CARACTERIZACION DE LA POBLACION

The general objective of this research was to elucidate specific physiological roles of AA and DHA and their underlying mechanisms. In order to achieve this objective, the research was divided into three parts: a) establishing a time course of D6D-/- pathology, b) determining specific HUFA physiological functions through dietary prevention of D6D-/- pathology, c) and elucidating the mechanism behind HUFA requirement for male fertility.

In chapter 4, a time course study of D6D-/- pathology was done to explain the delay in appearance of HUFA deficiency phenotype, specifically ulcerative dermatitis which occurs by 21 weeks of age. This study demonstrates the presence of HUFA stores in D6D-/- at weaning or 3 weeks of age, indicating HUFA is provided by the mother. These HUFA stores are rapidly depleted after weaning up to 6 weeks of age at which point the rate of depletion slows down.

The period of accelerated depletion coincides with the first observed HUFA deficiency

phenotype of hepatic lipidosis and disrupted spermatogenesis. In future studies, avoiding these HUFA stores in D6D-/- before weaning could help in accelerating HUFA depletion in tissue such as brain, which is abundant in HUFA and still at approximately 85% of +/+ at 5 months of age. This has been an important limitation in researching the physiological role of HUFA in brain function.

Specific functions for each HUFA were determined by preventing D6D-/- phenotype through dietary supplementation of either arachidonic acid (AA) or docosahexaenoic acid (DHA) (Table 10.1). In chapter 5, the dietary essential fatty acids linoleic and α-linolenic acid alone could not modulate liver lipid homeostasis resulting in hepatic lipidosis in D6D-/-. In this case, either AA or DHA supplementation was sufficient to prevent liver pathology. Therefore,

essentiality in liver does not seem to be specific to either HUFA. The mechanism behind HUFA

requirement in preventing accumulation of liver triglyceride is yet to be elucidated. This study indicates increased lipogenesis does not seem to play a role in this pathology. Future studies could focus on inefficient lipid secretion due to HUFA deficiency considering the presence of SNARE components during the VLDL secretion process and recent evidence of HUFA interactions with SNARE complex.

In chapter 6, an essential requirement for AA in skin function was demonstrated through prevention of D6D-/- ulcerative dermatitis by AA supplementation but not dietary DHA.

Previously, linoleic acid was considered the main polyunsaturated fatty acid required for skin physiology; however, its presence did not prevent D6D-/- skin pathology. Prevention of gastrointestinal ulceration was also specific to AA. The role of AA in skin and gastrointestinal tract is most likely related to prostaglandin production. Further research is required to determine which prostaglandins maintain epithelial integrity and how this is achieved. The trigger for excessive scratching behavior in D6D-/- prior to ulcerative dermatitis is yet to be elucidated.

In chapter 7, a specific role for DHA was determined for male fertility. The role for HUFA in male reproduction was previously only inferred, however, specific HUFA deficiency in the D6D-/- mouse allowed attribution of requirement for DHA in spermatogenesis since AA was not as successful in restoring all fertility parameters. Chapter 8 demonstrates a novel role for DHA in acrosome biogenesis, an important process in sperm cell maturation. Acrosome formation relies on vesicle fusion. Future research will consist in identifying vesicle fusion SNARE proteins which participate in the development of the acrosome and determine if DHA drives the formation of SNARE complex during spermiogenesis. It is also important to elucidate why AA fails to completely restore fertility parameters, as well as to identify specific steps that were rescued by its supplementation.

Several studies have demonstrated a role for HUFA in modulating the immune system through diet. Chapter 10 shows that HUFA not only modulate immune function but are essential in the immune system, specifically in the development of an antibody response to a T-cell

dependant antigen. The underlying mechanism behind impaired antibody response due to HUFA deficiency is yet to be elucidated. Decreased white pulp of D6D-/- spleen may be a focus area of future research since this is the site of immune cell interactions in the development of antibody.

Challenging the mouse with a T-cell independent antigen would also help elucidate which immune cells are compromised by HUFA deficiency. Macrophage from D6D-/- were shown to have altered metabolism with increased cholesterol synthesis and decreased paraoxynase

activity. Further macrophage characterization, specifically in relation to phagocytosis, would be important to elucidate a role for HUFA in immune function. Preliminary work suggests

impaired phagocytosis. This process relies on SNARE components for pseudopod formation and would most likely require HUFA. Neutrophil homeostasis would be another area of research to study HUFA function, considering the observed spleen myeloid hyperplasia, neutrophilia, and increase in bone marrow myeloid precursors, although this may be secondary to gastrointestinal ulceration.

In all, the two key findings in this research were AA essentiality in skin and a specific requirement for DHA in male fertility, specifically in acrosome biogenesis through vesicle fusion. Future research with the D6D-/- model will help elucidate the mechanism behind HUFA essentiality in testis as wells as other tissues, such as liver, skin, and the immune system. Further comprehension of the role of HUFA would help in the development of treatments for diseases that result from altered HUFA metabolism.