UK) in acetone. Slides were then dried at 50°C and placed in an incubator (80°C) overnight to allow sections to adhere to the slides. Slides were stored at -80°C until required. For each fixed tissue, used for either in situ hybridization or for immunocytolocalization of VTG, adjacent sections were stained with hematoxylin and eosin [31].
Expression of Trout VTGR mRNA
Total and poly A(+) mRNA extraction Total RNA was extracted from nonovarian and ovarian tissues at different stages of development, using the guanidinium thiocyanate-phenol-chloroform extraction method [32]. RNA extraction was carried out on the following ovarian tissues: 1) previtellogenic ovaries containing only ovarian follicles (OF) smaller than 300 µm in diameter; 2) previtellogenic ovaries containing only OF smaller than 400 µm; 3) ovaries at the onset of vitellogenesis with OF measuring 550 ± 50 µm; 4) three categories of vitellogenic ovaries with OF measuring 1500 ± 300 µm, 2500 ± 200 µm, and 3600 ± 300 µm; and 5) ovulated eggs of 5000 ± 200 µm. Poly A(+) mRNA was purified from these tissues using an Oligotex mRNA kit (Qiagen, Crawley, UK), according to the manufacturer's procedure.
Northern Blotting
Poly A(+) mRNA (15 µg/tissue) from ovarian and nonovarian tissues was denatured with glyoxal/dimethyl sulfoxide, separated by electrophoresis on a 1% agarose gel, and blotted onto Hybond-N+ membranes (Amersham, Little Chalfont, UK) using a vacuum blotter (Bio-Rad, Hemel Hampstead, UK). Membranes were prehybridized in hybridization buffer (40% formamide, 5-strength SSC [single-strength SSC is 0.15 M sodium chloride, 0.015 M sodium citrate], 50 mM sodium phosphate buffer [pH 6.5], 5-strength Denhardt's solution, 0.1% SDS, and 100 µg/ml yeast tRNA [Boehringer, Meylan, France]) for 5 h at 42°C and then hybridized in the same buffer containing 32P-labeled cDNA probe (Megaprime DNA labeling systems; Amersham, Les Ulis, France). The full-length rainbow trout VTGR cDNA (3940 bp) was used as probe. Washes were done in double-strength SSC, 0.1% SDS at 42°C for 4 x 15 min, and in 0.3-strength SSC, 0.1% SDS at 45°C for 3 x 30 min. Membrane blots were then exposed to Kodak X-OMAT film (Sigma, L'Isle D'Abeau Chesnes, France) for 24 h at -20°C. The signal intensities were quantified by video densitometric analysis using the program Bio 1D, v6.3 (Vilber Lourmat, Marne La Vallée, France). Membrane blots were stripped in 500 ml of 0.4 M NaOH at 42°C for 30 min and then agitated in 500 ml of 0.1-strength SSC, 0.1% SDS, 0.2 M Tris-HCl, pH 7.5, at 42°C for 30 min. The trout ß-actin cDNA was used as internal control. The stripped membranes were prehybridized in hybridization buffer containing 20 mM sodium phosphate buffer (pH 7.0), 5-strength Denhardt's solution, 1% SDS, 10% dextran sulphate, and 50% formamide at 42°C overnight, and then hybridized in the same buffer containing [32P]dCTP-labeled ß-actin probe (Rediprime DNA labeling systems; Amersham, UK). The membranes were washed twice in double-strength SSC, 0.1% SDS at 42°C for 5 and 10 min, respectively, washed again in single-strength SSC, 1% SDS at 42°C for 2 min, and then exposed to Hyperfilm-MP film (Amersham, UK) for 24 h at -20°C.
Probe Synthesis for In Situ Analysis
35S-labelled antisense and sense RNA probes were prepared by in vitro transcription using as template a 700-bp fragment from the 3' end of the full-length cDNA for the rainbow trout VTGR. The transcription was performed from 100 ng of linearized plasmid containing either a T3 or T7 RNA polymerase promoter upstream of the 700-bp fragment from trout cDNA VTGR using [35S]UTP (NEN, Paris, France; specific activity > 3.7 x 104 GBq/mmol) and T7 (antisense) or T3 (sense) RNA polymerases (Gibco BRL, Cergy Pontoise, France), as described by Le Moine and Bloch [33]. After alkaline hydrolysis to obtain 250-base cRNA fragments, the probes were purified on G50-Sephadex (Sigma, France) and precipitated in 3 M sodium acetate, pH 5.2 (0.1 vol)/absolute ethanol (2.5 vol). The quantity of labeled RNA synthesized under our conditions corresponds to a specific activity of 12 x 103 Bq/ng.
