102 DEVELOPMENTAL COMPETENCE OF BUFFALO (BUBALUS BUBALIS) OOCYTES VITRIFIED AT DIFFERENT STAGES OF MATURATION IN VITRO

2006 ◽  
Vol 18 (2) ◽  
pp. 159
Author(s):  
K. Loganathasamy ◽  
R. Rajhans ◽  
G. SaiKumar ◽  
G. T. Sharma
Keyword(s):  
Expression Pattern ◽  
Glucose Transporter ◽  
Storage Period ◽  
Subsequent Development ◽  
Developmental Competence ◽  
Control Group ◽  
Hsp 70 ◽  
Glucose Transporter 1 ◽  
Total Rna

Cryopreservation of unfertilized oocytes at very low temperature (-196�C) is carried out to ensure their continuous availability during different assisted reproductive techniques. However, various problems associated with the freezing of oocytes influence their developmental competence, resulting in suboptimal embryo production. The present study was planned to assess the developmental competence of buffalo oocytes vitrified at different meiotic stages of maturation. Expression profile of developmentally important genes, viz, heat shock protein 70 (HSP 70) and glucose transporter 1 (Glut1), was verified in these vitrified warmed oocytes. Buffalo cumulus-oocyte complexes were collected from slaughterhouse ovaries and divided into six groups: control (no vitrification); 0 h group (vitrified before maturation), and 6-, 12-, 18-, and 24-h groups [vitrified respectively at 6, 12, 18, and 24 h post-in vitro maturation (IVM)]. Vitrification solution consisted of propylene glycol (40% w/v), and trehalose (0.25 mol/L) in PBS + BSA (4% w/v) and vitrification was carried out by directly plunging 0.25-mL French mini-straws into liquid nitrogen. After a minimum storage period of 7 days, the straws were thawed at 37�C for 30 sec. In all groups, the oocytes completed 24-h of maturation. After 24 h maturation, a few oocytes from each of the six groups were stained with ethidium bromide to reveal their nuclear status. The remaining oocytes were co-incubated with frozen thawed buffalo semen in fertilization TALP with 6 mg/mL fatty acid free BSA and 10 �g/mL heparin sodium salt for 18 h. Presumptive zygotes were cultured in mSOF for 8 days. Vitrified warmed oocytes were subjected to total RNA isolation and RT-PCR for detection of mRNA transcripts of HSP 70 and Glut1 genes. Data were analyzed by one-way ANOVA and F-test analysis. Differences of P < 0.05 were considered significant. The percentage of oocytes recovered from all five vitrification groups varied from 89 to 92 out of which 84-91% of oocytes were morphologically normal. A higher proportion of nonvitrified control oocytes (72.8%; 40/55) reached the metaphase II stage than for the oocytes vitrified at 24 (60%; 36/60), 18 (54.4%; 31/57), 12 (42.3%; 22/52), 6 (33.3%; 20/60), and 0 (31.7%; 19/60) h of IVM. The cleavage rate of nonvitrified control oocytes was higher (36.8%) than that of oocytes vitrified at 0 (1.6%), 6 (2.0%), 12 (3.2%), 18 (5.3%), and 24 (5.2%) h of IVM. With regard to subsequent development, 0- and 6-h oocytes were blocked at 8 cells, whereas in other groups development reached the late morula (4.8%) and blastocyst (3.5%) stages, confirming that the stage of maturation at which oocytes are vitrified influenced the nuclear maturation and developmental competence. Total RNA content was 2.24 � 0.40 ng/oocyte in the control group and 2.11 � 0.22 ng/oocyte in the group vitrified after 24 h of IVM. The expression pattern of HSP 70 and Glut1 was identical in control and vitrified groups, indicating that the vitrification protocol did not alter the expression pattern of these genes.

