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Reproductive Technology |
Center for Regenerative Biology/Department of Animal Science,2 University of Connecticut, Storrs, Connecticut 06269
Reproductive Biology Associates,3 Atlanta, Georgia 30342
Clínica e Centro de Pesquisa em Reprodução Humana Roger Abdelmassih,4 São Paulo, Brasil
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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assisted reproductive technology, gamete biology, meiosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Despite the success in using germ cells to show that, in principle, haploidization is possible, attempts of haploidization using somatic cells have rarely been undertaken, and successful generation and fertilization of artificially haploidized gametes from somatic cells has not been reported. Most studies in which somatic haploidization were attempted employed cells at the G0/G1 stage and oocytes at the MII stage [4, 5, 13]. However, this donor cell-oocyte combination has been shown to result in the uneven segregation of chromosomes [13]. There have been two reports in which GV-stage oocytes were used in combination with somatic cells at the G0/G1 stage [1, 14]. However, these studies failed to generate cell-oocyte complexes that would undergo the first meiotic division.
Immature mammalian oocytes are arrested at the G2/M transition; therefore, combining somatic cells that are also arrested at the G2/M phase with GV ooplasts may provide a more compatible merger for the haploidization process. In the present study, we aimed to explore the possibility of improving somatic cell haploidization by the use of the third strategy discussed above, i.e., GV oocytes combined with G2/M-arrested somatic cells. Hence, we also investigated the nuclear morphology, cytokinesis, bipolar spindle formation, and pseudopronuclear formation during the haploidization process by this new strategy.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Unless otherwise indicated, all chemicals purchased were from Sigma Chemical Co. (St. Louis, MO). All media were prepared fresh and filter sterilized through a 0.22-µm filter (Acrodisc; Pall Gelman Laboratory, Ann Arbor, MI).
Animals and Recovery of GV Oocytes
The B6D2F1 (C57BL/6 x DBA/2) and CD-1 mice used were from Charles River Laboratories (Wilmington, MA). All animal treatments were approved by the Institutional Animal Care and Use Committee of the University of Connecticut, Storrs.
Ovaries were obtained from 8- to 12-wk-old CD-1 mice 44–48 h after eCG injection. Oocytes at the GV stage were retrieved from each ovary by puncturing the follicles with a sterile 25-gauge needle on a syringe containing M2 medium. The oocytes were stripped of cumulus cells by repeated aspiration through a glass pipette, the tip diameter of which was slightly larger than the diameter of an oocyte. The cumulus-denuded GV oocytes were then transferred into human tubal fluid (Specialty Media, Phillipsburg, NJ) with 10% fetal calf serum (FCS; Hyclone, Logan, UT) containing 50 µg/ml of 3-isobutyl-1-methylxanthine (IBMX) [15] and cultured for 3 h in 5% CO2 in air, 37°C. The 3-h exposure of IBMX was included to help oocytes develop a perivitelline space and to prevent GV breakdown during oocyte in vitro culture. Germinal vesicle-stage oocytes that had a visible perivitelline space were selected with the use of an inverted microscope (TE300; Nikon, Tokyo, Japan), and randomly assigned for either enucleation or in vitro maturation.
Somatic Cell Culture and Cell Cycle Synchronization
Ear biopsies of female B6D2F1 mice were cut into pieces of 1–2 mm2 and incubated as tissue explants in Dulbecco modified Eagle medium (DMEM; Gibco, Invitrogen, Carlsbad, CA) supplemented with 10% FCS. Fibroblast monolayers, which formed around the tissue explants, were harvested following incubation in PBS containing 0.25% trypsin and 0.75 mM EDTA. For cell passaging, confluent cells were detached and plated in two new dishes. For storage, confluent cells were detached, placed in DMEM with 20% FCS and 10% dimethyl sulfoxide, and frozen in liquid nitrogen.
To synchronize donor cells to G2/M phase, cells at passages 5–10 were cultured to approximately 50% confluency and then treated with 0.125 µg/ml nocodazole [16, 17] in DMEM supplemented with 10% FCS for 18 h at 37°C. The cell cycle stage and efficiency of synchronization were examined by flow cytometry analysis of the DNA content. Briefly, after synchronization treatment, a portion of the cells was fixed in cold ethanol overnight. The DNA was stained with 30 µg/ml propidium iodide in PBS. For each analysis, 10 000 cells were passed through a FACSscan cytometer (Becton-Dickinson, San Jose, CA). Each synchronization regimen was analyzed three times. In the population of large cells, approximately 70% of treated cells were at either G2 or metaphase stage, which has 4C of DNA and 2N of chromosomes [18].
