Postdoctoral Research Fellow University of Michigan Ypsilanti, Michigan, United States
Abstract Authors: Ritvija Agrawal1; Lindsay Moritz1; Mashiat Rabbani1; Sue Hammoud1
Abstract Text: Protamine-mediated DNA compaction in sperm, driven by electrostatic interactions between the arginine-rich core of protamines and DNA, is essential for maintaining sperm chromatin integrity, supporting development, and ensuring overall reproductive success. Protamines are subject to strong positive selection, exhibiting a distinctive form of purifying selection that preserves arginine/serine-rich regions. However, amino acid sequences outside the arginine core evolve rapidly, and the functional significance of this divergence remains poorly understood. To investigate the structure-function relationships of protamines, we introduced single and multiple amino acid substitutions at key positions within the protamine sequence—mutations that have arisen during evolution—and performed interspecies protamine swaps (e.g., mouse to rhesus macaque) to assess their impact on sperm function and zygote formation. Our results show that single mutations in the mouse P1 protamine sequence impair sperm motility, alter sperm morphology, and lead to subfertility. However, introducing multiple co-evolving amino acid substitutions can restore natural fertility in a non-competitive setting. Interestingly, when we directly competed sperm with wild-type (WT) protamines against sperm carrying swapped protamine sequences, the genetically modified sperm were less competitive in vitro. These findings suggest that while isolated point mutations are poorly tolerated, an alternative protamine from a different species can partially compensate for the loss of mouse PRM1, despite considerable sequence divergence. Notably, this interchangeability is observed for PRM1 but not for PRM2, as animals with substituted PRM2 are sterile. Moving forward, we aim to further investigate the compatibility of protamine variants both within and across species, as well as the effects of mutations on protein biophysical properties and embryonic development. We anticipate these experiments will provide valuable insights into the underlying principles of protamine sequence "grammar"—the rules governing DNA packaging and unpackaging during zygote formation.
