Journal of
Applied Ichthyology J. Appl. Ichthyol. 27 (2011), 501–504 2011 Blackwell Verlag, Berlin ISSN 0175–8659
Received: March 25, 2010 Accepted: December 20, 2010 doi: 10.1111/j.1439-0426.2010.01665.x
Molecular characterization of the growth hormone in Chinese sturgeon and its expression during embryogenesis and early larval stages By H. Cao1,2, R. J. Zhou1, Q. W. Wei3, C. J. Li3 and J. F. Gui2 1 College of Life Sciences, Wuhan University, Wuhan, China; 2State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China; 3Key Laboratory of Freshwater Biodiversity Conservation and Utilization of Agriculture Ministry of China, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Jingzhou, China
Summary Chinese sturgeon (Acipenser sinensis Gray) has a fast growth rate starting in early larval stage. Likely, the fast growth is attributed to the growth hormone (GH); an essential polypeptide required for growth and development of vertebrates. In this study, GH cDNA was cloned from a pituitary library. The AsGH cDNA consists approximately 954 bp in size including a 16 bp 5¢-untranslated region and 296 bp 3¢-untranslated region. The open reading frame (642 bp) encodes a 214 amino acids (aa), which represents the precursor composed of a 25 aa signal peptide followed by a 189 aa mature polypeptide. The polyclonal antibody was prepared from the in vitro expressed mature protein. Western blot analysis showed a constant GH expression of a monomeric form in the 13 embryo stages and in several larval stages. Interestingly, two dimeric bands of GH were detected since day 3 in the hatched larvae. Our findings suggest that the GH may play important physiological functions in embryonic and larval development of Chinese sturgeon. Introduction Chinese sturgeon is one of the Acipenseriformes, a group of Chondrichthyans with an evolutionary history of over 184 million years (Peng et al., 2007). This anadromous fish mainly lives in continental shelf of the Yellow Sea and the East China Sea and spawns in the upper Yangtze River. Moreover, it is one of the largest marine fish to enter freshwater. Since its reproduction migratory route was blocked in 1981 by the Gezhouba Dam, its population declined dramatically. Chinese sturgeon became a rare species. The Chinese sturgeon presents an obviously fast growth rate from embryo and larval stages throughout its life cycle allowing adults to reach up to 3 m sometimes 4 m. However, the underlying genetics of the growth process in sturgeon has not been understood so far. Growth hormone (GH) is a pluripotent hormone produced and secreted by the pituitary gland in vertebrates. It is an essential polypeptide required for growth and development of vertebrates. In fish, GH is also involved in many other physiological processes such as reproduction, carbohydrate metabolism, skeletal and soft tissue growth, lipid mobilization, nitrogen retention, foraging behaviour, aggression, predator avoidance, immune function and regulation of ionic and osmotic balance (Peter and Marchant, 1995; Bjo¨rnsson, 1997; Pe´rez-Sa´nchez, 2000). Despite a lot of knowledge which U.S. Copyright Clearance Centre Code Statement:
documents GH physiological function in fish, little is known of GH action in fish in early development stage. The research purpose was to broaden our principle knowledge on GH in sturgeon. Materials and methods Fish sampling
The embryo samples were collected at 1 and 24 h after fertilization from artificial spawning. The larvae were reared in indoor tanks and sampled at larvae 1–9 days after hatching (dph). All the samples were obtained from the Taihu Station, Yangtze River Fisheries Research Institute, Chinese Academy of Fisheries Science.
AsGH cloning and sequences analysis
In order to identify the AsGH, we constructed a SMART cDNA library from the pituitary of a wild mature female Chinese sturgeon. Briefly, the 3¢-end of cDNAs was added dATP-tails by incubating with dATP and Taq polymerase at 72C for 20 min. Then, the modified cDNAs were ligated to pMD-18T vector (Promega) and the plasmids were used to transform Escherichia coli DH5a super competent cells. The plasmid cDNA library was plated to appropriate density to pick individual colonies. DNA sequencing was performed using dRhodamine terminator cycle sequencing kit and ABI PRISMTM 310 Genetic Analyzer (Perkin Elmer). The randomly sequenced 2025 clones revealed the full-length cDNAs of AsGH. Nucleotide sequence identity analysis was performed using BLAST (GenBank, NCBI). The signal peptide and putative cleavage sites were detected using the software at ExPASy Molecular Biology Server (http://www.cbs.dtu.dk/ services/SignalP/). Amino acid alignment and similarity analysis of AsGH (Table 1) were carried out by Clustal multiple sequence alignment. Potential Nglycosylation sites were predicted by searching the motif Asn-Xaa-Ser ⁄ Thr.
Expression of fusion proteins and preparation of polyclonal antibodies
The AsGH cDNA coding mature protein was amplified using the primers: F: 5¢-ACGAATTCATGGCATCAGGTCT-3¢ and R: 5¢-GCCTCGAGCTACAGAGTACAGT-3¢ and digested with EcoRI and XhoI. The digested fragment was inserted in frame to the EcoRI and XhoI double-digested expression vector pET32a (+) vector (Novagen). After the
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Genus
Species
GH Identity (%)
GenBank
Chondrichthyes ⁄ Carcharhiniformes Dipnoi ⁄ Lepidosireniformes Chondrostei ⁄ Acipenseriformes Neopterygii ⁄ Semionotiformes Amiiformes Teleostei ⁄ Anguilliformes Cypriniformes Cypriniformes Siluriformes Salmoniformes Scorpaeniformes Perciformes Tetraodontiformes Pleuronectiformes
Prionace glauca Protopterus annectens Acipenser gueldenstaedtii Lepisosteus osseus Amia calva Anguilla japonica Carassius auratus gibelio Danio rerio Ictalurus punctatus Oncorhynchus tshawytscha Cottus kazika Epinephelus coioides Tetraodon nigroviridis Hippoglossus hippoglossus
64 60 98 79 79 63 47 46 45 42 42 41 40 38
P34006 AAC16496 AAX36064 AAB37388 AAB32122 AAA48535 AAP42272 CAI79040 AAF78944 AAB24612 BAC07248 AAO13496 AAR25694 BAC07253
Table 1 Amino acid identities between Chinese sturgeon and other fish species
recombinant constructs were confirmed by DNA sequencing, it was transformed into E. coli BL21 (DE3). A fusion protein Trx-AsGH was expressed in BL21 (DE3), and detected only in inclusion body. The protein was prepared and immunized rabbit and mouse as described previously (Cao et al., 2009).
Western blot analysis
The protein extracts prepared from Chinese sturgeon embryo and larvae samples were subjected to Western blot analysis. For the samples in different development stages, whole embryos or larvae were collected and determined the development stages under dissecting microscope (Nikon), and then homogenized in EB buffer. After they were separated on 15% SDS-PAGE gel, the proteins were electrophoretically blotted to PVDF membranes (Millipore). The membranes were blocked with 5% dry milk in TBS buffer (100 mM NaCl, 100 mM Tris–HCl, pH 7.5). The blocked membranes were incubated with rabbit antiserum AsGH at a dilution of 1 : 1000 in TBS buffer containing 1.0% dry milk at room temperature for 2 h. The membranes were washed three times for 10 min each in TBST buffer (TBS buffer and 0.1% Tween 20) and then incubated with 1 : 1000 diluted alkaline phosphatase conjugated goat anti-rabbit IgG. After washing three times for 10 min each in TBST buffer, detection was performed using BCIP ⁄ NBT. As a negative control, the pre-immuned sera were also used for Western blot detection, and no positive signal was detected (data not shown). Results Molecular characterization of AsGH cDNA
The cDNA sequences of AsGH have been deposited in GenBank (EU599640). The AsGH cDNA is 945 bp in total length with an open reading frame of 642 bp, starting with the start codon at position 17 and ending with a stop codon at position 659. It consists two untranslated regions (UTR) of 16 bp at the 5¢end as well as of 286 bp at the 3¢end. A consensus poly adenylation signal AATAAA is located 18 bp upstream from the polyA tail. The cleavage site for the putative signal peptide was predicted by means of Signal P and was located between amino acid position 25 and 26. It contains a putative N-linked glycosylation site at residue Asn186 (Fig. 1).
Phylogeny
Alignment of the aa sequences from Chinese sturgeon and Russian sturgeon (Acipenser gueldenstaedtii) revealed that
Fig. 1. The nucleotide and deduced amino acid sequences of cDNA encoding the GH subunit (EU599640). The nucleotide numbers are shown on both sides of the sequences. Putative N-glycosylation sites are marked by a triangle. The start and stop codons are indicated in grey, and the consensus sequence for the polyadenylation signal is boxed
there are only three amino acid residues are different from each other (Fig. 2a). As shown in Fig. 2b, the four cysteine residues and the N-glycosylation site are highly conserved. Table 1 shows the identities of aa sequences between Chinese sturgeon and other fishes ranging between 38 and 79%.
Expression of AsGH through embryo and larva development
We used the AsGH cDNA encoding mature protein to express recombinant proteins and to raise polyclonal antibodies. First of all, in the brain tissues of a 1-year old sample, only two bands can be detected in the pituitary, which were 23.5 and 45 kDa, respectively (Fig. 3). The predicted MW of 23.5 kDa Chinese sturgeon GH was consistent with that from Westernblot. This result verified the effectiveness of the polyclonal antibody. Subsequently, the protein extracts prepared from Chinese sturgeon embryo and larva samples were subjected to Western blot analysis. As shown in Fig. 4a, an equal size protein band of about 23.5 kDa was detected from the
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(a)
(b)
Fig. 2. Alignment of the amino acid sequences of the AsGH and GH of other fishes. (a) Alignment of the amino acid sequences A. sinensis (AsGH) and A. gueldenstaedtii (AgGH). (b) Amia calva (AcGH), Danio rerio (DrGH), Lepisosteus osseus (LoGH), Protopterus annectens (PaGH), Prionace glauca (PgGH). The four cysteine residues are marked by a reversed triangle. The putative N-linked glycosylation sites are boxed. The sequences were taken from GenBank and NCBI (accession no. in Table 1)
45 kDa 35 kDa
M
1
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3
4
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25 kDa Fig. 3. Western blot detection of AsGH in the different tissues of 1-year old Chinese sturgeon. (1: pituitary, 2: telencephalon, 3: midbrain, 4: cerebellum, 5: hypothalamus, 6: medulla oblongata, 7: olfactory bulb, 8: serum)
fertilized eggs to the hatched larvae during embryogenesis. Moreover, two protein bands, which are about 43 and 45 kDa respectively, were also detected from the third day larvae (Fig. 4b). Discussion Numerous GH coding sequences have been studied in teleosts, but only a few of them in primitive fishes, e.g. in blue shark (Prionace glauca) (Yamaguchi et al., 1989), bowfin (Amia calva) (Rubin and Dores, 1994), longnose gar (Lepisosteus osseus) (Rubin et al., 1996), alligator gar (Revol et al., 2005), and Russian sturgeon (Acipenser gueldenstaedtii) (Din et al., 2008). The GH of Chinese sturgeon has the highest degree of identity to GH sequences from Russian sturgeon. As in most teleost GH, it has also four cysteine residues and a putative glycosylation site. It has been demonstrated that N-linked glycosylation can serve as a signal for protein transport to the cell surface (Guan et al., 1985). GH is very important in the regulation of development and somatic growth in vertebrates. In this study, we detect the expression from fertilized eggs to 10 dph larvae when they begin to feed. However, this 23.5 kDa peptide diminishes in 10 dph with the onset of exogenous feeding (data not shown) indicating that the 23.5 kDa GH peptide is maternal factor. In
(a) 45 kDa 35 kDa 25 kDa 18 kDa
M
(b) 45 kDa 35 kDa 25 kDa 18 kDa
M 1 2
1 2
3 4
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8 9 10 11 12 13
GH
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9
GH dimer GH
Fig. 4. Western blot detection of AsGH in 13 typical embryo developmental stages (a) and larvae (b). (a) (1: 2-cell stage, 2: 4-cell stage, 3: 8-cell stage, 4: 32-cell stage, 5: blastulation, 6: gastrulation, 7: blastopore stage, 8: neurula stage, 9: heart primordia, 10: heart beating stage, 11: muscular effect stage, 12: organ formation stage, 13: hatching period). (b) 1: 1 dph, 2: 2 dph, 3: 3 dph, 4: 4 dph, 5: 5 dph, 6: 6 dph, 7: 7 dph, 8: 8 dph, 9: 9 dph)
fact, expression of GH in oocytes seems to occur in a wide range of vertebrates. In mammals, it has been previously reported in ovine (Lacroix et al., 1996), monkey (Golos et al., 1993), and humans (Frankenne et al., 1988), and the further results indicate that the endometrium, placenta and fetus are all potential targets for the placental GH (Lacroix et al., 1999). In fish, it has been detected the GH mRNA in mature oocytes in rainbow trout (Oncorhynchus mykiss) (Yang et al., 1999), orange-spotted grouper (Li et al., 2005) and alligator gar (Revol et al., 2005), as well as for mammals. Furthermore, GH expression was reported during larvae stage in some fishes. For example, GH immunoreactive cells were detected 1–2 days after hatching in sea bream (Sparus aurata) (Power and Canario, 1992) and sea bass (Dicentrarchus labrax) (Cambre´ et al., 1990) larvae. In orange-spotted grouper, GH mRNA were firstly detected in unfertilized eggs and then decreased sharply until the 1 dph (Li et al., 2005). In
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Chinese sturgeon, hatching occurs 2 days after fertilization of the eggs. Interestingly, in our study, two protein bands of about 43 and 45 kDa were detected since day 3 of the hatched larvae. Based on their size, the newly synthesized growth hormone proteins in larvae might be dimer of growth hormone. In the pituitary of 1-year old Chinese sturgeon, we detected a 45 kDa band. In fact, it has been previously reported that there are several forms of human growth hormone (hGH). In conclusion, our findings demonstrated the presence of GH in the early embryogenesis of Chinese sturgeon, which suggest that the GH may play a key role in its early development and this function may have appeared early during vertebrates evolution. Cloning of GH and the elucidation of its role during larval stages will contribute to the better understanding of Chinese sturgeon larval physiology. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (30900139). We appreciate the very helpful comments made by the anonymous reviewers. References Bjo¨rnsson, B. T. H., 1997: The biology of salmon growth hormone: from daylight to dominance. Fish Physiol. Biochem. 17, 9–24. Cambre´, M.; Mareels, G.; Corneillie, S.; Moons, L.; Ollevier, F.; Vandesande, F., 1990: Chronological appearance of the different hypophysial hormones in the pituitary of sea bass larvae (Dicentrarchus labrax) during their early development: an immunocytochemical demonstration. Gen. Comp. Endocrinol. 77, 408–415. Cao, H.; Zhou, L.; Zhang, Y. Z.; Wei, Q. W.; Chen, X. H.; Gui, J. F., 2009: Molecular characterization of Chinese sturgeon gonadotropins and cellular distribution in pituitaries of mature and immature individuals. Mol. Cell. Endocrinol. 303, 34–42. Din, S. Y.; Hurvitz, A.; Goldberg, D.; Jackson, K.; Levavi-Sivan, B.; Degani, G., 2008: Cloning of Russian sturgeon (Acipenser gueldenstaedtii) growth hormone and insulin-like growth factor I and their expression in male and female fish during the first period of growth. J. Endocrinol. Invest. 31, 201–210. Frankenne, F.; Closset, J.; Gomez, F.; Scippo, M. L.; Smal, J.; Hennen, G., 1988: The physiology of growth hormones (GHs) in pregnant women and partial characterization of the placental GH variant. J. Clin. Endocrinol. Metab. 66, 1171–1180. Golos, T. G.; Durning, M.; Fisher, J. M.; Fowler, P. D., 1993: Cloning of four growth hormone ⁄ chorionic somatomammotropin-related complementary deoxyribonucleic acids differentially expressed during pregnancy in the rhesus monkey placenta. Endocrinology 133, 1744–1752.
H. Cao et al. Guan, J. L.; Machamer, C. E.; Rose, J. K., 1985: Glycosylation allows cell-surface transport of an anchored secretory protein. Cell 42, 489–496. Lacroix, M. C.; Devinoy, E.; Servely, J. L.; Puissan, C.; Kann, G., 1996: Expression of the growth hormone gene in ovine placenta: detection and cellular localization of the protein. Endocrinology 137, 4886–4892. Lacroix, M. C.; Devinoy, E.; Cassy, S.; Servely, J. L.; Vidaud, M.; Kann, G., 1999: Expression of growth hormone and its receptor in the placental and feto-maternal environment during early pregnancy in sheep. Endocrinology 140, 5587–5597. Li, W. S.; Chen, D.; Wong, A. O.; Lin, H. R., 2005: Molecular cloning, tissue distribution, and ontogeny of mRNA expression of growth hormone in orange-spotted grouper (Epinephelus coioides). Gen. Comp. Endocrinol. 144, 78–89. Peng, Z.; Ludwig, A.; Wang, D.; Diogo, R.; Wei, Q.; He, S., 2007: Age and biogeography of major clades in sturgeons and paddlefishes (Pisces: Acipenseriformes). Mol. Phylogenet. Evol. 42, 854–862. Pe´rez-Sa´nchez, J., 2000: The involvement of growth hormone in growth regulation, energy homeostasis and immune function in the gilthead sea bream (Sparus aurata): a short review. Fish Physiol. Biochem. 22, 135. Peter, R. E.; Marchant, T. A., 1995: The endocrinology of growth in carp and related species. Aquaculture 129, 299–321. Power, D. M.; Canario, A. V., 1992: Immunocytochemistry of somatotrophs, gonadotrophs, prolactin and adrenocorticotropin cells in larval sea bream (Sparus auratus) pituitaries. Cell Tissue Res. 269, 341–346. Revol, A.; Rodrı´ guez, M. L. G.; Montenegro, V. H.; Aguilera, C.; Saldan˜a, H. B.; Mendoza, R., 2005: Cloning of the growth hormone cDNA of alligator gar Atractosteus spatula and its expression through larval development. Comp. Biochem. Physiol., Part A Mol. Integr. Physiol. 140, 423–429. Rubin, D. A.; Dores, R. M., 1994: Cloning of a growth hormone from a primitive bony fish and its phylogenetic relationships. Gen. Comp. Endocrinol. 95, 71–83. Rubin, D. A.; Youson, J. H.; Marra, L. E.; Dores, R. M., 1996: Cloning of a gar (Lepisosteus osseus) GH cDNA: trends in actinopterygian GH structure. J. Mol. Endocrinol. 16, 73–80. Yamaguchi, K.; Yasuda, A.; Lewis, U. J.; Yokoo, Y.; Kawauchi, H., 1989: The complete amino acid sequence of growth hormone of an elasmobranch, the blue shark (Prionace glauca). Gen. Comp. Endocrinol. 73, 252–259. Yang, B. Y.; Green, M.; Chen, T. T., 1999: Early embryonic expression of the growth hormone family protein genes in the developing rainbow trout, Oncorhynchus mykiss. Mol. Reprod. Dev. 53, 127–134. AuthorÕs address: Jian-fang Gui, State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China. E-mail:
[email protected] 