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Title: TGFβ signalling and cell cycle regulation during early follicle development
Author: Granados-Aparici, Sofia
ISNI:       0000 0004 6495 0515
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2017
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In the mammalian ovary, little is known about the mechanisms that maintain the relatively quiescent phenotype in primordial follicles or trigger primordial follicle activation (PFA). Previous studies in our lab showed the TGFβ mediator and transcription factor SMAD2/3 are detectable in granulosa cells (GCs) of primordial follicles. Since cell proliferation involves cell cycle progression, this thesis sought to investigate whether SMAD2/3 regulates the expression of MYC oncogene and the cell cycle genes CCND2 and p27 in GCs in the mouse ovary, and whether this regulation is associated with the maintenance and activation of primordial follicles. Therefore, the first part of this thesis focused on the expression and localisation of these factors throughout the stages of early follicle development. SMAD3, CCND2 and p27 proteins co-localised in GC nuclei of primordial follicles while expression of each decreased in growing follicles. Furthermore, using coimmunoprecipitation, CCND2 and p27 proteins associated in complexes in samples enriched in primordial follicles, and these complexes were lost in growing follicles, suggesting CCND2/p27 protein dynamics contributes to the maintenance of GC cell cycle arrest. Using ChIP-qPCR on immature mouse ovaries enriched with different proportions of small follicles, SMAD3 bound to the promoter of Ccnd2 and Myc, but not p27. Gene expression analysis using qPCR revealed that Ccnd2 was relatively higher in ovaries containing mostly primordial follicles, while Myc was relatively higher in older aged ovaries with many growing follicles. This suggests that SMAD3 promoted and repressed the expression of these genes, respectively. With this evidence, the effect of brief exposure to TGFβ1 ligand and TGFβ-receptor inhibitor on SMAD3-dependent regulation of cell cycle genes and PFA was examined in a validated neonatal mouse ovary culture model. TGFβ1 ligand increased Ccnd2 and Myc genes, while the inhibitor had no effect. SMAD3 binding to both genes was unchanged after any of the treatments; however, elevated pERK and pAKT indicated that changes in gene expression may be due to noncanonical TGFβ signalling. TGFβ1 ligand had a small influence on promoting PFA whereas the inhibitor caused a slight delay in GC proliferation and increased the presence of multi-oocyte follicles. Taken together, these findings suggest that basal TGFβ levels maintain the primordial follicle phenotype via the regulation of Ccnd2 and Myc in GCs. Altering the balance of TGFβ exposure influences the expression of these genes and PFA. Understanding these molecular events may shed some light on the mechanisms leading to age-related infertility and ovarian dysfunctions from conditions such as POI or PCOS.
Supervisor: Fenwick, Mark Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available