In Situ Hybridization
Paraffin sections were dewaxed by immersion in toluene (3 x 5 min) and then rehydrated in 100% ethanol (1 x 4 min), in 70% ethanol (1 x 4 min), and in water treated with DEPC (diethyl pyrocarbonate; Sigma, France; 3 x 5 min). Sections were then partially digested with proteinase K (2 µg/ml; Polylabo, Strasbourg, France) for 15 min at 37°C and with 0.2 N hydrochloric acid for 5 min at room temperature. After dehydration in graded ethanols, the sections were hybridized overnight at 55°C with 106 cpm of 35S-labeled probes (1–1.5 ng) in 50 µl of hybridization buffer containing 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, pH 8.0, 300 mM sodium chloride, 50% formamide, 10% dextran sulphate, single-strength Denhardt's solution, 0.1% SDS, 250 µg/ml yeast tRNA (Boehringer), 100 µg/ml salmon sperm DNA (Boehringer), and 0.1% sodium thiosulfate. After hybridization, the coverslips were floated off in 4-strength SSC on a rotary shaker at room temperature. Subsequently, the slides were treated with RNase A (20 µg/ml; Boehringer) for 15 min at 37°C; washed with double-strength SSC (2 x 5 min) at room temperature, double-strength SSC containing 50% formamide (2 x 15 min) at 65°C, double-strength SSC (2 x 5 min) at 65°C, and 0.1-strength SSC (5 min) at 65°C; and rinsed in 0.1-strength SSC (5 min) at room temperature before dehydration in graded alcohols. All SSC baths contained 1 mM dithiothreitol (Promega, Charbonnieres, France) except when formamide was present. Preincubation with RNase was used to assess the nonspecific binding of the probes to the sections.
Autoradiography and Image Analysis
After being air-dried, the slides were placed in x-ray cassettes and exposed to ßmax film (Amersham, France) for 2–7 days. They were then dipped into K5 emulsion (Ilford, Champs sur Marne, France), exposed for 15–20 days, developed, and stained with toluidine blue dye. Silver grains in cells and tissues in adjacent slides were counted by using an image analyzer RAG 200-BIOCOM (version 2.13; Biocom, Les Ulis, France) with an 80x objective and darkfield illumination, according to the method described by Le Moine et al. [34].
In the present study, image analysis of the in situ hybridization signals was used to determine the localization of VTGR mRNA and to quantify its expression through the density of silver grains per unit area of tissue, following the basic principles required for quantitative in situ hybridization histochemistry [34–36]. Data from 136 slides (4 slides per tissue per probe) were analyzed. The following measures were taken from the ovarian tissue: 1) OF and oocyte nucleus diameters and 2) OF, nucleus, and cytoplasm surface areas. For both ovarian and nonovarian tissues, the density of silver grains (number of grains/1000 µm2) was measured in at least 10 different subareas. Ovarian follicles were examined and used in the analyses only with sections cut through the nucleus, as this allowed us to determine the size of the oocytes accurately. The oocytes were then grouped into 8 different classes according to their cellular diameters: 35–49 µm; 50–99 µm, 100–199 µm; 200–299 µm; 300–499 µm; 500–699 µm; 700–999 µm and 
1000 µm.
Specific signals of hybridization were determined from differences between signals obtained using the VTGR sense probe. Those tissues hybridized with the antisense probe that contained a statistically higher number of silver grains than tissues of the same type hybridized with the sense probe were considered as labeled (specific signals).
Ovarian Uptake of VTG
Studies were undertaken to determine the concentration of VTG in the fish plasma and the timing of the onset of uptake of VTG into developing oocytes. The primary focus here was to determine how the synthesis of VTG and its endocytosis into the oocyte (and consequently, the time when the VTGR first became functional) related to the expression and localization of VTGR mRNA.
