130 THE SYNERGIC EFFECT OF NERVE GROWTH FACTOR AND VASCULAR ENDOTHELIAL GROWTH FACTOR ON IN VITRO MATURATION AND DEVELOPMENTAL COMPETENCE IN BOVINE OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 169
Author(s):  
B. Kim ◽  
I. M. Saadeldin ◽  
B. Lee ◽  
G. Jang
Keyword(s):  
Growth Factor ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Treated Group ◽  
Control Group ◽  
Dependent Manner ◽  
Vascular Endothelial ◽  
Treatment Groups ◽  
Endothelial Growth Factor

Nerve growth factor (NGF) has been reported to increase the mRNA expression of vascular endothelial growth factor (VEGF) in granulose cells of human, rat via TrkA signaling; VEGF has been shown to exert beneficial effects during bovine in vitro maturation (IVM) as well as early embryonic development. The aims of this study were 1) to investigate not only the direct effect of NGF but also the collaborative effect of NGF and VEGF during bovine in vitro maturation (IVM), in vitro culture (IVC), or both; and 2) to validate the correlation among transcript abundance of 7 genes (VEGF164, VEGF120, Flt-1, Flk-1, TrkA, PTGS2, and CYP11A1) in bovine cumulus cells and the results of IVM or IVC among the differently treated groups. In Experiment 1, concentrations of 0, 10, and 100 ng mL–1 NGF were added to our established IVM medium without serum, and in Experiment 2, control and treatment groups (concentration of 0, 10, and 100 ng mL–1 NGF with VEGF 100 ng mL–1) were added into chemically defined media. The oocytes of each group in Experiments 1 and 2 were determined by the proportion of MII oocytes after 24 h, and embryos were assessed after parthenogenetic activation. Cumulus cells from the differently treated matured cumulus cell–oocyte complexes (COC) were separated and synthesised into cDNA for RT-PCR and real-time PCR in order to measure relative abundance of 7 genes in a dose-dependent manner or a time-dependent manner. In Experiment 1, the concentration of 10 ng mL–1 (57.40%) and 100 ng mL–1 (62.75%) NGF treatment groups did not significantly increase the proportion of MII oocytes compared with the control group (55.06%). In Experiment 2, both the NGF 10 ng mL–1 with VEGF 100 ng mL–1 treated group (67.69%; P ≤ 0.01) and the NGF 100 ng mL–1 with VEGF 100 ng mL–1 treated group (72.24%; P ≤ 0.001) had a significantly higher percentage of polar body extrusion than control group (51.77%) and the group which was treated with VEGF 100 ng mL–1 (56.39%). The NGF treatment group with VEGF increased transcriptional level of VEGF164 and VEGF120 compared with the control group and only NGF- or VEGF-treated groups. In addition, either the NGF-treated group or NGF plus VEGF showed significantly increased mRNA abundance in VEGF164, VEGF120, Flt-1, Flk-1, and TrkA genes, whereas the NGF plus VEGF-treated group indicated the up-regulation of VEGF164, VEGF120, CYP11A1, and PTGS2 genes. In conclusion, NGF and exogenous VEGF have a synergic effect during bovine IVM and the early stage of embryo development; the elevated VEGF mRNA abundance in cumulus cells might contribute to the viability of bovine oocytes and early embryonic development. This study was supported by grants from IPET (#109023-05-1-CG000), NRF (#M10625030005-10N250300510), MKE (#2009-67-10033839, #2009-67-10033805), and BK21 program.

