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by Will W.M. Lee. / Thesis (M.Phil.) - Chinese University of Hong Kong. / Bibliography: leaves 101-110. / Chapter CHAPTER I --- GENERAL INTRODUCTION / Chapter A --- Spermatogenesis --- p.1 / Chapter B --- Sperm Maturation --- p.2 / Chapter C --- Ejaculation --- p.3 / Chapter D --- Sperms in Fertilization --- p.3 / Chapter E --- Aim of Spermatozoal Motility Studies --- p.7 / Chapter CHAPTER II --- AN ASSAY TO MEASURE THE SPERMATOZOAL PROGRESSIVE MOTION / INTRODUCTION --- p.8 / MATERIALS AND METHODS --- p.15 / Chapter A --- Sample Collection --- p.15 / Chapter B --- Cell Wash --- p.17 / Chapter C --- Media --- p.18 / Chapter D --- Motility Assay Chamber --- p.18 / RESULTS AND DISCUSSIONS --- p.22 / Chapter A --- Sperm Entry --- p.22 / Chapter B --- Sperm Motility --- p.32 / Chapter CHAPTER III --- EFFECT OF VARIOUS GROUPS OF CHEMICALS ON THE MOTILITY OF SPERMATOZOA / INTRODUCTION --- p.48 / MATERIALS AND METHODS --- p.55 / RESULTS AND DISCUSSIONS --- p.57 / Chapter A --- Energy Source --- p.57 / Chapter B --- "Phosphodiesterase Inhibitors and Cyclic 3',5'-Adenosine Monophosphate (cAMP)" --- p.59 / Chapter C --- p-Nitrophenyl Compounds --- p.64 / Chapter D --- Motility of Spermatozoa from Addicted Rat and Effect of Morphine on tozoa In VitroSperma- --- p.68 / Chapter E --- Metallic Ions and EDTA --- p.68 / Chapter CHAPTER IV --- EFFECT OF SEMINAL PLASMA ON SPERM MOTILITY --- p.77 / INTRODUCTION / MATERIALS AND METHODS --- p.79 / RESULTS --- p.82 / DISCUSSIONS --- p.98 / REFERENCES --- p.101 / Chapter APPENDIX I --- Spermatozoa Repellent as a Contraceptive --- p.111 / Chapter APPENDIX II --- Effect of p -Nitrophenylglycerol on Motility of Rat Epididymal Spermatozoa --- p.117

Bibliography: leaves 206-251. The motor and sensory interactions between nutrients and proximal gut in humans are not well understood, despite the pivotal importance of these interactions on appetite, absorption and thus, nutrition. In part, this lack of knowledge results from technical difficulties in studying motor function in the human gut. In particular, the inability to continuously measure intraluminal flow with any degree of temporal resolution, has impeded progress in this field. The studies described in this thesis focus on nutrient-gut interactions, and also on the development of novel methodologies aimed at advancing the understanding and interpretation of the relationships between intraluminal pressures and flows.

