Ovarian expression and localization of a vitellogenin receptor with eight ligand binding repeats in the cutthroat trout (Oncorhynchus clarki)

Comparative Biochemistry and Physiology, Part B 166 (2013) 81–90

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Ovarian expression and localization of a vitellogenin receptor with eight ligand binding repeats in the cutthroat trout (Oncorhynchus clarki) Hiroko Mizuta a, Wenshu Luo a, Yuta Ito a, Yuji Mushirobira a, Takashi Todo b, Akihiko Hara a, Benjamin J. Reading c, Craig V. Sullivan c, Naoshi Hiramatsu b,⁎ a b c

Division of Marine Life Science, Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1 Minato, Hakodate, Hokkaido, 041-8611, Japan Division of Marine Life Science, Faculty of Fisheries Sciences, Hokkaido University, 3-1-1 Minato, Hakodate, Hokkaido, 041-8611, Japan Department of Biology, North Carolina State University, Raleigh, NC 27695-7617, USA

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Article history: Received 7 April 2013 Received in revised form 12 July 2013 Accepted 12 July 2013 Available online 18 July 2013 Keywords: Ovarian development Salmonid Vitellogenin Vitellogenin receptor Yolk formation

a b s t r a c t A cDNA encoding a vitellogenin receptor with 8 ligand binding repeats (vtgr) was cloned from ovaries of the cutthroat trout, Oncorhynchus clarki. In situ hybridization and quantitative PCR analyses revealed that the main site of vtgr mRNA expression was the oocytes. Expression was strongly detected in perinucleous stage oocytes, gradually decreased as oocytes grew, and became hardly detectable in vitellogenic oocytes. A rabbit antibody (a-Vtgr) was raised against a recombinant Vtgr protein in order to immunologically detect and localize Vtgr within the ovarian follicles. Western blotting using a-Vtgr detected a bold band with an apparent mass of ~95–105 kDa in an ovarian preparation that also bound Sakhalin taimen, Hucho perryi, vitellogenin in ligand blots. Immunohistochemistry using a-Vtgr revealed that the Vtgr was uniformly distributed throughout the ooplasm of perinucleolus stage oocytes, subsequently translocated to the periphery of lipid droplet stage oocytes, and became localized to the oolemma during vitellogenesis. We provide the first characterization of Vtgr at both the transcriptional and the translational levels in the cutthroat trout, and our results suggest that this receptor is involved in uptake of Vtg by oocytes of this species. © 2013 Elsevier Inc. All rights reserved.

1. Introduction Oocyte growth in oviparous species is dependent on the uptake of maternal nutrients and their storage as yolk, whose constituents are subsequently used by the embryos and larvae during early development. Vitellogenin (Vtg), a major yolk precursor, is transported via the blood stream to the ovary where it is selectively accumulated by growing oocytes (Wallace, 1985; Mommsen and Walsh, 1988). Vitellogenin makes its way from the capillary network at the periphery of the ovarian follicle through the multiple cellular and extracellular layers surrounding the oocyte. After penetrating the basal lamina and passing between granulosa cells, Vtg traverses the zona radiata along the oocyte microvilli (Selman and Wallace, 1989). The Vtg is finally internalized at the oolemma by a selective mechanism via specific cell-surface receptors associated with coated pits and coated vesicles (Goldstein et al., 1985; Patiño and Sullivan, 2002). Proteins with high affinity for Vtg (Vtgr) have been identified and characterized in ovarian membrane preparations of chicken (Stifani et al., 1988), Xenopus (Stifani et al., 1990a), and several teleosts including, as examples, the following species: coho salmon, Oncorhynchus nerka (Stifani et al., 1990b); Nile tilapia, Oreochromis niloticus (Chan

⁎ Corresponding author. Tel./fax: +81 138 40 8878. E-mail address: naoshi@fish.hokudai.ac.jp (N. Hiramatsu). 1096-4959/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cbpb.2013.07.005

et al., 1991); rainbow trout, Oncorhynchus mykiss (Le-Menn and NuñezRodriguez, 1991; Tyler and Lancaster, 1993; Nuñez-Rodriguez et al., 1996; Tyler and Lubberink, 1996); Sakhalin taimen, Hucho perryi (Hiramatsu et al., 2001); and white perch, Morone americana (Tao et al., 1996; Hiramatsu et al., 2002; Reading et al., 2011). The numbers and apparent masses of Vtg-binding proteins detected by ligand blotting are not consistent among studies, perhaps due to differences between species, ovary maturational stages, and the methods used for extraction and detection of the ovarian membrane proteins in each study. A single form of Vtgr with a mass of ~100 kDa has typically been characterized in fishes; however, multiple ovarian membrane proteins that specifically bind Vtg also have been detected by ligand blotting in rainbow trout (Nuñez-Rodriguez et al., 1996; Tyler and Lubberink, 1996), Sakhalin taimen (Hiramatsu et al., 2001), and white perch (Reading et al., 2011). Molecular characterization of cDNA encoding the Vtgr (vtgr) has been performed in chicken (Bujo et al., 1994), Xenopus (Okabayashi et al., 1996), rainbow trout (Davail et al., 1998; Prat et al., 1998), blue tilapia, Oreochromis aureus (Li et al., 2003), and white perch (Hiramatsu et al., 2004). These studies revealed that the vtgr belongs to the low density lipoprotein receptor gene (ldlr) superfamily and encodes five constitutive protein domains: (1) ligand binding domain (LBD), (2) epidermal growth factor (EGF) precursor like domain, (3) O-linked sugar domain, (4) transmembrane domain, and (5) cytoplasmic domain. Among ldlr superfamily genes, vertebrate vtgrs are orthologous to mammalian very low-density lipoprotein receptor (vldlr), which also encodes