292 EXPRESSION PROFILES OF STRESS AND METABOLIC MARKER GENES DURING IN VITRO PRODUCTION OF BUFFALO (BUBALUS BUBALIS) EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 253
Author(s):  
R. Rajhans ◽  
G. S. Kumar ◽  
G. T. Sharma
Keyword(s):  
Bubalus Bubalis ◽  
Marker Genes ◽  
Hsp 70 ◽  
In Vitro Production ◽  
Glucose Transporter 1 ◽  
Total Rna ◽  
Gene Transcripts ◽  
Vitro Production ◽  
Glut1 Gene

An increased understanding of the pre-implantation embryo developmental stage, with respect to physiological interaction of embryo with its micromilieu both in vivo and in vitro, is imperative to comprehend the events of pre-implantation development.The objective of the present study was to examine the temporal expression of heat shock protein 70 (Hsp-70) and glucose transporter 1 (Glut1) genes in pre-implantation-stage buffalo embryos produced under the standard in vitro production (IVP) system. Embryos were produced from slaughterhouse ovaries employing standard in vitro embryo production protocol, and presumptive zygotes produced following IVM/IVF were cultured in vitro in mSOF under mineral oil; FCS (10%) was added at 48 h post-insemination (hpi).The time series of development at stages being zygote (18-20 hpi), 2-1 cell (48 hpi), 8-16 cell (94-96 hpi), morula (120-144 hpi), and blastocyst (168-192 hpi), pre-implantation embryos conforming to the above developmental pattern were considered as 'fast-cleaving embryos', and all the embryos that did not conform to the above developmental timing were regarded as 'slow-cleaving embryos'. Pools of immature oocytes (IM, 120), Matured oocytes (MO, 120), 8-16 cell stages (8-16, 70), morula (M, 28), and blastocyst (B, 9) were collected and prepared for total RNA isolation and RT-PCR for the specific transcripts, with �-actin as loading control. The total RNA content ranged from 2.5 to 5.0 ng per oocyte/embryo. Presence of Hsp 70 and Glut1 gene transcripts was assessed in different stages of buffalo pre-implantation embryos using primers designed from bovine Hsp 70 and Glut1 by using the OLIGO program. RT was standardized using the embryo equivalent of 1-10 oocytes/embryo as the template as described by Arcellana-Panlilio and Schultz 1993 (Methods Enzymol. 225, 303-328) with PCR conditions being 59�C and 62�C for 45 s with 39 cycles for Glut1 and Hsp 70 gene transcripts, respectively. Amplicons were subjected to restriction digestion and sequencing (Acc. No. AJ812563, AJ812564). The expression of Hsp 70 throughout pre-implantation development in the fast-cleaving embryos indicated their maternal and zygotic origin, but transcripts of the Hsp 70 gene, represented by a single 488-bp amplicon, were not detected in slow-cleaving embryos, suggesting altered zygotic expression. Glut1 expression was prominent from the 8-16 cell stage, indicating a metabolic shift from pyruvate to glucose after the pre-compaction stage. For slow-cleaving embryos, transcripts of the Glut1 gene, represented by a single 327-bp amplicon, were absent during morula- and blastocyst-stage embryos, indicating the poor developmental competence of these embryos, which morphologically appeared normal. These transcription patterns reflect the embryonic response to the in vitro culture conditions and also correlate with the embryo quality and the speed of development of the pre-implantation buffalo (Bubalus bubalis) embryos.

340 IN VITRO MATURATION OF PREPUBERTAL BOVINE OOCYTES ON A GRANULOSA CELL MONOLAYER FROM ADULT ANIMALS IMPROVES THEIR DEVELOPMENTAL COMPETENCE

2006 ◽  
Vol 18 (2) ◽  
pp. 277
Author(s):  
S. Ponebsek ◽  
C. Wrenzycki ◽  
K.-G. Hadeler ◽  
D. Herrmann ◽  
K. Korsawe ◽  
...  
Keyword(s):  
Granulosa Cell ◽  
Relative Abundance ◽  
Glucose Transporter ◽  
Cell Monolayer ◽  
Mrna Abundance ◽  
Developmental Competence ◽  
Hsp 70 ◽  
Developmental Capacity ◽  
Calf Oocytes

