<p>Apoptosis is a form of programmed cellular "suicide" which is activated in response to a variety of pro-death stimuli. Apoptotic cell death is orderly and energy-dependent, and cellular constituents are packaged into membrane-bound vesicles for consumption by phagocytes. Toxic intracellular signals are never exposed to neighboring cells or to the extracellular environment, and a host inflammatory response does not occur. Apoptosis is executed by the coordinated activation of caspase family proteins. Caspase-2 is an apical protease in this family, and promotes cell death after receipt of cues from intracellular stressor signals. Caspase-2 helps to initiate apoptosis by responding to cellular death stimuli and signaling for downstream cytochrome c release and executioner caspase activation.</p><p> Several years ago our lab determined that Xenopus laevis oocyte death is partly controlled by the activation of caspase-2. In the setting of oocyte or egg extract nutrient depletion, caspase-2 was observed to be activated upstream of mitochondrial cytochrome c. In fact, caspase-2 is suppressed in response to the nutrient status of the oocyte: nutrient-replete oocytes with healthy pentose phosphate pathway flux and abundant NADPH production are able to inhibit caspase-2 via S135 phosphorylation catalyzed by calcium/calmodulin-dependent protein kinase II. Phosphorylation of caspase-2 at S135 is critical in preventing oocyte cell death, and a caspase-2 mutant unable to be phosphorylated loses its ability to respond to suppressive NADPH signals. </p><p> In this dissertation we examine the converse mechanism of metabolically-regulated caspase-2 activation in the Xenopus egg extract. We now show that caspase-2 phosphorylated at S135 binds the interactor 14-3-3 zeta, thus preventing caspase-2 dephosphorylation. Moreover, we determined that S135 dephosphorylation is catalyzed by protein phosphatase-1, which directly binds caspase-2. Although caspase-2 dephosphorylation is responsive to metabolism, neither PP1 activity nor binding is metabolically regulated. Rather, release of 14-3-3 zeta from caspase-2 is the point of metabolic control and allows for caspase-2 dephosphorylation. Accordingly, a caspase-2 mutant unable to bind 14-3-3 zeta is highly susceptible to activation. Although this mechanism was initially established in Xenopus, we now demonstrate similar control of murine caspase-2 by phosphorylation and 14-3-3 binding in mouse eggs. </p><p> In the second part of this dissertation we examine the paradigm of caspase-2 metabolic regulation in a mammalian somatic cell context. We observed that mammalian caspase-2 is a metabolically-regulated phosphoprotein in somatic cells, and that the site of regulation is caspase-2 S164. Phosphorylation at S164 appears to inhibit mammalian caspase-2 by preventing its induced proximity oligomerization, thus also preventing procaspase-2 autocatalytic processing. We further identify some of the molecular machinery involved in S164 phosphorylation and demonstrate conservation with the validated Xenopus regulators. Interestingly, we extend the findings of caspase-2 phosphorylation to a study of ovarian cancer, and show that caspase-2 S164 phosphorylation might be involved in determining cancer cell chemosensitivity. We further provide evidence that chemosensitivity can be modulated by the cellular metabolic status in a caspase-2-dependent manner. Thus, we have identified a novel phosphorylation site on mammalian caspase-2 in somatic cells, and are working further to understand the implications of caspase-2 signaling in the context of cancer cell responsiveness to chemotherapeutic treatments.</p> / Dissertation

Cryopreservation of germplasm of aquatic species offers many benefits to the fields of aquaculture, conservation and biomedicine. Although successful fish sperm cryopreservation has been achieved with many species, there has been no report of successful cryopreservation of fish embryos and late stage oocytes which are large, chilling sensitive and have low membrane permeability. In the present study, the sensitivity to chilling and toxicity of cryoprotectants of early stage zebrafish ovarian follicles were studied before designing protocols for their cryopreservation using controlled slow cooling. The effect of cryoprotectant, freezing medium, cooling rate, method for cryoprotectant removal, post-thaw incubation time and ovarian follicle developmental stage were investigated. In vitro culture method for early stage zebrafish ovarian follicles were also developed. The studies showed that stage I and II ovarian follicles are less sensitive to chilling than stage III follicles and methanol was the least toxic cryoprotectant. 4M methanol in potassium chloride (KCl) buffer was found to be the optimal cryoprotective solution and the optimum cooling rate was 4 °C/min for stage I and II follicles. Although the highest survivals after 2 h post-thawed incubation were 50.7 ± 4.0% for stage II ovarian follicles obtained with FDA+PI staining, ADP/ATP ratios of the cryopreserved follicles were significantly increased indicating increased cell death. Furthermore, in vitro culture experiments showed that there was no growth for stage I and II ovarian follicles after cryopreservation, indicating that successful cryopreservation of early stage zebrafish ovarian follicles at liquid nitrogen still remains elusive. From in vitro culture study, 90% L-15 medium at pH 9.0 containing 10 IU/ml hCG was effective for in vitro culture of stage I and II ovarian follicles. Systematic study on cryopreservation of early stage fish ovarian follicles at liquid nitrogen temperature is reported ii here for the first time. The results will provide useful information on the future development of protocol design for successful cryopreservation of early stage fish ovarian follicles.

