Department of Obstetrics and Gynecology, New York University School of Medicine, New York, USA
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Abstract |
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Key words: activation/germinal vesicle transfer/maturation/oocyte/pronucleus transfer
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Introduction |
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There are many experimental options available to assess the embryonic potential of experimentally manipulated oocytes. Normal embryonic development and live offspring have been observed when in-vivo matured, metaphase II oocytes are artificially activated by ionophore-protein synthesis inhibition prior to transfer of a male pronucleus (Hagemann et al., 1995). Zygote reconstruction by pronucleus transfer, i.e. removing a haploid male or female pronucleus from a zygote and replacing it with a comparable pronucleus from a different zygote, has also resulted in normal offspring (Barton et al., 1984
; Surani et al., 1984
; Hagemann et al., 1995
). In this study we have incorporated these approaches into a sequential nuclear transfer procedure and describe early embryonic development when an oocyte's genome is subjected to GV transfer followed by pronucleus transfer. The long-term goal of such a procedure is to improve meiotic competence through GV transfer and to assess the developmental competence of the transferred GV genome through pronucleus transfer.
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Materials and methods |
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GV transfer
GV oocytes were incubated in HTF medium (Irvine Scientific, Santa Anna, CA, USA) supplemented with 10% fetal calf serum (FCS; HyClone, Logan, UT, USA) and 3-isobutyl-1-methylxanthine (IBMX; 50 µg/ml, Sigma) 46 h prior to micromanipulation. Oocytes of white (n = 10) and black (n = 10) mice were placed in a micro-droplet of HEPES-buffered HTF containing 10% FCS and cytochalasin B (7.5 µg/ml; Sigma) for 30 min at room temperature, and then the zona of each oocyte was opened with a sharp needle to facilitate GV removal by a pipette. The GV of each black mouse oocyte was removed and discarded. The GV from the oocytes of the white mice were then transferred to the perivitelline cavity of the enucleated oocytes of the black mice (Figure 2).
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Maturation and artificial activation of GV transferred oocytes
Maturation of GV transferred oocytes was evaluated after 14 h culture in vitro under 5% CO2, 37°C. Oocytes displaying a polar body were selected for further experimentation. Matured reconstructed oocytes were activated artificially as previously described except that anisomycin was used to inhibit protein synthesis (Hagemann et al., 1995). Preliminary studies indicated that both of the synthesis inhibitors anisomycin (2.5 µg/ml) and cycloheximide (5 µg/ml) were similarly effective in such a protocol. Oocytes were placed in phosphate buffered saline (PBS) containing 3 µm A23187 (Sigma) for 5 min at room temperature, washed in 2 ml HTF, and then cultured in HTF supplemented with 10% FCS and anisomycin for 45 h. The reconstituted oocytes were monitored 4 h later for activation as indicated by the presence of a female pronucleus (Figure 3a
).
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Haploid pronucleus transfer
Zygotes were reconstructed using the same micromanipulation and electrofusion procedures that were used for GV transfer. The pronuclei were identified, the female pronucleus being in close proximity to the second polar body (Figure 3b), and removed by an aspiration pipette. Two types of zygote reconstruction were performed. Type 1 zygotes were constructed by placing the male haploid nucleus of an in-vivo zygote into the cytoplasm of an oocyte that underwent GV transfer, in-vivo maturation and activation; type 2 zygotes were constructed by removing the female pronucleus from an in-vivo zygote and replacing it with the female haploid pronucleus of an oocyte subjected to GV transfer and activation (Figure 1
).
Cytogenetic analysis of activated oocytes and reconstructed zygotes
Activated oocytes with a single pronucleus and type 2 zygotes were cultured overnight at 37°C in HTF containing nocodazol (1 µg/ml; Sigma) to arrest the cells at metaphase. Cells were fixed for cytogenetic analysis (Tarkowski, 1966). Briefly, each oocyte or zygote was transferred into a 1% hypotonic trisodium citrate solution for 10 min and then fixed with methanol:acetic acid (3:1) on a clean glass slide. The chromosome spreads were then air-dried and stained with DAPI (3 ng/ml phosphate buffer) and then covered with thin glass slide. The number and the structure of the chromosomes were determined immediately under fluorescence microscopy.
Data analysis
Data were analysed using the 2 test with significance at P < 0.05.
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Results |
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Discussion |
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To assess the embryonic competency of reconstructed GV oocytes, it is necessary to stimulate them to complete the second meiotic division. Normally this is accomplished by the fertilizing spermatozoa. However, we experienced considerable difficulty fertilizing even in-vivo matured mouse oocytes either by exposure to spermatozoa in vitro or by intracytoplasmic sperm injection (ICSI) using piezo injector units. As a result, we artificially activated the reconstructed oocytes using brief Ca2+ ionophore treatment and protein synthesis inhibition. Previous studies report normal embryonic development and live offspring when in-vivo matured oocytes are activated in this fashion prior to transfer of a male pronucleus (Hagemann et al., 1995).