204 INADEQUACY OF VASCULAR ENDOTHELIAL GROWTH FACTOR IN CULTURE MEDIUM REDUCES THE VIABILITY OF CUMULUS CELLS AND PREVENTS IN VITRO MATURATION OF PORCINE OOCYTES

2016 ◽  
Vol 28 (2) ◽  
pp. 233
Author(s):  
T. T. M. Bui ◽  
P. P. Ferré ◽  
M. T. Tran ◽  
T. Wakai ◽  
H. Funahashi
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
In Vitro Maturation ◽  
Culture Medium ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Vegf Receptor ◽  
Vascular Endothelial ◽  
Endothelial Growth Factor

Recently, vascular endothelial growth factor (VEGF) has been regarded as an important factor associated with not only follicle development but also meiotic competence of oocytes. However, the mechanism of how VEGF works is poorly understood. In this study, we investigated in vitro maturation (IVM) of oocytes from different sizes in the absence or presence of a VEGF receptor inhibitor, Axitinib. Cumulus-oocyte complexes (COC) were obtained from small follicles (SF; l < 3 mm in diameter) and medium follicles (MF; 3–6 mm in diameter). Each group of 30–40 COC with at least 3 layers of clear and compact cumulus cells (CC) was cultured in 500 μL of modified porcine oocyte medium (POM-β-mercaptoethanol) supplemented with 10 IU mL–1 eCG, 10 IU mL–1 hCG and 1mM dibutyryl-cyclic-adenosine monophosphate (dbc-AMP) for the first 20 h and then without those supplements for another 24 h at 39°C, 5% CO2 in air. During the first 20 h of IVM, culture medium was also supplemented with or without 1.25 nM Axitinib. At 20 h and 44 h after the start of IVM, the oocytes were denuded and stained with 4′6-diamidino-2-phenylindole (DAPI) to observe the nuclear stages. At 20 h after the start of IVM, some COC were also stained with PI and SYBR Green I to evaluate the ratio of live/dead cumulus cells. Statistical analyses of data from 5 replications were analysed by ANOVA and Tukey’s multiple comparison test. As compared with controls at 20 h after the start of IVM, the number of dead cumulus cells increased significantly in the groups treated with Axitinib, regardless of COC derived from MF and SF (16.8 v. 43.1% in MF and 25.3 v. 57.7% in SF, P < 0.01, respectively). At that time, a majority of oocytes from MF and SF remained at the germinal vesicle (GV) stage in controls (89.8 and 84.6%, respectively), but the percentage significantly reduced in the presence of Axitinib (57.9 and 48.9% of oocytes from MF and SF, respectively) and proceeded around the metaphase-I stage (37.5 and 44.8% of oocytes from MF and SF, respectively). At 44 h after the start of IVM, lower maturation rates were observed in oocytes treated with Axitinib than controls (35.0 v. 81.2% in MF; 20.1 v. 49.0% in SF; P < 0.01). In conclusions, VEGF plays an important role in maitaining the viability of cumulus cells. The presence of VEGFR inhibitor caused the oocytes to develop uncontrollably, even in the presence of dbc-AMP. Moreover, the deficiency of VEGF prevented oocytes fully competent to resume meiosis and arrest to metaphase II.

scholarly journals Apoptosis in cumulus cells during in vitro maturation of bovine cumulus-enclosed oocytes

Reproduction ◽  
2003 ◽  
pp. 369-376 ◽  
Author(s):  
S Ikeda ◽  
H Imai ◽  
M Yamada
Keyword(s):  
Dna Fragmentation ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Treated Group ◽  
Developmental Competence ◽  
Pcr Analysis ◽  
Control Group ◽  
Dependent Manner

The aim of this study was to investigate whether apoptosis occurs in cumulus cells during in vitro maturation (IVM) of bovine cumulus-enclosed oocytes (CEOs). The bovine CEOs obtained from ovaries from an abattoir were cultured for 24 h in IVM medium in the presence or absence of 10% (v/v) fetal bovine serum. The developmental competence of enclosed oocytes, as assessed by the development of the blastocyst after IVF, was significantly higher in the serum-treated group than in the control group. The morphological features of apoptosis that were analysed by orcein staining were hardly detectable in the cumulus cells at the start (0 h) of IVM, but were evident at the end (24 h) of IVM both in the control and serum-treated groups. Genomic DNA was extracted from CEOs at 0, 6, 12, 18 and 24 h of IVM and subjected to ligation-mediated PCR (LM-PCR) to detect apoptotic internucleosomal DNA fragmentation. DNA fragmentation was hardly detectable at the start of IVM, but increased in a time-dependent manner as the IVM culture proceeded. DNA fragmentation was not observed in the oocytes, indicating that fragmentation occurs in cumulus cells. The degree of fragmentation was lower in the serum-treated group compared with the control group. The LM-PCR analysis of DNA extracted from CEOs at 24 h of IVM, in which the DNA had been pretreated with Klenow enzyme or T4 DNA polymerase, revealed that the characteristic forms of the DNA ends generated during cumulus cell apoptosis were mainly 3'-overhangs and blunt ends. In conclusion, the results of the present study demonstrate that cumulus cells in bovine CEOs spontaneously undergo apoptosis during IVM. The degree of apoptosis may be correlated with the developmental competence of the enclosed oocytes.

