Normal development and function of the male reproductive tract relies on the crucial balance between androgen and estrogen signalling, furthermore estrogens play an important role in the regulation of spermatogenesis and steroidogenesis. Tamoxifen (TAM) inducible Cre-loxP systems are widely used to study testicular function. TAM is a selective estrogen receptor modulator (SERM), thereby exerting anti- and pro-estrogenic effects. Therefore, it was hypothesised that acute (single dose) TAM administration to the postnatal testis has significant long-term effects on testicular development and adult testis function, questioning its utility in inducible transgenic systems. A suitable Cre line was first validated as a tool to target the postnatal adult Leydig cell (ALC) population. The Nestin-Cre expressing stem Leydig cells (LCs) were demonstrated as a source of a subset of the ALCs. Hence, a TAM inducible Nestin- Cre line was one of the mouse lines employed for further studies. A comprehensive investigation was carried out to assess any short-and long-term testicular phenotype upon administration of high (3mg) and low (1mg, 500ug and 250ug) doses of TAM. These studies were carried out in TAM-inducible Nestin- Cre/ERT2 and PDGFRA-Cre/ERT2 mouse lines as well as C57Bl/6 mice, to ensure that the observations made were independent of transgene effects. High dose TAM treatment resulted in transgene induction, however this also caused short-term spermatogenic arrest, alterations to steroidogenesis and LC number. Spermatogenesis recovers in young adults, but LCs show delayed maturation, suggesting changes in developmental programming of the ALC population. Thus it was concluded that a single dose of TAM in early postnatal life disrupts testicular function in adulthood. Single low doses of TAM did not induce the transgene, but surprisingly also had a long-term impact on ALC development, steroidogenesis and spermatogenesis. Severity of the phenotype worsened with dose concentration, indicating dose dependent impacts of TAM on the testis. Therefore, TAM has adverse impacts on the testis at doses below the threshold of Cre induction. In order to find a substitute for TAM in transgene induction studies, Raloxifene (RAL), another SERM, was hypothesised to induce transgenes with minimal disruption of testicular function. A 3mg dose of RAL did not show the adverse impacts of TAM. However, different dose regimens were assayed to induce the transgene without success, hence ruling out RAL as a substitute for TAM. Given the severity of previously undocumented TAM-induced phenotypes elucidated in these studies, it is evident that the off-target effects of TAM are severely underappreciated and can cause long-term programming effects. These off-target effects are likely to be present in other estrogen responsive tissues. Hence TAM-inducible Cre systems should be used with rigorous controls, to ensure correct conclusions are drawn from results obtained.

by Louisa Li-Ha Lo. / Bibliography: leaves 174-182 / Thesis (M.Ph.)--Chinese University of Hong Kong, 1986

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.

No description available.

Sy Chun Choi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 123-160). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / ACKNOWLEDGEMENT --- p.vi / Chapter 1. --- Introduction / Chapter 1.1 --- General review of the testis --- p.1 / Chapter 1.1.1 --- Structure and function of the testis --- p.1 / Chapter 1.1.2 --- Testicular vasculature --- p.2 / Chapter 1.1.3 --- Testicular angiogenesis --- p.3 / Chapter 1.2 --- Vascular endothelial growth factor (VEGF) --- p.4 / Chapter 1.2.1 --- Discovery of VEGF --- p.4 / Chapter 1.2.2 --- Organization of VEGF --- p.4 / Chapter 1.2.3 --- Properties of the VEGF isoforms --- p.5 / Chapter 1.2.4 --- VEGF receptors --- p.6 / Chapter 1.2.5 --- Functions of VEGF --- p.8 / Chapter 1.3 --- VEGF in the testis --- p.10 / Chapter 1.3.1 --- Localization of VEGF and VEGF receptors in the testis --- p.10 / Chapter 1.3.2 --- Postulated functions of VEGF in the testis --- p.11 / Chapter 1.4 --- Regulation of VEGF --- p.11 / Chapter 1.4.1 --- "VEGF, hypoxia and the testis" --- p.11 / Chapter 1.4.2 --- "VEGF, nitric oxide and the testis" --- p.14 / Chapter 1.4.3 --- Cadmium-induced testicular toxicity --- p.16 / Chapter 1.4.4 --- "VEGF, glucocorticoids and the testis" --- p.17 / Chapter 1.4.5 --- Hormonal regulation of VEGF expression 226}0ؤimportance of LH --- p.19 / Chapter 1.4.6 --- VEGF and Leydig cell - macrophage interaction --- p.21 / Chapter 1.5 --- Aims of the present study --- p.24 / Chapter 2. --- Materials and methods / Chapter 2.1 --- Animals --- p.26 / Chapter 2.1.1 --- Spermatic cord torsion --- p.26 / Chapter 2.1.2 --- Cadmium chloride treatment --- p.27 / Chapter 2.1.3 --- Leydig cell depletion and cadmium chloride treatment --- p.28 / Chapter 2.1.4 --- Dexamethasone pretreatment and cadmium chloride injection --- p.28 / Chapter 2.1.5 --- hCG injection --- p.29 / Chapter 2.2 --- Immunohistochemistry --- p.29 / Chapter 2.2.1 --- Perfusion fixation of the testes --- p.29 / Chapter 2.2.2 --- Processing of the testes for histological section --- p.29 / Chapter 2.2.3 --- Immunohistochemical staining for VEGF --- p.30 / Chapter 2.2.4 --- Photomicrograph --- p.32 / Chapter 2.3 --- Cell cultures --- p.32 / Chapter 2.3.1 --- Cell lines of mouse TM3 Leydig cells and TM4 Sertoli cells --- p.32 / Chapter 2.3.2 --- Tumour cell line of mouse MLTC-1 Leydig cells --- p.33 / Chapter 2.4 --- Cell treatments --- p.33 / Chapter 2.4.1 --- Hypoxic treatment --- p.34 / Chapter 2.4.2 --- Cadmium chloride treatment --- p.36 / Chapter 2.4.3 --- hCG treatment --- p.37 / Chapter 2.4.4 --- Activators of second messenger systems --- p.37 / Chapter 2.4.5 --- Effect of pro-inflammatory cytokines and angiogenic growth factors --- p.38 / Chapter 2.5 --- Preparation of cDNA probes --- p.39 / Chapter 2.5.1 --- VEGF cDNA probe preparation --- p.39 / Chapter 2.5.2 --- P-actin cDNA probe preparation --- p.42 / Chapter 2.5.3 --- Purification of PCR products --- p.44 / Chapter 2.5.4 --- Confirmation of PCR products --- p.45 / Chapter 2.5.5 --- cDNA probe labeling --- p.46 / Chapter 2.6 --- RNA extraction --- p.46 / Chapter 2.6.1 --- Extraction of total RNA from testicular cell lines --- p.46 / Chapter 2.6.2 --- Extraction total RNA from testicular tissues --- p.50 / Chapter 2.7 --- Northern blot analysis --- p.51 / Chapter 2.7.1 --- Measurement of total RNA concentration --- p.51 / Chapter 2.7.2 --- RNA gel electrophoresis --- p.52 / Chapter 2.7.3 --- Transfer of RNA to membrane --- p.53 / Chapter 2.7.4 --- Hybridization with [α-32P]dCTP-labelled probes --- p.53 / Chapter 2.7.5 --- Autoradiography and densitometric quantification --- p.54 / Chapter 2.8 --- Data and statistical analysis --- p.55 / Chapter 3. --- Results / Chapter 3.1 --- Effects of hypoxia and cobalt chloride treatment on VEGF expression in TM3 and TM4 cells --- p.57 / Chapter 3.2 --- Effects of testicular torsion on VEGF expression in adult rat testes --- p.61 / Chapter 3.3 --- Antagonism of hypoxic induction of VEGF expression in TM3 cells by nitric oxide --- p.66 / Chapter 3.4 --- Effect of cadmium on VEGF expression in TM3 and TM4 cells --- p.66 / Chapter 3.5 --- Effect of dexamethasone on Cd-induced increase in VEGF expression in TM3 cells --- p.73 / Chapter 3.6 --- Effect of cadmium treatment on VEGF expression in the adult rat testes --- p.73 / Chapter 3.7 --- Effect of Leydig cell depletion on basal and Cd-induced expression of VEGF in adult rat testes --- p.76 / Chapter 3.8 --- Effect of dexamethasone on basal and Cd-induced expression of VEGF in adult rat testes --- p.76 / Chapter 3.9 --- "Effects of hCG,forskolin and phorbol ester on VEGF expression in TM3 and TM4 cells" --- p.79 / Chapter 3.10 --- Effect of hCG on VEGF expression in MLTC-1 cells --- p.92 / Chapter 3.11 --- "Effect of EL-lα, IL-1β, IL-6, TNF- α and TNF- β on VEGF expression in TM3 cells" --- p.92 / Chapter 3.12 --- Effect of bFGF and TGF- β1 on VEGF expression in TM3 cells --- p.102 / Chapter 4. --- Discussion / Chapter 4.1 --- Upregulation of VEGF expression in TM3 and TM4 cells by hypoxia and cobalt chloride --- p.108 / Chapter 4.2 --- Effect of testicular torsion on VEGF expression in adult rat testes --- p.110 / Chapter 4.3 --- Antagonism of hypoxic induction of VEGF expression in TM3 cells by nitric oxide --- p.111 / Chapter 4.4 --- "Effect of cadmium on VEGF mRNA levels in TM3 and TM4 cells, and in adult rat testes" --- p.113 / Chapter 4.5 --- "Effect of hCG,forskolin and phorbol ester on VEGF expression in TM3 and TM4 cells" --- p.116 / Chapter 4.6 --- Effect of cytokines and growth factors on VEGF expression in TM3 cells --- p.119 / Chapter 5. --- References --- p.123

Thesis (Ph. D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 93-120).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Androgens are essential for the maintenance of male health and wellbeing. A disturbance in androgen signalling has been associated with a number of clinically relevant disorders such as cardiovascular disease, diabetes and metabolic disorders as well as infertility. Primarily produced in the testis in males, the actions of androgens are mediated through binding to androgen receptor (AR), a member of the nuclear receptor superfamily of ligand-activated transcription factors. The somatic cells of the testis are known to have a number of key roles in both testis function and development and the Sertoli, Leydig and Peritubular Myoid cells are known to express AR in adulthood. It is through AR that some testicular functions are mediated; for example, the Sertoli cells support of complete spermatogenesis with Sertoli cell androgen receptor knockout (SCARKO) testis demonstrating a halt of spermatogenesis before meiosis. However, how androgen signalling is impacting testicular function through each of the somatic cell types is not yet fully understood. Currently, treatments for male reproductive disorders such as hypogonadism (low androgens) and infertility are limited to treatment of the symptoms; using androgen replacement therapy and in vitro fertilisation techniques. This has been, up until recently, a result of a lack of understanding of the causes of these conditions and a lack of resources able to treat them, with research suggesting that a genetic component may be responsible in a number of cases. However, due to the limited genetic investigation diagnosis of men with male reproductive disorders, the wider understanding of the genetics underpinning male hypogonadism and infertility is incomplete. Developments in technology for the investigation and editing of the genetic code are triggering a surge in the exploration of genetic disorders and, in parallel, into the fields of gene delivery vectors and editing technologies. These technologies will allow an expansion into the knowledge and understanding of genetic disorders whilst simultaneously affording the opportunity to exploit this understanding for the development of therapeutics. There have been a small handful of previous studies using technologies such as viral vectors to target the testicular somatic cells and deliver exogenous transgenes with the purpose of both gene editing and repair, all with varying degrees of success. Here, techniques to introduce and target the Leydig and Sertoli cells were investigated to determine the most appropriate methodology for gene delivery to and manipulation of the testis. Refinement of injections into the interstitial compartment were carried out before introducing lentiviral vectors and targeting of Leydig cells was validated and optimised. Lentiviral vectors are able to permanently integrate into the host cell. Surprisingly, analysis of testis post lentiviral injection determined that the lentiviral targeted Leydig cells began to undergo apoptosis one week post injection and were subsequently cleared from the testis after ten days. Contrastingly, this was not the case when adenoviral vectors were introduced into the interstitial compartment, with Leydig cells continuing to express the delivered reporter transgene and, importantly, not expressing markers of apoptosis, ten days post injection. This would suggest that using adenoviral vectors to target the Leydig cell population in the adult testis would be more appropriate than using lentiviral vectors. Previous studies have successfully used lentiviral vectors to target the Sertoli cells in the adult testis via the introduction of the particles through the efferent duct. However, this can result in damage to efferent duct, resulting in blockages and subsequently the seminiferous tubules. To circumvent this, introduction of the lentiviral particles through the rete compartment of the testis at a range of lower injection pressures was examined and injecting at a lower pressure through the rete testis was found to reduce the likelihood of introducing negative impacts on testicular histology when targeting the seminiferous tubules. Using these refined methods of introducing lentiviral vectors, targeted Sertoli cells stably expressed the delivered transgene for up to one year post injection. Using viral vector delivered transgenes for both the investigation of testicular genetic disorders and for the development of therapeutics has great potential. To explore this potential, we first generated a mouse model in which AR was ablated from both the Leydig and Sertoli cells using Cre/LoxP technology, termed the SC-LC-ARKO. Alongside providing a potential model to 'repair' with viral vectors, the SC-LC-ARKO model also provided an additional model for comparison with other models exhibiting ablation of AR from both single somatic cell types and double somatic cell types. This further enabled a characterisation of the roles of AR in adult testicular function, with results suggesting that loss of AR from more than one cell type results in an additive phenotype when compared to single cell knock outs. Despite providing further insight into the roles of AR in the testis, further analysis of the Cre line used to generate the SC-LC-ARKO model indicated that a small number of Leydig cells were expressing the Cre recombinase, resulting in only a small population of Leydig cells with ablated AR. Considering this, to explore the potential of rescuing Sertoli cell AR using lentiviral vectors, we then utilised an already well characterised Sertoli Cell AR knockout (SCARKO) model. Lentiviral vectors expressing mouse AR and monomeric GFP (moeGFP) downstream of a CMV promoter were generated and injected into the rete testis of WT and SCARKO adult (day 100) males at low pressure. The contralateral testis was injected with a lentiviral vector expressing moeGFP alone (also downstream of a CMV promoter) using the same technique. Analysis of testis sections revealed a reintroduction of AR to Sertoli cells in 100% of SCARKO testis injected with lentivirus expressing mouse AR. As a result of this re-expression of AR in Sertoli cells, 66% of the testis injected with lentivirus expressing mouse AR had evidence of morphologically mature elongated spermatids, indicative of ongoing spermatogenesis. These results suggest that a rescue of the infertility phenotype reported in previous studies of SCARKO testis. Also demonstrated is the reversal of the SCARKO testicular phenotype in tubules targeted by the mAR expressing lentiviral vector. This suggests that absence Sertoli cell AR throughout development does not have a permanent impact on the Sertoli cells capacity to support spermatogenesis in adulthood following rescue of SC AR expression in adulthood. In summary, the results of these studies have provided a refinement in the methodologies for targeting the Sertoli and Leydig cells of the adult testis with viral vectors as well as demonstrating successful rescue of a previously reported mouse model exhibiting infertility through reintroduction of a functional gene. Alongside this, comparisons of AR knockout models have afforded insight into maintenance of testis function through AR.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Bibliography: leaves 169-205. This thesis suggests that the testicular macrophage exhibits characteristics similar to that of a suppressor macrophage phenotype. The inhibition of lymphocyte proliferation by the testicular macrophage, its unique cytokine profile, high basal production of GM-CSF and prostaglandins, and the refractoriness to LPS all suggests a role that contributes to the immune privilege that is afforded the testis. However, these aspects of testicular macrophage immuno-biology also support a role in local cell-cell communication and regulation of the normal physiology of the testis, and macrophages may be directly involved in Leydig cell steriogenesis.