Mature oocytes can be activated in vitro leading to the generation of parthenotes who will develop in culture forming blastocysts morphologically indistinguishable from those derived from fertilized eggs. Parthenotes have been used as a source of pluripotent cells that show the traditional features associated with their biparental counterpart, expression of totipotency markers, telomerase activity, embryoid body formation, in vitro differentiation and, in most cases, teratomas formation. However many aspects still need to be elucidated and, in particular little attention has been addressed so far to the incidence of aneuploidy in these cells. Limited data available for parthenotes derived from different mammalian species indicate a high rate of aneuploidy, which is considered to be caused by the lack of the paternal contribution, since alteration of the centrosome are known to lead to multipolar spindles that, in turn, cause aneuploid cells. In this study we analysed the rate of aneuploidy and centriole distribution (as a marker of centrosome anomalies) in cell lines derived from pig and sheep parthenotes and in sheep parthenotes foetus that reached their maximum development in vivo (24 days)., in order to separate the effect related to oocyte activation from those of the procedures used to derive pSC lines. Centriole number and distribution was assessed both by immunocytochemical analysis using an anti-Centrin-1 antibody (1:200, Abcam, UK) and an appropriate secondary antibody and with ultrastructural evaluation of thin sections, using a Jeol 1010 EX electron microscope. Karyotyping was performed on mitotically active cells. Metaphases were fully karyotyped under a Leica HC microscope. Images were then captured with digital camera Leica DC250 and cells karyotyped using the Leica CW4000 Karyo software. The results obtained indicate that cell lines of parthenogenetic origin have, in all examined cases, an incidence of aneuploidy significantly higher than that of their respective controls. In particular, although the diploid configuration represented the modal value, the majority of the cells displayed a steadily lower number of chromosomes, comprised between <1N (hypohaploid) and > 1N - <2N (hypodiploid). This result is possibly related to a loss of chromosomes during the mitotic process. A higher incidence of multiple centrioles was also detected, suggesting the hypothesis that aneuploidy may be related to the lack of paternal contribution that results in abnormal centrosome formation, incorrect control of the process of spindle rearrangement and consequent chromosomes malsegregation. Abnormal segregation and multicentriolar distribution was not limited to parthenogenetic cell lines but was observed in parthenogenetic fetus as well, indicating that culture artifacts are unlikely to be the cause.

Cell lines derived from mammalian parthenogenetic embryos display abnormal chromosome complements and aberrant centriole number / T. Brevini, G. Pennarossa, A. Vanelli, G. Tettamanti, L. Bogliolo, M. de Eguileor, S. Ledda, F. Gandolfi. - In: REPRODUCTION FERTILITY AND DEVELOPMENT. - ISSN 1031-3613. - 22:1(2010), pp. 324.318-324.318. ((Intervento presentato al convegno Annual conference of the International Embryo Transfer Society tenutosi a Córdoba, Argentina nel 2010 [10.1071/RDv22n1Ab324].

Cell lines derived from mammalian parthenogenetic embryos display abnormal chromosome complements and aberrant centriole number

T. Brevini;G. Pennarossa;F. Gandolfi
2010

Abstract

Mature oocytes can be activated in vitro leading to the generation of parthenotes who will develop in culture forming blastocysts morphologically indistinguishable from those derived from fertilized eggs. Parthenotes have been used as a source of pluripotent cells that show the traditional features associated with their biparental counterpart, expression of totipotency markers, telomerase activity, embryoid body formation, in vitro differentiation and, in most cases, teratomas formation. However many aspects still need to be elucidated and, in particular little attention has been addressed so far to the incidence of aneuploidy in these cells. Limited data available for parthenotes derived from different mammalian species indicate a high rate of aneuploidy, which is considered to be caused by the lack of the paternal contribution, since alteration of the centrosome are known to lead to multipolar spindles that, in turn, cause aneuploid cells. In this study we analysed the rate of aneuploidy and centriole distribution (as a marker of centrosome anomalies) in cell lines derived from pig and sheep parthenotes and in sheep parthenotes foetus that reached their maximum development in vivo (24 days)., in order to separate the effect related to oocyte activation from those of the procedures used to derive pSC lines. Centriole number and distribution was assessed both by immunocytochemical analysis using an anti-Centrin-1 antibody (1:200, Abcam, UK) and an appropriate secondary antibody and with ultrastructural evaluation of thin sections, using a Jeol 1010 EX electron microscope. Karyotyping was performed on mitotically active cells. Metaphases were fully karyotyped under a Leica HC microscope. Images were then captured with digital camera Leica DC250 and cells karyotyped using the Leica CW4000 Karyo software. The results obtained indicate that cell lines of parthenogenetic origin have, in all examined cases, an incidence of aneuploidy significantly higher than that of their respective controls. In particular, although the diploid configuration represented the modal value, the majority of the cells displayed a steadily lower number of chromosomes, comprised between <1N (hypohaploid) and > 1N - <2N (hypodiploid). This result is possibly related to a loss of chromosomes during the mitotic process. A higher incidence of multiple centrioles was also detected, suggesting the hypothesis that aneuploidy may be related to the lack of paternal contribution that results in abnormal centrosome formation, incorrect control of the process of spindle rearrangement and consequent chromosomes malsegregation. Abnormal segregation and multicentriolar distribution was not limited to parthenogenetic cell lines but was observed in parthenogenetic fetus as well, indicating that culture artifacts are unlikely to be the cause.
Settore VET/01 - Anatomia degli Animali Domestici
International Embryo Transfer Society
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/223218
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