Gastric emptying, and small intestinal glucose exposure and absorption, are potentially important determinants of postprandial blood glucose homeostasis and energy intake. The studies presented in this thesis were designed to provide novel insights into the interrelationships of upper gastrointestinal function with glycaemia and appetite in both health and type 2 diabetes. The issues which were addressed relate in particular to : ( i ) the physiology, regulation and measurement of gastric and small intestinal motility, ( ii ) the relationships between small intestinal glucose exposure, incretin hormone release, antropyloroduodenal motility and appetite, and ( iii ) the impact of gastric and small intestinal motility on glycaemia. The study reported in chapter 4 evaluated the effect of variations in small intestinal glucose delivery on blood glucose, plasma insulin, and incretin hormone ( GLP - 1 and GIP ) concentrations in healthy subjects. While initially rapid, and subsequently slower, duodenal glucose delivery potentiated incretin and insulin responses when compared to constant delivery of an identical glucose load, the overall glycaemic excursion was not improved. These observations add to the rationale for the use of dietary and pharmacological strategies designed to reduce postprandial glycaemic excursions in health and type 2 diabetes by slowing gastric emptying, rather than initially accelerating it. Fat is a potent inhibitor of gastric emptying. In chapter 5, the acute effect of slowing gastric emptying by fat, on postprandial glycaemia in type 2 diabetes, has been evaluated. Ingestion of a small amount of olive oil, as a 'preload' 30 min before a carbohydrate meal, was shown to markedly slow gastric emptying, affect intragastric meal distribution, delay the postprandial rises in blood glucose, plasma insulin, and GIP, and stimulate GLP - 1. In contrast, the effects of including the same amount of oil within the meal, on gastric emptying, as well as glycaemic and incretin responses, were relatively modest. As blood glucose levels had not returned to baseline by 210 min ( the end of each experiment ), effects on the overall glycaemic ( or insulinaemic ) response could not be determined ; this represents a priority for future studies. The energy content of a meal is a major determinant of its rate of gastric emptying. The study reported in chapter 6 demonstrated that the substitution of an artificial sweetener ( "diet" mixer ) for sucrose ( "regular" mixer ) in a mixed alcoholic beverage has a major impact on the rate of gastric emptying and alcohol absorption in healthy adults. A low calorie alcohol - containing drink ( made with "diet" mixer ) emptied from the stomach much more rapidly and resulted in higher blood alcohol concentrations when compared with a relatively high calorie alcoholic drink ( made with "regular" mixer ). These observations highlight the need for community awareness of factors, other than the alcohol content of a beverage, which should be taken into account in considering safe levels of consumption and the potential for inebriation. Upper gastrointestinal motor function and incretin hormone ( GLP - 1 and GIP ) secretion are known to be major determinants of postprandial glycaemia and insulinaemia, however, the impact of small intestinal flow events on glucose absorption and incretin release has not been evaluated. In the study reported in chapter 7, intraduodenal pressures and impedance signals were recorded simultaneously in healthy humans, while glucose was infused into the duodenum in the presence and absence of the anticholinergic drug, hyoscine butylbromide. The frequency of duodenal flow events ( evaluated by impedance ) was suppressed by hyoscine much more than that of duodenal pressure waves, or propagated pressure wave sequences ( evaluated by manometry ). Blood glucose and plasma 3 - OMG concentrations ( the latter provide an index of glucose absorption ) were lower during hyoscine than saline. Plasma insulin, GLP - 1, and GIP concentrations were initially lower during hyoscine. The disparity between impedance measurements and manometry in detecting alterations in flow during hyoscine infusion was marked and, accordingly, supports the potential utility of small intestinal impedance monitoring to evaluate alterations in gastrointestinal transit in various disease states. The observations also indicate that the frequency of small intestinal flow events is a determinant of both glucose absorption and incretin release. Intraduodenal administration of the local anaesthetic, benzocaine, has been shown to attenuate the release of cholecystokinin ( CCK ) by small intestinal lipid, and the perceptions of fullness, discomfort, and nausea induced by gastric distension during small intestinal lipid infusion, implying that local neural mechanisms may regulate CCK release in response to intraduodenal nutrients. In chapter 8, the effects of intraduodenal administration of benzocaine on : ( i ) blood glucose, incretin hormone and insulin concentrations ( ii ) antropyloroduodenal motility, and ( iii ) gut sensations and appetite, in response to an intraduodenal glucose infusion, were evaluated in healthy subjects. Benzocaine attenuated the perceptions of abdominal bloating and nausea, but had no effect on antro - pyloro duodenal motility, blood glucose concentrations, or incretin responses. These observations indicate that the induction of sensations by small intestinal glucose is mediated by local neural pathways. GLP - 1 is released from L - cells whose density is greatest in the distal jejunum and ileum, GIP predominantly from duodenal K cells, and cholecystokinin ( CCK ) from I cells, which appear confined to the duodenum and jejunum. The study reported in chapter 9 evaluated the effects of infusion of glucose into different gut regions ( mid - jejunal vs duodenal ) on incretin hormones, CCK, appetite and energy intake in healthy subjects. There was no difference in the incretin responses between infusion at the two sites ( 85 cm apart ), however the stimulation of CCK and suppression of hunger and energy intake, were greater with the duodenal compared to the jejunal infusion. These observations indicate that the site of small intestinal glucose exposure is a determinant of CCK release and appetite. Both glucose and fat are known to be potent stimuli for incretin secretion, but the effect of protein is uncertain. Protein may also stimulate insulin secretion directly via absorption of amino acids. In the study reported in chapter 10, gastric emptying, and the blood glucose, insulin and incretin responses, alter a 300 mL drink containing 50 g glucose, 25 g protein, or both 50 g glucose and 25 g protein, were evaluated in healthy subjects. This study established that the addition of protein to an oral glucose load improved the glycaemic response, predominantly by slowing gastric emptying. However, protein also stimulated incretin and insulin secretion. These observations have implications for the use of protein in the dietary management of type 2 diabetes. The relationship between glycaemia, incretin hormones, appetite suppression and modulation of antropyloroduodenal motility with duodenal glucose delivery is poorly defined. In chapter 11, the effects of intraduodenal glucose infusions at different caloric rates ( of 1 kcal / min, 2 kcal / min and 4 kcal / min, or control ( saline ) ) on antropyloroduodenal motility, plasma GLP - 1, GIP and CCK, appetite and energy intake have been evaluated in healthy subjects. While there was a rise in blood glucose in response to all the intraduodenal glucose loads, there was no significant difference in the response to infusions at 2 kcal / min and 4 kcal / min. An initial, transient, small rise in GLP - 1 was evident, in response to all glucose loads, but a sustained and progressive rise only occurred with the 4 kcal / min infusion. In contrast, a load - dependent stimulation of GIP occurred in response to all glucose infusions. The stimulation of CCK was much greater in response to the 4 kcal / min infusion. While antral pressures were suppressed by all rates of glucose infusion, the stimulation of basal pyloric pressure was load - dependent. Energy intake was suppressed only by the 4 kcal / min infusion. This may potentially reflect the substantially greater stimulation of CCK, consistent with the observations reported in chapter 9. This study establishes that there is a substantial discordance in the acute effects of small intestinal glucose on glycaemia, incretin hormones, CCK, motility and appetite. It is planned to perform measurements of plasma insulin on the stored samples - these results were, unfortunately, not available at the time of the submission of this thesis and are critical to the overall interpretation of the data. / Thesis (Ph.D.)--University of Adelaide, School of Medicine, Discipline of Medicine, 2007.

