Preliminary Results with Nuclear Transplantation

Mouse GV oocytes were retrieved by puncturing ovarian follicles of unstimulated B6D2F1 females, and cumulus-free oocytes were cultured in medium (M199) supplemented with a phosphodiesterase inhibitor (0.2 mM 3-isobutyl-1-methylxanthine; Sigma) to prevent spontaneous GV breakdown.

All the micromanipulation and electrofusion procedures were performed in a shallow plastic Petri dish on a heated stage, placed on an inverted microscope equipped with hydraulic micromanipulators (103). The zona pellucida was breached by a glass microneedle, then oocytes were exposed to 25 mg/ml of cytochalasin B. The GV surrounded by a small amount of cytoplasm (GV karyoplast) was removed by a micropipette with a 20 mm inner diameter. Next the GV karyoplast was inserted into the perivitelline space of a previously enucleated oocyte (GV cytoplast). Each grafted oocyte was aligned with a micromanipulator between two microelectrodes perpendicular to their axes. To induce fusion, a single or double 1.0 kV/cm direct current fusion pulse was delivered for 100 ms in an electrolyte medium (M2) by an Electro Cell Manipulator (BTX 200 and 2001, Genetronics, Inc., San Diego, CA). Then, after washing and culture for 30 min in a cytochalasin B-free medium, these oocytes were examined to confirm cell survival and fusion, cultured further, and then examined at 12-16 h after the fusion treatment. Finally, their nuclear maturation was evaluated, as evidenced by extrusion of the first polar body (PB).

For similar human studies, GV oocytes were obtained, as described earlier, from consenting patients undergoing ICSI (4, 10, 29, 36). Immediately before ICSI, corona cells were removed by enzymatic and mechanical treatments, and the denuded oocytes were examined under an inverted microscope to assess their integrity and maturation stages (4, 10, 29, 36). The nuclear transplantation procedure is shown in Figures 5.85.11. The reconstituted immature oocytes were cultured and were examined at 24 and 48 h after electrofusion to evaluate nuclear maturation, characterized by disappearance of the GV and extrusion of the first PB.

In the mouse, nuclear transplantation into GV-stage oocytes followed by extrusion of a PB revealed an overall efficiency of 80%. Interestingly, this aggressive technique appears not to increase the incidence of chromosomal abnormalities (103). Human oocytes were reconstituted with an efficiency of 73%, with a lower maturation rate of 62% following reconstitution, comparable to that of control human GV oocytes, as was the 20% incidence of aneuploidy among the constructed oocytes (104). Those reconstituted human oocytes were successfully fertilized by ICSI at a rate of 52%, but although they underwent early embryonic cleavage after ICSI, their survival rate and embryo quality appeared suboptimal compared to control oocytes matured in vivo (105).

In a limited number of transfers of aged GV oocyte into a younger ooplast, there was a normal haploidization during the first meiotic division accompanied by the extrusion of the first PB. In other studies, lower maturation rates and impaired embryo development have been the rule, probably attributable to the suboptimal in vitro culture conditions currently available for human oocyte maturation (102-107).

Figure 5.10. Cell alignment for electrofusion by microelectrodes under micromanipulator control (200x).

clei to metaphase II (108). Immature ooplasm is capable of inducing haploidization-like reduction division of transplanted somatic cell nuclei (107, 109), and we have proposed that the transfer of somatic nuclei to GV ooplasts and their ensuing haploidization may provide a source of viable mammalian oocytes. If oocytes can indeed be reconstituted by such techniques, this would greatly benefit patients who are candidates for oocyte donation.

To obtain somatic cell nuclei, endometrial stromal cells were collected from consenting patients undergoing endometrial cell coculture during IVF. Endometrial stromal and glandular cells were isolated by enzymatic digestion using 0.2% collagenase type II, separated by differential sedimentation (110), and cultured in a long-term culture medium supplemented with 10% FBS. In the case of the mouse, cumulus-oocyte complexes were obtained from B6D2F1 mice after ovarian stimulation with PMSG and hCG, and cumulus cells isolated by brief exposure to hyaluronidase were cultured for up to 30 days. GV oocytes were retrieved from the same strain of mouse by puncturing follicles of un-stimulated ovaries and were denuded by mechanical removal of cumulus cells.

All the micromanipulation and electrofusion procedures were carried out in a shallow plastic petri dish on a heated stage under an inverted microscope. A hole was made in the zona pellucida of the GV oocyte with a glass needle, and after short exposure to cytochalasin B, the GV surrounded by a small amount of ooplasm was removed with a glass micropipette (103). Cultured human endometrial stromal and mouse cumulus cells

Figure 5.11. Grafted oocyte 30 min after electrofusion (400x). Drifting of the transplanted nucleus (arrows) into the host ooplasm.

were isolated with trypsin-EDTA. Either a stromal or a cumulus cell was then inserted subzonally into an enucleated mouse GV oocyte. Each grafted oocyte was manually aligned between two microelectrodes, and cell fusion was induced by applying direct current. The resulting reconstituted oocytes were allowed to mature for 14-16 h until extrusion of the first PB (103). The distribution of nuclear chromatin between the ooplasm and the PB was evaluated by specific DNA staining. For this, some mature oocytes were stained with DAPI solution and evaluated under a fluorescent microscope, while others were anchored between a microslide and coverslip, fixed with methanol/ acetic acid (3:1; v/v), and stained with 1% aceto-orcein solution.

A total of 45 GV oocytes were enucleated, then fused with somatic cells. Staining showed the presence of metaphase chromosomes in the ooplasm and in the PB. The overall efficiency of the sequence from an intact GV oocyte to a reconstituted oocyte with an extruded PB was 60.0% for human endometrial cells and 42.8% for mouse cumulus cells. A longer period of in vitro culture did not induce any additional PB extrusions.

Other studies were performed to evaluate the karyotypes of reconstituted mature oocytes. Among a total of 78 intact GV oocytes, 77 were successfully enucleated and grafted with a single cumulus cell, 56 being reconstituted finally by electrofusion. Subsequently, 29 extruded the first PB during the 14-16 h of culture. Of the 13 oocytes that matured successfully and whose karyotypes were analyzable, 5 had 20 chromosomes, 5 were aneuploid, and 3 were diploid.

The observation that immature mouse ooplasts can accomplish haploidization of human or murine somatic cell nuclei suggests that this approach may provide an alternative source of viable human oocytes. However, more detailed cytogenetic information is needed, and the presence of the somatic cell centrosome in these manufactured oocytes means that the fertilization process and later embryo cleavage must be carefully investigated. It also remains to be established that genetic imprinting of the oocyte reconstructed from a cultured somatic cell is comparable to that in natural haploid.

Although this approach to haploidization of somatic cell nuclei may at first bring cloning to mind, the resulting haploid oocytes obviously still need the contribution of the paternal genome for further development. Nonetheless, these preliminary findings suggest that nuclear transplantation may provide a means of manufacturing viable oo-cytes from defective oocytes.

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