Program for In vitro Fertilization, Reproductive Surgery and Infertility, New York University School of Medicine, New York, NY 10016, USA
1 To whom correspondence should be addressed at: 660 First Avenue, Fifth Floor, New York, NY 10016, USA. e-mail: kreyivf{at}yahoo.com
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Abstract |
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Key words: germinal vesicle/in-vitro maturation/metaphase II nucleus/nuclear transfer/ooplasm
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Introduction |
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Euploidy in a mature oocyte is required for embryonic viability, but does not guarantee normal embryogenesis. In recent studies with human gametes, GV transferred oocytes that matured in vitro underwent fertilization and division but most arrested early in embryogenesis (Takeuchi et al., 2001). Because of the micromanipulation steps required, oocytes that receive a transferred GV are denuded and must undergo the final stages of maturation without contacts with their cumulus cells. Significantly, denuded, in-vitro matured oocytes are much less likely to complete embryonic development than in-vivo matured oocytes or cumulus-enclosed oocytes (Nogueira et al., 2000
). In previous studies with a mouse model of GV transfer, we reported similar consequences and suggested that such poor embryonic development is the result of pronucleus (PN) formation in an incompetent recipient ooplasm (Liu et al., 2000
).
Recently, we described sequential nuclear transfer procedures that improved the embryonic developmental potential of mouse oocytes that matured in vitro. These procedures included the following stepsMII spindle transfer into an ooplast that had matured in vivo and/or PN transfer into cytoplasts from enucleated, in-vivo fertilized oocytes (Liu et al., 2000; 2001). Using these procedures embryonic development improved significantly as judged by development to morphologically normal blastocysts. The present study was designed to examine whether we could couple these procedures with GV transfer to generate live-born offspring.
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Materials and methods |
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A second group of mice were killed 1618 h after hCG to harvest metaphase II (MII) stage oocytes. A final group was mated immediately after hCG injection and killed 22 h later to collect fertilized zygotes. MII oocytes and zygotes were harvested from the ampullae of excised Fallopian tubes in hTF medium supplied with HEPES-buffered hTF medium (MhTF, Irvine Scientific). Cumulus cells were removed by briefly exposing MII oocytes and zygotes to serum-free, MhTF medium containing 300 IU/ml hyaluronidase (Sigma). Collected oocytes were rinsed in three washes of MhTF prior to experimentation.
Nuclear transfer
The micromanipulation procedures for GV, MII nucleus and pronuclear transfer have been detailed elsewhere (Liu et al., 1999; 2000). Briefly, oocytes at GV, MII and PN stages are placed in a drop of MhTF containing 10% FCS and cytochalasin B (7.5 µg/ml; Sigma) for 15 min. The zona pellucida adjacent to the nucleus is slit with a sharp needle to facilitate the insertion of a transfer pipette to aspirate the nucleus and surrounding cytoplasm gently and slowly without rupturing the oolemma. The karyoplast is then transferred to the perivitelline space of an enucleated recipient oocyte. Fusion between the karyoplast and cytoplast takes place in a fusion chamber filled with medium (0.3 mol/l mannitol, 0.1 mmol/l CaCI2 and 0.05 mmol/l MgSO4 in H2O) and is initiated by a single pulse of direct current (1.82.5 kV/cm DC for 50 µs). Fused GV stage oocytes are placed in hTF drops for 1618 h in order to complete the first meiotic division. Fused MII stage oocytes are transferred to new HTF drops for 30 min prior to artificial activation or insemination. Fused reconstructed zygotes are removed to drops of G1 medium (IVF Science, Vitrolife AB, Gothenburg, Sweden) for overnight culture.
Artificial activation
MII stage oocytes were placed in PBS containing 3 µm ionophore A23187 (Sigma) for 5 min at 22°C, washed twice with MhTF, and then transferred to hTF supplemented with 10% FCS and cycloheximide (5 µg/ml; Sigma) for 45 h. Activated oocytes displayed a pronucleus (PN) and extruded second polar body at this time (Liu et al 2000).
Embryo transfer
Reconstructed zygotes were cultured in G1 medium for 1820 h in vitro. Those embryos that developed to the 2-cell stage, were surgically transferred into oviduct of day 1 pseudopregnant CD-1 foster mice as described previously (Liu et al., 2001). Pregnancy was checked by palpation at 11 days and thereafter. Whenever the foster mother carried a single fetus by palpation, Caesarean section was performed to deliver that pup at 20 days post coitus.
Experimental design
Four groups of zygotes were reconstructed to evaluate the development potential of a MII nucleus that had derived from a transferred GV. For the first groupGV-FPN zygotethe MII nucleus was activated artificially and the resultant PN transferred to the cytoplasm of an in-vivo fertilized oocyte from which the female PN had been removed (Figure 1a). In the second groupGV-S-FPN zygotethe MII nucleus was transferred into an enucleated oocyte that had matured in vivo. After artificial activation, the resultant PN was transferred into cytoplasm of an in-vivo fertilized oocyte from which the female PN had been removed (Figure 1b). In the third groupGV-S-MPN zygotethe MII nucleus was transferred into an enucleated oocyte that had matured in vivo. After artificial activation, the reconstructed oocyte was fertilized by fusing a male PN from an in-vivo zygote (Figure 1c). For the fourth groupGV-S zygotethe MII spindle was removed and transferred to an enucleated oocyte that had matured in vivo. This reconstructed oocyte was inseminated during a 6 h incubation in a droplet containing sperm harvested from the epididymis (Figure 1d).
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Results |
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The efficiency for each step in zygote reconstruction for the GV-FPN, GV-S-FPN, GV-S-MPN and GV-S groups are presented in Table I. No significant procedural variation was noted between groups and a 90% success rate was noted at each stage.
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Of the 49 GV-S-MPN zygotes created, 98% cleaved after overnight culture and were then transplanted to oviducts of foster mice. Three mice became pregnant and delivered six live offspring.
Of the 57 GV-S zygotes created, 66% were two cells after overnight culture and were then transplanted to oviducts of foster mice. Four mice became pregnant and delivered six live offspring.
After GV transfer and sequential nuclear transfer, 20 live offspring were born with a weight range from 1.92.1 g. Two pups were cannibalized by their foster mothers and two others died due to lack of breastfeeding. The remaining 16 pups grew normally to adulthood. The six male mice weighed 2225 g at 35 days of age; the 10 female mice weighed 1923 g. We monitored reproductive function in the six adult mice that originated from GV-S zygotes. All six (two male and four female) were fertile (Figure 3) and their offspring, in turn, also displayed normal fertility.
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Discussion |
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Live-birth outcomes have been reported previously following GV transfer in mice (Kono et al., 1996; Bao et al., 2000
). Significantly, these studies also employed sequential transfer following GV transfer; following blastocyst transfer a 30% live birth rate was observed. Live births have also been noted following GV transfer in rabbits (Li et al., 2001
) and cows (Kuwayama, 2002
); however, in these species the oocytes reconstructed by GV transfer were fertilized directly and the embryos transferred to recipient uteri. The resultant live birth rate in the rabbit was extremely low, <3% of the embryos resulting in a live birth; unfortunately the birth rate for the cow was not reported.
In previous studies with CB6F1 mouse oocytes, we reported that GV transfer resulted in reconstructed oocytes that, following artificial activation and male PN transfer, generated poor quality embryos that failed to generate a live birth (Liu et al., 2000). However, in order to perform GV transfer, the immature oocytes were denuded of their surrounding cumulus cells and subsequent studies in denuded oocytes not subjected to GV transfer produced similar findings (Liu et al., 2001
). Further studies using MII spindle transfer in denuded oocytes suggested that the source of the developmental incompetence originated in the cytoplasm and not the nucleus of oocytes reconstructed by GV transfer (Liu et al., 2001
). The present results in the GV-S-MPN group provide the most convincing evidence to support this view. Live births were only observed with embryos that developed with a maternal PN that formed following MII spindle transfer to in-vivo matured ooplasm. Significantly, similar birth rates were observed when fertilization was achieved by transfer of the female or male PN or by normal fertilization.
Another group of embryos (GV-FPN) were created by exchanging the PN that formed following GV transfer and artificial activation with the maternal PN of an in-vivo fertilized oocyte. Although this procedure improved embryo quality, no live births were observed following oviduct transfer. These observations point to cytoplasmic-induced changes in long-term nuclear function that normally occur during oocyte activation but do not take place in the ooplasm that matured in vitro. As a result the nucleus completes the second meiotic division as a euploid PN incapable of supporting long-term embryonic development even when placed in an in-vivo fertilized oocyte. As above, these effects can be circumvented by MII spindle transfer to ensure that activation and PN formation take place in cytoplasm that had completed maturation in vivo. Epigenetic mechanisms such as DNA methylation have been proposed to explain how cytoplasmic factors may influence long term nuclear function and have been demonstrated to take place during fertilization and early embryonic development (Renard et al., 1994).
In a series of follow-up studies we determined whether GV, MII spindle and/or PN transfer had any long-term effect on the resultant offspring. Not only were their birth size and growth rate normal, but these offspring, both male and female, displayed normal mating behaviour as adults. These matings resulted in normal offspring who were also fertile after reaching adulthood. Such a multi-generational follow-up is important considering the long-term safety concerns associated with these procedures and should be required for any future study involving nuclear transfer.
It is important to reiterate at this point that all of our studies were performed exclusively with oocytes from CB6/F1 mice cultured in hTF media supplemented with fetal calf serum. In contrast, Schroeder and Eppig (1984) noted that denuded oocytes of B6SJLF1 mice which had matured in vitro and were fertilized in MEM media similarly supplemented were competent to support embryonic growth to term and deliver live births even when matured in vitro. Whether this dichotomy is due to strain of mouse or culture system is currently under investigation.
GV transfer has been proposed as a potential procedure to circumvent the age-related increase in aneuploidy observed in the human female (Zhang et al., 1999). The concept is that by transferring the GV from an older womens oocyte into a donated ooplast from a young woman may reduce the aneuploidy rate in the first meiotic division. However, as with mouse oocytes, immature human oocytes must be stripped of their cumulus cells in order to perform the microsurgical transfer. Although cumulus-enclosed oocytes that mature in vitro are able to fertilize and support embryonic development to term (Chian et al., 1999
; Cha et al., 2000
; Child et al., 2002
), significantly lower rates of maturation and early embryonic development have been noted when immature, denuded, human oocytes are compared with cumulus-enclosed oocytes (Hwang et al., 2000
; Kim et al., 2000
; Nogueira et al., 2000
). Significantly, no births have been reported when embryos developing from denuded oocytes were transferred to the uterus. Thus, the impact of cumulusoocyte interactions on ooplasmic development may occur in the human as well as in the mouse. Several approaches may be taken to overcome this cytoplasmic effect. As reported here, sequential nuclear transfer could be used. However, this approach would require two different types of donated oocytes, immature oocytes for GV transfer and mature oocytes for MII spindle transfera clinically impractical arrangement. Another option would be to harvest immature oocytes immediately prior to GV breakdown; however, we must first learn when the important changes in cytoplasmic make-up take place during the final stages of maturation. A final possible option is to supplement the ooplasm of the reconstructed oocyte following GV transfer with the injection of ooplasm from an oocyte that matured normally in vivo. Such injections have been successful in treating women whose oocytes result in extensively fragmented embryos (Cohen et al., 1998
; Lanzendorf et al., 1999
). Although each of these options presents major problems including multiple oocyte donors, future work may uncover a more practical solution to this problem. Nonetheless, GV transfer, like all nuclear transfer procedures, is an important research tool to investigate the relationships between the ooplasm and maternal genome that are necessary for normal maturation, fertilization and embryonic and fetal development. Such analyses can then be used to develop models for molecular approaches that will identify the cellular factors underlying these relationships.
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References |
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Submitted on March 6, 2003; accepted on May 21, 2003.