• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.319-327
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• 2011

This study investigated whether the addition of porcine sperm cytosolic factor (SCF) at fusion/activation affects in vitro development of porcine parthenogenetic(PA) and nuclear transfer (NT) embryos. To determine the optimum concentration of SCF, control group of oocytes was activated with 0.3M mannitol (1.0 mM $CaCl_2{\cdot}2H_2O$), other three groups of oocytes were parthenogentically activated with the fusion medium (0.1mM $CaCl_2{\cdot}2H_2O$) supplemented with 100, 200 or 300 ${\mu}$g/ml SCF, respectively. Matured oocytes were activated with two electric pulses (DC) of 1.2 kv/cm for 30 ${\mu}$sec. The activated embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. Oocytes activated in the presence of SCF showed a significantly higher blastocyst rate than control (p<0.05). Apoptosis rate was significantly lower in 100 ${\mu}$g/ml SCF group than other groups (p<0.05). Cdc2 kinase activity in control and SCF treatment group of oocytes was determined using MESACUP cdc2 kinase assay kit at 1, 5, 10, 15, 30, 45 and 60 min after activation. Cdc2 kinase activity was significantly decreased (p<0.05) in SCF group than MII oocytes or control within 5 min. For NT embryo production, reconstructed oocytes were fused in the fusion medium supplemented with 0.1 mM $CaCl_2{\cdot}2H_2O$ (T1), 1.0 mM $CaCl_2{\cdot}2H_2O$ (T2) and 0.1 mM $CaCl_2{\cdot}2H_2O$ with 100 ${\mu}$g/ml SCF (T3). Fused embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. Developmental rate to blastocyst stage was significantly higher in T3 than other groups (23.0% vs. 13.5 to 15.2%) (p<0.05). Apoptosis rate was significantly lower in T3 than T1 or T2 (p<0.05). The relative abundance of Bax-${\alpha}$/Bcl-xl was significantly lower in in vivo or SCF group than that of control (p<0.05). Moreover, the expression of p53 and caspase3 mRNA was significantly lower in in vivo or SCF group than that of control (p<0.05). These results indicate that the addition of SCF at fusion/activation might improve in vitro development of porcine NT embryos through regulating cdc2 kinase level and expression of apoptosis related genes.

• Reproductive and Developmental Biology
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• v.33 no.2
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• pp.85-91
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• 2009

In this study, in vitro maturation system using fetal bovine serum (FBS) or porcine follicular fluid (pFF) was investigated to produce comparable oocytes to those derived from in vivo. Control group of oocytes was cultured in TCM 199 supplemented with 0.1% polyvinyl alcohol (PVA). Other three groups of oocytes were cultured in TCM 199 supplemented with 10% FBS, 10% pFF or 5% FBS + 5% pFF, respectively. After 44 h maturation, oocytes with the first polar body were activated with two electric pulses (DC) of 1.2 kv/cm for 30 ${\mu}sec$. Also, matured oocytes of four groups were reconstructed and fused. Reconstructed embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. The oocytes matured in the medium supplemented with FBS or/and pFF showed significantly higher maturation rates (64.0 vs. 73.9 to 85.2%). In PA embryos, cleavage rates (89.7 vs. 77.1 to 86.6%) and blastocysts rates (30.0 vs. 16.2 to 26.2%) were significantly higher in pFF group (p<0.05). In NT embryos, there was no difference among treatments in cleavage rate, but the blastocyst rates (28.5 vs. 15.5 to 24.6%) were significantly higher in pFF group (p<0.05). The apoptosis rate was significantly higher (p<0.05) in the control than other groups (10.8 vs. 4.9 to 8.2% for PA, 3.1 vs. 0.5 to 1.3% for NT). In order to select the comparable oocyte to in vivo oocytes, each group of oocytes was stained with Brilliant cresyl blue (BCB) after 42h maturation. The matured oocytes were separated according to color of cytoplasm; stained group (BCB+) and unstained group (BCB-). The oocytes matured in the presence of FBS or/and pFF showed significantly higher staining rates (70.3 to 72.7 vs. 35.1%) (p<0.05). To verify the fact that the supplementation of FBS or/and pFF can increase the maturation rates, cdc2 kinase activity, the catalytic subunit of MPF, was determined. The cdc2 kinase activity of the oocytes matured in the medium supplemented with FBS or/and pFF was significantly higher than control group (6.7 to 9.3 vs. 3.8). In conclusion, the supplementation of FBS or/and pFF can support in vitro maturation rate of porcine oocytes through the increment of cdc2 kinase activity level in the cytoplasm.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.12
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• pp.1695-1701
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• 2002

