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.
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 ig), 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 il 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.. 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.