Risto Santti

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.

Hydroxymatairesinol, a Novel Enterolactone Precursor With Antitumor Properties From Coniferous Tree (Picea abies)

Abstract: The potential for the extraction of the plant lignan hydroxymatairesinol (HMR) in large scale from Norway spruce (Picea abies) has given us the opportunity to study the metabolism and biological actions of HMR in animals. HMR, the most abundant single component of spruce lignans, was metabolized to enterolactone (ENL) as the major metabolite in rats after oral administration. The amounts of urinary ENL increased with the dose of HMR (from 3 to 50 mg/kg), and only minor amounts of unmetabolized HMR isomers and other lignans were found in urine. HMR (15 mg/kg body wt po) given for 51 days decreased the number of growing tumors and increased the proportion of regressing and stabilized tumors in the rat dimethylbenz[a]anthracene-induced mammary tumor model. HMR (50 mg/kg body wt) did not exert estrogenic or antiestrogenic activity in the uterine growth test in immature rats. HMR also showed no antiandrogenic responses in the growth of accessory sex glands in adult male rats. Neither ENL nor enterodiol showed estrogenic or antiestrogenic activity via a classical a- or b-type estrogen receptor-mediated pathway in vitro at <1.0 mM. HMR was an effective antioxidant in vitro.

Differential expression of estrogen receptors α and β in adult rat accessory sex glands and lower urinary tract

Abstract Estrogens induce pronounced structural and functional changes in male accessory sex glands and the lower urinary tract in both sexes, but the exact mechanisms of estrogen action are not fully understood. This study was undertaken to localise the tissue cell types that express estrogen receptor in adult rats, and to determine the receptor subtype (ERα and ERβ) in order to identify sites that may respond directly to estrogens. In the male accessory sex glands (seminal vesicles, prostatic lobes and ampullary glands), ERβ mRNA and protein were strongly expressed in the epithelium but not in the stroma, while ERα mRNA was present only in the fibromuscular tissue surrounding the prostatic collecting ducts in the posterior periurethral region and in ampullary gland stroma. In the epithelium of the urinary bladder and urethra of both sexes, high level of ERβ mRNA and protein, but no ERα mRNA, was detected. The connective tissue in urinary bladder of both males and females, as well as that in prostatic urethra in males expressed ERα mRNA. The neural cells in the autonomic ganglia of the prostatic plexus were strongly positive for ERβ mRNA, but were completely devoid of ERα. We conclude that ERβ is the predominant ER subtype in the epithelium of adult male rat accessory sex glands and the lower urinary tract of both males and females, as well as in the prostatic neural plexus regulating the function of the lower urinary tract in males, while ERα is present only in the stromal compartment of distinct sites. These results indicate that in these tissues in intact adults there are multiple targets for direct estrogen action. Furthermore, the differential or complementary expression of the two ER subtypes suggests that they may have specific functions, and may explain the complex structural and functional changes induced by estrogens.

GENISTEIN EXERTS ESTROGEN-LIKE EFFECTS IN MALE MOUSE REPRODUCTIVE TRACT

The aim of this study was to evaluate the estrogenicity of genistein in the neonatal and adult male mouse reproductive tract. In intact adults, genistein (2.5 mg s.c./kg of body weight/day for 9 days) reduced testicular and serum testosterone concentrations, pituitary LH-content and prostate weight. In castrated adults, genistein (0.025-2.5 mg s.c./kg of body weight) increased expression of c-fos gene in prostatic urethra. In adult, neonatally estrogenized mice showing an increased estrogen sensitivity, a 10-day treatment with genistein (2.5 mg s.c./kg of body weight) induced development of squamous epithelial metaplasia in prostatic collecting ducts. Neonatally, only a very high dose of genistein (1 mg/pup per day; i.e. approximately 500 mg/kg of body weight) induced persistent structural changes, similar to those seen in mice treated neonatally with diethylstilbestrol, in the urethroprostatic complex. These results suggest that in adult males, genistein induces the typical estrogenic effects in doses comparable to those present in soy-based diets, while in neonatal animals, considerably higher doses are required to show estrogen-like activity.

