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.
ABSTRACT 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.
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ABSTRACT: Successful antral formation in vitro from bovine preantral follicles (145–170 μm) has been described previously, but antrum formation from the primary follicle (50–70 μm) has not yet been achieved in vitro. The aim of the study was to establish an optimal culture system supporting the growth and maturation of bovine primary follicles (50–70 μm) in vitro. Bovine primary follicles were cultured in a three-dimensional culture system for 13 or 21 days in alpha-minimum essential medium. Various treatments including follicle stimulating hormone (FSH), luteinizing hormone (LH), 17β-estradiol (E2), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were tested. The follicular diameter and antrum formation rate were recorded, and follicular maturation markers (P450 aromatase, CYP19A1; anti-Mullerian hormone, AMH; growth differentiation factor-9, GDF9; bone morphogenetic protein-15, BMP15; and type III transforming growth factor β receptor, TGFβR3) were analyzed by real-time RT-PCR. After 21 days of culture under each treatment condition, the follicular diameter was significantly enlarged in the presence of FSH + LH + E2 + bFGF or FSH + LH + E2 + bFGF + EGF (p < 0.05). An addition of 50 ng/ml bFGF or bFGF + 25 ng/ml EGF initiated antrum formation by day 19 and day 17 of culture, and the antral cavity formation rate was 16.7% and 33.3% by 21 days of culture, respectively. The expression of follicular maturation markers (CYP19A1, AMH, GDF9, BMP15 and TGFβR3) was significantly altered. We conclude that addition of 50 ng/ml bFGF + 25 ng/ml EGF to media containing FSH + LH + E2 turned out to be the most effective optimized culture conditions to support the growth and maturation of bovine primary follicles in vitro.Reproductive biology 09/2013; 13(3):221–228. DOI:10.1016/j.repbio.2013.06.003 · 1.05 Impact Factor
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ABSTRACT: Cryopreservation of ovarian tissue or follicles has been proposed as an alternative method for fertility preservation. Although successful vitrification of follicles has been reported in several mammalian species, the survival is generally low. The aim of this study was to investigate the effects of FGF and EGF on in vitro preantral follicle development after vitrification. Preantral follicles with diameter of 150-180 μm were mechanically isolated from 18-21 days old NMRI mice ovaries. Follicles were vitrified and warmed then cultured in ɑ- minimal essential medium supplemented with 20 ng/ml FGF, 20 ng/ml EGF and 20 ng/ml FGF +20 ng/ml EGF simultaneously. After 12 days hCG/EGF was added to culture medium and after 18-20 h later the presence of cumulus oocyte complexes (COC) and oocyte maturation were assessed. χ2 test was used to analyse survival and ovulation rate of the follicles. our results showed that the rate of MII oocytes in FGF group increased in comparison with control and treatment groups (P<0.027), but there was no difference between control and EGF and EGF+FGF groups in oocyte maturation rate (P>0.05). There was significant decrease in survival rate of follicles in EGF-FGE group in comparison with other groups (P<0.008). After in vitro ovulation induction the follicles in EGF group showed a higher ovulation rate (P<0.008) than those cultured in other groups. FGF has beneficial effect on oocyte maturation and EGF increases COCs number in vitro. Combination of EGF and FGE decrease the number of survived follicles.Cell Journal 10/2013; 16(3). · 0.46 Impact Factor
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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 · 1.00 Impact Factor