Philosophiae Doctor - PhD / 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<0.05) were evident between the three groups. No significant differences (P>0.05) were found between the groups with respect to the APO-Direct assay. A significant difference (P<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. / South Africa

No / Further evidence for the rodent bone marrow micronucleus assay acting as a sensitive predictor of the possible germ cell mutagenicity of chemicals

No

Yes / Germ cell tumours (GCTs) are a heterogeneous group of rare neoplasms that present in different anatomical sites and across a wide spectrum of patient ages from birth through to adulthood. Once these strata are applied, cohort numbers become modest, hindering inferences regarding management and therapeutic advances. Moreover, patients with GCTs are treated by different medical professionals including paediatric oncologists, neuro-oncologists, medical oncologists, neurosurgeons, gynaecological oncologists, surgeons, and urologists. Silos of care have thus formed, further hampering knowledge dissemination between specialists. Dedicated biobank specimen collection is therefore critical to foster continuous growth in our understanding of similarities and differences by age, gender, and site, particularly for rare cancers such as GCTs. Here, the Malignant Germ Cell International Consortium provides a framework to create a sustainable, global research infrastructure that facilitates acquisition of tissue and liquid biopsies together with matched clinical data sets that reflect the diversity of GCTs. Such an effort would create an invaluable repository of clinical and biological data which can underpin international collaborations that span professional boundaries, translate into clinical practice, and ultimately impact patient outcomes. / ALF, JFA, and MJM declare funding from St Baldrick’s Foundation; grant reference number 358099.

Normal germ cell development in the human testis is crucial for subsequent fertility and reproductive health. Disruption of testis development in fetal life can result in deleterious health consequences such as testicular dysgenesis syndrome (TDS), which includes disorders, such as cryptorchidism, hypospadias, infertility and testicular germ cell tumours (TGCT). A rat model of TDS in which rats are exposed to phthalates in utero has been validated, but does result in the development of TGCT. In humans, TGCTs result from transformation of pre-neoplastic carcinoma in-situ (CIS) cells and these CIS cells are believed to arise from human fetal germ cells during their transition from gonocyte to spermatogonia, based on their morphology and protein expression profile. It has been proposed asynchronous differentiation of germ cells in the human fetal testis may predispose fetal germ cells to become CIS cells. Studying the development of these tumours in humans is difficult because of their fetal origins and prolonged duration from initiation of impaired development to invasive disease. For this reason the use of relevant animal models that can mimic normal and abnormal germ cell development may provide new insight into how TGCT develop. The Common Marmoset monkey, a New World primate exhibits many similarities to the human in terms of reproductive biology and could represent such a model. This thesis aimed to further characterise the origins of CIS cells in the human testis by investigating the protein expression profile of CIS cells in patients with TGCT and comparing them to established markers of human fetal germ cell types using immunohistochemistry and immunofluorescence. Quantification of the various subpopulations of CIS and proliferation within these populations was performed. The thesis also investigated the Common Marmoset monkey as a potential model of normal testis and germ cell development by comparing the differentiation and proliferation profile of germ cells with those of the human during fetal and early postnatal life. During the present studies methods were successfully developed that enabled us to use testicular xenografts to recapitulate normal development of immature testes from marmoset and human. This involved grafting pieces of testis tissue subcutaneously under the dorsal skin of immunodeficient mice and retrieving them several weeks later to investigate their development during the grafting period. Xenografts using tissue from fetal, neonatal and juvenile marmosets were performed in addition to testes from first and second trimester human fetuses. Finally the present studies aimed to use the marmoset and the xenografting approach as systems in which to examine the effects of gonadotrophin suppression and phthalate treatment on germ cell differentiation and proliferation, with particular attention to the potential for development of CIS and TGCT. Heterogeneous phenotypes of CIS cells were identified, mostly consistent with those seen in the normal human fetal testis, however some of these CIS cells did not exhibit the same phenotype as germ cells identified in normal fetal testes. In addition it was shown that some of the proteins considered to be ‘classical’ markers of CIS cells, such as the pluripotent transcription factor OCT4, were not expressed in a proportion of the CIS cells. The proliferation index of CIS cells is also significantly higher in those subpopulations with the most ‘undifferentiated’ phenotype (i.e. OCT4+/VASA-). The present studies have generated novel data showing that the marmoset is a good model of fetal and neonatal germ cell development, with similarities to the human in terms of an asynchronous and prolonged period of differentiation and proliferation of germ cells from gonocyte to spermatogonia. This feature is also common to the human, but not a characteristic of the rodent. Fetal, neonatal and pre-pubertal germ cell development can be re-capitulated by xenografting tissue from marmoset and human testes into nude mouse hosts. Human fetal testis grafts produced testosterone and were responsive to hCG stimulation. First trimester human testis xenografts that have not developed fully formed seminiferous cords prior to grafting can complete the process of cord formation whilst grafted in host mice. In addition, germ cells in fetal human and marmoset xenografts can differentiate and proliferate in a similar manner to that seen in the intact non-grafted testis. In the intact neonatal marmoset, suppression of gonadotrophins resulted in a 30% decrease in proliferation, however differentiation of gonocytes is not affected. In-utero treatment of neonatal marmosets with mono-n-butyl phthalate was associated with unusual ‘gonocyte’ clusters, however, di-n-butyl phthalate treatment of mice carrying fetal marmoset xenografts resulted in no visible effects on germ cell differentiation or proliferation and did not result in the development of CIS or TGCT. In conclusion, this thesis has shown that there are many subpopulations of CIS cells of which many have not been previously described. These subpopulations have different characteristics, such as variable proliferation rates and this may indicate the potential for progression or invasiveness. These subpopulations have similar protein expression phenotypes to normal human fetal germ cells although the present studies have identified some CIS cells with phenotypes that are not found in the normal human testis. This thesis has demonstrated that the marmoset is a comparable model to the human in terms of asynchronous fetal germ cell development, which may predispose this species to the development of CIS/TGCT. In addition to the use of intact marmosets, these studies have also demonstrated for the first time that testis xenografting provides a comparable system for testis cord formation, germ cell differentiation and proliferation in fetal/postnatal marmosets and fetal human testis. In addition the marmoset and xenografting models have indicated that phthalates may have minor effects on testis development in the human and marmoset but do not result in CIS or TGCT. These model systems are suitable for further investigation of normal and disrupted testis development.