Vascular endothelial growth factor synergistically enhances bone morphogenetic protein-4-dependent lymphohematopoietic cell generation from embryonic stem cells in vitro

Blood ◽  
2000 ◽  
Vol 95 (7) ◽  
pp. 2275-2283 ◽  
Author(s):  
Naoki Nakayama ◽  
Jae Lee ◽  
Laura Chiu
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Bone Morphogenetic Protein ◽  
Es Cells ◽  
Embryonic Stem ◽  
Dependent Manner ◽  
Vascular Endothelial ◽  
Morphogenetic Protein ◽  
Endothelial Growth Factor

Abstract The totipotent mouse embryonic stem (ES) cell is known to differentiate into cells expressing the β-globin gene when stimulated with bone morphogenetic protein (BMP)-4. Here, we demonstrate that BMP-4 is essential for generating both erythro-myeloid colony-forming cells (CFCs) and lymphoid (B and NK) progenitor cells from ES cells and that vascular endothelial growth factor (VEGF) synergizes with BMP-4. The CD45+ myelomonocytic progenitors and Ter119+ erythroid cells began to be detected with 0.5 ng/mL BMP-4, and their levels plateaued at approximately 2 ng/mL. VEGF alone weakly elevated the CD34+ cell population though no lymphohematopoietic progenitors were induced. However, when combined with BMP-4, 2 to 20 ng/mL VEGF synergistically augmented the BMP-4-dependent generation of erythro-myeloid CFCs and lymphoid progenitors from ES cells, which were enriched in CD34+ CD31lo and CD34+CD45− cell populations, respectively, in a dose-dependent manner. Furthermore, during the 7 days of in vitro differentiation, BMP-4 was required within the first 4 days, whereas VEGF was functional after the action of BMP-4 (in the last 3 days). Thus, VEGF is a synergistic enhancer for the BMP-4-dependent differentiation processes, and it seems to be achieved by the ordered action of the 2 factors.

312 EFFECTS OF TRANSFORMING GROWTH FACTOR ALPHA AND INSULIN-LIKE GROWTH FACTOR 1 SUPPLEMENTATION ON IN VITRO MATURATION OF CANINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 245
Author(s):  
A. Sato ◽  
B. Sarentonglaga ◽  
K. Ogata ◽  
M. Yamaguchi ◽  
A. Hara ◽  
...  
Keyword(s):  
Growth Factor ◽  
Synergistic Effect ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Transforming Growth Factor ◽  
Germinal Vesicle ◽  
Control Group ◽  
Insulin Like Growth Factor ◽  
Treatment Groups

