Testicular germ cell apoptosis is a process that begins in early development and continues in the adult testis. It is important during spermatogenesis for maintaining homeostasis of different types of germ cells. The number of sperm produced depends on the supportive capacity of surrounding Sertoli cells, which provide nutrition and an adaptive environment for growth and development of the germ cells. There are two major pathways that regulate germ cell apoptosis: extrinsic and intrinsic. We hypothesize that Sertoli cells use the extrinsic pathway to eliminate germ cells when exposed to phthalates, a common Sertoli cell toxicant. Death ligands, which are involved in the extrinsic pathway, were used in this research to test this hypothesis. Here, we demonstrate that: 1) the loss of FasL and TRAIL protein expression results in decreased production of mature spermatids in the adult testis, likely as a result of alterations in germ cell homeostatsis during the first wave of spermatogenesis. 2) The high baseline incidence of germ cell apoptosis in peripubertal FasL-/- and TRAIL-/- mice is correlated with increases in levels of TRAIL and FasL, respectively. 3) The decline in germ cell apoptosis observed after MEHP treatment in FasL-/- mice closely corresponds to the occurrence of increased levels of c-FLIP. 4) A more predominant role of FasL occurs in controlling the proper number of germ cells during the first wave of spermatogenesis in peri-pubertal mice. TRAIL is more critical for maintaining long-term homeostasis of the germ cell population in adult testis as well as in the reproductive function. 5) Several possible genes are involved in the altered spermatogenesis and development in the testis of gene-deficient mice. 6) Findings described in Chapter 6 indicate cellular mechanisms triggered by MEHP exposure that act to enhance tumor progression/metastasis in testicular embryonal carcinoma cells (NT2/D1). Taken together, these novel findings provide important mechanistic insights into the functional roles of FasL in the testis at distinct developmental periods and further indicate that FasL itself is required for the regulation of c-FLIP levels in the testis. Additionally, exposure to environmental toxicants, such as the phthalates, can enhance testicular cancer metastasis and invasion. / text

Spermatogenesis is an intricate process that strongly depends on the rapid turnover of short-lived proteins, both in the differentiating germ cells and in the supportive Sertoli cells. Recent evidence has demonstrated the importance of the ubiquitin-proteasome system for this turnover, with the final enzymatic E3 ligase providing the target specificity. One E3 ligase, Itch, has been well characterized in the immune system, but its role during spermatogenesis is not yet well understood. Mice lacking functional Itch protein display a late onset autoimmune disease characterized by severe inflammation, infiltration of immune cells into various organs, and most apparently chronic dermatitis, ultimately dying from pulmonary inflammation at 6 to 9 months of age. The work presented here evaluates the testes of itchy mice at two developmental time points, during the peri-pubertal period at postnatal day (PND) 28 and at adulthood, PND 56. Itchy mice are smaller in size and have lower spermatid head counts, most likely resulting from an increase in germ cell apoptosis rather than a decrease in Sertoli cell number. Litter sizes are reduced in the homozygous itchy colonies, with data suggesting a defect during fetal development and not in gamete production, although survival rates tend to be similar to that of wild type. At PND 28, itchy mice show a delay in spermatogenesis and an increase in meiotic figures, while PND 56 mice show alterations in germ cell layers, spermatid head formation, and irregular cell division. Examination of the previously identified targets of Itch revealed no significant increases in the testis, but led to discovery of immunoglobulin (IgG) deposits within the interstitial space. Changes in protein expression outside of the seminiferous epithelium suggest that cells of the immune system may be influencing proper development and functional spermatogenesis in the testis. While the previous studies using the itchy mice focused primarily on the late onset autoimmune dysfunction in these animals, increased spleen weights and changes in testicular protein are observed as early as PND 28, indicating that the loss of Itch impacts these animals much earlier during development. Taken together, these data indicate that Itch is required for functional spermatogenesis and that it may play different cellular roles depending on the developmental age of the animal. Future work is targeted at identifying the possible testis-specific targets of Itch and deciphering whether the observed phenotypes are the result of the primary loss of Itch or are a secondary effect from the overactive immune system. / text

Spermatogenesis is regulated by steroid hormones which induce expression of various genes responsible for the growth, proliferation and differentiation of spermatogonia to form mature haploid spermatozoa. The surrounding somatic cells including Leydig and Sertoli cells support the whole process in vivo. Previously, we used the post-vitamin A treated vitamin-A-deficient (PVA-VAD) rat model to study spermatogenesis, and identified 24 genes that are differentially up-regulated after retinol treatment. Vad1.2 is one of the up-regulated transcripts expressed in the rat testis from postnatal day 25. Vad1.2 transcript is localized to the round and elongating spermatids in the adult mouse testis. In silico analyses showed that Vad1.2 transcript is down-regulated in patients with teratozoospermia and non-obstructive azoospermia, suggesting that Vad1.2 may have important roles in spermatogenesis. However, how Vad1.2 affects spermatogenesis remains unclear. Therefore, the present study was designed to study the functional roles of Vad1.2 protein in mice using gene targeting approach and investigate the molecular changes in mice with Vad1.2 deficiency. Vad1.2 polyclonal antibody was raised against the full-length mouse Vad1.2 recombination protein and affinity purified. Vad1.2 protein was localized to the cytoplasm and flagellum of condensing spermatids, specifically to the fibrous sheath (FS) in cauda epididymal spermatozoa. Vad1.2 conditional knockout vector was constructed and used to generate Vad1.2 null mice. Vad1.2-/- male mice developed normally but were subfertile with reduced sperm count and motility. Vad1.2-/- male mice had smaller testis and higher incident of sloughing of immature germ cells into the seminiferous lumens when compared to the wild-type. Yet, the rates of germ cell proliferation and apoptosis were similar between the wild-type and the mutant testis. Interestingly approximately 50% of the mutant cauda epididymal spermatozoa showed deformed flagella and demonstrated structural defects typically associated with bending of flagellum at the principal piece or at the midpiece/principal piece junctions. The acrosome, nucleus and mitochondrial sheath of these spermatozoa appeared normal, while the flagellum displayed structural abnormalities including deformation of the two longitudinal columns of the FS and disruption of a portion of FS, suggesting that Vad1.2 might be involved in the biogenesis of FS in spermatogenesis. Furthermore, Vad1.2 interacted with Akap4 in vivo, and the two proteins were co-immunoprecipitated from the testis or cauda epididymal spermatozoa lysates. Akap4 and Vad1.2 were localized to the tail region of the testicular spermatids and cauda epididymal spermatozoa. The expression levels of pro- and mature Akap4 in Vad1.2-/- testes were markedly increased when compared with the wild-type mice. However, a significant decrease of Akap4 was found in the mutant cauda epididymal spermatozoa, suggesting that most of the mature Akap4 failed to incorporate into the FS. Taken together, Vad1.2 plays an important role in spermatogenesis and Vad1.2 deficiency leads to subfertility in mice with the deformed flagella in mature spermatozoa. Further studies on the regulation of FS formation may uncover the underlying molecular changes associated with Vad1.2 deficiency, and may provide fundamental information for treatment of infertile patients with FS defect in the spermatozoa. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Surgery / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

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Inactive mutants of the ped gene cause two phenotypes in Drosophila melanogaster: male sterility and the early translation of DHODH within spermatogenesis. Investigation of the PED amino acid sequence revealed an OTU domain and an ubiquitin interacting motif, suggesting that it is a member of the otubain sub-family of de-ubiqutinating enzymes. To test this, the putative active cysteine residue was mutated. Results show that this single cysteine residue is required for ped to confer male fertility. Purified wild type PED was also used to carry out in vitro deubiquitinating assays. These assays failed to show any ability for PED to cut ubiquitin chains of varying length or linkage type. Previously, a translational control element was identified in dhod mRNA which is required for its early translation phenotype in ped mutants. In an attempt to identify additional transcripts that have their translational timing affected by PED, the don juan-like 5′ UTR was inserted into a reporter gene and examined in a ped mutant background. No delay of this reporter gene was observed suggesting that don juan-like mRNA is not under the exact control pathway that dhod is.

Thesis (MScMed)--University of Stellenbosch, 2005. / Bibliography. Also available via the Internet.