314 MELATONIN ON IN VIVO AND IN VITRO MATURATION OF MOUSE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 246 ◽  
Author(s):  
H. Fernandes ◽  
L. Schefer ◽  
F. C. Castro ◽  
C. L. V. Leal
Keyword(s):  
Ovarian Stimulation ◽  
In Vitro Maturation ◽  
F1 Hybrids ◽  
Saline Control ◽  
Control Group ◽  
Mouse Oocytes ◽  
Maturation Rate ◽  
Different Doses

Melatonin is a pineal hormone related to the control of the circadian cycle, besides the reproductive seasonality of some animal species, and has shown positive effects on oocyte maturation and embryo development. The aim of this study was to assess the effects of melatonin on in vivo and in vitro maturation of mouse oocytes. Female F1 hybrids (C57BL/6 × CBA; n = 8 per group/treatment) were used in 3 different treatments (trt) groups: (I) in vivo trt: mice received 2 different doses of melatonin injections, 10 and 20 mg kg–1 per IP including a saline control dose (0 mg kg–1 per IP) for 4 days along with ovarian stimulation trt of 5 IU of eCG IP, followed by 5 IU of hCG IP 48 h later, and cumulus-oocyte complexes (COC) were collected 16 h after hCG; (II) mice received a similar in vivo melatonin trt, but ovarian stimulation trt was only 5 IU of eCG, no hCG, and COC were collected after 48 h and subsequently matured in vitro with 0.5 µg mL–1 of FSH for 16 h; (III) in vitro maturation of oocytes: COC were collected 48 h after 5 IU of eCG and maturated in the presence of 3 different doses of melatonin (10–9, 10–6, and 10–3 M) or 0.5 µg mL–1 of FSH (control) for 16 h. In vitro-maturing oocytes were in incubated at 37°C, 5% CO2, and 95% humidity. Maturation rates were evaluated according to the presence of the first polar body under an inverted microscope. Statistical analyses were performed by ANOVA followed by Tukey's test (4 replicates). In the first treatment, 20 mg kg–1 of melatonin showed the highest in vivo maturation rate, 80.3% (61/76), while 10 mg kg–1 of melatonin was 62.4% (53/85) and the saline control group was 69.4% (77/111), but differences were not significant (P > 0.05). For in vitro maturation of oocytes from animals previously treated with melatonin, the 10 mg kg–1 of melatonin group had the highest maturation rate, 53.2% (99/186), in comparison with the saline and 20 mg kg–1 of melatonin groups, which showed 46.6 (88/189) and 39.0% (85/218), respectively; again, no differences were detected (P > 0.05). In the last treatment, the maturation rates increased from 48.9 (43/88) to 53.7 (51/95) and 56.0% (56/100) as the melatonin concentrations decreased from 10–3, 10–6, and 10–9 M, respectively. The control group had the highest rate of 57.3% (55/96), but no statistical differences were observed (P = 0.706). In conclusion, under the conditions studied, melatonin was unable to improve the maturation rate neither after in vivo nor in vitro treatment. However, during in vitro maturation, melatonin alone was as efficient as FSH in promoting maturation in murine oocytes, indicating its potential effect on stimulating meiosis. Therefore, the role of melatonin in stimulating meiosis needs further investigation.Acknowledgments to FAPESP for fellowship (HF) and funding (CLVL).

157 EFFECT OF FSH STARVATION (COASTING) FOLLOWING SUPEROVULATION ON OOCYTE COMPETENCE AND CLONING EFFICIENCY IN GOATS

2017 ◽  
Vol 29 (1) ◽  
pp. 187 ◽  
Author(s):  
C. E. Méndez-Calderón ◽  
C. R. Lazzarotto ◽  
L. H. Aguiar ◽  
F. L. Ongaratto ◽  
K. C. S. Tavares ◽  
...  
Keyword(s):  
Pregnancy Outcome ◽  
In Vitro Maturation ◽  
Animal Health ◽  
Oocyte Quality ◽  
Control Group ◽  
In Vitro Production ◽  
Oocyte Competence ◽  
Maturation Rate

