Alejandro S Mechaly

Jordi Viñas

Colleen Murphy

Michael Reith

Dr Francesc Piferrer
email: piferrer@icm.csic.es

Gene Structure of the Kiss1 Receptor-2 (Kiss1r-2) in the Atlantic Halibut: Insights into the Evolution and Regulation of Kiss1r Genes

Keywords: Kisspeptin, Kiss1r, Kiss-1 receptor, gpr54, puberty, Atlantic halibut

Kisspeptin and its receptor, Kiss1r, play an essential role in the control of the onset of puberty in vertebrates. We characterized the cDNA and genomic DNA encoding Kiss1r in Atlantic halibut (Hippoglossus hippoglossus). The 1,146 bp open reading frame predicts a 381 amino acid protein with high homology to the Kiss1r-2 of other teleost fish. Phylogenetic analysis of Kiss1r sequences suggests that the mammalian Kiss1r-1 form arose by way of a gene duplication prior to the emergence of amphibians. Synteny analysis demonstrated the highly conserved nature of the Kiss1r-2 region in teleosts, suggesting that flanking regulatory sequences are also likely to be conserved. Bioinformatic analysis identified six conserved regions in piscine Kiss1r-2 upstream sequences, providing potential targets for future in-depth investigation of Kiss1r-2 regulation. Kiss1r-2 expression in the brain increased coinciding with the onset of puberty. Expression levels in the gonads were two orders of magnitude lower than those of the brain, a characteristic apparently conserved in other fishes, and expression in gonads was only detected in immature fish.

A c c e p t e d M a n u s c r i p t

Introduction

Puberty is a cascade of developmental events that culminates in a mature adult reproductive system [START_REF] Schulz | Puberty in male fish: concepts and recent developments with special reference to the African catfish (Clarias gariepinus)[END_REF]. This process is regulated by a series of actions along the hypothalamic-pituitary-gonadal (HPG) axis in mammals [START_REF] Ojeda | Minireview: the neuroendocrine regulation of puberty: is the time ripe for a systems biology approach?[END_REF] and fishes [START_REF] Weltzien | The brain-pituitary-gonad axis in male teleosts, with special emphasis on flatfish (Pleuronectiformes)[END_REF][START_REF] Taranger | Control of puberty in farmed fish[END_REF]. The hypothalamic neurons stimulate the production of the gonadotropin-releasing-hormone (GnRH) which, in turn, activates the pulsatile secretion of two gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) [START_REF] Funes | The KiSS-1 receptor GPR54 is essential for the development of the murine reproductive system[END_REF][START_REF] Gottsch | A role for kisspeptins in the regulation of gonadotropin secretion in the mouse[END_REF][START_REF] Messager | Kisspeptin directly stimulates gonadotropin-releasing hormone release via G protein-coupled receptor 54[END_REF]. A recently described system consisting of the peptide hormone kisspeptin and its receptor have been demonstrated to be crucial in the initiation of puberty in vertebrates [START_REF] De Roux | Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54[END_REF][START_REF] Funes | The KiSS-1 receptor GPR54 is essential for the development of the murine reproductive system[END_REF] by regulating the HPG. In mouse (Mus musculus) and rat (Rattus norvegicus), mutations of the receptor gene, Kiss1r, result in hypogonadotrophic hypogonadism, a syndrome characterized by reduced secretion of gonadotropins, and subsequent abnormal pubertal development [START_REF] De Roux | Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54[END_REF][START_REF] Seminara | The GPR54 gene as a regulator of puberty[END_REF].

Puberty regulation by the kisspeptin-Kiss1r system appears to be conserved in fish, amphibians and mammals (Roseweir and Mir, 2009).

The kisspeptin receptor (Kiss1r) was first identified in rat as an orphan G-protein coupled receptor [START_REF] Lee | Discovery of a receptor related to the galanin receptors[END_REF]. In 2001, the role of this protein as a receptor for kisspeptin was defined [START_REF] Kotani | The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54[END_REF][START_REF] Muir | AXOR12, a novel human G protein-coupled receptor, activated by the peptide KiSS-1[END_REF][START_REF] Ohtaki | Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor[END_REF]. This gene has been subsequently identified in many mammalian species, including human (Homo sapiens) [START_REF] Muir | AXOR12, a novel human G protein-coupled receptor, activated by the peptide KiSS-1[END_REF], mouse [START_REF] Stafford | Identification and characterization of mouse metastasis-suppressor KiSS1 and its G-protein-coupled receptor[END_REF], rhesus macaque (Macaca mulatta) [START_REF] Shahab | Increased hypothalamic GPR54 signaling: A potential mechanism for initiation of puberty in primates[END_REF] and pig (Sus scrofa) [START_REF] Li | Characterization of the porcine Kisspeptins receptor gene and evaluation as candidate for timing of puberty in sows[END_REF] among others. Kiss1r contains A c c e p t e d M a n u s c r i p t 4 seven transmembrane domains (TMDs) that are highly conserved among species, a feature that facilitates the cloning of the Kiss1r in additional taxa. This approach has been used to clone Kiss1r in lower vertebrates including an amphibian, the bullfrog (Rana catesbeiana) [START_REF] Moon | Molecular cloning of the bullfrog kisspeptin receptor GPR54 with high sensitivity to Xenopus kisspeptin[END_REF] and several fishes: Nile tilapia (Oreochromis niloticus) [START_REF] Parhar | Laser-captured single digoxigenin-labeled neurons of gonadotropin-releasing hormone types reveal a novel g protein-coupled receptor (Gpr54) during maturation in cichlid fish[END_REF], cobia (Rachycentron canadum) [START_REF] Mohamed | Developmental expression of the G protein-coupled receptor 54 and three GnRH mRNAs in the teleost fish cobia[END_REF], grey mullet (Mugil cephalus) [START_REF] Nocillado | Temporal expression of Gprotein-coupled receptor 54 (GPR54) gonadotropin-releasing hormones (GnRH), and dopamine receptor D2 (drd2) in pubertal female grey mullet, Mugil cephalus[END_REF], fathead minnow (Pimephales promelas) [START_REF] Filby | The kisspeptin/gonadotropinreleasing hormone pathway and molecular signaling of puberty in Fish[END_REF], zebrafish (Danio rerio) [START_REF] Biran | Molecular identification and functional characterization of the kisspeptin/kisspeptin receptor system in lower vertebrates[END_REF], Senegalese sole (Solea senegalensis) [START_REF] Mechaly | Identification of two isoforms of the kisspeptin-1 receptor (kiss1r) generated by alternative splicing in a modern teleost, the Senegalese Sole (Solea senegalensis)[END_REF] and goldfish (Carassius auratus) [START_REF] Li | Structural and functional multiplicity of the kisspeptin/GPR54 system in goldfish (Carassius auratus)[END_REF]. Additionally, Kiss1r has been identified in the genome sequences of other species including medaka (Oryzias latipes), three-spined stickleback (Gasterosteus aculeatus), pufferfish (Tetraodon nigroviridis), fugu (Takifugu rubripes), clawed frog (Xenopus tropicalis) and the lesser hedgehog (Echinops telfairi) [START_REF] Lee | Molecular evolution of multiple forms of kisspeptins and GPR54 receptors in vertebrates[END_REF][START_REF] Mechaly | Identification of two isoforms of the kisspeptin-1 receptor (kiss1r) generated by alternative splicing in a modern teleost, the Senegalese Sole (Solea senegalensis)[END_REF].

Recently, a second form of kisspeptin, Kiss2, which shows amino acid differences in three positions compared to Kiss1, has been identified in non-mammalian vertebrates (Felip et al., 2009;[START_REF] Kitahashi | Cloning and expression of kiss2 in the zebrafish and medaka[END_REF][START_REF] Lee | Molecular evolution of multiple forms of kisspeptins and GPR54 receptors in vertebrates[END_REF]. As well, multiple copies of Kiss1r have been detected in some species: two Kiss1r are present in zebrafish, medaka [START_REF] Lee | Molecular evolution of multiple forms of kisspeptins and GPR54 receptors in vertebrates[END_REF], goldfish [START_REF] Li | Structural and functional multiplicity of the kisspeptin/GPR54 system in goldfish (Carassius auratus)[END_REF] and platypus (Ornithorhynchus anatinus), while three are present in clawed frog [START_REF] Lee | Molecular evolution of multiple forms of kisspeptins and GPR54 receptors in vertebrates[END_REF]. Amino acid similarity and synteny analyses suggest that two Kiss1r lineages (Kiss1r-1 and Kiss1r-2) arose through gene or chromosome duplication with a second gene duplication occurring in the Kiss1r-1 lineage to produce the amphibian Kiss1r-1a and Kiss1r-1b genes [START_REF] Lee | Molecular evolution of multiple forms of kisspeptins and GPR54 receptors in vertebrates[END_REF]. Most fish Kiss1r characterized to date fall into the Kiss1r-2 class [START_REF] Mechaly | Identification of two isoforms of the kisspeptin-1 receptor (kiss1r) generated by alternative splicing in a modern teleost, the Senegalese Sole (Solea senegalensis)[END_REF]. The two paralogous Kiss1r genes detected in zebrafish, named Kiss1ra and Kiss1rb, showed differences in their pattern of gene expression during A c c e p t e d M a n u s c r i p t maturation. Thus, while Kiss1ra expression was higher during puberty and low in mature fish, Kiss1rb expression levels were consistent throughout these stages [START_REF] Biran | Molecular identification and functional characterization of the kisspeptin/kisspeptin receptor system in lower vertebrates[END_REF].

Pleuronectiformes, along with the order Tetraodontiformes, constitute a modern and derived group of teleosts. With the limited information available so far, it seems that in these orders the ligand and its receptor, Kiss1r-1 have been lost during evolution, but this needs confirmation in more species. In the Senegalese sole, only a single Kiss1r was identified.

However, two different transcript variants were detected [START_REF] Mechaly | Identification of two isoforms of the kisspeptin-1 receptor (kiss1r) generated by alternative splicing in a modern teleost, the Senegalese Sole (Solea senegalensis)[END_REF]. The two variants (named Ss Kiss1r_v1 and Ss Kiss1r_v2) originated from a single gene by alternative splicing that involved retention of the entire intron III. The two isoforms of Senegalese sole also showed differences in their expression pattern: Ss Kiss1r_v1 had a higher expression than the Ss Kiss1r_v2 in the brain while the opposite occurred in gonads [START_REF] Mechaly | Identification of two isoforms of the kisspeptin-1 receptor (kiss1r) generated by alternative splicing in a modern teleost, the Senegalese Sole (Solea senegalensis)[END_REF].

The different gene copies and isoforms of Kiss1r complicate the understanding of the roles of these genes in controlling the onset puberty in vertebrates. Whether the lack of two different genes in these fish is compensated by the appearance of splice isoforms is still not known.

