DETECTION OF GONADOTROPIN-RELEASING-HORMONE AND ITS RECEPTOR MESSENGER-RNA IN HUMAN PLACENTAL TROPHOBLASTS USING IN-SITU REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION

Neuropeptides such as gonadotrophin releasing hormone (GnRH) are presu med to play an important Pole in the regulation of the function and gr owth of human placenta. Knowledge about the placental site of GnRH exp ression and the eventual co-localization of its peptide with the GnRH receptor (GnRH-R) is crucial for a better understanding of possible au tocrine/paracrine mechanisms. We therefore investigated these question s by use of in-situ reverse transcription-polymerase chain reaction (R T-PCR) alone or in combination with immunocytochemistry in human first and third trimester placentae. Paraffin-embedded placental sections ( 7 Crm thick), or single trophoblasts in monolayer cultures for up to 3 days, were treated with proteinase K. Following RT with GnRH or GnRH- R specific oligoprimers, PCR was performed employing primers with exon -exon overlaps to exclude non-specific DNA amplification. Detection of the amplicons was accomplished by nested PCR which was performed with digoxigenin-labelled dUTP and nitroblue tetrazolium/5-bromo-4-chloro- 3-indoyl-phosphate (NBT/BCIP) for substrate visualization. The GnRH pe ptide was detected using a sandwich-antibody assay. GnRH and GnRH-R ge ne expression was found in all first and third trimester placentae, wi th abundant signals for the GnRH and GnRH-R message both in the cyto- and syncytiotrophoblasts. Single trophoblasts of different gestational ages in culture also displayed GnRH expression in individual cytotrop hoblasts and in syncytiotrophoblast-like fusionates. Additional immuno staining revealed GnRH peptide to be co-localized with GnRH-R message in trophoblast layers. Since messages for GnRH and GnRH-R were found i n virtually all trophoblasts, we infer that GnRH and GnRH-R are co-exp ressed in identical cells, These data strongly suggest that the tropho blasts are the source of GnRH, and that there is autocrine/paracrine r egulation by GnRH in human placenta.