Oocyte competence plays a key role in the overall efficiency of reproductive biotechnologies. In cattle, FSH starvation following superovulation (coasting) improves oocyte competence, blastocyst yield and pregnancy outcome when used in ovum pickup-in vitro production programs. The aim of this study was to compare the effect of coasting after exogenous FSH stimulation on goat oocyte quality and competence to support in vitro maturation and in vivo embryo development following cloning procedures in goats. Donor and recipient preparation, cumulus-oocyte complex (COC) retrieval and selection, IVM, cloning by somatic cell nuclear transfer, embryo transfer, and pregnancy diagnosis (Days 23–26) were performed according to our established procedures [Martins et al. 2016 doi: 10.1089/cell.2015.0082]. Cumulus-oocyte complexes were obtained in vivo from 71 cycling FSH-stimulated mature Nubian-crossed goats, combined or not with FSH starvation (coasting period). Donor females were oestrous synchronized with a progesterone intravaginal insert (Day 0). On Day 10, a 0.75-mg D-cloprostenol dose was given IM, with the onset of the superovulation treatment, composed of five 20-mg FSH doses (Folltropin®, Bioniche Animal Health, Pullman, WA, USA), via IM at 12-h intervals. Donors were subjected to laparoscopic ovum pickup either 9 h (control group, n = 36) or 21 h (coasting group, n = 35) after the last FSH dose, respectively. Skin fibroblast cell cultures from a male neonate were co-transfected with a mammary gland expression vector with the human lactoferrin (hLF) coding sequence and with CRISPR/Cas9 system either for the PRNP prion gene or the Rosa26 locus. A bi-allelic hLF-PRNP and a mono-allelic hLF-Rosa26 cell colony were used for cloning. Data were compared by ANOVA or the χ2 test (P < 0.05). No differences were observed between control and coasting for number of follicles (18.7 ± 1.4 v. 21.2 ± 1.7), and retrieved (17.3 ± 1.2 v. 20.7 ± 1.9), viable (15.9 ± 1.1 v. 19.6 ± 1.8), Grade I (1.5 ± 0.3 v. 2.5 ± 0.5), and Grades III+IV (6.0 ± 0.6 v. 5.7 ± 0.7) COC, as well as for COC retrieval (92.4%, 574/621 v. 94.5%, 685/725) and fusion (62.8%, 273/435 v. 61.3%, 311/507) rates, respectively, irrespective of the cell lines. However, the coasting group rendered higher number of Grade II COC (11.3 ± 1.2 v. 8.4 ± 0.7), number and proportion of Grades I+II COC (13.9 ± 1.5 v. 9.9 ± 0.9, 70.8% v. 62.4%), and maturation rate (70.9% v. 65.4%) than the control group, respectively, for a lower proportion of Grades III+IV (29.2% v. 37.6%, respectively). A total of 213 and 233 Day-1 cloned embryos from the control and the coasting groups were transferred to 18 (96/9 hLF-PRNP and 117/9 hLF-Rosa26 cells) and 19 (128/11 hLF-PRNP and 105/8 hLF-Rosa26 cells) female recipients, respectively, resulting in 1/9 (11.1%) and 4/11 (36.4%) pregnancies from the hLF-PRNP cells, and 3/9 (33.3%) and 3/8 (37.5%) from the hLF-Rosa26 cells, for the control (4/18, 22.2%) and coasting (7/19, 36.8%) groups, respectively, for an overall pregnancy rate of 29.7% (11/37). In conclusion, the use of coasting improved oocyte quality and in vitro maturation rate, also appearing to increase pregnancy outcome after goat cloning.

scholarly journals 46 Effect of the addition of α-tocopherol to in vitro maturation media of bovine oocytes

2020 ◽  
Vol 98 (Supplement_2) ◽  
pp. 2-3
Author(s):  
Theisy P Acosta Pérez
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Control Group ◽  
Chi Square ◽  
Cumulus Expansion ◽  
Minimum Concentration ◽  
Maturation Rate ◽  
Chi Square Test ◽  
System Data

Abstract α-tocopherol is known to be a powerful antioxidant, in this regard, it was added to bovine oocyte in vitro maturation media to evaluate its effect on oocyte maturation. Oocytes (n = 624) aspirated from ovaries of slaughtered cows were classified by quality and divided in four categories according to cytoplasm appearance and cumulus cells layers. Oocytes were washed in TCM-199 supplemented with fetal bovine serum (FBS) and FSH, then distributed in maturation media (TCM-199 supplemented with FBS, FSH and gentamicin). Three experimental groups of α-tocopherol (50, 100 and 200 mM) and a control group without α-tocopherol were used. Maturation was carried 22 h at 38.5°C in a 5% CO2 atmosphere. Oocytes were examined to determine cumulus expansion as categorical data (expansion or no expansion), as well as cumulus expansion Index (CEI). For CEI determination oocytes were graded 0 to 4, being 0 those with null expansion and 4 those with a noticeable cell expansion, then the number of oocytes were multiplied by the grade given and a sum of the totals was obtained, the new total was divided by the total of oocytes in the group and the result obtained corresponded to the CEI of the group. Results were analyzed with Chi Square test (for maturation rates) and an ANOVA (for the CEI) using the SAS system, data are presented as mean ± standard error. There was no statistical difference between control and α-tocopherol groups (P &gt;0.05). Numerically, the control group showed a higher maturation rate (100%) and obtained a higher CEI (2.44±0.20), followed by the 50 mM group (98.16%; 2.39±0.13), the groups 200 mM (97.40%; 2.00±0.14) and 100 mM (96.25%; 2.06±0.24) were the lowest. The addition of the minimum concentration (50 mM) of α-tocopherol to the maturation media could improve maturation rates without exposing oocytes to toxic effects.

scholarly journals Cilengitide Inhibits Neovascularization in a Rabbit Abdominal Aortic Plaque Model by Impairing the VEGF Signaling

2021 ◽  
Vol 2021 ◽  
pp. 1-18
Author(s):  
Fawang Zhu ◽  
Shuai Yuan ◽  
Jing Li ◽  
Yun Mou ◽  
Zhiqiang Hu ◽  
...  
Keyword(s):  
Umbilical Vein ◽  
Tube Formation ◽  
Control Group ◽  
In Vitro Experiments ◽  
Aortic Plaque ◽  
Abdominal Aortic ◽  
Aortic Plaques ◽  
Different Doses

Background. Cilengitide is a selective αvβ3 and αvβ5 integrin inhibitor. We sought to investigate the effect of cilengitide on the neovascularization of abdominal aortic plaques in rabbits and explore its underlying antiangiogenic mechanism on human umbilical vein endothelial cells (HUVECs). Materials and Methods. For the in vivo experiment, the abdominal aortic plaque model of rabbits was established and injected with different doses of cilengitide or saline for 14 consecutive days. Conventional ultrasound (CUS) and contrast-enhanced ultrasound (CEUS) were applied to measure the vascular structure and blood flow parameters. CD31 immunofluorescence staining was performed for examining neovascularization. Relative expressions of vascular endothelial growth factor (VEGF) and integrin of the plaque were determined. For in vitro experiments, HUVECs were tested for proliferation, migration, apoptosis, and tube formation in the presence of different doses of cilengitide. Relative expressions of VEGF, integrin, and Ras/ERK/AKT signaling pathways were determined for the exploration of underlying mechanism. Results. CEUS showed modestly increased size and eccentricity index (EI) of plaques in the control group. Different degrees of reduced size and EI of plaques were observed in two cilengitide treatment groups. The expressions of VEGF and integrin in the plaque were inhibited after 14 days of cilengitide treatment. The neovascularization and apoptosis of the abdominal aorta were also significantly alleviated by cilengitide treatment. For in vitro experiments, cilengitide treatment was found to inhibit the proliferation, migration, and tube formation of HUVECs. However, cilengitide did not induce the apoptosis of HUVECs. A higher dose of cilengitide inhibited the mRNA expression of VEGF-A, β3, and β5, but not αV. Lastly, cilengitide treatment significantly inhibited the Ras/ERK/AKT pathway in the HUVECs. Conclusions. This study showed that cilengitide effectively inhibited the growth of plaque size by inhibiting the angiogenesis of the abdominal aortic plaques and blocking the VEGF-mediated angiogenic effect on HUVECs.

scholarly journals Use of Melatonin in the In Vitro Production of Bovine Embryos

2020 ◽  
Vol 21 ◽  
Author(s):  
Alan da Silva LIRA ◽  
Ricardo de Macedo CHAVES ◽  
Felipe de Jesus MORAES JUNIOR ◽  
Sergio Henrique COSTA JUNIOR ◽  
Brenda Karine Lima do AMARAL ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Control Group ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Maturation Medium ◽  
Significant Difference ◽  
Maturation Rate ◽  
Vitro Production

ABSTRACT We aimed to assess the effects of melatonin in the in vitro production of bovine embryos. Our experiment was conducted at the Laboratório de Reprodução Animal of the Universidade Estadual do Maranhão. The cumulus-oocyte complexes (COCs) were distributed among treatments at concentrations of 0, 10-1, 10-3 and 10-5 µMol/L melatonin. Our experiment was further divided into two: the first was to assess the effect of different concentrations of melatonin (treatments) on the maturation rate of COCs, and the second was to assess the effects of melatonin treatments on the in vitro production of bovine embryos. The results from the first experiment demonstrated no significant difference between the in vitro maturation rate of the cultivated COCs in treatments with melatonin. In the second experiment, however, melatonin treatments yielded statistically higher cleavage, morula and blastocyst rates in the 10-5 µM group (52.9%, 52.9%, and 35.3%, respectively), and lower rates in the 10-1 µM group (19.5%, 19.5% and 7.8%, respectively), compared to the others. The control group (no melatonin) and the 10-3 µM group showed similar results. We concluded that supplementation of melatonin in the in vitro maturation medium resulted in no improvement in the oocyte maturation rate, but in the in vitro production of embryos at different concentrations, the 10-5 µM group displayed better results, but with no improvement in the variables (P < 0.05).

191 IN VITRO CULTURE DURING OOCYTE MATURATION AND FERTILIZATION INFLUENCES THE TRANSCRIPTOME PROFILES OF BOVINE BLASTOCYSTS

2015 ◽  
Vol 27 (1) ◽  
pp. 186
Author(s):  
A. Gad ◽  
U. Besenfelder ◽  
V. Havlicek ◽  
M. Hölker ◽  
F. Rings ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Oocyte Maturation ◽  
Microarray Data ◽  
In Vitro Maturation ◽  
Culture Conditions ◽  
Control Group ◽  
Bovine Embryos ◽  
Vitro Fertilization

Early embryonic development, the period from oocyte maturation until blastocyst formation, is the most critical period of mammalian development. It is well known that in vitro maturation, fertilization, and culture of bovine embryos is highly affected by culture conditions. However, the stage-specific effect of culture environment is poorly understood. Therefore, we aimed to examine the effect of in vitro culture conditions during oocyte maturation and fertilization on the transcriptome profile of the resulting blastocysts. Bovine oocytes were matured in vitro and then either directly transferred to synchronized recipients, fertilized, and cultured in vivo (Vitro_M), or transferred after in vitro fertilization (Vitro_F), or at zygote stage (Vitro_Z) and blastocysts were collected at Day 7 by uterine flushing. For in vivo or in vitro fertilization, the same frozen-thawed commercial bull semen has been used. Complete in vitro (IVP) and in vivo produced blastocysts were used as controls. Gene expression patterns between each blastocyst group and in vivo produced blastocyst group were compared using EmbryoGENE's bovine microarray (EmbryoGENE, Québec, QC, Canada) over six replicates of each group (10 blastocyst/replicate). Microarray data were statistically analysed using the Linear Models for Microarray Data Analysis (LIMMA) package under the R program (The R Project for Statistical Computing, Vienna, Austria). Results showed that, the longer the embryos spent under in vitro conditions, the higher was the number of differentially expressed genes (DEG, fold-change = 2 with adjusted P-value = 0.05) compared with in vivo control group. The Vitro_M group showed the lowest number of DEG (149); in contrast IVP group represented 841, DEG, respectively compared to in vivo control group. Ontological classification of DEG showed that lipid metabolism was the most significant function influenced by in vitro maturation conditions. More than 55% of DEG in the Vitro_M group were involved in the lipid metabolism process and most of them showed down-regulation compared to in vivo control group. On the other hand, Vitro_F and Vitro_Z groups showed nearly similar numbers of DEG (584 and 532, respectively) and the majority of these genes in both groups were involved in cell-death- and cell-cycle-related functions. Pathway analysis revealed that retinoic acid receptor activation pathways were the common ones in the Vitro_M and Vitro_F groups. However, different signalling pathways were commonly dominant in the Vitro_F and Vitro_Z groups. This study provides the transcriptome elasticity of bovine embryos exposed to different environments during maturation, fertilization, and culture periods of development.

Abstract P141: Cardiac TRH Partly Mediates Angiotensin II-induced Fibrotic and Hypertrophy Effects in "in vivo" and "in vitro" Models

Hypertension ◽  
2015 ◽  
Vol 66 (suppl_1) ◽  
Author(s):  
Silvia I García ◽  
Ludmila S Peres Diaz ◽  
Maia Aisicovich ◽  
Mariano L Schuman ◽  
María S Landa
Keyword(s):  
Gene Expression ◽  
Body Weight ◽  
Cardiac Hypertrophy ◽  
Structural Changes ◽  
In Vitro Models ◽  
Heart Weight ◽  
Saline Control ◽  
Control Group

Cardiac TRH (cTRH) is overexpressed in the hypertrophied ventricle (LV) of the SHR. Additionally in vivo siRNA-TRH treatment induced downregulation of LV-TRH preventing cardiac hypertrophy and fibrosis demonstrating that TRH is involved in hypertrophic and fibrotic processes. Moreover, in a normal heart, the increase of LV TRH expression alone could induce structural changes where fibrosis and hypertrophy could be involved, independently of any other system alterations. Is well-known the cardiac hypertrophy/ fibrotic effects induced by AII, raising the question of whether specific LV cTRH inhibition might attenuates AII induced cardiac hypertrophy and fibrosis in mice. We challenged C57 mice with AII (osmotic pumps,14 days; 2 mg/kg) to induce cardiac hypertrophy vs saline. Groups were divided and , simultaneously to pump surgery, injected intracardiac with siRNA-TRH and siRNA-Con as its control. Body weight, water consume and SABP were measured daily. As expected, AII significantly increased SABP (p<0.05) in both groups treated , although cardiac hypertrophy (heart weight/body weight) was only evident in the group with the cardiac TRH system undamaged, suggesting that the cardiac TRH system function as a necessary mediator of the AII-induced hypertrophic effect. As hypothesized, we found an AII-induced increase of TRH (p<0.05) gene expression (real-t PCR) confirmed by immunofluorescence that was not observed in the group AII+siRNA-TRH demonstrating the specific siRNA treatment efficiency. Furthermore, AII significantly increase (p<0.05) BNP (hypertrophic marker), III collagen and TGFB (fibrosis markers) expressions only in the group with AII with the cardiac TRH system intact. On the contrary, the group with AII and the cTRH system inhibited, shows genes expressions similar to the saline control group. We confirmed these results by immunofluorescence. Similar fibrotic results were observed with NIH3T3 cell culture where we demonstrated that AII induced TRH gene expression (p<0.05) and its inhibition impedes AII-induced increase of TGFB and III/I collagens expressions telling us about the role of the cTRH in the AII fibrosis effects. Our results point out that the cardiac TRH is involved in the AII-induced hypertrophic and fibrotic effects.

176 IN VITRO MATURATION OF DOG OOCYTES IN CANINE FOLLICULAR FLUID

2014 ◽  
Vol 26 (1) ◽  
pp. 202
Author(s):  
K. Reynaud ◽  
S. Canguilhem ◽  
S. Thoumire ◽  
S. Chastant-Maillard
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Assisted Reproductive Technologies ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Control Group ◽  
Limiting Factor ◽  
Cytoplasmic Maturation

In the canine species, assisted reproductive technologies, especially in vitro maturation (IVM) and IVF, are still ineffective. The main limiting factor remains the immaturity of the oocytes collected from anestrus ovaries. The ability of an oocyte to reach the MII stage in vitro is linked to the diameter of its follicle and anestrus oocytes, collected from small (<1 mm) follicles, are profoundly immature (De Lesegno et al. 2008). The objective of this study was to improve cytoplasmic quality by mimicking in vivo conditions; that is, to test the effect of pure preovulatory follicular fluid (FF) on survival and IVM rates of anestrus dog oocytes, in order to improve the nuclear and cytoplasmic maturation of these immature oocytes. Follicular fluids samples were collected from 54 Beagle bitches at 2 stages: before the LH peak (n = 23 bitches) and after the LH peak (n = 31 bitches). Only follicular fluid samples from large (>4 mm) follicles were collected and pooled by stage. Control oocytes were matured in 20% FCS/M199 medium. Groups of 5 oocytes were in vitro matured in 30 μL of follicular fluid, in half-area 96-well plates (5% CO2, 38°C). After 72 h of IVM, oocytes were denuded, fixed, and stained for DNA and tubulin before observation by confocal microscopy, and nuclear stages were classified as GV-A to GV-E, MI, and MII (Reynaud et al. 2012). A total of 460 oocytes were collected from 13 anestrus bitches and allocated to either the control medium (n = 155), the Pre-LH FF (n = 145) or the Post-LH FF (n = 160) groups. After 72 h of IVM, the morphology of the cumulus–oocyte complexes (COC) in the post-LH group was different from that of the others: cumulus cells appeared more compact and darker. Analysis of the nuclear stages showed that the degeneration rate was significantly higher (P < 0.05) in the post-LH group (58.7%) than in the pre-LH (40.9%) or in the control group (34.4%). No significant differences (P > 0.05) were observed between the 3 groups in the rate of immature GVA-B oocytes (36.4, 28.5, and 25.3% in the control, Pre-LH, and Post-LH groups, respectively), in the rate of meiotic resumption (GV-C/D/E, MI, MII stages, 44.4, 51.9, and 38.7% in the control, Pre-LH, and Post-LH groups, respectively). Metaphase II rates were not significantly different (12.1, 8.6, and 4.8% in the control, Pre-LH, and Post-LH groups, respectively). In conclusion, canine COC may survive when exposed to IVM in pure follicular fluid, but the degeneration rate was higher in the post-LH group. The presence of follicular fluid did not inhibit meiosis resumption, but did not significantly improve IVM rates. To better mimic in vivo conditions, IVM in a sequence of media, such as IVM in follicular fluid followed by IVM in oviducal fluid remains to be tested.

134 TRANSCRIPTOME PROFILE OF BOVINE BLASTOCYSTS DERIVED FROM ALTERNATIVE IN VIVO AND IN VITRO CULTURE CONDITIONS AT SPECIFIC PHASES OF EARLY EMBRYONIC DEVELOPMENT

2012 ◽  
Vol 24 (1) ◽  
pp. 179 ◽  
Author(s):  
A. Gad ◽  
U. Besenfelder ◽  
V. Havlicek ◽  
M. Hölker ◽  
M. U. Cinar ◽  
...  
Keyword(s):  
Embryonic Development ◽  
In Vitro Culture ◽  
In Vitro Maturation ◽  
Expression Patterns ◽  
Culture Conditions ◽  
Control Group ◽  
Transcriptome Profile ◽  
Vitro Fertilization

An understanding of gene expression patterns due to altered environmental conditions during different time points of the pre-implantation period would improve our knowledge on regulation of embryonic development and improve success of embryo culture. The aim of this study was to examine the effect of alternative in vivo and in vitro culture conditions at specific phases of early embryonic development on transcriptome profile of bovine blastocysts. Using nonsurgical endoscopic oviducal transfer technology, 5 different blastocyst groups were produced. The first 2 groups were matured in vitro and then either transferred after maturation or after in vitro fertilization to synchronized recipients. The other 3 groups were matured, fertilized and cultured in vitro until 4-cell, 16-cell and morula stage before transfer. Blastocysts from each group were collected by uterine flushing at Day 7 and pooled in groups of 10. Complete in vitro (IVP) and in vivo blastocysts were produced and used as controls. A unique custom microarray (Agilent) containing 42 242 oligo probes (60-mers) was used over 6 replicates of each group vs the in vivo control group to examine the transcriptome profile of blastocysts. Compared with the in vivo control group, clear dramatic shifts were found in the number of differentially expressed genes (DEG, fold change ≥2) at 2 different time points. The first shift occurred for blastocyst groups that were transferred after in vitro fertilization and before embryonic genome activation (EGA). The second shift occurred for blastocyst groups that were transferred after EGA, as well as for the IVP group. Ontological classification of DEG showed that the more time spent under in vitro conditions, the higher the percentage of DEG involved in cell death and apoptotic processes. Moreover, lipid metabolism was the most significant process affected in the blastocysts transferred after in vitro maturation and blastocysts transferred at 16-cell stage. Most DEG involved in this process were down-regulated. Pathway analysis revealed that signalling pathways were the dominant pathways in all groups except the group that was transferred after in vitro maturation. That group showed significant down-regulation for genes involved in retinoic acid receptors activation pathways. These results showed that fertilization and EGA were the most critical developmental stages influenced by in vitro culture conditions and subsequently affect blastocyst quality, as measured in terms of gene expression patterns. Moreover, we identified molecular mechanisms and pathways that were influenced by altered culture conditions. These findings will enable the examination of strategies for modifying in vitro culture conditions at critical stages that will allow more efficient production of developmentally competent blastocysts.

Overcoming Drug Resistance In Acute Lymphoblastic Leukemia by Inhibition of CBP/γ-Catenin

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3264-3264
Author(s):  
Enzi Jiang ◽  
Eugene Park ◽  
Cu Nguyen ◽  
James Yoon ◽  
Yao-Te Hsieh ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Western Blot ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Saline Control ◽  
Control Group ◽  
Apoptosis Protein

Abstract Abstract 3264 Survivin, an inhibitor of apoptosis protein (IAP) family, has been associated with poor prognosis in cancer including leukemia. Survivin can be downregulated in colon cancer cells by inhibition of the β-catenin/Creb-binding protein (CBP) interaction using ICG-001, a small molecule specific inhibitor of the β-catenin/CBP interaction. We have shown previously that combined ICG-001 and chemotherapy can downregulate Survivin and sensitize ALL cells to chemotherapy in vitro and in a pilot study in vivo. In this study, we determine the CBP interaction with ICG-001 in primary ALL cells and preclinically evaluate ICG-001 in vitro and in vivo as an adjuvant against primary ALL and. For this purpose, primary ALL cells were co-cultured with OP9 cells and treated for 4 days with ICG-001 (10mM, 20mM) or DMSO as vehicle control. Mean viability (trypan blue exclusion) of cells treated with ICG-001 was significantly lower (ICG-001 10mM: 75.12% ± 3.15%; 20mM: 41.18%± 7.88%) compared to cells treated with DMSO (84.99% ± 0.42%) (% cell viability relative to initial control) (p=0.03). Real time RT-PCR showed ICG-001 dose-dependent downregulation of Survivin in ALL compared to control (ICG10mM vs. control: p=0.0037 and 20mM vs. control: p=0.0031). Immunoblotting demonstrated reduction of Survivin after ICG-001 treatment. Primary ALL cells incubated with a combination of VDL (Vincristine, Dexamethasone and L-Asparaginase) and ICG-001 showed decreased viability (28.7%± 4.9%) versus VDL only (79.3%± 13.6%) (p=0.014) determined by MTT assay. To elucidate if ICG-001 interacts with β-catenin/CBP as shown previously in colon cancer, we analyzed ten primary pre-B ALL cells and found significantly greater γ-catenin and Survivin expression versus normal pre-B-Cells. β-catenin was absent or in some cases expressed only weakly. Expression of v-catenin and b-catenin in ALL xenograft cells were detected by Western blot. One primary ALL was selected and incubated with γ-catenin and β-catenin siRNA for 48hrs, followed by 6hrs incubation with Wnt3a. Wnt3a induced both of γ-catenin and β-catenin expression. Survivin was reduced by γ-catenin siRNA but not β-catenin siRNA treatment. Addition of Wnt3a partially recovered the decrease of Survivin. In addition, Survivin was knocked down in primary ALL using shRNA and non-silencing shRNA control or ICG-001 (2uM) and DMSO control. Western blot analysis showed that survivin shRNA or ICG-001 treatment lead to downregulation of Survivin and γ-catenin. Using a ChIP assay we could demonstrate occupancy of TCF4 and CBP association at the Survivin promoter, which was not altered by ICG-001 in primary ALL. Moreover, ICG-001 treatment of primary ALL cells prevents CBP but not p300 occupancy. For further preclinical in vivo evaluation of ICG-001, one Philadelphia chromosome positive ALLs (Ph+) and two Ph− primary ALL were injected into sublethally irradiated NOD/SCID IL2Rγ−/-mice and treated with ICG-001 (50mg or 100mg/kg/day per subcutaneous miniosmotic pump) with or without chemotherapy including VDL for Ph− ALL (per intraperitoneal injections) or Nilotinib for Ph+ ALL (per os). For analysis we pooled the survival of all three primary leukemias. The saline control group (n=10) (MST= 55.5.days) and the ICG-001 only groups (n=3) (MST=61 days) died rapidly. The group treated with chemotherapy (n=13) had a median survival time (MST) of 85 days. In marked contrast, the group treated with the combined chemotherapy+ICG-001 (n=15) lived significantly longer (MST=100) (p<0.05). Taken together, our data shows that Survivin transcription can be mediated by γ-catenin in primary ALL and that targeting CBP/γ-catenin by using ICG-001 ALL can sensitize ALL cells to chemotherapy in vitro and in vivo. Disclosures: No relevant conflicts of interest to declare.

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