Leena Strauss

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.

Increased Exposure to Estrogens Disturbs Maturation, Steroidogenesis, and Cholesterol Homeostasis via Estrogen Receptor α in Adult Mouse Leydig Cells

Deteriorated male reproductive health has been connected to overexposure to estrogens or to imbalanced androgen-estrogen ratio. Transgenic male mice expressing human aromatase (AROM(+) mice) serve as an apt model for the study of the consequences of an altered androgen-estrogen ratio. Our previous studies with AROM(+) mice showed that low androgen levels together with high estrogen levels result in cryptorchidism and infertility. In the present study, the AROM(+) mice were shown to have severe abnormalities in the structure and function of Leydig cells before the appearance of spermatogenic failure. Decreased expression of adult-type Leydig cell markers (Ptgds, Vcam1, Insl3, Klk21, -24 and -27, Star, Cyp17a1, and Hsd17b3) indicated an immature developmental stage of the Leydig cells, which appears to be the first estrogen-dependent alteration. Genes involved in steroidogenesis (Star, Cyp17a1, and Hsd17b3) were suppressed despite normal LH levels. The low expression level of kallikreins 21, 24, and 27 potentially further inhibited Leydig cell function via remodeling extracellular matrix composition. In connection with disrupted steroidogenesis, Leydig cells showed enlarged mitochondria, a reduced amount of smooth endoplasmic reticulum, and an accumulation of cholesterol and precursors for cholesterol synthesis. The results of studies with AROM(+) mice crossed with estrogen receptor alpha or beta (ERalpha and ERbeta, respectively) knockout mice lead to the conclusion that the structural and functional disorders caused by estrogen exposure were mediated via ERalpha, whereas ERbeta was not involved.

The diversity of sex steroid action: novel functions of hydroxysteroid (17β) dehydrogenases as revealed by genetically modified mouse models

Disturbed action of sex steroid hormones, i.e. androgens and estrogens, is involved in the pathogenesis of various severe diseases in humans. Interestingly, recent studies have provided data further supporting the hypothesis that the circulating hormone concentrations do not explain all physiological and pathological processes observed in hormone-dependent tissues, while the intratissue sex steroid concentrations are determined by the expression of steroid metabolising enzymes in the neighbouring cells (paracrine action) and/or by target cells themselves (intracrine action). This local sex steroid production is also a valuable treatment option for developing novel therapies against hormonal diseases. Hydroxysteroid (17β) dehydrogenases (HSD17Bs) compose a family of 14 enzymes that catalyse the conversion between the low-active 17-keto steroids and the highly active 17β-hydroxy steroids. The enzymes frequently expressed in sex steroid target tissues are, thus, potential drug targets in order to lower the local sex steroid concentrations. The present review summarises the recent data obtained for the role of HSD17B1, HSD17B2, HSD17B7 and HSD17B12 enzymes in various metabolic pathways and their physiological and pathophysiological roles as revealed by the recently generated genetically modified mouse models. Our data, together with that provided by others, show that, in addition to having a role in sex steroid metabolism, several of these HSD17B enzymes possess key roles in other metabolic processes: for example, HD17B7 is essential for cholesterol biosynthesis and HSD17B12 is involved in elongation of fatty acids. Additional studies in vitro and in vivo are to be carried out in order to fully define the metabolic role of the HSD17B enzymes and to evaluate their value as drug targets.

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.

Female Mice Expressing Constitutively Active Mutants of FSH Receptor Present with a Phenotype of Premature Follicle Depletion and Estrogen Excess

Strong gain-of-function mutations have not been identified in humans in the FSH receptor (FSHR), whereas such mutations are common among many other G protein-coupled receptors. In order to predict consequences of such mutations on humans, we first identified constitutively activated mutants of the mouse (m) Fshr and then expressed them under the human anti-Müllerian hormone promoter in transgenic mice or created knock-in mutation into the mouse genome. We show here that mutations of Asp580 in the mFSHR significantly increase the basal receptor activity. D580H and D580Y mutations of mFSHR bind FSH, but the activity of the former is neither ligand-dependent nor promiscuous towards LH/human choriogonadotropin stimulation. Transgenic expression of mFshr(D580H) in granulosa cells leads to abnormal ovarian structure and function in the form of hemorrhagic cysts, accelerated loss of small follicles, augmented granulosa cell proliferation, increased estradiol biosynthesis, and occasional luteinized unruptured follicles or teratomas. The most affected mFshr(D580H) females are infertile with disturbed estrous cycle and decreased gonadotropin and increased prolactin levels. Increased estradiol and prolactin apparently underlie the enhanced development of the mammary glands, adenomatous pituitary growth, and lipofuscin accumulation in the adrenal gland. The influence of the mFSHR(D580Y) mutation is milder, mainly causing hemorrhagic cysts in transgenic mFSHR(D580Y) and mFSHR(D580Y) -knock-in mice. The results demonstrate that gain-of-function mutations of the FSHR in mice bring about distinct and clear changes in ovarian function, informative in the search of similar mutations in humans.

Mast cell tryptase stimulates production of decorin by human testicular peritubular cells: possible role of decorin in male infertility by interfering with growth factor signaling

BACKGROUND Myofibroblastic, peritubular cells in the walls of seminiferous tubules produce low levels of the extracellular matrix (ECM) protein decorin (DCN), which has the ability to interfere with growth factor (GF) signaling. In men with impaired spermatogenesis, fibrotic remodeling of these walls and accumulation of tryptase-positive mast cells (MCs) occur. METHODS Human testicular biopsies with normal and focally impaired spermatogenesis (mixed atrophy) were subjected to immunohistochemistry and laser micro-dissection followed by RT-PCR. Primary human testicular peritubular cells (HTPCs), which originate from normal and fibrotically altered testes (HTPC-Fs), were studied by qRT-PCR, western blotting, enzyme-linked immunosorbent assay measurements and Ca(2+) imaging. Phosphorylation and viability/proliferation assays were performed. RESULTS Immunohistochemistry revealed DCN deposits in the walls of tubules with impaired spermatogenesis. Mirroring the situation in vivo, HTPC-Fs secreted more DCN than HTPCs (P < 0.05). In contrast to HTPCs, HTPC-Fs also responded to the main MC product, tryptase, and to a tryptase receptor (PAR-2) agonist by further increased production of DCN (P < 0.05). Several GF receptors (GFRs) are expressed by HTPCs and HTPC-Fs. DCN acutely increased intracellular Ca(2+)-levels and phosphorylated epidermal GF (EGFR) within minutes. Platelet-derived GF (PDGF) and EGF induced strong mitogenic responses in HTPC/-Fs, actions that were blocked by DCN, suggesting that DCN in the ECM interferes with GF/GFRs signaling of peritubular cells of the human testis. CONCLUSIONS The data indicate that the increase in testicular DCN found in male infertility is a consequence of actions of MC-derived tryptase. We propose that the increases in DCN may consequently imbalance the paracrine signaling pathways in human testis.