Global histone H3 and H4 acetylation plays a crucial role in the regulation of spatial and temporal control of chromosome segregation during meiosis. Previous studies indicate that the alteration of histone acetylation pattern during oocyte maturation induces aneuploidy and embryo death in mice (Akiyama et al., PNAS 2006, 103:7339-44). We recently observed that in vitro maturation (IVM)adversely affects the proper establishment of H4 acetylation at lysine 16 (AcH4K16) of equine oocytes compared to naturally ovulated oocytes (Franciosi et al., MHR 2012, 18:243-52). Horse was chosen as model of monovulatory species with a longer phase of follicular development and follicular wave pattern similar to women. The present study aimed to test the hypothesis that alteration of the acetylation pattern of H4K16 during IVM is associated with the formation of an irregular spindle and an unproper chromosome bipartition. Oocytes were collected by ultrasound guided aspiration. Metaphase II stage (MII) oocytes were isolated from preovulatory follicles (in vivo matured, N=16) and immature oocytes were aspirated from 5 to 25 mm follicles and subsequently matured in vitro (N=25). In vivo (N=4) and in vitro (N=14) MII oocytes were fixed and double-stained for alpha-tubulin and AcH4K16 determination. In vivo (N=12) and in vitro (N=11) MII oocytes were treated with Monastrol 100 μM, to collapse the bipolar spindle into a unipolar structure where chromosomes are dispersed and easily counted (Duncan et al., BOR 2009, 81:768-76) then fixed and stained for Aurora Kinase B phospho-Thr232, in order to visualize and count the centromeres. All samples were analyzed by confocal laser scanning microscopy. Image analysis was conducted with Image J software and centromere count was assessed by two independent operators. Data were analyzed by Fisher’s exact test. Immunofluorescence analysis showed that AcH4K16 staining was significantly higher in in vivo compared to in vitro matured oocytes, confirming our previous results. Low AcH4K16 pattern was associated with anomalies in spindle morphology, as multipolarity, flat poles and scattered chromosomes, in 78% of in vitro matured oocytes. On the contrary, anomalies in spindle morphology were not observed in in vivo matured oocytes. Moreover, in vivo matured oocytes showed spindles with a shorter pole to pole distance than in vitro ones (14.56±2.95 μm vs 19.89±3.81 μm, P=0.0283) and a smaller spindle equator length (13.55±1.83 μm vs 17.28±2.82 μm, P=0.0461). Both features have been associated with a correct spindle configuration in mice (Sanfins et al., BOR 2003, 69:2059-67). Finally, in vitro matured oocytes showed an incidence of aneuploidy significantly higher than in vivo matured oocytes (45% vs 0%, P=0.0137). Our study indicates that anomalies of chromosomal congression and spindle morphology can be related to alteration in histone acetylation pattern arisen during IVM. This study expands and supports the notion that assisted reproductive technologies can alter the normal epigenetic signature during crucial phases of oocyte development. Funded by L’Oreal Italia per le Donne e la Scienza 2012; by IFCE, France; by Postdoctoral School of Agriculture and Veterinary Medicine, POSDRU/89/1.5/S/ 62371, co-financed by the European Social Fund, Sectorial Operational Programme for Human Resource Development 2007-2013; by Dote Ricercatori FSE, Regione Lombardia; by CIG-Marie Curie Actions FP7-People (Contract 303640,Pro-Ovum).

In Vitro Oocyte Maturation in the Mare Is Associated with Decreased Acetylation of H4K16, High Incidence of Aneuploidy and Altered Spindle Morphology / F. Franciosi, G. Goudet, G. Duchamp, F. Reigner, S. Deleuze, C. Douet, I. Tessaro, I. Miclea, V. Lodde, A.M. Luciano. ((Intervento presentato al 46. convegno SSR’s Annual Meeting tenutosi a Montréal, Québec, Canada nel 2013.

