This thesis aimed at characterizing mRNA and miRNA expression profiles in the female gamete and its microenvironment, respectively, under physiological and non-physiological conditions in order i) to assess the effect of vitrification on the biomolecular profile of the oocytes, ii) to understand the molecular basis of reproductive ageing and iii) to identify miRNAs in FF to be used as non-invasive biomarkers of oocyte quality. We demonstrated that vitrification technique might be very helpful for preserving women s fertility, since it keeps unaltered oocyte molecular profile and does not cause degradation of mRNAs essential for oocyte development (i.e. BMP15, FIGLA, GDF9, OCT4, TAF4B). We determined Apoptotic Machinery (AM) transcriptome in mature MII human oocyte pools from women aged more than 38 years (old) and compared it to those of women aged up to 35 years (young). Subsequently, some AM candidate genes with a key role in apoptotic regulation were selected and analysed in single oocytes. These studies led us to identify AM transcripts never reported in human oocytes so far (BAG3, CD40, CFLAR, TNFRSF21, TRAF2, TRAF3) and to find out other differentially expressed genes in oocytes from older women. In fact, we found a significant upregulation of proapoptotic CD40, TNFRSF10A, TNFRSF21 and downregulation of antiapoptotic BCL2 and CFLAR. Our results demonstrate that during maturation old oocytes selectively accumulate mRNAs potentially able to trigger the extrinsic apoptotic pathway and express at low levels some survival factors: this condition could make old oocytes more inclined to apoptosis. Moreover, we found TP73 among differentially expressed genes in human MII oocyte pools during reproductive ageing, a process closely related to the production of oocytes with a reduced developmental competence whose main hallmark is aneuploidy. In order to verify the potential involvement of TP73 isoforms in reproductive ageing, we determined their expression in single mature MII oocytes from women younger than 35 and older than 38 years. We found out that TAp73 isoforms are significantly downregulated in oocytes from women of advanced reproductive age. There is evidence that TAp73 interacts with some kinetochore proteins in order to stop the anaphase if chromosomes are not properly attached to the meiotic spindle. The absence of TAp73 removes this cell cycle brake, so causing genomic instability. Consequently, TAp73 downregulation in old oocytes could lead to aneuploidy in the developing embryos, explaining both the reduction of fertility and the increase of newborns with chromosomal abnormalities. Finally, we profiled the expression of 384 miRNAs in human follicular fluid and its purified exosomal fraction with respect to plasma from the same women, providing the first molecular evidence of these bioactive vesicles inside ovarian follicle. Among the 37 miRNAs that we found upregulated in follicular microenvironment, the majority of them are carried by exosomes (exosome Follicular Fluid miRNAs eFF-miRNAs) and are involved in signaling pathways critically important for follicle growth, oocyte maturation, and early embryo development. Moreover, eFF-miRNAs are able to negatively regulate genes encoding inhibitors of follicle maturation and meiosis resumption as PTEN, MTOR, P21 and RB1. These data could reveal new actors in the molecular communication among cells of ovarian follicle and eFF-miRNAs may represent valuable biomarkers of oocyte quality and reproductive disorders.
Lo scopo di questa tesi è la caratterizzazione dei profili di espressione di mRNA e miRNA nel gamete femminile e nel suo microambiente, rispettivamente, in condizioni fisiologiche e non, al fine di i) valutare l'effetto della vitrificazione sul profilo biomolecolare degli ovociti, ii) comprendere le basi molecolari dell'invecchiamento riproduttivo, iii) identificare miRNA nel fluido follicolare da usare come biomarcatori non invasivi di qualità ovocitaria. Abbiamo dimostrato che il protocollo di vitrificazione potrebbe essere usato per preservare la fertilità delle donne, in quanto mantiene inalterato il profilo molecolare degli ovociti e non causa la degradazione di mRNA essenziali per il loro sviluppo. Abbiamo determinato il profilo di espressione dei geni del Macchinario Apoptotico (MA) in ovociti umani maturi in MII provenienti da donne di età superiore ai 38 anni (old) paragonandolo a quello di donne di età inferiore ai 35 anni (young). Successivamente, i dati sono stati confermati sui singoli ovociti. Questi studi ci hanno portato ad identificare alcuni trascritti del MA mai descritti finora negli ovociti umani e altri geni differenzialmente espressi negli ovociti old. Infatti, abbiamo riscontrato una significativa sovraespressione dei geni proapoptotici CD40, TNFRSF10A e TNFRSF21, ed una sottoespressione dei geni antiapoptotici BCL2 e CFLAR negli ovociti old. I nostri risultati dimostrano che, durante la loro maturazione, gli ovociti provenienti da donne in avanzata età riproduttiva accumulano mRNA potenzialmente in grado di attivare la via estrinseca dell apoptosi ed esprimono a bassi livelli alcuni fattori di sopravvivenza: tale condizione potrebbe rendere gli ovociti old più predisposti all apoptosi. Inoltre, tra i geni differenzialmente espressi negli ovociti umani di donne in avanzata età riproduttiva abbiamo riscontrato anche TP73. L invecchiamento riproduttivo è un processo strettamente legato alla produzione di ovociti con una ridotta competenza, la cui caratteristica principale è lo sviluppo di aneuploidie. Al fine di verificare il potenziale coinvolgimento delle isoforme di TP73 nell'invecchiamento riproduttivo, abbiamo determinato la loro espressione in singoli ovociti MII maturi provenienti da