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 go nadotropins LH and FSH. One of the actions of GnRH on the gonadotropin subunit genes (a lpha, LH beta, and FSH beta) is the regulation of transcription [messenger RNA (mRNA) synthesis]. Gonadotropin subunit transcription rates increase af ter gonadeetomy and following exogenous GnRH pulses. However, prior studies of subunit mRNA synthesis were limited by the available methodology that d id not allow simultaneous measurement of gene transcription and mature mRNA concentrations. The purpose of the current studies was to: 1) develop a re liable and sensitive method for assessing transcription rates by measuring gonadotropin subunit primary transcript RNAs (PT, RNA before intron splicin g); 2) investigate the PT responses to GnRH following castration or exogeno us GnRH pulses; 3) characterize the half-disappearance time for the three P T species after GnRH withdrawal; and 4) correlate changes in PT concentrati on with steady state gonadotropin subunit mRNA levels measured in the same pituitary RNA samples. Using oligonucleotide primers that flanked intron-exon boundaries, quantita tive 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 > LH beta congruent to FSH beta, similar to the relationship found previously for mRNA levels. Additionally, each PT speci es was only 1-2% as abundant as the corresponding mRNA. One week after cast ration, gonadotropin subunit PT levels were increased (alpha: 3-fold, LH be ta: 6-fold, and FSH beta: 8-fold) in a pattern similar to subunit mRNAs. Ad ministration of GnRH antagonist to 7-day castrate male rats resulted in a r apid decline in PT concentrations with a half-disappearance time of 2.7 h f or LH beta and 0.8 h for FSH beta, significantly faster than earlier measur ements of the half-disappearance time for mature mRNA. Finally, in a GnRH-d eficient male rat model, LH beta and FSH beta PT concentrations increased 4 - to 8-fold 5 min after a GnRH pulse and then declined toward levels seen i n 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. A dditionally, concentrations are elevated in long term castrates, and follow ing an exogenous GnRH pulse the transcriptional burst is rapid and brief.