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FGF2/FGFR signaling promotes cumulus-oocyte complex maturation in vitro. Du C et al. (2021) Fibroblast growth factor 2 (FGF2), a member of FGF family, binds with FGF receptors (FGFR) to initiate biological functions in various somatic cells. However, little is known regarding the role of FGF2/FGFR on oocyte meiosis. In this study, we investigated expression patterns and functions of FGF2/FGFR during in vitro maturation (IVM) of mouse cumulus-oocyte complexes (COCs). Among four FGFRs, Ffgr1 was the most abundant in COCs. The transcripts for Fgf2 and Ffgr1 in COCs increased during IVM. Ffgr1 was present in oocytes and cumulus cells, while Fgf2 was present in only cumulus cells. Treatment of COCs with the selective FGFR inhibitor SU5402 blocked oocyte meiotic progression and downregulated expression of Bmp15 and Gdf9. In contrast, supplement of FGF2 promoted oocyte meiotic progression and upregulated Bmp15 and Gdf9 expression. Inhibition of FGFR with SU5402 reduced cumulus expansion and expressions of Ptx3, Has2 and Tnfaip6. Treatment with FGF2 increased Ptx3 and Has2 expression. Inhibition of FGFR had no effect on meiotic progression of denuded oocytes (DOs). However, co-culture of DOs with COCs or supplementation with FGF2 promoted meiotic progression of DOs. Inhibition of FGF2/FGFR signaling also downregulated Ffgr1 expression, while supplemental FGF2 upregulated Fgfr1 expression. Furthermore, inhibition of FGFR in COCs interrupted the c-Mos/MAPK pathway and maturation-promoting factor (MPF), as indicated by downregulation of oocyte c-mos and Ccnb1 transcripts, respectively. Overall, this study suggests that FGF2 produced by cumulus cells, activates a FGF2/FGFR autocrine/paracrine loop within COCs to regulate cumulus expansion and oocyte meiosis. These findings reveal a novel role for FGF2/FGFR signaling during in vitro maturation of COCs.//////////////////
Effect of basic fibroblast growth factor (FGF2) on cumulus cell expansion, in vitro embryo production and gene expression in buffalo (Bubalus bubalis). Kumar S et al. (2020) The present study was undertaken to evaluate the effect of different concentration of FGF2 viz. 5 ng (T1), 10 ng (T2), and 20 ng/mL (T3) on cumulus cell expansion, oocyte maturation, in vitro embryo production, total cell number (TCN) of the blastocyst, and expression of the FGF2 and FGFR2 transcripts in buffalo oocytes and the embryos. Results showed that the effect of FGF2 on the diameter of buffalo COC was significantly higher (P < 0.05) in the T1 group than the other groups at 24h of maturation. The maturation and cleavage rate of oocytes was significantly higher (P < 0.05) in the T3 group than the control, however, the values did not different (P> 0.05) from other groups. The effect of FGF2 on morula and blastocyst yield did not different (P > 0.05) between treatment groups. However, the TCN of the blastocyst was slightly higher (P > 0.05) in the T3 group than the control and other groups. In subsequent trials, the expression of the FGF2 transcript was higher (P < 0.05) in A-grade of oocytes than the C- and D-grade of oocytes, but the expression was not different (P> 0.05) from the B-grade of oocytes. While the FGFR2 expression was higher (P < 0.05) in cumulus cells than any grades of oocytes. The relative abundance of FGF2 and FGFR2 transcripts was significantly higher (P < 0.05) in the 2-cell stage of the embryo than the other stages of embryos. This study was further extended to characterize the FGF2 ligand-binding site in the D3 domain of the buffalo FGF2 receptor. Bioinformatics analysis showed that the bovine FGF2 ligand-binding site in the D3 domain of buffalo was different from the D3 domain of the cattle.//////////////////
Utilising FGF2, IGF2 and FSH in serum-free protocol for long-term in vitro cultivation of primary human granulosa cells. Hensen K et al. (2020) Human granulosa cells acquired as leftover from IVF treatment can be used to study infertility problems and are a valuable tool in the research of follicle maturation and ovulation. There is a need for more defined and long-term culture protocols for studying the response of granulosa cells upon treatment with selected hormones/chemicals. In the current study, we tested the effect of adding FGF2, IGF2 and FSH into defined basal medium in order to find culture conditions that would support proliferation of cumulus and mural granulosa cells along with the expression of common granulosa cell type markers such as FSHR, AMHR2, LHR and CYP19A1. We found that FGF2, IGF2 together with FSH helped to retain granulosa cell marker expression while supporting cell survival at least for two weeks of culture. The defined serum-free culture conditions for long-term culturing will be valuable in providing new standards in the research of human granulosa cells.//////////////////