Human KISS1R is expressed in many tissues, including placenta, pituitary, spinal cord pancreas, brain, stomach, intestine, thymus, spleen, lung, testis, kidney and liver [START_REF] Kotani | The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54[END_REF] suggesting pleiotropic functions for this system. In puberty regulation, the focus has been the brain and the gonads, two organs that play crucial roles in the control of maturation during different stages of development [START_REF] Nocillado | Neuroendocrine regulation of puberty in fish: insights from the grey mullet (Mugil cephalus) model[END_REF]. Expression of Kiss1r during development in fishes shows a variety of patterns. For instance, in the brain of Nile tilapia Kiss1r expression was associated with three GnRH neuron types (GnRH1, GnRH2 and GnRH3) [START_REF] Parhar | Laser-captured single digoxigenin-labeled neurons of gonadotropin-releasing hormone types reveal a novel g protein-coupled receptor (Gpr54) during maturation in cichlid fish[END_REF] and showed a significant increase of expression during gonadal maturation [START_REF] Parhar | Laser-captured single digoxigenin-labeled neurons of gonadotropin-releasing hormone types reveal a novel g protein-coupled receptor (Gpr54) during maturation in cichlid fish[END_REF]. A similar pattern was observed in cobia, with an increase of A c c e p t e d M a n u s c r i p t 6 mRNA levels of Kiss1r in the brain during the early stages of puberty [START_REF] Mohamed | Developmental expression of the G protein-coupled receptor 54 and three GnRH mRNAs in the teleost fish cobia[END_REF]. However, Kiss1r expression in the brain of the grey mullet was elevated during early puberty but decreased during advanced stages [START_REF] Nocillado | Temporal expression of Gprotein-coupled receptor 54 (GPR54) gonadotropin-releasing hormones (GnRH), and dopamine receptor D2 (drd2) in pubertal female grey mullet, Mugil cephalus[END_REF]. In the brain of fathead minnow, Kiss1r mRNA peaked before the onset of puberty and decreased thereafter [START_REF] Filby | The kisspeptin/gonadotropinreleasing hormone pathway and molecular signaling of puberty in Fish[END_REF]. Finally, in the Senegalese sole, where two splice isoforms were detected, the expression in the brain was higher in pubertal fish than mature fish, and Ss Kiss1r_v1 expression was higher than Kiss1r_v2 in both stages [START_REF] Mechaly | Identification of two isoforms of the kisspeptin-1 receptor (kiss1r) generated by alternative splicing in a modern teleost, the Senegalese Sole (Solea senegalensis)[END_REF]. This variation in expression pattern between species may be due to species differences or, alternatively, to the presence of different genes or splice variants of the same gene [START_REF] Mechaly | Identification of two isoforms of the kisspeptin-1 receptor (kiss1r) generated by alternative splicing in a modern teleost, the Senegalese Sole (Solea senegalensis)[END_REF].

Nevertheless, this is another aspect of kisspeptin regulation that needs to be clarified.

The Atlantic halibut, Hippoglossus hippoglossus (Hh), belongs to the order Pleuronectiformes and thus presumably has only one Kiss1r gene, but whether it has a splice mechanism or not it not known. Atlantic halibut is the largest and longest-lived flatfish, capable of growing to sizes of over 2.2 m and of living for more than 50 years (Haug, 1990).

In addition, Atlantic halibut are slow to mature, requiring 5-7 years. Halibut females generally grow more quickly than males and attain a larger ultimate size [START_REF] Douglas | Length at age, sexual maturity and distribution of Atlantic halibut, Hippoglossus hippoglossus L., off the Northeast USA[END_REF]. Early maturation and reduced growth rates of males are one of the major problems for commercial halibut aquaculture [START_REF] Norberg | Effects of photoperiod on sexual maturation and somatic growth in male Atlantic halibut (Hippoglossus hippoglossus L.)[END_REF]. Thus, understanding the control of the initiation of puberty in this species not only has basic interest in knowing about the kisspetin-Kiss1r system in a flatfish, but may also have potentially significant implications for the halibut aquaculture industry. As a first step in better understanding the onset of puberty in the Atlantic halibut, Kiss1r was cloned and its expression analyzed in a variety of tissues at multiple stages of development. (Woods Harbour, NS). Fish were kept in tanks appropriate for their developmental stage under natural conditions of photoperiod and temperature and were fed twice a day with a commercial diet. These conditions allow Atlantic halibut to mature in captivity. Animals were treated according to the guidelines of the Canadian Council on Animal Care [START_REF] Demers | Animal research. Harmonization of animal care and use guidance[END_REF] and protocols were approved prior to experimentation by all relevant authorities. Four different year classes (one, three, five and seven year-old) were sampled. Fish were anesthetized with an overdose of MS-222 (Sigma-Aldrich, St. Louis, MO) and sacrificed by decapitation. Tissues were quickly removed under RNase-free conditions, flash frozen in liquid nitrogen and stored at -80ºC until used. Gonads (testis and ovaries) were fixed in 4% paraformaldehyde for further histological examination. Fin clips were taken and stored in 95% ethanol for DNA extraction. Biometric information including standard length (cm) and weight (g) were obtained for all fishes analyzed.

Histological analyses

After fixation in 4% paraformaldehyde for approximately 24 hrs at room temperature, gonads were washed for an additional 24 h in phosphate buffer (PB) (pH 7.4), dehydrated in an ascending series of ethanol concentrations, cleared in xylene (Fisher Scientific, WA, USA) and embedded in Paraplast (Fisher Scientific, Pittsburgh, PA). Sections (7 µm thick) were cut using a rotary microtome (Microm HM-355), float mounted on Colorfrost/Plus slides (Fisher A c c e p t e d M a n u s c r i p t 8 Scientific), warmed on a slide warmer for 1 h and stained with Instant Haematoxylin and Eosin (H-E) (Sigma). Stained sections were mounted in Permount (Fisher Scientific) under coverslips, examined on a DMRE Leica light microscope and photographed using Compix imaging software. Stages of gametogenesis were determined according to the different germ cell types present as previously described [START_REF] Hendry | Gonadal sex differentiation in Atlantic halibut[END_REF].

Partial cloning of Atlantic halibut Kiss1r gene

Total RNA was isolated from frozen tissue with TRIZOL Reagent (Invitrogen), its quality checked in a 1.5% agarose gel stained with SYBR safe (Syber Safe™, Invitrogen) and its quantity measured in a NanoDrop® ND-1000 spectrophotometer (NanoDrop® Technologies Inc, Wilmington, DE, USA). All RNAs were treated with DnaseI (Ambion) to avoid genomic contamination of the RNA samples. cDNA synthesis was performed following the manufacturer's instructions using 1 μg of RNA with Superscript III Reverse Transcriptase and random hexamer primers (Invitrogen) in a 20 μl reaction volume.

A partial cDNA sequence of Hh Kiss1r was first isolated by reverse transcription PCR (RT-PCR) using degenerate oligonucleotides (Hh_Kiss1r-F3 and Hh_Kiss1r-R3) (Table 1) that were designed on the basis of the previously published sequences of Nile tilapia (AB162143) [START_REF] Parhar | Laser-captured single digoxigenin-labeled neurons of gonadotropin-releasing hormone types reveal a novel g protein-coupled receptor (Gpr54) during maturation in cichlid fish[END_REF], cobia (DQ790001) [START_REF] Mohamed | Developmental expression of the G protein-coupled receptor 54 and three GnRH mRNAs in the teleost fish cobia[END_REF], Senegalese sole (EU136710) [START_REF] Mechaly | Identification of two isoforms of the kisspeptin-1 receptor (kiss1r) generated by alternative splicing in a modern teleost, the Senegalese Sole (Solea senegalensis)[END_REF], Atlantic croaker (Micropogonias undulates) (DQ347412) and grey mullet (DQ683737) [START_REF] Nocillado | Temporal expression of Gprotein-coupled receptor 54 (GPR54) gonadotropin-releasing hormones (GnRH), and dopamine receptor D2 (drd2) in pubertal female grey mullet, Mugil cephalus[END_REF]. PCR reactions were carried out using 1 µl of cDNA in a final volume of 25 µl containing 2.5 µl of 10X PCR reaction buffer (0.75 mM MgCl 2 ), 0.2 mM of each dATP, dCTP, dGTP, dTTP, 50 pmol of each primer and 0.2 µl of rTaq DNA polymerase (G.E. Healthcare). Cycling parameters A c c e p t e d M a n u s c r i p t consisted in an initial heat denaturation step at 94ºC for 5 min followed by 35 cycles of 30 s at 94ºC, 30 s at 60ºC and 30 s at 72ºC, and finished with a final extension step at 72ºC for 5 min.

Bands of the expected size were purified (Montage PCR, Millipore), ligated into pCR2.1-TOPO vector (Invitrogen), using the TOPO TA cloning method and transformed into E. coli competent cells following the manufacturer's instructions. White colonies were selected from X-Gal/IPTG ampicillin agar plates and grown in LB/ampicillin liquid media. Plasmid DNAs were purified with QIAprep Spin Miniprep (QIAGEN) and used as templates in sequencing reaction. The plasmid DNA was purified and sequenced with universal primers using an ABI PRISM 377 automatic sequencer (Applied Biosystems, Foster City, CA, USA). To avoid sequencing errors, ten different plasmids DNAs were sequenced in both directions. The resulting DNA sequences were compared by BLAST analysis to known piscine Kiss1r cDNA sequences available in GenBank.

BAC library screening

An Atlantic halibut BAC library composed of 35,328 clones with an average insert size of 100 kb was constructed by Lucigen Corp. (Middleton, WI). Based on the partial Hh Kiss1r sequence, primers for an overgo probe (Overgo-Kiss1r-F1 and Overgo-Kiss1r-R1) were designed using Soop (system for optimized overgo picking software; [START_REF] Thomas | Parallel construction of orthologous sequence -ready clone contig maps in multiple species[END_REF] for library screening. Overgo probes were labeled according to [START_REF] Ross | Nonradioactive detection of differentially expressed genes using complex RNA or DNA hybridization probes[END_REF].

Briefly, annealing of the overgo oligo pairs (10 pmol each) was done in 4.5 µl of water at 80°C for 5 min followed by 37°C for 10 min and then stored on ice. Labeling was carried out by adding 2.0 µl 5X OLB -{A, C}, 0.5 µl of 2 mg/ml acetylated BSA (New England Biolabs, Ipswich, MA), 0.5 µl each of [α-32 P] dATP and [α-32 P] dCTP(10 µCi/µl, ~3000Ci/mmol, Perkin Elmer, Shelton, Connecticut) and 1 µl (2U/µl) of Klenow enzyme (New England A c c e p t e d M a n u s c r i p t 10 Biolabs, Ipswich, MA). For ease of image alignment, an overgo probe from E. coli was also labeled. The reactions were incubated at room temperature for 1 h. Fifteen minutes before the end of the incubation 1 µl of a cold chase (200 µM each of dATP and dCTP) was added. Unincorporated dNTP's were removed using NICK Sephadex G50 columns (GE Healthcare Baie d'Urfe, Quebec). Filters were pre-hybridized in roller bottles in a hybridization oven using Church's hybridization buffer [START_REF] Ross | Nonradioactive detection of differentially expressed genes using complex RNA or DNA hybridization probes[END_REF] containing 0.5 ml sonicated salmon sperm DNA (10 mg/ml) (Ambion, Austin, Texas) that had been heated to 100°C for 5 min immediately before use. Pre-hybridization was carried out for 3 h at 55°C. The prehybridization solution was then replaced with 25 ml of fresh Church's buffer containing salmon sperm DNA to which was added the labeled denatured (95°C for 10 min) Kiss1r and E. coli (2% of the Kiss1r volume) overgo probes. Hybridization was carried out for 72 h at 55°C. Following hybridization the filters were rinsed once with 4X SSC containing 0.1% SDS at room temperature and then extensively washed at 55°C for 30 min: 2 X 1 liter each of 2X SSC/0.1% SDS, 1X SSC/0.1% SDS and 0.5X SSC/0.1% SDS. The filters were then exposed to Bio MS film (Kodak) with Biomax MS intensifying screens (Kodak) at -80°C for 24-48 h.

