Gonadal transcriptome profiling via RNA-seq during temperature-dependent sex determination and differentiation in the red-eared slider (Trachemys scripta elegans)

In species with temperature-dependent sex determination (TSD), incubation temperature during a critical embryonic window directs gonadal fate, yet the underlying molecular mechanisms remain poorly understood. Here, we performed transcriptome sequencing of embryonic gonads from the red-eared slider turtle (Trachemys scripta elegans) incubated under male- and female-producing temperatures (MPT and FPT). Our analyses revealed numerous stage-specific differentially expressed genes and identified key candidates consistently upregulated under MPT or FPT conditions. Gene co-expression network analysis highlighted DHRS11 as a potential thermosensitive hub gene at MPT, and ATP2B4, a calcium transporter, as a hub under FPT, implicating calcium signaling in early sex determination. In addition, we uncovered widespread alternative splicing, including intron retention in the chromatin modifiers JARID2 and KDM6B, with CIRBP, a cold-inducible RNA-binding protein, as a potential upstream regulator. These findings suggest a role for transcriptional and post-transcriptional mechanisms, including calcium signaling and temperature-responsive splicing, in mediating TSD in reptiles.