RBMOnline - Vol 5. No 1. 26–35 Reproductive BioMedicine Online; www.rbmonline.com/Article/383 on web 10 May 2002
Articles Ooplasmic transfer: animal models assist human studies Henry Malter is senior scientist at the Institute for Reproductive Medicine and Science at St Barnabas Medical Center. Dr Malter completed his PhD degree in cell and developmental biology at Emory University in 1998. He has worked in the field of mammalian embryology for over 15 years. He was instrumental in the development of human clinical micromanipulation leading to the first human births following the application of such techniques in 1988. He has published numerous papers, book chapters and co-authored a textbook dealing with clinical micromanipulation. His areas of interest include the genetics and cell biology of preimplantation development as well as the improvement of clinical embryology methodology. Dr HE Malter Henry E Malter1, Jacques Cohen The Institute for Reproductive Medicine and Science of Saint Barnabas Medical Center, 101 Old Short Hills Road, Suite 501, West Orange, NJ 07052, USA 1Correspondence: Tel: +1-973–3226303; e-mail:
[email protected]
Abstract Ooplasmic transplantation is based on the premise that ooplasmic components are compromised in some individuals. In theory, the transfer of small amounts of healthy ooplasm can correct such deficits, allowing for improved development and implantation. The technique is based on a well-established background of experimental embryology demonstrating that cytoplasmic manipulation in oocytes and early embryos can be entirely compatible with normal development. Cytoplasm has been manipulated via karyoplast and cytoplast transfer and by cytoplasmic injection. Term development has been obtained following such manipulations in a variety of mammalian species. While some manipulative scenarios have exhibited compromised development, others have exhibited improved development. Developmental problems involving specific epigenetic and mitochondrial incompatibilities have been observed in a very limited subset of animal studies. These studies are based on genetic and physical models that have little relation to the actual substance of ooplasmic transplantation in the human. In fact, the majority of animal studies suggest that ooplasmic transplantation is well-founded and unlikely to result in negative developmental consequences. Furthermore, there are considerable physical, physiological and developmental differences between human and rodent eggs and embryos. These differences suggest that potentially negative issues raised by rodent results may not be relevant in the human.
Keywords: cytoplasmic transfer, epigenetics, mitochondria, mouse, ooplasmic transplantation
Introduction The technique of ooplasmic transplantation is based on the specific premise that ooplasmic factors are compromised in some individuals (Cohen et al., 1997, 1998; Brenner et al., 2000; Barritt et al., 2001c). Ooplasmic transplantation involves the transfer of small volumes of cytoplasm from donor to patient oocytes. Patients for ooplasmic transfer present following multiple assisted reproduction treatment cycles that have failed due to poor preimplantation development and cryptic failure of implantation apparently resulting from oocyte-related deficits. At this point, such patients have no other treatment options besides the use of donated oocytes as gamete replacements.
26
Ooplasmic transplantation is not intended to address general issues such as maternal age. It is intended only as a methodology for improving the developmental potential of Paper based on contribution presented at the Alpha meeting in New York, USA, September 2001.
compromised patient oocytes via the introduction of a theoretically healthy infusion of ooplasmic components. The transferred ooplasm is derived from oocytes obtained in what are essentially standard donor egg cycles. Egg donors used in ooplasmic transplantation procedures are selected and screened using criteria appropriate to standard egg donation. Ooplasm is transferred using a minor modification of the wellestablished intracytoplasmic sperm injection (ICSI) procedure and survival and fertilization are commensurate with standard ICSI results. The overall results following ooplasmic transplantation have been promising and indicate a positive effect on implantation and term development (Barritt et al., 2001c). While some congenital anomalies have been observed following ooplasmic transfer, any direct connection between the essentially common abnormalities observed and the technique itself has yet to be established and seems unlikely (Barritt et al., 2000, 2001a).