Electron Microscopy
Microscopy was carried out on previtellogenic and vitellogenic OF to examine, at structural (semi-thin sections) and ultrastructural (ultra-thin sections) levels, the appearance of components associated with the endocytosis of the VTG. Small pieces of ovaries were fixed with 6% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) containing 0.5% calcium chloride for 24 h at room temperature. After being rinsed in the same buffer, the tissues were post-fixed in 1% buffered osmium tetroxide for 1 h at room temperature. After a further buffer rinse, the tissues were dehydrated in a graded series of ethanol and propylene oxide, and embedded in epoxy resin. Initially, semi-thin sections (1 µm) were prepared and stained with toluidine blue dye to examine OF in previtellogenic, early vitellogenic, and mid-vitellogenic development. Subsequently, ultra-thin sections were cut, mounted on copper grids, stained with uranyl acetate and lead citrate, and examined using a Philips (Eindowen, The Netherlands) CM 10 electron microscope.
Immunocytolocalization of VTG
Ovaries at different stages of development were fixed and the slides prepared as previously described. Paraffin-embedded sections were dewaxed in toluene (3 x 5 min), rehydrated in descending ethanol concentrations, and successively treated with 1% lithium carbonate, 0.1 M PBS, pH 7.4, and 0.1% hydrogen peroxide for 5 min each at room temperature. The slides were then incubated with 2.5% (v:v) normal pig serum in 0.1 M PBS for 1 h at 37°C, rinsed in the same buffer, and incubated with antiserum against rainbow trout VTG at a dilution of 1:500 in PBS for 2 h at 37°C. The anti-VTG was prepared and its specificity verified as described by Bon et al. [37]. The second antibody, swine anti-rabbit IgG-horseradish peroxidase (HRP; Dako, Glostrup, Denmark), was then applied at a dilution of 1:100 in PBS for 1 h at 37°C. After incubation, sections were thoroughly washed in PBS. The peroxidase activity was finally revealed using 3–3'diaminobenzidine tetrahydrochloride (DAB; Sigma, France). Control slides were treated with normal rabbit serum.
Measuring of Plasma VTG
Plasma titers of VTG in the fish were determined using a specific RIA for rainbow trout VTG [38].
Statistical Analysis
Differences in the in situ hybridization signals between the sense and antisense probes for the VTGR were determined for each tissue using the Mann-Whitney test. Similarly, differences between the specific signals in the different size classes of OF and between the ovarian and nonovarian tissues were determined using the Mann-Whitney test. GSI and plasma VTG data were transformed before statistical analysis using arc sine and logarithmic transformations, respectively. Data were analyzed by one-way ANOVA followed by Fisher's protected least-squares difference test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Morphometric measurements of the fish sampled and the status of ovarian development (categories O0–O6) are given in Table 1. Body weights of the fish sampled ranged from 10 ± 4.8 g in juvenile fish up to 2600 ± 232 g in fully mature fish. In the smallest fish sampled, ovaries (O0 and O1) were "immature"; the GSI was approximately 0.1% (n = 10), and the largest OF contained in these ovaries were < 300 µm in diameter. In the largest fish, the ovaries (O6) were close to full maturity, and OF were approaching ovulation, measuring 4900 ± 100 µm in diameter; GSI was 16 ± 1% (n = 10).
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Expression of VTGR mRNA
Northern blotting The expression of mRNA encoding for the VTGR from rainbow trout was analyzed by Northern blotting in ovarian tissues throughout development, as well as in various nonovarian tissues (Fig. 1a). The full-length VTGR cDNA probe was found to hybridize strongly in ovarian tissues to a single transcript of approximately 3.9 kb. The VTGR mRNA was expressed both in previtellogenic and vitellogenic ovaries (Fig. 1a).
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Densitometry conducted on the Northern blots revealed that the highest level of expression of VTGR mRNA was found in previtellogenic ovaries (O2; containing OF 
400 µm in diameter). The second highest expression (80% of that in O2) was detected in ovaries in which the pool of growing oocytes was at the onset of the cortical alveolus stage (O3; containing OF 600 µm). In previtellogenic ovaries (O1; containing OF < 300 µm), the transcripts were present but at a level around 50% lower than in O2. With the onset of full vitellogenesis, the VTGR mRNA levels decreased gradually (Fig. 1a), with O4 (OF 1500 µm) and O5 (OF 2500 µm) displaying an expression of 56% and 8%, respectively, in relation to O2. The VTGR transcript was not detected in ovaries in late vitellogenesis (O6; containing OF from 4800 to 5000 µm), or in ovulated eggs (E) (Fig. 1a). Very weak signals were found in the testis and muscle using the full-length VTGR cDNA probe (Fig. 1a).
In Situ Hybridization: Tissue Specificity and Cellular Localization of VTGR mRNA
The VTGR mRNA was predominantly found in the ovary, although weak signals of hybridization were also found in three nonovarian tissues, namely, the heart, muscle, and testis (Fig. 2). For reasons unknown, the testicular tissue showed a higher affinity for both sense and antisense probes than did the somatic tissues (Fig. 2).
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In the ovary, VTGR mRNA was localized uniquely in the cytoplasm of the oocyte hybridized with the antisense probe (Fig. 3e), where it was evenly distributed. No specific signal was detected in slides treated with the sense probe (Fig. 3f). Nevertheless, in oocytes at early stages of development (follicles measuring 60–70 µm in diameter), a heterogeneous distribution of mRNA was sometimes observed, with a higher concentration of VTGR mRNA around the nucleus (data not shown).
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In order to identify the onset and evolution of the transcription of VTGR cDNA in OF, we carried out in situ hybridization experiments on sections of ovaries isolated at different stages of development. Expression of VTGR mRNA was first quantified in OF of different sizes in ovaries at the same stage of development (in either O1 or O2 or O3, etc.; Fig. 4). The earliest stages of ovarian development examined were previtellogenic ovaries from small females weighing around 10 g (ovary category O0). No specific signal was detected in this ovary (Z = 0.640; p < 0.5224; Fig. 4). In ovaries derived from juvenile fish containing only pre-vitellogenic oocytes (O1, GSI = 0.1%), VTGR mRNA was expressed in all the size categories of follicles present in this ovary (OF measuring from 35 µm up to 199 µm in diameter). In O2 and O3, ovaries in which there was a predominance of pre-vitellogenic oocytes and oocytes just entering vitellogenesis, the pattern of VTGR mRNA expression was similar, with a high level of expression in OF measuring between 50 µm and 200 µm in diameter. In the later stages of ovarian development (O4), a similar pattern of expression of VTGR mRNA occurred, with specific signals in OF measuring up to 999 µm in diameter (Fig. 4).
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The expression of VTGR mRNA was then analyzed on the basis of OF size only regardless of the stage of development of the ovary (Fig. 2). The first transcripts were detected in small previtellogenic OF measuring around 45 µm in diameter, with a significant difference (Z = -3.421; p < 0.0006) between the signals obtained using the sense (Figs. 2 and 3b) and antisense probes (Figs. 2 and 3a). A dramatic increase in the mRNA density was then observed in all ovaries containing OF from 50 to 99 µm in diameter, with the highest density of grains (837 ± 36) found in OF measuring from 100 to 199 µm (Figs. 2 and 3a). Ovarian follicles measuring between 200 and 499 µm in diameter still showed high densities of transcripts when hybridized with the antisense probe (Figs. 2 and 3c), but specific signals were not detected using the sense probe (Figs. 2 and 3d). Thereafter, there was a gradual decrease in density in OF measuring up to 999 µm in diameter (Fig. 2). No expression of VTGR mRNA was detected in OF measuring above 1000 µm in diameter (Z = -1.836; p < 0.0663), corresponding to mid-vitellogenesis (Fig. 2; Fig. 3, g and h).
Ovarian Uptake of VTG
Microscopy of ovarian follicles and immunocytolocalization of VTG The staining procedures used for semi-thin ovary sections allowed us to distinguish between lipid globules, cortical alveoli, and yolk globules in the ooplasm. Ovarian follicles in early previtellogenic growth (measuring less than 150 µm in diameter) contained dense cytoplasm (Fig. 5a) largely devoid of inclusions. In the later stages of previtellogenic growth, numerous batches of Golgian multivesicular bodies (MVBs) developed. The nucleus in previtellogenic oocytes contained numerous nucleoli formed by amplification of the nucleolar organizer genes located in the nuclear envelope crests protruding towards the cytoplasm (Fig. 5a). At this stage, VTG was not detected in the ooplasm using immunocytochemistry (not shown). Concurrent with this, electron micrographs demonstrated that these oocytes were devoid of cellular structures associated with the uptake of VTG (Fig. 5d).