Although in vitro maturation (IVM) of oocytes has been successfully established for many species, the efficiency of IVM in canine oocytes is still very low. As growth factors have been shown to promote oocyte maturation in some species, we investigated whether use of transforming growth factor α (TGF-a) and insulin-like growth factor 1 (IGF-1) might overcome the difficulties of achieving meiotic maturation in cultured canine cumulus-oocyte complexes (COC). Ovaries were obtained from bitches at 6 months to 7 years of age by ovariohysterectomy and were sliced repeatedly to release COC. In the first experiment, the COC were cultured at 38.8°C for 48 h in 5% CO2 in air in medium 199 supplemented with either TGF-a (0, 1, 10, or 100 ng mL–1) or IGF-1 (0, 0.5, 5, 10, or 50 µg mL–1). In the second experiment, the synergistic effect of TGF-a and IGF-1 was investigated by culturing COC in medium 199 supplemented with both TGF-a (0, 1, 10, or 100 ng mL–1) and IGF-1 (0, 0.5, 5, 10, or 50 µg mL–1). At the end of the culture period, the oocytes were denuded of cumulus cells by pipetting with a fine bore glass pipette; the denuded oocytes were then fixed in Carnoy's solution and stained with Hoechst 33342. The nuclear configuration and chromatin morphology of the oocytes were evaluated under confocal laser scanning microscopy. The cells were assigned to 1 of the following meiotic stages: germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), or metaphase II (MII). Data were analysed by ANOVA with Fisher's PLSD test. In experiment 1, no significant difference were observed in the rates of cells maturing to the MI and MII stages, but that in the 10 ng mL–1 of TGF-a group (56.3%) were larger than in the other treatment groups (38.8–51.0%). The frequencies of MII stage cells in the 5, 10, and 50 µg mL–1 of IGF-1 treatment groups (9.8, 13.3, and 12.2%, respectively) were significantly higher than in the 0.5 µg mL–1 of IGF-1 group and the control group (5.3 and 2.2%, respectively). In experiment 2, the frequency of MI and MII cells in the control, 1 ng mL–1 of TGF-a plus 0.5 µg mL–1 of IGF-1, 10 ng mL–1 of TGF-a plus 5 µg mL–1 of IGF-1, 10 ng mL–1 of TGF-a plus 10 µg mL–1 of IGF-1, and 100 ng mL–1 of TGF-a plus 50 µg mL–1 of IGF-1 group were 44.1, 36.1, 63.5, 70.8, and 50.8%, respectively. The frequency of MII cells in the control group and the same treatment groups were 2.8, 7.2, 10.4, 15.3, and 10.8%, respectively. Both frequencies in the 10 ng mL–1 of TGF-a plus 10 µg mL–1 of IGF-1 group were significantly higher than in the control group. The TGF-a may act in a paracrine fashion on the surrounding granulosa cells, and IGF-1 may play multiple roles in cellular metabolism, proliferation, growth, and differentiation in canine oocyte maturation, as has been reported for many other species. In conclusion, these results demonstrate that a synergistic effect between TGF-a and IGF-1 produces an increased rate of in vitro maturation to the MI and MII stages in canine oocytes.

scholarly journals Follicle-Stimulating Hormone and Luteinizing Hormone/Chorionic Gonadotropin Stimulation of Vascular Endothelial Growth Factor Production by Macaque Granulosa Cells from Pre- and Periovulatory Follicles1

1997 ◽  
Vol 82 (7) ◽  
pp. 2135-2142
Author(s):  
Lane K. Christenson ◽  
Richard L. Stouffer
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Granulosa Cells ◽  
Dependent Manner ◽  
Vascular Endothelial ◽  
Progesterone Production ◽  
Endothelial Growth Factor ◽  
In Vitro Treatment