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eight ligand binding repeats in the primary protein sequence. The teleost vtgr expressed by COS-1 cells is ~97 kDa in size and specifically binds Vtg in ligand blots (Davail et al., 1998). Expression and localization of vtgr mRNA in the oocyte has been characterized in rainbow trout using in situ hybridization (Perazzolo et al., 1999). This analysis revealed high levels of vtgr mRNA in oocytes during pre-vitellogenesis, followed by a gradual decrease in vitellogenic oocytes. It is hypothesized that Vtgr proteins are recycled following receptor-mediated endocytosis by the vitellogenic oocytes without de novo synthesis of vtgr transcripts (Prat et al., 1998; Perazzolo et al., 1999; Hiramatsu et al., 2004). However, this hypothesis remains to be verified experimentally. The localization of Vtgr proteins within the ovarian follicle and oocyte has not previously been reported for any teleost. The first objective of the present study was to partially characterize the vtgr gene transcript and its encoded protein product in the cutthroat trout, Oncorhynchus clarki, our model salmonid species for basic research on teleost ovarian follicle growth. Our second objective was to evaluate changing patterns of ovarian expression and localization of vtgr gene transcripts and Vtgr proteins associated with ovarian follicle growth. 2. Materials and methods 2.1. Experimental animals and samples Fishes used in this study (cutthroat trout, O. clarki, and Sakhalin taimen, H. perryi) were reared at the Nanae Fresh-Water Laboratory, Field Science Center for Northern Biosphere, Hokkaido University (Hakodate, Japan). Ovaries of cutthroat trout were collected from vitellogenic females and stored at − 80 °C until used for preparing ovarian membranes. In order to investigate the tissue distribution of vtgr mRNA expression, pre-vitellogenic ovaries and somatic tissues (liver, heart, muscle, kidney, gill, stomach, Intestine, brain, and spleen) were collected from five cutthroat trout during April, immediately immersed in ice-cold RNA later (Ambion, CA, USA), incubated overnight at 4 °C, and stored at −30 °C before extraction of total RNA. In order to observe maturational changes of ovarian vtgr mRNA expression, ovaries or post-ovulatory follicles were sampled from five cutthroat trout every month from February 2009 to June 2009, and every other month from October 2009 to February 2010. Post-ovulatory follicles were obtained in February 2010 when fish ovulated. Ovarian samples were cut into small pieces by scissors and processed as described above before extraction of total RNA. Ovarian tissues for histological analyses (in situ hybridization and immunohistochemistry) were also excised from the females sampled at different maturational stages, fixed in 4% paraformaldehyde (PFA) in 0.1 M phosphatase buffer (PB; pH 7.4) with gentle shaking at 4 °C for 2 days, embedded in fresh filtered paraffin, and stored at 4 °C. Staging of follicles and oocytes in this study was performed according to Yamamoto et al. (1965). In order to purify Vtg used for ligand blotting, plasma samples were obtained from male Sakhalin taimen that were previously injected twice with estradiol-17β (E2; 5 mg/kg mass) at weekly intervals and bled 7 days after the second E2 injection, and the plasma was kept at −80 °C. 2.2. Molecular cloning of cutthroat trout vtgr Ovarian cDNA template was synthesized according to Luo et al. (2013). The first fragment of vtgr cDNA was amplified by PCR using primers (5′-ACTCTCACGCTCAGCTAC-3′ and 5′-GTTGGCAAGGAGACC AAAGC-3′), which were designed based on the known vtgr sequence of rainbow trout (GenBank accession no. Q90W12). The PCR amplification was performed using Advantage 2 Polymerase mix (Clontech, CA, USA) and the following thermal parameters: 1 cycle at 95 °C for

2 min, followed by 35 cycles at 94 °C for 45 s, 60 °C for 30 s, 68 °C for 3 min, and 1 final cycle at 72 °C for 10 min. The PCR products were separated by electrophoresis on a 1.5% agarose gel, excised from the gel, purified using GENECLEAN Turbo Kit (MP-Biochemicals, OH, USA), and ligated into pGEM-T Easy vector (Promega, WI, USA). The ligated products were transformed into high efficiency competent cells (XL1-Blue; Strategene, CA, USA). Positive colonies with a 2614-bp insert were cultured and plasmid DNA extracted using Wizard Plus SV Minipreps DNA Purification System (Promega). Plasmids were sequenced using BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems, CA, USA) and ABI PRISM 310 Genetic Analyzer (Applied Biosystems) according to the manufacturer's protocol. Sequence alignments and domain searches of the putative vtgr were performed using NCBI tools (http://www.ncbi.nlm.nih.gov). Sequence comparisons were performed using the Neighbor-Joining method by Genetyx Version 7 program (Genetyx Corporation, Tokyo, Japan).

