Isolation, cloning and characterization of the FSH beta gene promoter of the chinook salmon (oncorhynchus tshawytscha)

1 INTRODUCTION 1.1 The gonadotropins The gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are produced in the gonadotropes in the vertebrate pituitary gland These gonadotropins are heterodimers comprising a common α subunit and a unique, hormone specific β subunit (Pierce and Parsons, 1981) After their synthesis and release, FSH and LH bind to specific receptors in the testes and ovary to stimulate their growth and development, and to regulate their function In mammals, FSH is primarily involved in spermatogenesis in the male and folliculogenesis in the female In the female, it is also involved in the conversion of androgens to estrogens LH controls the synthesis of androgens as well as gamete release (spermiation in males and ovulation in females) In some species, it may also be responsible for regulating the synthesis and subsequent release of progesterone by the corpus luteum (Norris, 1997) In the teleost fish, it was initially thought that only a primordial gonadotropin (GtH) existed However in 1979, Ng and Idler reported the presence of two types of gonadotropin in plaice and flounder pituitaries Subsequently, two different forms of GtH, GtH-I and GtH-II, were isolated from salmon and shown to have different steroidogenic activities (Suzuki et al., 1988) The studies of the amino acid (Itoh et al., 1988) and the cDNA sequences (Sekine et al., 1989) revealed close similarity of GtH-I and GtH-II to the mammalian FSH and LH, respectively, and thus established the duality of gonadotropins in the teleost fish With additional information on the GtH structures from numerous other fish species, the mammalian nomenclature has been adopted (Quérat, 1994; 1995) The roles of the pituitary gonadotropins in controlling gonadal function and subsequent fertility in the teleosts are less clear than in mammals Nevertheless, studies have shown that in the salmonid species, the two gonadotropins have distinct roles in reproduction It has been suggested that FSH regulates the early stages of gonadal development, vitellogenesis and spermatogenesis, while LH is responsible for stimulating the events leading to final oocyte maturation and ovulation in females, and spermiation in males (Suzuki et al., 1988; Swanson et al., 1991; Gomez et al., 1999) This is in agreement with the observation that FSH levels in the pituitary and plasma increase with gonadal development and are also correlated to the levels of 11-ketotestosterone and estradiol in the male and female coho salmon (Swanson et al., 1991) FSH levels decrease while the LH levels begin to increase and reach a maximum during spawning (Swanson et al., 1991; Slater et al., 1994; Figure 1) This marked elevation of LH is most probably responsible for the single and synchronized spawning behavior of the salmonids This is further supported by electron microscopy studies which demonstrated the differential biosynthesis of FSH and LH during the reproductive cycle of the rainbow trout (Naito et al., 1991) Plasma levels of FSH and LH LH FSH Mar Apr May Jun July Aug vitellogenesis Sep Oct Nov ovulation Figure 1: Plasma levels of FSH and LH in coho salmon during reproductive maturation (modified from Dickhoff and Swanson, 1990) The expression and pulsatile secretion of these hormones are temporally regulated throughout the reproductive cycle and are subject to the complex control of hypothalamic releasing factors, paracrine factors from within the pituitary, and through the feedback actions of gonadal hormones The secretion of the hypothalamic factors in turn is also regulated by feedback actions of the gonadal hormones (summarized in Figure 2) Hypothalamus +ve GnRH inhibin, activin, follistatin - ve +ve -ve Pituitary -ve or +ve Gonadal peptides (inhibin, activin, follistatin) - ve +ve -ve +ve LH FSH Gonads Steriods (T, E, P) Figure 2: The regulation of gonadotropin gene expression in the hypothalamicpituitary-gonadal axis Gonadotropin releasing hormone (GnRH), synthesized in and released from the hypothalamus, binds to GnRH receptors on the surface of the pituitary gonadotrope This leads to the synthesis and secretion of LH and FSH which stimulate the production of steroid hormones in their gonads Testosterone (T), estrogen (E) and progesterone (P) negatively or positively regulate the synthesis of the gonadotropins directly at the pituitary, or indirectly by modulating GnRH secretion from the hypothalamus The gonadal peptides, inhibin, activin and follistatin, also regulate of gonadotropin gene expression by exerting positive or negative feedback (modified from Brown and McNeilly, 1999) Unlike the mammalian FSH and LH, which are largely co-expressed in the same cells of the mature pituitary, teleost FSH and LH are produced in distinct cells, in different locations within the proximal pars distalis (PPD) FSHβ is produced in cells in close association with the somatotrophs, surrounding the nerve ramifications, while the LHβ subunit is produced in the peripheral regions of the PPD The common α subunit, however, is produced in both types of cells (Nozaki et al., 1990 and Naito et al., 1991) 1.2 Genomic organization of the gonadotropins The gonadotropins are encoded by separate genes localized in different chromosomes (Naylor et al., 1983; Julier et al., 1984) The genomic sequences encoding the LHβ and FSHβ genes of several mammalian and a few teleost species have been elucidated, and analysis has revealed that like the α subunit genes, the genomic organization of β subunit genes is generally well conserved Unlike the α subunit gene however, which is composed of four exons and three introns (Fiddes and Goodman, 1979; Godine et al., 1982; Nilson et al., 1983; Gordon et al., 1988), all the β subunit genes comprise three exons and two introns Furthermore, the β genes are smaller due to the considerably smaller introns, with a total length of about 1.5 kb for LHβ and kb for FSHβ (Figure 3) The positions of the β subunit introns are also conserved The first intron interrupts the region of either the signal peptide or the 5’ untranslated sequences whereas the second intron is located three amino acids downstream from the fifth cysteine residue in the coding region (Jameson et al., 1984; Talmadge et al., 1984; Virgin et al., 1985; Gharib et al., 1989; Ezashi et al., 1990; Xiong et al., 1994a; Rosenfeld et al., 1997; Sohn et al., 1998) or the sixth cysteine residue in the goldfish (Yoshiura et al., 1997) However, compared to the LHβ subunit gene, the genomic structure of the FSHβ subunit gene is less conserved, particularly in regard to the size of introns and 3’ untranslated region (3’ UTR; Figure 3) Compared to the 0.3 – 0.35 kb size of intron of the LHβ genes, the size of the corresponding intron in the FSHβ genes is more variable, ranging from 0.28 kb to 1.35 kb (Figure 3) Furthermore, the mammalian FSHβ is different from other β subunit genes in that it possesses an extremely long 3’UTR that is over kb, as seen in the rat (1 kb), cow (1.2 kb) and human (1.5 kb) Within this 3’UTR, there are five highly conserved segments (Gharib et al., 1989) whose significance is presently not known but it is possible that they may be involved in determining RNA stability, as shown for other genes (Shaw and Kamen, 1986) Limited information is available for the teleost FSHβ genomic sequence as only two FSHβ genes (goldfish and tilapia) have been isolated and sequenced to date (Rosenfeld et al., 1997; Sohn et al., 1998) The genomic organization in both fish conforms to the three exons and two introns, as reported in the mammalian FSHβ genes, although the exon of the goldfish and tilapia FSHβ genes contains a much shorter 3’UTR than those of mammals (Figure 3) The location of the first and second introns in both fish shows a well-conserved pattern similar to that of mammalian FSHβ genes For the tilapia, the gene is found to be coded by a single copy gene In contrast, two distinct genes (GTHIβ-1 GTHIβ-2) encoding the FSHβ in the goldfish were isolated and sequence analysis revealed high sequence identity between the coding regions Initial studies revealed that both goldfish FSHβ genes are expressed, although one of these is expressed in higher proportion in sexually immature goldfish, while both are expressed in equal amounts in the mature fish This suggests differential regulation of the goldfish GTHIβ genes at various reproductive stages Comparison of the amino acid sequence of the tilapia FSHβ and LHβ with β subunits from other fish species showed that the FSHβ subunit protein sequence is less conserved than that of the LHβ gene The tilapia LHβ amino acid sequence shares the highest homology with the grouper at 96%, while the highest homology of the tilapia FSHβ amino acid sequence is with that of the bonito and is only 60% (Rosenfeld et al., 1997) LHβ subunit gene organization E1 Porcine Human Bovine E2 In 0.30 0.30 0.35 0.28 0.30 Rat Equine Tilapia E3 In 0.20 0.30 0.20 0.35 0.28 0.35 1.25 FSHβ subunit gene organization Goldfish Mouse Rat Ovine Bovine Porcine Human Tilapia 0.32 0.79 0.28 0.70 0.62 1.25 0.64 1.26 0.64 1.60 0.62 1.56 0.92 1.10 1.35 1.57 1.40 0.9 Figure 3: Comparison of the LHβ and FSHβ subunit genes The open reading frames are represented by yellow boxes and the untranslated region of exons (E) are represented by open boxes Blue lines between the boxes indicate introns (In) Numbers below the introns show the approximate length in kb References for LH β subunit genes: porcine (Ezashi et al., 1990); human (Talmadge et al., 1984); bovine (Virgin et al., 1985); rat (Jameson et al., 1984); equine (Sherman, 1992); tilalia (Rosenfeld et al., 1997) References for FSH β subunit genes: goldfish (Sohn et al., 1998); mouse (Kumar et al., 1995); rat (Gharib et al., 1989); ovine (Guzman et al., 1991); bovine (Kim et al., 1988); porcine (Hirai et al., 1990); human (Jameson et al., 1988); tilapia (Rosenfeld et al , 1997) (Modified from Bousfield et al., 1994) 1.3 Transcriptional regulation of the gonadotropin subunit genes The regulation of gonadotropin genes comprises basal gene expression which targets these genes specifically to the gonadotropes, and hormonal stimulation in which GnRH and gonadal steroids up-regulate expresson at puberty To date, much less is known about the regulation of these genes in teleost fish than in mammals and information regarding the regulation of the FSHβ subunit gene expression lags far behind what is known about the LHβ subunit 1.3.1 Regulation of gonadotropins by GnRH The decapeptide GnRH plays a critical role in reproductive development and function in vertebrates by stimulating the biosynthesis and secretion of the pituitary gonadotropins In the teleost, in vivo and transfection studies demonstrated the stimulatory effect of GnRH on LH and FSH gene expression, as seen in the goldfish (Khakoo et al., 1994; Klausen et al., 2001), tilapia (Melamed et al., 1996; Gur et al., 2002), striped bass (Hassin et al., 1995), sea bream (Kumakura et al., 2003) and salmonid species (Weil and Marcuzzi, 1990; Kitahashi, et al., 1998; Dickey and Swanson, 1998; Melamed et al., 2002) The degree of stimulatory effects of GnRH on the gonadotropin genes is dependent largely on the reproductive stage of the fish and the species, and to a lesser extent, the gender In the study of gonadotropin response to GnRH during sexual ontogeny of the common carp, gonadotropin levels in juvenile fish were unresponsive to GnRH administration In contrast, FSH- and LHβ mRNA of the maturing females increased up to three fold over controls, while there was only a slight increase of the LHβ mRNA in the male counterparts In the post-vitellogenic females, LHβ but not the FSHβ mRNA levels increased dramatically whereas in the male fish of the same age, increased FSHβ and LHβ mRNA levels were not observed (Kandel-Kfir et al., 2002) In immature striped bass, GnRH in combination with testosterone, stimulated the β subunit mRNA levels to various extents but had no effect in the maturing fish (Hassin et al., 2000) A study on female rainbow trout revealed that GnRH did not significantly stimulate FSHβ secretion at any stage of gametogenesis, even when the FSHβ levels increased after ovulation, whereas LHβ secretion increased following mid-vitellogenesis (Breton et al., 1998) Likewise, in the sham- and testosterone-implanted sea bass, GnRH had no effect on FSHβ mRNA levels, but increased LHβ mRNA levels (Mateos et al., 2002) In contrast, GnRH treatment induced the expression of all three gonadotropin subunit genes in the immature sea bream (Kumakura et al., 2003) In the goldfish, however, GnRH treatment increased both gonadotropin subunit mRNAs in the sexually matured fish but decreased the level of FSHβ mRNA in the sexually regressed fish (Khakoo et al., 1994; Sohn et al., 2001) Taken together, these studies indicate that the gonadotropin genes are regulated through various GnRH-induced signaling pathways and are also influenced by the reproductive stage of the fish 1.3.1.1 GnRH signaling pathway The stimulation of gonadotropin subunit genes by GnRH is activated through intracellular signaling transduction pathways following the binding of GnRH to G protein-coupled, seventransmembrane receptors (GnRHR) on the pituitary gonadotrope (Marshall and Kelch, 1986; Gharib et al., 1990) In mammals it has been shown that the receptor activates L-type calcium channels, allowing extracellular calcium into the cell (Naor, 1990) Phospholipase C (PLC) is also activated, leading to cleavage of phosphatidylinositol-diphosphate (PIP2) located in the cell membrane, into inositol 1,4,5-triphosphate (IP3), which mediates calcium release from 10 indicates a significant role of the BMP pathway in regulating the csFSHβ gene expression Due to the role of Smad4 as the functional partner in Smad-regulated transcription (Zawel et al., 1998; Howell et al., 1999; Masuyama et al., 1999; Zwijsen et al., 2003), including its known interaction with Smad1, it was rather surprising that the kb csFSHβ-CAT6 construct did not respond to Smad4 over-expression However, recent studies indicate exceptions from the current scheme of Smad function and other complex formation might exist R-Smads are reported to function without Co-Smads or with Co-Smads other than Smad4 (Massagué and Wotton, 2000: Moustakas et al., 2001) For example, mouse fibroblasts with a targeted disruption of Smad4 gene could still express the classical TGFβ response genes (Sirard et al., 2000) Consistent with this observation, the response of csFSHβ gene to TGFβ-signals may also exclude Smad4, providing a plausible explanation for the lack of response of the kb csFSHβ-CAT6 gene construct to Smad4 over-expression 128 4.8 Conclusion and Hypothesis In this research, about 8.6 kb the FSHβ subunit gene of the Chinook salmon was isolated and the transcription start site determined The genomic organization of this gene which is composed of three exons and two introns, conforms with that of other gonadotropin β-subunit genes To initiate the study of the transcriptional control of this gene, the promoter had to be isolated which proved to be a major challenge The 1.2 kb csFSHβ gene promoter was eventually isolated and initial transfection studies confirmed the functionality of the promoter, although its transcription activity appears to be repressed at the basal level and it requires activation by transcription factors A kb region within the first intron was initially isolated and cloned into the CAT6 and GFP reporter gene constructs, and unexpectedly exhibited transcriptional activities It could drive the CAT or EGFP reporter gene alone, or when induced by GnRH and activin treatment, or by various transcription factors Furthermore, a negative control element was also present At this juncture, the csFSHβ gene promoter is hypothesized to require the first intron or part thereof for efficient transcription activities but much work must be carried out to test this hypothesis Although this study has included some functional analysis on the putative regulatory elements in the promoter and in the first intron, it is at its infant stage More studies need to be carried out to determine the functionality of all of the putative regulatory sites and how their binding proteins may coordinate with each other to regulate the transcription of this gene The availability of the nucleotide sequence and the identification of a wealth of putative regulatory elements on the csFSHβ gene promoter and ntronic enhancer provide an excellent platform to continue these studies in the future 129 REFERENCES Abdollah, S., Macía-Silva, M., Tsukazaki, T., Hayashi, H., Attisano, L and Wrana, J.L 1997 TβRI phosphorylation of Smad2 on Ser 465 and 467 is required for Smad2/Smad4 complex formation and signalling J Biol Chem 272: 27678-27685 Andersen, B., Kennedy, G.C., Hamernik, D.L., Bokar, J.A., Bohinski, R and Nilson, 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multiple messenger ribonucleic acids Can J Zool 69:2572-2579 Xiong, F., Liu, D., Elsholtz, H.P and Hew, C.L 1994a The Chinook salmon gonadotropin IIβ gene contains a strong minimal promoter with a proximal negative promoter Mol Endocrinol 8:771-781 Xiong, F., Liu, D., Elsholtz, H.P and Hew, C.L 1994b Differential recruitment of steroid hormone response elements may dictate the expression of the pituitary gonadotropin IIβ subunit gene during salmon maturation Mol Endocrinol 8:782-793 Yaron, Z., Gur, G., Melamed, P., Rosenfeld, H., Levavi-Sivan, B., Elizur, A 2001 Regulation of gonadotropin subunit genes in tilapia Comp Biochem Physiol 129:489-502 Yen, F., Lee, Y., He, C., Huang, J., Sun, L., Dufour, S and Chang, C 2002 Estradiol-17β triggers Luteinizing hormone release in the Protandrous Black Porgy (Acanthopagrus schlegeli Bleeker) through multiple interactions with gonadotroin-releasing hormone control Biol Reprod 66:251-257 Yoshiura, Y., Kobayashi, M., Kato, Y and Aida, K 1997 Molecular cloning of the cDNAs encoding two gonadotropin β subunits (GTHIβ and IIβ) from the goldfish Gen Comp Endocrinol 105: 379-389 Zakaria, M.M., Jeong, K.H., Lacza, C and Kaiser, U.B 2002 Pituitary homeobox activates the rat FSH beta (rFSH beta) gene through both direct and indirect interactions with the rFSH beta gene promoter Mol Endocrinol 16:1840-1852 Zawel, L., Dai, J.L., Buckhaults, P., Zhou, S., Kinzler, K.W., Vogelstein, B and Kern, S.E 1998 Human Smad3 and Smad4 are sequence-specific transcription activators Mol Cell 1: 611-617 141 Zimmerman, C.M and Padgett, R.W 2000 Transforming growth factor β signaling mediators and modulators Gene 249: 17-30 Zwijsen, A., Verschueren, K and Huylebroeck, D 2003 New intracellular componenets of bone morphogenetic protein/Smad signaling cascades FEBS Letters 546: 133-139 142 [...]... are responsible for the cell-specific and basal expression of this subunit, and also those that mediate the stimulatory effects of GnRH and activin during puberty and sexual maturation 1.5 Aim The aim of this project is to initiate research on the mechanism of transcriptional regulation of the FSH subunit gene promoter of the Chinook salmon The Chinook salmon FSH subunit gene and 5’ flanking sequence... results were also obtained with the mouse FSH promoter using activin and follistatin in the LβT2 cells (Jacobs et al., 2003) In this respect, it is reasonable to assume a regulatory function of these extracellular proteins for the fish FSH subunit gene transcription, although the detailed mechanism remains to be elucidated 1.4 Hypothesis The promoter of the Chinook salmon FSH gene contains regulatory sequences... in oFSHβLuc transcription, and mutation of these AP-1 sites completely abolished the GnRH effect (Strahl et al., 1997, 1998) It was also shown that these AP-1 sites could support GnRH-mediated induction of oFSHβLuc in pituitary cultures derived from transgenic mice harboring the oFSHβLuc DNA constructs (Huang et al., 2001b) A Ptx-1 binding site on the rat FSH (rFSHβ) promoter that is similar to the. .. series of interconnecting signal transduction pathways, culminating with the concerted actions of the DNA-binding Smad proteins and other transcription factors and associated co-factors 21 It was reported that the Smad3 and Smad4 transcription factors are responsible for mediating the stimulation of the rat FSH promoter by activin in LβT2 cells Functional analysis identified the target site of the promoter. .. settle to the bottom of the tube before removing the ethanol by pipetting After the DNA was air dried for 15 s, it was dissolved in 400 µL of 8 mM NaOH The integrity of the genomic DNA (gDNA) was first determined One µg of gDNA and control human gDNA were loaded onto a 1.0 % agarose/EtBr gel to check for the size of the gDNA The quality of the genomic DNA was also analyzed by Dra I digestion The electrophoresis... complex is at least partly responsible for the activin-induced expression of the FSH gene via the Smad2 pathway 1.3.3.2 Hormonal modulators of activin action on the FSH subunit gene The actions of activin are regulated by the extracellular proteins inhibin and follistatin Inhibin interferes with the activin transduction pathway by disrupting the formation of functional activin-ActRII-ActRI complexes... parameters of PCR cycle 2.4.2 Cloning of the 1kb intronic enhancer into the vectors Both the amplified 1 kb fragment and the CAT6 and EGFP plasmid vectors were digested with HindIII and BamHI restriction enzymes (New England Biolabs) according to the manufacturer’s instructions The cut DNA fragments were analyzed by gel electrophoresis and gel purified A 1:3 molar ratio of vector : insert was used for the cloning. .. both basal and GnRH-stimulated expression of the gene Conversely, the GnRH-stimulated promoter activity was significantly reduced when the Ptx-1 site was mutated suggesting that Ptx-1 could mediate GnRH induction of the FSH gene (Zakaria et al., 2002) Ptx-1, a member of the Bicoid protein family of TFs (Lamonerie et al., 1996), is expressed in almost all cell-types in the pituitary including the gonadotropes... the specific signal pathways through which activin enhances FSH gene expression are at present unclear and have not been studied in the fish 1.3.3.1 Mechanisms of activin-stimulated gene expression Activin regulates FSH transcription through interaction with receptors that are members of the TGFβ family of transmembrane receptor kinases, resulting in both the elevation of FSH 19 mRNA levels and FSH. .. reveals a number of cis-acting elements, some of which have also been shown to be essential in the basal and GnRH-stimulated transcription in the mammalian homologs Since the first teleost genomic sequence for the LHβ gene was isolated from the Chinook salmon (csLHβ; Xiong and Hew, 1991), most functional studies on the putative elements have so far been restricted to this fish The csLHβ gene promoter contains ... the mechanism of transcriptional regulation of the FSHβ subunit gene promoter of the Chinook salmon The Chinook salmon FSHβ subunit gene and 5’ flanking sequence will be isolated, sequenced and. .. regard to the size of introns and 3’ untranslated region (3’ UTR; Figure 3) Compared to the 0.3 – 0.35 kb size of intron of the LHβ genes, the size of the corresponding intron in the FSHβ genes is... dC-tail of the cDNA and served as an extended template for RT The RT switched template from the mRNA to the SMARTII oligonucleotide and synthesize the complete cDNA copy of the original RNA The first-strand