MON-023 Dynamics of the Transcriptome in Rat Granulosa Cells Exposed to Different Follicle-Stimulating Hormone (FSH) Glycosylation Variants as Revealed by RNA-Seq/New Generation Sequencing (NGS)

Follicle-stimulating hormone exists as different major glycoforms defined by distinct glycosylation patterns of the hormone-specific β-subunit. It has been documented that variations in glycosylation confer differential biological effects to the glycoforms when multiple in vitro biochemical readings are analyzed. We here applied Next Generation Sequencing (NGS) to explore changes in the transcriptome of rat granulosa cells exposed for 0, 6, and 12 h to 100 ng/ml of four highly purified FSH glycoforms, each exhibiting distinctly different glycosylation patterns: human pituitary FSH21 and equine FSH (eFSH) (hypo-glycosylated), and human FSH24 and CHO cell-derived human recombinant FSH (recFSH) (fully-glycosylated). Total RNA from triplicate incubations was prepared from FSH glycoform-exposed cultured granulosa cells obtained from DES-pretreated immature female rats, and total RNA libraries were sequenced in a HighSeq 2500 sequencer (2 x 125 bp paired-end format, 10–15 x 106 reads/sample). The computational workflow was focused on investigating differences among the four FSH glycoforms at three levels: gene expression (Salmon and DESeq2 bioinformatic tools), enriched biological processes (DAVID tool), and perturbed pathways (GAGE tool). Among the top 200 differentially expressed genes, only 4 (0.6%) were shared by all 4 glycoforms at 6 h, whereas 118 genes (40%) were shared at 12 h. At 6 h, up-regulated genes in recFSH were associated with cell response, angiogenesis, extracellular matrix organization, and mitosis; eFSH with sex hormones (shared with FSH21); FSH21 with cellular response and response to drugs (shared with recFSH); and FSH24 with cAMP-related processes. There were more shared biological processes at 12 h, with fewer treatment-specific ones, except for recFSH, which exhibited stronger responses with more specifically associated processes. Similar results were found for down-regulated cell processes, with a greater number of processes at 6 h or 12 h, depending on the particular glycoform. In general, there were fewer down-regulated than up-regulated processes at both 6 h and 12 h, with FSH21 exhibiting the largest number of down-regulated associated processes at 6 h (10 vs 3 processes for eFSH, one process for FSH24, and one for recFSH), while eFSH exhibited the greatest number at 12 h (19 processes vs 4 for FSH21, 13 for FSH24, and 7 for recFSH). Two signaling cascades, largely linked to Rap-1 and cAMP pathways, were differentially activated by the glycoforms, with each glycoform exhibiting its own molecular signature. These transcriptomic data support previous biochemical observations demonstrating glycosylation-dependent differential regulation of intracellular signaling pathways triggered by FSH in granulosa cells.