Fish recombinant gonadotropins

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Pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play central roles in regulating gametogenesis and the production of gonadal hormones, in fish as in other vertebrates. Pituitary gonadotropins are composed of two non-covalently associated polypeptide subunits, which must be glycosylated, folded, and assembled as a heterodimer to be biologically active. Since biotechnology for the production of recombinant proteins has been greatly advanced, it is possible that fish FSH and LH are produced biotechnologically as an alternative way for various manipulations in modern aquaculture. This review discusses different platforms currently being applied for recombinant piscine gonadotropin production...

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  1. Fish recombinant gonadotropins by Berta Levavi-Sivan (1), Matan GoLan (1), Joseph aizen (1) & abigail eLizur (2) AbsTrAcT. - Pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play central roles in regulating gametogenesis and the production of gonadal hormones, in fish as in other vertebrates. Pituitary gonado- tropins are composed of two non-covalently associated polypeptide subunits, which must be glycosylated, folded, and assembled as a heterodimer to be biologically active. Since biotechnology for the production of recombinant proteins has been greatly advanced, it is possible that fish FSH and LH are produced biotechnologically as an alternative way for vari- ous manipulations in modern aquaculture. This review discusses different platforms currently being applied for recombi- nant piscine gonadotropin production. Key words. - expression system - Yeast - insect cells - estradiol - Follicle - Stimulating hormone - Luteinizing hormone. Introduction are being evaluated for the production of these valuable pro- it has been almost a century since the laboratories of teins. Although some have been used for many years, others Cushing and Aschner performed the first pituitary ablation are relatively new and still experimental. Factors such as studies, implicating this gland as a primary regulator of ver- scale-up, total annual production, speed of production set- tebrate reproduction. The gonadotropic effects of pituitary up, post-translational modifications and regulatory issues extracts were soon associated with two distinct endocrine come into play in choosing the system that is most suitable factors, known as gonadotropins (GTHs) that became desig- for any given target protein. This review discusses different nated as follicle-stimulating hormone (FSH) and luteinizing systems currently being applied for recombinant piscine hormone (LH) (Campbell, 2005). gonadotropin production. Phylogenetic analyses based on the amino acid sequenc- Recombinant fish gonadotropin production platforms es of vertebrate gonadotropin subunits, either derived from Thus far, recombinant gonadotropins have been produced chemically isolated peptides or deduced from cDnas, from 10 fish species, representing four teleostean orders, revealed monophylogenetic lineage for each subunit. The using various expression hosts: GPα of carp (Cyprinus car- observed common ancestral origin for piscine and tetrapod pio; Huang et al., 1991), FSH and LH of gilthead seabream gonadotropin subunits has justified the use of the terms FSH (Sparus aurata; Meiri et al., 2000), goldfish (Carassius and LH for the fish GtH I and GtH II, respectively (Yaron et auratus; Kobayashi et al., 2003, Kobayashi et al., 2006) al., 2003). In fishes, FSH is considered to regulate early Manchurian trout (Brachymystax lenok; Cui et al., 2007) and phases of gametogenesis, such as vitellogenesis and sperma- channel catfish (Ictalurus punctatus; zmora et al., 2003) by togenesis, whereas LH is responsible for the final maturation in vitro or in vivo insect cells; FSH and LH of African catfish processes, such as oocyte maturation, ovulation, spermia- Clarias gariepinus by amoeba Dictyostelium discoideum tion, and milt production (Yaron et al., 2003). (vischer et al., 2003); FSH of Japanese eel (Anguilla japoni- The gonadotropins are all glycoproteins comprised of ca; Kamei et al., 2003) and FSH and LH of tilapia (Oreo- dissimilar subunits designated α and β; the α subunit is the chromis mossambicus; Kasuto and Levavi-Sivan, 2005, same across hormones within a species, while the β subunits aizen et al., 2007a, 2007b) by yeast; Manchurian trout (Choi are unique to each hormone (Pierce and Parsons, 1981). et al., 2005) and Zebrafish (Danio rerio; So et al., 2005) by Since GTHs are heterodimeric glycoproteins, the prokaryot- mammalian cells; and LH and FSH of goldfish by Rainbow ic expression systems (bacteria) that do not carry out post- trout embryos (Morita et al., 2003,2004) (Tab. i). translational modifications of proteins are not appropriate for expression of biologically active gonadotropins. Howev- Yield er, eukaryotic cell culture systems (amoeba, yeast, insect and Bacteria have proven useful as bioreactors because they mammalian) have been established and small scale biologi- are grown easily at any scale. However, they are limited in cally active recombinant fish GTHs successfully produced in their ability to perform the post-translational protein modifi- these systems. cations necessary for the gonadotropin biological activity. The biotechnology industry is currently experiencing an Mammalian cells can perform complex post-translational extreme shortage of manufacturing capacity for recombinant modifications, although the costs associated with scaling proteins. as a result, a growing number of biological systems these systems up for mass-production purposes are extremely (1) Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University, PO Box 12, Rehovot 76100, Israel. [] (2) Faculty of Science, Health and Education, University of the Sunshine Coast, Maroochydore DC, Qld 4558, Australia. Cybium 2008, 32(2) suppl.: 17-21.
  2. Recombinant fish gonadotropins Levavi-Sivan et al. Table I. - Recombinant fish gonadotropins expressed at different platforms. high. Certain eukaryotic systems, such as yeast and insects, it is interesting to note that in the P. pastoris yeast sys- can be scaled-up with relative ease, are capable of post-trans- tem, the yield of the gonadotropin heterodimer was signifi- lational modifications, and may also offer adequate produc- cantly lower than that of the monomer, probably because of tion yields, but they have unique glycosylation patterns, the complex structure of the former (Kasuto and Levavi- which are not necessarily compatible with that of the native Sivan, 2005, Aizen et al., 2007a). However, in the drosophila protein/receptor. Transgenic fish have a potentially large pro- duction capacity at lower costs than mammalian cell culture cells the monomer was toxic to the cells and only the het- but involve relatively slow and expensive production set-up erodimer could be produced at high levels (Zmora et al., and have yet to cross many regulatory hurdles. 2003). 18 Cybium 2008, 32(2) suppl.
  3. Levavi-Sivan et al. Recombinant fish gonadotropins Table I. - Continued. Structural integrity of recombinant gonadotropins molecular mass estimates may result from differences in the The molecular mass estimates of recombinant FSH and degree of glycosylation. LH vary considerably, however were in the range of those Glycosylation of recombinant gonadotropins reported for native teleost species; between 15 kDa and 28 among gonadotropin subunits, the sequences of the com- kDa for the subunits and between 29 kDa and 55 kDa for the mon α-subunit show the highest degree of conservation intact hormones (Weltzien et al., 2004 and Tab. I). Because across vertebrates. Sequence comparisons exhibited similar- the amino acid composition of individual subunits is similar ities ranging from 60% to 90%, largely due to the carboxyl- for of all teleosts species (Yaron et al., 2003) the different terminus half, which is the most conserved part of the mole- Cybium 2008, 32(2) suppl. 19
  4. Recombinant fish gonadotropins Levavi-Sivan et al. cule (Yaron et al., 2003). Fish FSHβ-subunits are more divergent than the LHβ-subunits (average identities of 53% and 67%, respectively) mainly due to a rapid change which occurred during the evolution of teleosts in general, and that of perciform species in particular. The N-terminus of teleosts FSHβ sequences exhibits an unexpected divergence at asparagine (N), putative glyco- sylation sites, which are most conserved in FSHβ of other vertebrates. The teleost GPα subunit, FSHβ subunit and LHβ subunit carries two, one or two N-linked oligosaccha- ride, respectively, and that consists of a heterogeneous array of neutral, sulfated, and sialylated structures. The sugars, amounting to more than 30% of the gonadotropin mass, form the major chemical basis of the charge heterogeneity of GTH isoforms, in the pituitary and bloodstream of mammals and are essential for GTH intracellular folding, secretion, clear- Figure 1. - Secretion of estradiol from previtellogenic tilapia ova- ance from blood, binding and signaling at the target-cell ries (GSI = 0.004% ± 0.0002%) in response to various concentra- tions of recombinant tilapia FSH or LH, in response to tilapia pitu- level. itary extract (TPE), or human chorionic gonadotropin (hCG). Deglycosylation of recombinant fish FSH and LH with PnGase F, which hydrolyzes all types of n-glycan chains, in vitro to gonadal fragments (Tab. i), showing that the reduced the glycosylated forms to the nascent translated pro- recombinant proteins (with all the necessary modifications) tein. This implies that in Japanese eel, tilapia, goldfish and were able to activate the proper gonadotropin receptor. trout, the carbohydrate modifications on the mature protein Most of the species that their gonadotropins were studied occur exclusively through N-linked, and not at all through posses synchronous ovaries (catfish, eel, trout, and grouper) o-linked, glycosylation (Kamei et al., 2003, Kasuto and meaning that their ovaries contain, at the same time, only Levavi-Sivan, 2005, Aizen et al., 2007a, Morita et al., 2004, one generation of follicles, all at the same stage. We used Ko et al., 2007). Deglycosylation experiments examining tilapia that posses asynchronous ovaries, thus their ovaries other species of teleosts are needed in order to determine if contain several generations of follicles, at different stages, at in all fish GTHs the glycosylation is exclusively through the same time. in order to elucidate the differential role of N-linked or in some species it might have also O-linked oli- each of the gonadotropins on the secretion of estradiol along gosaccharides. in contrast, mammalian glycoprotein hor- the reproductive cycle, we have exposed tilapia previtello- mones carry both o-linked and n-linked oligosaccharides. genic ovarian fragments (GSI = 0.004% ± 0.0002%; The role of glycosylation in the piscine gonadotropins is still BW = 24.1 gr ± 2.23), to various doses of recombinant tFSH unclear and should be further elucidated. or tLH according to (aizen et al., 2007a). As shown in figure Gene expression in response to application of recombinant 1 estradiol was increased in a dose dependent manner in gonadotropins response to both gonadotropins, with a marked increase in The mRNA levels of Cyp19a1a and Cyp19a1b (the ovar- response to FSH. Both tilapia pituitary extract (TPe) and ian and brain forms of aromatase) rose dramatically after hCG, a mammalian gonadotropin that is commonly used in injecting recombinant grouper LH, concomitantly with induced spawning of various fish species, were also potent secretion of estradiol, while mRNA levels of GnRH dropped in the induction of E2 secretion (Fig. 1). We have recently and that of LHβ and GPα increased (Cui et al., 2007). found that both GnrH-stimulated and basal secretion of The gene expression of different enzymes along the ster- 11-KT is inhibited after injection of anti-recombinant FSHβ, oid biosynthesis pathway should be elucidated in several implicating the involvement of FSH in 11-KT secretion in other teleost groups, especially in fish bearing asynchronous tilapia (aizen et al., 2007a). Taken together these results are ovaries. Using recombinant gonadotropins which can be in accord with the role of FSH in the regulation of early administered together or separately, can aid in elucidating phases of gametogenesis (Yaron et al., 2003). the physiological events associated with secretion of each of ELISA using recombinant gonadotropins the different steroids. recombinant tilapia gonadotropins were recently used to Sex steroid production develop specific and homologous competitive ELISAs for all the recombinant LH and/or FSH, prepared by the dif- measurements of FSH and LH in the plasma and pituitary of ferent systems, increases the secretion rates of e2 and/or the fish, using primary antibodies against rtLHβ or rtFSHβ, androgen in females and males, respectively, when applied respectively, rtLHβα or rtFSHβα for the standard curves, 20 Cybium 2008, 32(2) suppl.
  5. Levavi-Sivan et al. Recombinant fish gonadotropins and rtLHβ or rtFSHβ for coating. The sensitivity of the assay KASUTO H. & B. LEVAVI-SIVAN, 2005. - Production of biologi- was 15.84 pg/ml for tLH and 0.24 pg/ml for tFSH measure- cally active tethered tilapia LHβα by the methylotrophic yeast Pichia pastoris. Gen. Comp. Endocrinol., 140: 222-232. ments in the plasma, whereas for the measurements in the KO H., PARK W., KIM D.-J., KOBAYASHI M. & Y.C. SOHN, pituitary, the sensitivity was 2.43 ng/ml and 1.52 ng/ml for 2007. - Biological activities of recombinant Manchurian trout tLH and tFSH, respectively. The standard curves for tFSH FSH and LH: Their receptor specificity, steroidogenic and vitel- logenic potencies. J. Mol. Endocrinol., 38: 99-111. and tLH paralleled those of serially diluted pituitary extracts KOBAYASHI M., MORITA T., IKEGUCHI K., YOSHIZAKI G., of other cichlids, as well as of serially diluted pituitary SUZUKI T. & S. WATABE, 2003. - Production of recombinant extract of seabream, european seabass and hybrid bass goldfish gonadotropins by baculovirus in silkworm larvae. Fish (aizen et al., 2007b). Physiol. Biochem., 28: 469-471. KOBAYASHI M., MORITA T., IKEGUCHI K., YOSHIZAKI G., Conclusion SUZUKI T. & S.WATABE, 2006. - In vivo biological activity of recombinant goldfish gonadotropins produced by baculovi- The glycoprotein hormones are an interesting group of rus in silkwonn larvae. Aquaculture, 256: 433-442. molecules not only because of their importance in reproduc- MEIRI I., ZMORA N. & A. ELIZUR, 2000. - Functional expres- tion and overall physiology but also because of their unique sion of recombinant seabream FSH and LH in baculovirus structural features. The mammalian gonadotropins have infected insect cells. In: Sixth International Symposium on Reproductive Physiology of Fish. (Norberg B. et al., eds), p. proven to be useful models for understanding protein folding 488. Univ. of Bergen. and protein-protein interactions, as well as for studying the MORITA T., YOSHIZAKI G., KOBAYASHI M. & T. TAKEUCHI, role of carbohydrates in protein function. The availability of 2003. - Production of biologically-active recombinant goldfish gonadotropins in transgenic rainbow trout. Fish Physiol. Bio- recombinant fish FSH and LH in sufficient quantities should chem., 28: 473-474. enable us to better define their physiological role and pitui- MoriTa T., YoSHizaKi G., KoBaYaSHi M., WaTaBe S. & T. tary control as well as to further explore the usefulness of TAKEUCHI, 2004. - Fish eggs as bioreactors: the production fish as a model system for understanding gonadotropin func- of bioactive luteinizing hormone in transgenic trout embryos. Transgenic Res., 13: 551-557. tion in vertebrates. PIERCE J.G. &T.F. PARSONS, 1981. - Glycoprotein hormones: structure and function. Annu. Rev. Biochem., 50: 465-495. References SO W.-K., KWOK H.-F. & W. GE, 2005. - Zebrafish gonadotropins aizen J., KaSuTo H., GoLan,M., zaKaY H. & B. Levavi- and their receptors: II. Cloning and characterization of zebrafish SIVAN, 2007a .- Expression and characterization of biologi- follicle-stimulating hormone and luteinizing hormone subunits cally active recombinant tilapia FSH: Immunohistochemistry, their spatial temporal expression patterns and receptor specifi- stimulation by GnrH and effect on steroid secretion. Biol. city. Biol. Reprod., 72: 1382-1396. Reprod., 76: 692-700. VISCHER H.F., GRANNEMAN J.C.M., LINSKENS M.H.K., AIZEN J., KASUTO H. & B. LEVAVI-SIVAN, 2007b. - Develop- SCHULZ R.W. & J. BOGERD, 2003. - Both recombinant Afri- ment of specific enzyme-linked immunosorbent assay for deter- can catfish LH and FSH are able to activate the African catfish mining LH and FSH levels in tilapia, using recombinant gona- FSH receptor. J. Mol. Endocrinol., 31: 133-140. dotropins. Gen Comp Endocrinol, 153: 323-332. 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TRANT, 2003. - (Epinephelus coioides) luteinizing hormone in insect cells by Production of channel catfish (Ictalurus punctatus) recom- the baculovirus expression system and its biological effect. binant gonadotropins using the S2 Drosophila cell line system. Biol. Reprod., 76, 74-84. Fish Physiol. Biochem., 28: 475-477. HUANG C., HUANG F., CHANG G., CHANG Y., LO C., FRAS- er M. & T.B. Lo, 1991. - expression of two forms of carp gonadotropin alpha subunit in insect cells by recombinant bac- ulovirus. Proc. Natl. Acad. Sci. USA, 88(17): 7486-7490. KAMEI H., OHIRA T., YOSHIURA Y., UCHIDA N., NAGASAWA H. & K. AIDA, 2003. - Expression of a biologically active recombinant follicle stimulating hormone of Japanese eel Anguilla japonica using methylotropic yeast, Pichia pastoris. Gen. Comp. Endocrinol., 134: 244-254. Cybium 2008, 32(2) suppl. 21



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