Granulosa cells in the ovulatory follicle express messenger ribonucleic acid encoding vascular endothelial growth factor (VEGF), an agent that may mediate the neovascularization of the developing corpus luteum, but it is not known whether luteinizing granulosa cells synthesize and secrete VEGF during the periovulatory interval. Studies were designed to evaluate the effects of an in vivo gonadotropin surge on VEGF production by macaque granulosa cells (study 1) and to test the hypothesis that gonadotropins act directly on granulosa cells to regulate VEGF production (study 2). Monkeys received a regimen of exogenous gonadotropins to promote the development of multiple preovulatory follicles. Nonluteinized granulosa cells (i.e. preovulatory; NLGC) and luteinized granulosa cells (i.e. periovulatory; LGC) were aspirated from follicles before and 27 h after an ovulatory gonadotropin bolus, respectively. Cells were either incubated for 24 h in medium with or without 100 ng/mL hCG (study 1) or cultured for 6 days in medium with or without 100 ng/mL hCG or 0.1, 1, 10, and 100 ng/mL of recombinant human LH (r-hLH) or r-hFSH (study 2). Culture medium was assayed for VEGF and progesterone. In study 1, LGC produced 8-fold greater levels of VEGF than NLGC (899 ± 471 vs. 111 ± 26 pg/mL, mean ± sem; P &lt; 0.05). In vitro treatment with hCG increased (P &lt; 0.05) VEGF production by NLGC to levels that were not different from the LGC incubated under control conditions. In vivo bolus doses of r-hCG (100 and 1000 IU) and r-hFSH (2500 IU) were equally effective in elevating granulosa cell VEGF production. In study 2, in vitro treatment with r-hFSH, r-hLH, and hCG markedly increased (P&lt; 0.05) VEGF and progesterone production by the NLGC in a dose- and time-dependent manner. By comparison, the three gonadotropins (100 ng/mL dose) only modestly increased VEGF and progesterone production by LGC. These experiments demonstrate a novel role for the midcycle surge of gonadotropin (LH/CG or FSH) in primates to promote VEGF production by granulosa cells in the periovulatory follicle. Further, the data demonstrate that FSH-like as well as LH-like gonadotropins directly stimulate VEGF synthesis by granulosa cells.

321 OOCYTE DIAMETER INFLUENCES THE MEIOTIC RESUMPTION AND PROGRESSION INDUCED BY OKADAIC ACID IN DOG

2006 ◽  
Vol 18 (2) ◽  
pp. 268
Author(s):  
F. Ariu ◽  
L. Bogliolo ◽  
I. Rosati ◽  
M. T. Zedda ◽  
S. Pau ◽  
...  
Keyword(s):  
Okadaic Acid ◽  
In Vitro Maturation ◽  
Mammalian Species ◽  
Cumulus Cells ◽  
Meiotic Maturation ◽  
Control Group ◽  
Oocyte Diameter ◽  
Treatment Groups ◽  
Different Diameters

The acquisition of meiotic competence, in the bitch as in many other mammalian species, is related to the oocyte diameter. This study was designed to determine the effect of okadaic acid (OA), a potent inhibitor of seronine/threonine 1 and 2A phosphatases, on meiotic resumption and progression in canine oocytes with different diameters. In two experiments, healthy cumulus-oocytes complexes were collected from ovaries of bitches at various stages of the estrous cycle and divided, by diameters, into three treatment groups for in vitro maturation: <110 �m, 110-120 �m, and >120 �m. In Experiment 1, oocytes were pre-incubated for 1 h in TCM-199 + 20% estrous canine serum (SCE) + cysteamine + OA (0.5 �M). Then, oocytes were cultured for 48 h in the same medium without OA at 38.5�C, 5% CO2 in air. As a control group, oocytes were matured in vitro under the same conditions but without pre-incubation with OA. In Experiment 2, to determine if the effect of OA is mediated by cumulus cells, >120 �m oocytes were denuded from cumulus cells, incubated with or without OA, and cultured in vitro as previously described. At 48 h, all oocytes were stained and fixed with glycerol-Hoechst 33342 to assess the stage of meiotic maturation. In Experiment 1, OA induced a significantly higher incidence of meiotic resumption in oocytes <110 �m (16/108, 14.8%; P < 0.05) and 110-120 �m (70/130, 53.8%; P < 0.01) as compared to that of oocytes in the <110 �m and 110-120 �m control groups (2/58, 3.4%; 24/82, 29.3%). The percentage of oocytes in the 110-120 �m OA group that underwent in vitro maturation to metaphase II (MII) was significantly higher than in the 110-120 �m control group (18/130, 13.8% vs. 4/82, 4.9%, respectively; P < 0.05). In contrast, smaller oocytes (<110 �m) did not develop to MII with or whitout OA. Meiotic resumption rate of >120 �m OA group (64/78, 82.0%) was similar to the >120 �m control group (56/72, 77.8%), but a significantly higher proportion of the oocytes pre-incubated with OA progressed to MII than did the control oocytes (40/78, 51.3% vs. 12/72, 16.7%, respectively; P < 0.01). Low rates of meiotic resumption were observed in denuded >120-�m oocytes with (7/63, 11.1%) or without OA (7/55, 12.7%) and none of them progressed to MII. In conclusion, the results of the present study indicate that treatment of fully grown (>120 �m) oocytes with okadaic acid at the onset of in vitro maturation can result in a higher frequency of meiotic maturation than previously reported. Also, we determined that the beneficial effect of okadaic acid was mediated by cumulus cells.

