Spatiotemporal Expression of ARRDC5 in Mammalian Spermatogenesis

Spermiogenesis is the final phase of sperm development during which haploid round spermatids transform into specialized spermatozoa and occurs across distinct stages of sperm production in the seminiferous epithelium. Abnormalities in this process lead to malformations and an inability of sperm to fertilize eggs. Previous studies demonstrated that the alpha-arrestin family molecule alpha-arrestin domain-containing 5 (Arrdc5) is required for normal spermiogenesis in mice; however, the mechanism of action is undefined.

To further describe the role ARRDC5 plays in spermiogenesis, a description of the expression profile is needed.

Here, single cell RNA-sequencing and single cell ATAC-sequencing datasets generated from testicular tissue of mice and men were computationally integrated to define cell-type expression profiles and chromatin accessibility of ARRDC5 and other alpha-arrestin family member loci. To validate computational data, in situ hybridization of both mouse and human testicular tissue was probed for ARRDC5 transcript abundance. Subsequently, immunohistochemistry was used on an Arrdc5-eGfp mouse model to measure ARRDC5 protein expression. Lastly, the motif-based sequence analysis tool MEME was used to predict RNA-binding protein (RBP) motifs in the mouse and human ARRDC5 transcripts.

Of the six alpha-arrestin family members, the ARRDC5 locus was found to be uniquely in a heterochromatin state in testicular somatic cells but remodeled into an open euchromatin state specifically in spermatids. Concomitantly, Arrdc5 transcripts are uniquely detected in spermatids of both mice and humans. Additionally, in mice, transcript abundance peaks in stages VI-VIII of the seminiferous epithelium, and subsequent ARRDC5 protein abundance was found to be present in all steps of spermatid development with peaks in stages IX-XII of the seminiferous cycle. Outcomes of RNA motif analysis identified several conserved testis-specific RBPs with predicted binding to both mouse and human ARRDC5 transcripts.

Collectively, these findings provide evidence that spermatid-specific transcription of the Arrdc5 locus is driven by remodeling of the chromatin landscape. Furthermore, the temporal peak of ARRDC5 expression in specific stages during spermiogenesis coincides with critical events in spermatid development, including the histone-to-protamine exchange, acrosome formation, and cytoplasmic droplet biogenesis, implicating ARRDC5 in orchestrating these processes.