ELUCIDATING THE GENETIC BASES OF HUMAN INFERTILITY USING MOUSE MODELS

Infertility is a relatively common health condition that affects upwards of ~15% of couples in the United States. Approximately ¼ of these cases are considered idiopathic, but many are believed to have a genetic origin. Over the last several years, I have worked on an association- and linkage-free approach to identify segregating infertility alleles. With the use of CRISPR/CAS9 genome editing, I have modeled nine putatively deleterious, nonsynonymous single nucleotide polymorphisms (SNPs) in mouse orthologs of fertility genes. Of the nine variants, five cause fertility defects in mice. This dissertation focuses on human alleles SPO11 P306T, DMC1 M200V, and SEPT12 G169E. SPO11 initiates recombination by creating double-strand breaks (DSBs) at several hundred sites across the genome, a fraction of which are repaired by crossover recombination, which is essential for proper disjunction at the first meiotic division. I found that SPO11P306T/P306T mice are subfertile due to deficient SPO11P306T DSB activity. But unlike typical hypomorphs, the activity of SPO11P306T occurs around the elusive DSB threshold. This causes an unexpected array of defects, including some cells surviving quality checkpoints to become viable gametes. DMC1 initiates repair of SPO11-catalyzed DSBs by mediating homology search for and strand invasion of donor duplexes. DMC1 null mutants exhibit meiotic failure, which makes DMC1 an obvious candidate for studying infertility. Previous publications have implicated the allele DMC1M200V as the causal mutation in a case of premature ovarian insufficiency. However, work to experimentally validate DMC1M200V fell short of evaluating in vivo effects, which proved to be necessary because DMC1M200V/M200V mice are fertile and lack obvious meiotic defects. This work underscores the importance of going beyond association- and in vitro-based methods to validate potential infertility alleles. SEPT12 is a testis-specific GTP-binding protein that is critical for sperm formation and motility. Recent studies have implicated multiple SEPT12 alleles as the primary factor behind teratozoospermia cases, but again, often lack proper experimental validation. Here, I assessed three SEPT12 alleles in mice and found that SEPT12G169E/G169E males sired significantly less offspring. Upon closer examination of sperm, motility was severely weakened in the mutant