Biol Reprod Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print June 6, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.061598
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow [Supplemental Figure]
Right arrow All Versions of this Article:
77/3/504    most recent
biolreprod.107.061598v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Agulleiro, M. J
Right arrow Articles by Babin, P. J
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Agulleiro, M. J
Right arrow Articles by Babin, P. J
Agricola
Right arrow Articles by Agulleiro, M. J
Right arrow Articles by Babin, P. J
BIOLOGY OF REPRODUCTION 77, 504–516 (2007)
DOI: 10.1095/biolreprod.107.061598
© 2007 by the Society for the Study of Reproduction, Inc.

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)1

Maria J Agulleiro 3, Michèle André 4, Sofia Morais 4, Joan Cerdà 5, and Patrick J Babin 2 4

Institut de Recerca i Tecnologia Agroalimentàries Center of Aquaculture,3 Sant Carles de la Ràpita, Tarragona 43540, Spain Génomique et Physiologie des Poissons,4 Université Bordeaux 1, UMR NuAGe, 33405 Talence, France Institut de Recerca i Tecnologia Agroalimentàries Lab IRTA-ICM (Consejo Superior de Investigaciones Científicas),5 08003 Barcelona, Spain

ABSTRACT

Transcripts encoding a fatty acid-binding protein (FABP), Fabp11, and two isoforms of very low-density lipoprotein receptor (Vldlr; vitellogenin receptor) were characterized from the ovary of Senegalese sole (Solea senegalensis). Phylogenetic analyses of vertebrate FABPs demonstrated that Senegalese sole Fabp11, as zebrafish (Danio rerio) homologous sequences, is part of a newly defined teleost fish FABP subfamily that is a sister clade of tetrapod FABP4/FABP5/FABP8/FABP9. RT-PCR revealed high levels of vldlr transcript splicing variants in the ovaries and, to a lesser extent, in somatic tissues, whereas fabp11 was highly expressed in the ovaries, liver, and adipose tissue. In situ hybridization analysis showed vldlr and fabp11 mRNAs in previtellogenic oocytes, whereas no hybridization signals were detected in the larger vitellogenic oocytes. Transcript expression of fabp11 was strongly upregulated in somatic cells surrounding atretic follicles. Real-time quantitative RT-PCR demonstrated that ovarian transcript levels of vldlr and fabp11 had a significant positive correlation with the percentage of follicles in previtellogenesis and atresia, respectively. These results suggest that the expression level of vldlr transcripts may be used as a precocious functional marker to quantify the number of oocytes recruited for vitellogenesis and that fabp11 mRNA may be a very useful molecular marker for determining cellular events and environmental factors that regulate follicular atresia in fish.

follicular development, gametogenesis, gene regulation, oocyte development, ovary

INTRODUCTION

Atresia, a follicular degeneration and resorptive process, is a normal physiologic event of vertebrate ovarian morphogenesis. In teleost fish, atresia is involved in normal ovarian growth and postovulatory regression, mostly in females that are not able to carry out maturation or ovulation after vitellogenesis. The presence of atretic follicles is also frequently associated with environmental stress or changes in hormone levels during vitellogenesis [1–3]. For example, ovarian atresia may be caused by hypophysectomy [1, 4] or exposure to environmental contaminants [5, 6]. It is well established in mammals that ovarian follicular degeneration is a hormonally controlled apoptotic process and is an event of programmed cell death [7–10]. While apoptosis has been demonstrated in the ovary of several fish species [11, 12] and in response to the presence of endocrine-disrupting chemicals [13, 14], a relationship between programmed cell death and atresia has not been firmly established in fish. In addition, induced proteolysis of oocyte yolk proteins may lead to follicular atresia [15]. Hormonal factors produced in fish gonads seem to play a central role in ovarian follicle growth, maintenance of ovarian synchronicity, and the process of ovarian follicular atresia [16, 17].

In teleost fish, as in other oviparous vertebrates, female germinal cells accumulate the informational components and nutritional reserves needed for embryo development [18–21]. Oocyte growth occurs through the uptake of plasma egg yolk precursor proteins, predominantly vitellogenin (Vtg). This glycolipophosphoprotein is synthesized mainly in the liver under estrogenic control; is specifically incorporated in the oocyte by receptor-mediated endocytosis through the very low-density lipoprotein (VLDL) receptor precursor (VLDLR), also named in oviparous animals Vtg receptor (VtgR); and is further cleaved and processed into yolk proteins. The VLDLR gene (vldlr) belongs to the supergene family of low-density lipoprotein receptor (LDLR)-related proteins [22]. These plasma membrane receptors have co-evolved in egg laying and viviparous animals to support ligand transport inside the cell and to sustain the reproductive effort of oviparous species [23, 24]. Fatty acid-binding proteins (FABPs) are highly conserved cytoplasmic proteins that bind long-chain fatty acids and other hydrophobic ligands. It is thought that FABPs roles include fatty acid uptake, transport, and metabolism. These may also be responsible in the modulation of cell growth and proliferation [25, 26]. Different FABP types have been immunodetected in rodent ovaries at different physiologic stages [27–30]. It has also been demonstrated that the expression of some genes coding for proteins related to lipid and lipoprotein metabolism, including FABPs and LDLR, are among the most regulated in the rat ovary after human chorionic gonadotropin administration [31]. Previtellogenic fish oocytes express high amounts of vldlr and fabp3 transcripts [32, 33], and the reabsorption of the yolk in atretic follicles produces the appearance of egg yolk proteins associated with plasma high-density lipoproteins [34], which enlighten the importance of lipid metabolism and its related genes in follicular growth and degeneration.

Histology of teleost fish gonadal tissue is routinely used to describe and quantify morphologic stages, including the number of vitellogenic and atretic follicles [1, 2, 35], but no molecular markers are currently available to study the mechanisms underlying or regulating the balance between follicular development and atresia in teleosts. Here we describe the molecular characterization from Senegalese sole (Solea senegalensis) ovary of cDNAs of VLDLR, including two transcript variants, and of an Fabp that was demonstrated to be part of a new type, named Fabp11, specific to teleost fishes. The results presented indicate that these transcripts were expressed in previtellogenic oocytes, and fabp11 transcript expression was strongly upregulated in somatic cells surrounding atretic follicles. Furthermore, we provide evidence that ovarian transcript levels of vldlr and fabp11 had a significant positive correlation with the percentage of follicles in previtellogenesis and atresia, respectively.

MATERIALS AND METHODS

Procedures relating to the care and use of animals were approved by the Ethics Committee from Institut de Recerca i Tecnologia Agroalimentàries (Spain) in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.

Animals

Adult Senegalese soles were raised as previously described [36], and females were killed during three consecutive years at different times of the reproductive cycle, corresponding to different folliculogenesis stages [36, 37]. The body and gonads of each animal were weighed to calculate the gonadosomatic index (GSI: gonad weight/body weight x 100). A piece of the gonad adjacent to the piece sampled for subsequent RNA extraction was fixed in modified Bouin solution (75% picric acid and 25% formalin) for histologic analysis.

Histologic Analysis

Ovaries fixed in modified Bouin solution for 3–4 h were dehydrated, embedded in paraplast, sectioned at 5 µm, and stained with hematoxilin-eosin. The percentages of previtellogenic, early vitellogenic, vitellogenic, and atretic oocytes were calculated by counting 100–150 total oocytes in at least three different histologic sections from the same ovary, as previously described [36].

Construction of an Ovarian cDNA Library and Isolation of Senegalese Sole vldlr and fabp11 cDNAs

Total RNAs were extracted from vitellogenic ovaries using the Qiagen RNeasy Maxi Kit (Qiagen, Courtaboeuf, France), and a cDNA library was constructed in Uni-ZAP XR vector (Stratagene, Amsterdam, The Netherlands) following the manufacturer's instructions. Full-length vldlr cDNA was obtained after screening with a 32P-labeled rainbow trout (Oncorhynchus mykiss) vldlr cDNA fragment. The 794-bp probe used was generated after cleavage of trout vldlr cDNA (GenBank accession number OMY417877) [38] with SmaI and XbaI restriction enzymes at 1063 and 1857 bp, respectively.

Three positive clones were identified as vldlr cDNA: S9A5, corresponding to the full-length vldlr transcript, S5A, a truncated vldlr transcript sequence, and S8A2, a chimeric sequence including 5' 579 bp that contained the 402-bp full-length translated region of a cDNA encoding a protein subsequently named Fabp11 (see below), as well as part of the 5' end of the vldlr transcript sequence. The missing 107-bp 3' untranslated region of the fabp11 transcript was amplified by PCR with the SHF3-S1 sense primer (5'-ACGACAGGAAGACCAAGACCG-3') and 3' rapid amplification of cDNA ends (RACE) primer, using the previously described procedure [39] and PCR reaction profile [40].

The VLDLR protein sequence deduced from clone S9A5 lacked the O-linked sugar domain and was, therefore, classified as a VLDLR– isoform. The cDNA encoding the VLDLR+ isoform was amplified with S-og-S1 primer (5'-AGCTCATCCAGCTGTCAG-3'), located 169 bp upstream, and S-og-AS1 primer (5'-TCCACATGAGGAAACCAC-3'), located 122 bp downstream from the alternative splicing site of primary transcripts, leading or not to a putative O-linked sugar domain.

