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