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Available online at www.sciencedirect.com

Theriogenology 77 (2012) 766–772

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Review

Parthenogenesis in non-rodent species: developmental competenceand differentiation plasticity

T.A.L. Brevini*, G. Pennarossa, A. Vanelli, S. Maffei, F. GandolfiUnistem, Centre for Stem Cell Research, Laboratory of Biomedical Embryology, Università degli Studi di Milano, Italy

Received 4 March 2011; received in revised form 19 October 2011; accepted 24 November 2011

Abstract

An oocyte can activate its developmental process without the intervention of the male counterpart. This form of reproduction, known asparthenogenesis, occurs spontaneously in a variety of lower organisms, but not in mammals. However, it must be noted that mammalian oocytescan be activated in vitro, mimicking the intracellular calcium wave induced by the spermatozoon at fertilization, which triggers cleavageivisions and embryonic development. The resultant parthenotes are not capable of developing to term and arrest their growth at different stages,epending on the species. It is believed that this arrest is due to genomic imprinting, which causes the repression of genes normally expressedy the paternal allele. Human parthenogenetic embryos have recently been proposed as an alternative, less controversial source of embryonictem cell lines, based on their inherent inability to form a new individual. However many aspects related to the biology of parthenogeneticmbryos and parthenogenetically derived cell lines still need to be elucidated. Limited information is available in particular on the consequencesf the lack of centrioles and on the parthenote’s ability to assemble a new embryonic centrosome in the absence of the sperm centriole. Indeed,n lower species, successful parthenogenesis largely depends upon the oocyte’s ability to regenerate complete and functional centrosomes inhe absence of the material supplied by a male gamete, while the control of this event appears to be less stringent in mammalian cells. In anttempt to better elucidate some of these aspects, parthenogenetic cell lines, recently derived in our laboratory, have been characterized for theirluripotency. In vitro and in vivo differentiation plasticity have been assessed, demonstrating the ability of these cells to differentiate into cellypes derived from the three germ layers. These results confirmed common features between uni- and bi-parental embryonic stem cells.owever data obtained with parthenogenetic cells indicate the presence of an intrinsic deregulation of the mechanisms controlling proliferations. differentiation and suggest their uni-parental origin as a possible cause.

2012 Elsevier Inc. All rights reserved.

Keywords: Parthenogenesis; Mammalian; Pluripotency; Differentiation; Centriole

Contents

1. Introduction .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7672. Parthenogenesis, asymmetric imprinting and development .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7673. Differences between IVF-embryos and parthenotes as a source of stable pluripotent

cell lines .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768. Self-renewal of parthenogenetic cell lines .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768. Differentiation ability of parthenogenetic

cell lines .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 769

* Corresponding author. Tel: 0039(8) 02 503179880; fax: 0039(8) 02 50317989.

www.theriojournal.com

E-mail address: tiziana.brevini@unimi.it (T.A.L. Brevini).

093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved.oi:10.1016/j.theriogenology.2011.11.010

767T.A.L. Brevini et al. / Theriogenology 77 (2012) 766–772

6. Parthenogenetic cells show a deregulation of de-novo centriole assembly .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7697. Parthenogenetic cells and mitotic spindle checkpoint molecules .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7708. Conclusions .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 770

Acknowledgments .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 770References .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 770

1. Introduction

Parthenogenesis is the process by which an oocytecan begin development without the involvement of themale gamete. While Mammals are not spontaneouslycapable of this form of reproduction, a variety of lowerorganisms, such as fish, ants, flies, honeybees, amphib-ians, lizards and snakes may routinely reproduce in thismanner. However, it must be noted that mammalianoocytes can be successfully activated in vitro, inducingthem to perform several cleavage divisions and thusinitiating embryonic development. These events can beobtained by mimicking the intracellular calcium waveinduced by the spermatozoon at fertilization, a basicmechanism which controls many cellular events. Inparticular, oscillations of this ion causes cortical gran-ule exocytosis, decreasing MPF and MAP kinase ac-tivities with cell cycle resumption and the recruitmentof maternal mRNAs which regulate embryonic devel-opment [1]. In the past years, parthenogenetic develop-ment has been investigated in connection with repro-ductive cloning, since oocyte activation is an essentialpart of the procedure. As a result it is well known thatactivated mammalian oocytes can develop in vitro atrates comparable to IVF oocytes. However, parthenotestransferred in vivo can reach a variable level of devel-opment, depending on the species, but are inherentlyunable to develop to term [2] because of the epigeneticmodification of mammalian DNA, known as imprinting[3], which is responsible for silencing of genes[4] thatare normally expressed by the paternal allele.