The studies in this thesis examined piglet gonocyte identification, isolation, purification, preservation and potential for initiation of spermatogenesis after transplantation into irradiated recipient testes. As a first step, we characterized a previously non-described auto-fluorescence in the piglet testis tissue. This auto-fluorescence mainly originated from granules among the testis interstitial cells, and we found that its interference with immuno-fluorescence can be overcome using Sudan black staining. We also showed that porcine gonocytes can be specifically labelled with the lectin Dolichos biflorus agglutinin (DBA). To optimize gonocyte isolation, we found that ~9-fold more live cells could be harvested by enzymatic digestion of testis tissues than with mechanical methods. However, the proportion of gonocytes (~7%) did not differ between the mechanical and enzymatic methods of testis cell isolation. We then developed a novel three-step strategy for isolation of gonocytes by combining enzymatic digestion and vortexing, resulting in a gonocyte proportion of ~40% (~5-fold more than that from conventional methods). For short-term preservation of testis cells, we found that the survival of testis cells under hypothermic conditions was dependent on the cell type, and affected by storage duration, temperature and medium used. More than 80% of live testis cells survived the 6-day hypothermic preservation period in 20% FBS-L15, without visible changes to the cell culture potential or gonocyte proportion. In another experiment where testis tissues were maintained under hypothermic conditions, we found that ~25% of testis cells could survive for 6 days if preserved in HypoThermosol-FRS solution (HTS-FRS), without morphological changes. To purify gonocytes, we showed that centrifugation of testis cells using 17% Nycodenz can lead to precipitation of gonocytes in pellets (with a purity of > 80%). We also found that pre-coating tissue culture plates with both fibronectin and poly-D-lysine can result in the negative selection of gonocytes (with a purity of up to 85%). We subsequently showed that further purification of gonocytes (to > 90%) could be achieved by combining the two latter approaches. To prepare recipients for germ cell transplantation, we used local irradiation of piglet testes which reduced testis growth, decreased seminiferous tubule diameters and completely eliminated spermatogenesis at 4 months post-irradiation. Compared with the absence of endogenous spermatogenesis in the control testes, spermatogenesis up to elongating spermatids was observed in the irradiated testes after gonocyte transplantation. In summary, we investigated several critical elements in the study and manipulation of gonocytes in a large animal model.

The studies in this thesis examined piglet gonocyte identification, isolation, purification, preservation and potential for initiation of spermatogenesis after transplantation into irradiated recipient testes. As a first step, we characterized a previously non-described auto-fluorescence in the piglet testis tissue. This auto-fluorescence mainly originated from granules among the testis interstitial cells, and we found that its interference with immuno-fluorescence can be overcome using Sudan black staining. We also showed that porcine gonocytes can be specifically labelled with the lectin Dolichos biflorus agglutinin (DBA). To optimize gonocyte isolation, we found that ~9-fold more live cells could be harvested by enzymatic digestion of testis tissues than with mechanical methods. However, the proportion of gonocytes (~7%) did not differ between the mechanical and enzymatic methods of testis cell isolation. We then developed a novel three-step strategy for isolation of gonocytes by combining enzymatic digestion and vortexing, resulting in a gonocyte proportion of ~40% (~5-fold more than that from conventional methods). For short-term preservation of testis cells, we found that the survival of testis cells under hypothermic conditions was dependent on the cell type, and affected by storage duration, temperature and medium used. More than 80% of live testis cells survived the 6-day hypothermic preservation period in 20% FBS-L15, without visible changes to the cell culture potential or gonocyte proportion. In another experiment where testis tissues were maintained under hypothermic conditions, we found that ~25% of testis cells could survive for 6 days if preserved in HypoThermosol-FRS solution (HTS-FRS), without morphological changes. To purify gonocytes, we showed that centrifugation of testis cells using 17% Nycodenz can lead to precipitation of gonocytes in pellets (with a purity of > 80%). We also found that pre-coating tissue culture plates with both fibronectin and poly-D-lysine can result in the negative selection of gonocytes (with a purity of up to 85%). We subsequently showed that further purification of gonocytes (to > 90%) could be achieved by combining the two latter approaches. To prepare recipients for germ cell transplantation, we used local irradiation of piglet testes which reduced testis growth, decreased seminiferous tubule diameters and completely eliminated spermatogenesis at 4 months post-irradiation. Compared with the absence of endogenous spermatogenesis in the control testes, spermatogenesis up to elongating spermatids was observed in the irradiated testes after gonocyte transplantation. In summary, we investigated several critical elements in the study and manipulation of gonocytes in a large animal model.

No description available.

No description available.

Yes / In vivo tests for male reproductive genotoxicity are time consuming, resource-intensive and their use should be minimised according to the principles of the 3Rs. Accordingly, we investigated the effects in vitro, of a variety of known, phase-specific germ cell mutagens, i.e. pre-meiotic, meiotic, and post-meiotic genotoxins, on rat spermatogenic cell types separated using Staput unit-gravity velocity sedimentation, evaluating DNA damage using the Comet assay. N-ethyl-N-nitrosourea (ENU), N-methyl-N-nitrosourea (MNU) (spermatogenic phase), 6-mercaptopurine (6-MP) and 5-bromo-2'-deoxy-uridine (5-BrdU) (meiotic phase), methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS) (post-meiotic phase) were selected for use as they are potent male rodent, germ cell mutagens in vivo. DNA damage was detected directly using the Comet assay and indirectly using the TUNEL assay. Treatment of the isolated cells with ENU and MNU produced the greatest concentration-related increase in DNA damage in spermatogonia. Spermatocytes were most sensitive to 6-MP and 5-BrdU while spermatids were particularly susceptible to MMS and EMS. Increases were found when measuring both Olive tail moment (OTM) and % tail DNA, but the greatest changes were in OTM. Parallel results were found with the TUNEL assay, which showed highly significant, concentration dependent effects of all these genotoxins on spermatogonia, spermatocytes and spermatids in the same way as for DNA damage. The specific effects of these chemicals on different germ cell types matches those produced in vivo. This approach therefore shows potential for use in the detection of male germ cell genotoxicity and could contribute to the reduction of the use of animals in such toxicity assays.