Vad1.3 is an evolutionarily-conserved, testis-specific gene identified from a retinol-treated Vitamin A-deficiency (VAD) rat model. VAD1.3 is expressed throughout spermiogenesis at the acrosome of spermatids and epididymal spermatozoa, suggesting a role in acrosome biogenesis or acrosome reaction. The present study aimed to explore the functional role of VAD1.3 in spermatogenesis and sperm functions by the cellular and gene-knockout approaches. 　　Double immunofluorescent microscopy confirmed the co-localization of VAD1.3 and syntaxin 1 in mouse spermatids and spermatozoa. Deletion analysis of the Vad1.3 gene in transfected mouse spermatocyte GC2-spd and human cervical cancer HeLa cells revealed a polarized peri-nuclear/Golgi expression pattern for the N-terminal GFP-VAD fusion proteins which contain a bipartite nucleus localization (BNL) motif, but a nuclear expression pattern for the C-terminal GFP-VAD. The N-terminal sequences of VAD1.3 mediated its interaction with syntaxin 1, as demonstrated by both co-localization and co-immunoprecipitation studies. The full-length GFP-VAD co-localized with the Golgi markers and was redistributed into the endoplasmic reticulum after brefeldin A treatment, suggesting that VAD1.3 was recruited through the ER-Golgi-acrosome pathway. 　　Vad1.3+/- mice was previously generated by the conventional knockout approach. The heterozygous mice had normal spermatogenesis during postnatal days and adulthood (6-8 weeks). At the age of 8-19 months, 6 out of 17 heterozygous mice but no wild-type exhibited a decrease in the epididymal sperm count and testicular weight (p < 0.05). Histological analyses unveiled disarrangement of the seminiferous epithelium and sloughing of germ cells, predominantly spermatids, which was mediated partially by apoptosis as a higher percentage of TUNEL-positive cells were detected in these heterozygous mice (p < 0.05). This phenotype was associated with a decrease in the mRNA (p < 0.05) and protein levels of VAD1.3 in the testis. 　　Crossing of the Vad1.3+/- mice produced wild-type and heterozygous offspring in a ratio of 1:3, but no Vad1.3-/- mice were found. There was no significant difference between the heterozygous intercrosses and the wild-type intercrosses in the number of oocytes ovulated, the developmental rate of embryos from zygotes to blastocysts, the number of implantation site, resorption site or the offspring could result from defective fertilization between Vad1.3 null gametes rather than developmental lethality. The role of VAD1.3 in fertilization was supported by the inhibitory effects of the anti-VAD1.3 antibody on in vitro fertilization and progesterone-induced acrosome reaction. Immuno-staining revealed that VAD1.3 was present in the acrosome-intact spermatozoa but not in acrosome-reacted spermatozoa, indicating a role of VAD1.3 in ZP-binding or acrosome reaction rather than sperm-egg fusion. In oocytes VAD1.3 was distributed in the cytoplasm near the cortex. litter size. Only a few Vad1.3-/- embryos were found at the zygotic (3.7%) and 2-cell (3%) stages in the heterozygous intercrosses. These findings suggested that the absence of the Vad1.3-/- 　　In sum, VAD1.3 may play important roles in fertilization and spermatogenesis in mice. The BNL motif of VAD1.3 directs its Golgi expression and the N-terminal sequence of the protein mediates its interaction with syntaxin 1. The use of tissue-specific knockout approach may help to answer the functional role of VAD1.3 in future. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

 In rat testes, at stage VIII of the epithelial cycle of spermatogenesis, two cellular events, namely blood-testis barrier (BTB) restructuring and spermiation, take place simultaneously but at the opposite ends of the seminiferous epithelium. BTB is constituted by tight junctions (TJs), basal ectoplasmic specializations (ES), gap junctions and desmosomes, which must disassemble intermittently at stage VIII to facilitate preleptotene spermatocyte migration across the barrier. Synchronously, spermiation occurs at the luminal edge of the tubule lumen, involving the disruption of the apical ES, the only anchoring device there, and the release of sperm. The mechanism coordinating these events is not well understood. In this dissertation, I provide evidence that intercellular adhesion molecule (ICAM)-1 and -2, are working in concert with sarcoma (Src) family kinases to regulate these events. ICAMs comprise an immunoglobulin subfamily of cell adhesion proteins expressed by hematopoietic, endothelial and epithelial cells. They are known to function in the transendothelial migration of leukocytes. In the rat testis, ICAM-1 was shown to localize to both BTB and apical ES stage-specifically, with its immunoreactivity highest at stage VIII at the BTB. Besides co-immunoprecipitation and co-localization with BTB proteins, such as occludin and N-cadherin, ICAM-1 was found to promote BTB integrity in that its over-expression (O-E) in Sertoli cells in vitro increased transepithelial electrical resistance (TER). However, O-E of a truncated form of ICAM-1 (sICAM-1) that only consisted of the extracellular domain resulted in decreased TER and down-regulation of several BTB constituent proteins, possibly via the Src/Pyk2 signaling pathway. O-E of sICAM-1 in vivo also compromised the BTB integrity. These findings illustrate that ICAM-1 is an important regulator of the BTB. On the other hand, the localization of ICAM-2 was restricted to the Sertoli-germ cell interface and absent from the BTB, and associated with β1-integrin, nectin-3 and F-actin at the apical ES. Further, ICAM-2 was shown to interact with Src and Pyk2, as well as annexin II, a phospholipid-binding protein. Intriguingly, ICAM-2, Src and annexin II were specifically up-regulated during CdCl2-induced germ cell loss. These results reveal that ICAM-2 actively participates in the restructuring of apical ES based on studies using the cadmium model. The function of c-Yes, a member of the Src family, was also investigated. It was found to be stage-specifically expressed at the BTB and the apical ES, and it structurally associated with BTB components (e.g., occludin and N-cadherin) and with the apical ES proteins (e.g., β1-integrin, laminin β3 and γ3). In the study, the knockdown of c-Yes by RNAi in vitro and in vivo affected BTB and apical ES function, causing changes in the distribution/localization of adhesion proteins at the BTB and the apical ES, inducing germ cell loss from the seminiferous epithelium, possibly via an interference with the F-actin network. These findings implicate that ICAMs and c-Yes are regulatory molecules of cell adhesion at the BTB and the apical ES, and are biomarkers for male contraceptive development. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Surgery / Master / Master of Philosophy

FER-1 is required for fusion of specialized vesicles, called membranous organelles, with the sperm plasma membrane during Caenorhabditis elegans spermiogeneis. To investigate the role of FER-1 in membranous organelle fusion, I first examined ten fer-1 mutations and found that they all cause the same defect in membrane fusion. FER-1 and the ferlin protein family are membrane proteins with four to seven C2 domains which commonly mediate Ca2+-dependent lipid-processing events. Most of the fer-1 mutations fall within these C2 domains, showing that they have distinct, non-redundant functions. I found that membranous organelle fusion requires intracellular Ca2+ and that C2 domain mutations alter Ca2+ sensitivity. This suggests that the C2 domains are involved in Ca2+ sensing and further supports their independent function. Using two immunological approaches we found three FER-1 isoforms, two of which may arise from FER-1 by proteolysis. By both light and electron microscopy these FER-1 proteins are localized to membranous organelle membranes. Together, these results suggest that the ferlin family members may share a conserved mechanism to regulate cell-type specific membrane fusion.In Chapter III, I present additional results toward studying the function of FER-1 using several broad-based approaches. First, I present a bioinformatics analysis of FER-1 C2 domains and the preliminary results of their calcium-dependent phospholipid binding capabilities. Second, preliminary interactions found with individual FER-1 functional domains by a yeast-two hybrid screen are discussed. Lastly, I present results from a candidate-gene approach to identify additional regulators of MO fusion, the sperm-specific synaptobrevins.

No description available.

