Sulfogalactosylglycerolipid (SGG) is a sulfoglycolipid present specifically at a substantial level in mammalian male germ cells. It has been shown to function as an adhesion molecule important for sperm-egg interaction and a structural lipid involved in formation of sperm lipid rafts during capacitation in vitro. Due to the unique characteristics and functions, SGG can potentially serve as a biomarker for sperm fertility as well as a target for development of a non-hormonal contraceptive. To confirm the in vivo roles of SGG, we sought for transgenic mice with reduced amounts of sperm SGG. Cgt knockout male mice, transgenetically deficient in UDP-galactose:ceramide galactosyltransferase (CGT), an enzyme involved in SGG synthesis, are infertile due to spermatogenesis disruption. However, the Cgt+/- males can still produce sperm and sire offspring. We hypothesized that Cgt+/- males, expected to have reduced SGG amounts, would have compromised fertilizing ability and could serve as in vivo models for studying roles of SGG in fertilization and spermatogenesis. Unexpectedly, our results revealed that Cgt+/- males exhibited unimpaired spermatogenesis and fecundity. Moreover, the levels of SGG as well as lipid profiles of sperm and testes of Cgt+/- mice were similar to those of the wild type, suggesting that compensatory mechanisms must have occurred to maintain SGG levels in the Cgt+/- mice. Although these results revealed that Cgt+/- mice could not be used as the animal models, they implicated significance of normal testis and sperm SGG levels in maintaining normal spermatogenesis and fertility. The possible compensatory mechanisms regulating SGG levels were further investigated in Cgt+/- mice. As expected, only half of Cgt mRNA expression level of the wild type was transcribed in the Cgt+/- testes; however, testicular CGT polypeptides as well as their enzymatic activities in the Cgt+/- mice were found at a comparable level to those of the wild type. On the other hand, no change was found in terms of mRNA levels, polypeptide levels or enzymatic activities of arylsulfatase A (ASA), the enzyme responsible for SGG degradation in the testis. In conclusion, the compensatory mechanisms for SGG level adjustment in Cgt +/- mice occurred through the biosynthetic pathway, rather than the degradation pathway, by increasing the CGT polypeptide expression level. Therefore, identification of specific spermatogenic cell stages, contributing to normal expression levels of CGT and SGG in the Cgt+/- testes warrants further studies, as these studies should provide useful information regarding CGT and SGG importance during male germ cell development. In addition, a new approach to produce the animal models that can produce sperm with reduced SGG levels should be attempted. The RNA interference (RNAi) techniques may be tried to achieve this goal.

This thesis research characterized apoptosis in the adult goldfish pituitary. Apoptosis is a form of programmed cell death that plays key roles in many biological processes by selectively removing old or unwanted cells. Morphological and biochemical signs of apoptosis established its presence within the gland. The pituitary gonadotrophs secrete gonadotropin-II to regulate sex steroid production, ovulation and sperm production. It was possible to combine TUNEL with immunocytochemistry and demonstrate apoptosis in gonadotropin-II positive cells in the pars distalis of the pituitary, allowing for the fist time the identification of apoptotic gonadotrophs in situ. Other studies suggest that pituitary cell populations and function fluctuate through the seasonal reproductive cycle. Apoptosis could play a role in regulating secretion by regulating the cell number. Treatments with the dopamine agonist apomorphine, and with a gonadotropin-releasing hormone agonist failed to modulate the apoptosis levels measured by DNA laddering or by TUNEL. Finally, this study demonstrated that the calcium ionophore A23187, significantly increases apoptosis in whole pituitary gland cultures. These studies established a new method to identify apoptotic hormone-secreting cells.

Experiments were undertaken to evaluate the factors that control the re-absorptive salvage of high density lipoproteins (HDL) in the kidney. HDL is readily taken up at the apical surface of polarized human proximal tubule epithelial cells (HKC-8). HKC-8 cells do not degrade HDL apolipoproteins, but instead transport and re-secrete the lipoprotein from the opposite, basolateral surface. Only ∼10% of the HDL lipids taken up are re-secreted, while ∼60% of the internalized HDL proteins are re-secreted. The composition and charge of HDL directly affects their ability to be internalized and transported through HKC-8 cells. HDL-apolipoproteins stimulate the transport of HDL components to the basolateral surface, while HDL-lipids inhibit the process. Enrichment of HDL with phosphatidylinositol (PI) increases HDL negative charge and inhibits its transport and secretion from the basolateral surface. Enrichment with phosphatidylcholine (PC) decreases HDL charge and enhances the transcytosis of HDL. These results show that HDL composition and charge regulate the re-absorptive salvage of HDL apolipoproteins in the kidney by controlling the intracellular metabolism of this lipoprotein. Our data suggest that apical to basolateral transport within proximal tubule cells could serve to recover HDL from the glomerular filtrate and return it back to the circulation. A stimulation of this process may decrease the loss of HDL from the circulation and therefore be anti-atherogenic.

