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Distinct DNA replication patterns in mouse embryos from in vitro fertilization, parthenogenesis, and somatic cell nuclear transfer

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Abstract:

Epigenetic modifications of DNA have been shown to affect DNA replication during cell divisions. Proliferating cell nuclear antigen (PCNA), a co-factor of DNA polymerases, played an essential role in DNA replication, repair, cell cycle control and epigenetic maintenance by recruiting various protein factors at the replication folks. To understand the mechanisms of nuclear reprogramming in mouse cloned embryos, we compared the spatio-temporal DNA replication foci distribution and chromosomal morphology during the first cell cycle of mouse embryos derived from in vitro fertilization (IVF), parthenogenesis (PA) and somatic cell nuclear transfer (SCNT).
By immuno-staining PCNA with antibody and DNA with propidium iodide (PI), we observed that the three types of embryos have distinct changes of PCNA foci localization and associated with different chromatin regions. In IVF embryos, PCNA signals started to appear approximately 4 hour post fertilization (hpf) in male pronuclei, and 6 hpf in female pronuclei. Additionally, PCNA foci started to associate with dense PI signal at 6 and 8 hpf in male and female pronuclei, respectively. This asymmetric replication progression continued to end of the DNA replication. On the other hand, PA embryos showed symmetric replication patterns between the two pseudopronuclei, both initiated replication at 4 hour post activation (hpa), and PCNA started to be associated with dense PI stain at 6 hpa. Interestingly, although SCNT embryos initiated replication process at 4 hpa as in PA embryos, PCNA foci were not associated with dense PI foci until 10 hpa. These observations suggest that the SCNT embryos have a 2-hour delay in the dynamic changes and association of PCNA stain and PI-dense signals compared to PA embryos.
Because dense PI signals indicate heterochromatin, we subsequently stained PA and SCNT embryos with both PCNA and heterochromatin protein β1 (HP1β) antibodies to confirm the association of PCNA foci with heterochromatin. HP1β were strongly labeled at nucleolus precursor bodies (NPBs) and nuclear periphery regions where heterochromatin localized. PCNA signals stated to associate with HP1β at 6 hpa in PA embryos; while PCNA and HP1β stains remained in distinct locations in SCNT embryos at this time point. Taken together, we observed specific DNA replication patterns in IVF, PA and SCNT embryos. The delay in replication of heterochromatin in SCNT embryos may be caused by the unique epigenetic modifications of DNA in somatic donor cells DNA compared to the DNA in oocyte or sperm.
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Association:
Name: Connecticut's Stem Cell Research International Symposium
URL:
http://stemconn.org


Citation:
URL: http://citation.allacademic.com/meta/p319184_index.html
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MLA Citation:

Lin, Chih-Jen., Chen, Ching-Chien., Amano, Tomokazu., Sung, Li-Ying., Yang, Jerry. and Tian, Cindy. "Distinct DNA replication patterns in mouse embryos from in vitro fertilization, parthenogenesis, and somatic cell nuclear transfer" Paper presented at the annual meeting of the Connecticut's Stem Cell Research International Symposium, Omni Hotel, New Haven, CT, Mar 23, 2009 <Not Available>. 2014-11-29 <http://citation.allacademic.com/meta/p319184_index.html>

APA Citation:

Lin, C. , Chen, C. , Amano, T. , Sung, L. , Yang, J. and Tian, C. , 2009-03-23 "Distinct DNA replication patterns in mouse embryos from in vitro fertilization, parthenogenesis, and somatic cell nuclear transfer" Paper presented at the annual meeting of the Connecticut's Stem Cell Research International Symposium, Omni Hotel, New Haven, CT <Not Available>. 2014-11-29 from http://citation.allacademic.com/meta/p319184_index.html

Publication Type: Poster
Review Method: Peer Reviewed
Abstract: Epigenetic modifications of DNA have been shown to affect DNA replication during cell divisions. Proliferating cell nuclear antigen (PCNA), a co-factor of DNA polymerases, played an essential role in DNA replication, repair, cell cycle control and epigenetic maintenance by recruiting various protein factors at the replication folks. To understand the mechanisms of nuclear reprogramming in mouse cloned embryos, we compared the spatio-temporal DNA replication foci distribution and chromosomal morphology during the first cell cycle of mouse embryos derived from in vitro fertilization (IVF), parthenogenesis (PA) and somatic cell nuclear transfer (SCNT).
By immuno-staining PCNA with antibody and DNA with propidium iodide (PI), we observed that the three types of embryos have distinct changes of PCNA foci localization and associated with different chromatin regions. In IVF embryos, PCNA signals started to appear approximately 4 hour post fertilization (hpf) in male pronuclei, and 6 hpf in female pronuclei. Additionally, PCNA foci started to associate with dense PI signal at 6 and 8 hpf in male and female pronuclei, respectively. This asymmetric replication progression continued to end of the DNA replication. On the other hand, PA embryos showed symmetric replication patterns between the two pseudopronuclei, both initiated replication at 4 hour post activation (hpa), and PCNA started to be associated with dense PI stain at 6 hpa. Interestingly, although SCNT embryos initiated replication process at 4 hpa as in PA embryos, PCNA foci were not associated with dense PI foci until 10 hpa. These observations suggest that the SCNT embryos have a 2-hour delay in the dynamic changes and association of PCNA stain and PI-dense signals compared to PA embryos.
Because dense PI signals indicate heterochromatin, we subsequently stained PA and SCNT embryos with both PCNA and heterochromatin protein β1 (HP1β) antibodies to confirm the association of PCNA foci with heterochromatin. HP1β were strongly labeled at nucleolus precursor bodies (NPBs) and nuclear periphery regions where heterochromatin localized. PCNA signals stated to associate with HP1β at 6 hpa in PA embryos; while PCNA and HP1β stains remained in distinct locations in SCNT embryos at this time point. Taken together, we observed specific DNA replication patterns in IVF, PA and SCNT embryos. The delay in replication of heterochromatin in SCNT embryos may be caused by the unique epigenetic modifications of DNA in somatic donor cells DNA compared to the DNA in oocyte or sperm.


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