Hormonal status of male reproductive system: androgens and estrogens in the testis and epididymis. In vivo and in vitro approaches

The purpose of this article was to summarize our results on the role of androgens and estrogens in human, rodent and equine testes and epididymides, in both, physiological and patological conditions, obtained in the space of the Solicited Project (084/PO6/2002) financially supported by the State Committee for Scientific Research during the last three years. Testosterone produced by Leydig cells of the testes is clearly the major androgen in the circulation of men and adult males of most mammalian species. However, androgen metabolites make up a significant fraction of total circulating steroids. Moreover, androgen metabolism may proceed to amplify the action of testosterone through its conversion to dihydrotestosterone (DHT) or its aromatization to estradiol. The distribution of androgen and estrogen receptors (ARs and ERs) within male reproductive tissues is important because of their crucial role in mediating androgen and/or estrogen action. Attempts were undertaken to discuss not only the role of aromatase and ERs in mediating the action of estrogens in the male, but also the importance of DHT in hormonal regulation of the epididymis. In the latter, alterations caused by finasteride treatment and lead-induced oxidative stress are described. Male reproductive function of the testis and epididymis reflected by the alterations in enzymatic activity, distribution of steroid hormone receptors, differences in steroid hormone levels and altered gene expression of antioxidant enzymes are also discussed.     

Effects of 3-beta-diol, an androgen metabolite with intrinsic estrogen-like effects, in modulating the aquaporin-9 expression in the rat efferent ductules

Background  

Fluid homeostasis is critical for normal function of the male reproductive tract and aquaporins (AQP) play an important role in maintenance of this water and ion balance. Several AQPs have been identified in the male, but their regulation is not fully comprehended. Hormonal regulation of AQPs appears to be dependent on the steroid in the reproductive tract region. AQP9 displays unique hormonal regulation in the efferent ductules and epididymis, as it is regulated by both estrogen and dihydrotestosterone (DHT) in the efferent ductules, but only by DHT in the initial segment epididymis. Recent data have shown that a metabolite of DHT, 5-alpha-androstane-3-beta-17-beta-diol (3-beta-diol), once considered inactive, is also present in high concentrations in the male and indeed has biological activity. 3-beta-diol does not bind to the androgen receptor, but rather to estrogen receptors ER-alpha and ER-beta, with higher affinity for ER-beta. The existence of this estrogenic DHT metabolite has raised the possibility that estradiol may not be the only estrogen to play a major role in the male reproductive system. Considering that both ER-alpha and ER-beta are highly expressed in efferent ductules, we hypothesized that the DHT regulation of AQP9 could be due to the 3-beta-diol metabolite.     

Detection of estrogen receptors ER-alpha and ER-beta in human ejaculated immature spermatozoa with excess residual cytoplasm

Background  

A key role of estrogens in human sperm biology has been recently suggested by aromatase and estrogen receptor detection in human testicular germ cells and ejaculated spermatozoa. However, the involvement of these hormones in the sperm maturation process is still not defined. The aim of this work was to investigate the expression of estrogen receptors, ER-alpha and ER-beta, in human ejaculated immature spermatozoa with excess residual cytoplasm.     

Estrogen receptor-alpha and estrogen receptor-beta are present in the human growth plate in childhood and adolescence,in identical distribution

OBJECTIVE: To localize estrogen receptor-alpha (ER-alpha) and estrogen receptor-beta (ER-beta) within the growth plate and adjacent bony tissue of children in the prepubertal and pubertal age period. METHODS: Tissue was taken during orthopedic surgery (epiphysiodesis) for correction of congenital or traumatic leg length difference in 2 prepubertal females and 2 adolescent males. Immunohistochemistry was performed on paraffin-embedded or cryostat sections by using commercially available rabbit polyclonal antibodies for ER-alpha and ER-beta. RESULTS: Both ER-alpha and ER-beta were detected within the growth plate in all sections investigated. Immunostaining was restricted to hypertrophic chondrocytes. In the bony tissue adjacent to the growth plate, osteoblasts stained positive for both ER-alpha and ER-beta, whereas osteocytes and osteoclasts were negative. Staining with ER-alpha was mainly nuclear but some cells also showed cytoplasmic signals, while ER-beta staining was predominantly cytoplasmic, only few nuclei stained positive. There was no difference in the local distribution of both ERs between tissue from prepubertal and pubertal patients. CONCLUSION: Our findings indicate that the hypertrophic chondrocyte is the main target cell for estrogen action within the growth plate. The presence of ER in prepubertal children suggests that estrogens play a role in skeletal maturation under physiological conditions also in this age-group.     

Cellular localization of estrogen receptor beta messenger ribonucleic acid in cynomolgus monkey reproductive organs.

There is now evidence that the recently identified estrogen receptor (ER) beta is more widely distributed in the body than is ER-alpha. In order to gain more information about the role of ER-beta in reproduction, we have investigated by in situ hybridization the localization of mRNA expression of this ER subtype in adult monkey reproductive organs. In the pituitary gland of animals of both sexes, in both the anterior and intermediate lobes, a large number of cells were positive. No specific signal was observed in the posterior lobe. In the ovary, granulosa cells in primary and growing follicles highly expressed ER-beta mRNA. The theca interna cells were also strongly labeled. In some corpora lutea, the luteal cells were strongly labeled, while in other ones, the signal was weak. A hybridization signal was also detected in the ovarian surface epithelium. In the uterus, ER-beta mRNA was found in high concentration in glandular epithelial cells and stromal cells of the endometrium, while weaker labeling was consistently observed in smooth muscle cells. In the mammary gland, labeling was detected in the epithelial cells of acini and interlobular ducts as well as stromal cells. In the testis, specific labeling was detected in the seminiferous epithelium whereas the interstitial Leydig cells were unlabeled. Although it was not possible to clearly identify all the positive cell types, it appears that Sertoli cells as well as the vast majority of germinal cells express ER-beta mRNA. In the prostate, the secretory epithelial cells exhibited a specific autoradiographic reaction while the stromal cells did not show mRNA expression. The epithelial cells of the prostatic urethra showed a strong labeling. No hybridization signal was detected in the seminal vesicles. It then appears quite clear that ER-beta is expressed in a cell-specific manner in all the monkey reproductive organs studied. In the female, the wide distribution of these receptors in the ovary and uterus suggests that ER-beta may play an important role in the mediation of the known effects of estrogen in reproduction functions. In the male testis and prostate, ER-beta has been found in cells that contain very little or no ER-alpha. The role of circulating or locally produced estrogens in the male reproductive system remains to be clarified.     

Evidence for sulfatase and 17beta-hydroxysteroid dehydrogenase type 1 activities in equine epididymis and uterus

Our previous work showed that stallion testis produces high amounts of estrogens which are subsequently found in the ejaculate. These estrogens are mainly synthesized by testicular aromatase, and the major estrogen produced is estrone sulfate (E1S). The objective of this study was to investigate the potential role of E1S as a source of estrogens in the male and female horse reproductive tracts by determining whether both estrone sulfatase (Sulf) and 17beta-hydroxysteroid dehydrogenase type I (17beta-HSD1) activities were present in equine testes, epididymis and uterus. We assessed E1S bioconversion into estrone (E1) and estradiol (E2) in these tissues. Both Sulf and 17beta-HSD1 activities were well detected in the cauda epididymis and uterus. Additionally, Sulf activity was present in the distal corpus of the epididymis, and 17beta-HSDI in the proximal corpus. In contrast, aromatase gene expression, measured as an internal control of endogenous estrogen production, had high activity only in the testis. We found that seminal E1S of testicular origin can be metabolized to E2, especially in the cauda epididymis and uterus. Because E2 appears to play a major role in male and female reproduction, we propose that the bioconversion of seminal E1S could affect male and female fertility.     

