SMAD3 directly regulates cell cycle genes to maintain arrest in granulosa cells of mouse primordial follicles

Sofia Granados-Aparici,Isam B Sharum,Mark A Fenwick,Sarah L Waite,Kate Hardy,Stephen Franks

doi:10.1038/s41598-019-42878-4

Abstract

Primordial follicles, consisting of granulosa cell (GC)-enveloped oocytes are maintained in a state of developmental arrest until activated to grow. The mechanism that operates to maintain this arrested state in GCs is currently unknown. Here, we show the TGFβ-activated transcription factor SMAD3 is expressed in primordial GC nuclei alongside the cell cycle proteins, cyclin D2 (CCND2) and P27. Using neonatal C57/Bl6 mouse ovaries densely populated with primordial follicles, CCND2 protein co-localised and was detected in complex with P27 by immunofluorescence and co-immunoprecipitation, respectively. In the same tissue, SMAD3 co-precipitated with DNA sequences upstream of Ccnd2 and Myc transcription start sites implicating both as direct SMAD3 targets. In older ovaries follicle growth was associated with nuclear exclusion of SMAD3 and reduced P27 and CCND2 in GCs, alongside elevated Myc expression. Brief (2 H) exposure of neonatal ovaries to TGFβ1 (10 ng/ml) in vitro led to immediate dissociation of SMAD3 from the Ccnd2 and Myc promoters. This coincided with elevated Myc and phospho-S6, an indicator of mTOR signalling, followed by a small increase in mean primordial GC number after 48 H. These findings highlight a concentration-dependent role for TGFβ signalling in the maintenance and activation of primordial follicles, through SMAD-dependent and independent signalling pathways, respectively.

Highlights

Primordial follicles, each consisting of a central oocyte surrounded by a single layer of supporting granulosa cells (GCs) are held in a relative state of developmental arrest until activated to grow[1,2,3]
We recently showed that in the mouse ovary, the TGFβ-mediated transcription factors SMAD2/3 are detectable in nuclei of primordial GCs, suggesting that this pathway is active in these cells[6]
Immunofluorescent localisation of SMAD2 was very weak in ovaries at each age, while SMAD3 was expressed in GCs of small follicles, notably in the earliest primordial stages as exemplified in sections from day 4 ovaries (Fig. 1B)

Summary

Introduction

Primordial follicles, each consisting of a central oocyte surrounded by a single layer of supporting granulosa cells (GCs) are held in a relative state of developmental arrest until activated to grow[1,2,3]. We recently showed that in the mouse ovary, the TGFβ-mediated transcription factors SMAD2/3 are detectable in nuclei of primordial GCs, suggesting that this pathway is active in these cells[6]. It is well established that TGFβ signalling is important for maintaining growth arrest in a range of cell types by directly inhibiting Myc[7,8,9], as well as regulating the expression of other cell cycle proteins[10,11]. Mouse as a model, we show that SMAD3 localises to GCs of small follicles where it directly promotes the expression of Ccnd[2] and represses Myc. The CCND2 protein is detectable in complex with the inhibitory factor P27 and together highlights a mechanism that potentially maintains primordial GC arrest, whilst ensuring they are poised ready to proliferate. The loss of SMAD3 in GCs of growing follicles is associated with elevated mTOR signalling, potentially explaining a dual mechanism of TGFβ signalling in follicle growth and arrest

Methods

Results

Conclusion