2.3. Real-time quantitative reverse transcription PCR (rtqRT-PCR) Transcript abundance of vtgr and elongation factor 1 (ef1) alpha (internal control) was quantified by rtqRT-PCR in ovarian follicles and other somatic tissues obtained from female cutthroat trout. We chose ef1 alpha as the internal control gene due to its stable expression profile reported in the following studies of closely related salmonid species: (1) ef1 alpha has been evaluated as the most reliable (stable) reference gene when 6 different housekeeping genes were tested for 8 distinct tissue in the Atlantic salmon (Olsvik et al., 2005); (2) the mRNA abundance of ef1 alpha did not exhibit any significant difference over the preovulatory period of the rainbow trout when it was normalized to the abundance of 18S rRNA (Bobe et al., 2006); and (3) ef1 alpha transcript levels were not different across various ovarian stages when mRNA prepared from the ovaries of rainbow trout was used as template (Luckenbach et al., 2008). First-strand cDNA templates for rtqRT-PCR were synthesized from 1250 ng of total RNA extracted from the ovarian and somatic tissue samples using SuperScript® VILO cDNA Synthesis kit (Invitrogen). Reverse transcribed cDNA of pre-vitellogenic ovarian tissue (collected in May) was used as an inter-assay standard (IAS) to normalize between plates. In addition, reactions without reverse transcriptase enzyme were used as negative controls (NRT). The cutthroat trout vtgr sequence obtained in this study was used as the template to design gene-specific primers for amplification of a 125-bp vtgr mRNA fragment (5′-GACAGAATGTGAGCCCAGTC-3′ and 5′-AGGTCTTCCGAA CGCAGGTG-3′). Amplification of ef1 alpha mRNA was performed by use of the primer set designed in our previous study (Luo et al., 2013). All rtqRT-PCR reactions were carried out in a volume of 20 μL. Primers were added to reactions at final concentration of 100 nM for both vtgr and ef1 alpha. The PCR amplifications and fluorescence detection were performed using the ABI prism model 7300 sequence detector (Applied Biosystems) and the manufacturer's universal thermal cycling conditions. The first-strand cDNA samples (equivalent to 1.25 ng total RNA per reaction) were amplified using SYBR Green PCR Master Mix (Applied Biosystems). In addition, duplicate standard curves were generated using a serial dilution of plasmid DNA containing the target gene (10 to 10,000,000 copies per reaction). Primer specificity was confirmed by dissociation curve analysis of PCR products. The expression levels (copy number per reaction) of the target gene (vtgr) were normalized to the expression levels of the reference gene (ef1 alpha). Results were reported as a fold change in abundance relative to the values obtained for cDNA reverse transcribed from the IAS RNA sample [i.e., relative value = (sample vtgr copy number/sample ef1 alpha copy number) / (IAS vtgr copy number/IAS ef1 alpha copy number]. Calculations of copy number were performed as described in our previous study (Luo et al., 2013).

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2.4. In situ hybridization In situ hybridization and corresponding probe synthesis were carried out according to our previous report (Luo et al., 2013). Digoxigenin-labeled antisense or sense RNA probes were prepared by in vitro transcription of a 554-bp portion of the vtgr cDNA that was amplified by PCR using gene specific primers (5′-GAGCTGTGG TAACATCACGTGC-3′ and 5′-TACACTCTCCACTCCGACAC-3′). In addition to in situ hybridization, the prepared sections were also stained with hematoxylin and eosin for morphological observation. Photographs were taken with a light microscope (Model 80iTUW-31-1, Nikon, Tokyo, Japan). 2.5. Production of recombinant Vtgr protein 2.5.1. Construction of vtgr ligand binding domain (vtgr-lbd)—pET 302 plasmid Sub-cloning of a cDNA fragment encoding the ligand binding domain of vtgr (vtgr-lbd) into the pET302 NT/His expression vector (Invitrogen) was performed by use of In-Fusion™ Advantage PCR Cloning Kit (Clontech) according to the manufacturer's protocol. Briefly, the PCR reaction was performed with primers 5′-CACGTGAATTCGCTCTCAAAG ACAGAATGTGAGC-3′ and 5′-AATATCATCGATCTCGCACTCATTGATGGG CTC-3′, which contained adaptor sequences corresponding to the terminal 15 bp sequences of the expression vector (underlined) and amplified a 981-bp vtgr-lbd fragment. The PCR product was separated by 1.5% agarose gel, purified, and cloned into pET302 expression vector which had been modified to provide a His-tag (6 × His) at the N-terminal region of the resulting recombinant protein. This construct was confirmed by sequencing as described above. 2.5.2. Expression and purification of recombinant Vtgr-lbd Expression and purification of recombinant Vtgr-lbd was performed according to manufacturer's protocol (User protocol: TB055 10th Edition Rev. B 0403, TB245 Rev. E 0304, TB054 Rev. D 0303; Novagen) unless otherwise stated below. Briefly, the constructed plasmid (vtgrlbd—pET302) was transformed into the Escherichia coli strain Origami (DE3) pLysS (Novagen, WI, USA). A transformant was cultured in LB medium containing ampicillin (LB-amp) at 37 °C and expression of recombinant Vtgr-lbd was induced by the addition of isopropyl-βD-thiogalactoside (IPTG) to a final concentration of 1 mM. The cell pellet collected after centrifugation was resuspended with 5 mL of Bugbuster Protein Extraction Reagent (Novagen) containing 0.2% lysonase and 1% cocktail protease inhibitor (Novagen)/1 g of bacterial pellet. Recombinant Vtgr-lbd appeared to be dominant in the soluble fraction and was purified by His-Bind Resin (Novagen) chromatography. After binding of recombinant Vtgr-lbd to Ni-charged His-Bind Resin, the column was washed with a buffer containing 60 mM imidazole, and samples were subsequently eluted with a buffer containing 1 M imidazole. This crude Vtgr-lbd was dialyzed with ~1 L of 20 mM Tris–HCl (pH 8.0) containing 2% NaCl and 0.1% NaN3. Following concentration by Amicon Ultra centrifuge filter (UFC801024; Millipore, MA, USA), the crude preparation was subjected to Superdex 200 gel filtration column chromatography (GE Healthcare, Buckinghamshire, UK) connecting to FPLC system (GE healthcare). Recombinant Vtgr-lbd was eluted from the column according to the method described by Hong et al. (2009). A single symmetric peak of absorbance at 280 nm was observed in the elution pattern and fractions around this peak were collected as purified recombinant Vtgr-lbd. 2.6. Production of specific antibody to Vtgr An antiserum against recombinant Vtgr-lbd (a-Vtgr) was raised in a rabbit by intradermal injection of a sample emulsified in an equal volume of Freund's complete adjuvant (Merck, Darmstadt, Germany).