Nearly fifty percent of North American myocarditis cases are associated to coxsackievirus group B, type 3 (CVB3) infection. CVB3 infection of mice provides a useful model to study pathogenic mechanism of myocarditis. The objective of this study is to test the hypothesis that susceptibility, during the acute CVB3 infection, is under polygenic control including H2 as well as the non H-2 genes. To identify differential parameters of the disease, and if they are influenced by the H-2 haplotype, several phenotypic traits were characterized. Three inbred strains of mice and two congenic strains were infected with CVB3. Differences in survival, body weight loss, quantification of myocarditis and quantification of sarcolemmal disruption were found by comparing three sets of mice sharing the same H-2 haplotype but not the same background. It was determined that host susceptibility to CVB3-induced myocarditis is mainly controlled by "background" genes. Moreover, because there is a naturally occurring variability among inbred mice, ten inbred strains of mice were used for the genetic analysis of four CVB3-induced phenotypes: survival, body weight loss, heart viral load and quantification of sarcolemmal disruption. It was concluded that the strains could be divided into three groups: the highly resistant, the resistant to intermediate strains and the highly susceptible strains. This phenotypic data on commonly used and genetically diverse inbred mouse strains sets up the platform for a detailed analysis of the genetic basis of susceptibility to CVB3.

A high-salt diet raises the sodium concentration in the cerebrospinal fluid (CSF [Na+]) in salt-sensitive individuals, an effect that can be mimicked by intracerebroventricular (icv) infusion of NaCl. Increasing CSF [Na+] increases the concentration of an endogenous brain ouabain-like substance(s) (OLS) that inhibits brain Na, K-ATPases, which in turn activates the brain renin-angiotensin system, augmenting sympathetic nervous system activity and blood pressure. It is unknown which of the ouabain-sensitive Na, K-ATPase alpha subunit isoforms (alpha2 or alpha3) in the brain mediates the pressor responses to increased CSF [Na+]. We hypothesize that the alpha2 isoform mediates the pressor responses to elevated CSF [Na +], such that reduced expression of the alpha2 isoform in mice, via heterozygous gene-targeted knockout, should enhance the pressor response to icv infusion of Na+ or ouabain, compared to wildtype litter mates. These studies suggest a critical role for the alpha2 subunit isoform of the Na, K-ATPase in the pressor response to increased CSF [Na+]. (Abstract shortened by UMI.)

Indirect calorimetry and nitrogen excretion measurements allow quantifying dynamic changes in the rates of carbohydrate, lipid and protein oxidation over time. Although measuring oxygen consumption and carbon dioxide production is relatively simple, only a few studies presented these two measurements in avian models, but none used the data to calculate rates of metabolic fuel oxidation. Therefore, the first goal of this study was to use indirect calorimetry and nitrogen excretion measurements to quantify the rates of lipid, carbohydrate and protein oxidation over time in ruff sandpiper during cold exposure and terrestrial locomotion. Even though the data obtained does not allow me to test these hypotheses, one finding is really interesting. The ruff sandpiper may use intramuscular lipid reserves to power muscle contractions, but these small reserves would probably be depleted within a short period of time, especially during flight. Therefore, this species may have the capacity to use a secondary mechanism to carry lipids from the adipocytes to the working muscles. Because plasma albumin is present in limited amounts, and because each albumin molecule can only bind a limited number of fatty acids, VLDL-TAG from the liver may be a good way to insure that the working muscles do not run out of fuel. (Abstract shortened by UMI.)

Epidemiological studies have associated cardiopulmonary morbidity and mortality with air pollution. Inhalation of pollutants increases plasma levels of the vasoconstrictor peptide endothelin (ET)-1 and its precursor bigET-1 in experimental animals and human subjects, and induces an oxidative stress in the lungs. Changes of circulating ET-1 is attributed to increased de novo synthesis in lung endothelial cells and spillover in the systemic circulation. Clinical studies indicate that excess ET-1 can be detrimental to individuals with cardiovascular and pulmonary diseases. My hypothesis is that oxidative stress pathways in the alveoli mediate the regulation of the endothelinergic system in response to inhalation of air pollutants. I have tested this hypothesis in male Fischer-344 rats by blocking a potential superoxide surge during or after inhalation of pollutants with a superoxide dismutase (SOD) mimetic drug, AEOL 10150. Rats were injected with 2mg/kg of AEOL 10150 two hours prior to inhalation exposure for four hours to pollutants, and sacrificed immediately or 24 hours post exposure. Treatment with the SOD mimetic abrogated the increase in expression of preproET-1 mRNA and ECE-1 mRNA and plasma ET-1 levels caused by the air pollutants. This suggests that oxidative stress pathways contribute to the regulation of endothelinergic system.

It was previously unknown what the significance and location of intracellular lipidation of newly synthesized apoA-I in hepatocytes were in plasma HDL formation. By labeling primary mouse hepatocytes with 3H-choline, we showed that phospholipidation of apoA-I is most significant in endoplasmic reticulum (ER) and medial Golgi compartments with minor lipidation upon export from the cell. Intracellular LDL-cholesterol lipidation of apoA-I is absent, with rapid cholesterol accumulation at the plasma membrane. De novo synthesized cholesterol was able to lipidate apoA-I intracellularly to a small but significant level. In hepatocytes lacking ABCA1, phospholipidation and lipidation by de novo cholesterol were both reduced in Golgi, while ER lipidation remained mostly unchanged. Plasma membrane lipidation by LDL-cholesterol was also significantly reduced. This implies that HDL formation begins with apoA-I phospholipidation in the ER, followed by modest cholesterol lipidation in the Golgi, dependent on ABCA1, with the bulk of cholesterol lipidation occurring at the plasma membrane.

Uncoupling protein 3 (UCP3) is an integral mitochondrial membrane protein thought to disassociate fuel substrate oxidation by allowing proton re-entry into the mitochondrial matrix. Expression of UCP3 has been correlated with fatty acid and glucose metabolism, and reactive oxygen species (ROS) formation. To improve our understanding of the potential involvement of UCP3 in such pathways we investigated the effects of a UCP3 overexpression (2.2-2.5 fold) in the L6 muscle cell line. These findings were compared to those of UCP2 overexpression and DNP exposure. Palmitate oxidation was significantly increased by overexpressing UCP3 but unaffected by the other treatment conditions. Both glucose oxidation and oxygen consumption were unaffected by UCP2 and UCP3 overexpression but were significantly increased by DNP treatment. ROS production was decreased by UCP2, UCP3 and DNP treatment. These findings suggest a role for UCP3 in the regulation of fatty acid oxidation and ROS formation but not in glucose oxidation.

The aim of this thesis was to study aspects of the neuronal control of catecholamine secretion in a teleost, the rainbow trout. The development and validation of a nerve stimulation technique made it possible to determine that a portion of the neuronal control of catecholamine release, which prevailed at low frequency stimulation, could be attributed to vasoactive intestinal polypeptide (VIP) and/or pituitary adenylate cyclase activating polypeptide (PACAP). On the other hand, cholinergic stimulation predominated during higher levels of neuronal activity. Fluorescent histochemical techniques in combination with pharmacological approaches provided direct evidence that VIP and PACAP can elicit the secretion of adrenaline, only, from the chromaffin tissue via specific VIP binding sites that exhibited properties of VPAC receptors. Using in situ perfused posterior cardinal vein preparations, evidence was provided that while the nicotinic receptor appears to be the predominant pathway mediating the effects of acetylcholine on catecholamine secretion, muscarinic receptor stimulation may augment the cellular response to nicotinic receptor activation. Under extreme conditions, muscarinic receptors may directly elicit the secretion of catecholamines. The impact of extracellular catecholamines on catecholamine secretion from chromaffin cells was also investigated. Results revealed that the mechanisms of adrenergic inhibition of catecholamine secretion in response to cholinergic stimulation include activation of chromaffin cell membrane beta2-receptors and presynaptic alpha2-adrenergic receptors. However, catecholamine release in response to VIP appears to be insensitive to the adrenergic negative feedback mechanisms. Finally, despite the rapid progress in cDNA cloning, molecular information on the receptors mediating the effects of VIP and PACAP in fish is scant. In this thesis, I report preliminary findings of the cloning of the trout PAC1, VPAC1, and VPAC2 receptors from brain cDNA. In summary, VIP and PACAP appear to function as neurotransmitters in the neuronal regulation of catecholamine release in this species. The apparent complexity of the mechanisms regulating the secretion of catecholamines from trout chromaffin cells may reflect the precise control required for these hormones to play their role in physiological and biochemical homeostasis in these organisms.