Transcriptional profiling of Nlrp5 +/+, -/- and +/- germinal vesical murine oocytes

Nlrp5 encodes a core component of the subcortical maternal complex (SCMC) a cytoplasmic protein structure unique to the mammalian oocyte and cleavage-stage embryo. NLRP5 mutations have been identified in patients presenting with early embryo arrest, recurrent molar pregnancies and imprinting disorders. Correct patterning of DNA methylation over imprinted domains during oogenesis is necessary for faithful imprinting of genes. It was previously shown that oocytes with mutations in the human SCMC gene KHDC3L had globally impaired methylation, suggesting a role for the SCMC in the establishment of DNA methylation at imprinted regions. Here, we present a multi-omic analysis of an Nlrp5-null mouse model, which displays a global misregulation of maternal proteins in Nlrp5-null oocytes, likely underlying oocyte developmental incompetence. We also show that de novo DNA methylation is altered in Nlrp5-null oocytes, and that epigenetic modifiers such as DNMT3L are significantly reduced. This provides evidence for mechanisms leading to downstream misregulation of imprinted genes, which in turn, may result in imprinting syndromes, multi-locus imprinting disturbances (MLID), and hydatidiform moles. Overall design: To determine the effects of an Nlrp5 inactivating mutation on transcriptional regulation in the murine oocyte, we generated a novel Nlrp5 mutant mouse line using CRISPR/Cas9 gene editing, and performed single cell transcriptome sequencing (scRNA-seq) on germinal vesicle stage oocytes from female Nlrp5 +/+, -/- and +/- mice. Oocytes were collected at 3 weeks old. Nuclear maturation staging (i.e. non-surrounded nucleous (NSN), or surrounded nucleolus (SN)) was determined post scRNA-sequencing, using a list of NSN-SN differentially expressed genes.