Fibroblast growth factor 2 regulates cumulus differentiation under the control of the oocyte. Barros RG et al. (2019) We first assessed regulation of FGF2 expression in cumulus cells by FSH and oocyte-secreted factors during in vitro maturation (IVM). Then, we tested the hypothesis that FGF2 regulates meiotic progression, cumulus expansion, and apoptosis in cumulus-oocyte complexes (COC) undergoing IVM. In vitro maturation of bovine COC was utilized as a model to assess regulation of FGF2 expression by FSH and oocyte-secreted factors (via microsurgical removal of the oocyte), as well as effects of graded doses of FGF2 on meiotic progression, degree of cumulus expansion, dissociation of cumulus cells, and cumulus cells apoptosis. Expression of genes regulating functional endpoints altered by FGF2 treatment was assessed in cumulus cells by real-time PCR. Cultures were replicated 4-5 times and effects of treatments were tested by ANOVA. FGF2 mRNA expression was increased by FSH and oocyte-secreted factors during IVM. Addition of FGF2 to the IVM medium advanced meiosis resumption, decreased the ease with which cumulus cells were dissociated, and inhibited cumulus cells apoptosis. Decreased cumulus dissociation was accompanied by decreased expression of TNFAIP6. This is the first study showing that FGF2 expression is regulated by the oocyte in cumulus cells. Moreover, we report novel effects of FGF2 on cumulus cell survival and extracellular matrix (ECM) quality during IVM that may favor acquisition of developmental competence and suggest physiological roles during the final steps of COC differentiation.//////////////////
Quadrupling efficiency in production of genetically modified pigs through improved oocyte maturation. Yuan Y et al. (2017) Assisted reproductive technologies in all mammals are critically dependent on the quality of the oocytes used to produce embryos. For reasons not fully clear, oocytes matured in vitro tend to be much less competent to become fertilized, advance to the blastocyst stage, and give rise to live young than their in vivo-produced counterparts, particularly if they are derived from immature females. Here we show that a chemically defined maturation medium supplemented with three cytokines (FGF2, LIF, and IGF1) in combination, so-called "FLI medium," improves nuclear maturation of oocytes in cumulus-oocyte complexes derived from immature pig ovaries and provides a twofold increase in the efficiency of blastocyst production after in vitro fertilization. Transfer of such blastocysts to recipient females doubles mean litter size to about nine piglets per litter. Maturation of oocytes in FLI medium, therefore, effectively provides a fourfold increase in piglets born per oocyte collected. As they progress in culture, the FLI-matured cumulus-oocyte complexes display distinctly different kinetics of MAPK activation in the cumulus cells, much increased cumulus cell expansion, and an accelerated severance of cytoplasmic projections between the cumulus cells outside the zona pellucida and the oocyte within. These events likely underpin the improvement in oocyte quality achieved by using the FLI medium.//////////////////
Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA) synergistically promote steroidogenesis and survival of cultured buffalo granulosa cells. Mishra SR et al. (2017) The present study investigated the combined effect of fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGF-A) on estradiol (E2) secretion and relative abundance of mRNA for aromatase enzyme (CYP19A1), proliferating cell nuclear antigen (PCNA) and BCL-2 associated X protein (BAX) in cultured buffalo granulosa cells (GCs). Follicles were isolated and classified into four groups based on size and E2 concentration in follicular fluid (FF): Small, 4-6mm diameter, E2<0.5ng/ml; Medium, 7-9mm, E2=0.5-5ng/ml; Large, 10-13mm, E2=5-40ng/ml; Preovulatory (PFs), >14mm, E2>180ng/ml. The GCs of PF were cultured in 24 well cell