2014 journal article
Lrp13 is a novel vertebrate lipoprotein receptor that binds vitellogenins in teleost fishes
JOURNAL OF LIPID RESEARCH, 55(11), 2287–2295.
open access via www.jlr.org (publisher PDF)
Transcripts encoding a novel member of the lipoprotein receptor superfamily, termed LDL receptor-related protein (Lrp)13, were sequenced from striped bass (Morone saxatilis) and white perch (Morone americana) ovaries. Receptor proteins were purified from perch ovary membranes by protein-affinity chromatography employing an immobilized mixture of vitellogenins Aa and Ab. RT-PCR revealed lrp13 to be predominantly expressed in striped bass ovary, and in situ hybridization detected lrp13 transcripts in the ooplasm of early secondary growth oocytes. Quantitative RT-PCR confirmed peak lrp13 expression in the ovary during early secondary growth. Quantitative mass spectrometry revealed peak Lrp13 protein levels in striped bass ovary during late-vitellogenesis, and immunohistochemistry localized Lrp13 to the oolemma and zona radiata of vitellogenic oocytes. Previously unreported orthologs of lrp13 were identified in genome sequences of fishes, chicken (Gallus gallus), mouse (Mus musculus), and dog (Canis lupus familiaris). Zebrafish (Danio rerio) and Nile tilapia (Oreochromis niloticus) lrp13 loci are discrete and share genomic synteny. The Lrp13 appears to function as a vitellogenin receptor and may be an important mediator of yolk formation in fishes and other oviparous vertebrates. The presence of lrp13 orthologs in mammals suggests that this lipoprotein receptor is widely distributed among vertebrates, where it may generally play a role in lipoprotein metabolism. Transcripts encoding a novel member of the lipoprotein receptor superfamily, termed LDL receptor-related protein (Lrp)13, were sequenced from striped bass (Morone saxatilis) and white perch (Morone americana) ovaries. Receptor proteins were purified from perch ovary membranes by protein-affinity chromatography employing an immobilized mixture of vitellogenins Aa and Ab. RT-PCR revealed lrp13 to be predominantly expressed in striped bass ovary, and in situ hybridization detected lrp13 transcripts in the ooplasm of early secondary growth oocytes. Quantitative RT-PCR confirmed peak lrp13 expression in the ovary during early secondary growth. Quantitative mass spectrometry revealed peak Lrp13 protein levels in striped bass ovary during late-vitellogenesis, and immunohistochemistry localized Lrp13 to the oolemma and zona radiata of vitellogenic oocytes. Previously unreported orthologs of lrp13 were identified in genome sequences of fishes, chicken (Gallus gallus), mouse (Mus musculus), and dog (Canis lupus familiaris). Zebrafish (Danio rerio) and Nile tilapia (Oreochromis niloticus) lrp13 loci are discrete and share genomic synteny. The Lrp13 appears to function as a vitellogenin receptor and may be an important mediator of yolk formation in fishes and other oviparous vertebrates. The presence of lrp13 orthologs in mammals suggests that this lipoprotein receptor is widely distributed among vertebrates, where it may generally play a role in lipoprotein metabolism. The LDL receptor (LDLR) gene family is comprised of different genes encoding membrane receptors involved in endocytosis of a variety of ligands, most notably plasma lipoproteins. Characterized members of this family in vertebrates include LDLR (1Yamamoto T. Davis C.G. Brown M.S. Schneider W.J. Casey M.L. Goldstein J.L. Russel D.W. The human LDL receptor: a cysteine-rich protein with multiple Alu sequences in its mRNA.Cell. 1984; 39: 27-38Abstract Full Text PDF PubMed Scopus (979) Google Scholar), LDLR-related protein (LRP)1 (2Herz J. Hamann U. Rogne S. Myklebost O. Gausepohl H. Stanley K.K. 