495 The effects of targeted deletion of the aromatase enzyme on prostatic contractile responses to noradrenaline in mice Katherine T Gray, Jennifer L Short, Evan R Simpson1 and Sabatino Ventura Prostate Research Co-operative, Victorian College of Pharmacy, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia 1Prince Henry’s Institute of Medical Research, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia (Correspondence should be addressed to S Ventura; Email: [email protected]) Abstract This investigation aimed to see whether a change in the concentration-dependent contractions. Prazosin (0.3 mM) oestrogen to androgen ratio alters prostate contractility. Isolated attenuated the responses induced by noradrenaline and EFS in organ bath studies using prostates from aromatase knockout all mice (P%0.019, nZ5–7), while cocaine (10 mM) attenuated (ArKO) mice which were homozygous (ArKOK/K)and the responses evoked by tyramine (P!0.001, nZ6). There were heterozygous (ArKOC/K) for the disrupted aromatase cyp19 no genotype differences in EFS- and noradrenaline-induced gene and wild-type littermates (ArKOC/C) were conducted. responses (PR0.506, nZ10–13). Prostates from ArKOK/K The distribution of noradrenergic nerves was visualized using and ArKOC/K mice were more sensitive to tyramine than the sucrose–potassium phosphate–glyoxylic acid method. prostates from ArKOC/C mice (P!0.001, nZ11–13). Dense ArKOK/K mice had increased prostate weights compared adrenergic innervation of the prostate was similar in all mice. with ArKOC/C mice. Frequency–response curves to electrical These results suggest that although the absence of aromatase field stimulation (EFS; 0.5 ms pulse duration, 60 V,0.1–20 Hz) increases prostatic growth, this translates only to a subtle and yielded frequency-dependent contractions, while noradrenaline selective increase in contractility in mature mice. (10 nM–1 mM) and tyramine (1 mM–1 mM) produced Journal of Endocrinology (2007) 195, 495–502 Introduction oestradiol to dihydrotestosterone; concurrently, there is a significant increase in prostate size and the proportion of stroma Benign prostatic hyperplasia (BPH) is predominantly a to epithelium (Krieg et al.1993, Farnsworth 1999, Shibata et al. stromal disease resulting in a non-malignant enlargement of 2000). Whether this increased stroma translates to an increase in the prostate gland (Bartsch et al. 1979). BPH has both static contractility is not known. Oestrogen receptor numbers have and dynamic components associated with its development. also been shown to change with the onset of BPH (Schulze & The static component arises from hyperplasia of both the Claus 1990). Low numbers of oestrogen receptors are seen in the glandular and stromal tissue, while the dynamic component stroma of normal prostates, high concentrations in stromal tissue arises from increased stromal contractility mediated through from patients with non-obstructive BPH and no oestrogen increased noradrenergic activation of a1-adrenoceptors. Both receptors in the stroma of prostates from men with severe components of BPH increase prostatic pressure on the urethra obstructive BPH (Schulze & Claus 1990). This study concluded and bladder, resulting in troublesome lower urinary tract that oestrogen receptors may not mediate the stromal growth symptoms. Androgen-induced prostate growth is well known observed in BPH, however, the increase in oestrogen receptors to induce the static component of BPH but the role that in the stroma of non-obstructive BPH tissues suggests that androgens play in mediating the dynamic component is less oestrogen receptors may be important in the initiation of BPH certain. An androgen-induced change in a1-adrenoceptor (Schulze & Claus 1990). density and/or function has been postulated to contribute to The role that oestrogens play in prostate contractility is the development of the dynamic component (Yamada et al. unclear. Sex hormones have been shown to have varied effects 1987, Walden et al. 1999). To the contrary, it has been shown in cell lines derived from individuals suffering BPH. Stromal that androgen deprivation does not alter a1-adrenoceptor cells from some patients were unchanged after treatment with density in the rat ventral prostate (Lacey et al. 1996). oestradiol and testosterone, while stromal cells from other The relative oestrogen dominance observed in older males, patients displayed a more contractile phenotype (Smith et al. compared with younger males, has been hypothesized to be a 2000). Previously, it has been shown that the expression of causative factor in the development of BPH. With increasing smooth muscle cell markers is influenced by oestradiol age, it is widely accepted that there is an increase in the ratio of treatment, however, the same treatment had little impact on Journal of Endocrinology (2007) 195, 495–502 DOI: 10.1677/JOE-07-0411 0022–0795/07/0195–495 q 2007 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 10/01/2021 03:28:57AM via free access 496 K T GRAY and others . Prostate contractility in ArKO mice