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Di-(2-ethylhexyl) phthalate (DEHP) is a ubiquitous environmental toxicant. The active metabolite of DEHP, mono-(2-ethylhexyl) phthalate (MEHP), is ultimately responsible for disrupting the process of spermatogenesis and promoting germ cell death. In addition, this toxicant has been positively correlated with developmental problems such as cryptorchidism, a derangement of the seminiferous tubules, and a syndrome called testicular dysgenesis, leading to reduced sperm number. The potential impact of MEHP on human fertility justifies a detailed investigation into the mechanisms by which this agent causes germ cell death. MEHP is known to directly target and damage the Sertoli cell, a testicular cell whose main function is to support the development of the principle germ cell types from the earliest stem cell to the most mature spermatozoa. This dissertation examines the downstream effect of Sertoli cell damage on germ cell homeostasis and the proteins that modulate the sensitivity of germ cells to undergo apoptotic elimination. Specifically, the stabilization of the p53 protein is proposed to be an important upstream determinant of Fas-mediated apoptosis in germ cells following MEHP exposure. Furthermore, that the resulting cell death is the result of increased death receptor expression and c-FLIPL ubiquitinylation. The mechanism is speculated to reside in the spermatocyte sub-population of germ cells, which appears to be most responsive to changes in apoptosis. Exposures of wild type mice to MEHP caused an increased p53 stability and elevated protein levels of the membrane-bound death receptors Fas and DR5 in testicular spermatocytes. The expression of these proteins occur coincident with increases in spermatocyte apoptosis and are driven by p53 activity. To further assess the mechanisms responsible for the sensitivity of germ cells to undergo p53-mediated apoptosis, we used the germ cell line GC-2spd(ts) (a p53 temperature sensitive spermatocyte-like cell line that allows for p53 nuclear localization at 32°C but not 37°C). Induction of the p53 protein led to higher levels of the death receptors DR5 and Fas, activation of caspase-8, and decreases in c-FLIPL. Addition of TRAIL (the cognate ligand for DR5) and the agonistic DR5 agonistic antibody MD5-1, triggered a robust synergistic increase of apoptosis in GC-2 cells maintained at the p53 permissive temperature (32°C). DR5 levels on the germ cell plasma membrane were considerably enhanced following these treatments. Immunoprecipitation of c-FLIPL suggests that the protein is ubiquitinylated after cellular stress and concomitant with p53 activity. Experiments also reveal that c-FLIPL levels may be influenced by Itch, a regulatory protein able to label targets for the proteasomal degradation using a ubiquitinylating E3 ligase. Immunohistochemical detection in adult wild type mouse testis show robust increases in Itch protein levels upon MEHP treatment (1g/kg) and subsequently localization to the cytoplasm of meiotic spermatocyte germ cells. Western blot analysis of testis from MEHP treated mice also show a correlation between the reduction of c-FLIPL and an increase in Itch threonine-222 phosphorylation, a necessary modification for its E3 ligase function. These results provide a possible model in which the removal of Sertoli cell support promotes germ cell death through the extrinsic pathway, ultimately leading to disruption of spermatogenesis and testicular dysgenesis in mammals. However, removal of Itch also show increases in apoptosis and Itch protein deficient mice demonstrate defects in meiosis. Thus, Itch may also play a novel role in the cell cycle.

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published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

In multicellular organisms, apoptosis plays an important role in maintaining cellular homeostasis and removing damaged cells that present a threat to the integrity of the organism. Several lines of evidence suggest that apoptosis might also play a role in the normal aging process. In Drosophila, inhibition of apoptosis in muscle tissues results in lifespan extension. The magnitude of lifespan extension depends on the nature of the UAS-DIAP1 transgene. These results led to the working hypothesis that there might be an optimal level of apoptosis needed to maximally extend lifespan, and was tested with two experimental approaches. First, in order to determine the correlation between longevity and the level of apoptosis in four different genotypes that overexpress DIAP1 using the DJ694-GAL4 driver, DIAP1 expression and caspase activity were examined by way of western blots and caspase assays, respectively. Second, the hypothesis was also tested through the manipulation of DIAP1 expression levels. The number of the UAS-DIAP1 transgene was doubled in the genome, which resulted in an increase in DIAP1 expression levels. The DJ694 driver is able to drive the expression of the UAS-DIAP1 transgene, which resulted in DIAP1 overexpression during adult life as confirmed by western blots. The elevated DIAP1 protein levels resulted in lower caspase activity in animals with one copy of the UAS-DIAP1 transgene and a DJ694 driver. Lifespan extension was observed in these animals overexpressing DIAP1. In animals possessing two copies of the UAS-DIAP1 transgene and a DJ694-GAL4 driver, there was a two-fold increase at transcriptional level. Elevated levels of the DIAP1 protein and a reduction in caspase activity were observed in these animals, but these animals did not exhibit a greater extension in lifespan as compared to the animals driving the expression of only one UAS-DIAP1 transgene. Based on the results obtained, three models for the relationship between longevity and inhibition of apoptosis were proposed. The first model predicts that lifespan is expected to increase with apoptosis inhibition. According to the second model, a specific level of apoptosis inhibition is needed to achieve an optimal extension in lifespan. Inhibition of apoptosis outside of this specific level results in a shortened lifespan. The third model hypothesizes that there is a maximum lifespan that can be achieved by the presence of apoptosis inhibition, however further inhibition beyond this point will not result in an additional increase in longevity. / Thesis (Master, Biology) -- Queen's University, 2008-08-09 03:57:11.27

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Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (M.S.)--Villanova University, 2008. / Biology Dept. Includes bibliographical references.

Thesis (M.S.)--Marshall University, 2009. / Title from document title page. Includes abstract. Document formatted into pages: contains 102 p. Includes bibliographical references p. 44-50.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xv, 115 p.; also includes graphics (some col.) Includes bibliographical references (p. 108-115). Available online via OhioLINK's ETD Center