Tomi Streng

Altered Structure and Function of Reproductive Organs in Transgenic Male Mice Overexpressing Human Aromatase1

Aromatization of androgens is a key step in estrogen production, and it regulates the delicate balance between estrogens and androgens in the gonads and sex steroid target tissues. In the present study, we generated transgenic mice (AROM(+)) bearing the human ubiquitin C promoter/human P450 aromatase fusion gene. AROM(+) male mice are characterized by an imbalance in sex hormone metabolism, resulting in elevated serum E(2) concentrations, combined with significantly reduced testosterone and FSH levels, and elevated levels of PRL and corticosterone. AROM(+) males present a multitude of severe structural and functional alterations in the reproductive organs, such as cryptorchidism associated with Leydig cell hyperplasia, dysmorphic seminiferous tubules, and disrupted spermatogenesis. The males also have small or rudimentary accessory sex glands with abnormal morphology; a prominent prostatic utricle with squamous epithelial metaplasia, and edema in the ejaculatory ducts and vas deferens. In addition, the abdominal muscle wall is thin, and the adrenal glands are enlarged, with cortical hyperplasia. Some of the abnormalities, such as undescended testes and undeveloped prostate, resemble those observed in animals exposed perinatally to high levels of exogenous estrogen, indicating that the elevated aromatase activity results in excessive estrogen exposure during early phases of development. Some of the disorders in the reproductive organs, furthermore, can be explained by the fact that AROM(+) males are hypoandrogenic, and have elevated levels of serum PRL and corticosterone. Thus, the AROM(+) mouse model provides a novel tool to investigate the consequences of a prolonged increase in conversion of androgens to estrogens which results in complex hormonal disturbances altering the structure and function of various male reproductive organs.

Dietary phytoestrogens and their role in hormonally dependent disease

Epidemiological studies suggest that diets rich in phytoestrogens (plant estrogens), particularly soy and unrefined grain products, may be associated with low risk of breast and prostate cancer. It has also been proposed that dietary phytoestrogens could play a role in the prevention of other estrogen-related conditions, namely cardiovascular disease, menopausal symptoms and post-menopausal osteoporosis. However, there is no direct evidence for the beneficial effects of phytoestrogens in humans. All information is based on consumption of phytoestrogen-rich diets, and the causal relationship and the mechanisms of phytoestrogen action in humans still remain to be demonstrated. In addition, the possible adverse effects of phytoestrogens have not been evaluated. It is plausible that phytoestrogens, as any exogenous hormonally active agent, might also cause adverse effects in the endocrine system, i.e. act as endocrine disrupters.

Phasic non-micturition contractions in the bladder of the anaesthetized and awake rat

To characterize the contractile activity that occurs in the bladder during the filling phase of the micturition cycle (non‐micturition contractions, NMCs), which generate transient rises in intravesical pressure not associated with urine flow.

Transient receptor potential A1 (TRPA1) activity in the human urethra--evidence for a functional role for TRPA1 in the outflow region.

BACKGROUND A role for the transient receptor potential (TRP) A1 ion channel in rat lower urinary tract (LUT) sensation and disease has been proposed, but in the human LUT no information on TRPA1 activity is available. OBJECTIVES To investigate the distribution of TRPA1 in the human urethra and to study the effect of TRPA1 agonists on isolated urethral strip preparations. DESIGN, SETTINGS, AND PARTICIPANTS Urethral specimens were obtained preoperatively from 10 patients and were freshly prepared for Western blot, immunohistochemistry, and functional in vitro investigations. MEASUREMENTS The expression patterns of TRPA1 were studied with Western blot and immunohistochemistry. The effects of allyl isothiocyanate (AI), cinnamaldehyde (CA), and NaHS (donor of H(2)S) on tension of urethral strips were investigated in tissue baths. RESULTS AND LIMITATIONS TRPA1 immunoreactivity (-IR) was found in nerve fibres in the suburothelial space and was also located to nerve fibres of the muscle layer. Single TRPA1-IR nerves extended into the urothelium. A majority, but not all TRPA1-IR nerves also expressed immunoreactivity for CGRP or TRPV1. In the urothelium, TRPV1 was located to the outer layers whereas TRPA1 was observed in basal urothelial cells. Interspersed between strands of smooth muscle cells of the urethral wall, TRPA1- and vimentin-IR cells containing central nuclei and slender cytoplasmatic extensions were observed. In functional experiments, TRPA1-agonists had no contractile effect in urethral preparations. After precontraction with phenylephrine, AI, CA, and NaHS caused concentration-dependent relaxations of urethral strip preparations. CONCLUSIONS The localization of TRPA1 to nerves that also express TRPV1 and CGRP, and in urothelial cells and interstitial cells, as well as the findings that TRPA1 agonists can modify tone of urethral preparations, propose a role for TRPA1 in afferent and efferent sensory signaling of the human outflow region.

