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 and teleosts. 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. 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.
Our further study of the effects of rgLh (0.5, 2.5, 5, 10 ig) 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.
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. 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.
Baculoviruses offer many advantages over other expression vector systems, including safety, ease of scale-up, high levels of recombinant gene expression, and accuracy. 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.
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 . 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. Recombinant gonadotropins should be a useful tool to resolve these problems.
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 ig/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.
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 ig/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.
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).
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).
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).
Effect of rgLh on Testosterone (T) and 17^-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.. 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.