Follicular antral oxygen tension is thought to influence subsequent oocyte developmental competence. Despite this, in vitro maturation (IVM) is routinely performed in either 5 or 20% oxygen and while low oxygen has been shown to be beneficial to embryo development in many species, the effects of altering oxygen concentration during IVM have not been adequately investigated. Here we investigated the effects of a range of oxygen concentrations (2, 5, 10 & 20% oxygen) during IVM of mouse oocytes on a range of oocyte and embryonic parameters as well as fetal/placental outcome measures and cumulus cell gene expression. While common short term measures of oocyte developmental competence such as maturation, fertilisation, and embryonic development rates were not affected over the range of oxygen levels used, more in depth investigations found several striking differences. Following IVM at 5% oxygen, the oocyte mitochondria were found to have altered patterns of both membrane potential (a measure of mitochondrial activity) and distribution suggesting altered oocyte metabolism. Following IVF, the cellular make up of embryos was investigated. In blastocysts derived from low IVM oxygen (2%) there was found to be an increased number of trophectoderm cells, an increased level of apoptosis (although this was not of sufficient magnitude to account for the cell number difference) and more cells positive for both Cdx2 and Oct4 (markers of trophectoderm and inner cell mass cell types respectively) suggesting a less differentiated cell type. Furthermore, following embryo transfer, the ability of the embryos to implant or develop was not altered by IVM oxygen concentration; however, fetal and placental weights were reduced in the 5% oxygen group. Cumulus cell gene expression was also examined and was found to be altered both across IVM oxygen treatment groups and when compared to cells isolated from in vivo derived complexes. This change in gene expression elucidates some of the many ways in which oxygen concentration during IVM may be affecting the cumulus-oocyte complex (COC) and its future development. Together, this data highlights the importance of looking past common outcome measures when determining the effects of IVM culture conditions. The results of this study also suggest that while IVM oxygen concentration contributes to the perturbing nature of current IVM systems, it is only one of many constituents that require proper investigation, understanding and optimisation. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1368831 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2009

The pharmacological development of non-steroidal contraceptives has yet to be achieved. Arresting oocyte maturation without blocking ovulation has been evaluated using different inhibitors of the phosphodiesterase 3A (PDE3A). Unfortunately, PDE3A is also expressed in the heart and blood vessels, and inhibition of PDE3A in oocytes can produce cardiovascular side effects. We reviewed the literature on available PDE3 inhibitors and selected cilostazol (CLZ), which is an FDA approved therapeutic. CLZ has the ability to decrease cellular adenosine uptake and consequently antagonizes side effects of PDE3A inhibition in vital organs. CLZ inhibited oocyte meiotic maturation in vitro. CLZ has more degenerative impact on arrested oocytes than matured oocytes, indicating that prolonged meiotic arrest of oocytes is harmful. Administration of CLZ any time from 9h before the ovulatory stimulus to 4h after the stimulus resulted in ovulation of immature oocytes. Controlling CLZ dose, time of CLZ administration, and time of oocyte collection resulted in ovulation of oocytes at different meiotic stages. Oral administrations of CLZ in naturally cycling mice were also observed to block pregnancy whereas remating of those previously treated females resulted in normal offspring and litter sizes. Therefore, CLZ does not only have a wide margin of contraception but also is reversible. Ovulated immature oocytes were observed to have higher rates of advanced chromatin configuration and cortical granule distribution, normal spindle and chromosomal organization, maturation, and in vitro fertilization (IVF) than ovarian immature oocytes. Ovulated metaphase I oocytes that were matured in vitro or in vivo had higher IVF rates than ovulated mature oocytes. Ovulated germinal vesicle (GV) oocytes that were in vitro matured also showed higher IVF rates but when in vivo matured, they had lower IVF rates than ovulated mature oocytes because of the high degeneration and low fertilization rates associated with in vivo maturation of GV oocytes. In summary, CLZ merits further evaluation as a non-steroidal contraceptive and is capable of producing oocytes of various meiotic stages with advanced developmental features.

During vertebrate oocyte maturation, the chromosomes progress to and arrest at metaphase of meiosis II in preparation for fertilization. This process includes emission of the first polar body. The second polar body is emitted after fertilization. A number of proteins are accumulated during oocyte maturation. Inhibition of this de novo translation does not appear to affect the progression of meiosis during oocyte maturation. The role of these pools of proteins has yet to be elucidated. Curiously, several of the upregulated proteins are key players in mitosis, including INCENP, a subunit of the chromosome passenger complex implicated in chromosome segregation and cytokinesis. During early stages of development in Xenopus laevis, the embryo cycles through mitosis, also known as embryo cleavage, every 30min with little to no time for transcription/translation. Our goal is to determine if the de novo translation of these mitotic proteins during oocyte maturation has a role in early embryogenesis. We used morpholino oligonucleotides antisense to INCENP mRNA (INCENPmorpho) to inhibit de novo translation during oocyte maturation. Using confocal imaging and the host transfer technique, these injected oocytes were matured, fertilized and assessed for developmental competency. INCENPmorpho and a control morpholino (ctrlmorpho) had no discernable effect on 1st or 2nd polar body emission. Whereas ctrlmorpho embryos developed normally, INCENPmorpho embryos did not cleave. Thus, de novo translation of INCENP during oocyte maturation is necessary for embryogenesis. Specifically, accumulation of INCENP and other mitotic proteins during oocyte maturation may be a common strategy in this species to prepare for the rapid and synchronous mitoses during early embryogenesis.

