• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.287-294
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• 2011

Autophagy is a process of intracellular bulk protein degradation, in which the accumulated proteins and cytoplasmic organelles are degraded. It plays important roles in cellular homeostasis, apoptosis, and development, but its role during early embryo development remains contentious. Therefore, in the present study, we investigated the effects of 3-methyladenine (3-MA) on early embryonic development in pigs, we also investigated several indicators of developmental potential, including mitochondrial distribution, genes expressions (autophagy-, apoptosis- related genes), apoptosis and ER-stress, which are affected by 3-MA. After in vitro maturation and fertilization, presumptive pig embryos were cultured in PZM-3 medium supplemented with 3-MA for 2 days at $39^{\circ}C$ 5% $CO_2$ in air. Developmental competence to the blastocyst stage in the presence of 3-MA was gradually decreased according to increasing concentration. Thus, all further experiments were performed using 2 mM 3-MA. Blastocysts that developed in the 3-MA treated group decreased LC3-II intensity and expressions of autophagy related genes than those of the untreated control, resulting in down-regulates the autophagy. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 3-MA treated group compared with control ($6.0{\pm}1.0$ vs $3.3{\pm}0.6$, p<0.05). Also, the expression of the pro-apoptotic gene Bax increased in 3-MA treated group, whereas expression of the anti-apoptotic gene Bcl-XL decreased. Mito Tracker Green FM staining showed that blastocysts derived from the 3-MA treated group had lower mitochondrial integrity than that of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. Then, the expression of the spliced form of pXBP-1 product (pXBP-1s) increased in 3-MA treated group, resulting increase of ER-stress. Taken together, these results indicate that inhibition of autophagy by 3-MA is closely associated with apoptosis and ER-stress during preimplantation periods of porcine embryos.

• Journal of Embryo Transfer
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• v.23 no.4
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• pp.263-267
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• 2008

The purpose of this study was to determine toxic effect of sucrose and trehalose prior to cryopreservation on nuclear maturation and embryonic development in immature bovine oocytes. All cryoprotectant was prepared in tissue culture medium 199-HEPES (TCM 199-HEPES) with 10% fetal bovine serum (FBS). Immature oocytes were exposed to 1.2M ethylene glycol (EG) and 0.1M sucrose or 1.2M EG and 0.1M trehalose for 3 min and then were exposed to 3.2 M EG and 0.25 M sucrose or 3.2 M EG and 0.25 M trehalose for 1 min. Oocytes treated with cryoprotectants were exposed to 0.25 M sucrose or 0.25 M trehalose for 5 min and then 0.1 M sucrose or 0.1 M trehalose for 5 min. Depending on type of sugar added to cryopreservation solution, oocytes were allocated to sucrose group and trehalose group, respectively. Oocytes exposed to TCM 199-HEPES with 10% FBS were considered as control. Oocytes were cultured in TCM 199 supplemented with 10% FBS, 5 ng/ml epidermal growth factor, 0.01 IU/ml luteinizing hormone, and $1\;{\mu}g/ml$ estradiol for 24 h in $39^{\circ}C$, 5% $CO_2$. Nuclear maturation was assessed by staining oocytes with 1% aceto-orcein. Oocytes were fertilized in vitro and were cultured in TCM 199 supplemented with 10% FBS, 5 mM sodium pyruvate, and antibiotics in $39^{\circ}C$, 5% $CO_2$. The rates of cleavage and blastocyst, and cell number in blastocyst were assessed. Metaphase II rates were not different among experimental groups regardless of type of sugar. The cleavage rate of trehalose group (73.3%) was significantly higher (p<0.05) than those of sucrose group (62.8%) and control group (60.8%). The blastocyst rate was significantly higher in trehalose group (p<0.05). Mean cell number in blastocyst were not different among experimental groups, although cell number of blastocyst in trehalose group was significantly higher on day 7 (p<0.05). In conclusion, sucrose and trehalose were not toxic to immature bovine oocytes prior to cryopreservation. In particular, trehalose was more effective on embryonic development.

• Clinical and Experimental Reproductive Medicine
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• v.42 no.2
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• pp.62-66
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• 2015

Objective: To evaluate the effect of a gonadotropin-releasing hormone (GnRH) antagonist protocol using corifollitropin alfa in women undergoing assisted reproduction. Methods: Six hundred and eighty-six in vitro fertilization-embryo transfer (IVF)/ intracytoplasmic sperm injection (ICSI) cycles were analyzed. In 113 cycles, folliculogenesis was induced with corifollitropin alfa and recombinant follicle stimulating hormone (rFSH), and premature luteinizing hormone (LH) surges were prevented with a GnRH antagonist. In the control group (573 cycles), premature LH surges were prevented with GnRH agonist injection from the midluteal phase of the preceding cycle, and ovarian stimulation was started with rFSH. The treatment duration, quality of oocytes and embryos, number of embryo transfer (ET) cancelled cycles, risk of ovarian hyperstimulation syndrome (OHSS), and the chemical pregnancy rate were evaluated in the two ovarian stimulation protocols. Results: There were no significant differences in age and infertility factors between treatment groups. The treatment duration was shorter in the corifollitropin alfa group than in the control group. Although not statistically significant, the mean numbers of matured (86.8% vs. 85.1%) and fertilized oocytes (84.2% vs. 83.1%), good embryos (62.4% vs. 60.3%), and chemical pregnancy rates (47.2% vs. 46.8%) were slightly higher in the corifollitropin alfa group than in the control group. In contrast, rates of ET cancelled cycles and the OHSS risk were slightly lower in the corifollitropin alfa group (6.2% and 2.7%) than in the control group (8.2% and 3.5%), although these differences were also not statistically significant. Conclusion: Although no significant differences were observed, the use of corifollitropin alfa seems to offer some advantages to patients because of its short treatment duration, safety, lower ET cancellation rate and reduced risk of OHSS.

• Journal of Embryo Transfer
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• v.16 no.3
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• pp.223-231
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• 2001

The success of nuclear transplantation with mammalian oocytes depends critically on the potential of oocytes activation, which mainly caused to prevent the re-accumulation of maturation promoting factor (MPF). This study was conducted to compare the effect of combined treatment of lonomycin with a Hl-histone kinase inhibitor (dimethylaminopurine, DMAP) or cdc2 kinase inhibitor (sodium pyrophosphate, SPP) on activation of bovine oocytes. In vitro matured bovine oocytes with the first polar body (PB) and dense cytoplasm were assigned to 3 experimental groups. For activation treatment, oocytcs were exposed to 5 $\mu$M lonomycin for 5 min (Group 1), and followed by 1.9 mM dimethylaminopurine (DMAP) for 3 h (Group 2) or followed by 2 mM sodium pyrophosphate (SPP) for 3 h (Group 3). The activation effects in the three treatments and the control group (untreated) were judged by the extrusion of the second PB and formation of a pronucleus (PN). Differences among groups were analysed using one-way ANOVA after arc-sine transformation of proportional data. All three treatments led to high activation rates (90% to 95%), with significant difference from the control. However, the extrusion of the second PB and the rate of PN formation differed remarkably among treatments. In Group I and 3, about 95% of the oocytes had extruded the second polar body, but one PN had formed in a higher proportion of oocytes in Group 3 than in Group 1 (90% vs. 5%). In experiment 2, the rates of cleavage and development into blastocysts in Group 1 were significantly lower than those of Group 2 and 3 (8.7% and 0% vs. 50.5% and 11.6%, and 44.6% and 7.2%, respectively, P<0.05). In experiment 3, ~80% of parthenotes in Group 1 were developed with haploid chromosomal sets. However, when ionomycin was followed immediately by DMAP (Group 2). only 20% of parthenotes were haploid. In Group 3, combined treatment with ionomycin and SPP, the appearance of abnormal chromosomal tracts was significantly (P〈0.05) reduced and the proportion of haploid parthenotes was increased to 85% (17/20) than in Group 2. These results demonstrate that SPP acted as a cdc2 kinase inhibitor and formed the haploidy in oocyte activation. Thus, the present study suggests that cdc2 kinase inhibitor, such as sodium pyrophosphate, may have an effective role in oocyte activation for the production of cloned embryos/animals by nuclear transplantation.

• Journal of Embryo Transfer
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• v.29 no.1
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• pp.59-66
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• 2014

Transcription activator like effector nucleases (TALENs) are artificial restriction enzymes generated by fusing a TALE DNA binding domain to a DNA cleavage domain which remove and introduce specific genes to produce transgenic animals. To investigate the efficient laboratory techniques for the injection of TALEN mRNA, pEGFP-N1 commercial plasmid were microinjected into porcine parthenogenetic and in vitro fertilization (IVF). In Experiment 1, to investigate injection time, compared 4 different time durations (2 hr, 4 hrs, 6 hrs & 8 hrs) after post activation of parthenogenetic embryos and after 6 hrs of co-incubation with sperms in IVF embryos. There were significant difference (P<0.05) in development to the blastocysts (4.4, 8.9, 3.9, 0.6%), GFP expression in blastocysts (1.3, 5.7, 2.3, 0.0%) which injected after post activation of 4 hrs compared with other 3 groups. IVF embryos after 2 hrs and 4 hrs injected were expressed GFP significantly higher than rest of two groups (P<0.05). In Experiment 2, compared development of 2 different concentrations ($20ng/{\mu}l$ and $50ng/{\mu}l$) of EGFP injection. There were significant difference (P<0.05) between two treatments which has higher cleavage (58.8 vs 41.9%), blastocysts development rate (13.0 vs 11.1%) and GFP expressed blastocysts (5.7 vs 0.0%) in $20ng/{\mu}l$ than the $50ng/{\mu}l$ in parthenogenetic embryos. In IVF embryos, only $20ng/{\mu}l$ injected embryos were expressed GFP (4.2%) after 7 days of incubation and 77.3 vs 64.7% of cleavage, 26.4 vs 23.5% development to blastocysts. In Experiment 3, three different volumes (5, 10 and 20 pl) were microinjected into porcine embryos to determine the most appropriate volume. Out of 3 groups, significantly higher development rates of cleavage (68.3, 58.0, 29.3%), blastocysts (11.7, 12.7, 0.5%) and GFP expressed blastocysts (2.9, 7.8, 0.0%) were shown in the 10 pl group (P<0.05). In conclusion, these results imply that $20ng/{\mu}l$ concentration, 10 pl of volume and injection at 4 hrs after post activation for parthenogenetic and 2~4 hrs after IVF, $20ng/{\mu}l$ concentration and 10 pl volume for IVF embryos were more effective microinjection conditions.

• Journal of Embryo Transfer
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• v.25 no.1
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• pp.9-14
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• 2010

This study was conducted to find out the effects of artificial shrinkage (AS) on post-thaw development of bovine embryos. The blastocoelic cavity of blastocyst was punctured to remove its fluid contents and then incubated in the holding medium (HM) for 10 min. The punctured and non-punctured (control) blastocysts were equilibrated in vitrification solution 1 (VS1; TCM-199+20% FBS+10% EG) for 5 min and vitrification solution 2 (VS2; TCM199+20% FBS+35% EG+5% PVP+0.5 M Sucrose) for 1 min and vitrified by direct dropping into the liquid nitrogen. Vitrified blastocysts (punctured and control) were thawed and cultured in vitro (12 hr) for studying survival and hatching rates. The levels of shrinkage were measured by the volume of the blastocyst during equilibration in VS1 (at 1, 3 and 5 min of equilibration) and VS2 (at 30 and 60 sec of equilibration) that was considering the volume of non-punctured blastocyst in HM as 100%. The levels of shrinkage were higher in punctured group (62.4, 64.6, 64.3% at 1, 3 and 5 min in VS1; 50.6 and 52.7% at 30 and 60 sec in VS2) than control group (84.8, 86.6, 86.4% at 1, 3 and 5 min in VS1; 72.1 and 68.8% at 30 and 60 sec in VS2), but within each group the levels of shrinkage were similar. The survival (90.9%) and hatching (50.0%) rates of vitrified blastocysts at 12 hr post-thaw were higher in punctured group than that in control group (76.9% and 0.0% respectively). We confirmed that vitrification solutions (VS1 and VS2) have no toxic effect on the survival of blastocysts because the survival rates of blastocysts exposed to VS1 and VS2 for 24 hr were similar between punctured and control groups (94.3 vs. 96.0%; p>0.05). In conclusion, the preliminary data show that AS of blastocyst may improve survival and hatching rate after thawing.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.2
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• pp.137-147
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• 1994

Controlled ovarian hyperstimulation(COH) for in vitro fertilization and embryo transfer(IVFET) often results in the production of more embryos than can be efficaciously transferred at one time. However, embryo cryopreservation provides a mechanism by which additional embryos can be stored for later thawing and transfer. From November, 1990 to October, 1992, we completed 42 transfer cycles of cryopreserved pronucleus(PN) l-cell embryos using the fixed protocol of hormonal replacement therapy in a physiological manner regardless of individual ovarian function. Artificial endometrial stimulation was performed with only exogenous estradiol and progesterone(E-P) in 36 transfer cycles (Group I) and with gonadotropin-releasing hormone agonist(GnRHa) and exogenous estradiol and progesterone(GEEP) in 6 transfer cycles(Group II ). The results were as follows. 1. The Survival rate of total cryopreserved-thawed embryos was 64.9%(198/305): 64.9% (172/265) in Group I and 65.0% (26/40) in Group II. 2. Total 168 embryos were transferred with an average of 4.7 per ET in Group I and total 26 embryos were transferred with an average of 4.3 per ET in Group II. 3. The pregnancy rate(PR) per cryopreserved-thawed ET and the implantation rate was 33.3 %(14/42) and 6.7%(13/194), respectively. The PRs per cryopreserved-thawed ET were 30.6% (11/36) in Group I and 50.0% (3/6) in Group II without significant difference. 4. The take home baby rate was 11.1%(4/36) in Group I and 33.3% (2/6) in Group II.

• Development and Reproduction
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• v.17 no.3
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• pp.207-213
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• 2013

This study was performed to investigate the effect of peroxisome proliferators activated receptor-${\gamma}$ (PPAR-${\gamma}$) ligand, pioglitazone, on production of regulated upon activation normal T-cell expressed and secreted (RANTES) and in vitro fertilization (IVF) outcome in infertile patients with endometriosis. Sixty-four infertile patients with stage III or IV endometriosis undergoing IVF were randomly allocated to the study or the control group. The long protocol of GnRH agonist (GnRH-a) was used for controlled ovarian stimulation (COS) in all patients. Patients in the study group were treated with pioglitazone at a dose of 15 mg/day orally from the starting day of GnRH-a treatment to the day of hCG injection. Blood samples were drawn for serologic assay of RANTES on the first day of GnRH-a treatment and the day of hCG injection. There were no differences between the study and control groups in patient characteristics. There were also no differences between the two groups in COS duration, and the numbers of retrieved oocytes, fertilized oocytes and embryos transferred. The clinical pregnancy rate per cycle was higher in the study group, but this difference was not statistically significant. However, embryo implantation rate was significantly higher in the study group of 12.5% compared with 8.6% in the control group (P<0.05). The serum RANTES levels after pioglitazone treatment were significantly lower than those before pioglitazone treatmen in the study group (P<0.05). Our data suggest that pioglitazone treatment can suppress RANTES production and improve the embryo implantation rate in patients with endometriosis undergoing IVF.

• Journal of Embryo Transfer
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• v.25 no.3
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• pp.171-177
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• 2010

Plasminogen activators (PAs) are serine proteases that convert plasminogen to plasmin. The PA/plasmin system has been associated with a number of physiological processes such as fibrinolysis, ovulation and fertilization. Although correlations have been reported between reactive oxygen species (ROS) and oocyte maturation, the relationship between PA activity and ROS is unknown. The present study was undertaken to determine the effects of cumulus cells on PA activity in matured porcine oocytes under xanthine (X)-xanthine oxidase (XO) system. When oocytes were matured under the X-XO system, the proportion of oocytes remaining GV stage was higher (p<0.05) in oocytes without cumulus cells. The incidence of degenerated oocytes was higher (p<0.05) in the X+XO ($11.1{\pm}6.1$ and $21.6{\pm}3.4%$) than in the control group ($2.9{\pm}1.8$ and $4.0{\pm}1.6%$). The proportion of TUNEL-positive oocytes and activity of caspase-3 were higher (p<0.05) in cumulus-free oocytes and oocytes exposed to ROS. Tissue-type plasminogen activator-plasminogen activator inhibitor (tPA-PAI) and tissue-type plasminogen activator (tPA) activity were detected in oocytes that were separated from cumulus-oocytes complexs (COCs) at 44 h of maturation culture, and only tPA was produced in oocytes that were denuded before the onset of maturation culture. On the other hand, the activities of PA were increased (p<0.05) when oocytes were cultured under the X-XO system. The higher activity of tPA was observed in denuded oocytes (DOs) underwent apoptotic changes by oxidative stress. In COCs, however, tPA-PAI as well as tPA activity was detected and apoptotic changes such as DNA cleavage or caspase-3 activation were not observed. These results suggest that tP A may be relevant to apoptotic cell death in porcine oocytes by oxidative stress.

• Journal of Embryo Transfer
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• v.16 no.2
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• pp.91-98
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• 2001

We evaluated the effects of the substrate and inhibitors related to phosphatidylinositol metabolism on in vitro maturation and fertilization of porcine oocytes. Cumulus-oocyte complexes were cultured in mTLP-PVA medium supplemented with or without inositol (250 mM) fur 46h. Subsequently, these oocytes were inseminated with fresh boar semen in mTALP-PVA medium for 6h. At 6h after insemination, oocytes were cultured for further 12 h in TCM-199 supplemented with 10% FBS (fetal bovine serum). The higher percentage of oocytes in inositol-supplemented medium reached metaphase of the second meiotic division compared to those in control (81.4% vs. 67.3%; P<0.()5). following 18 h of insemination, more number of male pronuclei were formed in the oocytes matured in inositol-supplemented medium than in those of control experiment (42.0% vs. 27.3%; P<0.05). When oocytes were cultured in medium with 10mM LiCl (chloride lithium) or 0.5mM dbcAMP (dibutyryl cyclic adenosine monophosphate) to determine the role of inositol on the maturation of oocytes, these two drugs inhibited the meiotic division of oocytes (P<0.05). However, addition of inositol to the culture medium did overcome the inhibitory effect of these drugs on the oocyte maturation. DbcAMP and verapamil supplemented synergistically arrested the meiotic division of oocytes. Addition of verapamil did not inhibit germinal vesicle breakdown, but it severly inhibited the second meiotic division of oocytes. These results suggest that inositol exert its improving effects on maturation, by activating the PI (phosphatidylinositol) cycle and causing beneficial changes in both cytoplasm and membrane of oocytes.