Distribution and Function of Cannabinoid Receptors 1 and 2 in the Rat, Monkey and Human Bladder

PURPOSE We investigated the distribution of cannabinoid receptor subtypes 1 and 2 in the detrusor of different species and studied the effects of cannabinoid receptor 1 and 2 agonists on bladder function. MATERIALS AND METHODS Cannabinoid receptor 1 and 2 expression was studied with Western blot and immunohistochemistry in rat, monkey and human detrusors. Co-staining was done for markers of sensory nerves using calcitonin gene-related peptide (Euro-Diagnostica, Malmö, Sweden) and transient receptor potential vanilloid 1, and for cholinergic nerves using VAChT (Santa Cruz Biotechnology, Santa Cruz, California). Actions of the endogenous cannabinoid receptor-1 and 2 agonist anandamide (Sigma(R)), and the cannabinoid receptor 1 and 2 agonist CP55,940 (Sigma) on isolated detrusor and during cystometry in conscious rats were recorded. RESULTS Higher expression of cannabinoid receptor 2 but not cannabinoid receptor 1 was noted in the mucosa than in the detrusor. Compared to the detrusor larger amounts of cannabinoid receptor 2 containing nerves that also expressed transient receptor potential vanilloid 1 or calcitonin gene-related peptide were observed in the suburothelium. Nerve fibers containing cannabinoid receptor 2 and VAChT were located in the detrusor. Neither anandamide nor CP55,940 affected isolated detrusor carbachol (Sigma) contractions. Nerve contractions were enhanced by 10 muM anandamide and decreased by 10 muM CP55,940 (p<0.05). In vivo CP55,940 increased the micturition interval by 46% and threshold pressure by 124% (p <0.05). Anandamide increased threshold pressure by 26% and decreased the micturition interval by 19% (p <0.05 and <0.01, respectively). CONCLUSIONS The distribution of cannabinoid receptor 2 on sensory nerves and in the urothelium, and effects by CP55940 on the micturition interval and threshold pressure suggest a role for cannabinoid receptor 2 in bladder afferent signals. Co-expression of VAChT and cannabinoid receptor 2, and effects by CP55940 on nerve contractions suggest a cannabinoid receptor 2 mediated modulatory effect on cholinergic nerve activity. Anandamide may not be a good tool for cannabinoid receptor studies due to its activity at other receptors.

The role of corticotropin releasing factor and its antagonist, astressin, on micturition in the rat

The purpose of this investigation was to evaluate the role of corticotropin releasing factor (CRF) on micturition. CRF is involved in the endocrine and central nervous system responses to stress and is also expressed in sites responsible for the control of micturition. In this investigation, cystometric experiments were performed in awake and unrestrained Wistar rats and on Spontaneous Hypertensive Rats, which are used as a rodent model of detrusor overactivity and anxiety. In vitro effects of CRF were evaluated using strips of detrusor muscle in an organ bath preparation. CRF (6.0 microg) administered via intrathecal and intraperitoneal routes, but not intracerebroventricularly, lowered the micturition threshold. CRF reduced the intercontraction interval by 28% and 26% after intrathecal or intraperitoneal administration, respectively, and reduced micturition volume by 34.7% and 30.2%, respectively. In Wistar-Kyoto rats, 6.0 microg intrathecal CRF significantly reduced intercontraction interval (423 +/- 79 vs. 669 +/- 59 s) and micturition volume (0.30 +/- 0.04 vs. 0.69 +/- 0.07 ml) compared to controls that received saline vehicle. These effects were blocked by pretreatment with 6.0 mug intrathecal astressin, a potent CRF antagonist, demonstrating that the effects are CRF receptor mediated. In Spontaneous Hypertensive Rats, 6.0 mug intrathecal CRF was found to have minimal stimulatory effects on the bladder, whereas astressin reduced baseline detrusor overactivity. CRF had no direct contractile effects on detrusor muscle strips. These results demonstrate that in the absence of detrusor overactivity, CRF stimulates micturition when administered via the intrathecal or intraperitoneal routes. Further studies are needed to explore the possibility whether CRF antagonists are effective for detrusor overactivity and the overactive bladder syndrome.

