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.
GTP-binding proteins (GTPases) have been detected in the mitochondria of human placenta. It has been proposed that porin interacts with GTPases in the mitochondrion to modulate contact site function, however, their identity and location is not known. In this study, we investigated the location of GTPases in mitochondria from term placentae as well as the expression of mitochondrial GTPases in mid-term placentae. Mitochondria obtained from human term and mid-term placentae were purified by sedimentation. Sub-mitochondrial vesicles prepared from ruptured and sonicated mitochondria were separated by ultracentrifugation in sucrose density gradients. The location of membrane vesicles was determined using marker enzymes. Mitochondrial proteins were separated by SDS-PAGE. Western blots were incubated in [alpha-(32)P]-GTP and detected using autoradiography or antibodies against known GTPases and porin followed by enhanced chemiluminescence. [alpha-(32)P]-GTP bound 24 and 28 kDa proteins located in the outer membrane. The G(salpha)antibody detected 42.5, 53 and 67 kDa proteins. The G(ialpha)antibody identified a 40.5 kDa band in contact sites and the outer membrane, as well as 55 and 105 kDa proteins in contact site vesicles. The Ran antibody detected a 28 kDa protein, mainly in the outer membrane. Porin migrated at 30 kDa. G(ialpha)and Ran were detected in mitochondria from both term and mid-term placentae. The location of porin and GTPases leave open the possibility that these proteins interact in contact sites and may also be responding to extra-mitochondrial signals. Ran and G(ialpha)are expressed by mid-term in human placentae and may be necessary for placental functions at this stage of development. It will be important in future experiments to characterise the physiological functions of these GTP-binding proteins in the mitochondria of human placenta.
GDF-9 and -9B (BMP-15) are oocyte-derived members of the TGF-β superfamily. In the mouse ovary, the absence of GDF-9 leads to an arrest of follicle development at the primary/preantral stage. As a result GDF9 deficient mice are infertile1 because follicle development does not reach a stage where ovulation and oocyte release can occur. In contrast, GDF-9B knockout mice are subfertile2. GDF-9 was shown to act via TGFβRI and BMPRII and GDF-9B via BMPRIB and BMPRII. We have much to learn about what regulates the production of GDF-9, GDF-9B and the expression of its receptors. These studies investigated whether FSH, an important mediator of folliculogenesis, plays a role in this regulation. Preantral follicles (110–135 µm in diameter) were isolated from 18 day old C57BL/6 mice using fine needles. Follicles were cultured (30–35 per well) for 7 days with varying doses of FSH (0–100ng/ml). RNA was extracted, reverse transcribed and real time PCR was carried out with primer sets for GDF-9, GDF-9B, TGFβRI, BMPRII and GAPDH. Immunohistochemistry was conducted on sections of formalin-fixed 18 day old mouse ovary using antisera directed at TGFβRI and BMPRII. GDF-9 and GDF-9B mRNAs were downregulated by FSH treatment (compared to untreated control). There was no effect of FSH on the expression of either receptor. TGFβRI and BMPRII receptor proteins were localised to the cytoplasm of oocytes and granulosa cells in 18 day old mouse ovary. Both were mostly localised to secondary follicles, lighter TGFβRI staining was found in less mature follicles. Receptors for GDF9 signalling were both present consistent with direct effects of GDF9 on ovarian function. GDF9B might also have an effect although it remains to be seen if the type 1 receptor is localised to the mouse ovary. Further studies are required to investigate receptor regulation.
Nuclear factor-κB (NF-κB) designates a family of transcription factors that have been shown to modulate antiviral, inflammatory and immune responses. Activation of NF-κB is dependent on IKKβ a component of the IκB kinase (IKK) complex which promotes degradation of IκB inhibitory proteins and allows nuclear translocation of NF-κB. Our studies in ovarian granulosa cell tumour cell lines (COV434 and KGN) indicate that NF-κB signalling is constitutively activated. FSH has been reported to increase XIAP expression through NFκB activity in granulosa cells, but beyond that the role of NFκB signalling in folliculogenesis has not been elucidated. To establish the significance of NF-κB signalling in the ovary (and testis), we have generated a gonadal specific IKKbeta conditional knockout mouse. A transgenic mouse line containing floxed IKKβ alleles (gift of M Karin, UCSD) was crossed with a cre mouse line (gift of M Matzuk, BCM) expressing the recombinase in anti-Müllerian hormone receptor expressing cells (granulosa cells or Sertoli cells). The resulting mice will not express IKKβ in granulosa cells or Sertoli cells and thus cannot activate the classical NFκB signalling pathway. On histological assessment, the ovaries and testes from flox x cre (heterogenous) mice appear normal with follicles of all developmental stages and corpora lutea. Preliminary data suggests that breeding with the heterogenous females resulted in increased litter sizes. The histology of the testes is also unremarkable. The mice homozygous for the deletion of IKKβ in the granulosa cells appear healthy and a preliminary assessment does not reveal gross morphological abnormalities of the ovaries. The results of detailed histological and overall assessment of these mice will be presented. These IKK conditional knockout mice should provide insights into the role of NFκB signalling in gonadal function.
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