Because of their inability to develop to term, par-thenotes may represent a promising tool for studies ofthe mechanisms driving early embryogenesis and forthe preclinical testing of experimental protocols in hu-man assisted reproduction (i.e., different oocyte cryo-preservation procedures, oocyte in vitro maturation orpolar body genetic screening) that would otherwiseimply the destruction of a viable embryo.

Moreover, research in mouse [5,6] and non-humanprimate models [7,8] it has recently demonstrated that,although unable to form a new individual, parthenotesdevelop enough to allow the successful derivation of

stable pluripotent cell lines and therefore they can rep-

resent a stimulating research tool as well as an alterna-tive, less controversial source of embryonic stem celllines in the human [2–10]. Parthenogenetic stem cellshave been derived in human [3,9–12] as well as non-human primates [13,14], pig [15,16], cow [17–19] andbuffalo [20] (Table 1).

These exciting perspectives and applications are,however, hampered by the fact that many aspects re-lated to the biology of parthenogenetic embryos andparthenogenetic derived cell lines are largely unknownand still need to be elucidated. In an attempt to betterunderstand some of these aspects, parthenogenetic celllines, recently derived from different species in ourlaboratory [3,15,16,21], were studied and characterizedfor their pluripotency and differentiation plasticity,both in vitro and in vivo. Here we describe some fea-tures of these cell lines that are common to biparentalembryonic stem cells. By contrast, we also report thepresence of an intrinsic deregulation of molecules con-trolling cell proliferation and cell adhesion and suggestthat the uniparental origin of these cells may representone of the possible cause.

2. Parthenogenesis, asymmetric imprinting anddevelopment

Genomic imprinting is an epigenetic mechanism bywhich a relatively small subset of genes are expressedfrom only one parental allele, either of paternal ormaternal origin [22,23]. This selective expression andrepression of genes is controlled by DNA methylationand histone modifications, and determines the specificdifferentiation and properties of individual cells. Ma-ternal and paternal ‘imprints’ are retained after fertil-ization by embryos and are required for normal mam-

Table 1Parthenogenetic cell lines derived in non-rodent species.

Species References

Human [2,3,9,10,11,12]Non-human primates [13,14]Pig [15,16]Cow [17,18,19]

Buffalo [20]

8wa

tmcf

M

768 T.A.L. Brevini et al. / Theriogenology 77 (2012) 766–772

malian development [24]. It is now known that at least0 genes are imprinted in humans and mice, many ofhich are involved in embryonic and placental growth

nd development [25–28].The parthenogenetic blastocyst genome is consti-

uted by a double set of an epigenetically imprintedaternal genome this implies that duplications of spe-

ific chromosomal regions may lead to detrimental ef-ects on the developing embryo. Furthermore, the ab-

sence of paternal contribution in mammalian parthenotescauses significant adjustments during their development.Placental deficiency has been described as one of thesealterations since placental development requires inputfrom both paternally and maternally imprinted genes [29].Its abnormal regulation is considered the cause for thearrest of parthenogenetic embryos at different stages afteractivation, depending on the species (i.e., Day 10 in themouse, Day 21 in sheep and Day 29 in pigs) [30–32].

More detailed morphologic analysis of parthenoge-netic embryos revealed that the failure of parthenotes todevelop to term may be due to abnormal proliferationand differentiation in both embryonic and extraembry-onic lineages. In particular Fundele, et al demonstratedthat murine parthenotes show cell-autonomous defectsthat involve specific embryo tissues, namely skeletalmuscle, liver and pancreas [33,34]. Furthermore, tran-scriptional profile studies comparing parthenote andcontrol fetus genomes in swine recently demonstrateddifferential expression for at least 25 imprinted genes[35]. All the alterations described above have clearlybeen correlated with genomic imprinting that appears tobe restricted to eutherian mammals, where maternaland paternal genomes are complementary but notequivalent [36]. Mammalian oocytes are not consideredas totipotent [4] and a paternal genome is thereforeessential to ‘rescue’ the female gamete at fertilization,re-establishing the correct imprinting in the developingembryo.

