Reproductive Biology and Endocrinology 2005, 3:37

cDNA library screening A unidirectional cDNA library, against mature male kidney cDNA, constructed in Lambda ZAP Express?(Stratagene, La Jolla, CA, USA) was used to screen for AR cDNA using a DIG-labeled AR cRNA probe. The screening was conducted according to the Lambda ZAP Express?manual (Stratagene). DIG labeled anti-sense RNA probes were generated using the DIG RNA Labeling Kit (Roche, Mannheim, Germany). Hybridization was performed at 45癈 overnight (O/N) in hybridization buffer (5 ?SSC, 50% formamid, 0.02% SDS (w/v), 0,1% N-laurylsarcosine (w/v) and 2% blocking solution (w/v)) (Roche). Membranes were washed for 2 ?5 min in 2 ?SSC and 0.1% (w/v) SDS at room temperature, and for 2 ?15 min in 0.2 ?SSC, 0.1% (w/v) SDS at 68癈. Signals were detected using CSPD (Roche) and exposure of Hyperfilm?MP film (Amersham Pharmacia Biotech, Buckinghamshire, England) and hybridization signals were visualized using a CURIX 60 Film Developer (AGFA-Gevaert AB, Kista, Sweden). Positive plaques were purified through four successive hybridization rounds, and individual clones were isolated by phagemid excision. Following sequence identification of the clones as AR, they were sequenced to completion by Cybergene AB (Huddinge, Sweden).

Reproductive Biology and Endocrinology 2008, 6:66

cDNA microarray We used a custom-made utero-placental cDNA microarray that was developed in our laboratory as previously described [21,22]. In brief, a cDNA library was constructed from mRNA isolated from endometrial (caruncular and intercaruncular endometrium) and placental tissues (cotyledonary and intercotyledonary fetal membrane) of Japanese black cows. The PCR products of about 4,000 clones from the cDNA library were robotically spotted onto glass slides. The clones were simultaneously sequenced using the MegaBACE 1000 DNA Sequencing System (Amersham Pharmacia Biotech, Piscataway, NJ). The array contained 3,955 spots that were clustered into 1,738 unique genes on the basis of sequence analysis. An additional 35 genes that were not included in the cDNA library but had also been spotted onto the cDNA microarray were used for the analysis since these genes have been shown to be characteristically expressed during gestation in bovines and humans [4,11,19,23-27]. MMP related genes made up the bulk of this group. The cDNA microarray hybridization procedures were described in previous reports [21,22]. Briefly, two μg of poly (A)+ RNA were reverse transcribed in the presence of cyanine 3 (Cy3) or Cy5 fluorescence dye (Amersham Biosciences, Buckinghamshire, UK) using SuperScript II reverse transcriptase (Invitrogen) to make the hybridization probes. Identical samples were labeled separately with either Cy3- or Cy5-dye. Thus, two hybridization reactions could be carried out with the same sample. The arrays were sequentially washed with different concentrations of SSC solutions after 16 hr incubation at 65°C. The arrays were dried by centrifugation at 1,000 × g. Hybridization images were immediately scanned by a GenePix 4000B laser scanner (Axon Instrument, Union City, CA, USA) and analyzed with GenePix Pro 4.0 software. Data normalization was performed by previously described procedures [26,27]. The local background intensity of each array spot was smoothed by local weight regression (Lowess) and subtracted from the spot intensity data. The subtracted intensity data were subjected to non-parametric regression and local variance normalization since non-parametric regression can reduce intensity-dependent biases. The accuracy is improved over that of linear regression if the points in the scatter plot of Cy3 versus (vs.) Cy5 are not distributed around a straight line. Data for individual genes were obtained by averaging the intensity values of analogous spots on the microarray. Data were log2 transformed and used for cluster analysis. The Cluster 3.0 program was used for the hierarchical clustering. The hierarchically clustered data were visualized using the TreeView 0.99 program (M.B. Eisen's based clustering program [28],