The purposes of this study were to optimize the in vitro maturation (IVM) and culture (IVC) systems of rabbit oocytes. Cytoskeletal structures in the germinal vesicle stage (GV) and during IVM are also investigated. Ovaries were transported from local slaughterhouses and the cumulus-oocyte complexes (COCs) were collected from ovarian follicles (${\geq}1mm$). COCs were randomly allocated to TCM199-based medium ($T_1$, TCM-199) supplemented with $NaHCO_3$, glucose, sodium pyruvate and FSH ($T_2$), $T_2+E_2+LH$ ($T_3$), $T_3+FBS$ ($T_4$), or $T_1+E_2+LH+FSH+FBS$ ($T_5$), for IVM. In Experiment 1, COCs were retrieved from the follicles and 51 GV oocytes were fixed in the fixative (MTSB-XF) for nuclear and cytoplasmic examinations. In Experiment 2, progressive changes of both the nucleus and the cytoskeleton were examined at 0, 6, 16, and 20 h after IVM. Maturation (MR) and developmental rates were assessed in Experiment 3. Cytoplasmic microtubules (MT) were clearly observed in rabbit GV oocytes. To our knowledge, this is the first report that describes the appearance of MT structures in the GV stage ooplasm. Tremendous variations in cytoskeletal alterations were observed among treatments with the exception of the vitelline ring (VR), which is constantly visible and unchanged during maturation. Germinal vesicle breakdown (GVBD) does not occur at 6 h after onset of maturation culture. When the oocytes for IVM were collected within 2 h, results from Experiment 3 showed that rates of nuclear maturation were 42, 8, 42, 37 and 65% at 16 h of IVM for $T_1$ through $T_5$, respectively, in which $T_1$, $T_4$ and $T_5$ had significantly greater MR than those in other groups (p<0.05). Morula/blastocyst development after parthenogenetic activation ranged from 20 to 63% with significantly greater rates in $T_3$, $T_4$ and $T_5$ (p<0.05). These results suggested that oocytes recovered from slaughterhouse ovaries can be matured and parthenogenetically activated in vitro, but the MR remained low in this study. Addition of $E_2$ and LH in the medium may be beneficial for cytoplasmic maturation, but FBS exerts a nega- tive role in the subsequent development of parthenogenetic embryos when energy substrates are provided in the IVC media. More studies are required for improving the MR and further development of the GV stage rabbit oocytes.

• Journal of Embryo Transfer
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• v.31 no.1
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• pp.33-38
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• 2016

In this study, to improve the in vitro development of various cells including cloned embryos, the effects that isoproterenol and melatonin have on in vitro development of porcine parthenogenetic oocytes were investigated. Parthenogenetic activation was induced with electrical stimulation, BSA and 6-DMAP treatment. $10^{-7}M$ of melatonin and isoproterenol ($10^{-10}$, $10^{-12}$ and $10^{-14}M$) were supplemented for in vitro maturation (IVM) and in vitro culture (IVC) medium, with different concentrations. When isoproterenol and melatonin were supplemented in IVM medium with different concentrations, there was no significant (P<0.05) difference of maturation rate in the treatment groups as well as in that of only melatonin. As isoproterenol and melatonin were supplemented in IVM medium with different concentrations, blastocyst rates of isoproterenol $10^{-12}M$ treatment group (37.1%) were significantly (P<0.05) higher than control group (26.0%). Isoproterenol and melatonin were supplemented in IVC medium with different concentrations, then the cleavage rate of $10^{-12}M$ isoproterenol treatment group (82.2%) was significantly (P<0.05) higher than the group that melatonin was only supplemented (70.9%). There was no difference of blastocyst rate between the treatment groups. When isoproterenol and melatonin were supplemented for IVM+IVC medium with different concentrations, the cleavage rate of $10^{-12}M$ isoproterenol treatment group (92.5%) was significantly (P<0.05) higher than the control group (82.8%) and the group that melatonin was only treated (81.6%). The blastocyst rate of $10^{-12}M$ as 45.6% was significantly (P<0.05) higher than control group (25.2%) and melatonin treatment group (31.2%). The cell number of blastocyst in $10^{-12}M$ isoproterenol treatment group $35.5{\pm}3.4$ was significantly (P<0.05) highest. The results of this study showed that the development rate of IVC when both isoproterenol and melatonin were supplemented was higher than when melatonin was only supplemented. Therefore, it is concluded that isoproterenol is rather effective in the activation of melatonin. $10^{-7}M$ melatonin and $10^{-12}M$ isoproterenol were considered suitable concentration.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.8
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• pp.1095-1101
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• 2016

Ginsenoside Rg1 is a natural compound with various efficacies and functions. It has beneficial effects on aging, diabetes, and immunity, as well as antioxidant and proliferative functions. However, its effect on porcine embryo development remains unknown. We investigated the effect of ginsenoside Rg1 on the in vitro development of preimplantation porcine embryos after parthenogenetic activation in high-oxygen conditions. Ginsenoside treatment did not affect cleavage or blastocyst formation rates, but did increase the total cell number and reduced the rate of apoptosis. In addition, it had no effect on the expression of four apoptosis-related genes (Bcl-2 homologous antagonist/killer, B-cell lymphoma-extra large, Caspase 3, and tumor protein p53) or two metabolism-related genes (mechanistic target of rapamycin, carnitine palmitoyltransferase 1B), but increased the expression of Glucose transporter 1 (GLUT1), indicating that it may increase glucose uptake. In summary, treatment with the appropriate concentration of ginsenoside Rg1 ($20{\mu}g/mL$) can increase glucose uptake, thereby improving the quality of embryos grown in high-oxygen conditions.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.9
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• pp.1354-1360
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• 2007

The aim of this study was to evaluate if oocytes, aspirated from postovulatory ovarian follicles of superovulated rabbits 14 h post-hCG administration, could be efficiently used as ooplasm recipients for somatic cell nuclear transfer (SCNT). Within a common SCNT protocol, a comparison between oocytes recovered by direct aspiration (aspirated) from available ovarian follicles and oocytes flushed out from oviducts (flushed) was carried out. The results showed that maturation and enucleation rates of aspirated oocytes were 70.7% and 69.2%, significantly lower than 95.3% (p<0.01) and 83.6% (p<0.05), respectively, from flushed oocytes. However, following enucleation of matured oocytes as ooplasm recipients for SCNT, no difference was recorded in fusion and cleavage rates, as well as blastocyst development from cleaved embryos or hatching of blastocysts between aspirated and flushed groups. Additionally, some matured aspirated and flushed oocytes were also used for immediate parthenogenetic activation and the resulting embryo development was not significantly different. Results from this study show the following: i) the majority of oocytes aspirated from postovulatory ovarian follicles of superovulated rabbits 14 h post-hCG administration are matured and can be used directly as ooplasm recipients for SCNT; ii) the reconstructed embryos derived from these oocytes have similar in vitro developmental ability to those flushed from the oviducts.

• Korean Journal of Veterinary Research
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• v.42 no.1
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• pp.29-33
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• 2002

