These study was carried out to investigate the effects of the supplementation with sodium nitroprusside (SN) and nitric oxide (NO) of canine oocytes on IVM rates. Oocytes were incubated in TCM-199 supplement with at 0.03～0.10 mM SN and 0.3～1.0 mM NO for 48 hrs. Oocytes were transferred to 50 ul drops of maturation medium covered mineral oil and cultured in a CO2 incubator (5% CO2, 95% air, 38℃). The in vitro maturation rate of oocytes cultured for 48 hrs in TCM-199 medium supplement with 0.03, 0.05, 0.07, 0.10 mM SN were 25.9±3.5%, 36.4±3.2%, 33.3±3.5%, 28.8±3.2%, respectively. The in vitro maturation rate of oocytes cultured for 48 hrs in TCM-199 medium supplement with 0.03～0.07 mM SN were significantly increased compare to the control (26.0±2.2%). The in vitro maturation rates of oocytes cultured for 48 hrs in TCM-199 medium supplement with 0.3, 0.5, 0.7, 1.0 mM NO were 28.0±4.2%, 36.5± 3.6%, 30.0±3.8%, 19.2±3.5%, respectively. The in vitro maturation rate of oocytes in TCM-199 medium supplemented with 0.3 and 0.5 mM NO were significantly increased compare to the control (26.0±2.2%). The in vitro maturation rates of oocytes cultured for 12～48 hrs in TCM-199 medium supplement with 0.05 mM SN were 26.0±3.2%, 28.0±3.4%, 38.0±3.2%, respectively. The in vitro maturation rate of oocytes cultured for 12～48 hrs in TCM-199 medium supplement with 0.5 mM NO were 22.0±3.0%, 30.0±3.8%, 36.0±4.2%, respectively. These result was significantly increased compare to the control.

Presently, the effect of 0.5 mM dibutyryl cAMP (dbcAMP)-supplemented maturation medium during different incubation time on meiotic arrest (germinal vesicle) and resumption (metaphase II) of porcine oocytes and embryonic development of porcine oocytes following in vitro fertilization (IVF) or parthenogenetic activation (PA) was determined. Porcine cumulus oocyte complexes (COCs) were cultured in 0.5 mM dbcAMP for 17, 22, 27, or 42 h, and an additional 22 h without 0.5 mM dbcAMP. The nuclear status was examined at each time point. Oocytes cultured from 39~49 h displayed more than 80% meiotic resumption. More than 85 % of meiotic arrest was presented at 17~22 h. Oocytes were cultured for 22 h with 0.5 mM dbcAMP and additional 22 h without dbcAMP to assess developmental potential following IVF or PA. There were no significant differences in blastocyst rates among the dbcAMPIVF, IVF, dbcAMP-PA, and PA groups, although cleavage rate of IVF group was significantly higher than those of dbcAMP-PA, and PA groups. In conclusion, 0.5 mM dbcAMP influenced meiotic maturation of porcine oocytes depending on incubation time of oocyte, although embryonic development was not improved in both IVF and PA.

Acteoside (verbascoside) is a typical phenylethanoid glycoside, extracted from various plants. It has various biological functions such as anti-oxidant, anti-inflammation, and anti-hypertension. Specially, it was powerful anti-oxidants either by direct scavenging of reactive oxygen and nitrogen species, or by acting as chain-breaking peroxyl radical scavengers. We examined the role of acteoside in IVM medium on the morphological progress of meiosis, developmental competence, and ROS in porcine oocytes. And we investigated effect of acteoside on the oocytes condition represented by cytoplasmic maturation by homogeneous distribution and formation of cytoplasmic organelles and regulation of apoptosis-related genes. The selected COCs were cultured in TCM-199 with various concentration of acteoside: 0 (control), 10, 30, and 50 μM. After 22 h of maturation with hormones, the oocytes were washed twice in a fresh maturation medium before being cultured in hormone-free medium for additional 22 h. The oocytes maturation rates of supplemented with acteoside were no significantly different compared with control group (71.13, 75.96, 72.95 and 73.68%, respectively). Level of ROS was significantly decreased in acteoside treated group. Furthermore, the parthenogenetic blastocyst rate was significantly improved in 10 μM acteoside treated group compared with control group (40.03 vs. 22.95%). During IVM, 10 μM acteoside treated oocytes showed that the mitochondria and lipid droplet were smaller and homogeneous distribution in cytoplasm compare with non-treated control oocytes. And reverse transcription polymerase chain reaction (RT-PCR) witarthenogenetic blstocysts revealed that acteoside increased the anti-apoptoticgenes, otherwise reibued pro-apoptotic genes. In conclusion, our results represents that addition of acteoside to the IVM medium has a beneficial effect in physiology of porcine oocytes such as viability and activation, providing a improved method for porcine oocytes in vitro.

The study investigated the effects of trans-ε-viniferin on in vitro maturation (IVM) and gene expression. Three experiments were conducted. Firstly the trans-ε-viniferin was purified from the leaves and stems of the Vitis amurensis , a common wild grape found in Korea, Japan, and China. In the first experiment, a total of 594 cumulus oocytes complexes (COCs) were used for the evaluation of the nuclear maturation. COCs were matured with various concentrations of trans-ε-viniferin (0, 0.1, 0.5, 1.0 and 5.0 μM). After IVM 42 44 h, the nuclear maturation was evaluated. In the second experiment, a total of 300 matured oocytes were used to examine the effects of different trans-ε-viniferin concentrations (0, 0.5 and 5.0 μM) on porcine oocyte intracellular GSH and ROS levels. In the third experiment, the gene expression of oocytes matured with trans-ε-viniferin (0.5 μM) and the untreated group were evaluated after IVM. As results, we observed that trans-ε- viniferin treatment during IVM did not improve the nuclear maturation. But significantly increased (p<0.05) intracellular GSH levels in 0.5 μM group (0 μM vs. 0.5 μM; 14.6 vs. 16.8 pmol/oocyte) and reduced ROS levels (0 μM vs. 0.5 μM and 50 μM; 174.6 vs. 25.7 and 23.8 pixel/oocyte). The trans-ε-viniferin treatment during IVM of recipient oocytes promoted higher (p<0.05) expression of Dnmt1 mRNA in 0.5 μM treatment group than in the control group. But, the other gene expressions (PCNA, OCT4, caspase3, BAK, BAX and sit1) did not significantly differ from the control. In conclusion, these results indicated that the trans-ε-viniferin treatment during porcine IVM increased the cytoplasmic maturation through increasing the intracellular GSH synthesis, reducing ROS levels and increasing the Dnmt1 gene expression.

The present study investigated the effects of resveratrol (a phytoalexin with various pharmacological activities) during in vitro maturation (IVM) of porcine oocytes on nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels, gene expressions in matured oocytes, cumulus cells, and IVF-derived blastocysts, and subsequent embryonic development after parthenogenetic activation (PA) and in vitro fertilization (IVF). In the nuclear maturation after 44 h IVM, the groups of 0.1, 0.5, and 2.0 μM (83.0%, 84.1%, and 88.3%, respectively) had no significant difference compared to the control (84.1%), but the group of 10.0 μM decreased the nuclear maturation (75.0%) significantly (p<0.05). The groups of 0.5 and 2.0 μM showed a significant (p<0.05) increase in intracellular GSH levels compared to the control and 10.0 μM groups. Intracellular ROS level of oocytes matured with 2.0 μM resveratrol was significantly (p<0.05) decreased compared to the other groups. Oocytes treated with 2.0 μM resveratrol during IVM had significantly higher blastocyst formation rate, and total cell numbers after PA (62.1% and 49.1 vs. 48.8%, and 41.4, respectively) and IVF (20.5% and 54.0 vs. 11.0% and 43.4, respectively) compared to the control group. Cumulus-oocytes complex (COCs) treated with 2.0 μM resveratrol were showed lower (p<0.05) expressions of apoptosis-related genes in both matured oocytes (Bax, Bak, and Caspase-3) and cumulus cells (Bax). In IVF-derived blastocysts derived from 2.0 μM resveratrol treated oocytes had also decreased (p<0.05) expression of Bak compared to the control. In conclusion, the 2.0 μM resveratrol supplementation during IVM improved the developmental potential of PA and IVF in porcine embryos by increasing the intracellular GSH level, decreasing ROS level, and regulating apoptosis-related genes expression during oocyte maturation.

X‐box binding protein‐1 (XBP‐1) is an important regulator of a subset of genes active during endoplasmic reticulum (ER) stress. In the present study, we analyzed XBP‐1 level and location to explore the effect of ER stress on oocyte maturation and developmental competency of porcine embryos in an in vitro culture system. First, we examined the localization of XBP‐1 at different meiotic stages of porcine oocytes and at early stages of parthenogenetic embryo development. Fluorescence staining showed that expression of functional XBP‐1 was weak in mature oocytes and at the one‐cell, two‐cell, and eight‐cell stages of embryos, but abundant at the GV oocyte, four‐cell, morula, and blastocyst stages. In addition, RT‐PCR revealed that both spliced XBP‐1 (XBP‐1s ) and unspliced XBP‐1 (XBP‐1u) were expressed at the GV oocyte, four‐cell, morula, and blastocyst stages. Tunicamycin (TM), an ER stress inducer, blocked porcine embryonic development at the four‐cell stage, exhibiting the effect on embryonic genome activation. Next, porcine embryos cultured in the presence of tauroursodeoxycholate (TUDCA), an ER stress inhibitor, were studied. Total cell numbers and the extent of the ICM increased (p<0.05), whereas the rate of nuclear apoptosis decreased (p<0.05). Moreover, expression of the anti‐apoptotic gene Bcl‐2 increased whereas expression of the pro‐apoptotic genes Bcl‐xl and p53 decreased. The results indicated that inhibition of ER stress enhanced porcine oocyte maturation and embryonic development by preventing ER stress‐mediated apoptosis in vitro.

This study was undertaken to evaluate the relationship between in vitro maturation and plasminogen activators (PAs) activity on porcine cumulus-oocytes complexes (COCs) exposed to oxidative stress. When COCs were cultured in maturation medium with hydrogen peroxide (H2O2), the proportion of the germinal vesicle breakdown (GVBD) and oocytes maturation were decreased with addition of H2O2, and were significantly (p<0.05) lower in medium with 0.1 mM H2O2 than control group. Also, the rate of degenerated oocytes was increased in as H2O2 concentration increased. When COCs were cultured for 48 h, three plasminogen-dependent lytic bands were observed: tissue-type PA (tPA); urokinase-type PA (uPA); and tPA-PA inhibitor (tPA-PAI). PA activity was quantified using SDS-PAGE and zymography. When H2O2 concentration was increased, tPA and tPA-PAI activities also increased in porcine oocytes cultured for 48 h, but not uPA. In other experiment, embryos were divided into three groups and cultured in (1) control medium, (2) control medium with 1.0 mM H2O2 and (3) control medium with 1.0 mM H2O2 along with catalase in concentrations of 0.01, 0.1, and 1.0 mg/ml, respectively. H2O2 decreased the rate of GVBD and maturation in porcine COCs but catalase revealed protective activity against oxidative stress caused by H2O2. In this experiment, tPA and tPA-PAI activities were higher in media with 1.0 mM H2O2 alone. Increasing concentration of catalase decreased tPA and tPA-PAI activities in porcine oocytes. These results indicate that the exposure of porcine follicular oocytes to ROS inhibits oocytes maturation to metaphase-II stage and increase the oocytes degeneration. Also, we speculated that increased ROS level may trigger tPA and tPA-PAI activities in porcine oocytes matured in vitro.

The in vitro maturation rate of vitrified-thawed canine oocytes was 30.8±3.4%. The in vitro maturation rate of vitrified oocytes was lower than that of the control (52.0±2.5%, p<0.05). The in vitro maturation rate of vitrified-thawed oocytes were significantly (p<0.05) lower than those of fresh oocytes. The in vitro maturation and developmental rates of the vitrified-thawed oocytes were 17.5±2.5% and 8.8±3.4%, respectively. This results were lower than the control group (43.6±3.2% vs 20.0±3.0%). SOD1 gene expression of 1～2 mm of follilce size were higher than those of above 6 mm follicle size. SOD2 gene expression of 1～2 mm of follicle size were significantly higher than those of above 6 mm follicle size (p<0.01). The expression pattern of SOD1, 2 was constantly expressed in both groups but strongly expressed in follicles (1～2 mm) group when compared to the above 6 mm follicles. SOD gene expression between groups the fresh and vitrified oocytes groups were significant differences in rates. However, RGS gene expression between groups the fresh and vitrified oocytes groups were no significant differences in rates.

The aim of this study was to investigate what protein(s) of porcine epididymal fluid (pEF) are able to enhance the nuclear maturation of porcine germinal vesicle (GV) oocytes in vitro. Proteins of pEF were fractionated by affinity, ion exchange, and gel filtration chromatography. Porcine cumulus-oocytes complexes (COC) from follicles were cultured in tissue culture medium (TCM 199) containing various fractions obtained by chromatography. Porcine COCs were also cultured in TCM 199 containing various meiosis inhibitors and pEF. After 24 or 48 h culture, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. When porcine COCs were cultured in the medium with meiosis inhibitor such as, dibutyryl cAMP (dbcAMP) and forskolin (Fo), more than 80% of oocytes were unable to resume meiosis. However, porcine COCs supplemented with pEF were able to overcome the inhibitory effect of dbcAMP and Fo. Maturation rate of oocytes was significantly (p<0.05) increased in the media supplemented with cationic protein(s) during in vitro maturation than in those with anionic protein(s) (44.1% vs 20.0%). When oocytes were cultured in the TCM 199 with fractions obtained by gel filtration, the maturation rate of oocytes was significantly (p<0.05) higher in fraction 11 containing 18 kDa than other fractions. The present study suggests that 1) dbcAMP and Fo prevent the spontaneous maturation of oocyte after isolation from follicles, and that pEF contain a substance(s) that improves meiosis resumption in vitro of porcine COCs, 2) cationic 18 kDa protein(s) are responsible for promotion of MⅡ stage.

The objective of this study was to evaluate the effects of co-culture of bovine oocytes with cumulus cells on in vitro maturation and development following in vitro fertilization in bovine oocytes. Bovine cumulus-oocyte complexes (COCs) and denuded oocytes (DO) were co-cultured with the cumulus cells in TCM199 for 20~22 hr, and evaluated the nuclear type of oocyte. After in vitro maturation, oocytes were coincubated for in vitro fertilization with frozen-thawed spermatozoa selected by 65% percoll in DM-Heparin and DM-Caffeine for 15~18 hr. Presumptive zygotes were cultured for 48 hr in CR1aa in vitro culture medium with 10% FBS, and evaluated the cleavage rates. The results confirmed that the highest percentage of metaphase II (M-II) stage was observed in COCs (30.1±3.5%, 24.2±1.8%) as compared to DO (7.1±1.3%, 17.4±13.9%) (p<0.05). In addition, the increased cleavage rates were obtained from COCs (69.6±2.1%, 75.6±2.9%) when compared to DO (21.6±7.5%, 29.5±12.6%) (p<0.05). In conclusion, this study suggested that cumulus cells secreted positive factors during in vitro maturation of oocytes and early embryonic development after in vitro fertilization of bovine oocytes.

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 ( and ) than in the control group ( and ). 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.

This study was carried out to assess the effect of vitamin E against the reactive oxygen species (ROS) on chemical activation of in vitro matured oocytes. Bovine oocytes were aspirated from slaughtered ovaries and transferred to maturation medium with or without vitamin E (). After 22 hours of culture, oocytes with polar bodies were selected and submitted to activation treatments with or without vitamin E. After activation, oocytes were cultured in mSOF medium and rate of development was monitored. For ROS () detection, in vitro matured and activated oocytes were selected and stained with DCFDA and observed under fluorescence microscope. The ROS contents were not significant differences in IVM rate, activation process and embryonic development to blastocysts with or without vitamin E. The cell number of blastocyst showed significant difference (p<0.05) in embryos matured and activated with vitamin E. The results of the present study demonstrated that the exposure of vitamin E in IVM and activation process improved the quality of embryos evaluated by the cell number of blastocysts.