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The smallest size of OF in which VTG was detected was around 300 µm in diameter (Fig. 5, b and c) illustrating the onset of VTG endocytosis. VTG and its yolk products appeared in the peripheral ooplasm as brown precipitates within both MVBs and small yolk granules (Fig. 5c). The appearance of MVBs and yolk globules was associated with a marked change in the ultrastructure of the peripheral region of the OF. Elements associated with endocytic activity were observed (e.g., clathrin-coated pits and vesicles in the oolemma and the onset of deposition of the external zona radiata; Fig. 5e).
During the so-called cortical alveolus stage of development, the OFs measuring from 300 to 800 µm in diameter were characterized by the appearance of cortical alveoli and lipid globules in the periphery of the ooplasm (Fig. 6, a and c). Immunostaining of oocytes at this stage revealed the presence of VTG and/or yolk proteins derived from VTG in MVBs and yolk globules (Fig. 6c). The endocytic events associated with VTG and lipid internalization were concomitant with the synthesis of cortical alveoli in the oocytes. The build-up of the external zona radiata occurred during this stage, concomitantly with the active endocytosis of VTG (Fig. 6e).
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During active vitellogenesis, there was a dramatic increase in the number and size of the lipid and yolk globules (Fig. 6b). Cortical alveoli were located at the periphery of the ooplasm (Fig. 6b). The considerable increase in endocytosis of VTG associated with this phase of ovarian development was clearly defined by the presence of a high concentration of yolk globules and MVBs containing VTG (Fig. 6d) and, ultrastructurally, the prevalence of numerous clathrin-coated pits and vesicles in the oolemma (Fig. 6f), just under the well-developed internal zona radiata (Fig. 6g).
Plasma Concentrations of VTG
Vitellogenin was detected in the plasma of female trout throughout all stages of reproductive development, although the concentrations varied considerably. Concentrations of plasma VTG in the "immature" females (O1 and O2), in which the maximum-sized OF measured 250 µm (O1) and 400 µm in diameter (O2), were 6 ± 2 µg/ml and 16 ± 1 µg/ml, respectively. The development of the ovary from O2 to O3 was associated with a marked increase in the concentration of circulating plasma VTG, from 16 ± 1 µg/ml to 460 ± 90 µg/ml (p < 0.001). Correspondingly, there was a rapid increase in the sequestration of VTG into the ovary. During subsequent development of the ovary, there was a progressive increase in the concentration of VTG in the plasma with increasing OF size. Maximal concentrations of VTG (15 mg/ml) occurred shortly before ovulation, when vitellogenic OF measured 4900 ± 100 mm in diameter (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The data obtained by Northern analysis corroborated previous studies in the rainbow trout [27, 28], in which a single transcript was strongly expressed in the ovary. The highest VTGR mRNA level occurred in early vitellogenic ovaries in which the largest OF measured 600 µm in diameter. However, the presence of transcripts in previtellogenic ovaries from females weighing just 15 g (in which the largest OF measured less that 250 µm) suggests that the synthesis of receptors occurs very early in ovary development, before VTG uptake into the ovary is initiated. On the other hand, mRNA levels decreased at the same time that vitellogenesis progressed, with the presence of transcripts no longer detected in vitellogenic ovaries containing OF more than 2500 µm in diameter.
In situ hybridization studies confirmed the Northern analyses, showing that the expression of VTGR mRNA was initiated very early in oocyte development. The first transcripts appeared in OF measuring 45–50 µm at the nucleolar amplification stage of development. Similarly, in chickens, high levels of VLDL/VTG receptor mRNA were detected in very small previtellogenic OF (60–80 µm in diameter) [25]. In Xenopus, an early synthesis of maternal mRNA in the cytoplasm of young previtellogenic oocytes has also been reported [39]. In the present study, the transcripts were exclusively localized in the oocyte cytoplasm, where they were evenly distributed. Nevertheless, transcripts observed in some previtellogenic OF (60–70 µm) were predominantly distributed in the area surrounding the nucleus, which may reflect the translocation of the first mature messengers from the nucleus towards the cytoplasm, after splicing has taken place in the nucleus.
