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BOR - Papers in Press, published online ahead of print October 4, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.004770
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Biology of Reproduction 67, 1790-1795 (2002)
DOI: 10.1095/biolreprod.102.004770 © 2002 Society for the Study of Reproduction, Inc.

Embryo

Serial Pronuclear Transfer Increases the Developmental Potential of In Vitro-Matured Oocytes in Mouse Cloning1

Björn Heindryckx2,a, Andreï Rybouchkina, Josiane Van der Elsta, and Marc Dhonta

a Infertility Center, Department of Obstetrics and Gynaecology, Ghent University Hospital, 9000 Ghent, Belgium


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In vitro-matured germinal vesicle oocytes are an interesting source of cytoplast recipients in both animal and human nuclear transfer (NT) experiments. We investigated two technical aspects that might improve the developmental potential of nuclear transfer mouse embryos constructed from in vitro-matured germinal vesicle oocytes. In a first step, the effect of two maturation media on the embryonic development of NT embryos originating from in vitro-matured oocytes was compared. Supplementation of the oocyte maturation medium with serum and gonadotrophins improved the developmental rate of NT embryos constructed from in vitro-matured oocytes, but it was still inferior to that obtained with in vivo-matured metaphase II (MII) oocytes. Second, we investigated the effect of serial pronuclear transfer from NT zygotes originating from both in vitro- and in vivo-matured oocytes to in vivo-fertilized zygotic cytoplasts. Blastocyst quality was evaluated by counting nuclei from trophectoderm and inner cell mass cells using a differential staining. Sequential pronuclear transfer significantly improved the blastocyst formation rate of NT embryos originating from in vitro-matured oocytes up to the rate obtained with in vivo-matured MII oocytes. We conclude that the developmental potential of NT embryos constructed from in vitro-matured oocytes can be optimized by serial pronuclear transfer to in vivo-produced zygotic cytoplasts.

embryo


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
For somatic nuclear transfer (NT) experiments, mature oocytes arrested at metaphase of the second meiotic division (MII) have become the recipient cells of choice. Many cloned animals have been created by transfer of differentiated nuclei at G0/G1 or M phase of the cell cycle into MII oocytes [1–11]. Although it has been reported that enucleated zygotes and blastomeres of two-cell embryos might be the best recipients of donor nuclei [12], it has been suggested that the use of MII oocytes may improve reprogramming of the donor genetic material because of the induction of nuclear envelope breakdown and premature chromosome condensation, thus exposing the donor chromatin to maternally derived oocyte factors necessary for early development [11, 13, 14].

However, there are limitations in using in vivo-matured oocytes as recipients in cloning experiments. There is a lack of availability in some animal species in which MII oocytes can only be recovered under expensive surgical conditions and where immature germinal vesicle (GV) oocytes are used as an alternative. In humans, mature MII oocytes are retrieved as part of an infertility treatment with in vitro fertilization and are exclusively used for this purpose. Immature human GV oocytes are a potential source of oocytes for scientific research provided this is approved by the ethical committee and the patient has given informed consent. Because immature oocytes are abundantly available from slaughterhouses [15], in vitro-matured oocytes have been used for embryo cloning in cattle and sheep [16–18].

Few controlled studies have been carried out, but there is general consensus that the developmental potential of in vitro-matured oocytes is compromised compared with in vivo-matured oocytes [19]. Several studies have shown that oocyte maturation conditions can affect embryonic development of in vitro fertilization (IVF)-derived embryos. It has been clearly demonstrated that, in ideal conditions, in vitro maturation (IVM) has no effect on subsequent developmental potential of mouse IVF embryos [20], but other studies indicate that in vitro-matured mouse oocytes are more sensitive to suboptimal culture conditions than in vivo-matured oocytes [21]. Moreover, embryonic development of IVF embryos is affected by the choice of oocyte maturation media in mouse [22, 23] and bovine [24]. It is still unknown whether development of NT embryos, starting from immature oocytes, can also be affected by the choice of the oocyte maturation medium.

For NT embryos, development to the blastocyst stage tended to be higher with in vivo-derived oocytes than with in vitro-matured oocytes in sheep NT experiments [25, 26] and was significantly better when in vivo-matured instead of in vitro-matured oocytes were used as recipients in pig NT experiments [27]. Serial nuclear transfer, passing the donor nucleus twice through a mature cytoplasm, was found to improve development of cloned pig and mouse embryos [28, 29] starting from in vivo-matured oocytes in cloning experiments. This led us to the idea that serial transfer of donor nuclei from reconstructed NT zygotes from in vitro-matured oocytes to in vivo-fertilized zygote cytoplasts may be a way to promote development of NT embryos starting from immature oocytes.

In the present study, we investigated two experimental ways to circumvent the possible decreased developmental potential related to the use of in vitro-matured oocytes as recipients in mouse NT. In a first set of experiments, we investigated whether the type of oocyte in vitro-maturation medium can influence preimplantation development of mouse NT embryos reconstructed from immature GV oocytes. A comparison was made with development of NT embryos derived from in vivo-matured oocytes. In a second set of experiments, we examined the effect of serial NT of pronuclei from reconstructed NT zygotes to in vivo-derived zygotic cytoplasts. We compared serial transfer of pronuclei (PN) of reconstructed NT embryos originating from in vivo- versus in vitro-matured oocytes. Finally, quality analysis of the obtained NT blastocysts in the different reconstructed groups was done using differential staining of trophectoderm (TE) and inner cell mass (ICM).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

All mice were treated according to the guidelines of the Laboratory Animal Ethical Committee of the Ghent University Hospital. Mice were kept under controlled temperature and lighting conditions during experiments and were given water and food ad libitum.

Recipient Oocytes and Donor Cell Nuclei

Female B6D2 F1 mice 7–14 wk of age (hybrid of inbred strains C57BL/6J x DBA/2J), purchased from Iffa Credo (Brussels, Belgium), were stimulated with 10 IU eCG (Folligon, Intervet, The Netherlands) followed by 10 IU of hCG (Chorulon; Intervet) 48 h later.

In vivo-matured metaphase II oocytes were recovered 12–13 h after hCG injection while immature GV oocytes were collected by puncturing ovarian follicles at 3 h after hCG injection. Healthy-looking GV cumulus-oocyte complexes with a round and centrally located oocyte were collected and matured in either {alpha}-minimum essential medium ({alpha}-MEM; Gibco BRL, Life Technologies, Merelbeke, Belgium) supplemented with 4 mg/ml BSA (Gibco BRL), hereafter called {alpha}-MEM1, or in {alpha}-MEM supplemented with 100 m IU/ml recombinant FSH (rFSH; Puregon, Organon, Oss, The Netherlands), 5% heat-inactivated fetal bovine serum (FBS; Gibco BRL), 5 µg/ml insulin-10 µg/ml transferrin-5 µg/ml selenium (Boehringer Mannheim, Mannheim, Germany), and 10 IU/ml hCG, hereafter called {alpha}-MEM2. After 14–15 h of culture, maturation status of GV oocytes was evaluated. Oocytes displaying a first polar body (PB) were selected for further experiments. Cumulus cells were dispersed of both in vitro- and in vivo-matured MII oocytes by treatment with 200 IU/ml hyaluronidase in KSOM-Hepes, prepared in the laboratory. The cumulus cells originating from in vivo-matured oocytes served as nuclei donors and were washed two times by centrifugation at 3200 rpm for 10 sec and were finally kept in KSOM-Hepes in 5-µl droplets under mineral oil in the manipulation dish at room temperature.

Nuclear Transfer Procedure and Embryo Culture

The nuclear transfer procedure was carried out as described in Heindryckx et al. [30].

Briefly, after a tangential slit was made in the zona pellicuda with a sharp needle, the oocyte chromosome-spindle complex, visible as a translucent region in the ooplasm, was removed from MII oocytes, incubated in KSOM-Hepes supplemented with 1 µg/ml cytochalasine D. Injection of cumulus cell nuclei was carried out in KSOM-Hepes plus 20% (v/v) FBS using a blunt pipette (6- to 7-µm inner diameter) on an inverted microscope stage cooled to 15–17°C. Up to 3 h after injection, reconstructed oocytes were activated in Ca2+-free KSOM, prepared in the laboratory, containing 10 mM SrCl2 and 2 µg/ml cytochalasine D for 6 h. Sr2+-ions were used to induce activation and cytochalasine D to prevent extrusion of the second PB in order to maintain a correct ploidy. Following activation, oocytes showing two pronuclei were cultured in KSOM [31] prepared in the laboratory for the first 60–72 h postactivation and then embryos were transferred to G2 medium (Vitrolife, Götheburg, Sweden). In vitro- and in vivo-matured nonmanipulated parthenogenetically activated oocytes served as controls for culture conditions. Embryo development was assessed at 24 h (two-cell), 48 h (three + four cell), 72 h (morula/early blastocyst), and 96 h (blastocyst) postactivation time.

