2026 Theses Doctoral

Semen-derived amyloid fibrils as carriers of RNA to sperm and the early embryo

Mallen, William S.

Paternal contributions to embryonic development extend beyond transmission of DNA sequence and include diverse RNA species delivered by sperm; however, the mechanisms by which extracellular RNA in seminal fluid is transferred to sperm remain poorly understood. This dissertation identifies functional amyloid fibrils in seminal fluid as previously unrecognized carriers that bind, transport, and deliver RNA to mammalian sperm and ultimately to the early embryo. Using an integrative approach combining biophysical reconstitution, proteomics, RNA sequencing, advanced electron microscopy, and functional fertilization assays, it is investigated how seminal fluid amyloid fibrils interact with RNA and participate in intercellular RNA trafficking.

It is shown that highly cationic amyloidogenic peptides derived from seminal fluid phase separate with RNA to form condensates that mature into amyloid fibrils, establishing a mechanistic framework in which electrostatic condensation promotes amyloid assembly and RNA capture. Analysis of endogenous human seminal fluid demonstrates that amyloid-enriched, sarkosyl- insoluble fractions contain diverse small RNA populations, including tRNA-derived fragments, and molecular chaperones that could regulate fibril dynamics. Cryo-electron tomography and complementary volume electron microscopy reveal that amyloid fibrils are internalized by sperm and associate with intracellular membranes and subcellular structures, supporting a pathway for RNA cargo entry into sperm cells. Biophysical and murine in vitro fertilization assays support a model in which amyloid-associated RNA is delivered to the zygote, where chaperone-regulated disassembly can enable RNA release and translation.

Together, these findings establish a mechanistic model in which semen-derived amyloid fibrils act as dynamic extracellular RNA-binding scaffolds that bridge seminal fluid, sperm, and the embryo. This work expands current understanding of sperm RNA biology, links functional amyloids to epigenetic inheritance mechanisms, and suggests that naturally occurring amyloid assemblies may serve as adaptable platforms for RNA delivery during fertilization.