Pulsatile gonadotropin-releasing hormone modifies polyadenylation of gonadotropin subunit messenger ribonucleic acids

Abstract

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 a and LHβ 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 LHβ, FSHβ, and α-subunit mRNAs. However, the LHβ and α-subunit 3′-fragments were increased in size, indicating a shift to more adenylated forms of the LHβ and a transcripts. For FSHβ, the 3′-fragment bands were more diffuse than for LHβ or α-subunit, and no alteration in the lengths of FSHβ 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 LHβ and α-subunit mRNAs were observed in GnRH-treated cells compared to cells receiving no GnRH. Changes in the lengths of LHβ and α-subunit mRNAs were shown to be due to increased polyadenylation, and there was no observable change in polyadenylation of FSHβ mRNA. In addition, no changes were observed in the size of the 3′-fragments of PRL or β-actin mRNAs. These data demonstrate that pulsatile GnRH administered in vitro elicits specific increases in the lengths of the LHβ and α-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.