Articles - Animal models for ooplasmic transfer - HE Malter, J Cohen
In theory, factors are present in the overtly ‘healthy’ transferred ooplasm that, when introduced into compromised patient oocytes, have positive effects on the events of early development. This theory is based on a large body of embryological research demonstrating apparently dominant effects following ooplasmic manipulation (Muggleton-Harris et al., 1982; Gulyas et al., 1984; Pratt and Muggleton-Harris, 1988; Flood et al., 1990; Smith et al., 1991; Kono et al. 1992, 1996; Reik et al., 1993; Smith and Alcivar, 1993; Levron et al., 1996; Meirelles and Smith, 1998; Van Blerkom et al., 1998). Such effects have been attributed, in some cases, to specific messenger RNAs, proteins and mitochondria. However, in many studies, the source of these effects remains obscure, as is currently the case with the clinical technique. In some cases, manipulation of the oocyte and early embryo cytoplasm has resulted in negative developmental effects. These effects have been connected with the unique combination of nuclear, cytoplasmic, and mitochondrial components created by such manipulations (Latham and Sapienza, 1998; Nagao et al., 1998). However, these developmental and physiological effects have been observed under unique conditions in rodent models. Such models, based on highly artificial inbred and congenic strains, have little relation to the genetic and developmental characteristics of normal mammalian species, particularly the highly randombred human. They are also based on manipulative scenarios and outcomes that, in fact, have no direct relation to the specifics of the ooplasmic transplantation procedure. Considering the entirety of current knowledge on cytoplasmic manipulation and the interaction between nuclear, cytoplasmic, and mitochondrial components in development, it can be argued that the concept of ooplasmic transplantation is well founded. This paper constitutes a balanced review of embryology and developmental biology research related to the ooplasmic transplantation technique.
Basic experiments related to ooplasmic transplantation The creation of unique combinations of ooplasm is a wellestablished scenario in mammalian experimental embryology. While in a limited number of cases, highly specific nuclear/cytoplasmic combinations have resulted in aberrant developmental consequences, for the most part these types of manipulations have been entirely compatible with normal development. Furthermore, in some cases, such manipulation has resulted in apparently improved development.
Nuclear transfer Mammals including mice, sheep, bovines, goats, and rhesus monkeys have been produced following the transfer of nuclei from a variety of sources into enucleated oocytes and zygotes (McGrath and Solter, 1983; Willadsen, 1986; Kwon and Kono, 1996; Meng et al., 1997; Wilmut et al., 1997; Kato et al., 1998; Wakayama et al., 1998; Baguici et al., 1999). Nuclei have been transferred via both karyoplast fusion and direct injection. In karyoplast fusion, a bolus of cytoplasm is transferred along with the nucleus. Even in the case of direct nuclear injection some cytoplasmic components are probably transferred as well. Thus, these experiments create novel combinations of cytoplasm during early development and can be considered as
cytoplasmic manipulation. For the most part, development following nuclear transfer experiments has been compromised. This is no doubt related to deficits in artificial activation protocols and the, often substantial, differences in genetic background, developmental and/or cell cycle state that exists between the transferred nucleus and recipient cytoplasm particularly during ‘cloning’ scenarios (Kono et al., 1992; Fissore et al., 1999; Piotrowska et al., 2000). However, even following cases of extensive and invasive nucleo/cytoplasmic manipulation, development can be highly satisfactory when these issues are optimized. Nuclear transfer mouse embryos (enucleated ooplasts received a 4-cell stage nucleus and the resulting diploid genome re-transferred into enucleated zygote) developed in vitro to the blastocyst stage at a rate of 83% (Kwon and Kono, 1996). Following embryo transfer, 57% developed to term and a healthy second generation was produced following breeding of these founder nuclear-transfer animals (Kwon and Kono, 1996). Healthy mice have recently been produced following a drastic exchange of cytoplasm via reciprocal karyoplast transfer between synchronous cohorts of CB6 F1 hybrid mouse zygotes (Malter et al., 2001, in preparation). These mice have currently been bred to the third generation and show no signs of developmental or physiological problems.
Cytoplast transfer Other experiments, primarily in mouse zygotes, have involved the direct transfer of cytoplasm (in the form of cytoplasts) between different developmental stages and across separate strains and subspecies (Smith et al., 1991; Jenuth et al., 1996; Levron et al. 1996; Meirelles and Smith, 1997, 1998). While a complete evaluation of development was not a primary goal of these experiments, their results indicate that cytoplasmic transfer per se has no detrimental effect on development. In some experiments, the rate of preimplantation development to the blastocyst stage was identical between control embryos and those in which cytoplast transfer occurred (Smith et al., 1991; Meirelles and Smith, 1998). Overtly normal and healthy mice have been efficiently produced from such cytoplast transfer experiments and maintained over 15 generations (Meirelles and Smith, 1997; Takeda et al., 2000; LC Smith, personal communication). Preliminary work has demonstrated that the transfer of modest cytoplasts between metaphase II (MII) mouse eggs and mouse zygotes led to preimplantation development rates that were identical to those of control embryos (Levron et al., 1996). In these experiments, MII cytoplast transferred embryos actually exhibited implantation and day 10 viability rates that significantly exceeded those of non-manipulated embryos. This result not only clearly demonstrated the lack of a detrimental effect for cytoplasmic transfer, but suggested the presence of a positive effect under some scenarios.