Effects of ghrelin on activation of Akt1 and ERK1/2 pathways during in vitro maturation of bovine oocytes

Zygote ◽  
2017 ◽  
Vol 25 (2) ◽  
pp. 183-189 ◽  
Author(s):  
Thomas-Markos Chouzouris ◽  
Eleni Dovolou ◽  
Fotini Krania ◽  
Ioannis S. Pappas ◽  
Konstantinos Dafopoulos ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Cumulus Cells ◽  
Treated Group ◽  
Control Group ◽  
Dependent Manner ◽  
Nuclear Maturation ◽  
Bovine Oocytes ◽  
Phosphorylation Rate ◽  
Matured Oocyte

SummaryThe purpose of this study was to investigate the possible molecular pathways through which ghrelin accelerates in vitro oocyte maturation. Bovine cumulus–oocyte complexes (COCs), after 18 or 24 h maturation in the absence or the presence of 800 pg ml–1 of acylated ghrelin were either assessed for nuclear maturation or underwent in vitro fertilization in standard media and putative zygotes were cultured in vitro for 8 days. In a subset of COCs the levels of phosphorylated Akt1 and ERK1/2 (MAPK1/3) were assessed at the 0th, 6th, 10th, 18th and 24th hours of in vitro maturation (IVM). At 18 and 24 h no difference existed in the proportion of matured oocytes in the ghrelin-treated group, while in the control group more (P < 0.05) matured oocyte were found at 24 h. Oocyte maturation for 24 h in the presence of ghrelin resulted in substantially reduced (P < 0.05) blastocyst yield(16.3%) in comparison with that obtained after 18 h (30.0%) or to both control groups (29.3% and 26.9%, for 18 and 24 h in maturation, respectively). Ghrelin-treated oocytes expressed lower Akt1 phosphorylation rate at the 10th hour of IVM, and higher ERK1/2 at the 6th and 10th hours of IVM compared with controls. In cumulus cells, at the 18th and 24th hours of IVM Akt1 phosphorylation rate was higher in ghrelin-treated oocytes. Our results imply that ghrelin acts in a different time-dependent manner on bovine oocytes and cumulus cells modulating Akt1 and ERK1/2 phosphorylation, which brings about acceleration of the oocyte maturation process.

343 EFFECTS OF EPIDERMAL GROWTH FACTOR SUPPLEMENTATION ON IN VITRO MATURATION AND GENE EXPRESSION OF CANINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 279
Author(s):  
H.-J. Song ◽  
S.-H. Lee ◽  
G.-H. Maeng ◽  
J.-G. Kim ◽  
S. Balasubramanian ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
In Vitro Maturation ◽  
Luteinizing Hormone Receptor ◽  
Control Group ◽  
Dependent Manner ◽  
Junction Protein ◽  
Epidermal Growth

