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

• Published in: Scientific reports 2019-04, Vol.9 (1), p.6513, Article 6513
• Main Authors: Granados-Aparici, Sofia, Hardy, Kate, Franks, Stephen, Sharum, Isam B., Waite, Sarah L., Fenwick, Mark A.
• Format: Article
• Language: English
• Subjects: 13/51; 38/77; 631/136/83/2359; 631/443/494; 82/80; Animals; Animals, Newborn; Cell cycle; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Cyclin D2; Cyclin D2 - genetics; Cyclin D2 - metabolism; Cyclin-Dependent Kinase Inhibitor p27 - genetics; Cyclin-Dependent Kinase Inhibitor p27 - metabolism; Female; Follicles; Gene Expression Regulation - drug effects; Granulosa cells; Granulosa Cells - cytology; Granulosa Cells - metabolism; Humanities and Social Sciences; Immunofluorescence; Immunoprecipitation; Mice, Inbred C57BL; multidisciplinary; Myc protein; Neonates; Nucleotide sequence; Oocytes; Oocytes - cytology; Oocytes - metabolism; Ovarian Follicle - cytology; Ovarian Follicle - metabolism; Ovaries; Ovary - cytology; Ovary - drug effects; Ovary - metabolism; Population density; Proto-Oncogene Proteins c-myc - genetics; Proto-Oncogene Proteins c-myc - metabolism; Science; Science (multidisciplinary); Signal transduction; Smad protein; Smad3 protein; Smad3 Protein - genetics; Smad3 Protein - metabolism; TOR protein; Transforming Growth Factor beta1 - pharmacology; Transforming growth factor-b1
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-019-42878-4

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.