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Department of Molecular and Cell Biology and Biotechnology Center, University of Connecticut, Storrs, Connecticut 06269-3044
Address all correspondence and requests for reprints to: Dr. Thomas T. Chen, Department of Molecular and Cell Biology, University of Connecticut, 75 North Eagleville Road, U-3044, Storrs, Connecticut 06269-3044. E-mail: thomas.chen{at}uconn.edu.
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Abstract |
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Introduction |
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Our previous studies on the rainbow trout (rt) (Oncorhynchus mykiss) SL (rtSL) have revealed that three additional hybridization signals with lesser intensities were detected in addition to the SL mRNA, when the C terminus of the rtSL cDNA was used as a probe under low stringency conditions (2). These weak hybridization bands were smaller than the 2.3-kb rtSL mRNA. This observation could suggest the presence of different forms of rtSL, because rainbow trout has two forms of GH (3) and two forms of PRL (4). It is equally possible that these weaker hybridization signals may suggest the presence of unknown species of SL-related mRNA in the trout pituitary gland.
To determine whether any rtSL-related mRNA may be present in rainbow trout, we screened a rainbow trout pituitary cDNA library. A cDNA clone with a high degree of homology to SL was isolated, and the nucleic acid sequence was determined. By conducting 5'- and 3'-rapid amplification of cDNA ends (RACE), the full-length cDNA was obtained. Using antiserum raised against a synthetic peptide designed from the deduced amino acid sequence of rtSL-like protein (rtSLP) as a probe in immunoblot analysis, it was confirmed that this SLP was a secretory protein consistent with other members of the GH family proteins. In this paper, we report the complete nucleotide sequence of rtSLP, and patterns of the expression of the gene in different tissues and developmental stages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gt10 vector. The 500-bp probe was prepared by amplification of rainbow trout pituitary first-strand cDNA with a pair of primers (C57: 5'-TGTTTGAGGAGATGTTTGTCC-3'; and C58: 5'-TGGAGCAGCTTGAGGAAG-3') designed from the highly conserved regions of the published SL amino acid sequences. The probe was labeled with 
-[32P]deoxy-CTP by random priming method (5). Hybridization was carried out in a solution containing 5x Denhardt’s solution, 6x saline sodium citrate (SSC), 0.1% sodium dodecyl sulfate (SDS), and 50% formamide at 42 C over night. The membranes were washed in a solution containing 0.5x SSC/0.1% SDS at 52 C. After autoradiography, the positive clones were purified and subcloned into the M13 mp18 vector, and the nucleotide sequence was determined by the dideoxynucleotide chain termination method (6). Full-length SLP cDNA sequence was determined by combining the 5'- and 3'-RACE. One microgram of rainbow trout pituitary gland total RNA was used in the synthesis of the first-strand cDNA using a mixture of 3'-RACE (T)17-adapter primer [5'-GACTCGAGTCGACATCGA(T)17-3'] and 5'-RACE switch primer (5'-AAGCAGTGGTATCAACGCAGACTCGAGGGG-3'). The method of 5'-RACE was modified from Schramm (7). The first-strand cDNA was then amplified by PCR using Taq DNA polymerase and primer UPM-XhoI (5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTAACAACGCAGACT-3' and 5'-CTAATACGACTCACTATAGGGC-3') and an SLP-specific primer 5'RACE-C (5'-CGAGTCAGATCTTAAAAGAGAGCAGCC-3'). The PCR products were further amplified by 5'RACE-N (5'-AAGCAGTGGTAACAACGCAGACT-3') and 5'RACE-C. The PCR products were digested by BglII and XhoI and cloned into pBluescriptII SK(-) at the BamHI/XhoI site.
The method of 3'-RACE was modified from Frohman (8). The first-strand cDNA was amplified by an SLP-specific primer 3'RACE-N (5'-AGAGTGAAGCTTTCACCTCAAAACC-3') and 3'RACE-C (5'-GAGAGAGACTCGAGTCGACATCGA-3'). The PCR products were digested with BamHI and XhoI and cloned into pBluescriptII SK(-) at the BamHI/XhoI site. The nucleotide sequence was determined by using the BigDye terminator cycle sequencing method (Applied Biosystems, Foster City, CA).
Tissue collection and RNA preparation
Fertilized rainbow trout eggs obtained from Angelo’s Trout Farm (Normalville, PA) were incubated in running water at 10 C in the University of Maryland Aquaculture Research Center (Baltimore, MD) until hatching, and heads of fish at embryonic d 20, 27, and 47 were collected and stored at -80 C. Pituitary glands of fingerlings (17.5–25 cm in length, 57.6–149 g in weight; Qunnibaug State Hatchery, Plainfield, CT; September, 2001) and 2-yr-old (Albert Powell Trout Hatchery, Hagerstown, MD; August, 1993) rainbow trout were collected for determining the expression of the SLP gene in different developmental stages. Brain, gill, heart, kidney, liver, skeletal muscle, pyloric caeca, spleen, immature egg, ovary, and testes were collected from 2-yr-old trout. Individual pituitary gland and tissue were placed into a microfuge tube and immediately frozen in liquid nitrogen. The tissues were kept at –80 C until used. Total RNA was isolated by disrupting the samples in a lysis buffer containing 4 M guanidine isothiocynate, 25 mM sodium citrate, 0.5% sarcosyl, 1% ß-mercaptoethanol, and 200 mM sodium acetate, and followed by phenol/chloroform extraction (9). RNA samples were quantified by determining the absorbance at 260 nm in a spectrophotometer, and their qualities were assessed by electrophoresis on a 1.2% agarose gel.
RNA blot analysis
Five micrograms of pituitary total RNA were separated by electrophoresis on a formaldehyde-1.2% agarose gel and then transferred to a nylon membrane. Probes specific for SLP [nucleotide position (nt) 240–699], SL (nt 1–669), GH (nt 65–697), and PRL (nt 234–662) were labeled with -[32P]deoxy-CTP by the random priming method (5). Hybridization was carried out in a solution containing 5x Denhardt’s solution, 6x SSC, 0.1% SDS, and 50% formamide at 42 C overnight. Membranes were washed in a solution containing 0.1x SSC and 0.1% SDS at 52 C. After autoradiography, the membranes were boiled in H2O for 1 min to remove the probe, and then rehybridized to a rainbow trout 18S rRNA probe (5'-CGGCATGTATTAGCTCTAGAATTACCA CAG-3') (10). Hybridization was carried out in a solution containing 5x Denhardt’s solution, 6x SSC, 0.1% SDS, and 0.05% sodium pyrophosphate, at 42 C for 16 h. Membranes were washed in 6x SSC/0.1% SDS at 42 C and subjected to autoradiography. The levels of SLP hybridization signals were quantified in a gel documentation system (Gel Doc 1000; Bio-Rad Laboratories, Inc.) and then normalized against the amount of 18S rRNA.
RT-PCR and hybridization
One microgram of total RNA from the pituitary gland or 2 µg total RNA from trout head or tissues were used for the first-strand cDNA synthesis. Two pairs of primers SLP1/SLP2 (SLP1, 5'-TTCCTAATCAAGTTCTGG-3'; and SLP2, 5'-ACAGGGTAGTCTGCCAGG-3') and actin1/actin2 (actin1, 5'-CCTCACAGTGATCAATG-3'; and actin2, 5'-TCACCAACTGGGATGACA TG-3') were used for amplifying SLP and ß-actin cDNA, respectively. PCR was carried out in a 50-µl vol using standard buffer conditions described by the manufacturer (Promega Corp., Madison, WI). The first-strand cDNA was preheated at 95 C for 5 min. Five microliters of 15-mM MgCl2 were then added. The reaction was carried out at 94 C for 10 sec, 50 C for 10 sec, and 72 C for 15 sec for 40 cycles. The PCR products were resolved on agarose gels, and DNA bands were transferred to a nylon membrane and hybridized to 
-[32P] end-labeled SLP-specific oligomer SLP3 (5'-GCGTTGTCATAGCGATCC-3'). Hybridization was carried out at 42 C for 3 h in a solution containing 6x SSC, 0.1% SDS, 5x Denhardt’s solution, and 0.05% sodium pyrophosphate. Membranes were washed at 42 C in a solution containing 0.1% SDS and 6x SSC, and hybridization signals were detected in a phosphoimager (GS525; Bio-Rad Laboratories, Inc.).
Pituitary organ culture
Rainbow trout pituitary glands were cultured in vitro following conditions modified from Yada et al. (11). Pituitary glands were collected from female rainbow trout (23–28 cm in length and 200–250 g in weight). Fish were obtained from Qunnibaug State Hatchery and maintained in the fish culture facility at the University of Connecticut. Fish were killed by decapitation before collection of the pituitary glands. An individual gland was placed in a single well of a 96-well culture plate containing 200 µl Eagle’s MEM (M3024; Sigma, St. Louis, MO) minus L-glutamine, supplemented with 100 U penicillin G, 10 µg/ml streptomycin, and 2.2 g Na bicarbonate/liter, at pH 7.5 and 290 mOSm/kg H2O. Cultures were kept at 13 C in an orbital shaker. The medium in each well was changed daily, and the harvested medium was stored at -20 C until used.
Anti-rtSLP antiserum and immunoblotting analysis
Anti-rtSLP antibody (rtSLPab) was raised in rabbit against a synthetic peptide (GEGAVM LEQNNKNLA) corresponding to amino acids 166–180 in rtSLP. Individual pituitary glands were disrupted in 100–300 µl of a lysis buffer (10 mM Tris, pH 8.0; 150 mM NaCl; 10 mM EDTA; 0.1% SDS with 1x protease inhibitor cocktail). SDS-PAGE was performed on 12% acrylamide gels, and proteins were transferred electrophoretically onto a polyvinylidene difluoride microporous membrane (Immobilon-P; Millipore Corp., Bedford, MA). Before immunostaining, the Immobilon blots were blocked for 1 h at room temperature in TBS (10 mM Tris-HCl, pH 8.0; 150 mM NaCl) containing 10% nonfat milk. The proteins on Immobilon blots were reacted with rtSLPab (1:800 dilution), GHab (1:2,000 dilution) (12), PRLab (1:1,000 dilution) (12), or rtSLab (1:4,000 dilution) (2) in TBST (TBS with 0.05% Tween 20) for 2 h at room temperature. The membranes were washed three times (5 min each time) with TBST. The bound antibodies were detected with alkaline phosphatase-conjugated goat antirabbit IgG (H+L) (1:20,000 dilution; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) as secondary antibody and color developed with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium as substrates.
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Results |
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, A and B). Two 3'-RACE PCR products (f1 and f2) were obtained. Although the shorter 3'-RACE PCR product (1262 bp) contains a nucleotide sequence identical with that of the longer form, this cDNA clone does not contain a polyadenylation signal. Thus, we believe that this cDNA clone may not be derived from a separate SLP mRNA species. The full-length cDNA consists of 1653 bp, with an open reading frame of 687 bp (Fig. 1C). The putative polyadenylation signal (AATAA) was present at nt 1643–1648. The cDNA encoded a protein with 229 amino acid residues, including a putative signal peptide of 26 amino acid residues (13) and a mature peptide of 203 amino acid residues. The calculated molecular mass of the mature peptide is 23.2 kDa, and a theoretical isoelectric point of 7.0. A potential N-glycosylation site (Asn141-Lys-Thr-Lys144) and a potential CAAX-box (Cys226-Ser-Leu-Leu229) were found in the deduced amino acid sequence. The amino acid sequence identity between this protein and that of rtSL is 56%, and the similarity is 66.5% (Table 1, Fig. 2A). Because this protein contained most of the conserved amino acid residues found in all known SLs of different species (Fig. 2A), it was therefore designated as trout SL-like protein (rtSLP).
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summarizes the comparison of the amino acid sequence similarities and identities of rtSLP with those of SL from a wide variety of fish species, and with those of rtGH1/2 and rtPRL published previously. Whereas rtSLP shared 41–57% identity and 54.3–67.9% similarity with SL from many fish species, rtSL shared a much wider range of identity (49–90%) and similarity (65.2–99.5%) with those same fish species. The amino acid sequence identities and similarities of rtSL and rtSLP with those of rtGH1/2 and rtPRL are 26.1% and 21.1%, respectively. Notably, the conserved somatotropin (GH family protein) domains were also present in the C terminus of rtSLP (Fig. 1C). In addition, six cysteine residues in rtSLP were found to be located at the conserved positions among all SLs. Most notably, the four cystein residues capable of forming two disulfide bonds, as those in GH and PRL, were also localized at the regions corresponding to those in GH and PRL. On the basis of these characteristics, we believed that rtSLP is a member of the GH family proteins.
Presence of SLP in trout pituitary glands
The expression of SLP gene in rainbow trout pituitary gland was examined by RNA hybridization using rtSLP cDNA fragment as a probe as well as by immunoblot analysis. A unique hybridization band was detected in the total RNA prepared from the pituitary gland of either male or female trout (Fig. 3A). This hybridization band was smaller than rtSL mRNA but larger than the rtGH or rtPRL mRNA. The relative levels of SLP mRNA in pituitary glands of 2-yr-old trout of both sexes were determined by RNA blot analysis, and the data were normalized to the amount of 18S rRNA in each sample. As shown in Fig. 3B, the levels of SLP mRNA in the 2-yr-old males are about 2-fold higher than those in the 2-yr-old females.
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). Although the peptide recognized by rtSLPab comigrated with rtGH on SDS-polyacrylamide gels, the rtSLPab did not cross-react with rtGH (Fig. 4B, lane 1). Furthermore, SLP was detected in both male and female trout pituitary glands (Fig. 4B, lanes 3 and 4).
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. Comparison of both profiles revealed that both proteins shared a very high degree of similarity. In addition to having a hydropathy profile similar to that of rtSL, rtSLP contains a hydrophobic region commonly found in the proximal amino termini of secretary proteins and a C226SLL229 box at its carboxyl terminus matching with the known functional CAAX motif (14). To determine whether rtSLP could still be secreted out of the pituitary gland after its synthesis, an immunoblot assay was conducted. Pituitary glands were cultured in a serum-free medium, and the culture medium was collected daily and analyzed by SDS-PAGE and immunoblotting. Lane 2 of Fig. 4B shows the result of the immunoblot assay using medium collected on the third day of the pituitary culture. A single immunoreactive band with the size corresponding to rtSLP was detected in the culture medium. This result confirms that, once rtSLP is synthesized, it is secreted out of the pituitary gland, as are other GH family proteins.
SLP gene is specifically expressed in pituitary glands
The tissue distribution of SLP mRNA in rainbow trout was determined by RT-PCR. Total RNA was prepared from brain, gill, heart, kidney, liver, skeletal muscle, pituitary gland, pyloric caeca, spleen, immature egg, ovary, and testis of 2-yr-old rainbow trout. The first-strand cDNA was amplified using primer pairs either specific for rtSLP (Fig. 5A) or ß-actin, as a control (Fig. 5C). A rtSLP-specific PCR product (216 bp) was detected only in the RNA sample prepared from pituitary glands. No positive signal was detected in any other tissue samples examined, even after the RT-PCR products were hybridized with an rtSLP-specific oligomer (Fig. 5B). These results strongly suggest that the SLP gene is specifically expressed in the pituitary gland of rainbow trout.
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, A and C) or ß-actin (Fig. 6, A and E), and the RT-PCR products were transferred to nylon membranes and hybridized to an SLP-specific oligomer (Fig. 6, B and D). The SLP transcripts were detected in both males and females of 2-yr-old fish. Only a very weak hybridization signal was detected in one out of four fingerlings examined, and none was detected in the embryos of all stages. These results showed that SLP gene is expressed in the reproductive mature adult fish but not, or very little, in immature fish or embryos.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several studies conducted using SDS–PAGE analysis showed that SL migrated as two protein bands with slightly different molecular sizes (24 and 28 kDa in rabbitfish, 23 and 26 kDa in Atlantic cod, 25 and 28 kDa in gilthead sea bream, and 24 and 27 kDa in Atlantic halibut and sole). Both protein bands were stained positive with antiserum of Japanese flounder or Atlantic cod SL but not with antisera of GH and PRL (15, 16, 17, 18, 19). These results suggested that the fast migrating band was likely the deglycosylated form of SL. However, in our studies, either rtSLab or rtSLPab recognized only a single band on the immunoblots of trout pituitary extracts. Although rtSLab and rtSLPab are polyclonal antibodies, they were raised in rabbits against synthetic peptides specific for rtSL and rtSLP, respectively, and they were shown to be monospecific to each other. Therefore, the peptide that was recognized by the rtSLPab in trout pituitary extracts was not likely the deglycosylated form of rtSL.
The expression pattern of SLP gene is completely different from that of SL gene, although SLP is highly homologous to SL. In trout, the zygotic expression of SL gene starts at the early stages of embryonic development, before the formation of the pituitary gland, and its expression continues to adulthood (20). On the contrary, the expression of SLP gene determined in this study was only restricted to adult fish. This observation provides additional evidence substantiating the fact that rtSLP is different from rtSL. However, we cannot completely rule out the possibility that the absence of rtSLP mRNA in young trout was caused by the seasonal variation of the rtSLP levels, because pituitary samples used in this study were collected only in one time point of the seasonal cycle. A thorough analysis of samples collected from animals at different developmental stages in various seasonal cycles will help to answer this question.
In trout, the expressions of GH family genes are not restricted to the pituitary gland alone (2, 20). As reported by Yang et al. (2, 20), whereas the expression of known GH family protein genes was detected in the pituitary gland and other extrapituitary tissues, the expression of SLP gene was only detected in the pituitary gland, as determined in this study. The transcriptional regulatory factor Pit-1 is known to play an essential role in regulating tissue-specific expression of GH family protein genes in the pituitary gland in both mammals and fish (21, 22, 23, 24). Whether the pituitary-specific expression of the rtSLP gene is also under the regulation of Pit-1 remains to be determined. In an immunohistochemical analysis reported by Takeo et al. (25), antisera raised against Pit-1 stained not only the cell types producing GH, PRL, or SL but also other cell types in the trout pituitary gland. It is conceivable that Pit-1 might also regulate the expression of pituitary peptides other than GH, PRL, and SL. Although the exact cell type in the pituitary gland that rtSLP is synthesized awaits determination, it is possible that this peptide might be coproduced in the same cell type that produces GH, PRL or SL. If it were indeed the case, the question of why Pit-1 in the same cell type does not turn on the SLP gene in certain developmental stages would be worth investigating.
Like other GH family proteins, rtSLP contains a hydrophobic N-terminal signal peptide and is secreted out of the pituitary cells after its intracellular processing. A sequence resembling a CAAX box (14) is also found in the C terminus of rtSLP. In many cases, CAAX-containing proteins are covalently attached to a fatty acid chain, which inserts into the cytoplasmic leaflet of the lipid bilayer, anchoring the protein to the membrane. To date, none of the GH family proteins have been shown to anchor to the membrane. Furthermore, the enzymes that attach a fatty acid chain to the CAAX motif are present in the cytoplasm. Therefore, it is unlikely the CAAX motif in the C terminus of rtSLP is a function motif.
In summary, we have identified a new GH family protein, SLP, in the pituitary gland of rainbow trout. Although the function of SLP has not been determined, it is highly possible that this protein might act as a hormonal factor, because a signal peptide sequence was identified in the N terminus of the putative protein. In addition, the notion of SLP as a hormonal factor was further substantiated by the fact that SLP was detected in the medium of pituitary gland cultured in vitro. However, to confirm that SLP is a true hormonal factor in fish, identification of the physiological function, as well as its true target tissue, will be indispensable. Results of RT-PCR analysis and RNA blot hybridization showed that SLP mRNA was primarily detected in pituitary glands of 2-yr-old fish and that the level of mRNA in the male fish was 2-fold higher than that of the female fish. Therefore, it is tempting to speculate that SLP might regulate some physiological function relating to reproductive maturation. However, the exact physiological function of rtSLP requires a thorough investigation.
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Acknowledgments |
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Footnotes |
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Abbreviations: ab (suffix), Antibody; nt, nucleotide position; PRL, prolactin; rt, rainbow trout; RACE, rapid amplification of cDNA ends; SDS, sodium dodecyl sulfate; SL, somatolactin; SLP, SL-like protein; SSC, saline sodium citrate.
Received September 25, 2002.
Accepted for publication November 20, 2002.
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, conserved cysteine residues.
, conserved cysteine residues in SLs; 
, conserved cysteine residues in GH, PRL, and SL. B, Hydropathy profiles of rtSLP and rtSL. Positive values of hydropathy index are hydrophobic; negative values are hydrophilic.
