Chromosome number and development of artificial mouse oocytes and zygotes

B. Heindryckx1, S. Lierman, J. Van der Elst and M. Dhont

Infertility Centre, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium

1 To whom correspondence should be addressed. e-mail: Bjorn.Heindryckx{at}Ugent.be


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Infertility due to the absence of gametes is one of the last frontiers in reproductive medicine. Sperm or oocyte donation is currently the only treatment option but this approach lacks the genetic contribution of both partners. Artificial production of gametes through haploidization may offer an alternative strategy. The aim of this study was to evaluate the efficiency of producing artificial oocytes and zygotes with correct chromosome number. METHODS and RESULTS: Somatic cumulus cell nuclei were injected into non-enucleated oocytes to produce artificial zygotes and into enucleated mature mouse oocytes to produce artificial oocytes. The expected chromosome number of artificial zygotes and oocytes is 40 and 20 chromosomes respectively. Fertilization and developmental potential of artificial zygotes and oocytes inseminated by IVF or ICSI were investigated. The expected chromosome numbers were found in 12% of artificial zygotes and 15% of artificial oocytes. Varying the time interval between injection of the somatic nucleus and activation (3, 5, 8 h) tended to increase the efficiency up to 18 and 23% for zygotes and oocytes respectively. Two-cell formation rates were 90% for artificial zygotes and 37% for artificial oocytes after IVF and 53% for artificial oocytes after ICSI. Blastocyst formation rates were 15, 8 and 9% respectively. CONCLUSIONS: Chromosome number analysis shows that the efficiency of obtaining artificial zygotes and oocytes with correct chromosome number was low and that developmental potential was severely hampered. These observations question the possibility of obtaining chromosomally normal embryos from artificial oocytes or zygotes.

Key words: artificial oocytes/blastocyst/chromosome number/haploidization/mouse


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In vitro reproductive technologies are effective in overcoming male and female infertility. Ovarian stimulation combined with IVF or ICSI has become standard treatment in cases of ovarian cycle anomalies, tubal pathology, sperm antibodies, oligoasthenoteratozoospermia and even azoospermia. Only for patients without gametes are there currently no treatment options leading to their own genetic children. Women with ovarian failure or defective oocytes or men with spermatogenic failure are sterile and can only be treated by using donor gametes or embryos or by adoption.

Novel technology derived from cloning may offer an alternative strategy for treatment of male and female sterility by creating gametes artificially (Tsai et al., 2000Go; Tesarik et al., 2001Go; Trounson, 2001Go). A cloned embryo is usually produced by transferring an adult diploid somatic nucleus into an enucleated oocyte while polar body (PB) extrusion is prevented. The oocyte cytoplasm has the capacity to reprogram the somatic nucleus into a totipotent state. Reprogrammed nuclei have been shown to be able to support full term development in several animals (Li et al., 2003Go). The artificial creation of a gamete involves the transfer of a diploid somatic nucleus into an enucleated oocyte followed by induction of PB extrusion to reduce artificially the diploid chromosome number to the haploid status in the absence of meiosis (Trounson, 2001Go; Tesarik, 2002Go).

Artificial haploidization of somatic nuclei can be obtained via two different experimental approaches (Kubiak and Johnson, 2001Go; Fulka et al., 2002aGo). G2-stage somatic cells (diploid, double-chromatid chromosomes, 4c DNA) can be transferred into an immature germinal vesicle (GV) stage oocyte to be initiated to undergo reductional division in the absence of recombination and meiosis until metaphase II (MII)-like arrest, in order to extrude half of the chromosomes into the first PB. After activation of this artificial MII oocyte, half of the chromatids can be extruded in the second PB, leaving a haploid set of chromatids in the ooplasm. A second approach is transfer of a G0/G1-stage somatic nucleus (diploid, single-chromatid chromosomes, 2c DNA) into a mature MII recipient oocyte. The MII oocyte is capable of forcing a G0/G1-stage somatic nucleus into a premature M-phase without previous S-phase. This may result in extrusion of a pseudo-second PB, assumed to contain one set of single chromatid chromosomes, leaving a haploid set of chromosomes in the reconstructed oocyte (haploid, 1c DNA) (Tesarik et al., 2001Go).

The number of reports on haploidization of somatic nuclei to produce artificial oocytes or zygotes is steadily increasing. Tesarik et al. (2001Go) have shown the production of a limited number of artificial human oocytes where haploidization was confirmed by fluorescence in situ hybridization (FISH) for five chromosomes. Lacham-Kaplan et al. (2001Go) injected diploid somatic cells into non-enucleated mouse oocytes to create artificial zygotes, but very few blastocysts and no offspring were obtained. Tateno et al. (2003Go) described the inability of mature mouse oocytes to induce haploidization of somatic cumulus cells with only 8.9% of oocytes containing the correct chromosome number. Fulka et al. (2002aGo,b) suggested that immature mouse GV oocyte cytoplasm is unable to sustain somatic cell nuclei to proceed through reduction division. Palermo et al. (2002aGo) have shown on the contrary that immature mouse GV ooplasm can support the separation of somatic chromosomes to haploid numbers.

In this study we created artificial zygotes and oocytes by injection of a somatic cumulus cell nucleus in non-enucleated and enucleated mouse MII oocytes respectively. Polar body extrusion was induced by artificial activation in a Ca-free medium supplemented with Sr ions. The primary goal of our study was to verify whether segregation of chromosomes can take place in a numerically correct manner in artificially constructed zygotes and oocytes. We also checked whether chromosome segregation can be manipulated by varying the time interval between nuclear injection and oocyte activation. In this respect we analysed the chromosome number of artificial zygotes and oocytes after three different time intervals between injection and activation (3, 5, 8 h). Finally the preimplantation developmental potential of artificial zygotes or oocytes fertilized by IVF or ICSI was investigated.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
Mice were purchased from Iffa Credo (Brussels, Belgium) and handled according to the guidelines of the Animal Ethical Committee of the Ghent University Hospital. Mice were kept under controlled temperature and lighting conditions. Food and water were available ad libitum.

Recipient oocytes and donor somatic cells
Female B6D2 F1 (C57 Bl/6JxDBA/2) hybrid mice aged 7–14 weeks were stimulated with 8–10 IU pregnant mare’s serum gonadotrophin (Folligon; Intervet, The Netherlands) followed by 8–10 IU of hCG (Chorulon; Intervet) 48 h later. In vivo-matured MII oocytes were collected 13 h–13 h 30 min after hCG administration and transferred to home-made Potassium (K+) Simplex Optimized Medium (KSOM)–HEPES supplemented with 200 IU hyaluronidase (type VIII) (Sigma–Aldrich Chemie, Belgium) to disperse cumulus cells. The oocytes obtained served as recipient oocytes and were kept in the incubator (37°C, 6% CO2) while the cumulus cells served as donor somatic cells to be injected into recipient oocytes. Cumulus cells were washed twice by centrifugation at 500 g for 8 s and were finally kept in KSOM–HEPES in 5 µl droplets under mineral oil in the manipulation dish at room temperature.

Haploidization technology
One hour before nuclear injection, freshly thawed polyvinylpyrrolidone solution (Vitrolife Sweden AB, Sweden) was added to the droplet with cumulus cells for spreading and attachment to the surface of the manipulation dish. Only cumulus cells estimated as middle-sized (9–13 µm) were used for injection. Chromosome analysis has shown that >90% of cumulus cells of this size show elongated single chromatid chromosomes in G0–G1 phase (Wakayama et al., 1998Go; Heindryckx et al., 2001Go).

Three experimental groups were created: group A, artificial zygotes produced by injection of a somatic nucleus in a non-enucleated recipient oocyte; group B, artificial oocytes produced by injection of a somatic nucleus in an enucleated recipient oocyte; group C, artificially activated, non-manipulated parthenogenetic control oocytes.

A tangential slit was made in the zona pellucida of recipient oocytes with a sharp needle. For production of enucleated recipient oocytes, the oocyte chromosome–spindle complex, visible as an opaque non-granulated area, was aspirated with a blunt polished pipette in KSOM–HEPES supplemented with 1 µg/ml cytochalasine D (Sigma–Aldrich Chemie). Injection of somatic cumulus cell nuclei into non-enucleated or enucleated oocytes was carried out in KSOM–HEPES plus 20% fetal bovine serum (FBS, no. 10120-152. Invitrogen, Belgium) using a blunt pipette (6–7 µm inside diameter) on an inverted microscope stage cooled to 15–17°C. Up to 5 h after injection, reconstructed artificial zygotes (group A) and oocytes (group B) and parthenogenetic control oocytes (group C) were activated in home-made Ca2+-free KSOM, containing 10 mmol/l SrCl 2 (Sigma–Aldrich Chemie) during 4 h. After artificial activation, the number of oocytes showing pronuclei (PN) and extruded second polar bodies (PB) was noted in all groups. The expected pattern is 2PN and two second PB for artificial zygotes. For artificial oocytes and parthenogenetic control oocytes, 1PN and one second PB are expected. In a separate set of experiments, the effect of different time intervals (3, 5 and 8 h) between injection and activation was verified in both non-enucleated and enucleated injected oocytes.

Cytogenetic chromosome number analysis
After PN and second PB evaluation, zygotes were put in KSOM supplemented with 1 µg/ml nocodazole (Sigma–Aldrich Chemie) to induce metaphase arrest. At 8–12 h later, the zona pellucida of artificial oocytes and zygotes with vanished PN was removed with 0.5% (w/v) pronase (Sigma–Aldrich Chemie). Oocytes and zygotes were transferred to a hypotonic 0.75 mmol/l KCl (Sigma–Aldrich Chemie) solution for 8 min and fixed with methanol/acetic acid (v/v; 3/1). Zygote spreads were stained with Giemsa and numbers of chromosomes were counted at x1000 magnification. The expected chromosome number is 40 in artificial zygotes and 20 in artificial oocytes. In the control group, MII oocytes were activated and a chromosome number of 20 is expected.

Development of artificial zygotes
Following nucleus injection, the number of surviving oocytes was recorded. After activation, artificial zygotes were cultured in KSOM (Erbach et al., 1994Go) containing low glucose (0.2 mmol/l) and 0.4% BSA (Euro Biochem, Belgium). Sixty to 70 h post start activation, embryos were transferred to G2 medium (Vitrolife) (Heindryckx et al., 2001Go, 2002). Parthenogenic diploid embryos were created by artificial activation of MII oocytes in Ca2+-free KSOM containing 10 mmol/l SrCl 2 and 2 µg/ml cytochalasin D to prevent PB extrusion, and served as medium controls. Embryo development was assessed at 24 h (2-cell), 48 h (3–4-cell), 72 h (morula/early blastocyst) and 96 h (blastocyst) post activation time.

Fertilization and development of artificial oocytes
The number of oocytes that survived the injection of a cumulus cell nucleus was recorded. Up to 4 h 30 min after injection, artificial oocytes were fertilized in two different ways, either by IVF or by ICSI.

For IVF, the procedure was carried out as described in Liu et al. (2001Go). Inseminated oocytes were put into sequential culture in KSOM with low glucose (0.2 mmol/l) + 0.4% BSA (w/v) followed by G2 media (Heindryckx et al., 2001Go). Embryo development was assessed at 24 h (2-cell), 48 h (3–4-cell), 72 h (morula/early blastocyst) and 96 h (blastocyst) post activation time. As controls for IVF on artificial oocytes, we carried out IVF on zona-dissected MII oocytes (IVF-dissected oocyte) and on denuded MII oocytes (IVF MII) at 21 h post hCG, the analogue time when artificial oocytes were inseminated.

