난자의 성숙과정과 노화에 관한 이해는 인공수정과 체외수정 최적기를 판단하기 위하여 가장 중요한 연구내용으로 알려져 있다. 이러한 기작은 번식 호르몬들에 의하여 조절되는 것으로 알려져 있으나 난자 세포질 변화에 관한 내용은 잘 알려져 있지 않다. 본 연구에서는 산화질소물(nitric oxide, NO)이 난자 성숙과정에서 증가하는 것을 밝혔으며 난자의 미성숙단계(germinal vesicle stage, GV)와 난자핵막붕괴단계(germinal vesicle breakdown, GVBD) 및 성숙완료단계(metaphase II, MII)단계에서 생산되는 NO의 양을 비교하였다. 또한, 난자를 체외에서 배양할 때, MII단계로 성숙되지 않는 성장 단계의 난자에서는 NO의 증가 현상을 관찰할 수 없었고, 세포질이 불균일한 노화된 난자에서는 NO가 증가된 상태로 유지되는 특성이 있음을 밝혔다. 이러한 결과는 NO의 작용이 난자의 성숙과정과 난자 노화과정에서 중요한 기능을 담당하고 있음을 보여주고 있다.

In vitro maturation (IVM) of oocytes is the procedure where the immature oocytes are cultivated in a laboratory until they are mature. Since IVM oocytes generally have low developmental competence as compared to those matured in vivo, development of an optimal IVM culture system by fine-tuning culture conditions is crucial to maintain high quality. In-depth knowledge and a deep understanding of the in vivo physiology of oocyte maturation are pre-requisites to accomplish this. Within ovarian follicles, various signaling pathways that drive oocyte development and maturation regulate interaction between oocytes and surrounding somatic cells. This review discusses the sonic hedgehog (SHH) signaling pathway, which has been demonstrated to be intimately involved in folliculogenesis and oocyte maturation. Advances in elucidating the role of the SHH signaling pathway in oocyte maturation will aid attempts to improve the current inferior in vitro oocyte maturation system.

Camel (camelus dromedarius) is a unique large mammalian species that can survive harsh environmental conditions and produce milk, meat, and wool. Camel reproduction is inferior when compared to other farm animal species such as cattle and sheep. Several trials have been reported to increase camel reproduction and production through assisted reproductive techniques (ARTs) such as in vitro fertilization and cloning. For these reasons, obtaining enough mature oocytes is a cornerstone for ARTs. This demand would be improved by the oocyte in vitro maturation (IVM) systems. In this review, the current approaches and views from different laboratories using ARTs and the IVM to produce embryos in vitro in camel species. For the last two decades, conventional IVM system was the common approach, however, recently the bi-phasic IVM system has been introduced and showed promising improvement in IVM of camel oocytes. Detailed studies are needed to understand camel meiosis and IVM to efficiently increase the production of this species.

This study aimed to recover the ovarian function through in vitro culture of preantral follicles from aged mice. First, we isolated the preantral follicles from ovaries of sixty-seven-week old B6D2F1 mice with decreased fecundity to know how many follicles were present in them, which was 6 preantral follicles including 2 primary, 2 early secondary and late secondary follicles from 8 aged mice. It was confirmed that a few follicles (~2) were present in aged mice through histological analysis compared to adult mice as control. The 9 days of in vitro culture of preantal follicles showed in vitro growth and induced maturation after treatment with hCG (2.5 IU/mL) and EGF (5 ng/mL). Cumulus cells in the cumulus-oocyte complexes (COCs) were removed using hyaluronidase and oocytes at the germinal vesicle (GV) and GV breakdown (GVBD) were obtained from preantral follicle culture of aged mice in vitro. In conclusion, these observations demonstrated that there still were a few preantral follicles in the ovaries of 67 week-old mice, which we were able to culture in vitro and oocytes were obtained from them. This study proposed an in vitro culture system using preantral follicle as a therapeutic strategy for fertility preservation in humans for assisted reproductive medicine.

Morphology of cumulus-oocyte-complexes (COCs) at germinal vesicle (GV) stage as one of the evaluation criteria for oocyte maturation quality after in vitro maturation (IVM) plays important roles on the meiotic maturation, fertilization and early embryonic development in pigs. When cumulus cells of COCs are insufficient, which is induced the low oocyte maturation rate by the increasing of reactive oxygen species (ROS) in porcine oocyte during IVM. The ROS are known to generate including superoxide and hydrogen peroxide from electron transport system of mitochondria during oocyte maturation in pigs. To regulate the ROS production, the cumulus cells is secreted the various antioxidant enzymes during IVM of porcine oocyte. Our previous study showed that Mito-TEMPO, superoxide specific scavenger, improves the embryonic developmental competence and blastocyst formation rate by regulating of mitochondria functions in pigs. However, the effects of Mito-TEMPO as a superoxide scavenger to help the anti-oxidant functions from cumulus cells of COCs on meiotic maturation during porcine oocyte IVM has not been reported. Here, we categorized experimental groups into two groups (Grade 1: G1; high cumulus cells and Grade 2: G2; low cumulus cells) by using hemocytometer. The meiotic maturation rate from G2 was significantly (p < 0.05) decreased (G1: 79.9 ± 3.8% vs G2: 57.5 ± 4.6%) compared to G1. To investigate the production of mitochondria derived superoxide, we used the mitochondrial superoxide dye, Mito-SOX. Red fluorescence of Mito-SOX detected superoxide was significantly (p < 0.05) increased in COCs of G2 compared with G1. And, we examined expression levels of genes associated with mitochondrial antioxidant such as SOD1, SOD2 and PRDX3 using a RT-PCR in porcine COCs at 44 h of IVM. The mRNA levels of three antioxidant enzymes expression in COCs from G2 were significantly (p < 0.05) lower than COCs of G1. In addition, we investigated the anti-oxidative effects of Mito-TEMPO on meiotic maturation of porcine oocyte from G1 and G2. Meiotic maturation and mRNA levels of antioxidant enzymes were significantly (p < 0.05) recovered in G2 by Mito-TEMPO (0.1 μM, MT) treatment (G2: 68.4 ± 3.2% vs G2 + MT: 73.9 ± 1.4%). Therefore, our results suggest that reduction of mitochondria derived superoxide by Mito-TEMPO may improves the meiotic maturation in IVM of porcine oocyte.

Ganglioside GM3 is known as an inhibition factor of cell differentiation and proliferation via inhibition of epidermal growth factor receptor (EGFR) phosphorylation. Our previous study showed that the exogenous ganglioside GM3 reduced the meiotic maturation of porcine oocytes and induced apoptosis at 44 h of in vitro maturation (IVM). However, the role of ganglioside GM3 in the relationship between EGFR signaling and apoptosis during porcine oocyte maturation has not yet been studied. First, porcine cumulus-oocyte complexes (COCs) were cultured in the NCSU-23 medium with exogenous ganglioside GM3 according to maturation periods (non-treated, only IVM I: 0 - 22 h, only IVM II: 22 - 44 h and IVM I & II: 0 - 44 h). We confirmed that the proportion of germinal vesicle breakdown (GVBD) increased significantly in the IVM I treated group than in the control group. We also confirmed that the meiotic maturation until M II stage and polar body formation decreased significantly in the only IVM I treated group. Cumulus cell expansion and mRNA levels of the expansion-related factors (HAS2, TNFAIP6 and PTX3) decreased significantly in the IVM I treated group than in the control group. Protein levels of EGFR, p-EGFR, ERK1/2, and p-ERK1/2 decreased significantly in the GM3-treated groups, during the IVM I period. In addition, cellular apoptosis, determined using TUNEL assay, and protein levels of Cleaved caspase 3, were increased significantly in the GM3-treated COCs during the IVM I period. Based on these results, ganglioside GM3 exposure of porcine COCs during the IVM I period reduced meiotic maturation and cumulus cell expansion via inhibition of EGFR activity in pigs.

Sex hormones including progesterons, androgens, and estrogens are influential in differentiation of ovarian tissues and competence of fertility. These steroid hormones derived from cholesterol are required for cumulus-oocyte complexes(COCs) during oocyte maturation. COCs is a total functional and active entity playing a central role in oocyte. Lipid metabolism in the mammalian COCs is controlled by environmental factors. The intracellular cholesterol contents go through remarkable changes. It plays an important part of oocyte developmental competence. However, heat stress affects steroid hormone by decreasing progesterone, estrogen concentrations, and resumption of meiosis in COCs maturation. Reduction of the hormone and meiotic resumption might lead to the decline of ovarian function, follicle maturation, and subsequent embryogenesis. In the same vein, heat stress also influence on germinal vesicle breakdown, lipolytic variations, and loss of the nuclear envelope in the course of maturation of oocytes. In summary, we examined the effects of thermal stress on oocyte maturation through steroid hormone contents of change identifying the molecular mechanisms of lipids metabolism. It may have the solution to further the therapy methods for disorders regarding sterility.

In general, the shape of cumulus-oocyte-complexes (COCs) at germinal vesicle (GV) stage is important roles on meiotic maturation of porcine oocyte during in vitro maturation (IVM). Then, mitochondria produce reactive oxygen species (ROS) such as superoxide from electron transport system during oocyte maturation. ROS levels on oocytes are regulated by various antioxidant enzymes in cumulus cells (CCs). However, the effect of mitochondria derived superoxide production from CCs during IVM of porcine oocyte has not been reported. Firstly, we divided groups according to large number of CCs (Grade 1: G1) and small number of CCs (Grade 2: G2). Then, we counted cumulus cells of G1 and G2 oocyte by using haemocytometer. The oocyte maturation rate was significant decreased (p < 0.05) in G2 oocytes than that of G1 oocytes. We measured mitochondria derived superoxide in G1 and G2 COCs by using Mito-SOX staining. Mitochondrial superoxide was higher in G2 COCs than G1 COCs. Then, the mRNA expression levels of antioxidant enzymes (SOD1, SOD2 and PRDX3) in G2 COCs were decreased compared to G1 COCs. To reduce mitochondria derived superoxide, we used Mito-TEMPO as mitochondrial superoxide scavenger. Oocyte maturation rates in both G1 and G2 groups treated with Mito-TEMPO were increased than that of non-treated groups. Mitochondrial superoxide was lower in G1 and G2 groups treated with Mito-TEMPO than that of non-treatment groups. The mRNA expression levels of antioxidant enzymes in G1 and G2 COCs treated with Mito-TEMPO were increased compared to non-treated groups. Based on these findings, we suggest that reduction of mitochondria derived superoxide by Mito-TEMPO assists maturation competence in porcine oocytes.

U0126 is a highly selective inhibitor of both MEK1 and MEK2, a type of MAPK/ERK kinase. This study was conducted to evaluate the effect of U0126 treatment during in vitro maturation (IVM) on nuclear maturation, intra-oocyte glutathione content, and embryonic development after parthenogenesis (PA). U0126 (5 μM) was supplemented to IVM medium during the first 0 (control), 2, and 4 h. The basic medium used for IVM was medium-199 supplemented with 10% (v/v) porcine follicular fluid (standard), 0.6 mM cysteine, 0.91 mM pyruvate, 75 μg/ml kanamycin, and 1 μg/ml insulin. Immature pig oocytes were matured for 44 h and then oocytes reached metaphase II stage were electrically activated to induce PA. The in vitro culture medium for embryonic development was porcine zygote medium-3 containing 0.3% (w/v) fatty acid-free BSA. When immature oocytes were treated with U0126 during the first 0, 2, 4 h of IVM culture, nuclear maturation was significantly (P < 0.05) increased by the U0126 treatment for 4 h (96.2 ± 1.3%) compared to standard IVM (90.6 ± 2.1%). Cleavage of PA embryos was significantly increased by 4 h- treatment (90.6 ± 2.2%) compared to standard medium (83.9 ± 1.8%). In addition, blastocyst formation of PA embryos was significantly (P < 0.05) increased by the treatment for 4 h (55.8 ± 5.7%) compared to 2 h (38.1 ± 6.1%). The glutathione contents in IVM oocytes were not altered by the U0126 treatments for 0, 2, and 4 h (1.28 ± 0.10, 1.16 ± 0.09, and 1.10 ± 0.09, respectively). Our results demonstrated that 5 μM U0126 treatment during the first 4 h of IVM showed positive effects on nuclear maturation, cleavage, and embryonic development in pigs.

The aim of this study was to investigate the role of Src homology 2-containing phosphotyrosine phosphatase SHP2 in intricate signaling network invoked by oocyte to achieve cytoplasmic maturation and also blastocyst development. Activation of SHP2 regulates multicellular differentiation, proliferation and survival through numerous signal pathways. The most prominent pathway is RAS/PI3K and p-AKT signaling cascade, as a result mitogenic effect become enhanced. Oocytes were cultured in cisplatin an anticancer drug, but selective activator of SHP2 and our grouping were SOF medium alone, SOF + EGF, SOF + CISPLATIN 0.3 μM, and SOF + EGF + CISPLATIN 0.3 μM. We evaluated that EGF neutralizes the apoptotic effect of cisplatin as well as maintain the high expression of SHP2, as a result blastocyst development become boosted up. We also found that inhibition of SHP2 with its specific inhibitor PHPS1 5 μM decreases the blastocyst development and neutralizes growth factors effect. The developmental ability and quality of bovine embryos were determined by assessing their cell number, gene expression, immunofluorescence, and immunoblot. The differences in embryo development between experimental groups were analyzed by one-way ANOVA. Our results show that SHP2 have significant effect on MAP kinase pathways which expand the cumulus cells during oocyte maturation and blastocyst development as compare to inhibition of SHP2 with PHPS1. SHP2 not only transduce the signaling of epidermal growth factor but it also has a role in signal transduction of FGF and IGF. The expression of ERK, PI3K/p-AKT and mTOR was increased with EGF, but with the treatment of SHP2 inhibitor the expression of these genes become drop done. So we can conclude from these results that SHP2 is important for oocyte maturation as well as for blastocyst development.

This study was designed to determine the effect of monosodium glutamate (MSG) on in vitro maturation (IVM) of oocytes and early development of parthenogenesis (PA) embryos in pigs. Each IVM and IVC medium was supplemented with various concentrations (0, 0.1, 0.5 and 5 mM) of MSG and non-essential amino acids (NEAA) depending on the experimental design. Immature pig oocytes were matured for 44 h and then oocytes reached metaphase II (MII) stage were electrically activated to induce parthenogenesis (PA). When immature oocytes were treated with MSG in the absence of NEAA during IVM, nuclear maturation (83.1-87.1%), intra-oocyte glutathione content, cumulus expansion, and cleavage (91.4-93.4%) of PA embryos were not influenced by MSG treatment at all concentrations. However, blastocyst formation of PA embryos was significantly increased by 5.0 mM MSG (45.3 ± 6.2%) compared to control (25.6 ± 3.4%). MSG treatment during IVM in the presence of NEAA did not show significant effect on nuclear maturation of oocytes and blastocyst formation after PA while 0.5 mM MSG (89.3 ± 1.9%) decreased (P < 0.05) cleavage of PA embryos compared to 0.1 mM MSG (94.6 ± 1.1%). When PA embryos were treated for 7 days with MSG during IVC, 5.0 mM MSG significantly decreased blastocyst formation (27.8 ± 4.9%) compared to no treatment (41.4 ± 1.9%) while no decrease in blastocyst formation was observed in 0.1 and 0.5 mM (37.4 ± 3.4% and 34.4 ± 2.6%, respectively). Our results demonstrated that 5 mM MSG in a NEAA-free chemically defined maturation medium showed positive effect on PA embryonic development while 5 mM MSG treatment during IVC was deleterious to PA embryonic development in pigs.

Mitochondrial dysfunction is found in oocytes and transmitted to offspring due to maternal obesity. Treatment of obese mothers with endoplasmic reticulum (ER) stress inhibitors such as salubrinal (SAL) can reverse the mitochondrial dysfunction and result in normal embryonic development. Pig oocytes have also shown ER stress mostly in metaphase II stage. ER stress in oocytes may hinder the in vitro production of pig embryos. This study investigated the effect of ER stress inhibition by SAL treatment during in vitro maturation (IVM) of porcine oocytes at 1, 10, 50 and 100 nM concentrations. Firstly, we tested various concentrations of SAL. SAL at 10 nM showed higher (P < 0.05) developmental competence to the blastocyst stage (55.6%) after parthenogenesis (PA) than control (44.2%) while not different from other concentrations (49.2, 51.6, and 50.8% for 1, 50, and 100 nM, respectively). Secondly, we performed time-dependent treatment at 10 nM of SAL for IVM of oocytes. It revealed that treatment with SAL during 22 to 44 h of IVM significantly improved PA embryonic development to the blastocyst stage compared to control (40.5, 46.3, 51.7 and 60.2% for control, 0 to 22 h, 22 to 44 h and 0 to 44 h of IVM, respectively, P < 0.05). Glutathione (GSH) content is an indicator of cytoplasmic maturation of oocytes. Reactive oxygen species (ROS) have a harmful effect on developmental 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 decreased 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 (39.6% vs. 24.7%, P < 0.05). Our results indicate that treatment of pig oocytes with ER stress inhibitor SAL during IVM improves preimplantation development PA and cloned pig embryos by influencing cytoplasmic maturation in terms of increased GSH content and decreased ROS level in IVM pig oocytes.

The objective of this study was to determine the effect of fructose that was supplemented to a chemically defined in Vitro maturation (IVM) medium on oocyte maturation and embryonic development after parthenogenesis in pigs. The base medium for in Vitro maturation (IVM) was porcine zygote medium (PZM) that was supplemented with 0.05% (w/v) polyvinyl alcohol (PVA) or 10% (v/v) porcine follicular fluid (pFF). In the first experiment, when immature pig oocytes were matured in a chemically defined medium that was supplemented with 5.5 mM glucose or with 1.5, 3.0 and 5.5 mM fructose, 3.0 mM fructose resulted in a higher nuclear maturation (91.5%) than 1.5 and 5.5 mM fructose (81.9 and 81.9%, respectively) but showed a similar result with 5.5 mM glucose (94.2%). However, there was no significant differences among groups in the embryo cleavage (89.4-92.4%), blastocyst formation (37.5-41.1%), and mean cell number of blastocyst (30.8-34.2 cells). Fructose at the concentration of 3.0 mM (1.08 pixels/oocyte) resulted in a higher intra-oocyte glutathione (GSH) content than 1.5 and 5.5 mM fructose (1.00 and 0.87 pixels/oocytes, respectively) while the cumulus cell expansion was not influenced. In the second experiment, effect of individual and combined supplementation of a chemically defined maturation medium with 5.5 mM glucose or 3.0 mM fructose was examined. No significant effect was found in the nuclear maturation (86.3-92.6%). Embryo cleavage was significantly increased by the combined supplementation with glucose and fructose (95.2%) compared to that with 3.0 mM fructose only (85.7%) while blastocyst formation (37.3-42.8%) and embryonic cell number (33.3-34.1 cells) were not altered. Effect of supplementation of pFF-containing medium with glucose and fructose + glucose was examined in the third experiment. No significant effect by the supplementation with glucose and fructose or glucose alone was observed in the nuclear maturation of oocytes (90.7-94.1%) and blastocyst formation (51.0-56.5%). Our results demonstrate that 3.0 mM fructose was comparable to 5.5 mM glucose in supporting in Vitro oocyte maturation and embryonic development after parthenogenesis and could be used as an alternative energy source to glucose for in Vitro maturation of pig oocytes.

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.

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.

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.

Zinc (Zn2+) is one of essential factors during mammalian oocyte maturation and fertilization. Previous studies showed that depletion of cellular Zn by metalion chelator impair asymmetric division of oocyte. But the detailed mechanism of these phenomena is unclear. We found that depletions of zinc by cell-permeable heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) caused the decrease of cytoplasmic actin mesh level. Spire2-GFP is co-localized with zinc at the cortex and intracellular vesicle. By the treatment of TPEN, number of Spire2-GFP decorated vesicle is drastically decreased, indicating that Zn2+is essential for the localization of the spire in mouse oocyte. Two putative zinc-binding regions were located in the C-terminal part of Spire2. Mutations of zinc binding site on spire abolish its localization at the intracellular vesicle. Over expression of C-terminal region containing zinc binding site of spire impair oocyte maturations and decrease cytoplasmic actin mesh. Taken together, these results suggest that intracellular zinc is crucial for the proper localizations of spire in the mouse oocyte, and unraveling the novel regulatory mode of actin nucleator spire by Zn2+.