PMON183 Impact of Small Negative Allosteric Modulators (NAM) of the Follicle-stimulating Hormone (FSH) Receptor on the Dynamics of the Transcriptome in Rat Granulosa Cells Exposed to Different FSH Glycosylation Variants.

Follicle-stimulating hormone exists as different major glycoforms defined by distinct glycosylation patterns. It has been documented that variations in glycosylation confer differential biological effects to FSH when multiple in vitro biochemical changes are considered. In fact, transcriptomic data from cultured granulosa cells support those observations, showing glycosylation-dependent differential regulation of gene expression triggered by distinct FSH glycoforms. In the present study, RNA-Seq/Next Generation Sequencing (NGS) was applied to analyze the effects of NAMs of the FSH receptor [ADX68692 (full NAM) and ADX68693 (partial/biased NAM)] on changes in the transcriptome of granulosa cells exposed for 0, 6h, and 12h to 100 ng/ml of hypo- (FSH(21)) or fully- (FSH(24)) glycosylated human FSH glycoforms. Total RNA was prepared from cultured granulosa cells (obtained from DES-pretreated immature female rats) exposed to NAMs as well as from control cells with no NAM, and treated with each FSH glycoform. RNA libraries were sequenced in a HighSeq 2500 Illumina sequencer (2×125 bp paired-end format, 10-15×10(6) reads/sample). The computational workflow was focused on investigating differential gene expression (Salmon and DESeq2) and its association with sets of enriched biological processes (EBP)(Gene Ontology and KEGG) of cells exposed to the FSH glycoforms and the NAMs. Differential gene expression analysis showed that the presence of NAMs alone stimulated differential expression of a number of FSH-regulated genes, whereas in their absence mRNA expression exhibited time- and glycoform-dependent changes. In the presence of NAMs, FSH-stimulated cells expressed a similar number but different set of genes. There was a small subset of overexpressed genes that were differentially expressed, independent of experiment or time of FSH exposure; overexpressed genes such as Atg1, Upf1, or Psmd1 were ubiquitous in all experiments, whereas Smarce or Gspt1 were common in experiments involving both NAMs, suggesting that gene overexpression was due to the action of FSH. Meanwhile, decreased gene expression was more closely related to the effect of the NAMs. Striking differences in EBP were detected in the presence of NAMs, with marked disparities in the number and types of EBP between both molecules: ADX68692 inhibited more processes stimulated by FSH(21) than ADX68693 at 6h but not at 12h, whereas for FSH24 the number of EBP were lower in the presence of ADX68693 than of ADX68692 at 6h and 12h. These data indicate that small modifications in small molecule NAM structure are sufficient to impact on temporal gene expression and biological effects in cells exposed to distinct FSH glycoforms, likely related to cAMP independent effects of FSH. (The authors thank Addex Therapeutics LTD, Geneve, Switzerland, for providing the NAMs). Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.