Sequence Data Sets

Deduced protein sequences were extracted from GenBank/EBI (http://www.ncbi.nlm.nih.gov/) or UniProt (http://www.ebi.uniprot.org/index.shtml) databases or, in a few cases, were deduced from Ensembl Takifugu rubripes (ETR) (http://www.ensembl.org/Takifugu_rubripes/index.html) or Tetraodon Genome Browser (TGB) (http://www.genoscope.cns.fr/externe/tetranew/). Accession numbers (the number in parentheses after the accession number refers to the code number used for sequence designation) for vertebrate FABPs were UniProt:P07148 (1) for FABP1, UniProt:P12104 (2) for FABP2, UniProt:P05413 (3) for FABP3, UniProt:P15090 (4) for FABP4, UniProt:Q01469 (5) for FABP5, UniProt:P51161 (6) for FABP6, UniProt:O15540 (7) for FABP7, and UniProt:P02689 (8) for FABP8 of human (Homo sapiens); UniProt:P12710 (9) for FABP1, UniProt:P55050 (10) for FABP2, UniProt:P11404 (11) for FABP3, UniProt:P15090 (12) for FABP4, UniProt:Q05816 (13) for FABP5, UniProt:P51162 (14) for FABP6, UniProt:P51880 (15) for FABP7, UniProt:P24526 (16) for FABP8, and UniProt:O08716 (17) for FABP9 of mouse (Mus musculus); UniProt:P02693 (18) for FABP2, UniProt:P07483 (19) for FABP3, UniProt:P70623 (20) for FABP4, UniProt:P55053 (21) for FABP5, UniProt:P80020 (22) for FABP6, UniProt:P55051 (23) for FABP7, and UniProt:P55054 (24) for FABP9 of rat (Rattus norvegicus); UniProt:P49924 (25) for FABP1, UniProt:Q45KW7 (26) for FABP2, UniProt:O02772 (27) for FABP3, UniProt:O97788 (28) for FABP4, UniProt:Q2EN74 (29) for FABP5, and UniProt:P10289 (30) for FABP6 of pig (Sus scrofa); UniProt:Q90WA9 (31) for FABP1, UniProt:Q7ZZZ5 (32) for FABP2, UniProt:Q6DRR5 (33) for a protein annotated here FABP3, UniProt:Q90X55 (34) for FABP4, UniProt:Q5ZIR7 (35) for a protein annotated here FABP5, UniProt:Q05423 (36) for a protein annotated here FABP7, GenBank:XP_418309 (37) for FABP8, and UniProt:P80226 (38) for FABP10 of chick (Gallus gallus); deduced from clone GenBank:CN098757 (39) for a protein annotated here FABP2, and UniProt:Q28CE2 (40) for FABP7 of Western clawed frog (Xenopus tropicalis); UniProt:Q6PGR8 (41) for a protein annotated here FABP3a, UniProt:Q6GPY9 (42) for a protein annotated here FABP3b, UniProt:Q5PPW3 (43) for a protein annotated here FABP7, and UniProt:Q6GPT0 (44), UniProt:Q5FWM7 (45), UniProt:Q66L00 (46), and UniProt:Q6P705 (47) for deduced unclassified FABPs of African clawed frog (Xenopus laevis); GenBank:BC095259 (48) for Fabp1a, GenBank:XM_680590 (49) for Fabp1b, UniProt:Q9PRH9 (50) for Fabp2, UniProt:Q8UVG7 (51) for Fabp3, GenBank:NP_001002076 (52) for Fabp6, UniProt:Q9I8N9 (53) for Fabp7a, UniProt:Q6U1J7 (54) for Fabp7b, UniProt:Q9I8L5 (55) for Fabp10, UniProt:Q66I80 (56) for a protein annotated here Fabp11a, and UniProt:Q503X5 (57) for a protein annotated here Fabp11b of zebrafish (Danio rerio); UniProt:Q90W92 (58) for Fabp3, UniProt:Q645P9 (59) for a protein annotated here Fabp10, and deduced from clone GenBank:CN985071 (60) for a protein annotated here Fabp11 of killifish (Fundulus heteroclitus); deduced from clone GenBank:CA845456 (61) for a protein annotated here Fabp2a, ETR:NEWSINFRUT00000154534 (62) for a protein annotated here Fabp2b, ETR:NEWSINFRUT00000161229 (63) for a protein annotated here Fabp10, and deduced from clone GenBank:CA847283 (64) for a protein annotated here Fabp11 of torafugu (T. rubripes); GenBank:AAM22208 (65) for a protein annotated here Fabp11 of orange-spotted grouper (Epinephelus coioides); deduced from clone GenBank:CK890396 (66) for a protein annotated here Fabp1, and UniProt:Q6R758 (67) for a protein annotated here Fabp3 of Atlantic salmon (Salmo salar); deduced from clone GenBank:BX866673 (68) for a deduced protein annotated here Fabp2, UniProt:O13008 (69) for Fabp3, deduced from clone GenBank:CX251727 (70) for a deduced protein annotated here Fabp10, deduced from clone GenBank:CA357251 (71) for a deduced protein annotated here Fabp11a, and deduced from clone GenBank:CF752694 (72) for a deduced protein annotated here Fabp11b of rainbow trout (Oncorhynchus mykiss); UniProt:Q4S6K4 (73) for a protein annotated here Fabp2, UniProt:Q4RMM1 (74) for a protein annotated here Fabp7a, UniProt:Q4T8P8 (75) for a protein annotated here Fabp7b, TGB:GSTENT00003005001 (76) for a protein annotated here Fabp10, and GenBank:CR733066 (77) for a protein annotated here Fabp11 of green puffer (Tetraodon nigroviridis); deduced from clone GenBank:AM144272 (78) for a protein annotated here Fabp3, UniProt:Q2PHF0 (79) for Fabp7, deduced from clone GenBank:BJ910672 (80) for a protein annotated here Fabp10, and deduced from clone GenBank: BJ875298 (81) for a protein annotated here Fabp11 of medaka (Oryzias latipes); UniProt:O57668 (82) for Fabp3, and UniProt:O57691 (H6-FABP; 83) for a protein annotated here Fabp11 of crocodile icefish (Cryodraco antarcticus); UniProt:O57669 (84) for Fabp3, and UniProt:O57663 (H6-FABP; 85) for a protein annotated here Fabp11 of black rockcod (Notothenia coriiceps); UniProt:O57670 (86) for Fabp3, and UniProt:O57665 (H6-FABP; 87) for a protein annotated here Fabp11 of humped rockcod (Gobionotothen gibberifrons); UniProt: O57666 (H6-FABP) (88) for a protein annotated here Fabp11 of an Antarctic dragonfish (Parachaenichthys charcoti); GenBank:BAA92355 (89) for Fabp3 of Japanese eel (Anguilla japonica); deduced from clone GenBank:BM028330 (90) for a protein annotated here Fabp3, deduced from clone GenBank:BE574176 (91) for a protein annotated here Fabp7, and deduced from clone GenBank:BM438483 (92) for a protein annotated here Fabp10 of channel catfish (Ictalurus punctatus); deduced from clone GenBank:BJ696441 (93) for a protein annotated here Fabp3 of redtail sheller (Ptyochromis sp.); deduced from clone GenBank: CF661735 (94) for a protein annotated here Fabp11 of carp (Cyprinus carpio); GenBank:AM501530 (95) for a protein annotated here Fabp11 of Senegalese sole (S. senegalensis). Accession numbers for vertebrate VLDLR/VtgR sequences used were UniProt:P98155 of human (Homo sapiens), UniProt:P98156 of mouse (Mus musculus), UniProt:P98165 of chick (Gallus gallus), UniProt:Q90W12 and UniProt:O73921 of rainbow trout (Oncorhynchus mykiss), UniProt:Q6NS01 of African clawed frog (Xenopus laevis), UniProt:Q7ZTG7 of tilapia (Oreochromis aureus), UniProt:Q6Y857 of white perch (Morone americana), and UniProt: Q2L4C1 of Senegalese sole (S. senegalensis). Accession numbers for Senegalese sole (S. senegalensis) bactin mRNAs sequences used were GenBank:DQ485686 and GenBank:AM501529.

Sequence Analyses

Sequences were aligned using EMBOSS needle program and Blosum62 matrix or ClustalW (http://www.ebi.ac.uk/). The signal peptide sequence of the deduced protein was predicted using the SignalP 3.0 prediction tool (http://www.cbs.dtu.dk/services/SignalP/) using hidden Markov models trained on eukaryotes. The putative GalNAc O-glycosylation sites were predicted using NetOGlyc 3.1 server (http://www.cbs.dtu.dk/services/NetOGlyc/). The phylogenetic tree and branch support values were estimated using three different methodologies of phylogenetic reconstruction: the neighbour joining (NJ), the maximum likelihood (ML), and the Bayesian inference (BI) methods according to the procedures previously described with modifications [41]. NJ algorithm was based on the number of amino acid substitutions per site using the Poisson correction distance method. Bootstrap support values were obtained with 5000 pseudoreplicates. ML analysis was carried out with PHYML v2.4.4 [42] starting from the BIONJ tree, and the gamma distribution for rate heterogeneity across sites ({Gamma}) was modeled with a four-category {Gamma} distribution and a shape parameter equal to 2. The WAG substitution model was selected by ProtTest v1.3 [43], following the Akaike information criterion, as the best-fitting model among the models tested that could be used in PHYML. Bootstrap values were based on 500 pseudoreplicates to estimate support for the nodes of the ML tree. In the nonparametric bootstrap trees, bootstrap confidence level values of 80 or greater were accepted as significant. BI was performed using MrBayes v3.1.2 [44] with the WAG model of amino acid substitution provided in the package. Two simultaneous runs, each with four simultaneous Markov Chain Monte Carlo (MCMC) chains, were performed for 1 600 000 generations, after which the average standard deviation of split frequencies was 0.009795, saving the current tree to file every 100 generations for a total of 16 001 trees in the initial sample. The first 4000 trees prior to log likelihood stabilization were discarded (as burn-in), and the following 12 001 tree samples were used to estimate topology and tree parameters. A graphic representation of the majority rule consensus tree was generated, and the clades were accepted as significant at ≥0.90 posterior probability.

Northern Blot Analysis

Total RNAs were extracted from a previtellogenic (GSI: 1.5%; percentage of previtellogenic oocytes: 99%; percentage of atretic oocytes: 1%) or a vitellogenic (GSI: 11.3%, percentage of vitellogenic oocytes: 10%, percentage of atretic oocytes: 5%) ovary using WIZ RNA isolation reagent without DNAse treatment (Ambion, Huntingdon, United Kingdom), according to the manufacturer's instructions. Each RNA sample (10 µg) was denatured at 70°C for 15 min in 50% formamide, 10% formaldehyde, 5% borax buffer 10x, 50 ng/µl ethidium bromide, and 5% bromophenol blue, separated by electrophoresis on a 1.2% agarose denaturing gel containing 16.8% formaldehyde and 10% borax 10x for several hours at 50 V under cold conditions to avoid denaturing the RNAs, and was then transferred to a Hybond-N nylon membrane (GE Healthcare, Orsay, France).