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Injections were conducted four times at intervals of 7 days. Specificity of a-Vtgr to the antigen was confirmed by Western blotting. The antiserum was further purified to produce specific IgG antibody using affinity column chromatography. Briefly, Sepharose 4B (GE Healthcare) was coupled with ~ 150 mg of the crude Vtgr-lbd (i.e., Bugbuster soluble fraction) according to the method described by Amano et al. (2007). The gel was poured into a glass column (2.5 cm × 3.0 cm) and equilibrated with phosphate buffered saline (PBS; 0.01 M sodium phosphate buffer, pH 7.0, containing 0.25 M NaCl). The antiserum (~20 mL) was initially subjected to a precipitation using 40% saturated ammonium sulfate (SAS), and the resulting precipitate was dissolved in and dialyzed against PBS. This crude antibody was then subjected to purification on the affinity column. Samples were first eluted with PBS at a flow rate of 30 mL/h and then with 8 M urea at a flow rate of 60 mL/h. The latter fractions eluted with 8 M Urea were collected as specific a-Vtgr IgG, dialyzed against PBS, and stored at −30 °C. 2.7. Western blotting Proteins were separated by SDS–PAGE under reducing or nonreducing conditions according to Laemmli (1970) and blotted onto polyvinylidene difluoride (PVDF) membranes (Millipore) according to Towbin et al. (1979). The apparent molecular weights of proteins on Western blots were estimated using broad-range molecular mass standards (10–250 kDa) (Bio-Rad, CA, USA). In Western blotting using aVtgr, the blotted membrane was blocked with 5% non-fat skim milk in Tris buffered saline (TBS: 0.02 M Tris, 0.5 M NaCl, pH 7.5) for 1 h at room temperature and incubated with a-Vtgr (1:500 or 1:2000) overnight at 15 °C, followed by rinsing with TBS for 5 min × 2 and TBS containing 0.025% Tween-20 (TBS-T) for 5 min × 1. The membrane was then incubated in a 1:2000 dilution of goat anti-rabbit IgG (H + L) conjugated to horseradish peroxydase (Bio-Rad) for 90 min at room temperature, followed by rinsing as described above. Detection was performed with color development solution (20 mL of TBS containing 15 mg 4-chloro-1-naphthol, 5 mL of methanol and 15 μL of 30% hydrogen peroxide (H2O2). In Western blotting using Ni-NTA HRP conjugate (QIAGEN, Tokyo, Japan), which specifically binds to His-tag (6 × His), the blotted PVDF membrane was blocked with 3% bovine serum albumin (BSA) in TBS for 1 h at room temperature, followed by incubating with the Ni-NTA HRP conjugate (1:2,000) for 1.5 h at room temperature. After rinsing with T-TBS for 10 min × 3, detection was performed as described above. 2.8. Immunohistochemistry Paraffin sections of 5 μm thickness were mounted onto MAS coat Super-frost slides (Matsunami, Osaka, Japan), and the slides were dried on a hotplate overnight at 37 °C. The sections were deparaffinized in xylene for 10 min × 2, then hydrated through a series of 100%, 90%, 80%, and 70% ethanol washes for 10 s each. After rinsing with PBS for 5 min, the sections were incubated in PBS containing 0.05% proteinase K for 30 min at 37 °C and then rinsed with PBS for 10 min. Blocking of non-specific binding reactions was performed by incubation in PBS containing 1% BSA for 1 h at room temperature. Primary antibody incubation (10 μg IgG/mL) was performed in PBS overnight at 4 °C. Slides were then washed with PBS for 5 min × 3 and incubated in a 1:200 dilution of Alexa 594 labeled donkey anti-rabbit IgG (Invitrogen) in PBS for 2 h at room temperature in the dark. After washing with PBS for 5 min × 3, sections were enclosed in ProLong® Gold antifade reagent (Invitrogen). Fluorescence confocal microscopy was performed using a TCS-SP5 spectral confocal microscope (Leica, Tokyo, Japan) based on a DMI6000B inverted microscope (Leica) equipped with either HCX PL APO CS 10 × 0.4 dry objective lens or HCX PL APO CS 63 × 1.4 oil immersion objective lens. The fluorescence of Alexa 594 was excited with a green He/Ne laser at 543 nm and detection wavelength range

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was set to 581–680 nm with a beam splitter (488/543). Laser attenuation, pinhole diameter, gain, and offset were kept constant for confocal imaging. All overlay images of the fluorescent and phase contrast images were obtained using Photoshop 5.0 (Adobe, Edinburgh, UK) software. 2.9. Ligand blotting Ovarian membrane extracts were prepared from vitellogenic ovaries of cutthroat trout according to our previous report (Hiramatsu et al., 2002). Biotinylated taimen Vtg was prepared as described in our previous study (Hiramatsu and Hara, 1997). Taimen Vtg was used instead of cutthroat trout Vtg for ligand blotting because, due to its large size, we preferentially utilize this primitive salmonid as donor of blood plasma for routine purification of salmonid Vtg. The ability of the Vtgr of one species to bind Vtg from distantly related species, even those of a different vertebrate class, has been verified previously (appropriate citations are given in Hiramatsu et al., 2002). For example, we previously showed that Vtgs from cutthroat trout and Sakhalin taimen have equivalent ability to displace white perch (Order Perciformes) Vtg from its receptor (Hiramatsu et al., 2002). SDS–PAGE (under non-reducing condition) and electro-blotting were performed as described above. After blocking with 5% non-fat skim milk in 0.02 M Tris–HCl (pH 8.0) containing 2% NaCl and 0.01% NaN3 for 4 h at room temperature, membranes were incubated in blocking buffer containing biotinylated Vtg (5 μg/mL) with or without a 100-fold excess of cold (non-biotinylated) Vtg over night at 4 °C. After three washes with Tris–HCl buffer containing 0.25% Triton X-100 for 5 min each, membranes were incubated with ABC-AP Reagent (VECTASTAIN ABC-AP Rabbit IgG Kit; Vector Laboratories, CA, USA) for 30 min at room temperature. After washing as described above, the membrane proteins coupled to biotinylated Vtg were visualized by Vector Red Alkaline Phosphatase Kit (Vector Laboratories) according to the manufacturer's protocol.

3.3. Ovarian localization of vtgr mRNA Ovarian localization of vtgr expression was confirmed by in situ hybridization (Fig. 2). The most intense signal of vtgr expression was seen in the ooplasm of pre-vitellogenic, perinucleolus stage oocytes. As follicular growth progressed, vtgr expression in the ooplasm decreased and became hardly detectable in the vitellogenic oocytes. No specific signals were present in the oocyte nucleus or in the surrounding follicular cell layers (i.e., theca and granulosa). Detectable signals were not observed in sections using the negative control (sense) probe (data not shown). 3.4. Production of recombinant Vtgr-lbd and its antibody Recombinant Vtgr-lbd appeared as a protein band with an apparent mass of ~50 kDa in SDS–PAGE of IPTG-induced bacteria extracts (Fig. 3), and this band exhibited a positive reaction in Western blotting using Ni-NTA conjugate (data not shown). Specificity of a-Vtgr was confirmed by Western blotting of purified Vtgr-lbd and bacteria extracts (with

A

Differences in ovarian vtgr mRNA levels between fish sampled on different dates (N = 5 in March, June, August, October; N = 4 in February 2009, April, December, February 2010; N = 3 in May) were evaluated by One-way analysis of variance (ANOVA) followed by Tukey–Kramer honestly significant difference (HSD) test. Differences between tissues of pre-vitellogenic females (N = 5) sampled in April were evaluated by one-way ANOVA followed by HSD test. For all statistical tests, the level of statistical significance was set at P b 0.05.

vtgr mRNA (relative value)

2.10. Data analyses

revealed in our previous study (Luo et al., 2013). The highest level of vtgr mRNA expression was observed in ovaries collected in March, during pre-vitellogenesis (Fig. 1A). The ovarian vtgr mRNA expression gradually and significantly decreased as vitellogenesis progressed, reaching the lowest levels in October and December. The vtgr mRNA levels began rising again in postovulatory ovaries in February. Pre-vitellogenic ovaries were found to be the major site of vtgr mRNA expression when compared to somatic tissues in female cutthroat trout (Fig. 1B). The vtgr transcripts also were expressed in brain, however the brain vtgr mRNA levels were significantly lower than in pre-vitellogenic ovaries.

3. Results

B

3.2. Tissue distribution and maturational changes of ovarian vtgr mRNA expression Annual changes in gonad somatic index (GSI), oocyte diameter, and oocyte stage in cutthroat trout appeared to be similar to the patterns

vtgr mRNA (relative value)

A cDNA encoding the cutthroat trout vtgr was sequenced (GenBank accession no. KC771282) and consisted of a 26-bp 5′-untranslated region (UTR) followed by a 2529-bp open reading frame (ORF) and a 59-bp 3′UTR. The ORF encoded a polypeptide consisting of 843 amino acids (aa) with a predicted mass of 93.2 kDa. The encoded cutthroat trout Vtgr exhibited domain features typical of lipoprotein receptors and contained 8 ligand binding repeats similar to Vtgr and Vldlr in other vertebrates. The vtgr/Vtgr sequences shared the highest identity (98%) with vtgr/Vtgr sequences of the rainbow trout (GenBank accession nos. NM_001124375. 1 for nucleic acid and NP_001117847 for amino acid).

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3.1. Molecular cloning of cutthroat trout vtgr

II

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Fig. 1. Changes in expression of vitellogenin receptor (vtgr) mRNA in female cutthroat trout. (A) Changes in ovarian vtgr mRNA expression. Ovarian follicles were divided into four stages based on histological observations: pre-vitellogenic (I), vitellogenic (II), ovarian follicle maturation (III), and post-ovulation (IV). (B): Tissue distribution of vtgr mRNA. Samples include the following: Ov, ovary; Li, liver; He, heart; Mu; muscle; Ki, kidney; Gi, gill; St, stomach; In, intestine; Br, brain; Sp, spleen. Vertical brackets indicate standard errors (N = 5). Mean values bearing different letter superscripts are significantly different (P b 0.05).