Drosophila Lasp contains a LIM domain, two nebulin repeats, and a SH3 domain, and exhibits high homology with mammalian Lasp family proteins. Vertebrate Lasp localizes to focal adhesions and to the leading edge of migrating cells and binds filamentous actin. To investigate Drosophila Lasp in vivo, we generated a Lasp null mutant, named Laspl, and showed that Laspl is male sterile. We observed two major functions of Lasp during Drosophila spermatogenesis. First, in the stem cell niche, hub cells fail to localize to the apical end of Drosophila testis in Laspl mutant. Hub cell anchoring is dependent on cell adhesion between cells and extracellular matrix (ECM), which is mediated by integrins. Lasp genetically interacts with betaPS integrin showing complete hub cell mislocalization. This indicates that Lasp is involved in an integrin-dependent process. However, hub cell anchoring is not required for fertility or stem cell maintenance. Secondly, we observe that actin cones, a unique actin structure during spermatid individualization, are perturbed in Laspl. Our data for Lasp expression in actin cones and incomplete individualization indicate that Lasp may play a role in tethering actin to the plasma membrane.

The role of male germ cell death in spermatogenesis is an important one as it removes dysfunctional or genetically damaged germ cells and is necessary to maintain an optimal germ cell to Sertoli cell ratio. The formation of the bloodtestis barrier requires the elimination of excessive germ cells and a surge of germ cell apoptosis occurs prior to puberty regulating the ratio of germ cells to Sertoli cells. The aim of this study was to evaluate the presence of four apoptotic markers on sperm from patients with various grades of fertility using flow cytometry. Furthermore, any correlations between the apoptotic marker assays and the standard semen analysis results were identified. This study compares early and late parameters of apoptosis with morphological features in spermatozoa in the same samples. The three sample groups were identified as: teratozoospermic [G-pattern] (n=26), teratozoospermic [P-pattern] (n=98) and oligoteratozoospermic [Ppattern] (n=36). Standard semen analysis was conducted on the semen samples according to the WHO guidelines. Four apoptotic marker assays using flow cytometry was applied in this study to examine the apoptotic alterations in ejaculate sperm. These assays included the Annexin-V staining for the determination of phosphatidylserine exposure, APO-Direct to identify DNA fragmentation, caspase-3 to detect expression of this active protease during early apoptosis and Fas expression. For the Annexin-V and caspase-3 assays, statistically significant differences (P&lt / 0.05) were evident between the three groups. No significant differences (P&gt / 0.05) were found between the groups with respect to the APO-Direct assay. A significant difference (P&lt / 0.05) was found when comparing the teratozoospermic [G-pattern] group and the oligoteratozoospermic [P-pattern] group for the Fas assay. A strong positive correlation was evident between the Fas and the caspase-3 assays in the teratozoospermic [G-pattern] group. For the teratozoospermic [P-pattern group] the following positive correlations existed between the APO-Direct and the Fas assays, APO-Direct and caspase-3 assays and between caspase-3 and Fas assays. The only strong positive correlation was between the caspase-3 and APO-Direct assays in the oligoteratozoospermic [P-pattern] group. The presence of spermatozoa showing microscopic features resembling apoptosis has been identified in ten human ejaculate samples per sample group. Electron microscopy was used to identify morphological features of apoptosis in these human sperm samples. Classical apoptosis as observed in diploid cells could be identified in sperm and these included: loose fibrillarmicrogranular chromatin network, presence of vacuoles in the nuclear chromatin, membranous bodies within the vacuoles of the chromatin, partially disrupted nuclear membranes, plasma membrane protuberances and apoptotic bodies containing cytoplasmic vacuoles and dense masses. This study has confirmed that semen samples with abnormal semen parameters exhibit the presence of apoptotic markers in sperm. The identification of apoptotic markers on the sperm suggests that abnormalities occur during their developmental process, however, the exact mechanism thereof remains unclear. These findings may suggest that certain apoptotic markers may be an indicator of abnormal sperm function and possibly indicative of male infertility.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 138-155) Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 138-155) Also available online.

Thesis (Ph.D.)--University of Melbourne, Dept. of Paediatrics, The Bruce Lefroy Centre for Genetic Health Research, The Murdoch Childrens Research Institute, 2009. / Typescript. Includes bibliographical references (leaves 183-207)