Inhibition of the forkhead transcription factor, FOXO3a, can promote the transition from primordial to primary follicle and subsequent follicle development in mammalian ovaries. Stem cell factor (SCF) initiates anti-apoptotic signaling from its membrane receptor, c-kit, to Bcl-2 family members through PI3K/AKT in oocytes of primordial follicles. However, whether FOXO3a mediates the apoptosis of naked oocytes and oocytes of primordial follicles remains unknown. In the present study, oocytes from nests and primordial follicles from neonatal rat ovaries were cultured, and oocyte apoptosis was examined using the TUNEL technique. The pro-apoptotic action of FOXO3a and the potential signal transduction pathways were investigated using RT-PCR, Western blot, and immunocytochemistry. Culturing oocytes in the presence of SCF did not affect the level of total FOXO3a protein, but rapidly elevated the level of phosphorylated FOXO3a (indicating functional suppression). As phosphorylated FOXO3a increased, oocyte apoptosis was inhibited. The specific PI3K/Akt inhibitor, LY 294002, abolished the phosphorylation of FOXO3a and the anti-apoptotic action of SCF. SCF down-regulated the expression of p27KIP1 and pro-apoptotic factors such as Bim, Bad, and Bax, and this activity was reversed by LY 294002. SCF up-regulated the expression of MnSOD, which was also inhibited by LY 294002. However, SCF had no effect on Bcl-2 protein. These results suggest that FOXO3a is involved in oocyte apoptosis in the neonatal rat ovary, and the SCF-PI3K/Akt-FOXO3a signaling pathway mediates oocyte apoptosis and primordial follicle formation.
"Studies have shown that TGF-β1 expression in MCs is increased under high glucose conditions20,21,22,24,25 and that TGF-β1 participates in activation of the PI3K/Akt pathway26,27,28,29. PI3K/Akt pathway activation by TGF-β1 induces the phosphorylation and inactivation of FoxO3a and decreases expression of its downstream target genes, Bim and MnSOD30,31,32,33,34,35,36. Consequently, both the cell proliferation rate and ROS production increase37. "
[Show abstract][Hide abstract] ABSTRACT: Aim:
To investigate whether NO over-production in rat mesangial cells cultured in high glucose (HG) is related to activation of the TGF-β1/PI3K/Akt pathway.
Rat mesangial cells line (HBZY-1) was exposed to HG (24.44 mmol/L) or H2O2 (10 μmol/L) for 16 h. NO release was quantified using the Griess assay. The TGF-β1 level was measured using ELISA. The protein expression of p-Akt, t-Akt, Bim, and iNOS was examined by Western blotting. The mRNA levels of TGF-β1 and Bim were measured using RT-PCR. The cell proliferation rate was estimated using a BrdU incorporation assay.
Treatment of the cells with HG, H2O2, or TGF-β1 (5 ng/mL) significantly increased the NO level that was substantially inhibited by co-treatment with the NADPH oxidase inhibitor diphenylene iodonium (DPI), TGF-β1 inhibitor SB431542, or PI3K inhibitor LY294002. Both HG and H2O2 significantly increased the protein and mRNA levels of TGF-β1 in the cells, and HG-induced increases of TGF-β1 protein and mRNA were blocked by co-treatment with DPI. Furthermore, the treatment with HG or H2O2 significantly increased the expression of phosphorylated Akt and iNOS and cell proliferation rate, which was blocked by co-treatment with DPI, SB431542, or LY294002. Moreover, the treatment with HG or H2O2 significantly inhibited Bim protein and mRNA expression, which was reversed by co-treatment with DPI, SB431542, or LY294002.
The results demonstrate that high glucose causes oxidative stress and NO over-production in rat mesangial cells in vitro via decreasing Bim and increasing iNOS, which are at least partially mediated by the TGF-β1/PI3K/Akt pathway.
"The Forkhead transcription factor 3a (FOXO3a) halts the transition from primordial to primary follicle in rodents (Reddy et al., 2005), by inhibiting cyclin-dependent kinases, thus decreasing proliferation (Kops et al., 2002) and stimulating apoptosis of the oocyte and follicular cells (Liu et al., 2009). FOXO3a is phosphorylated by KL (Liu et al., 2009) and testosterone (Yang et al., 2010) inactivating the protein and preventing cell apoptosis allowing the transition from primary to secondary follicle. The anti-Müllerian hormone (AMH), synthesized by granulosa cells (Sadeu et al., 2008), is another member of the TGF-β family known to inhibit the development of primordial follicles (Moniruzzaman and Miyano, 2010): for example, in rat ovarian cultures, the addition of AMH reduces the transition from primordial to primary follicles even in the presence of KL (Nilsson et al., 2007). "
[Show abstract][Hide abstract] ABSTRACT: Knowledge on the physiological processes that control follicular development may allow for the development of strategies to increase reproductive efficiency in domestic ruminants. Follicle development depends on the balance between survival factors, proliferation and cell death, which determine whether the follicle starts and continues to grow or is removed from the ovary. During fetal development of the female, primordial germinal cells proliferate by mitosis to reach the gonadal ridge, where the oogonia are surrounded by flattened cells to assemble the primordial follicles. Kit ligand, BMP-15 and GDF-9 stimulates a group of primordial follicles to begin their growth until they reach preantral development. The growth to the antral stage seems to be independent of gonadotrophins and promoted by growth factors. In gonadotrophin suppressed animals follicles will grow up to a diameter of 2 mm in sheep and 4mm in cattle. The cyclic secretion of FSH and LH promotes the recruitment and growth to the large antral stage and the eventual ovulation if luteolysis occurs. Follicular development in cows, sheep and goats during the estrous cycle occurs in a pattern like-wave, where groups of follicles begin their growth in response to an increase of FSH, but only some (sheep and goats) or one (cows) is selected as the dominant, and ovulates if its dominance coincides with the lysis of the CL and the reduction of progesterone. Among the factors that determine whether a large antral follicle starts, continues and completes its development are its responsiveness to gonadotropins, its steoidogenic capability and the presence of survival and proliferating factors such as IGF-I and VEGF.
Tropical and Subtropical Agroecosystems 01/2012; 15(1):147-160.
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