High quality fish oocytes are essential for in vitro maturation (IVM), in vitro fertilization (IVF) protocols, and for use in cryopreservation. It is important to develop methods for assessing oocyte quality for applications in aquaculture, the preservation of endangered species and managing fish models used in biomedical research. The lack of reliable methods of evaluating oocyte quality limits progress in these areas. The present study was undertaken to develop new methods to assess ovarian follicle viability and quality of stage III zebrafish (Danio rerio) ovarian follicles. The methods developed were then applied to study the impact of cryoprotectant and/or cryopreservation procedures. A vital staining procedure, not previously used with zebrafish oocytes, has been investigated. FDA-PI (Fluorescein diacetate-Propidium Iodide) staining was found to be a more sensitive then currently used viability tests and it could also be applied to all ovarian follicles developmental stages. Mitochondrial activity and distribution as biological markers was investigated with the mitochondrial membrane potentialsensitive dye JC-1- (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide). Confocal microscopy, Cryo-scanning and electron microscopy studies were undertaken to determine mitochondria distributional arrangement within the ovarian follicle. This provided new information on zebrafish ovarian follicle structure, and showed that mitochondria exhibited a contiguous distribution at the margin of the granulosa cell layer surrounding stage III zebrafish oocytes. Cryoscanning results showed a polygonal structure of the vitelline envelope, which is reported here for the first time with the mitochondrial distributional arrangement in the granulosa cell layer. Mitochondrial distribution and the evaluation of mitochondrial activity proved to be sensitive markers for ovarian follicle quality, providing more detailed information on cryoprotectant impact. The measurement of ATP levels, ADP/ATP ratio and mtDNA copy number were also undertaken following cryoprotectant exposure. These findings, together with the observation of mitochondrial distribution, suggested that even cryoprotectant treatments that are considered to have little or no toxicity can have a deleterious effect on mitochondrial activity, potentially compromising oocyte growth and embryo development. Therefore, a further optimization of the currently used protocol may need to be considered. The study of organelle distribution and organisation would support in vitro maturation and oocyte development fields, as well as their use as biological markers for quality determination. These findings will contribute to a better understanding of oogenesis/folliculogenesis processes in fish.

No description available.

Primordial follicles are the first small follicles to appear in the mammalian ovary. Women are born with a fixed number of primordial follicles in the ovaries. Once formed, the pool of primordial follicles serves as a source of developing follicles and oocytes. The first aim of this thesis was to investigate the functional role of the intra-oocyte signaling pathways, especially the phosphatidylinositol-3 kinase (PI3K) and mammalian target of rapamycin complex 1 (mTORC1) pathways in the regulation of primordial follicle activation and survival. We found that a primordial follicle remains dormant when the PI3K and mTORC1 signaling in its oocyte is activated to an appropriate level, which is just sufficient to maintain its survival, but not sufficient for its growth initiation. Hyperactivation of either of these signaling pathways causes global activation of the entire pool of primordial follicles leading to the exhaustion of all the follicles in young adulthood in mice. Mammalian oocytes, while growing within the follicles, remain arrested at prophase I of meiosis. Oocytes within the fully-grown antral follicles resume meiosis upon a preovulatory surge of leutinizing hormone (LH), which indicates that LH mediates the resumption of meiosis. The prophase I arrest in the follicle-enclosed oocyte is the result of low maturation promoting factor (MPF) activity, and resumption of meiosis upon the arrival of hormonal signals is mediated by activation of MPF. MPF is a complex of cyclin dependent kinase 1 (Cdk1) and cyclin B1, which is essential and sufficient for entry into mitosis. Although much of the mitotic cell cycle machinery is shared during meiosis, lack of Cdk2  in mice leads to a postnatal loss of all oocytes, indicating that Cdk2 is important for oocyte survival, and probably oocyte meiosis also. There have been conflicting results earlier about the role of Cdk2 in metaphase II arrest of Xenopus  oocytes. Thus the second aim of the thesis was to identify the specific Cdk that is essential for mouse oocyte meiotic maturation. We generated mouse models with oocytespecific deletion of Cdk1  or Cdk2  and studied the specific requirements of Cdk1 and Cdk2 during resumption of oocyte meiosis. We found that only Cdk1 is essential and sufficient for the oocyte meiotic maturation. Cdk1 does not only phosphorylate the meiotic phosphoproteins during meiosis resumption but also phosphorylates and suppresses the downstream protein phosphatase 1, which is essential for protecting the Cdk1 substrates from dephosphorylation.