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Evolution of oogenesis: the receptor for vitellogenin from the rainbow trout.J. Lipid Res. 1998; 39: 1929-1937Abstract Full Text Full Text PDF PubMed Google Scholar). These receptors typically consist of unique configurations of epidermal growth factor precursor, class b YWxD (LDLb), and O-linked sugar domains, which define their identities, and class A ligand binding (LDLa) repeats, which determine their particular ligand specificities. Exceptional receptors also contain complement C1r/C1s, Uegf, Bmp1 (CUB) domains (i.e., LRP3, LRP10, LRP12) or motif at the N terminus with seven cysteines (MANEC) and polycystic kidney disease (PKD) domains (i.e., LRP11). With the exception of LRP4, LDLR family receptors are type I membrane proteins and all members contain transmembrane and cytoplasmic domains. Acanthomorph fishes express three distinct lipoprotein yolk precursors, vitellogenins (Vtgs) (VtgAa, VtgAb, and VtgC) (15Finn R.N. Kristoffersen B.A. Vertebrate vitellogenin gene duplication in relation to the "3R hypothesis": correlation to the pelagic egg and the oceanic radiation of teleosts.PLoS ONE. 2007; 2: e169Crossref PubMed Scopus (173) Google Scholar, 16Reading B.J. Hiramatsu N. Sawaguchi S. Matsubara T. Hara A. Lively M.O. Sullivan C.V. Conserved and variant molecular and functional features of multiple egg yolk precursor proteins (vitellogenins) in white perch (Morone americana) and other teleosts.Mar. Biotechnol. (NY). 2009; 11: 169-187Crossref PubMed Scopus (67) Google Scholar). These Vtgs are produced by the liver, released into the circulatory system, and taken up specifically by growing oocytes via receptor-mediated endocytosis. We discovered four Vtgr proteins in white perch (Morone americana) ovary: a receptor greater than 212 kDa that binds only VtgAa (VtgAar), two receptors (116 kDa and 110.5 kDa) that preferentially bind VtgAb (VtgAbr), and a 150 kDa putative LDLR (pLDLR) that weakly and indiscriminately binds both VtgAa and VtgAb (17Reading B.J. Hiramatsu N. Sullivan C.V. Disparate binding of three types of vitellogenin to multiple forms of vitellogenin receptor in white perch.Biol. Reprod. 2011; 84: 392-399Crossref PubMed Scopus (38) Google Scholar). The VtgC does not bind ovary Vtgr proteins in this species. We reported the molecular identity of a white perch Vtgr that is orthologous to mammalian VLDLR (18Hiramatsu N. Chapman R.W. Lindzey J.K. Haynes M.R. Sullivan C.V. Molecular characterization and expression of vitellogenin receptor from white perch (Morone americana).Biol. Reprod. 2004; 70: 1720-1730Crossref PubMed Scopus (66) Google Scholar). This Vtgr is termed "Lr8−", because it is a spliced variant gene transcript of vldlr that does not encode the O-linked sugar domain, as is characteristic of this form of Vldlr in fishes and chickens (19Bujo H. Lindstedt K.A. Hermann M. Dalmau L.M. Nimpf J. Schneider W.J. Chicken oocytes and somatic cells express different splice variants of a multifunctional receptor.J. Biol. Chem. 1995; 270: 23546-23551Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 20Prat F. Coward K. Sumpter J.P. Tyler C.R. Molecular characterization and expression of two ovarian lipoprotein receptors in the rainbow trout, Oncorhynchus mykiss.Biol. Reprod. 