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(English) Oocyte-to-embryo transition (OET) is one of the most complex developmental events where a differentiated oocyte gives rise to a totipotent zygote. During the growth phase an oocyte prepares for fertilization and progression to zygotic genome activation. It does so by transcribing and storing the necessary mRNAs till a fully-grown oocyte attains transcriptional quiescence. Therefore, transcriptome regulation in a fully-grown oocyte is of utmost importance. Study of post-transcriptional regulatory pathways revealed that the small-RNA mediated regulatory pathways exist in a unique conformation in mouse oocytes. Endogenous RNAi pathway is essential for mouse female germline while miRNA pathway which is ubiquitously present in most cell types is dispensable for oocyte maturation and fertilization. My PhD project was aimed at understanding the constraints of the miRNA pathway in the oocyte which makes it non-functional. As a fully-grown oocyte is a huge cell with a proportionally large maternal transcriptome we analysed the miRNA: mRNA stoichiometry changes that occur from growing to the fully-grown mouse oocyte. Inability of miRNAs to accumulate during oocyte growth phase leads to their dilution in fully-grown oocyte rendering them inactive. Low miRNA concentrations were also observed in rat,...

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The intra-oocyte signaling pathways that control oocyte growth and early follicular development are largely unknown. The aim of this thesis was to investigate the regulation and functions of phosphatidylinositol 3 kinase (PI3K) pathway in the oocyte, focusing in the roles of Foxo3a, p27, and Pten (phosphatase and tensin homolog deleted on chromosome ten). The physiological significance of Foxo3a in oocytes had been investigated by generating a transgenic mouse, whereby constitutively active Foxo3a is maintained in oocytes using the oocyte-specific ZP3 (Zona pellucida) promoter. The expression of the constantly active “negative” molecule Foxo3a in mouse oocytes was found to cause retardation of oocyte growth, resulting in a significant reduction in oocyte volume in secondary follicles. The transgenic mice also showed arrested follicular development and were infertile. In addition, when Foxo3a was overexpressed in oocytes of primary follicles, oocyte growth and follicular development were retarded. One of the causes of this phenotype may be the retained expression of the cyclin-dependent kinase (Cdk) inhibitor 1B (Cdkn1b), commonly known as p27kip1 or p27, in the nuclei of oocytes. The role and related mechanisms of p27 in controlling early follicular development and oocyte growth were then investigated using wild-type and p27-deficient (p27-/-) mice, and we demonstrated that (i) p27 suppresses follicle endowment/formation and activation, (ii) p27 induces follicle atresia that occurs prior to sexual maturity, and (iii) the overactivated follicles in p27-/- ovaries are depleted in early adulthood, causing premature ovarian failure (POF). In this thesis, we also provide genetic evidence that in mice with conditional deletion of Pten a major negative regulator of PI3K in oocytes, the entire pool of primordial follicles becomes activated, and subsequently all activated follicles are depleted in young adulthood, causing POF. Further mechanistic studies revealed that loss of Pten in oocytes resulted in elevated Akt signaling, which led to upregulation of both expression and activation of ribosomal protein S6 (rpS6) in oocytes. The results thus show that the mammalian oocyte serves as the headquarters of programming of the occurrence of follicle activation, and that the PI3K pathway of the oocyte governs follicle activation through control of initiation of oocyte growth.

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

Thesis (M.Sc. (Anatomy)) -- Mahidol University, 1969.

(English) Oocyte-to-embryo transition (OET) is one of the most complex developmental events, during which a differentiated oocyte gives rise to a totipotent zygote. During OET a transcriptionally silent oocyte undergoes massive reprogramming of gene expression, which transforms it into a transcriptionally active zygote. Although numerous studies have contributed to understanding the mechanism of OET, many genes involved in OET are yet to be identified. A whole new level of possible regulation of OET came with the discovery of long non-coding RNAs (lncRNA). LncRNAs are pol II transcripts longer than 200 nucleotides, that are typically spliced and polyadenylated but do not encode proteins. While lncRNAs have been studied in many model systems including embryonic stem cells, their expression in oocytes and early embryos and contribution to OET were largely unexplored at the beginning of this project. In my PhD project, I aimed to identify, annotate, and analyze lncRNAs expressed during OET. First, using RNA-Seq, 1600 highly reliable lncRNAs were identified and annotated in mouse oocytes and early embryos. Majority of lncRNAs were novel with expression exclusively at OET stages. A significant fraction of these lncRNAs was found associated with LTR retrotransposons, contributing to their novelty and...

Cryopreservation of oocytes is a challenge. Studies were conducted to vitrify mouse zygotes and cumulus-intact bovine oocytes from follicles of different diameters, small (≤ 4 mm) and medium (4 to 10 mm), using nylon mesh. The specific goals were to assess changes in apoptotic gene expression (Fas-FasL, Bax, Bcl-2, and survivin) in conjunction with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and caspase assays. Mouse zygotes were exposed to increasing concentrations of ethylene glycol (EG), Ficoll-70 and sucrose in phosphate buffered saline (PBS) for vitrification on nylon mesh and plunged into liquid nitrogen. Warming resulted in 81.7% morphological survival. The rate of blastocyst formation was 59.9% for vitrified zygotes but, this was significantly lower than that of non vitrified embryos (66.2%). There was no difference in the hatching rates between groups. Both Fas and FasL mRNA were detected at the 4-cell and morula stages, suggesting Fas signaling was operational in early embryos. The level of expression of Bax mRNA tended to increase, while expression of survivin mRNA was not different for 2- and 4-cell embryos. Fragmented embryos showed an increase in Bax mRNA levels, while survivin mRNA level was reduced. In the second experiment, vitrification of bovine oocytes was carried out. Pre-cooled cryovials resulted in 98.9% morphological survival. The oocytes from small and medium size follicles had a significant impact on cleavage (53.7 ± 1.6% vs. 43.8 ± 1.6%, respectively) and blastocyst rates were 16.9 ± 1.0% and 11 ± 1.2%, respectively. Follicle size for oocytes had no impact on the expression of apoptotic genes. The Fas-FasL and Bax-Bcl-2 mRNA showed increased expression after vitrification, but tended to decrease after 9 h of maturation. Yet, results from TUNEL and caspase assays did not support the evidence of the downstream apoptotic signaling pathway in embryos. The semen utilized for in vitro fertilization in both vitrified and control oocytes responded differently in the 4 tested bulls than their field fertility record. The altered transcriptional activities of apoptotic genes, Fas-FasL and Bax-Bcl-2, and survivin were indicative of possible apoptotic activity in vitrified embryos and oocytes subjected to in vitro fertilization. / Ph. D.

Many attempts have been made to improve the in vitro production of cattle embryos by refining in vitro maturation (IVM) and culture systems. Cysteine supplementation to IVM media of bovine oocytes increases cellular glutathione production, which reduces reactive oxygen species (ROS). Similarly, beneficial effects of growth factors for improving the rate of blastocyst development have been reported, but combined effects are unknown. This study was conducted to determine the additive effect of the antioxidant cysteine with epidermal growth factor (EGF) and/or insulin-like growth factor-I (IGF-I) on subsequent embryo development. Bovine oocytes from slaughterhouse ovaries were matured in TCM-199 (control), with or without the addition of 0.6 mM cysteine (C) at 0 or 12 h of maturation. After in vitro fertilization, embryos were allocated to culture treatments containing synthetic oviductal fluid medium. Culture treatments included fetal calf serum (FCS, 4%) alone; IGF-I (100 ng/mL); EGF (10 ng/mL); and IGF-I+EGF (100 ng/mL+10 ng/mL) for all IVM treatments. Although rates for blastocysts development were not different among treatments, an increased proportion of embryos attaining morula formation was achieved when cysteine was added to the IVM media (12 h C IGF-I+EGF, 41.4%; 0 h C EGF, 40.0%) as compared to control (FCS: 34.6%). When cysteine treatments were combined, percent cleavage was greater for IGF-I+EGF (70.8%) compared to FCS (61.2%). The abundance of mRNA from the apoptotic genes, Bax and Bcl-2, and the oxidative stress genes, copper (Cu)-zinc (Zn) superoxide dismutase (SOD; SOD1) and manganese (Mn) SOD (SOD2) in embryos was assessed. No significant treatment effect was observed on the expression of apoptotic and oxidative stress genes. Bax was expressed strongly (4-fold) in morulae with the addition of IGF-I, but was less prevalent in all other morula and blastocyst groups relative to FCS. There was slightly less expression of both SOD1 and SOD2 with treatments compared to FCS in morulae and blastocysts, indicative of low mitochondrial activity and/or a low level of oxidative stress in treatments. There was no significant treatment effect on total cell number, apoptotic nuclei, or apoptotic index. In conclusion, supplementation of cysteine during IVM of oocytes, in conjunction with growth factors could effectively be used as a replacement for FCS. / Master of Science

