Throughout folliculogenesis, primordial follicles, blocked at the diplotene stage of prophase I, develop into antral follicles. In this latter, oocytes present two distinct chromatin organization: surrounding nucleolus (SN) or not-surrounding nucleolus (NSN). Although both can be fertilized in vitro, only SN-types develop beyond the 2-cell stage, reach blastocyst. Cumulus cells (CCs) surround the oocyte during its growth and establish a bidirectional exchange with the gamete. The purpose of this study was to investigate the contribution of CCs to oocyte maturation and to the following embryo preimplantation development. To this purpose, feeder-layers of CCs were prepared using CCs derived from developmentally competent mouse oocyte (FL-SN-CCs) or developmentally incompetent mouse oocyte (FL-NSN-CCs). Denuded, fully grown oocytes (DOs) were in vitro matured (IVM) for 15 hours to metaphase II on either FL-SN-CCs or FL-NSN-CCs, inseminated with spermatozoa and cultured throughout preimplantation development. Media derived from the two types of feeder-layers were extracted and examined by Nanoparticle Tracking Analysis (NTA), western-blot, fluorimetric analysis, and confocal microscopy. The main results obtained are: (I) DOs matured upon FL-SN-CCs develop to blastocyst stage; (II) DOs matured upon FL-NSN-CCs arrest their development before reaching the blastocyst stage, mainly at the 2-cell stage; (III) both FL-SN-CCs and FL-NSN-CCs release extracellular vesicles (EVs), the majority of which are exosomes (EXOs); (IV) the majority of EVs are released between 9 and 15 hours of IVM; (V) EVs cross the oocyte zona pellucida and the oolemma, reaching the ooplasm. NGS analysis on miRNAs extracted from EVs of the two experimental conditions detected a total of 70 miRNAs differentially expressed. Of these, 15 miRNAs resulted significantly upregulated and 12 significantly down-regulated in the comparison between FL-SN-CCs vs FL-NSN-CCs. Amongst these miRNAs, mmu-miR-122-5p and mmu-miR-126a-5p (upregulated) and mmu-miR-28a-5p and mmu-miR-28c (downregulated) are well known for their involvement in oocytes maturation. The next experiments will be focused first on the validation of the NGS results; then, we will try to inactivate the action of specific miRNAs carried by the EVs and follow, using our culture platform, oocytes maturation and developmental competence.
La folliculogenesi è quel processo attraverso il quale i follicoli primordiali, bloccati allo stadio di diplotene della profase I, si sviluppano in follicoli antrali. In quest'ultimi, l'oocita presenta due distinte organizzazioni di cromatina: surrounding nucleolus (SN) o not-surrounding nucleolus (NSN). Sebbene entrambi i suddetti tipi possano essere fecondati in vitro, solo gli SN sono superano lo stadio a due cellule e raggiungono la blastocisti. Le cellule cumulo (CC), circondano l’oocita durante la sua crescita e stabiliscono uno scambio bidirezionale con il gamete. Questo lavoro ha intenzione di studiare il contributo delle CC alla maturazione degli oociti e alla fase di sviluppo preimpianto dell'embrione. A tal fine, sono stati preparati feeder-layer di CC derivate da oociti di topo competenti (FL-SN-CC) o incompetenti (FL-NSN-CC) allo sviluppo. Gli oociti antrali denudati (DO) sono stati maturati in vitro (IVM) per 15 ore allo stadio di metafase II su entrambi i feeder-layer, inseminati con spermatozoi e seguiti durante lo sviluppo preimpianto. I terreni di coltura derivati sono stati estratti ed esaminati mediante Nanoparticle Tracking Analysis (NTA), western-blot, analisi fluorimetrica e microscopio confocale. I principali risultati ottenuti sono: (I) i DO maturati su FL-SN-CC si sviluppano allo stadio di blastocisti; (II) i DO maturati su