Female gametes, or oocytes grow and mature in a niche environment maintained by the somatic cells of the ovarian follicle. At ovulation ovarian follicle cells respond to the luteinising hormone (LH) surge coordinating the final maturation, meiotic resumption and release of oocytes. Simultaneously, production of a unique “mucified” extracellular matrix surrounding the oocyte through synthesis of Hyaluronan (HA) and HA cross-linking proteins produces an “expanded” and stabilised cumulus oocyte matrix with a specific composition, structure and function. In vitro maturation (IVM) of oocytes is a procedure by which cumulus oocyte complexes (COCs) are stimulated to produce cumulus matrix and undergo oocyte maturation ex vivo. In vitro maturation is a useful procedure for studying oocyte competence as well as offering health benefits for patients undergoing assisted reproduction. Oocytes derived from IVM have much lower developmental competence than in vivo matured oocytes, likely as a result of altered environmental conditions and gene expression leading to suboptimal maturation and/or inappropriate metabolic control in oocytes. Cumulus matrix expansion is widely used as an indicator of good oocyte developmental potential, however, the mechanism(s) that endow oocyte quality and how these may be influenced by the cumulus matrix are poorly understood. To better understand the process by which cumulus matrix is linked to the final stages of oocyte maturation, I undertook investigation of mouse COC matrix composition and function after in vivo maturation in comparison to IVM. The gene responsible for Hyaluronan synthesis, Has2, was not impaired under IVM conditions. In contrast, two key extracellular matrix proteins; Versican and Adamts1, which are normally selectively incorporated into periovulatory COCs in vivo, were greater than 10-fold reduced in IVM whether stimulated with Egf and/or FSH. This work is the first to show that commonly used IVM conditions result in altered gene expression in cumulus cells. Furthermore, the absence of Adamts1 and Versican suggest that COC matrix may be functionally insufficient. Although associated with good developmental potential, the function of the COC matrix in oocyte maturation is unknown. I assessed the properties of COC matrix that control metabolite supply to oocytes by examining transport of fluorescently labelled glucose and cholesterol across mouse COCs. Profound differences in the control of metabolite supply to oocytes in IVM were observed. In vivo matured complexes were capable of excluding glucose from the entire COC and cholesterol was excluded from oocytes. Conversely IVM COCs were more permissive to rapid equilibration of glucose and cholesterol concentrations across the complex and in oocytes. In fact both metabolites accumulated rapidly in IVM oocytes resulting in inverse gradient patterns of glucose and cholesterol abundance with highest concentrations accumulating in the oocyte after IVM vs highest concentrations surrounding the COC after in vivo maturation conditions. As oocytes are highly sensitive to high glucose my results indicate that metabolic balance in IVM may be disrupted due to impaired molecular filtration properties of the mucified COC matrix that controls supply of hydrophilic and lipophylic substrates. Importantly these novel findings can explain the glucose sensitivity of IVM oocytes and identifies a mechanism by which IVM may lead to poorer oocyte developmental competence. To translate these findings into the improvement of IVM I generated recombinant expression plasmid constructs for several Adamts1 and Versican functional domains. The efficacy of Versican as an IVM supplement that activates cumulus cell signal transduction was proved in principle, by showing enhanced COC matrix expansion when added to mouse IVM cultures. Similar mechanisms are likely to be functional in human COCs since I demonstrated VERSICAN and ADAMTS1 expression in human in vivo matured cumulus and granulosa cells. This work has advanced our understanding of oocyte maturation and will lead to improvements in IVM and healthier outcomes from reproductive therapies. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1342419 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