In Vitro Oocyte Maturation in the Mare Is Associated with Decreased Acetylation of H4K16, High Incidence of Aneuploidy and Altered Spindle Morphology

F. Franciosi;I. Tessaro;V. Lodde;A.M. Luciano
2013

Abstract

Global histone H3 and H4 acetylation plays a crucial role in the regulation of spatial and temporal control of chromosome segregation during meiosis. Previous studies indicate that the alteration of histone acetylation pattern during oocyte maturation induces aneuploidy and embryo death in mice (Akiyama et al., PNAS 2006, 103:7339-44). We recently observed that in vitro maturation (IVM)adversely affects the proper establishment of H4 acetylation at lysine 16 (AcH4K16) of equine oocytes compared to naturally ovulated oocytes (Franciosi et al., MHR 2012, 18:243-52). Horse was chosen as model of monovulatory species with a longer phase of follicular development and follicular wave pattern similar to women. The present study aimed to test the hypothesis that alteration of the acetylation pattern of H4K16 during IVM is associated with the formation of an irregular spindle and an unproper chromosome bipartition. Oocytes were collected by ultrasound guided aspiration. Metaphase II stage (MII) oocytes were isolated from preovulatory follicles (in vivo matured, N=16) and immature oocytes were aspirated from 5 to 25 mm follicles and subsequently matured in vitro (N=25). In vivo (N=4) and in vitro (N=14) MII oocytes were fixed and double-stained for alpha-tubulin and AcH4K16 determination. In vivo (N=12) and in vitro (N=11) MII oocytes were treated with Monastrol 100 μM, to collapse the bipolar spindle into a unipolar structure where chromosomes are dispersed and easily counted (Duncan et al., BOR 2009, 81:768-76) then fixed and stained for Aurora Kinase B phospho-Thr232, in order to visualize and count the centromeres. All samples were analyzed by confocal laser scanning microscopy. Image analysis was conducted with Image J software and centromere count was assessed by two independent operators. Data were analyzed by Fisher’s exact test. Immunofluorescence analysis showed that AcH4K16 staining was significantly higher in in vivo compared to in vitro matured oocytes, confirming our previous results. Low AcH4K16 pattern was associated with anomalies in spindle morphology, as multipolarity, flat poles and scattered chromosomes, in 78% of in vitro matured oocytes. On the contrary, anomalies in spindle morphology were not observed in in vivo matured oocytes. Moreover, in vivo matured oocytes showed spindles with a shorter pole to pole distance than in vitro ones (14.56±2.95 μm vs 19.89±3.81 μm, P=0.0283) and a smaller spindle equator length (13.55±1.83 μm vs 17.28±2.82 μm, P=0.0461). Both features have been associated with a correct spindle configuration in mice (Sanfins et al., BOR 2003, 69:2059-67). Finally, in vitro matured oocytes showed an incidence of aneuploidy significantly higher than in vivo matured oocytes (45% vs 0%, P=0.0137). Our study indicates that anomalies of chromosomal congression and spindle morphology can be related to alteration in histone acetylation pattern arisen during IVM. This study expands and supports the notion that assisted reproductive technologies can alter the normal epigenetic signature during crucial phases of oocyte development. Funded by L’Oreal Italia per le Donne e la Scienza 2012; by IFCE, France; by Postdoctoral School of Agriculture and Veterinary Medicine, POSDRU/89/1.5/S/ 62371, co-financed by the European Social Fund, Sectorial Operational Programme for Human Resource Development 2007-2013; by Dote Ricercatori FSE, Regione Lombardia; by CIG-Marie Curie Actions FP7-People (Contract 303640,Pro-Ovum).
Settore VET/01 - Anatomia degli Animali Domestici
http://www.ssr.org/archives/13Meeting.shtml
In Vitro Oocyte Maturation in the Mare Is Associated with Decreased Acetylation of H4K16, High Incidence of Aneuploidy and Altered Spindle Morphology / F. Franciosi, G. Goudet, G. Duchamp, F. Reigner, S. Deleuze, C. Douet, I. Tessaro, I. Miclea, V. Lodde, A.M. Luciano. ((Intervento presentato al 46. convegno SSR’s Annual Meeting tenutosi a Montréal, Québec, Canada nel 2013.
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