1998; 58: 1146-1153Crossref PubMed Scopus (67) Google Scholar). Herein, we refer to these Vldlr forms as Lr8+ and Lr8−, based on the presence or absence of the O-linked sugar domain, respectively. We have suggested that white perch Lr8− corresponds to one or both VtgAbr proteins based on the predicted molecular mass of the protein and on prior reports of fish and chicken Lr8− (17Reading B.J. Hiramatsu N. Sullivan C.V. Disparate binding of three types of vitellogenin to multiple forms of vitellogenin receptor in white perch.Biol. Reprod. 2011; 84: 392-399Crossref PubMed Scopus (38) Google Scholar). However, the molecular identity of VtgAar remains unclear. Here, we describe the structure, expression, subcellular localization, and Vtg-binding properties of a novel lipoprotein receptor named 'Lrp13' that corresponds to VtgAar and show that the Lr8− corresponds to the VtgAbr. All experiments were conducted according to the Guide for the Care and Use of Laboratory Animals (21National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory AnimalsGuide for the Care and Use of Laboratory Animals. 8th edition. National Academies Press, Washington, DC2011Google Scholar) and the procedures were approved by the North Carolina State University (NCSU) Institutional Animal Care and Use Committee. White perch (weight 425 ± 113 g; total length 265 ± 25 mm; all values are reported as mean ± standard deviation) were reared at NCSU (Raleigh, NC). Blood plasma was sampled from male white perch injected with estradiol-17β, as described previously (22Heppell S.A. Jackson L.F. Weber G.M. Sullivan C.V. Enzyme-linked immunosorbent assay (ELISA) of vitellogenin in temperate basses (genus Morone): plasma and in vitro analyses.Trans. Am. Fish. Soc. 1999; 128: 532-541Crossref Scopus (22) Google Scholar), for purification of Vtgs. Ovaries were excised from vitellogenic white perch (n = 3; maximum oocyte diameter 536–552 μm) (23Jackson L.F. Sullivan C.V. Reproduction of white perch (Morone americana): the annual gametogenic cycle.Trans. Am. Fish. Soc. 1995; 124: 563-577Crossref Google Scholar), for preparation of ovary membranes. Striped bass (Morone saxatilis) were reared at the NCSU Pamlico Aquaculture Field Laboratory (Aurora, NC). Ovary tissues from striped bass (n = 3) were collected by dissection or biopsy using a plastic cannula inserted through the urogenital pore at four time points: August (weight 1.90 ± 0.61 kg; total length 512 ± 18.0 mm), November (3.80 ± 0.14 kg; 625 ± 6.0 mm), February (4.40 ± 0.94 kg; 626 ± 46.6 mm), and April (4.54 ± 1.37 kg; 663 ± 74.5 mm). The most advanced oocytes during these time points represented one of four oocyte growth stages (24Reading B.J. Williams V.N. Chapman R.W. Williams T.I. Sullivan C.V. Dynamics of the striped bass (Morone saxatilis) ovary proteome reveal a complex network of the translasome.J. Proteome Res. 2013; 12: 1691-1699Crossref PubMed Scopus (23) Google Scholar): early secondary growth (ESG, oocyte diameter 310 ± 21.8 μm), mid-vitellogenic growth (MVG, 503 ± 60.1 μm), late-vitellogenic growth (LVG, 831 ± 276 μm), and post-vitellogenic growth (PVG, 986 ± 33.0 μm), respectively. Ovary and liver samples were preserved in RNALater (Ambion, Austin, TX) for real-time quantitative RT-PCR. Ovary tissue was frozen in liquid nitrogen for quantitative tandem mass spectrometry. Ovary tissue from MVG and LVG striped bass was fixed for in situ hybridization in 0.2 M sodium phosphate buffer (pH 7.4) containing 4% paraformaldehyde, and for immunohistochemistry in Bouin's solution (Sigma, Saint Louis, MO). Brain, heart, liver, ovary, foregut, muscle, and adipose from MVG striped bass were preserved in RNALater for semi-quantitative RT-PCR. Primers (SB618 F1 and SB618 R1; see supplementary Table I for all primer sequences used in this study) were designed for striped bass contig 00618 (25Reading B.J. Chapman R.W. Schaff J.E. Scholl E.H. Opperman C.H. Sullivan C.V. An ovary transcriptome for all maturational stages of the striped bass (Morone saxatilis), a highly advanced perciform fish.BMC Res. Notes. 