Construction and fertilization of reconstituted human oocytes

doi:10.1016/S1472-6483(10)60838-3

Reproductive BioMedicine Online

Volume 11, Issue 3, 2005, Pages 309-318

https://doi.org/10.1016/S1472-6483(10)60838-3 Get rights and content

Abstract

Construction of artificial gametes may be made possible by transferring somatic cells into enucleated oocytes and inducing chromosomal halving of their nuclei. This study examines the possibility of constructing viable human gametes, and their potential for participation in normal fertilization. Spare germinal vesicle-stage oocytes were donated by consenting patients undergoing intracytoplasmic sperm injection (ICSI). Approximately 62% of in-vitro matured oocytes survived enucleation and subsequent cumulus cell injection. Following micromanipulation and subsequent activation, about 40% of the reconstituted oocytes yielded two pronuclear-like entities. This was not accompanied by extrusion of a polar body, but resulted in the formation of two ‘putative haploid’ pronuclei. Therefore selective removal of a female pronucleus marker was required to restore a balanced ploidy. Male pronuclei were identified by association with sperm mitochondria. Additional pronuclei were then removed, allowing further cleavage. Zygotes derived were ‘putatively haploid’ in ~38% of cases with a limited number of chromosomes assessed. However, on karyotypic analysis, blastomeres isolated from cleaving embryos showed a chaotic distribution of chromosomes. Oocytes could induce ‘putative haploidization’ of transplanted somatic cell nuclei independently of donor cell gender. Fertilization of artificial oocytes was followed by embryonic cleavage despite blastocyst development and chromosomal content possibly being compromised.

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References (73)

JA Barritt et al.
Mitochondrial DNA point mutation in human oocytes is associated with maternal age
Reproductive BioMedicine Online
(2000)
D Birnbacher
Human cloning and human dignity
Reproductive BioMedicine Online
(2005)
S Brunet et al.
Bipolar meiotic spindle formation without chromatin
Current Biology
(1998)
V Galat et al.
Cytogenetic analysis of human somatic cell haploidization
Reproductive BioMedicine Online
(2005)
ME Gaulden
Maternal age effect: the enigma of Down syndrome and other trisomic conditions
Mutation Research
(1992)
L Gianaroli et al.
Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for which it should be proposed
Fertility and Sterility
(1999)
L Gianaroli et al.
Preimplantation genetic diagnosis increases the implantation rate in human in vitro fertilization by avoiding the transfer of chromosomally abnormal embryos
Fertility and Sterility
(1997)
JB Gurdon
Reproductive cloning: past, present and future
Reproductive BioMedicine Online
(2005)
DL Keefe et al.
Mitochondrial deoxyribonucleic acid deletions in oocytes and reproductive aging in women
Fertility and Sterility
(1995)
SS Kim et al.
Follicular development, ovulation, and corpus luteum formation in cryopreserved human ovarian tissue after xenotransplantation
Fertility and Sterility
(2002)

O Lacham-Kaplan et al.

Fertilization of mouse oocytes using somatic cell as male germ cells

Reproductive BioMedicine Online

(2001)

S Munné et al.

Embryo morphology, developmental rates and maternal age are correlated with chromosome abnormalities

Fertility and Sterility

(1995)

ZP Nagy

Haploidization to produce human embryos: a new frontier for micromanipulation

Reproductive BioMedicine Online

(2004)

ZP Nagy

Micromanipulation of the human oocyte

Reproductive BioMedicine Online

(2003)

K Oktay et al.

Embryo development after heterotopic transplantation of cryopreserved ovarian tissue

Lancet

(2004)

K Oktay et al.

Cryopreservation of immature human oocytes and ovarian tissue: an emerging technology?

Fertility and Sterility

(1998)

GD Palermo et al.

Oocyte-induced haploidization

Reproductive BioMedicine Online

(2002)

GD Palermo et al.

Intracytoplasmic sperm injection: a novel treatment for all forms of male factor infertility

Fertility and Sterility

(1995)

E Porcu et al.

Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes

Fertility and Sterility

(1997)

JM Shaw et al.

Evaluation of the long-term function of cryopreserved ovarian grafts in the mouse, implications for human applications

Molecular Cell Endocrinology

(2000)

ES Sills et al.

Genetic and epigenetic modifications associated with human ooplasm donation and mitochondrial heteroplasmy – considerations for interpreting studies of heritability and reproductive outcome

Medical Hypothesis

(2004)

C Strong

The ethics of human reproductive cloning

Reproductive BioMedicine Online

(2005)

T Takeuchi et al.

Implications of cloning technique for reproductive medicine

Reproductive BioMedicine Online

(2004)

T Takeuchi et al.

Identification of the male pronucleus during fertilization of artificial oocytes

Fertility and Sterility

(2002)

T Takeuchi et al.

The efficiency of ‘production’ of mammalian haploid oocytes as a function of the cell cycle stage of the transferred somatic karyoplasts

Fertility and Sterility

(2000)

T Takeuchi et al.

An alternative source of oocytes

Human Reproduction

(1999)

H Tateno et al.

Inability of mature oocytes to create functional haploid genomes from somatic cell nuclei

Fertility and Sterility

(2003)

J Tesarik et al.

Fertilizable oocytes reconstructed from patient's somatic cell nuclei and donor ooplasts

Reproductive BioMedicine Online

(2001)

C Tietze

Reproductive span and rate of reproduction among Hutterite women

Fertility and Sterility

(1957)

JF van Uem et al.

Birth after cryopreservation of unfertilized oocytes

Lancet

(1987)

J Zhang et al.

In vitro maturation of human preovulatory oocytes reconstructed by germinal vesicle transfer

Fertility and Sterility

(1999)

DE Battaglia et al.

Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women

Human Reproduction

(1996)

F Beermann et al.

Maternal modulation of the inheritable meiosis I error Dipl I in mouse oocytes is associated with the type of mitochondrial DNA

Human Genetics

(1988)

G Capmany et al.

The timing of pronuclear formation, DNA synthesis and cleavage in the human 1-cell embryo

Molecular Human Reproduction

(1996)

CC Chang et al.

Nuclear and microtubule dynamics of G2/M somatic nuclei during haploidization in germinal vesicle-stage mouse oocytes

Biology of Reproduction

(2004)

C Chen

Pregnancies after human oocyte cryopreservation

Annals of the New York Academy of Sciences

(1988)

Cited by (23)

