Placenta
Volume 29, Supplement 2 , Pages 121-125, October 2008

Parthenogenetic Activation: Biology and Applications in the ART Laboratory

  • A. Paffoni

      Affiliations

    • Fondazione IRCCS Ospedale Maggiore Policlinico Mangiagalli e Regina Elena, Infertility Unit, via M. Fanti 6, 20122 Milan, Italy
    • Corresponding Author InformationCorresponding author. Tel.: +39 02 55034305; fax: +39 02 55034302.
    • ,
  • T.A.L. Brevini

      Affiliations

    • Laboratory of Biomedical Embryology, Center for Stem Cell Research, University of Milan, via Celoria 10, 20133 Milan, Italy
    • ,
  • F. Gandolfi

      Affiliations

    • Laboratory of Biomedical Embryology, Center for Stem Cell Research, University of Milan, via Celoria 10, 20133 Milan, Italy
    • ,
  • G. Ragni

      Affiliations

    • Fondazione IRCCS Ospedale Maggiore Policlinico Mangiagalli e Regina Elena, Infertility Unit, via M. Fanti 6, 20122 Milan, Italy

Accepted 7 August 2008. published online 09 September 2008.

Article Outline

Abstract 

Parthenogenesis is a reproductive strategy typical of lower species where a female gives birth to offsprings without a paternal contribution. On the contrary, parthenogenesis is not a form of natural reproduction in mammals even if mammalian oocytes, under appropriate stimuli, can undergo to parthenogenetic activation. This review describes the biological mechanisms regulating parthenogenetic activation in mammals and illustrates the fundamental differences between embryos and parthenotes. Ethical, legal and political concerns on the value of human embryos regulate and limit human embryological studies founded on the widespread belief that human embryos should not be created and studied for research purposes only. Based on the differences between parthenotes and embryos the use of parthenogenesis is proposed as an experimental tool to investigate embryo development which may solve many of the ethical concerns associated with the use of human embryos for experimental purposes. Examples of the possible uses of parthenotes in many field of research such as in vitro assays aimed to study some aspects of assisted reproductive technologies (ART), toxicology or stem cell are described and their validity is discussed.

Keywords: Parthenogenesis, IVF, Embryo, Experimental model, Ethical tool

 

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1. Introduction 

Parthenogenesis is a reproductive strategy in some species of insects such as flies, ants, and honeybees and vertebrates such as lizards, snakes, fish and amphibians [1] where a female gives birth to offsprings without a paternal contribution. In this process, a single egg can develop to term in the absence of a male genetic counterpart and without meiotic chromosome reduction.

Parthenogenesis is not reported as a form of natural reproduction in mammals even if mammalian oocytes, under appropriate stimuli, can undergo to parthenogenetic activation in vivo or in vitro which mimics embryonic development in its early phases. Thus far, parthenotes obtained in vitro have been studied and transferred in the uterus of recipient females in a variety of mammals including mice [2], sheep [3], cows [4], pigs [5], rabbits [6] and monkeys [7]. These studies evidenced that mouse parthenotes can develop beyond implantation until the forelimb bud stage [8]; rabbit parthenotes until day 11 postactivation [6] while parthenotes from primates have only been shown to reach the implantation stage [7].

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2. Mechanisms inhibiting parthenote development 

The failure of mammal parthenotes to develop to term is due to the absence of paternal genome: this peculiarity leads to an abnormal regulation of differentiation and proliferation mainly in extra-embryonic lineages, resulting in a poor support of embryonic growth [9]. Studies with mouse chimeras and teratomas evidenced that parthenogenetic cells lines do not contribute efficiently to mesodermal lineages and to extra-embryonic tissues, such as trophectoderm and primitive endoderm lineages [10].

