Re-visiting the Protamine-2 locus: deletion, but not haploinsufficiency, renders male mice infertile

Abstract

Protamines are arginine-rich DNA-binding proteins that replace histones in elongating spermatids. This leads to hypercondensation of chromatin and ensures physiological sperm morphology, thereby protecting DNA integrity. In mice and humans, two protamines, protamine-1 (Prm1) and protamine-2 (Prm2) are expressed in a species-specific ratio. In humans, alterations of this PRM1/PRM2 ratio is associated with subfertility. By applying CRISPR/Cas9 mediated gene-editing in oocytes, we established Prm2-deficient mice. Surprisingly, heterozygous males remained fertile with sperm displaying normal head morphology and motility. In Prm2-deficient sperm, however, DNA-hypercondensation and acrosome formation was severely impaired. Further, the sperm displayed severe membrane defects resulting in immotility. Thus, lack of Prm2 leads not only to impaired histone to protamine exchange and disturbed DNA-hypercondensation, but also to severe membrane defects resulting in immotility. Interestingly, previous attempts using a regular gene-targeting approach failed to establish Prm2-deficient mice. This was due to the fact that already chimeric animals generated with Prm2+/− ES cells were sterile. However, the Prm2-deficient mouse lines established here clearly demonstrate that mice tolerate loss of one Prm2 allele. As such they present an ideal model for further studies on protamine function and chromatin organization in murine sperm.

Prm2deficiency triggers a Reactive Oxygen Species (ROS)-mediated destruction cascade during epididymal sperm maturation in mice

“…This prohibited detailed functional studies on protamine function in mice. Recently, we reported the successful generation and establishment of Prm2-deficient mouse lines using CRISPR/Cas9-mediated gene-editing in oocytes (27). Interestingly, Prm2 +/males displayed normal fecundity with sperm being morphologically and functionally indistinguishable from wildtype sperm.…”

“…To verify that other secondary sperm defects can be also assigned to the proposed ROS pathway, these defects are expected to appear and intensify during epididymal sperm maturation as well. The initial phenotypical characterization showed severe acrosomal malformations in Prm2-deficient cauda epididymal sperm (27). Here, periodic-acid-Schiff (PAS) staining, which stains glycoprotein-rich acrosomal structures pink, was performed on testicular tissue sections.…”

“…All animal experiments were conducted according to German law of animal protection and in agreement with the approval of the local institutional animal care committees (Landesamt für Natur, Umwelt und Verbraucherschutz, North Rhine-Westphalia, approval ID: AZ84-02.04.2013.A429). Prm2-deficient mice (MGI: 5760133; 5770554) were generated and characterized as published previously (27).…”

“…Since cell culture models for human spermatogenesis are scarce and inefficient, mouse models have contributed to a broader understanding of protamine function. Several knockout studies for both, Prm1 as well as Prm2, have shown that both isoforms are indispensable for male fertility in mice as well (24), (25), (26), (27). Similar to human sperm, impaired protamination was linked to faulty DNA compaction and increased DNA fragmentation.…”

“…targeted treatment options. Here, our Prm2-deficient mouse lines serve as an ideal model for functional investigations, as they enable for the first time studies on a genetically and phenotypically uniform sperm population(27). Protamine-induced DNA hypercondensation is proposed to protect the paternal genome from damaging environmental insults.…”