Oocytes from prepubertal calves have a decreased developmental competence compared with oocytes from adult animals. The goal of this study was to improve the developmental competence of juvenile oocytes by maturation on granulosa cell (GC) monolayers from adult animals. Oocytes were recovered by ovum pickup (OPU) from 48 Holstein Friesian calves at 7-8 months of age and 18 adult cows. Animals received intramuscular injections of 60 mg FSH 48 h prior to each OPU session. Follicles were punctured twice per week in six consecutive OPU sessions. Cumulus oocyte complexes (COCs) recovered from calves were divided into three quality groups (classes I-III) and were then randomly distributed into three maturation groups: COCs were matured for 24 h on either GC or fibroblasts or without co-culture. Cow oocytes were matured without co-culture. TCM-199 supplemented with BSA (0.1%), hCG (5 IU/mL), and eCG (10 IU/mL) served as the medium in all groups. After maturation, all COCs were fertilized in vitro; after 18 h, presumptive zygotes were cultured in SOF+BSA for 8 days (37�C, 5% CO2). On Day 3, cleavage rates and, on Day 8, blastocyst rates were determined. The relative mRNA abundance of the following transcripts, critically involved in early embryonic development was determined: growth differentiation factor-9 (GDF-9), heat shock protein 70 (Hsp-70), and glucose transporter-3 (Glut-3). Single immature and matured oocytes (for GDF-9 and Hsp-70) and 8-16-cell embryos and expanded blastocysts (for Hsp-70 and Glut-3) from calves and cows were examined by semiquantitative RT-PCR. Cleavage and blastocyst rates were similar in oocytes derived from cows and calves matured on GC (74.3% vs. 70.0% and 22.3% vs. 22.3%, respectively), but were significantly higher (P < 0.05; one way ANOVA, Student-Newman-Keuls Method) than in the group without co-culture on fibroblasts (55.2% vs. 53.6% and 11.7% vs. 5.5%, respectively). GDF-9 expression was similar in immature calf and cow oocytes. After maturation, a significant decrease in GDF-9 expression was observed in calf oocytes. Matured cow oocytes showed a significantly higher mRNA abundance of GDF-9 than matured calf oocytes. The relative abundance of Hsp-70 was decreased in matured oocytes of all groups. Expanded blastocysts derived from adult oocytes expressed Hsp-70 significantly higher than blastocysts derived from oocytes of the control calves. The relative abundance of Glut-3 mRNA was similar in 8-16 cell embryos and expanded blastocysts in all groups. Overall, mRNA expression pattern for Hsp-70 and Glut-3 in blastocysts from GC matured oocytes were similar to that of cow blastocysts. Results indicate that maturation of juvenile calf oocytes on granulosa cells from adult animals improves their developmental competence. These findings provide clues toward identification of factors critically involved in acquiring full developmental capacity at puberty.

346 BOVINE EMBRYO DEVELOPMENT AFTER IVM/IVF/IVC OF OOCYTES STORED FOR 22 H IN VARIOUS MEDIA

2007 ◽  
Vol 19 (1) ◽  
pp. 288
Author(s):  
C. Kubota ◽  
T. Kojima ◽  
T. Nagai ◽  
X. Tian ◽  
X. Yang
Keyword(s):  
Subsequent Development ◽  
Industrial Applications ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Bovine Embryo ◽  
Becton Dickinson ◽  
In Vitro Storage ◽  
Bovine Oocytes