Female gametes, or oocytes grow and mature in a niche environment maintained by the somatic cells of the ovarian follicle. At ovulation ovarian follicle cells respond to the luteinising hormone (LH) surge coordinating the final maturation, meiotic resumption and release of oocytes. Simultaneously, production of a unique “mucified” extracellular matrix surrounding the oocyte through synthesis of Hyaluronan (HA) and HA cross-linking proteins produces an “expanded” and stabilised cumulus oocyte matrix with a specific composition, structure and function. In vitro maturation (IVM) of oocytes is a procedure by which cumulus oocyte complexes (COCs) are stimulated to produce cumulus matrix and undergo oocyte maturation ex vivo. In vitro maturation is a useful procedure for studying oocyte competence as well as offering health benefits for patients undergoing assisted reproduction. Oocytes derived from IVM have much lower developmental competence than in vivo matured oocytes, likely as a result of altered environmental conditions and gene expression leading to suboptimal maturation and/or inappropriate metabolic control in oocytes. Cumulus matrix expansion is widely used as an indicator of good oocyte developmental potential, however, the mechanism(s) that endow oocyte quality and how these may be influenced by the cumulus matrix are poorly understood. To better understand the process by which cumulus matrix is linked to the final stages of oocyte maturation, I undertook investigation of mouse COC matrix composition and function after in vivo maturation in comparison to IVM. The gene responsible for Hyaluronan synthesis, Has2, was not impaired under IVM conditions. In contrast, two key extracellular matrix proteins; Versican and Adamts1, which are normally selectively incorporated into periovulatory COCs in vivo, were greater than 10-fold reduced in IVM whether stimulated with Egf and/or FSH. This work is the first to show that commonly used IVM conditions result in altered gene expression in cumulus cells. Furthermore, the absence of Adamts1 and Versican suggest that COC matrix may be functionally insufficient. Although associated with good developmental potential, the function of the COC matrix in oocyte maturation is unknown. I assessed the properties of COC matrix that control metabolite supply to oocytes by examining transport of fluorescently labelled glucose and cholesterol across mouse COCs. Profound differences in the control of metabolite supply to oocytes in IVM were observed. In vivo matured complexes were capable of excluding glucose from the entire COC and cholesterol was excluded from oocytes. Conversely IVM COCs were more permissive to rapid equilibration of glucose and cholesterol concentrations across the complex and in oocytes. In fact both metabolites accumulated rapidly in IVM oocytes resulting in inverse gradient patterns of glucose and cholesterol abundance with highest concentrations accumulating in the oocyte after IVM vs highest concentrations surrounding the COC after in vivo maturation conditions. As oocytes are highly sensitive to high glucose my results indicate that metabolic balance in IVM may be disrupted due to impaired molecular filtration properties of the mucified COC matrix that controls supply of hydrophilic and lipophylic substrates. Importantly these novel findings can explain the glucose sensitivity of IVM oocytes and identifies a mechanism by which IVM may lead to poorer oocyte developmental competence. To translate these findings into the improvement of IVM I generated recombinant expression plasmid constructs for several Adamts1 and Versican functional domains. The efficacy of Versican as an IVM supplement that activates cumulus cell signal transduction was proved in principle, by showing enhanced COC matrix expansion when added to mouse IVM cultures. Similar mechanisms are likely to be functional in human COCs since I demonstrated VERSICAN and ADAMTS1 expression in human in vivo matured cumulus and granulosa cells. This work has advanced our understanding of oocyte maturation and will lead to improvements in IVM and healthier outcomes from reproductive therapies. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1342419 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

Research Doctorate - Doctor of Philosophy (PhD) / Membrane fusion events are a fundamental aspect of cellular biology and underpin important processes such as organ formation and fertilisation. Within the latter, proteins that are expressed on the egg surface which are responsible for mediating sperm recognition, binding and fusion to the egg, are yet to be fully determined. Evidence does however suggest that egg surface glycophosphatidylinositol (GPI)-anchored proteins play a role in sperm binding, whilst another class of proteins, known as tetraspanins, appear to be important in downstream events of membrane fusion. Of the tetraspanins, CD9 and CD81 have been identified as fulfilling roles in membrane fusion; identifications are however yet to obtained for the important GPI-anchored protein(s). This research aimed to identify and characterise egg surface proteins implicated in sperm-egg interaction, and embodied attempts to both identify the important GPI-anchored protein(s) as well as expand upon tetraspanin studies through investigations into mice lacking the tetraspanin CD151. Throughout this research, it was hypothesised that membrane fusion events of fertilisation parallelled those of enveloped virus – host cell fusion, for which rearrangement of surface protein thiols is essential. In vitro binding and fusion experiments were utilised as