Transducin-like enhancer protein 1(TLE-1) is protein associated with cell proliferation. This study analyzed change of TLE-1 mRNA expression during in vivo and in vitro maturation in porcine oocytes. Oocytes and granulose cells were collected from follicles of <2 mm, 2～6 mm and >6 mm in diameter in slaughtered pig’s ovaries. Oocytes collected from follicles of 2～6 mm in diameter were used after in vitro maturation for 0, 10, 20 and 44 h. Cumulus cells and granulose cells were collected after treatment with hyaluronidase. In results, TLE-1 mRNA expression in oocytes collected from follicle >6 mm in diameter is increased, TLE-1 RNA expression in cumulus cells and granulosa cells from follicles <2 mm, 2 mm 6 mm and >6 mm in diameter. However, there is no significant difference. On the other hand, TLE-1 mRNA expression from oocytes cultured for 10 h and 44 h is increased, TLE-1 mRNA in cumulus cells cultured for 10 h is significant increased(p<0.05) than other culture periods. In conclusion, these results show that TLE-1 is expressed in all cell types of oocytes, cumulus cells and granulose cells, and associated with oocyte maturation.

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.

In the present study, effects of concentration and time of culture in presence of roscovitine on nuclear maturation and meiotic spindle configuration, chromosomal alignment were examined in porcine oocytes. In experiment 1, porcine cumulus oocyte complexes (COCs) were cultured at in a 5% atmosphere in North Carolina State University 23 (NCSU-23) supplemented with 25, 50, 75 or roscovitine for 22 h and then were cultured for additional 22 h after removal of roscovitine. Nuclear maturation and morphology of the meiotic spindle and chromosomal alignment were examined to determine the optimal concentration of roscovitine in oocyte maturation. In experiment 2, COCs were cultured in NCSU-23 supplemented with roscovitine for 17, 20, 27 or 42 h and then an additional 22 h without roscovitine was followed to determine the optimal time of culture. The optimal concentration of roscovitine to arrest and resume meiosis of porcine oocyte was by examining nuclear status (p<0.05) and normal spindle and chromosome configuration. The optimal time of culture in presence of roscovitine to arrest meiosis of porcine oocyte was 17 h (p<0.05), although MII rates and normal morphology of the meiotic spindle and chromosomal alignment were not significantly different among various times of culture. In conclusion, the optimal concentration and time of culture in presence of roscovitine to arrest porcine oocytes are and 17 h, respectively.

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.

These study was carried out to investigate the effects of the recovery time, diameter of oocytes on in vitro fertilization or intracytoplasmic sperm injection (ICSI). The in vitro maturation rates to MII stage of oocytes recovered at the inactive, follicular and luteal stages matured for 72 h were 1.4±0.0%, 43.4±3.2% and 10.8±2.7%, respectively. The fertilization rates of in vitro cultured oocytes recovered from ovaries at the in active, follicular and luteal stages were 0.0±0.0%, 15.7±3.4% and 7.6±3.5%, respectively. The in vitro maturation rate of oocytes recovered from ovaries at the follicular stage of the reproductive cycle was significantly higher than those at the inactive and luteal stages (p<0.05). The penetration rate determined that the percentages of oocytes with diameters in the <100 μm, 100 to 100 μm and 110 to 120 μm ranges were 17.5±4.7%, 43.9±4.5%, 21.3±3.4%, respectively. The penetration rate of oocytes with diameters between 100 to 110 μm was significantly higher than that of oocytes whose diameters were 100< μm and 110～120 μm (p<0.05). The penetration rate of oocytes determined that the percentages of ovaries with diameters between 1 to 5 mm and 6 to 10 mm were 32.9±3.2% and 17.5±3.7%, respectively. Thus, the diameters of the ovaries were significantly higher at 1 to 5 mm (p<0.05). A total of 264 oocytes were fixed and stained after co-incubation with sperm, of which 72 had identifiable nuclear material. After in vitro fertilization for 20 hrs, 27.3% of oocytes were penetrated by spermatozoas. Oocytes were fixed and stained after ICSI, of which 38 oocytes contained identifiable nuclear material. After in vitro fertilization and ICSI for 20 hrs, to 27.3% and 67.9% of oocytes were penetrated by spermatozoas. The in vitro fertilization rates by ICSI was significantly higher than that in vitro fertilization method (p<0.05).

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.

Correlations between cumulus cells and germinal vesicle (GV) chromatin configuration were examined in porcine oocytes. Cumulus-oocyte complexes (COCs) were collected from 2～6 mm follicles and divided into three categories according to cumulus cell morphology. "A" group was compacted COCs with more than three cumulus cell layers. "B" group was COCs with less cumulus cell layers than "A" group. "C" group was COCs with one or less layer of cumulus cells. Cumulus cells were removed 0.1% hyaluronidase, and denuded oocytes were stained with Hoechst 33342. GV chromatin configuration was classified into GV-Con and GV-Dis. GV-Con meant that a nucleus was surrounded by condensed chromatin in a ring. GV-Dis meant that filamentous chromatin clumps were distributed in nucleus. The proportion (80.2%) of GV-Con in "A" group was significantly higher than "B" (62.0%) or "C" (44.9%). The proportion (55.1%) of GV-Dis in "C" group was significantly higher than "A" (19.8%) or "B" (38.0%). The meiotic competence of COCs was examined after 44 h culture. The proportion (90.0%) of oocytes reaching to metaphase II (M-II) in "A" group was significantly higher than "B" (76.5%) or "C" (45.5%). In conclusion, oocytes with good quality cumulus cell layers are synchronized early GV stage, and early GV stage is important for meiotic competence in pigs.

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.

The purpose of this study was to investigate the effects of the collection time, co-culture and sperm penetration of canine oocytes on in vitro maturation and fertilization. The oocytes were cultured in TCM-199 media containing hormonal supplements (10% FCS, 10 IU/ml HCG, 10 IU/ml PMSG) at 5% CO2, 95% air, 38℃. The in vitro maturation rate to MⅡ stage of in vitro oocytes recovered from ovaries that collected at follicular, luteal and inactive phases of the reproductive phase for 44～72 hrs were 19.2%, 12.2%, and 6.0%, respectively. Follicular phases oocytes had a significantly higher in vitro maturation rate than oocytes collected at luteal and anestrus stage (p<0.05). The in vitro maturation rates to the MII stage of canine oocytes after 48 hrs of culture with glutathione, pyruvate, or glutathione + pyruvate were 12.5%, 10.7%, and 17.5%, respectively. This was higher than that in both alone or the combination of the two compared to the control group (19.0%). The sperm penetration rates of in vitro matured oocytes by fresh and frozen semen were 29/80 (36.3%) and 18/80 (22.5%), respectively. Although there are limited reports about canine oocytes co-culture and in vitro fertilization, our results on in vitro maturation is comparable to the results from other researches.