Natriuretic peptide type C (CNP) and its receptor natriuretic peptide receptor 2 (NPR2) contribute to maintaining oocyte meiotic arrest. In mice CNP is produced by granulosa cells (GC) and binds to its receptor NPR2, a guanylyl-cyclase that mediates cGMP delivery to the oocyte through gap junction mediated coupling (GJC). CNP/NPR2 signaling inhibits oocyte Phosphodiesterase 3A (PDE3A), and thus maintains oocyte meiotic arrest. In mice, exogenous CNP maintains oocyte meiotic arrest only in the presence of estradiol (E2) that induces the expression of NPR2. We investigated the role of CNP in bovine cumulus enclosed oocytes (CEOs) in 1) preventing spontaneous oocyte meiotic resumption upon removal from follicle, 2) sustaining GJC and 3) promoting chromatin configuration changes (transition from GV1 to GV3) required to achieve developmental potential before germinal vesicle (GV) breakdown. Experiments were repeated 3 times. Data were analyzed by Fisher’s exact test or ANOVA. In preliminary dose-response experiments, 325 CEOs were cultured with 0-500 nM CNP ± 100 nM E2 for 14 h. Compared to control group, 100 nM CNP +100 nM E2 was the most effective treatment in maintaining meiotic arrest but only transiently even in presence of E2. Successively, 900 CEOs were cultured for 6-8 h either in the absence or presence of CNP or 10 μM Cilostamide, a specific PDE3A inhibitor used as a control of efficient meiotic arrest. Both treatments sustained GJC during 6-8 h of culture, when compared to control group (P<0.01) as assessed by Lucifer Yellow-dye spreading in surrounding cells after ooplasmic injection. However, while CNP arrested significantly more oocytes at the GV stage compared to control (85% vs 59% after 6 h and 75% vs 49% after 8 h, respectively, P<0.005), Cilostamide more efficiently maintained meiotic arrest for 6-8 h (96-98%, P<0.01). Progressive chromatin configuration transition from GV1 to GV3 stages was observed, by DAPI staining, during meiotic arrest in both treatments. This was confirmed by immunolocalization of G2/M mitosis-associated ser/thr phospho-proteins (MPM2) used as a marker of meiotic resumption. MPM2 signal increased in the GV along with changes in chromatin configurations and in the ooplasm during meiotic resumption but ooplasmic MPM2 augmentation was delayed during meiotic arrest with either CNP or Cilostamide. Finally, reversibility of meiotic arrest was assessed on a total of 458 CEOs. After 6h of culture with either CNP or Cilostamide, the percentage of oocytes reaching MII after 22h of IVM was similar to control for 24 h (76%, 82% and 84%, respectively). This is the first report on the putative role of CNP on bovine meiotic progression and its involvement in GJC control and large-scale chromatin remodeling. Importantly, our study indicates striking differences compared to the mouse since CNP only transiently arrests meiotic progression even in presence of E2. Moreover, RT-PCR showed the presence of Npr2 in both oocyte and GCs, while mice only expressed Npr2 in GC. Interestingly CNP represent a ‘physiological’ option instead of pharmacological inhibition of meiotic resumption for the development of culture systems that can sustain GJC and promote large-scale chromatin remodeling and oocyte differentiation. Funded by: L’Oreal Italia per le Donne e la Scienza 2012; Marie Curie Actions FP7-CIG (Contract: 303640,"Pro-Ovum"); Dote Ricerca-FSE, Regione Lombardia.

Role of Natriuretic Peptide Type C (CNP) in Maintaining Meiotic Arrest and Sustaining Gap Junction Mediated Communications and Chromatin Configuration Changes in Bovine Cumulus Enclosed Oocytes / I. Tessaro, F. Franciosi, G. Coticchio, V. Lodde, S.C. Modina, R. Fadini, M. Dal Canto, M. Mignini Renzini, D.F. Albertini, A.M. Luciano. ((Intervento presentato al 46. convegno SSR’s Annual Meeting tenutosi a Montréal, Québec, Canada nel 2013.