Phytoestrogens: Potential Endocrine Disruptors in Males

Exposure to diethylstilbestrol (DES) induces persistent structural and functional alterations in the developing reproductive tract of males. It is possible that xenoestrogens other than DES alter sexual differentiation in males and account for the increasing incidence of developmental disorders of the reproductive tract in men and wild animals. Phytoestrogens (coumestans, isoflavonoids, flavonoids, and lignans) present in numerous edible plants are quantitatively the most important environmental estrogens when their hormonal potency is assessed in vitro. They exert their estrogenic activity by interacting with estrogen receptors (ERs) in vitro. They may also act as antiestrogens by competing for the binding sites of estrogen receptors or the active site of the estrogen biosynthesizing and metabolizing enzymes, such as aromatase and estrogen-specific 17β-hydroxysteroid oxidoreductase (type 1). In theory, phytoestrogens and structurally related compounds could harm the reproductive health of males also by acting as antiestrogens. There are very little data on effects of phytoestrogens in males. Estrogenic effects in wildlife have been described but the evidence for the role of phytoestrogens is indirect and seen under conditions of excessive exposure. In doses comparable to the daily intake from soy- based feed, isoflavonoids such as genistein were estrogen agonists in the prostate of adult laboratory rodents. When given neonatally, no persistent effects were observed. In contrast, the central nervous system (CNS)-gonadal axis and the male sexual behavior of the rat appear to be sensitive to phytoestrogens during development. The changes were similar but not identical to those seen after neonatal treatment with DES, but higher doses of phytoestrogens were needed.There are no data on effects of phytoestrogens given as pure compounds to humans, and all evidence currently available is indirect and based on experiments with phytoestrogen- rich diets. The hormonal effects have so far been marginal. It is known that the intake of phytoestrogens is higher in countries where the incidence rates of clinical conditions linked to estrogen exposure, such as hypospadia or testicular and prostatic cancers, are low. This makes it unlikely that phytoestrogens, or structurally related compounds in amounts present in Asian diets, would have DES-like actions. This does not exclude possibilities that they influence concentrations of endogenous sex hormones and interact with the ER, and that through these mechanisms they alter male sex differentiation, and consequently increase the risks of male genital tract tumors or developmental disorders, particularly in doses exceeding the daily intake of phytoestrogens in Asian diets.

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.

Erratum to “Differential expression of estrogen receptors α and β in adult rat accessory sex glands and lower urinary tract”[Mol. Cell. Endocrinol. 164 (2000) 109–116]

Estrogens induce pronounced structural and functional changes in male accessory sex glands and the lower urinary tract in both sexes, but the exact mechanisms of estrogen action are not fully understood. This study was undertaken to localise the tissue cell types that express estrogen receptor in adult rats, and to determine the receptor subtype (ERa and ERb) in order to identify sites that may respond directly to estrogens. In the male accessory sex glands (seminal vesicles, prostatic lobes and ampullary glands), ERb mRNA and protein were strongly expressed in the epithelium but not in the stroma, while ERa mRNA was present only in the fibromuscular tissue surrounding the prostatic collecting ducts in the posterior periurethral region and in ampullary gland stroma. In the epithelium of the urinary bladder and urethra of both sexes, high level of ERb mRNA and protein, but no ERa mRNA, was detected. The connective tissue in urinary bladder of both males and females, as well as that in prostatic urethra in males expressed ERa mRNA. The neural cells in the autonomic ganglia of the prostatic plexus were strongly positive for ERb mRNA, but were completely devoid of ERa. We conclude that ERb is the predominant ER subtype in the epithelium of adult male rat accessory sex glands and the lower urinary tract of both males and females, as well as in the prostatic neural plexus regulating the function of the lower urinary tract in males, while ERa is present only in the stromal compartment of distinct sites. These results indicate that in these tissues in intact adults there are multiple targets for direct estrogen action. Furthermore, the differential or complementary expression of the two ER subtypes suggests that they may have specific functions, and may explain the complex structural and functional changes induced by estrogens.

