PULSATILE GONADOTROPIN-RELEASING-HORMONE MODIFIES POLYADENYLATION OF GONADOTROPIN SUBUNIT MESSENGER RIBONUCLEIC-ACIDS

Gonadotropin subunit mRNA levels rise after castration, coincident with a period of increased GnRH input to the pituitary. In addition to increased levels of gonadotropin mRNAs, we observed that the sizes of the alpha and LH-beta mRNAs were increased after ovariectomy (OVX) of rats. To determine whether these changes occurred in the 5' (alternate transcriptional start site or splicing)- or 3' (altered polyadenylation)-end of the molecules, mRNAs were cleaved using oligonucleotide-directed RNase-H digestion, and the fragments were analyzed by Northern blot, using probes specific to the 5'- and 3'-segments of each transcript. After OVX, there was no change in the sizes of the 5'-segments of LH-beta, FSH-beta, and alpha-subunit mRNAs. However, the LH-beta and alpha-subunit 3'-fragments were increased in size, indicating a shift to more adenylated forms of the LH-beta and alpha transcripts. For FSH-beta, the 3'-fragment bands were more diffuse than for LH-beta or alpha-subunit, and no alteration in the lengths of FSH-beta poly(A) tails were detected. A perifused pituitary cell system was used to determine whether pulses of GnRH were sufficient to cause modifications of polyadenylation. GnRH was administered as hourly 10-nM pulses for 4-12 h. Time-dependent increases in the sizes of LH-beta and alpha-subunit mRNAs were observed in GnRH-treated cells compared to cells receiving no GnRH. Changes in the lengths of LH-beta and alpha-subunit mRNAs were shown to be due to increased polyadenylation, and there was no observable change in polyadenylation of FSH-beta mRNA. In addition, no changes were observed in the size of the 3'-fragments of PRL or beta-actin mRNAs. These data demonstrate that pulsatile GnRH administered in vitro elicits specific increases in the lengths of the LH-beta and alpha-subunit mRNA poly(A) tails. Similar changes occur after OVX. Thus, in addition to transcriptional stimulation of the gonadotropin gene, GnRH modifies gonadotropin mRNAs at a posttranscriptional level.