The objective of this study was to examine the effects of high concentrations of glucose on porcine parthenotes developing in vitro. Addition of 55 mM glucose to the culture medium of embryos at the four-cell-stage significantly inhibited blastocyst formation, resulting in fewer cells in blastocyst-stage embryos and increased levels of apoptosis and autophagy compared to control. Quantitative reverse transcriptase (RT) PCR analysis revealed that the expression of pro-apoptotic genes (Caspase 3, Bax and Bak) and autophagy genes (Atg6 and Atg8/Lc3) were increased significantly by the addition of 55 mM glucose to the culture medium compared to control. MitoTracker Green fluorescence revealed a decrease in the overall mitochondrial mass compared to control. However, the addition of 55 mM glucose had no effect on mRNA expression of the nuclear DNA-encoded mitochondrial-related genes, cytochrome oxidase (Cox) 5a, Cox5b and Cox6b1. These results suggest that hyperglycemia reduced the mitochondrial content of porcine embryos developing in vitro and that this may hinder embryonic development to the blastocyst stage and embryo quality by increasing apoptosis and autophagy in these embryos.

Autophagy is conserved response to starvation by which cells catabolize their components to create an internal supply of essential nutrients. Ceramide is known to induce autophagy in many cells through down-regulation of amino acid and glucose transporters. The mechanism of starvation induced-autophagy in mouse embryo remains unclear. In order to understand the mechanism by which starvation regulates autophagy, in this study, we investigated nutrient transporters expression and the effect of c2-ceramide on the in vitro development, apoptosis and autophagy via starvation in mouse embryo. Glucose transporters (Glut1 and Glut 3), high levels of transcript were expressed from 1 to 2 cells and gradually decreased through the morula and blastocyst (BL) stages. Amino acid transporters (LAT-1 and 4F2hc) gradually decreased from the zygote to the BL stage. Furthermore, the expression of nutrient transporters (Glut1, 3, LAT-1 and 4F2hc) were significantly reduced at the BL stage after ceramide treatment. Especially, mTOR expression after ceramide treatment of embryos was significantly higher than controls. Ceramide treated embryos exhibited significantly reduced developmental rates and total cell numbers, and increased apoptotic cell death at the BL stage. Consequently, we next evaluated the effect of ceramide treatment on mitochondrial number and morphology. There was a significant decrease in the average mtDNA copy number and the mitochondrial area in ceramide treated BL stage embryos. Both the expression of autophagy-related genes, Lc3, Gabarap, Atg4A and Atg4B, and the synthesis of LC3 were significantly induced at the BL stage. These results suggest that autophagy under starvation condition influences the in vitro development and apoptosis and autophagy, and may play a role in early mouse embryogenesis.

It is well established that mitochondrial genome is strictly maternally inherited in mammalian, despite the fact that paternal mitochondria enter into oocyte during fertilization. To date, although some mechanisms have been extrapolated to interpret the elimination of paternal mitochondria, the exact mechanism still is unclear. Recent studies suggest that autophagy process and the ubiquitin-mediated degradation pathway may be involved in elimination of paternal mitochondria. However, the dynamic profiles of autophagy and ubiquitination associated with paternal mitochondria degradation have not been determined in mouse model. Through immunostaining with specific antibody LC3 and Ubiquitin and confocal microscopy, we investigated the dynamic profiles of LC3 and Ubiquitin signals in mouse embryos during preimplantation development. In addition, embryos were stained with MitoTracker Red for tracking the degradation process of paternal mitochondria. Our results showed that paternal mitochondria gradually degraded during postfertilization development, and sporadic paternal mitochondria were found at least in 16 cell embryos. LC3 and Ubiquitin signals appeared in the midpiece of sperm at 3 h postfertilization, and they were strictly colocalizated with paternal mitochondria from zygote to 2 cell embryo. Nevertheless, the colocalization became loose at 4 cell embryos, and gradually disappeared beyond 4 cell embryos. Our results confirmed that autophagy process and the ubiquitin-mediated degradation pathway may take part in the postfertilization remove of paternal mitochondria.

The coupling of autophagy and endoplasmic reticulum (ER) stress has been implicated in a variety of biological processes. However, little is known regarding the involvement of the autophagy/ER stress pathway in early embryogenesis or the underlying mechanism (s). Here, we showed that the developmental competence of in vitro-produced (IVP) bovine embryos was highly dependent on the autophagy/ER stress balance. Although relative abundances of autophagy-associated gene transcripts, including LC3, Atg5, and Atg7 transcripts, were high in oocytes and throughout the early stages of preattachment development, extensive autophagosome formation was only detected in fertilized embryos. Using inducer and inhibitor of autophagy, we showed that transient elevation of autophagic activity during early preattachment development greatly increased the blastocyst development rate, trophectoderm cell numbers, and blastomere survival; these same parameters were reduced by both inhibition and prolonged induction of autophagy. Interestingly, the induction of autophagy reduced ER stress and associated damage, while the developmental defects in autophagy-inhibited embryos were significantly alleviated by ER stress inhibitor treatment, indicating that autophagy is a negative regulator of ER stress inearly embryos. Collectively, these results suggest that early embryo genesis of IVP bovine embryos depends on an appropriate balance between autophagy and ER stress. These findings may increase our understanding of important early developmental events by providing compelling evidence concerning the tight association between autophagy and ER stress, and may contribute to the development of strategies for the production of IVP bovine blastocysts with high developmental competence.

Autophagy is known to be involved in a variety of biological processes. However, relatively a little is known regarding oocyte maturation and preimplantation development in mammals. Thus, the current study was conducted to investigate the role of autophagy in oocyte maturation and subsequent preimplantation development in pigs. Porcine oocytes were matured in the presence or absence of 1 μM rapamycin, an autophagy inducing agent, fertilized in vitro, and cultured to blastocyst stage. From Western blotting analysis, we found that active form LC3 was detected during in vitro maturation (IVM) period, suggesting the possible role of autophagy in oocyte maturation. Interestingly, treatment of rapamycin during IVM significantly increased nuclear maturation compared to control group. Importantly, rapamycin-assisted IVM greatly improved monospermic fertilization and blastocyst development rates compared to control embryos. In addition, we also found that cell number and blastomere survival in blastocysts were markedly increased in rapamycin treatment group, which was further evidenced by both elevation of anti-apoptotic transcript Bcl-XL and decrease of pro-apoptotic transcript Bax. Collectively, these results strongly suggest that induction of autophagy may contribute to the completion of nuclear and cytoplasmic maturation of porcine oocytes.

The purpose of this thesis is to examine the effect of hormone treatment in blastocyst development of in vitro cultured porcine oocyte. Oocytes used in the study was matured in vitro in the presence of 10% FBS or 10% pFF, and treated with FSH, LH or FSH+ LH, and the rate of blastocyst development was assessed based on the expression of autophagic genes. There was no significant differences in blastocyst development between oocytes maturaed in 10% FBS or 10% pFF. In vitro matured oocytes treated with FSH+LH showed blastocyst development rate as high as that of untreated oocytes, while groups treated with LH only showed a decrease in blastocyst development. About the expression of cell death assosiated factors, mRNA levels of autophagy and apoptosis genes were increased in oocytes matured in 10% FBS and treated with LH. Oocytes that did not receive hormone treatment showed low expression of most cell death genes except ATG5. When oocytes were matured in 10% pFF, ATG5 expression was the highest in FSH treated group, while LC3 showed strong expression in all hormone treated groups. On the other hand, the expression level of mTOR and caspase-3 did not show significant differences between groups. We also examined the protein level of apoptotic genes in the blastocyst. The amount of caspase-3 protein was similar between groups matured in 10% FBS and 10% pFF, but was the highest when treated with LH. Blastocysts treated with FSH and FSH+LH showed similar level of caspase-3 protein, while the level was the lowest when hormone treatment was not given. Within the blastocyst, caspase-3 was mostly expressed in trophoblast cells when matured in 10% FBS, while maturation in 10% pFF caused expression of this protein in the inner cell mass (ICM). Expression of MAP1LC3A was higher in groups matured in 10% pFF than groups matured in 10% FBS in all types of hormone treatment. Among the blastocysts matured in 10% pFF, MAP1LC3A level increased in the order of untreated < FSH < FSH+ LH. Expression of MAP1LC3A within the FBS-matured blastocyst was concentrated to the trophoblast, while pFF-matured blastocyst showed expression in both trophoblast and ICM. The expression of mTOR showed a pattern opposite to that of MAP1LC3A. That is, its expression was the lowest in 10% FBS group without hormone treatment. In both FBS and pFF group, and in all three combinations of hormone treatment, mTOR expression was ovserved mostly in ICM. Together, these results indicated that hormone treatments tend to induce expression of genes associated with programmed cell death. We suggest that proper induction of programmed cell death by FSH and LH treatment would increase the rate of blastocyst development. * This work was supported by BioGreen 21 Program (No. PJ008029). Rural Development Administation, Republic of Korea.

There are diverse methods of cryopreservation of mammalian embryos with variable degrees of success. Although cryopreservation technique of mammalian embryos has been advanced, freezing stress affect to cellular event such as apoptosis and autophage in embryos. The objective of the study is to investigate the affection of to survival, development, live offspring, apoptosis and autophagy on embryo. Mouse embryos were vitrified and thawed using normal straw and modified cut standard straw (M-CSS), then in vitro cultured until blastocyst stage and transferred to recipient. Recovery rates (100 vs 99.2%), survival rates (99.2 vs 78.6%), developmental rates (18.4 vs 10.7%), total cell numbers (45 vs 37), preganacy rates (34.5 vs 25%) and offspring numbers (10.1 vs 4.9 %) of M-CSS group are significantly higher than those of normal straw vitrified group. Also, rate of apoptosis in blastocysts developed using M-CSS (1.9%) was significantly lower than using normal straw vitrification (2.7%). Apoptosis-related gene, caspase 3, was expressed at the highest level in blastocysts derived from normal straw group. However, no differences of autophagy related gene, Atg6 and expression of LC3 between normal straw and M-CSS groups were observed. In conclusion, the standard vitrification procedure induces mitochondrial apoptosis in zygotes in an autophagy-independent manner, whereas the novel M-CSS procedure may improve embryo vitrification.