Excessive expression and activity of cathepsin B in sheep cumulus cells compromises oocyte developmental competence
2017, Small Ruminant Research
Citation Excerpt :
Cumulus cells play an important role in the nuclear and cytoplasm maturation of oocytes (Tanghe et al., 2002) and protect oocytes against oxidative stress-induced apoptosis (Tatemoto et al., 2000; Wongsrikeao et al., 2005). In addition, cumulus cells participate in mechanisms controlling sperm access, the selection, capacitation, and acrosome reactions of sperm, and the formation of male pronuclei (Liu et al., 2004; Rijsdijk and Franken, 2007; Takeuchi et al., 2005; Tanghe et al., 2002; Van et al., 2002). Because of the critical role of cumulus cells, high level of apoptosis in cumulus cells would compromise the quality and developmental competence of oocytes (Albertini et al., 2001; Corn et al., 2005).
In vitro developmental competence of sheep oocytes is still relatively low. In this study, we investigated cathepsin B activity in sheep cumulus-oocyte complexes, the expression of cathepsin B and apoptosis rates in sheep cumulus cells, and the correlations between these indicies and in vitro developmental competence of sheep oocytes. The present result suggested that the active form of cathepsin B in cumulus-oocyte complexes was predominantly present in cumulus cells rather than oocytes. Cathepsin B activity in cumulus-oocyte complexes was strongly inhibited by cathepsin inhibitor E-64. RT-PCR analysis showed that cathepsin B expression in cumulus cells decreased during in vitro maturation. TUNEL staining and flow cytometry of cumulus cells showed that the apoptosis rate increased after in vitro maturation, and treatment with E-64 reduced both cathepsin B expression and apoptosis levels. Furthermore, E-64 treatment increased blastocyst rates of sheep.
Cumulus cell apoptosis changes with exposure to spermatozoa and pathologies involved in infertility
2009, Fertility and Sterility
To determine whether the incidence of apoptosis in mature oocyte cumulus cells changes after insemination related to infertility.
Prospective study.
Public hospital and university.
One hundred women undergoing in vitro fertilization and embryo transfer (IVF-ET).
Collection of cumulus cells from IVF-ET cycles with different infertility etiologies.
Detection of apoptosis in cumulus cells; fertilization, embryo quality, and pregnancy rate.
The incubation of cumulus–oocyte-complexes with spermatozoa led to an increase in cumulus cell apoptosis from 34.2 ± 3.7 to 44.5 ± 6.3%. After insemination, cumulus cells of poor quality embryos showed a statistically higher apoptotic rate versus cumulus cells of good quality embryos (61.5 ± 6.4 vs. 40.6 ± 3.9%). Cumulus cells arising from oocytes with ≥50% fertilization rates after insemination showed higher apoptosis rates did cumulus cells from oocytes with <50% fertilization rates (46.0 ± 3.7 vs. 33.8 ± 4.0%). Patients with endometriosis presented higher apoptotic rates before insemination (77.6 ± 9.06%). Cumulus cells obtained after aspiration showed no differences in their apoptosis rates for the following factors: age of women, aspirated oocytes, estradiol level, fertilization rate, and embryo quality or pregnancy. The apoptotic profile from pregnant women was less than (but not statistically significantly different from) profiles from nonpregnant women.
These results suggest that the incidence of apoptosis in cumulus cells is associated with exposure to spermatozoa and the cause of infertility.
Development of artificial gametes
2008, Reproductive BioMedicine Online
Virtually all normal cells can reproduce themselves. The germ cells, however, can initiate reproduction of the entire organism. A special sequence of events, meiosis, is responsible for generating mature germ cells bearing a haploid genome. The basic features of meiosis, i.e. two cell divisions with no intervening DNA replication, resulting in a halving of the chromosome complement, are evident throughout evolution. The idea of generating an artificial gamete was recently put forward to provide an ultimate solution for the treatment of infertility. Currently, there are different strategies to create artificial gametes in vitro, such as converting somatic cells from mitotic division to meiotic division directly (somatic cell haploidization), or de-differentiating somatic cells into embryonic stem (ES) cells and re-differentiating ES cells into gametes, or extracting adult stem cells and re-differentiating them into gametes.
Duplication of the sperm genome by human androgenetic embryo production: Towards testing the paternal genome prior to fertilization
2007, Reproductive BioMedicine Online
There is currently no technique for evaluating the sperm genome before fertilization. However, sperm genome duplication could offer a way forward, whereby one of the sister blastomeres of a 2-cell haploid androgenetic embryo could be analysed. A method was developed for production of human androgenotes by enucleation of oocytes at telophase II (TII) after intracellular sperm injection (ICSI). The results were compared with those obtained via the more usual procedure of oocyte enucleation at metaphase II (MII) prior to ICSI. TII enucleation led to an improvement in the rate of embryo survival, increased the production rate of 1PN-embryos, and also the production of 2- to 8-cell-stage embryos (85.0, 74.9 and 65.8% in TII enucleation, versus 73.8, 48.9 and 33.3% in MII enucleation). Fluorescence in-situ hybridization (FISH) analysis of 30 2- to 5-cell androgenic embryos for two to seven chromosomes revealed the correct chromosome distribution in 76.7% of haploid human androgenotes.
Butyrolactone I reversibly alters nuclear configuration, periooplasmic microtubules and development of porcine oocytes
2007, Theriogenology
In the present study, we investigated the effects of specific cdc2 kinase inhibitor, butyrolactone I (BL I) on the prevention of germinal vesicle breakdown, changes of microtubular structures, and development of porcine oocytes after removal of the drug. In Experiment 1, cumulus–oocyte complexes (COCs) were cultured (44 h) in NCSU-23 medium containing different concentrations of BL I. The percentages of oocytes remaining at GV stage were 0, 0, 32, 80, and 84% (P < 0.05), and the maturation rates were 86, 63, 30, 0, and 0% (P < 0.05) for oocytes treated with 0, 10, 20, 40, and 80 μM of BL I, respectively. When oocytes were released from BL I incubation (Experiment 2) and cultured for an additional 44 h, 79, 84, and 83% of oocytes resumed meiosis, but only 52, 38 and 17% of oocytes reached normal metaphase II (MII) stage in the groups treated with 20, 40 and 80 μM BL I, respectively. In Experiments 3–5, reversibility and development of oocytes and embryos were evaluated after removal of the inhibitor. A reduced duration of BL I incubation (22 h) at 20 μM increased the percentage of oocytes remaining at the GV stage compared to the control group (85% versus 9%, P < 0.05). Blastocyst rates were lower in treatment groups than in the control (44 h) group (0–14% versus 24%; P < 0.05). However, all developing blastocysts possessed similar cell numbers, regardless of the drug-treated or non-treated controls. Taken together, treatment with 20–80 μM of BL I effectively prevented the resumption of meiosis and polymerization of periooplasmic microtubules. Furthermore, reversibility of the oocytes after reduced duration of BL I treatment was satisfactory.
The Use of Micromanipulation Methods as a Tool to Prevention of Transmission of Mutated Mitochondrial DNA
2007, Current Topics in Developmental Biology
Citation Excerpt :
The morphology of human zygotes is, however, completely different because pronuclei are clearly visible in living zygotes (Fulka et al., 2004). In fact, the correction of polyploidy, when one pronucleus is removed from tripronuclear zygotes, uses some of the steps described above (Takeuchi et al., 2005b). Thus, in theory, we can expect that the pronuclear exchange between zygotes could be successfully used also in humans for the elimination of certain detrimental cytoplasmic defects.
The introduction of different micromanipulation techniques into reproductive and developmental biology has helped us not only to answer many essential biological questions but it is now evident that these techniques also have wide practical applications. In human‐assisted reproduction, the most commonly used approach is the injection of a donor sperm into the oocyte cytoplasm—intracytoplasmic sperm injection. It is, however, speculated that with these techniques it would be also possible to improve the oocyte developmental potential especially in those cases when the quality of the cytoplasm is rather poor and thus its function is compromised. Another important application would be the elimination of mutated mitochondrial DNA (mtDNA) by transferring the nuclear material from an abnormal oocyte into a healthy donor oocyte cytoplast. Some of these techniques were already successfully tested in experimental animals, but it is evident that before their introduction into human medicine many questions must be answered, and we must be sure that these approaches are absolutely or almost absolutely safe. In our contribution, we will be specifically oriented to the nuclear (nuclear material) replacement approaches that could be potentially used to prevent the transmission of mutated mtDNA from mother to offspring. Because these techniques are very delicate, some training with oocytes from other species other than human is strongly recommended. © 2007, Elsevier Inc.