Genomic structure of Hh Kiss1r gene

Positive clones were isolated, tested by PCR and purified using a Qiagen Large Construct Kit (Qiagen). The BAC DNAs were sequenced on a Roche GS-FLX by the McGill University and Genome Quebec Innovation Centre. The contigs returned from the GS-FLX sequencing had a gap in intron I of the Hh Kiss1r gene. To obtain the complete gene sequence, genomic DNA was amplified with primers located in intron I (Intron I-F1 and Intron I-R1) (Table 1). PCR reactions were carried out in a final volume of 25 µl containing 2.5 µl of 10x PCR reaction buffer with 0.75 mM of MgCl 2 , 0.2 mM of each dATP, dCTP, PCR was carried out with an initial heat denaturation at 94ºC for 5 min, followed by 35 cycles of 30 s at 92ºC, 30 s at 55ºC and 30 s at 72ºC, and finished with the final extension step at 72ºC for 5 min. PCR products were analyzed on 1.5% agarose gels and bands of the expected size were excised and purified by gel extraction (Qiaquick-Qiagen). The PCR products were directly sequenced by Genewiz Inc. (South Plainfield, NJ). Partial sequences were assembled and edited using Bioedit software version 7.0.5.3 [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF].

DNA sequence and phylogenetic analysis

The location of the putative signal peptide cleavage site of halibut Kiss1r was predicted using the SignalP server of the Center for Biological Sequence Analysis (http://www.cbs.dtu.dk/services/SignalP/). The seven TMDs helices were predicted by the HMMTOP Server [START_REF] Tusnády | The HMMTOP transmembrane topology prediction server[END_REF]. Upstream sequences of fish Kiss1r-2 were analyzed with Meme [START_REF] Bailey | Fitting a mixture model by expectation maximization to discover motifs in biopolymers[END_REF] and conserved regions were compared to the Transfac database (Wingender et al., 1996) with Tomtom [START_REF] Gupta | Quantifying similarity between motifs[END_REF]. Multiple alignments and phylogenetic analysis was performed using the amino acid sequences of the seven TMDs of Kiss1r available from GenBank and Ensembl. The sequence sources and GenBank accession numbers were as follow: dog (Canis lupus familiaris) (XM_850105), sheep (Ovis aries) (ABY61032), rhesus macaque (XP_001117198), mouse (NP_444474), rat (AAD19664), pig (ABE73453), human (AAK83235), chimpanzee (Pan troglodytes) (Microcebus murinus). Sequences were aligned using Clustal W [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[END_REF] and edited in Bioedit [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF]. Sequence alignment was based on the TMDs because adjacent regions were difficult to align and therefore to assign homology among the amino acid residues. A phylogenetic tree was constructed using the neighbor-joining method [START_REF] Saitou | The Neighbor-Joining method -a new method for reconstructing phylogenetic trees[END_REF] using amino acid Poisson distance. Evaluation of statistical confidence in nodes was based on 500 nonparametric bootstrap replicates [START_REF] Felsenstein | Confidence limits on phylogenies -an approach using the bootstrap[END_REF]. All phylogenetic analysis were conducted on MEGA version 4 [START_REF] Tamura | MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) Software Version 4.0[END_REF]

Tissue-specific expression of Hh Kiss1r

Total RNA isolation and cDNA synthesis from brain, ovary, testis, liver, muscle, stomach, heart, intestine and kidney, from three and seven year old males and females were carried out following the protocol described in section 2.3. The resulting cDNAs were used to amplify Hh Kiss1r by RT-PCR using the RT Hh Kiss1r-F2 and -R2 primers (Table 1). The PCR cycling conditions for Hh Kiss1r were: 5 min at 94°C; 35 cycles of 30 s at 94°C, 30 s at A c c e p t e d M a n u s c r i p t 13 55°C, 30 s at 72°C and a final extension of 5 min at 72°C. Amplification of β-actin RNA was done as a control using the RT Hh βactin-F1 and -R1 primers (Table 1) under the same conditions. A no-template control (NTC) was included to ensure that contamination of reagents had not occurred. PCR products were resolved on a 1% agarose gel stained with ethidium bromide.

Microsatellite analysis

For immature fish with undeveloped gonads, sex was determined by genotyping with five microsatellite markers (Hhi4IMB, Hhi39IMB, Hhi52IMB, Hhi62IMB, Hhi68IMB; Reid RT Hh Kiss1r-R2) (Table 1).

The qPCR amplification reaction mixture contained 2 µl of diluted cDNA (1:3) (freshly synthesized from 1 µg of RNA), 4 µM of each primer, and 10 µl of SYBR Green PCR Master Mix (Roche) in a final volume of 20 µl. Thermal cycling conditions comprised heating to 95°C for 10 min to activate the FastStart Taq DNA Polymerase (Qbiogene, Inc), 40 cycles at 95°C for 10 s and 60°C for 30 s. At the end of the PCR cycles, the qPCR products were immediately analyzed using a dissociation curve step to confirm that only a single PCR product was amplified. No-template control reactions for every primer pair were also included on each reaction plate to check for external DNA contamination. The amplification efficiency (E) of each primer set/gene target was determined in validation assays containing serially diluted cDNA templates (1:3, 1:10, 1:30, 1:100, 1:300 and 1:1000). Efficiency was estimated Sequence assembly resulted in a small number (6-12) of contigs for each BAC clone, with Hh Kiss1r-2 split between two contigs in both cases. One contig contained upstream sequences, exon I and the beginning of intron I, while the second contig contained the end of intron I, the remaining coding region and downstream sequences. PCR reactions using primers flanking the gap between the Kiss1r-2 contigs (Intron I-F1 and R1) were used to complete the sequence of intron I. The complete Hh Kiss1r-2 sequence was assembled and deposited in GenBank (accession number GQ330487).

Characterization of the Hh Kiss1r-2

The coding region of the Hh Kiss1r-2 spans 9,338 bp, which includes Figure 2), as well as a 47 amino acid signal peptide (Figure 2).

Synteny analysis of Hh Kiss1r-2

The larger of the two BAC clones encoding Hh Kiss1r-2 contained sequences encoding several flanking genes, including a partial atp6v0b (encoding V-type proton ATPase 21 kDa proteolipid subunit), three microRNA genes (miR-23a, miR-27a and miR-24), rabL5

(encoding rab-like protein 5) and a partial odf2L (encoding sperm tail outer dense fiber protein 2-like). The organization of genes around the Hh Kiss1r-2 locus was compared to that of other fish Kiss1r-2 with available genome sequences (Figure 3). Interestingly, the atp6v0b~Kiss1r-2~miR-23a~miR-27a~miR-24 gene organization is conserved in all six species, with b4galt2 located upstream of atp6v0b in the species with full genome sequences.

Downstream of the microRNA genes, rabL5~odf2L is conserved in medaka, three-spined stickleback and Atlantic halibut, while atp5L is present in both pufferfish and fugu. The conservation of flanking genes among fish Kiss1r-2 clearly supports a common ancestry, with subsequent taxa-specific rearrangements downstream of micro RNA genes.

Phylogenetic analysis of Kiss1r

The high conservation in the TMDs, together with new information for Kiss1r from a large number of species, facilitated the alignment of 42 Kiss1r amino acid sequences. Of the 36 species analyzed, five of them contained more than one paralogous gene, with the clawed frog being the only species with three genes and medaka, zebrafish, goldfish and platypus with two genes. The phylogenetic analysis (Figure 4A) clustered all Kiss1r sequences from Similarly, a strongly supported (97%) cluster groups all fish Kiss1r sequences along with those from some reptiles and amphibia. Following the scheme of [START_REF] Lee | Molecular evolution of multiple forms of kisspeptins and GPR54 receptors in vertebrates[END_REF], the cluster containing all mammalian plus the clawed frog-1A sequences is designated Kiss1r-1, while the cluster containing all piscine sequences plus sequences from lizard and some amphibia is designated Kiss1r-2. In turn, the Kiss1r-2 cluster could be subdivided into two. One, named Kiss1r-2A contained all piscine species that have only one Kiss1r gene plus two species of amphibia, and the other, named Kiss1r-2B, containing one reptile (anole lizard), amphibian (clawed frog) and those fish with a second Kiss1r gene (which were originally named medaka-1, goldfish-B, zebrafish-B). Kiss1r-2B sequences were intermediate between the Kiss1r-2A sequences and mammalian sequences (Kiss1r-1).

Analysis of the putative Hh Kiss1r-2 promoter region

The 980 bp upstream sequence from Hh Kiss1r-2 along with 1500 bp of sequence upstream of the ATG start from the zebrafish, medaka, three-spined stickleback, fugu and pufferfish Kiss1r-2 were analyzed for conserved sequence regions using Meme [START_REF] Bailey | Fitting a mixture model by expectation maximization to discover motifs in biopolymers[END_REF]. Six conserved regions were identified on the coding strand that ranged in size from 11 to 29 bp (Figure 5). All conserved regions fall within an 1100 bp region upstream of the Kiss1r-2 ATG. Two of these regions showed considerable similarity to the binding sites for the transcription factors MIF-1 (black boxes in Figure 5) and estrogen receptor (grey boxes). Notable are the conserved location of the estrogen receptor-like sequences near -100 in five of six species and the conserved, adjacent arrangement of the regions represented by the black and white boxes in four of the species.

A c c e p t e d M a n u s c r i p t

Determination of the sex and development stages of Atlantic halibut

Sex was determined and fish were classified to their stage of maturation based on the different types of germinal cells present in gonads (Weltzien et al., 2002;[START_REF] Hendry | Gonadal sex differentiation in Atlantic halibut[END_REF].

Sex in several immature fish with undeveloped gonads was determined by pedigree analysis and the inheritance of a sex-specific microsatellite marker (D. Reid, M. Reith, unpublished).

The histological analysis (Figure 6) confirmed that all fish from the one-year class were immature, with spermatogonia present in the males and primordial oocytes in females. The three-year-old fish were entering the early stages of puberty, as determined by the presence of spermatocytes and previtellogenic oocytes. The five-year-old fish were in middle of sexual development, showing spermatids in males, and vitellogenic oocytes in females. Finally, the seven-year class fish were sexually mature with late spermatogenesis evident in males and late vitellogenesis in females.

Tissue distribution of Hh Kiss1r-2 in early puberty and adult fish

The expression of Hh Kiss1r-2 was analyzed by RT-PCR in different tissues from fish from two year-classes (three-and seven-year-old fish), which corresponded to two different maturation stages, early puberty and mature, respectively. Hh Kiss1r-2 was most highly expressed in the brain in all four combinations of sex and year class (Figure 7). In the threeyear-old fish, Hh Kiss1r-2 expression was detectable in most tissues at low levels, with intestine and stomach showing intermediate expression levels in the male and stomach and kidney having intermediate levels in the female. In the seven-year-old fish, Hh Kiss1r-2 expression could only be detected in the brain and stomach in both sexes. and gonads from the different year classes. For all four year-classes, Hh Kiss1r-2 expression in the brain was not significantly different between the two sexes (P = 0.981, P = 0.719, P = 0.170, and P = 0.555), and thus values were pooled together. One-way ANOVA showed a significant increase in the expression of Hh Kiss1r-2 in the brain (Figure 8A) coinciding with the onset of puberty (three-year-old class) in comparison with the rest of the year-classes (P < 0.05). On the other hand, in gonads (Figure 8B), Hh Kiss1r-2 expression was only detected in individuals at an extremely low level compared to the brain (>100-fold lower expression) and only in fish from the one-year-old class. In this class, no differences were detected between sexes (P = 0.945). No expression was detected in the rest of the year classes.

Discussion

Kiss1r is a G-protein-coupled receptor highly expressed in the brain [START_REF] Kuohung | GPR54 and KiSS-1: Role in the regulation of puberty and reproduction[END_REF]) and found to be associated with GnRH neurons in different animals, including rat, mice and Nile tilapia (Irwing et al., 2004;[START_REF] Messager | Kisspeptin directly stimulates gonadotropin-releasing hormone release via G protein-coupled receptor 54[END_REF][START_REF] Parhar | Laser-captured single digoxigenin-labeled neurons of gonadotropin-releasing hormone types reveal a novel g protein-coupled receptor (Gpr54) during maturation in cichlid fish[END_REF].

Kiss1r has been linked to the regulation of the initiation of puberty in vertebrates [START_REF] Smith | Regulation of the neuroendocrine reproductive axis by kisspeptin-GPR54 signaling[END_REF][START_REF] Tena-Sempere | GPR54 and kisspeptin in reproduction[END_REF], although it may have additional physiological functions [START_REF] Makri | The kisspeptin (KiSS1-1)/Gpr54 system in cancer biology[END_REF][START_REF] Roa | New frontiers in kisspeptin/GPR54 physiology as fundamental gatekeepers of reproductive function[END_REF].

The analysis of the genomic structure of the Hh Kiss1r-2 gene revealed the presence of five exons and four introns, a gene organization that is conserved from teleost fish to amphibians and mammals [START_REF] Van Aerle | Evidence for the existence of a functional Kiss1/Kiss1 receptor pathway in fish[END_REF]. Comparison of the coding sequence with An exhaustive phylogenetic analysis of Kiss1r provided insights into its evolutionary history. The phylogenetic relationships described here (Figure 4) are in general in agreement with the evolutionary tree constructed in our previous study [START_REF] Mechaly | Identification of two isoforms of the kisspeptin-1 receptor (kiss1r) generated by alternative splicing in a modern teleost, the Senegalese Sole (Solea senegalensis)[END_REF], although a close inspection of the newer tree, which contains more species (36 vs 20 in [START_REF] Mechaly | Identification of two isoforms of the kisspeptin-1 receptor (kiss1r) generated by alternative splicing in a modern teleost, the Senegalese Sole (Solea senegalensis)[END_REF] reveals some unexpected relationships and suggests that gene duplications have occurred in this lineage. Placental mammalian Kiss1r sequences cluster tightly together (Kiss1r-1 cluster) with the marsupial and monotreme sequences branching from the base of this cluster. The observation that the platypus-1 and clawed frog-1A sequences share a well- Reptile and amphibian sequences cluster between these branches and distance analysis suggests that the clawed frog-2 and bullfrog sequences are most closely related to the main fish group (Kiss1r-2A group), while the clawed frog-1B and anole lizard sequences group with the three outlying medaka-1, zebrafish-B and goldfish-B sequences (Kiss1r-2B group).

Thus, Kiss1r-2B do not cluster with the Kiss1r-1 of mammals, as surmised earlier [START_REF] Biran | Molecular identification and functional characterization of the kisspeptin/kisspeptin receptor system in lower vertebrates[END_REF][START_REF] Li | Structural and functional multiplicity of the kisspeptin/GPR54 system in goldfish (Carassius auratus)[END_REF], but are an additional, non-mammalian form of Kiss1r. Our interpretation of Kiss1r evolution (Figure 4B) is that an ancestral gene duplication occurred prior to the emergence of fish, leading to the 2A and 2B groups, and that an additional duplication of the 2B gene, prior to the emergence of amphibians, resulted in the ancestor of clawed frog-1A. This latter gene appears to be the only Kiss1r version retained in higher vertebrates, assuming it is present in reptiles. A duplication of Kiss1r-1 appears to have occurred within the monotremes. Thus, the evolutionary relationships within the fish Kiss1r-2A group and within the placental mammalian Kiss1r-1 group does not reflect the expected organismal evolutionary relationships but rather the evolution of this gene lineage. In this regard, it would be interesting to know the structure and sequence of the Kiss1r-1 gene(s) in other vertebrates, including Sarcopterygian fishes. Also, as of today, the are still no avian Kiss1r sequences that can be identified in public databases, despite several attempts.

Among fish, many species appear to have lost Kiss1r-2B. Genome sequences from fugu, pufferfish and three-spined stickleback demonstrate the presence of only the 2A form of Kiss1r-2. Extensive searches with multiple degenerate primers have failed to detect a second Kiss1r-2 in Senegalese sole [START_REF] Mechaly | Identification of two isoforms of the kisspeptin-1 receptor (kiss1r) generated by alternative splicing in a modern teleost, the Senegalese Sole (Solea senegalensis)[END_REF] and Atlantic halibut (this study). These results suggest that Kiss1r-2B could be lost in some of the most derived teleost orders, including the Pleuronectiformes and Tetraodontiformes, as well as some other Acanthopterygian fish. It is not yet clear whether the loss of Kiss1r-2B was a single event in the evolution of fish or has independently occurred multiple times.

Recent studies have shown that Kiss1 also underwent a duplication in some fishes [START_REF] Felip | Evidence for two distinct KiSS genes in non-placental vertebrates that encode kisspeptins with different gonadotropin-releasing activities in fish and mammals[END_REF], a situation similar to the one described for the Kiss1r gene, whereas in mammals, with the exception of the platypus, only one Kiss1 gene is observed. Interestingly, the three teleost species in which two Kiss1 genes have been reported so far, i.e., zebrafish (Kitahashi et al., 2008), medaka (Kanda et al., 2009) and goldfish (Yang et al., 2009), also have two Kiss1r genes. To the best of our knowledge, there are no species with only one Kiss1 gene and two Kiss1r genes or vice versa.

This study also provides the first synteny analysis of the Kiss1r-2 region. Results

clearly support a common ancestry of fish Kiss1r-2 with subsequent rearrangement of the genes downstream of the three miRNA genes showing taxa-specific organization. The highly conserved nature of the Kiss1r-2 region suggests that the flanking regulatory sequences are also likely to be conserved. As a first attempt at defining potential transcription factor binding sites, bioinformatic analysis has identified six conserved regions in fish Kiss1r-2 upstream sequences, two of which show some similarity to known transcription factor binding sites. We are hesitant to suggest that these transcription factors bind to these sites and regulate Kiss1r-2 without further experimental analysis. However, they provide potential targets for future indepth investigation of Kiss1r-2 regulation in different species.

A c c e p t e d M a n u s c r i p t

Analysis by qPCR of Kiss1r expression in different developmental stages revealed no major differences in expression between male and female Atlantic halibut. A similar absence of sexual dimorphism in the expression of Kiss1r was detected in the brain of fathead minnow (Filby et al., 2008). The Hh Kiss1r-2 expression level in the brain was significantly higher in early puberty stages than the other stages investigated. A similar pattern of expression throughout development was observed in some teleost fish such as the fathead minnow (Filby et al., 2008) and for the zebrafish-A gene [START_REF] Biran | Molecular identification and functional characterization of the kisspeptin/kisspeptin receptor system in lower vertebrates[END_REF]. However, in other species such as Nile tilapia and cobia, Kiss1r expression continues to increase as puberty advances [START_REF] Parhar | Laser-captured single digoxigenin-labeled neurons of gonadotropin-releasing hormone types reveal a novel g protein-coupled receptor (Gpr54) during maturation in cichlid fish[END_REF]Mohamed et al., 2006).

The expression of Hh Kiss1r-2 in gonads of 1-year-old halibut was 400-fold lower than brain, which is in the same order of magnitude as the 100-fold difference observed in Nile tilapia (Martínez-Chávez et al., 2008), but higher than the 14-fold difference observed in fathead minnow (Filby et al., 2008) or the 10-fold difference observed in mature female grey mullet (Nocillado et al., 2006). Thus, it seems that regardless of species, the relationship between Kiss1r-2 expression in the brain and the gonads is in the range of 1-2 orders of magnitude. In the 3, 5 and 7 year classes, Ct values in gonads were too high to accurately determine Hh Kiss1r-2 expression levels, while the expression levels in ovaries and testis in the 1 year-old-class were not statistically different. Similar results were observed in studies carried out in rats, where the ovarian expression of Kiss1r did not fluctuate during ovulation [START_REF] Castellano | Expression of KiSS-1 in rat ovary: putative local regulator of ovulation?[END_REF]. However, in the Senegalese sole, the Kiss1r_v1 isoform showed higher expression in the testis than the ovaries [START_REF] Mechaly | Identification of two isoforms of the kisspeptin-1 receptor (kiss1r) generated by alternative splicing in a modern teleost, the Senegalese Sole (Solea senegalensis)[END_REF].

A c c e p t e d M a n u s c r i p t

In aquaculture, precocious sexual maturation, usually of males, has significant negative economic impact since early maturing fish are smaller and may have diminished flesh quality. Understanding the hormonal signals of puberty, their detection and the genes they regulate should provide tools to monitor the sexual maturation of fish at a molecular level and eventually develop management practices to avoid triggering this process. The isolation of Hh Kiss1r-2 and analysis of its gene expression will aid in understanding puberty in this species and provide insights into the evolution and regulation of this developmentally important gene.

