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Physiology and Pharmacology of Androgens

Clinical Pharmacology of Testosterone

A Conway, C Fennell, S Savkovic, L Turner, DJ Handelsman

The Department of Andrology at Concord Hospital provides testosterone treatment for men who have testosterone deficiency. As an international leader in research into the physiology and pharmacology of androgens, we continue to research the best and most acceptable forms of delivery of testosterone treatment for men who genuinely need this treatment. Our extensive research into various depot forms of testosterone have helped define the best ways to use these treatments to improve quality of life for hormone deficient men.

Measuring Steroids in Serum and Biological Samples

R Desai, T Harwood, P Keski-Rahkonen and DJ Handelsman

Accurate measurement of steroid hormones from research_andro_proj1clinical and biological samples is essential for the diagnosis and monitoring of reproductive disorders as well as for experimental laboratory studies. For the last few decades, either radioimmunoassay (RIA) or gas chromatography mass spectrometry (GC/MS) have been the standard methods used for these measurements. However, their limitations such as low sensitivity (GC/MS) and non-specificity (immunoassays) together with development of bench-top liquid chromatography (LC) mass spectrometry (MS) methods to measure steroid hormones from biological samples are now accurate and affordable.

We have developed an ultra-sensitive LC-MS/MS method (funded by an ARC LIEF grant). To measure accurately and sensitively androgens (testosterone, dihydrotestosterone and androstanediol isomers) and estrogens (estradiol and estrone) to extremely low levels efficiently and within a single run (Fig 1). The lab now provides the only Australian steroid reference laboratory for highly sensitive analysis of serum androgens and estrogens in serum samples from a wide variety of human and animal studies. The lab has analysed over 15,000 serum and tissue samples form collaborating groups around Australia and overseas and is among the leading labs in the world for steroid MS analysis.

Using our MS estradiol assay as a reference, we have conducted a performance evaluation of the 5 most commonly used direct estradiol immunoassays. We found all 5 were significantly suboptimal in sensitivity and/or accuracy and highlighted the need for improved MS-based estradiol assays in clinical and research practice.

Most recently the lab was awarded an NHMRC grant to develop an ultrasensitive estradiol using LC-MS assays. Dr Keski-Rahkonen has identified a series of promising novel derivatization chemistries to achieve a 10-20 fold boost in sensitivity of our existing sensitive estradiol assay. This new methodology holds great promise to improve the applicability of estradiol assays in clinical and research applications where the existing estradiol assays have proved inadequate.

Androgen Misuse: Testosterone Overprescribing

DJ Handelsman

Androgens play a major role in muscle strength, energy and quality of life in men. This can be dramatic in the small minority of men with genuine testosterone deficiency where testosterone replacement therapy often provides striking benefits. In addition however there is a widespread mistaken belief that testosterone might reverse features of male ageing especially decline in sexual function and energy levels. As a result there is widespread and increasing overuse of testosterone as an anti-ageing and sexual tonic among older men and for performance or image enhancement among young men.

We have continued to undertake national surveillance of testosterone prescribing patterns using both national health service (PBS) as well as commercial wholesales data (IMS) to chart the progress of the epidemic of testosterone overprescribing in Australia and globally. These patterns of over-use call for heightened vigilance and increased professional and public education to reduce wasteful, misguided and possibly harmful over-use of testosterone.

Androgen Abuse: Testosterone & Sports Doping

A Idan, C Fennell, M Jimenez, DJ Handelsman in collaboration with A Death, L McRobb, K McGrath (Heart Research Institute) and C Goebel, A Cawley, R Kazlausakas, G Trout, C Howe (National Measurement Institute)

Androgens, synthetic forms of testosterone, are the most effective and popular drugs abused in sports doping. In recent years new designer androgens and indirect forms of androgen doping have been developed to evade detection of androgen doping. Maintaining effective bans on androgens requires continual vigilance in detection of illicit androgens and of indirect androgen doping. We continue to undertake World Anti-Doping Agency (WADA) and Australian Sports Anti-Doping Authority (ASDA) supported clinical and laboratory studies to develop new and more powerful detection tests for such novel androgens and other means to evade detection of androgen abuse.

Healthy Male Ageing: The Health Man Study

G Sartorious, S Savkovic, AJ Conway, DJ Handelsman with Prof RI McLachlan and Dr C Allan (Prince Henry’s Institute of Medical Research, Melbourne)

Why do some men remain healthy well into old age and others do not? Our Healthy Man study aimed to determine the role of circulating androgen levels in maintaining or reflecting good health and to explore the reasons why testosterone concentrations vary in one man compared with another. Through analysis of over 300 very healthy ageing men, this study evaluated the prospects for age-specific reference ranges for testosterone in an “elite” healthy male population. The findings were published in a leading international Endocrinology journal showed that there was no apparent decrease in serum testosterone and even mild increases in testosterone metabolites, dihydrotestosterone and estradiol among older men who maintain excellent health. The findings showed that it is most likely that ageing itself does not produce any lowering of blood testosterone as men grow older but that the decreases seen in larger populations of older men are due to their acquisition of age-related diseases (co-morbidities) of ageing.

Measuring Progress of Puberty

G Singh, T Sivananthan, F Bathur, A Idan, A Conway, DJ Handelsman

Male sexual development and fertility develop relatively rapidly over a few years during adolescence, a period of time known as puberty. The triggers for puberty remain a mystery and the age at which it starts and its rate of progression vary widely between individuals for largely unknown reasons and have hardly ever been studied in the community. The failure of male puberty to occur when expected can cause deep and lasting effects on a developing man’s psyche because of the difficulties it creates in “fitting-in”, being perceived as immature, creating difficulties in finding a social niche and forming life-long partnerships.

The Andrology department is participating in several studies related to male puberty including improving treatment of boys who fail to undergo puberty as well as studying the normal evolution of puberty on the health and wellbeing of young adults in the community. In addition Gurmeet Singh has developed methods to measure the progress of puberty using urine steroid hormone excretion and these methods are being applied to the ARCHER study, a NHMRC funded longitudinal cohort study of adolescents passage through puberty.

Androgens and the Prostate

Intraprostatic Androgen Signalling and Androgen Sensitivity of the Prostate

U Simanainen, F Suarez, K McNamara, M Jähne, B Zhao, DJ Handelsman
Collaboration: Prof Diane Robins (University of Michigan, Ann Arbor USA); Prof Janet Keast (Kolling Institute of Medical Research, University of Sydney); Dr Stephen McPherson (Australian Prostate Cancer Research Centre, Queensland University of Technology).

The androgen receptor (AR) has a crucial role in both normal prostate development and the emergence and progression of prostate cancer. We have created a model targeting AR in the prostate epithelium to explore the role of androgen in the prostate development, as well as in prostate proliferative diseases of benign prostate hyperplasia and cancer (transgenic prostate cancer models) that develop in later life. We have demonstrated that while androgens are assumed pro-proliferative in the prostate, the epithelial AR suppresses cell proliferation by keeping the epithelial cells differentiated. In addition, we have shown that the epithelial AR modifies the prostate steroidal sensitivity and intraprostatic steroid signalling. Our ongoing research will also investigate the influence prostate disease initiation/progression on steroidal sensitivity and regulation of intraprostatic steroids of the prostate, noting that intraprostatic steroids may have essential roles in the development, but also in the treatment of the prostate cancer later in life. In collaboration with Prof Diana Robins and Prof Janet Keast we have explored the influence of CAG repeat of AR as well as neurotrophic factor Neurturin on androgen sensitivity. Our research may provide new clues for targets for prevention, screening and/or treatment for prostate diseases including prostate cancer. An additional important and novel finding was our proof, for the first time, that the CAG triplet repeat (polyglutamine tract repeat polymorphism) of exon 1 of the androgen receptor does directly modulate androgen sensitivity.