3. Differences between IVF-embryos andparthenotes as a source of stable pluripotentcell lines

Results obtained in our laboratory demonstrate that celllines obtained from IVF embryos and from parthenotesdisplay several common regulatory pathways. Strikingdiversities are, however, evident in other aspects. In pigs,parthenogentic activation generated a significantly highernumber of blastocysts than IVF (23.1% vs 8.93%) and hada significantly higher ability to form outgrowths (22.16%

vs 4.82%) and to generate pluripotent cell lines[16]. This

observation suggests a better ability of parthenogeneticembryos to adhere to the feeder cells and to form out-growths. Although, further studies are needed to betterelucidate this aspect, we think that one of the possibleexplanations of this trend may be found in the higherexpression levels of beta integrin-1 and vitronectin, thatwas consistently detected in parthenogenetic inner cellmasses (ICMs) [16]. Because both these molecules areinvolved in the implantation cascade and stabilize cell tocell adhesion [37], their increased expression in partheno-genetic ICMs may be one of the possible factors account-ing for the significantly higher number of outgrowthsobtained from parthenotes.

The higher ability of parthenogenetic ICM to attachand form outgrowths is common to many species (Ta-ble 2), such as the mouse [38], the human [11] and therabbit [39], and suggest that similar regulatory path-ways are likely to exist among different species andwhich modulate many of the mechanisms involved inthe control of cell adhesion, outgrowth formation andcell line generation.

4. Self-renewal of parthenogenetic cell lines

Parthenogenetic cell lines derived in our laboratory,both in the pig and human, have been growing for twoand a half years. These cells display the main featuresof biparental cells (Fig. 1) and are capable of self-renewal; they remain undifferentiated and do not showsigns of senescence. Indeed, parthenogenetic cells arecharacterized by high telomerase activity, which is of-ten correlated with unlimited regeneration and immor-tality and is typically expressed in germ cells and em-bryonic stem cells [40,41]. Interestingly, when thesecells were subjected to specific stimuli to induce tissuedifferentiation, telomerase activity was turned downand could not be detected, demonstrating that a physi-ologically normal control of telomerase activity is pres-ent in the parthenogenetic cells that we tested. Theexpression of transcription factors as well as surface

Table 2Parthenogenetic blastocysts have a higher ability to formoutgrowths in mammalian species.

Outgrowth fromparthenogenetic

ICM

Outgrowth frombi-patental

embryo ICM

Ratio

ouse [38] 65.00% 15.00% 4.30Rabbit [11] 75.00% 25.00% 3.00Pig [16] 22.16% 4.82% 4.60

Human [11] 66.70% 15.00% 4.40

XiicoaddqtatnutppccTlprtfhn

769T.A.L. Brevini et al. / Theriogenology 77 (2012) 766–772

markers distinctive of pluripotent cells were detected inparthenogenetic lines, indicating that their self-renewalsignature is not influenced by the exclusive presence ofthe maternal genome [16,21].

5. Differentiation ability of parthenogeneticcell lines

Culture in hanging droplets with a medium devoidof Leukemia inhibitory factor (LIF) and Fibroblastgrowth factor (FGF), which have been shown to beinvolved in the maintenance of the pluripotent statusof porcine embryonic cells [16], induces partheno-genetic cells to form embryoid bodies (EBs). Molec-ular studies have demonstrated that these newlyformed structures actively transcribe RNAs involvedin specification of the three embryonic germ layers.These data indicate that parthenogenetic cells havethe ability to differentiate into the main tissue typesof the body [16]. This is confirmed by results ob-tained in experiments where EBs were disaggregatedand transferred to gelatin precoated tissue culturedishes. Shortly after plating, cells grew in monolayercultures that were immune-positive for vimentin,desmin and cytokeratin-17, indicating that ecto-derm-, mesoderm- and endoderm-related cells hadbeen generated (Fig. 2). Although further assays totest the real extent of cellular functionality areneeded, these results suggest parthenogenetic celllines differentiation potential. Furthermore, thesefindings indicate that, outside the normal develop-mental paradigm, the differentiation potential of uni-parental cells may be much less restricted than that

Fig. 1. Parthenogenetic Cell Lines have self-renewal ability andtelomerase activity. They actively transcribe for the main pluripo-tency related transcription factors and surface markers. They have ahigh differentiation ability.

of parthenogenetic cells in chimeras and that these [

cells can be an interesting and relevant model for thestudy of fundamental mechanisms involved in humanlineage determination.