The cDNAs used for probe synthesis were obtained by PCR with the following primers: VTGR-S2 (5'-TGGCWCTGGATGCAGAC-3') and VTGR-AS2 (5'-CACATGTAGSNACAGCCTCC-3') for vldlr, SHF3-S4 (5'-ATGGTTGAGAGTTTTGTTGGGAC-3') and SHF3-AS1 (5'-GTACGTCCTCACTGCGAC-3') for fabp11, using S9A5 and S8A2 clones, respectively, as templates. PCR products of 748 bp for vldlr, from nucleotides +1415 to +2162 (numbered from the translation initiator codon), and 390 bp for fabp11, from nucleotides –30 to +360, were used as probes after purification, subcloning, and radiolabeling.

RT-PCR Analysis

For 1 µg total RNAs isolated from various tissues with WIZ RNA isolation reagent (Ambion, Huntingdon, United Kingdom) RT-PCR was performed in a final volume of 20 µl using the M-MLV Superscript II Reverse Transcriptase RNase H Minus, 500 ng/µl universal Pt RACE primer, and 500 ng/µl hexamer random primers (Promega, Charbonnières les Bains, France) following the manufacturer's instructions. The females used, and thus the percentage of stage-frequency oocytes in histologic sections, are the same as those used for Northern blot analysis. The resulting total cDNAs were used to amplify vldlr, fabp11, and bactin transcript fragments by PCR in a final volume of 25 µl. S-og-S1 and S-og-AS1 primers were used for the vldlr+ transcript variant, and SHF3-S4 and SHF3-AS1 were used for fabp11. To design specific primers for a S. senegalensis bactin transcript, a fragment was amplified by standard PCR using ovary cDNAs as a template and degenerated FishBA1 (5'-ACATGGAGAAGATCTGGC-3') and FishBA2 (5'-GCRTACAGRTCCTTACGGA-3') primers, determined after alignment of teleost fish bactin transcripts currently available in GenBank (data not shown). After PCR product purification, subcloning in pGEMT-easy vector, and sequencing, the two specific primers were designed: S-BAC-S (5'-GACCTTCAACACTCCTGC-3') and S-BAC-AS (5'-GTCACACTTCATGATGCTG-3'). RT-PCR was performed under standard conditions, with 34 amplification cycles instead of 30 for vldlr. Annealing temperature was 57°C for bactin, 59°C for vldlr, and 62°C for fabp11 transcripts.

In Situ Hybridization Studies

Both antisense and sense digoxigenin-labeled RNA probes were synthesized as previously described [45], with the So-VLDLR and So-FABP11 clones also used for Northern blot analysis. The probes were purified on ProbeQuant G50 microcolumns (GE Healthcare, Orsay, France) and checked for purity by denaturing agarose gel electrophoresis.

The sample treatment and sectioning procedure were as previously described [46], avoiding the acetylating step. The digoxigenin-labeled RNA probe was detected as previously described [47], and the resulting dark blue to purple color indicated localization of the transcripts. The sections were photographed using a digital DMX1200 camera and Eclipse E1000 microscope (Nikon, Champigny sur Marne, France).

Real-Time Quantitative RT-PCR

Total RNAs were extracted from ovaries of 16 females sampled at different times of the year, corresponding to various GSIs and percentages of atresia, using Qiagen RNeasy Lipid Tissue Mini Kit (Qiagen, Courtaboeuf, France), including a DNaseI treatment step, according to the manufacturer's instructions. RNA was quantified using a NanoDrop ND-1000 spectrophotometer (Nyxor Biotech, Paris, France), with values of A260/A280 between 2.00 and 2.06, and total RNA integrity was checked by ethidium bromide staining in agarose borax gel. RT reactions were performed with 0.1 µg total RNA using the StrataScript qPCR cDNA Synthesis Kit (Stratagene), following the manufacturer's instructions. One RT negative control (without reverse transcriptase) was carried out for each sample.

Real-time quantitative RT-PCR (qPCR) amplifications were performed in triplicate for each sample in a final volume of 20 µl with 10 µl Brilliant SYBR Green qPCR master mix (Stratagene), 2 µl diluted (1:300) cDNA, and 0.5 µM (for vldlr and bactin transcripts) or 0.3 µM (fabp11 transcripts) oligodeoxyribonucleotide primers. Primers designed to amplify the 161-bp bactin, 169-bp vldlr, 219-bp fabp11 transcript sequences were Bact-So-S3 (5'-ACTGCTGCCTCCTCCTCCT-3') and Bact-So-AS3 (5'-ATGCTGTTGTAGGTTGTCTCGTG-3'), VTGR-So-S1 (5'-CTGTGTTTGAGGACCGAGTGTT-3') and VTGR-So-AS1 (5'-GAGCACCAGTTAGTTCCTGACA-3'), FABP11-So-S4 (5'-ATTTGATCCTGAGCGTGGAC-3') and FABP11-So-AS3 (5'-CGTCTGAGATCTCCCTCTCG-3'), respectively. The bactin primer sequences were designed from a published sequence [48]. Sequences were amplified using the MX 3000P qPCR thermal cycler instrument (Stratagene). The two-step qPCR profiles consisted of an initial denaturation step at 95°C for 10 min, followed by 30 (bactin) or 35 (vldlr and fabp11) cycles, as follows: 30 sec at 95°C, 30 sec at the specific primer pair-annealing Tm (61°C for bactin and fabp11 and 63°C for vldlr), and 30 sec at 72°C. After the amplification phase, 1 min of incubation at 95°C and 30 sec at 55°C, a ramp up to 95°C, at 0.01°C/sec, was performed in which data were collected in continuum to obtain a dissociation curve. The qPCR product sizes were checked on 2% agarose gel, purified, and sequenced. No amplification was observed in RT negative controls without enzymes, and no primer-dimer formation occurred in the nontemplate control, containing no cDNA.

In order to assess interrun reproducibility, three independent standard curves were generated, including one in the same run as the templates, using nine serial dilutions (from 4.0 to 0.016 ng) of a pool of first-strand cDNA template from all samples. Standard curves represented the cycle threshold (Ct) value as a function of the logarithm of the number of copies generated, defined arbitrarily as one copy for the most diluted standard. Efficiency of qPCR was always above 97.6%, and the correlation coefficient ≥0.992 for all target genes. In addition, a data quality control was performed [49] to determine whether differences in target transcript concentrations between samples resulted in different amplification efficiencies, thus biasing relative quantification. Nine samples representative of all stages of gonad development were amplified, in duplicate, at three serial dilutions for each gene (1:31, 1:310, and 1:3100), and the correlation between Ct and the logarithm (base 2)-transformed concentration of the template was examined.

To normalize the qPCR results, genomic DNA and total RNA concentrations were determined from 2 µl of the 1:100 dilution of the total tissue homogenate generated by the Qiagen RNeasy Lipid Tissue Mini Kit (Qiagen, Courtaboeuf, France). Before their extraction, genomic DNA and total RNAs were measured by Quant-iT dsDNA HS and Quant-iT RNA assay kits, respectively, using a Qubit Fluorometer (Invitrogen, Cergy Pontoise, France), following the manufacturer's instructions. Results were normalized by the RNA:DNA ratio and expressed as copy number of transcript per nanogram of genomic DNA. The amount of bactin transcript was also used to express the results as the number of copies of each target genes divided by the number of copies of this normalizing gene.

Statistical Analysis

To analyze the qPCR data quality control results, the correlation model and SAS program (http://www.biomedcentral.com/content/supplementary/1471-2105-7–85-S1.sas [49]) were used with SAS statistical software (SAS Institute Inc, Cary, NC). Variations in normalized transcript levels were analyzed for statistical significance by linear regression and correlation using SigmaStat 3.1 software (Systat Software Inc, San Jose, CA). The significance level was set at 0.05.

RESULTS

Molecular Characterization of Two Splicing Variants of vldlr from S. senegalensis

The full-length Solea vldlr cDNA sequence, deduced from clone S9A5 and corresponding to VLDLR– splicing variant, was deposited in the GenBank/EBI Data Bank under accession number GenBank:AJ879619. The 3968-bp cDNA consisted of a 173-bp 5'-untranslated region, a 2547-bp open reading frame (ORF), and a 1248-bp 3'-untranslated region, including the poly(A) tail at position 3951. The ORF contained a deduced non-O-linked glycosylated VLDLR isoform. The predicted sequence of the deduced protein was 848 amino acids in length. A 27-amino acid signal peptide was predicted, resulting in a mature protein of 821 amino acids with a theoretical molecular mass of 90 595 Daltons. PCR on total cDNAs synthesized from a vitellogenic ovary with primers flanking the putative O-linked sugar domain region amplified a VLDLR+ transcript variant with an additional 60 bp in the open reading frame, leading to a 4028-bp cDNA. This resulted in a mature protein of 841 amino acids with a theoretical molecular mass of 92 612 Daltons (Fig. 1). Five potential GalNAc O-glycosylation sites were predicted in the putative O-linked sugar domain region.


Figure 01
View larger version (110K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 1 Alignment of S. senegalensis (Ss) VLDLR with homologous protein sequences from G. gallus (Gg) and H. sapiens (Hs). Amino acid sequences are numbered from the initiator methionine. Residues identical to Senegalese sole sequence are colored gray. Gaps inserted to optimize alignments are indicated by dashes. The receptor is composed of domains that are indicated above the multiple alignment. The eight cysteine-rich repeats, also named LA repeat clusters and numbered I to VIII, make up the ligand-binding domain. Asterisks below the human sequence indicate conserved cysteine residues in LA and A, B, and C EGF-type repeats. The repeats with six cysteines of the ligand-binding domain presumably mediate the folding of the domain into a rigid structure with clusters of negatively charged residues on its surface that contain the signature SDE. These residues are underlined below the human sequence. The YW(T)D sequence found in multiple tandem repeats and predicted to form a beta-propeller structure is double underlined. The facultative O-linked sugar domain found in Senegalese sole VLDLR is double upperlined. Black arrowheads above the alignment indicate five potential GalNAc O-glycosylation sites in the sole sequence. The highly conserved FDNPVY motif of the cytoplasmic domain, putatively involved in endocytosis of the receptor after ligand binding, is underlined below the human sequence.

