Background: Ovarian sex differentiation network involves a panoply of interacting factors. Yet, no single sex-determining factor has been identified to be an equivalent of SRY or SOX9 in the testis. Recently, data suggested CBX2 as a pioneer regulator promoting testis development. In addition to its implication in ovary pathway differentiation which remains unclear.
Objective and hypotheses: To deepen our understanding of the regulatory network that underpins the molecular basis of ovary development. Within, we light up whether CBX2 is a putative regulator.
Method: i)We evaluated genes modulation effects on specific female-markers by RT-qPCR following WT/MTCBX2 forced-expression and knock-down assay. ii) DamID/RNAseq approaches performed under overexpression and down-regulation of CBX2 to screen for new female-determining genes.
Results: We established an in vitro cell system suitable for the screening of new ovarian-determining genes. In addition, forced-expression and RNA interference of CBX2 isoforms (CBX2.1 and CBX2.2) showed divergent effects on the expression of FOXl2/WNT4/RSPO1/FST ovarian-specific genes. CBX2.1 was a repressive actor vis-à-vis the female cluster. However, a preferential bidirectional interaction relating CBX2.2 and RSPO1 has been highlighted. Likewise, we assume a positive regulatory feed-forward loop relating the two markers. CBX2.2 expression seems to be enhanced upon WNT4 overexpression, suggesting that CBX2.2 may be positively regulated through the WNT4 pathway. In the same context, we showed an antagonistic interaction between FOXL2 and the two CBX2 isoforms mirroring the FOXl2 vs SOX9 picture. Of utmost importance, substantial number of novel CBX2 targets has been identified. Eight candidates (DKK1, DOK5, BMP5, SIRT5, BMP2, AMIGO2, FZD7, and RSPO3) have been selected basing on their potential link to sex process. Surprisingly, DKK1 an identified mice pro-male marker, turned out to be preferentially expressed in human KGN and found to be positively regulated by the female players.
Conclusion: Few steps have been made in the human ovary differentiation-regulatory network. Further information of this molecular pathway will doubtlessly be provided by the ongoing genome-wide high throughput analysis to rationalise the potential role of CBX2 and its partners within the ovary gene repertoire.
Funding: Swiss National Science Foundation Grant Nr 320030_130645/1 to ALB.
01 - 03 Oct 2015
European Society for Paediatric Endocrinology