Multi‐stage transcriptome analysis reveals genetic orchestration of rat testis development
Chew Chin Chan, Sajad Jamalpour, Chandramathi Samudi, Hasmahzaiti Omar, Subha BhassuAbstract
Background
Testicular development is a highly orchestrated process essential for male fertility, but its temporal genetic regulation remains incompletely resolved in many mammalian models. This study re‐analyses rat testis transcriptomes across 15 stages from embryonic day 12 to postnatal week 16 using bulk RNA‐sequencing data from a multispecies organ development atlas, generating a continuous, testis‐focused temporal framework, and pinpointing key developmental transition windows and regulatory modules.
Results
Of 23,748 annotated genes, 17,717 were differentially expressed (DEGs), and hierarchical plus co‐expression analyses identified four principal expression modules and five clusters capturing spermatogenic, morphogenetic, immune, and neurone‐like programs. Four major transcriptional transition windows, embryonic day 14, embryonic day 19, postnatal week 2, and postnatal week 6, were defined, with the largest remodeling between 2 and 6 weeks coinciding with the onset of robust spermatogenesis and marking a particularly vulnerable window for environmental or toxicant‐induced perturbation. Stage‐specific spermatogenesis‐associated gene sets at embryonic day 19 ( n = 28), postnatal week 2 ( n = 61), and week 6 ( n = 735) showed minimal overlap, supporting sequential regulatory waves driving germ cell commitment, early meiosis, and terminal differentiation. Target enrichment analyses highlighted five conserved microRNAs (rno‐miR‐151‐5p, rno‐miR‐29b‐3p, rno‐miR‐384‐5p, rno‐miR‐500‐5p, rno‐miR‐672‐5p) whose predicted and validated targets form modules related to extracellular matrix remodeling, apoptosis and cell‐cycle control, ion‐channel signaling, and neuronal‐like pathways, with high sequence conservation to human homologs. Immune‐related genes and blood–testis barrier components displayed coordinated expression dynamics, and a conserved core of 487 spermatogenesis genes showed strong cross‐mammalian orthology, indicating a deeply shared transcriptional backbone on which species‐specific regulatory nuances are layered.
Conclusions
This multistage transcriptomic analysis delineates major developmental transitions, co‐expression modules, and regulatory miRNA–mRNA networks that orchestrate rat testis maturation and highlight a critical P2W–P6W transition window. The resource complements existing multiorgan and single‐cell datasets by providing a testis‐focused temporal framework and identifies conserved gene sets and regulatory candidates of direct relevance to male infertility, toxicology, and cross‐species translational studies.