(272) Paternal Contribution Mediated by the Sperm-Borne circRNA NAPE-PLD

C. García-Merino^1*, P. Navarrete-López^1*, M. Maroto¹, R. Fernández-González¹, A. Gutiérrez- Adán¹

1. Department of Animal Reproduction, INIA-CSIC, Madrid, Spain

*These authors contributed equally to this work.

Abstract Text:

Circular RNAs (circRNAs) play a key role in the regulation of gene expression. The presence of both coding and non-coding RNAs in mammalian sperm has attracted increasing interest in recent decades. Spermatozoa carry various classes of RNA, predominantly non-coding RNAs with regulatory functions that influence sperm function, fertilization success, early embryonic development, and paternal transgenerational inheritance. Among these, circRNAs are a particularly stable RNA class, known to function as microRNA (miRNA) sponges, among other roles. Here, we investigate the function of circNAPE-PLD, a circRNA expressed in both human and murine spermatozoa, with potential involvement in the transmission of paternal RNA and epigenetic marks to the offspring. Its linear counterpart, NAPE-PLD, is expressed in the testis and is involved in the biosynthesis of the endocannabinoid anandamide (AEA), which plays a role in early pregnancy modulation. We conducted total RNA analysis in sperm from circNAPE-PLD-deficient mice to assess the impact of its absence on sperm function and post-fertilization processes.

Mutant mice were generated using CRISPR-Cas9 technology in zygotes by excising the NAPE-PLD gene region between exons 2 and 4 with two sgRNAs, removing the intron containing the circRNA Nape-Pld. Notably, a significant reduction in multiple mRNAs and lncRNAs was observed in sperm from mutant mice. These alterations may reflect a regulatory role of linear NAPE-PLD, whose expression is disrupted or diminished as a result of circNAPE-PLD ablation, in male germ cell differentiation. The affected biological processes include cilium movement, sperm motility, spermatid differentiation, and fertilization. Despite these molecular changes, no overt phenotypic consequences were detected in fertility or sperm function, likely due to incomplete suppression of linear NAPE-PLD expression or compensatory mechanisms.

Small RNA-seq analysis revealed altered miRNA profiles in mutant mice, including the downregulation of miR-339-5p, miR-144-5p, miR-122-5p, and miR-669-5p. These miRNAs may be sequestered by circNAPE-PLD, contributing to their stabilization during sperm maturation and facilitating their delivery to the oocyte upon fertilization. The predicted target genes of these miRNAs are involved in key developmental processes such as RNA splicing, chromosome segregation, translation regulation, muscle cell differentiation, and nervous system development. Specifically, targets of miR-122-5p and miR-660-5p are closely associated with early embryonic development, with enriched GO terms including cell cycle regulation, nuclear division, gastrulation, embryo implantation, and placental development.

Although no apparent defects in early embryonic development were observed, the altered miRNA landscape suggests potential impacts on epigenetic inheritance in the offspring. These findings support the role of the affected miRNAs as paternal contributors to the embryonic epigenome, potentially mediated by circNAPE-PLD. Further studies are needed to determine whether these miRNAs are indeed absent in embryos derived from mutant sperm, compared to those from wild-type counterparts.