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

Ladministration de spermine aux rats non sevrés, dans des conditions expérimentales précises, constitue un modèle détude de la maturation postnatale de lintestin grêle. Lanalyse des variations survenant au sein du protéome cellulaire, trois jours après lingestion de spermine, met en évidence des protéines impliquées dans le processus de maturation de lintestin grêle. La même étude, réalisée quelques heures après lingestion, précise la phase de desquamation cellulaire initialement observée. Les analyses effectuées révèlent que, six heures après le traitement des ratons à la spermine, la phase de desquamation touche à sa fin et lépithélium se régénère tandis que, dix-huit heures après ce traitement, les résultats indiquent un début de maturation des entérocytes, signalé, notamment, par lapparition, dans liléon, dune isoforme de la phosphatase alcaline, qui est spécifique de lépithélium intestinal mature. Des protéines impliquées dans la conformation de la chromatine et, par là, dans la régulation de lexpression de gènes spécifiques, sont identifiées six heures après lingestion de la polyamine. Devant la diversité des protéines identifiées trois jours après le traitement des animaux, nous nous sommes plus particulièrement intéressée à des modifications survenant au niveau de la cathepsine D et des enzymes du cycle de lurée. Des propriétés des enzymes de ce cycle sont effectivement modifiées lors de la maturation postnatale spontanée de lintestin grêle. Lors de lingestion de spermine, on observe des modifications au niveau de la transcription de gènes codant pour certaines de ces enzymes. Dans le cas de la cathepsine D, lexpression du gène codant pour lenzyme et lactivité de celle-ci sont diminuées dans les entérocytes de liléon suite à lingestion de spermine. Laction de cette polyamine a été reproduite, in vitro, à partir dune culture de cellules de tumeurs mammaires surexprimant le gène de la cathepsine D. Par ailleurs, nos résultats montrent que, dans certains cas, la spermine nagit pas au niveau de lexpression génique mais que des modifications post-traductionnelles des protéines sont à lorigine des variations dactivités enzymatiques observées, comme cest le cas de la phosphatase alcaline intestinale. Les protéines identifiées par lanalyse protéomique, et létude théorique de leur régulation, révèlent que plusieurs voies de signalisation intracellulaire sont affectées par lingestion de spermine : cette polyamine active la voie de signalisation faisant intervenir la PLC ainsi que celle dépendant des glucocorticoïdes. A linverse, la signalisation intracellulaire impliquant la PKA est inhibée. En conclusion, notre approche globale, considérant les protéines dont des propriétés varient de manière majeure dans les entérocytes de rats immatures soumis à laction de la spermine, ouvre plusieurs pistes pour des recherches futures ciblées. La poursuite des investigations, limitées cette fois à considérer une voie métabolique particulière ou des protéines spécifiques, conduira à une compréhension plus complète des modifications survenant dans les entérocytes du rat au moment du sevrage.

