Amelia Mohamadi

We analyzed the transcriptome data of wildtype and estrogen receptor β knockout (Erβ^KO) rat ovaries during the early postnatal period and detected remarkable changes in epigenetic regulators and transcription factors. Compared with postnatal day (PD) 4.5 ovaries, PD 6.5 wildtype ovaries possessed 581 differentially expressed downstream transcripts (DEDTs), including 17 differentially expressed epigenetic regulators (DEERs) and 23 differentially expressed transcription factors (DETFs). Subsequently, compared with PD 6.5 ovaries, PD 8.5 wildtype ovaries showed 920 DEDTs, including 24 DEERs and 68 DETFs. The DEDTs, DEERs, and DETFs in wildtype ovaries represented the gene expression during primordial follicle activation and the gradual development of primary follicles of first-wave origin because the second-wave follicles remained dormant during this developmental period. When we compared the transcriptome data of age-matched Erβ^KO ovaries, we observed that PD 6.5 Erβ^KO ovaries had 744 DEDTs compared with PD 4.5 ovaries, including 46 DEERs and 55 DETFs. The loss of ERβ rapidly activated the primordial follicles of both first- and second-wave origin on PD 6.5 and showed a remarkable increase in DEDTs (744 vs. 581). However, compared with PD 6.5 ovaries, PD 8.5 Erβ^KO ovaries showed only 191 DEDTs, including 8 DEERs and 10 DETFs. This finding suggests that the PD 8.5 Erβ^KO ovaries did not undergo remarkable ovarian follicle activation greater than that had already occurred in PD 6.5 Erβ^KO ovaries. The results also showed that the numbers of DEERs and DETFs were associated with increased changes in DEDTs; the greater the number of DEERs or DETFs, the larger the number of DEDTs. In addition to the quantitative differences in DEERs and DETFs between the wildtype and Erβ^KO ovaries, the differentially expressed regulators showed distinct patterns. We identified that 17 transcripts were tied to follicle assembly, 6 to follicle activation, and 12 to steroidogenesis. Our observations indicate that a loss of ERβ dysregulates the epigenetic regulators and transcription factors in Erβ^KO ovaries, which disrupts the downstream genes in ovarian follicles and increases follicle activation. Further studies are required to clarify if ERβ directly or indirectly regulates DEDTs, including DEERs and DETFs, during the neonatal development of rat ovarian follicles.

RNA sequencing (RNA-Seq) has become a widely adopted technique for studying gene expression. However, conventional RNA-Seq analyses rely on gene expression (GE) values that aggregate all the transcripts produced under a single gene identifier, overlooking the complexity of transcript variants arising from different transcription start sites or alternative splicing. Transcript variants may encode proteins with diverse functional domains, or noncoding RNAs. This study explored the implications of neglecting transcript variants in RNA-Seq analyses. Among the 1334 transcription factor (TF) genes expressed in mouse embryonic stem (ES) or trophoblast stem (TS) cells, 652 were differentially expressed in TS cells based on GE values (365 upregulated and 287 downregulated, ≥absolute 2-fold changes, false discovery rate (FDR) p-value ≤ 0.05). The 365 upregulated genes expressed 883 transcript variants. Further transcript expression (TE) based analyses identified only 174 ( 0.05) between ES and TS cells expressed 2215 transcript variants. These included 477 (>21%) differentially expressed transcripts (276 upregulated and 201 downregulated, ≥absolute 2-fold changes, FDR p-value ≤ 0.05). Hence, GE based RNA-Seq analyses do not represent accurate expression levels due to divergent transcripts expression from the same gene. Our findings show that by including transcript variants in RNA-Seq analyses, we can generate a precise understanding of a gene’s functional and regulatory landscape; ignoring the variants may result in an erroneous interpretation.