culture plates and allowed to become 75-80% confluent. Then cultured GCs were treated with FGF2 (200ng/ml) and VEGF-A (100ng/ml) separately and in combination for three incubation periods (24, 48 and 72h). Estradiol secretion was greater in GCs treated with FGF2+VEGF-A compared to FGF2 or VEGF-A at all incubation periods and was greatest (P<0.05) at 72h of incubation. The relative abundance of CYP19A1 and PCNA mRNA were relatively consistent with the amount E2 secretion. In contrast, the relative abundance of Bax mRNA was less in GCs treated with the combination of FGF2 and VEGF-A as compared to either FGF2 or VEGF-A alone and the least concentration (P<0.05) was at 72h of incubation. Findings with use of immunocytochemistry of cells treated with these factors were consistent to the relative abundance of mRNA transcript for the factor. The present findings indicate that FGF2 and VEGF-A may function in a synergistic manner to promote steroidogenesis and survival of cultured buffalo GCs.//////////////////
Expression and localization of fibroblast growth factor (FGF) family in buffalo ovarian follicle during different stages of development and modulatory role of FGF2 on steroidogenesis and survival of cultured buffalo granulosa cells. Mishra SR et al. (2016) The present study investigated the expression and localization of FGF and its functional receptors in the follicle of buffalo and the treatment of FGF2 on mRNA expression of CYP19A1 (aromatase), PCNA, and BAX (BCL-2 associated X protein) in cultured buffalo granulosa cells (GCs). Follicles were classified into four groups based on size and E2 level in follicular fluid (FF): F1, 4-6mm diameter, E2<0.5ng/ml of FF; F2, 7-9mm, E2=0.5-5ng/ml; F3, 10-13mm, E2=5-40ng/ml; F4, >14mm, E2>180ng/ml. The qPCR studies revealed that the mRNA expression of FGF1, FGF2 and FGF7 were maximum (P<0.05) in theca interna (TI) whereas the transcripts of FGFR1, FGFR2, FGFR2IIIB and FGFR2IIIC were up-regulated (P<0.05) in GCs of F4 follicles. Protein expression of most members were maximum (P<0.05) in F4 follicles except FGFR3 and FGFR4. All members were localized in GC and TI with a stage specific immunoreactivity. Primary culture of GCs with treatment of FGF2 at different dose-time combinations revealed that the mRNA expression and immunoreactivity of CYP19A1 and PCNA were maximum (P<0.05) whereas BAX was minimum (P<0.05) with 200ng/ml at 72h of incubation. The findings indicate that FGF family members are expressed in a regulated manner in buffalo ovarian follicles during different stages of development where FGF2 may promote steroidogenesis and GC survival through autocrine and paracrine manner.//////////////////
VEGF and FGF2 Improve Revascularization, Survival, and Oocyte Quality of Cryopreserved, Subcutaneously-Transplanted Mouse Ovarian Tissues. Li SH et al. (2016) This study was conducted to investigate the effect of the vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) on revascularization, survival, and oocyte quality of cryopreserved, subcutaneously-transplanted mouse ovarian tissue. Autologous subcutaneous transplantation of vitrified-thawed mouse ovarian tissues treated with (experimental group) or without (control group) VEGF and FGF2 was performed. After transplantation to the inguinal region for two or three weeks, graft survival, angiogenesis, follicle development, and oocyte quality were examined after gonadotropin administration. VEGF coupled with FGF2 (VEGF/FGF2) promoted revascularization and significantly increased the survival rate of subcutaneously-transplanted cryopreserved ovarian tissues compared with untreated controls. The two growth factors did not show long-term effects on the ovarian grafts. In contrast to the untreated ovarian grafts, active folliculogenesis was revealed as the number of follicles at various stages and of mature oocytes in antral follicles after gonadotropin administration were remarkably higher in the VEGF/FGF2-treated groups. Although the fertilization rate was similar between the VEGF/FGF2 and control groups, the oocyte quality was much better in the VEGF/FGF2-treated grafts as demonstrated by the higher ratio of blastocyst development. Introducing angiogenic factors, such as VEGF and FGF2, may be a promising strategy to improve revascularization, survival, and oocyte quality of cryopreserved, subcutaneously-transplanted mouse ovarian tissue.//////////////////
