Abstract
Mammalian sexual development hinges on sexually dimorphic genetic networks operating within the embryonic gonads. These differential networks streamline development of the undifferentiated bipotential gonad into the ovary in females and testis in males. A major bottleneck in our understanding of gonad development are the regulatory mechanisms underlying the transcriptional variability in both sexes. A comprehensive understanding of these sex-dimorphic networks is crucial for increasing our knowledge of Differences of Sex Development (DSDs), infertility and reproductive disorders, which are currently poorly categorised.
Recent studies have discovered widespread alternative splicing, a mechanism allowing a single gene to express multiple transcript isoforms, within the developing gonads. It is hypothesised that the sex-specific expression of isoforms can fine-tune sex-specific gonad development. Studies have also revealed that epigenetic modifications like DNA methylation can regulate transcript isoform expression. Herein, we investigated methylation-regulated splicing mechanisms in candidate genes that are important for reproductive development and show sex-biased isoform expression during gonad development.
Using published RNAseq data, the sex-specific transcript isoform expression of Lhx9, Lef1, WT1, Fgf9 and Sox9 was characterised from E10.5 – E13.5. Bisulfite amplicon sequencing was then used to analyse methylation patterns at CpG islands (CGIs) associated with splice sites of these genes between E11.5- E13.5, encompassing the critical period of sex determination and early gonad differentiation. Significant sex-dimorphic and stage-specific methylation was observed at target CGIs of Lhx9, Lef1, WT1 and Fgf9. This suggested that sex-dimorphic methylation patterns could potentially regulate differential splicing during gonad development.
To explore the mechanisms behind these methylation patterns, the expression and binding of WT1 transcription factor was explored. Analysis of published single cell-RNA sequencing data revealed that WT1 transcription factor was widely expressed during gonad development. WT1 was also co-expressed with the candidate genes in sex-specific somatic cells of the gonads. As WT1 is a key regulator of gonad development and DNA methylation state, it was hypothesised that differential WT1 binding would be found at candidate CGIs. Chromatin immunoprecipitation followed by qPCR analysis revealed significantly higher WT1 binding at Sox9 CGI 22 in males compared to females during sex determination. This suggested a potential role of WT1-mediated regulation of methylation state at CGIs.
These findings provide further insights into the sexually dimorphic genetic and epigenetic programs operating during gonad development by characterising sex-dimorphic patterns of transcript isoform expression, DNA methylation patterns and WT1 binding at CGIs. Future studies should aim to determine the functional significance of these sex differences and their relevance in the context of DSDs, infertility and disease states.