Micromanipulation of GV Oocytes
The GV oocytes were incubated in a microdroplet of M2 containing cytochalasin B (CCB, 7.5 µg/ml) and IBMX (50 µg/ml) for 30 min at room temperature (25°C), and then a slit was made through the zona pellucida (ZP) of each oocyte with a sharp needle. Micromanipulations were performed using an inverted microscope (Nikon TE300) equipped with two microinjectors (IM-6; Narishige, East Meadow, NY) and two oil hydraulic micromanipulators (Narishige). An enucleation pipette (25 µm) was inserted into the cytoplasm to remove the GV nucleus through the slit in the ZP. The synchronized donor cells were screened and the larger cells (
28 µm), presumably at G2/M, were selected for nuclear transfer [16]. After insertion of a donor cell into the perivitelline space, cell-ooplast complexes were washed thoroughly in M2 to remove CCB and IBMX before electrofusion. Two microelectrodes, 100 µm in diameter, were applied to align the cell-ooplast complexes (Fig. 1A). Then the complexes were placed into electrofusion medium (0.28 M mannitol, 100 µM CaCl2, 100 µM MgSO4, and 0.005% BSA) and subjected to two pulses of 2.0 kV/cm direct current (BTX 200; BTX Inc., San Diego, CA) for 15 µsec. The cell-ooplast complexes were then washed in M2 and, 30 min following the electrical pulses, were examined for fusion (Fig. 1B).
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Oocyte In Vitro Maturation and Activation
The fused cell-ooplast complexes were incubated with in vitro maturation medium containing human tubal fluid and 10% FCS in 5% CO2 in air, 37°C, and after 17 h of maturation, the reconstructed oocytes were observed for polar body extrusion. The reconstructed and intact oocytes were then subjected to activation by exposure to activation medium containing calcium-free human tubal fluid, 10 mM Sr2+, and 5 µg/ml CCB for 6 h.
Fluorescence Microscopy
To examine the nuclear morphology of oocytes during maturation and after activation, control and micromanipulated oocytes were fixed at the expected MI, MII (17 h postfusion), and pronuclear (PN) stages (6 h postactivation; Fig. 2) by transferring them into a fixative solution containing 2.5% paraformaldehyde. For MI stage, the control and manipulated oocytes were fixed at 9–10 h after onset of maturation and 4–6 h postfusion, respectively. Fixed oocytes were stained for DNA with 10 µg/ml Hoechst 33342 in M2 medium. The positions of the chromosomes, the nucleus, or the first polar body were confirmed under ultraviolet and regular light microscopy using an Olympus AX70 (Olympus, Tokyo, Japan) epifluorescence microscope.
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Immunohistochemistry and Laser-Scanning Confocal Microscopy
For examination of microtubules, oocytes were fixed in a microtubule stabilizing buffer containing 2% formaldehyde, 0.5% Triton X-100, 1 µM taxol, 10 U/ml aprotinin, and 50% deuterium oxide at 37°C for at least 30 min. They were then washed in washing buffer (PBS containing 3 mM NaN3, 0.01% Triton X-100, 0.2% nonfat dry milk, 2% normal goat serum, 0.1 M glycine, and 2% BSA) three times and left in washing buffer overnight at 4°C for blocking and permeabilization [19]. Oocytes were then double stained to visualize microtubules and DNA. Briefly, samples were incubated in mouse anti-
-tubulin antibody (1:200) for 4 h at 37°C or overnight at 4°C. After three washes in washing buffer, the oocytes were incubated in fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin G (1:200) for 1 h at 37°C. Finally, the oocytes were washed, stained for DNA with 7.5 µM propidium iodide, mounted in PBS containing 50% glycerol as an antifading reagent and 25 mg/ml NaN3, and examined with a laser-scanning confocal microscope (Leica TCS SP2; Mannheim, Germany).
Statistical Analyses
The data were analyzed using chi-square tests in the Statistical Analysis System (SAS Inc., Cary, NC).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We transferred G2/M-arrested mouse fibroblast cells into 262 enucleated GV oocytes. Among these reconstructed oocytes, 150 (57.3%) had successfully fused 30 min after electrofusion (Fig. 1B and Table 1). Following culture in in vitro maturation conditions for 17 h, 84.7% (138/163) of control oocytes extruded the first PB (Fig. 3A). Among the manipulated oocytes that did fuse successfully, 52.1% (62/119) extruded pseudo-PBs (Table 1). However, large variations in the size of the first pseudo-PB were observed in manipulated oocytes. Both normal-sized pseudo-PB (similar size to that in control oocytes; Fig. 3B) and large pseudo-PB, up to four times the size of normal, were noted (Fig. 3C). Symmetrical cell division, which resulted in two cells of equal size, was also seen, although this was rare. Over half of the manipulated oocytes (41/62, 66.1%) had normal-sized pseudo-PB while the occurrence of the abnormally large pseudo-PBs was 33.9% (21/62) (Table 1). Furthermore, we observed a difference in the timing of first PB extrusion. For control GV oocytes, extrusion of the first PB occurred 9–10 h after the release from IBMX. However, the reconstructed oocytes extruded their first pseudo-PBs 5–7 h after fusion, which was approximately 3–4 h earlier than controls (Fig. 2).
