Reconstruction of enucleated mouse germinal vesicle oocytes with blastomere nuclei

Abstract

We have investigated the possibility that mitotic nuclei originating from preimplantation stage embryos and placed in the oocyte cytoplasm can undergo remodelling that allows them to undergo meiosis in the mouse. To address this question, we have used enucleated germinal vesicle (GV) ooplasts as recipients and blastomeres from the 2-, 4-or 8-cell stage as nuclear donors. We employed two methods to obtain ooplasts from GV oocytes: cutting and enucleation. Although efficiency of the reconstruction process was higher after enucleation than after cutting (90% and 70% respectively), the developmental potential of the oocytes was independent of how they had been produced. Nuclei from the 2-, 4-, or 8-cell stage embryos supported maturation in about 35%, 55% and 60% of cases, respectively. The time between nuclear envelope breakdown and the first meiotic division was shortened by up to 5 h in reconstructed oocytes, a period equivalent to the mitotic division of control blastomeres. About onethird of oocytes reconstituted with blastomere nuclei divided symmetrically instead of extruding a polar body; however, in the majority of them metaphase plates were found, suggesting that reconstructed oocytes (cybrids) underwent a meiotic rather than mitotic division. The highest percentage of asymmetric divisions accompanied by metaphase plates was found in cybrids with 8-cell-stage blastomere nuclei, suggesting that the nuclei from this stage appear to conform best to the cytoplasmic environment of GV ooplasts. Our results indicate that the oocyte cytoplasm is capable of remodelling blastomere nuclei, allowing them to follow the path of the meiotic cell cycle.

Reconstruction of mammalian oocytes by germinal vesicle transfer: A systematic review

“…To imagine the nuclear material (in prophase1oocytes) and meiotic spindle (in MI oocytes) localization, oocytes could be incubated in the enucleating medium with or without 2% sucrose for 1/2 hr ( 9 , 37 , 38 ). Indirect enucleation ( 9 , 10 , 18 , 23 ), direct enucleation ( 4 , 7 , 9 - 12 , 17 - 21 , 32 , 39 , 40 ) and oocyte fraction ( 6 , 38 ) are three enucleation methods for GV oocytes. In the first method, enucleation was performed ‘indirectly’, to eject karyoplast via a slot made in the ZP by rising inside holding pipette pressure ( 9 , 10 , 18 , 23 ).…”

“…In the first method, enucleation was performed ‘indirectly’, to eject karyoplast via a slot made in the ZP by rising inside holding pipette pressure ( 9 , 10 , 18 , 23 ). GV- or MI-oocyte enucleation was accomplished using this way ( 9 , 40 ).…”

“…The enucleation pipette with inner diameter (ID) of 10-15 µm ( 9 , 23 ) or 20-25 µm was inserted moderately into the perivitelline space (PVS) via the ZP ( 4 , 7 , 10 - 12 , 17 - 21 , 32 , 39 ) ProMI- or MI-karyoplast was aspirated with the smallest possible volume of surrounding ooplasm by smooth suction and the pipette was moderately withdraw from the oocyte ( 9 ). Then karyoplast was inserted inside the PVS of enucleated oocyte against the holding pipette ( 4 , 7 , 10 - 12 , 17 - 21 , 32 , 39 ) through the slit was made in the ZP with the same pipette ( 9 , 40 ) or another pipette (ID: 25µm) ( 22 , 23 ). In the second method, to speed removing and replacing the GV karyoplast and determine the size of it, the process was done ‘directly’ by a tapered enucleation pipette with an inner diameter of about 20 μm ( 4 , 7 , 9 - 12 , 17 - 21 , 32 , 33 , 37 , 39 , 40 ).…”

Reconstruction of mammalian oocytes by germinal vesicle transfer: A systematic review

“…In the first method, enucleation was performed "indirectly", to eject karyoplast via a slot made in the ZP by rising inside holding pipette pressure (9,10,18,23). GV-or MI-oocyte enucleation was accomplished using this way (9,40).…”

Experimental embryology of mammals at the Jastrzebiec Institute of Genetics and Animal Breeding

“…Also, we were able to produce mouse (Mohammed et al, 2008). When tested as the recipients of embryonic nuclei, classically enucleated GV oocytes have proved capable of remodeling blastomere nuclei, so that they followed the path of meiotic cell cycle (Grabarek et al, 2004). The influence of early embryonic environment on somatic cells is another goal of our team and the embryonic-somatic chimaeras are the model used.…”