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Acquisition of the oocyte developmental competence
No full textOocyte developmental competence: in search of a transcriptional signature
Maurizio Zuccotti1, Valeria Merico2, Martina Belli2, Giulia Vigone2, Silvia Garagna2
1. Dipartimento di Scienze Biomediche, Biotecnologiche e Traslazionali (S.Bi.Bi.T.), Sezione di Anatomia, Istologia ed Embriologia, University of Parma, Italy.
2. Dipartimento di Biologia e Biotecnologie ‘Lazzaro Spallanzani’, Laboratorio di Biologia dello Sviluppo, University of Pavia, Italy.
In our laboratory we try to unravel the molecular signature that lays behind the developmental competence of a mammalian egg, aiming at finding markers that could help to the evaluation of the quality of the female gamete. Here, we will present a series of studies that led to the identification of transcriptional networks (TN) differentially expressed in developmentally competent or incompetent oocytes, and also we will show that one of these TNs is maintained during preimplantation and in embryonic stem cells (ESCs), representing a fil rouge of developmental continuity that links the egg to the blastocyst.
To this end, we made use of a model study in which a type specific of metaphase II (MII) oocyte ceases development at the 2-cell stage. Based on their chromatin organisation – as observed after staining with the Hoechst 33342 supravital fluorochrome - fully-grown mouse antral oocytes are classified into surrounded nucleolus (SN) or not surrounded nucleolus (NSN) oocytes (Debey et al., 1992; Mattson and Albertini, 1990; Zuccotti et al., 1995). Following a different time-course and chromatin rearrangements (Belli et al., 2014) both oocytes mature in vitro to MII (MIISN and MIINSN), but, when fertilised, only MIISN may reach full-term development, whereas MIINSN arrest at the 2-cell stage (Zuccotti et al., 1998, 2005).
To understand the developmental incompetence of MIINSN oocytes, we investigated into their transcriptional legacy. Gene expression was compared by whole-transcriptome microarrays analysis, which brought up 380 differentially expressed genes, 77 down-regulated and 303 up-regulated. Further bioinformatics, RT-PCR and immunocytochemistry analyses of these differentially expressed genes and proteins showed an emerging network of 25 genes mostly up-regulated in MIINSN oocytes and assigned to adverse biochemical pathways such as apoptosis and mitochondrial dysfunction (Zuccotti et al., 2008; 2009, 2011a). Importantly, most of these genes are known to be regulated by the transcription factor OCT4, one of a handful of master transcription factors that regulate cell pluripotency.
In a next step, we compared the whole-transcriptional profile of developmentally competent vs. incompetent oocytes and that of their derived 2-cell embryos; the OCT4-TN was further expanded to 80 transcripts, mostly expressed in cancer cells and 37 notable companions of the OCT4 transcriptome in ESCs (Zuccotti et al., 2011b; 2012). For the first time, these results indicate that the OCT4-TN may represent a developmental link between eggs, early preimplantation embryos and ESCs, indicating that the molecular signature that characterises the ESCs pluripotency may be rooted in oogenesis.
Bibliography
Mattson BA, Albertini DF: Oogenesis: chromatin and microtubule dynamics during meiotic prophase. Mol Reprod Dev 25: 374-383, 1990.
Debey P, Szöllösi MS, Szöllösi D, Vautier D, Girousse A, Besombes D. Competent mouse oocytes isolated from antral follicles exhibit different chromatin organization and follow different maturation dynamics. Mol Reprod Dev 36: 59-74, 1993.
Zuccotti M, Piccinelli A, Giorgi Rossi P, Garagna S, Redi C: Chromatin organization during mouse oocyte growth. Mol Reprod Dev 41: 479-485, 1995.
Zuccotti M, Giorgi Rossi P, Martinez A, Garagna S, Forabosco A, Redi CA. Meiotic and developmental competence of mouse antral oocytes. Biol Reprod 58: 700-704, 1998.
Zuccotti M, Garagna S, Merico V, Monti M, Alberto Redi C: Chromatin organisation and nuclear architecture in growing mouse oocytes. Mol Cell Endocrinol 234: 11-17, 2005.
Zuccotti M, Merico V, Sacchi L, Bellone M, Brink TC, Stefanelli M, Redi CA, Bellazzi R, Adjaye J, Garagna S. Oct-4 regulates the expression of Stella and Foxj2 at the Nanog locus: implications for the developmental competence of mouse oocytes. Hum Reprod. 24: 2225-2237, 2009.
Zuccotti M, Merico V, Cecconi S, Redi CA, Garagna S. What does it take to make a developmentally competent mammalian egg? Hum Reprod Update 17: 525-540, 2011a.
