Tameka C Phillips1,2, David E Wildt1,2, Pierre Comizzoli3,4. 1. Smithsonian Conservation Biology Institute, National Zoological Park, Washington, DC, 20008, USA. 2. Smithsonian Conservation Biology Institute, National Zoological Park, Front Royal, VA, 22630, USA. 3. Smithsonian Conservation Biology Institute, National Zoological Park, Washington, DC, 20008, USA. comizzolip@si.edu. 4. Smithsonian Conservation Biology Institute, National Zoological Park, Front Royal, VA, 22630, USA. comizzolip@si.edu.
Abstract
PURPOSE: This study aims to characterize the regulations of histone methylations, key epigenetic markers of oocyte competence, in germinal vesicle (GV) from different follicles (preantral, early, small, or large antral stage) using the domestic cat model. METHODS: In Experiment 1, the incidence of H3K4me3 or H3K79me2 was determined in GVs from the diverse follicle stages directly or after exposure to (1) a methyltransferase inhibitor, (2) sonication to fracture the cytoplasmic membranes and wash away the cytoplasmic content, or (3) methyltransferase inhibitor followed by sonication. In Experiment 2, the presence and maintenance of nuclear methyltransferases SMYD3 and DOT1L (regulating H3K4me3 and H3K79me2, respectively) was characterized in separate GV stages before and after sonication. Functionality of GVs from the various follicle stages (with or without transient isolation from the cytoplasm) then was assessed in Experiment 3 by transfer into recipient competent oocytes. RESULTS: The incidence of histones H3K4me3 and H3K79me2 within the GV were influenced by the cytoplasmic environment at all stages except at the transition to the early antral stage where nuclear regulating factors appeared to be mainly involved. The methyltransferase SMYD3 and DOT1L also appeared tightly bound to the nucleus at that transition. Interestingly, oocytes reconstructed with a GV isolated from the cytoplasm for a prolonged period had the capacity to form an embryo after fertilization which proved that communication between the donor GV and the host cytoplasm (likely including the regulation of epigenetic factors) could be restored. CONCLUSIONS: Histone methylation apparently becomes regulated by specific nuclear factors at the acquisition of competence during the folliculogenesis and does not seem to be disrupted by prolonged isolation from the surrounding cytoplasm.
PURPOSE: This study aims to characterize the regulations of histone methylations, key epigenetic markers of oocyte competence, in germinal vesicle (GV) from different follicles (preantral, early, small, or large antral stage) using the domestic cat model. METHODS: In Experiment 1, the incidence of H3K4me3 or H3K79me2 was determined in GVs from the diverse follicle stages directly or after exposure to (1) a methyltransferase inhibitor, (2) sonication to fracture the cytoplasmic membranes and wash away the cytoplasmic content, or (3) methyltransferase inhibitor followed by sonication. In Experiment 2, the presence and maintenance of nuclear methyltransferases SMYD3 and DOT1L (regulating H3K4me3 and H3K79me2, respectively) was characterized in separate GV stages before and after sonication. Functionality of GVs from the various follicle stages (with or without transient isolation from the cytoplasm) then was assessed in Experiment 3 by transfer into recipient competent oocytes. RESULTS: The incidence of histones H3K4me3 and H3K79me2 within the GV were influenced by the cytoplasmic environment at all stages except at the transition to the early antral stage where nuclear regulating factors appeared to be mainly involved. The methyltransferase SMYD3 and DOT1L also appeared tightly bound to the nucleus at that transition. Interestingly, oocytes reconstructed with a GV isolated from the cytoplasm for a prolonged period had the capacity to form an embryo after fertilization which proved that communication between the donor GV and the host cytoplasm (likely including the regulation of epigenetic factors) could be restored. CONCLUSIONS: Histone methylation apparently becomes regulated by specific nuclear factors at the acquisition of competence during the folliculogenesis and does not seem to be disrupted by prolonged isolation from the surrounding cytoplasm.