FL-NSN-CC non raggiugono lo stadio di blastocisti, terminando lo sviluppo principalmente allo stadio di due cellule; (III) entrambi i feeder-layer rilasciano vescicole extracellulari (EV), la cui maggioranza sono esosomi (EXO); (IV) la maggior parte delle EV viene rilasciata dal feeder-layer tra le 9 e le 15 ore di IVM; (V) Le EV attraversano la zona pellucida dell'oocita e l’oolemma, raggiungendo l'ooplasma. L’analisi NGS sui miRNA estratti dalle EV di entrambi due tipi di feeder-layer ha rilevato un totale di 70 miRNA differentemente espressi. Di questi, dalla comparazione tra FL-SN-CCs vs FL-NSN-CCs, 15 miRNA sono risultati significativamente sovra-regolati e 12 significativamente sotto-regolati. Tra questi miRNA, mmu-miR-122-5p e mmu-miR-126a-5p (sovra-regolati) e mmu-miR-28a-5p e mmu-miR-28c (sotto-regolati) sono già conosciuti per il loro contributo nella maturazione dell’oocita. I prossimi esperimenti si focalizzeranno prima sulla validazione dei risultati NGS; quindi, proveremo a inattivare l'azione di miRNA specifici trasportati dalle EV e seguiremo, utilizzando la nostra piattaforma di coltura, la maturazione degli oociti e la loro competenza allo sviluppo.
Contributo delle cellule del cumulo all’acquisizione della competenza allo sviluppo dell’oocita di topo
BOSI, DAVIDE
2019/2020
Abstract
Throughout folliculogenesis, primordial follicles, blocked at the diplotene stage of prophase I, develop into antral follicles. In this latter, oocytes present two distinct chromatin organization: surrounding nucleolus (SN) or not-surrounding nucleolus (NSN). Although both can be fertilized in vitro, only SN-types develop beyond the 2-cell stage, reach blastocyst. Cumulus cells (CCs) surround the oocyte during its growth and establish a bidirectional exchange with the gamete. The purpose of this study was to investigate the contribution of CCs to oocyte maturation and to the following embryo preimplantation development. To this purpose, feeder-layers of CCs were prepared using CCs derived from developmentally competent mouse oocyte (FL-SN-CCs) or developmentally incompetent mouse oocyte (FL-NSN-CCs). Denuded, fully grown oocytes (DOs) were in vitro matured (IVM) for 15 hours to metaphase II on either FL-SN-CCs or FL-NSN-CCs, inseminated with spermatozoa and cultured throughout preimplantation development. Media derived from the two types of feeder-layers were extracted and examined by Nanoparticle Tracking Analysis (NTA), western-blot, fluorimetric analysis, and confocal microscopy. The main results obtained are: (I) DOs matured upon FL-SN-CCs develop to blastocyst stage; (II) DOs matured upon FL-NSN-CCs arrest their development before reaching the blastocyst stage, mainly at the 2-cell stage; (III) both FL-SN-CCs and FL-NSN-CCs release extracellular vesicles (EVs), the majority of which are exosomes (EXOs); (IV) the majority of EVs are released between 9 and 15 hours of IVM; (V) EVs cross the oocyte zona pellucida and the oolemma, reaching the ooplasm. NGS analysis on miRNAs extracted from EVs of the two experimental conditions detected a total of 70 miRNAs differentially expressed. Of these, 15 miRNAs resulted significantly upregulated and 12 significantly down-regulated in the comparison between FL-SN-CCs vs FL-NSN-CCs. Amongst these miRNAs, mmu-miR-122-5p and mmu-miR-126a-5p (upregulated) and mmu-miR-28a-5p and mmu-miR-28c (downregulated) are well known for their involvement in oocytes maturation. The next experiments will be focused first on the validation of the NGS results; then, we will try to inactivate the action of specific miRNAs carried by the EVs and follow, using our culture platform, oocytes maturation and developmental competence.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/11870