This study was carried out to investigate the optimal activation condition for parthenogenetic development. In order to activate oocytes at 24 hrs post onset of maturation, the oocytes were cultured $3{\sim}13{\mu}M\;Ca^{2+}$ for 5 min., $5-8{\mu}g/ml$ cytoclacin for 6 hrs, 0.5~2.0 mM 6-dimethylaminopurine(DMAP) for 3 hrs alone or combination. The activated oocytes were cultured in TCM-199 media at 5% $CO_2$, 95% air, $38^{\circ}C$. The cleavage rate after 48 hrs culture of oocytes treated with $3-13{\mu}M\;Ca^{2+}$, $5-8{\mu}g/ml$ cytoclacin and 0.5~2.0 mM DMAP for 5 min., 6 hrs and 3 hrs were 9.6%~20.0%, 0.0%~7.3% and 9.4%~21.8%, 0.0%~7.3% and 9.1%~21.8% and 0.0%~7.3%, respectively. When oocyte were treated with $10{\mu}M\;Ca^{2+}$, $10{\mu}g/ml$ cytoclacin and 2.0 mM DMAP the blastocyst formation rate was significantly higher than other group. The cleavage rate after 48 hrs culture of oocytes treated with $Ca^{2+}$ + cytoclacin, $Ca^{2+}$ + DMAP, cytoclacin + DMAP were 75.9%~93.5% and 9.7%~19.0%, respectively. When oocytes were treated with $Ca^{2+}$ followed by DMAP, the blastocyst formation rate was significantly higher than other group(p<0.05). When necleus transferred embryos co-cultured with bovine serum albumin(BSA), epithemal growth factor(EGF) and calf serum(CS), the developmental rate to blastocyst were higher than control group.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.2
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• pp.121-129
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• 2022

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) is an N6-methyladenosine (m⁶A) RNA modification regulator and a key determinant of prem-RNA processing, mRNA metabolism and transportation in cells. Currently, m⁶A reader proteins such as hnRNPA2/B1 and YTHDF2 has functional roles in mice embryo. However, the role of hnRNPA2/B1 in porcine embryogenic development are unclear. Here, we investigated the developmental competence and mRNA expression levels in porcine parthenogenetic embryos after hnRNPA2/B1 knock-down. HhnRNPA2/B1 was localized in the nucleus during subsequent embryonic development since zygote stage. After hnRNPA2/B1 knock-down using double stranded RNA injection, blastocyst formation rate decreased than that in the control group. Moreover, hnRNPA2/B1 knock-down embryos show developmental delay after compaction. In blastocyste stage, total cell number was decreased. Interestingly, gene expression patterns revealed that transcription of Pou5f1, Sox2, TRFP2C, Cdx2 and PARD6B decreased without changing the junction protein, ZO1, OCLN, and CDH1. Thus, hnRNPA2/B1 is necessary for porcine early embryo development by regulating gene expression through epigenetic RNA modification.

• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.127-134
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• 2010

This study was to investigate the effect of flavonoid treatment on in vitro development of bovine somatic cell nuclear transfer (SCNT) embryos, and their pregnancy and delivery rate after embryo transfer into recipient. In experiment 1, to optimize the flavonoid concentration, parthenogenetic day 2 ($\geq$ 2-cell) embryos were cultured in 0 (control), 1, 10 and $20\;{\mu}M$ flavonoid for 6 days. In the results, in vitro development rate was the highest in $10\;{\mu}M$ flavonoid group (57.1%) among treatment groups (control, 49.5%; $1\;{\mu}M$, 54.2%; $20\;{\mu}M$, 37.5%), and numbers of total and ICM cells were significantly (p<0.05) higher in $10\;{\mu}M$ flavonoid group than other groups. We found that $10\;{\mu}M$ flavonoid treatment can significantly (p<0.05) decrease the apoptotic index and derive high expression of anti-oxidant, anti-apoptotic, cell growth and development marker genes such as Mn-SOD, Survivin, Bax inhibitor, Glut-5, In-tau, compared to control group. In experiment 2, to produce the cloned Jeju Black Cattle, beef quality index grade 1 bull somatic cells were transferred into enucleated bovine MII oocytes and reconstructed embryos were cultured in $10\;{\mu}M$ flavonoid added medium. When the in vitro produced day 7 or 8 SCNT blastocysts were transferred into a number of recipients, $10\;{\mu}M$ flavonoid treatment group presented higher pregnancy rate (10.2%, 6/59) than control group (5.9%, 2/34). Total three cloned Jeju Black calves were born. Also, two cloned calves in $10\;{\mu}M$ flavonoid group were born and both were all healthy at present, while the one cloned calf born in control group was dead one month after birth. In addition, when the result of short tandem repeat marker analysis of each cloned calf was investigated, microsatellite loci of 11 numbers matched genotype between donor cell and cloned calf tissue. These results demonstrated that the flavonoid addition in culture medium may have beneficial effects on in vitro and in vivo developmental capacity of SCNT embryos and pregnancy rate.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2005.10a
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• pp.39-40
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• 2005