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- Luteinizing Hormone in Insect Cells: RESULTS(3)

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.

Effect of rgLh on the mRNA Expression Level of cyp19a1a and cyp19a1b

The ratios of cyp19a1b mRNA to 18S RNA of 5X10~2 ig/ 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).
Fig4Production of Recombinant-4
FIG. 4. SDS-PAGE and PAGE analysis of rgLh expressed by Sf9 insect cells. A) M, protein molecular weight marker; 1, negative control; 2, expressed rgLh after 24 h of transfection; 3, expressed rgLh after 48 h of transfection; 4, expressed rgLh after 72 h of transfection; 5, expressed rgLh after 96 h of transfection; 6, expressed rgLh after 120 h of transfection. B) PAGE with silver staining.

Fig5Production of Recombinant-5
FIG. 5. Effects of rgLh (5 X 10—2 ig/bw-g) on gnrh mRNA level in pituitary (A), hypothalamuss (B), and gonad (C) after interperitoneal injection. PC: physiological salt solution; NC: the negative control, derived from insect cells transformed with an expression vector containing no rgLh. RT-PCR was repeated three times. The gnrh mRNA levels were expressed as the ratio between gnrh mRNA and the mRNA of 18S RNA (internal control) with the same sample. Data are presented as means 6 SEM. Different lowercase letters indicate statistically significant differences (P < 0.05 by Dunnett test, n = 3).

Fig6Production of Recombinant-6
FIG. 6. Effects of rgLh (5 X 10~2 ig/bw-g) on cyp19a1b mRNA level in pituitary (A), hypothalamus (B), and gonad (C) after interperitoneal injection. RT-PCR was repeated three times. The cyp19a1b mRNA levels were expressed as the ratio between cyp19a1b 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).

Fig7Production of Recombinant-7
FIG. 7. Effects of rgLh (5 X 10~2 ig/bw-g) on cyp19a1a mRNA level in pituitary (A), hypothalamus (B), and gonad (C) after interperitoneal injection. RT-PCR was repeated three times. The cyp19a1a mRNA levels were expressed as the ratio between cyp19a1a mRNA and the mRNA of IBS 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).

TABLE 1. Oligonucleotide sequences used in this study.

Gene Primer Primer sequencea
cga P1 5′ -CTAGCTAGCATGGGCTCAGTGAGATCAG-3′
P2 5′ -GGGGTACCTCATATCTTGTGGAAATAGCAG-3′
lhb P3 5′ -GGAATTCATGATGGCTGTACAGGTTG-3′
P4 5 -GCCC AAGCTTCTAGTAGTAAAAAGGTATGTC-3′
gnrh P5 5 -GCAGACAAGAACAAATCAGAGAC-3′
P6 5 -TGTCCATTTCCCCTGTCGGT-3
cyp19a1aP7 5 -GGAGACATTGTGAGAGTCTGGATC-3′
P8 5 -TGACAGGTACATCCAGGAAGAGTC-3′
cyp 19a 1bP9 5 -TGACACCTGGCAAACAGTTC-3′
P10 5 -GATGGTGTCGTCTTCCAGAG-3
18S RNA18SU 5 -CCTGAGAAACGGCTACCACATCC-3′
18SD 5 -AGCAACTTTAGTATACGCTATTGGAG-3′
pUC/M13(+) 5′ -GTTTTCCCAGTCACGAC-3′
pUC/M13(—)5′ -CAGGAAACAGCTATGAC-3′

a Bold indicates the sites of restriction enzyme digestion.

June 2, 2014 Hormone
Tags: hormone mechanisms of hormone action testosterone