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C IV II

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Fig. 2. Localization of vitellogenin receptor (vtgr) transcripts in cutthroat trout ovarian follicles by in situ hybridization (ISH). Panels A–C represent hematoxylin-eosin staining of ovarian sections, and panels D–F represent their corresponding ISH sections. Panels G, H, and I represent enlarged images of the corresponding boxed areas in panels D, E, and F, respectively. Numbers (I–IV) in panels represent stages of the oocytes in each of the follicles: perinucleolus stage (I), lipid droplet stage (II and III), and vitellogenic stage (IV).

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3.5. Detection of Vtgr in ovarian membrane extracts

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or without IPTG induction). The antiserum specifically reacted to the major (~50 kDa) Vtgr-lbd band and some minor bands, as well as these corresponding bands in Western blots of IPTG-induced bacteria extracts, but not in the non-induced bacterial sample (Fig. 3). Specificity of the affinity-purified anti-Vtgr (specific a-Vtgr IgG) was shown to be identical to that of the original antiserum by Western blotting (data not shown).

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a-Vtgr

Fig. 3. Expression of recombinant protein corresponding to a portion of the ligand binding domain of the cutthroat trout vitellogenin receptor (Vtgr-lbd) and generation of antiserum against the purified recombinant product (a-Vtgr). SDS–PAGE and corresponding Western blotting were performed under reducing conditions for the following samples: 1, bacterial extracts without induction of recombinant Vtgr; 2, bacterial extracts with induction of recombinant Vtgr; 3, purified recombinant Vtgr. The SDS–PAGE gel was stained with Coomassie Brilliant Blue (CBB), while the corresponding blot was probed with a-Vtgr.

The Vtgr was identified in cutthroat trout ovarian membrane extracts by Western blotting done using a-Vtgr in conjunction with ligand blotting performed using biotinylated taimen Vtg (Fig. 4). A bold band with an apparent mass of ~95–105 kDa was detected in Western blotting (Fig. 4A), while the ligand blotting revealed two additional positive bands with apparent masses of N250 kDa and ~210 kDa, in addition to the ~95- to 105-kDa band (Fig. 4B). All of these bands disappeared when non-biotinylated (unlabeled) Vtg was added at 100-fold molar excess. Biotinylated Vtg also bound to a pair of lower molecular mass bands (~70–72 kDa), but this binding was non-specific as evidenced by the lack of displacement of the binding by unlabeled Vtg. 3.6. Ovarian localization of Vtgr protein Localization of Vtgr protein in cutthroat trout ovary was confirmed by immunohistochemisty performed using the affinity-purified specific

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A

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b-Vtg + unlabeled Vtg

Fig. 4. Detection of vitellogenin receptor (Vtgr) in the cutthroat trout ovarian membrane preparation by Western blotting (A) and by ligand blotting (B). Blots were probed either with a-Vtgr or biotin-labeled taimen vitellogenin (b-Vtg), with the latter being performed in the presence or absence of a 100-fold molar excess of unlabeled Vtg (b-Vtg + unlabeled Vtg).

a-Vtgr IgG (Figs. 5 and 6). The positive fluorescent signal was weakly distributed throughout the ooplasm of perinucleolus stage oocytes (Fig. 5F). In lipid droplet stage follicles of diameter ≧600 μm, the positive signal translocated toward the periphery of oocytes (Figs. 5G, H and 6A, B). When follicles reached vitellogenesis (primary and secondary yolk stages), the Vtgr signal was strongly localized near the oolemma (Figs. 5I, J and 6C, D), but this signal became weak in the tertiary yolk stage follicles (Fig. 6E). 4. Discussion In the present study, a putative vtgr cDNA was cloned from the cutthroat trout ovary. The obtained cDNA sequence encoded a protein that was 98% identical to the Vtgr of the rainbow trout, whose primary structure exhibited typical domains and motifs reported for the Vtgr in the trout and other oviparous vertebrates (Bujo et al., 1994; Okabayashi et al., 1996; Davail et al., 1998; Prat et al., 1998; Li et al., 2003; Hiramatsu et al., 2004). The protein whose primary sequence was encoded by this clone was hereby identified as the cutthroat trout Vtgr. Quantification of mRNA by rtqRT-PCR revealed predominant vtgr expression in pre-vitellogenic ovaries (sampled at April) as similarly described for other oviparous species (Bujo et al., 1994, 1995a; Prat et al., 1998; Hiramatsu et al., 2004). Significant expression of vtgr also was observed in brain, and the average vtgr mRNA level was about 4fold lower than in pre-vitellogenic ovaries while it was equivalent to or exceeded the levels seen in vitellogenic ovaries. When ldlr expression was quantified in the cutthroat trout, the brain was also the dominant expression site after the ovaries (Luo et al., 2013). In mammals, VLDLR (LR8) is predominantly expressed in the heart, brain, skeletal muscle, and adipose tissues (Takahashi et al., 1992; Jokinen et al., 1994; Oka et al., 1994). VLDLR in brain may participate in cellular signaling and/ or regulate development and functional maintenance of the nervous system (Trommsdorff et al., 1999). Dominant expression of lipoprotein receptors in the trout brain may relate to active signal transduction in the nervous system or general lipid metabolism, although this hypothesis remains to be verified.

