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.

Yeung Lam. / Thesis submitted in: December 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 134-150). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 摘要 --- p.III / ACKNOWLEDGMENT --- p.V / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- General review of angiogenesis --- p.1 / Chapter 1.2 --- Vascular endothelial growth factors (VEGFs) --- p.2 / Chapter 1.2.1 --- VEGF-A --- p.2 / Chapter 1.2.2 --- P1GF --- p.4 / Chapter 1.2.3 --- VEGF-B --- p.5 / Chapter 1.2.4 --- VEGF-C and VEGF-D --- p.6 / Chapter 1.3 --- VEGF receptors (VEGFRs) --- p.9 / Chapter 1.3.1 --- VEGFR-1 (or flt-1) --- p.9 / Chapter 1.3.2 --- VEGFR-2 ( or flk-1) --- p.10 / Chapter 1.3.3 --- VEGFR-3 ( or flt-4) --- p.11 / Chapter 1.4 --- Hormonal regulation of VEGFs by LH/hCG --- p.14 / Chapter 1.5 --- General review of the testis --- p.17 / Chapter 1.5.1 --- Structure and function of the testis --- p.17 / Chapter 1.5.2 --- Testicular vasculature --- p.18 / Chapter 1.5.3 --- Testicular angiogenesis --- p.19 / Chapter 1.6 --- Localization of VEGF and VEGF receptors in the testis --- p.20 / Chapter 1.7 --- Aims of the present study --- p.21 / Chapter 2. --- Materials and methods --- p.23 / Chapter 2.1 --- Animals --- p.23 / Chapter 2.1.1 --- Depletion of Leydig cell --- p.23 / Chapter 2.1.2 --- Suppression of Leydig cell and stimulation by hCG --- p.24 / Chapter 2.1.3 --- Collection of tissue --- p.25 / Chapter 2.2 --- Preparation of primary cells from rat testes --- p.27 / Chapter 2.2.1 --- Sertoli cell preparation --- p.27 / Chapter 2.2.2 --- Germ cell preparation --- p.29 / Chapter 2.2.3 --- Interstitial cell and Leydig cell preparation --- p.30 / Chapter 2.3 --- Cell cultures --- p.32 / Chapter 2.3.1 --- Reagents and cell lines --- p.32 / Chapter 2.3.2 --- "Mouse Leydig cell line, TM3 and Sertoli cell line, TM4" --- p.33 / Chapter 2.3.3 --- "Mouse tumor Leydig cell line, MLTC-1" --- p.34 / Chapter 2.3.4 --- "Rat tumor Leydig cell line, R2C" --- p.34 / Chapter 2.3.5 --- "Rat tumor Leydig cell line, LC540" --- p.35 / Chapter 2.4 --- Reverse-transcription polymerase chain reaction (RT-PCR) and semi-quantitative RT-PCR --- p.35 / Chapter 2.4.1 --- Extraction of total RNA --- p.35 / Chapter 2.4.2 --- Quantitation of total RNA --- p.37 / Chapter 2.4.3 --- RT-PCR --- p.37 / Chapter 2.4.4 --- Purification and authentication of PCR products --- p.47 / Chapter 2.5 --- Immunohistochemical staining --- p.48 / Chapter 2.5.1 --- Perfusion and processing of testes for histological sections --- p.48 / Chapter 2.5.2 --- Immunohistochemical staining of tissue sections --- p.50 / Chapter 2.6 --- Western immunoblotting --- p.52 / Chapter 2.6.1 --- Extraction and quantitation of total protein --- p.52 / Chapter 2.6.2 --- SDS-PAGE --- p.53 / Chapter 2.6.3 --- Immunoblotting --- p.55 / Chapter 2.7 --- Statistical analyses --- p.57 / Chapter 3. --- Results --- p.58 / Chapter 3.1 --- Expression and localization of VEGFs in the rat testis --- p.58 / Chapter 3.1.1 --- VEGF-A --- p.58 / Chapter 3.1.2 --- VEGF-B --- p.64 / Chapter 3.1.3 --- VEGF-C --- p.69 / Chapter 3.1.4 --- VEGF-D --- p.73 / Chapter 3.1.5 --- P1GF --- p.77 / Chapter 3.2 --- Effect of Leydig cell depletion on VEGFs expression in the rat testis --- p.81 / Chapter 3.2.1 --- Effect on VEGF-A --- p.81 / Chapter 3.2.2 --- Effect on VEGF-B --- p.82 / Chapter 3.2.3 --- Effect on VEGF-C --- p.88 / Chapter 3.2.4 --- Effect on VEGF-D --- p.91 / Chapter 3.2.5 --- Effect on P1GF --- p.94 / Chapter 3.3 --- Effect of Leydig cell suppression and hCG stimulation on VEGFs expression in the rat testis --- p.97 / Chapter 3.3.1 --- Effect on VEGF-A --- p.97 / Chapter 3.3.2 --- Effect on VEGF-B --- p.107 / Chapter 3.3.3 --- Effect on VEGF-C --- p.113 / Chapter 3.3.4 --- Effect on VEGF-D --- p.119 / Chapter 4. --- Discussion --- p.126 / Chapter 5. --- References --- p.134