Basic Fibroblast Growth Factor Promotes Macaque Follicle Development In Vitro. Lu C et al. (2015) Fertility preservation is an important type of frontier scientific research in the field of reproductive health. The culture of ovarian cortices to (1) initiate primordial follicle growth and (2) procure developing follicles for later oocyte maturation is a promising fertility preservation strategy, especially for older women or cancer patients. At present, this goal remains largely unsubstantiated in primates, because of the difficulty in attaining relatively large follicles via ovarian cortex culture. To overcome this hurdle, we cultured macaque monkey ovarian cortices with follicle-stimulating hormone (FSH), kit ligand (KL), basic fibroblast growth factor (bFGF), and/or epidermal growth factor (EGF). The various factors and factor combinations promoted primordial follicle development to different extents. Notably, both bFF (bFGF, 100 ng/ml and FSH, 50 ng/ml) and KF (KL, 100 ng/ml and FSH, 50 ng/ml) contributed to the activation of primordial follicles at day 12 (D12) of culture, whereas at D18, the proportions of developing follicles were significantly higher in the bFF and KF groups relative to the other treatment groups, particularly in the bFF group. Estradiol and progesterone production were also highest in the bFF group, and primary follicle diameters were the largest. Up until D24, the bFF group still exhibited the highest proportion of developing follicles. In conclusion, the bFGF/FSH combination promotes non-human primate primordial follicle development in vitro, with the optimal experimental window within 18 days. These results provide evidence for the future success of human ovarian cortex culture and the eventual acquisition of mature human follicles or oocytes for fertility restoration.//////////////////
Immunohistochemical Localization of Fibroblast Growth Factor-2 in the Sheep Ovary and its Effects on Pre-antral Follicle Apoptosis and Development In Vitro. Santos J 2014 et al.
Studies with sheep are important to improve our knowledge about the factors that control folliculogenesis in mammals and to explore possible physiological differences among species. The aims of this study were to characterize FGF-2 protein expression in ovine ovaries and to verify the effect of FGF-2 on the morphology, apoptosis and growth of ovine pre-antral follicles cultured in vitro. After collection, one fragment of ovarian tissue was fixed for histological analysis and TUNEL analysis (fresh control). The remaining fragments were cultured for 7days in control medium (a-MEM(+) ) alone or supplemented with FGF-2 at different concentrations (1, 10, 50, 100 or 200ng/ml). After culturing, ovarian tissue was destined to histology and TUNEL analysis, and oocyte and follicle diameters were measured. The immunostaining for FGF-2 was observed in oocytes from primordial, primary and secondary follicles, as well as in granulosa cells of secondary and antral follicles. The percentage of normal follicles was similar among control medium, 1 and 10ng/ml FGF-2, and significantly higher than those observed in 50, 100 or 200ng/ml FGF-2. A significant increase in follicle diameter was observed when tissues were cultured in 10, 50, 100 or 200ng/ml FGF-2 compared with the fresh control and the other treatments. Similar results were observed for oocyte diameter in tissues cultured with 50, 100 or 200ng/ml FGF-2 (p<0.05). However, the percentage of apoptotic cells only decreased (p<0.05) in ovarian tissues cultured in 1 or 10ng/ml FGF-2 compared with the control medium and other FGF-2 treatments. In conclusion, this study demonstrated the presence of FGF-2 in ovine ovaries. Furthermore, 10ng/ml FGF-2 inhibits apoptosis and promotes ovine follicle growth. As the sheep ovary is more similar to that of humans, the culture system demonstrated in this work seems to be an appropriate tool for studies towards human folliculogenesis.
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Basic fibroblast growth factor promotes the development of human ovarian early follicles during growth in vitro. Wang TR 2014 et al.
STUDY QUESTION
What is the effect of basic fibroblast growth factor (bFGF) on the growth of individual early human follicles in a three-dimensional (3D) culture system in vitro?
SUMMARY ANSWER
The addition of 200 ng bFGF/ml improves human early follicle growth, survival and viability during growth in vitro.
