Effects of IGF- 1, TGF-alpha plus TGF-beta1 and bFGF on in vitro survival, growth and apoptosis in FSH-stimulated buffalo (Bubalis bubalus) preantral follicles
Investigate the effect of various growth factors viz. IGF-I, TGF-alpha + TGF-beta1 and bFGF either alone or in combination, with FSH on in vitro growth, survival, antrum formation, steroidogenesis and apoptosis of buffalo preantral follicles (PFs).
Buffalo ovaries were collected from abattoir; PFs were isolated and divided into five treatment groups. TCM-199 supplemented with 10% FBS, 1% ITS+EGF+FSH control (group a), control+IGF-I (group b), control + TGF-alpha + TGF-beta1 (group c), control + IGF-I + TGF-alpha + TGF-beta1 (group d) and control+bFGF (group e). Progesterone (P4) and 17beta-estradiol (E2) concentrations were evaluated by RIA and apoptosis by TUNEL assay.
TGF-alpha + TGF-beta1 inhibited follicular survival and induced oocyte apoptosis, while IGF-I + TGF-alpha + TGF-beta1 suppressed this inhibitory action. IGF-I significantly (P < 0.05) enhanced the follicle survival, growth and induced antrum formation. FGF had greater effects on both survival and growth rate of oocytes than other treatment groups. Progesterone and estradiol accumulation was significantly (P < 0.05) greater in presence of FGF and IGF-I than TGF-alpha + TGF-beta1.
Survival, growth, antrum formation and steroidogenesis are stimulated by IGF-I and bFGF, whereas TGF-alpha + TGF-beta1 inhibited growth and survival of PFs which led to induced oocyte apoptosis in buffalo PFs.
Available from: José Ricardo Figueiredo
- "Considering that IGF-1 stimulated the antrum formation and steroidogenesis after in vitro culture of buffalo preantral follicles (Sharma et al., 2010) and that antral follicles of cattle had greater IGF binding protein gene expression than secondary follicles (Wandji et al., 1992), the greater relative amounts of mRNA for IGF1 with use of MEM-S in the present study may have been due to the presence of antral follicles and the greater estradiol concentrations after in vitro culture. However, the accumulation of estradiol with the small supplementation medium replacement method (MEM-S and TCM-S) in the present study may have inhibited the VEGF production because VEGF gene expression was not detected. "
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ABSTRACT: Two culture media and replacement methods were compared during long-term in vitro culture of secondary follicles of cattle using α-MEM(+) or TCM-199(+) as base media. The medium replacement methods were: Conventional - removal and subsequent addition of the same amount (60μl) in a 100μl aliquot (MEM-C and TCM-C), and Small Supplementation - addition of 5μl of fresh medium to an initial small aliquot (50μl), resulting in a final volume of 125μl on the last day of culture (MEM-S and TCM-S). A total of 207 secondary follicles were cultured individually for 32 days at 38.5°C in 5% CO2 and medium replacement was performed every other day. The MEM-S treatment resulted in a larger (P<0.01) follicular diameter, greater (P<0.02) growth rate, greater (P<0.02) antrum formation, as well as greater (P<0.0001) estradiol concentrations when compared with the MEM-C treatment. The medium change methods did not affect (P>0.05) the follicular and estradiol end points for TCM-199(+). The expression of the FSHR gene was greater (P<0.03) with the TCM-C than TCM-S treatment, while the relative amounts of mRNA for IGF1 was greater (P<0.02) with MEM-S than TCM-S treatments and for VEGF was greater (P<0.02) with MEM-C than TCM-C treatment. In conclusion, the type of base medium and the effect of periodic addition of medium differentially affected follicle development, estradiol production, and gene expression. Furthermore, α-MEM(+) can be used to replace TCM-199(+) for culture of preantral follicles of cattle if progressive addition of medium is used for medium change.
Copyright © 2015 Elsevier B.V. All rights reserved.