For ICSI, sperm were recovered from the caudae epididymae of 4–12 month old CD-1 males in KSOM–HEPES medium and only motile sperm were selected. Sperm heads were separated from tails and subsequently injected into the artificial oocytes. The number of artificial oocytes that survived the sperm injection was recorded. Embryo development was assessed at 24 h (2-cell), 48 h (3–4-cell), 72 h (morula/early blastocyst) and 96 h (blastocyst) post activation time. Zona-dissected MII oocytes injected with isolated sperm heads served as ICSI manipulation control (ICSI-dissected oocyte).

Parthenogenic diploid embryos were created by artificial activation of MII oocytes and served as medium controls.

Statistics
In the first set of experiments where cytogenetic chromosome number analysis was performed in artificially created oocytes and zygotes, six replicates were done. Five replicate experiments were done when different time intervals between cumulus nucleus injection and artificial activation were examined. Developmental potential of non-enucleated MII oocytes injected with cumulus cell nuclei was verified in three replicates. Developmental potential of enucleated somatic nuclear injected MII oocytes fertilized with ICSI versus IVF was verified in eight replicates. All data were analysed by contingency table analysis followed by {chi}2-test for independence. The level of significance was set at P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Analysis of chromosome number
Chromosome number analysis was done on fixed chromosome spreads of artificial oocytes and zygotes. Spreads were discarded when the number of chromosomes was 20 in the group of non-enucleated oocytes (group A) and 0 in the group of enucleated oocytes (group B), because this outcome was due to technical failure of somatic nuclear injection. The frequency of non-successful injection was 8% in group A and 13% in group B.

Results on chromosome number analysis of successfully injected non-enucleated (group A) and enucleated (group B) oocytes injected with cumulus cell nuclei are given in Table I. Survival rate after cumulus nuclear injection is comparable in both groups A and B (69 and 74% respectively). Of 104 oocytes that survived cumulus cell nucleus injection in group A, 50 zygotes (48%) displayed the expected number of two PN and 49 (47%) zygotes showed the expected two second PB. Of the 123 oocytes that survived cumulus nuclear injection in group B, 79 zygotes (64%) showed the expected formation of one pronucleus and 73 zygotes (59%) showed one second PB. The expected chromosome numbers of 40 and 20 were found in 12% (reconstructed zygotes) and 15% (reconstructed oocytes) respectively. Correct chromosome number was found in 62% (81/131) of control non-manipulated oocytes which was significantly higher than in groups A and B (P < 0.0001).


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Table I. Chromosome number analysis of artificial mouse zygotes and oocytes created by injection of a somatic mouse cumulus cell nucleus into non-enucleated and enucleated metaphase II oocytes
 
Different time intervals between injection and activation
Data on chromosome number analysis after different time intervals between nucleus injection and oocyte activation are given in Table II.


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Table II. Chromosome number analysis of artificial mouse zygotes and oocytes created by injection of a somatic mouse cumulus cell nucleus into non-enucleated and enucleated metaphase II oocytes after different time intervals between nucleus injection and artificial activation
 
There was no significant difference in PN formation rate using three different time intervals in both groups A and B. In group A, 47, 50 and 53% showed 2PN formation in the 3, 5 and 8 h time-interval respectively. In group B, 67, 80 and 77% showed 1PN formation in the 3, 5 and 8 h time interval respectively. The PB extrusion pattern was also similar between the different time intervals within both groups A and B. The percentages of artificial oocytes and zygotes showing correct chromosome number were comparable for the three time intervals within groups A (3 h: 15%, 5 h: 18%, 8 h: 13%) and B (3 h: 17%, 5 h: 23%, 8 h: 19%). The percentage of oocytes with the correct chromosome number (53%) was significantly higher in the parthenogenetic haploid control oocytes from group C (P < 0.01 at least).

Preimplantation development of artificial zygotes and oocytes
Preimplantation development of non-enucleated oocytes injected with a cumulus cell nucleus is given in Table III. A high percentage of these artificial zygotes reached the compacted morula stage but blastocyst formation was low (15%) compared to the parthenogenetic diploid control embryos (71%) (P < 0.0001).


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Table III. Developmental potential of non-enucleated metaphase II oocytes injected with cumulus cell nucleus (artificial zygotes)
 
Artificial oocytes were fertilized by IVF or ICSI and fertilization and developmental rates are compared in Table IV. The percentage of oocytes that survived nucleus injection was similar in both IVF and ICSI groups (73 and 75% respectively) but many oocytes lysed during the second injection of sperm in the ICSI group (final survival rate 48%). While 53% of the surviving oocytes in the ICSI group reached the 2-cell stage, only 37% of oocytes in the IVF group formed 2-cell embryos (P < 0.01). The capacity of 2-cell embyos to reach the 4-cell, morula and blastocysts stages were low but comparable in both IVF and ICSI groups. Only 9% of 2-cell embryos reached the blastocyst stage in the ICSI group and 8% in the IVF group. In all control groups, blastocyst formation was significantly higher than for the fertilized artificial oocyte group (P < 0.0001). Blastocyst rate was 84% in the parthenogenetic diploid control embryos, 67 and 82% in the denuded and dissected IVF control respectively, and 93% in the ICSI control group.


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Table IV. Developmental potential of enucleated somatic nuclear injected oocytes (artificial oocytes) fertilized with ICSI versus IVF
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A somatic cumulus cell nucleus transferred in the cytoplasm of a MII oocyte can progress to a meiotic form of cell division, thus halving the original number of chromosomes. We created artificial zygotes and oocytes by injecting a cumulus cell nucleus into non-enucleated and enucleated MII oocytes respectively. The chromosome number was taken as main outcome parameter. The expected chromosome numbers of 40 and 20 were found in low percentages of artificial zygotes and oocytes (12 and 15% respectively). Attempts to influence the efficiency of segregation by increasing the time interval between nuclear injection and artificial activation did not significantly increase the outcome. Maximum percentage of zygotes and oocytes with correct chromosome number were 18 and 23% respectively, although this does not mean that each chromosome was present or contained in only one copy. The developmental capacity of artificial zygotes was severely hampered, showing a blastocyst formation rate of 15 versus 71% in a parthenogenetic diploid control group. IVF of artificially created oocytes by IVF or ICSI showed that 2-cell formation was disturbed and that blastocyst formation rate was <10%. These results question the possibility of obtaining chromosomally normal embryos from artificial oocytes or zygotes.

Successful development and haploidization technology require the syngamy between a somatic injected nucleus and a gamete nucleus (Tesarik, 2002Go). Previous attempts to create artificial zygotes in the mouse showed that the efficiency of producing blastocysts using cumulus cells as sperm substitutes was very low and no offspring were obtained (Lacham-Kaplan, 2001Go). In the present study, preimplantation development of artificial zygotes was satisfactory up till the 2-cell and morula stages. The near-failure to form blastocysts may reflect the abnormal chromosome nature of the reconstructed embryos. Indeed cytogenetic data showed that a maximum of 18% of the reconstructed zygotes had normal chromosomal numbers in the present study. Development of artificial oocytes was investigated after IVF or ICSI. The second injection needed in case of ICSI is reducing the oocyte survival rate to <50%. Two-cell formation on the other hand is significantly lower with IVF than ICSI, probably due to oocyte ageing, manipulation and/or zona hardening, preventing sperm penetration during IVF. This is further illustrated by the lowest degree of 2-cell formation in non-zona-manipulated denuded control oocytes after IVF. The final outcome parameter of blastocyst formation fertilized by IVF or ICSI per original artificial oocyte is comparable.

Haploidization strategy based on the injection of diploid somatic nuclei into enucleated mature MII oocytes has also been described in several recent studies. Tateno et al. (2003Go) showed only 8.9% correct chromosome numbers after injection of cumulus cells into enucleated mature mouse oocytes. Unfortunately no controls were used to verify the chromosome segregation. In our study, a maximum of 23% of artificial oocytes showed correct chromosome number when a 5 h interval was used between cumulus nucleus injection and activation. However, we did not check assortment of chromosome segregation by karyotyping or FISH. Using hamster cumulus cell nuclei and karyotyping, Tateno et al. (2003Go) revealed zero normal haploid complements of chromosomes of the 14.1% that showed the correct haploid number. So far, results on haploidization of human oocytes are not conclusive since few oocytes or chromosomes were analysed (Takeuchi et al., 2001Go; Tesarik et al., 2001Go).

