Very small testes are seen in boys with ATRX syndrome. To study this, we deleted Atrx from mouse testicular Sertoli cells; knockout mice develop small testes with discontinuous unbranched tubules and apoptosis of Sertoli cells during fetal life.
Aims: To investigate the mechanism underlying the Atrx syndrome gonadal phenotype.
Methods: Mouse embryonic gonads were processed sectioned and co-immunofluorescence (Co-IF) or immuno-FISH performed followed by quantification using ImageJ.
Results: Knockout embryos were studied at E16.5 when testes development is first arrested. In wildtype embryos, co-IF with ATRX and somatic cell marker GATA4 co-localized at a single nuclear speckle in Sertoli cells, which also co-strained for PML with a diameter typical of a PML nuclear body (1mm). In knockout mice, in ~30%% Sertoli cells, these ‘Gata4 PML nuclear bodies’ were 2-3 times larger. Also, 84% of knockout Sertoli cells (vs 4.1% in control) showed DNA damage either within the ATRX-deficient speckle or becoming widespread throughout the nucleus of apoptotic cells. ATRX-deficient speckles also lacked ATRX-binding partners DAXX, H3.3 and HP1a, suggesting heterochromatin condensation failure. Immuno-FISH with BAC chromosome as well as centromere and telomere probes identified a single Yp signal inside the GATA4 PML speckle in both control and ScAtrxKO cells, whereas Yq signal localized outside the speckle.
Conclusion: A single novel nuclear body was identified in Sertoli cells, bearing GATA4 and the short arm of the Y chromosome, that requires ATRX for condensation of the Y chromosome needed for Sertoli cell survival, and testis development.