In the present study we characterized GnRH gene expression in intact female rats across the estrous cycle and during a steroid-induced LH surge in ovariectomized (OVX) rats, using the quantitative ribonuclease protection assay. We measured cytoplasmic messenger RNA (mRNA) levels and nuclear primary transcript levels as an index of transcription. In Exp I, cycling rats were killed at 1100 or 1800 h on estrus, diestrus day 1, or diestrus day 2 (D2) or at 1100, 1500, 1800, or 2100 h on proestrus (P). In Exp II, proestrous rats were killed at the same time points or injected with pentobarbital (Pb) at the onset of the LH surge and killed on that day or the following day. In Exp III, a LH surge was induced in OVX rats treated with estradiol benzoate plus progesterone. Rats were killed at 1200, 1500, 1600, 1700, or 2100 h on the day of the surge. For all experiments, blood samples were collected and frozen for quantitation of LH by RIA. The preoptic area-anterior hypothalamus was dissected, and cytoplasmic and nuclear RNA were extracted and assayed separately by ribonuclease protection assay. In Exp I, cytoplasmic mRNA levels exhibited two significant peaks, one on D2 and another at 1500h on P. Primary transcript levels were significantly elevated only at 1500 h on P. In Exp II, proestrous rats and rats given Pb and killed the next day had a peak in cytoplasmic mRNA levels at 1500 h, which was blocked in rats given Pb and killed the same day. In Exp III with OVX rats, no significant changes in mRNA or primary transcript levels were observed between steroid or control groups. We hypothesize that the increase in cytoplasmic mRNA levels in cycling rats on D2 is probably due to a posttranscriptional mechanism, because it was not paralleled by changes in primary transcript levels, which would be expected if a transcriptional mechanism were involved. On P, both cytoplasmic mRNA and primary transcript levels changed, suggesting a transcriptional mechanism at this time.
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