Deletion of Orc4 during oogenesis severely reduces polar body extrusion and blocks zygotic DNA replication†.

Origin recognition complex subunit 4 (ORC4) is a DNA-binding protein required for DNA replication. During oocyte maturation, after the last oocyte DNA replication step and before zygotic DNA replication, the oocyte undergoes two meiotic cell divisions in which half the DNA is ejected in much smaller polar bodies. We previously demonstrated that ORC4 forms a cytoplasmic cage around the DNA that is ejected in both polar body extrusion (PBE) events. Here, we used ZP3 activated Cre to delete exon 7 of Orc4 during oogenesis to test how it affected both predicted functions of ORC4: its recently discovered role in PBE and its well-known role in DNA synthesis. Orc4 deletion severely reduced PBE. Almost half of Orc4-depleted germinal vesicle (GV) oocytes cultured in vitro were arrested before anaphase I (48%), and only 25% produced normal first polar bodies. This supports the role of ORC4 in PBE and suggests that transcription of the full-length Orc4 during oogenesis is required for efficient PBE. Orc4 deletion also abolished zygotic DNA synthesis. Fewer Orc4-depleted oocytes developed to the metaphase II (MII) stage, and after activation these oocytes were arrested at the two-cell stage without undergoing DNA synthesis. This confirms that transcription of full-length Orc4 after the primary follicle stage is required for zygotic DNA replication. The data also suggest that MII oocytes do not have a replication licensing checkpoint as cytokinesis progressed without DNA synthesis. Together, the data confirm that oocyte ORC4 is important for both PBE and zygotic DNA synthesis.

The role of ORC4 in enucleation of Murine Erythroleukemia (MEL) cells is similar to that in oocyte polar body extrusion.

The Origin Replication Complex subunit 4 (ORC4) is one in six subunits of the Origin Replication Complexes (ORCs) which is essential for initiating licensing at DNA replication origins and recruiting adaptor molecules necessary for various cellular processes. Previously, we reported that ORC4 also plays a vital role in polar body extrusion (PBE) during oogenesis in which half the chromosomes are extruded from the oocyte. We hypothesized that ORC4 might play a broader role in chromatin elimination. We tested its role in enucleation during the development of erythrocytes. Murine erythroleukemia (MEL) cells can be propagated in culture indefinitely and can be induced to enucleate their DNA by treatment with Vacuolin-1, thereby mimicking normal erythrocyte enucleation. We found that ORC4 appeared around the nuclei of the MEL cells with Vacuolin-1 treatment, gradually increasing in thickness before enucleation. We then tested whether ORC4 was required for MEL enucleation by down regulating ORC4 with siRNA-ORC4 during Vacuolin-1 treatment and found that this prevented MEL enucleation. These data are consistent with the model that ORC4 is required for erythroblast enucleation just as it is for oocyte PBE. They suggest a new model in which ORC4 expression is a marker for the initiation to the enucleation pathway.