Fertilization with Protamine-2 deficient sperm triggers abnormal pronucleus development and zygotic cleavage†

Spermatozoan DNA is hyper-condensed by protamines, which are essential for sperm motility and function. Since defects in this process impact natural fertilization, little is known about how changes to protamine-mediated condensation impact the oocyte's ability to recognize and reprogram the paternal genetic material for the early stages of embryogenesis. Here, we performed intracytoplasmic sperm injection (ICSI) with sperm lacking Protamine 2 (Prm2 KO) and tracked preimplantation development in mice. We found that ICSI with Prm2 KO sperm leads to embryo fragmentation and arrest at the 2-cell stage. Surprisingly, injected Prm2 KO sperm DNA is rapidly depleted from the oocyte during the completion of maternal meiosis II, leading to a zygote with one morphologically abnormal pronucleus. Direct induction of DNA damage in wild type sperm did not recapitulate the pronuclear abnormalities found in Prm2 KO-derived zygotes. Co-injection with wild type sperm failed to rescue these defects, indicating that they were not caused by absence of a normally protaminated paternal genome. Finally, we find that testicular Prm2 KO sperm support progression to the blastocyst stage, suggesting a model where fertility-compromising factors are acquired during epididymal maturation. Our results demonstrate that Protamine 2 is necessary for correct maturation of epididymal sperm and essential for their ability to form a functional zygote at fertilization.