Sperm mobility denotes the net movement of a sperm population. Previous work implicated mitochondrial function as a basis underlying phenotypic variation in this quantitative trait. Our objective was to determine if mitochondrial function was indeed critical to expression of phenotype. Phenotype was assigned to roosters within a random bred population (n=242). A representative subpopulation (n=40) was used to correlate sperm mobility with oxygen consumption (r=0.83). In contrast, sperm mobility was independent of mitochondrial helix length in a sample of males (n=7) representing the range of phenotype observed within the population. Thus, mitochondrial function rather than number appeared to be critical to expression of phenotype. This hypothesis was tested by ultrastructural analysis of sperm midpieces. Males from the lower and upper tails of the distribution were characterized with high and low proportions of sperm containing aberrant mitochondria in 47 and 4% of the cells respectively. When sperm from average males were allowed to segregate into immobile and mobile subpopulations, 40% of immobile sperm contained aberrant mitochondria. In contrast, only 9% of sperm from the same males contained aberrant mitochondria in non-segregated populations. In conclusion, the mitochoridrion is an organelle that may account for phenotypic differences in sperm mobility. / Graduation date: 2002

Polo-like kinase 4 (Plk4) has been identified as a molecular marker of resistance to therapy in cancer. Our laboratory has recently shown a motility defect in Plk4+/- murine embryonic fibroblasts (MEFs) compared to Plk4+/+. I hypothesized that Plk4 augments cancer cell motility. Plk4 depletion with siRNA in Plk4+/+ MEFs and HeLa cells suppressed invasion compared to control. Transient over-expression of Flag-Plk4 in cancer cell lines did not consistently increase invasion. However, modest Plk4 over-expression using stable clones showed higher invasion rates than non-induced. The RhoGEF Ect2, an important Plk4 substrate, transiently localized to protrusions in MEFs, suggesting a RhoA-based signalling cascade in motility. The effect of Plk4 heterozygosity on metastasis was tested in a transgenic mouse model but there was no significant difference in developing metastasis compared to wild type. Further studies are required to characterize the effect of Plk4 on motility, and its potential as a therapeutic cancer target.

Polo-like kinase 4 (Plk4) has been identified as a molecular marker of resistance to therapy in cancer. Our laboratory has recently shown a motility defect in Plk4+/- murine embryonic fibroblasts (MEFs) compared to Plk4+/+. I hypothesized that Plk4 augments cancer cell motility. Plk4 depletion with siRNA in Plk4+/+ MEFs and HeLa cells suppressed invasion compared to control. Transient over-expression of Flag-Plk4 in cancer cell lines did not consistently increase invasion. However, modest Plk4 over-expression using stable clones showed higher invasion rates than non-induced. The RhoGEF Ect2, an important Plk4 substrate, transiently localized to protrusions in MEFs, suggesting a RhoA-based signalling cascade in motility. The effect of Plk4 heterozygosity on metastasis was tested in a transgenic mouse model but there was no significant difference in developing metastasis compared to wild type. Further studies are required to characterize the effect of Plk4 on motility, and its potential as a therapeutic cancer target.

In vitro fertilization is not efficient in the horse, and this may be due to a failure of induction of hyperactivated motility in stallion sperm in vitro. Hyperactivated motility is characterized by high curvilinear velocity and amplitude of lateral head movement, and is required for the sperm to penetrate the zona pellucida of the oocyte in order to achieve fertilization. In mice, hyperactivated motility is induced by calcium influx into the flagellum of the sperm through the CatSper channel, a tetrameric cation channel made up of CatSper proteins 1 to 4. The CatSper channel is located specifically in the principal piece of the sperm tail, and opens in response to an increase in intracellular pH. Factors associated with the induction of hyperactivated motility of sperm have been reported in most domestic and laboratory species, with the notable exception of the horse. In addition, presence of CatSper proteins has not been demonstrated in the horse. Our objective was to determine the presence and location of the CatSper1 protein on stallion sperm, and to determine whether alkalinization of the intracellular pH induces hyperactivated motility in stallion sperm. Presence of the CatSper1 protein on the principal piece of the flagellum of stallion sperm was confirmed by immunocytochemistry with an anti-human CatSper1 C-terminus antibody. Incubation of stallion sperm with the cell-permeant weak base NH4Cl increased curvilinear velocity and amplitude of lateral head movement values in stallion sperm as measured by computer-assisted sperm analysis. These measures are indicative of hyperactivated motility in other species. Hyperactivated-like motility in response to NH4Cl was dependent upon the CO2 atmosphere in which the sperm were incubated and was enhanced by presence of glucose in the medium. Maximum motility values were reached with 25 mM NH4Cl at 60 minutes in a 5% CO2 atmosphere. These results indicate that the CatSper1 protein, and thus presumably the CatSper channel, is present on the principal piece of stallion sperm and that treatments inducing a rise in intracellular pH increase hyperactivated-like motility. These findings represent a basis for establishment of in vitro fertilization protocols that include induced hyperactivated motility to ensure zona penetration.

published_or_final_version / Mechanical Engineering / Master / Master of Philosophy

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