Matters of Life and Death: Mechanisms of Spermatogonial Stem Cell Selection in the Mouse

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 male’s reproductive lifespan, passing the genome on to the next generation. SSCs are derived from primordial germ cells (PGCs), which are specified early in embryonic development. After their specification, PGCs undergo genome-wide DNA demethylation as they migrate through the gut mesentery to the developing gonad. Once inside the gonad, PGCs receive signals from the surrounding soma that initiate the acquisition of sex-specific characteristics in a process known as germ cell sex determination. In the testis, male germ cells (MGCs) undergo several rounds of mitotic divisions before entering a prolonged period of G0 cell cycle arrest. In mice, G0 arrest lasts from embryonic day (E) 14.5 until postnatal day (P) 1. During this time, MGCs undergo intense epigenetic remodeling that is necessary for the specification of SSC fate. After G0 exit, a select population of MGCs migrates to the basement membrane of the testis cords and establishes the SSC population. Nearly every step of germ cell development is conducive to DNA damage. However, despite their hazardous developmental path, SSCs maintain a very low mutational load compared to somatic cells. According to the literature, the majority of MGCs exit G0 arrest and begin to differentiate, constituting the first wave of spermatogenesis. Only a small proportion of MGCs migrate to the basement membranes and take up SSC fate. Previous studies have found that the MGCs that become SSCs also have a lower mutational load than those that enter the first wave, and both these germ cell populations have lower mutational rates than somatic cells. This suggests that there must be stringent quality control mechanisms in place that safeguard MGC development and govern SSC selection. We have utilized a mouse model to study how events during G0 arrest impact SSC fate specification. Previous work in the Capel lab identified differential expression of the germ cell-specific RNA-binding protein, Dead End 1 (DND1), within the MGC population. Beginning at E16.5, DND1-hi and DND1-lo cell populations could be distinguished through FACS or transcriptome analysis. DND1 is known to be a necessary factor for MGC development, but the functional significance of the DND1-hi and -lo cell populations remained unclear. In this study, we describe a novel wave of MGC death beginning at E16.5 and corresponding with low DND1 expression. By late gestation, we show that 70% of MGCs are positive for propidium iodide, a hallmark of cell death. The DND1-lo dying MGCs display an increase in dsDNA breaks and exhibit signs of mitochondrial dysfunction. Notably, markers of apoptosis such as TUNEL staining and cPARP are absent during this developmental window. Instead, we found evidence that dying MGCs are removed at the time of birth through a unique mechanism resembling necroptosis. This work identifies a novel mechanism of MGC culling that occurs during G0 arrest and shows hallmarks of necroptotic cell death. We hypothesize this wave of MGC death functions as a quality control mechanism to remove damaged MGCs from the testis prior to SSC specification.