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.

Nuclear Hormone Receptors (NRs) are hormone dependent DNA binding proteins that translate physiological signals into gene expression. Gene products have been identified that belong to the NR superfamily on the basis of homology. However, the endogenous and /or synthetic ligands that regulate their activity remain unknown, consequently, this subgroup of proteins are designated as orphans). Retinoic acid receptor related orphan receptors alpha and gamma(RORα and γ) are orphan NRs, and are preferentially expressed in skeletal muscle a major metabolic tissue and other tissues including pancreas, thymus, prostate, liver, adipose and testis. Surprisingly, the specific roles of ROR α and γ in skeletal muscle, a peripheral tissue, have not been examined. Muscle is one of the most energy demanding tissues which accounts for ~40% of the total body mass and energy expenditure, ~75% of glucose disposal and relies heavily on β-oxidation of fatty acids. We hypothesize that ROR α and γ regulates metabolism in this major mass lean tissue. Initially, this hypothesis was examined by “gain and loss” of function studies in an in-vitro mouse skeletal muscle cell culture model. Previous in vitro studies analyzed the role of RORα in the regulation of lipid homeostasis in skeletal muscle cells. We similarly conducted in vitro RORγ gain and loss of function studies in skeletal muscle cells to understand the role of this isoform in metabolism. We utilized stable ectopic over-expression of VP16-RORγ (gain of function), native RORγ and RORγΔH12 (loss of function) vectors to modulate RORγ mRNA expression and function. Candidate driven expression profiling of lines that ectopically express the native and variant forms of RORγ suggested that this orphan NR has a function in regulating the expression of genes that control lipid homeostasis (fatty acid-binding protein 4), CD36 (fatty acid translocase), lipoprotein lipase and uncoupling protein 3), carbohydrate metabolism (GLUT5 (fructose transporter), adiponectin receptor 2 and interleukin 15 (IL-15)) and muscle mass (including myostatin and IL-15). Interestingly, our study revealed a function for RORγ in the pathway that regulates production of reactive oxygen species which was also correlated with increased expression of UCP3 mRNA. Subsequently, we conducted in vivo studies with mouse models displaying global and muscle specific perturbation in RORα expression and function to elucidate the physiological role of this orphan NR in the context of metabolism.Along these lines, we characterized homozygous staggerer mice (sg/sg) in the context of lipid, carbohydrate and energy homeostasis. Staggerer mice were characterized by decreased and dysfunctional retinoic acid receptor-related orphan receptor alpha (RORα) expression. We observed decreases in serum (and liver) triglycerides and total and high density lipoprotein serum cholesterol in staggerer mice. Moreover, the staggerer mice were associated with reduced adiposity, decreased fat pad mass and adipocyte size. Candidate-based expression profiling demonstrated that the dyslipidemia in staggerer mice was associated with decreased hepatic expression of SREBP-1c, and the reverse cholesterol transporters, ABCA1 and ABCG1. This was consistent with the reduced serum lipids. Furthermore, the lean phenotype in staggerer mice was also characterized by significantly increased expression of PGC-1α, PGC-1β, and lipin1mRNAin liver and white and brown adipose tissue from staggerer mice. In addition, we observed a significant 4-fold increase in β2-adrenergic receptor mRNA in brown adipose tissue. Finally, dysfunctional RORα expression protects against diet-induced obesity. Following a 10-week high fat diet, wild-type (but not sg/sg) mice exhibited a ~20% weight gain, increased hepatic triglycerides, and notable white and brown adipose tissue accumulation. In summary, these changes in gene expression (that modulate lipid homeostasis) in metabolic tissues were involved in decreased adiposity and resistance to diet induced obesity in the sg/sg mice, despite hyperphagia. Finally, we specifically modulated RORα signaling in skeletal muscle by the targeted over-expression of truncated RORαΔDE (lacking the ligand binding domain) driven by a myogenic specific promoter, to investigate the contribution of this peripheral tissue to the RORα phenotype. Interestingly, transgenic heterozygous animals exhibit increased fasting blood glucose levels and mild glucose intolerance. Expression profiling (and western analysis) identified perturbations in the insulin signaling cascade. For example, we observed attenuation of p85alpha (PI3K) and Akt2 (mRNA and protein) expression; and insulin dependent induction of phospho-Akt2. In concordance, significantly increased levels of active phospho-AMPK were detected in the muscle of transgenic mice (relative to wt littermates). The increase in phospho-AMPK correlated with: (i) the suppression of lipogenic gene expression; and (ii) increased phospho-ACC and activation of genes involved in fatty acid oxidation in the skeletal muscle of transgenic animals. In conclusion, we suggest these orphan nuclear receptors (RORα and γ) are key modulators of fat and carbohydrate homeostasis in skeletal muscle tissue. Specifically, we propose that, RORα plays vital role in fat accumulation in adipose tissue and insulin mediated glucose homeostasis in skeletal muscle. Therefore we suggest that selective muscle specific RORα modulators may have utility in the treatment of type2 diabetes and obesity.

