Developmental potential of human oocytes reconstructed by transferring somatic cell nuclei into polyspermic zygote cytoplasm

The aim of the study was to determine the in vitro developmental potential of caprine cloned embryos following pseudophysiological (transcytoplasmic) transcomplementary activation of oocytes reconstructed with ear skin-derived fibroblast cell nuclei. The source of nuclear recipient cells were IVM doe oocytes. The reconstruction of the previously enucleated oocytes (i.e. ooplasts) was performed by microinjection of either the somatic cell-derived karyoplasts or intact whole tiny nuclear donor cells directly into the cytoplasm. The reconstructed oocytes were incubated in Upgraded B2 INRA medium for 30 min to 1 h before their pseudophysiological activation. The activation was achieved by electrofusion of clonal cybrids with the allogeneic cytoplasts isolated from caprine IVF-created zygotes, which led to the formation of triple allocytoplasmic hybrids (allocybrids). These originate from 3 sources: (1) homologous whole nuclear donor fibroblast cells or their karyoplasts; (2) enucleated oocytes (ooplasts), and (3) zygote-derived cytoplasts. Single zygote-descended cytoplasts (the so-called zygoplasts) were inserted into the perivitelline space of previously reconstituted oocytes. The resulting zygoplast-clonal cybrid couplets were subsequently subjected to electrofusion, which was induced by application of a single DC pulse of 2.4 kV cm-1 for 15 μs. The electrofusion of zygoplast and reconstructed oocyte plasma membranes occurred in an isotonic dielectric solution deprived of Ca2+ ions. The transcytoplasmically activated clonal cybrids were cultured in vitro in Upgraded B2 INRA medium for 48 h at 38.5°C in a 100% water-saturated atmosphere of 5% CO2 and 95% air. Afterward, cleaved embryos were co-cultured with Vero cells in medium supplemented with 10% fetal bovine serum for an additional 96 to 144 h up to morula and blastocyst stages under the same thermal and atmospheric conditions. A total of 53/78 (67.9%) oocytes reconstructed with fibroblast cell nuclei were successfully fused with zygoplasts. From among 53 cultured cloned embryos, 34 (64.2%) cleaved. The rates of embryos that reached the morula and blastocyst stages were 21/53 (39.6%) and 11/53 (20.8%), respectively. In conclusion, the relatively high percentages of morulae and blastocysts were noticed among in vitro-cultured caprine cloned embryos produced by the strategy of pseudophysiological transcytoplasmic activation of oocytes reconstructed with adult dermal fibroblast cell nuclei. Therefore, the use of cytoplasmic components originating from zygotes as the stimuli for activation of nuclear-transferred oocytes appeared to be an effective procedure in the generation of goat blastocysts by somatic cell cloning.

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Microtubular spindle dynamics and chromosome complements from somatic cell nuclei haploidization in mature mouse oocytes and developmental potential of the derived embryos

Human Reproduction ◽

10.1093/humrep/deh168 ◽

2004 ◽

Vol 19 (5) ◽

pp. 1181-1188 ◽

Cited By ~ 18

Author(s):

S.-U. Chen

Keyword(s):

Somatic Cell ◽

Cell Nuclei ◽

Developmental Potential ◽

Mouse Oocytes ◽

Spindle Dynamics ◽

Mature Mouse

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Effect of delayed enucleation on the developmental potential of nuclear-transferred oocytes receiving adult and fetal fibroblast cells

Zygote ◽

10.1017/s0967199402002289 ◽

2002 ◽

Vol 10 (3) ◽

pp. 217-222 ◽

Cited By ~ 15

Author(s):

Xi Jun Yin ◽

Yoko Kato ◽

Yukio Tsunoda

Keyword(s):

Somatic Cell ◽

Cell Number ◽

Heart Beat ◽

Fibroblast Cells ◽

Cell Nuclei ◽

Average Cell ◽

Developmental Potential ◽

Membrane Protrusions ◽

Implantation Sites

To enhance the probability of reprogramming somatic cell nuclei, fibroblast cells from an adult male rabbit and a 12-day-old fetus were fused with oocytes at the second metaphase. The chromosomes of recipient oocytes were then removed by treatment with demecolcine for 1 or 2 h after fusion. Demecolcine treatment of fused oocytes induced membrane protrusions that contained all the maternal chromosomes, thus making it possible to remove the chromosomes. The potential of nuclear-transferred oocytes to develop into blastocysts was high (48% and 59%) and the average cell number of the blastocysts was large (149 and 159) 96 h after in vitro culture. The proportions of nuclear-transferred oocytes enucleated 1 h after fusion and implanted after transfer to pseudopregnant recipients were relatively high (2.8% and 4.9%) compared with our previous reports (1.7%: Yin et al., 2000; 0.6% and 1.0%: Yin et al., 2002a) where donor cells were fused with previously enucleated oocytes. Of 34 adult somatic cell implantation sites, 6 had fetuses on day 12 or 14 of pregnancy, but none of the fetuses had a heart beat or developed to term. None of the nuclear-transferred oocytes whose chromosomes were removed 2 h after demecolcine treatment implanted after transfer to recipients. The possible reasons why the high-quality nuclear-transferred oocytes did not develop to term are discussed.

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The effect of the time interval between injection and parthenogenetic activation on the spindle formation and the in vitro developmental potential of somatic cell nuclear-transferred rat oocytes

Zygote ◽

10.1017/s0967199409990025 ◽

2009 ◽

Vol 18 (1) ◽

pp. 9-15 ◽

Cited By ~ 4

Author(s):

Shigetoshi Mizumoto ◽

Yoko Kato ◽

Yukio Tsunoda

Keyword(s):

Somatic Cell ◽

Time Interval ◽

Cell Nuclei ◽

Spindle Formation ◽

Developmental Potential ◽

Developmental Rates ◽

Culture Duration ◽

Oocyte Cytoplasm ◽

Preincubation Time

SummaryWe examined the optimal conditions for somatic cell nuclear transfer (SCNT) in the rat. First, we examined the effect of preincubation time before activation on SCNT rat oocytes produced in the presence of MG132 with regard to spindle formation and the potential to develop into blastocysts. The spindles of SCNT oocytes continued to elongate with an increase in the culture duration and, in approximately half of oocytes, the chromosomes were distributed along the spindles at 120 min after incubation. Such abnormal spindle formation in SCNT oocytes is a possible reason for the low developmental potential of SCNT rat oocytes. To inhibit the formation of abnormal spindle formation, we examined secondly the developmental potential of rat SCNT oocytes that had been preincubated with nocodazole and demecolcine instead of MG132. The developmental rates in SCNT oocytes, however, were decreased. For successful rat somatic cell cloning, two steps might be required: (1) to culture the somatic cell nuclei for a sufficient time in MII oocyte cytoplasm to enhance nuclear reprogramming; and (2) to induce normal spindle formation with normal chromosomal construction.

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Developmental ability of cloned embryos from neural stem cells

Reproduction ◽

10.1530/rep.1.01010 ◽

2006 ◽

Vol 132 (6) ◽

pp. 849-857 ◽

Cited By ~ 42

Author(s):

Eiji Mizutani ◽

Hiroshi Ohta ◽

Satoshi Kishigami ◽

Nguyen Van Thuan ◽

Takafusa Hikichi ◽

...

Keyword(s):

Somatic Cell ◽

Success Rate ◽

Sertoli Cells ◽

Donor Cell ◽

Cumulus Cells ◽

Cloning Efficiency ◽

Cell Nuclei ◽

Developmental Potential ◽

Undifferentiated State

The success rate is generally higher when cloning mice from embryonic stem (ES) cell nuclei than from somatic cell nuclei, suggesting that the embryonic nature or the undifferentiated state of the donor cell increases cloning efficiency. We assessed the developmental ability of cloned embryos derived from cultured neural stem cell (NSC) nuclei and compared the success rate with that of embryos cloned from other donor cells such as differentiated NSCs, cumulus cells, Sertoli cells and ES cells in the mouse. The transfer of two-cell cloned embryos derived from cultured NSC nuclei into surrogate mothers produced five live cloned mice. However, the success rate (0.5%) was higher in embryos cloned from cultured NSC nuclei than from differentiated NSCs (0%), but lower than that obtained by cloning mice from other cell nuclei (2.2–3.5%). Although thein vitrodevelopmental potential to the two-cell stage of the cloned embryos derived from NSC nuclei (73%) was similar to that of the cloned embryos derived from other somatic cell nuclei (e.g., 85% in Sertoli cells and 75% in cumulus cells), the developmental rate to the morula–blastocyst stage was only 7%. This rate is remarkably lower than that produced from other somatic cells (e.g., 50% in Sertoli cells and 54% in cumulus cells). These results indicate that the undifferentiated state of neural cells does not enhance the cloning efficiency in mice and that the arrest point forin vitrodevelopment of cloned embryos depends on the donor cell type.

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Human artificial oocytes from patients’ somatic cells: past, present and future

Reproduction and Fertility ◽

10.1530/raf-20-0039 ◽

2021 ◽

Vol 2 (1) ◽

pp. H1-H8

Author(s):

Jan Tesarik ◽

Carmen Mendoza ◽

Raquel Mendoza-Tesarik

Keyword(s):

Somatic Cell ◽

Cell Types ◽

Ips Cells ◽

Cell Nuclei ◽

Human Oocytes ◽

Age Related ◽

Human Ips Cells ◽

Ovarian Diseases ◽

Induced Pluripotent ◽

The One

The first attempts at generating functional human oocytes by using the transfer of patients’ somatic cell nuclei, as DNA source, into donor enucleated oocytes date back to the early 2000s. After initial attempts, that gave rather encouraging results, the technique was abandoned because of adverse results with this technique in the mouse model. Priority was then given to the use of induced pluripotent stem (iPS) cells, based on excellent results in the mouse, where mature oocytes and live healthy offspring were achieved. However, these results could not be reproduced in humans, and oogenesis with human iPS cells did not continue beyond the stage of oogonium. These data suggest that the use of enucleated donor oocytes will be necessary to achieve fertilizable human oocytes with somatic cell-derived DNA. The main problem of all these techniques is that they have to meet with two, sometimes contradictory, requirements: the haploidization of somatic cell-derived DNA, on the one hand, and the remodeling/reprogramming of DNA of somatic cell origin, so as to be capable of supporting all stages of preimplantation and postimplantation development and to give rise to all cell types of the future organism. Further research is needed to determine the optimal strategy to cope with these two requirements. Lay summary The recourse to artificial oocytes, generated by using the patient’s own DNA derived from cells of somatic origin, represents the ultimate opportunity for women who lack healthy oocytes of their own but yearn for genetically related offspring. Many different pathologies, such as ovarian cancer, premature ovarian failure, other ovarian diseases and natural, age-related ovarian decay can cause the absence of available oocytes. The demand for artificial oocytes is increasing continuously, mainly because of the tendency to postpone maternity to still more advanced ages, when the quantity and quality of oocytes is low. This minireview focuses on the generation of artificial oocytes using different strategies and scenarios, based on the accumulated experience in humans and experimental animals.

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P-20. Haploidization of somatic cell nuclei by cytoplasm of human oocytes

Reproductive BioMedicine Online ◽

10.1016/s1472-6483(12)60103-5 ◽

2002 ◽

Vol 4 ◽

pp. 48-49 ◽

Cited By ~ 3

Author(s):

V Galat ◽

Y Verlinsky

Keyword(s):

Somatic Cell ◽

Cell Nuclei ◽

Human Oocytes

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Faculty Opinions recommendation of Visualization of actin filaments and monomers in somatic cell nuclei.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717982313.793476991 ◽

2013 ◽

Author(s):

Jan Lammerding

Keyword(s):

Somatic Cell ◽

Actin Filaments ◽

Cell Nuclei

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Introduction of Mouse Embryonic Fibroblasts into Early Embryos Causes Reprogramming and (Con)fusion

Cells ◽

10.3390/cells10040772 ◽

2021 ◽

Vol 10 (4) ◽

pp. 772

Author(s):

Pierre Savatier

Keyword(s):

Somatic Cell ◽

Cell Nuclei ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts ◽

Early Embryos

The reprogramming of somatic cell nuclei to achieve pluripotency is one of the most important biological discoveries of the last few decades [...]

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Effects of caffeine treatment on aged porcine oocytes: parthenogenetic activation ability, chromosome condensation and development to the blastocyst stage after somatic cell nuclear transfer

Zygote ◽

10.1017/s0967199405003370 ◽

2005 ◽

Vol 13 (4) ◽

pp. 335-345 ◽

Cited By ~ 23

Author(s):

Masaki Iwamoto ◽

Akira Onishi ◽

Dai-ichiro Fuchimoto ◽

Tamas Somfai ◽

Shun-ichi Suzuki ◽

...

Keyword(s):

Somatic Cell ◽

Blastocyst Stage ◽

The Other ◽

Control Group ◽

Blastocyst Formation ◽

Cell Nuclei ◽

Somatic Nucleus ◽

Membrane Breakdown ◽

Caffeine Treatment ◽

M Phase

The possibility of using aged porcine oocytes treated with caffeine, which inhibits the decrease in M-phase promoting factor activity, for pig cloning was evaluated. Cumulus–oocyte complexes (COCs) were cultured initially for 36h and subsequently with or without 5mM caffeine for 24h (in total for 60h: 60CA+ or 60CA– group, respectively). As a control group, COCs were cultured for 48h without caffeine (48CA–). The pronuclear formation rates at 10h after electrical stimulation in the 60CA+ and 60CA– groups decreased significantly (p<0.05) compared with the 48CA– group. However, the fragmentation rate was significantly higher (p<0.05) in the 60CA– group than in the 60CA+ and 48CA– groups. When the stimulated oocytes were cultured for 6 days, the 60CA+ group showed significantly lower blastocyst formation and higher fragmentation or degeneration rates (p<0.05) than the 48CA– group. However, the number of total cells in blastocysts was not affected by maturation period or caffeine treatment. When somatic cell nuclei were injected into the non-enucleated oocytes and exposed to cytoplasm for a certain duration (1–11h) before the completion of maturation (48 or 60h), the rate of nuclear membrane breakdown after exposure to cytoplasm for 1–2h in the 60CA– oocytes was significantly lower (p;<0.05) than in the other experimental groups. The rate of scattered chromosome formation in the same 60CA– group tended to be lower (p=0.08) than in the other groups. After the enucleation and transfer of nuclei, blastocyst formation rates in the 60CA+ and 60CA– groups were significantly lower (p<0.05) than in the 48CA– group. Blastocyst quality did not differ among all the groups. These results suggest that chromosome decondensation of the transplanted somatic nucleus is affected by both the duration of exposure to cytoplasm and the age of the recipient porcine oocytes, and that caffeine treatment promotes nuclear remodelling but does not prevent the decrease in the developmental ability of cloned embryos caused by oocyte aging.

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