WHAT IS KNOWN ALREADY
It has been demonstrated that bFGF enhances primordial follicle development in human ovarian tissue culture. However, the growth and survival of individual early follicles in encapsulated 3D culture have not been reported.
STUDY DESIGN, SIZE, DURATION
The maturation in vitro of human ovarian follicles was investigated. Ovarian tissue (n= 11) was obtained from 11 women during laparoscopic surgery for gynecological disease, after obtaining written informed consent. One hundred and fifty-four early follicles were isolated by enzymic digestion and mechanical disruption. They were individually encapsulated into alginate (1% w/v) and randomly assigned to be cultured with 0, 100, 200 or 300 ng bFGF/ml for 8 days.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Individual follicles were cultured in minimum essential medium a (aMEM) supplemented with bFGF. Follicle survival and growth were assessed by microscopy. Follicle viability was evaluated under confocal laser scanning microscope following Calcein-AM and Ethidium homodimer-I (Ca-AM/EthD-I) staining.
MAIN RESULTS AND THE ROLE OF CHANCE
After 8 days in culture, all 154 follicles had increased in size. The diameter and survival rate of the follicles and the percentage with good viability were significantly higher in the group cultured with 200 ng bFGF/ml than in the group without bFGF (P < 0.05). The percentage of follicles in the pre-antral stage was significantly higher in the 200 ng bFGF/ml group than in the group without bFGF (P < 0.05), while the percentages of primordial and primary follicles were significantly lower (P < 0.05).
LIMITATIONS, REASONS FOR CAUTION
The study focuses on the effect of bFGF on the development of individual human early follicles in 3D culture in vitro and has limited ability to reveal the specific effect of bFGF at each different stage. The findings highlight the need to improve the acquisition and isolation of human ovarian follicles.
WIDER IMPLICATIONS OF THE FINDINGS
The in vitro 3D culture of human follicles with appropriate dosage of bFGF offers an effective method to investigate their development. Moreover, it allows early follicles to be cultured to an advanced stage and therefore has the potential to become an important source of mature oocytes for assisted reproductive technology; particularly as an option for fertility preservation in women, including patients with cancer.
STUDY FUNDING/COMPETING INTEREST(S)
This work was supported by the National Basic Research Program of China (2011|CB944504, 2011CB944503) and the National Natural Science Foundation of China (81200470, 81000275, 31230047, 8110197). There are no conflicts of interest to declare.
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Gene bionetworks that regulate ovarian primordial follicle assembly. Nilsson E 2013 et al.
BACKGROUND
Primordial follicle assembly is the process by which ovarian primordial follicles are formed. During follicle assembly oocyte nests break down and a layer of pre-granulosa cells surrounds individual oocytes to form primordial follicles. The pool of primordial follicles formed is the source of oocytes for ovulation during a female's reproductive life.
RESULTS
The current study utilized a systems approach to detect all genes that are differentially expressed in response to seven different growth factor and hormone treatments known to influence (increase or decrease) primordial follicle assembly in a neonatal rat ovary culture system. One novel factor, basic fibroblast growth factor (FGF2), was experimentally determined to inhibit follicle assembly. The different growth factor and hormone treatments were all found to affect similar physiological pathways, but each treatment affected a unique set of differentially expressed genes (signature gene set). A gene bionetwork analysis identified gene modules of coordinately expressed interconnected genes and it was found that different gene modules appear to accomplish distinct tasks during primordial follicle assembly. Predictions of physiological pathways important to follicle assembly were validated using ovary culture experiments in which ERK1/2 (MAPK1) activity was increased.
CONCLUSIONS
A number of the highly interconnected genes in these gene networks have previously been linked to primary ovarian insufficiency (POI) and polycystic ovarian disease syndrome (PCOS). Observations have identified novel factors and gene networks that regulate primordial follicle assembly. This systems biology approach has helped elucidate the molecular control of primordial follicle assembly and provided potential therapeutic targets for the treatment of ovarian disease.