Androgens and the Testis

The Department of Andrology is interested in researching all available avenues to help those men seeking fertility but also the development of safe effective male contraception.

Male Hormonal Contraception

L Turner, C Fennell, AJ Conway, PY Liu, M Jimenez, DJ Handelsman

A major practical application of knowledge about how hormones control sperm production is the development of a male hormonal contraceptive. Following a decade of preliminary feasibility and path-findings studies, in 2003 the Andrology Department published a proof of principle study establishing very high reliability of a depot combined hormonal male contraceptive. Through many preliminary studies using a depot form of testosterone, we defined the lowest effective dose of testosterone having sufficient suppression but avoiding undesirable side effects and tested it with a progestin to identify the best combination. The excellent result for our prototype hormonal combination was a major advance and made international headline news. These path-finding studies have led progress in optimising the approach to develop a practical hormonal male contraceptive regimen. Currently, based on our 2003 study, we are extending our clinical experience with the combined depot approach in providing first medical male hormonal contraceptive service offered anywhere in the world. Furthermore, a major CONRAD and WHO sponsored international multicentre trial is using a similar injectable depot androgen-progestin combination to extend and refine the findings on contraceptive effectiveness for this “leading candidate” approach for a marketable male hormonal contraceptive.

Hormonal Control of Sertoli Cell Function and Spermatogenesis

R Hazra, D Upton, T Rastegar, L Corcoran, J Spaliviero, M Jimenez, DJ Handelsman, CM Allan
Collaboration: P Stanton (Prince Henry’s Institute of Medical Research)

Reproductive hormones such as sex steroids (eg. androgen) and gonadotrophins control testis development, sperm production (spermatogenesis) and male fertility. These major hormones converge upon unique testis cells called Sertoli cells, which are vital for sexual development and spermatogenesis. A major research focus is to understand how different hormones control postnatal Sertoli cell development and function. Specifically, we created novel genetic models to study the role of the androgen receptor (AR) and follicle-stimulating hormone (FSH) in Sertoli cells.Collaboration with Peter Stanton (PHRI) showed the importance of androgen and FSH actions for the formation of the blood-testis barrier, which is essential for functional spermatogenesis. To target AR actions, we selectively disrupted AR DNA binding in Sertoli cells, which showed DNA (genomic) AR interaction is vital for sperm development. A visiting PhD student Tina Rastegar (Tehran, Iran) showed that Sertoli cell AR DNA binding is vital for the final stages of spermatogenesis. We also investigated the role of estradiol (the classic female sex steroid) in testis function, and showed that the paradoxical induction of sperm production by estradiol requires Sertoli cell AR (ScAR). A new gain-of-function model targeted premature AR expression in Sertoli cells to identify its role during pre-pubertal/pubertal development. Using this model, Rasmani Hazra (PhD student) showed that atypical ScAR expression provides a direct molecular mechanism for premature testicular development, leading to reduced adult testis size and altered spermatogenesis. We also revealed that ScAR activity controls the pubertal maturation of Leydig cells, which is vital for normal androgen production and male fertility. Our studies continue to identify key hormonal-regulated biological pathways in testis cell populations that are crucial for functional testicular development and sperm production. These research projects increase our fundamental knowledge of underlying biological pathways that control (or inhibit) spermatogenesis and male fertility, predicted to provide valuable genetic targets for therapy (eg. infertility), or to develop novel strategies for male contraception.