A dramatic increase was observed in the mRNA density in OF from 50 to 199 µm in diameter, indicating that the density of transcripts is maximal at the onset of previtellogenesis. However, a marked decline of this density was found in the subsequent size classes of OF, just before and at the onset of the VTG endocytosis into the oocytes. Recent studies on another gene in the rainbow trout that also plays a role in oocyte growth/embryo development (cathepsin D, which mediates yolk storage/mobilization) [40] have similarly demonstrated that the transcription of the gene occurs very early in oocyte development (previtellogenesis), a considerable time before the functional enzyme is present or required.
Using the technique of in situ hybridization, VTGR mRNA was not detected in OF greater than 1000 µm. In contrast, the Northern blot analysis detected messengers in vitellogenic ovaries containing OF up to 2500 µm in diameter, although the signals were weak. We have frequently observed a high heterogeneity in OF size in the ovaries, with considerable numbers of previtellogenic oocytes (< 400 µm) being found among the predominant populations of vitellogenic oocytes (Fig. 3, c–h). Thus, the in situ results suggest that the VTGR messengers detected by Northern analysis in vitellogenic ovaries (OF up to 2500 µm) were mostly, if not entirely, derived from the smaller-sized follicles in that ovary. Alternatively, the level of expression of VTGR mRNA in OF greater than 1000 µm in diameter may have been below the threshold for detection in the adopted in situ protocol. Indeed, it is well known that some of the histological procedures that precede in situ hybridization (the chemical treatments, etc.) can lead to a reduction in the subsequent hybridization signal by as much as 25% [41]. This loss of signal intensity, however, is considered acceptable for tissues of which frozen sections are not practically feasible (which is the case for oocytes, in which the very high deposits of yolk in the cytoplasm make the histological sectioning extremely difficult) [42].
The end of VTGR mRNA synthesis in such a precocious stage suggests that the major synthesis of the protein corresponding to this transcript is likely to occur between the previtellogenic phase and early vitellogenesis. Consequently, one can suppose that only a highly efficient recycling process involving the VTGR could ensure the uptake of VTG during the entire process of vitellogenesis, which begins in OF from 300 µm in diameter and continues until the end of yolk accumulation, just before oocyte maturation (when oocytes measure 5000 µm).
An interesting hypothesis, initially proposed by Shen et al. [43] for the chicken VLDL/VTG receptor, is that most receptors are synthesized predominantly at the early stages of oocyte development and then stored inside the cytoplasm (perhaps in a nonfunctional form) to be gradually mobilized according to the evolution of the vitellogenesis process. In addition, minimal degradation of the receptor would contribute to optimizing such a mechanism.
It is interesting to note that the VTGR messengers were clearly detected in trout juvenile females with body weights of 15 g (GSI = 0.1%), but not in juvenile females of ~10 g with a GSI < 0.1%. The absence of VTGR mRNA in these fish might suggest a modulation of the VTGR gene by some regulating factors.
Weak hybridization signals observed in some of the trout somatic tissues and in the testis (in both the Northern and the in situ analysis) are likely to represent hybridization of the VTGR probe with either the so-called rainbow trout lipoprotein receptor [27], or to another member of the VLDL receptor (VLDLR) family. In the chicken, VTGR mRNA is expressed at low levels in muscle and heart [25]. Little is known about expression of the mRNAs encoding VLDLR in fish, but in mammals, the heart, brain, skeletal muscle, and adipose tissue are the predominant sites for their expression [44–48]. Receptor binding studies in the rainbow trout, however, suggest that liver, muscle, and spleen are sites of expression of VLDLR since these tissues display a high binding affinity for trout VLDL and LDL [49].
The smallest OF size in which VTG (or yolk proteins derived from VTG) could be detected immunologically was about 300 µm in diameter. At this stage of ovary development, the concentration of plasma VTG was very low (Fig. 7). Previous studies on the rainbow trout have indicated that the onset of vitellogenesis occurs when OF measure between 400 µm and 600 µm in diameter [24, 50]. Our results, however, demonstrate that the uptake of VTG is initiated somewhat earlier in oocyte development. The findings of the immunological studies are supported by the ultrastructure data presented here, in which the onset of deposition of the external zona radiata also occurred in OF measuring approximately 300 µm in diameter. These observations are in agreement with previous data by Hyllner et al. [51], which suggest that the synthesis of vitelline proteins, integrated in the build-up of the zona radiata by oocytes, occurs in hepatocytes under estrogenic induction, as it happens with VTG. Thus, the onsets of both external zona radiata deposition and VTG endocytosis seem to be strongly associated.