The VLDLR/VtgR showed a high degree of conservation among different vertebrate species. Comparison of deduced mature sole VLDLR+ amino acid sequence with human, chicken, and Xenopus laevis VLDLR/VtgR mature sequences revealed 69.5%, 69.8%, and 67% identity, respectively, whereas 83.4%–89.8% identity was found between sole VLDLR– sequence and homologous sequences from rainbow trout, tilapia, and white perch. Comparison with specific domains revealed a high conservation in the vertebrate lineage of the ligand-binding domain comprising type A ligand-binding repeats, also named LA repeat clusters and containing eight repeats with six cysteines each, which presumably mediate the folding of the domain into a rigid structure with clusters of negatively charged residues, which include the signature tripeptide SDE, on its surface. Also conserved are the EGF precursor domain consisting of type A, B, and C repeats; the LDLR repeat class B containing the YW(T)D repeat found in multiple tandem repeats and predicted to form a beta-propeller structure; a facultative O-linked sugar domain, a less conserved transmembrane domain, and a highly conserved cytoplasmic domain including the conserved FDNPVY motif putatively involved in the endocytosis of this receptor after ligand binding (Fig. 1).

Molecular Characterization and Phylogenetic Relationships of Fabp11 from S. senegalensis

The full-length sole fabp11 cDNA sequence, deduced from clone S8A2 and extended with 3'-RACE-PCR, was deposited in the GenBank/EBI Data Bank under accession number GenBank:AM501530. The 839-bp cDNA consisted of a 47-bp 5'-untranslated region, a 405-bp open reading frame, and a 370-bp 3'-untranslated region. The predicted sequence of the deduced protein was 134 amino acids with a theoretical molecular mass of 15 044 Daltons (Fig. 2).


Figure 02
View larger version (50K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 2 Comparison of amino acid sequences of Solea senegalensis (Ss) Fabp11 with selected H. sapiens (Hs) FABPs. Amino acid sequences are numbered from the initiator methionine. Residues identical to Senegalese sole sequence are colored gray. Gaps inserted to optimize alignments are indicated by dashes. Sites of identical amino acids in all sequences are indicated by an asterisk below the alignment. The amino acid insertion at position 46 of Senegalese sole Fabp11, which is found in all teleost fish Fabp11 sequences currently available in sequence databases and not identified in FABP3/FABP4/FABP5/FABP7/FABP8/FABP9 and FABP2 clusters of vertebrate species (see Supplemental Figure 1), is indicated by an arrow below the alignment.

Alignment of the amino acid sequence of Solea Fabp11 with selected human FABPs (Fig. 2) and vertebrate FABPs previously characterized or extracted from sequence databases (see Supplemental Figure 1, available online at www.biolreprod.org), revealed 75.6%–85.1% identity with teleost fish species Fabp11, whereas 63.4% and 82.1% identity was recovered with the duplicated Fabp11 forms found in zebrafish. There was a much lower degree of identity with selected tetrapod FABP4/FABP5/FABP8/FABP9 (e.g., 54.5% and 52.2% with human FABP4 and FABP8, respectively) and vertebrate FABP3/FABP7 (e.g., 57.4% and 60.4% with human FABP3 and FABP7, respectively), and even less with vertebrate FABP1/FABP6/FABP10 (e.g., 27.1% and 23% with human FABP1 and FABP6, respectively) and FABP2 (e.g., 21.6% with human FABP2) types. The evolutionary relationship of vertebrate genes in the FABP family was evaluated after aligning selected protein sequences of each subfamily, and the phylogenetic analyses were conducted using NJ, ML, and BI (Fig. 3) methods. The phylogenetic tree separated with confidence into separate clusters FABP1/FABP6/FABP10 (group 1), FABP2 (group 2), and FABP3/FABP4/FABP5/FABP7/FABP8/FABP9/FABP11 (group 3) (NJ: 100; ML: 100; BI: 1.00). Significant internal branches of group 3 defined three FABP sequence subgroups. The first one was FABP3 subfamily (NJ: 80; ML: 82; BI: 1.00), and the second one was FABP7 subfamily (NJ: 99; ML: 92; BI: 0.97). The third subgroup formed a monophyletic branch, suggesting that these proteins arose from a common ancestror, and contained FABP4/FABP5/FABP8/FABP9/FABP11 subfamilies. Fabp11 was an additional FABP subfamily that seemed to be specific to teleost fish species (NJ: 97; ML: 97; BI: 1.00). Monophyly of teleost fish Fabp11 was also supported by the presence of an amino acid insertion at position 46 of Senegalese sole Fabp11 that is found in all teleost fish Fabp11 sequences currently available in sequence databases and not identified in the sequence of the other subfamilies of group 3 (i.e., FABP3/FABP4/FABP5/FABP7FABP8/FABP9). It should be noted that a similar unique amino acid insertion was present in group 1 but not in group 2 sequences. BI method supported with confidence that FABP11 subfamily was a sister clade of tetrapod FABP4/FABP5/FABP8/FABP9 cluster (BI: 0.97). This cluster contained tetrapod sequences only and has been split (BI: 1.00) into two significant internal branches, corresponding to FABP5 subfamily (NJ: 99; ML: 100; BI: 1.00) on one side and FABP4/FABP8/FABP9 (BI: 0.93) on the other side. It should be noted that identified duplicated zebrafish Fabp isoform sequences (i.e., Fabp1, Fabp7, and Fabp11) possibly resulting from the ancestral teleostean fish genomewide duplication were clustered with their respective subfamilies.


Figure 03
View larger version (35K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 3 BI phylogenetic reconstruction of the FABP family in vertebrates. The tree was based on the alignment (see Supplemental Figure 1) of amino acid sequences of all teleost fish Fabp sequences currently available in sequence databases and a selection of tetrapod sequences. Sequences are indicated as numbers according to the code number used for sequence designation and detailed in Materials and Methods. Teleost fish or Senegalese sole Fabp11 sequence numbers are highlighted in green and red, respectively. Branch lengths are proportionate to BI estimates of numbers of amino acid substitutions, and numerals at each node indicate the posterior probability estimated by MrBayes under the model, summed over 12 001 tree samples. Posterior probabilities ≥0.90 are shown. Due to graphic space limitation, an asterisk within FABP subfamilies indicates that the posterior probability value was 1.00. Numbers in circles indicate the three separate FABP family groups.

Expression of vldlr, fabp11, and bactin in Adult S. senegalensis Tissues

Northern blot analysis of RNAs extracted from a previtellogenic ovary revealed an intense vldlr transcript band of around 4.0 kb, which corresponds to the size of the VLDLR+ and VLDLR– transcript variants (Fig. 4A). The same methodology applied to RNAs extracted from a vitellogenic ovary revealed a fabp11 transcript band of around 0.8 kb, corresponding to the size of the recovered transcript.


Figure 04
View larger version (48K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 4 Expression of vldlr, fabp11, and bactin in adult S. senegalensis tissues. A) Northern blot hybridization of total RNAs isolated from an ovary of a vitellogenic (V) or a previtellogenic (P) female. Samples (10 µg) of purified RNAs were resolved by electrophoresis through agarose formaldehyde gels, and blots were hybridized with 32P-labeled sole fabp11 or vldlr probes. Size of hybridizing species shown is relative to mobility of RNA standards. B) Transcripts of vldlr, fabp11, and bactin that were detected by RT-PCR in RNAs extracted from adult tissues. RT-PCR products were generated from total RNAs and were extracted from a previtellogenic (P) or a vitellogenic (V) female and from a male using cDNA-specific primers. The various adult tissues used were: liver (L), kidney (K), adipose tissue (A), muscle (M), testicle (T), heart (H), and ovary (O). A negative control (C) lacking reverse transcriptase generated no RT-PCR products. The two alternative splicing variants of primary transcripts leading to a putative (O+) or not putative (O-) O-linked sugar domain are indicated on the right.

RT-PCR using specific vldlr primers flanking the deduced O-linked sugar domain detected both mRNA transcript variants in ovaries in both previtellogenic and vitellogenic stages (Fig. 4B). However, there was a higher proportion of the smaller, 291-bp, amplified band, corresponding to the VLDLR– isoform, than of the 351-bp amplified band, corresponding to the additional 60-bp O-linked sugar domain of the VLDLR+ isoform (Fig. 4B). These two variants were also detected to a lesser extent in heart and adipose tissue, whereas the short transcript variant was also found in trace amount in liver, kidneys, muscles, and testicles. Tissue expression of fabp11 and bactin transcripts was also investigated by RT-PCR and showed that the specific products were amplified from the total RNAs of all tissues examined (Fig. 4B). Transcript levels of fabp11 were remarkably high in liver, adipose tissue, and vitellogenic ovaries, whereas a small amount was amplified from previtellogenic ovaries, heart, kidneys, and muscles, and trace amount was found in testicles. In the conditions used, bactin transcript was highly amplified from ovaries, kidneys, testicles, and heart, and to a lesser extent from liver, adipose tissue, and muscles.

Cellular Localization of vldlr and fabp11 Transcripts in S. senegalensis Ovaries

Ovarian development in Senegalese sole was group synchronous, with two groups of oocytes being observed: one developing and the second in a previtellogenic, resting stage. In situ hybridization studies were conducted on serial sections at different ovarian developmental stages, using an antisense probe able to detect both VLDLR+ and VLDLR– splicing variants. A strong vldlr hybridization signal was localized in the cytoplasm and nucleoli of previtellogenic oocytes (Fig. 5, A, C, and E), whereas hybridization with a sense probe resulted in no signal (Fig. 5, B, D, and F). The staining signal was also absent in vitellogenic oocytes, as well as in the surrounding granulosa and theca layers (Fig. 5E).