Research Doctorate - Doctor of Philosophy / On leaving the testis, spermatozoa can neither swim nor fertilize the oocyte. These functional properties are acquired as spermatozoa engage in a process of post-testicular maturation in the epididymis. The studies described in this thesis were designed to elucidate some of the fundamental mechanisms associated with the regulation of epididymal maturation in mouse spermatozoa. The initial studies described in this thesis investigated the expression of a cAMP/PKAdependent, tyrosine phosphorylation signaling pathway that becomes activated during epididymal sperm maturation. It was demonstrated that the entry of spermatozoa into the epididymis was accompanied by the sudden stimulation of this pathway, initially in the principal piece of the cell and subsequently in the midpiece. The competence of these cells to phosphorylate the entire sperm tail, particularly the mitochondria, was accompanied by a capacity to exhibit hyperactivated motility on stimulation with cAMP. A distinctly different pattern of tyrosine phosphorylation involving the acrosomal domain of the sperm head was provoked as spermatozoa entered the caput epididymis and then remained high until these cells entered the distal corpus and cauda. However, tyrosine dephosphorylation of the sperm acrosomal domain during epididymal transit did not appear to be functionally involved in controlling the acrosome reaction. Research into the biochemical basis of sperm epididymal maturation revealed that this process was associated with the activation of sperm mitochondria, leading to the creation of a mitochondrial membrane potential (MMP) and activation of mitochondrial free radical generation. Immature caput spermatozoa displayed a low MMP whereas mature caudal spermatozoa actively maintained a high MMP. Moreover mitochondrial generation of reactive oxygen species (ROS) could be triggered by antimycin A in mature caudal spermatozoa but not in immature caput spermatozoa, suggesting a lack of electron flux in the latter. The molecular mechanisms responsible for regulating mitochondrial function were also found to be reversible, as washing the cells free of epididymal fluid allowed caput spermatozoa to acquire a high MMP and generate ROS while incubating caudal spermatozoa in caput epididymal fluid, suppressed MMP and their ability to generate ROS. Pharmacological suppression of mitochondrial activity was subsequently found to be associated with the inhibition of hyperactivated motility. These results strongly suggested that fluid from the caput epididymis contained a mitochondrial inhibitor and that activation of mitochondrial activity was due to the removal or inactivation of this inhibitor during epididymal transit. This causative factor was not species specific. Incubation of ejaculated human spermatozoa in murine epididymal fluid systematically suppressed their MMP. The characterization of caput epididymal fluid suggested that the putative mitochondrial inhibitor is a heat-resistant protein with a molecular weight larger than 30 kDa. The final results presented in this thesis demonstrate that a full-length Riken protein is a potential candidate for the putative mitochondrial inhibitor that switches off mitochondrial function in caput spermatozoa. Indeed, these results represent the first report suggesting that the epididymal maturation is associated with activation of sperm mitochondria and the first study of a testis specific protein that could be a regulator of mitochondrial function in the male germ line. Further characterization of the mechanisms by which epididymal spermatozoa control mitochondrial function may hold the key to our understanding of sperm maturation. It may also lead us to a clear exposition of the molecular basis of human male infertility, potentially serve as a target for infertility treatment and possibly contribute to the development of novel contraceptive agents.

Pendant la maturation des graines, la germination, tolérance à la dessication et longévité sont acquises de manière séquentielle. La maturation s’achève par la dessication qui amène l’embryon à l’état de quiescence. La maturité des graines à la récolte est le premier facteur qui in¿ uence la longévité et l’établissement de la culture lors du semis. On ne comprend pas comment la longévité est installée pendant la maturation et comment un séchage prématuré in¿ uence la longévité et la reprise des activités cellulaires pendant l’imbibition. L’objectif de la thèse était de répondre à ces questions en comparant les transcriptomes de graines immatures et matures de soja et Medicago truncatula pendant la dessication et l’imbibition. Les graines immatures furent récoltées après le remplissage avant la dessiccation, lorsque la longévité n’est pas encore acquise.Chez le soja, la comparaison des transcriptomes des graines immatures et matures montre que le séchage forcé n’est pas identique à la dessication in planta qui se caractérise par la synthèse de protéines chaperones. Plus de 89% des gènes différentiellement exprimés après 18 h d’imbibition présentent des pro¿ ls d’expression identiques dans les graines immatures et matures, en accord avec la germination comparable de celles-ci. L’analyse des transcrits dont la teneur augmente uniquement pendant l’imbibition des graines mature suggère la mise en place de mécanismes de réparation. La comparaison de ces données avec Medicago montre que l’imbibition des graines matures se caractérise par une sur-représentation des gènes liés au / During seed maturation, germination, desiccation tolerance and longevity are acquired sequentially. Seed maturation is terminated by a desiccation phase that brings the embryo to a quiescent state. Seed maturity at harvest in¿ uences seed longevity and crop establishment. After harvest, seeds are usually dried to water content compatible with long term storage and post-harvest treatments. However, there is a lack of understanding of how seed longevity is acquired during seed maturation and how premature drying impacts longevity and resumption of cellular activities during imbibition. This was addressed here by comparing transcriptome changes associated with maturation drying and imbibition of seeds of soybean and Medicago truncatula, harvested at an immature stage and mature dry stage.The immature stage corresponded to end of seed ¿ lling when longevity was not acquired while other vigor traits were acquired. Transcriptome characterization in soybean revealed that enforced drying was not similar to maturation drying in planta, which stimulated degradation of chlorophyll and synthesis of protective chaperones. Eighty-nine % of the differentially expressed genes during a 18h-imbibition period showed a similar pattern between immature and mature seeds, consistent with a comparable germination between stages. An analysis of the 147 transcripts that increased during imbibition of mature seeds but not in immature seeds suggested an activation of processes associated with shoot meristem development and DNA repair. These data were compared with imbibing immature and mature seeds

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Previous reports have demonstrated that running economy (RE), a measure of efficiency of locomotion, is superior in adults than in children; however, it is unclear how these differences come to be. Purpose: To identify the effect of maturity status, physical activity and various other anatomical and physiological factors on RE development in boys aged 8 to 16 years. Methods: Data were collected as part of the Saskatchewan Growth and Development Study (SGDS; 1964-1973). Using a pure longitudinal study design, anthropometric, maturity, physiological characteristics (treadmill run) and physical activity were assessed annually for nine consecutive years. Two-hundred and two eight year-old males were measured in 1965; by 1973, complete longitudinal data were available for 63 participants. During the treadmill run, a measure of submaximal oxygen consumption (VO2) was recorded, an index of RE. Four approaches of normalizing VO2 to body size were investigated. Maturity status was determined based upon chronological age at peak height velocity (PHV). Physical activity was assessed by two teacher ratings and two questionnaires. Results: Normalizing VO2 to body surface area was found to be the most appropriate body size adjustment. Submaximal VO2 (ml/m^2/min) at 9.6 km/h decreased with increasing chronological age (p<0.05). At common chronological age bands, late-maturing boys demonstrated superior RE than early-maturing boys from ages 10-14 years (p<0.05); average-maturing boys were also found to be more efficient than early-maturers at 12 and 13 years of age (p<0.05). Physical activity was not found to have any significant effect on the development of RE (p>0.05). A series of age-specific regression analyses identified body surface area and respiratory exchange ratio (RER) as variables which account for a significant portion of the variance in absolute VO2 (0.619<R^2<0.903); RER was not significant (p>0.05) at all chronological ages. Conclusion: Determining an appropriate approach for normalizing VO2 values is essential to allow for reliable investigation into factors other than size that affect RE. Maturity status was found to significantly affect RE development; however, only during the circumpubertal years. No effect of physical activity was found on RE development in boys 8-16 years. The relative influence of maturity status and RER are variable across different ages.

Previous reports have demonstrated that running economy (RE), a measure of efficiency of locomotion, is superior in adults than in children; however, it is unclear how these differences come to be. Purpose: To identify the effect of maturity status, physical activity and various other anatomical and physiological factors on RE development in boys aged 8 to 16 years. Methods: Data were collected as part of the Saskatchewan Growth and Development Study (SGDS; 1964-1973). Using a pure longitudinal study design, anthropometric, maturity, physiological characteristics (treadmill run) and physical activity were assessed annually for nine consecutive years. Two-hundred and two eight year-old males were measured in 1965; by 1973, complete longitudinal data were available for 63 participants. During the treadmill run, a measure of submaximal oxygen consumption (VO2) was recorded, an index of RE. Four approaches of normalizing VO2 to body size were investigated. Maturity status was determined based upon chronological age at peak height velocity (PHV). Physical activity was assessed by two teacher ratings and two questionnaires. Results: Normalizing VO2 to body surface area was found to be the most appropriate body size adjustment. Submaximal VO2 (ml/m^2/min) at 9.6 km/h decreased with increasing chronological age (p<0.05). At common chronological age bands, late-maturing boys demonstrated superior RE than early-maturing boys from ages 10-14 years (p<0.05); average-maturing boys were also found to be more efficient than early-maturers at 12 and 13 years of age (p<0.05). Physical activity was not found to have any significant effect on the development of RE (p>0.05). A series of age-specific regression analyses identified body surface area and respiratory exchange ratio (RER) as variables which account for a significant portion of the variance in absolute VO2 (0.619<R^2<0.903); RER was not significant (p>0.05) at all chronological ages. Conclusion: Determining an appropriate approach for normalizing VO2 values is essential to allow for reliable investigation into factors other than size that affect RE. Maturity status was found to significantly affect RE development; however, only during the circumpubertal years. No effect of physical activity was found on RE development in boys 8-16 years. The relative influence of maturity status and RER are variable across different ages.