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.

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.

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.

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 plastic monomer bisphenol A (BPA) is well known as a representative environmental hormones. Recent studies showed that the BPA exposure induced mitochondrial dysfunction and mitochondrial derived reactive oxygen species (mito-ROS). However, changes of antioxidant enzymes expression and ROS production from mitochondria according to the BPA exposure on in vitro maturation (IVM) of porcine oocytes have not been studied. We hypothesized that regulation of ROS production from mitochondria by BPA may play a critical role in meiotic maturation or expansion of cumulus cells in cumulus-oocyte complexes (COCs). To investigate the negative effects of BPA exposure on oocyte maturation, immature pig oocytes were matured in NCSU-23 medium supplemented with BPA (50, 75 and 100 μM) for 44 h. Expectedly, the rates of meiotic maturation and cumulus cell expansion of COCs in the BPA (75 μM) treated group was significantly lower than those of control group (p<0.01). Most of secretion factors expressions from COCs were significantly decreased (p<0.05) in the BPA treated COCs. Next, we investigated the intracellular ROS and mitochondrial specific superoxide production according to the BPA exposure using DCF-DA and mito-SOX staining, respectively. BPA exposure were showed that increasing of both intracellular ROS and mito-ROS, as well as mitochondrial related antioxidant enzymes (sod2, prdx3, prdx5) mRNA expression significantly increased (p<0.01) in COCs. And then, mitochondria membrane potential (MMP) dramatically reduced, and mitochondrial-derived apoptotic factors (bax, bcl-xl, caspase 3) mRNA expressions were increased (p<0.01) in BPA treated COCs. In additon, protein levels of mitochondrial-derived apoptosis genes (AIF, cleaved parp1 and caspase 3) were significantly increased (p<0.05) by BPA exposure. To confirm the reduction of BPA-induced mito-ROS, we used to the mitochondrial-targeted ROS scavenger, mito-TEMPO. Interestingly, addition of mito-TEMPO (0.1 μM) to the BPA pre-treated COCs recovered in meiotic maturation of porcine oocytes. These results demonstrated that BPA exposure was induced increasing of mitochondrial dysfunction, mito-ROS and mitochondrial-mediated apoptosis on pig oocyte maturation. Therefore, we suggest that controlling of mito-ROS plays a critical role in pig oocyte maturation in vitro. These findings will be helpful to solve causes of mitochondrial-related infertility.

Melatonin has an important role as anti-oxidative effect and reducing of endoplasmic reticulum(ER)-stress on oocyte maturation and embryo development. Under ER-stress condition, unfolding protein response (UPR) is a defence mechanism in mammalian cells. Recently, regulation of UPR signaling genes are involved in oocyte maturation, embryo development and female reproduction. However, there is no report on the role of melatonin for UPR signaling and ER-stress mediated apoptosis during pig oocyte maturation progression. Moreover, the changes of UPR genes expression according to the porcine oocyte maturation is not yet fully understood. Here, we investigated the changes of UPR signal (BIP/GRP78, ATF4, p90/p50ATF6, and XBP1) and ER-stress apoptotic factor CHOP genes expressions in porcine oocyte maturation by Western blot and RT-PCR analysis. During oocyte maturation, UPR marker and CHOP genes expressions were significantly increased in matured oocytes or cumulus-oocyte complexes (COCs). UPR markers expressions were significantly increased by ER-stress inducer, tunicamycin (Tm), treated (1, 5, 10 μg/ml) groups in a dose-dependent manner compared with control group. To confirm the reducing of ER-stress by melatonin (0.1 μM), we were compared to the effects of ER-stress inhibitor, TUDCA (200 μM), after pre-treated Tm (5 μg/ml) for 22 h maturation. Expressions of UPR markers and meiotic maturation were recovered by melatonin (0.1 μM) in COCs. And, we observed the role of Grp78/Bip as UPR signaling beginning marker using siRNA. In result, reduction of Grp78/Bip gene expression by siRNA was induced the inhibition of oocyte maturation (32.5±10.1 vs control; 77.8±5.3), and p50ATF6 protein level was significantly decreased (p<0.001) in cultured COCs for 44 h. In addition, these results were recovered through the addition of melatonin (0.1 μM) or TUDCA (200 μM) in maturation medium. These results demonstrated that the regulation of UPR signaling via Grp78/Bip gene induction plays a critical role in porcine oocyte maturation in vitro. Furthermore, this present study first confirmed a functional link between inhibition effect of ER-stress by melatonin and regulating of UPR signaling in porcine oocyte maturation. In conclusion, melatonin improves the oocyte maturation and cumulus cells expansion of COCs through the regulation of UPR signal pathway by BIP/GRP78 against the ER-stress during porcine oocyte maturation periods.

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, and protein kinase B (PKB) expression and activation levels in matured CCs by western blotting. Each concentration (0, 1, 10, and 100 ng/ml) of GDF8 was treated in maturation medium (TCM199) while 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 each group of oocytes and CCs were sampled. To assess the effect of GDF8 on specific gene transcription level changes as a dose response during IVM, the realtime PCR was performed. In CCs, all of GDF8 treatment groups showed significantly higher CREB transcription regulator cbp mRNA and the 1- and 10 ng/ml treatment groups observed significantly increased cumulus expansion related genes areg, cox-2, has2, ptx3 and tnfaip6 transcription levels after IVM. In matured oocytes, the maternal factors jmjd3 and zar1, transcriptional regulator foxo1 and sirt1, mitochondrial activity factor sirt3 and acadl, and anti-apoptosis gene bcl-2 mRNA transcription levels were significantly increased in 1- and10 ng/mL of GDF8 treatment groups compared with control. To determine effect of GDF8 treatment during IVM, translation regulator PKB protein expression and phosphorylation levels were analyzed in CCs by western blotting. The 10 ng/ml treatment group showed significantly increased phosphorylated PKB (1.4 times higher than control) protein levels (P < 0.05). In conclusion, treatment 10 ng/ml of GDF8 during IVM activates CREB related transcription and induced cumulus cells expansion via activation of PKB signaling in CCs. The transcriptional landscape changes in CCs result maternal factors accumulation and mitochondrial activation in oocytes during IVM.

(-)-Epicatechin gallate (ECG) is a polyphenol compound of green tea exhibiting biological activities, such as antioxidant and anticancer effects. To examine the effect of ECG on porcine oocytes during in vitro maturation (IVM), oocytes were treated with 0-, 5-, 15-, and 25 μM ECG. After maturation, we investigated nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels and subsequent embryonic development after parthenogenetic activation (PA) and in vitro fertilization (IVF). After 42 hours of IVM, the 5 μM group exhibited significantly increased (p< 0.05) nuclear maturation (89.8%) compared with the control group (86.1%). However, the 25 μM group observed significantly decreased (p< 0.05) nuclear maturation (83.5%). In intracellular maturation assessment the 5-, 15-, and 25 μM groups had significantly increased (p< 0.05) GSH levels and decreased ROS levels compared with the controls. The 5- and 15 μM group showed significantly increased (p< 0.05) embryo formation rates and total cell number of blastocysts after PA (18% and 68.9, 15% and 85.1 vs. 12% and 59.5, respectively) compared with controls. Although the 25 μM group observed significantly lower blastocyst formation rates after PA (27.6% vs. 23.2%) than control group, the 5 μM group showed significantly increased blastocyst formation rates after PA (37.2% vs. 23.2%) compared to the control group. Furthermore, the 5 μM group measured significantly increased blastocyst formation rates (20.7% vs. 8.6%) and total cell number after IVF (88.3±1.5 vs. 58.0±3.6) compared to the control group. The treatment of 5 μM ECG during IVM affectively improved the porcine embryonic developmental competence by regulating intracellular oxidative stress during IVM.

Gangliosides exist in glycosphingolipid-enriched domains on the cell membrane and regulate various functions such as adhesion, differentiation, and receptor signaling. Ganglioside GM3 by ST3GAL5 enzyme provides an essential function in the biosynthesis of more complex ganglio-series gangliosides. However, the role of gangliosides GM3 in porcine oocytes during in vitro maturation and early embryo development stage has not yet understood clear. Therefore, we examined ganglioside GM3 expression patterns under apoptosis stress during maturation and preimplantation development of porcine oocytes and embryos. First, porcine oocytes cultured in the NCSU-23 medium for 44 h after H2O2 treated groups (0.01, 0.1, 1 mM). After completion of meiotic maturation, the proportion MII (44 h) was significantly different among control and the H2O2 treated groups (76.8±0.3 vs 69.1±0.4; 0.01 mM, 55.7±1.0; 0.1 mM, 38.2±1.6%; 1 mM, P<0.05). The expressions of ST3GAL5 in H2O2 treated groups were gradually decreased compared with control group. Next, changes of ST3GAL5 expression patterns were detected by using immunofluorescene (IF) staining during preimplantation development until blastocyst. As a result, we confirmed that the expressions of ST3GAL5 in cleaving embryos were gradually decreased (P<0.05) according to the early embryo development progress. Based on these results, we suggest that the ganglioside GM3 was used to the marker as pro-apoptotic factor in porcine oocyte of maturation and early embryo production in vitro, respectively. Furthermore, our findings will be helpful for better understanding the basic mechanism of gangliosides GM3 regulating in oocyte maturation and early embryonic development of porcine in vitro.

The present study assessed the effect of FSH and LH on oocyte meiotic, cytoplasmic maturation and on the expression level and polyadenylation status of several maternal genes. Cumulus-oocyte complexes were cultured in the presence of FSH, LH, or the combination of FSH and LH. Significant cumulus expansion and nuclear maturation was observed upon exposure to FSH alone and to the combination of FSH and LH. The combination of FSH and LH during entire IVM increased the mRNA level of four maternal genes, C-mos, Cyclin B1, Gdf9 and Bmp15, at 28 h. Supplemented with FSH or LH significantly enhanced the polyadenylation of Gdf9 and Bmp15; and altered the expression level of Gdf9 and Bmp15. Following parthenogenesis, the exposure of oocytes to combination of FSH and LH during IVM significantly increased cleavage rate, blastocyst formation rate and total cell number, and decreased apoptosis. In addition, FSH and LH down-regulated the autophagy gene Atg6 and upregulated the apoptosis gene Bcl-xL at the mRNA level in blastocysts. These data suggest that the FSH and LH enhance meiotic and cytoplasmic maturation, possibly through the regulation of maternal gene expression and polyadenylation. Overall, we show here that FSH and LH inhibit apoptosis and autophagy and improve parthenogenetic embryo competence and development.

It is well established that mammalian cumulus cell (CC) expansion requires BMP15 (bone morphogenetic protein bone morphogenetic protein 15) and GDF9 (growth differentiation factor 9). However, the mechanisms of the factors in CC expansion are largely unclear. This study was conducted to examine the two paracrine factors and their receptor SMAD intracellular signaling mechanism of mediating porcine CC expansion and oocyte maturation, and to compare COCs (Cumulus–oocyte complexes) maturation to DOs (Denuded oocytes). COCs and DOs were in vitro matured in medium with FSH, LH and TGFB superfamily antagonists. Our results showed that the expansion of COCs was unaffected by addition of GDF9 and BMP15 recombinant protein, but cumulus cell proliferation and DOs maturation rate were enhanced. The mRNA expressions of SMAD receptor confirmed that oocytes secreted factors that activate SMAD3,4 and SMAD1 in granulosa cells and oocytes, but unaffected SMAD2. Treatment of COCs with a SMAD2/3 phosphorylation inhibitor (SB431542) inhibited CC expansion and expression of TNFAIP6. SB431542 also was revealed to inhibit DOs maturation. The activation of CC SMAD signaling by oocytes, and the requirement of SMAD2/3 signaling for expansion and oocyte maturation were studied in pig. Nonetheless, porcine oocyte maturation without SMAD2/3 signaling is likely to be needed for optimal matrix formation, but also BMP15 and GDF9 is likely to be needed in oocyte.

The objective of the present study was to investigate the effects of different concentrations of sorbitol supplementation for in vitro maturation medium and in vitro culture medium, on porcine cumulus oocyte complexe(COC) maturation and subsequent developmental capacity after parthenogenetic activation. Porcine COC were cultured for 44 h(0～ 22 h termed MI stage and 22～44 h termed MII stage) in TCM199 without(— ) or with(+) sorbitol (20 μM, 100 μM, 200 μM), and the resultant metaphase II oocytes cultured in PZM-3 for 7 days following activation. Our results showed that supplementation with appropriate concentrations of sorbitol (20 μM) during full term maturation culture(MI+/MII+) significantly(p<0.05) improved blastocyst formation rates and total cell number. When the concentration of sorbitol were increased to 100 μM and 200 μM during maturation culture, the maturation rate of COC were significantly reduced compared with 20 μΜ or control groups. Also blastocyst formation rates significantly(p<0.05) reduced with increasing concentration of sorbitol(200 μM). Supplementation with sorbitol(20 μM, 50 μM, 100 μM) into PZM-3 for in vitro culture significantly(p<0.05) inhibited blastocyst formation compared with control group. However, the blastocyst formation rates start to rise again when 50 μ M sorbitol was used for the first 48 hours and then cultured in PZM-3 without sorbitol. There was no significant difference in cell number between control and sorbitol treated groups. When the activated oocytes were cultured in PZM-3 for 48h and then cultured in PZM-3 with sorbitol, interestingly, the blastocyst formation rate was similar to that of PZM-3 with sorbitol for in vitro culture and significantly lower than control group. These results suggest that addition of low concentrations of sorbitol(20 μM) during oocyte maturation is beneficial for subsequent blastocyst development and improved embryo quality. However, treatment with sorbitol supplementation during in vitro culture medium is negative effect to blastocyst formation.

In all the studies of mammalian species, chromatin in the germinal vesicle (GV) is initially decondensed with the nucleolus not surrounded by heterochromatin (the NSN configurations). During oocyte growth, the GV chromatin condenses into perinucleolar rings (the SN configurations) or other corresponding configurations with or without the perinucleolar rings, depending on species. During oocyte maturation, the GV chromatin is synchronized in a less condensed state before germinal vesicle breakdown (GVBD) in species that has been minutely studied. As not all the species show the SN configuration and gene transcription always stops at the late stage of oocyte growth, it is suggested that a thorough condensation of GV chromatin is essential for transcriptional repression. Because the GV chromatin status is highly correlated with oocyte competence, oocytes must end the NSN configuration before they gain the full meiotic competence and they must take on the SN or corresponding configurations to stop gene transcription before they acquire the competence for early embryonic development. In this study, we firstly investigated whether the follicle size could determine chromatin configuration in porcine oocyte. For this experiment, follicles was divided into three groups (<1 mm follicle, 1～3 mm follicle and 3～6 follicle). Using DAPI staining, the GV nucleolus and chromatin of porcine oocytes was classified into SN, SN-NSN and NSN configurations. MⅠ and M Ⅱ of three groups's Mature oocytes by staining was confirmed the configuration of chromatin. The maturation rate and parthenogenetic development potential were significant different between the SN and NSN configurations oocytes. These results indicated that chromatin changes in GV oocytes affect the development potential of porcine embryos.

Despite of the presence of estradiol-17β (E2) in ovarian follicles, its role(s) in in vitro maturation (IVM) is still largely unknown, especially in pigs. Thus, the current study was conducted to investigate the effect of E2 on in vitro maturation (IVM) of porcine oocytes and subsequent preimplantation development using in vitro fertilization (IVF)- or somatic cell nuclear transfer (SCNT)-derived embryos. To define the effects of E2 on IVM and early embryogenesis, porcine oocytes were matured in the presence or absence of E2, fertilized in vitro and cultivated to blastocyst stage. Compared to control group, the production of MII oocytes was significantly increased by treatment with E2, accompanying with the increase in MPF content and ERK phosphorylation, and monospermic fertilization and blastocyst development rates were also greatly elevated in the E2-treated oocytes. In addition, the advantageous role of E2 was also found in blastomere survival, which was further evidenced by both elevation of anti-apoptotic transcript Bcl-XL and decrease of pro-apoptotic transcript Bax. Furthremore, these positive effects of E2 were highly reproducible in early development of SCNT embryos. Collectively, the current study strongly suggests that E2 can be used as a efficient IVM supplement leading to successful nuclear/cytoplasmic maturatioin in pigs.

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.