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.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.

Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Science, 2003. / "April 2002" Includes bibliographical references (leaves 200-249).

Thesis (M.S.)--University of Michigan, 1988.

During gonad development and fetal life, the germ cells (GC) undergo a range of different developmental processes necessary for correct postnatal gametogenesis and the production of the next generation. If these fetal events are disrupted by genetic or environmental factors, there could be severe consequences that may not present until adulthood. This is of particular importance in relation to human testicular GC tumours (TGCT), the most common cancer of young men, as TGCT is thought to arise from fetal GCs that have failed to differentiate normally during development and thus persist into adulthood, eventually becoming tumourigenic. TGCT is one of several related disorders of male reproductive health thought to comprise a Testicular Dysgenesis Syndrome (TDS), in which faulty testis development in fetal life may predispose to the development of cryptorchidism, hypospadias, reduced sperm count and TGCT. Currently there is no accepted animal model for TGCT, but some insight into human TDS has been gained through the use of a rat model using in utero Di (n-Butyl) Phthalate (DBP) exposure to induce cryptorchidism, hypospadias, low sperm count and reduced fertility (but not TGCT). However, a previous study suggested that DBP exposure can disrupt GC differentiation, resulting in significantly reduced GC number prior to birth and postnatal consequences. This thesis has been directed at investigating the normal process of GC development in the fetal rat and how this is altered by DBP exposure; such understanding may give insights into the origins of human TGCT by showing how and when disruption of normal fetal GC differentiation can occur. The first objective was to characterise GC development in both the rat testis and ovary to understand the normal events that occur between embryonic day (e)13.5 and e21.5, as most data in the literature is based on the mouse. Analysis by immunohistochemical, stereological and mRNA expression indentified that during this time period, a GC will undergo a dynamic sequence of changes involving migration, proliferation followed by differentiation (manifested by loss of specific protein markers), whilst undergoing germ-line specific remethylation. Whilst whole gonad development is vastly different between testes and ovaries, GC development was broadly the same with only minor differences up to the point where GCs in the ovary enter meiosis. Having established the normal process of GC development in the fetal rat testis, the effects of in utero DBP exposure was then investigated. DBP exposure reduced GC number at all ages investigated even after only 24 hours of exposure and simultaneously prolonged GC proliferation. As apoptosis was unaltered by DBP exposure, the consistent reduction in GC number was suggested to be due to an initial reduction in GC number that does not recover to control levels. GC differentiation was assessed by the expression and localization of specific protein markers (OCT4, DMRT1 and DAZL). The pattern of expression of OCT4 and DMRT1 was altered by DBP exposure. GCs in DBP exposed animals also showed a delay in disaggregation from within the centre of seminiferous cords. These results suggested that a delay in GC differentiation was occurring with DBP exposure. This delay in GC development persisted into early postnatal life, following cessation of DBP exposure. Thus at postnatal day (D)6, GC specific re-expression of DMRT1, GC migration to the basal lamina and resumption of GC proliferation all showed a delay. DBP also induced an increase in the presence of multinucleated gonocytes. DNA methylation in the fetal rat testis was also investigated as a mechanism that could be disrupted by DBP exposure. DNA methylation of GCs increased during the last week of fetal life by global methylation of the GC genome and the increased expression of DNA methyl transferases. No effect of DBP exposure was detected. Inhibition of methylation by 5-aza-2’deoxycytidine was then investigated as a way to block GC differentiation in fetal rat testes and this resulted in a similar transient delay in GC differentiation but was perinatally lethal to the fetus. Bisulphite sequencing of the OCT4 promoter was also performed but proved inconclusive. Methylation patterns may be being altered by DBP exposure, but such changes could not be identified in this thesis. To complement the in vivo DBP exposure studies, an in vitro testis explant system using e14.5 testes was investigated. These in vitro testis explants showed some GC effects with MBP, the active metabolite of DBP, and also suggested a novel role for Hedgehog signalling in GC survival in the fetal rat testis. The studies in this thesis have characterised several aspects of fetal GC development in the rat and identified which of these are affected by DBP exposure, resulting in a delay in GC development. As DBP exposure delays but does not block GC differentiation, this may explain why TGCT is not induced in the DBP exposure rat model for TDS.