In conclusion, in this study we have described the genomic organization of Atlantic halibut Kiss1r-2 gene. We identified only one Kiss1r gene, Hh Kiss1r-2, and proposed that the flatfish, as well as several other orders of fish, have lost the second gene during the evolution of teleost fish. Further, new phylogenetic relationships were proposed based on the analysis of all available Kiss1r sequences. Synteny analysis demonstrates a conserved gene order around the Kiss1r-2 locus in many teleosts and suggests the likelihood of conservation of regulatory regions. Furthermore, the concordant expression patterns of Senegalese sole Kiss1r_v1 and Atlantic halibut Kiss1r-2, which show higher expression in brain, demonstrates the importance of the kisspeptin-Kiss1r system in the brain, compared to the gonads, during early puberty. Abbreviations: b4galt2, β-1,4-galactosyltransferase 2; atp6v0b, V-type proton ATPase 21 kDa proteolipid subunit; hyp. prot., conserved hypothetical protein; miRNA, three predicted A c c e p t e d M a n u s c r i p t

ATGTTTTCCTCCGAGGAACCGTGGAACTCCACCGAGCAGGTCTGGCTCAATGGCTCGGAG 60 M F S S E E P W N S T E Q V W L N G S E AGGGCGAACTTCTCCCTGGGAAGACGCGGGAGCGACGACCACGAGGAGGAGGAGGACGGG 120 R A N F S L G R R G S D D H E E E E D G GATCAGCACCCCTTCCTCACAGACGCCTGGCTGGTGCCCCTCTTGTTCTCCCTCATCATG 180 D Q H P F L T D A W L V P L L F S L I M CTGGTGGGTGTGGTGGGCAACTCGCTGGTCATCTATGTCATCTCCAAACACAGGCAGATG 240 L V G V V G N S L V I Y V I S K H R Q M AGGACGGCGACTAACTTCTACGCAGCAAACCTGGCTGCCACTGACATCATCTTCTTGGTG 300 R T A T N F Y A A N L A A T D I I F L V TGCTGCGTCCCCTTCACTGCCACCCTCTACCCTCTCCCCGGATGGATCTTTGGCAACTTC 360 C C V P F T A T L Y P L P G W I F G N F ATGTGCAAGTTTGTCGCCTTTCTACAGCAGGTGACGGTCCAGGCCACTTGTATCACCCTG 420 M C K F V A F L Q Q V T V Q A T C I T L ACTGCCATGAGTGGAGACCGCTGTTACATCACCATCTACCCTCTGAAATCCCTCCGCCAC 480 T A M S G D R C Y I T I Y P L K S L R H CGCACCCCGAGAGTAGCCATGATCGTCAGCATCTGCATTTGGATCGGCTCCTTCATCCTG 540 R T P R V A M I V S I C I W I G S F I L TCCACCCCCATTTTGATGTACCAGCGCATAGAGGAGGGTTACTGGTACGGTCCCAGACAA 600 S T P I L M Y Q R I E E G Y W Y G P R Q TACTGCATGGAGAGATTTCCCTCTAAAACACACGAGAGGGCTTTCATCCTCTACCAGTTC 660 Y C M E

  of Atlantic halibut were obtained from Scotian Halibut Ltd.

  s c r i p t dGTP, dTTP, 50 pmol of each primer and 0.2 µl of rTaq DNA polymerase (G.E. Healthcare).

(

  XR_020820) opossum (Monodelphis domestica) (XM_001374715), platypus (platypus-1 Ultra_266:390150;395924; platypus-1B, Contig_15629:1100:6789), clawed frog (clawed frog-1A, EU85678; clawed frog-1B EU853679; clawed frog-2, zebrafish (zebrafish-A, ABV44612; zebrafish-B, ABV44613); goldfish (goldfish-A, ACK77792; goldfish-B, ACK77793); bullfrog (ACD44939), fathead minnow (ABV45419), Nile tilapia (BAD34454), Senegalese sole (ABW96362), grey mullet (ABG76790), cobia (ABG82165), Atlantic croaker (ABC75101) and Atlantic halibut (GQ330487). Additionally, sequences for several species were retrieved from genome sequences (http://www.ensembl.org): three-spined stickleback, pufferfish, fugu, lesser hedgehog[START_REF] Mechaly | Identification of two isoforms of the kisspeptin-1 receptor (kiss1r) generated by alternative splicing in a modern teleost, the Senegalese Sole (Solea senegalensis)[END_REF] and Anole lizard (Anolis carolinensis), pika (Ochotona princeps), guinea pig (Cavia porcellus), megabat (Pteropus vampyrus), microbat (Myotis lucifugus), kangaroo rat (Dipomomys ordii), gorilla (Gorilla gorilla), bushbaby (Otolemur garnettii), dolphin (Tursiops truncates), orangutan (Pongo pygmaeus) and mouse lemur

  et al., 2007) to establish their pedigree and sex was subsequently inferred from the inheritance of paternal alleles (D.Reid, M. Reith, unpublished). In this case, genomic DNA was extracted from fin clips (8-10 mg) using the Puregene DNA purification/extraction kit (Gentra Systems, USA) as per the solid-tissue extraction method. The standard PCR cocktail included: 10X high fidelity buffer (20mM Tris-HCl, 50 mM KCl, 1.5mM MgCl 2 ) (Larova GmbH, Germany), 200 μM of each dNTP; 0.4 to 0.8 pmol each of forward and reverse primer, 0.25 units of high fidelity Taq DNA polymerase (Larova GmbH, Germany) and 0.05 µl of BSA. The PCR reactions contained 9.5 µl (8.4 µl PCR cocktail and 1.1 µl of 50 ng/µl DNA) reactions in a 96 well polypropylene microplate and were run on a Techne TC-412 (Techne, USA). PCR conditions were an initial denaturing of 95 o C for 5 min, followed by 35 cycles of 95 o C for 30 s, 56 o C (for markers 3 and 5) or 58 o C (for markers 1, 2 and 4) for 30 s and 72 o C for 45 s and finally 5 min. at 72 o C. Markers 1, 2 and 4 were run in a single reaction and markers 3 and 5 were run in a second reaction. For each sample, 1.5 µl of the first PCR and 2.5 µl of the second PCR were combined with 35 µl CEQ sample loading solution and 0.5 µl of internal DNA size standard (CEQ) into a CEQ sample plate. Pedigree analysis was A c c e p t e //www.uoguelph.ca/~rdanzman/software/PROBMAX/) using previously collected genotypes from potential parents.2.9. Determination of Hh Kiss1r mRNA levels in brain and gonadsThe expression pattern of Hh Kiss1r was analyzed in whole brain and ovaries and testis from four different year-classes (1, 3, 5 and 7 years old) by real-time quantitative PCR (qPCR). RNA was isolated as indicated above and it was further purified with a RNA cleanup column (Qiaprep, Qiagen) and a second DNAse treatment, in which 20 µg of purified RNA were treated with 2 µl of DnaseI (Ambion) and 1 µl of DnaseI Reaction Buffer in a final volume of 25 µl. Aliquots of the RNA were analyzed with the Agilent 2100 Bioanalyzer (Agilent Technologies) to check the quality of the samples. The Hh Kiss1r primers used for qPCR were located in exons 3 and 4 and designed to span intron III (RT Hh Kiss1r-F2 and

  A c c e p t e d M a n u s c r i p t as E = 10 (-1/slope). The amplification efficiencies were 1.959 and 1.989 for the Hh Kiss1r and β-actin, respectively. The slopes of the curve were -3.424 and -3.349 and the melting temperatures (T M ) were 86.4 and 82, respectively. A single peak was obtained from each gene during melt curve analysis, demonstrating specificity of the primers. PCR products were resolved on 1% agarose gel stained with ethidium bromide to verify the expected size of the band.Data were analyzed using the comparative Ct method[START_REF] Bustin | Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays[END_REF] (also known as the DDCt method) to calculate relative changes in gene expression using the Light Cycler 480 Software (release 1.5.0, Roche). Fold change (or relative quantification, RQ) was calculated from the DDCt. The LightCycler Relative Quantification Software (Roche) was used for determining calibrator-normalized target/reference ratios with automated efficiency-corrected quantification according to the recommendations of the manufacturer. Fold change was normalized to the endogenous reference gene β-actin. Determinations were carried out in technical triplicates for the Hh Kiss1r and β-actin genes. The RQ values for each sample were averaged and the standard error was calculated, yielding the average fold change of the target gene.2.10. Statistical analysesThe results are presented as mean ± standard error of the mean (S.E.M). Statistical analyses were performed using the SPSS v16 Software. The significance of pairwise comparisons of the mRNA levels of Hh Kiss1r in brain of males and females was determined using a Student's t-test. Expression levels of Hh Kiss1r in brain during maturation were analyzed by a one-way ANOVA. Differences were accepted as significant when P<0.05.

  Cloning and sequencing of the full-length Hh Kiss1r-2 gene Using degenerate primers located within exon 5, a 202 bp fragment of Hh Kiss1r-2 was obtained by PCR. The full-length Hh Kiss1r-2 gene was obtained by screening a BAC library with an overgo probe based on the initial Hh Kiss1r-2 partial sequence. Two positive clones, both containing the complete Hh Kiss1r-2, were isolated, purified and sequenced.

  1,146 bp for the five exons of the open reading frame and 8,192 bp for the four introns(2,425, 5,308, 94 and 365 bp for introns I, II, III and IV, respectively). A putative poly-A signal, AAAAAAACAAAAA, was found in the presumptive 3′ UTR. Alignment of the Hh Kiss1r-2 sequence with those of other teleost fish demonstrated a high degree of nucleotide identity (> 86%). The sizes of the five exons of the Kiss1r-2 gene from the two pleuronectiformes fishes, Atlantic halibut and Senegalese sole were very similar in length, whereas the sizes of the introns varied (Table2). Translation of the complete Hh Kiss1r-2 coding region predicts a protein that has a high degree of sequence similarity to Kiss1r proteins from other fish. As in other Kiss1r, seven transmembrane domains were detected (Figure1 and

  a single group. Monotreme (platypus) and marsupial (opossum) mammalian Kiss1r-1 sequences are represented by branches at the base of this cluster.

  Expression of Hh Kiss1r-2 in brain and gonads during development Real time PCR (qPCR) was used to quantify the expression of Hh Kiss1r-2 in brain

  a high degree (>90%) of homology. Comparison to the gene organization in a closely related species, the Senegalese sole[START_REF] Mechaly | Identification of two isoforms of the kisspeptin-1 receptor (kiss1r) generated by alternative splicing in a modern teleost, the Senegalese Sole (Solea senegalensis)[END_REF], revealed substantial length differences in introns II and IV. Interestingly, intron III of Atlantic halibut and Senegalese sole exhibited a considerable degree of sequence conservation (50% identity) and a similar length, 94 and 81 bp, respectively. In Senegalese sole, intron III missplicing results in a second isoform with a different expression pattern than the full-length form. Similar to the Senegalese sole, the Hh Kiss1r-2 intron III has a branch point with low sequence conservation compared to the consensus sequence. Despite numerous attempts, alternative Kiss1r isoforms could not be detected in a variety of Atlantic halibut tissues. However, since not all tissue-developmental stages combinations could be tested, alternative splicing of Hh Kiss1r-2 remains a possibility. Alternative splicing of Kiss1r has also been detected at intron I in sheep (Genbank accessions EU272411 and EU272412) and with the deletion of the entire TMD5 in mouse (EU532436 and EU532437), both of which result in proteins with altered TMDs. Alternative splicing may provide an additional mechanism for regulating the expression of Kiss1r or for directing it to an alternative subcellular location.

  suggests that the clawed frog-1A Kiss1r shares a common ancestor with the mammalian Kiss1r. Most of the fish sequences cluster in a tight group at the end of the tree while the medaka-1, zebrafish-B and goldfish-B are located on a separate branch.

  . A.,Taranger, G. L., Karlsen, O., Norberg, B., 2002. Spermatogenesis and related plasma androgen levels in Atlantic halibut (Hippoglossus hippoglossus L.

Figure 1 .

 1 Figure Legends

Figure 2 .

 2 Figure 2. Amino acid alignment of Atlantic halibut (Hh Kiss1r-2) with selected fish Kiss1r-2

Figure 3 .

 3 Figure 3. Synteny of Kiss1r-2 in teleost fish. Genomic organization of the zebrafish,

  -23a, miR-27a, miR-24); b0s7g2, novel kinesin-like protein; atp5L, ATP synthase subunit g; rabL5, rab-like protein 5; odf2L, sperm tail outer dense fiber protein 2like.

Figure 4 .

 4 Figure 4. Phylogenetic and evolutionary relationships of vertebrate Kiss1r sequences. (A)

Figure 5 .

 5 Figure 5. Conserved upstream regions in fish Kiss1r-2. The positions of six conserved

Figure 6 .

 6 Figure 6. Essential biometry and gonadal development in Atlantic halibut. Total length (TL)

Figure 7 .

 7 Figure 7. Tissue distribution of Hh Kiss1r-2 at two developmental stages, early puberty (three

Figure 8 .

 8 Figure 8. Hh Kiss1r-2 expression in (A) brain and (B) gonads from different year-class fish

  Figure1.[START_REF] Mechaly | Identification of two isoforms of the kisspeptin-1 receptor (kiss1r) generated by alternative splicing in a modern teleost, the Senegalese Sole (Solea senegalensis)[END_REF]

Table 1 .

 1 ). Comp. Degenerate and specific primers used in the Hh Kiss1r-2 cloning, screening, Intron I isolation, qPCR and RT-PCR

	Biochem. Physiol. A Mol. Integr. Physiol. 132, 567-575.
	Wingender, E., Dietze, P., Karas, H., Knuppel, R., 1996. TRANSFAC: A database on
	transcription factors and their DNA binding sites. Nucl. Acids Res. 24, 238-241.
	Yang, B., Jiang, Q., Chan, T., Ko, W. K., Wong, A. O. 2009. Goldfish kisspeptin: Molecular
	cloning, tissue distribution of transcript expression, and stimulatory effects on
	prolactin, growth hormone and luteinizing hormone secretion and gene expression via
	direct actions at the pituitary level. Gen. Comp. Endocrinol. doi;
	10.1016/j.ygcen.2009.06.001.

Table 2 .

 2 Intron and exon size in Atlantic halibut Kiss1r-2 gene (Hh Kiss1r-2) and Senegalese sole (Ss Kiss1r)

		Hh Kiss1r-2	Ss Kiss1r
	Exon 1	261 bp	252 bp
	Intron I	2.5 kb	2.5 kb
	Exon 2	129 bp	129 bp
	Intron II	5.3 kb	0.2 kb
	Exon 3	136 bp	131 bp
	Intron III	0.9 kb	0.8 kb
	Exon 4	239 bp	244 bp
	Intron IV	0.4 kb	1.3 kb
	Exon 5	381 bp	381 bp

Acknowledgements

Thanks are due to B. Blanchard and the staff of Scotian Halibut Ltd. for providing fish, D. Reid for help with the fish sampling and for the microsatellite analysis, C. Leggiadro for help with the histology and to Dr. L. C. Knickle for technical advice on qPCR.

A.S.M. was supported by a predoctoral scholarship from the Spanish Ministry of Education and Science (MEC)


Minna Jääskeläinen

Renata Prunskaite-Hyyryläinen

Florence Naillat

Helka Parviainen

Mikko Anttonen

Markku Heikinheimo

Annikki Liakka

Roxana Ola

Seppo Vainio

Tommi E Vaskivuo

Juha S Tapanainen
email: juha.tapanainen@oulu.fi

WNT4 is Expressed in Human Fetal and Adult Ovaries, and Its Signaling Contributes to Ovarian Cell Survival

Keywords: WNT4, apoptosis, oocyte, granulosa cell, (human ovary)

WNT4 plays an important role in female sexual development and ovarian function. WNT4deficiency leads disturbed development of the internal genitalia in mouse and human, and to a dramatic reduction of mouse oocytes. However, the expression and role of WNT4 in human ovaries is yet unknown. The expression of WNT4 mRNA and protein was studied in human adult and fetal ovaries (gestational ages 12-41 weeks), and the role of Wnt4 in oocyte apoptosis was investigated in Wnt4-deficient mice. WNT4 mRNA and protein were present in human ovaries throughout fetal development and in different follicular stages in adult ovaries. Compared with wild-type mice, Wnt4-deficient mice had a markedly enhanced rate of oocyte apoptosis, with the highest values at gestational ages of 14.5 and 16.5 days post-coitum. The current results support a view that WNT4 may have a role in oocyte selection and follicle formation and maturation in human ovaries.

Introduction:

The locally acting excreted glycoprotein WNT4 is a member of the WNT (wingless-type MMTV integration site family) family of extracellular ligands [START_REF] Cadigan | Wnt signaling: a common theme in animal development[END_REF][START_REF] Vainio | Female development in mammals is regulated by Wnt-4 signalling[END_REF]. The action of the WNT4 ligand is transmitted through receptors located on the cellular membrane, most importantly those of the Frizzled family [START_REF] Cadigan | Wnt signaling: complexity at the surface[END_REF]. This canonical Wnt signaling pathway eventually leads to nuclear accumulation of β-catenin and transcriptional regulation of target genes [START_REF] Cadigan | Wnt signaling: complexity at the surface[END_REF]. Wnt genes have been highly conserved throughout evolution and Wnt signaling has been observed to play a role in a wide variety of physiological and pathological events from embryogenesis to the onset and development of cancer.

Wnt4 has an important role in female sexual differentiation [START_REF] Vainio | Female development in mammals is regulated by Wnt-4 signalling[END_REF][START_REF] Jeays-Ward | Endothelial and steroidogenic cell migration are regulated by WNT4 in the developing mammalian gonad[END_REF][START_REF] Kim | Fgf9 and Wnt4 act as antagonistic signals to regulate mammalian sex determination[END_REF][START_REF] Ottolenghi | Loss of Wnt4 and Foxl2 leads to female-to-male sex reversal extending to germ cells[END_REF]. It is downregulated in male gonads after the initiation of testicular development (11.5 days post-coitum [dpc]) while it is persistent in the developing ovary [START_REF] Vainio | Female development in mammals is regulated by Wnt-4 signalling[END_REF]. Mice with homozygous mutation in the Wnt4 gene have severely disturbed development of the internal genitalia and ovaries. Wnt4-deficient ovaries are masculinized, with a round and non-encapsulated form and they develop in close association with a fat body structure. Female mutant mice lack Müllerian ducts and have male-type Wolffian ducts instead [START_REF] Vainio | Female development in mammals is regulated by Wnt-4 signalling[END_REF]. In addition to their masculinized appearance, mutant ovaries express many Leydig and Sertoli cell markers and secrete testosterone and Müllerian-inhibiting substance (MIS) [START_REF] Heikkila | Wnts and the female reproductive system[END_REF][START_REF] Heikkila | The partial female to male sex reversal in Wnt-4-deficient females involves induced expression of testosterone biosynthetic genes and testosterone production, and depends on androgen action[END_REF].

WNT4 plays an important role also in human sex determination. Female patients with loss-of-function mutation in the WNT4 gene have developmental abnormalities mimicking the phenotype observed in Wnt4 mutant mice. They are partially masculinized, with clinical features of primary amenorrhea, androgen excess, regressed Müllerian duct derivatives and anomalous location of the ovaries [START_REF] Biason-Lauber | A WNT4 mutation associated with Mullerian-duct regression and virilization in a 46,XX woman[END_REF][START_REF] Biason-Lauber | WNT4 deficiency--a clinical phenotype distinct from the classic Mayer-Rokitansky-Kuster-Hauser syndrome: a case report[END_REF].

By the time of birth, the number of oocytes in Wnt4 mutant mouse ovaries is abnormally low [START_REF] Vainio | Female development in mammals is regulated by Wnt-4 signalling[END_REF]). Wnt4 has not been shown to affect the number of primordial germ cells in the undifferentiated gonad, but it is thought to act as an oocyte survival factor during female embryogenesis [START_REF] Vainio | Female development in mammals is regulated by Wnt-4 signalling[END_REF][START_REF] Heikkila | Wnts and the female reproductive system[END_REF]. The massive germ cell loss in Wnt4-deficient mice occurs most rapidly at 16.5 dpc, when more than 90% of the oocytes are being depleted [START_REF] Yao | Follistatin operates downstream of Wnt4 in mammalian ovary organogenesis[END_REF]. At this time, apoptotic oocytes can be detected in the central region of both wild-type and mutant ovaries, but in contrast to the normal ovary, rapid apoptosis takes place in the ovarian cortices of Wnt4-deficient mice [START_REF] Yao | Follistatin operates downstream of Wnt4 in mammalian ovary organogenesis[END_REF].

During normal ovarian development, Bcl-2 family genes are central in the regulation of oocyte apoptosis. The family contains several anti-and pro-apoptotic factors which have been shown to be important in the regulation of apoptosis in mouse ovaries. Oocytes of BclX L -deficient mice undergo massive apoptosis [START_REF] Rucker | Bcl-x and Bax regulate mouse primordial germ cell survival and apoptosis during embryogenesis[END_REF], while knockout of pro-apoptotic Bax results in a decreased rate of oocyte apoptosis [START_REF] Perez | Prolongation of ovarian lifespan into advanced chronological age by Bax-deficiency[END_REF][START_REF] Taketo | Switch from BAX-dependent to BAX-independent germ cell loss during the development of fetal mouse ovaries[END_REF]). In addition to the Bcl-2 family, numerous other factors are associated with the regulation of ovarian apoptosis. For instance, TRAIL, a tumor necrosis factor-related apoptosis-inducing ligand, has been shown to participate in the regulation of apoptosis in mammalian ovaries [START_REF] Inoue | Roles of tumor necrosis factor-related apoptosis-inducing ligand signaling pathway in granulosa cell apoptosis during atresia in pig ovaries[END_REF][START_REF] Jaaskelainen | TRAIL pathway components and their putative role in granulosa cell apoptosis in the human ovary[END_REF]. Moreover, several transcriptional factors such as GATA-4 and its co-factor FOG-2 participate in the regulation of oocyte survival in mice and men [START_REF] Heikinheimo | Expression and hormonal regulation of transcription factors GATA-4 and GATA-6 in the mouse ovary[END_REF][START_REF] Vaskivuo | Survival of human ovarian follicles from fetal to adult life: apoptosis, apoptosis-related proteins, and transcription factor GATA-4[END_REF][START_REF] Anttonen | FOG-2 and GATA-4 Are coexpressed in the mouse ovary and can modulate mullerian-inhibiting substance expression[END_REF].

