Towards elucidating the function of the oocyte Subcortical Maternal Complex: A multi-omic analysis of an Nlrp5-null murine model

Abstract

The subcortical maternal complex (SCMC) is composed of some of the most abundant proteins in the oocyte. SCMC mutations have been linked to early embryo arrest, recurrent molar pregnancies and imprinting disorders. Genomic imprinting depends upon correct patterning of DNA methylation over imprinted domains during oogenesis. It was previously shown that oocytes deficient in the human SCMC gene KHDC3L had globally impaired methylation, thus suggesting a role for the SCMC in the establishment or maintenance of DNA methylation marks at imprinted regions. This thesis aims to identify the functional role of the most abundant SCMC protein NALP5 and, by extension, the SCMC, in oocyte developmental competence. This study focuses primarily on elucidating the role of NALP5 in the context of DNA methylation patterning in the oocyte. This was done by establishing a null mouse line for Nlrp5, the gene encoding NALP5. A phenotypic analysis of the Nlrp5 mutant confirmed that embryos derived from Nlrp5-null oocytes arrest before or at the 2-cell stage of development. Proteomic analysis of Nlrp5-null oocytes shows significantly reduced abundances of other SCMC proteins, demonstrating that the Nlrp5 knockout model can effectively interrogate the function of the complex at large. GDF9 and BMP15, regulators of ovarian follicle development via paracrine signalling, were also notably reduced in Nlrp5-null oocytes. Other proteins altered in Nlrp5-null oocytes include epigenetic modifiers DNMT3L and UHRF1, both of which are reduced in the mutant, the former by over 75%, and the latter by 40%. Immunofluorescence analysis of Nlrp5-null oocytes indicates an altered DNMT3L localisation, but no change in DNMT1 localisation. Single-cell transcriptomic analysis of Nlrp5-null oocytes detected over 580 significant differentially expressed genes when compared with wild type oocytes, but no robust enrichment in any molecular pathways. Parallel single-cell methylation analysis indicates a slight global reduction in DNA methylation, which could reflect the strong reduction in the de novo methyltransferase DNMT3L. Some methylation differences overlapped with imprinted genes. Methylation profiling of Nlrp5-null murine oocytes does not indicate an impairment in methylation to the same degree as that of the human KHDC3L mutant but is still at a level that may cause transcriptomic dysregulation.