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

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.

SUBMITTER: Granados-Aparici S

PROVIDER: S-EPMC6478827 | biostudies-other | 2019 Apr

REPOSITORIES: biostudies-other

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

Granados-Aparici Sofia S Hardy Kate K Franks Stephen S Sharum Isam B IB Waite Sarah L SL Fenwick Mark A MA

Scientific reports 20190424 1

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-loca ...[more]

PMID: 31015579

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Project description:BackgroundFor the first time, thorough morphometrical measurements of primordial ovarian follicles were performed and their age-related changes were investigated in Lithuanian women of the reproductive age.MethodsOvaries of dead women (n = 30) were divided into six age groups: 15-20 years old, 21-25 years old, 26-30 years old, 31-35 years old, 36-40 years old and 41-46 years old. Histological slides of left and right ovaries were stained using haematoxylin-eosin and periodic acid-Schiff (PAS) staining methods. The morphometrical measurements of 10 primordial ovarian follicles of the left and right ovary of each woman were made from microphotographs.ResultsThe diameter of primordial ovarian follicles increased in groups of women from 15 years old to 35 years old and decreased in the groups from 36 years old to 46 years old. The area of primordial ovarian follicles increased in the groups of women until 35 years old. It decreased in the groups of women older than 36 years. The follicular basement membrane thickened from 1.29 ± 0.11 μm to 1.43 ± 0.18 μm with increasing age of women. The diameter of primary oocytes enlarged until 35 years and then began to decrease. The area of primary oocytes increased in women until 35 years. It decreased in groups of women aged 36-40 and 41-46 years old. The diameter and the area of primary oocytes nuclei increased in women aged 15-30 years old; later, it began to decrease. The length of follicular cells varied from 8.56 ± 0.43 μm to 8.72 ± 0.27 μm (p > 0.05). The height of follicular cells varied from 2.59 ± 0.27 μm to 2.7 ± 0.21 μm (p > 0.05). The diameter, the area and the basement membrane thickness of primordial ovarian follicles and the diameter and the area of primary oocytes and their nuclei differed insignificantly in left and right ovaries in all age groups of women (p > 0.5). The length and height of follicular cells were similar in left and right ovaries of the same age group (p > 0.5).ConclusionsThe age decreasing of morphometrical parameters begins in primordial ovarian follicles and their primary oocytes in Lithuanian women older than 35 years. The thickness of the follicular basement membrane increased with increasing age of women. No significant differences were found in the morphometrical parameters in primordial follicles of left and right ovaries in the same age group of women.

Dataset Information

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

Publications

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

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