Zuccotti M, Merico V, Bellone M, Mulas F, Sacchi L, Rebuzzini P, Prigione A, Redi CA, Bellazzi R, Adjaye J, Garagna S. Gatekeeper of pluripotency: a common Oct4 transcriptional network operates in mouse eggs and embryonic stem cells. BMC Genomics 12: 1-13, 2011.
Zuccotti M, Merico V, Belli M, Mulas F, Sacchi L, Zupan B, Redi CA, Prigione A, Adjaye J, Bellazzi R, Garagna S. OCT4 and the acquisition of oocyte developmental competence during folliculogenesis. Int J Dev Biol. 56: 853-858, 2012.
Belli M, Vigone G, Merico V, Redi CA, Garagna S, Zuccotti M. Time-lapse dynamics of the mouse oocyte chromatin organisation during meiotic resumption. Biomed Res Int. 2014:207357, 2014
Time-lapse imaging of chromatin and cytoplasmic movements occurring during the GV-to-MII transition: in search for markers of mouse oocytes developmental competence
No full textThe transition from the germinal vesicle to the metaphase II stage (GV-to-MII transition) is crucial to the acquisition of the oocyte developmental competence. Here, using live, time-lapse, imaging we describe the movements occurring during the GV-to-MII transition to the chromatin (CHR-MOV) and to the cytoplasm (CYTO-MOV) of mouse oocytes of known developmental competence or incompetence.
Fully-grown cumulus-oocyte-complexes were punctured from the ovarian surface, the GV oocytes isolated and stained with the supravital Hoechst 33342 fluorochrome (Ho) which allowed the identification of gametes whose nucleolus is surrounded by a ring of Ho-positive chromatin (surrounded nucleolus, SN, oocytes) from those that lack this ring (not surrounded nucleolus, NSN, oocytes). Importantly, when in vitro cultured to MII and inseminated with sperm, whilst SN oocytes may develop to term, NSN oocytes arrest development at the 2-cell stage.
The time-lapse observation of CHR-MOV describe distinct chromatin changes in NSN compared to SN oocytes, with a longer GV-to-MII transition in NSN oocytes that reach the M-phase without the gathering of heterochromatin regions around the nucleolus.
Furthermore, by coupling bright-field time-lapse observations with the Particle Image Velocimetry method, we analysed the CYTO-MOV of these two types of oocytes. We showed that SN and NSN oocytes exhibit distinct profiles and, at four main time-frame intervals, their CYTO-MOV velocity is significantly different. In addition, we integrated the information of the CYTO-MOV profile of each single oocyte with an artificial neural network analysis that blindly identified the oocyte as SN or NSN with a robust probability.
The presence of SN and NSN oocytes in all mammals, including humans, extends the interest of these results to the field of assisted reproductive technologies (ART)
Time-lapse imaging of chromatin and cytoplasmic movements occurring during the GV-to-MII transition: in search for markers of mouse oocytes developmental competence
No full textThe transition from the germinal vesicle to the metaphase II stage (GV-to-MII transition) is crucial to the acquisition of the oocyte developmental competence. Here, using live, time-lapse, imaging we describe the movements occurring during the GV-to-MII transition to the chromatin (CHR-MOV) and to the cytoplasm (CYTO-MOV) of mouse oocytes of known developmental competence or incompetence.
Fully-grown cumulus-oocyte-complexes were punctured from the ovarian surface, the GV oocytes isolated and stained with the supravital Hoechst 33342 fluorochrome (Ho) which allowed the identification of gametes whose nucleolus is surrounded by a ring of Ho-positive chromatin (surrounded nucleolus, SN, oocytes) from those that lack this ring (not surrounded nucleolus, NSN, oocytes). Importantly, when in vitro cultured to MII and inseminated with sperm, whilst SN oocytes may develop to term, NSN oocytes arrest development at the 2-cell stage.
The time-lapse observation of CHR-MOV describe distinct chromatin changes in NSN compared to SN oocytes, with a longer GV-to-MII transition in NSN oocytes that reach the M-phase without the gathering of heterochromatin regions around the nucleolus.
Furthermore, by coupling bright-field time-lapse observations with the Particle Image Velocimetry method, we analysed the CYTO-MOV of these two types of oocytes. We showed that SN and NSN oocytes exhibit distinct profiles and, at four main time-frame intervals, their CYTO-MOV velocity is significantly different. In addition, we integrated the information of the CYTO-MOV profile of each single oocyte with an artificial neural network analysis that blindly identified the oocyte as SN or NSN with a robust probability.
The presence of SN and NSN oocytes in all mammals, including humans, extends the interest of these results to the field of assisted reproductive technologies (ART)
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Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
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koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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