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research-article |
State Key Laboratory of Biocontrol,4 Sun Yat-sen University, Guangzhou 510275, People's Republic of China
Institute of Aquatic Economic Animals and Guangdong Provincial Key Laboratory for Aquatic Economic Animals,5 School of Life Science, Sun Yat-sen University, Guangzhou 510275, People's Republic of China
ABSTRACT
The cDNA sequence encoding orange-spotted grouper lhb (LHbeta) and cga (GTHalpha) subunits were cocloned into baculovirus transfer vectors and expressed in insect Sf9 cells. The results showed that two bands of 15.6 kDa and 11.4 kDa could be detected by SDS-PAGE and a band of 30 kDa could be detected by native PAGE. The recombinant grouper Lh (rgLh) could stimulate the secretion of testosterone (T) and estradiol-17beta (E2) from the gonad in a static incubation system in a time-dependent, but not a dose-dependent, manner. Using in vivo bioassay, the mRNA levels of two aromatases (cyp19a1a [P450aromA] and cyp19a1b [P450aromB]), gnrh (GnRH), lhb, and cga in the pituitary, gonad, and hypothalamus were determined in different groups of orange-spotted groupers treated respectively with rgLh, human chorionic gonadotropin (hCG), and a culture medium of insect cells transformed with an expression vector without lhb and cga subunits. The mRNA levels of cyp19a1a and cyp19a1b rose dramatically after injecting rgLh intraperitoneally, which was consistent with the secretion of sex steroid hormones. Interestingly, the mRNA levels of gnrh dropped in the pituitary, hypothalamus, and gonad, and the mRNA levels of lhb and cga in the pituitary of the experimental group expressed at a higher level than that of the hCG group. These results are in accord with the long positive feedback loop of Lh on gonad sex steroid hormones and the short negative feedback loop of Lh on gnrh mRNA levels. These results indicate that the rgLh is successfully expressed by the baculovirus-insect expression system and that the rgLh has biological activity.
baculovirus expression system, estradiol, follicle-stimulating hormone, grouper gonadotropin-releasing hormone, insect cell, luteinizing hormone, mechanisms of hormone action, testosterone
Earlier work suggested that the pituitary of fish synthesizes a single gonadotropin, called maturational gonadotropin, which regulates all processes of reproduction [1]. However, the existence of two gonadotropins was established in a salmonid species with the discovery of a new gonadotropin, called Fsh (formerly GTH-I), chemically different from the previously known maturational gonadotropin, or Lh (formerly GTH-II) [2]. Lh and Fsh were shown to be heterodimeric glycoproteins hormones, each consisting of a common 
and hormone-specific ß subunit which are noncovalently linked [3]. The amino acid sequence of the Lhb subunit is highly conserved, particularly in regions thought to be important for receptor interaction [4], while the primary structure of the Fshb subunit is more variable than that of the Lhb subunit even in regions thought to confer ligand specificity. Further sequence analysis demonstrated that fish GTH-I and GTH-II were homologous to tetrapod FSH and LH, respectively [5]. This homology was supported by physiological studies showing that Fsh (formerly GTH-I) levels are higher at the beginning of the reproductive cycle, whereas Lh (formerly GTH-II) increases later in the cycle [6]. These studies indicated that Fsh is involved in the control of puberty and gametogenesis, whereas Lh mainly regulates final gonadal maturation and spawning [7, 8].
The control of reproductive functions in lower vertebrates has a number of significant differences from that in mammals. Thus, studies of the hormonal regulation of sex gland functions in fish are vital for understanding how these mechanisms develop during reproduction. Gonadotropin could activate the hormone-sensitive adenylate cyclase (AC) via Gs protein leading to formation of cAMP, activation of PKA and CRE [9], and modulation of gene expression associated with enhanced steroidogenesis [10–11]. In vitro experiments on the vitellogenesis of common carp (Cyprinus carpio), tuna (Thunnus thunnus), and chum salmon (Oncorhynchus keta) have shown that Fsh and Lh have equivalent functions in stimulating ovarian steroid production [12]. Sex steroid hormones play important roles at all stages of the reproductive cycle in vertebrates; several studies on their structures and roles in mediating various stages of gonadal development, maturation, and synthesis of Lh in teleost fishes have been reported [13, 14]. The results have shown a close relationship between aromatase activity and gonadal estradiol (E2) levels [15]. However, only limited data have been available for the grouper.
The baculovirus expression system is a eukaryotic expression system that can produce posttranslational modifications of recombinant protein. Since human beta interferon was first expressed with a baculovirus expression vector in 1983 [16], more than 500 genes from viruses, bacteria, epiphytes, plants, invertebrates, and vertebrates have been expressed by the baculovirus expression system [17]. Recombinant grass carp Gh is able to produce immunological and biological activities similar to the native hormone in the baulovirus-silkworm larvae system [18]. Recombinant goldfish gonadotropin has been expressed by baculovirus, and the results suggest that the biological activities of goldfish recombinant Fsh and Lh might be used to induce gonadal development in aquaculture fishes as a substitute for pituitary extract [19].
The orange-spotted grouper, Epinephelus coioides, is a marine fish widely cultured in Southern China. It is a protogynous hermaphroditic fish. Females reach sexual maturity at about 5 years of age when they reach approximately 55–75 cm in body length; the transition of the sex gland from female to male begins at about 7 years of age. To study the biological activities and physiological relevance of the two gonadotropins, the availability of homologous hormones was essential. As a first step in unraveling the mechanism involved in the sex change and gonadotropin function of the orange-spotted grouper, rgLh was expressed in insect cells by the baculovirus expression system, and the biological effects of the recombinant protein were determined with in vitro and in vivo approaches.
Two-year-old orange-spotted groupers with body weights (bw) of about 420–670 g and gonadosomatic index (the ratio of gonad to body weight without the visceral mass) of 0.2% or less were obtained from Guangdong Daya Bay Fishery Development Center (Huizhou City, Guangdong, PR China) and raised in indoor water tanks at room temperature under a natural photoperiod for approximately 5 days until use. All experiments were performed under license from the Government of the People's Republic of China and endorsed by the Animal Experimentation Ethics Committee of Sun Yat-sen University.
Total RNA isolation, Gene Cloning, and Construction of the Baculovirus Transfer Vector
Total RNA of grouper pituitary was isolated using TRIzol (Invitrogen; http://www.invitrogen.com/) according to the manufacturer's protocol, and quantified based on the absorbance at 260 nm. The integrity of the RNA was checked with agarose gel electrophoresis. To synthesize cDNA from the total RNA, the mRNA was enriched using oligo(dT) cellulose, and then reverse transcribed for 90 min at 48°C in a reaction of 20 µl containing 1 µg RNA, 0.2 mM dNTP, 2 pM T12 adapter, 5U RNAsin, 0.01 M DTT, and 10 U of Superscript reverse transcriptase (Invitrogen). The polymerase chain reaction (PCR) primers used for amplification of grouper lhb and cga subunits were based on the sequences of GenBank (Accession No. AF507939/AY186243 and AY129308). The primer sequences of the oligonucleotides are listed in Table 1. The primers P1 and P3 contained an EcoRI site and an Nhe I site upstream of the ATG start codon, respectively, and the primers P2 and P4 had a HindIII site and a Kpn I site after the stop codon. The PCR was carried out under a cycle protocol of pre-denaturation at 94°C for 4 min, 30 cycles of denaturation at 94°C for 1 min, annealing at 61°C for 30 sec, and extension at 72°C for 45 sec, and a final elongation at 72°C for 10 min. The PCR products were then purified on 1.5% agarose gel and the DNA bands were extracted using E.Z.N.A. Gel Extraction Kit (Omega Biotek). The lhb fragment was digested with double restriction endonuclease EcoRI and HindIII, and cloned into corresponding double restriction endonuclease-digested pFastBacDual bacmid transfer vector (Invitrogen) to generate the construct of gpFast-lhb, and then the cga cDNA treated with Nhe I and Kpn I double restriction endonuclease digestion was cloned into the constructed gpFast-lhb, which had been double restriction endonuclease digested. This vector contained a mini-Tn7 element, an E. coli origin of replication, and an amplicillin marker. The mini-Tn7 contained an expression cassette consisting of gentamycin resistance marker, polyhedron promoter, a multicloning site, and an SV40 PolyA signal inserted between the left and right arms of Tn7. Initial transformation and screening were done in Blue-gal, and the positive clones were confirmed by dideoxy sequencing and used for transformation of Escherichia coli DH10Bac.
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The Bac-to-Bac baculovirus was a derivative of Autographa californica nuclear polyhedrosis virus (AcMNPV). The recombinant virus was propagated in a monolayer culture of Spodoptera frugiperda cells, Sf9, which were grown in a monolayer culture at 27°C in Grace insect medium containing antibiotics (streptomycin, 100µg/ml; penicillin, 50 U/ml), and 10% FBS according to the manufacturer's protocol (Invitrogen).
Generation of Recombinant Baculovirus and Transposition
Generation of recombinant baculovirus expressing rgLh in Sf9 cells was carried out using the Bac-to-Bac baculovirus expression system kit (Invitrogen). DNA of the recombinant pFastBac transfer vector containing cDNA corresponding to the grouper lhb and cga sequence was introduced into E. coli DH10Bac for the transposition of the sequence into baculovirus genomic DNA (bacmid) according to the manufacturer's protocol. Colonies containing recombinant bacmids were identified by disruption of the lacZ gene, and the white colonies were chosen for recombinant bacmid DNA isolation. DNA was isolated using a Qiagen plasmid isolation kit (Qiagen Inc.) specific for DNA over 135 kb long. The recombinant bacmid DNAs were analyzed on 0.5% agarose gel and followed by PCR analysis using pUC/M13 forward and reverse primers (Table 1) to confirm the presence as well as the correct insert size of the transposed grouper lhb and cga cDNA within the bacmid. To confirm the authenticity of the bacmid, Southern blot analysis was conducted. After capturing the image of the agarose gel electrophoresis, the bacmids were transferred by vacuum and cross-linked to Hybond-N nylon paper (B.M.) following the instruction manual. After overnight hybridization at 42°C the blot was washed with 2xSSC, 0.1% SDS for 5 min twice at room temperature and then with 0.5xSSC, 0.1% SDS for 15 min twice at 65 °C. After the membrane was submerged into the detection buffer with CDP-Star substrate, the chemiluminescent signals from the membrane were detected with the GeneGenome System (SynGene, UK) [20]. The recombinant bacmid DNA was then used to transfect Sf9 cells grown in Grace's medium. At a density of 1x106 cells per 35-mm well, the cells were transfected with the bacmid DNA using Cellfectin reagent (Invitrogen) according to the manufacturer's protocol. The recombinant virus was harvested at 72 h posttransfection. According to the manual of the kit, plaque assays were performed to determine the titer of recovered recombinant virion particles, and each recombinant viral stock was made for expression studies.
Production of Recombinant Protein
To analyze the production of the rgLh, Sf9 cells were grown in 75-mm tissue culture flasks until 70%–80% confluency was reached. The cells were infected with recombinant virus at 10 multiplicities of infecton (moi) and the infection was carried out at 27°C for 0 h, 24 h, 48 h, 72 h, 96 h, and 120 h. To examine the intracellular production, cytoplasmic extract was prepared from the infected cells as described by Mathur et al. [21]. Briefly, cells were washed with phosphate-buffered saline and suspended in 0.5 ml of homogenization buffer containing 10 mM Tris-HCl (pH 8.0) and 25 mM NaCl, after which they were disrupted with a Dounce homogenizer. The lysate was centrifuged at 10 000 rpm at 4°C for 5 min and the supernatant was collected for rgLh determination in series at 0 h, 24 h, 48 h, 72 h, 96 h, and 120 h postinfection. Using BCA methods (Biorad), the rgLh in supernatant was quantitated and then used for in vivo and in vitro assays. It was also analyzed on the SDS-PAGE and PAGE; the PAGE was stained for glycoprotein using the silver staining method [22].
Detection of gnrh, cyp19a1a and cyp19a1b, lhb and cga mRNA Expression
The hCG (1 IU/bw·g), rgLh (5x10–3, 5x10–2, and 5x10–1µg/bw·g), physiological salt solution (PS) (1 µl/bw·g), and the negative control (NC) (0 µg/bw·g), which derived from the insect cells transformed with an expression vector not containing rgLh, were injected intraperitoneally into 2-yr-old orange-spotted groupers (six fish per treatment), respectively. At 18 h postinjection, fish were first anesthetized by ice, blood was collected from caudal vein, and the serum was stored at –20°C for radioimmunoassay. After that, total RNA sample was harvested from pituitary, gonad, and hypothalamus using Trizol reagent (Invitrogen) for the further determination of mRNA levels of gnrh, cyp19a1a and cyp19a1b, cga, and lhb by RT-PCR. The total RNA (2 µg) was reverse transcribed into single-stranded cDNA using the oligo(dT) primer and SuperScriptTM first-strand synthesis system for RT-PCR (Invitrogen). Each RNA sample was treated with RNase-free DNase I (Invitrogen) to remove any genomic DNA contamination. The integrity of all RNA samples was verified by the successful amplification of 18S RNA. The following gene-specific primers (Table 1) were used for PCR detection: P5 and P6 for grouper gnrh, P7 and P8 for grouper cyp19a1a, P9 and P10 for grouper cyp19a1b, P1 and P2 for grouper cga, P3 and P4 for grouper lhb, and 18SU and 18SD for the 18S RNA as a positive control in the RT-PCR. The cycling conditions were as follows: 95°C for 4 min; 35, 33, 33, 30, and 30 cycles of denaturation at 94°C for 30 sec; 56°C, 55°C, 55°C, 64°C, and 58°C for 30 sec for gnrh, cyp19a1a, cyp19a1b, cga, and lhb, respectively; and 72°C for 1 min; followed by a final extension at 72°C for 7 min. The RT-PCR products were separated on agarose gels, followed by ethidium bromide staining, and then detected under ultraviolet light with Gel Doc 2000 (BioRAD). The cDNA band densities were measured using Quantity One software (BioRAD).
Effect of rgLh on Testosterone (T) and 17ß-Estradiol (E2) Concentration of Orange-Spotted Grouper Gonad and Serum
Gonad fragments from orange-spotted grouper were prepared according to the procedures for pituitary fragment preparation described by Li et al. [23]. Gonad fragments were suspended in medium (M199 medium with Earle salts, Gibco-BRL, supplemented with 25 mmol/L Hepes, 2.2 g/L NaHCO3, 100 000 U/L penicillin, 100 mg/L streptomycin) and cultured in 24-well Falcon plates. After overnight pre-incubation (18~24 h) at 25°C under 5% CO2 and saturated humidity, the wells were replaced with 1 ml fresh medium. The fragments were then allowed to rest for at least 30 min until the experiment. The rgLh (0.5, 2.5, 5, and 10 µg), hCG (1, 10, 100, and 1000 IU) were then added to the desired final concentrations, and an identical amount of M199 was added in the control group. After incubation for 2, 6, 12, and 24 h, the test media were carefully collected and centrifuged at 10 000x g for 10 min at 4°C. The supernatants were lyophilized and resolved with 50 µl homogenization buffer for radioimmunoassay (RIA). T and E2 contents of the supernatant and the serum collected from the above injection treatment were measured by RIA, which was modified from the method reported by Nishihara et al. [24]. The tracers and reagents for the assay were purchased from the Beijin North Biology Company. All samples for measurement were run in a single assay; the sensitivities of T and E2 for RIA were 1 pg/RIA tube and 0.05pg/RIA tube, respectively, and the intra-assay coefficient of variation was less than 10%.
The relative mRNA levels of cga, lhb, gnrh, cyp19a1a, and cyp19a1b were expressed as the ratio of the target gene mRNA level to that of 18S ribosomal RNA. Statistical analysis was determined using one-way analysis of variance (ANOVA) followed by Dunnett test. Data were represented as mean ± SEM (n = 3), and the differences were considered significant at P < 0.05.
Complementary DNA Cloning and Construction of Recombinant Baculovirus Transfer Vector
In the present study, the ORF cDNAs encoding grouper cga and lhb were subcloned using RT-PCR. The recombinant transfer vector was constructed and digested by double restriction endonuclease digestion analysis. Several aim fragments were released in double restriction endonuclease digestion analysis (EcoRV + EcoRI; EcoRI + HindIII; HindIII + Nhe I; Nhe I + EcoRV; Kpn I and EcoR I; HindIII and EcoRV), which were used to prove the correct construction of the recombinant transfer vector (Fig. 1).
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PCR and Southern Blot Analysis of Recombinant Bacmid-lh
The bacmid DNA was >135 kb. It was difficult to verify the insertion of the specific gene with the typical restriction endonuclease digestion analysis. Therefore, PCR was used to confirm transposition of the target gene to the bacmid. The pUC/M13 amplification primers were directed at sequences on either side of the miniattTn7 site within the lacZ-complementation region of the bacmid. The PCR product of successful transposition was about 3361 bp, while the PCR fragment without transposition was about 273 bp (Fig. 2C). The result of Southern blot indicated that orange-spotted grouper lhb and cga had been transposed into the baculovirus genomic DNA (bacmid) (Fig. 2, A and B).
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Transfection of Sf9 Insect Cells
At early stages of infection, cell diameter increased and nuclei began to fill the cells (Fig. 3A, B, C). Cells stopped growing and appeared vesicular. Budded virus was released into the medium 72 h after transfection (Fig. 3D) at late stage infection, and cells released from the plate, lysed and appeared clear in the monolayer in very late stage infection (Fig. 3, E and F).
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Production of rgLh in Insect Cells
To analyze the production of rgLh, the Sf9 cell monolayer was infected with recombinant virus at 10 multiplicities of infection. The medium was collected at the appropriate time (i.e., 0, 24, 48, 72, 96, and 120 h postinfection) and separated by SDS-PAGE and non-denaturing PAGE. The results in Figure 4 show that a band at 15.6 kDa and a product of 11.4 kDa could be detected by SDS-PAGE while a band in the range of about 30 kDa was detected by native PAGE under non-reducing conditions. The concentration of rgLh was about 50 mg/L.
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Effect of rgLh on the mRNA Expression Level of gnrh
To test for the actions of rgLh on gnrh gene expression in the pituitary, gonad, and hypothalamus, 2-yr-old groupers were injected intraperitoneally with 5x10–2 µg/bw·g rgLh, and gnrh mRNA levels were examined by a semiquantitative RT-PCR after 18 h. In this case, rgLh was effective in reducing gnrh mRNA levels. For the rgLh treatment group, the ratio of gnrh mRNA to 18S RNA was 0.12 (Fig. 5A), 0.065 (Fig. 5B), and 0.096 (Fig. 5C) in pituitary, hypothalamus, and gonad, respectively, which were significantly lower than that of the hCG group.
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Effect of rgLh on the mRNA Expression Level of cyp19a1a and cyp19a1b
The ratios of cyp19a1b mRNA to 18S RNA of 5x10–2 µg/ bw·g rgLh group were 1.7, 1.4, and 1.1 times greater than those of the hCG group in pituitary, hypothalamus, and gonad, respectively, and reached 0.88, 0.57, and 0.31, respectively (Fig. 6A, B, C). Moreover, the ratios of cyp19a1a mRNA to 18S RNA of the rgLh group also reached 1.1, 1.1, and 1.8 times greater than those of the hCG group in pituitary, hypothalamus, and gonad, respectively (Fig. 7A, B, C).
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Effect of rgLh on the mRNA Expression Level of cga and lhb
The mRNA levels of cga (Fig. 8A, B, C) and lhb (Fig. 9) were also tested after 5x10–2 µg/bw·g rgLh injected intraperitoneally. The ratios of cga mRNA to 18S RNA were significantly higher than those of the hCG group and reached 1.20, 0.95, and 0.92 in pituitary, hypothalamus, and gonad, respectively. The mRNA level of lhb in pituitary was similar to that of cga.
