In mice, a population of male germ cells show characteristics of non-apoptotic cell death during G0 arrest

Abstract

In mammals, a small population of spermatogonial stem cells (SSCs) is established shortly after birth. These cells self-renew and produce sperm for the entirety of a males reproductive lifespan, passing the genome on to the next generation. Thus, establishment of a population of SSCs with high genomic integrity is essential. SSCs are derived from a much larger precursor population of male germ cells (MGCs) that differentiate during fetal life. During the last third of gestation, MGCs undergo a prolonged period of G0 cell cycle arrest during which they sustain high levels of transcription and acquire epigenetic programming for SSC fate. Although these differentiation steps can cause cellular and genomic damage, it has been unclear whether selection for germ cell quality occurs during G0 arrest since no classic markers of cell death have been detected. In this study, we utilize a mouse model to characterize a population of MGCs that begin to accumulate markers if cell death, such as AnnexinV (AnV) and propidium iodide (PI), at E16.5. The AnV- and PI-positive MGC population is characterized by low expression of the RNA-binding protein, Dead End 1 (DND1), and exhibit dsDNA breaks and mitochondrial dysfunction. Interestingly, we do not see evidence of an active cell death cascade until the time of birth, where we see phosphorylation of MLKL, a hallmark of a necroptotic cell death mechanism. Based on these findings, we propose that variable cellular health is an important basis for selection of the SSC precursors. Significance StatementSpermatogonial stem cells (SSCs) are essential for reproductive fitness, yet how their precursors are selected during development is not known. Utilizing a mouse model, this study describes high levels of cellular damage within a subset of male germ cells (MGCs) during G0 arrest. The damaged MGC population was marked by low expression of the RNA-binding protein, DND1, and was strongly associated with mitochondrial dysfunction and dsDNA breaks. We observed signs of non-apoptotic cell death by embryonic day (E)16.5 and the appearance of necroptotic markers in MGCs at the time of birth. This study uncovers previously unknown heterogeneity in the MGC pool and points to MGC health as an important source of selection during G0 arrest.