Regulation of gonadotropin subunit gene transcription by gonadotropin-releasing hormone: measurement of primary transcript ribonucleic acids by quantitative reverse transcription-polymerase chain reaction assays.

GnRH regulates the synthesis and secretion of the pituitary gonadotropins LH and FSH. One of the actions of GnRH on the gonadotropin subunit genes (alpha, LHbeta, and FSHbeta) is the regulation of transcription [messenger RNA (mRNA) synthesis]. Gonadotropin subunit transcription rates increase after gonadectomy and following exogenous GnRH pulses. However, prior studies of subunit mRNA synthesis were limited by the available methodology that did not allow simultaneous measurement of gene transcription and mature mRNA concentrations. The purpose of the current studies was to: 1) develop a reliable and sensitive method for assessing transcription rates by measuring gonadotropin subunit primary transcript RNAs (PT, RNA before intron splicing); 2) investigate the PT responses to GnRH following castration or exogenous GnRH pulses; 3) characterize the half-disappearance time for the three PT species after GnRH withdrawal; and 4) correlate changes in PT concentration with steady-state gonadotropin subunit mRNA levels measured in the same pituitary RNA samples. Using oligonucleotide primers that flanked intron-exon boundaries, quantitative RT-PCR assays for each subunit PT species were developed. These assays require only ng amounts of RNA to measure each gonadotropin subunit PT and allow us to measure both PTs and steady-state mRNAs in a single pituitary RNA sample. Primary transcript concentrations in intact male rats showed a relative abundance of alpha > LHbeta congruent with FSHbeta, similar to the relationship found previously for mRNA levels. Additionally, each PT species was only 1-2% as abundant as the corresponding mRNA. One week after castration, gonadotropin subunit PT levels were increased (alpha: 3-fold, LHbeta: 6-fold, and FSHbeta: 3-fold) in a pattern similar to subunit mRNAs. Administration of GnRH antagonist to 7-day castrate male rats resulted in a rapid decline in PT concentrations with a half-disappearance time of 2.7 h for LHbeta and 0.8 h for FSHbeta, significantly faster than earlier measurements of the half-disappearance time for mature mRNA. Finally, in a GnRH-deficient male rat model, LHbeta and FSHbeta PT concentrations increased 4- to 6-fold 5 min after a GnRH pulse and then declined toward levels seen in control animals. These data indicate that the effects of GnRH on subunit gene transcription are an important determinant of gonadotropin regulation. The appearance and disappearance of PT RNA occurs more rapidly than changes in mature mRNA. Additionally, concentrations are elevated in long term castrates, and following an exogenous GnRH pulse the transcriptional burst is rapid and brief.