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Effect of rgLh on T and E2 Concentration of Gonad and Serum
To test whether rgLh had an effect on T and E2 secretion, time-course and dose-dependent studies were conducted by static incubation of gonad with 1, 10, 100, and 1000 IU hCG for 2, 6, 12, and 24 h, respectively. Compared with the time-matched controls, hCG induced a time-dependent increase in T and E2 release from 2 to 12 h, then a reduction at 24 h. hCG-induced T and E2 secretion were further confirmed by subsequent dose-response studies. In this case, gonads were incubated for 12 h with increasing levels of hCG (1, 10, 100, and 1000 IU), and T and E2 secretion were not elevated in a dose-related fashion (Fig. 10, A and C). The effects of rgLh on T and E2 secretion were similar to those in hCG time-course studies (Fig. 10, B and D), and 2.5 µg rgLh significantly increased T and E2 secretion when compared with hCG (10 IU).
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After treatment with hCG and rgLh, serum levels of T an E2 were found to increase significantly with dose dependence while no significant effects were observed in the PS and NC groups. Serum T concentrations of 0.1, 1, and 10 IU/bw·g hCG treatment group reached 201.78, 266.32, and 372.27 pg/ml (Fig. 11A), and E2 concentration were 98.24, 145.47, and 256.33 pg/ml (Fig. 11C). Serum T levels of 0.05, 0.5, and 5 µg rgLh group reached 212.35, 426.85 and 972.83 pg/ml (Fig. 11B) and E2 levels was 99.86, 182.10, and 523.56 pg/ml, respectively (Fig. 11D). However, the glycoprotein fraction derived from the insect cell medium that transformed with an expression vector not containing the rgLh gene did not stimulate steroid production.
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The functional features of fish gonadotropins have remained unclear. This situation can be ascribed largely to the difficulty of obtaining sufficient quantities of purified gonadotropins, since many animals are needed for the collection of pituitaries and purification of gonadotropins [19]. In addition, the structural and chemical properties between Fsh and Lh are similar, which makes it difficult to separate them and produce a specific antiserum. Consequently, it has not been possible to distinguish the two hormone-producing cells by immunohistology in many families of teleosts [4]. Recombinant gonadotropins should be a useful tool to resolve these problems. At present, abundant biologically active recombinant mammalian gonadotropins or their analogs have been successfully expressed in different expression systems, including yeast (Pichia pastoris) [25–26], insect cells [27], and mammalian cells [28]. The Bac-to-Bac baculovirus expression system provides a rapid and efficient method to generate recombinant protein, and the baculovirus system has become one of the most versatile and powerful eukaryotic vector systems for recombinant protein expression [16]. Since 1985, when the first protein (IL-2) was produced on a large scale from a recombinant baculovirus, use of the baculovirus expression system has increased dramatically; more than 600 recombinant genes have been expressed in baculoviruses to date [16]. Baculoviruses offer many advantages over other expression vector systems, including safety, ease of scale-up, high levels of recombinant gene expression, and accuracy [29]. In the present study, Lh of the orange-spotted grouper was synthesized using the Bac-to-Bac baculovirus expression system.
It has been accepted that FSH is involved in the control of puberty and gametogenesis and LH regulates the maturation of the ovary and spermatogenesis in mammals [30]. Gonadotropins stimulate androgen synthesis from cholesterol by binding to the receptor on the oocyte in the ovary, and then androgen is converted into estrogen, catalyzed by aromatase, and finally facilitates the synthesis of hepatic vitellogenesis [31]. Aromatase P450 is an essential component of the important enzyme complex which catalyzes the conversion of androgen into estrogen. As in some other teleost fishes, the orange-spotted grouper has two distinct cDNA forms of aromatase P450-cyp19a1a and cyp19a1b-which mainly express in the ovary and the brain, respectively [32]. Gonadotropins may activate the hormone-sensitive adenylate cyclase (AC) via Gs protein leading to formation of cAMP, activation of PKA and CRE [9], and modulation of gene expression associated with enhanced steroidogenesis [10]. In vitro experiments on the vitellogenesis of common carp, tuna, and chum salmon show that Fsh and Lh have equivalent function in stimulating ovarian steroid production [12]. Experiments have shown that sex steroids differentially regulate gonadotropin gene expression in the pituitary of goldfish: a strong in vivo inhibitory effect on fshb mRNA production, but a weak stimulatory effect on lhb in sexually immature and recrudescent fish. T treatment in vitro does not significantly decrease fshb mRNA levels, but increases that of lhb only in the cells of immature fish [33]. Human CG was used in this study because it is commercially available, very stable in solution, and has a relatively high binding affinity for gonadotropin receptors [34]. More important, it is biologically active in fish, such as goldfish (Carassius auratus) [35], yellow perch (Perca flavescens) [36], and channel catfish (Ictalurus punctatus ) [37]. In our in vitro bioassays, rgLh significantly stimulated cyp19a1a and cyp19a1b gene expression and the secretion of T and E2. These results indicate that hCG can significantly increase T and E2 secretion, and that rgLh not only mimics the effect of hCG but also displays more potent action than hCG. Our further study of the effects of rgLh (0.5, 2.5, 5, 10 µg) on T and E2 secretion with different treatment time (2, 6, 12, and 24 h) suggests that rgLh can stimulate sex steroid hormone secretion in a time-dependent manner.
Gonadotropin secretion in vertebrates is mainly regulated by the brain-pituitary-gonadal axis. It is produced in the pituitary by the stimulation of GnRH produced in the hypothalamus and acts on the gonad to produce androgens and estrogens. The whole metabolic process is regulated by neural and endocrine mechanisms, including negative and positive feedback loops, to maintain the dynamic and physiological balance among various hormones [38]. In the present in vivo study, marked increases were observed in the mRNA levels of lhb in the pituitary, hypothalamus, and gonad of immature groupers. These results are consistent with the findings that sex steroids exert a strong inhibitory effect on lhb mRNA levels in sexually immature and maturing goldfish [39]. It is possible that the in vivo effect of sex steroids is mediated via their effect on the brain to modulate the secretion of Gnrh or dopamine [40]. In addition, aromatase activity may reflect the response of Lhb in the pituitary in vivo.
The effects of GnRH and its various analogs on gonadotropin have been extensively studied in mammals [41] and teleosts [42]. It has been determined that GnRH is secreted from the hypothalamus in a pulsatile fashion, which parallels the pulsatile release of LH and FSH, and that a feedback regulation exists between them [43]. It has also been demonstrated that GnRH is capable of stimulating cga, fshb, and lhb mRNA levels in the goldfish pituitary following 24 h of treatment in vivo [44]. In studies on rats, treatment of pituitary cells with GnRH increased LHß mRNA levels between 12 and 24 h [45]. In this study, the mRNA level of gnrh decreased in the hypothalamus after intraperitoneal injection of rgLh , which suggests that there is a negative feedback regulation in Gnrh secretion.
In conclusion, the present study has shown that biologically active rgLh can be produced using the baculovirus expression system in insect cells. This recombinant hormone could be employed to improve artificial production in the aquaculture industry by replacing current methods that rely on hormones such as hCG, which show low activity in stimulating sex steroid hormone synthesis and secretion. It is also expected to be helpful in elucidating the physiological function of rgLh in gonadal development and the mechanism of lhb and cga gene expression and regulation in the orange-spotted grouper.
ACKNOWLEDGMENTS
We thank Ms. Miner He for her technical help with Sf9 insect cell culture.
FOOTNOTES
3These authors contributed equally to this work. 
1Supported by the grants from the Major State Basic Research Development Program of China (973 Program) (No. 2004CB117402), the National High Technology Research and Development Program of China (863 Program) (No. 2003AA603011), and the Guangdong Province National Science Foundation (No. 20023002) to W.S.L.
Correspondence: 2 FAX: 86 20 8411 3717; e-mail: ls32{at}mail.sysu.edu.cn
Received: 5 January 2006.
First decision: 24 January 2006.
Accepted: 25 September 2006.
REFERENCES
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DNA/HindIII marker; 1, no transposition bacmid DNA; 2,3,4,5, transposition bacmid-lh DNA. C) M, 1 kb DNA ladder marker; 1, no recombinant bacmid; 2, 3, 4, 5 recombinant bacmid-lh DNA.
