Gene expression in sheep cumulus-oocyte complexes meiotically inhibited with roscovitine
Autor(a) principal: | |
---|---|
Data de Publicação: | 2015 |
Outros Autores: | , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.1016/j.smallrumres.2015.10.014 http://hdl.handle.net/11449/172229 |
Resumo: | The majority of mammalian oocytes destined for in vitro maturation (IVM) have not undergone all molecular and structural changes necessary for competence acquisition to support the fertilization and early embryogenesis. In this context, different methods able to provide a transient arrest of meiosis resumption have been tested in order to improve the in vitro developmental potential of oocytes. Based on that, our study aimed to evaluate the effect of temporary meiosis inhibition using roscovitine on gene expression in sheep oocytes and cumulus cells. For this, cumulus-oocyte complexes (COCs) were cultured for 6 h in modified TCM199 medium with (Rosco) and without (Control) 75 μM roscovitine. Subsequently, they were in vitro matured for a further 18 h in inhibitor-free TCM199 medium supplemented with gonadotropins. At 0, 6 and 24 h of culture, nuclear status of oocytes and expression of selected genes were evaluated by Hoescht staining and qRT-PCR, respectively. The analysis of oocyte chromatin organization revealed that roscovitine efficiently inhibited the meiosis of sheep oocytes for 6 h and its action was completely reversed after 18 h of in vitro maturation in inhibitor-free medium. Besides, no detrimental effect on cumulus expansion was observed. The expression profile of most investigated genes in cumulus cells (PTX3, GREM1, GLUT1, PTGS2, ALK5, ALK6) and oocytes (ZAR1, NLRP5, SOD1, BMP15, GDF9) was similar between Control and Rosco treatments and the ratio BCL2/BAX was maintained in both cell types even in the presence of roscovitine. These results indicate that reversible meiotic arrest promoted by roscovitine, at the concentration and exposure time studied, neither impaired nor improved the expression of investigated genes in sheep oocytes and cumulus cells. Moreover, the efficiency of temporary meiotic arrest and the absence of deleterious effect on COCs suggest that roscovitine provides a useful method for transportation or manipulation of sheep oocytes at onset of maturation. However, further investigations are necessary to evaluate the benefits of roscovitine treatment on in vitro development of sheep embryos and its effects on cellular ultrastructure. |
id |
UNSP_719aa3c1706087d40400f8ebb7b0a41e |
---|---|
oai_identifier_str |
oai:repositorio.unesp.br:11449/172229 |
network_acronym_str |
UNSP |
network_name_str |
Repositório Institucional da UNESP |
repository_id_str |
2946 |
spelling |
Gene expression in sheep cumulus-oocyte complexes meiotically inhibited with roscovitineCumulus expansionMeiosis inhibitormRNANuclear statusOvineqRT-PCRThe majority of mammalian oocytes destined for in vitro maturation (IVM) have not undergone all molecular and structural changes necessary for competence acquisition to support the fertilization and early embryogenesis. In this context, different methods able to provide a transient arrest of meiosis resumption have been tested in order to improve the in vitro developmental potential of oocytes. Based on that, our study aimed to evaluate the effect of temporary meiosis inhibition using roscovitine on gene expression in sheep oocytes and cumulus cells. For this, cumulus-oocyte complexes (COCs) were cultured for 6 h in modified TCM199 medium with (Rosco) and without (Control) 75 μM roscovitine. Subsequently, they were in vitro matured for a further 18 h in inhibitor-free TCM199 medium supplemented with gonadotropins. At 0, 6 and 24 h of culture, nuclear status of oocytes and expression of selected genes were evaluated by Hoescht staining and qRT-PCR, respectively. The analysis of oocyte chromatin organization revealed that roscovitine efficiently inhibited the meiosis of sheep oocytes for 6 h and its action was completely reversed after 18 h of in vitro maturation in inhibitor-free medium. Besides, no detrimental effect on cumulus expansion was observed. The expression profile of most investigated genes in cumulus cells (PTX3, GREM1, GLUT1, PTGS2, ALK5, ALK6) and oocytes (ZAR1, NLRP5, SOD1, BMP15, GDF9) was similar between Control and Rosco treatments and the ratio BCL2/BAX was maintained in both cell types even in the presence of roscovitine. These results indicate that reversible meiotic arrest promoted by roscovitine, at the concentration and exposure time studied, neither impaired nor improved the expression of investigated genes in sheep oocytes and cumulus cells. Moreover, the efficiency of temporary meiotic arrest and the absence of deleterious effect on COCs suggest that roscovitine provides a useful method for transportation or manipulation of sheep oocytes at onset of maturation. However, further investigations are necessary to evaluate the benefits of roscovitine treatment on in vitro development of sheep embryos and its effects on cellular ultrastructure.Department of Animal Reproduction and Veterinary Radiology School of Veterinary Medicine and Animal Science UNESP, Distrito de Rubião Junior s/noDepartment of Physiology Institute of Biosciences UNESP, Distrito de Rubião Junior s/noDepartment of Animal Reproduction and Veterinary Radiology School of Veterinary Medicine and Animal Science UNESP, Distrito de Rubião Junior s/noDepartment of Physiology Institute of Biosciences UNESP, Distrito de Rubião Junior s/noUniversidade Estadual Paulista (Unesp)Crocomo, Letícia Ferrari [UNESP]Marques Filho, Wolff Camargo [UNESP]Ackermann, Camila Louise [UNESP]Lima, Paula Fernanda de [UNESP]Buratini Junior, José [UNESP]Landim-Alvarenga, Fernanda da Cruz [UNESP]Bicudo, Sony Dimas [UNESP]2018-12-11T16:59:16Z2018-12-11T16:59:16Z2015-01-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article115-122application/pdfhttp://dx.doi.org/10.1016/j.smallrumres.2015.10.014Small Ruminant Research, v. 132, p. 115-122.0921-4488http://hdl.handle.net/11449/17222910.1016/j.smallrumres.2015.10.0142-s2.0-849479302612-s2.0-84947930261.pdfScopusreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengSmall Ruminant Research0,485info:eu-repo/semantics/openAccess2024-01-28T06:47:28Zoai:repositorio.unesp.br:11449/172229Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462024-01-28T06:47:28Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
Gene expression in sheep cumulus-oocyte complexes meiotically inhibited with roscovitine |
title |
Gene expression in sheep cumulus-oocyte complexes meiotically inhibited with roscovitine |
spellingShingle |
Gene expression in sheep cumulus-oocyte complexes meiotically inhibited with roscovitine Crocomo, Letícia Ferrari [UNESP] Cumulus expansion Meiosis inhibitor mRNA Nuclear status Ovine qRT-PCR |
title_short |
Gene expression in sheep cumulus-oocyte complexes meiotically inhibited with roscovitine |
title_full |
Gene expression in sheep cumulus-oocyte complexes meiotically inhibited with roscovitine |
title_fullStr |
Gene expression in sheep cumulus-oocyte complexes meiotically inhibited with roscovitine |
title_full_unstemmed |
Gene expression in sheep cumulus-oocyte complexes meiotically inhibited with roscovitine |
title_sort |
Gene expression in sheep cumulus-oocyte complexes meiotically inhibited with roscovitine |
author |
Crocomo, Letícia Ferrari [UNESP] |
author_facet |
Crocomo, Letícia Ferrari [UNESP] Marques Filho, Wolff Camargo [UNESP] Ackermann, Camila Louise [UNESP] Lima, Paula Fernanda de [UNESP] Buratini Junior, José [UNESP] Landim-Alvarenga, Fernanda da Cruz [UNESP] Bicudo, Sony Dimas [UNESP] |
author_role |
author |
author2 |
Marques Filho, Wolff Camargo [UNESP] Ackermann, Camila Louise [UNESP] Lima, Paula Fernanda de [UNESP] Buratini Junior, José [UNESP] Landim-Alvarenga, Fernanda da Cruz [UNESP] Bicudo, Sony Dimas [UNESP] |
author2_role |
author author author author author author |
dc.contributor.none.fl_str_mv |
Universidade Estadual Paulista (Unesp) |
dc.contributor.author.fl_str_mv |
Crocomo, Letícia Ferrari [UNESP] Marques Filho, Wolff Camargo [UNESP] Ackermann, Camila Louise [UNESP] Lima, Paula Fernanda de [UNESP] Buratini Junior, José [UNESP] Landim-Alvarenga, Fernanda da Cruz [UNESP] Bicudo, Sony Dimas [UNESP] |
dc.subject.por.fl_str_mv |
Cumulus expansion Meiosis inhibitor mRNA Nuclear status Ovine qRT-PCR |
topic |
Cumulus expansion Meiosis inhibitor mRNA Nuclear status Ovine qRT-PCR |
description |
The majority of mammalian oocytes destined for in vitro maturation (IVM) have not undergone all molecular and structural changes necessary for competence acquisition to support the fertilization and early embryogenesis. In this context, different methods able to provide a transient arrest of meiosis resumption have been tested in order to improve the in vitro developmental potential of oocytes. Based on that, our study aimed to evaluate the effect of temporary meiosis inhibition using roscovitine on gene expression in sheep oocytes and cumulus cells. For this, cumulus-oocyte complexes (COCs) were cultured for 6 h in modified TCM199 medium with (Rosco) and without (Control) 75 μM roscovitine. Subsequently, they were in vitro matured for a further 18 h in inhibitor-free TCM199 medium supplemented with gonadotropins. At 0, 6 and 24 h of culture, nuclear status of oocytes and expression of selected genes were evaluated by Hoescht staining and qRT-PCR, respectively. The analysis of oocyte chromatin organization revealed that roscovitine efficiently inhibited the meiosis of sheep oocytes for 6 h and its action was completely reversed after 18 h of in vitro maturation in inhibitor-free medium. Besides, no detrimental effect on cumulus expansion was observed. The expression profile of most investigated genes in cumulus cells (PTX3, GREM1, GLUT1, PTGS2, ALK5, ALK6) and oocytes (ZAR1, NLRP5, SOD1, BMP15, GDF9) was similar between Control and Rosco treatments and the ratio BCL2/BAX was maintained in both cell types even in the presence of roscovitine. These results indicate that reversible meiotic arrest promoted by roscovitine, at the concentration and exposure time studied, neither impaired nor improved the expression of investigated genes in sheep oocytes and cumulus cells. Moreover, the efficiency of temporary meiotic arrest and the absence of deleterious effect on COCs suggest that roscovitine provides a useful method for transportation or manipulation of sheep oocytes at onset of maturation. However, further investigations are necessary to evaluate the benefits of roscovitine treatment on in vitro development of sheep embryos and its effects on cellular ultrastructure. |
publishDate |
2015 |
dc.date.none.fl_str_mv |
2015-01-01 2018-12-11T16:59:16Z 2018-12-11T16:59:16Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.1016/j.smallrumres.2015.10.014 Small Ruminant Research, v. 132, p. 115-122. 0921-4488 http://hdl.handle.net/11449/172229 10.1016/j.smallrumres.2015.10.014 2-s2.0-84947930261 2-s2.0-84947930261.pdf |
url |
http://dx.doi.org/10.1016/j.smallrumres.2015.10.014 http://hdl.handle.net/11449/172229 |
identifier_str_mv |
Small Ruminant Research, v. 132, p. 115-122. 0921-4488 10.1016/j.smallrumres.2015.10.014 2-s2.0-84947930261 2-s2.0-84947930261.pdf |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Small Ruminant Research 0,485 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
115-122 application/pdf |
dc.source.none.fl_str_mv |
Scopus reponame:Repositório Institucional da UNESP instname:Universidade Estadual Paulista (UNESP) instacron:UNESP |
instname_str |
Universidade Estadual Paulista (UNESP) |
instacron_str |
UNESP |
institution |
UNESP |
reponame_str |
Repositório Institucional da UNESP |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
repository.mail.fl_str_mv |
|
_version_ |
1797790419343376384 |