Figure 05
View larger version (139K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 5 Cellular localization of vldlr transcripts in Solea senegalensis ovaries. In situ hybridization on ovarian histologic sections, of a previtellogenic (A–D; GSI: 1.5%; percentage of previtellogenic oocytes: 99%; percentage of atretic oocytes: 1%) or a vitellogenic (E and F; GSI: 11.3%; percentage of vitellogenic oocytes: 10%; percentage of atretic oocytes: 5%) female. The sections were hybridized with antisense (A, C, E) or sense (B, D, F) digoxigenin-labeled riboprobes recognizing both splicing variants. The hydridization signal is colored dark-blue to purple. No staining signal was observed using the sense probe. Transcripts are localized in high concentrations in the cytoplasm (c) and nucleoli (n) of previtellogenic oocytes (p). a, atretic oocyte; nu, nucleus; v, vitellogenic oocyte. Bar = 100 µm (A, B, E, F) and 16 µm (C and D).

The distribution of fabp11 mRNA in the ovaries revealed a hybridization signal in the cytoplasm of previtellogenic oocytes (Fig. 6, A, C, and E). A very strong signal was also observed in somatic cells of atretic follicles in both the previtellogenic (Fig. 6A) and vitellogenic (Fig. 6, C, E, G, and I) ovarian stages. Atretic follicles were characterized by the disintegration of the nucleus (Fig. 6E), zona radiata breakdown (Fig. 6G), and modification of the appearance of yolk globules (Fig. 6, E and G), as well as by an increase in the number and size of extra-oocyte follicular cells (Fig. 6, G and I), resulting in residual atretic follicles that strongly expressed fabp11 transcripts (Fig. 6, A and I). No staining signal was detected in vitellogenic oocytes (Fig. 6, C, E, G, and I) or in parallel sections treated with the sense probe (Fig. 6, B, D, F, H, and J).


Figure 06
View larger version (80K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 6 Cellular localization of fabp11 transcripts in S. senegalensis ovaries. In situ hybridization on ovarian histologic sections from a previtellogenic (A and B; the same animal as used in Fig. 5) or a vitellogenic (C–J; GSI: 4%; percentage of vitellogenic oocytes: 6%; percentage of atretic oocytes: 5%) female. The sections were hybridized with antisense (A, C, E, G, I) or sense (B, D, F, H, J) digoxigenin-labeled riboprobes. No staining signal was observed using the sense probe. A moderate hybridization signal is localized in the cytoplasm (c) of previtellogenic oocytes (p). High fabp11 transcript levels are detected in the somatic cells (sc) surrounding atretic oocytes (a). e, early vitellogenic oocyte; nu, nucleus; v, vitellogenic oocyte; zr, zona radiata. Bar = 250 µm (C, D), 100 µm (A, B, E, F), 50 µm (I, J), and 25 µm (G, H).

Differential Expression of vldlr, bactin, and fabp11 During Oogenesis as Evaluated by qPCR

The correlation model used for qPCR data quality control of nine representative samples revealed: first, a simple linear relationship for each gene and sample combination, with slopes not significantly different from –1; and second, very similar slopes for all three combinations of genes and samples that were not significantly different from each other (data not shown). This test confirmed the acceptability of the qPCR data and the absence of a sample dilution effect [49].

The expression level of bactin, vldlr, and fabp11 transcripts was determined by qPCR analysis of ovarian samples obtained from 16 females at different stages of gonad development. The level of bactin transcripts was initially determined with the intention of using it as a reference normalizing gene. However, it was later found that the quantity of transcripts fluctuated, depending on the oogenesis stage frequency profile. Expression of bactin had a significant positive correlation with the percentage of previtellogenic oocytes (data not shown) and a negative correlation with the percentage of vitellogenic oocytes (data not shown), thus hampering its use as a reference gene under these conditions. Other authors had already noticed that housekeeping genes are not always good as normalizing genes [50], and proposed other methods for normalizing qPCR or microarray data [51–53]. Consequently, in these experimental conditions, qPCR data (i.e., Ct converted into copy number through a dilution series standard curve) were normalized by the genomic RNA:DNA ratio. As assessed by visual counts on adjacent histologic ovarian sections, our results demonstrated a significant positive correlation between vldlr transcript levels and the percentage of previtellogenic oocytes (Fig. 7A), and a negative correlation with the percentage of vitellogenic oocytes (data not shown). In contrast, fabp11 transcript levels were not significantly affected by the percentage of previtellogenic (Fig. 7B) and vitellogenic (data not shown) oocytes in the samples.


Figure 07
View larger version (11K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 7 Relationship between the copy number of vldlr (A) or fabp11 (B) transcripts and the percentage of previtellogenic oocytes in S. senegalensis ovary. The percentage of oocytes in previtellogenesis was counted on histologic sections, and the amount of vldlr or fabp11 transcripts was quantified by real-time quantitative RT-PCR from 16 females. Transcript levels were normalized by the RNA:DNA ratio in the sample, and the number of copies of selected transcripts was expressed as copy number of transcript per nanogram of genomic DNA. The linear regression in A was: y = 1.47x – 67.54; r2 = 0.586; slope was significantly different from zero at P = 0.0005; and correlation between the two variables was significant, with nonparametric Spearman r = 0.521, P (two-tailed) = 0.039.

Probably due to the high amount of somatic cells around the atretic oocyte (Fig. 6, E, G, and I), normalization by the genomic RNA:DNA ratio did not give any strong correlation between vldlr or fabp11 transcript levels and the percentage of atretic follicles in histologic samples. However, bactin could be used as a reference gene for atresia, as no correlation was observed between bactin transcript levels and the percentage of atretic follicles in the ovary (data not shown). No change in vldlr expression was detected in relation to the percentage of atretic follicles (Fig. 8A). In contrast, fabp11 transcript levels were correlated significantly with the percentage of atretic follicles in ovarian histologic sections (Fig. 8B). It should be noted that in farmed Senegalese sole, atresia occurred at all stages of follicle development, but its prevalence was significantly correlated with the number of vitellogenic oocytes (data not shown).


Figure 08
View larger version (11K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 8 Relationship between the expression level of vldlr (A) or fabp11 (B) transcripts and the percentage of atretic oocytes in S. senegalensis ovary. The percentage of oocytes in atresia was counted on histologic sections, and the amount of vldlr or fabp11 transcripts was quantified by qPCR from 16 females. Transcript level of bactin was also quantified to use as a normalizing gene, and the results shown are the number of copies of vldlr or fabp11 transcripts divided by the number of copies of bactin transcripts. The linear regression in B was: y = 0.08x + 0.63; r2 = 0.334; slope was significantly different from zero at P = 0.019; and correlation between the two variables was significant, with nonparametric Spearman r = 0.755, P (two-tailed) = 0.0007.

DISCUSSION

Oocyte growth, particularly in oviparous species, is characterized by the intense deposition of products (e.g., RNAs, proteins, lipids, vitamins, and hormones) that enable the subsequent development into a viable embryo. The avian VLDLR, also termed LDLR relative with 8 binding repeats (LR8) or VtgR, is a receptor of VLDL and of Vtg, as documented by both biochemical and genetic evidence in chicken [54, 55]. These lipoproteins are selectively sequestered by growing oocytes due to receptor-mediated endocytosis [55, 56]. The overall modular structure of mammalian VLDLR is virtually superimposable with that of human LDLR, except that its ligand-binding domain contains eight LA repeats rather than seven [57]. The vertebrate VLDLR exists in variant forms arising from differential splicing. The longer and predominant splice form in mammalian tissues contains an additional exon specifying an O-linked sugar domain similar to that always present in LDLRs (28–30 amino acids long). Chicken oocytes, on the other hand, express a shorter variant, named LR8–, coding a receptor without the O-linked sugars, whereas the somatic cells and tissues, particularly the granulosa cells surrounding the oocytes, express predominantly LR8+ [55, 58, 59]. The function of the O-linked sugar domain is unclear, although it has been suggested to be responsible for controlling receptor recycling and degradation [60, 61].

VLDLRs of a few teleost fish species were previously characterized and showed a very high degree of amino acid identity to Xenopus [62] or chicken [54] VLDLR sequences. Coho salmon (Oncorhynchus kisutch) VLDLR was identified by ligand blotting and showed cross-reactivity with the chicken and Xenopus systems, both in terms of ligand recognition and immunoreactivity. In addition, its ligand-binding activity is inhibited by the polyanionic drug suramin [63], a property also demonstrated by other receptors of the lipoprotein receptor superfamily in oviparous animals [23]. The biochemical characterization of VLDLR from white perch revealed a specific and saturable binding of autologous Vtg to extracts of ovarian membranes displaceable by chicken egg yolk VLDL and having sensitivity to suramin [64]. Molecular cloning of VLDLR cDNA from rainbow trout resulted in the characterization of a 97-kDa protein containing a ligand-binding domain with eight LA repeats, lacking an O-linked sugar domain, and putatively endocytically active via an FDNPVY motif in the cytoplasmic domain [38]. The molecular characterization of a homologous receptor in both white perch [64, 65] and tilapia [66] demonstrated, as in rainbow trout, the presence of an ovarian lipoprotein receptor with eight LA repeats, lacking an O-linked sugar domain, and highly expressed in the ovaries, as previously demonstrated in chicken. A second cDNA, differing by a stretch of 105 nucleotides encoding an O-linked sugar domain, has been identified in rainbow trout and was expressed, in addition to the ovary, in muscles, liver, spleen, heart, and intestine [67]. PCR-based evidence of the expression in several tissues of the two splice variants lacking or containing 20 residues in the region normally corresponding to an O-linked sugar domain was obtained in tilapia [66]. Consistent with these findings, our data demonstrated the presence, in Senegalese sole ovaries, of two VLDLR isoforms with high sequence similarity to human VLDLR and chicken VLDLR/VtgR/LR8, one containing the O-linked sugar domain and the other not containing it. There was a higher proportion of the shorter transcript variant in the ovaries, a variant that was detected in low amounts in adipose tissue, heart, liver, kidneys, muscles, and testicles, whereas the longer variant was also significantly found in ovaries, adipose tissue, and heart. The isoform with a putative O-linked sugar domain found in ovarian and extraovarian tissues might contribute to the clearance of VLDL, a lipoprotein class containing apolipoprotein B (apoB) in teleost fish [68, 69], similarly to tetrapod vertebrate species, and present at high plasma concentrations in previtellogenic and early vitellogenic stages in rainbow trout [70]. The ligand-binding properties of the LA repeats of the tilapia VLDLR were tested in a semiquantitative yeast two-hybrid protein-protein interaction system that revealed the critical properties of the first three LA repeats in the Vtg binding region containing a motif known to be homologous to positively charged LR-binding motifs in mammalian apoB-100 and apolipoprotein E (apoE) [66]. These LA clusters are thought to participate in the binding of lipoprotein(s) via positively charged residues on apoB-100 or apoE [22]. The lack of the O-linked sugar domain did not affect the binding of the receptor to its ligands [66]. This receptor-binding domain was also detected by sequence homology in teleost fish apoB [71] and apoE [72, 73], the latest apolipoprotein not being detected in birds [23].

