Osmolarity at early culture stage affects development and expression of apoptosis related genes (Bax-alpha and Bcl-xl) in pre-implantation porcine NT embryos.
This study examined whether high osmolarity of culture medium at the early culture stage affects development and expression of apoptosis related genes (Bax-alpha and Bcl-xl) of porcine nuclear transfer (NT) and in vitro fertilization (IVF) embryos. NT and IVF embryos were divided into three groups and the basic medium was PZM-3 (260-270 mOsmol, control group). The control group of embryos was cultured in PZM-3 for whole culture period. Other two groups of embryos were cultured in a modified PZM-3 with 0.05 M sucrose (300-320 mOsmol, sucrose group) or increased NaCl to 138 mM (300-320 mOsmol, NaCl group) for the first 2 days, and then cultured in PZM-3 for 4 days. NT embryos cultured in NaCl group showed a significantly higher developmental rate to the blastocyst stage with a decreased apoptosis rate compared to the control (P < 0.05). There was no difference in blastocyst formation and apoptosis incidence among the three culture treatments for IVF-derived embryos. Bax-alpha mRNA expression was significantly higher in the control than sucrose or NaCl group for both NT and IVF embryos (P < 0.05). Moreover, the relative abundance of Bax-alpha/Bcl-xl was higher in the control than the treatment groups. These results indicate that the higher osmolarity at the early embryonic stage of porcine NT and IVF embryos can improve the in vitro development with reduced apoptosis through regulating the Bax-alpha/Bcl-xl gene expression.
Hwang IS
，Park MR
，Moon HJ
，Shim JH
，Kim DH
，Yang BC
，Ko YG
，Yang BS
，Cheong HT
，Im GS
... -
《-》
Effects of vascular endothelial growth factor on porcine preimplantation embryos produced by in vitro fertilization and somatic cell nuclear transfer.
This study examined the effects of vascular endothelial growth factor (VEGF) on porcine embryos produced by in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) at different developmental stages. Four sets of experiments were performed. In the first, supplementation of the in vitro culture medium with 5 ng/mL VEGF was suitable for porcine IVF embryo development, and the blastocyst formation rate was significantly higher than the control and other groups (57.73 ± 6.78% (5 ng/mL VEGF) vs. 43.21 ± 10.22% (control), 42.16 ± 10.24% (50 ng/mL VEGF) and 41.91 ± 11.74% (500 ng/mL VEGF); P < 0.05). The total cell number after supplementation with 5 ng/mL VEGF was significantly higher than the control and other groups (151.85 ± 39.77 (5 ng/mL VEGF) vs. 100.00 ± 34.43 (control), 91.2 ± 31.51 (50 ng/mL VEGF), and 112.53 ± 47.66 (500 ng/mL VEGF); P < 0.05). In the second experiment, when VEGF was added at different developmental stages of IVF derived embryos (early stage, days 1-3, late stage, days 4-7), the blastocyst formation rate and total cell number were significantly higher at the late stage (47.71 ± 9.13% and 131.5 ± 20.70, respectively) than in the control (34.32 ± 7.44% and 85.50 ± 20.41, respectively) and at the early stage (33.60 ± 5.78% and 86.75 ± 25.10, respectively; P < 0.05). There was no significant difference in the blastocyst development rate or total cell number between the whole culture period (days 1-7) and the late stage culture period after supplementation with 5 ng/mL VEGF (P > 0.05). In the third experiment, the cleavage rate was significantly higher when SCNT embryos were cultured with VEGF during the whole culture period than in the late stage (63.56 ± 15.52% vs. 39.72 ± 4.94%; P < 0.05), but there was no significant difference between the control and the early stage culture period (P > 0.05). The blastocyst formation rate was significantly higher at the late stage culture period with VEGF than at the early stage culture period (34.40 ± 15.06% vs. (16.07 ± 5.01%; P < 0.05). There was no significant difference in the total cell number between the groups (P > 0.05). In experiment 4, using real-time PCR, VEGF mRNA expression was detected in all the developmental stages of IVF and SCNT embryos, but the expression level varied according to the developmental stage. VEGF receptor, KDR mRNA was detected in all stages IVF and SCNT embryos. However, flt-1 mRNA was not expressed in all embryonic stages of IVF and SCNT embryos. These data suggest that VEGF supplementation at the late embryonic developmental stage might improve the developmental potential of both IVF and SCNT preimplantation porcine embryos through its receptors.
Biswas D
，Jung EM
，Jeung EB
，Hyun SH
... -
《-》
In vitro development of preimplantation porcine nuclear transfer embryos cultured in different media and gas atmospheres.
This study investigated the effect of culture media and gas atmospheres on the development of porcine nuclear transfer embryos. Oocytes derived from a local abattoir were matured for 42-44 h and enucleated. Fetal fibroblasts were prepared from a Day 35 porcine fetus. Confluent stage fetal fibroblasts were introduced into the perivitelline space of enucleated oocytes. Fusion and activation were induced simultaneously with two direct current (1.2 kV/cm for 30 micros) in 0.3 M mannitol medium. For parthenogenetic activation, the same pulses were used. In Experiment 1, parthenogenetically activated oocytes were cultured in North Carolina State University-23 (NCSU-23), Porcine Zygote Medium-3 (PZM-3), or Beltsville Embryo Culture Medium-3 (BECM-3). Parthenogenetically activated oocytes cultured in PZM-3 had a higher (P < 0.05) developmental rate to the blastocyst stage (15.2% versus 3.7-9.6%) as compared to BECM-3 or NCSU-23. The number of nuclei in Day 6 blastocysts was higher (P < 0.05) in PZM-3 (23.6) and NCSU-23 (21.4) than BECM-3 (14.2). In Experiment 2, parthenogenetically activated oocytes were cultured in NCSU-23 under a gas atmosphere of 5% CO(2) in air for 6 days (T1), 5% CO(2), 5% O(2), 90% N(2) for 6 days (T2), 5% CO(2) in air for 3 days, then 5% CO(2), 5% O(2), 90% N(2) for 3 days (T3), or 5% CO(2), 5% O(2), 90% N(2) for 3 days, then 5% CO(2) in air for 3 days (T4). Blastocyst formation rates were not different among treatments (12.9 =/-3.6 %, 13.5 +/- 4.2%, 10.8+/-2.4%, and 12.6+/-2.7%, respectively). However, T2 (36.7+/-2.9) and T3 (33.8+/-3.0) resulted in more nuclei per blastocyst than T1 (23.2+/-2.1) or T4 (26.0+/-2.1 ). In Experiment 3, reconstructed porcine nuclear transfer (NT) embryos were cultured in NCSU-23 or PZM-3 under a gas atmosphere of 5% CO(2) in air or 5% CO(2), 5% O(2), 90% N(2). Developmental rates to blastocyst stage for porcine NT embryos cultured in NCSU-23 under a gas atmosphere of 5% CO(2) in air or 5% CO(2), 5% O(2), 90% N(2) were 7.2+/-1.4% and 12.3+/-1.4%, and the number of nuclei was 12.2=/-0.8% and 19.4+/-1.0, respectively. NT embryos cultured in PZM-3 under a gas atmosphere of 5% CO(2) in air or 5% CO(2), 5% O(2), 90% N(2) had developmental rates to blastocyst stage of 18.8+/-1.9 %, and 17.8+/-3.8% the nuclei number was 20.9 +/- 1.9 and 21.9+/-3.3, respectively. NT embryos cultured in NCSU-23 had a higher developmental rate to the blastocyst stage in 5% CO(2), 5% O(2), 90% N(2) than in 5% CO(2) in air (P < 0.05). Regardless of gas atmospheres, NT embryos cultured in PZM-3 had a higher developmental rate (18.3 =/- 1.7% versus 16.9 +/- 1.2%) and nuclei number (21.4 +/-1.8 versus 16.9 +/- 1.2) than in NCSU-23 (P < 0.05). In conclusion, a gas atmosphere of 5% CO(2), 5% O(2), 90% N(2) supported a higher development rate of porcine NT embryos than 5% CO(2) in air when the porcine NT embryos were cultured in NCSU-23. Furthermore, regardless of atmosphere, PZM-3 supported a higher development rate of porcine nuclear transfer embryos than NCSU-23.
Im GS
，Lai L
，Liu Z
，Hao Y
，Wax D
，Bonk A
，Prather RS
... -
《THERIOGENOLOGY》
Anti-apoptotic effect of insulin-like growth factor (IGF)-I and its receptor in porcine preimplantation embryos derived from in vitro fertilization and somatic cell nuclear transfer.
Insulin-like growth factor (IGF)-I is a receptor-mediated autocrine and/or paracrine growth and/or survival factor for mammalian embryo development. It is known to promote the growth and development of mouse preimplantation embryos. The present study was designed to investigate the effects of IGF-I (50 ng/ml), anti-IGF-I receptor antibody (50 ng/ml) and their combination on porcine preimplantation embryo development. Furthermore, the mechanism underlying the embryotropic effects of IGF-I was evaluated by monitoring the incidence of apoptosis and expression of apoptosis-related genes. In both in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos, culturing with IGF-I increased the rate of blastocyst formation and this embryotrophic effect was neutralized by culturing with IGF-I along with anti-IGF-I receptor (IGF-IR) antibody. Culturing IVF and SCNT embryos with IGF-I significantly increased the number of total cells in blastocysts and decreased the number of apoptotic nuclei. These effects of IGF-I were also neutralized by culturing with IGF-I along with anti-IGF-IR antibody. Expression of the anti-apoptotic Bcl-2 gene was increased, while expression of the pro-apoptotic Bax was decreased in both IVF and SCNT embryos cultured with IGF-I. In both IVF and SCNT embryos, anti-IGF-IR antibody along with IGF-I neutralized the effect of IGF-I on expression of Bcl-2 and Bax genes. In conclusion, the present study demonstrated that IGF-I through its specific receptors improved the developmental competence of IVF and SCNT embryos by decreasing the incidence of apoptosis and regulating apoptosis-related genes in porcine preimplantation embryos.
Kim S
，Lee SH
，Kim JH
，Jeong YW
，Hashem MA
，Koo OJ
，Park SM
，Lee EG
，Hossein MS
，Kang SK
，Lee BC
，Hwang WS
... -
《MOLECULAR REPRODUCTION AND DEVELOPMENT》
ResearchGate
(全网免费下载)
钛学术
(全网免费下载)
