Male Mice Heterozygous for Protamine 1 and Protamine 2 Are Infertile, Displaying Sperm Damage and Retention of Protamine 2 Precursors, Transition Proteins, and Histones.

BACKGROUND: During spermiogenesis, protamines (PRMs) replace histones to hypercondense and protect sperm DNA. Rodents and primates express two PRMs (PRM1 and PRM2). Alterations of species-specific PRM ratios are associated with sub- or infertility in mice and humans. Prm1- and Prm2-deficient mice are infertile, while Prm1+/- males are subfertile showing an altered PRM ratio. Prm2± males are fertile displaying PRM ratio comparable to WT. OBJECTIVES: Here, we investigated whether heterozygosity of both PRMs affects male fertility. MATERIAL AND METHODS: Double heterozygous (dHET) mice lacking one allele of each PRM were generated and analyzed. Fertility, sperm DNA compaction, and morphology were analyzed. RESULTS: dHET males were infertile, showing sperm histone and TNP retention. Protamine 2 precursor (pre-PRM2) levels were increased, while mature PRM2 levels were decreased. Ratio and total PRM content of mature sperm remained equal. However, CMA3 staining revealed incomplete protamination. Rounded and bigger sperm nuclei indicated impaired DNA-hypercondensation. DNA degradation was apparent, albeit less compared to Prm1- and Prm2-deficient males. Increased 8-OHdG levels suggested oxidative stress in epididymides of dHETs. Yet, a fraction of sperm was capable of fertilization, with embryonic development reaching 8-cell stage. DISCUSSION AND CONCLUSION: Our results suggest that male factor infertility detection requires analysis of protamination levels and pre-PRM2 retention, instead of solely measuring PRM ratio.