2012; 5: 111Crossref PubMed Scopus (45) Google Scholar). All DNA oligos were designed with Mac Vector (Accelrys Software, San Diego, CA) and obtained from Integrated DNA Technologies (Coralville, IA). Products were cloned from a Stratagene Uni-Zap XR cDNA library (La Jolla, CA) constructed from pooled white perch ovaries (18Hiramatsu N. Chapman R.W. Lindzey J.K. Haynes M.R. Sullivan C.V. Molecular characterization and expression of vitellogenin receptor from white perch (Morone americana).Biol. Reprod. 2004; 70: 1720-1730Crossref PubMed Scopus (66) Google Scholar), and those from four colonies were bi-directionally sequenced according to our prior studies (16Reading B.J. Hiramatsu N. Sawaguchi S. Matsubara T. Hara A. Lively M.O. Sullivan C.V. Conserved and variant molecular and functional features of multiple egg yolk precursor proteins (vitellogenins) in white perch (Morone americana) and other teleosts.Mar. Biotechnol. (NY). 2009; 11: 169-187Crossref PubMed Scopus (67) Google Scholar). Total RNA was extracted from pooled white perch ovaries using TRIzol reagent (Invitrogen, Carlsbad, CA) (25Reading B.J. Chapman R.W. Schaff J.E. Scholl E.H. Opperman C.H. Sullivan C.V. An ovary transcriptome for all maturational stages of the striped bass (Morone saxatilis), a highly advanced perciform fish.BMC Res. Notes. 2012; 5: 111Crossref PubMed Scopus (45) Google Scholar) and 3′ and 5′ rapid amplification of cDNA ends (RACE) were performed using FirstChoice RLM-RACE kit (Ambion). Primers for 5′RACE (WP618 R1 O and WP618 R1 N) and 3′RACE (WP618 F1 O, SB618 F0 O, WP618 F1 N, WP618 F2 N, and WP618 F3 N) with the suffix "O" or "N" were paired with outer and inner RACE primers, respectively. Sequences were assembled using MacVector and polypeptide domains were characterized using SMART (26Letunic I. Doerks T. Bork P. SMART 7: recent updates to the protein domain annotation resource.Nucleic Acids Res. 2012; 40: D302-D305Crossref PubMed Scopus (1301) Google Scholar). Eukaryotic Linear Motif (27Gould C.M. Diella F. Via A. Puntervoll P. Gemünd C. Chabanis-Davidson S. Michael S. Sayadi A. Bryne J.C. Chica C. et al.ELM: the status of the eukaryotic linear motif resource.Nucleic Acids Res. 2010; 38: D167-D180Crossref PubMed Scopus (208) Google Scholar) was used to identify putative functional motifs within the polypeptide domains. The NCBI databases were queried for lrp13 orthologs and Lrp13 polypeptide sequences were collected from fishes, birds, and mammals along with sequences of other representative lipoprotein receptors. Sequences were aligned by ClustalW (28Thompson J.D. Higgins D.G. Gibson T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55720) Google Scholar) to generate a dendrogram. Synteny of lrp13 and vldlr (lr8) were compared using zebrafish (Danio rerio) chromosome 5_9,505,781-9,678,348 and chromosome 10_15,283,756-15,333,718 (v. Zv9) and Nile tilapia (Oreochromis niloticus) scaffold GL831141.1_3,169,873-3,288,125 (reverse complemented) and scaffold GL831199.1_1,600,394-1,676,338 (v. Orenil1.0) available from Ensembl (29Flicek P. Amode M.R. Barrell D. Beal K. Brent S. Carvalho-Silva D. Clapham P. Coates G. Fairley S. Fitzgerald S. et al.Ensembl 2012.Nucleic Acids Res. 2012; 40: D84-D90Crossref PubMed Scopus (768) Google Scholar). A mixture of VtgAa and VtgAb (VtgAa/b) was purified according to our studies (17Reading B.J. Hiramatsu N. Sullivan C.V. Disparate binding of three types of vitellogenin to multiple forms of vitellogenin receptor in white perch.Biol. Reprod. 2011; 84: 392-399Crossref PubMed Scopus (38) Google Scholar, 30Hiramatsu N. Matsubara T. Hara A. Donato D.M. Hiramatsu K. Denslow N.D. Sullivan C.V. Identification, purification and classification of three forms of vitellogenin from white perch (Morone americana).Fish Physiol. Biochem. 2002; 26: 355-370Crossref Scopus (63) Google Scholar) and 20 mg of VtgAa/b was coupled to an equal volume (12 ml) of Affi-Gel 15 activated immunoaffinity support (Bio-Rad, Hercules, CA) following procedures adapted from Roehrkasten et al. (31Roehrkasten A. Ferenz H. Buschmann-Gebhardt B. Hafer J. Isolation of the vitellogenin-binding protein from locust ovaries.Arch. Insect Biochem. Physiol. 1989; 10: 141-149Crossref Scopus (37) Google Scholar). Affinity medium was poured into a 25 mm internal diameter column (12 ml) and the chromatography system was initialized with 10 column volumes of coupling buffer [20 mM HEPES, 150 mM NaCl, 1 mM CaCl2 (pH 7.4)] and equilibrated with 10 column volumes of binding buffer [20 mM Tris-HCl, 2 mM CaCl2, and 150 mM NaCl (pH 8.0), containing 1 mM phenylmethyl-sulfonyl fluoride and 4 IU/l aprotinin] at 4°C. Solubilized membrane proteins prepared from 25 g of vitellogenic white perch ovaries, according to our studies (17Reading B.J. Hiramatsu N. Sullivan C.V. Disparate binding of three types of vitellogenin to multiple forms of vitellogenin receptor in white perch.Biol. Reprod. 2011; 84: 392-399Crossref PubMed Scopus (38) Google Scholar, 32Hiramatsu N. Hara A. Hiramatsu K. Fukada H. Weber G.M. Denslow N.D. Sullivan C.V. Vitellogenin-derived yolk proteins of white perch, Morone americana: purification, characterization and vitellogenin-receptor binding.Biol. Reprod. 2002; 67: 655-667Crossref PubMed Scopus (82) Google Scholar), were recycled through the affinity media for 4.5 h at 4°C (1.0 ml/min flow rate). The column was washed with 15 vol of binding buffer at 4°C and buffer in the top reservoir was decanted and replaced with elution buffer [20 mM Tris-HCl, 10 mM suramin, 5 mM EDTA, 150 mM NaCl (pH 6.0)]. The column was equilibrated for 30 min at 4°C before eluting proteins with 5 vol of elution buffer. Fractions were collected and assayed for protein concentration using a BCA protein assay kit (Pierce, Rockford, IL) and a Bio-Rad 3550 microplate reader. Peak fractions were pooled and concentrated to 100 μg protein/ml with a Centricon YM-30 (Millipore, Billerica, MA). Affinity purified Vtgrs diluted 1:1 in Laemmli buffer were electrophoresed through 5% acrylamide precast Tris-HCl Ready Gels, as we described (17Reading B.J. Hiramatsu N. Sullivan C.V. Disparate binding of three types of vitellogenin to multiple forms of vitellogenin receptor in white perch.Biol. Reprod. 2011; 84: 392-399Crossref PubMed Scopus (38) Google Scholar), and stained with Silver Stain Plus (Bio-Rad). Aliquots of solubilized ovary membrane proteins, affinity purified Vtgrs, and purified VtgAa/b were processed for LC-ESI-MS/MS using an LTQ linear ion trap mass spectrometer (Thermo, San Jose, CA) at the NCSU Genomic Sciences Laboratory (Raleigh, NC). Samples were analyzed in quadruplicate and ion fragmentation spectra were queried by MASCOT (Matrix Science, Boston, MA) against white perch VtgAa, VtgAb, VtgC (16Reading B.J. Hiramatsu N. Sawaguchi S. Matsubara T. Hara A. Lively M.O. Sullivan C.V. Conserved and variant molecular and functional features of multiple egg yolk precursor proteins (vitellogenins) in white perch (Morone americana) and other teleosts.Mar. Biotechnol. (NY). 2009; 11: 169-187Crossref PubMed Scopus (67) Google Scholar), white perch Lr8− (18Hiramatsu N. Chapman R.W. Lindzey J.K. Haynes M.R. Sullivan C.V. Molecular characterization and expression of vitellogenin receptor from white perch (Morone americana).Biol. Reprod. 2004; 70: 1720-1730Crossref PubMed Scopus (66) Google Scholar), and the translated striped bass ovary transcriptome (24Reading B.J. Williams V.N. Chapman R.W. Williams T.I. Sullivan C.V. Dynamics of the striped bass (Morone saxatilis) ovary proteome reveal a complex network of the translasome.J. Proteome Res. 2013; 12: 1691-1699Crossref PubMed Scopus (23) Google Scholar, 25Reading B.J. Chapman R.W. Schaff J.E. Scholl E.H. Opperman C.H. Sullivan C.V. An ovary transcriptome for all maturational stages of the striped bass (Morone saxatilis), a highly advanced perciform fish.BMC Res. Notes. 2012; 5: 111Crossref PubMed Scopus (45) Google Scholar). A portion of cutthroat trout (Oncorhynchus clarki) CtLR13+1 (Lrp13) (33Hiramatsu N. Luo W. Reading B.J. Sullivan C.V. Mizuta H. Ryu Y-W. Nishimiya O. Todo T. Hara A. Multiple ovarian lipoprotein receptors in teleosts.Fish Physiol. Biochem. 2013; 39: 29-32Crossref PubMed Scopus (26) Google Scholar) was cloned into pET302/NT-His expression vector (Invitrogen) using In-Fusion Advantage PCR cloning kit (Clontech, Mountain View, CA) and primers CtLR13+1F and CtLR13+1R. Recombinant CtLR13+1 His-fusion proteins were expressed in Rosetta-gami B (DE3) pLysS (Novagen, Madison, WI) and recovered using BugBuster protein extraction reagent and Ni-charged His-Bind resin chromatography (Novagen). Superdex 200 gel purified recombinant CtLR13+1 was used to raise polyclonal α-CtLrp13 in rabbit as described by Hong et al. (34Hong L. Fujita T. Wada T. Amano H. Hiramatsu N. Zhang X. Todo T. Hara A. Choriogenin and vitellogenin in red lip mullet (Chelon haematocheilus): purification, characterization, and evaluation as potential biomarkers for detecting estrogenic activity.Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 2009; 149: 9-17Crossref PubMed Scopus (21) Google Scholar). Synthetic white perch Lrp13 peptides (CSLGYSGDSCQDHLLKT and TTLNESSQLRNLATQDC) and Lr8− peptides (CRPEANVSTSIQVDSTARGSA and CSVDLNGDNRKKVLQS) were used to raise polyclonal α-WpLrp13 and α-WpLr8− in chickens (GeneTel Laboratories, Madison, WI). Solubilized white perch ovary membrane proteins were separated by electrophoresis through 7.5 and 5% acrylamide precast Tris-HCl ready gels and subjected to ligand blotting with digoxigenin (DIG)-labeled VtgAa/b or Western blotting with α-WpLrp13 or α-WpLr8− at 1:10,000, as we previously described (17Reading B.J. Hiramatsu N. Sullivan C.V. Disparate binding of three types of vitellogenin to multiple forms of vitellogenin receptor in white perch.Biol. Reprod. 2011; 84: 392-399Crossref PubMed Scopus (38) Google Scholar, 35Mizuta H. Luo W. Ito Y. Mushiroba Y. Todo T. Hara A. Reading B.J. Sullivan C.V. Hiramatsu N. Ovarian expression and localization of vitellogenin receptor with eight ligand binding repeats in the cutthroat trout (Oncorhynchus clarki).Comp. Biochem. Physiol. B. 2013; 166: 81-90Crossref PubMed Scopus (40) Google Scholar). Primers were designed for striped bass lrp13 (SB618For1 and SB618Rev1) and ribosomal protein L9 (rpl9) [contig 10830 (25Reading B.J. Chapman R.W. Schaff J.E. Scholl E.H. Opperman C.H. Sullivan C.V. An ovary transcriptome for all maturational stages of the striped bass (Morone saxatilis), a highly advanced perciform fish.BMC Res. Notes. 