FIG. 1.

LHβ and FSHβ gene expression is repressed in immature gonadotropes by HDACs, and this is overcome by GnRH. RT-PCR analysis of LHβ and FSHβ mRNA levels was carried out in immature αT3-1 (A) and mature LβT2 (B) gonadotrope cells following a 24-h exposure to TSA (20, 100, or 200 ng/ml) and/or GnRH (d-Ala6-LHRH; 100 nM) in three repeats for each treatment. Total RNA was isolated from the cells, and cDNA was synthesized and used as template for PCR. Control reactions were carried out in which the reverse transcriptase was omitted to ensure that the amplicon did not result from genomic DNA contaminations (not shown). The primers were designed to amplify fragments of the LHβ and FSHβ cDNAs, while amplification of a fragment of the mouse 60sRP cDNA is shown as a control. Total RNA was extracted from αT3-1 or LβT2 cells treated with TSA (C) or GnRH (D), and quantitative real-time PCR was performed. Changes in cycle threshold values were normalized to those of 60sRP and compared to the levels in untreated controls. Values are means ± standard errors of the means (n = 3). *, P < 0.05; **, P < 0.005; ***, P < 0.0001.