It is well documented that maternal and paternal genomes are quantitatively identical but not functionally equivalent, due to complementary epigenetic modifications which lead to differential roles and gene expression in the embryo [9], [11], therefore the unbalanced development typical of parthenotes is due to the lack of expression of the paternal copies of some genes which are crucial for development of extra-embryonic tissues [12]. The separate contribution of the maternal and paternal inherited alleles of some genes to the correct development of the embryo is mediated by an epigenetic phenomenon that selectively silences or promotes the expression of one of them named ‘genetic imprinting’. Gene imprinting does not limit its effect to early embryonic development but imprinted genes are associated with human diseases, including disorders affecting development and behaviour such as Beckwith–Wiedemann, Prader–Willi, Angelman and Russel–Silver Syndromes [13], [14].

Kono et al. [15], with some elegant manipulations of mouse parthenotes, provided the formal evidence that imprinting is the phenomenon limiting the development of mammalian embryos containing only female-derived chromosomes.

H19 and Igf2 are two imprinted genes whose coordinate regulation by cis-acting elements is crucial in murine embryogenesis [16], [17]. These neighbouring genes share an enhancer but expression is only from the maternal copy of H19 and only from the paternal copy of Igf2. On the maternal chromosome, the H19 differentially methylated domain (DMD) is unmethylated and allows the binding of the protein CTCF; this complex prevents enhancers from interacting with Igf2, instead promotes their interaction with H19 and subsequent transcription. On the paternal allele, DMD is methylated and therefore the CTCF protein is unable to bind the sequence: this condition allows enhancers to bind Igf2 and promotes its transcription and, at the same time, prevents transcription of the downstream H19 gene [18]. In conventional parthenotes with two maternal genomes, Igf2 is not expressed and development is impaired. Kono et al. [15] obtained parthenotes combining chromosomes from mature oocytes of normal mice, bearing all maternal imprints, with chromosomes obtained from a non-growing oocytes, thus not imprinted, from mutant mice, whose DMD and H19 regions were deleted. These deletions resembled the paternal imprinting of H19 and enabled therefore Igf2 transcription. This approach allowed the authors to obtain a viable parthenogenetic mouse from a reconstructed oocyte with two maternal genomes.

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3. Parthenogenetic activation and genetic make-up of parthenotes 

Parthenotes can be efficiently obtained in vitro with a variety of mechanical, chemical, and electrical stimuli using oocytes of several species at different stages along oocyte meiosis resulting in parthenotes with different chromosome complements. According to the method of production, mammalian parthenotes can be either haploid or diploid. The developmental capacity of parthenotes is influenced by the resulting ploidy and genetic information. In some species, the development of both haploid and diploid parthenotes has been reported. These studies demonstrated that the haploid condition can impair cleavage at early stages [19], [20], [21], [22].

Parthenogenetic activation can be performed in oocytes at the second metaphase resulting in the extrusion of the second polar body and leading to the formation of a haploid parthenote. This method is rarely used since, in this case, the developmental competence is reduced compared to normal embryos and to diploid parthenotes [23].

Diploid parthenotes are easily obtained: they can be derived from metaphase-2 oocytes whose sister chromatids of the chromosome segregate without being extruded into the second polar body: thus the oocyte retains its diploid status, either homozygous or including cross-over associated heterozygosity in a single pronucleus. However, even the failure of diploid parthenotes to sustain a prolonged development highlights the need for bi-parental chromosome complement and, indirectly, the different and sometimes opposite developmental role of imprinted genes. Diploid parthenotes can be obtained in two main different ways. The most common one consists in combining the activation of metaphase-2 oocytes with exposure to an inhibitor of the extrusion of the second polar body without affecting the formation and movement of pronuclei [24]. Alternatively, a diploid parthenote can be generated by treating the oocyte with cytochalasin D during in vitro maturation before activation. This drug binds to the positive end of F-actin and blocks further addition of G-actin monomers preventing the extrusion of the first polar body. This protocol leads to the formation of tetraploid oocytes [25]. The diploid status is then re-established at the end of oocyte maturation with the extrusion of the second polar body.