In situ hybridization studies performed on ovarian sections of Senegalese sole showed a high vldlr transcript level during previtellogenesis, and qPCR data demonstrated a significant positive correlation between vldlr transcript level and percentage of previtellogenic oocytes. Expression of bactin also had a significant positive correlation with the percentage of previtellogenic oocytes, thus hampering its use as a reference gene under these conditions. The nucleoli storage of vldlr mRNA during previtellogenesis, the disappearance of the hybridization signal in vitellogenic oocyte, and the significant negative correlation between vldlr transcript levels and the percentage of vitellogenic oocytes indicate, as previously suggested in rainbow trout [32] and chicken [74], that VLDLR is translated predominantly at early stages of oocyte development and is stored inside the cytoplasm to be gradually mobilized and recycled to the oocyte surface after the appearance of Vtg in the plasma (i.e., during the vitellogenic growth phase). The very low level of vldlr mRNA at the end of folliculogenesis was supported by qPCR data obtained in white perch [65] and by the absence of such transcript from the transcriptome of zebrafish fully grown ovarian follicles as determined by serial analysis of gene expression [47]. Data obtained in the present study also revealed that vldlr transcript hybridization signal was not detected in atretic follicles nor their levels, assessed by qPCR, correlated with percentage of atresia. These results indicate that vldlr transcript level may be used as a precocious functional molecular marker of the number of oocytes recruited for vitellogenesis.

Follicle atresia in diverse vertebrate species has been shown to result from apoptotic cell death initiated within the granulosa cell population [7, 8], or it may originate from apoptosis initiated first in the oocyte [8, 75]. It has been demonstrated that fish oocytes produce factors that modulate follicular functions and that proteolysis through lysosomal enzymes of oocyte yolk proteins may lead to follicular atresia [12, 76] without a widespread follicular apoptosis [12]. There is a preliminary report of choriolysin expression during atresia of white perch, which might be related to degradation of the zona radiata [77]. As atresia advances, the yolk content of the atretic follicles is gradually digested and reabsorbed, resulting in the formation of corpora atretica and aggregates primarily composed of pigments and oxidized lipids [1, 4, 35]. A massive transfer of some yolk proteins and possibly lipids, combined with high-density lipoproteins in the bloodstream, has been demonstrated in rainbow trout [34] and chicken [78] during the course of follicular atresia.

FABPs are a group of low-molecular weight (14–15 kDa) cytoplasmic proteins belonging to the conserved multigene family of intracellular lipid-binding proteins. Together with two other families of ligand-binding proteins, lipocalins and avidins, FABPs form part of an overall structural superfamily: calycins [79]. In mammals, at least nine different FABP types have been identified, with tissue-specific distribution [25, 26, 80, 81] and distinct patterns of fatty acid interactions [82, 83]. They are generally named after the tissue in which they were discovered or are prominently expressed. However, their expression is not exclusive, and more than one FABP may be found in a given cell or tissue, so a numerical nomenclature has been introduced to distinguish among the various FABP genes [84]. An additional type (FABP10) was recently characterized in nonmammalian species, including teleost fish [26, 85]. Our BI analysis demonstrated that all FABP protein subfamilies found in teleost fish correspond to the human subfamilies, with the exception of a single FABP subfamily, clustered in a sister group of tetrapod FABP4/FABP5/FABP8/FABP9, which is found only in teleost fish and is named here FABP11. The S. senegelensis Fabp sequence characterized in this study is part of this newly defined FABP11 subfamily, which also includes sequences previously characterized from Antartic teleost fish species [86].

FABPs are ubiquitously expressed in vertebrate and nonvertebrate tissues, with distinct expression patterns for individual FABPs [25, 87]. The primary role of all FABP family members is to regulate fatty acid uptake and intracellular transport. Other known functions are targeting fatty acids to specific metabolic pathways and involvement in regulating gene expression and cell growth [25, 26]. Rat ovaries express FABP2 and FABP3, as demonstrated by their immunohistochemical localization in interstitial cells of the theca interna [27], in somatic cells during postnatal development and in immature ovaries treated with gonadotropins [28]. In this species, fabp3 expression was downregulated and fabp5 expression was upregulated in theca cells of the rat ovary after human chorionic gonadotropin administration [31]. Immunodetection performed on mice ovaries revealed FABP3 in interstitial gland cells and theca cells [29], FABP4 in apoptotic granulosa cells of atretic antral follicles [30], and FABP5 in a specific population of ovarian macrophages in advanced atretic follicles [29].

It has been shown that zebrafish fabp3 mRNA was abundant and restricted to the ooplasm of previtellogenenic oocytes [33], whereas a low level of this transcript was detected in the transcriptome of fully grown ovarian follicles [47]. Our data indicated that sole fabp11 transcript was highly expressed in the ovaries, liver, and adipose tissue, and to a lower extent in heart, muscles, kidneys, and testicles. In situ hybridization analysis demonstrated that sole fabp11 transcript is expressed in previtellogenic oocytes, whereas no hybridization signal was detected in the larger oocytes. However, contrary to vldlr mRNA, a very strong fabp11 hybridization signal was detected in the somatic layers of atretic follicles. Furthermore, a significant linear-positive correlation between sole fabp11 transcript level and the percentage of atresia in ovarian samples was demonstrated by qPCR. This high transcript level has been identified in hypertrophied somatic cells during oocyte fragmentation, follicular cell invasion of the space occupied by the oocyte and elimination of cellular remnants, and degeneration of atretic follicles. These cells may have also a high content of cellular nucleic acid-binding protein, as previously shown in degenerating and atretic follicles of zebrafish ovary [88]. During the process of follicular atresia in mice, the invasive granulosa cells found between the zona pellucida and the oocyte may have a macrophage-like cell function for the elimination of oocytes from atretic follicles [89]. Additional studies are needed to determine the nature and origin of the numerous invasive fabp11-expressing cells found into the Senegalese sole atretic follicles. FABPs undoubtedly play an essential role in cellular fatty acid transport and utilization. In the vertebrate ovary, a differential role of FABP subtypes may be related to the changes in the efficient uptake, intracellular transport, and utilization of fatty acids accompanying folliculogenesis, atresia, and hormone synthesis. High expression levels of fabp11 transcripts in somatic cells of atretic follicles suggest the possible importance of lipid-metabolic processes and involvement of Fabp11 and/or its ligand fatty acids in the process of follicular atresia.

In conclusion, the present study has shown that the quantification of vldlr and fabp11 transcripts by qPCR may allow, through linear regression analysis, determination of the percentage of oocytes recruited for vitellogenesis and those undergoing atresia. These markers may undoubtedly help to identify factors that may be involved in the regulation of follicular growth and degeneration in the teleost fish ovary and would help improve breeding conditions to reduce stress leading to atresia in female ovaries, as well as improve the understanding of the reproductive biology of reared stocks.

ACKNOWLEDGMENTS

We wish to thank Anja Knoll-Gellida for advice. Fish maintenance by Josep Ll. Celades and Joaquín Canoura is gratefully acknowledged.

FOOTNOTES

1Supported by the French Ministry of Research and Education to P.J.B, and by the Junta Asesora de Cultivos Marinos (JACUMAR, Spain) and Reference Center in Aquaculture (Spain) to J.C. M.J.A. was supported by a predoctoral fellowship from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA, Spain). S.M. was supported by a postdoctoral fellowship from the French government. Back

Correspondence: 2Patrick J. Babin, Génomique et Physiologie des Poissons, Université Bordeaux 1, UMR NuAGe, Avenue des Facultés, Bât. B2, 33405 Talence Cedex, France. FAX: 33 540008915; e-mail: p.babin{at}gpp.u-bordeaux1.fr

Received: 16 March 2007.

First decision: 16 April 2007.

Accepted: 25 May 2007.

