Research

Image of macrophage (green) reaching through the basement membrane (white) of a testis cord containing yH2AX (yellow) positive germ cells (pink).

Germ cells are arguably some of the most interesting and important cells in the human body. They are responsible for passing on their DNA to the next generation. Despite their importance, germ cells face many unique hurdles in their development: they must migrate across the embryo in order to colonize the genital ridge, maintain fate plasticity while avoiding inappropriate differentiation, undergo multiple rounds of epigenetic reprogramming, and undergo intensional DNA breaks during meiotic recombination. However, because there is no stronger selective pressure for the survival of a sexually reproducing species than viable gamete formation, germ cells have evolved special mechanisms to overcome these challenges.

How do male germ cells develop?

During my Ph.D., I have focused on understanding the development of the male germline. In both mice and humans, male germ cell development is marked by a prolonged period of G0 cell cycle arrest, which in mice lasts from embryonic day (E) 14.5 until postnatal day (P) 2. After G0 exit, there is a split in MGC fate: The majority of MGCs differentiate into type II spermatogonia and enter the first wave of spermatogenesis, while only a small subset of MGCs migrate to the basement membrane and become spermatogonial stem cells (SSCs). SSCs are are the resident stem cells of the male germline and are responsible for supplying sperm for the entirety of a male’s reproductive lifespan. Despite the importance of proper SSC specification, the process that determines which MGCs will be lost in the first wave of spermatogenesis and which will become SSCs is not well understood.

Schematic of MGC development beginning at E14.5 until SSC fate establishment (P4).

How are SSC specified?

The majority of my thesis work has been to characterize a previously unknown period of MGC death that occurs during G0. Unlike other known periods of germ cell death, MGCs dying during G0 are negative for TUNEL staining and instead show characteristics of necrosis. We observe that ~60% of MGCs are positive for Annexin-V at E16.5. However, we do not see a significant loss in MGC number until P0. Interestingly, we found phosphorylation of the necroptotic effector protein, RIPK3, as well as its downstream target, MLKL, coinciding with the onset of germ cell loss. Based on this, I hypothesize that G0 MGCs undergo a unique form of necroptosis mediated by RIPK3 during the neonatal period. This suggests a novel mechanism at play which negatively selects MGCs against an SSC fate.

Time-course of yH2AX staining (yellow) and progression of MGC death as measured by FACS analysis with Annexin-V and propidium iodide.

How does G0 arrest prepare cells for SSC specification?

Cross section of an E16.5 mouse testis. Germ cells are expressing DND1 tagged with a GFP fusion protein. DND1-GFP localizes in the cytoplasm, perinuclear germ granules, and in the nucleus, althought the nuclear function of DND1 is not yet understood.

Previously, the field has examined markers for positive SSC selection. They have done this by looking for genes that are differentially expressed during the G0 period of cell cycle arrest, and therefore may mark germ cells that are preselected to become SSCs. Previous work from the Capel lab demonstrated that the RNA-binding protein DND1 is differentially expressed between MGCs as early as E14.5. However, the importance of DND1-high and DND1-low protein levels in SSC specification are unclear.

While DND1 is best known for acting on RNAs in the cytoplasm to either promote or inhibit their translation, I have found data suggesting novel role(s) for DND1 in the nucleus. We hypothesize that DND1 may play multiple roles throughout G0 to safeguard MGC development towards a SSC fate.

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