View all citing articles on Scopus

Dr Takumi Takeuchi completed his MD and PhD training at Gunma University, Japan. At the same university, from June 1993 to March 1997, Dr Takeuchi was co-responsible for the ART programme. In April 1997, he was awarded a research fellowship at the Weill Medical College of Cornell University where together with Gianpiero Palermo he established and pioneered nuclear transplantation procedures in mouse and human oocytes. In September 2002, due to his outstanding research work, he became Assistant Professor of Reproductive Medicine and was also recruited into the Embryology laboratory. Dr Takeuchi routinely performs ICSI as well as genetic testing and epigenetic analysis on gametes and embryos. He has devoted himself to devising novel therapeutic options to treat age-related infertility, such as germinal vesicle transplantation and somatic cell haploidization. On this topic, Dr Takeuchi has published many reports and delivered several lectures before international audiences. Recently, he is working on embryonic stem cell harvesting and differentiation.

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ArticleConstruction and fertilization of reconstituted human oocytes

Abstract

Section snippets

Reproductive BioMedicine Online

Reproductive BioMedicine Online

Current Biology

Reproductive BioMedicine Online

Mutation Research

Fertility and Sterility

Fertility and Sterility

Reproductive BioMedicine Online

Fertility and Sterility

Fertility and Sterility

Reproductive BioMedicine Online

Fertility and Sterility

Reproductive BioMedicine Online

Reproductive BioMedicine Online

Lancet

Fertility and Sterility

Reproductive BioMedicine Online

Fertility and Sterility

Fertility and Sterility

Molecular Cell Endocrinology

Medical Hypothesis

Reproductive BioMedicine Online

Reproductive BioMedicine Online

Fertility and Sterility

Fertility and Sterility

Human Reproduction

Fertility and Sterility

Reproductive BioMedicine Online

Fertility and Sterility

Lancet

Fertility and Sterility

Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women

Human Reproduction

Maternal modulation of the inheritable meiosis I error Dipl I in mouse oocytes is associated with the type of mitochondrial DNA

Human Genetics

The timing of pronuclear formation, DNA synthesis and cleavage in the human 1-cell embryo

Molecular Human Reproduction

Nuclear and microtubule dynamics of G2/M somatic nuclei during haploidization in germinal vesicle-stage mouse oocytes

Biology of Reproduction

Pregnancies after human oocyte cryopreservation

Annals of the New York Academy of Sciences

Article
Construction and fertilization of reconstituted human oocytes