Sperm Lacking Protamine 2 Fail to Support Normal Fertilization and Embryogenesis in Mouse

Bluma J. Lesch^1,2,3,4, Shannon R. Rainsford^1,3, Kaelyn Sumigray^1,3,4, Zachary D. Smith^1,3,5

1. Department of Genetics, Yale School of Medicine, New Haven, CT, USA

2. Department of Obstetrics, Gynecology & Reproductive Medicine, Yale School of Medicine, New Haven, CT, USA

3. Yale Stem Cell Center, Yale School of Medicine, New Haven, CT USA

4. Yale Cancer Center, Yale School of Medicine, New Haven, CT, USA
5. Max Planck Institute for Molecular Genetics, Berlin, Germany

Abstract Text: Protamines are essential for sperm head condensation and male fertility in mammals. In naturally mated protamine knockout (KO) mice, infertility is attributable to severe sperm motility defects that prevent the sperm from reaching the oocyte. However, correct packaging of the sperm nuclear material is also important for mediating reprogramming of the paternal nucleus at fertilization and supporting the transition to embryogenesis. In addition to sperm motility, we hypothesized that protamine packaging plays an essential role in reprogramming paternal chromatin for normal fertilization after the sperm enters the oocyte and is important for early embryogenesis. To test this hypothesis, we generated mice carrying a genetic deletion of protamine 2 (Prm2 KO) and bypassed the sperm motility defect using intracytoplasmic sperm injection (ICSI). After Prm2 KO ICSI, embryos arrested at the 2-cell stage. A transient fragmentation phenotype was often observed at the first cell division. The paternal pronucleus uniformly failed to decondense and could not be detected in oocytes by two hours after injection, suggestive of either degradation or eviction due to failure to reprogram. The maternal pronucleus was morphologically abnormal, with aberrant nuclear membrane shape and reduced levels of lamin A/C. We confirmed previous reports of elevated DNA damage in Prm2 KO sperm, but found that sperm with DNA damage induced by DNAse did not fully recapitulate the effects of Prm2 KO knockout sperm following ICSI. Prm2 KO sperm was sufficient to induce the observed embryogenesis phenotypes even when co-injected with normal sperm, indicating that the presence of the abnormal sperm, rather than the absence of normal sperm, is responsible for the phenotype. We conclude that Protamine 2 is essential for sperm function not only prior to fertilization, but also for enacting a normal fertilization process after entering the oocyte. Our findings have implications for assisted reproduction, as men with fertility defects induced by protamine dysfunction may be poor candidates for ICSI.