Using one or the other method has important consequences on the genetic make-up of the parthenotes. In fact, performing the oocyte activation before the inhibition of the second polar body extrusion determines the formation of highly homozygous parthenotes, since the diploid status of the parthenotes is obtained after the segregation of sister chromatids. In this case, the degree of heterozygosity depends only on the extent of crossing over taking place during the prophase of the first meiotic division which is very limited in most species [26]. On the contrary, extrusion of homologous chromosomes does not take place when the first polar body extrusion is inhibited. In this case activation is induced in a tetraploid oocyte and the diploid status is reached again after the extrusion of the second polar body. As a consequence, the segregation of sister chromatids occurs only at this stage, therefore the parthenotes have the same proportion of chromosomes derived from the mother and the father of the donor as the oocyte and its donor. Parthenotes generated in this way, not only are genetically identical to each other but have the same heterozygosity of their mother [25].

During fertilization, sperm entry triggers intracellular calcium oscillations causing activation of the oocyte. After a few hours, a series of events makes fertilization complete, through the inactivation of maturation promoting factor (MPF) and of mitogen-activated protein kinase that leads to resumption and completion of meiosis, DNA synthesis and pronuclei formation. Intracellular calcium rise can be induced in the oocytes without a spermatozoa, using several activating agents, such as ethanol, Ca++ ionophores, and electroporation: as a consequence, oocyte is released from metaphase arrest but levels of MPF are not properly reduced and further development is impaired [27]. In order to overcome this limitation, additional agents inhibiting protein synthesis or protein phosphorylation can be added and higher activation and in vitro development rates are observed in several species including primates [28].

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4. Parthenogenetic activation of human oocytes 

In humans, spontaneous in vivo parthenogenesis is supposed to be the origin of a subgroup of ovarian teratomas [29] and a parthenogenetic chimaerism was demonstrated in a young patient suffering for developmental and sexual disturbances [30]. In vitro parthenogenesis has been described as an occasional phenomenon among oocytes retrieved during in vitro fertilization (IVF) cycles [31] and can be intentionally induced for research purposes [32]. The exposure of metaphase-2 human oocytes to a calcium ionophore alone or followed by a protein synthesis inhibitor leads to high activation rates but poor parthenote development [33]. However, if a protein kinase inhibitor is added to the culture medium after activation, better developmental rates of parthenotes can be obtained [34]. Elevation of intracellular Ca++ levels with ionomycin followed by inhibition of protein phosporylation with 6-dimethylaminopurine (6-DMAP) results in efficient oocyte activation and better development of parthenotes, even to the blastocyst stage [34], [35], [36]. One possible explanation is that 6-DMAP causes the inhibition of MPF reactivation, triggering a kinetic similar to that occurring after fertilization in bovine [37] and pig oocytes [38]. According to these observations, an effective parthenogenetic activation protocol can be conducted on viable metaphase-2 oocytes through sequential exposure to 5μM ionomycin in fertilization medium, followed by an incubation in 2mM 6-DMAP in cleavage medium. Oocytes are then cultured in standard conditions and after 18–20h (day 1), considering the exposure to ionomycin as time 0, oocytes can be evaluated for signs of activation. Oocytes showing one enlarged pronucleus and no extrusion of the second polar body are considered activated [36]. Parthenotes can be kept in culture in fresh cleavage medium for up to 5 or 6 days, when blastocysts are expected to form. Such evidence highlights a certain developmental and differentiative potential of human parthenogenetic embryos; however, as any other mammalian parthenote, they would fail to develop much further because of the genetic constraints detailed above.

Nevertheless, the ability of human parthenotes to sustain early embryological development until the blastocyst stage, makes them an interesting experimental model in many research fields related to embryology, stem cells and regenerative medicine [39].

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5. Parthenogenetic activation as an experimental model in human ART 

Research in the field of human embryology presents a series of specific limits related to ethical concerns. Many people regard in vitro-obtained embryos as human beings even though there is a complete lack of consensus on when the moral and legal status of the developing conceptus should be recognized. Numerous national legislatures or guidelines have therefore banned the use of embryos, even supernumerary or discarded, for research purposes. The International Federation of Fertility Societies (IFFS) Surveillance 2007 reports that about half of the 57 surveyed nations indicated that experimentation on human “pre-embryos” is unacceptable [40]. Authorized researches are always subjected to time limits with respect to embryonic developmental stage and special cautions are mandatory for studies on embryos. Ethical, legal and political concerns on the value of human embryos regulate embryological studies and, similarly to any other study involving patients, impose that research projects are preliminary examined by Institutional Review Boards, use informed consent forms, follow international guidelines, such as the Declaration of Helsinky [41] or World Health Organization Ethical Guidelines [42], founded on the widespread belief that human embryos should not be created and studied for research purposes only.

In vitro fertilization (IVF) consists of a series of techniques in which egg cells are fertilized by sperm outside the woman's body, with the aim of obtaining viable developing embryos. Many variables contribute to the success or the failure of IVF procedures. A significant part of them belongs to the early embryological phases of the process but pathophysiologic conditions of the patients and clinical aspects of the entire process also play important roles. Therefore, the optimal outcome for studies comparing different approaches or techniques related to IVF is the live birth rate. However, due to the fact that live birth rate is generally between 20% and 30% per procedure, studies in this field need large cohorts of patients in order to reach enough statistical power to demonstrate expected variations of a few percentage points. On the contrary, limiting the outcome of experimental studies to in vitro development or to morphological and genetic analysis of could be an option but the design of such studies is limited by the above mentioned ethical concerns.

On this basis, parthenogenesis can be proposed as a possible embryological model since parthenote development is virtually identical to embryo development as observed in several animal models [28], [43], [44]. Experimental models based on parthenogenetic activation [36] would overcome many of the ethical limitations to research on human embryos and offer an interesting and valuable tool to evaluate the developmental potential of oocytes exposed to experimental conditions. In the recent years a few studies described different procedures for the parthenogenetic activation of human oocytes which allow the development of the parthenotes to the blastocyst stage with a satisfactory efficiency that ranged from 16.6% [45] to 28.6% [34]. However evidences that primate parthenote development could reflect embryo development was available only in rhesus monkey [28]. Therefore we recently tried to validate the use of human parthenotes as experimental model performing a direct comparison of the in vitro development between fertilized and activated oocytes [45]. Our results indicated that activation rate was not statistically different from fertilization rate and no significant differences in embryonic development at 42–48 and 66–68hours postinsemination or activation were observed between the two randomized groups. Furthermore, blastomeres number was not different between parthenotes and embryos. However, based on embryological criteria of integrity and size of blastomeres, parthenotes showed a lower average morphological grade on day 3 postactivation. This is consistent with the suboptimal morphology documented among parthenotes in other mammalian species [28], [46], [47]. In our study, however, blastocyst rate on day 5 after activation, was lower than previously reported [34], [45]. We believe that the reason of this difference probably relies on the relatively high mean age (35.2 years) of the infertile donor patients recruited for this study which is known to be an important factor, strongly influencing the success rate of both in vitro fertilization and parthenogenetic development [43], [44]. This, in a way, further confirms that parthenogenetic activation closely mimics the results obtainable with normal fertilization. At the same time it points to the limits inherent to the use of discarded, unfertilized or aged oocytes as this material cannot be considered appropriate to evaluate developmental potential [48].

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6. Experimental applications of human parthenogenesis 

According to these principles, we recently designed a randomized in vitro study aimed to compare oocytes survival following cryopreservation with slow freezing or vitrification as well as their subsequent in vitro developmental competence after parthenogenetic activation. The rate of supernumerary metaphase-2 oocytes which survived after slow freezing or vitrification was comparable as it was the activation rate. However, although the first developmental stages were comparable between the two groups, a higher cleavage rate was documented three days after activation in parthenotes obtained from oocytes allocated to slow freezing [49]. Besides our specific results it is noteworthy that in this study we were able to compare a standardized slow freezing protocol with a more experimental vitrification procedure without using any embryo, indicating that this experimental approach can be useful and relevant for the further refinement of oocyte cryopreservation protocols.

Consistent with our findings, a recent paper [48] showed that non-inseminated cryopreserved human oocytes, undergo very satisfactorily to artificial activation (86.1%) and subsequent parthenogenetic embryos (96.8%) and blastocysts development (16.7%), similarly to fresh oocytes. Authors suggest that parthenogenesis with non-inseminated cryopreserved oocytes could represent for scientists an opportunity to work in the field of embryology in countries with restrictions on the use of human embryos.