Effect of overexpression of estrogen receptors in osteoblasts

Summary Our study focused on investigating the mechanism of action of estrogen in regulating p53 levels within osteoblasts. In the studies reported here, we attempted to understand the role of estrogen receptors, ER-alpha and ER-beta, in the regulation of p53 and osteoblast differentiation. We stably expressed ER-alpha and ER-beta in ROS 17/2.8 cells and isolated several single cell clones. These clones were initially characterized for expression of the exogenous receptors, and representative clones from each type were chosen for further analyses. Cell proliferation, alkaline phosphatase activity, and the viability of these clones in culture were tested. The cells expressing exogenous ER-alpha exhibited more differentiated characteristics than cells expressing ER-beta. Morphologically, ER-beta-overexpressing cells were more rounded than the ER-alpha-overexpressing cells, which were more elongated and fibroblastic in appearance. The ER-beta-expressing cells had a higher survival and growth rate when compared with ER-alpha cells. The ER-alpha clones were not as viable as ER-beta clones, and some of the ER-alpha cell lines showed signs of senescence, with an increase in senescence-associated (SA) galactosidase activity. The basal levels of p53 functional activity were higher in cells expressing ER-alpha as was protein expression of the p53-regulated gene p21. The significance of these receptors to osteoblast differentiation and p53 regulation is discussed.     

Selective estrogen receptor-alpha and estrogen receptor-beta agonists rapidly decrease pulmonary artery vasoconstriction by a nitric oxide-dependent mechanism

Both endogenous and exogenous estrogen decrease pulmonary artery (PA) vasoconstriction. Whether these effects are mediated via estrogen receptor (ER)-alpha or ER-beta, and whether the contribution of ERs is stimulus-dependent, remains unknown. We hypothesized that administration of the selective ER-alpha agonist propylpyrazole triol (PPT) and/or the selective ER-beta agonist diarylpropiolnitrile (DPN) rapidly decreases PA vasoconstriction induced by pharmacologic and hypoxic stimuli via a nitric oxide (NO)-dependent mechanism. PA rings (n = 3-10/group) from adult male Sprague-Dawley rats were suspended in physiologic organ baths. Force displacement was measured. Vasoconstrictor responses to phenylephrine (10(-8)M - 10(-5)M) and hypoxia (Po(2) 35-45 mmHg) were determined. Endothelium-dependent and -independent vasorelaxation were measured by generating dose-response curves to acetylcholine (10(-8)M - 10(-4)M) and sodium nitroprusside (10(-9)M - 10(-5)M). PPT or DPN (10(-9)M - 5 x 10(-5)M) were added to the organ bath in the presence and absence of the NO-synthase inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME) (10(-4)M). Selective ER-alpha activation (PPT, 5 x 10(-5)M) rapidly (<20 min) decreased phenylephrine-induced vasoconstriction. This effect, as well as PPT's effects on endothelium-dependent vasorelaxation, were neutralized by l-NAME. In contrast, selective ER-beta activation (DPN, 5 x 10(-5)M) rapidly decreased phase II of hypoxic pulmonary vasoconstriction (HPV). l-NAME eliminated this phenomenon. Lower PPT or DPN concentrations were less effective. We conclude that both ER-alpha and ER-beta decrease PA vasoconstriction. The immediate onset of effect suggests a nongenomic mechanism. The contribution of specific ERs appears to be stimulus specific, with ER-alpha primarily modulating phenylephrine-induced vasoconstriction, and ER-beta inhibiting HPV. NO inhibition eliminates these effects, suggesting a central role for NO in mediating the pulmonary vascular effects of both ER-alpha and ER-beta.     

Expression and cellular localization of naturally occurring beta estrogen receptors in uterine and mammary cell lines

The protein ER-alpha has been exhaustively characterized in estrogen-sensitive tissues and cell lines. However, little is known regarding the expression and cellular distribution of the newly identified ER-beta protein. We first quantified the specific estradiol binding site content in the estrogen-responsive cell lines MCF-7 (mammary) and SHM (myometrial). In the two cell types, these sites were associated to the expression of both ER-alpha and -beta isoforms. Native ER-beta was visualized to reside inside the nucleus by means of conventional indirect immunofluorescence. The cells expressed ER-beta as a tight approximately 50 kDa triplet when resolved by sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and blotted using antibodies mapping different domains of the cloned ER-beta version. When the cells were subjected to homogenization and differential centrifugation, a substantial proportion of ER-beta immunolabeling was localized at membrane subfractions. ER-beta expression and partitioning was confirmed by Ligand blotting assays using estrogen derivatives coupled to different macromolecular tags. However, ER-alpha was expressed as the major estrogen binding protein in both cell lines. Similar localization experiments were performed on HeLa cells (cervix). Though usually considered ER-negative, this cell line displayed basal significant estrogen binding capacity and co-expression of both ER isoforms. Taken as a whole, the results indicate that ER-beta could be expressed as functional estrogen binding proteins among a dominant population of ER-alpha sites in the cell lines under study.     

