Uncovering Novel Cytogenetic and Molecular Etiologies for Male Infertility

Abstract

Infertility affects 10-15% of couples, making it one of the most frequent health disorders for reproductive aged individuals. Over 20% of cases are idiopathic, and it is commonly thought that many individuals have an underlying genetic etiology. Identifying genes involved in unexplained infertility provides information to support diagnosis, genetic counseling and potential therapeutic intervention. One approach to identifying genes involved in infertility is to examine the phenotype-genotype correlation in subjects with a clinical phenotype accompanied by a balanced chromosomal aberration (BCA), as is the foundation of gene discovery in the Developmental Genome Anatomy Project (DGAP). Participant DGAP230 has severe oligospermia and 46,XY,t(20;22)(q13.3;q11.2). While BCAs may reduce fertility by production of unbalanced gametes, a chromosomal rearrangement may also disrupt or dysregulate genes important in fertility. Using large-insert genome sequencing, chromosomal breakpoints were determined with nucleotide-level precision. Investigation of genes in the topologically associated domains at the sites of the rearrangement and subsequent use of the novel 3C-PCR technique revealed exclusive dysregulation of synaptonemal complex protein 2 (SYCP2) from the derivative chromosome 20 (der(20)) allele, which resides 1.5 Mb centromeric to the der(20) breakpoint. 4C-seq from the SYCP2 promoter revealed interactions eight Mb downstream of the der(20) breakpoint in chromosome 22, supporting a model of enhancer adoption. SYCP2 encodes synaptonemal complex protein 2, a member of the synaptonemal complex involved in male meiosis I homologous chromosome synapsis. To assess its impact on meiosis, we misexpressed RED1, the functional axial element homolog of SYCP2 in Saccharomyces cerevisiae. By performing Red1 immunolocalization on surface-spread meiotic nuclei, we discovered that excess Red1 forms polycomplexes, disrupting the structural integrity of the synaptonemal complex by preventing incorporation of the transverse filament Zip1. This model highlights aberrant meiosis as the defect in spermatogenesis leading to severe oligospermia. Finally, we reveal two novel frameshift mutations in SYCP2 identified in azoospermic men, which further support a mechanism of SYCP2-mediated infertility. This thesis provides the first evidence of SYCP2-mediated infertility in humans and illuminates that rearrangement breakpoints should be considered as an alternative etiology to that of segregation of unbalanced gametes in infertile men harboring BCAs.