The view of androgens as a chiefly male- associated hormone is long outdated, and there is an increasing wealth of evidence for the role of androgens in women. Androgens, in addition to estrogen and other hormones, play several various and critical roles in females, particularly in the development and function of the female genital tract. Countless, targeted studies have been conducted in efforts to elucidate how androgens affect the development and function of the female genitalia, most notably in regards to the vagina, uterus, and ovaries. However, though the significance of female androgens is relatively well established, knowledge on this subject is still developing and somewhat fragmented. The objective of this review is to present a comprehensive view of the role of androgens in the development and function of the female genital tract, summarize new studies, and integrate the most current information in order to gain a broadened and enhanced understanding of the importance and implications of androgens in females.

Thesis (Ph. D.)--University of Virginia, 2007. / Includes bibliographical references. Also available online through Digital Dissertations.

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Fracture healing requires a series of events including inflammatory response and callus formation, callus remodeling and bone healing. Fracture healing is a complex process, there are several overlapping phases , including inflammation , cartilage formation and bone remodeling, there are many internal or external factors could impact on fracture healing, leading to delayed bone healing or non healing. The global androgen receptor knockout (GARKO) mice has been know to reduce bone mass in endochondral bone and osteoblast mineralization, but the impact for callus formation in fracture healing is still unclear. The goals of study is to investigate the role of androgen and androgen receptor in wild-type (WT) mice and GARKO mice after fracture healing during callus formation and bone mineralization and bone remodeling. Therefore, long-term animal experiments observed by micro-computed tomography to study the roles of androgen and androgen receptor on the process and mechanisms of fracture healing is necessary. We applied in vivo micro-computer tomography (Micro-CT) to build up the three-dimensional model images at different time points for wild-type mice and GARKO mice after fracture healing and observe the bone healing process of micro-structure of the development of callus during fracture healing. The callus tissue morphology observed by histological staining to study the proportion and position of collagen, fibrous tissue and bone. The results show that the healing of WT mice is better than GARKO mice. GARKO mice develop smaller callus size and less bone volume and show delayed healing. In general, orchiectomy (ORX) decreases callus size in WT mice but not in GARKO mice. However, the healing rate of elderly GARKO mice is not obvious in comparison with young GARKO mice. Together, our study demonstrated that the androgen and androgen receptor regulate fracture healing and play an important role in bone repair and healing. Our mouse model may be used for the therapeutic drug screening of bone fractures caused by osteoporosis.

Background: The first-line treatment for patients with advanced prostate cancer (PC) is androgen deprivation therapy. This therapy is initially effective, but after some time tumors relapse, predominantly within the bone, and are then termed castration-resistant prostate cancer (CRPC). The majority of CRPC tumors show androgen receptor (AR) activity despite castrate levels of circulating testosterone. AR activity could be caused by several mechanisms including; intratumoral androgen synthesis, AR amplification, AR mutations and expression of AR splice variants. The mechanisms controlling CRPC growth in the clinically most relevant metastatic site, the bone, are not fully identified. The purpose of this thesis was therefore to explore AR expression and possible mechanisms behind CRPC growth in PC bone metastases in order to find mechanisms that could be targeted for treatment and/or predict response to certain therapies. Materials and Methods: We have examined hormone-naïve and CRPC bone metastases samples obtained from patients at metastasis surgery, non-malignant and malignant prostate samples obtained from patients at radical prostatectomy, and PC cell lines cultured in vitro. Analysis has been performed using RT-PCR, whole-genome expression arrays, immunohistochemistry, western blotting, FISH, copy number assays and gene ontology analysis. Functional studies have been made by protein overexpression and knock-down in PC cells in vitro and effects studied by evaluation of cell viability, migration, and invasion. Results: We found that high nuclear AR immunostaining (presumed to reflect high AR activity) in bone metastases from CRPC patients was associated with a particularly poor prognosis, while no difference in AR staining was observed between hormone-naïve and CRPC metastases. Further, expression of AR splice variants (AR-V7, AR-V567es) was associated with a high nuclear AR immunostaining score and shown to be increased in CRPC compared to hormone-naïve bone metastases. High levels (levels in the upper quartile) of AR splice variants in CRPC bone metastases was related to disturbed cell cycle regulation and short patients survival. No differences in steroidogenic enzyme levels were detected between CRPC and hormone-naïve bone metastases. Higher levels of enzymes involved in late steps of androgen synthesis (adrenal gland steroid conversion) were observed in bone metastases than in non-malignant and/or malignant prostate tissue, while the enzyme levels in earlier steps (de novo steroidogenesis) were lower in bone metastases. A subgroup of metastases expressed very high levels of AKR1C3, indicating that this group may have an induced capacity of converting adrenal-gland derived steroids into more potent androgens. This was not associated to CRPC but merely with the advanced stage of metastasis. High protein levels of AR splice variants were found in bone metastases with low AKR1C3 levels, while metastases with high AKR1C3 levels primarily contained low AR variant levels. Furthermore, about half of the CRPC bone metastases showed androgen receptor gene amplification which was associated with co-amplification of YIPF6, and a gene expression pattern that pointed at decreased osteoclast activity, and consequently decreased bone resorption. Conclusions: The majority of CRPC bone metastases show high nuclear AR immunostaining that seems to be associated with a particularly unfavorable outcome after metastasis surgery. Subgroups of CRPC bone metastases could be identified according to presence of AR amplification and expression levels of AKR1C3 or AR splice variants, which might have clinical relevance for treatment of PC patients. / <p>Författaren är även publicerad med efternamnet Hörnberg.</p>

Androgen receptor (AR) signalling is essential for the development and function of the male reproductive system. Conditional gene ablation using the Cre-loxP system has previously assisted in the elucidation of the role of AR in different cell types. The aim of this study was to examine the effects of the ablation of AR in previously untargeted cell types, with the hypothesis that this will have significant and novel effects on reproductive development and function that have not been previously documented by current models of androgen disruption. In these studies, three Cre recombinase lines were empirically validated for action in the male reproductive system, before being used to ablate AR and the phenotypes of the resulting lines were characterised. Endothelial-specific receptor tyrosine kinase (Tie2)-Cre was shown to target the vascular and endothelial cells of the testis, and used to ablate AR in these cells. The testes of the resulting Tie2-ARKO line were morphologically similar to controls, with normal spermatogenesis and mature spermatozoa present in the cauda epididymis. Aquaporin 2 (Aqp2)-Cre was shown to target the post-meiotic germ cells of the testis, and was used to ablate AR in these cells. The testes of the resulting Aqp2-ARKO line were morphologically similar to controls, with normal spermatogenesis and mature spermatozoa present in the cauda epididymis. It was concluded that the Ar gene was dispensable in the endothelial cells and post-meiotic germ cells of the testis for normal spermatogenesis. Forkhead box protein G1 (FoxG1)-Cre was shown to target the caput epididymal epithelium and pituitary, and used to ablate AR in these cells. d100 FoxG1-ARKO mice had a severe testicular phenotype, with sloughing of the seminiferous epithelium, atrophy of some seminiferous tubules and distension of the rete testis with spermatozoa. Despite the severe testis phenotype, ablation in the testis was incomplete and restricted to a small percentage of Leydig cells, with no ablation in Sertoli cells. Ablation of AR in the embryonic pituitary did not cause adult serum testosterone or LH concentrations to change, nor did it cause changes in other pituitary hormone transcripts. Mosaic ablation of AR in the caput epididymal epithelium was shown to impair epididymal development, with failure of initial segment (segment I) development and a significant decrease in epithelial cell height and lumen diameter in the remaining proximal caput epididymis (segment II). Dysfunction of the caput epididymis resulted in the failure of spermatozoa to transit the efferent ducts into the epididymis correctly: instead they were found to stall in the efferent ducts and produce a block. The testicular phenotype could be explained as the result of fluid backpressure effects resulting from the efferent duct block. Consequently, low concentration of spermatozoa in the cauda epididymis resulted in infertility in the FoxG1-ARKO, which represents a new model of obstructive azoospermia.

Menopause is associated with disorders such as osteoporosis and ovarian cancer. It is unclear whether the postmenopausal ovary retains steroidogenic capacity and how it can impact the development of these disorders. The present studies used the VCD-treated follicle-depleted mouse model of menopause to test the hypothesis that residual ovarian tissue retains steroidogenic capacity following ovarian failure and, thus, affects the development of these disorders. Microarray technology was used to evaluate gene expression in residual ovarian tissue of follicle-depleted mice compared to that in ovaries from cycling animals. Among the genes identified were those encoding proteins for synthesis of androgens. Steroidogenic capacity of residual ovarian tissue was further evaluated by determining the expression of genes and proteins involved in ovarian steroidogenesis, and by measuring levels of circulating and rostenedione and gonadotropins. Follicle-depleted ovaries were enriched in mRNAs for androgenic enzymes, receptors involved in the internalization of cholesterol, and luteinizing hormone receptor. Increased circulating levels of FSH and LH and detectable and rostenedione were measured throughout the study. Protein for 3β-hydroxysteroid dehydrogenase, 17α- hydroxylase/17,20-lyase and luteinizing hormone receptor was detected in follicledepleted ovaries by Western blot analysis and localized by immunofluorescence staining. The contribution of retaining residual ovarian tissue to accelerated bone loss following ovarian failure was evaluated by comparing bone mineral density from young and aged VCD-treated mice to that in age-matched ovariectomized (OVX) animals. Retaining residual ovarian tissue resulted in protection against accelerated bone loss in young but not aged VCD-treated mice. Whether residual ovarian tissue is more susceptible to development of ovarian neoplasms compared to ovaries from cycling animals was addressed by combining the VCD-treated mouse with the DMBA model of ovarian carcinogenesis. VCD-treated follicle-depleted mice that received DMBA developed Sertoli-Leydig cell tumors while no tumors were observed in cycling animals. Residual ovarian tissue following ovarian failure appears to have a protective effect against loss of bone integrity, but a detrimental effect on development of ovarian neoplasms. Findings from these studies: provided evidence of a physiological role for residual ovarian tissue following ovarian failure, and furthered the use of the VCD-treated mouse as a relevant model for menopause and associated disorders.

The endometrium undergoes recurrent cycles of dynamic remodelling, involving breakdown and scarless repair, proliferation and differentiation, including decidualisation of the stroma, during the menstrual cycle. Extensive studies have characterised how the steroid hormones oestrogen and progesterone acting via their nuclear receptors coordinate these remarkable changes. Although a few previous studies have postulated a role for androgens the impact of androgens on endometrial function remains understudied. The studies described in this thesis aimed to 1) identify cellular processes, pathways and networks regulated by androgens in human androgen receptor-positive endometrial stromal cells (hESCs), 2) investigate the potential for regulation and determine the regulation of putative dihydrotestosterone (DHT)-regulated gene expression by androgen in hESCs, 3) investigate the expression and regulation of putative androgen-regulated genes in the human endometrium across the menstrual cycle and in early pregnancy and 4) explore the role of androgens in modulating metformin-induced gene expression associated with decidualisation of hESCs. Analysis of data from a whole genome array conducted previously in the laboratory using primary hESCs treated with DHT for 2 or 8 hours identified time dependant putative androgen-regulated mRNAs (34 and 268 genes, respectively). Thereafter, all work was completed by the author. Gene ontology and functional based bioinformatic analyses of the putative androgen-regulated gene sets revealed potential androgen regulation of a variety of cell processes, pathways and networks including those associated with gene transcription, signal transduction pathways (such as phosphatidylinositol, oestrogen receptor alpha (ERα) and Wnt signalling), cancer pathways, metabolism, cell cycle, development, apoptosis/survival. In addition, various transcription factors (e.g. AR, c-Myc, SP1, ERα, p53, E2F1, RUNX2, CREB1 and STAT3) were associated with androgen regulation in hESCs. Consensus androgen receptor binding sites were identified in the promoter sequences of 18 genes by transcription factor binding site sequence analysis. Direct DHT regulation of ten of 15 of these genes was validated in endometrial stromal cells using qRTPCR. Of these genes, RGS2, SIK1, and SNCAIP mRNAs were confirmed as DHT-regulated in hESCs by use of an AR inhibitor (flutamide) and in addition, were not found to be regulated by oestradiol. Discovery bioinformatics predicted these genes may interact in a gene network involving AR and the cAMP transduction pathway. Expression of the 15 putative androgen-regulated genes was confirmed by qRTPCR in intact human endometrial tissue (13 novel) and 9 of these genes were regulated in association with decidualisation i.e. either in the secretory phase, the time at which decidualisation begins and/or in first trimester decidua. Protein expression of RGS2, SIK1 and Synphilin-1 (encoded by SNCAIP) was confirmed by immunohistochemistry in endometrial tissues and protein expression also appeared greater in decidua. Regulation of putative androgen-regulated gene expression by decidualisation was confirmed in 4 out of 8 genes by employing a model of reduced in vivo decidualisation i.e. decidua from ectopic pregnancies. Regulation of 5 out of 7 genes was confirmed in decidualised hESCs (RGS2, SIK1, SLC6A6, SNCAIP and AXIN2) but expression of these genes was not altered by DHT inclusion during decidualisation. Finally, only a high metformin concentration enhanced hESC decidualisation and putative androgen-regulated gene expression (4 genes) in decidualised hESCs. In comparison, in the presence of DHT, a lower clinically relevant metformin concentration (100μM) did enhance decidualisation marker expression but did not alter expression of putative androgen-regulated genes. In summary, these studies have revealed new insights into androgen action in the human endometrium. Studies in hESCs 1) predicted the pathways and interacting transcription factor regulatory networks that may be androgen-dependent in this cell type, these were associated with cell differentiation, apoptosis and proliferation, 2) identified novel putative androgen-regulated genes expressed in hESCs and in endometrial tissues, 3) showed putative androgen-regulated genes are regulated by DHT (possibly via AR) in endometrial stromal cells, some of which are also regulated in association with decidualisation and 4) showed that androgens may enhance decidualisation during exposure to the commonly used drug metformin. Collectively, these new findings support a physiological role for androgens in endometrial function and provide a series of new avenues for further studies of the regulation of differentiation and proliferation.

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