No description available.

The unique microenvironment structure of the testis affects the function of Leydig Cells (LCs) both physically and physiologically. The testicular macrophages are located adjacent to the LCs in the interstitial space and the two cell types share a close physiological and functional relationship. Macrophages first appear in the testis in prenatal life, and increase in number during both prenatal and postnatal development when they support the development and function of the testis. The dynamics of macrophage population expansion correlates with generation of the adult Leydig cell population in postnatal life. From these observations I hypothesise that testicular macrophage numbers have a consistent ratio to the number of LCs, and therefore manipulating testicular macrophage numbers may modulate LC number and testosterone (T) production by LCs. As such, manipulation of testicular macrophages represents a viable and novel mechanism by which LC function can be improved. To test this, markers for distinct macrophage populations in the testes were identified, namely c-fms-GFP, Mac2 and CD163. The number of either Mac2+ or CD163+ cell populations was determined at key stages throughout postnatal life, and the ratios of these cells to LC number were calculated at each age. This showed a consistent ratio between macrophages and LCs in the testis throughout postnatal life. The stimulatory effect of macrophages during LC development was then determined, by increasing the number of macrophages through cytokine treatment with recombinant CSF1-Fc. This model was then analysed for changes in testicular macrophage number, LC function and LC number. CSF1-Fc increased macrophage numbers in the developing testis. Macrophage number was increased following CSF1-Fc treatment at stem LC, progenitor LC and immature LC stages of LC development, and in adulthood. Importantly, increasing macrophage number during development led to early maturation of the LC population, suggesting macrophages may function as a driver of LC maturation. In adulthood, testicular macrophage numbers were reduced via treatment with an anti-CSF1 antibody to further determine the role of testicular macrophages in LC number and function. Whilst CD163+ macrophage number was reduced, no change in LH or T was observed. In contrast CSF1-Fc treatment induced an increase in macrophage number and LC number, with an elevated T level. Results suggest that macrophage support of steroidogenesis in adulthood is dispensable or can be compensated through LH/T feedback, but CSF1-Fc can contribute to LC function, LC number and T production through action at the level of the brain and the testis. Finally, to determine the potential clinical significance of increasing testicular macrophage support, experiments were completed on animals with pathological conditions: LC androgen receptor knockout mice (LCARKO) (LCs fail to fully mature) and ageing mice (cumulative free radical damage). Delivery of CSF1-Fc was observed to improve LC maturation in LCARKO mice, but failed to modulate LCs in ageing animals, suggesting CSF1-Fc may have clinical application in specific pathologies related to LC dysfunction. In summary, these studies further define the testicular macrophage population as important supporting cell types for LC development, function and maturation, and identifies possible mechanisms by which enhancing macrophage action can support or improve poor LC development and function.

Includes bibliographical references (leaves 150-183). / xii, 183 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Considers the tubule and capillary barriers from the point of view of anatomy, physiological function and possible factors which may cause the tubule barrier to be breached or influence substance exchange across the capillary wall. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Sciences, 1994