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Fibroblast growth factor (FGF) 2 is a key determinant of vascular sprouting during bovine luteal angiogenesis. Woad KJ et al. Fibroblast growth factor (FGF) 2 and vascular endothelial growth factor (VEGF) A are thought to be key controllers of luteal angiogenesis; however, their precise roles in the regulation and co-ordination of this complex process remain unknown. Thus, the temporal and spatial pattern of endothelial network formation was determined by culturing mixed cell types from early bovine corpora lutea on fibronectin in the presence of FGF2 and VEGFA (6h to 9 days). Endothelial cells, as determined by von Willebrand factor imunohistochemistry, initially grew in cell islands (days 0-3), before undergoing a period of vascular sprouting to display a more tubule-like appearance (days 3-6), and after 9 days in culture had formed extensive intricate networks. Luteal cells were treated with SU1498 (VEGF receptor 2 inhibitor) or SU5402 (FGF receptor 1 inhibitor) or control on days 0-3, 3-6, or 6-9 to determine the role of FGF2 and VEGFA during these specific windows. The total area of endothelial cells was unaffected by SU1498 treatment during any window. In contrast, SU5402 treatment caused maximal reduction in the total area of endothelial cell networks on days 3-6 versus controls (mean reduction 81%; P<0.001) during the period of tubule initiation. Moreover, SU5402 treatment on days 3-6 dramatically reduced the total number of branch points (P<0.001) and degree of branching per endothelial cell island (P<0.05) in the absence of changes in mean island area. This suggests that FGF2 is a key determinant of vascular sprouting and hence critical to luteal development.
Gene Expression and Immunolocalization of Fibroblast Growth Factor 2 in the Ovary and Its Effect on the In Vitro Culture of Caprine Preantral Ovarian Follicles. Almeida A et al. This study quantified Fibroblast growth factor 2 (FGF-2) mRNA and localized FGF-2 protein in different categories of follicles isolated from goat ovaries. In addition, we verified the effects of this factor on the in vitro culture of preantral follicles isolated from goats. For mRNA quantification, we performed real-time PCR using primordial, primary and secondary follicles, as well as cumulus-oocyte complexes (COCs) and mural granulosa and theca cells of small and large antral follicles. For FGF-2 protein localization, the ovaries were subjected to conventional immunohistochemical procedures. Preantral follicles were isolated and cultured in vitro for 12 days in either control (basic) or supplemented with FGF-2 medium. The expression of FGF-2 mRNA was detected in all categories of follicles and there was no difference in preantral follicles and COCs or granulosa/theca cells from small and large antral follicles. However, in large antral follicles, COCs showed expression levels significantly lower than in granulosa/theca cells (p < 0.05). We observed moderate expression of FGF-2 protein in preantral follicles but not in granulosa cells of primordial follicles and theca cells of secondary follicles. In both small and large antral follicles, strong, moderate and weak staining was observed in oocytes, granulosa and theca cells, respectively. The addition of FGF-2 caused a significant increase in the daily follicular growth rate compared to the control group. We conclude that FGF-2 mRNA is expressed throughout follicular development and that its protein can be found in different patterns in preantral and antral follicles. Furthermore, FGF-2 increases the follicular growth rate in vitro.
Fibroblast growth factor-2 regulation of sprouty and NR4A genes in bovine ovarian granulosa cells. Jiang ZL et al. Fibroblast growth factors (FGFs) alter ovarian function, at least in part by inhibiting steroid hormone secretion and affecting survival of granulosa cells. The mechanism of action of FGFs in ovarian follicle cells is largely unknown; in the present study we identified the major pathways used by FGF2 in non-luteinizing granulosa cells cultured under serum-free conditions. FGF2 increased abundance of mRNA encoding SPRY1, 2, and 4, but not SPRY3. Common pathways employed by FGF2 in the regulation of SPRY1, 2, and 4, as demonstrated by immunoblot and inhibitor studies, included ERK1/2 and Akt signaling. In contrast, PKC activation was necessary for FGF2-stimulated expression of SPRY1 and 4, but not for SPRY2. Intracellular calcium flux is critical and sufficient for SPRY2 expression, but not for SPRY1 and 4. We also identified the orphan nuclear receptor NR4A1 as a potential early response gene in FGF2 signaling, whose expression, like that of SPRY2, is critically dependent on calcium signaling. Together, these data identify FGF2-target genes in follicular granulosa cells, and demonstrate alternative pathway use for the differential control of SPRY genes. J. Cell. Physiol. 226: 1820-1827, 2011. ? 2010 Wiley-Liss, Inc.
