Human Reproduction, Vol. 15, No. 9, 1997-2002,
September 2000
© 2000 European Society of Human Reproduction and Embryology
In-vitro development of mouse zygotes following reconstruction by sequential transfer of germinal vesicles and haploid pronuclei
Department of Obstetrics and Gynecology, New York University School of Medicine, New York, USA
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Abstract |
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We evaluated whether mouse oocytes reconstructed by germinal vesicle (GV) transfer can develop to blastocyst stage. The oocytes were artificially activated with sequential treatment of A23187 and anisomycin; fertilization was then established by transfer or exchange of pronuclei with those of zygotes fertilized in vivo. Type 1 zygotes were constructed by placing the male haploid pronucleus from a zygote into the cytoplasm of an oocyte that underwent GV transfer, in-vitro maturation and activation; for type 2 zygotes, the female pronucleus was removed from a zygote and replaced with the female pronucleus of an oocyte subjected to GV transfer, in-vitro maturation and activation. Karyotypes of activated oocytes and type 2 zygotes were also subjected to analysis. When cultured in human tubal fluid (HTF) medium, reconstructed oocytes matured and, following artificial activation, consistently developed a pronucleus with a haploid karyotype; the activation rate for this medium was two- to three-fold higher than that of oocytes cultured in M199 (87% versus 30% respectively). Following transfer of a male pronucleus, only 47% of the type 1 zygotes developed to morula or blastocyst stage and embryo morphology was poor. In contrast, 73% of the type 2 zygotes developed to morula or blastocyst stage, many even hatching, with few morphological anomalies. Normal karyotypes were observed in 88% of the type 2 zygotes analysed. These observations demonstrate that the nucleus of a mouse oocyte subjected to sequential nuclear transfer at GV and pronucleus stages is, nonetheless, capable of maturing meiotically, activating normally and supporting embryonic development to hatching blastocyst stage. In contrast, the developmental potential of the cytoplasm of such oocytes appears to be compromised by these procedures.
Key words: activation/germinal vesicle transfer/maturation/oocyte/pronucleus transfer
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Introduction |
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Germinal vesicle (GV) transfer involves the transfer of the GV of an immature mouse or human oocyte into an enucleated egg at the same cell cycle stage. The resultant heterologous oocyte then completes the first meiotic division normally, extruding the first polar body (Liu et al., 1999
; Zhang et al., 1999). Using GV transfer, one may examine whether cytoplasmic defects in certain oocytes compromise normal meiotic maturation and even attempt to `rescue' normal maturation of such genomes. However, at this time little is known about the developmental competency of reconstructed GV oocytes beyond their maturation to metaphase II. One might expect that the developmental capacity of such reconstructed oocytes might be compromised because the oocytes must be stripped of cumulus cells to permit visualization of the cell organelles for transfer and subsequently they must be matured in vitro.
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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Recovery of fully grown GV stage oocytes and zygotes
Mice were purchased from Charles River Laboratories (Wilmington, MA, USA). Two strains of mice were used: FVB/N mice (white colour coat) supplied the GV nuclei for transfer from immature oocytes and, following maturation and activation, the female pronuclei for transfer as well as male pronuclei derived from in-vivo zygotes; C57BL/6 mice (black colour coat) were used to generate enucleated ooplasm of GV-stage oocytes as well as partially enucleated cytoplasm of in-vivo zygotes (Figure 1
).
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Female mice (6–8 weeks old) were injected i.p. with 5 IU pregnant mare serum gonadotrophin (PMSG; Sigma, St Louis, MO, USA) and, 48 h later, with 5 IU human chorionic gonadotrophin (HCG; Sigma) i.p. One group of mice was killed 1 h later to collect immature oocytes at the GV stage. These oocytes were released from the ovary by puncturing the follicles with a needle and any attached cumulus cells were dissociated by repeated pipetting. Another group of mice was placed with a male immediately after the HCG injection and then killed 22 h later to harvest zygotes by opening the ampullae of the Fallopian tubes. Cumulus cells were also removed from the zygotes by a brief exposure to serum-free modified human tubal fluid (HTF) medium containing 300 IU/ml hyaluronidase (Sigma).
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) 4–6 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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Fusion of GV karyoplast and cytoplast was initiated with direct current as previously described (Liu et al., 1999
). After alignment with an AC pulse of 6–8 V for 5–10 s, fusion was achieved with electrical pulses (1.8–2.5 kV/cm DC for 50 µs) delivered by a Model 2001 Electro Cell Manipulator® (BTX Inc., San Diego, CA, USA).
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 4–5 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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In an initial study, we compared the maturation and activation rates of the reconstructed oocytes in three different culture media: S1 (IVF Science, Vero Beach, FL, USA), M199 (Sigma) and HTF (Irvine Scientific). Each medium was supplemented with 10% fetal bovine serum (FBS).
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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Influence of media on maturation and activation of GV transferred oocytes
Electrofusion, maturation and activation success rates for oocytes cultured in S1, M199 and HTF are presented in Table I
. Electrofusion rates were equivalent irrespective of the culture medium used. However, whereas only 55% of the reconstructed oocytes cultured in S1 medium extruded the first polar body, the maturation rates of reconstructed oocytes cultured in M199 and HTF were significantly higher, 78% and 83% respectively. The ability of the matured oocytes for activation also varied between the different culture media. When cultured in M199, only 30% of the matured oocytes developed a pronucleus. In contrast, activation was observed in 90% and 87% of the mature oocytes cultured in S1 and HTF respectively. In view of these results we chose to use HTF as the culture medium in all further studies.
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Cytogenetic analysis of activated, GV transfer oocytes
When activated GV transfer oocytes (n = 20) were exposed to nocodazol overnight, they all arrested at metaphase. Following oocyte spread, 75% showed a haploid complement of 20 chromosomes (Figure 4a
). A complement of 40 chromosomes was observed in three arrested oocytes presumably because the activated oocyte failed to extrude the second polar body. Fewer than 20 chromosomes were observed in only two oocytes.
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Development of in-vivo fertilized zygotes in M199, HTF, S1 and S1–S2
We also investigated the influences of culture medium on the early embryonic development of zygotes fertilized in vivo (Table II
). When cultured in M199, the zygotes displayed a significantly lower division rate than those cultured in HTF, S1 or S1-S2 (71 versus 98, 100 and 97% respectively; P < 0.05); moreover, development of virtually all of these zygotes arrested at the 2-cell stage. Zygotes cultured in HTF or S1 continued development, 51 and 70% respectively reaching morula-blastocyst stage within 5 days. The greatest rate of blastocyst development (83%) was seen with zygotes cultured in S1 for 2 days and then S2 for 3 days. In view of these data, S1–S2 medium was used in the study to assess the in-vitro development of reconstructed zygotes.
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Development of zygotes reconstructed by sequential GV and haploid pronucleus transfer
Electrofusion treatment was routinely successful for type 1 and type 2 zygotes (97 and 100% respectively). The rates of progression to 2-cell, 4–8-cell and morula–blastocyst stages of development for the type 2 zygotes were indistinguishable from those of non-manipulated zygotes (Table III
); 73 and 83% respectively of these zygotes developed into morphologically indistinguishable morulae or blastocysts with clearly visible inner cell masses (Figure 5b). However, the rates of progression for the type 1 zygotes were consistently lower at the 2-cell, 4–8-cell and morula–blastocyst stages of development (P < 0.05 compared with in-vivo or type 2 zygotes). Moreover, although the 2-cell embryos appeared normal, the morphology of the morulae and blastocysts that developed from the type 1 zygotes was poor (Figure 5a).
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Cytogenetic analysis of embryos following pronuclear transfer
The karyotypes of type 2 zygotes (n = 24) were also analysed to determine if the transfer of the female pronucleus or electrofusion procedures affected chromosome number. Two sets of a haploid complement of 20 chromosomes were observed in each pronucleus in 21 (88%) of these zygotes (Figure 4b
). Two of the remaining three zygotes had fewer chromosomes, the other displayed additional chromosomes. |
Discussion |
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We have previously reported that following GV transfer, reconstructed immature mouse oocytes complete the first meiotic division normally and arrest at metaphase II (Liu et al., 1999
). Significantly, these events occur only when a GV is transferred into an enucleated GV egg; when transferred into post-meiotic ooplasm, the GV fails to mature further. At this time we know little about the developmental competency of reconstructed GV oocytes beyond metaphase II. However, two conditions for this procedure suggest that post-fertilization development of such oocytes may be compromised. The first is that GV oocytes, i.e. immature oocytes, must be harvested and used. Thus, the oocytes are removed from the normal physiological site in the Graafian follicle prior to exposure to the peri-ovulatory gonadotrophin surge and its direct and indirect actions that normally trigger the final stages of oocyte maturation. Secondly, the oocytes must be stripped of cumulus cells; recent studies suggest that cumulus cell–oocyte contacts and interactions are important for several aspects of oocyte development and function (Eppig, 1991; Fagbohun and Downs, 1992; Eppig et al., 1993; Xia et al., 1994).
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 14–19% 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 S1–S2 media. One might expect similar results when adapting these procedures for clinical use.
Nuclear–cytoplasmic 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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1 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, New York University School of Medicine, 660 First Avenue, Fifth Floor, New York, NY 10016, USA. E-mail: kreyivf{at}yahoo.com
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Submitted on March 27, 2000; accepted on June 13, 2000.
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