The elevated temperature and high humidity has been known as main reason for heat stress on animals and cause detrimental effects on productivity of organisms and physiological conditions of normal bioactivities. The aims of this study were to evaluate the relationship between time of heat shock simulation during in vitro maturation and developmental competence of subsequent embryo after in vitro fertilization. Heat shocked cumulus-oocyte complexes (COCs) of Korean native cattle were subjected to normal conditions for 22, 21, 18 and 12 h respectively and transferred to heat stress inducing condition at 40.5 °C in other incubator for 0 (control), 1 and 4 h. After maturation for 22 h, the oocytes were fertilized and cultured in mSOF media for 8 d and examined the developmental capacity of embryos. There were no differences in maturation and cleavage rates between 0, 1 and 4 h heat socked oocytes, but blastocysts formation were lower in the 4 h heat stressed oocytes. The apoptotic cells of developed blastocysts were also increased in at day 8 with 4 h heat shocked oocytes. These results indicate that heat shock on oocytes during maturation could cause negative effects on the developmental competence of embryos.

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. The purpose of this study is to investigate the effects of GDF8 on porcine oocytes during in vitro maturation (IVM). We investigated a specific gene transcription levels in oocytes and cumulus cells (CC) after IVM by realtime PCR arry, and specific protein expression and activation levels in matured CCs by western blotting. Each concentration (0, 1, 10, and 100 ng/ml) of GDF8 was added in maturation medium (TCM199) during process of IVM. Data were analyzed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science). Data are presented as the mean and Differences were considered significant at P < 0.05. After 44 h of IVM, oocytes are mechanically denuded from CCs with 0.1% of hyaluronidase, and then the separated oocytes and CCs were sampled following each group. To assess the effect of GDF8 on specific gene transcription level changes as a dose response during IVM, the realtime PCR array was performed. In CCs the 1- and 10 ng/ml of GDF8 supplement group showed the transcription co-factors CBP and SP1, cell metabolic regulator MAPK1, and cumulus expansion related genes Has2, Cox-2, Ptx3 and Areg transcription levels were significantly distinguished with control when hierarchically clustered by Euclidean distance with average linkage method after IVM. In matured oocytes the 10- and 100 ng/ml of GDF8 supplement group showed the maternal factors JMJD3 and Zar1, transcriptional regulator FOXO1, Sirt1 and Sirt2, mitochondrial activity factor Sirt3, ACSL3 and ACADL, anti-apoptosis gene BCL-2, and oocyte secrete factor BMP15 mRNA transcription levels were significantly distinguished compared with control. To determine effect of GDF8 supplement during IVM, the GDF8 down steam canonical regulator SMAD2/3 protein phosphorylation levels analyzed in CCs by western blotting. The 10- and 100 ng/ml supplement groups showed significantly increase phosphorylated (P)-SMAD3 (1.56 and 1.34 times higher than control) protein levels (P < 0.05). In conclusion, supplement of GDF8 during IVM activates FOXO homolog transcription and induced cumulus cells expansion via activation of SMAD3 signaling in CCs. While process of IVM, the transcriptional landscape changes in CCs may consequently result maternal factors accumulation and mitochondrial activation in oocytes.

Ganglioside GT1b, glycosphigolipids with three sialic acid, is known to play an important role in signal transduction such as epidermal growth factor receptor (EGFR). EGF is also known to induce resumption of meiosis and cumulus cells expansion during porcine oocyte maturation. Therefore, this study was conducted to evaluate the effects of ganglioside GT1b on resumption of meiosis and cumulus cells expansion in porcine oocyte maturation. First, porcine cumulus-oocyte complexes were cultured in NCSU-23 medium supplemented with GT1b (0, 1, 2 and 4 μM) at 44 h. We observed that the proportion of the metaphase II (M II) stage was significantly increased in the 2 μM GT1b (78.0 ± 2.3) treated group than in the other groups. Furthermore, expression of cumulus cells expansion factor genes (Has2, TNFAIP6, Ptx3) were significantly increased in the 2 μM GT1b treated group than in the other groups. Next, we investigated the meiotic maturation and the expressions of cumulus cells expansion factor genes after GT1b and/or EGF treatment. The proportion of the M II stage was significantly higher in the GT1b+EGF (90.1 ± 2.3) treated group than in the other groups. Moreover, expressions of cumulus cells expansion factor genes were significantly increased in the GT1b+EGF treated group than in the control group. After in vitro fertilization, fertilization rate, preimplantation development competence and quality of blastocyst were improved in oocytes derived from GT1b+EGF treated group. Taken together, these results suggest that exogenous ganglioside GT1b improving the developmental competence of porcine embryos via increase of resumption of meiosis and cumulus cells expansion during in vitro maturation of porcine oocytes.

Several species show low sensitivity to IVM, and the development of optimized medium possible oocyte quality and stable growth. Furthermore, adding additive to the medium can effectively reducing development cost and leads to easy handling of oocytes. Isoliquritigenin and formononetin are extracts found in licorice. Previous studies reported that isoliquritigenin and formononetin affected the activity of sperm, but the oocytes are unknown. This study adds isoliquritigenin or formononetin to αMEM to mature oocytes under simple IVC conditions. Recovered oocytes are cultured in αMEM, isoliquritigenincontaining medium and formononetin-containing medium. In study we proved that in addition to the medium, above the quality of oocytes cultured when specific additives were added, more stable growth is possible. collection and IVM of oocyte. SD rats at 6 to 8 wks of age are injected is intraperitoneal with 30 IU/mL of PMSG and 48 hrs later, HCG 50 IU/mL is intraperitoneal injected. Oocytes are collected ovary after 17 hrs. Collected oocytes are cultured for 16 hrs with 200 μL αMEM and 200 μL αMEM containing isoliquritigenin or formononetin at 0, 0.01, 0.02, 0.04, 0.1 mg/mL. Also, isoliquritigenin and formononetin were mixed with 200 μL αMEM at a ratio of 0.25: 0.75, 0.50: 0.50, and 0.75: 0.25 mg/mL respectively. Oocytes supplemented with isoliquritigenin and Formononetin had high quality than oocytes cultured with αMEM and showed an increase in the IVF fertility rate. Our experimental results indicate that using isoliquritigenin, formononetin when cell culture, rather than used only in medium, more effective oocyte quality and stable growth.

In vitro maturation (IVM) systems have become indispensable for the production of large numbers of competent oocytes in domestic species. The quality of in vitro matured oocyte is one of the important factors determining the success of assisted reproductive technologies (ARTs) including intracytoplasmic sperm injection (ICSI), in vitro fertilization (IVF), and somatic cell nuclear transfer (SCNT) in human and livestock. Incomplete cytoplasmic maturation of oocytes can lead not only to a failure of fertilization but also to a developmental arrest after ARTs. Thus, establishment of a stable IVM system to produce a large number of high quality oocytes, especially in domestic animals, is essential for improvement of ARTs efficiency by producing high quality embryos. The morphological characteristics are commonly used to predict the developmental potential of oocytes and embryos. Usually, normal oocytes shrink when exposed to a hypertonic medium, and recover their morphology when returned to an isotonic medium. During this process, oocytes show various morphologic changes, such as shrinkage in spherical (SSP) or irregular shapes (SIR). In the first study, we investigated whether the shrinkage pattern of oocytes that was observed after hyperosmotic treatment could be used as a morphologic characteristic to predict the quality of IVM oocytes in pigs. We found that SSP oocytes showed improved developmental competence after PA and SCNT. This improved embryonic development was most likely because of the more advanced nuclear and cytoplasmic maturation in SSP oocytes compared with SIR oocytes. Pig oocytes shows a wide variation in the size of perivitelline space (PVS) after IVM. Based on this finding, we examined in the second study whether or not there was any correlation between the PVS size of IVM oocytes and their developmental competence after PA and SCNT. Our results demonstrated that in vitro developmental competence to the blastocyst stage positively correlated with the size of the PVS of oocytes after IVM. In addition, we observed that mature oocytes with a larger PVS showed higher levels of intracellular GSH content and transcription factor expression. Furthermore, enlargement of the PVS by culturing in reduced NaCl medium improves the embryonic development after PA and SCNT. In the third study, we investigated the effects of a hypotonic medium with reduced NaCl (61.6 mM) compared with an isotonic medium (108.0 mM NaCl) on oocyte maturation and embryonic development after PA and SCNT. In addition, we attempted to optimize our IVM system using a hypotonic maturation medium by examining the effects of hypotonic medium during various stages of IVM on oocyte maturation and subsequent embryonic development. Our results demonstrated that maturation of pig oocytes in hypotonic medium with reduced NaCl during the last 11 hr of IVM increased the developmental competence of oocytes after PA and SCNT. These beneficial effects was also shown in a commercial medium (a minimum essential medium; aMEM) in which the NaCl concentration was reduced to 61.6 mM. In addition, IVM of oocytes in medium with reduced NaCl increases the proportion of SSP oocytes in pigs. In summary, our results demonstrate that IVM of pig oocytes in a hypotonic medium with low-NaCl is better able to support embryonic development after PA and SCNT, most likely by improving the cytoplasmic maturation via increased intraoocyte GSH content and widened PVS. Based on these results, the newly developed IVM system using a hypotonic medium with reduced NaCl can produce high quality oocytes and be considered a new strategy for improving ARTs efficiency in pigs.

Ganglioside GD1a is specifically formed by the addition of sialic acid to ganglioside GM1a by ST3 β- galactoside α -2,3-sialyltransferase 2 (ST3GAL2). Above all, GD1a are known to be related with the functional regulation of several growth factor receptors, including activation and dimerization of epidermal growth factor receptor (EGFR) in tumor cells. The activity of EGF and EGFR is known to be a very important factor for meiotic and cytoplasmic maturation during in vitro maturation (IVM) of mammalian oocytes. However, the role of gangliosides GD1a for EGFR-related signaling pathways in porcine oocyte is not yet clearly understood. Here, we investigated that the effect of ST3GAL2 as synthesizing enzyme GD1a for EGFR activation and phosphorylation during meiotic maturation. To investigate the expression of ST3GAL2 according to the EGF treatment (0, 10 and 50 ng/ml), we observed the patterns of ST3GAL2 genes expression by immunofluorescence staining in denuded oocyte (DO) and cumulus cell-oocyte-complex (COC) during IVM process (22 and 44 h), respectively. Expression levels of ST3GAL2 significantly decreased (p<0.01) in an EGF concentration (10 and 50 ng/ml) dependent manner. And fluorescence expression of ST3GAL2 increased (p<0.01) in the matured COCs for 44 h. Under high EGF concentration (50 ng/ml), ST3GAL2 protein levels was decreased (p<0.01), and their shown opposite expression pattern of phosphorylation-EGFR in COCs of 44 h. Phosphorylation of EGFR significantly increased (p<0.01) in matured COCs treated with GD1a for 44 h. In addition, ST3GAL2 protein levels significantly decreased (p<0.01) in GD1a (10 μM) treated COCs without reference to EGF pre-treatment. These results suggest that treatment of exogenous ganglioside GD1a may play an important role such as EGF in EGFR-related activation and phosphorylation in porcine oocyte maturation of in vitro.

The oocyte undergoes various events during In vitro maturation (IVM) and subsequence development. One of the events is production of reactive oxygen species (ROS) that is a normal process of cell metabolism. But imbalances between ROS production and antioxidant systems induce oxidative stress that negatively affect to mammalian reproductive process. In vitro environments, In vitro matured oocytes have many problems, such as excessive production of ROS and imperfect cytoplasmic maturation. Therefore, In vitro matured oocytes still have lower maturation rates and developmental competence than in vivo matured oocytes. In order to improve the IVM and In vitro culture (IVC) system, antioxidants, vitamins were added to the IVM, IVC medium. Antioxidant supplementation was effective in controlling the production of ROS and it continues to be explored as a potential strategy to overcome mammalian reproductive disorders. Based on these studies, we expect that the use of antioxidants in porcine oocytes could improved maturation and development rates.

본 연구는 방사무늬김(Pyropia yezoensis) 사상체의 생장에 대한 단색광의 영향을 연구하기 위해 blue light(480 ㎚), green light(550 ㎚), yellow light(600 ㎚) and red light(730 ㎚)에서 방사무늬김(P. yezonesis)의 유리 사상체를 배양한 후 colony 직경, chlorphyll a 함량, 각포자낭 형성률을 측정하여 생장특성을 비교 분석하였다. 그 결과, colony 직경과 chlorophyll a 함량이 480(blue light) ㎚에서 각각 2,472.6±27.0 ㎛ and 1.55±0.03 mg g.dw-1로 최대값이 나타났고 각포자낭 형성률 역시 37.87±1.08 %로 blue light에서 최대값으로 확인되었다. 따라서 blue light는 다른 단색광(green, yellow, red light)에 비해 김 사상체의 성장 및 성숙을 촉진하는 데에 효과적으로 작용하며, 본 연구결과 는 향후 김의 실내 양식 기술 개발에 활용될 수 있을 것이라 사료된다

‘Palbangmi’ is a super high-yield rice cultivar bred by interspecies cross between Tongil type and Africa rice. But, ‘Palbangmi’ has a problem in grain maturation which results in rice yield decreases. Because ‘Palbangmi’ is Tongil and Africa rice interspecies-crossed cultivar, it would be predicted to require higher temperature for grain maturation than japonica rice. Therefore, ‘Palbangmi’ would be required to determine Late Marginal Heading Date (LMHD) for normal maturation. In this study, experiment of transplanting date was carried out to determine LMHD of ‘Palbangmi’ in Suwon and Iksan for 3 years. When daily mean temperature was below 23oC, grain filling ratio (%) increased about 8% for each 1oC. On the other hands, grain filling ratio (%) didn't increase when the daily mean temperature was above 23oC. Therefore, it is determined that 23oC is adequate for minimum threshold temperature for grain maturation. The LMHD of ‘Palbangmi’ was determined from calculated heading dates for each year which could satisfy threshold temperature for 40 days after heading during last 15 years. As a results, LMHD was earlier than August 1 in 16 regions where it is inadequate for ‘Palbangmi’. Continually, LMHD was August 1 ~ August 5 in 13 regions, August 6 ~ August 10 in 14 regions and August 11 ~ August 15 in 21 regions. Finally, LMHD was later than August 15 in 14 regions. Considering that heading date of ‘Palbangmi’ is August 13 as mid maturing ecotype, it is suggested that earlier transplanting required for normal maturing in almost areas except south plain area and some areas of central plain area.

Crocin is a carotenoid that may protect cells against oxidative stress by scavenging free radicals particularly superoxide anions. It has been reported that oocyte maturation is influenced by the free radicals generated during in vitro culture (IVC) process. The objective of study was to examine the effect of crocin in in vitro maturation (IVM) medium as an antioxidant on oocyte maturation and embryonic development after parthenogenesis (PA). Cumulus-oocyte complexes (COCs) were collected from ovaries of prepubertal gilts. The basic medium for IVM was medium-199 containing 10% pig follicular fluid, cysteine, pyruvate, epidermal growth factor, kanamycin, insulin, and hormones. Oocytes were treated for 44 hours with crocin at 0, 25, 50, and 100 μg/ml during IVM. Oocytes reached the metaphase II stage were induced for PA and cultured for 7 days in porcine zygote medium-3. Nuclear maturation of oocytes was not influenced by various concentrations of crocin (89.0, 87.3, 84.3, and 94.1% for control, 25, 50, and 100 μg/ml crocin, respectively). IVM oocytes treated with 50 μg/ml crocin showed a higher (P<0.05) intraoocyte glutathione (GSH) contents than untreated oocytes (1.00 vs. 1.29 pixels/oocyte). Blastocyst formation of PA embryos treated with 50 (42.9%) and 100 μg/ml crocin (43.8%) was significantly higher (P<0.05) than oocytes treated with 25 μg/ml crocin (30.5%) but not different from that (35.2%) of untreated oocytes. In summary, crocin increases cytoplasmic maturation in terms of intraoocyte GSH content which may be beneficial for later embryonic development by protecting from harmful effect of reactive oxygen species. Further studies are needed to determine whether the beneficial effect of crocin treatment during IVC would be shown in embryonic development after in vitro fertilization and somatic cell nuclear transfer.

In mature oocytes, maturation promoting factor (MPF) activity is playing important roles in arrest at M-phase and its continuous phenomenon, oocyte aging. In most mammals, metaphase II oocytes show high MPF activity and have been used as ooplasts in somatic cell nuclear transfer (SCNT). Caffeine has been found to regulate MPF activity in mammalian oocytes. Caffeine inhibits p34cdc2 phosphorylation and increases MPF activity. The present study investigated the effects of caffeine treatment during last 4 hours of in vitro maturation (IVM) on oocyte maturation and embryonic development after parthenogenesis (PA) and SCNT. The IVM medium was medium-199, 10% (v/v) PFF, cysteine, pyruvate, epidermal growth factor, kanamycin, insulin, and hormones. Immature oocytes were matured in IVM medium without or with 2.5 mM caffeine during the last 4 hours of IVM. The in vitro culture medium for embryonic development was porcine zygote medium-3 containing 0.3% (w/v) bovine serum albumin. Nuclear maturation (83.6–87.2%) and intraoocyte glutathione contents (0.9–1.0 pixels/oocyte) of oocytes were not influenced by the caffeine treatment. The membrane fusion of cell-cytoplast couplets (75.5–76.5%) and cleavage (85.4–86.2%) were also not altered by the caffeine treatment. However, caffeine-treated oocytes showed higher (P<0.05) blastocyst formation after SCNT (47.5 vs. 34.3%) than untreated oocytes. Our results demonstrate that caffeine treatment during last 4 hour of IVM improves the developmental competence of SCNT embryos probably by influencing MPF activity.

Crocin is a carotenoid that may protect cells against oxidative stress by scavenging free radicals particularly superoxide anions. It has been reported that oocyte maturation is influenced by the free radicals generated during in vitro culture (IVC) process. The objective of study was to examine the effect of crocin in in vitro maturation (IVM) medium as an antioxidant on oocyte maturation and embryonic development after parthenogenesis (PA). Cumulus-oocyte complexes (COCs) were collected from ovaries of prepubertal gilts. The basic medium for IVM was medium-199 containing 10% pig follicular fluid, cysteine, pyruvate, epidermal growth factor, kanamycin, insulin, and hormones. Oocytes were treated for 44 hours with crocin at 0, 25, 50, and 100 μg/ml during IVM. Oocytes reached the metaphase II stage were induced for PA and cultured for 7 days in porcine zygote medium-3. Nuclear maturation of oocytes was not influenced by various concentrations of crocin (89.0, 87.3, 84.3, and 94.1% for control, 25, 50, and 100 μg/ml crocin, respectively). IVM oocytes treated with 50 μg/ml crocin showed a higher (P<0.05) intraoocyte glutathione (GSH) contents than untreated oocytes (1.00 vs. 1.29 pixels/oocyte). Blastocyst formation of PA embryos treated with 50 (42.9%) and 100 μg/ml crocin (43.8%) was significantly higher (P<0.05) than oocytes treated with 25 μg/ml crocin (30.5%) but not different from that (35.2%) of untreated oocytes. In summary, crocin increases cytoplasmic maturation in terms of intraoocyte GSH content which may be beneficial for later embryonic development by protecting from harmful effect of reactive oxygen species. Further studies are needed to determine whether the beneficial effect of crocin treatment during IVC would be shown in embryonic development after in vitro fertilization and somatic cell nuclear transfer.

Mitochondrial dysfunction is found in oocytes and transmitted to the offspring due to maternal obesity. This is curable by endoplasmic reticulum (ER) stress inhibitors such as salubrinal (SAL). Recently pigs are considered as a model animal for biomedical research due to its physiological similarity with human. Pig oocytes have shown ER stress mostly in metaphase II stage. ER stress is hindering the in vitro embryo production (IVP). This study investigated the effect of ER stress inhibition by using SAL during 44 h of in vitro maturation (IVM) of oocytes at 1, 10, 50 and 100 nM concentrations. Firstly, we defined the concentration of SAL during IVM of pig oocytes. SAL at 10 nM showed higher (44.2 to 55.6%, P<P0.05) development competence to the blastocyst state than control and other concentrations after parthenogenetic activation (PA). Secondly, we sorted out the time-dependent treatment at 10 nM of SAL for IVM of oocytes. It revealed that treatment with SAL during 22 to 44 h and 0 to 44 h of IVM improved PA embryonic development significantly (40.5, 51.7 and 60.2% for control, 22 to 44 h and 0 to 44 h of IVM, respectively, P<0.05). Glutathione (GSH) level is an indicator of cytoplasmic maturation of oocytes. Reactive oxygen species (ROS) have a harmful effect on development competence of oocytes. For this, we determined the intraoocyte levels of GSH and ROS after 44 h of IVM. It was found that SAL increased intraoocyte GSH level and also decrease ROS level (P<0.05). Finally, we performed somatic cell nuclear transfer (SCNT) after treating oocytes with 10 nM SAL during IVM. SAL treatment significantly improved blastocyst formation of SCNT embryos compared to control (24.7 vs. 39.6%, P<0.05). Our results indicate that treatment of pig oocytes with ER stress inhibitor SAL during IVM improves preimplantation development cloned pig embryos by influencing cytoplasmic maturation in terms of increased GSH content and decreased ROS level in IVM pig oocytes.

Although in vitro production (IVP) techniques of porcine follicular oocytes have progressed and are well studied, the developmental potential of porcine oocytes matured in vitro remains low compared with those matured in vivo. It is well known that one of the reason occurred impair in vitro maturation (IVM) of porcine oocytes is the oxidative stress. Oxidative stress is mainly caused by reactive oxygen species (ROS) generation formed during cellular metabolism. β-cryptoxanthin (BCX) is one of the carotenoid pigment and possesses strong anti-oxidative and free radical scavenging activities and suppresses lipid peroxidation and nitrogen oxide production. The objective of this study was to examine the effects of BCX treatment on porcine oocyte during IVM and their in vitro developmental potential. The follicular oocytes were cultured in IVM medium supplemented with 0, 0.1, 1, 10 and 100 μM BCX (control, 0.1 B, 1 B, 10 B and 100 B). In analysis of intracellular ROS expression level after IVM, 1 B group was the lowest among all groups (p<0.05), while other BCX treated groups are similar to control group. Also, 1 B group was significantly decreased during the classified oocyte maturation stage (GVBD, MⅠ and MⅡ) than control (p<0.05). In addition, the relative mRNA expression level of antioxidant gene (superoxide dismutase-2 and peroxiredoxin-5) was significantly higher in 1 B group than control (p<0.05). After parthenogenetic activation, there was no different in the cleavage rate between two groups, however, the blastocyst formation rate was significantly higher in 1 B group than in control (p<0.05). In embryo quality, the total cell number and DNA fragmentation of blastocysts were no different between two groups. These results demonstrate that BCX is helpful for decreasing ROS level of porcine follicular oocytes and improves their developmental potential.

For useful research animal to study human’s disease and for xenotransplantation donor, pig was studied to improve the quality of in vitro production (IVP). But, still the developmental ability of in vitro porcine embryos is still lower than in vivo embryos. Using a antioxidant is one of the strategy to overcome the drawback of in vitro producted embryos by protecting the oocyte from free radicals during in vitro maturation (IVM). Resveratrol, one of the plant-derived polyphenol antioxidants, have been used as effective antioxidants. Therefore, resveratrol treatment during IVM of porcine oocytes is expected to improve efficiency of the IVP by reducing free radical accumulation. In this study, we designed control (no treated) and resveratrol treatment groups (0, 2 and 4uM), evaluated maturation rate, cleavage rate, blastocyst formation rate and total cell number. Additionally GSH and ROS accumulation levels were measured via staining oocytes. In the results, maturation rate had not shown significant difference among the groups. However, in further development, not only the results of cleavage rate (0uM : 84.64±2.65 vs 4uM : 93.67±2.36, p<0.05) and blastocyst formation rate (0uM : 6.39± 0.90, vs 4uM : 13.67±2.32, P<0.05) were significantly increased in 4uM resveratrol treated group, and result of total cell number (0uM : 22.47±0.76 vs 2, 4uM : 30.35±1.76, 27.65±1.23, P<0.05) also shown significant difference in 2, 4uM resveratrol groups with control. GSH accumulated levels of matured oocytes in resvetrol treated groups were significantly higher than control. Meanwhile ROS levels of treated groups were significantly reduced [GSH (0uM : 142±10.49 vs 2, 4uM : 163.2±3.29, 169.7±0.94, P<0.05), ROS (0uM : 170.2±7.76 vs 2, 4uM : 118.6±7.90, 130±7.07, P<0.05)]. From these results, we conclude the treatment of resveratol improved further development of porcine embryos by regulating intracellular GSH, ROS levels during porcine IVM. Therefore, exogenous antioxidants such as a resveratol can be supportive substances for obtaining the improved quality of IVP.

In the present study, we examined potential roles of glucose and pyruvate in nuclear and cytoplasmic maturation of porcine oocytes. In the presence and absence of 10% porcine follicular fluid (PFF), either 5.6 mM glucose or 2mM pyruvate effect on meiotic maturation and followed development ability. However, DOs doesn't take full advantage of the glucose in medium, only pyruvate can increase MII rate and follow early embryo development ability significance. COCs were matured with 200 uM pentose phosphate pathway (PPP) inhibitor (dehydroepiandrosterone, DHEA) or 2 μM glycolysis inhibitor (iodoacetate, IA), significantly lower levels of GHS in the DHEA an IA treated oocytes and the levels of ROS were higher significantly in the DHEA treated oocytes, treatment with DHEA significantly reduced the intra-oocyte ATP and NADPH level. Blastocysts from DHEA or IA treated group also presented higher apoptosis levels, meanwhile, the percentage of proliferating cells was dramatically lower than the non-treated group. In conclusion, our results suggest that 10% PFF promoted oocytes make full use of energy, glucose metabolism during in vitro maturation inseparable from the cumulus cells, PPP and glycolysis promoted porcine oocytes cytoplasmic maturation by supplying energy and reducing oxidative stress.

Spindlin1(Spin1), a meiotic spindle-binding protein that is highly expressed in cancer cells. Spindle-binding was dependent on its phosphorylation status, which was partially regulated by Mos/MAP kinase pathway. Nevertheless, the biologic roles of Spin1 in oocytes maturation are still largely unknown. For exploring the function of Spin1 in porcine oocyte maturation, Knockdown and overexpression methods were employed to the present study. Spin1 mRNA were enriched in maternal stages, from germinal vesicle - to 2 cell - stage, but sharply decreased after 4 cell stage, zygotic genome activation. Protein of SPIN1 was localized in spindle-chromatin complex during the metaphase I and metaphase II stages. Knockdown of Spin1 did not affect the first polar body extrusion, however, Spin1 depletion caused mitotic spindle defects, chromosome instability and pronuclear formation in metaphase II stage. Percentage of 2cell, 4cell embryos and blastocyst formation were significantly reduced in knockdown group compared with control, but cell numbers in blastocyst were no difference between control and knockdown groups. Another hand, Oocyte failed to maturation and induced metaphase I arrest following Spin1 over-expression. In conclusion, Spin1 is involved in the spindle formation and maintenance during oocytes meiotic maturation in pigs.

The periods of elevated temperature and high humidity has been longer since last ten years and cause problems in program of artificial insemination or at the efficiency of in vitro production of transferable embryos. The aims of this study were to evaluate the relationship between time of heat shock (0, 1, 2 and 4), during in vitro maturation and developmental competence of subsequent embryo after in vitro fertilization. The develpmentat rate and percetage of apoptotic cells were evaluated on matured oocyte and day 8. 41℃ Heat treatment after IVM culture significantly decreased the developmental capacity of IVF embryos. Also the number of apoptotic cell in COCs, morula and blatostcysts was started to increase at 2 hr heat treatment but did not affect on the rate of maturation. These results indicate that heat treatment for 2 to 4 hr at 41℃ have negative effects on maturation rate of COCs and lower the developmental competence of heat shocked oocyte derived embryos.