Despite many efforts to improve canine in vitro maturation (IVM), the efficiency is still low compared to that of other mammalian species (Marie et al. 2004). Epidermal growth factor (EGF) has stimulatory effects on the resumption of oocyte maturation and cumulus expansion in vitro and on prei-mplantation embryonic development in mammals by either an autocrine or a paracrine pathway, or a combination of both systems (Paria et al. 2001 PNAS 98, 1047-1052). The present study investigated the effects of EGF supplementation on in vitro maturation and gene expression of canine oocytes. Oocytes were recovered by slicing ovaries recovered from 40 bitches after ovariohysterectomy at random stages of the estrous cycle. Cumulus-oocyte complexes (COCs) were matured in TCM-199 containing 10% FBS, 1 �g/mL FSH and LH, and EGF (0, 10, or 30 ng/mL) for 48 or 72 h at 39�C in a humidified atmosphere of 5% CO2 in air. In Experiment I (n = 2520 oocytes), the nuclear maturation status was assessed by fluorescence microscopy after bisbenzimide (Hoechst 33342) staining (10 �g/mL) at 0, 48, and 72 h of incubation. In Experiment II (n = 90 oocytes), expression of transcripts such as EGF receptor (EGFR), luteinizing hormone receptor (LHR), and gap junction protein (GJA5) were determined in 10 intact COCs each at 0, 48, and 72 h, respectively, by reverse transcription-polymerase chain reaction (RT-PCR). At 0 h 10-20% of the oocytes had undergone resumption of meiosis (GVBD<MII). After 48 h of IVM, rate of meiotic resumption for 0, 10, and 30 ng/mL EGF were 28, 35, and 30%, respectively. At 72 h of IVM, oocytes in the 10 ng/mL EGF group had resumed meiosis at a higher frequency (55%; P < 0.05) than in the 30 ng/mL EGF or the control group (39 and 42%, respectively). At 72 h of IVM, the frequency of maturation to the MII stage was significantly higher in the 10 ng/mL EGF group (9.6%) than in the 30 ng/mL EGF or the control group (4.2 and 3.3%, respectively). The expression of EGFR was significantly higher (P < 0.05) in 0 h oocytes than in the 48- or 72-h oocytes. Further EGFR expression levels were decreased in the presence of EGF in a dose dependent manner. Transcripts for LHR were detected at all maturation intervals and its expression patterns were not altered by supplementation with 10 ng/mL EGF. Expression of GJA5 was observed only after 48 h of IVM, and levels of expression were similar in oocytes supplemented with both 10 and 30 ng/mL EGF. In summary, our results indicate that supplementation of canine IVM medium with 10 ng/mL EGF had a positive influence on the progression of maturation to MII at 72 h. The effect may not be related to the alteration of mRNA expression of genes analyzed in the present study, due to the complex patterns regulating meiotic arrest in canine oocytes. This work was supported by Grant no. 204119-03-1-LG000 from ARPC, Republic of Korea.

Vascular endothelial growth factor synergistically enhances bone morphogenetic protein-4-dependent lymphohematopoietic cell generation from embryonic stem cells in vitro

Blood ◽  
2000 ◽  
Vol 95 (7) ◽  
pp. 2275-2283 ◽  
Author(s):  
Naoki Nakayama ◽  
Jae Lee ◽  
Laura Chiu
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Bone Morphogenetic Protein ◽  
Es Cells ◽  
Embryonic Stem ◽  
Dependent Manner ◽  
Vascular Endothelial ◽  
Morphogenetic Protein ◽  
Endothelial Growth Factor

The totipotent mouse embryonic stem (ES) cell is known to differentiate into cells expressing the β-globin gene when stimulated with bone morphogenetic protein (BMP)-4. Here, we demonstrate that BMP-4 is essential for generating both erythro-myeloid colony-forming cells (CFCs) and lymphoid (B and NK) progenitor cells from ES cells and that vascular endothelial growth factor (VEGF) synergizes with BMP-4. The CD45+ myelomonocytic progenitors and Ter119+ erythroid cells began to be detected with 0.5 ng/mL BMP-4, and their levels plateaued at approximately 2 ng/mL. VEGF alone weakly elevated the CD34+ cell population though no lymphohematopoietic progenitors were induced. However, when combined with BMP-4, 2 to 20 ng/mL VEGF synergistically augmented the BMP-4-dependent generation of erythro-myeloid CFCs and lymphoid progenitors from ES cells, which were enriched in CD34+ CD31lo and CD34+CD45− cell populations, respectively, in a dose-dependent manner. Furthermore, during the 7 days of in vitro differentiation, BMP-4 was required within the first 4 days, whereas VEGF was functional after the action of BMP-4 (in the last 3 days). Thus, VEGF is a synergistic enhancer for the BMP-4-dependent differentiation processes, and it seems to be achieved by the ordered action of the 2 factors.

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