In the present work the expression of WNT4 mRNA and protein was studied for the first time during human fetal development and human adult follicular maturation. In addition, apoptosis and the expression of key apoptosis-regulating factors in Wnt4-deficient and in wild-type mouse ovaries were analyzed in detail to evaluate the contribution of Wnt4 signaling to survival of the maturing oocytes.

Human ovarian tissue samples

Fetal ovarian tissue samples were obtained from 7 fetuses (aged 12-23 wk) after spontaneous or therapeutic abortion and from 9 fetuses (aged 22-41 wk) after intrauterine death followed by spontaneous or induced delivery or cesarean section. In addition, ovaries from 6 neonates (gestational age 23, 26, 27, 31, 35 and 38 weeks) who died because of perinatal asphyxia or infection within 30 min-3 days after birth were studied. The obduction data of all female fetuses during the years 2000-2007 at Oulu University Hospital were examined and on the basis of the information all samples with chromosomal abnormalities, significant anomalies or autolysis were excluded. Thereafter, a pediatric pathologist analyzed the morphology of the remaining samples, and samples with signs of autolysis in HE-staining were further excluded. Three fetuses included in the study were diagnosed as having chorioamnionitis. However, their ovaries were morphologically intact and did not have any signs of infection. All fetuses and neonates had normal karyotypes. Adult ovarian tissues were obtained from 19 premenopausal patients aged 22-49 years undergoing ovariectomy because of endometriosis. In addition, human adrenal gland and testis were used as control tissues. All samples were fixed in 4% buffered formaldehyde for 24 hours and embedded in paraffin. Histological sections (4 μm) were cut and processed for immunohistochemistry and in situ hybridization. The study was approved by the Ethics Committees of Oulu University Hospital and the University of Oulu. A permit to study human autopsy tissues and resection material was obtained from the Finnish National Authority for Medicolegal Affairs.

Generation of Wnt4 mutant mice:

Wnt4 +/-mice in SV129 background were crossed to obtain -/-embryos and the genotypes were analyzed as described earlier [START_REF] Heikkila | The partial female to male sex reversal in Wnt-4-deficient females involves induced expression of testosterone biosynthetic genes and testosterone production, and depends on androgen action[END_REF][START_REF] Stark | Epithelial transformation of metanephric mesenchyme in the developing kidney regulated by Wnt-4[END_REF]. Female wild-type and Wnt4-deficient mouse embryos aged 12.5, 14.5 and 16.5 dpc and newborn animals were studied.

Immunohistochemistry:

Immunohistochemistry for WNT4 was performed on human ovarian paraffin-embedded tissues. Paraffin sections were first incubated at 37 ºC for 30 min and then at 60 ºC for 30 min. Deparaffinization was carried out in xylene and the tissues were hydrated gradually through a series of graded alcohols. Pretreatment in Tris-HCl in a microwave oven (700 W for 2 min and 300 W for 15 min) was performed. Tissues were then cooled at RT for 20 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol. The sections were then washed in dH 2 O and PBS. Diluted normal serum provided with the immunostaining kit (Vectastain Elite ABC Kit, Vector laboratories, Burlingame, CA) was used to block nonspecific binding sites. Primary antibody against WNT4 protein (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; cat. no. sc-5214) at a concentration of 1:50 in background-reducing antibody diluent (Dako, Glostrup, Denmark) was added to the samples and they were incubated under plastic cover slips in a humidified chamber overnight at +4 ºC. A commercially available avidin-biotin immunoperoxidase system was used to visualize bound antibodies (Vectastain Elite ABC Kit, Vector laboratories, Burlingame, CA).

Immunohistochemistry involving mouse ovarian tissues was performed following a standard protocol. Primary antibodies against Bcl-X L (1:800), Bok (1:100), TRAIL (1:50), GATA-4 (1:200) and FOG-2 (1:200) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; cat. nos. sc-7122, sc-11424, sc-6079, sc-1237 and sc-10755, respectively) and aromatase (1:500) (Acris Antibodies, Hiddenhausen, Germany, cat. no. BP278), diluted in PBS, were used. A commercially available avidin-biotin immunoperoxidase system was used to visualize bound antibodies (Vectastain Elite ABC Kit, Vector laboratories, Burlingame, CA). Probes for WNT4 in situ hybridization were prepared from a commercially available WNT4 cDNA clone in pBluescriptR vector (Image clone 30330746, Genbank accession no BC057781, MCR geneservice, Cambridge, UK). Human tissue sections were hybridized with single-stranded antisense and sense RNA probes of human WNT4. The plasmid containing WNT4 cDNA was linearized with EcoRI for the antisense and BamHI for the sense RNA probes. The RNA probes were labeled with digoxigenin-UTP by in vitro transcription with T3/T7 polymerase using a DIG RNA Labeling Kit (Roche Diagnostics GmbH, Mannheim, Germany). 200 ng of the probe was used in the hybridization, and the sense probe was used as a negative control. In situ hybridization was carried out with Ventana Discovery automatic staining instrument using commercial buffers (Roche Diagnostics GmbH, Mannheim, Germany). The DIG labelled probe was detected with monoclonal biotinylated anti-digoxigenin antibody (Jackson ImmunoResearch Laboratories Inc., PA) used at 1:2000 dilution and with the BlueMap kit (Roche Diagnostics GmbH, Mannheim, Germany).

In situ DNA 3´ end-labeling:

Apoptosis was studied by using an Apoptag ® Peroxidase In Situ Apoptosis Detection Kit (Millipore, Billerica, MA) as previously described [START_REF] Vaskivuo | Survival of human ovarian follicles from fetal to adult life: apoptosis, apoptosis-related proteins, and transcription factor GATA-4[END_REF]. The instructions of the manufacturer were followed. Briefly, paraffin-embedded tissues were deparaffinized in xylene and rehydrated through a series of graded alcohols. The tissues were pretreated in proteinase K (4 mg/ml) and 3% hydrogen peroxide before applying the enzyme deoxynucleotidyl transferase (TdT) to the samples. Incubation took place under plastic cover slips in a humidified chamber at 37 ºC for one hour. Bound enzyme was visualized with anti-digoxigenin antibody and diaminobenzidine tetrahydrochloride (15 μl/ml) (DAB, DakoCytomation Ltd., Ely, UK). The samples were counterstained with hematoxylin.

Sample analysis:

Immunohistochemical and in situ hybridization stainings were analyzed by two independent observers. For analysis of apoptosis the ovarian samples from Wnt4-deficient and wild-type animals were photographed using Qimaging MicroPublisher 5.0 RTV and Qcapture-Pro software. The size of each tissue section was then measured (ImageJ program) and apoptotic cells were counted and the results adjusted to section size. The data was analysed by using SPSS 15.0 software (SPSS, Chicago, IL, USA). The data is presented as arithmetic mean plus SD. Student's t test were used for analyses of signicance and a value of p < 0.05 was considered significant.

A c c e p t e d M a n u s c r i p t 6

Results

WNT4 mRNA and protein are expressed in human fetal ovaries

The in situ hybridization analyses showed strong expression of WNT4 mRNA in human ovaries at all fetal ages studied. Both granulosa cells and oocytes expressed WNT4 mRNA (Fig. 1 a). Similarly, immunostaining of WNT4 protein was detected in human ovaries throughout fetal development (Fig. 2A a-h). During early ovarian development (12-18 weeks), moderate WNT4 protein expression was mainly localized to pregranulosa and granulosa cells and only weak protein expression could be detected in oocytes, recognized by their round appearance and large amount of cytoplasm (Fig. 2A, a andb). As follicle formation proceeded, staining intensity tended to increase, but more samples would be needed to confirm this observation (Fig 2A c-f). During the latter half of fetal development strong WNT4 mRNA and protein expression was observed mainly in oocytes, but granulosa cells in primordial and primary follicles were also moderately stained (Figs. 1 a; 2A, e-h).

WNT4 mRNA and protein are present during follicular maturation in human adult ovaries WNT4 mRNA was expressed in granulosa cells and oocytes of primordial/primary and secondary follicles (Fig. 1 c) and in granulosa and theca cells of the antral follicles (Fig. 1 e). Immunohistochemical staining of WNT4 protein was observed at all stages of follicular maturation. In small primordial/primary follicles the oocytes were strongly positive for WNT4 (Fig. 2B,a). Greater magnification showed that granulosa cells in these follicles were also positively stained (Fig. 2B,c). Interestingly, in antral follicles, moderate WNT4 staining was detected in theca cells while granulosa cells were stained weakly (Fig. 2B,e). In all samples staining was located mainly in the cytoplasm. Human adrenal gland and testis were used as control tissues in immunohistochemistry and in situ hybridizations. Adrenal gland showed strong WNT4 mRNA and protein expression (Fig. 1 g; 2B, g and h). Only weak expression was observed in human adult testis, mainly localized to Leydig cells (Fig. 1 i; 2B, i and j). Similar Wnt4 expression pattern has been previously detected in marsupial adult testis [START_REF] Yu | Differential expression of WNT4 in testicular and ovarian development in a marsupial[END_REF].

Increased oocyte apoptosis in Wnt4-deficient mice

Apoptosis was detected in the ovaries of fetal and newborn wild-type and mutant mice (Fig. 3, A-H). Most of the apoptotic cells presented typical morphological features of germ cells including large size and round cytoplasm. In both genotypes the apoptosis rate increased after mid-pregnancy, reaching a maximum at 16.5 dpc (Fig. 3, E andF) and then declining towards birth. When comparing the numbers of apoptotic cells in Wnt4 mutant and wild-type mice, a significantly higher number of apoptotic cells was observed in Wnt4-deficient ovaries at 14.5 dpc (Fig. 3, C andD) and the difference was most pronounced at 16.5 dpc (Fig. 3, E andF). In the ovaries of newborn mice the rate of apoptosis was similar in Wnt4 wild-type and mutant mice (Fig. 3, G andH).