The cellular mechanisms underlying oocyte activation have been studied extensively in mice since previous observations (Siracusa et al., 1978) that the metaphase II oocyte contains protein factor(s) that maintain the meiotic block at this stage. Meiosis-promoting factor activity is maintained at a high level by a continuous equilibrium between cyclin B synthesis and degradation that is stabilized by C-mos activation of MAP kinase pathways (Kubiak et al., 1993
; Colledge et al., 1994
; Araki et al., 1996
; Verlhac et al., 1996
). The earliest events that occur following sperm entry into the oocyte are an increase in intracellular Ca2+ concentrations followed by a series of oscillations in intracellular free Ca2+ concentrations (Shen, 1992
; Miyazaki, 1995
; Swann and Lai, 1997
). Other events occur subsequently, including declines in mitogen-activated protein kinase (Sun et al., 1998
) and cdc2/cyclin B kinase activities (Moos et al., 1996
); significantly such declines can also be induced by protein synthesis inhibition. A similar pattern of intracellular events is generated by the sequential A23187 + anisomycin activation procedure that we employed. In fact, the high activation rate that we observed in reconstructed oocytes is comparable to that previously reported (Hagemann et al., 1995
) for oocytes that matured in vivo. Significantly, the activation rate is only 60% less following either ionophore or protein synthesis inhibitor treatment alone (Hagemann et al., 1995
). Activation of human oocytes by Ca2+ ionophore or protein synthesis inhibition has also been reported (Winston et al., 1991
; Balakier and Casper, 1993
).
The 90% success rates for activation and pronuclear transfer that we experienced in this study have made it possible to study sufficiently large numbers of zygotes and reach meaningful conclusions about embryonic competence. Cytogenetic analyses of the female genome following oocyte reconstruction, maturation and activation consistently revealed normal chromosome numbers in 75% of the eggs tested; in the remainder 15% failed to extrude the second polar body and 10% were aneuploid with fewer than 20 chromosomes. However, an even higher percentage (88%) were euploid after these procedures and subsequent pronuclear transfer to an in-vivo matured zygote. Taken together, these observations suggest that few anomalies in ploidy are associated with these approaches. Previous work (O'Neill et al., 1991) also reported no significant increases in chromosome segregation errors following strontium-induced parthenogenesis. However, this author did report a 1419% rate of aneuploidy in ethanol-induced single-pronucleus parthenogenones derived from metaphase II oocytes that matured in vivo (O'Neill et al., 1989
).
As part of these studies we assessed whether tissue culture media had any effect on the development of the reconstructed oocytes and zygotes. Recent studies have described that stage-specific media can optimize embryo growth and development (Gardner, 1998; Bavister, 1999
). Our results clearly indicate that the success of these transfer procedures can be influenced by the choice of culture media. Maturation, activation rates were significantly higher in HTF and S1 medium than in M199, a surprising result considering the extensive use of M199 in studies on oocyte maturation in vitro (Tonetta et al., 1988
; Yang et al., 1993
; Zhang et al., 1995
; Liu and Moor, 1997
). Moreover, M199 failed to support embryo development of zygotes fertilized in vivo to blastocyst stage. Not surprisingly, the highest rate of growth to blastocyst stage was observed following sequential exposure to S1S2 media. One might expect similar results when adapting these procedures for clinical use.
Nuclearcytoplasmic interactions are thought to play an important role in oocyte development and maturation. Although more extensive studies are needed, recent chromosome analyses have suggested that GV transfer might be a means of rescuing genomes from the maternal age-related increase in chromosome non-disjunction during the first meiotic division in human oocytes (Zhang et al., 1999). However, if this rescue is to be relevant the reconstructed oocytes must be capable of fertilization and subsequent embryonic growth. The present observations suggest that such is the case in mice. Following transfer to and maturation and artificial activation in a heteroplasmic environment, the genome of the reconstructed oocyte undergoes normal chromosome segregation and division and then generates a schedule of gene expression and differentiation necessary for embryonic development through the hatching blastocyst stage. In fact, a second transfer to a third cytoplasmic milieu, one that had the opportunity to mature completely in vivo, actually results in more active growth and development. However, although able to support activation and embryonic development despite extensive physical manipulation and exposure to harsh chemicals, including a temporary inhibition of protein synthesis, the cytoplasm of the reconstructed oocyte does appear to be affected adversely resulting in the generation of poor quality embryos that may have difficulty establishing a viable pregnancy. We are currently assessing the potential of type 1 and type 2 zygotes to support late embryonic development through to birth. We are also assessing whether media supplements influence the functional competence of the ooplasm of eggs matured in vitro. One candidate would be oestradiol, a steroid reported to improve cytoplasmic maturation of immature human oocytes (Tesarik and Mendoza, 1995
).
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Notes |
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References |
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Submitted on March 27, 2000; accepted on June 13, 2000.