Correct chromosome segregation is crucial in artificial haploidization. Meiotic chromosomes in an oocyte have a different behaviour compared to mitotic chromosomes in somatic cells (Fulka et al., 2002aGo). In meiosis, the MII chromosomes each consist of two chromatids which are physically attached to each other at their centromere, while G0/G1 cumulus cells contain single-stranded chromatid chromosomes. The correct chromosome position and attachment on the spindle as well as a distinctive regulation of the cohesion between sister chromatids seem to be crucial for correct chromosome reduction (Paliulis and Nicklas, 2000Go; Fulka et al., 2002aGo,b). When G0/G1 somatic chromosomes are transferred into MII ooplasm, they lack any physical association between their homologous single-chromatid chromosomes. In the absence of any cohesion at all, reduction division may be totally random (Tesarik, 2002Go). The mechanism for correct separation of these single chromatids, present in the G0/G1 cumulus cell, remains to be determined.

Nevertheless, some artificially created oocytes are apparently chromosomally normal, as proven by FISH with a limited number of chromosome-specific probes (Palermo et al., 2002aGo,b). How may we explain this unexpected behaviour of presumably correct haploidization of diploid somatic cells possessing unattached chromatids? According to Eichenlaub-Ritter (2003Go), ooplasm, even in the absence of chromosomes, has the capacity to organize bipolar spindles, which requires expression of microtubule motor proteins, tubulin, and cell extracts with active maturation promoting factor and cytosolic factor. Some back-up mechanisms are at the basis of pairs of non-exchange univalent chromosomes to segregate to opposite poles rather than the same pole during oogenesis in some species (Karpen et al., 1996Go). These mechanisms present in the oocyte may contribute to some percentage of artificially created oocytes showing the correct ploidy.

Except for ploidy disturbance, several other perturbations have to be addressed in artificial haploidization technology including the outcome of possibly excessively short telomere length in the somatic nucleus, heteroplasmy (host mitochondria), the co-existence of somatic cell- and sperm-derived centrosomes, incomplete somatic nuclear reprogramming, and genomic imprinting abnormalities (Tesarik, 2002Go; Eichenlaub-Ritter, 2003Go). In somatic cells, only one allele is imprinted, either maternally or paternally. In the case of haploidization, there is a 50% chance that it contains the wrong imprint, leading to bi-allelic expression or repression of imprinted regions similar to that which gives rise to congenital abnormalities and genetic disease in cases of uniparental disomy (e.g. Prader–Willi or Angelman syndrome) (Eichenlaub-Ritter, 2003Go). Even when a haploid set of chromosomes is artificially created by haploidization, it is extremely unlikely that such a chromosome complement would contain exclusively maternally or paternally imprinted chromosomes (Tateno et al., 2003Go), which makes the haploidization theory even more difficult to succeed.

We can conclude from our results that artificial oocytes and zygotes have a very high level of chromosomal number abnormalities with a likelihood of decreased development as a consequence. Substantial genetic analysis of chromosome segregation by FISH or by full karyotyping has to be done to verify the real assortment of chromosomes. It must be remembered that the consequences of autosomal aneuploidy in the mouse are preterm lethalities, but in the human, some autosomal aneuploidies are viable to term with consequently severe medical problems.


    Acknowledgements
 
The authors wish to thank Ms S.Lissens for assistance in lay-out and Ms V.David for taking care of the mice used in these experiments. Supported by a research grant from the Bijzonder Onderzoeksfonds of the Ghent University, Belgium (grant no. BOF 01110301).


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on November 26, 2003; resubmitted on December 23, 2003; accepted on February 18, 2004.





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