Research in the field of human embryology not only involves IVF procedures but also has gained great interest since human embryonic stem-cell lines have been shown to have enormous therapeutic and research potential (for a review, see Ref. [50]); however, the derivation of embryonic stem cells currently implies the destruction of living embryos. In order to overcome the opposition to the use of human embryonic stem cell, many groups worldwide have tried to develop new strategies to replace the current method for deriving such cell lines. Among the available alternatives, parthenogenesis is one of the possible approaches [51], [52], which include the use of embryos after irreversible arrest of cellular division [53]; of chromosomally abnormal embryos, incapable of developing to term [54]; the biopsy of single blastomere from early embryos [55]; the de-differentiation of somatic cell lines [56]; or the epigenetic reprogramming of adult cells with the transfection of factors, that seems to play a crucial role in the induction and maintenance of pluripotency [57], [58]. Each of these techniques presents various degrees of practical and ethical limitations as well as many scientific challenges.

Parthenotes have been shown to be a reliable source of pluripotent cell lines and different groups have recently reported the derivation of parthenogenetic cell lines that exhibit features typical of bi-parental stem cells and that stably maintain them in culture. These cells exhibited high plasticity when subjected to differentiation protocols, and the ability to form embryoid bodies in hanging drops culture [51], [52], [59], [60].

As parthenotes originate from single eggs, they represent a potential source of histocompatible stem cells that should be isogenic with the oocyte donor and considerably reduce the complexity of tissue matching for transplant purposes [34]; therefore they are suggested as highly suitable for applications in cell or tissue replacement therapy and could be used to establish a bank of histocompatible cell lines for a broad spectrum of patients. Homozygosity can be seen as a potential benefit when the reduction of immunogenicity of a stem-cell derivative is considered [61], [62]. The possibility to generate stem cells which are homozygous for all three sets of HLA (A, B and DR), would exponentially increase the number of phenotypes a graft can fully match. Furthermore, homozygosity has also been suggested to be an advantage to be exploited for selecting cell lines carrying drug response genes, a disease gene or a cancer gene providing a useful research tool for drug testing and development [34]. At the same time, it must be remembered that homozygosity can represent a severe risk, accompanied with chromosome aneuploidy and carcinogenesis in humans, being the result of a uniparental disomy associated with loss of imprinting. Loss of imprinting as loss of heterozygosity, in fact, may have unpredictable effects and can be associated with increased risk of cancer. Loss of imprinting of Igf2 has been shown to be a risk factor for colorectal cancer both in humans and in animal models (for a review, see Ref. [63]).

Epigenetic instability, due to their exclusively maternal origin, is therefore a major safety issue for the use of these cell lines in regenerative medicine and substantial studies are mandatory in order to better elucidate all these aspects prior to advocate a possible use of parthenogenesis as a source of stem cells in humans. At present, a more realistic application of parthenotes and derived stem lines could be the use as surrogate embryos for pharmacological and toxicological research in its in vitro phases.

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7. Conclusions 

Using unfertilized eggs, for research, is commonly judged less controversial than using embryos, as many ethical concerns are avoided. However, parthenotes are not entirely free from ethical controversy since are considered by some as artificial entities challenging natural rules. These contradictions await resolution in a broad ethical framework [1].

Nevertheless, during their in vitro development to the blastocyst stage, parthenotes are comparable to embryos and therefore are useful tools for any research aimed at investigating culture conditions, different treatment options, exposure to chemicals and many variables of the laboratory routine.

Parthenotes can also be an interesting source of stem cells due to the unique advantage of homozygosity, which renders them less immunogenic. The occurrence of a high degree of homozygosity has been evaluated in contrasting ways in the perspective of using these entities as a source of embryonic stem cells.

The use of parthenotes as experimental models in human reproduction is at its early days. Future studies will define more in detail its real advantages and limitations.

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8. Conflict of interest 

The authors have no conflict of interest.

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PII: S0143-4004(08)00242-7

doi:10.1016/j.placenta.2008.08.005

Placenta
Volume 29, Supplement 2 , Pages 121-125, October 2008

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