The enigmatic process of fetal oocyte attrition (FOA) is responsible for the selective elimination of more than two-thirds of meiotic prophase I (MPI) oocytes before birth in mice. While numerous hypotheses have been considered for explaining this massive oocyte loss, the underlying mechanism remains unknown. A role for LINE-1 retrotransposon activity has been recently described in FOA (Malki et al., 2014) upon showing that increased L1 expression in oocytes correlates with their preferential elimination. This data suggests that the process of L1 retrotransposition results in a genotoxic effect that triggers FOA. To identify this trigger, we will consider the intrinsic reverse transcriptase (RT) activity of L1 ORF2p. It has been shown that by blocking RT activity using the nucleoside RT inhibitor AZT, FOA is greatly attenuated, suggesting that intermediates of L1 reverse transcription - RNA:DNA hybrids and ssDNA - are dangerous to the cell. I will develop a method to detect these intermediates and identify which is the driver of FOA using a mutant mouse model that fails to hydrolyze RNA:DNA hybrids. In MPI, the DNA damage response (DDR) is known to sense programmed DNA breaks and eliminates oocytes with irreparable damage at a mid-pachynema DNA damage checkpoint. Since we know that FOA occurs primarily in earlier stages of MPI (leptonema and zygonema), we propose that DDR machinery can also detect signals from L1 TPRT as dangerous and cull oocytes with excess L1 TPRT intermediates or TPRT- associated damage prior to the canonical mid-pachynema checkpoint. I intend to implicate the DDR in FOA by independently disrupting activity of the core sensor of DNA breaks, ATM kinase and downstream effector checkpoint kinase 2 and monitor effects on downstream signaling of the DDR and oocyte number in early stages of MPI. Our findings will uncover a new function of the DDR in oocyte elimination during early stages of MPI rather than solely at the mid-pachytene checkpoint. Further, by identifying the genotoxic trigger of FOA, we are making significant advances in our understanding FOA that have great implications for human reproductive health and fertility.
Fertility and reproductive lifespan, both of which rely on the quality and quantity of oocytes that comprise the ovarian reserve, are concerns for the entire female population. For this reason, understanding the processes involved in the establishment of the ovarian reserve are of critical importance, one of which being the selective elimination of ~80% of oocytes before birth. I aim to build upon a recently described role for mobile DNA elements known as transposons in this massive oocyte loss by investigating how transposon activity leads to genome instability and cell death.
