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Am J Physiol Endocrinol Metab 295: E463-E476, 2008. First published June 3, 2008; doi:10.1152/ajpendo.90385.2008
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Grass carp somatolactin: I. Evidence for PACAP induction of somatolactin-{alpha} and -β gene expression via activation of pituitary PAC-I receptors

Quan Jiang,1 Wendy K. W. Ko,1 Ethan A. Lerner,2 K. M. Chan,3 and Anderson O. L. Wong1

1School of Biological Sciences, University of Hong Kong, Hong Kong, China; 2Department of Dermatology, Massachusetts General Hospital, Charlestown, Massachusetts; and 3Department of Biochemistry, Chinese University of Hong Kong, Shatin, Hong Kong, China

Submitted 22 April 2008 ; accepted in final form 29 May 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Somatolactin (SL), the latest member of the growth hormone/prolactin family, is a novel pituitary hormone with diverse functions. At present, SL can be identified only in fish but not in tetrapods and its regulation at the pituitary level has not been fully characterized. Using grass carp as a model, we examined the direct effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on SL secretion and synthesis at the pituitary cell level. As a first step, the structural identity of grass carp SL, SL{alpha} and SLβ, was established by 5'/3'-rapid amplification of cDNA ends. These two SL isoforms are single-copy genes and are expressed in two separate populations of pituitary cells located in the pars intermedia. In the carp pituitary, PACAP nerve fibers were detected in the nerve tracts of the neurohypophysis and extended into the vicinity of pituitary cells forming the pars intermedia. In primary cultures of grass carp pituitary cells, PACAP was effective in stimulating SL release, cellular SL content, and total SL production. The increase in SL production also occurred with parallel rises in SL{alpha} and SLβ mRNA levels. With the use of a combination of molecular and pharmacological approaches, PACAP-induced SL release and SL gene expression were shown to be mediated by pituitary PAC-I receptors. These findings, as a whole, suggest that PACAP may serve as a hypophysiotropic factor in fish stimulating SL secretion and synthesis at the pituitary level. Apparently, PACAP-induced SL production is mediated by upregulation of SL{alpha} and SLβ gene expression through activation of PAC-I receptors.

pituitary adenylate cyclase-activating polypeptide; grass carp pituitary cells

SOMATOLACTIN(SL), the latest member of growth hormone (GH)/prolactin (PRL) family, is a glycoprotein hormone secreted from the pars intermedia of the fish pituitary (17). It was first isolated from the pituitary of flounder (27) and Atlantic cod (33). In subsequent studies, the protein was also cloned in salmon (39), sole (30), rainbow trout (47), European eel (24), goldfish (3), red drum (54), and zebrafish (52). The protein structure of SL is comparable but yet distinct from GH and PRL, and the amino acid sequences of SL are highly conserved in fish models (38). Two SL isoforms, SL{alpha} and SLβ, have been reported and phylogenetic analysis reveals that they are paralogues that arose by genome duplication in bony fish (52). In general, it is commonly accepted that SL and PRL were derived from the ancestral GH during early stages of gnathostome evolution (19). Subsequent duplication of GH genes in primates and PRL genes in rodents and ruminants have given rise to placental lactogen in mammals (6). Apparently, SL is unique to fish models, as the gene could not be identified in tetrapods and might have been lost during the evolution of land vertebrates (7).

Although the biological functions of SL have not been fully characterized, recent studies have revealed that SL may be involved in gonadal maturation (35), steroidogenesis (32), stress responses (34), background adaptation (53), immune functions (1), energy mobilization (18), lipid metabolism (9), ion transport (20), and acid-base balance (16). In medaka, deletion in SL gene can be detected in "color-interfere" mutants, implying that SL may play a role in the proliferation and/or morphogenesis of epidermal chromatophores (8). In zebrafish, the development and subsequent inflation of the swim bladder can be inhibited by gene silencing of SLβ but not SL{alpha} (51), indicating that the two forms of SL may have different functions in embryonic development. Although not much information is available for SL regulation, it has been shown that SL release in rainbow trout can be differently regulated by neurotransmitters (e.g., dopamine) and neuropeptides (e.g., corticotropin-releasing hormone and gonadotropin-releasing hormone; Ref. 15). In salmon pituitary cells, SL gene expression can be induced by treatment with gonadotropin-releasing hormone and sex steroids (e.g., estradiol and 11-ketotestosterone; Ref. 28). In recent studies (31), in vitro treatment with leptin can also trigger SL secretion in sea bass and this stimulatory effect is dependent on the reproductive status of the fish. These findings indicate that both central and peripheral signals can modulate SL expression at the pituitary level. Although the two SL isoforms may have different functions in fish models, neuroendocrine regulation of SL{alpha} and SLβ expression in the same species has not been examined.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic hormone with diverse functions and is widely expressed in the central nervous system as well as in peripheral tissues (5). Its NH2-terminal amino acid sequence is highly homologous to that of vasoactive intestinal polypeptide (VIP; Ref. 41). The biological actions of PACAP are mediated through three receptor subtypes, namely PAC-I, VPAC-I, and VPAC-II receptors. In general, PAC-I receptors have a binding affinity 10- to 100-fold higher for PACAP than the structurally related peptide VIP, whereas the VPAC receptors can bind PACAP and VIP with equal affinity. VPAC-I and -II receptors can be further differentiated by their binding affinity for the lizard venom helodermin (11). In mammals, PACAP produced in the hypothalamus can be released into hypophysial portal blood and serves as a hypophysiotropic factor in the pituitary (4) mainly through activation of cAMP production (25). Under certain conditions, PACAP can trigger the release of LH, GH, PRL, and ACTH (36), and the functional role of PACAP as a hypophysiotropic factor is well conserved in fish models (45). In representative fish species, PACAP immunoreactivity can be detected in the posterior pituitary, e.g., in goldfish (43) and European eels (26). In stargazer, PACAP nerve fibers are located in close proximity to SL cells in the pars intermedia (22), suggesting that the peptide may play a role in SL regulation. In a recent study (23) in goldfish using a cell immunoblot assay, PACAP treatment was shown to increase the immunoblot area for SL immunoreactivity in individual pituitary cells, implying that PACAP may act as a stimulator to trigger SL release at the pituitary level. Given that functional studies to clarify the receptor specificity for PACAP action have not been performed, the receptor subtype responsible for PACAP-induced SL secretion is still unknown.

In our recent studies (44) in grass carp, we have demonstrated that the carp pituitary is under the direct innervation of PACAP nerve fibers originated from the hypothalamus. Grass carp PACAP has been cloned and found to stimulate GH release and GH gene expression at the pituitary level (37). To further investigate the pituitary functions of PACAP in the carp species, we sought to examine the effects of PACAP on SL{alpha} and SLβ gene expression and characterize the receptor subtype(s) responsible for PACAP actions. As a first step, the structural identity of grass carp SL{alpha} and SLβ were established by molecular cloning. Their gene copy number, tissue distribution patterns, and transcript expression at the pituitary level were characterized by genomic Southern, RT-PCR, and Northern blot, respectively. Based on the sequences obtained, slot blot assays were set up for quantitative measurement of SL{alpha} and SLβ mRNA expression. With the use of primary cultures of grass carp pituitary cells, the effects of PACAP on SL{alpha} and SLβ gene expression was examined at the pituitary level and the receptor specificity for PACAP actions was determined using a combination of pharmacological and molecular approaches. For the first time, we provided evidence that PACAP stimulates SL production via upregulation of SL{alpha} and SLβ gene expression through PAC-I receptor activation at the pituitary level.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. One-year-old grass carps (Ctenopharyngodon idellus) with body weight ranging from 1.5 to 2.0 kg were acquired from local markets. After acclimation in the laboratory, the fish were killed for tissue collection and pituitary cell preparation by anesthesia in 0.05% tricaine methanesulfonate (Sigma, St. Louis, MO) according to the regulations for animal use at the University of Hong Kong.

Test substances. Ovine PACAP6-38, cod VIP, and (4-Cl-D-Phe6,Leu17)VIP were purchased from Bachem Fine Chemicals (La Jolla, CA), whereas grass carp PACAP38 (purity: 93%) was synthesized at the HSC Biotechnology Services Center of the Hospital for Sick Children (University of Toronto, Toronto, ON, Canada). Recombinant proteins of Maxadilan and M65 were produced in E. coli, purified, and functionally characterized as described previously (40). These peptide hormones and their functional analogs (including agonist and antagonists) were dissolved in double-distilled deionized water and stored frozen at –80°C as 0.1-mM stocks in small aliquots. Stock solutions of test substances were diluted with culture medium to appropriate concentrations 15 min before drug treatment. For the experiments with VIP and PACAP antagonists, the antagonists were applied at least 5 min before subsequent addition of grass carp PACAP38.

Molecular cloning of grass carp SL. Total RNA was extracted from carp pituitaries using Trizol (Invitrogen, San Diego, CA) and reverse transcribed using SuperScript II (Invitrogen). Nested PCR were then performed to pull out the partial fragments of carp SL{alpha} and SLβ cDNA using primers designed based on the conserved regions of zebrafish SL{alpha} and SLβ, respectively. Based on the cDNA sequences obtained, gene-specific primers were designed and 5'/3'-rapid amplification of cDNA ends (RACE) was conducted using a GeneRacer kit (Invitrogen). PCR products were purified for DNA sequencing using a BigDye sequencing kit (Applied Biosystems, Foster City, CA). The full-length cDNAs for grass carp SL{alpha} (GenBank Accession No: EF372074) and SLβ (GenBank Accession No: EF372075) were compiled from their 5'- and 3'-sequences and analyzed with MacDNASIS (Hitachi, San Bruno, CA) and MacVector V.9.5.2 programs (Acclelrys, San Diego, CA). With the use of crystal structure of human GH as a template, the three-dimensional (3D) models of grass carp SL{alpha} and SLβ were deduced with the knowledge-based modeling program ProMod II provided by the SWISS-MODEL server (www.expasy.org/swissmod). Phylogenetic analysis of SL{alpha} and SLβ with respect to the evolution of GH and PRL was also performed by PHYLIP and TreeView programs provided by the Taxonomy and Systematics Server (www.taxonomy.zoology.gla.ac.uk/rod/treeview).

Tissue distribution of SL expression. Tissue distribution of SL{alpha} and SLβ expression was examined using RT-PCR. Briefly, total RNA was isolated from various tissues and selected brain areas of the grass carp using TRIZOL. These RNA samples were digested with RNase-free DNase I (Roche, Mannheim, Germany) and reverse transcribed using SuperScript II. The RT samples obtained were used as the template for PCR using primers specific for grass carp SL{alpha} and SLβ, respectively. After that, PCR products were resolved in 1% agarose gel, visualized by ethidium bromide staining, and transblotted onto a positively charged nylon membrane. Southern blot was then conducted to check for the authenticity of PCR products using digoxigenin (DIG)-labeled cDNA probes for carp SL{alpha} and SLβ, respectively. In these studies, RT-PCR of β-actin was used as an internal control. To further characterize the transcript expression of SL{alpha} and SLβ expressed in the pituitary, Northern blot was performed using the standard procedures in our laboratory (14). In this case, total RNA isolated from the carp pituitary was size fractionated in 1% agarose gel containing 1x MOPS and 1% formaldehyde. After transblotting, the membrane with RNA samples was UV cross-linked, prehybridized with blocking solution (Roche), and hybridized with the DIG-labeled probes for grass carp SL{alpha} and SLβ, respectively. Hybridization signals were detected using a DIG luminescent detection kit (Roche) and visualized in an IC440 Image Station (Kodak, New Haven, CT). To ensure the absence of RNA degradation in our samples, parallel blotting of β-actin mRNA was also conducted in these experiments.

Genomic southern for SL{alpha} and SLβ. To determine the gene copy number for SL{alpha} and SLβ, Southern blot was performed with genomic DNA prepared from the grass carp as described previously (13). Briefly, whole blood was obtained from grass carp and digested for 15 h with proteinase K (Roche). After phenol:choloroform extraction and RNA clearance by DNase-free RNase (Sigma), genomic DNA obtained was digested overnight at 37°C with restriction enzymes, including EcoR V, Pst I, BamH I, Bgl II, Sty I, and Hind III, respectively. On the following day, the digested products were size fractionated in a 0.7% agarose gel, transblotted onto a positively charged nylon membrane, and hybridized with the DIG-labeled probes for grass carp SL{alpha} and SLβ, respectively. After that, hybridization signals were detected as described for Northern blot.

In situ hybridization of SL{alpha} and SLβ. Pituitaries were excised from grass carps, fixed in 4% paraformaldehyde, and embedded in paraffin wax. Pituitary sections of 5 µm in thickness were prepared and mounted onto slides precoated with 2% 3-aminopropyltriethoxy silane (Sigma). For in situ hybridization, pituitary sections were dewaxed with xylene, rehydrated with decreasing levels of ethanol, and postfixed in 4% paraformaldehyde. After that, the sections were digested with proteinase K (Roche), washed, and incubated at 37°C for 10 min with hybridization solution (4x SSC, 1x Denhardt's solution, 10% dextran sulfate, 10 mM DTT, 40% formamide, 1 mg/ml calf thymus DNA, 500 µg/ml yeast tRNA, 5 µg/ml polydeoxyadenylic acid, and 100 µg/ml polyadenylic acid). DIG-labeled antisense riboprobes for grass carp SL{alpha} and SLβ prepared by in vitro transcription were added, and the pituitary sections were incubated at 55°C overnight in a humidified chamber. On the following day, posthybridization washing was performed in decreasing dilutions of SSC solution at 42°C. After the clearance of unbound riboprobes by RNase A (Invitrogen) digestion, signal development was conducted with anti-DIG antibody (1:500, Roche) using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as the substrates. In these experiments, hybridization with the sense strands of the SL{alpha} and SLβ riboprobes was used as the negative control.

Measurement of SL{alpha} and SLβ mRNA expression. Primary cultures of grass carp pituitary cells were prepared by the trypsin/DNase digestion method as described previously (46). After enzyme digestion, pituitary cells were seeded in 24-well clustered plates at a density of ~2.5 x 106 cells·ml–1·well–1 and cultured overnight at 28°C in carp MEM (MEM Eagle Medium supplemented with 26 mM NaHCO3, 25 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml fungizone, pH 7.7) with 5% FBS. On the following day, culture medium was replaced with carp MEM with appropriate levels of test substances and the cells were allowed to incubate at 28°C for 48 h. Based on our validation, a 48-h incubation is the optimal duration for PACAP treatment to trigger SL{alpha} and SLβ gene expression. After drug treatment, total RNA was extracted from pituitary cells using TRIZOL and was subjected to slot blot assays (49) using the DIG-labeled cDNA probes for grass carp SL{alpha} and SLβ, respectively. The probes were prepared by a PCR DIG probe synthesis kit (Roche) using primers franking position 109–392 of grass carp SL{alpha} and positions 197–486 of grass carp SLβ cDNA, respectively. To ensure equal loading of RNA for individual samples in slot blots, parallel probing for 18S rRNA was also conducted to serve as an internal control.

Western blot for SL immunoreactivity. Pituitary cells (~2.5 x 106 cells/well) were incubated at 28°C for 48 h in carp MEM containing increasing levels of PACAP or VIP. After drug treatment, Western blot for SL immunoreactivity was performed as described previously (13). Briefly, culture medium was harvested for detection of SL release. After being rinsed, pituitary cells were lysed in RIPA buffer (50 mM Tris·HCl, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 0.25% Na deoxycholate) for monitoring of cellular SL content. Culture medium and cell lysate from individual wells were mixed pro rata to prepare the samples for total SL production. These samples were resolved by SDS-PAGE in 10% gel followed by electroblotting onto a nitrocellulose membrane at 65 V for 2.5 h. The membrane was blocked by 2% nonfat dried milk and incubated overnight at 4°C with an antiserum (1:10,000) raised against goldfish SL (3). After that, horseradish perioxidase-conjugated anti-rabbit IgG (1:15,000; Bio-Rad, Hercules, CA) was added and the Immobilon Western chemiluminescent substrate (Millipore, Billerica, MA) was used for signal development. In these experiments, Western blot of β-actin using an actin Ab-1 kit (Oncogene, Boston, MA) was used an internal control.

RT-PCR of immunoidentified SL cells. Immunohistochemical staining was performed in grass carp pituitary sections prefixed in Bouin's fixative with a Vectastain ABC kit (Vector Laboratory, Burlingame, CA) using the antiserum for goldfish SL (1:50,000). SL immunostaining was conducted in the carp pituitary to confirm that the antiserum could be used for localization of SL cells in neurointermediate lobe (NIL) with no cross-reactivity with GH or PRL cells located in the pars distalis. In this study, immunostaining with an antiserum raised against ovine PACAP38 (1:2,000; Peninsula Laboratories Belmont, CA) was also performed with parallel staining of normal rabbit serum as a negative control. For RT-PCR of immunoidentified SL cells, the NIL was manually dissected from individual pituitaries under a stereomicroscope and dispersed using trypsin/DNase digestion (46). The cells prepared (designated as "NIL cells") were evenly spread onto glass slides using an Autosmear CF centrifuge (Sakura Fine Technical, Nagand, Japan). After that, the cytospin preparation of NIL cells was fixed in Bouin's fixative, stained with SL antiserum, and subjected to laser capture microdissection (LCM) using a PixCell eII cell isolation system (Arcturus, Mountain View, CA; Ref. 50). NIL cells with SL immunoreactivity (referred to as "SL cells") were captured on Capsure HS LCM Caps (~200 cells/Cap) and used for RT-PCR with primers for grass carp PAC-I receptor (Accession No. EU305549). Since the SL antierum used in the present study does not differentiate SL{alpha} and SLβ isoforms, the results based on immunoidentified SL cells were further confirmed with single-cell RT-PCR with LCM-captured NIL cells tested positive for SL{alpha} and SLβ gene expression, respectively.

Data transformation and statistics. For slot blot assays, SL{alpha} and SLβ mRNA levels were quantified in terms of "arbitrary density unit" and normalized against 18S RNA expression in the same sample. Since no significant changes could be detected for 18S RNA expression in these experiments, the normalized data were simply transformed as a percentage of the mean value in the control group for statistical analysis (referred to as "%Ctrl"). For dose-response studies, ED50 values were deduced using the four-parameter logistic equation with GraphPad Prism (GraphPad, San Diego, CA). Data presented, expressed as means ± SE, are the results pooled from four to six experiments and were analyzed using ANOVA followed by Fisher's least significant difference test. Differences were considered significant at P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
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Molecular cloning and sequence analysis of grass carp SL. With the use of 5'/3'-RACE, the full-length cDNAs for SL{alpha} and SLβ were isolated from the carp pituitary. The SL{alpha} cDNA is 1,259 bp in size with two polyadenylation signals in 3'-UTR and an open reading frame of 708 bp encoding a 235 aa precursor for carp SL{alpha} (Fig. 1A). The SL{alpha} precursor is composed of a 24 aa signal peptide followed by a 211 aa mature protein with a deduced molecular weight of 26.8 kDa. Seven conserved Cys residues and an N-linked glycosylation site can be located in the mature protein of grass carp SL{alpha}. Sequence alignment also reveals that the amino acid sequence of grass carp SL{alpha} is highly homologous to that reported in zebrafish (76%), salmon (74%), medaka (65%), and cod (63%) (Fig. 2A). Similarly, the SLβ cDNA pulled out from the carp pituitary is 869 bp in size with two polyadenylation signals in 3'-UTR and an open reading frame of 687 bp encoding a 228 aa precursor for grass carp SLβ (Fig. 1B). The SLβ precursor has a 203 aa mature protein with a deduced molecular weight of 25.4 kDa preceded by a 25 aa signal peptide. Unlike SL{alpha}, the third conserved Cys is missing and only six conserved Cys residues can be identified in grass carp SLβ. Nevertheless, a N-linked glycosylation site can still be noted in the mature protein of SLβ. Similar to the case of SL{alpha}, the amino acid sequence of carp SLβ is highly homologous to that reported in goldfish (76%), zebrafish (68%), and catfish (51%) (Fig. 2B).


Figure 1
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  		Fig. 1. Nucleotide and deduced amino acid sequences of grass carp somatolactin (SL)-{alpha} (A) and SLβ cDNA (B). The full-length cDNA for SL{alpha} and SLβ were constructed using the MacDNASIS Pro and MacVector 6.5.3 programs. The amino acid sequences deduced from the open reading frame are presented along with the corresponding DNA sequence. The signal peptides were underlined, conserved Cys residues were boxed, and N-linked glycosylation sites ("NKTK" and "NVSS") were double underlined for identification in the protein sequences for SL{alpha} and SLβ, respectively. For the nucleotide sequences, the polyadenylation signals in the 3'-UTR ("aattaa," "aatata," "aacaaa," and "aattaaa") were underlined in bold type. *Stop codon located at the end of the coding sequences.

 

Figure 2
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  		Fig. 2. Alignment of carp SL{alpha} (A) and SLβ (B) with the amino acid sequences for the respective SL isoforms reported in other species. Sequence alignment was conducted using the Clustal W algorithm with MacVector V.9.5.2. The conserved amino acid residues in these sequences are boxed in gray, whereas the conserved Cys residues are marked by inverted triangles. The sequences of SL{alpha} and SLβ for other species were downloaded from the GenBank (http://www.ncbi.nih.gov).

 
Given that SL is a novel member of the GH/PRL family (19), phylogenetic analysis was performed to examine its evolutionary relationship with GH and PRL reported in other fish models (Fig. 3A). With the use of unrooted analysis based on PHYLIP and TreeView programs, the nucleotide sequences of grass carp SL{alpha} and SLβ can be grouped within the clades of SL{alpha} and SLβ respectively, which are distally related to the corresponding members in GH and PRL families. Similar to GH and PRL, in silico protein modeling confirms that the 3D structures of grass carp SL{alpha} and SLβ are composed of a central core with four {alpha}-helices (H1–H4) arranged in an antiparallel manner (Fig. 3B). Apparently, the H2 and H3 helices in SL{alpha} are noticeably shorter than that of the β-isoform. Although the amino acid sequences of the newly cloned SL{alpha} and SLβ are highly comparable to the corresponding isoforms in related species (e.g., zebrafish or goldfish), the sequence homology between grass carp SL{alpha} and SLβ is only 57.1% at the protein level with the highly conserved regions clustered around the four helical domains (Fig. 3C).


Figure 3
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  		Fig. 3. Phylogenetic analysis, protein modeling, and sequence comparison of grass carp SL{alpha} and SLβ. A: unrooted analysis based on the nucleotide sequences of SL{alpha}, SLβ, growth hormone (GH), and prolactin (PRL) reported in fish species was performed using PHYLIP and TreeView 3.2 programs. The guide tree was constructed using the neighbor-joining method after 500 bootstraps (bootstrap value: 459–500). Scale bar on right represents the genetic distance for evolution. B: 3-dimensional (3D) protein modeling of grass carp SL{alpha} and SLβ was performed using the ProMod II program using the crystal structure of human GH as the template. Ribbon plots of the 3D structures for SL{alpha} and SLβ reveals the presence of four {alpha}-helices (H1–H4) arranged in an antiparallel manner. Apparently, the H2 and H3 helices of SL{alpha} are noticeably shorter than that of SLβ. C: alignment of protein sequences of grass carp SL{alpha} and SLβ using the Clustal W algorithm. The conserved amino acid residues are boxed in gray, whereas the H1–H4 helices for the 2 SLs are marked by the horizontal lines.

 
Gene copy number and tissue expression of SL{alpha} and SLβ. In this study, the gene copy number of SL{alpha} and SLβ was examined in grass carp using Southern blot (Fig. 4A). As revealed by the hybridization signals using DIG-labeled cDNA probes for SL{alpha} and SLβ, a single band was consistently observed in grass carp genomic DNA with prior digestion of BamHI, EcoRI, PstI, HindIII, and StyI, respectively. These results indicate that the newly cloned SL{alpha} and SLβ are single-copy genes in the carp genome. To characterize the tissue expression profile of SL{alpha} and SLβ, Northern blot was performed in RNA samples prepared from various tissues of the grass carp. However, hybridization signals were detected only in the carp pituitary (Fig. 4B) but not in other tissues (data not shown). At the pituitary level, single transcripts of SL{alpha} and SLβ with the size of 2.0 and 1.6 kb, respectively, could be recognized. As revealed by the intensity of hybridization signals, the 2.0 kb SL{alpha} transcript appeared to be the major form of SL mRNA expressed in the carp pituitary. To further evaluate the possibility of extrapituitary expression of SL, a more sensitive method, namely RT-PCR, was used for PCR detection of SL{alpha} and SLβ expression in selected tissues including the brain, gills, heart, intestine, kidney, liver, gonad, pituitary, and muscle (Fig. 4C). Similar to the results of Northern blot, the pituitary was confirmed to be the location with the highest levels of SL{alpha} and SLβ mRNA expression. However, faint bands for SL{alpha} were also detected in all the tissues examined while low levels of SLβ signals could be noted in the brain and liver only. Given that the 282-bp PCR product for β-actin was observed in all the samples examined, the lack of PCR signals for SLβ in other tissues could not be due to RNA degradation during sample preparation.


Figure 4
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  		Fig. 4. Gene copy number, transcript characterization, and tissue expression of grass carp SL{alpha} and SLβ. A: determination of gene copy number for SL{alpha} and SLβ in the carp genome by Southern blot. Grass carp genomic DNA was digested with BamHI, EcoRV, HindIII, PstI, and StyI, respectively. After size fractionation by gel electrophoresis, positive signals for SL{alpha} and SLβ genes were detected by hybridization using DIG-labeled cDNA probes for SL{alpha} and SLβ, respectively. B: characterization of SL{alpha} and SLβ transcripts expressed in the carp pituitary by Northern blot. Total RNA was isolated from the carp pituitary, resolved in 1% agarose gel, and transblotted onto a nylon membrane. SL{alpha} and SLβ transcripts were detected by hybridization using the DIG-labeled cDNA probes as described for Southern blot. Parallel blotting for β-actin mRNA was used as an internal control. C: tissue expression profiles for SL{alpha} and SLβ. RT-PCR of SL{alpha} and SLβ was performed in RNA samples prepared from the brain, gills, gonad, heart, intestine, kidney, liver, muscle, and pituitary. The PCR products were size fractionated and visualized by ethidium bormide staining. The authenticity of PCR products for SL{alpha} and SLβ was confirmed by Southern blot, and RT-PCR of β-actin was also conducted to serve as an internal control.

 
In situ hybridization of SL{alpha} and SLβ. To further investigate the pituitary expression of SL{alpha} and SLβ, in situ hyridization was performed in grass carp pituitary sections using DIG-labeled antisense riboprobes for SL{alpha} and SLβ, respectively (Fig. 5). Hybridization signals for SL transcripts were detected in the NIL but not in the rostral and proximal pars distalis. Within the NIL, hybridization signals were located mainly in the cytoplasm of pituitary cells forming the pars intermedia but not in the nerve tracts forming the neurohypophysis. Apparently, SL{alpha} and SLβ hybridization signals were detected in two distinct populations of pituitary cells, with SL{alpha}-positive cells as the dominant form occupying the anterior two thirds of the NIL and SLβ-positive cells as the minor form locating in the more posterior region. Given that parallel hybridization with the sense strand of SL riboprobes did not yield any detectable signals, the possibility of nonspecific binding of hybridization probes (e.g., with GH and PRL transcripts) was unlikely.


Figure 5
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  		Fig. 5. In situ hybridization of SL{alpha} and SLβ in the carp pituitary. A: zonation of the grass carp pituitary. The carp pituitary can be divided into three major areas, namely the rostral pars distalis (RPD), proximal pars distalis (PPD), and neurointermediate lobe (NIL). The NIL is composed of pituitary cells forming the pars intermedia (PI) and nerve tracts/fibers from the hypothalamus forming the neurohypophysis (NHP). B: in situ hybridization of grass carp pituitary sections with DIG-labeled antisense riboprobe for grass carp SL{alpha} and SLβ mRNA, respectively. C: negative control for SL{alpha} and SLβ in situ hybridization. Hybridization was conducted using DIG-labeled sense riboprobe for SL{alpha} and SLβ, respectively. For in situ hybridization of SL{alpha} and SLβ transcripts, histological examination was performed under bright-field illumination with a magnification at x25 (1) x40 (2), x100 (3), and x400 (4), respectively. Hybridization signals for SL{alpha} and SLβ mRNA were detected in different areas of the NIL but not in RPD or PPD. Within the NIL, the hybridization signals were observed mainly in the cytoplasm of pituitary cells located in the PI but not in the nerve tracts of the NHP.

 
PACAP-induced expression of SL immunoreactivity. To provide the anatomical basis for PACAP regulation of SL expression, immunostaining was performed in carp pituitary sections using an antiserum raised against ovine PACAP38. The antiserum has been validated in our previous studies and proven to be useful in probing PACAP nerve fibers in the carp pituitary (44). In this study, PACAP immunostaining signals could be recognized in the NIL (Fig. 6A, 1) as well as in the anterior pituitary (data not shown). Within the NIL, PACAP immunoreactivity was detected in the nerve tracts forming the neurohypophysis (Fig. 6A, 2) and extensions of PACAP nerve fibers could also be located in the vicinity of pituitary cells forming the pars intermedia (Fig. 6A, 3). Parallel staining with the same dilution of normal rabbit serum, however, did not reveal any detectable signals in grass carp pituitary sections (data not shown).


Figure 6
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  		Fig. 6. Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulation of SL release, SL production, and SL gene expression at the pituitary level. A: PACAP immunostaining in grass carp pituitary sections. Immunohistochemical staining was performed using the antiserum for ovine PACAP38 (1:2,000). PACAP immunoreactivity was detected in the neurointermediate lobe of the carp pituitary (1: bright field, x40 magnification). Immunostaining signals were localized mainly in the nerve tracts of the neurohypophysis (2: bright field, x100 magnification) with nerve fibers extending into the vicinity of pituitary cells forming the pars intermedia (3: phase contrast, x400 magnification). B: effects of PACAP and VIP treatment on SL secretion and SL protein expression in grass carp pituitary cells. Pituitary cells were incubated for 48 h with increasing concentrations (0.1–100 nM) of grass carp PACAP (top) or cod VIP (bottom). After that, SL release, cellular SL content, and total SL production were monitored by Western blot using goldfish SL antiserum (1:10,000) and parallel blotting for β-actin was used as an internal control. C: effects of PACAP and VIP on SL{alpha} and SLβ gene expression at the pituitary level. Pituitary cells were challenged for 48 h with increasing levels (0.1–100 nM) of grass carp PACAP or cod VIP. After drug treatment, total RNA was isolated and slot blots for SL{alpha} and SLβ mRNA were performed as described in MATERIALS AND METHODS. In these experiments, parallel blotting of 18S RNA was used as an internal control. Data presented are expressed as means ± SE and are the pooled results from 4 separate experiments. Experimental groups denoted by the same letter represent a similar level of transcript expression [P > 0.05, ANOVA followed by Fisher's least significant difference (LSD) test]. Representative results of slot blots are also included for reference.

 
To examine the direct effects of PACAP on SL expression at the pituitary level, primary cultures of carp pituitary cells were incubated for 48 h with increasing levels of PACAP or VIP (0.1–1,000 nM). After drug treatment, SL protein expression was monitored by Western blot using an antiserum for goldfish SL (3). Based on our validation, the antiserum cross-reacts with SL{alpha} and SLβ (data not shown) and SL immunoreactivity reported in the present study refers to the sum total of the two SL isoforms. As shown in Fig. 6B (top), grass carp PACAP was effective in elevating SL secretion, cellular SL content, as well as total SL production. The minimal effective doses for PACAP to trigger these stimulatory effects could be noted in the 0.1–1 nM dose range. In parallel experiments with similar doses of cod VIP (Fig. 6B, bottom), VIP was found to stimulate SL release and SL production but with no effects on SL content. Unlike PACAP treatment, the stimulatory actions of cod VIP could be observed only with VIP doses reaching the level of 100 nM.

PACAP induction of SL{alpha} and SLβ mRNA expression. To further investigate the mechanisms for PACAP-induced SL expression, the effects of PACAP treatment on SL{alpha} and SLβ gene expression were tested in grass carp pituitary cells under static incubation (Fig. 6C). In this case, increasing concentrations of grass carp PACAP (0.1–100 nM) could elevate basal levels of SL{alpha} and β mRNA in a dose-dependent manner. The minimal effective doses for PACAP stimulation could be observed at 0.1 nM level, while the maximal responses were noted in the 10- to 100-nM dose range. The ED50s for PACAP-induced SL{alpha} and β mRNA expression were estimated to be 1.6 and 2.1 nM, respectively. In parallel experiments, increasing doses of cod VIP (0.1–100 nM) were not very effective in stimulating SL gene expression in carp pituitary cells and significant rises in SL{alpha} and -β mRNA levels could be detected only at a dose of 100 nM.

To confirm the differential actions of PACAP and VIP on SL gene expression, SL{alpha} and SLβ mRNA expression induced by grass carp PACAP (1 nM) were tested in the presence of the PACAP antagonist PACAP6-38 (100 nM; Fig. 7A) and VIP antagonist (4-Cl-D-Phe6,Leu17)VIP (100 nM; Fig. 7B), respectively. A dose of 1 nM was routinely used as PACAP treatment at this concentration could induce SL gene expression close to the half-maximal responses, whereas a similar dose of VIP did not alter SL{alpha} and β mRNA levels. In these experiments, PACAP consistently elevated basal levels of SL{alpha} and β mRNA in carp pituitary cells. These stimulatory effects could be suppressed by simultaneous treatment with PACAP6-38 but (4-Cl-D-Phe6,Leu17)VIP was not effective in these regards. The differential selectivity for PACAP stimulation is consistent with the pharmacological properties of mammalian PAC-I receptors. Therefore, the involvement of PAC-I receptors in PACAP-induced SL gene expression was also tested. In this case, increasing concentrations of the PAC-I receptor agonist Maxadilan (0.001–0.1 nM) were effective in elevating SL{alpha} and SLβ mRNA levels in a dose-dependent manner (Fig. 7C). However, the stimulatory actions of grass carp PACAP (1 nM) on SL{alpha} and SLβ mRNA expression could be reduced or totally abolished by cotreatment with the PAC-I receptor antagonist M65 (100 nM; Fig. 7D).


Figure 7
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  		Fig. 7. Receptor specificity for PACAP-induced SL gene expression at the pituitary level. Effects of PACAP6-38 (A) and (4-Cl-D-Phe6,Leu17)VIP (B; VIP antag) on PACAP-induced SL{alpha} and SLβ mRNA expression in grass carp pituitary cells. Pituitary cells were exposed to grass carp PACAP (1 nM) for 48 h in the presence or absence of PACAP6-38 (100 nM) or VIP antag (100 nM). C: PAC-I receptor activation on SL{alpha} and SLβ mRNA expression at the pituitary level. Pituitary cells were incubated for 48 h with increasing concentrations of the PAC-I receptor agonist Maxadilan (0.001–0.1 nM). D: PAC-I receptor antagonism on PACAP-induced SL{alpha} and SLβ mRNA expression. Pituitary cells were treated with grass carp PACAP (1 nM) for 48 h with or without cotreatment of the PAC-I receptor antagonist M65 (100 nM). In these experiments, total RNA was extracted for the measurement of SL{alpha} and -β mRNA expression and parallel blotting of 18S RNA was used as an internal control. Data presented are means ± SE and are the pooled results from 6 experiments. Groups denoted by the same letter represent a similar level of transcript expression (P > 0.05; ANOVA followed by Fisher's LSD test).

 
PAC-I receptor expression in grass carp SL cells. Given that the pharmacological profiles of PACAP-induced SL{alpha} and -β gene expression were highly comparable with that reported for PAC-I receptors, PAC-I receptor expression in grass carp SL cells was also examined by RT-PCR coupled to LCM. With the use of the antiserum for goldfish SL, SL immunoreactivity was detected exclusively in the NIL of the carp pituitary (Fig. 8A). Similar to the results of in situ hybridization, immunostaining signals were located in the cytoplasm of the pituitary cells within the pars intermedia but not in the nerve tracts forming the neurohypophysis. Pituitary cells with SL immunostaining (refer to as "SL cells") were isolated by LCM (Fig. 8B) and subjected to RT-PCR with primers for the grass carp PAC-I receptor (Fig. 8C). In this case, the 216-bp PCR product for PAC-I receptor was consistently detected in SL cells as well as in mixed populations of grass carp pituitary cells. The PCR product could not be the result of genomic DNA contamination, as no detectable bands were noted in parallel PCR without reversed transcription. Since the antiserum used in this study did not differentiate SL{alpha} and SLβ in grass carp, single-cell PCR was performed in dispersed pituitary cells prepared from the NIL of the carp pituitary (as "NIL cells"). The identity of NIL cells with SL{alpha} (as "SL{alpha} cells") and SLβ expression (as "SLβ cells") was confirmed by RT-PCR using primers specific for SL{alpha} and SLβ, respectively. In the samples prepared, the PCR product for SL{alpha} was not detected in SLβ cells and vice versa (data not shown). Similar to the results based on mixed populations of NIL cells, the 216-bp PCR product for PAC-I receptor was also detected in the RT samples prepared from individual SL{alpha} (Fig. 8D, left) and SLβ cells (Fig. 8D, right).


Figure 8
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  		Fig. 8. PAC-I receptor expression in grass carp SL cells. A: immunohistochemical staining of SL in grass carp pituitary sections using goldfish SL antiserum. Histological examination was conducted under bright field with a magnification at x25 (1), x40 (2), x100 (3), and x400 (4), respectively. In this study, SL immunoreactivity was detected in the cytoplasm of pituitary cells located in the neurointermediate lobe but not in the regions covering the pars distalis. B: isolation of immunoidentified SL cells from mixed populations of grass carp pituitary cells. SL cells were identified by immunostaining and captured on a Capsure HS PCR Cap by laser capture microdissection (LCM). C: RT-PCR of PAC-I receptor in grass carp pituitary cells with SL immunoreactivity. About 200 immunoidentified SL cells were LCM captured on a single cap. Total RNA was extracted and reversely transcribed in the presence (+RT) or absence of Superscript II (–RT). After that, RT-PCR was performed using primers for grass carp PAC-I receptors. The RT sample prepared from mixed populations of pituitary cells was used as a positive control, and parallel RT-PCR for GAPDH was used as an internal control. D: single-cell RT-PCR for PAC-I receptor in grass carp pituitary cells with SL{alpha} and SLβ gene expression. Dispersed pituitary cells were prepared from the NIL of the carp pituitary (as "NIL cells"). These NIL cells were captured individually (with 1 cell per cap) by LCM and subjected to single-cell PCR using primers for PAC-I receptors. The identity of NIL cells with PAC-I receptor expression was later confirmed by PCR using primers for grass carp SL{alpha} and SLβ, respectively. In these studies, RT-PCR of β-actin was used as the internal control.

 

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As a first step to examine SL gene expression in the carp pituitary, grass carp SL{alpha} and SLβ were cloned by nested PCR coupled to 5'/3'-RACE. The results of the Southern blot reveal that SL{alpha} and SLβ are single-copy genes in the carp genome. The amino acid sequences of grass carp SL{alpha} and SLβ are similar and yet distinct from that of GH and PRL (20–25% sequence homology). However, they are highly comparable with the corresponding SL{alpha} and SLβ sequences reported in other species (45–76% sequence homology). Unlike GH and PRL, the mature proteins for SL{alpha} and SLβ may be glycosylated, as a N-linked glycosylation site can be located in the coding sequences of the grass carp SLs. This idea is supported by the findings that SL cells located in the pars intermedia of fish species are sensitive to periodic acid Schiff staining, which is commonly used for glycoprotein detection at the pituitary level (17). Phylogenetic analysis based on nucleotide sequences also confirms that grass carp SL{alpha} and SLβ can be grouped within the clades of SL{alpha} and SLβ, respectively, but are distally related to GH and PRL. These results are in agreement with the recent proposal that SL{alpha} and SLβ represent two distinct SL families that have arisen by genome duplication in bony fish before the divergence of cyprinids (carps) and salmonids (52). Similar to the protein structures for the members of the GH/PRL family (12), 3D protein modeling revealed that grass carp SL{alpha} and SLβ are composed of four antiparallel {alpha}-helixes (H1-H4) arranged in a up-up-down-down topography. The highly conserved regions between the two SLs can be found in H1-H4 helical domains, although the size of H2 and H3 helixes in SL{alpha} are much smaller than those of SLβ. Besides, the third conserved Cys residue in grass carp SL{alpha} is missing in the corresponding position of SLβ. A similar loss of the third conserved Cys has been reported in SLβ of the goldfish (3), zebrafish (52), and trout (48), which constitutes a major structural difference between the two SL isoforms. In fish models, SL{alpha} is known to have seven conserved Cys residues with the last four (1 in the central region and 3 in the COOH terminus) corresponding to the four conserved Cys residues reported in GH and PRL families (2). These seven Cys residues, except for the third one, can form three intramolecular loops in SL{alpha} by disulfide bonding (19). Apparently, the loss of the third conserved Cys residue does not affect the pattern of disulfide loop formation in SLβ and the functional relevance of this structural change still remains to be determined.

In grass carp, SL{alpha} and SLβ were found to express predominately in the pituitary with single transcripts of 2.0 and 1.6 kb, respectively. However, low levels of SL{alpha} transcripts were also detected in other tissues, including the brain, heart, gills, kidney, intestine, liver, muscle, and gonad. In contrast to SL{alpha} with a wide range of tissue distribution, SLβ transcripts were recognized only at low levels in the brain and liver, indicating that the two forms of SLs are differentially expressed in a tissue-specific manner. In fish models, extrapituitary expression of SL has been reported in rainbow trout (47) and perch (21). In rainbow trout, SL transcripts are ubiquitously expressed and extrapituitary expression of SL can be observed even at very early developmental stages (47). To our knowledge, differential expression of SL{alpha} and SLβ in peripheral tissues has not been previously demonstrated and the functional role of these extrapituitary SLs is still unknown. Although the pituitary represents the major site of SL{alpha} and SLβ production, the results based on in situ hybridization reveal that the two forms of SL are expressed in different cell populations located in the pars intermedia of the carp pituitary, with the cells expressing SL{alpha} as the dominant form. These findings are in agreement with the results of the Northern blot showing that SL{alpha} mRNA expression was much higher than that of SLβ at the pituitary level. In a recent study with zebrafish, SL{alpha} and SLβ expression was located in different regions of the posterior pituitary but the population size of the two cell types appeared to be comparable (52). The cause for the discrepancy on relative abundance of the two cell populations is unclear and may be related to species-specific variations. In chum salmon, SL gene expression in the posterior pituitary is activated during the course of sexual maturation (29). Furthermore, an increase in pituitary cells with SL immunostaining can be noted in both male and female pejerrey during gonadal development (42). Given that the carps used in our study were 1-yr-old prepubertal fish, which are different from the adult fish used in the zebrafish study, we do not exclude the possibility that the relative abundance of SL{alpha} and SLβ cells can be modified by subsequent gonadal maturation in the carp species.

In modern-day bony fish, the brain-pituitary axis is unique for the lack of a hypophysial portal blood system and the pituitary is under the direct innervation of the hypothalamus (10). In our recent study (44) on the functional role of PACAP as a GH-releasing factor in grass carp, nerve fibers with PACAP immunoreactivity were found to be widely distributed in various regions of the anterior pituitary. As revealed by the results of PACAP immunostaining in this study, PACAP fibers were also detected in the nerve tracts of the neurohypophysis and spread to the pituitary cells forming the pars intermedia. These anatomical findings raise the possibility that SL expression in the pars intermedia may be under the modulation of PACAP released from the nerve fibers originated from the hypothalamus. This idea is supported by our in vitro studies with grass carp pituitary cells in which PACAP was found to elevate SL release, cellular SL content, and total SL production. In these experiments, the increase in SL production also occurred with parallel rises in SL{alpha} and SLβ mRNA expression, suggesting that PACAP can not only induce SL secretion but can also trigger SL synthesis via upregulation of SL gene expression. It is worth mentioning that the minimal effective doses for PACAP-induced SL release, SL production, and SL gene expression were in the lower nanomolar dose range (0.1–1 nM), which is 100- to 1,000-fold lower than that required for VIP (100 nM) to trigger significant rises in SL responses. Furthermore, transcript expression of SL{alpha} and SLβ induced by PACAP was sensitive to the blockade by the PACAP antagonist PACAP6-38 but not by the VIP antagonist (4-Cl-D-Phe6,Leu17)VIP. These pharmacological properties are similar to that of mammalian PAC-I receptors that are known to have a strong preference for PACAP but with little/no binding affinity for VIP (11). PAC-I receptors have been cloned in fish models (e.g., goldfish) and confirmed to be expressed at the pituitary level (43). With the use of RT-PCR coupled to LCM isolation techniques, PAC-I receptor expression was detected in grass carp NIL cells with SL immunoreactivity. Similar results were also obtained by single-cell PCR in individual NIL cells with SL{alpha} and SLβ transcript expression. In parallel studies with carp pituitary cells, PACAP-stimulated SL{alpha} and SLβ mRNA expression was mimicked by the PAC-I receptor agonist Maxadilan and suppressed by simultaneous treatment with the PAC-I receptor antagonist M65. These results, as a whole, provide evidence for the first time that PACAP can act at the pituitary level to trigger SL{alpha} and SLβ gene expression via activation of PAC-I receptors.

In summary, we have cloned SL{alpha} and β in grass carp and confirmed that they are single-copy genes encoding two SL isoforms with 3D structures comparable with the members of the GH/PRL family. In grass carp, SL{alpha} and SLβ transcripts were detected mainly in the pituitary with low levels of expression in peripheral tissues. At the pituitary level, SL{alpha} and SLβ were found to express in two separate cell populations located in different regions of the pars intermedia. In grass carp, PACAP fibers could be detected in the neurohypophysis and spread to the pars intermedia, in which SL cells were shown to be a major component. Using primary cultures of grass carp pituitary cells, we have shown that PACAP could trigger SL secretion, SL production, and SL gene expression via direct action at the pituitary level. With the use of a combination of molecular and pharmacological techniques, the stimulatory effects of PACAP on SL{alpha} and SLβ gene expression were confirmed to be mediated by pituitary PAC-I receptors. These findings, taken together, strongly suggest that PACAP may serve as a hypophysiotropic factor stimulating SL secretion and synthesis in fish models.


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The project was supported by grants from the Research Grant Council (Hong Kong; to A. O. L. Wong), National 973 Program (China), and University Research Committee (University of Hong Kong). Financial support was also provided from the School of Biological Sciences (University of Hong Kong) in the form of a postgraduate studentship (to Q. Jiang).


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. O. L. Wong, Endocrinology Division, School of Biological Sciences, 4S-12 Kadoorie Biological Sciences Bldg., Univ. of Hong Kong, Pokfulam Road, Hong Kong SAR, P.R. China (e-mail: olwong{at}hkucc.hku.hk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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