Estrogènes et spermatozoïdes

Aromatase is the terminal enzyme responsible for estrogen biosynthesis; it is present in the endoplasmic reticulum membrane of steroidogenic cells in vertebrates. This enzyme functions with the ubiquitous reductase as the electron donor. The aromatase gene is unique and its expression is regulated in a tissue and more precisely in a cell-specific fashion via the alternative use of several promoters located in the first exons. This enzymatic complex is generally involved in development, reproduction, sexual differentiation and behaviour, but also in bone and lipid metabolism, brain functions and diseases such as breast and testicular tumors. The aromatase gene expression and its transduction in a fully active protein in testicular somatic cells and germ cells together with the widespread distribution of estrogen receptors (ERα & β) in the testis and the genital tract of the male, are clearly in favor of a physiological role for estrogens in the spermatogenesis processings especialy in sperm maturation. Therefore, we begin to understand the physiopathological roles of the estrogens in males indeed, the aromatase deficiency is associated, with severe bone maturation problems and sterility in man. Conversely, it is also obvious that estrogens in excess are responsible of the impaired spermatogenesis. These female hormones (or the ratio androgens/estrogens) do play a physiological role in the development and maintenance of male gonadal functions and obviously, several steps are concerned especially the sperm production and maturation.     

Functional characterization and sex differences in small mesenteric arteries of the estrogen receptor-beta knockout mouse

The role of the estrogen receptor (ER) subtypes in the modulation of vascular function is poorly understood. The aim of this study was to characterize ex vivo the functional properties of small arteries and their response to estrogens in the mesenteric circulation of female and male ER-beta knockout mice (beta-ERKO) and their wild-type (WT) littermates. Responses to changes in intraluminal flow and pressure were obtained before and after incubation with 17beta-estradiol or ER-alpha agonist propyl-pyrazole-triol (3 h; 10 nM). Cumulative concentration-response curves to acetylcholine, norepinephrine, and passive distensibility were compared with respect to sex and genotype. The collagen and elastin content within the vascular wall and ER expression were also determined. Endothelial morphology was visualized by scanning electron microscopy. 17beta-Estradiol and propyl-pyrazole-triol-treated arteries from female beta-ERKO and WT mice showed enhanced flow-mediated dilation, but this was not evident in males. Distensibility was decreased in arteries from beta-ERKO females. Sex differences in myogenic tone were observed in 17beta-estradiol-treated arteries, but were similar between beta-ERKO and WT mice. Acetylcholine- and norepinephrine-induced responses were similar between groups and sexes. ER-alpha was similarly expressed in the endothelium and media of arteries from all groups studied, as well as ER-beta in WT animals. Endothelial morphology was similar in arteries from animals of both sexes and genotype; however, arterial elastin content was decreased, and collagen content was increased in beta-ERKO male compared with WT male and with beta-ERKO female. We suggest that ERs play a sex-specific role in estrogen-mediated flow responses and distensibility, and that deletion of ER-beta affects artery structure but only in male animals. Further studies in beta-ERKO mice with established hypertension and in alpha-ERKO mice are warranted.     

Aromatase messenger RNA is derived from the proximal promoter of the aromatase gene in Leydig, Sertoli, and germ cells of the rat testis

