In numerous mammalian species, immature oocytes at the Germinal Vesicle (GV) stage display different chromatin configurations, associated with crucial roles in the development of the oocyte competence. In particular, in mouse ovaries it is possible to distinguish two different populations of GV oocytes with different frequencies and developmental potential: Surrounded Nucleus (SN) and Not Surrounded Nucleus (NSN) oocytes. The major difference between these two cell types lies in their heterochromatin configuration after DAPI staining: while SN oocytes’ heterochromatin forms a positive stained ring surrounding the nucleolus, the NSN’s is more lax and clustered in spots of various sizes. This difference also reflects the oocyte’s developmental potential since NSN oocytes, unlike SNs, cannot develop after the 4-cells stage, after maturation and fertilization. Even though the reasons behind this difference are still unknown, researches on this topic have uncovered many cytological and molecular factors that may concur to cause it. This work of thesis concentrates on the relation (if exists) between nutrients internalization and oocyte maturation, investigating the role that Clathrin, one of the proteins that has been found to be upregulated in NSN oocytes and one of the main players of endocytosis, might have in these processes. Immunofluorescence results showed the presence of three different profiles of Clathrin’s localization in both SN and NSN oocytes and therefore named complete, incomplete and half-moon. Qualitative and semi-quantitative analyses of the three profiles showed no significant differences between SN and NSN oocytes, thus demonstrating no correlation between chromatin configuration and Clathrin localization. While the complete (35.8% of SN, 25.7% of NSN) and incomplete configurations (42.6% of SN, 46.6% of NSN) are the most recurrent and are presumably characterized by Clathrin plaques on the cell membrane in a more-or-less continuous way, the half-moon one was less frequent (21.6% of SN, 25.7% of NSN), less intense and represented by a peculiar localization of Clathrin plaques in one half of the cell. This interesting pattern may indicate those oocytes that, if they had not been isolated, would have likely undergone Germinal Vesicle Breakdown, MI and MII. During the phases behind the maturation process, organelles and factors are arranged in one side of the cell, ready to be recruited. To better visualize the endocytosis process and evaluate the presence of Clathrin coated structures at the membrane level, presumable on the cell area opposite to spindle formation, Time-lapse analyses with a membrane marker were made. Clathrin is a well-known moonlighting protein that, in addition to its role in endocytosis, becomes a spindle stabilizer in MII oocytes, where it locates at the spindle level. To understand if and how Clathrin-mediated endocytosis’ inhibition could affect the progression of the cell cycle by interfering with Clathrin’s recruitment at the spindle level further analyses were made. Organelles positioning and cytoplasmic rearrangements were followed in vivo to understand better the maturation process and, in particular, endo-lysosomal movements were observed through TimeLapse experiments to look for predictive models of polar body extrusion side and spindle positioning. Taken all together, these results contribute to shed light on Clathrin’s localization and function during oocyte’s maturation, emphasizing its moonlighting nature. They also revealed an important feature of this process: Clathrin asymmetric localization predicts the site of spindle formation in mouse oocytes. If we consider the importance of the endocytosis process it is clear that this type of analysis may be essential to unveil the still unknown mechanisms behind the maturation of gametes in humans and, for example, in species of economic interest.
In numerose specie animali, gli oociti antrali allo stadio di GV (Germinal Vesicle) presentano diverse conformazioni cromatiniche, suddivisibili in classi, associate a differenze nelle loro capacità di sviluppo. Diversi studi pubblicati in letteratura hanno collegato tali differenze all’espressione genica e al contenuto citoplasmatico di questi oociti, che sono attualmente oggetto di studio. In particolare, due diverse popolazioni di oociti antrali sono state identificate nell’ovario di topo, attraverso la colorazione con il DAPI o l’Hoechst33342: gli oociti SN (Surrounded Nucleus), caratterizzati dalla presenza di un anello DAPI positivo attorno al nucleo, e gli oociti NSN (Not Surrounded Nucleus), caratterizzati da una conformazione più irregolare e diffusa, a spot. Tale differenza nella loro configurazione cromatinica riflette anche le loro diverse capacità di sviluppo: mentre gli oociti SN sono in grado di svilupparsi, in seguito ad ovulazione e fecondazione, in un organismo completo, gli NSN non sono in grado di differenziarsi oltre lo stadio di 4 cellule, anche se ovulati e fecondati. Benché i meccanismi alla base di questa differenza nelle capacità di sviluppo siano ancora sconosciuti, nell’ultimo decennio sono stati identificati molti fattori molecolari e citoplasmatici che potrebbero contribuire a generarla. Questo progetto di tesi si pone l’obiettivo di verificare se esista o meno una relazione fra l’internalizzazione dei nutrienti e la maturazione degli oociti, esplorando il ruolo che la Clatrina, una delle proteine up-regolate negli oociti antrali NSN, potrebbe avere nell’avanzamento della meiosi. La localizzazione della Clatrina è stata analizzata in un pool di oociti GV (sia SN che NSN) da un punto di vista qualitativo e semi-quantitativo attraverso una serie di esperimenti di immunofluorescenza. Tre diversi profili di localizzazione sono stati identificati e nominati completo, incompleto e a mezza luna. Mentre il segnale completo (35.8% degli SN, 25.7% degli NSN) e quello incompleto (42.6% degli SN, 46.6% degli NSN) si sono rivelati essere i più frequenti, il segnale a mezza luna è meno diffuso (21.6% degli SN, 25.7% degli NSN). Durante le fasi che precedono la divisione cellulare, organelli ed altre molecole portate nella cellula grazie all’endocitosi mediata da Clatrina, necessarie per la maturazione dell’oocita, si posizionano in una delle due metà della cellula pronte ad essere reclutate. Analisi in Time Lapse con un marcatore di membrana (un colorante lipofilo) sono state utilizzate per visualizzare il processo di endocitosi e localizzare le strutture formate dalla Clatrina a livello di membrana, nell’area presumibilmente opposta a quella di assemblaggio dello spindle. Infine, il posizionamento di alcuni organelli ed i riarrangiamenti citoplasmatici sono stati seguiti in vivo attraverso una serie di esperimenti in Time Lapse. In particolare, i movimenti endo-lisosomiali sono stati analizzati usando due coloranti, il Ds-Red ed il LysoTracker, per visualizzare la distribuzione dei lisosomi nel citoplasma e la loro dinamica durante la maturazione degli oociti. Questi risultati aiutano a spiegare i profili di localizzazione della Clatrina identificati e la sua funzione nel processo di maturazione dell’oocita, enfatizzando la sua natura moonlighting e predicendo il sito della formazione dello spindle. Nonostante nessuna differenza sia stata riscontrata fra i pattern di endocitosi di oociti antrali SN e NSN, studi successivi serviranno per individuare i fattori sottesi alla differente capacità di sviluppo riscontrata fra questi due tipi cellulari, che potrebbe avere un importante riscontro anche per le specie di interesse economico e per la salute umana.
The asymmetric localization of Clathrin heavy chain predicts the site of spindle formation in mouse oocytes
TODISCO, ELENA
2020/2021
Abstract
In numerous mammalian species, immature oocytes at the Germinal Vesicle (GV) stage display different chromatin configurations, associated with crucial roles in the development of the oocyte competence. In particular, in mouse ovaries it is possible to distinguish two different populations of GV oocytes with different frequencies and developmental potential: Surrounded Nucleus (SN) and Not Surrounded Nucleus (NSN) oocytes. The major difference between these two cell types lies in their heterochromatin configuration after DAPI staining: while SN oocytes’ heterochromatin forms a positive stained ring surrounding the nucleolus, the NSN’s is more lax and clustered in spots of various sizes. This difference also reflects the oocyte’s developmental potential since NSN oocytes, unlike SNs, cannot develop after the 4-cells stage, after maturation and fertilization. Even though the reasons behind this difference are still unknown, researches on this topic have uncovered many cytological and molecular factors that may concur to cause it. This work of thesis concentrates on the relation (if exists) between nutrients internalization and oocyte maturation, investigating the role that Clathrin, one of the proteins that has been found to be upregulated in NSN oocytes and one of the main players of endocytosis, might have in these processes. Immunofluorescence results showed the presence of three different profiles of Clathrin’s localization in both SN and NSN oocytes and therefore named complete, incomplete and half-moon. Qualitative and semi-quantitative analyses of the three profiles showed no significant differences between SN and NSN oocytes, thus demonstrating no correlation between chromatin configuration and Clathrin localization. While the complete (35.8% of SN, 25.7% of NSN) and incomplete configurations (42.6% of SN, 46.6% of NSN) are the most recurrent and are presumably characterized by Clathrin plaques on the cell membrane in a more-or-less continuous way, the half-moon one was less frequent (21.6% of SN, 25.7% of NSN), less intense and represented by a peculiar localization of Clathrin plaques in one half of the cell. This interesting pattern may indicate those oocytes that, if they had not been isolated, would have likely undergone Germinal Vesicle Breakdown, MI and MII. During the phases behind the maturation process, organelles and factors are arranged in one side of the cell, ready to be recruited. To better visualize the endocytosis process and evaluate the presence of Clathrin coated structures at the membrane level, presumable on the cell area opposite to spindle formation, Time-lapse analyses with a membrane marker were made. Clathrin is a well-known moonlighting protein that, in addition to its role in endocytosis, becomes a spindle stabilizer in MII oocytes, where it locates at the spindle level. To understand if and how Clathrin-mediated endocytosis’ inhibition could affect the progression of the cell cycle by interfering with Clathrin’s recruitment at the spindle level further analyses were made. Organelles positioning and cytoplasmic rearrangements were followed in vivo to understand better the maturation process and, in particular, endo-lysosomal movements were observed through TimeLapse experiments to look for predictive models of polar body extrusion side and spindle positioning. Taken all together, these results contribute to shed light on Clathrin’s localization and function during oocyte’s maturation, emphasizing its moonlighting nature. They also revealed an important feature of this process: Clathrin asymmetric localization predicts the site of spindle formation in mouse oocytes. If we consider the importance of the endocytosis process it is clear that this type of analysis may be essential to unveil the still unknown mechanisms behind the maturation of gametes in humans and, for example, in species of economic interest.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/20.500.14239/13925