Animal reproduction science 07/2015; DOI:10.1016/j.anireprosci.2015.07.006 · 1.51 Impact Factor
Available from: Vicente J Freitas
- "Rosairo et al. (2008) observed a significant reduction in the number of murine preantral follicles after 10 d of culture in the presence of TGF-β and FSH, with a concomitant increase in atretic follicles, suggesting an inhibitory role of this association on follicular development and progression. In buffaloes, TGF-β associated with TGF-α inhibited the follicular growth and survival (Sharma et al. 2010). In cattle, it drastically reduced the survival rates of preantral follicles (Wandji et al. 1996). "
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ABSTRACT: This study aimed to evaluate the immunolocalization and messenger RNA (mRNA) expression for transforming growth factor-beta (TGF-β) and its receptors (TGF-βRI and RII), as well as mRNA expression for P450 aromatase and FSH receptor in caprine preantral follicles. The effects of TGF-β, FSH alone, or in association on the in vitro follicular development were also assessed. Immunohistochemical analyses showed the expression of TGF-β and its receptors in oocytes of all follicle stages and granulosa cells of primary and secondary follicles. mRNA for TGF-β receptors and for FSH receptor (FSHR) was present in preantral follicles as well as in oocytes and granulosa cells of antral follicles. Isolated secondary follicles were cultured in α-minimum essential medium (MEM) alone or supplemented with either FSH (100 ng/ml), TGF-β (10 ng/ml), or TGF-β + FSH for 18 d. TGF-β increased significantly oocyte diameter when compared to FSH alone and control. After 18 d of culture, all groups showed a significant reduction in P450 aromatase and FSHR mRNA levels in comparison to fresh control. In contrast, treatment with FSH significantly increased the mRNA expression for TGF-β in comparison to fresh control and other treatments. In conclusion, the findings showed that TGF-β and its receptors are present in caprine ovarian follicles. Furthermore, they showed a positive effect on oocyte growth in vitro.
In Vitro Cellular & Developmental Biology - Animal 05/2014; 50(8). DOI:10.1007/s11626-014-9775-9 · 1.15 Impact Factor
Available from: zju.edu.cn
- "Basic fibroblast growth factor (bFGF or FGF2), as a prominent member of the FGF family, is one of the potent mitogens regulating proteins which take part in stimulating cellular functions and control various aspects of morphogenesis, proliferation, migration and differentiation of a variety of cells (Zhou et al. 2009; Boosani et al. 2010; Kwan et al. 2011). It was observed that bFGF played pivotal role in ovarian functions, such as survival and growth of oocytes, development of follicles, endocrine of hormone and inhibitory action on the production of nitric oxide (Grasselli et al. 2002; Ergin et al. 2008; Sharma et al. 2010). Moreover, bFGF was related to the process of ovarian disease, such as ovarian cancer (Li and Jiang 2010). "
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ABSTRACT: The proliferating effect of basic fibroblast growth factor (bFGF) on granulosa cells from ovarian pre-hierarchical follicles was evaluated in the laying chickens. Expression of bFGF receptor 1 (FGFR1) from small yellow follicles was determined by immunohistochemistry and RT-PCR. The FGFR1 protein and mRNA were intensively expressed in the granulosa layer. After 8- to 24-h treatment with bFGF (0.1-100 ng/ml), the proliferation of cultured granulosa cells was remarkably enhanced in a dose- and time-dependent manner. The FGFR1 antagonist SU5402 inhibited bFGF-induced cell proliferation. This stimulating effect was further confirmed by 5-bromo-2-deoxyuridine incorporation and terminal transferase dUTP nick end-labelling assay. Immunocytochemistry of protein kinases A (PKA) and C (PKC) showed that the pro-proliferation action of bFGF predominantly activated PKC expression. Meanwhile, the bFGF-induced cell proliferation was significantly promoted by PKC activator PMA and inhibited by PKC inhibitor H(7) (p < 0.05). In addition, the bFGF-elicited cell proliferation was accompanied with increased mRNA expression of the cell cycle-regulating genes including cyclins D1 and E1, cyclin-dependent kinases 2 and 6. In conclusion, bFGF promoted the proliferation of ovarian granulosa cells through binding with FGFR1 and involving PKC pathway in the pre-hierarchical follicles of the laying chickens.
Reproduction in Domestic Animals 05/2011; 47(1):135-42. DOI:10.1111/j.1439-0531.2011.01813.x · 1.52 Impact Factor
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