Involvement of Pro-Inflammatory Cytokines, Mediators of Inflammation, and Basic Fibroblast Growth Factor in Prostaglandin F2{alpha}-Induced Luteolysis in Bovine Corpus Luteum.
Neuvians TP,et al .
The process of luteolysis requires very subtly modulated coordination of different factors and regulation systems. Immune cells and cytokines were shown to be relevant for bovine luteolysis. The aim of this study was to investigate the detailed pattern of mRNA expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNFalpha), TNF receptor type 1 (TNF-R1), interleukin 1beta (IL-1beta), and interferon gamma (IFNgamma), and of the inducible nitric oxide synthase (iNOS) and the basic fibroblast growth factor (FGF-2) during prostaglandin (PG) F(2alpha)-induced luteolysis in the bovine corpus luteum (CL). In addition, the mRNA expression for the LH-receptor (LH-R) and the steroidogenic enzyme P450scc was determined. Cows in the midluteal phase (Days 8-12) were injected with the PGF(2alpha) analogue cloprostenol, and CL were collected by transvaginal ovariectomy before and 2, 4, 12, 48, and 64 h after PGF(2alpha) injection. Conventional and real-time reverse transcription polymerase chain reaction RT-PCR (LightCycler) using SYBR Green I detection were employed to determine the mRNA expression for the investigated factors. All cytokines were significantly up-regulated during induced luteolysis. LH-R and P450scc mRNA were down-regulated (P < 0.05) during structural luteolysis (after 12 h), and P450scc in addition at 2 h after PGF(2alpha) (P < 0.05). FGF-2 expression increased (P < 0.001) during functional luteolysis (until 12 h after PGF(2alpha)) and diminished thereafter. The mRNA expression for iNOS decreased (P < 0.05) after induction of luteolysis. In conclusion, cytokines may be involved not only in structural but also in functional luteolysis and the deprivation of luteal survival factors, leading to a situation where apoptosis can occur. FGF-2 may participate in the suppression of cytokine-induced iNOS mRNA expression and in the prevention of an inflammatory reaction in the surrounding tissues.
Follicle stimulating hormone and fibroblast growth factor-2 interact and promote goat primordial follicle development in vitro. Matos MH et al. The aims of the present study were to investigate the effects of the interaction between follicle stimulating hormone (FSH) and fibroblast growth factor-2 (FGF-2) on survival, follicular growth initiation and further growth of caprine preantral follicles. Pieces of caprine ovarian cortex were cultured for 1 or 7 days in minimum essential medium (MEM) supplemented with FSH, FGF-2 or FSH + FGF-2. Small fragments from non-cultured ovarian tissue and from those cultured for 1 or 7 days were processed for classical histology and transmission electron microscopy (TEM) to verify follicular morphology and growth. The results showed that, after 7 days culture, the highest percentages of normal follicles were observed in medium supplemented with FSH. After 7 days culture, the interaction between FSH and FGF-2 was most effective to promote the initiation of primordial follicles growth and oocyte growth. TEM showed ultrastructural integrity of follicles after 1 day of culture in MEM and after 7 days in all treatments, except in those follicles cultured for 7 days in MEM. In conclusion, this study demonstrated that the interaction between FSH and FGF-2 stimulates the initiation of primordial follicles growth and the subsequent growth of developing follicles. Furthermore, these data showed that FSH is important to maintain follicular integrity after 7 days culture.