Apoptosis regulating factors in Wnt4-deficient mouse ovaries

Immunohistochemical staining of Bcl-X L, Bok, TRAIL, GATA-4, FOG-2 and aromatase showed that these proteins were similarly expressed in the ovaries of mutant and wild-type mice during fetal development (Fig 4). Expression of the pro-apoptotic Bcl-2 family member Bok was restricted mainly to oocytes ( In the present study we demonstrate the expression of WNT4 mRNA and protein in human ovaries during fetal development and fertile life. These results support the role of WNT4 in human ovarian development and its importance in normal ovarian function [START_REF] Biason-Lauber | A WNT4 mutation associated with Mullerian-duct regression and virilization in a 46,XX woman[END_REF][START_REF] Biason-Lauber | WNT4 deficiency--a clinical phenotype distinct from the classic Mayer-Rokitansky-Kuster-Hauser syndrome: a case report[END_REF]). The expression of WNT4 protein in human fetal ovaries was high during mid-pregnancy, when new follicles are also rapidly being formed. This implies that WNT4 may have a substantial role in regulation of the follicle formation in human ovaries, as also demonstrated in Wnt4-deficient animals, which lack proper follicle structures [START_REF] Vainio | Female development in mammals is regulated by Wnt-4 signalling[END_REF]. Moreover, WNT4 mRNA and protein were present in ovaries later during fetal life and in adult ovaries, supporting a view that WNT4 may also have a role in follicular development and maturation in human ovaries [START_REF] Philibert | Identification and functional analysis of a new WNT4 gene mutation among 28 adolescent girls with primary amenorrhea and mullerian duct abnormalities: a French collaborative study[END_REF].

Wnt4 mRNA has previously been shown to be expressed in rodent ovaries during fetal development [START_REF] Vainio | Female development in mammals is regulated by Wnt-4 signalling[END_REF][START_REF] Barrionuevo | Homozygous inactivation of Sox9 causes complete XY sex reversal in mice[END_REF]) and its expression increases in young females after the primordial follicles are formed [START_REF] Hsieh | Regulated expression of Wnts and Frizzleds at specific stages of follicular development in the rodent ovary[END_REF]. In human ovaries a somewhat similar tendency was observed. WNT4 was detected throughout the fetal development and the strongest expression of WNT4 took place after follicular formation when oocyte apoptosis starts to decrease [START_REF] Vaskivuo | Survival of human ovarian follicles from fetal to adult life: apoptosis, apoptosis-related proteins, and transcription factor GATA-4[END_REF]. The observed WNT4 expression pattern in human ovaries and the markedly increased apoptosis rate in Wnt4deficient mouse ovaries suggest that WNT4 may be an important factor in the selection of human follicles that either survive or become deleted from the ovary.

In adult human ovaries WNT4 mRNA and protein were expressed in the small primary and growing secondary follicles. Antral follicles expressed WNT4 variably and in addition to granulosa cells the theca cells surrounding the follicles were also positive. This suggests that WNT4 may be involved in the regulation of all cell compartments. Similarly to our finding in human ovaries, Wnt4 mRNA is expressed in small growing follicles and in preovulatory and ovulatory follicles in mouse ovaries [START_REF] Hsieh | Regulated expression of Wnts and Frizzleds at specific stages of follicular development in the rodent ovary[END_REF].

In adult and fetal human ovaries WNT4 mRNA and protein were detected both in the granulosa cells and in the oocytes. There has not been general agreement on the cellular localization of WNT4 in the ovary. The results of previous studies on mice suggest that Wnt4 is expressed in granulosa cells rather than germ cells [START_REF] Vainio | Female development in mammals is regulated by Wnt-4 signalling[END_REF][START_REF] Yu | Differential expression of WNT4 in testicular and ovarian development in a marsupial[END_REF]). On the other hand, Wnt4 mRNA has been detected in oocytes in postnatal chick ovaries [START_REF] Oreal | Different patterns of anti-Mullerian hormone expression, as related to DMRT1, SF-1, WT1, GATA-4, Wnt-4, and Lhx9 expression, in the chick differentiating gonads[END_REF]. Although oocytes are known to show nonspecific immunostaining, the present results raise the possibility that WNT4 expression also takes place in human germ cells. However, comparison of results from mice and humans might also point towards species-specific differences in the expression pattern of Wnt4.

The expression of apoptosis-regulating factors was studied in the ovaries of Wnt4-deficient and wild-type mice by immunohistochemistry. Several analyses were performed with primary antibodies against TRAIL, Bok, Bcl-X L , GATA-4, FOG-2 and aromatase. No significant differences were observed in the expression patterns of these proteins between Wnt4-deficient and wild-type animals. Thus our results suggest that increased apoptosis in the absence of Wnt4 action is not directly caused by a significant change in the expression levels of the factors known to regulate apoptosis in ovaries and other organs. Further studies are, however, needed to comprehend fully the mechanisms of WNT4 in regulating apoptosis in ovaries.

To conclude, WNT4 mRNA and protein are present in human fetal and adult ovaries and ovarian apoptosis is significantly enhanced in Wnt4 mutant mice when compared with wild-type animals. The results support a view that WNT4 may have a role of in the regulation of oocyte survival, follicle formation and follicle maturation in human ovaries. More robust studies on the molecular mechanisms are needed to understand the protective role of WNT4 in oocyte demise. In adult human ovaries WNT4 mRNA was expressed in oocytes and granulosa cells of primordial/primary follicles (c). In growing antral follicles strong WNT4 labeling was detected in granulosa and theca cells (e). Sense controls (d and f). Human adrenal gland tissue was used as positive control and showed strong WNT4 expression (g). Weak WNT4 mRNA expression could also be detected in Leydig cells of adult human testes (i). Sense controls (h and j). . WNT4 protein expression in human fetal (A) and adult (B) ovaries. WNT4 protein was detected in human ovaries at all fetal ages studied. During early development, staining was mainly present in granulosa cells, while the oocytes (with round shape and a large cytoplasm) showed weak expression (A: a and b). From fetal age 19 weeks onwards, staining intensity in oocytes tended to increase (A: c and d). After midgestation strong/moderate WNT4 protein expression was detected in most of the follicle-encapsulated oocytes, and granulosa cells were also moderately stained (A; e-h). Negative control using PBS instead of primary antibody (A: i and j). Staining of WNT4 was intense in primary and secondary follicles of adult human ovaries and was detected mainly in oocytes, but also granulosa cells were moderately stained (B: a and c). In antral follicles granulosa cells were weakly stained and moderate staining was observed in theca cells (B: e). Negative control using PBS instead of primary antibody (B: b, d and f). Human adrenal gland (B: g and h) and testis (B: i and j) were used as control tissues.

  Fig 4, e and f). In both genotypes Bok was observed in the oocytes of the medullar and cortex regions of the ovary. The cytokine-like apoptosis-inducing ligand TRAIL (Fig 4, a and b), the antiapoptotic mitochondrial membrane protein Bcl-X L (Fig 4, c and d) and aromatase (Fig 4, k and l) were expressed in oocytes and somatic cells in both Wnt4-deficient and wild-type ovaries throughout fetal development. GATA-4 (Fig 4, g and h) and its cofactor FOG-2 (Fig 4, i and j) were expressed mainly in pregranulosa and granulosa cells of mutant and wild-type ovaries.

Figure 1 .

 1 Figure LegendsFigure 1. WNT4 mRNA expression in human fetal and adult ovaries. WNT4 mRNA expression was observed in all of the fetal ovaries studied (a). Sense control (b).In adult human ovaries WNT4 mRNA was expressed in oocytes and granulosa cells of primordial/primary follicles (c). In growing antral follicles strong WNT4 labeling was detected in granulosa and theca cells (e). Sense controls (d and f). Human adrenal gland tissue was used as positive control and showed strong WNT4 expression (g). Weak WNT4 mRNA expression could also be detected in Leydig cells of adult human testes (i). Sense controls (h and j). Blue color indicates mRNA expression. *= Oocyte, arrows indicate granulosa cells. Scale bar 100 μm.

  Figure 1. WNT4 mRNA expression in human fetal and adult ovaries. WNT4 mRNA expression was observed in all of the fetal ovaries studied (a). Sense control (b).In adult human ovaries WNT4 mRNA was expressed in oocytes and granulosa cells of primordial/primary follicles (c). In growing antral follicles strong WNT4 labeling was detected in granulosa and theca cells (e). Sense controls (d and f). Human adrenal gland tissue was used as positive control and showed strong WNT4 expression (g). Weak WNT4 mRNA expression could also be detected in Leydig cells of adult human testes (i). Sense controls (h and j). Blue color indicates mRNA expression. *= Oocyte, arrows indicate granulosa cells. Scale bar 100 μm.

  Figure 2. WNT4 protein expression in human fetal (A) and adult (B) ovaries. WNT4 protein was detected in human ovaries at all fetal ages studied. During early development, staining was mainly present in granulosa cells, while the oocytes (with round shape and a large cytoplasm) showed weak expression (A: a and b). From fetal age 19 weeks onwards, staining intensity in oocytes tended to increase (A: c and d). After midgestation strong/moderate WNT4 protein expression was detected in most of the follicle-encapsulated oocytes, and granulosa cells were also moderately stained (A; e-h). Negative control using PBS instead of primary antibody (A: i and j). Staining of WNT4 was intense in primary and secondary follicles of adult human ovaries and was detected mainly in oocytes, but also granulosa cells were moderately stained (B: a and c). In antral follicles granulosa cells were weakly stained and moderate staining was observed in theca cells (B: e). Negative control using PBS instead of primary antibody (B: b, d and f). Human adrenal gland (B: g and h) and testis (B: i and j) were used as control tissues. Strong staining was detected in adrenals while in human testis tubular cells showed only negligible and Leydig cells moderate staining. Brown color indicates protein expression. *= Oocyte, arrows indicate granulosa cells. Scale bar 100 µm.

  Figure 2. WNT4 protein expression in human fetal (A) and adult (B) ovaries. WNT4 protein was detected in human ovaries at all fetal ages studied. During early development, staining was mainly present in granulosa cells, while the oocytes (with round shape and a large cytoplasm) showed weak expression (A: a and b). From fetal age 19 weeks onwards, staining intensity in oocytes tended to increase (A: c and d). After midgestation strong/moderate WNT4 protein expression was detected in most of the follicle-encapsulated oocytes, and granulosa cells were also moderately stained (A; e-h). Negative control using PBS instead of primary antibody (A: i and j). Staining of WNT4 was intense in primary and secondary follicles of adult human ovaries and was detected mainly in oocytes, but also granulosa cells were moderately stained (B: a and c). In antral follicles granulosa cells were weakly stained and moderate staining was observed in theca cells (B: e). Negative control using PBS instead of primary antibody (B: b, d and f). Human adrenal gland (B: g and h) and testis (B: i and j) were used as control tissues. Strong staining was detected in adrenals while in human testis tubular cells showed only negligible and Leydig cells moderate staining. Brown color indicates protein expression. *= Oocyte, arrows indicate granulosa cells. Scale bar 100 µm.

F

Acknowledgements:

Special thanks go to Petri Itäranta and Kirsi Sainio for help with the in situ hybridization analyses. We thank Dr. Riitta Herva for collecting fetal tissues and Johanna Arola for providing human adrenal gland tissue. We also thank Nick Bolton for revising the language.

Grants: The project was supported by grants from the Academy of Finland (M.H., S.V., T.E.V., J.S.T.), the Sigrid Jusélius Foundation (M.H., S.V., J.S.T.), Oulu University Hospital (J.S.T.) and The Europian Union (S.V., LSHG-CT-2004-005085).