Serial NT

For the serial NT experiments, B6D2 F1 female mice were induced to superovulate and to be fertilized to obtain in vivo-derived zygotic cytoplasts. Immediately after hCG injection, they were mated to a CD1 male and then killed 19 h later to harvest zygotes by opening the ampullae of the excised oviducts. After removal of cumulus cells by brief exposure to hyaluronidase, fertilized zygotes showing two PN were enucleated in KSOM-Hepes supplemented with 2 µg/ml cytochalasine D and 1 µg/ml nocodazole using a 25-µm glass pipette by aspirating both PN (and the second PB).

In a first setting, serial nuclear transfer was done from an in vivo-matured oocyte to an in vivo-produced zygotic cytoplast. Karyoplasts containing the two PN of NT embryos originating from parthenogenetically activated in vivo-matured MII oocytes after NT (MII-NT) were aspirated in KSOM-Hepes plus 2 µg/ml cytochalasine D and 1 µg/ml nocodazole and were transferred into the perivitelline space of previously enucleated fertilized zygotes. Fusion of the PN karyoplasts and the zygote cytoplasts was induced by application of two direct-current pulses of 60 V for 40 µsec delivered by a Model 830 Electro Cell Manipulator (Merck-Eurolab, Leuven, Belgium) after manual alignment. Couplets of fused PN karyoplasts and zygote cytoplasts were then washed and cultured in KSOM/G2 with follow-up of embryonic development (Fig. 1, MII-SNT). As a procedure control, NT embryos originating from in vivo-matured MII oocytes without serial transfer were put in culture in KSOM/G2 (Fig. 1, MII-NT). Nonmanipulated parthenogenetically activated in vivo-matured MII oocytes served as controls for culture environment (Fig.1, MII-control).



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  		FIG. 1. Construction of NT and serial NT embryos starting from both in vitro- and in vivo-matured oocytes. After the NT procedure and artificial activation, one part of the NT zygotes was kept for culture (IVM MII-NT: NT zygote originating from an in vitro-matured oocyte; MII-NT: NT zygote originating from an in vivo-matured MII oocyte) while the rest underwent a serial NT to in vivo-produced zygotic cytoplasts (IVM MII-SNT: NT zygote originating from an in vitro-matured oocyte that underwent a second transfer of PN to in vivo-produced enucleated zygotes; MII-SNT: NT zygote originating from an in vivo-matured MII oocyte that underwent a second transfer of PN to in vivo-produced enucleated zygotes). Parthenogenetically activated in vitro-matured oocytes (GV-control) and in vivo-matured oocytes (MII-control) served as controls for culture conditions. All embryos were cultured in KSOM/G2 media and daily follow-up to the blastocyst stage was done

In a second setting, serial nuclear transfer was done from an in vitro-matured oocyte to an in vivo-produced zygotic cytoplast. The serial NT procedure was done with karyoplasts containing the PN of NT embryos reconstituted from in vitro MII-matured oocytes, whose maturation from the GV stage was accomplished in {alpha}-MEM2 medium (Fig. 1, IVM MII-SNT). As a procedure control, NT embryos from in vitro-matured oocytes without serial transfer were put in culture in KSOM/G2 (Fig. 1, IVM MII-NT). Nonmanipulated parthenogenetically activated oocytes from in vitro-matured oocytes served as controls (Fig. 1, GV-control).

Blastocyst Analysis (Differential Staining)

The TE and ICM cells of blastocysts were differentially labeled with polynucleotide-specific fluorochromes using the method of Hardy et al. [32], which was slightly modified [33, 34]. This staining procedure was done between 110–115 h after the start of activation. First, the zona was removed by exposing the embryos to prewarmed acid Tyrode solution (pH 2.1) for a few seconds, monitored under the stereomicroscope, and then washed in KSOM-Hepes. The zona-free blastocysts were incubated and labeled with trinitrobenzene sulfonic acid (TNBS; Sigma P-2297, Sigma-Aldrich, Bornem, Belgium) for 10 min at 4°C. Excess TNBS was washed away with KSOM-Hepes before exposing the blastocysts to antidinitrophenol in KSOM-Hepes at 37°C for 10 min. After washing shortly in KSOM-Hepes, the blastocysts were incubated in guinea-pig complement diluted 1/4 in KSOM-Hepes with 2 µg/ml propidium iodide (PI) for 10 min at 37°C. Then blastocysts were quickly washed in BSA-free KSOM-Hepes medium supplemented with 5 µg/ml PI and then fixed in ice-cold ethanol for 5 min. Finally, embryos were transferred to 10 µg/ml Hoechst 33258 in ethanol for at least 10 min at 4°C. The stained blastocysts were mounted in 100% glycerol and evaluated by fluorescence microscopy (Axioplan 2, Zeiss, Zaventem, Belgium). Blue nuclei were considered as inner cells, while red nuclei were counted as trophoblast cells.

Statistics

In the first set of experiments where development of NT embryos originating from in vitro- versus in vivo-matured oocytes was compared, four replicate experiments were done. Five replicate experiments were done in each SNT experiment. All data were analyzed by contingency table analysis followed by chi-square for independence. The level of significance was set at P <= 0.05.

The mean numbers of TE and ICM cells were compared between experimental groups using one-way analysis of variance followed by Newman-Keuls posttest when the level of significance reached P <= 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Table 1 documents the preimplantation development of NT embryos derived from in vivo- versus in vitro-matured MII mouse oocytes. Comparison of two different oocyte maturation media was done. NT embryos originating from in vitro-matured oocytes showed decreased development compared with NT embryos from in vivo-matured oocytes from the two-cell stage on when maturation was in {alpha}-MEM1 (P <= 0.0006) and from the three + four-cell stage on when maturation was in {alpha}-MEM2. Three-cell arrest was significantly higher in the NT embryos derived from in vitro-matured oocytes in {alpha}-MEM1 (P <= 0.003) and tended to be higher when in vitro maturation was done in {alpha}-MEM2 compared with three-cell arrest in NT embryos derived from in vivo-matured oocytes. Within the group of NT embryos starting from in vitro-matured oocytes, development to the blastocyst stage was significantly better for oocytes in vitro matured in {alpha}-MEM2 versus {alpha}-MEM1 (P <= 0.02). Also a significantly higher proportion of GV oocytes matured to the MII stage in {alpha}-MEM2 compared with {alpha}-MEM1 (P <= 0.0005). Nonmanipulated parthenogenetically activated control embryos reached the blastocyst stage at significantly higher rates (92%) than that observed in all NT embryos (P <= 0.0001).


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  		TABLE 1. Preimplantation development of mouse nuclear transfer (NT) embryos derived from in vitro- versus in vivo-matured MII oocytes under the influence of different oocyte maturation media

Preimplantation development of serial NT embryos created by transfer of PN of NT zygotes from in vivo-matured MII oocytes to in vivo-fertilized enucleated zygotes (MII-SNT) is presented in Table 2. Development was compared between serial and single nuclear transfer, referred to as MII-SNT and MII-NT, respectively. The lower two-cell formation in the MII-SNT group compared with the single MII-NT group was the result of a higher degree of fragmentation following SNT. Three-cell arrest was significantly higher in the MII-NT group compared with the MII-SNT group (P <= 0.007). A significantly higher proportion of MII-SNT embryos reached the compacted morula stage in comparison with the MII-NT group (P <= 0.04) while two-cell to blastocyst formation was comparable in both groups. The used media KSOM/G2 sustained blastocyst formation in the parthenogenetic control embryos very well, and blastocyst rates were significantly higher than in the two NT groups (P <= 0.0001).


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  		TABLE 2. Preimplantation development of mouse serial nuclear transfer (NT) embryos starting from in vivo-matured oocytes as recipients

Preimplantation development of serial NT embryos created by transfer of PN of NT zygotes from in vitro-matured MII oocytes matured in {alpha}-MEM2 to enucleated in vivo-fertilized zygotes (IVM MII-SNT group) is presented in Table 3. Development was compared between serial and single nuclear transfer, referred to as IVM MII-SNT and IVM MII-NT, respectively. Blastocyst formation was significantly better in the IVM MII-SNT group compared with the IVM MII-NT group (P <= 0.003) and was comparable with the MII-NT group, which served as an additional control. Parthenogenetic embryos originating from IVM oocytes (GV-control) showed equal blastocyst developmental rates as parthenogenetic embryos from in vivo-matured oocytes (MII-control).


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  		TABLE 3. Preimplantation development of mouse serial nuclear transfer (NT) embryos starting from in vitro-matured oocytes as recipients

The results of blastocyst quality analysis after SNT are presented in Table 4. Total cell number of all obtained NT blastocysts was significant lower compared with all control groups (P <= 0.05). Total cell number in the IVM MII-SNT group was comparable with that in the MII-NT group, which was significantly higher than the TCN in the IVM MII-NT group (P <= 0.05).


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  		TABLE 4. Blastocyst quality analysis by differential staining of inner cell mass (ICM) and trophectoderm (TE) cells of blastocysts after single (NT) or serial nuclear transfer (SNT) starting from IVM oocytes, compared with NT blastocysts starting from in vivo-matured oocytes (MII-NT) and parthenogenetically activated embryos (MII-control and GV-control)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we first investigated whether in vitro- versus in vivo-matured MII oocytes differ in their capacity to support preimplantation development of NT mouse embryos. We found that the developmental potential of NT embryos originating from in vitro-matured oocytes was significantly less than that of NT embryos from in vivo-matured oocytes. We further observed that embryonic development of NT embryos derived from immature oocytes was affected by the choice of the medium for in vitro maturation of oocytes. Supplementation of the {alpha}-MEM medium in our study with gonadotropins and serum gave a higher maturation rate and subsequently better embryonic development of NT embryos than {alpha}-MEM supplemented with BSA only. Addition of gonadotropins to the oocyte maturation medium has been shown to be beneficial for cytoplasmic maturation in vitro in a variety of species [35]. Moreover, previous studies in the mouse revealed that the inclusion of FSH in maturation medium improves fertilization and increases the frequency of preimplantation and fetal development [19, 36, 37]. Also, cumulus cells respond to gonadotropins and are known to secrete various substances that not only control nuclear maturation but also play an important role in cytoplasmic maturation [38]. In the present study, GV oocytes were surrounded by cumulus cells, of which the beneficial effects on early development have been reported for rabbit, mouse, rat, bovine, and human [35].

Second, we investigated whether sequential nuclear transfer from in vitro-matured oocytes to cytoplasts of in vivo-fertilized zygotes had a beneficial effect. This second transfer was done for reconstructed NT zygotes originating both from in vivo- and in vitro-matured MII oocytes. Striking was the fact that the decreased development associated with in vitro-matured oocytes could be overcome by transferring PN to in vivo-fertilized cytoplasts. Studies in pigs and sheep have shown that GV-matured cytoplasm is less capable of supporting NT development compared with in vivo-matured cytoplasm [21, 22]. It is furthermore known that different culture conditions for oocyte in vitro maturation can affect fertilization and embryonic development of IVF embryos [26, 35]. Studies in bovine [27] and mice [25] have shown that maturation media can improve the capacity of the oocyte to develop to the blastocyst stage after IVF.

Our results show that a significant loss of NT embryos originating from both in vivo-matured but in particular from in vitro-matured oocytes begins at the two- to three + four-cell transition where the switch to zygotic genomic control occurs. Also, the significantly higher arrest at the three-cell stage in the in vitro-matured NT group can possibly explain the decreased blastocyst formation. Previous study has shown that practically all these three-cell NT embryos arrest with signs of binucleation or give rise to semideveloped poor-quality morulas [30].

Another study showed that development of IVF embryos from in vitro-matured oocytes was restricted to the early cleavage divisions (two- to four-cell stage) while 78% of in vivo-matured IVF embryos formed blastocysts [19]. This suggests that the conditions during IVM sensitized the embryos to the culture conditions. This in vitro developmental block of embryos from IVM oocytes is reminiscent of the so-called in vitro two-cell block that is apparent in the majority of mouse inbred strains [39]. Clearly, there is potential for IVM oocytes to be rendered sensitive to culture conditions [24, 40].

One can argue that the cytoplasm of in vitro-matured oocytes is compromised and is not capable of supporting embryonic development equally to in vivo-matured oocytes. To shed some light on this interesting topic, we have applied a second nuclear transfer to a new cytoplasmic milieu, one that had the opportunity to mature completely in vivo till the zygotic stage. Liu et al. [41] demonstrated that the nucleus of a mouse oocyte subjected to sequential transfer at the GV and PN stages is capable of supporting normal embryonic development of noncloned embryos, but in contrast, the cytoplasm of such oocytes appears to be compromised. It is known that PN exchange between zygotes does not prevent development of the reconstructed embryos [11]. Furthermore, two studies applying SNT at the two-cell stage in mouse showed significantly better developmental rates in the SNT group [42, 43]. Transfer of PN from NT embryos originating from in vivo-matured MII oocytes to in vivo-produced enucleated zygotes resulted in significantly better compacted morula formation compared with single NT embryos in our study, but overall blastocyst formation was not increased by this serial NT. It has been suggested that additional reprogramming of the donor nucleus takes place by transferring it to in vivo-produced zygotic cytoplasm. The equal blastocyst formation rate of MII-NT and MII-SNT does not support this hypothesis for mice in this study.

Serial NT was also recently applied for mice embryos by Ono et al. [29]. These authors showed significantly better morula/blastocyst formation in the single NT group than in the serial NT group starting from in vivo-matured MII oocytes, but live offspring was only obtained in the serial NT group. Their study differed from our study in several aspects. In vivo-matured MII oocytes were used, while we compared in vitro- and in vivo-matured oocytes; as a donor cell nucleus, they used fibroblast cells arrested at metaphase instead of cumulus cells in the G0/G1 stage, as in our study; also, the procedure for embryo reconstruction was different (electrofusion for single NT versus direct mechanical injection into the oocyte cytoplasm); and finally, they used IVF zygotes as cytoplasts, while we used in vivo-fertilized zygotes. As shown by Kwon and Kono [44], the ability of cytoplasts to support development differs markedly between fertilized and parthenogenetic one-cell embryos, the origin of the recipient cytoplasm thus being crucial. Moreover, the quality of the recipient cytoplast affects gene expression during nuclear reprogramming in the mouse [45]. The lower rates of blastocyst production and fetal survival from the in vitro-matured cytoplasts reported by Jinno et al. [21] most probably relate to the poorer quality of the ooplasm.

Our data on serial transfer of PN from reconstructed NT oocytes to in vivo-enucleated zygotes show significantly better developmental rates in the IVM MII-SNT group compared with the group of NT embryos from GV oocytes not having received a second transfer to a fertilized cytoplast (IVM MII-NT). Equal blastocyst formation rates were observed between the IVM MII-SNT and MII-NT groups, indicating that, when in vitro-matured oocytes are used as recipients, the quality of cytoplasm seems to be crucial for supporting development of NT embryos under these circumstances. Although further experiments are required to clarify the beneficial effect of SNT, several lines of possibilities may be suggested. The cytoplasmic environment after fertilization leads to gene expression capable of supporting development to term, while that of parthenogenetic embryos does not. One possible explanation is the recent finding that mRNA synthesis occurs first in the male PN starting in early S phase of the first cell cycle. It may be that these early transcripts are important for directing later development. Otherwise, donor cell-derived products in the constructed eggs are diluted by the second NT, making them less harmful for further development [29].

Blastocyst quality analysis revealed that parthenogenetically activated embryos have a much higher total cell number (TCN) than all NT groups. Although the TCN of NT embryos derived from IVM oocytes (IVM MII-NT) was significantly lower than the TCN of NT embryos derived from in vivo-matured oocytes (MII-NT), this inferior blastocyst quality can be overcome by application of a serial NT, which gave comparable total cell numbers between IVM MII-SNT and MII-NT. In another study of our group [46], it has been shown that NT blastocysts show some retardation in development, only a minority of NT embryos had started cavitation at 72–74 h poststart activation time, in contrast with parthenogenetic activated control embryos. Also it was shown in this article that NT blastocysts had significantly reduced quality compared with parthenogenetic and ICSI blastocysts in terms of cell numbers. The proportion of dead cells in the TE and ICM was significantly higher for NT blastocysts than for parthenogenetic or intracytoplasmic sperm injection blastocysts. Mitotic cell indices for all three groups were equal.

In conclusion, we have shown that the decreased developmental potential when using in vitro-matured oocytes as recipients in NT can be circumvented by serial NT to in vivo-fertilized cytoplasm. Whether postimplantation development can also be affected by using this SNT technique remains to be investigated.


   ACKNOWLEDGMENTS
 
The authors wish to thank Ms. S. Lissens for assistance in layout and Ms. V. David for taking care of the mice used in these experiments. The authors wish to express their gratitude to the Merck-Eurolab Company (Leuven, Belgium), which provided the Electro Cell Manipulator Model 830 for use in these experiments.


   FOOTNOTES
 
1 Supported by a research grant from the Bijzonder Onderzoeksfonds of the Ghent University, Belgium (grant BOF 01110301). Back

2 Correspondence: Björn Heindryckx, Infertility Center, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium. FAX: 32 9 240 4972; bjorn.heindryckx{at}rug.ac.be Back

Received: 20 February 2002.

First decision: 8 March 2002.

Accepted: 1 July 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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