Mitochondria are well known to regulate the mammalian embryo development. Recent studies showed that the mitochondrial dynamics responses are mainly generated through mitochondrial membrane potential (MMP) and cellular ATP production, which is dependent on mitochondrial reactive oxygen species (ROS). However, these mechanisms are unclear on development process of preimplantation porcine embryos. The aim of this study was to evaluate that difference of the mitochondrial dynamics-derived various functions on the embryo development according to lipid composition of zygote. First, zygote was classify two groups (high lipid, grade 1: G1 and low lipid, grade 2: G2) by lipid composition of cytoplasm. And, we performed the in vitro culture (IVC) using zygote of divided groups. The nuclei numbers and developmental rates of blastocysts were lower in G2 than those of G1 embryos. Next, we investigated the intracellular ROS and mitochondrial derived superoxide production in porcine embryos by using DCF-DA and Mito-SOX staining. As expected, both intracellular ROS and mitochondrial derived superoxide were significantly increased (p<0.05) in the preimplantation stage embryos of G2 group compared with G1 group. In addition, to observe difference of the mitochondrial functions, we investigated the mitochondrial membrane potential (MMP, ΔΨ) and contents of ATP in the preimplantation stage embryos by using JC-1 kit and ATP determination kit. These functions of mitochondria were dramatically reduced in cleavage stage embryos or blastocysts of G2 group. Finally, to verify the difference of the mitochondrial dynamics-derived various functions, we investigated the expressions of mitochondrial fission (Drp1, pDrp1-616) and fusion (Mfn1, Mfn2) factors by Western blotting analysis. Interestingly, the protein levels of pDrp1-616 in embryos of G1 group were continuously increased until blastocyst stage. Whereas, the expression patterns of Mfn1/2 in embryos of G2 group were significantly reduced during IVC progression. The expression patterns of mitochondria dynamic between the two groups were shown opposite. These results demonstrated that the lipid contents of zygote were related the positive-correlation with mitochondrial dynamics-derived functions in porcine embryos. Moreover, we suggest that lipid of zygote is play a important role on mitochondrial functions and dynamics during preimplantation embryos development in pigs.

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+.

During the oocyte maturation, antioxidants may be beneficial for futher developmental competence against reactive oxygen species (ROS) because the media for oocytes lack boiomolecules that serve as scavengers. In this study, N-acetyl-L-cysteine (NAC), N-acetyl-L-cysteine amide (NACA), glutathione (GSH) and cysteamime were compared to determine the effects of protection for ROS from GV to MII stage when supplemented during in vitro maturation (IVM) and in vitro culture (IVC) of bovine oocytes. NAC is one of well known ROS scavanger and NACA is modified form of NAC to help permeation into cytosolic area of oocytes. Significant improvement on the development undergoing blastocysts (32%, vs 18%, 22%) were found when cysteamine (0.1mM) was added to the maturation medium than NAC (0.3 mM), NACA (0.2mM) or GSH (0.5 mM) as compared to control medium with antioxydents. However, the addition of NAC(18%) or NACA(21%) to media did not improve the proportion of oocytes undergoing development to morula and blastocysts than control (24%) and GSH (26%). Our study showed that medium supplementation with cysteame during IVM and in vitro culture (IVC) improved the rate of bovine embryo development, in contrast to extracellular antioxidants like NAC, NACA and GSH that caused no improvement.

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.

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.

CDK2 inhibition plays a central role in DNA damage–induced cell cycle arrest and DNA repair. However, whether CDK2 also influences early porcine embryo development is unknown. In this study, we examined whether CDK2 is involved in the regulation of oocyte meiosis and early embryonic development of porcine. We found that disrupting CDK2 activity with RNAi or an inhibitor did not affect meiotic resumption or MII arrest. However, CDK2 inhibitor-treated embryos showed delayed cleavage and ceased development before the blastocyst stage. Disrupting CDK2 activity is able to induce sustained DNA damage as demonstrated by the formation of distinct γH2AX foci in nuclei of day 3- and day 5-embryos. Inhibiting CDK2 triggers a DNA damage checkpoint by activating of the ATM-P53-P21 pathway. However, the mRNA expression of genes involved in non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways for double strand break (DSB) repair reduced after administering CDK2 inhibitor to 5-day-old embryos. Furthermore, CDK2 inhibition caused apoptosis in day 7 blastocysts. Thus, our results indicate that an ATM-P53-P21 DNA damage checkpoint is intact in the absence of CDK2; however, CDK2 is important for proper repair of the damaged DNA by either directly or indirectly influencing DNA repair-related gene expression.

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) have a important role in influence of pre-messenger RNA (pre-mRNA) processing and mRNA metabolism and transportation in cells. Recently, hnRNP A2/B1 can recognize m6A modifications on pre-mRNA or pre-miRNA and affect alternative splicing and miRNA processing in HeLa Cells. However, roles of hnRNP A2/B1 in various cells and tissues, especially in elary embryo development, are unclear. Here, we investigated the temporal and spatial expression patterns of hnRNPA2B13 during mammalian early embryo development. In mouse, hnRNPA2B1 was localized at the nucleus after 1-cell stage, however, hnRNPA2B1 was expressed after 2-cell stage in pig. Then, knockdown of hnRNP A2/B1 induced by RNA interference (RNAi) was used to analyze the effect of hnRNP A2/B1 in preimplantation develop in pigs. Knockdown of hnRNP A2/B1 delayed embryo development. Interestingly, ICM marker OCT4 and Sox2 was significantly decreased in blastocyst stage. mRNA expression show that transcription factors which is Pou5f1, Sox2, Nanog, Cdx2 and AP2γwas decreased the transcription levels without the changing of junction protein, ZO-1, occludin, and CXADR. Outgrowth results indicated that knock-down of hnRNPA2B1 embryos cannot format the colony. Knock-down of Methyltransferase like 3(METTL3) embryos mislocalized the hnRNPA2/B1 at the nucleus. In summary, the expression patterns of hnRNPA2/B1 differ between mouse and porcine embryos, and these differences may reflect species-specific functions during preimplantation embryo development. Our results suggested that hnRNPA2/B1 is necessary for newly synthesis of mRNA related with transcription factor, and early embryo development by the RNA epigenetic modification.

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.

Unlike somatic cells mitosis, germ cell meiosis consists of two consecutive rounds of divisions that segregate homologous chromosomes and sister chromatids, respectively. The meiotic oocyte is characterized by an absence of centrioles and asymmetric divisions. Centriolin is a relatively novel centriolar protein that functions in mitotic cell cycle progression and cytokinesis. Here, we explored the function of centriolin in meiosis and showed that it was localized to meiotic spindles, and concentrated at the spindle poles and midbody during oocyte meiotic maturation. Unexpectedly, knockdown of centriolin in oocytes with either siRNA or Morpholino micro-injection, did not affect meiotic spindle organization, cell cycle progression, or cytokinesis (as indicated by polar body emission), but led to a failure of peripheral meiotic spindle migration; and symmetric division or large polar body emission. These data suggest that, unlike in mitotic cells, the centriolar protein centriolin does not regulate cytokinesis, but plays an important role in regulating asymmetric division of meiotic oocytes.

This study was conducted to examine the effects of activation methods on the ER stress induction and subsequent apoptosis and in vitro development of porcine parthenogenetic embryos. Porcine in vitro matured oocytes were activated by four activation methods; 1) electric stimulus(ES) with two DC pulses of 1.25 kV/cm, for 30 ㎲ (E), 2) ES + 10 μM Ca-ionophore (A23187) treatment for 5 min (EC), 3) ES + 2 mM 6-dimethylaminopurine treatment for 3 h (ED), or 4) ES + A23187 + 6-DMAP (ECD). After activation, parthenogenetic embryos were in vitro cultured in PZM-3 medium and sampled to analyze the x-box binding protein 1 (Xbp1) mRNA, ER stress-associated genes and apoptotic genes at 3 h post ES and the 1-cell and blastocyst stages. The un-spliced and spliced x-box binding protein 1 (Xbp1) mRNA were confirmed by RT-PCR. Also ER stress-associated genes, such as the C/EBP homologous protein (CHOP), binding protein (BiP), activating transcription factor 4 (ATF4) and glucose-regulated protein 94 (GRP94), and apoptotic genes were analyzed by real-time quantitative RT-PCR (RT-qPCR). The band intensities of spliced Xbp1 (Xbp1s) mRNA was higher in the EC group than other three groups at 3 h and the 1-cell stage, while it was higher in the ED groups compared with E group at the blastocyst stage. Four ER stress-associated genes were showed the highest expression in the EC group and weakly expressed in the ED group at 3 h. However, most of those genes were highly expressed in EC and ECD groups at the 1-cell and blastocyst stages with some variation. The expressions of Bcl-2-associated X protein (Bax) and caspase-3 mRNAs were significantly higher in EC group than other three groups at all stages. The developmental rate to the blastocyst stage was higher (p<0.05) in ED and ECD groups (32.1±3.8 to 34.6±2.2%) than that of E group (26.1±3.9%). These results suggest that the intracellular ER stress of parthenogenetic porcine embryos is affected by activation method and subsequently lead to the apoptosis of embryos.

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.

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.

Lysophosphatidic acid (LPA) is a member of the phospholipid autacoid family and has growth factor and hormone-like activities on various animal cells. In this study, we investigated the effect of LPA on porcine embryo development. Porcine parthenogenetic embryos were treated into various concentrations of 0 (control), 0.1, 1 and 10 μM LPA (0 LPA, 0.1 LPA, 1 LPA and 10 LPA) during in vitro culture for 7 days or cultured in basic culture medium until day 4 and treated LPA from day 4 to day 7. In the LPA treatment for culturing from day 0 to day 7, there was no significant difference on cleavage and blastocyst formation rate. In addition, the blastocyst development proportion which was classified as expanded, hatching, or hatched blastocystshas was no significant difference among all groups. In the LPA treatment for culturing from day 4 to day 7, 0.1 and 1 LPA groups were presented increased blastocyst formation compared to other groups, but cleavage rate and over-expanded blastocyst formation rate were not significantly different among all LPA treated groups. The total cell number was not different but apoptosis was reduced when 1 LPA treated from day 4 to day 7. The relative mRNA expression level of anti-apoptosis gene, BCL2L1 was higher and pro-apoptosis gene, BAK was lower in the 1 LPA treated group than the control. In comparison with the control and the 1 LPA treated group using time-lapse monitoring system, 1 LPA treated embryo was accelerated developmental speed via morula compaction and expanded blastocyst. The 1 LPA treated group significantly increased the relative expression levels of gap junction and tight junction related genes, GJD1, CDH1 and ZO-1 compared to the control. These results indicated that 1 μM LPA supplementation for culturing from day 4 to day 7 post activation is efficient in blastocyst formation and LPA may be helpful for embryo developmental capacity.

Allicin (AL) regulates the cellular redox, proliferation, viability, and cell cycle of different cells against extracellular-derived stress. This study was to investigate the effect of allicin treatment during in vitro maturation (IVM) on porcine oocyte maturation and developmental competence. Porcine follicular oocytes were cultured in 0 (control), 0.01, 0.1, 1, 10, and 100 μM AL added IVM media. The rate of polar body emission was higher in the 0.1 μM AL-treated group (74.5% ± 2.3%) than in the control (68.0% ± 2.6%). After parthenogenetic activation, the rates of cleavage and blastocyst formation were significantly higher in the 0.1 μM AL-treated group than in the control (p < 0.05). The reactive oxygen species level at metaphase II was not significantly different among all groups. In matured oocytes, the relative mRNA expression of both BAK and CASP3, and BIRC5 were significantly lower and higher, respectively, in the 0.1 AL-treated group than in the control. Also, the mRNA expression of BMP15 and cyclin B, and the activity of phospho-p44/42 MAPK, was significantly increased. These results indicate that supplementation of oocyte maturation medium with allicin during IVM improves the maturation of oocytes and the subsequent developmental competence of porcine oocytes.

PP2A-B55α, a regulatory subunit of PP2A plays an important roles in regulating cell proliferation and survival. However, the functions for PP2A-B55α in mouse early embryo development is not clear. The objective of present were to investigate the expression patterns and to explore its biological function during mouse early development. Thetranscripts of PP2A-B55α were detected at all developmental stages in mouse embryo and decreased during embryo development. Immunostaining revealed that PP2A-B55α was present in both the nucleus and cytoplasm in early cleavage stage embryos. In the late embryonic development, PP2A-B55α was predominantly located in the cytoplasm. Knockdown (KD) of PP2A-B55α using double strand RNA not affect the proportion of cleaved embryos, but resulted in significantly decreased development to blastocyst stage and reduced total cell number in blastocyst. KD PP2A-B55α is able to induce sustained DNA damage and reduced the transcripts of non-homologous end joining (NHEJ) or homologous recombination (HR) pathways relative genes in mouse early embryo. KD PP2A-B55αcaused apoptosis and increase the transcripts of pro-apoptotic genes in blastocyst. Furthermore, The KDPP2A-B55α showed significantly lower cell proliferating rates (from 5-Bromo-deoxyuridineassayresults) in blastocysts and to talareas of out growth potential was decreased. These observation provide novel in sight into PP2A-B55α expression patterns in mouse early embryos and down-regulation of PP2A-B55α negatively impacted blastocyst development, total cell number, DNA damage, apoptosis, and proliferation and post-hatchingevents.

Fatty acid synthesis (FASN) is an enzyme responsible for the de novo synthesis of long-chain fatty acids. During oncogenesis, FASN plays a role in growth and survival rather than acting within the energy storage pathways. Here, the function of FASN during early embryonic development was studied using its specific inhibitor C75. We found that the presence of the inhibitor reduced blastocyst hatching. FASN inhibition decreased Cpt1 expression, leading to a reduction in mitochondrial copy numbers and ATP content. This inhibition of FASN results in the down-regulation of the AKT pathway, thereby triggering apoptosis through the activation of the p53 pathway. Activation of the apoptotic pathways also leads to increased accumulation reactive oxygen species and autophagy. In addition, the FASN inhibitor can impair cell proliferation, a parameter of blastocyst quality for outgrowth. The level of OCT4, an important factor in embryonic development, decreased after treatment with the FASN inhibitor. These results show that FASN exerts an effect on the early embryonic development by regulation of both fatty acid oxidation and the AKT pathway in pigs.

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.

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.

Oocytes from small antral follicles (< 3 mm in diameter; SAFO) show lower developmental competence compared to those from medium antral follicles (3-8 mm in diameter; MAFO) in pigs. This study was designed to evaluate the effect of various macromolecules such as fetal bovine serum (FBS), porcine follicular fluid (PFF), bovine serum albumin (BSA) and polyvinyl alcohol (PVA) in in vitro growth (IVG) medium on oocyte growth, maturation, and embryonic development after parthenogenesis (PA). The base medium for IVG was α-MEM supplemented with dibutyryl cyclic AMP, pyruvate, kanamycin, hormone. This medium was further supplemented with 10% FBS, 10% PFF, 0.4% BSA, or 0.1% PVA. The in vitro maturation (IVM) medium was medium-199 supplemented with 10% PFF, cysteine, pyruvate, epidermal growth factor, kanamycin, insulin, and hormones. SAFO were cultured for 2 days for IVG and then cultured for 44 h for IVM. After IVG, the mean diameter of SAFO treated with FBS, PVA, and no IVG-MAFO (114.1, 113.0, and 114.8 μm, respectively) was significantly larger (P<0.01) than that of no IVG-SAF (111.8 μm). Oocyte diameter after IVM was greater (P<0.01) in SAFO treated with FBS, BSA and PVA (112.8, 112.9 and 112.6 μm, respectively) than other groups (110.4, 109.6, and 109.8 μm for no IVG-MAFO, no IVG-SAFO and PFF, respectively). Intraoocyte GSH content was not influenced by the macromolecules in IVG medium (0.92, 0.93, 1.05, and 1.12 pixels/oocyte for FBS, PFF, BSA and PVA, respectively). The proportion of oocytes reached the metaphase II stage was higher in PFF (73.6%) than in BSA (43.5%) and PVA (53.7%) but not different from that of FBS treatment (61.5%). The cumulus expansion score of oocytes after IVG was significantly influenced (P<0.01) by the macromolecules (2.94, 2.24, 1.84, and 1.38 for PFF, FBS, PVA, and BSA treatments, respectively). Blastocyst formation of PA oocytes that were treated with FBS (51.8%), PFF (50.4%), and PVA (45.2%) during IVG was higher (P<0.05) than that of BSA-treated oocytes (20.6%) but was not significantly different from that (54.8%) of no IVG-MAFO oocytes. Our results demonstrated that growth, maturation, and embryonic development of SAFO are greatly influenced by macromolecules in IVG medium and that PFF or FBS can be replaced with a chemically defined synthetic macromolecule PVA.