the growth of smooth muscle cell cultures (Zhang et al. 1997). was carefully dissected and placed in a Petri dish containing A recent study, also using cell culture techniques, showed that Krebs–Henseleit solution (mM: NaCl 118.1, KCl 4.69, at certain concentrations, testosterone, oestradiol and dihydro- KH2PO4 1.2, NaHCO3 25.0, glucose 11.7, MgSO4 1.1and testosterone, either alone or in combination, influenced the CaCl2 2.5)whereexcessfatandconnectivetissuewereremoved. agonist-induced activity of human cultured prostatic stromal Prostates were weighed at the conclusion of experiments. cells through regulation of a-andb-adrenoceptors and/or signal transduction pathways (Nguyen et al.2007). Isolated organ bath studies This study investigates the role of oestrogen modulation on prostate contractility in vitro using prostate tissue taken from Due to the size of the mouse prostate, the results presented in this aromatase knockout (ArKO) mice. Oestrogen synthesis from work were obtained from whole prostate tissues rather than any androgens is catalysed by the enzyme aromatase cytochrome specific individual lobes. Whole prostate tissues were mounted in P450 (Means et al. 1989). Disruption of the aromatase gene 10 ml water-jacketed glass organ baths containing Krebs– prevents the biosynthesis of endogenous oestrogens and Henseleit solution, maintained at 37 8Candbubbledwith5% allows investigation into whether the absence of oestrogen CO2 in O2. Due to the multi-directional alignment of the smooth alters prostate contractility in mature mice. It is well muscle fibres within the prostate, it was not possible to orientate documented that homozygous null ArKO mice have no the tissue in a specific direction; rather, one end of the prostate was detectable oestradiol, increased testosterone and dihydro- attached to a Perspex tissue holder and the other to a Grass testosterone levels and exhibit an increase in prostate size, FTO3C transducer for recording of isometric contractions of the compared with wild-type controls (Fisher et al. 1998, prostatic smooth muscle. Developed force was recorded via a McPherson et al. 2001). Despite this, it is still not known PowerLab data acquisition system (Chart 3.6)whichwasrunon whether this increase in size corresponds to any change in an HP Compaq dc7100 personal computer. Preparations were prostate contractility. This study aims to determine whether equilibrated for 60 min under a resting tension of 0.4–0.7 g.To the absence of oestrogen through disruption of the aromatase ensure tissue viability, prostatic tissues were electrically stimulated gene leads to a change in prostate contractility. during the equilibration period via two vertical parallel platinum electrodes incorporated in the tissue holder, which was connected to a Grass S88 stimulator. Stimulation parameters during equilibration were 0.5 ms pulse duration, 60 Vat 0.01 Hz. Materials and Methods Electrical field stimulation To assess nerve-mediated Animals contractions, frequency–response curves (0.5ms pulse Adult male ArKO mice were generated as previously duration, 60 V, 0.1–20 Hz) to electrical field stimulation described (Fisher et al. 1998). Briefly, exon 9 of the mouse were constructed using a frequency progression ratio of cyp19 gene was selected for disruption by the insertion of a approximately one-third of a log unit. The endogenous firing neomycin-resistant cassette between the EcoRV and XhoI rates of the nerves innervating the mouse prostate are not restriction sites. Heterozygous breeding pairs were used to known. Thus, using a wide range of frequencies (0.1–20 Hz) produce mice which were homozygous (ArKOK/K) and ensures any endogenous firing frequencies are covered in our heterozygous (ArKOC/K) for the gene disruption, as well as experimental protocol. Furthermore, we have previously age-matched wild-type (ArKOC/C) littermate controls. shown these parameters to be tetrodotoxin sensitive and Mice were obtained and bred on a mixed C57Bl6J and J129 therefore specific for depolarizing nerves (Gray & Ventura background and were routinely genotyped by PCR using 2005). Trains of pulses were delivered at intervals of 10 min. genomic DNA extracted from tail tissue biopsies. Mice were Each train consisted of 10 pulses at low frequencies (%1 Hz) or housed in the Victorian College of Pharmacy animal house at was for 10 s duration at higher frequencies (R1 Hz). An initial 22 8C and exposed to a photoperiod of 12 h light:12 h frequency–response curve was constructed to determine the darkness. Mice had free access to water and soy-free rat and contractile response of the tissue at each frequency. Following mouse chow (Specialty Feeds, Glen Forrest, Australia). At the initial frequency–response curve, tissues were washed and 8–16 weeks of age, mice were weighed before being killed by exposed to the a1-adrenoceptor antagonist, prazosin (0.3 mM) cervical dislocation. Prior approval for animal experi- for 60 min before a second frequency–response curve was mentation was obtained from the Monash University constructed.