During vertebrate oocyte maturation, the chromosomes progress to and arrest at metaphase of meiosis II in preparation for fertilization. This process includes emission of the first polar body. The second polar body is emitted after fertilization. A number of proteins are accumulated during oocyte maturation. Inhibition of this de novo translation does not appear to affect the progression of meiosis during oocyte maturation. The role of these pools of proteins has yet to be elucidated. Curiously, several of the upregulated proteins are key players in mitosis, including INCENP, a subunit of the chromosome passenger complex implicated in chromosome segregation and cytokinesis. During early stages of development in Xenopus laevis, the embryo cycles through mitosis, also known as embryo cleavage, every 30min with little to no time for transcription/translation. Our goal is to determine if the de novo translation of these mitotic proteins during oocyte maturation has a role in early embryogenesis. We used morpholino oligonucleotides antisense to INCENP mRNA (INCENPmorpho) to inhibit de novo translation during oocyte maturation. Using confocal imaging and the host transfer technique, these injected oocytes were matured, fertilized and assessed for developmental competency. INCENPmorpho and a control morpholino (ctrlmorpho) had no discernable effect on 1st or 2nd polar body emission. Whereas ctrlmorpho embryos developed normally, INCENPmorpho embryos did not cleave. Thus, de novo translation of INCENP during oocyte maturation is necessary for embryogenesis. Specifically, accumulation of INCENP and other mitotic proteins during oocyte maturation may be a common strategy in this species to prepare for the rapid and synchronous mitoses during early embryogenesis.

Kyoto University (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 / 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

During vertebrate oocyte maturation, the chromosomes progress to and arrest at metaphase of meiosis II in preparation for fertilization. This process includes emission of the first polar body. The second polar body is emitted after fertilization. A number of proteins are accumulated during oocyte maturation. Inhibition of this de novo translation does not appear to affect the progression of meiosis during oocyte maturation. The role of these pools of proteins has yet to be elucidated. Curiously, several of the upregulated proteins are key players in mitosis, including INCENP, a subunit of the chromosome passenger complex implicated in chromosome segregation and cytokinesis. During early stages of development in Xenopus laevis, the embryo cycles through mitosis, also known as embryo cleavage, every 30min with little to no time for transcription/translation. Our goal is to determine if the de novo translation of these mitotic proteins during oocyte maturation has a role in early embryogenesis. We used morpholino oligonucleotides antisense to INCENP mRNA (INCENPmorpho) to inhibit de novo translation during oocyte maturation. Using confocal imaging and the host transfer technique, these injected oocytes were matured, fertilized and assessed for developmental competency. INCENPmorpho and a control morpholino (ctrlmorpho) had no discernable effect on 1st or 2nd polar body emission. Whereas ctrlmorpho embryos developed normally, INCENPmorpho embryos did not cleave. Thus, de novo translation of INCENP during oocyte maturation is necessary for embryogenesis. Specifically, accumulation of INCENP and other mitotic proteins during oocyte maturation may be a common strategy in this species to prepare for the rapid and synchronous mitoses during early embryogenesis.

During vertebrate oocyte maturation, the chromosomes progress to and arrest at metaphase of meiosis II in preparation for fertilization. This process includes emission of the first polar body. The second polar body is emitted after fertilization. A number of proteins are accumulated during oocyte maturation. Inhibition of this de novo translation does not appear to affect the progression of meiosis during oocyte maturation. The role of these pools of proteins has yet to be elucidated. Curiously, several of the upregulated proteins are key players in mitosis, including INCENP, a subunit of the chromosome passenger complex implicated in chromosome segregation and cytokinesis. During early stages of development in Xenopus laevis, the embryo cycles through mitosis, also known as embryo cleavage, every 30min with little to no time for transcription/translation. Our goal is to determine if the de novo translation of these mitotic proteins during oocyte maturation has a role in early embryogenesis. We used morpholino oligonucleotides antisense to INCENP mRNA (INCENPmorpho) to inhibit de novo translation during oocyte maturation. Using confocal imaging and the host transfer technique, these injected oocytes were matured, fertilized and assessed for developmental competency. INCENPmorpho and a control morpholino (ctrlmorpho) had no discernable effect on 1st or 2nd polar body emission. Whereas ctrlmorpho embryos developed normally, INCENPmorpho embryos did not cleave. Thus, de novo translation of INCENP during oocyte maturation is necessary for embryogenesis. Specifically, accumulation of INCENP and other mitotic proteins during oocyte maturation may be a common strategy in this species to prepare for the rapid and synchronous mitoses during early embryogenesis.

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

No description available.

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.