functional bioassays in the investigation of factors affecting sperm-egg interaction, such as tetraspanin deletion and the xenobiotic modification of cell surface thiols, while mass spectrometry (MS)-based proteomics and bioinformatics-based analyses were employed to compile oocyte protein databases and to identify candidate proteins responsible for mediating sperm-egg interaction, such as GPI-anchored proteins. It was determined that exposing oocytes to compounds with a capacity to alkylate cell surface thiols strongly inhibited sperm-egg binding. Additionally, while CD151 deletion had no effect on sperm-egg binding, the downstream events of membrane fusion were significantly impaired. Ovaries from CD151 null mice also exhibited abnormal phenotypes. In addition, a total of 11 identifications were obtained in the search for the GPI-anchored proteins expressed within eggs, however only 6 of these were deemed to be potential mediators of sperm-egg interaction. In conclusion, the experiments outlined herein demonstrate a novel inhibitory effect for specific xenobiotics on sperm-egg interaction, and correlate the inhibitory action of these compounds with their capacity to reduce cell surface thiol labelling. A novel role for CD151 in the mediation of sperm-egg fusion was also discovered, while at the same time the important GPI-anchored protein(s) implicated in sperm-egg binding may be among 6 identified potential candidates. Together the findings reiterate the consensus that oocytes possess a cell surface protein complex responsible for mediating sperm binding and fusion as separate events, and in light of the demonstrated importance of surface thiols, that events of sperm-egg membrane fusion parallel those of enveloped virus – host cell fusion.

Research Doctorate - Doctor of Philosophy (PhD) / Membrane fusion events are a fundamental aspect of cellular biology and underpin important processes such as organ formation and fertilisation. Within the latter, proteins that are expressed on the egg surface which are responsible for mediating sperm recognition, binding and fusion to the egg, are yet to be fully determined. Evidence does however suggest that egg surface glycophosphatidylinositol (GPI)-anchored proteins play a role in sperm binding, whilst another class of proteins, known as tetraspanins, appear to be important in downstream events of membrane fusion. Of the tetraspanins, CD9 and CD81 have been identified as fulfilling roles in membrane fusion; identifications are however yet to obtained for the important GPI-anchored protein(s). This research aimed to identify and characterise egg surface proteins implicated in sperm-egg interaction, and embodied attempts to both identify the important GPI-anchored protein(s) as well as expand upon tetraspanin studies through investigations into mice lacking the tetraspanin CD151. Throughout this research, it was hypothesised that membrane fusion events of fertilisation parallelled those of enveloped virus – host cell fusion, for which rearrangement of surface protein thiols is essential. In vitro binding and fusion experiments were utilised as functional bioassays in the investigation of factors affecting sperm-egg interaction, such as tetraspanin deletion and the xenobiotic modification of cell surface thiols, while mass spectrometry (MS)-based proteomics and bioinformatics-based analyses were employed to compile oocyte protein databases and to identify candidate proteins responsible for mediating sperm-egg interaction, such as GPI-anchored proteins. It was determined that exposing oocytes to compounds with a capacity to alkylate cell surface thiols strongly inhibited sperm-egg binding. Additionally, while CD151 deletion had no effect on sperm-egg binding, the downstream events of membrane fusion were significantly impaired. Ovaries from CD151 null mice also exhibited abnormal phenotypes. In addition, a total of 11 identifications were obtained in the search for the GPI-anchored proteins expressed within eggs, however only 6 of these were deemed to be potential mediators of sperm-egg interaction. In conclusion, the experiments outlined herein demonstrate a novel inhibitory effect for specific xenobiotics on sperm-egg interaction, and correlate the inhibitory action of these compounds with their capacity to reduce cell surface thiol labelling. A novel role for CD151 in the mediation of sperm-egg fusion was also discovered, while at the same time the important GPI-anchored protein(s) implicated in sperm-egg binding may be among 6 identified potential candidates. Together the findings reiterate the consensus that oocytes possess a cell surface protein complex responsible for mediating sperm binding and fusion as separate events, and in light of the demonstrated importance of surface thiols, that events of sperm-egg membrane fusion parallel those of enveloped virus – host cell fusion.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 5 uppsatser.