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- Luteinizing Hormone in Insect Cells: RESULTS(4)

RESULTS(4)

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 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 1000IU 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 ig rgLh significantly increased T and E2 secretion when compared with hCG (10 IU).

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 ig 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.
Fgh8Production of Recombinant-8
FIG. 8. Effects of rgLh (5 X 10~2 ig/bw-g) on cga mRNA level in pituitary (A), hypothalamus (B), and gonad (C) after inter-peritoneal injection. RT-PCR was repeated three times. The cga mRNA levels were expressed as the ratio between cga mRNA and the mRNA of 18S RNA (internal control) with the same sample. Data are presented as means ± SEM. Different lowercase letters indicate statistically significant differences (P < 0.05 by Dunnett test, n = 3).

Fig9Production of Recombinant-9
FIG. 9. Effects of rgLh (5 X 10~2 ig/bw-g) on lhb mRNA level in pituitary after inter-peritoneal injection. RT-PCR was repeated three times. The lhb mRNA levels were expressed as the ratio between lhb mRNA and the mRNA of 18S RNA (internal control) with the same sample. Data are presented as means ± SEM. Different lowercase letters indicate statistically significant differences (P < 0.05 by Dunnett test, n = 3).

Fig10Production of Recombinant-10
FIG. 10. Effects of rgLh on testosterone (T) and estradiol-17b (E2) secretion in grouper gonad fragments with static incubation. Each value represents the mean ± SEM; different lowercase letters indicate statistically significant differences (P < 0.05 by Dunnett test, n = 6).

Fig11Production of Recombinant-11
FIG. 11. Testosterone (T) and estradiol-17b (E2) levels of grouper serum after rgLh interperitoneal injection. Each value represents the mean ± SEM;different lowercase letters indicate statistically significant differences (P < 0.05 by Dunnett test, n = 6).

June 4, 2014 Hormone
Tags: hormone mechanisms of hormone action testosterone