donne di età inferiore ai 35 anni e di età superiore ai 38 anni. I risultati mostrano che l isoforma TAp73 è sottoespressa in maniera significativa negli ovociti provenienti da donne di avanzata età riproduttiva. Dati di letteratura mostrano che la TAp73 interagisce con alcune proteine del cinetocore per bloccare l anafase nel caso in cui i cromosomi non sono correttamente attaccati al fuso meiotico. L'assenza della TAp73 rimuove questo freno del ciclo cellulare, causando così instabilità genomica. Di conseguenza, la sottoespressione della TAp73 in ovociti old potrebbe portare ad aneuploidie nell embrione in via di sviluppo, spiegando così sia la riduzione della fertilità che l'aumento di neonati con anomalie cromosomiche che si riscontra man mano che aumenta l età materna. Infine, abbiamo caratterizzato il profilo di espressione di 384 miRNA nel fluido follicolare umano e negli esosomi da esso isolati lo abbiamo paragonato a quello del plasma prelevato dalle stesse donne, dimostrando per la prima volta la presenza di queste vescicole bioattive all'interno del follicolo ovarico. I risultati mostrano che la maggior parte dei 37 miRNA che abbiamo trovato sovraespressi nel microambiente follicolare sono trasportati dagli esosomi (eFF-miRNA) e che essi controllano delle pathway estremamente importanti per la crescita del follicolo, la maturazione ovocitaria e le prime fasi dello sviluppo embrionale. Inoltre, gli eFF-miRNA sono in grado di regolare negativamente geni codificanti inibitori della maturazione del follicolo e della ripresa della meiosi come PTEN, MTOR, P21 e RB1. Questi dati potrebbero rivelare nuovi attori molecolari nella comunicazione tra le cellule del follicolo ovarico.
Gene expression and human oocyte maturation: transcriptional control and post-transcriptional mechanisms based on intercellular signalling via exosomal miRNAs in follicular microenvironment / Santonocito, Manuela. - (2013 Dec 09).
Gene expression and human oocyte maturation: transcriptional control and post-transcriptional mechanisms based on intercellular signalling via exosomal miRNAs in follicular microenvironment
SANTONOCITO, MANUELA
2013-12-09
Abstract
This thesis aimed at characterizing mRNA and miRNA expression profiles in the female gamete and its microenvironment, respectively, under physiological and non-physiological conditions in order i) to assess the effect of vitrification on the biomolecular profile of the oocytes, ii) to understand the molecular basis of reproductive ageing and iii) to identify miRNAs in FF to be used as non-invasive biomarkers of oocyte quality. We demonstrated that vitrification technique might be very helpful for preserving women s fertility, since it keeps unaltered oocyte molecular profile and does not cause degradation of mRNAs essential for oocyte development (i.e. BMP15, FIGLA, GDF9, OCT4, TAF4B). We determined Apoptotic Machinery (AM) transcriptome in mature MII human oocyte pools from women aged more than 38 years (old) and compared it to those of women aged up to 35 years (young). Subsequently, some AM candidate genes with a key role in apoptotic regulation were selected and analysed in single oocytes. These studies led us to identify AM transcripts never reported in human oocytes so far (BAG3, CD40, CFLAR, TNFRSF21, TRAF2, TRAF3) and to find out other differentially expressed genes in oocytes from older women. In fact, we found a significant upregulation of proapoptotic CD40, TNFRSF10A, TNFRSF21 and downregulation of antiapoptotic BCL2 and CFLAR. Our results demonstrate that during maturation old oocytes selectively accumulate mRNAs potentially able to trigger the extrinsic apoptotic pathway and express at low levels some survival factors: this condition could make old oocytes more inclined to apoptosis. Moreover, we found TP73 among differentially expressed genes in human MII oocyte pools during reproductive ageing, a process closely related to the production of oocytes with a reduced developmental competence whose main hallmark is aneuploidy. In order to verify the potential involvement of TP73 isoforms in reproductive ageing, we determined their expression in single mature MII oocytes from women younger than 35 and older than 38 years. We found out that TAp73 isoforms are significantly downregulated in oocytes from women of advanced reproductive age. There is evidence that TAp73 interacts with some kinetochore proteins in order to stop the anaphase if chromosomes are not properly attached to the meiotic spindle. The absence of TAp73 removes this cell cycle brake, so causing genomic instability. Consequently, TAp73 downregulation in old oocytes could lead to aneuploidy in the developing embryos, explaining both the reduction of fertility and the increase of newborns with chromosomal abnormalities. Finally, we profiled the expression of 384 miRNAs in human follicular fluid and its purified exosomal fraction with respect to plasma from the same women, providing the first molecular evidence of these bioactive vesicles inside ovarian follicle. Among the 37 miRNAs that we found upregulated in follicular microenvironment, the majority of them are carried by exosomes (exosome Follicular Fluid miRNAs eFF-miRNAs) and are involved in signaling pathways critically important for follicle growth, oocyte maturation, and early embryo development. Moreover, eFF-miRNAs are able to negatively regulate genes encoding inhibitors of follicle maturation and meiosis resumption as PTEN, MTOR, P21 and RB1. These data could reveal new actors in the molecular communication among cells of ovarian follicle and eFF-miRNAs may represent valuable biomarkers of oocyte quality and reproductive disorders.File | Dimensione | Formato | |
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