• Development and Reproduction
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• v.3 no.1
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• pp.75-85
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• 1999

To investigate the origin and action mechanism of cytoplasmic factors as regulators of morphogenesis, the embryonic development, RNA synthesis and protein phosphorylation were examined in reconstituted embryos. A half of 1-cell mouse embryo with both pronuclei was electrofused with the enucleated cytoplasm of 1- or 2-cell embryos which were cultured for 24 hrs from post 20 hrs hCG in CZB with or without cycloheximide (CHX, an inhibitor of protein synthesis; P+P-CHX group), genistein (Gen, an inhibitor of tyrosine protein kinase; P+2-Gen group) and 6-dimethylaminopurine (6-DMAP, an inhibitor of serine-threonine protein kinase; P＋2-DMAP group), and co-cultured with Vero cells for 5 days. And their development, cell numbers at compaction, [5, 6-$^3$H]-uridine incorporation into RNA and the pattern of protein phosphorylation after labeling of [$^{32}$ P] orthophosphate were compared with that of the reconstituted embryos such as P+2 or P+P (control group). Embryonic development and the time of RNA synthesis in P+P-CHX were similar to those in P+P. But the time and the cell stages of embryonic compaction in P+P-CHX were similar to those in P+2. The compaction was initiated at 4-cell in P+2 and P+2-Gen, but at 8-cell in P+P and P+2-DMAP. On a two-dimensional gel electrophoresis, phosphorylation of 80KD and 110KD proteins were inhibited after 3 hrs of reconstruction in the embryo of P+2-DMAP when compared with that of P+2 and P+2-Gen. These results suggest that protein synthesis between 1- and 2-cell stage affects the timing of embryonic genome activation, and that cytoplasmic factors derived from oocyte or their modification regulates the time schedule of embryonic compaction in mouse. Also, serine-threonine protein kinase has an important role on the regulation of compaction.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.213-220
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• 2009

The objective of this study was to examine the effect of eCG and various concentrations (20, 40, and 80 ${\mu}g/ml$) of porcine FSH on nuclear maturation and intracellular glutathione (GSH) level of oocytes, and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Immature pig oocytes were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones (10 IU/ml hCG and 10 IU/ml eCG or $20{\sim}80{\mu}g/ml$ FSH) for the first 22 h and then further cultured in hormone-tree medium for an additional 22 h. Nuclear maturation of oocytes ($85{\sim}89%$) was not influencem foreCG and various concentrations FSH. Embryonic development to the cleavage stage ($86{\sim}94%$) and mean number of cells in blastocyst ($33{\sim}37$ cells) after PA were not altered but blastocyst formation e-treignificaddlor(p<0.05) improvem forthe supplementation eith 80 ${\mu}g/ml$ FSHr(64%) compared to 47%, io8%, iand 47% in oocytes that were treated with eCG, 20,i and 40 ${\mu}g/ml$ FSH,i numectivelo. In SCNT, fusion ($78{\sim}83%$) of cell-cytoplast couplets and siosequent embryo cleavage ($82{\sim}88%$) were not influencem fordifferent gonadotropins but blastocyst formation tended to increase forthe supplementation eith 80 ${\mu}g/ml$ FSHr(25% vs. $11{\sim}18%$). Our nuults demonstrated that oocyte maturation and embryonic development after PA and SCNT e-frinfluencem fortype of gcem fortype of gits concentration. In this study, supplementation of maturation medium eith 80 ${\mu}g/ml$ FSHrimproved preimplantation development of PA and SCNT pig embryos, probably by increasing intracellular GSH concentration of matured oocytes.

• Development and Reproduction
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• v.16 no.2
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• pp.95-100
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• 2012

Bisphenol A (BPA), a chemical with weak estrogenic activity is reported to affect preimplantation embryos, fetuses and alter their postnatal development. This study amied to determine the relation between the levels of BPA in the amniotic fluid and pregnancy outcomes. ELISA was used to measure amniotic fluid BPA in 120 pregnant women who underwent genetic amniocentesis at 15~20 weeks gestation. The most common indication for amniocentesis was advanced maternal age (35 yrs or older). BPA was detected in all amniotic fluid. The range of amniotic fluid BPA concentrations was from 0.89 ng/mL to 37.13 ng/mL with a mean level of 7.24 ng/mL. We compared the means of amniotic fluid BPA concentrations according to maternal age (${\geq}35$ vs. <35 yrs), fetal sex (male vs. female), gestational age at birth (${\geq}37$ vs. <37 weeks), and infant birth weight (${\geq}2.5$ vs. <2.5 kg). No significant differences were found in these outcomes. This is the first report of amniotic fluid BPA levels in Korean pregnant women. Our findings suggest that BPA may not affect the pregnancy outcomes such as fetal sex, preterm delivery and low birth weight. Whether prenatal exposure to BPA can have teratogenic effect on developing embryo needs to be studied.

• Korean Journal of Animal Reproduction
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• v.27 no.2
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• pp.169-177
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• 2003

This study was conducted to determine effects of polyvinyl alcohol (PVA), fetal bovine serum (FBS) bovine serum albumin (BSA) and epidermal growth factor (EGF) on blastocoel formation, total cell number, apoptosis and apoptosis-related gene expression of porcine diploid parthenotes developing in vitro. The addition of 0.4% BSA to the culture medium enhanced the development of 2-cell stage parthenotes to the blastocysts stage (P<0.01). FBS reduced cell numbers of blastocysts (P<0.01) and increased percentage of apoptosis in the blastocysts (P<0.001). However, while BSA increased cell numbers, it did so only when EGF was present. Either agent on its own had no effect. Similarly, apoptosis in the blastocysts was not influenced by either agent on its own but was reduced when both BSA and EGF were present. Furthermore, semi-quantitative reverse-transcriptase polymerase chain reaction revealed that EGF enhanced the mRNA expression of Bcl-xL in the presence of 0.4% BSA but BSA and EGF alone had no effect, and EGF and/or BSA did not influence Bak gene expression in the blastocyst stage parthenotes. However FBS reduced Bcl-xL mRNA expression (P <0.05) and enhanced Bak expression. This result suggests that apoptosis related genes expression is significantly affected by supplements, which may result in alteration of apoptosis and embryo viability of porcine embryos developing in vitro.

• Korean Journal of Animal Reproduction
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• v.26 no.2
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• pp.173-182
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• 2002

The mechanisms underlying of the visual assessment and resulting in optimum embryonic development following in vitro maturation, fertilization, and culture are unclear, It was known that in vitro produced embryos show more frequent occurrence of fragmentation, which resulted in poor developmental potential and decreased implantation rate. The objective of this study was to investigate the apoptotic rates in bovine blastocyst derived from in vitro fertilization (IVF) and nuclear transfer (NT). In addition, the expression levels of Bcl-2 and Bax gene were investigated in the blastocyst to confirm their potential roles in the regulation of apoptosis during preimplantation embryonic development. Analysis of apoptosis was carried out by using terminal deoxynucleotidyl transferase mediate dUTP nick end labeling (TUNEL) method. The levels of Bcl-2 and Bax gene in the blastocyst derived from IVF and NT were determined by RT-PCR. The proportion of TUNEL positive signal in blastocyst derived from NT was significantly higher than that in blastocyst derived from IVF (p<0.001). Bcl-2 expression level of blastocyst derived from IVF was higher than that of blstocyst derived from NT. However, high expression level of Bax was observed in the blastocyst derived from NT. These results indicates that apoptosis is more responsible for fiagmentation in bovine blastocyst derived from NT than IVF. These results suggested that the increase of developmental failure followed by NT could be caused by nuclear fragmentation as apoptosis.

• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.65-70
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• 2006

This study investigated apoptosis and in vitro development of parthenogenetic preimplantation porcine embryos. In vitro matured oocytes for $42{\sim}44h$ were used. Apoptotic cell death was analyzed by using a terminal deoxynucleatidyl transferase mediated deoxyuridine 5-triphosphate nick-end tabling (TUNEL) assay. In experiment 1, oocytes were activated with two electric pulses (CH) of 1.2 kV/cm for $30{\mu}sec$ (E), E + 6-dimethylaminopurine (6-DMAP) or E + cycloheximide (CH) and cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$. In experiment 2, oocytes were activated by E and cultured in PZM-3 or NCSU-23 under a gas atmosphere of 20% $O_2$ ($5%\;CO_2$, in air) or 5% $O_2$ $(5%\;CO_2,\;5%\;O_2\;90%\;N_2)\;at\;38.5^{\circ}C$. Oocytes activated with E+6-DMAP or E+CH showed higher blastocyst rates (36.3% and 32.5%) compared to E alone (27.7%). The frequency of apoptosis according to treatments were 5.3%, 7.7% and 7.1% respectively. Oocytes activated with E alone showed lower (P<0.05) frequency of apoptosis compared to other groups. In experiment 2, parthenotes cultured in PZM-3 showed slightly higher blastocyte rates (28.2% and 29.7%) compared to NCSU-23 (22.6% and 24.4%) regardless of atmosphere. Blastocysts generated in PZM-3 showed lower (P<0.05) apoptosis rate under 20% $O_2$ (9.2% vs 16.9%), whereas those in NCSU-23 had slightly lower apoptosis rate under 5% $O_2$ (14.0% vs 18.4%). This result represents that activation method and culture condition could affect the frequency of apoptosis as well as in vitro developmental rate.

• Korean Journal of Veterinary Research
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• v.32 no.1
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• pp.15-24
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• 1992

The effects of CC the ovulatory response, oocyte normality, ovarian steroidogenesis and subsequent embryo developmental potential were examined in PMSG-treated rats. On Days of 25~27 of age, immature female Sprague Dawley rats were treated with three different doses(0.05, 0.1 or 1.0mg /day) of clomiphene citrate or vehicle. The females subsequently received 4IU PMSG on Day 28 and/or 10IU hCG on Day 30, and were killed on Day 31. Some females given 0.1mg CC or vehicle with 4IU PMSG were then mated and killed on Days 2, 3, 4 and 5 of pregnancy. Compared to vehicle(control) group, by increasing the doses of CC, there were a significant decrease in the ovulatory response as judged by both the proportion of rats ovulating and the mean number of oocytes per rat and a marked reduction of ovarian weight. The increasing doses of CC substantially promoted the degeneration(%) of oocytes ovulating in a dose-dependent manner. The CC-mediated inhibitions of the ovulatory response and ovarian weight were oompletely overcome by a subsequent treatment of hCG. Increasing doses of CC resulted in a siginificant elevation of serum estradiol with the decreased levels of progesterone and androgens. The additive treatment with hCG was effective to reduce the elevation of estradiol and to increase the reduction of progesterone produced by high dose(1.0mg) of CC. The preimplantation embryos recovered from 0.1mg CC-treated pregnant rats demonstrated a progressive early loss from Day 3 of pregnancy with a significant increase in the percentage of degeneration during all periods examined, compared to controls. The rate of progressive embryo cleavage in the CC-treated rats were slower than that in controls from Day 3 of pregnancy. Additionally, the percentage of the cleaved embryos recovered from the CC-treated rats remained significantly lower consistently from Day 2 of pregnancy, compared to control regimen. These results demonstrate a possible mechanism of CC-mediated inhibition of ovulatory response in the rats which may include the attenuation or blockade of the endogenous secretion of gonadotropins and also suggest that its detrimental effects observed on oocyte normality and embryonic development may be caused by abnormal follicular steroidogenesis( especially elevated estradiol) preceding fertilization.