Cytoplasmic injection The above mouse experiments are based on the isolation and transfer of membrane bound cytoplasm via cytoskeletal relaxant-facilitated manipulation and cell fusion. Other experiments have been reported in the mouse based on the isolation and transfer by ‘injection’ of oocyte or 2-cell embryo cytoplasm following treatment with cytochalasin D
27
Articles - Animal models for ooplasmic transfer - HE Malter, J Cohen
(Muggleton-Harris et al., 1982; Pratt and Muggleton-Harris, 1988). In these experiments, the development of outbred mouse embryos exhibiting the ‘2-cell block’ was in fact substantially improved following transfer of F1 hybrid cytoplasm. The synchronous exchange of homologous cytoplasm between F1 embryos and even the injection of outbred ‘blocking’ strain cytoplasm into F1 embryos resulted in preimplantation development that was essentially identical with controls. A limited set of experiments involving direct ooplasmic aspiration and injection was reported in Cynomolgous monkeys (Flood et al., 1990). In these experiments, cytoplasm was directly transferred between metaphase II and prophase I oocytes. Transfer of embryos derived from such cyto-transferred oocytes resulted in a pregnancy rate that was not significantly different from those of control transfers and several live births were obtained.
the mouse (Kimura and Yanagimachi, 1995). A piezomanipulator based ooplasmic transfer procedure which mimics the human procedure is currently being developed in the mouse (Malter et al., unpublished data). Current results indicate that while the smaller, more delicate mouse egg is more sensitive than human eggs to such manipulations, the aspiration/injection of cytoplasm between mouse eggs appears compatible with viability. Ooplasmic transfer recipient mouse eggs can be fertilized in vitro and exhibit satisfactory preimplantation development. Ongoing research will further expand on this system and address several issues including physical aspects of the ooplasmic transfer procedure and mitochondrial heteroplasmy. Experiments involving cytoplast transfer and direct injection are outlined in Figure 1. These experiments, as well as the human experimental trials involving ooplasmic transfer, are further summarized in Table 1.
Techniques such as cytoplast transfer via cell fusion and the transfer of cytochalasin D-treated cytoplasm are physically quite different from the ICSI-based ooplasmic transfer technique applied clinically in the human oocyte. Until recently, this type of technique was technically impossible in
Epigenetic aspects While these and other related experimental results have demonstrated that cytoplasmic transfer can be compatible with normal development, other experiments have suggested that
Table 1. Summary of animal experiments and human experimental trials involving ooplasmic transfer.
28
Donor cell
Recipient cell
Technique
Outcome
References
F1 hybrid mouse zygotes/2-cell embryos
Random-bred mouse zygotes/2-cell embryos
Cytochalasin D and injection
Improved preimplantation development
Muggleton-Harris et al., 1982
Cynomolgous monkey MII oocytes
Cynomolgous monkey prophase oocytes
Direct injection
Successful in-vitro maturation/live births
Flood et al., 1990
F1 hybrid and inbred mouse zygotes – differing mitochondrial backgrounds
F1 hybrid and inbred mouse zygotes – differing mitochondrial backgrounds
Cytoplast and karyoplast transfer + electrofusion
Normal development (depending on background) live births + creation of multiple multi-generational lines
Smith et al., 1991; Meirelles and Smith, 1997, 1998
F1 hybrid mouse MII oocytes
F1 hybrid mouse zygotes
Cytoplast transfer + electrofusion
Normal preimplantation development, improved implantation/viability (depending on volume/ stage of cytoplast)
Levron et al., 1996
Human MII oocytes from standard oocyte donors
Human MII oocytes from patients with repeated implantation failure
Direct injection via modified ICSI procedure
Several live births, apparently improved rate of implantation (over expectation from such patients)
Cohen et al., 1997, 1998
Human cryopreserved MII oocytes from standard oocyte donors
Human MII oocytes from patients with poor embryo quality or age >40 years
Direct injection via modified ICSI procedure
Live birth – twins (poor embryo quality group)
Lanzendorf et al., 1999
Human tri-pronucleate zygotes from random IVF patients
