Unfertilized or parthenogenetically non-activated porcine oocytes matured in vitro conditions are subjected to a process known as aging. During such development, porcine oocytes undergo the complex of the structural and functional changes, which can result in spontaneous parthenogenetic activation, fragmentation or lysis. After three days of culture in our condition, 23% of oocytes remained at the stage of metaphase II, 48% of oocytes were spontaneously parthenogenetically activated, 26% of oocytes were subjected to fragmentation and 3% of oocytes were lysed. The complete suppression of porcine oocyte fragmentation during the process of aging occurred during oocytes cultured in medium with sulphide donor Na2S in concentrations 150 µM and 300 µM. Inhibition of enzymes catalyzing the synthesis of hydrogen sulfide in the oocytes during the process of aging (cystathionine-gamma-lyase and cystathionine beta-synthase), iniciates earlier onset of oocytes fragmentation. The effect of both inhibitors could be completely reversed by using sulphide donor Na2S. The process of aging in porcine oocytes significantly reduces the success of the activation processes. Parthenogenetic activation occurred in 94% of pig oocytes, which were not subjected to aging. The proportion of activated oocytes after exposure to 24...

Fully grown oocytes undergo their further development in the absence of transcription. Completion of meiosis and early embryo development rely on the maternal mRNAs synthetized and stored during earlier development. Thus, the regulation of gene expression in oocytes during that period is controlled almost exclusively at the level of mRNA stabilization and translation. In the same vein, any mRNA metabolism could play a critical function at this stage of development. RNA localization followed by a local translation is a mechanism responsible for the control of spatial and temporal gene expression in the cell. We focused on visualization of mRNA and in situ translation in the mammalian oogenesis and embryogenesis. We characterized localization of global RNA population in the oocyte and early embryo nucleus together with RNA binding proteins. Additionally we visualized specific ribosomal proteins that contribute to translation in the oocyte and embryo. We have shown that the key player of cap-dependent translation mTOR becomes highly active post nuclear envelope breakdown (NEBD) and in turn its substrate, translational repressor 4E-BP1 becomes inactive. Precise localization of inactivated 4E-BP1 at the newly forming spindle of the oocyte indicates the ongoing translation in this area. Furthermore, from...

Eye tracking is the process of measuring where people are looking at with an eye tracker device. Eye tracking has been used in many scientific fields, such as education, usability research, sports, psychology, and marketing. Eye tracking data are often obtained from a static eye tracker or are manually extracted from a mobile eye tracker. Visualization usually plays an important role in the analysis of eye tracking data. So far, there existed no software package that contains a whole collection of eye tracking data processing and visualization tools. In this dissertation, we review the eye tracking technology, the eye tracking techniques, the existing software related to eye tracking, and the research on eye tracking for posters and related media. We then discuss the three main goals we have achieved in this dissertation: (i) development of a Matlab toolbox for automatically extracting mobile eye tracking data; (ii) development of the linked microposter plots family as new means for the visualization of eye tracking data; (iii) development of an R package for automatically extracting and visualizing data from mobile and static eye trackers.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19026号 / 農博第2104号 / 新制||農||1030(附属図書館) / 学位論文||H27||N4908(農学部図書室) / 31977 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 今井 裕, 教授 久米 新一, 教授 松井 徹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第21400号 / 生博第401号 / 新制||生||53(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 松崎 文雄, 教授 石川 冬木, 教授 松本 智裕 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

N,N,N’,N’-tetrakis-(2-Pyridylmethyl) ethylenediamine (TPEN) is a heavy metal chelator with high affinity for zinc. TPEN causes important responses in mammalian eggs. For example, these eggs are arrested at the MII stage by the Endogenous Mitotic Inhibitor 2 (Emi2), which prevents activation of the Anaphase Promoting Complex (APC) and degradation of Cyclin B. By chelating zinc, TPEN inactivates Emi2 and eggs undergo spontaneous exit of meiosis and egg activation. TPEN chelates Ca2+ with lower affinity, although in the Endoplasmic Reticulum (ER), where Ca2+ concentrations are high, TPEN may sequester Ca2+ preventing release into the cytoplasm. Initial exposure of TPEN to MII eggs failed to cause spontaneous intracellular Ca2+ release from the ER. Interestingly, in the case of GV oocytes, addition of TPEN caused Ca2+ influx. This influx could be blocked via the addition of 2-APB, a plasma membrane Ca2+ channel blocker. To determine the possible role of TPEN on chelation of ER Ca2+, MII and GV cells were incubated in TPEN and ER Ca2+ released was by exposure to Cyclopiazonic Acid (CPA), a sarco/endoplasmic reticulum (SERCA) pump inhibitor, or Ionomycin (IO), a Ca2+ ionophore. In MII oocytes, the amplitude of the rises caused by CPA and IO, in TPEN-treated oocytes, was smaller than controls and experienced a delay in return to baseline. In GV oocytes, TPEN enhanced rather than reduced Ca2+ responses to CPA and IO. Given its inability to fully chelate ER Ca2+, the use of TPEN as a tool to study Ca2+ homeostasis in mouse oocytes needs additional investigation.

The development of a preimplantation embryo is a stepwise process consisting of morphological, biochemical, and genomic changes. Much remains unknown about the attainment of embryo competency to develop and establish pregnancy. To investigate this, we compared methods of selection at the oocyte or embryo level for improved blastocyst production. Brilliant cresyl blue staining was used to sort oocytes by their growth status (not fully grown vs. fully grown) and the timing of the first embryonic cell division to sort embryos. We found that an embryo's cleavage kinetics are more indicative of their competency than the growth status of the oocyte that gave rise to that embryo. We further investigated the cryopreservation survival of embryos with fast or slow cleavage kinetics and found no significant differences in their ability to hatch post-thawing. Next, we used the complete sequence of the cattle Y chromosome to identify oligonucleotides for efficient sexing of samples. These materials may be used to understand sexual dimorphism as a biological factor in future experiments. Finally, we designed a new method to induce targeted DNA sequence deletions and mRNA cleavage in zygotes using CRISPR-Cas. We targeted the gene OCT4, since the literature shows variable knockout outcomes. Our method improved deletion efficiency while accounting for preexisting or maternally inherited mRNA of the target gene. Our findings can be used to better understand early embryo development and biological drivers of quality, which can be leveraged to improve embryo production and transfer outcomes. / Master of Science / The development of an early embryo involves many biological and structural changes. Much remains unknown about the influences on embryo quality and ability to successfully develop. To investigate this, we compared methods for selecting the highest quality cattle eggs or embryos. We found that the observation of an embryo's development speed is better for selecting high quality embryos than egg quality. We further investigated the freezing survival of embryos with fast or slow growth. We found that the freezing survival of fast and slow growing embryos is not different. Next, we used the complete sequence of the cattle Y chromosome to identify PCR primers for determining sample sex. These resources can help us understand how an individual's sex can influence biological differences. Finally, we designed a new method for removing the total function of a gene in embryos. For this, we deleted segments of DNA and cut RNAs. Our findings can be used to better understand early embryo development and biological drivers of quality, which can be leveraged to improve embryo production and transfer outcomes.

Profiles of plasma estradiol and testosterone concentrations, cathepsin D, L, and B activities, and quantitative and qualitative protein content were developed and evaluated in four commercial strains of channel catfish, Gold Kist (2), Thompson and NWAC-103 for one year (age 2 to age 3). Great variation between individuals of the same strain precluded the identification of any significant, strain-specific differences for the variables under investigation. When variables from fish of all strains were collectively evaluated over time, both estradiol and testosterone concentrations significantly increased in July and then later from February to April. The increase in hormone concentration was accompanied by oocyte growth and increases in proteolytic activity of cathepsins D, L, and B, supporting the role of estradiol in regulating vitellogenesis. Vitellogenin was enzymatically broken down into smaller protein units by cathepsins L, D, and B that were separately predominant at different stages of oocyte development. During oocyte development, there were sequential relationships among hormone concentration, cathepsin activity, protein content, and predominant oocyte proteins. This observation was associated with high levels of activity of cathepsin L in February, suggesting an important role in protein degradation during that time, while high activity of cathepsin B occurred, stimulating during November to January. Cathepsin B is more important in oogenesis or early vitellogenesis, and cathepsin L assumes a principal role during middle vitellogenesis. Twenty hours subsequent to the injection of fish with either carp pituitary hormone or luteinizing hormone releasing hormone, increases in the concentration of plasma estradiol and testosterone, activities of cathepsins L, D, and B, egg size, and egg protein content occurred, stimulating the process of oocyte maturation. The percentages of spawning obtained were 18.8% of LHRH injected fish, 12.4% of CPE injected fish, 9.4% of fish not injected, and 0% of saline injected fish. Injection of females with LHRH can potentially serve as a tool to increase spawning success in appropriate commercial settings, particularly for improving three year old catfish spawning success early in the spawning season. Low estradiol levels in all three-year-old fish suggest that insufficient stimulation of vitellogenin production by estradiol may underlie the lack of vitellogenin incorporation into developing oocytes. In the present study, the measurement of the activities of the cathepsins and their relationships to other parameters were evaluated for the first time. This is also the first study to report plasma estradiol and testosterone concentrations, protein content, and egg size in 2 to 3-year old channel catfish. All of the parameters collectively evaluated may serve to assist in the selection of the best 2- year old channel catfish female broodstock, and to determine the optimal timing of treatments of hormone injection to increase reproductive performance.

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Many primate populations face the threat of extinction due to habitat loss, intensive agriculture, hunting for meat, the pet trade and/or use in traditional medicines. An alternative approach to in situ conservation includes gene banking and the use of assisted reproductive technologies (ART), such as oocyte in vitro maturation (IVM) and in vitro fertilization (IVF). Although many of these 'high-tech' solutions have not yet been proven viable for pragmatic wildlife conservation, basic research and development of these emerging tools can provide necessary information needed to optimize these techniques and institute ART as a routine practice in conservation efforts. A severely limiting factor in the successful application of ARTs is the availability of mature developmentally competent oocytes. Oocyte maturation involves many nuclear and cytoplasmic factors, which can be affected by maturation conditions and female age. In vitro maturation does not have the same success rate across species studied. In primates especially, IVM oocytes exhibit reduced developmental capacity upon fertilization when compared to in vivo matured (IVO) oocytes. This study aimed to investigate possible causes of reduced developmental capacity of primate IVM oocytes using the rhesus macaque (Macaca mulatta) as a model. Research efforts included investigation of ovarian senescence, oocyte karyotype and spindle morphology, and establishment of an optimal sperm cryopreservation protocol for use in IVF. Histological examination of the rhesus ovary demonstrated an age-related pattern of follicle depletion similar to that described in the human ovary. Oocyte karyotype analysis revealed a significant effect of IVM on the frequency of hyperhaploidy. In addition, immunostaining and confocal microscopy demonstrated a significant increase of anomalous chromosome congression on the oocyte metaphase II spindle equator in relation to IVM and donor female age. These results indicate that IVM can produce serious, if not lethal consequences for embryo development. This study presents baseline data on ovarian aging in the rhesus macaque and aspects of nuclear maturation during macaque IVM that may contribute to the design of primate oocyte recovery plans. Implementation of either of two sperm cryopreservation methods originally developed for rhesus and vervet monkeys will aid future investigation of the developmental capacity of IVM oocytes.