Autophagy, the process of bulk degradation and recycling of long-lived proteins, macromolecular aggregates, and damaged intracellular organelles, has recently been shown to be important for pre-implantation development and cavitation in mouse embryos. This study investigated the occurrence of autophagy and its importance in determining the in vitro development of pig embryos produced by in vitro fertilization (IVF) or parthenogenetic activation (PA). Western blot analysis for autophagy marker, microtubule associated protein light chain 3 (MAP-LC3), revealed the temporal pattern of LC3-conversion with intense changes during 10 20 h post-insemination and at morula-blastocyst transition in pig embryos. Specific inhibition of autophagy in 2 4 cell stage pig embryos, by treatment with 3-methyladenine (3MA), did not affect their embryonic development up to morula stage (p>0.05) but completely blocked their progression to the blastocyst stage (0.0±0.0 vs. 28.5±1.7% p<0.05). On the other hand, autophagy-inhibition in morula stage embryos significantly inhibited the formation of blastocoel (14.9±3.6 vs. 37.5±7.2%) and reduced the proportion of expanded blastocysts (5.6±2.6 vs. 29.6± 4.6% p<0.05). TUNEL assay revealed that autophagy-inhibited embryos had significantly increased indices of apoptosis (10.2±0.4 vs. 2.3±0.2) and DNA fragmentation (0.8± 0.1 vs. 0.3±0.1) than those of controls (p<0.05). Interestingly, while anti-oxidants reduced (p<0.05) the apoptosis and improved the blastocyst formation rate in pig embryos, it had no influence (p>0.05) on the expression of MAP-LC3. These data therefore, suggest that autophagy may have essential role during blastocyst formation in pig embryos.

Autophagy is a process of intracellular bulk protein degradation, in which the accumulated proteins and cytoplasmic organelles are degraded. It plays important roles in cellular homeostasis, apoptosis, and development, but its role during early embryo development remains contentious. Therefore, in the present study, we investigated the effects of 3-methyladenine (3-MA) on early embryonic development in pigs. we also investigated several indicators of developmental potential, including mitochondrial distribution, genes expressions (autophagy-, apoptosis- related genes), apoptosis and ER-stress, which are affected by 3-MA. After in vitro maturation and fertilization, presumptive pig embryos were cultured in PZM-3 medium supplemented with 3-MA for 2 days at 39℃, 5% CO2 in air. Developmental competence to the blastocyst stage in the presence of 3-MA was gradually decreased according to increasing concentration. Thus, all further experiments were performed using 2 mM 3-MA. Blastocysts that developed in the 3-MA treated group decreased LC3-II intensity and expressions of autophagy related genes than those of the untreated control, resulting in down-regulates the autophagy. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 3-MA treated group compared with control (6.0±1.0 vs 3.3±0.6, p<0.05). Also, the expression of the pro-apoptotic gene Bax increased in 3-MA treated group, whereas expression of the anti-apoptotic gene Bcl-XL decreased. Mito Tracker Green FM staining showed that blastocysts derived from the 3-MA treated group had lower mitochondrial integrity than that of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. Then, the expression of the spliced form of pXBP-1 product (pXBP-1s) increased in 3-MA treated group, resulting increase of ERstress. Taken together, these results indicate that inhibition of autophagy by 3-MA is closely associated with apoptosis and ER-stress during preimplantation periods of porcine embryos.

The life cycle of ticks is characterized by alternate off-host and on-host conditions. The life span is estimated at several years and most ixodid ticks spend more than 95% of their life off the host. They seem to have a unique strategy to endure the off-host state for a long period. By electron microscopy, isolation membrane-, autophagosome- and autolysosome-like structures were found in the midgut epithelial cells of unfed ticks. Therefore, we focused on autophagy which is well-conserved from yeast to higher eukaryotes and induced by starvation. We have identified homologues of autophagy-related (ATG) genes (ATG3, ATG4 and ATG8) from cDNA libraries of the 3-host tick Haemaphysalis longicornis. Each expression profile of H. longicornis ATG (HlATG) genes and HlAtg proteins at the stages of nymph and adult were examined by real-time PCR and immunoblotting. Moreover, autophagy is known to be induced by inactivation of target of rapamycin (TOR), a phosphatidylinositol kinase. To examine the effect of TOR function on the expression of HlAtg protein(s), rapamycin, a specific inhibitor of the signal transduction mediated by TOR, was injected to unfed adults. It was revealed that the expression of HlAtg protein(s) was enhanced in response to the rapamycin. This result indicates that tick have the nutrient-sensitive TOR signaling pathway which regulate autophagy.