Oxidative stress contributes to inadequate in vitro maturation of porcine oocytes which leads to a failure of successful fertilization and embryo development. Therefore, the overall objective of this research was to characterize the mechanisms of oxidative stress in maturing oocytes and determine how oocytes alleviate oxidative stress with the assistance of supplemental antioxidants. A preliminary study was conducted to evaluate the effects of glutathione (GSH), N-acetyl-cysteine (NAC), and N-acetyl-cysteine-amide (NACA) supplemented to the maturation medium on intracellular GSH concentrations, nuclear maturation, fertilization success and embryo development. Antioxidants GSH, NAC and NACA (1.0 mM) were supplemented to the media during oocyte maturation. Intracellular GSH concentrations were recorded at 48 h of maturation and nuclear maturation and fertilization were analyzed 12 h after IVF. Embryo development was analyzed at 48 h and 144 h after IVF or intracytoplasmic sperm injection (ICSI). Supplementation of antioxidants had no effect on intracellular levels of GSH, nuclear maturation or fertilization traits. Blastocyst formation for NAC (35.0 ± 7.4%) and NACA (40.0 ± 7.4%) supplementation were higher (P < 0.05) than the control (20.0 ± 7.4%) and GSH supplemented (20 ± 7.4%) oocytes. The same pattern was seen for ICSI-derived embryos: blastocyst formation for NAC (22.0 ± 5.9%) and NACA (25.0 ± 4.6%) supplementation were higher (P < 0.05) than the un-supplemented (10.0 ± 6.0%) oocytes. There were no differences between NAC and NACA supplementation and there were no differences between the cleavage rates for any of the treatment groups. These results indicate that supplementing 1.0 mM of NAC or NACA to the oocyte maturation medium and the ICSI medium increased the percentage of viable embryos reaching the blastocyst stage of development, and could warrant further investigation. The next study was conducted to evaluate the effects of different concentrations of NAC supplemented to the maturation medium on embryo development. Comparisons of significant concentrations of NAC and NACA on embryo development were evaluated for nuclear maturation, fertilization success and embryo development. Concentrations of NAC (0, 0.5, 1.0, 1.5, 2.0, 2.5, 5.0 mM) were supplemented to maturing oocytes and embryo development was analyzed at 48 h and 144 h post-fertilization. There were no differences between cleavage rates for any of the treatment groups. Blastocyst formation for 1.5 mM NAC (56.5 ± 9.2%) was significantly higher (P < 0.05) than all other supplementations. There were no differences in nuclear maturation or fertilization when comparing 1.5 mM NAC and 1.5 mM NACA supplementation to the maturation media. There was no difference between cleavage rates of 1.5 NAC and 1.5 mM NACA supplementation to the maturation media. Blastocyst formation for 1.5 mM NAC (44.4 ± 4.7%) and 1.5 mM NACA (46.2 ± 3.4%) supplementation were significantly higher (P < 0.05) than the control (32.1 ± 6.2%) oocytes. These results indicate that supplementing 1.5 mM of NAC or NACA to the oocyte maturation medium increased the percentage of viable embryos reaching the blastocyst stage of development and could be used during the oxidative stress experiments. In the final study, the mechanisms of oxidative stress in maturing oocytes were studied in addition to evaluating the effects of antioxidant supplementation to the media. This study focused on superoxide dismutase (SOD), GSH peroxidase, catalase and intracellular GSH concentrations with respect to DNA fragmentation evaluated using the single cell Comet assay. Results indicate that when SOD was inhibited, the GSH peroxide levels and length of DNA migration significantly increased (P < 0.05). Catalase levels significantly decreased (P < 0.05) and intracellular GSH remained unchanged. When GSH peroxidase was inhibited, the SOD levels and catalase levels significantly decreased (P < 0.05) but the intracellular GSH and DNA migration length significantly increased (P < 0.05). The supplementation of 1.5 mM NAC and 1.5 mM NACA had multiple effects on the enzyme levels. Specifically, supplementation of 1.5 mM NAC or 1.5 mM NACA significantly decreased (P < 0.05) the length of DNA migration when other enzymes were inhibited compared to no antioxidant supplementation. These results indicate that antioxidant supplementation may alleviate the free radicals associated with oxidative stress in the maturing porcine oocyte. In conclusion, supplementing the antioxidants NAC or NACA to the oocyte maturation media does not have negative effects on IVF or embryo culture. Supplementation of NACA increases the number of oocytes reaching the blastocyst stage of development. Glutathione, SOD, catalase, and GSH peroxidase are all required to be functional during oocyte development to alleviate oxidative stress on the oocyte. Antioxidants enhance the enzyme activity during oocyte maturation and may even contribute to protecting the oocyte when enzyme activity is impaired. / Ph. D.