REFERENCES

  1. Saidapur SK. Follicular atresia in the ovaries of nonmammalian vertebrates Int Rev Cytol 1978 54225–244[Medline]
  2. Fish Physiology, vol. IX, chapter 6. Reproduction, Part A: Endocrine Tissues and Hormones Nagahama Y. The functional morphology of the teleost gonad 1983 Academic Press223–275 In:
  3. Nagahama Y. Endocrine regulation of gametogenesis in fish Int J Dev Biol 1994 38217–229[Medline]
  4. Monographs in Developmental Biology, vol. 18, chapter VIII Guraya SS. The cell and molecular biology of fish oogenesis 1986 Karger, Basel169–180 In:
  5. Kling D. Total atresia of the ovaries of Tilapia leucosticta (Cichlidae) after intoxication with the insecticide Lebaycid Experientia 1981 3773–74[CrossRef][Medline]
  6. Van Den Belt K, Wester PW, Van Der Ven LTM, Verheyen R, Witters H. Effects of ethynylestradiol on the reproductive physiology in zebrafish (Danio rerio): time dependency and reversibility Environ Toxicol Chem 2002 21767–775[CrossRef][Medline]
  7. Kaipia A and Hsueh AJ. Regulation of ovarian follicle atresia Annu Rev Physiol 1997 59349–363[CrossRef][Medline]
  8. Morita Y, Perez GI, Maravei DV, Tilly KI, Tilly JL. Targeted expression of Bcl-2 in mouse oocytes inhibits ovarian follicle atresia and prevents spontaneous and chemotherapy-induced oocyte apoptosis in vitro Mol Endocrinol 1999 13841–850[Abstract/Free Full Text]
  9. Matova N and Cooley L. Comparative aspects of animal oogenesis Dev Biol 2001 231291–320[CrossRef][Medline]
  10. Hussein MR. Apoptosis in the ovary: molecular mechanisms Hum Reprod Update 2005 11162–177[Abstract/Free Full Text]
  11. Janz DM and Van Der Kraak G. Suppression of apoptosis by gonadotropin, 17beta-estradiol, and epidermal growth factor in rainbow trout preovulatory ovarian follicles Gen Comp Endocrinol 1997 105186–193[CrossRef][Medline]
  12. Wood AW and Van Der Kraak GJ. Apoptosis and ovarian function: novel perspectives from the teleosts Biol Reprod 2001 64264–271[Abstract/Free Full Text]
  13. Janz DM, McMaster ME, Munkittrick KR, Van der Kraak G. Elevated ovarian follicular apoptosis and heat shock protein-70 expression in white sucker exposed to bleached kraft pulp mill effluent Toxicol Appl Pharmacol 1997 147391–398[CrossRef][Medline]
  14. Uchida D, Yamashita M, Kitano T, Iguchi T. An aromatase inhibitor or high water temperature induce oocyte apoptosis and depletion of P450 aromatase activity in the gonads of genetic female zebrafish during sex-reversal Comp Biochem Physiol A Mol Integr Physiol 2004 13711–20[CrossRef][Medline]
  15. Wood AW and Van Der Kraak G. Yolk proteolysis in rainbow trout oocytes after serum-free culture: evidence for a novel biochemical mechanism of atresia in oviparous vertebrates Mol Reprod Dev 2003 65219–227[CrossRef][Medline]
  16. Andreu-Vieyra CV and Habibi HR. Factors controlling ovarian apoptosis Can J Physiol Pharmacol 2000 781003–1012[CrossRef][Medline]
  17. Perspective in Comparative Endocrinology: Unity and Diversity Habibi HR, Andreu-Vieyra C, Mirhadi E. Functional significance of gonadal gonadotropin-releasing hormone 2001Bologna, Italy Monduzzi Editore959–968 In:
  18. Developmental Biology Wallace RA. Vitellogenesis and oocyte growth in non-mammalian vertebrates 1985New York Plenum Press127–177 In:
  19. Fish Physiology, vol. 11A Mommsen PT and Walsh PJ. Vitellogenesis and oocyte assembly 1988New York Academic Press347–406 In:
  20. Tyler CR and Sumpter JP. Oocyte growth and development in teleosts Rev Fish Biol Fish 1996 6289–318
  21. Wiegand MD. Composition, accumulation and utilization of yolk lipids in teleost fish Rev Fish Biol Fish 1996 6259–286
  22. Jeon H and Blacklow SC. Structure and physiologic function of the low-density lipoprotein receptor Annu Rev Biochem 2005 74535–562[CrossRef][Medline]
  23. Schneider WJ, Nimpf J, Brandes C, Drexler M. Low-density lipoprotein receptor family: genetics, function, and evolution Curr Atheroscler Rep 1999 1115–122[Medline]
  24. Schneider WJ and Nimpf J. LDL receptor relatives at the crossroad of endocytosis and signaling Cell Mol Life Sci 2003 60892–903[CrossRef][Medline]
  25. Zimmerman AW and Veerkamp JH. New insights into the structure and function of fatty acid-binding proteins Cell Mol Life Sci 2002 591096–1116[CrossRef][Medline]
  26. Haunerland NH and Spener F. Fatty acid-binding proteins—insights from genetic manipulations Prog Lipid Res 2004 43328–349[CrossRef][Medline]
  27. Watanabe M, Ono T, Kondo H. Immunohistochemical studies on the localisation and ontogeny of heart fatty acid binding protein in the rat J Anat 1991 17481–95[Medline]
  28. Iseki I, Amano O, Fujii H, Kanda T, Ono T. Inmunohistochemical localization of two types of fatty acid-binding protein in rat during postnatal development and in immature rat ovaries treated with gonadotropins Anat Rec 1995 241235–243[CrossRef][Medline]
  29. Nourani MR, Owada Y, Kitanaka N, Abdelwahab SA, Iwasa H, Sakagami H, Spener F, Kondo H. Localization of epidermal-type fatty acid binding protein in macrophages in advanced atretic follicles of adult mice J Mol Hist 2005 36391–400[CrossRef]
  30. Nourani MR, Owada Y, Kitanaka N, Sakagami H, Hoshi H, Iwasa H, Spener F, Kondo H. Occurence of inmunoreactivity for adipocyte-type fatty acid binding protein in degenerating granulosa cells in atretic antral follicles of mouse ovary J Mol Hist 2005 36491–497[CrossRef]
  31. Leo CP, Pisarska MD, Hsueh AJ. DNA array analysis of changes in preovulatory gene expression in the rat ovary Biol Reprod 2001 65269–276[Abstract/Free Full Text]
  32. Perazzolo LM, Coward K, Davail B, Normand E, Tyler CR, Pakdel F, Schneider WJ, Le Menn F. Expression and localization of messenger ribonucleic acid for the vitellogenin receptor in ovarian follicles throughout oogenesis in the rainbow trout, Oncorhynchus mykiss Biol Reprod 1999 601057–1068[Abstract/Free Full Text]
  33. Liu RZ, Denovan-Wright EM, Wright JM. Structure, linkage mapping and expression of the heart-type fatty acid-binding protein gene (fabp3) from zebrafish (Danio rerio) Eur J Biochem 2003 2703223–3234[Medline]
  34. Babin PJ. Apolipoproteins and the association of egg yolk proteins with plasma high density lipoproteins after ovulation and follicular atresia in the rainbow trout (Salmo gairdneri) J Biol Chem 1987 2824290–4296
  35. Blazer VS. Histopathological assessment of gonadal tissue in wild fishes Fish Physiol Biochem 2002 2685–101[CrossRef]
  36. Agulleiro MJ, Anguis V, Cañavate JP, Martínez-Rodríguez G, Mylonas CC, Cerdà J. Induction of spawning of captive-reared Senegal sole (Solea senegalensis) using different administration methods for gonadotropin-releasing hormone agonist Aquaculture 2006 257511–524[CrossRef]
  37. García-López A, Couto E, Canario AVM, Sarasquete C, Martínez-Rodríguez G. Ovarian development and plasma sex steroid levels in cultured female Senegalese sole Solea senegalensis Comp Biochem Physiol A Mol Integr Physiol 2007 146342–354[CrossRef][Medline]
  38. Davail B, Pakdel F, Bujo H, Perazzolo LM, Waclawek M, Schneider WJ, Le Menn F. Evolution of oogenesis: the receptor for vitellogenin from the rainbow trout J Lipid Res 1998 391929–1937[Abstract/Free Full Text]
  39. André M, Ando S, Ballagny C, Durliat M, Poupard G, Briançon C, Babin PJ. Intestinal fatty acid binding protein gene expression reveals the cephalocaudal pattering during zebrafish gut morphogenesis Int J Dev Biol 2000 44249–252[Medline]
  40. Marza E, Barthe C, Andre M, Villeneuve L, Helou C, Babin PJ. Developmental expression and nutritional regulation of a zebrafish gene homologous to mammalian microsomal triglyceride transfer protein large subunit Dev Dyn 2005 232506–518[CrossRef][Medline]
  41. Avarre JC, Lubzens E, Babin PJ. Apolipocrustacein, formerly vitellogenin, is the major egg yolk precursor protein in decapod crustaceans and is homologous to insect apolipophorin II/I and vertebrate apolipoprotein B BMC Evol Biol 2007 73[CrossRef][Medline]
  42. Guindon S, Lethiec F, Duroux P, Gascuel O. PHYML Online-a web server for fast maximum likelihood-based phylogenetic inference Nucleic Acids Res 2005 33W557–W559[Abstract/Free Full Text]
  43. Abascal F, Zardoya R, Posada D. ProtTest: selection of best-fit models of protein evolution Bioinformatics 2005 212104–2105[Abstract/Free Full Text]
  44. Ronquist F and Huelsenbeck JP. MrBayes 3: Bayesian phylogenetic inference under mixed models Bioinformatics 2003 191572–1574[Abstract/Free Full Text]
  45. Morais S, Knoll-Gellida A, André M, Barthe C, Babin PJ. Conserved expression of alternative splicing variants of peroxisomal acyl-CoA oxidase 1 in vertebrates and developmental and nutritional regulation in fish Physiol Genomics 2007 28239–252[Abstract/Free Full Text]
  46. Bogerd J, Babin PJ, Kooiman FP, André M, Ballagny C, Van Marrewijk WJA, Van der Horst D. Molecular characterization and gene expression in the eye of the apolipophorin II/I precursor from Locusta migratoria J Comp Neurol 2000 427546–558[CrossRef][Medline]
  47. Knoll-Gellida A, André M, Gattegno T, Forgue J, Admon A, Babin PJ. Molecular phenotype of zebrafish ovarian follicle by serial analysis of gene expression and proteomic profiling, and comparison with the transcriptomes of other animals BMC Genomics 2006 746[CrossRef][Medline]
  48. Fernandez-Trujillo MA, Porta J, Borrego JJ, Alonso MC, Alvarez MC, Bejar J. Cloning and expression analysis of Mx cDNA from Senegales sole (Solea senegalensis) Fish Shellfish Immunol 2006 21577–582[CrossRef][Medline]
  49. Yuan JS, Reed A, Chen F, Steward CN Jr. Statistical analysis of real-time PCR data BMC Bioinformatics 2006 785[CrossRef][Medline]
  50. Lee PD, Sladek R, Greenwood CMT, Hudson TJ. Control genes and variability: absence of ubiquitous reference transcripts in diverse mammalian expression studies Genome Res 2002 12292–297[Abstract/Free Full Text]
  51. Hill AA, Brown EL, Whitley MZ, Tucker-Kellogg G, Hunter CP, Slonim DK. Evaluation of normalization procedures for oligonucleotides array data based on spiked cRNA controls Genome Biol 2001 20055.1–0055.13
  52. Van de Peppel J, Kemmeren P, van Bakel H, Radonjic M, van Leenen D, Holstege FC. Monitoring global messenger RNA changes in externally controlled microarrays experiments EMBO Rep 2003 4387–393[CrossRef][Medline]
  53. Kanno J, Aisaki K, Igarashi K, Nakatsu N, Ono A, Kodama Y, Nagao T. "Per cell" normalization method for mRNA measurement by quantitative PCR and microarrays BMC Genomics 2006 764[CrossRef][Medline]
  54. Bujo H, Hermann M, Kaderli MO, Jacobsen L, Sugawara S, Nimpf J, Yamamoto T, Schneider WJ. Chicken oocyte growth is mediated by an eight ligand binding repeat member of the LDL receptor family EMBO J 1994 135165–5175[Medline]
  55. Bujo H, Yamamoto T, Hayashi K, Hermann M, Nimpf J, Schneider WJ. Mutant oocytic low density lipoprotein receptor gene family member causes atherosclerosis and female sterility Proc Natl Acad Sci U S A 1995 929905–9999[Abstract/Free Full Text]
  56. Perry MM and Gilbert AB. Yolk transport in the ovarian follicle of the hen (Gallus domesticus): lipoprotein-like particles at the periphery of the oocyte in the rapid growth phase J Cell Sci 1979 39257–272[Abstract/Free Full Text]
  57. Takahashi S, Kawarabayasi Y, Nakai T, Sakai J, Yamamoto T. Rabbit very low density lipoprotein receptor: a low density lipoprotein receptor-like protein with distinct ligand specificity Proc Natl Acad Sci U S A 1992 899252–9256[Abstract/Free Full Text]
  58. Bujo H, Lindstedt KA, Hermann M, Dalmau LM, Nimpf J, Schneider WJ. Chicken oocytes and somatic cells express different splice variants of a multifunctional receptor J Biol Chem 1995 27023546–23551[Abstract/Free Full Text]
  59. Nimpf J and Schneider WJ. The VLDL receptor: an LDL receptor relative with eight ligand binding repeats, LR8 Atherosclerosis 1998 141191–202[CrossRef][Medline]
  60. Iijima H, Miyazawa M, Sakai J, Magoori K, Ito MR, Suzuki H, Nose M, Kawarabayasi Y, Yamamoto TT. Expression and characterization of a very low-density lipoprotein receptor variant lacking the O-linked sugar region generated by alternative splicing J Biochem 1998 124747–755[Abstract/Free Full Text]
  61. Magrané J, Casaroli-Marano RP, Reina M, Gåfvels M, Vilaró S. The role of O-linked sugars in determining the very low-density lipoprotein receptor stability or release from the cell FEBS Lett 1999 45156–62[CrossRef][Medline]
  62. Okabayashi K, Shoji H, Nakamura T, Hashimoto O, Asashima M, Sugino H. cDNA cloning and expression of the Xenopus laevis vitellogenin receptor Biochem Biophys Res Commun 1996 224406–413[CrossRef][Medline]
  63. Stifani S, Le Menn F, Rodriguez JN, Schneider WJ. Regulation of oogenesis: the piscine receptor for vitellogenin Biochim Biophys Acta 1990 1045271–279[Medline]
  64. Tao Y, Berlinsky DL, Sullivan CV. Characterization of a vitellogenin receptor in white perch (Morone americana) Biol Reprod 1996 55646–656[Abstract]
  65. Hiramatsu N, Chapman RW, Lindzey JK, Haynes MR, Sullivan CV. Molecular characterization and expression of vitellogenin receptor from white perch (Morone americana) Biol Reprod 2004 701720–1730[Abstract/Free Full Text]
  66. Li A, Sadasivam M, Ding JL. Receptor-ligand interaction between vitellogenin receptor (VtGR) and vitellogenin (Vtg), implications on low-density lipoprotein receptor and apolipoprotein B/E J Biol Chem 2003 2782799–2806[Abstract/Free Full Text]
  67. Prat F, Coward K, Sumpter JP, Tyler CR. Molecular characterization and expression of two ovarian lipoprotein receptors in the Rainbow Trout, Oncorhynchus mykiss Biol Reprod 1998 581146–1153[Abstract/Free Full Text]
  68. Babin PJ. Plasma lipoproteins and apolipoproteins distribution as a function of density in the rainbow trout (Salmo gairdneri) Biochem J 1987 246425–429[Medline]
  69. Babin PJ and Vernier JM. Plasma lipoproteins in fish: a review J Lipid Res 1989 30467–489[Medline]
  70. Wallaert C and Babin PJ. Age-related, sex-related and seasonal changes of plasma lipoprotein concentrations in trout J Lipid Res 1994 351619–1633[Abstract]
  71. Babin PJ, Deryckere F, Gannon F. Presence of an extended duplication in the low density lipoprotein receptor-binding domain of apolipoprotein B. Cloning and characterization of the domain in salmon Eur J Biochem 1995 23045–51[Medline]
  72. Durliat M, André M, Babin PJ. Conserved protein motifs and structural organization of a fish gene homologous to mammalian apolipoprotein E Eur J Biochem 2000 267549–559[Medline]
  73. Poupard G, André M, Durliat M, Ballagny C, Bœuf G, Babin PJ. Apolipoprotein E gene expression correlates with endogenous lipid nutrition and yolk syncytial layer lipoprotein synthesis during fish development Cell Tissue Res 2000 300251–261[CrossRef][Medline]
  74. Shen X, Steyrer E, Retzek H, Sanders EJ, Schneider WJ. Chicken oocyte growth: receptor-mediated yolk deposition Cell Tissue Res 1993 272459–471[CrossRef][Medline]
  75. Eppig JJ. Oocyte control of ovarian follicular development and function in mammals Reproduction 2001 122829–838[Abstract]
  76. Wang Y and Ge W. Spatial expression patterns of activin and its signaling system in the zebrafish ovarian follicle: evidence for paracrine action of activin on the oocytes Biol Reprod 2003 691998–2006[Abstract/Free Full Text]
  77. Donato DM, Hiramatsu N, Arey KM, Hiramatsu K, Donato DM, Kennedy AM, Morton CL, Hara A, Sullivan CV. Atresia in temperate basses: cloning of hatching enzyme (choriolysin) homologues from atretic ovaries Fish Physiol Biochem 2003 28329–330[CrossRef]
  78. Walzem RL, Davis PA, Hansen RJ. Overfeeding increases very low density lipoprotein diameter and causes the appearance of a unique lipoprotein particle in association with failed yolk deposition J Lipid Res 1994 351354–1366[Abstract]
  79. Flower DR. The lipocalin protein family: structure and evolution Biochem J 1996 3181–14[Medline]
  80. Storch J and Thumser AEA. The fatty acid transport function of fatty acid-binding proteins Biochim Biophys Acta 2000 148628–44[Medline]
  81. Chmurzynska A. The multigene family of fatty acid-binding proteins (FABPs): function, structure and polymorphism J Appl Genet 2006 4739–48[Medline]
  82. Richieri GV, Ogata RT, Zimmerman AW, Veerkamp JH, Kleinfeld AM. Fatty acid binding proteins from different tissues show distinct patterns of fatty acid interactions Biochem 2000 397197–7204[CrossRef][Medline]
  83. Hanhoff T, Lücke C, Spener F. Insights into binding of fatty acids by fatty acid binding proteins Mol Cell Biochem 2002 23945–54[CrossRef][Medline]
  84. Hertzel AV and Bernlohr DA. The mammalian fatty acid-binding protein multigene family: molecular and genetic insights into function Trends Endocrinol Metab 2000 11175–180[CrossRef][Medline]
  85. Sharma MK, Liu RZ, Thisse C, Thisse B, Denovan-Wright EM, Wright JM. Hierarchical subfunctionalization of fabp1a, fabp1b and fabp10 tissue-specific expression may account for retention of these duplicated genes in the zebrafish (Danio rerio) genome FEBS J 2006 2733216–3229[CrossRef][Medline]
  86. Vayda ME, Londraville RL, Cashon RE, Costello L, Sidell BD. Two distinct types of fatty acid-binding protein are expressed in heart ventricle of Antartic teleost fishes Biochem J 1998 330375–382[Medline]
  87. Esteves A and Ehrlich R. Invertebrate intracellular fatty acid binding proteins Comp Biochem Physiol C Toxicol Pharmacol 2006 142262–274[CrossRef][Medline]
  88. Liu JX and Gui JF. Expression pattern and developmental behaviour of cellular nucleic acid-binding protein (CNBP) during folliculogenesis and oogenesis in fish Gene 2005 356181–192[CrossRef][Medline]
  89. Kamo A, Araki Y, Maeda K, Watanabe H. Characteristics of invasive cells found in between zona pellucida and oocyte during follicular atresia in mice Zygote 2004 12269–276[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow [Supplemental Figure]
Right arrow All Versions of this Article:
77/3/504    most recent
biolreprod.107.061598v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Agulleiro, M. J
Right arrow Articles by Babin, P. J
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Agulleiro, M. J
Right arrow Articles by Babin, P. J
Agricola
Right arrow Articles by Agulleiro, M. J
Right arrow Articles by Babin, P. J

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS