Expression of the melatonergic system during meiotic maturation of cynomolgus monkey cumulus-oocyte complexes.

BACKGROUND: Melatonin is a multifunctional hormone synthesized in the pineal gland and peripheral reproductive tissues that regulates many biological processes. There is increasing evidence for a role of melatonin in oocyte maturation and embryonic development in various mammals. However, no study has reported evidence for the existence of melatonergic system, such as melatonin synthesis enzymes, melatonin membrane receptors, or melatonin binding sites in non-human primate cumulus-oocyte complexes (COCs). METHODS: Reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry were performed to detect transcripts and proteins of the rate-limiting enzyme in melatonin synthesis (arylalkylamine N-acetyltransferase, AANAT), melatonin membrane receptors (MT1 and MT2), and a melatonin binding site (NRH: quinone oxidoreductase 2, NQO2) in cynomolgus monkey COCs. RESULTS: RT-PCR analyses revealed the presence of AANAT, MT1, MT2, and NQO2 transcripts in granulosa cells, germinal vesicle (GV)- and metaphase II (MII)-stage cumulus cells, and oocytes. Immunocytochemistry revealed the presence of AANAT, MT1, MT2, and NQO2 proteins in GV- and MII-stage COCs. CONCLUSIONS: Our results provide the first evidence for the existence of the rate-limiting enzyme required for melatonin synthesis, melatonin membrane receptors, and a melatonin binding site in non-human primate COCs.

Enhancement of target specificity of CRISPR-Cas12a by using a chimeric DNA-RNA guide.

The CRISPR-Cas9 system is widely used for target-specific genome engineering. CRISPR-Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR-Cas12a that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR-Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.

Induction of autophagy protects against extreme hypoxia-induced damage in porcine embryo.

In the mammalian female reproductive tract, physiological oxygen tension is lower than that of the atmosphere. Therefore, to mimic in vivo conditions during in vitro culture (IVC) of mammalian early embryos, 5% oxygen has been extensively used instead of 20%. However, the potential effect of hypoxia on the yield of early embryos with high developmental competence remains unknown or controversial, especially in pigs. In the present study, we examined the effects of low oxygen tension under different oxygen tension levels on early developmental competence of parthenogenetically activated (PA) and in vitro-fertilized (IVF) porcine embryos. Unlike the 5% and 20% oxygen groups, exposure of PA embryos to 1% oxygen tension, especially in early-phase IVC (0-2 days), greatly decreased several developmental competence parameters including blastocyst formation rate, blastocyst size, total cell number, inner cell mass (ICM) to trophectoderm (TE) ratio, and cellular survival rate. In contrast, 1% oxygen tension did not affect developmental parameters during the middle (2-4 days) and late phases (4-6 days) of IVC. Interestingly, induction of autophagy by rapamycin treatment markedly restored the developmental parameters of PA and IVF embryos cultured with 1% oxygen tension during early-phase IVC, to meet the levels of the other groups. Together, these results suggest that the early development of porcine embryos depends on crosstalk between oxygen tension and autophagy. Future studies of this relationship should explore the developmental events governing early embryonic development to produce embryos with high developmental competence in vitro.

SPAM1/HYAL5 double deficiency in male mice leads to severe male subfertility caused by a cumulus-oocyte complex penetration defect.

The glycosylphosphatidylinositol-anchored sperm hyaluronidases (Hyals), sperm adhesion molecule 1 (SPAM1) and HYAL5, have long been believed to assist in sperm penetration through the cumulus-oocyte complex (COC), but their role in mammalian fertilization remains unclear. Previously, we have shown that mouse sperm devoid of either Spam1 or Hyal5 are still capable of penetrating the COC and that the loss of either Spam1 or Hyal5 alone does not cause male infertility in mice. In the present study, we found that Spam1/Hyal5 double knockout (dKO) mice produced significantly fewer offspring compared with wild-type (WT) mice, and this was due to defective COC dispersal. A comparative analysis between WT and Spam1/Hyal5 dKO epididymal sperm revealed that the absence of these 2 sperm Hyals resulted in a marked accumulation of sperm on the outside of the COC. This impaired sperm activity is likely due to the deficiency in the sperm Hyals, even though other somatic Hyals are expressed normally in the dKO mice. The fertilization ability of the Spam1/Hyal5 dKO sperm was restored by adding purified human sperm Hyal to the in vitro fertilization medium. Our results suggest that Hyal deficiency in sperm may be a significant risk factor for male sterility.-Park, S., Kim, Y.-H., Jeong, P.-S., Park, C., Lee, J.-W., Kim, J.-S., Wee, G., Song, B.-S., Park, B.-J., Kim, S.-H., Sim, B.-W., Kim, S.-U., Triggs-Raine, B., Baba, T., Lee, S.-R., Kim, E. SPAM1/HYAL5 double deficiency in male mice leads to severe male subfertility caused by a cumulus-oocyte complex penetration defect.

Exposure of Triclosan in Porcine Oocyte Leads to Superoxide Production and Mitochondrial-Mediated Apoptosis During In Vitro Maturation.

While triclosan (TCS) exerts detrimental effects on female reproduction, the effect of TCS-derived toxins on porcine oocytes during in vitro maturation (IVM) is unclear. This study investigated the effects of TCS on mitochondrion-derived reactive oxygen species (ROS) production and apoptosis pathways during porcine oocyte maturation. Porcine oocytes were treated with TCS (1, 10, and 100 µM) and triphenylphosphonium chloride (Mito-TEMPO; 0.1 µM), and matured cumulus oocyte complexes (COCs) were stained with orcein, dichlorofluorescein diacetate (DCF-DA), and Mito-SOX. Proteins and mRNA levels of factors related to cumulus expansion and mitochondrion-mediated apoptosis and antioxidant enzymes were analyzed by western blotting and reverse-transcription polymerase chain reaction (RT-PCR), respectively. Meiotic maturation and cumulus cell expansion significantly decreased for COCs after TCS treatment along with an increase in mitochondrial superoxide levels at 44 h of IVM. Further, mitochondrion-related antioxidant enzymes and apoptosis markers were significantly elevated in porcine COCs following TCS-mediated oxidative damage. The protective effect of Mito-TEMPO as a specific superoxide scavenger from TCS toxin improved the maturation capacity of porcine COCs. Mito-TEMPO downregulated the mitochondrial apoptosis of TCS-exposed porcine COCs by reducing superoxide level. In conclusion, our data demonstrate that TCS mediates toxicity during porcine oocyte maturation through superoxide production and mitochondrion-mediated apoptosis.

Chaetocin Improves Pig Cloning Efficiency by Enhancing Epigenetic Reprogramming and Autophagic Activity.

Efficient epigenetic reprogramming is crucial for the in vitro development of mammalian somatic cell nuclear transfer (SCNT) embryos. The aberrant levels of histone H3 lysine 9 trimethylation (H3K9me3) is an epigenetic barrier. In this study, we evaluated the effects of chaetocin, an H3K9me3-specific methyltransferase inhibitor, on the epigenetic reprogramming and developmental competence of porcine SCNT embryos. The SCNT embryos showed abnormal levels of H3K9me3 at the pronuclear, two-cell, and four-cell stages compared to in vitro fertilized embryos. Moreover, the expression levels of H3K9me3-specific methyltransferases (suv39h1 and suv39h2) and DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) were higher in SCNT embryos. Treatment with 0.5 nM chaetocin for 24 h after activation significantly increased the developmental competence of SCNT embryos in terms of the cleavage rate, blastocyst formation rate, hatching rate, cell number, expression of pluripotency-related genes, and cell survival rate. In particular, chaetocin enhanced epigenetic reprogramming by reducing the H3K9me3 and 5-methylcytosine levels and restoring the abnormal expression of H3K9me3-specific methyltransferases and DNA methyltransferases. Chaetocin induced autophagic activity, leading to a significant reduction in maternal mRNA levels in embryos at the pronuclear and two-cell stages. These findings revealed that chaetocin enhanced the developmental competence of porcine SCNT embryos by regulating epigenetic reprogramming and autophagic activity and so could be used to enhance the production of transgenic pigs for biomedical research.

Butylparaben Is Toxic to Porcine Oocyte Maturation and Subsequent Embryonic Development Following In Vitro Fertilization.

Parabens are widely used in personal care products due to their antimicrobial effects. Although the toxicity of parabens has been reported, little information is available on the toxicity of butylparaben (BP) on oocyte maturation. Therefore, we investigated the effects of various concentrations of BP (0 µM, 100 µM, 200 µM, 300 µM, 400 µM, and 500 µM) on the in vitro maturation of porcine oocytes. BP supplementation at a concentration greater than 300 µM significantly reduced the proportion of complete cumulus cell expansion and metaphase II oocytes compared to the control. The 300 µM BP significantly decreased fertilization, cleavage, and blastocyst formation rates with lower total cell numbers and a higher rate of apoptosis in blastocysts compared to the control. The BP-treated oocytes showed significantly higher reactive oxygen species (ROS) levels, and lower glutathione (GSH) levels than the control. BP significantly increased the aberrant mitochondrial distribution and decreased mitochondrial function compared to the control. BP-treated oocytes exhibited significantly higher percentage of γ-H2AX, annexin V-positive oocytes and expression of LC3 than the control. In conclusion, we demonstrated that BP impaired oocyte maturation and subsequent embryonic development, by inducing ROS generation and reducing GSH levels. Furthermore, BP disrupted mitochondrial function and triggered DNA damage, early apoptosis, and autophagy in oocytes.

Effect of Oocyte Quality Assessed by Brilliant Cresyl Blue (BCB) Staining on Cumulus Cell Expansion and Sonic Hedgehog Signaling in Porcine during In Vitro Maturation.

Brilliant cresyl blue (BCB) staining is used to select developmentally competent cumulus-oocyte complexes (COCs) for in vitro maturation (IVM). However, limited attention has been paid to what drives the higher developmental competence of BCB+ COCs. Sonic hedgehog signaling (SHH) is an important signaling pathway for ovarian follicular development and oocyte maturation. Therefore, this study investigated the effect of oocyte quality assessed by BCB staining on cumulus cell expansion, oocyte nuclear maturation, subsequent embryo development, apoptosis levels, and SHH signaling protein expression, in porcine COCs. After IVM, BCB+ COCs exhibited a significantly higher proportion of complete cumulus cell expansion and metaphase II rate in oocytes than BCB- COCs. After in vitro fertilization, the BCB+ group showed a significantly higher monospermy rate, fertilization efficiency, percentage of cleavage and blastocyst formation, with a higher total cell number and a lower apoptosis in blastocysts as compared with the BCB- group. Furthermore, significantly lower apoptosis levels and a higher expression of SHH-signaling proteins in COCs were observed, before and after IVM. In conclusion, high-quality oocytes had a greater potential to expand their surrounding cumulus cells with active SHH signaling and a lower apoptosis. This could provide COCs with a proper environment for maturation, thereby leading to a better subsequent embryo development.

Effect of Triclosan Exposure on Developmental Competence in Parthenogenetic Porcine Embryo during Preimplantation.

Triclosan (TCS) is included in various healthcare products because of its antimicrobial activity; therefore, many humans are exposed to TCS daily. While detrimental effects of TCS exposure have been reported in various species and cell types, the effects of TCS exposure on early embryonic development are largely unknown. The aim of this study was to determine if TCS exerts toxic effects during early embryonic development using porcine parthenogenetic embryos in vitro. Porcine parthenogenetic embryos were cultured in in vitro culture medium with 50 or 100 µM TCS for 6 days. Developmental parameters including cleavage and blastocyst formation rates, developmental kinetics, and the number of blastomeres were assessed. To determine the toxic effects of TCS, apoptosis, oxidative stress, and mitochondrial dysfunction were assessed. TCS exposure resulted in a significant decrease in 2-cell rate and blastocyst formation rate, as well as number of blastomeres, but not in the cleavage rate. TCS also increased the number of apoptotic blastomeres and the production of reactive oxygen species. Finally, TCS treatment resulted in a diffuse distribution of mitochondria and decreased the mitochondrial membrane potential. Our results showed that TCS exposure impaired porcine early embryonic development by inducing DNA damage, oxidative stress, and mitochondrial dysfunction.

Establishment and Characterization of Immortalized Miniature Pig Pancreatic Cell Lines Expressing Oncogenic K-RasG12D.

In recent decades, many studies on the treatment and prevention of pancreatic cancer have been conducted. However, pancreatic cancer remains incurable, with a high mortality rate. Although mouse models have been widely used for preclinical pancreatic cancer research, these models have many differences from humans. Therefore, large animals may be more useful for the investigation of pancreatic cancer. Pigs have recently emerged as a new model of pancreatic cancer due to their similarities to humans, but no pig pancreatic cancer cell lines have been established for use in drug screening or analysis of tumor biology. Here, we established and characterized an immortalized miniature pig pancreatic cell line derived from primary pancreatic cells and pancreatic cancer-like cells expressing K-rasG12D regulated by the human PTF1A promoter. Using this immortalized cell line, we analyzed the gene expression and phenotypes associated with cancer cell characteristics. Notably, we found that acinar-to-ductal transition was caused by K-rasG12D in the cell line constructed from acinar cells. This may constitute a good research model for the analysis of acinar-to-ductal metaplasia in human pancreatic cancer.

Transient meiotic arrest maintained by DON (6-diazo-5-oxo-l-norleucine) enhances nuclear/cytoplasmic maturation of porcine oocytes.

The developmental competence of in vitro-matured oocytes is still lower than that of the in vivo-matured oocytes due to precocious meiotic resumption and inappropriate cytoplasmic maturation. Although numerous efforts have been attempted to accomplish better in vitro maturation (IVM) condition, only limited progress has been achieved. Thus, a current study was conducted to examine the effects of 6-diazo-5-oxo-l-norleucine (DON, an inhibitor of hyaluronan synthesis) during the first half period of IVM on nuclear/cytoplasmic maturation of porcine oocytes and subsequent embryonic development. Based on the observation of the nucleus pattern, metaphase II (MII) oocyte production rate in 1 µM DON group was significantly higher than other groups at 44 h of IVM. The 1 µM of DON was suggested to be optimal for porcine IVM and was therefore used for further investigation. Meiotic arrest effect of DON was maximal at 6 h of IVM, which was supported by the maintenance of significantly higher intra-oocyte cAMP level. In addition, increased pERK1/2 levels and clear rearrangement of cortical granules in membrane of MII oocytes matured with DON provided the evidence for balanced meiosis progression between nuclear and cytoplasmic maturation. Subsequently, DON significantly improved blastocyst formation rate, total cell numbers, and cellular survival in blastocysts after parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer. Altogether, our results showed for the first time that 1 µM DON can be used to increase the yield of developmentally competent MII oocytes by synchronizing nuclear/cytoplasmic maturation, and it subsequently improves embryo developmental competence.

Melatonin improves the meiotic maturation of porcine oocytes by reducing endoplasmic reticulum stress during in vitro maturation.

Under endoplasmic reticulum (ER)-stress conditions, the unfolded protein response (UPR) generates a defense mechanism in mammalian cells. The regulation of UPR signaling is important in oocyte maturation, embryo development, and female reproduction of pigs. Recent studies have shown that melatonin plays an important role as an antioxidant to improve pig oocyte maturation. However, there is no report on the role of melatonin in the regulation of UPR signaling and ER-stress during in vitro maturation (IVM) of porcine oocytes. Therefore, the objective of this study was to investigate the antioxidative effects of melatonin on porcine oocyte maturation through the regulation of ER-stress and UPR signaling. We investigated the changes in the mRNA/protein expression levels of three UPR signal genes (Bip/Grp78, ATF4, P90/50ATF6, sXbp1, and CHOP) on oocytes, cumulus cells, and cumulus-oocyte complexes (COCs) during IVM (metaphase I; 22 hours and metaphase II; 44 hours) by Western blot and reverse transcription-polymerase chain reaction analysis. Treatment with the ER-stress inducer, tunicamycin (Tm), significantly increased expression of UPR markers. Additionally, cumulus cell expansion and meiotic maturation of oocytes were reduced in COCs of Tm-treated groups (1, 5, and 10 µg/mL). We confirmed the reducing effects of melatonin (0.1 µmol/L) on ER-stress after pretreatment with Tm (5 µg/mL; 22 hours) in maturing COCs. Addition of melatonin (0.1 µmol/L) to Tm-pretreated COCs recovered meiotic maturation rates and expression of most UPR markers. In conclusion, we confirmed a role for melatonin in the modulation of UPR signal pathways and reducing ER-stress during IVM of porcine oocytes.

Iloprost supports early development of in vitro-produced porcine embryos through activation of the phosphatidylinositol 3-kinase/AKT signalling pathway.

Despite evidence of the presence of prostaglandin (PG) I2 in mammalian oviducts, its role in early development of in vitro-produced (IVP) embryos is largely unknown. Thus, in the present study we examined the effects of iloprost, a PGI2 analogue, on the in vitro developmental competence of early porcine embryos and the underlying mechanism(s). To examine the effects of iloprost on the development rate of IVF embryos, iloprost was added to the in vitro culture (IVC) medium and cultured for 6 days. Supplementation of the IVC medium with iloprost significantly improved developmental parameters, such as blastocyst formation rate, the trophectoderm:inner cell mass ratio and cell survival in IVF and parthenogenetically activated (PA) embryos. In addition, post-blastulation development into the expanded blastocyst stage was improved in iloprost-treated groups compared with controls. Interestingly, the phosphatidylinositol 3-kinase (PI3K)/AKT signalling pathway was significantly activated by iloprost supplementation in a concentration-dependent manner (10-1000nM), and the beneficial effects of iloprost on the early development of porcine IVF and PA embryos was completely ablated by treatment with 2.5µM wortmannin, a PI3K/AKT signalling inhibitor. Importantly, expression of the PI3K/AKT signalling pathway was significantly reduced in somatic cell nuclear transfer (SCNT) compared with IVF embryos, and iloprost supported the early development of SCNT embryos, as was the case for IVF and PA embryos, suggesting a consistent effect of iloprost on the IVC of IVP porcine embryos. Together, these results indicate that iloprost can be a useful IVC supplement for production of IVP early porcine embryos with high developmental competence.

Superovulatory responses in cynomolgus monkeys (Macaca fascicularis) depend on the interaction between donor status and superovulation method used.

The current study was performed to investigate the effect of oocyte donor status, including age and body weight, on metaphase II (MII) oocyte recovery using two superovulation methods in cynomolgus monkeys. The use of Method A [recombinant gonadotrophin (75 IU/kg, 3 ×, 3-day intervals) and human chorionic gonadotropin (hCG)] led to great increases in ovary size and the mean number of MII oocytes retrieved in age- and body-weight-dependent manner; in contrast, both the parameters were similar in Method B [recombinant gonadotrophin (60 IU, twice daily, 6 days), recombinant gonadotropin and recombinant human luteinizing hormone (rhLH) (60 IU, twice daily, 3 days), and hCG]. Importantly, Method A showed maximal MII oocyte recovery rate in > 60-month-old or 4.5-5.0-kg female monkeys, whereas Method B was equally effective regardless of the donor age and body weight. These results indicate that superovulatory responses depend on the interaction between oocyte donor status and the superovulation method used in cynomolgus monkeys.

Native plants (Phellodendron amurense and Humulus japonicus) extracts act as antioxidants to support developmental competence of bovine blastocysts.

OBJECTIVE: Phellodendron amurense (P. amurense) and Humulus japonicus (H. japonicus) are closely involved in anti-oxidative response and increasing antioxidant enzymes activities. However, the effects of their extracts on development of preimplantation bovine embryos have not been investigated. Therefore, we investigated the effects of P. amurense and H. japonicus extracts on developmental competence and quality of preimplantation bovine embryos. METHODS: After in vitro fertilization, bovine embryos were cultured for 7 days in Charles Rosenkrans amino acid medium supplemented with P. amurense (0.01 µg/mL) and H. japonicus (0.01 µg/mL). The effect of this supplementation during in vitro culture on development competence and antioxidant was investigated. RESULTS: We observed that the blastocysts rate was significantly increased (p<0.05) in P. amurense (28.9%±2.9%), H. japonicus (30.9%±1.5%), and a mixture of P. amurense and H. japonicus (34.8%± 2.1%) treated groups compared with the control group (25.4%±1.6%). We next confirmed that the intracellular levels of reactive oxygen species (ROS) were significantly decreased (p<0.01) in P. amurense and/or H. japonicus extract treated groups when compared with the control group. Our results also showed that expression of cleaved caspase-3 and apoptotic cells of blastocysts were significantly decreased (p<0.05) in bovine blastocysts derived from both P. amurense and H. japonicus extract treated embryos. CONCLUSION: These results suggest that proper treatment with P. amurense and H. japonicus extracts in the development of preimplantation bovine embryos improves the quality of blastocysts, which may be related to the reduction of ROS level and apoptosis.

Exogenous γ-tocotrienol promotes preimplantation development and improves the quality of porcine embryos.

γ-tocotrienol (GTT), an isomer of vitamin E, has been the subject of increasing interest due to its strong anti-oxidant effects. Therefore, in this study, the effects of GTT on blastocyst development, expression levels of reactive oxygen species (ROS) and apoptotic index were investigated in preimplantation porcine embryos. After in vitro maturation and fertilisation, porcine embryos were cultured for 6 days in porcine zygote medium 3 supplemented with or without GTT (200µM) under oxidative stress conditions (200µM hydrogen peroxide (H2O2)). Blastocyst development was significantly improved in the GTT-treated group when compared with the H2O2-treated group (P<0.05). Subsequent evaluation of the intracellular levels of ROS and numbers of apoptotic nuclei in GTT-treated blastocysts revealed that ROS levels of GTT-treated porcine blastocysts were decreased (P<0.05) and the numbers of apoptotic nuclei were reduced by GTT treatment in porcine embryos. Moreover, the total cell numbers of blastocysts were significantly increased in the GTT-treated group relative to the untreated group under H2O2-induced oxidative stress (P<0.05). The expression levels of apoptosis-related genes (BCL-XL, BAX) in GTT-treated blastocysts were then investigated using real-time reverse transcription polymerase chain reaction. Expression of the anti-apoptotic BCL-XL gene was shown to be increased in the GTT-treated blastocyst group, whereas expression of the pro-apoptotic BAX gene was decreased. Taken together, these results suggest that GTT (200µM) under H2O2-induced oxidative stress, thereby improving the developmental competence of porcine embryos via modulation of intracellular levels of ROS and the apoptotic index during the preimplantation stage.

Quantitative expression analysis of APP pathway and tau phosphorylation-related genes in the ICV STZ-induced non-human primate model of sporadic Alzheimer's disease.

The accumulation and aggregation of misfolded proteins in the brain, such as amyloid-ß (Aß) and hyperphosphorylated tau, is a neuropathological hallmark of Alzheimer's disease (AD). Previously, we developed and validated a novel non-human primate model for sporadic AD (sAD) research using intracerebroventricular administration of streptozotocin (icv STZ). To date, no characterization of AD-related genes in different brain regions has been performed. Therefore, in the current study, the expression of seven amyloid precursor protein (APP) pathway-related and five tau phosphorylation-related genes was investigated by quantitative real-time PCR experiments, using two matched-pair brain samples from control and icv STZ-treated cynomolgus monkeys. The genes showed similar expression patterns within the control and icv STZ-treated groups; however, marked differences in gene expression patterns were observed between the control and icv STZ-treated groups. Remarkably, other than ß-secretase (BACE1) and cyclin-dependent kinase 5 (CDK5), all the genes tested showed similar expression patterns in AD models compared to controls, with increased levels in the precuneus and occipital cortex. However, significant changes in gene expression patterns were not detected in the frontal cortex, hippocampus, or posterior cingulate. Based on these results, we conclude that APP may be cleaved via the general metabolic mechanisms of increased α- and γ-secretase levels, and that hyperphosphorylation of tau could be mediated by elevated levels of tau protein kinase, specifically in the precuneus and occipital cortex.

Developmental competence of bovine early embryos depends on the coupled response between oxidative and endoplasmic reticulum stress.

The stress produced by the coupling of reactive oxygen species (ROS) and endoplasmic reticulum (ER) has been explored extensively, but little is known regarding their roles in the early development of mammalian embryos. Here, we demonstrated that the early development of in vitro-produced (IVP) bovine embryos was governed by the cooperative action between ROS and ER stress. Compared with the tension produced by 5% O2, 20% O2 significantly decreased the blastocyst formation rate and cell survival, which was accompanied by increases in ROS and in levels of sXBP-1 transcript, which is an ER stress indicator. In addition, treatment with glutathione (GSH), a ROS scavenger, decreased ROS levels, which resulted in increased blastocyst formation and cell survival rates. Importantly, levels of sXBP-1 and ER stress-associated transcripts were reduced by GSH treatment in developing bovine embryos. Consistent with this observation, tauroursodeoxycholate (TUDCA), an ER stress inhibitor, improved blastocyst developmental rate, trophectoderm proportion, and cell survival. Moreover, ROS and sXBP-1 transcript levels were markedly decreased by supplementation with TUDCA, suggesting a possible mechanism governing the mutual regulation between ROS and ER stress. Interestingly, knockdown of XBP-1 transcripts resulted in both elevation of ROS and decrease of antioxidant transcripts, which ultimately reduced in vitro developmental competence of bovine embryos. Based on these results, in vitro developmental competence of IVP bovine embryos was highly dependent on the coupled response between oxidative and ER stresses. These results increase our understanding of the mechanism(s) governing early embryonic development and may improve strategies for the generation of IVP embryos with high developmental competence.

Valproic acid enhances early development of bovine somatic cell nuclear transfer embryos by alleviating endoplasmic reticulum stress.

Despite the positive roles of histone deacetylase inhibitors in somatic cell nuclear transfer (SCNT), few studies have evaluated valproic acid (VPA) and its associated developmental events. Thus, the present study was conducted to elucidate the effect of VPA on the early development of bovine SCNT embryos and the underlying mechanisms of action. The histone acetylation level of SCNT embryos was successfully restored by VPA, with optimal results obtained by treatment with 3mM VPA for 24h. Importantly, the increases in blastocyst formation rate and inner cell mass and trophectoderm cell numbers were not different between the VPA and trichostatin A treatment groups, whereas cell survival was notably improved by VPA, indicating the improvement of developmental competence of SCNT embryos by VPA. Interestingly, VPA markedly reduced the transcript levels of endoplasmic reticulum (ER) stress markers, including sXBP-1 and CHOP. In contrast, the levels of GRP78/BiP, an ER stress-alleviating transcript, were significantly increased by VPA. Furthermore, VPA greatly reduced cell apoptosis in SCNT blastocysts, which was further evidenced by the increased levels of the anti-apoptotic transcript Bcl-xL and decreased level of the pro-apoptotic transcript Bax. Collectively, these results suggest that VPA enhances the developmental competence of bovine SCNT embryos by alleviating ER stress and its associated developmental damage.

Induction of autophagy during in vitro maturation improves the nuclear and cytoplasmic maturation of porcine oocytes.

While a critical role of autophagy in mammalian early embryogenesis has been demonstrated, few studies have been conducted regarding the role of autophagy in in vitro maturation (IVM) of immature oocytes. In the present study we investigated the effect of rapamycin, a chemical autophagy inducer, on the nuclear and cytoplasmic maturation of porcine oocytes. Rapamycin treatment led to increased expression of LC3-II, an autophagy marker. Compared with the control group, as well as the 5 and 10nM rapamycin treatment groups, the rate of MII oocyte production was higher in the 1nM rapamycin treatment group, indicating improvement in nuclear maturation. In the analyses of cytoplasmic maturation, we found that the level of p34(cdc2), a cytoplasmic maturation marker, and the monospermic fertilisation rate were higher in the 1nM rapamycin treatment group than in the other groups. Moreover, the beneficial effect of 1nM rapamycin on cytoplasmic maturation of MII oocytes was further evidenced by increases in blastocyst formation rate, total cell number and cell survival. In the blastocyst embryos, anti-apoptotic Bcl-xL transcript levels were elevated in the 1nM rapamycin-treated group, whereas pro-apoptotic Bax transcript levels were decreased. Collectively, these results suggest that induction of autophagy during IVM contributes to enhancement of the nuclear and cytoplasmic maturation of porcine oocytes.