Fibroblast growth factor requirements for in vitro development of bovine embryos. Fields SD et al. The overall goal was to describe the importance of fibroblast growth factors (FGFs) during development of the bovine embryo. An inhibitor of FGF receptor kinase activity (SU5402) was used to examine whether FGF signaling is required for embryo development. Addition of 20 ?M SU5402 on Day 0 (Day of IVF) reduced (P = 0.04) the percentage of oocytes becoming blastocysts on Day 7 compared to controls (5.9 ? 2.1 vs 16.9 ? 2.4; average ? SEM). Also, Day-8 blastocysts placed into individual culture drops of medium containing SU5402 tended to have decreased (P = 0.08) blastomere numbers at Day 11 (211.1 ? 27.5 vs 297.8 ? 25.0). A second series of studies determined if supplemental FGF2 enhances development in vitro. There was no effect of FGF2 on cleavage or blastocyst development rates when 5 or 100 ng/mL FGF2 was provided immediately after fertilization. Also, FGF2 supplementation beginning on Day 5 post-fertilization did not significantly affect blastocyst rates or the number of trophoblast and inner cell mass cells. However, addition of 500 ng/mL FGF2 at both Day 0 and Day 4 increased (P = 0.03) the percentage of oocytes that became blastocysts on Day 7 compared with controls (27.4 ? 1.3 vs 19.7 ? 1.3). In a final study, the thermal-protective ability of FGF2 was examined by adding FGF2 1 h before exposing Day 5 embryos to heat shock. Addition of FGF2 did not significantly influence embryo thermal-tolerance. In conclusion, FGF receptor activation was important for optimal blastocyst formation and FGF2 supplementation increased bovine blastocyst formation when provided at high concentrations.
276 fibroblast growth factor 2 promotes bovine oocyte meiotic maturation and developmental competence. Zhang K et al. Oocyte competency is acquired during the course of folliculogenesis and is controlled by various endocrine and paracrine signals. One of these is fibroblast growth factor 2 (FGF2). Its expression is up-regulated in theca and granulosa cells during final maturation of a bovine follicle, and its cognate receptors are expressed in cumulus cells and oocytes throughout the final stages of oocyte maturation. The overall goal of this work was to describe how supplementing FGF2 during oocyte maturation in vitro affects oocyte maturation and subsequent embryo development. Cumulus-oocyte complexes (COC) were collected from bovine ovaries obtained from a local abattoir and cultured in defined TCM-based oocyte maturation medium. Depending on the study, oocytes were examined either during (6h) or after (21h) maturation or were fertilized in vitro and examined throughout in vitro embryo development in modified SOFF. Data were analysed with least-squares ANOVA using GLM of SAS. Adding 0.5 to 50ngmL(-1) of FGF2 did not affect cleavage rate or the percentage of 8 to 16 cell embryos at day 3 post-IVF. However, the blastocyst rate at day 7 was greater when oocytes were exposed to 0.5ngmL(-1) of FGF2 during maturation [30.0?1.9% (17/109) v. 16.0?2.6% (23/77) for nontreatment control; 4 replicates; P<0.05], whereas higher doses of FGF2 did not affect blastocyst rates when compared with controls. Total cell number per blastocyst was not affected by FGF2 addition. The effects of FGF2 on oocyte maturation and cumulus expansion were examined to better understand how FGF2 improves oocyte competency. Adding 0.5ngmL(-1) of FGF2 did not affect the percentage of oocytes containing condensed chromatin after 6h IVM or metaphase II (MII) rate after 21h IVM, but 0.5ngmL(-1) of FGF2 treatment increased the cumulus expansion index score after 21h IVM (P<0.05). Interestingly, adding 5ngmL(-1) but not 50ngmL(-1) of FGF2 increased MII rate [61.5?4.3% (53/120) for 5ngmL(-1) of FGF2 v. 46.9?5.9% (64/104) for nontreatment controls; 7 replicates; P<0.05], but neither FGF2 affected rates of chromatin condensation and cumulus expansion. Changes in the relative abundance for several putative oocyte competency markers and maternal genes (CTSB, Sprouty2, EGFR, FSHR, Has2, BMP15, GDF9, JY-1, Follistatin, H2A) were examined at 6 and 21h after treatment with 0.5ngmL(-1) of FGF2 by quantitative RT-PCR. Relative amounts of 18S RNA was used as an internal control, and 2-??CT was used to quantify relative gene expression. The relative abundance of most of the transcripts examined was not affected by FGF2, but EGFR mRNA levels were greater after 6h but not 21h IVM in cumulus cells isolated from FGF2-supplemented COC (P=0.057). In summary, improvements in blastocyst development were achieved by FGF2 treatment during oocyte maturation. The reason for the enhanced oocyte competency remains unclear, but it may occur in part because of improvements in cumulus expansion and production of EGFR.
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