Two forms of LR8 transcript, which appear to be produced by differential splicing of the O-linked sugar domain, have been identified in the chicken (Bujo et al., 1994, 1995a) and the rainbow trout (Prat et al., 1998). In these species, the form lacking the O-linked sugar domain (LR8−) was predominantly expressed in the ovary, whereas the form that contained the O-linked sugar domain (LR8+) was expressed in the somatic tissues. We performed RT-PCR and agarose gel electrophoresis using primers designed upstream and downstream of the O-linked sugar domain to detect the presence of these two splice variants of vtgr in cutthroat trout (data not shown). The results revealed that LR8− is predominantly expressed in the ovary (pre-vitellogenic stage), although the liver, kidney, gill, stomach, intestine, brain, and spleen also expressed the LR8− variant with less intensity. Predominant expression of LR8+ was found in the muscle. Like other species, these results confirmed that there are two receptor variants (LR8+ and LR8−) and that the LR8− variant is the ovarian type, which is responsible for Vtg internalization (i.e., Vtgr) in the cutthroat trout. Transcripts of ovarian lipoprotein receptors including vtgr are expressed during early reproductive phases in teleosts, well before their encoded proteins are reported to be functional (Prat et al., 1998; Perazzolo et al., 1999; Hiramatsu et al., 2004; Luo et al., 2013). The patterns of ovarian vtgr mRNA expression associated with ovarian growth in cutthroat trout are similar to those observed in other oviparous species. The vtgr is highly expressed in ovaries during pre-vitellogenesis and the expression gradually decreases during vitellogenesis (Perazzolo et al., 1999; Hiramatsu et al., 2004). These changes in vtgr expression in whole ovarian tissues are in agreement with the results of in situ hybridization, which also revealed an ovarian growth-dependent decrease in vtgr expression in the ooplasm. In contrast, the results of immunocytochemistry revealed that, while some Vtgr could already be detected in the ooplasm of pre-vitellogenic oocytes, it was most intensely evident in the periphery of vitellogenic oocytes, in association with the oolemma. These observations suggest that de novo transcription of vtgr occurs mainly during pre-vitellogenesis and does not actively occur in vitellogenic oocytes, and that synthesis of Vtgr protein, beginning in pre-vitellogenesis is associated with depletion of the vtgr mRNA pool. After synthesis in the ooplasm, the Vtgr proteins translocate to the oolemma and are continuously recycled to the oolemma following endocytosis during vitellogenesis (Goldstein et al., 1985; Shen et al., 1993; Perazzolo et al., 1999). The subsequent increase in vtgr mRNA expression in postovulatory follicles of cutthroat trout seen during February could be due to active expression in the next clutch of oocytes recruited into pre-vitellogenic growth, as this salmonid species is iteroparous (Johnson et al., 1999). The molecular mass of Vtgr was estimated to be 100 kDa in coho salmon by ligand blotting performed using radiolabeled Vtg (Stifani et al., 1990b). In the rainbow trout, Tyler and Lubberink (1996) detected 4 receptor proteins (220, 210, 110, and 100 kDa) by ligand blotting, with predominant Vtg binding being associated with the 210-kDa form, whereas Nuñez-Rodriguez et al. (1996) also showed 4 receptor protein bands (173, 168, 113, and 99 kDa); however, Vtg binding was primarily associated with the 113-kDa form. Thus, the molecular mass and number of Vtgrs remains to be definitively verified in salmonids. In the present study, ligand blotting of cutthroat trout ovarian membrane proteins revealed 3 bands (N250, ~210, and ~95–105 kDa) that bound Vtg, with predominant Vtg binding being associated with the ~95- to 105-kDa form. This binding of Vtg tracer was specifically displaced by excess unlabeled Vtg. Additionally, the molecular weight of vtgr predicted from the polypeptide sequence (93.2 kDa) is similar to this size. It is unclear why this receptor resolves as a broad band with a 10-kDa range in size in ligand blots; however, it may indicate the presence of two similarly sized receptors located in close proximity to the 100-kDa position. The presence of a double receptor band located near this position (~116 kDa and ~110.5 kDa) has been reported in white perch (Reading et al., 2011). These receptors preferentially bind VtgAb, whereas a third, large receptor (N 212 kDa) preferentially binds

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Fig. 5. Confocal laser scanning microscopic images of cutthroat trout ovarian follicles showing localization of vitellogenin receptor (Vtgr) protein. Panels represent the fluorescent images (A–E) and their overlay with images obtained by phase contrast microscopy (F–J). Numbers I–V in panels F–J represent stages of the oocytes in each of the numbered follicles: perinucleolus stage (I), lipid droplet stage (II and III), primary yolk stage (IV), and secondary yolk stage (V). The 10 × 0.4 dry objective lens was used for generating all images. Horizontal bar = 250 μm.

VtgAa in the white perch. Additionally, splice variants have been reported for vtgr in the ovary of the rainbow trout (Prat et al., 1998) and other species (Bujo et al., 1995a; Okabayashi et al., 1996; Li et al., 2003). Therefore, further investigation will be required to address the possibility of more than one Vtgr or Vtgr-related receptor in the cutthroat trout. In teleosts, only two reports have shown that a cloned vtgr actually encoded a functional Vtg-binding protein (Davail et al., 1998; Li et al.,

2003). The present study supports these previous reports as a-Vtgr raised against recombinant cutthroat trout Vtgr-lbd specifically detected a ~95- to 105-kDa protein revealed by Western blotting that specifically bound Vtg as shown by ligand blotting. The recombinant Vtgr-lbd was designed to correspond to the ligand binding domain of Vtgr, as this region is responsible for selective binding of Vtg but not other plasma lipoproteins in salmonid species (Tyler and Lubberink, 1996). This

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Fig. 6. Localization of vitellogenin receptor (Vtgr) near the oolemma in cutthroat trout oocytes. Panels A, B, C, and D represent enlarged images of the boxed areas indicated in panels G, H. I, and J in Fig. 5, respectively. Panel E represents Vtgr localization near the oolemma of a tertiary yolk stage oocyte. 63 × 1.4 oil immersion objective lens was used for generating all images. O, oocyte; ZR, zona radiata; GC, granulosa cell layer; TC, thecal cell layer.

property indicates that the ligand binding domain of Vtgr is unique among lipoprotein receptors as the resulting antibody is specific to Vtgr. We conducted immunohistochemistry with a-Vtgr in order to characterize ovarian localization of Vtgr for the first time in any teleost. Collectively, the results of in situ hybridization and immunohistochemistry in cutthroat trout suggest that vtgr mRNA expressed in perinucleolus stage oocytes is gradually translated into Vtgr protein. The Vtgr also is detected in oocytes before the onset of vitellogenesis in the rainbow trout (Lancaster and Tyler, 1994) and in the ovaries of immature hens (Bujo et al., 1995b). During the lipid droplet stage in cutthroat trout, the Vtgr proteins subsequently migrate toward the periphery of the oocytes and become localized near the oolemma. In chicken oocytes, Vtgr proteins are similarly localized centrally and then redistribute peripherally at the onset of Vtg uptake (Shen et al., 1993). These data suggest that Vtgr proteins are synthesized and stored in the ooplasm and that receptor translocation is essential for Vtg uptake and begins before the onset of vitellogenesis. Further investigation is required to identify factors that might regulate this translocation process. The Vtgr gene transcripts and proteins were not detected in zona radiata, theca, or granulosa of cutthroat trout follicles. The ultrastructure of mummichog, Fundulus heteroclitus, ovarian follicles has been reported (Selman and Wallace, 1989) and, as extracellular egg envelope materials accrue, the microvillar processes extending from both the oocyte and granulosa cells increase in length within pore canals that penetrate the zona radiata. In addition, heterologous gap junctions have been identified between the distal ends of microvillar processes. Such a structure suggests that Vtg may pass through an extracellular space between zona radiata and microvilli of teleost ovarian follicles to bind the Vtgr on the oolemma. Aside from the present study, the pathway of Vtg internalization has been visualized by in vitro incubation of colloidal goldconjugated Vtg in the Xenopus oocyte (Busson et al., 1989). In Xenopus, Vtg localized in coated pits at the oolemma, but not within microvilli or the zona radiata, suggesting that Vtg passes through pore canals of the zona radiata before binding Vtgr on the oocyte surface. Therefore, in additional to translocation of Vtgr to the oocyte surface, morphological

changes in the zona radiata also may be required to make the oolemma accessible to Vtg for internalization. The results of the present study reveal the changes in expression patterns and localization of ovarian vtgr/Vtgr in the cutthroat trout during oogenesis that suggest sequential mechanisms of Vtg internalization via the Vtgr. Based on our findings, we propose a model for Vtgrmediated endocytosis in salmonids (Fig. 7). First, vtgr transcripts are expressed in perinucleolus stage oocytes and are gradually translated to form Vtgr proteins (Fig. 7, steps 1 and 2). As oocytes reach the lipidic stage, Vtgr translocates to the oocyte oolemma in preparation for Vtg internalization (Fig. 7, step 3). When follicles reach the vitellogenic stage, Vtg passes through the gap space between the zona radiata and microvilli (Fig. 7, step 4) and binds the Vtgr on the oolemma (Fig. 7, step 5). The Vtg–Vtgr complex is internalized through clathrin-coated pits (Fig. 7, step 6), and Vtg is proteolytically processed before storage in yolk granules (Fig. 7, step 7). The internalized Vtgr is then recycled back to the surface of the oolemma (Fig. 7, step 8). The present study was conducted to provide basic knowledge on the mechanisms underlying vitellogenesis in teleosts, with special focus on the expression and localization of vtgr/Vtgr associated with ovarian follicle growth in the cutthroat trout. Ovarian localization of Vtgr protein was directly confirmed for the first time in any fish species and this observation provides the basis for a new model of vitellogenesis. Future studies on post-translational changes of Vtgr related to receptor translocation and recycling in developing follicles will further aid in our understanding of fish oocyte growth. Acknowledgement We thank E. Yamaha and S. Kimura, Nanae Fresh-Water Laboratory, Field Science Center for Northern Biosphere, Hokkaido University, for the maintenance of fish during this study. We also thank M. Shimizu and H. Kudo, Faculty of Fisheries sciences, Hokkaido University for helpful discussion. This work was supported in part by the Grant-in-Aid for JSPS Fellows (JSPS KAKENHI grant number 24-1961), the Grant-in-Aid

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Gap junction Fig. 7. Proposed model for vitellogenin (Vtg) internalization via its receptor (Vtgr) in cutthroat trout. Numbers on the figure represent sequential processes in this model (for details, see Discussion). N, nucleus; GC, granulosa cell; MV, microvillus; ZR, zona radiata; PV, perivitellin space; YG, yolk granule.

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