The role of the rhabdosphincter in female rat voiding

To obtain information on the mechanisms of female rat micturition using a model in which pressure was measured in the bladder and distal part of the urethra corresponding to the location of the rhabdosphincter, providing information on the role of the sphincter in opening and closing the urethral lumen.

Prostatic inflammation and obstructive voiding in the adult noble rat: Impact of the testosterone to estradiol ratio in serum

The age‐related decline of the testosterone to estradiol (T‐to‐E2) ratio in serum is associated with the increased prevalence of prostatic inflammation and lower urinary tract symptoms suggesting obstructive voiding. The impact of the T‐to‐E2 ratio on the development and reversal of non‐bacterial prostatic inflammation and obstructive voiding was tested in adult Noble rats.

Multiple Structural and Functional Abnormalities in the P450 Aromatase Expressing Transgenic Male Mice Are Ameliorated by a P450 Aromatase Inhibitor

The present study was undertaken to analyze the effect of a P450 aromatase inhibitor (finrozole) on 4-month-old transgenic mice expressing human P450 aromatase (P450arom) under the human ubiquitin C promoter (AROM+). AROM+ mice present several dysfunctions, such as adrenal and pituitary hyperplasia, cryptorchidism, Leydig cell hypertrophy and hyperplasia, and gynecomastia. The present study demonstrates that these abnormalities were efficiently treated by administration of a P450arom inhibitor, finrozole. The treatment normalized the reduced intratesticular and serum testosterone levels, while those of estradiol were decreased. The body weight and several affected organ weights were normalized with the treatment. Histological analysis revealed that both the pituitary and adrenal hyperplasia were diminished. Furthermore, the cryptorchid testes present in the untreated AROM+ males descended to scrotum, 4 to 15 days after inhibitor treatment. In addition, the disrupted spermatogenesis was recovered and qualitatively complete spermatogenesis appeared with the inhibitor treatment. This was associated with normalized structure of the interstitial tissue, as analyzed by immunohistochemical staining for Leydig cells and macrophages. One of the features was that the Leydig cell hypertrophy was markedly diminished in the treated mice. AROM+ mice also present with severe gynecomastia, while the development and differentiation of the mammary gland in AROM+ males was markedly diminished with the inhibitor treatment. Interestingly, the mammary gland involution was associated with the induction of androgen receptor in the epithelial cells, while estrogen receptors were still detectable in the epithelium. The data show that AROM+ mouse model is a novel tool to further analyze the use of P450arom inhibitors in the treatment of the dysfunctions in males associated with misbalanced estrogen to androgen ratio, such as pituitary adenoma, testicular dysfunction, and gynecomastia.

TRPA1 Receptor Induced Relaxation of the Human Urethra Involves TRPV1 and Cannabinoid Receptor Mediated Signals, and Cyclooxygenase Activation

PURPOSE We studied whether TRPA1 agonists interact with sensory and inflammatory signals to relax human urethral smooth muscle. MATERIALS AND METHODS Urethral specimens were obtained perioperatively from 19 patients, and prepared for immunohistochemistry and functional experiments. The effects of allyl isothiocyanate, cinnamaldehyde and NaHS were studied in phenylephrine activated preparations combined with capsaicin, capsazepine, N omega-nitro-L-arginine, indomethacin or CP55940. RESULTS TRPA1, cannabinoid 1 and cannabinoid 2 immunoreactivity was colocalized in nerve fibers of the human urethra. All TRPA1 agonists produced relaxation of phenylephrine contracted urethral preparations. Capsaicin increased relaxant responses to all TRPA1 agonists. It increased the mean +/- SEM -logIC50 of cinnamaldehyde and NaHS from 4.91 +/- 0.26 to 5.15 +/- 0.22 and 3.27 +/- 0.14 to 3.79 +/- 0.35, and the -logIC30 of allyl isothiocyanate from 3.11 +/- 0.24 to 3.41 +/- 0.26 (each p <0.05). Capsazepine in 5 preparations, indomethacin in 6 and CP55940 in 5 decreased cinnamaldehyde mediated relaxation by up to 39%, 88% and 89%, respectively. Nomega-nitro-L-arginine and urothelial removal had no effect on relaxation by cinnamaldehyde in 5 preparations. CONCLUSIONS Relaxation to TRPA1 agonists in human urethral preparations seem to work in cooperation with TRPV1 mediated signals, are negatively coupled via cannabinoid receptor activation and involve cyclooxygenase products. Urothelial TRPA1 signals may not be important to regulate normal human urethral smooth muscle tone. This does not exclude a role in the initiation of afferent activity normally and in disease states.