2012; 5: 111Crossref PubMed Scopus (45) Google Scholar)] (RPl9For and RPl9Rev). The quality of total RNA extracted from brain, heart, liver, ovary, foregut, muscle, and adipose tissue was evaluated by NanoDrop ND-1000 (Thermo Scientific, Wilmington, DE) OD260/OD280 and OD260/OD230 and agarose electrophoresis. Extracts were treated with DNA-Free (Applied Biosystems, Grand Island, NY) and cDNA was synthesized using SuperScript First-Strand synthesis system (Invitrogen). Amplifications were performed using PCR SuperMix (Invitrogen) and no template and no reverse transcription controls were incorporated into the assay. Primers for lr8− variant of vldlr (SBLR8For and SBLR8Rev) and lrp13 (SBLRX+1For and SBLRX+1Rev) were designed from striped bass contigs 04238 and 00618 (25Reading B.J. Chapman R.W. Schaff J.E. Scholl E.H. Opperman C.H. Sullivan C.V. An ovary transcriptome for all maturational stages of the striped bass (Morone saxatilis), a highly advanced perciform fish.BMC Res. Notes. 2012; 5: 111Crossref PubMed Scopus (45) Google Scholar). Priming sites of low complementarity between these striped bass contig sequences were chosen to ensure primer specificity. Total ovary and liver RNA was extracted and evaluated as described above and cDNA was synthesized with a high capacity cDNA synthesis kit (Applied Biosystems). Absolute real-time quantitative PCR assays were performed as previously described (36Tipsmark C.K. Baltzegar D.A. Ozden O. Grubb B.J. Borski R.J. Salinity regulates claudin mRNA and protein expression in the teleost gill.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2008; 294: R1004-R1014Crossref PubMed Scopus (74) Google Scholar, 37Williams V.N. Reading B.J. Amano H. Hiramatsu N. Schilling J. Salger S.A. Islam Williams T. Gross K. Sullivan C.V. Proportional accumulation of yolk proteins derived from multiple vitellogenins is precisely regulated during vitellogenesis in striped bass (Morone saxatilis).J. Exp. Zool. A Ecol. Genet. Physiol. 2014; 321: 301-315Crossref PubMed Scopus (36) Google Scholar) using Brilliant II SYBR Green QPCR Master Mix (Agilent Technologies, Santa Clara, CA). Samples were measured in triplicate using a 7300 real-time PCR system (Applied Biosystems) and gene expression was reported as copy number calculated from serially diluted plasmid DNA standard curves. Picha and colleagues (38Picha M.E. Silverstein J.T. Borski R.J. Discordant regulation of hepatic IGF-I mRNA and circulating IGF-I during compensatory growth in a teleost, the hybrid striped bass (Morone chrysops x Morone saxatilis).Gen. Comp. Endocrinol. 2006; 147: 196-205Crossref PubMed Scopus (56) Google Scholar, 39Picha M.E. Turano M.J. Tipsmark C.K. Borski R.J. 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Melting curve analysis and agarose gel electrophoresis were performed to verify primer specificity and no template and no reverse transcription controls were employed in the assay. DIG-labeled antisense and sense RNA probes were prepared by in vitro transcription of striped bass lrp13 using primers SB618ishF and SB618ishR (Roche, Indianapolis, IN). In situ hybridization of DIG-labeled probes was performed for 40 h at 65°C according to our previous report (40Luo W. Ito Y. Mizuta H. Massaki K. Hiramatsu N. Todo T. Reading B.J. Sullivan C.V. Hara A. Molecular cloning and partial characterization of an ovarian receptor with seven ligand binding repeats, an orthologue of low-density lipoprotein receptor, in the cutthroat trout (Oncorhynchus clarki).Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2013; 166: 263-271Crossref PubMed Scopus (18) Google Scholar). Sections were photographed using a DXM1200F camera cou