Role of Natriuretic Peptide Type C (CNP) in Maintaining Meiotic Arrest and Sustaining Gap Junction Mediated Communications and Chromatin Configuration Changes in Bovine Cumulus Enclosed Oocytes

I. Tessaro
Primo
;
F. Franciosi
Secondo
;
V. Lodde;S.C. Modina;A.M. Luciano
Ultimo
2013

Abstract

Natriuretic peptide type C (CNP) and its receptor natriuretic peptide receptor 2 (NPR2) contribute to maintaining oocyte meiotic arrest. In mice CNP is produced by granulosa cells (GC) and binds to its receptor NPR2, a guanylyl-cyclase that mediates cGMP delivery to the oocyte through gap junction mediated coupling (GJC). CNP/NPR2 signaling inhibits oocyte Phosphodiesterase 3A (PDE3A), and thus maintains oocyte meiotic arrest. In mice, exogenous CNP maintains oocyte meiotic arrest only in the presence of estradiol (E2) that induces the expression of NPR2. We investigated the role of CNP in bovine cumulus enclosed oocytes (CEOs) in 1) preventing spontaneous oocyte meiotic resumption upon removal from follicle, 2) sustaining GJC and 3) promoting chromatin configuration changes (transition from GV1 to GV3) required to achieve developmental potential before germinal vesicle (GV) breakdown. Experiments were repeated 3 times. Data were analyzed by Fisher’s exact test or ANOVA. In preliminary dose-response experiments, 325 CEOs were cultured with 0-500 nM CNP ± 100 nM E2 for 14 h. Compared to control group, 100 nM CNP +100 nM E2 was the most effective treatment in maintaining meiotic arrest but only transiently even in presence of E2. Successively, 900 CEOs were cultured for 6-8 h either in the absence or presence of CNP or 10 μM Cilostamide, a specific PDE3A inhibitor used as a control of efficient meiotic arrest. Both treatments sustained GJC during 6-8 h of culture, when compared to control group (P<0.01) as assessed by Lucifer Yellow-dye spreading in surrounding cells after ooplasmic injection. However, while CNP arrested significantly more oocytes at the GV stage compared to control (85% vs 59% after 6 h and 75% vs 49% after 8 h, respectively, P<0.005), Cilostamide more efficiently maintained meiotic arrest for 6-8 h (96-98%, P<0.01). Progressive chromatin configuration transition from GV1 to GV3 stages was observed, by DAPI staining, during meiotic arrest in both treatments. This was confirmed by immunolocalization of G2/M mitosis-associated ser/thr phospho-proteins (MPM2) used as a marker of meiotic resumption. MPM2 signal increased in the GV along with changes in chromatin configurations and in the ooplasm during meiotic resumption but ooplasmic MPM2 augmentation was delayed during meiotic arrest with either CNP or Cilostamide. Finally, reversibility of meiotic arrest was assessed on a total of 458 CEOs. After 6h of culture with either CNP or Cilostamide, the percentage of oocytes reaching MII after 22h of IVM was similar to control for 24 h (76%, 82% and 84%, respectively). This is the first report on the putative role of CNP on bovine meiotic progression and its involvement in GJC control and large-scale chromatin remodeling. Importantly, our study indicates striking differences compared to the mouse since CNP only transiently arrests meiotic progression even in presence of E2. Moreover, RT-PCR showed the presence of Npr2 in both oocyte and GCs, while mice only expressed Npr2 in GC. Interestingly CNP represent a ‘physiological’ option instead of pharmacological inhibition of meiotic resumption for the development of culture systems that can sustain GJC and promote large-scale chromatin remodeling and oocyte differentiation. Funded by: L’Oreal Italia per le Donne e la Scienza 2012; Marie Curie Actions FP7-CIG (Contract: 303640,"Pro-Ovum"); Dote Ricerca-FSE, Regione Lombardia.
Settore VET/01 - Anatomia degli Animali Domestici
http://www.ssr.org/archives/13Meeting.shtml
Role of Natriuretic Peptide Type C (CNP) in Maintaining Meiotic Arrest and Sustaining Gap Junction Mediated Communications and Chromatin Configuration Changes in Bovine Cumulus Enclosed Oocytes / I. Tessaro, F. Franciosi, G. Coticchio, V. Lodde, S.C. Modina, R. Fadini, M. Dal Canto, M. Mignini Renzini, D.F. Albertini, A.M. Luciano. ((Intervento presentato al 46. convegno SSR’s Annual Meeting tenutosi a Montréal, Québec, Canada nel 2013.
Conference Object
File in questo prodotto:
File Dimensione Formato  
2013_SSR_Tessaro.pdf

accesso riservato

Tipologia: Publisher's version/PDF
Dimensione 101.36 kB
Formato Adobe PDF
101.36 kB Adobe PDF   Visualizza/Apri   Richiedi una copia
Pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/248661
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact