Acquisition of the oocyte developmental competence

Oocyte 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.