Androgens, Ageing and Female Reproductive Physiology

Androgens and the Ovary

K Walters, A Caldwell, L Middleton, C Cerna, CM Allan, DJ Handelsman
Collaborations: Dr Jeremy Smith (University of Western Australia)


Enhanced understanding of ovarian function is of

great importance as infertility occurs in 1 in 6 Australian couples. Androgens are essential for male reproduction, however, in recent years, we and others have shown experimentally in mouse models, that androgen actions mediated by the androgen receptor (AR) have a previously unrecognized influence on female fertility. Improving our understanding in to the role of androgens in follicle development may provide long overdue new insights into androgen associated female reproductive disorders such as polycystic ovary syndrome (PCOS). Polycystic ovary syndrome (PCOS) is associated with hyperandrogenism and is one of the most common causes of anovulation and infertility in women, affecting between 5-10% of women of reproductive age worldwide. Despite substantial research trying to define the cause of PCOS its origins are unknown.

To identify the precise AR-mediated mechanisms involved in normal ovarian function and the development of PCOS, we are using our novel androgen resistant female androgen receptor knockout (ARKO) mouse models alone, or combined with established rodent models of PCOS. Dr Walters has revealed an important role for AR-mediated actions in ovulation (Fig. 1), follicle health and female fertility. Our long term goals are to further enhance our understanding of how androgens regulate female reproductive function, and unravel disruptions in androgenic mechanisms which may be involved in the establishment of androgen-associated reproductive disorders, such as PCOS.

FSH and Female Reproductive Ageing

D Upton, K Walters, S Lamb, DJ Handelsman, CM Allan.
Collaborations: Dr Viive Howell (Kolling Institute, University of Sydney)

In women, reproductive ageing (declining fertility) coincides with an accelerated loss of ovarian follicles (developing eggs). An early sign of reproductive ageing is increasing levels of circulating FSH. High FSH levels are associated with premature ovarian failure or onset of menopause, and may accelerate the loss, or decrease the quality, of ovarian eggs. Dr Allan established a genetic model with rising FSH levels that displays premature female infertility, despite the presence of maturing eggs. Furthermore, we revealed that embryos derived from eggs exposed to high FSH levels displayed normal uterine implantation rates and unexpectedly had increased survival levels during development. This beneficial effect of high FSH levels may have relevance to FSH treatments during assisted reproduction technology (ART). We also plan to use this model to examine the long-term effects of elevated FSH on genetic abnormalities (aneuploidy) in eggs, which increases as egg quality declines during reproductive ageing in women.

We found that higher levels of FSH produced earlier onset of infertility as well as ovarian hemorrhagic cysts. Dannielle Upton (University of Sydney) recently commenced her PhD candidature to investigate FSH actions using these models. Our work has identified FSH-regulated factors which may play a role in the hemorrhagic process, and may provide important insight into the rare but potentially fatal ovarian hyperstimulation syndrome (OHSS) during ART, or due to rare mutations in FSH signalling. High FSH levels may also contribute to ovarian cancer, and a collaboration with Dr Howell has created new models to study elevated FSH actions in combination with the mutation of potential factors (Brca1, Pten, p53) associated with ovarian tumorigenesis. Initial work using these complex genetic combinations shows that the ovary is remarkably resistant to direct or local changes to potential cancer-causing factors, and supports emerging evidence that many ovarian cancers may originate in other tissues or organs.

Androgens and the Mammary Gland

U Simanainen, K Walters, E Gao, P Choi, B Psarommatis, DJ Handelsman

One in nine Australian women will develop breast cancer within their lifetime. Yet for this common, fatal and feared disease, the causes and mechanisms remain elusive. The strongest clues are from sex hormones as epidemiological risk factors with estrogen exposure being widely recognised while the role of androgens, while assumed protective, remains controversial. Our ongoing research utilizes the female androgen resistant mouse models in combination with transgenic and chemical carcinogenesis allowing a direct and versatile experimental approach for analysis of androgen actions in mammary gland development, function (lactation) and tumorigenesis. The knowledge of AR functions at the physiological, cellular and molecular level, modifying breast hormonal sensitivity, will be pivotal to designing novel biomarkers and rational therapeutic or preventative approaches of importance for women’s health, like breastfeeding and breast cancer.

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