(268) Revealing RNA Poly(A) Tail Dynamics During Spermiogenesis And Its Function To Support Male Fertility

Marine Baptissart¹; Ankit Gupta¹; Alexander C. Poirot¹; Brian Papas² and Marcos Morgan¹

1. Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Durham, North Carolina 27709, USA

2.Integrative Bioinformatics, Biostatistics and Computational Biology Branch, National Institute of Environmental Health Sciences, National Institutes of Health, Durham, North Carolina 27709, USA

Abstract Text:

Spermiogenesis is the final process of spermatogenesis where round spermatids are elongating to shape the future sperm. Because spermatids are silenced transcriptionally, their differentiation relies exclusively on post-transcriptional regulations. In the 80s, changes in mRNA poly(A) tail length have been shown to control the translation of Transition proteins and Protamines transcripts during spermiogenesis.

Since, our understanding of how poly(A) tail processing might shape the transcriptome of spermatids remains limited. Here, we characterize the whole transcriptome poly(A) tail dynamics during spermiogenesis and its impact on mRNA metabolism and fertility.

We used FACS sorting to isolate round and elongated spermatids from mice testis. Long read RNA sequencing was adapted to profile poly(A) tails in both cell types. As the germ cells enter spermiogenesis, we show a global increase in poly(A) tail length associated with transcript stabilization. During spermiogenesis progression, the poly(A) tail of transcripts is shortened to 60-nts leading to translational activation. In addition, unstable transcripts show high proportion of uridylated poly(A) tails, a 3’-end modification commonly associated with mRNA decay. TENT5C and TUT7 are terminal nucleotidyltransferases responsible for mRNA polyadenylation and 3'-end uridylation, respectively. They both are required for spermatogenesis, but their role in shaping the poly(A) tail of the spermatid's transcriptome remains limited. We demonstrate for the first time that the poly(A) polymerase activity of TENT5C is required for fertility and spermiogenesis: male mice expressing catalytically dormant TENT5c (TENT5C dCat) are sterile and produce headless spermatozoa - a phenotype associated with a handful of transcripts showing abnormally short poly(A) tails in elongated spermatids. With the analysis on TUT7-depleted spermatids, we revealed a global loss of uridylated transcripts in elongated spermatids; however, this process is not required for fertility as TUT7 null mice remain fertile. In summary, we comprehensively characterize the dynamics of poly(A) tail during spermiogenesis and its role to support mRNA metabolism and fertility. This study opens new perspectives with the potential to exploit poly(A) tailing to develop male contraceptive and therapeutics for infertility.