There was a marked increase in the concentration of VTG in the plasma of fish containing ovaries in which the maturing oocytes were in the cortical alveolus stage of development, a period characterized by the formation of cortical alveoli and lipid globules in the oocyte [52]. Associated with the increase in plasma VTG at this time was an increase in the content of VTG or VTG degradation products in the oocytes (in the MVBs and yolk globules). Full vitellogenic development was characterized by a rapid rise in the concentration of plasma VTG, similar to that reported previously [37, 53], and by specific structural changes in the oocyte. These structural changes involved the deposition of internal zona radiata, the presence of numerous clathrin-coated pits and vesicles, and high concentrations of MBVs and yolk globules containing VTG and its yolk products (Fig. 6).
In summary, the data presented have shown that the synthesis of the VTGR transcript is initiated in early previtellogenic development, before large quantities of VTG are sequestered by the ovary. Indeed, the onset of vitellogenesis was marked by a concomitant decrease of VTGR mRNA, whereas full vitellogenesis was characterized by the absence of these transcripts (Fig. 7). The pattern of expression of VTGR mRNA in the rainbow trout supports the concept of a recycling of the functional VTGR in fish.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: F. Le Menn: Laboratoire de Biologie de la Reproduction des Poissons, Unité Associée INRA, Université Bordeaux I, Avenue des Facultés, 33405 Talence, France. FAX: 33 5 56 84 89 15; lemenn{at}ubrpinra.u-bordeaux.fr
Accepted: November 24, 1998.
Received: June 10, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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vaert D, Feldmann G. Detection of albumin mRNAs in rat liver by in situ hybridization: usefulness of paraffin embedding and comparison of various fixation procedures. J Histochem Cytochem 1987; 35:453–459.[Abstract]This article has been cited by other articles:
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  D. Han, N. H. Haunerland, and T. D. Williams Variation in yolk precursor receptor mRNA expression is a key determinant of reproductive phenotype in the zebra finch (Taeniopygia guttata) J. Exp. Biol., May 1, 2009; 212(9): 1277 - 1283. [Abstract] [Full Text] [PDF]
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 M. J Agulleiro, M. Andre, S. Morais, J. Cerda, and P. J Babin High Transcript Level of Fatty Acid-Binding Protein 11 but Not of Very Low-Density Lipoprotein Receptor Is Correlated to Ovarian Follicle Atresia in a Teleost Fish (Solea senegalensis) Biol Reprod, September 1, 2007; 77(3): 504 - 516. [Abstract] [Full Text] [PDF]
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 S. Asahina, C. Sato, and K. Kitajima Developmental Expression of a Sialyltransferase Responsible for Sialylation of Cortical Alveolus Glycoprotein during Oogenesis in Rainbow Trout (Oncorhynchus mykiss) J. Biochem., August 1, 2004; 136(2): 189 - 198. [Abstract] [Full Text] [PDF]
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N. Hiramatsu, R. W. Chapman, J. K. Lindzey, M. R. Haynes, and C. V. Sullivan Molecular Characterization and Expression of Vitellogenin Receptor from White Perch (Morone americana) Biol Reprod, June 1, 2004; 70(6): 1720 - 1730. [Abstract] [Full Text] [PDF]
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N. Hiramatsu, A. Hara, K. Hiramatsu, H. Fukada, G. M. Weber, N. D. Denslow, and C. V. Sullivan Vitellogenin-Derived Yolk Proteins of White Perch, Morone americana: Purification, Characterization, and Vitellogenin-Receptor Binding1 Biol Reprod, August 1, 2002; 67(2): 655 - 667. [Abstract] [Full Text] [PDF]
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A. Menuet, I. Anglade, G. Flouriot, F. Pakdel, and O. Kah Tissue-Specific Expression of Two Structurally Different Estrogen Receptor Alpha Isoforms along the Female Reproductive Axis of an Oviparous Species, the Rainbow Trout Biol Reprod, November 1, 2001; 65(5): 1548 - 1557. [Abstract] [Full Text] [PDF]
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C. R. McCudden, W.H. Tam, and G. F. Wagner Ovarian Stanniocalcin in Trout Is Differentially Glycosylated and Preferentially Expressed in Early Stage Oocytes Biol Reprod, September 1, 2001; 65(3): 763 - 770. [Abstract] [Full Text] [PDF]
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