During spermatogenesis, developing germ cells must migrate across the blood-testis barrier (BTB) and enter the adluminal compartment for further development. Throughout this process, extensive junction restructuring occurs at Sertoli-Sertoli and Sertoli-germ cell interfaces. Cytokines are known to play crucial roles in regulating testicular cell junction dynamics at different regulatory levels. However, the mechanism of cytokine-mediated regulation on newly identified junction molecules remains unclear. In this dissertation, the molecular mechanisms on how cytokines regulate the junction proteins of immunoglobulin superfamily (IgSF) including coxsackievirus and adenovirus receptor (CAR), nectin-like molecule-2 (Necl-2) and Necl-4 in testicular cells were studied. CAR is expressed on Sertoli and germ cells. It mediates both homophilic and heterophilic interaction for Sertoli-germ cell adhesion. It was found that combined treatment of interferon-γ (IFN-γ) and tumor necrosis factor α (TNFα) reduced CAR mRNA and protein levels, and caused the disappearance of CAR from germ cell interface. IFN-γ+TNFα promoted CAR protein degradation via ubiquitin-proteasome pathway. In addition, IFN-γ+TNFα reduced CAR mRNA through regulating the binding of NF-κB subunits and SP/KLF proteins to CAR promoter. Collectively, these results demonstrated for the first time the potential mechanism utilized by IFN-γ+TNFα to exert their effects during testicular inflammation. Necl-2 is exclusively expressed by spermatogenic cells in the testis. In this study, it was demonstrated that transforming growth factor-β1 (TGF-β1) down-regulated Necl-2 mRNA and protein levels, and caused the disappearance of Necl-2 from cell surface. Using inhibitors and shRNAs, it was found that TGF-β1 induced Necl-2 protein degradation through clathrin-dependent endocytosis. Endocytosis assay further confirmed that TGF-β1 accelerated the internalization of Necl-2 to cytosol. Moreover, TGF-β1 repressed Necl-2 gene transcription in Smad-dependent manner. Taken together, these results unraveled the mechanism of how TGF-β1 regulates Necl-2 expression to achieve timely junction restructuring during spermatogenesis. Necl-4 has been detected in Sertoli cells, but little is known about its regulation in the testis. It was found that TNFα down-regulated Necl-4 mRNA and protein levels. Inhibitor studies suggested that both caveolin-dependent endocytosis and ubiquitin-proteasome pathway were involved in TNFα-induced Necl-4 protein degradation. Co-immunoprecipitation indicated that Necl-4 was physically associated with Par3, Par6, aPKC and Cdc42 which are the major components of polarity complex in mouse testis. Further study was shown that TNFα reduced the expression of Par3, and altered the binding between Necl-4 and Par complex in Sertoli cells. These results suggested that Necl-4-mediated cell adhesion could be disrupted by TNFα via reducing its expression and altering its interaction with Par complex. Studies reported herein suggest that junction proteins of the IgSF are precisely regulated by cytokines at transcriptional and post-translational levels. These results further enrich current understanding on how junction dynamics are regulated during spermatogenesis. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Further investigation of possible regulatory pathway of NYD-SP24 demonstrated that the cell cycle of NYD-SP24 overexpressing cells was incompletely blocked at G2/M phase, and one of cell cycle-related genes, p21, shown to be an inducer of differentiation in different types of cell system, was found upregulated in a p53-independent manner, consistent with a role of NYD-SP24 in differentiation. (Abstract shortened by UMI.) / Spermatogenesis is a unique cell differentiation process consisting of three main phrases, namely mitosis, meiosis and postmeiosis. The differentiation of germ cells in the process involves distinct transcriptional programs, each under control of different transcription factor network. Many testis-specific transcription factors have been reported previously, however, few detailed studies have been done. This thesis describes the characterization and functional studies of a newly discovered testis-specific transcription factor, NYD-SP24, and the investigation of the possible regulatory pathway of NYD-SP24 in spermatogenesis. / The results demonstrated that in normal human tissues, NYD-SP24 was specifically and highly expressed in the testis but not in other somatic tissues, indicating its possible role in spermatogenesis. In the mouse model, mRNA and protein of NYD-SP24 mouse homolog gene (mNYD-SP24) were increased in the first wave of spermatogenesis. During the differentiation of germ cells, mNYD-SP24 mRNA and protein were confined to spermatocyte, round spermatid and spermatozoa. In hyperthermic mouse model, expression of mNYD-SP24 was decreased following the damage to germ cells by heat shock, but returned to normal level upon recovery of spermatogenesis. These results suggest the involvement of NYD-SP24 in spermatogenesis. / To identify the possible downstream targets of NYD-SP24, microarray and two-dimension gel technologies were performed in NYD-SP24 overexpressing cells and control cells. The results of microarray showed that 16 genes were upregulated and 4 genes were downregulated in NYD-SP24 overexpressing cells. In the two-dimension gel analysis, 12 protein spots were found to be altered significantly, with 8 increased and 4 decreased. Among these proteins, 3 were successfully identified by mass spectrometry. The nuclei localization in germ cells and the ability of NYD-SP24 to alter gene expression profile suggest that NYD-SP24 may be a testis-specific transcription factor, involved in gene regulation in spermatogenesis. / Zhu Hu. / "June 2005." / Adviser: Chan Hsiao Chang. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3607. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 136-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

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.

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.

published_or_final_version / abstract / Zoology / Doctoral / Doctor of Philosophy