Daktaro disertacijos „Nėštumo laukimo laiką prognozuojančių veiksnių tyrimas“ tikslas - įvertinti nėštumo laukimo laiko sąsajas su demografiniais, socialiniais, gyvensenos, darbo, aplinkos ir genetiniais veiksniais ir sudaryti prognostinius jų įtakos modelius. Tyrimo uždaviniai: 1.Įvertinti demografinių, socialinių, gyvensenos, darbo ir gyvena–mosios aplinkos veiksnių sąsajas su nėštumo laukimo laiku. 2.Sudaryti svarbiausių demografinių, socialinių, gyvensenos, darbo ir gyvenamosios aplinkos veiksnių, kurie nulemia 12 mėnesių ir ilgesnį nėštumo laukimo laiką, prognostinį įvertinimo modelį. 3.Įvertinti FSH receptoriaus geno polimorfizmo variantų įtaką nėš–tumo laukimo laikui. 4.Sudaryti FSH receptoriaus geno polimorfizmo įtakos svarbiausiems demografiniams, socialiniams, gyvensenos, darbo ir gyvenamosios aplinkos veiksniams, kurie nulemia 12 mėnesių ir ilgesnį nėštumo laukimo laiką, prognostinį modelį. Nustatėme, kad svarbiausi nepriklausomi 12 mėnesių ir ilgesnį nėštumo laukimo laiką nulemiantys prognostiniai veiksniai yra 30 metų ir vyresnis amžius, anksčiau gydyti vaisingumo sutrikimai, ginekologinės ligos, kontracepcijos priemonių naudojimas iki nėštumo planavimo pradžios ir FSH receptoriaus geno SER/SER variantas, kurie pastojimo po 12 ir daugiau mėnesių tikimybę didino, atitinkamai, 1,95, 1,57, 2,21, 1,87 ir 1,68 kartus. / “Study of time to pregnancy prognostic factors”. The aim of the study was to investigate the relation between various factors and female fecundity, which was expressed as time to pregnancy (TTP) and to create prognostic models. Tasks of the study were: 1.Estimate the relation between socioeconomic, demographic, life-style, environmental and job-related factors and time to pregnancy; 2.Create prognostic valuation model for the most important social, demographic, life-style, environmental and job-related factors what are associated with 12 month or longer time to pregnancy; 3.Estimate the impact of FSH receptor gene polymorphism variant on time to pregnancy; 4.Create prognostic model for the most important factors what are associated with 12 month or longer time to pregnancy under the influence of FSH receptor gene polymorphism. We established, that the most important independent risk factors prognoses time to pregnancy of 12 or more months in women analyzed for FSH receptor gene polymorphism group were older age, having gynecological diseases or fertility problems in the past, the use of contraception prior to conception and SER/SER polymorphism variant, what increased the probability of conceiving after 12 or more months 1.95, 1.57, 2.21, 1.87 and 1.68 times respectively.