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Nuclear Progression During In Vitro Maturation
In this component of our study, the nuclear morphology of manipulated oocytes was observed at the expected MI and MII stages of oocyte maturation. These stages occurred in control oocytes at 8–10 h and 17 h after the onset of maturation for MI and MII, respectively. In manipulated oocytes, the timing was 4–6 and 17 h after fusion for MI and MII, respectively (Fig. 2). At both MI and MII stages, the control oocytes had condensed chromosomes lining up at the metaphase plate (Fig. 4, A and E). However, in the manipulated oocytes, various abnormal nuclear morphologies were observed at the expected MI stage. These nuclear configurations fell into three general categories: 1) condensed chromosomes (Fig. 4B); 2) partially condensed chromosomes or an uneven distribution of chromatin (Fig. 4C); and 3) uncondensed chromatin or an interphase-like nucleus (Fig. 4D). Thirty-one manipulated oocytes were examined at the expected MI stage and the majority of these oocytes, 61.3% (19/31; Table 2), had a degree of chromosome condensation (Fig. 4, B and C). Among these, 38.7% (12/31) had condensed chromosomes and 22.6% had an uneven distribution of chromatin (7/31). The remainder had an interphase-like nucleus (12/31, 38.7%).
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At the expected MII stage (17 h after in vitro maturation), we examined 59 manipulated oocytes. Among these, 29 were from the group that had extruded the first polar bodies. More than half of these oocytes were arrested at the condensed chromosome stage (Fig. 4F; 16/29, 55.2% and Table 2), presumably MII-like, while 37.9% (11/29) contained an uneven distribution of chromatin (Fig. 4G) or an interphase-like nucleus (Fig. 4H; 2/29, 6.9%). For the 30 observed oocytes that did not extrude their first PB during maturation, 40% of them (12/30) were arrested at the condensed chromosome stage, while 9 (9/30, 30%) showed an uneven distribution of chromatin, and another 9 were arrested with interphase-like nuclei (9/30, 30%).
Microtubule Networks During In Vitro Maturation
Immunohistochemistry revealed that, at both the expected MI and MII stages, bipolar spindle-like microtubule networks were formed in reconstructed oocytes in the vicinity of the condensed somatic cell chromosomes in the enucleated GV ooplasts (Fig. 5). However, at both stages, the somatic chromosomes did not align properly in the newly established microtubule networks or spindles.
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Nuclear Progression after Activation
To convert a 4C and 2N somatic nucleus into 1C and 1N, reconstructed oocytes that had extruded the first pseudo-PB require activation to induce the second meiotic division. However, activating the reconstructed oocytes consistently caused fragmentation (data not shown). When we included CCB in the activation medium, oocyte survival was improved. The inclusion of CCB in the activation medium prevented the extrusion of the second PB, but we were still able to observe the nuclear separation within the activated oocytes. Activated control oocytes divided their chromosomes into two sets in the presence of CCB, and each was observed to form two PN with visible pronuclear membrane 6 h postactivation (45/45, 100%; Fig. 6, A and B and Table 3). In the manipulated oocytes, the pseudopronuclear membrane was not visible under light microscopy (Fig. 6C). However, pseudo-PNs were visualized under an epifluorescence microscope after DNA staining. In oocytes that extruded the first pseudo-PBs, more than half (63.6%; 21/33, Fig. 6E) had two pseudo-PNs, with the remainder having either a single pseudo-PN (Fig. 6D; 18.2%) or uneven condensed chromosomes (Fig. 6F; 18.2%). Interestingly, for those oocytes that did not extrude the first pseudo-PBs, nuclear DNA still separated in 44.4% of them (Table 3). The nuclear configuration of the remaining activated oocytes without pseudo-PBs was a single pseudo-PN (8/27, 29.6%) or an uneven distribution of chromatin (7/27, 26.0%; Table 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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However, we observed an accelerated rate of pseudo-PB extrusion in these somatic cell-ooplasm complexes. In control oocytes, the first PB extrusion was about 9–10 h following the removal of maturation inhibitor, while it took the oocytes with somatic nuclei only 5–7 h to extrude their pseudo-PBs. It is unclear what caused such a hastening of the cell-cycle progression. Nevertheless, the acceleration of PB extrusion has also been found in hybrids of MI oocytes with zygotes, presumably because the MI oocyte supplied the maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) to the zygotes [20]. Similarly, because a large percentage of the synchronized somatic cells used here were in metaphase, it is likely that they provided the MPF/MAPK that induced the enucleated GV oocytes to enter MI at a faster pace.
In the present study, we observed abnormally large pseudo-PBs in about one third of our reconstructed oocytes as compared with PB from control oocytes after 17 h of in vitro maturation. We surmise that this resulted from a lack of proper cellular polarity in the manipulated oocytes. In order for the oocytes to have an asymmetrical division resulting in a normal-sized PB, the MI spindle has to be physically located at the periphery of the oocyte. It has been shown in the mouse that the spindle, as well as the chromosomes, is physically attached to the oocyte cortex [21]. Specifically, the proximal and distal poles of the spindle are attached to the cortex by microfilaments and nonspindle microtubules, respectively. Furthermore, the chromosomes are not only secured to the cortex by the spindles, they are also attached to the cortex by elements such as actin cable and nonspindle microtubules [21, 22]. As noted, the removal of the GV and introduction of a somatic nucleus hastened the cell-cycle progression and, thus, likely there was insufficient time for some of the somatic nuclei to establish these complex connections in the enucleated GV oocytes. This would result in cytokinesis occurring while the somatic nuclei were still in the process of migrating to the cortex. These events would lead to the partially asymmetrical cell division, resulting in the generation of larger pseudo-PBs. Large PBs have also been observed in mos-/- knockout mice, where approximately 20% of matured oocytes have large PBs [23]. Mos is known to activate the MAPK pathway, which plays an important role in cytoskeleton changes during meiotic maturation [24, 25]. Both Mos and MAPK are found in the nucleus as well as in the cytoplasm of oocytes. The removal of the GV nucleus and a small amount of associated cytoplasm during enucleation in the present study likely reduced the total amount of Mos/MAPK in the oocytes and slowed the processes of polarization, which requires cytoskeletal changes. This would also contribute to the formation of large pseudo-PBs.
In the present study, about half of the reconstructed oocytes that successfully extruded a pseudo-PB were arrested at the condensed chromosome stage after 17 h of in vitro maturation. These oocytes were possibly at the preprogrammed MII arrest. This indicates that oocytes with somatic nuclei were able to progress through the complex meiotic division cycle. This was confirmed by our immunohistochemical staining of the microtubules where bipolar spindle-like structures were formed in the reconstructed oocytes at the expected stage of MII. In our study, the bipolar spindle-like microtubule networks also formed around the transferred chromosomes and separated the somatic cell chromosomes by releasing pseudo-PBs. Therefore, the use of G2/M somatic cells together with GV cytoplasm improved the ability of somatic cells to undergo meiotic division. However, because of the scattering of somatic chromosomes on the microtubule network, as revealed by immunohistochemical staining of the reconstructed oocytes, it is likely that errors in chromosome separation occurred in most oocytes after extrusion of the first pseudo-PB. Even though the use of CCB prevented the extrusion of the second PB during oocyte activation, we were able to observe the second meiosis-like division with nuclear stains in the reconstructed oocytes. The fact that division of the nuclei occurred in nearly 64% of oocytes that extruded the first pseudo-PBs suggested that the chromosomes were attached to the spindles despite a lack of complete chromosome condensation in some oocytes. Additionally, the fact that the pronuclear membrane was absent in these oocytes suggests that the removed GV materials may be important for the formation of the pronuclear membrane.
To date, few studies have clarified the details of the relationships between the nuclear configuration of somatic cells and their recipient GV ooplasts throughout meiosis during somatic haploidization. In the present study, we showed that a large percentage of oocytes containing G2/M somatic cells progressed through the first meiotic division and were arrested at an MII-like stage, suggesting communication of cell-cycle control factors between the somatic nuclei and the oocytes' cytoplasm. In summary, we have significantly improved the rates of first pseudo-PB extrusion and MII arrest compared with prior studies and provided detailed nuclear progression analysis, including the microtubule dynamics of haploidization. The information provided here is valuable for further improvement of this newly emerged challenging technology.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received: 14 June 2003.
First decision: 10 July 2003.
Accepted: 7 November 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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