Reproductive Biology and Endocrinology 2008, 6:42

Somatolactin (Sl) is a fish specific adenohypophyseal peptide hormone related to growth hormone (Gh). Some species, including salmonids, possess two forms: Sl alpha and Sl beta. The somatolactin receptor (slr) is closely related to the growth hormone receptor (ghr). Sl has been ascribed many physiological functions, including a role in sexual maturation. In order to clarify the role of Sl in the sexual maturation of female Atlantic salmon (Salmo salar), the full length cDNAs of slr, Sl alpha and Sl beta were cloned and their expression was studied throughout a seasonal reproductive cycle using real-time quantitative PCR (RTqPCR).

Reproductive Biology and Endocrinology 2008, 6:42

Sequencing of Atlantic salmon Slα and Slβ Total RNA was extracted from six pooled pituitaries of juvenile Atlantic salmon stored in RNAlater (Ambion, Austin, TX) using the RNeasy Mini kit (Qiagen, Germantown, MD) with treatment with RNase free DNase I (Qiagen). Three micrograms of this total RNA was reverse transcribed into cDNA using Power Script Reverse Transcriptase (Clontech, Palo Alto, CA) using oligo (dT)12. Two primers based on the rainbow trout Slα cDNA [21] were used to obtain Atlantic salmon Slα open reading frame (ORF) of 721 nucleotides: SlαF1 and SlαR1 (Table 1). A primer pair based on the rainbow trout Sl-like protein (rtSLP), the putative Slβ form [22], was used to obtain the partial Atlantic salmon Slβ ORF of 627 nucleotides: SlβF1 and SlβR1 (Table 1). PCR was carried out for 30 cycles of 94°C for 15 s, 55°C for 30 s and 68°C for 60 s using Platinum Pfx DNA polymerase (Invitrogen, Carlsbad, CA). The resulting bands, 720 bp for Slα and 610 bp for Slβ as seen on 1.2% agarose gel, were purified and directly sequenced by an ABI capillary sequencer (MWG Biotech, Ebersberg, Germany). The corresponding 5' and 3' ends were obtained by RACE with the SMART RACE cDNA Amplification Kit (Clontech) using gene specific primers: Slα5RACErev, Slα3RACEfor, Slβ5RACErev and Slβ3RACEfor (Table 1). All RACE products were gel purified, subcloned and sequenced and yielded the complete Atlantic salmon Slα and Slβ cDNA.

Reproductive Biology and Endocrinology 2008, 6:42

Phylogenetic analysis Protein sequence alignments were carried out with ClustalW (1.83) software [23]. Phylogenetic analysis of putative fish slr and ghr sequences and Sl sequences were made by the maximum likelihood method using the PHYLIP 3.6a3 package [24]. Branch lengths show genetic change and bootstrap values are shown as % on the branches (100 datasets). The analysis using parsimony and distance-matrix methods such as UPGMA and neighbour joining generated trees of similar topology and lower bootstrap values.

Reproductive Biology and Endocrinology 2008, 6:42

Histological analysis of oocyte maturation At the time of sampling, a piece of ovarian lamella was isolated by transversal cuts with a scalpel blade from each female and fixed in Bouin's fixative for histological analysis. Fixed samples were transferred to ethanol (70%), and subsequently dehydrated and embedded in paraffin according to conventional techniques. Histological examination was carried out as described previously [27] using 5 μm sections after hematoxylin-eosin staining. Animals considered as immature, i.e. showing primary growth phase follicles only, were not found in the present study. As salmon females are not fully synchronous in their sexual maturation, females sampled at a certain date may have reached different levels of maturity. Thus, it is important not only to analyze the data in relation to date, but also in relation to maturational status. This was determined for each sampled female according to the most advanced type of follicles as follows: (i) oocytes with cortical alveoli and perinuclear oil droplets (oil drop), or (ii) showing follicles with oocytes in true vitellogenesis. The latter were further differentiated in oocytes showing a limited number of small eosinophilic yolk globules in the periphery of the oocyte (primary yolk stage), with numerous but still small yolk globules distributed throughout the oocyte (secondary yolk stage), or with oocytes filled with large yolk globules (tertiary yolk stage). This analysis was carried out until all ovaries were found to have reached tertiary yolk state (June). Both in the time-based (n = 6–10) and in the maturation-based (n = 14–24) analyses, the data were tested for homogeneity of variance using Levene's test, and where appropriate, the data were log-transformed or arctan-transformed before analysis of variance (ANOVA with P < 0.0166 to compensate for comparison of three variables) using SPSS version 15.0 (SPSS Inc., Chicago, IL). Tukey's multiple means comparison test was performed as post-hoc test. Correlation analysis using two-tailed Spearman's ρ was carried out at each sampling date and for each maturational category. All data are presented as means ± standard error of the mean (SEM).

Reproductive Biology and Endocrinology 2008, 6:42

Atlantic salmon Slα cDNA sequence [GenBank:EU255779]. Nucleotides and amino acids are positively numerated starting with the initiation methionine. Signal peptide (underlined with solid line); Cysteine residues c; Stop (*); Polyadenylation signal (boxed). Benedet et al. Reproductive Biology and Endocrinology 2008 6:42

Reproductive Biology and Endocrinology 2008, 6:42

Phylogenetic analysis of Sl sequences. Phylogram of fish Sl amino acid sequences showing genetic change made by maximum likelihood method. Atlantic salmon Gh1 (Salmo salar [GenBank:X14305]) is the rooting out-group and bootstrap values as % are shown on the branches (100 datasets). European eel (Anguilla Anguilla [GenBank:U633884]), rainbow trout Slβ (Oncorhynchus mykiss; [22]), Atlantic salmon Slβ [GenBank:EU255780], channel catfish Slβ (Ictalurus punctatus [GenBank:AF267991]), grass carp Slβ (Ctenopharyngodon idella [GenBank:EF372075]), zebrafish Slβ (Danio rerio [GenBank:AY221126]), goldfish (Carassius auratus [GenBank:U72940]), white sturgeon (Acipenser transmontanus [GenBank:AB017200]), African lungfish (Protopterus annectens [GenBank:AB017766]), zebrafish Slα [GenBank:AY376857], grass carp Slα [GenBank:EF372074], rainbow trout Slα ([21]), chum salmon Slα (Oncorhynchus keta [GenBank:D10638]), cod (Gadus morhua [GenBank:D10639]), Senegalese sole (Solea senegalensis [GenBank:U06753]), Atlantic halibut (Hippoglossus hippoglossus [GenBank:L02117], Bastard halibut (Paralichthys olivaceus [GenBank:M33696]), medaka (Oryzias latipes [GenBank:NM_001104790]), acarα (Cichlasoma dimerus [GenBank:EF192603]), yellow perch (Perca flavescens [GenBank:AY332490]), lumpfish (Cyclopterus lumpus [GenBank:L02118]), orange spotted grouper (Epinephellus coioides [GenBank:AY169406]), tuna (Thunnus thynnus [GenBank:AB222036]), European sea bass (Dicentrarchus labrax [GenBank:AJ277390]), pufferfish (Tetraodon miurus [GenBank:AF253066]), red drum (Sciaenops ocellatus [GenBank:AF062520]), rabbitfish (Siganus guttatus [GenBank:AB026186]), red sea bream (Pagus major [GenBank:AB219244), black porgy (Acanthopagrus schlegelii [GenBank:AY714370]). Benedet et al. Reproductive Biology and Endocrinology 2008 6:42

Reproductive Biology and Endocrinology 2008, 6:42

Relative mRNA expression of Slα (○) and Slβ (●) in the pituitary and slr (▼) in the ovary of Atlantic salmon female broodstock from August 2003 to December 2004. Each point represents mean ± SEM of n = 6–10 per date and * denotes significance at P < 0.0166. Relative transcription levels were normalized to ef1α and compared to the initial August sample. Benedet et al. Reproductive Biology and Endocrinology 2008 6:42