CO-LOCALIZATION OF ANDROGEN RECEPTOR WITH ESTROGEN RECEPTOR β IN THE LOWER URINARY TRACT OF THE MALE RAT

PURPOSE Androgens and estrogens influence voiding. In this study their target sites in the lower urinary tract of the male rat were identified. MATERIALS AND METHODS Cryosections of the bladder body, bladder neck, prostatic urethra, mid proximal urethra and prostatic autonomic ganglia of adult male rats were immunostained with specific estrogen receptor alpha, estrogen receptor beta (ERbeta) or androgen receptor (AR) antibodies. The sections were then examined under conventional, fluorescence or confocal fluorescence microscopy. RESULTS Co-expression of AR and ERbeta in the urothelium, bladder smooth muscle cells, proximal urethra striated muscle cells and neurons in the autonomic ganglia of the prostatic plexus suggests that estrogen and androgen have direct effects in the lower urinary tract. CONCLUSIONS The local interaction of AR and ERbeta in the hormonal control of voiding is an intriguing possibility.

Prostatic dysplasia associated with increased expression of C-MYC in neonatally estrogenized mice

Neonatal estrogenization of the mouse with diethylstilbestrol (DES; 2 micrograms./pup/day for days 1 to 3) or 17 beta-estradiol (200 micrograms./pup/day for days 1 to 3) resulted in epithelial dysplasia in the posterior periurethral region of the prostate at the age of 1 year. The dysplastic lesions ranged from mild to severe and, in addition to emergence of nuclear anaplasia, the architectural pattern of the glands was disturbed. Prenatal estrogenization (100 micrograms./kg. of maternal body weight on days 13 and 15 of gestation) only resulted in mild epithelial hyperplasia and occasional dysplasia in the ventral lobe of the prostate, but not in the posterior periurethral region. When neonatally estrogenized mice were allowed to grow until the age of 18 months, the degree and extent of the dysplasia of the posterior periurethral region was increased, but no frank invasion or metastases could be demonstrated. Combined estrogen and androgen treatment of neonatally estrogenized mice for 3 months (between 9 and 12 months of age) augmented nuclear dysplasia, but no invasive growth was seen in this group, either. Mild epithelial dysplasia was found in the dorsolateral lobes and coagulating glands of similarly treated control animals. A relation between the activation of certain proto-oncogenes and the development of several cancers has been shown in humans and experimental animals. In the present study, Northern blot analysis of total RNAs showed that the levels of c-myc mRNA were increased in the ventral and dorsolateral lobes, coagulating glands and prostatic urethra of neoDES mice at the age of 9 months. However, it remains to be determined whether the increase in c-myc expression is involved in the development of hyperplastic and dysplastic changes in the prostate of neoDES mice.

Inflammation and epithelial alterations in rat prostate: impact of the androgen to oestrogen ratio.

Chronic non-bacterial prostatitis may offer new insights into the pathogenesis of human benign prostatic hyperplasia and prostate cancer and the strategies for their treatment and prevention. The potential significance of androgen replacement therapy in terms of the reversal of oestradiol (E(2))-induced inflammatory reaction was studied in the dorsolateral prostate (DLP) of the Noble rat. Castrated Noble rats were treated with E(2) and different doses of androgens [dihydrotestosterone (DHT) and testosterone (T)] to achieve an elevated concentration of E(2) and a wide range of the androgen-to-oestradiol ratios in serum. After the 3-week treatment, inflammatory changes in the DLP were classified and counted. Oestrogen receptor alpha (ER alpha), progesterone receptor (PR), fos-related antigen-2 (Fra2), Ki-67 and P63 were immunocytochemically stained. T, E(2) and prolactin concentrations in serum were measured and the relative weights of the seminal vesicles and pituitary glands and microscopic structures of the DLP and seminal vesicle ducts were determined. Hypoandrogenic doses of DHT (judged on the basis of seminal vesicle weight gain), dose-dependently increased the number of perivascular and stromal inflammatory infiltrates. T and DHT were anti-inflammatory at the doses which normalized or over stimulated the growth of the seminal vesicles. As signs of anti-oestrogenicity, androgens dose-dependently decreased the number and distribution of the ER alpha and PR-positive cells at proinflammatory concentrations. Anti-inflammatory concentrations were needed to reduce the expression of Fra2, E(2)-increased prolactin concentration in serum and pituitary weight. The androgen concentrations required to prevent proinflammatory and epithelial responses to E(2) in the presence of elevated E(2) concentrations may subject the accessory sex glands to more intense androgenic stimulation than is normal for the male. The androgen-resistant endpoints of oestrogen action (body weight reduction and hyperplasia of seminal vesicle ducts) further indicate limitations in the possible preventive effects of androgen-replacement therapy.