Vitamin D is central in calcium turnover, and adequate levels are important for skeletal health. It is not clear how large contributions from food and sunlight are in Swedish primary care patients, considering the low radiation of UVB in Sweden and fortification of some foods, and whether differences exist between patients of immigrant and Swedish origin. Increasing incidence of osteoporosis-related fractures is a major global health problem. Genetic variations in metabolising enzymes and in the Vitamin D receptor (VDR) have also been shown to be of importance to the overall effect of vitamin D. Polymorphic variation in the gene CYP2R1 encoding the 25-hydroxylase has previously been reported to correlate with circulating levels of 25(OH)D3. Results of association studies between genetic variants of the VDR and muscle strength, as well as falls have been contradictory. The purposes of this thesis were to examine possible differences in plasma-25(OH)D3 levels and intake of vitamin D between Swedish and immigrant female primary care patients, to estimate what foods contribute the most, and to identify contributors to vitamin D status (Paper I-II). Furthermore, the relationship between polymorphisms in the CYP2R1 gene and levels of 25(OH)D3 as well as other biochemical parameters (parathyroid hormone, calcium, phosphate and fibroblast growth factor 23) of skeletal homeostasis, bone mineral density and incidence of fractures was investigated (Paper III). Also, the association between genetic variations in the gene for the vitamin D receptor and measures of muscle strength, physical performance and falls (Paper IV), was investigated by using data from a Swedish multicenter study of elderly men (MrOS). Most important results: Vitamin D deficiency was common, with significant difference between Swedish born and immigrant patients (Paper I). Food intake of vitamin D is associated with circulating vitamin D, but the factors most strongly affecting vitamin D levels were reported sun holiday and origin (Paper II). CYP2R1 polymorphisms are associated with circulating levels of 25(OH)D3 and bone mineral density (Paper III). VDR genetic variants do not appear to have a direct effect on muscle strength or physical performance and incidence of falls in elderly Swedish men (Paper IV).

The mechanisms that govern the repression of 5-HT1A receptor gene expression mediated by a novel mental retardation gene, Freud-1, were examined in HEK293 and SKNSH cells. This study provides a possible mechanism of 5-HT1A receptor gene regulation by Freud-1, which, to mediate its action, recruits Swi/Snf and Sin3A/histone deacetylase (HDAC) complexes in non-neuronal HEK293 cells and Swi/Snf only in neuronal, 5-HT1A receptor-expressing SKNSH cells. Thus, Freud-1 has a dual mechanism of repression depending on cell type: HDAC dependent in HEK293 cells and HDAC independent in SKNSH cells. In addition, I present evidence that Freud-1 is not sumoylated at its consensus sumoylation sites and I present the lipid binding properties of Freud-1 and Freud-1 mutants.

The mechanisms that govern the repression of 5-HT1A receptor gene expression mediated by a novel mental retardation gene, Freud-1, were examined in HEK293 and SKNSH cells. This study provides a possible mechanism of 5-HT1A receptor gene regulation by Freud-1, which, to mediate its action, recruits Swi/Snf and Sin3A/histone deacetylase (HDAC) complexes in non-neuronal HEK293 cells and Swi/Snf only in neuronal, 5-HT1A receptor-expressing SKNSH cells. Thus, Freud-1 has a dual mechanism of repression depending on cell type: HDAC dependent in HEK293 cells and HDAC independent in SKNSH cells. In addition, I present evidence that Freud-1 is not sumoylated at its consensus sumoylation sites and I present the lipid binding properties of Freud-1 and Freud-1 mutants.

The mechanisms that govern the repression of 5-HT1A receptor gene expression mediated by a novel mental retardation gene, Freud-1, were examined in HEK293 and SKNSH cells. This study provides a possible mechanism of 5-HT1A receptor gene regulation by Freud-1, which, to mediate its action, recruits Swi/Snf and Sin3A/histone deacetylase (HDAC) complexes in non-neuronal HEK293 cells and Swi/Snf only in neuronal, 5-HT1A receptor-expressing SKNSH cells. Thus, Freud-1 has a dual mechanism of repression depending on cell type: HDAC dependent in HEK293 cells and HDAC independent in SKNSH cells. In addition, I present evidence that Freud-1 is not sumoylated at its consensus sumoylation sites and I present the lipid binding properties of Freud-1 and Freud-1 mutants.

The mechanisms that govern the repression of 5-HT1A receptor gene expression mediated by a novel mental retardation gene, Freud-1, were examined in HEK293 and SKNSH cells. This study provides a possible mechanism of 5-HT1A receptor gene regulation by Freud-1, which, to mediate its action, recruits Swi/Snf and Sin3A/histone deacetylase (HDAC) complexes in non-neuronal HEK293 cells and Swi/Snf only in neuronal, 5-HT1A receptor-expressing SKNSH cells. Thus, Freud-1 has a dual mechanism of repression depending on cell type: HDAC dependent in HEK293 cells and HDAC independent in SKNSH cells. In addition, I present evidence that Freud-1 is not sumoylated at its consensus sumoylation sites and I present the lipid binding properties of Freud-1 and Freud-1 mutants.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21995号 / 医博第4509号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 戸井 雅和, 教授 万代 昌紀, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19545号 / 理博第4205号 / 新制||理||1603(附属図書館) / 32581 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 村山 美穂, 教授 幸島 司郎, 教授 伊谷 原一 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM