The total RNA (2 ig) 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 Ihb, and 18SU and 18SD for the 18S RNA as a positive control in the RT-PCR.
The lysate was centrifuged at 10 000 rpm at 4°C for 5 min and the supernatant was collected for rgLh determination in series at0h,24h,48h,72h,96h, and 120h 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.
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.
Colonies containing recombinant bacmids were identified by disruption of the lacZa 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 Ihb and cga cDNA within the bacmid.
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.
Cell Culture and Virus
The Bac-to-Bac baculovirus was a derivative of Autographa californica nuclear polyhedrosis virus (AcMNPV).
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).
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.
The results have shown a close relationship between aromatase activity and gonadal estradiol (E2) levels. 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.
These studies indicated that Fsh is involved in the control of puberty and gametogenesis, whereas Lh mainly regulates final gonadal maturation and spawning.
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, and modulation of gene expression associated with enhanced steroidogenesis.
Earlier work suggested that the pituitary of fish synthesizes a single gonadotropin, called maturational gonadotropin, which regulates all processes of reproduction. 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). Lh and Fsh were shown to be heterodimeric glycoproteins hormones, each consisting of a common a and hormone-specific p subunit which are noncovalently linked.