The timing of IVM–IVF–IVC is restricted by the onset of oocyte maturation, and sometimes oocytes must be treated at midnight. If we could regulate the timing of IVM of oocytes without decreasing their developmental competence, the IVM–IVF–IVC system could be a more applied technology. The present study was performed to examine the effects of in vitro storage of bovine oocytes in simple media prior to maturation culture to manipulate the start of IVM. Bovine follicular fluid (bFF), Dulbecco&apos;s PBS (PBS), M199 Earle salts (M199), and Earle salts supplemented with 5 mM NaHCO3 (M199A) were used as the fundamental media, after an addition of antibiotics, for in vitro storage of bovine cumulus&ndash;oocyte complexes (COCs) collected from ovaries obtained at the slaughterhouse. The fundamental media except for bFF were supplemented with 10&percnt; fetal bovine serum (FBS) or 1 mg mL&minus;1 polyvinyl alcohol (PVA). COCs were collected from follicles (3&ndash;8 mm in diameter) and washed twice in each medium; then approximately 50 COCs were submerged in 1 mL of each medium in cryotubes (Falcon #2812, 2.5 mL; Becton Dickinson Labware, Lincoln, NJ, USA), which were stored in a container kept at 38.5&deg;C for 22 h under air-closed condition (in vitro storage: IVS). Subsequently, the stored COCs were in vitro-matured (IVM) for 22 h in M199 with 10&percnt; FBS and 20 &micro;g mL&minus;1 estradiol, fertilized (IVF), and cultured in CR1aa (IVC) for examination of their development to the blastocyst stage (Kubota et al. 1998 Mol. Reprod. Dev. 51, 281&ndash;286). Fresh oocytes without IVS were used as controls. The nuclear status of oocytes after IVS&ndash;IVM was compared to that of control oocytes by aceto-orcein stain. Their developmental rates to the blastocyst stage after IVM&ndash;IVF&ndash;IVC were compared between experimental and control groups. The experiment was repeated more than 3 times, and results were statistically analyzed using Student&apos;s t-test. When bFF and PBS supplemented with FBS or PVA were used for IVS, the rates of survived COCs after IVS and the development to the blastocyst stage after IVM&ndash;IVF&ndash;IVC (bFF (n &equals; 87): 0&percnt;, 0&percnt;; PBS/FBS (n &equals; 72): 84&percnt;, 1&percnt;; and PBS/PVA (n &equals; 81): 89&percnt;, 6&percnt;, respectively) were significantly lower than those of the control group (n &equals; 406; 97&percnt; and 29&percnt;, respectively). On the other hand, when M199A supplemented with FBS or PVA was used for IVS, the survival rate after IVS and the developmental rate to the blastocyst stage after IVS&ndash;IVM&ndash;IVF (M199A/FBS (n &equals; 97): 82&percnt;, 28&percnt;; and M199A/PVA (n &equals; 111): 98&percnt;, 31&percnt;, respectively) did not differ from those of the control group. After IVS, cumulus expansion was not seen and most of the oocyte nuclei reached the GVBD stage. These results suggest that the nuclear maturation progress of bovine oocytes can be regulated for at least 22 h in M199A without any deleterious influence on the number of oocytes surviving at an immature state after the storage and their subsequent development to the blastocyst stage after IVM&ndash;IVF&ndash;IVC. The delayed maturation allows a flexible fertilization schedule which is advantageous in research and industrial applications.

scholarly journals 121 HEAT SHOCK TO PIG OOCYTES DOES NOT INDUCE APOPTOSIS BUT REDUCES EMBRYO DEVELOPMENT

2005 ◽  
Vol 17 (2) ◽  
pp. 211
Author(s):  
J.-K. Tseng ◽  
P.-C. Tang ◽  
J.-C. Ju
Keyword(s):  
Heat Shock ◽  
Apoptotic Cell ◽  
Electric Pulse ◽  
Annexin V ◽  
Developmental Competence ◽  
Control Group ◽  
Hsp 70 ◽  
Cell Numbers ◽  
Significant Difference

Oocytes are susceptible to heat shock (HS) during the maturation process. It has been demonstrated that HS induces apoptosis and/or the expression of HS protein 70 (hsp 70) in in vitro-produced oocytes and embryos. The objectives of this study were to analyze the effects of HS on the development and apoptosis of pig oocytes and embryos. Porcine ovaries were collected from a local slaughterhouse and the cumulus-oocyte complexes (COCs) were aspirated from follicles 3–6 mm in diameter and subjected to standard in vitro maturation procedures at 39°C for 42 h. The in vitro matured oocytes were then randomly allocated to different HS treatments at 41.5°C for 0 (control, C0h), 1 (HS1h), 2 (HS2h), or 4 h (HS4h). An additional control group of oocytes was cultured for 4 h without HS (C4h). Data were analyzed by chi-square test. In Experiment 1, anti-hsp 70 (SPA-810AP, Stressgen, San Diego, CA, USA) and Western blotting were used to examine the expression of hsp 70. Results indicated that no significant difference of hsp 70 expression in metaphase II porcine oocytes occurred between controls and HS groups (P > 0.05, 7 replicates). In Experiment 2, apoptosis of metaphase II oocytes after HS was identified by annexin V-FITC (Sigma, St. Louis, MO, USA) staining and TUNEL (Roche, Indianapolis, IN, USA). No significant apoptotic signal was detected in the HS groups compared to the controls. The intensity of annexin V staining was not affected by HS, but it increased with the time of culture (P < 0.05, n = 24–37). In Experiment 3, the apoptotic rate and developmental competence of the HS-oocytes were evaluated by TUNEL assay (n = 123–137, 4 replicates). Parthenogenetic activation (n = 123–137) was performed by an electric pulse (2.2 kV cm−1) combined with 6-dimethyaminopurine treatment (6-DMAP, 2.5 μM, 4 h, Sigma). The cleavage rates in HS2h (43 ± 29%) and HS4h (35 ± 28%) decreased (P < 0.05) compared to those in C0h (62 ± 12%) and C4h (66 ± 8%). In addition, the blastocyst formation rates and total cell numbers reduced (P < 0.05) after 2 h (11 ± 10%, 20 ± 16) and 4 h (11 ± 8%, 19 ± 8) of HS treatments compared to those in C0h (23 ± 14%, 32 ± 22) and C4h (21 ± 11%, 27 ± 17), all respectively. The numbers of blastocysts with TUNEL-positive signals were not significantly different between the HS and control groups, but the signals increased (P < 0.05) before the 8-cell stage in HS groups (22–24%) compared to the C0h and C4h controls (16 and 11%), respectively. These results indicate that reduction in developmental competence of in vitro-matured pig oocytes after heat shock is not closely correlated to the expression of hsp 70 in the oocytes and to the apoptotic cell numbers in the blastocyst. Whether detection of apoptosis by TUNEL or annexin V-FITC in oocytes is a good indicator requires further investigation.

114 PROGESTERONE CONCENTRATIONS DURING IVM AFFECT BOVINE OOCYTE QUALITY AT THE MOLECULAR LEVEL

2014 ◽  
Vol 26 (1) ◽  
pp. 171 ◽  
Author(s):  
N. Schlüter ◽  
A. Kassens ◽  
H. Stinshoff ◽  
K. Knauer ◽  
S. Wilkening ◽  
...  
Keyword(s):  
Progesterone Receptor ◽  
Glucose Transporter ◽  
Vehicle Control ◽  
Developmental Competence ◽  
Control Group ◽  
P Value ◽  
Glucose Transporter 1 ◽  
Developmental Rates ◽  
Further Development

Progesterone (P4) is important for the developmental competence of cumulus–oocyte complexes (COC) used for in vitro maturation (IVM). In a recent study, we were able to show that circulating P4 concentrations had an effect on the molecular quality of COC recovered during repeated ovum pickup sessions, which might affect further development. (Schlüter et al. 2013 Reprod. Fertil. Dev. 25, 250). The aim of the present study was to determine the influence of different P4 concentrations during IVM on the molecular quality of bovine COC. The COC were collected from slaughterhouse ovaries and cultured as described recently (Stinshoff et al. 2011 Theriogenology 76, 1433–1441). The IVM medium was supplemented with 0, 50, 150, 300, and 450 ng mL–1 P4. Ethanol served as vehicle control. After IVF with a bull of proven fertility, the presumptive zygotes were cultured in SOF under 5% oxygen (Stinshoff et al. 2011). Cleavage and developmental rates were determined at Day 3 and Day 7/8 (Day 0: IVF). Additionally, maturation rates were assessed. For mRNA analysis, immature and matured denuded COC (n = 5) were individually frozen at –80°C to analyse the relative transcript abundance using RT-qPCR. The transcripts studied play important roles during oocyte development [growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), glucose transporter 1 (SCL2A1), hypoxia inducible factor 2α (HIF2α), nuclear progesterone receptor (PGR), progestin and adipoQ receptor 5 (PAQR5), progesterone receptor membrane component 1 and 2 (PGRMC1, PGRMC2)]. Data were tested using ANOVA followed by multiple pairwise comparisons using Tukey's test. A P-value of <0.05 was considered significant. The percentage of oocytes that reached the MII stage was similar in oocytes from all treatment groups (82.2–88.1%). Despite similar cleavage rates across all groups, the developmental rates did show significant differences. More embryos developed to the blastocyst stage stemming from oocytes cultured without any supplement or cultured only with alcohol compared to oocytes stemming from the group cultured with less than 50 ng mL–1 P4 (25.4 ± 5.7 and 27.9 ± 7.2 v. 15.8 ± 2.6). The relative abundance of SCL2A1, BMP15, and PGRMC1transcripts in single oocytes did not show differences related to the supplementation of the IVM medium, whereas GDF9, HIF2α, and PAQR5 mRNA was reduced in oocytes of all groups compared with immature ones. The PGR and PGRMC2 transcripts were increased in matured oocytes of the control group and the vehicle control group (PGRMC2). In summary, supplementation of the IVM medium with different P4 concentrations had an effect on the molecular quality of oocytes after IVM, which might affect further development. The financial support of the FBF (Förderverein Biotechnologieforschung) e.V. is gratefully acknowledged.

scholarly journals Rosmarinic acid treatment during porcine oocyte maturation attenuates oxidative stress and improves subsequent embryo development in vitro

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6930
Author(s):  
Yan Zhang ◽  
Jing Guo ◽  
Xiao Wei Nie ◽  
Zi Yue Li ◽  
Yu Meng Wang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Oocyte Maturation ◽  
Rosmarinic Acid ◽  
Reactive Oxygen Species Generation ◽  
Subsequent Development ◽  
Developmental Competence ◽  
Control Group ◽  
Porcine Oocyte ◽  
Number Of Cells

Background In vitro maturation (IVM) of oocytes has been widely used in the field of assisted reproductive technology. However, oocytes can be injured by oxidative stress during the process of IVM. Methods The present study was designed to evaluate the influences of rosmarinic acid (RA) on the IVM of porcine oocytes and the subsequent development of early-stage embryos as well as its underlying mechanisms. Various concentrations of RA (5 µM, 10 µM, and 25 µM) were treated with porcine oocyte maturation medium during the period of IVM. Results and Discussion The results showed that 5 µM RA treatment during the period of porcine oocyte IVM improves blastocyst quality and hatching ability after parthenogenetic activation. Furthermore, the presence of RA during the period of IVM dramatically improved the total number of cells after somatic cell nuclear transfer compared to the number of cells in the control group. Notably, RA treatment during the period of porcine oocyte IVM decreased intracellular reactive oxygen species generation not only in oocytes but also in cumulus cells. Further analysis showed that the intracellular free thiols levels in the oocytes were enhanced by treatment with RA during the period of porcine oocyte IVM compared to the free thiols levels in the control groups. These results indicate that RA improves the developmental competence of porcine oocytes during the IVM period by attenuating oxidative stress.

Supplementation of c-type natriuretic peptide during in vitro growth period benefits the development of murine preantral follicles

Zygote ◽  
2020 ◽  
pp. 1-5
Author(s):  
Li Ang ◽  
Cao Haixia ◽  
Li Hongxia ◽  
Li Ruijiao ◽  
Guo Xingping ◽  
...  
Keyword(s):  
Natriuretic Peptide ◽  
Granulosa Cells ◽  
Culture System ◽  
Early Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Follicle Development ◽  
Control Groups ◽  
Preantral Follicles

Summary The present study investigated the effects of c-type natriuretic peptide (CNP) on the development of murine preantral follicles during in vitro growth (IVG). Preantral follicles isolated from ovaries of Kunming mice were cultured in vitro. In the culture system, CNP was supplemented in the experimental groups and omitted in the control groups. In Experiment 1, CNP was only supplemented at the early stage and follicle development was evaluated. In Experiments 2 and 3, CNP was supplemented during the whole period of in vitro culture. In Experiment 2, follicle development and oocyte maturity were evaluated. In Experiment 3, follicle development and embryo cleavage after in vitro fertilization (IVF) were assessed. The results showed that in the control groups in all three experiments, granulosa cells migrated from within the follicle and the follicles could not reach the antral stage. In the experimental groups in all three experiments, no migration of granulosa cells was observed and follicle development was assessed as attaining the antral stage, which was significantly superior to that of the control group (P < 0.0001). Oocyte meiotic arrest was effectively maintained, hence giving good developmental competence. In conclusion, CNP supplementation in the culture system during IVG benefited the development of murine preantral follicles.

Cotreatment with RepSox and LBH589 improves the in vitro developmental competence of porcine somatic cell nuclear transfer embryos

2018 ◽  
Vol 30 (10) ◽  
pp. 1342 ◽  
Author(s):  
Zhao-Bo Luo ◽  
Long Jin ◽  
Qing Guo ◽  
Jun-Xia Wang ◽  
Xiao-Xu Xing ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Epigenetic Reprogramming ◽  
Developmental Competence ◽  
Abnormal Development ◽  
Control Group ◽  
Blastocyst Formation

Accumulating evidence suggests that aberrant epigenetic reprogramming and low pluripotency of donor nuclei lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). The present study demonstrates that treatment with the small molecule RepSox alone upregulates the expression of pluripotency-related genes in porcine SCNT embryos. Treatment with the histone deacetylase inhibitor LBH589 significantly increased the blastocyst formation rate, whereas treatment with RepSox did not. Cotreatment with 12.5 μM RepSox and 50 nM LBH589 (RepSox + LBH589) for 24 h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9% vs 8.5% respectively; P < 0.05). Furthermore, the expression of pluripotency-related genes octamer-binding transcription factor 4 (NANOG) and SRY (sex determining region Y)-box 2 (SOX2) were found to significantly increased in the RepSox + LBH589 compared with control group at both the 4-cell and blastocyst stages. In particular, the expression of NANOG was 135-fold higher at the blastocyst stage in the RepSox + LBH589 group. Moreover, RepSox + LBH589 improved epigenetic reprogramming. In summary, RepSox + LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming and improves the in vitro development of porcine SCNT embryos.

Influence of glucose transporter 1 activity inhibition on neuroblastoma in vitro

Gene ◽  
2019 ◽  
Vol 689 ◽  
pp. 11-17 ◽  
Author(s):  
Yan Peng ◽  
Si-ning Xing ◽  
Hu-ying Tang ◽  
Chang-dong Wang ◽  
Fa-ping Yi ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Glucose Transporter 1 ◽  
Activity Inhibition

Modification of ovary stock solution with magnesium and raffinose improves the developmental competence of oocytes after long preservation

Zygote ◽  
2005 ◽  
Vol 13 (4) ◽  
pp. 303-308 ◽  
Author(s):  
H. Iwata ◽  
T. Hayashi ◽  
H. Sato ◽  
K. Kimura ◽  
T. Kuwayama ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Germinal Vesicle ◽  
Storage Period ◽  
Cell Number ◽  
Developmental Competence ◽  
Germinal Vesicle Breakdown ◽  
Storage Solutions ◽  
Stock Solutions ◽  
Phosphate Buffered Saline

During ovary storage oocytes lose some of their developmental competence. In the present study, we maintained storage solutions of phosphate-buffered saline (PBS) at various temperatures (20 or 35 °C) or supplemented them with magnesium (Mg), raffinose and sucrose. Subsequently, we examined the kinetics of electrolytes in the follicular fluid (FF) during the ovary storage period (9h), the survival rate of granulosa cells in the follicles, and the developmental competence of oocytes after the storage. Lowering the temperature from 35 to 20 °C increased the total cell number of blastocysts that developed at 7 days after in vitro maturation and in vitro fertilization of oocytes. In stock solution with supplements of 15 mM Mg or a combination of 5 mM Mg and 10 mM raffinose or sucrose, a significantly higher number of oocytes developed into blastocysts with a large number of cells in each blastocyst, and a significantly higher number of living granulosa cells were obtained as compared with stock solutions without any supplements. During ovary storage, the concentrations of potassium and chloride in the FF were increased, and the addition of Mg to the stock solution increased the concentration of Mg in the FF. Germinal vesicle breakdown in oocytes that were collected from ovaries stored in the solution supplemented with 15 mM Mg or a combination of 5 mM Mg and 10 mM of raffinose occurred at a slower rate than that in oocytes collected from ovaries stored in PBS alone. On the other hand, the oocytes collected from ovaries stored in the solution supplemented with 15 mM Mg or a combination of 5 mM Mg and 10 mM raffinose reached the metaphase II (MII) stage more rapidly than the oocytes collected from ovaries stored in the PBS alone. In conclusion, the modification of stock solution by the addition of Mg and raffinose improved the developmental competence of oocytes obtained from ovaries preserved for a long period.

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