6. Parthenogenetic cells show a deregulation ofde-novo centriole assembly

Centrioles degenerate during the process of oogen-esis and, while oogonia and growing oocytes displaynormal centrioles until the pachytene stage, these or-ganelles are absent in the mature oocytes [42]. Thisdegenerative process has been demonstrated in rhesusmonkeys [43], rabbits [44], cows [45], sea urchins [46],

enopus [47], and many other species [48] while theres no experimental evidence of centriole degenerationn mouse oogenesis [49]. Owing to the absence ofentrioles, the oocyte centrosomal material does notrganize into unified foci and is not capable of gener-ting astral microtubules nor a correctly oriented spin-le, unless rescued by a spermatozoon. Parthenogeneticevelopment is a very good model to study the conse-uence of the lack of centrioles in the early embryo andhis aspect has been studied in lower species wherenastral, abnormal spindles have been observed [50]. Inhese species successful development of parthenoge-etic embryos has been demonstrated to depend largelypon the oocyte’s ability to form complete and func-ional centrosomes, in the absence of the material sup-lied by a male gamete. In sea urchin and insects,arthenogenetically activated eggs generate multipleentrioles, possibly because of the absence of a correctontrol along the process of spindle formation [51–53].hese observations on the parthenogenetic process in

ower species agree with our findings in pig and humanarthenogenetic cells, that, similar to what has beeneported in sea urchins and many insects, display mul-iple centrioles and suggest that the ability to rearrangeunctional centrosomes is altered in uniparental cells. Itas been proposed that centriole de novo assembly isormally turned off when centrioles are present in a cell

Fig. 2. Culture in hanging droplet conditions and in the absence ofLIF and bFGF induces parthenogenetic cells to differentiate and formembryoid bodies (EBs) that contain cells belonging to the three germlayers (ectoderm, endoderm and mesoderm).

54]. This implies that the absence of the sperm-im-

770 T.A.L. Brevini et al. / Theriogenology 77 (2012) 766–772

ported centriole in parthenotes may erase the negativeregulatory mechanism that normally suppresses denovo centriole assembly and may account for the pres-ence of multicentriolar structures, such as those de-scribed in parthenotes from lower species or those wedetect in the cells derived in our laboratory.

Centriole abnormalities do not seem to limit cellproliferation, self-renewal and correct differentiationinto a variety of tissues, indicating that the require-ment of a paternal centrosome described in loweranimals appears to be less stringent in mammaliancells, at least in in vitro controlled conditions, suchas the ones used in our experiments. One explanationfor this may be found in the hypothesis that genomicimprinting may represent the main mechanism thatensures biparental fertilization in higher mammals[55]. By contrast, centriole abnormalities may ex-plain the high percentage of cells showing the highincidence of polyploid and mixoploid chromosomalcomplements that have been reported in parthenotesderived from bovine and porcine activated oocytesand the multiple chromosome malsegregations thathave been previously described in human oocytesafter parthenogenetic activation, either spontane-ously or induced by puromycin [56]. It is interestingto note that these studies report the occurrence ofabnormal chromosomal complements as early ascompletion of the first cell cycle and link them to theabsence of a paternally supplied centrosome [57,58].

7. Parthenogenetic cells and mitotic spindlecheckpoint molecules

Molecular studies carried out in parthenogenetic celllines that we derived from human oocytes demonstratealtered expression levels of mitotic check point mole-cules [21]. In particular, when we compared partheno-genetic cells with biparental embryonic stem cell lineswe could detect a much higher level of expression ofMad-1, and the related molecules MAX and SIN3 inthe former. Since Mad-1 is a central component of thespindle assembly checkpoint and recruitment of kinet-ochores [59–61], and deregulation of its expression hasbeen shown to affect cell cycle progression [62], thisfinding indicates that anomalies in the control of pro-liferation and differentiation of these cells are verylikely.

Disturbances in the control of spindle formation andcell division are also consistent with the observation oflow transcription for TTK and CENP-E, molecules

normally involved in kinetochore-microtubule binding,

correct chromosome congression and alignment as wellas segregation, during mitosis. These findings fit wellwith the high percentage of cells showing high inci-dences of polyploid and mixoploid chromosomal com-plements, previously reported in parthenotes derivedfrom bovine and porcine as well as in human afterspontaneous or induced activation [56].

8. Conclusions

Parthenotes have recently been proposed as a lessquestionable source of embryonic stem cells that pos-sess most of the main features of biparental cell lines.Parthenogenetic cells display the typical morphology,actively transcribe for pluripotency markers, and areable to differentiate into mature cell types in responseto appropriate conditions. However, molecules relatedto spindle formation and cell cycle check points areexpressed at aberrant levels in these cells and are likelycorrelated to the abnormal number of centrioles andchromosome malsegregation detected in parthenoge-netic cells. We hypothesize that their uniparental originmay represent one of the possible cause for the altera-tions described and suggest great caution if a therapeu-tic use of these cells is to be considered.

Acknowledgments

GP and AV were supported by Istituto Nazionale diGenetica Molecolare (INGM).

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