Ovarian follicular growth and differentiation in response to transforming growth factor-b (TGFB) was investigated using postnatal and immature ovarian models. TGFB ligand and receptor mRNAs were present in the rat ovary 4-12 days after birth and at day 25. In order to assess the impact of TGFB1 on follicle growth and transition from the primordial through to the primary and preantral stages of development, we established organ cultures with 4-day-old rat ovaries. After 10 days in culture with FSH, TGFB1, or a combination of the two, ovarian follicle numbers were counted and an assessment of atresia was undertaken using TUNEL. Preantral follicle numbers declined significantly when treated with the combination of FSH and TGFB1, consistent with our morphological appraisal suggesting an increase in atretic primary and preantral follicles. To investigate the mechanisms behind the actions of TGFB1, we isolated granulosa cells and treated them with FSH and TGFB1. Markers of proliferative, steroidogenic, and apoptotic capacity were measured by real-time PCR. Cyclin D2 mRNA expression by granulosa cells was significantly increased in response to the combination of FSH and TGFB. The expression of forkhead homolog in rhabdomyosarcoma (Foxo1) mRNA by granulosa cells was significantly reduced in the presence of both FSH and TGFB1, individually and in combination regimes. By contrast, the expression of steroidogenic enzymes/proteins was largely unaffected by TGFB1. These data suggest an inhibitory role for TGFB1 (in the presence of FSH) in follicle development and progression.
The role TGF-β plays in ovarian follicular growth and differentiation was investigated using a ‘physiological' culture system. TGF- β ligand and receptors are present in the rat ovary from 4 days after birth. Therefore we established organ cultures with these ovaries in order to assess the potential impact of TGF- β1 on follicle growth and transition from the primordial through to the primary and preantral stages of development. Whole ovaries were isolated and cultured for 10 days on floating filters with the addition of supplemented DMEM/Hams F-12 media and either FSH (100ng/mL), TGF- β1 (10ng/mL), or a combination of the two. Media as well as treatments were refreshed every second day. At the end of the culture period, ovaries were fixed in 10% formalin, embedded in paraffin and sectioned at 5µm. Sections were used for morphological assessment and ovarian follicle counting with three serial sections mounted/slide and every alternate slide used for counting of primordial, primary and preantral follicles. An evaluation of atresia by the detection of apoptotic cells was undertaken using terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick-end labelling (TUNEL) via the ApopTag® Peroxidase in situ apoptosis detection kit. Results gathered from this study show preantral follicle numbers declined significantly when treated with the combination of FSH and TGF- β1, consistent with our morphological appraisal of atresia where the combined treatment appeared to produce more apoptotic follicles than healthy follicles, suggesting an increase in atretic primary and preantral follicles. These preliminary findings suggest an inhibitory role for TGF- β1 in the presence of FSH, resulting in fewer follicles making the transition from the primary to the preantral stage. Further studies are required to test the effects of other TGF-β superfamily members on follicle transition in vitro. Supported by the NHMRC of Australia (Regkeys 241000, 441101, 465415, 198705)
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