It has long been recognized that individual cell types within the testes possess the capacity to synthesize estrogen. A number of studies on different species have demonstrated that the levels of aromatase expression and the patterns of regulation are distinct between the different cell types of the testes. Whereas a variety of promoters have been shown to contribute to the patterns of aromatase expression in different cell lineages, studies using ovarian RNA, testis RNA, and Leydig cell tumor lines have demonstrated that the same promoter (promoter II) was used in each. Recent experiments using potent aromatase inhibitors or analysis of animals in which the genes encoding the estrogen receptor-alpha (ER-alpha) or the aromatase, P450, are defective, have confirmed the importance of local estrogen formation in normal testicular function. In order to permit experiments to identify the elements controlling aromatase expression in the individual cell compartments of the testes, we prepared RNA from purified preparations of Leydig, Sertoli, and germ cells. Using specific oligonucleotide primers, the sites of initiation of the aromatase mRNA were determined using rapid amplification of cDNA ends (RACE) and nucleotide sequence analysis of the resulting cDNA fragments. Our results indicate that aromatase mRNA is derived from the proximal promoter (PII) of the aromatase gene in each of the major cell types of the rat testes.     

Lindane may modulate the female reproductive development through the interaction with ER-beta: an in vivo-in vitro approach

Lindane (gamma-HCH) is a persistent environmental pollutant that may act as endocrine disrupter, affecting the nervous, immune and reproductive system, possibly through endocrine-mediated mechanisms. Since both estrogen receptors (ER-alpha and -beta) have shown to be target for endocrine disruption, we investigated the role of gamma-HCH on the development of female reproductive system. For an in vivo evaluation of gamma-HCH effects during prenatal period, pregnant CD1 mice were treated p.o. on gestational days 9-16 with 15 mg/kg bw/day of gamma-HCH and vehicle. The in vivo findings in treated F1 pups - in the absence of signs of systemic toxicity - included increase in the absolute and relative and absolute uterus weight revealed on post-natal day 22, earlier vaginal patency and reduced diameters of primary oocytes at fully sexual maturity. No effects on steroid hormone metabolism (aromatase, testosterone catabolism) were observed. Thus, gamma-HCH elicited subtle effects on female reproductive development likely mediated by ER-beta-mediated pathway(s), without a concurrent impairment of steroid hormone metabolism. Furthermore, to verify whether the endocrine interference of gamma-HCH is attributable to stimulation of ER-beta-mediated pathway(s), its effect has been evaluated in vitro on a cell line, LNCaP, expressing only functional ER-beta. In vitro treatments revealed a concentration-related effect on LNCaP cell viability and proliferation. Significantly, the contemporary addition of a pure anti-estrogen, the ER antagonist ICI 182,780, completely reversed gamma-HCH effects indicating an ER-beta-mediated action. Our findings indicate that gamma-HCH may act as endocrine disruptor during the female reproductive system development and ER-beta as a potential target for this compound and other endocrine disrupting chemicals as well.     

雌激素受体α和β亚型在文昌鱼神经系统、哈氏窝和性腺中的定位

用兔抗人ER-α和ER-β多克隆抗体对文昌鱼神经系统、轮器哈氏窝和性腺进行免疫细胞化学的定位研究。结果揭示幼年和成年两性不同发育时期文昌鱼在这些部位分布ER-α和ER-β蛋白。ER-α定位在端脑、中脑、后脑和神经管中大多数神经细胞核,少数在胞质及其突起和神经纤维,ER-β则定位在细胞质或细胞膜上,少数在核内。ER—α免疫阳性物质主要分布在哈氏窝下层的上皮细胞核,少数在上层细胞质,β受体则在上层细胞核。在性腺,ER-α分布在卵巢中卵原细胞和小生长期卵母细胞胞质与核仁,生发泡(核)显免疫阴性,在大生长期卵母细胞核膜和核仁的免疫阳性显著增强,成熟期则在卵细胞生发泡表达,ER-β免疫阳性物质分布在卵原细胞和早期卵母细胞质以及成熟卵细胞的卵被膜检测到,生发泡显免疫阴性。在精巢,这两种ER亚型均定位在精原细胞、初级与次级精母细胞和足细胞质,精子细胞在胞核,精子显免疫阴性。另外,双染结果还揭示ER-α和ER-β在上述部位多数共存于同一细胞,少数在不同细胞表达,且在细胞定位有不同。首次发现这两种雌激素受体亚型在文昌鱼有广泛分布,它们介导雌激素对文昌鱼神经内分泌组织的调节作用。α和β受体在靶细胞定位的不同,提示两者在介导雌激素信号路线和基因转录机制可能有不同生理作用。