Localization of type 8 17beta-hydroxysteroid dehydrogenase mRNA in mouse tissues as studied by in situ hybridization.

The enzyme type 8 17beta-hydroxysteroid dehydrogenase (17beta-HSD) selectively catalyzes the conversion of estradiol (E2) to estrone (E1). To obtain detailed information on the sites of action of type 8 17beta-HSD, we have studied the cellular localization of type 8 17beta-HSD mRNA in mouse tissues using in situ hybridization. In the ovary, hybridization signal was detected in granulosa cells of growing follicles and luteal cells. In the uterus, type 8 17beta-HSD mRNA was found in the epithelial (luminal and glandular) and stromal cells. In the female mammary gland, the enzyme mRNA was seen in ductal epithelial cells and stromal cells. In the testis, hybridization signal was observed in the seminiferous tubule. In the prostate, type 8 17beta-HSD was detected in the epithelial cells of the acini and stromal cells. In the clitoral and preputial glands, labeling was detected in the epithelial cells of acini and small ducts. The three lobes of the pituitary gland were labeled. In the adrenal gland, hybridization signal was observed in the three zones of the cortex, the medulla being unlabeled. In the kidney, the enzyme mRNA was found to be expressed in the epithelial cells of proximal convoluted tubules. In the liver, all the hepatocytes exhibited a positive signal. In the lung, type 8 17beta-HSD mRNA was detected in bronchial epithelial cells and walls of pulmonary arteries. The present data suggest that type 8 17beta-HSD can exert its action to downregulate E2 levels in a large variety of tissues.     

Cellular localization of mRNA expression of enzymes involved in the formation and inactivation of hormonal steroids in the mouse prostate.

It is well documented that several tissues, including the prostate, are actively involved in the local formation and inactivation of hormonal steroids. To identify the cell types involved in the formation and inactivation of androgens and estrogens in the ventral lobe prostate, we have localized by in situ hybridization (ISH) a large number of steroidogenic as well as steroid-inactivating enzyme mRNAs in the adult mouse prostate. In parallel studies, we also measured enzyme mRNA levels by quantitative real-time PCR (RT-PCR) in ventral lobe prostates. From the results obtained with quantitative RT-PCR, it appears that, with a few exceptions, the enzyme with low mRNA expression could not be detected by ISH. The following enzymes have been localized by ISH: 17beta-hydroxysteroid dehydrogenase (17beta-HSD) types 1, 2, 3, 4, 7, 8, 9, 10, and 11; 5alpha-reductase type 2; 5beta reductase type 1; P450 7alpha hydroxylase; estrogen sulfotransferase type 1; 11beta-HSD types 1 and 2; and UDP-glucuronosyltransferase 1A6. All of these mRNAs are expressed in the epithelial cells of prostatic acini. Several enzyme mRNAs were also localized in stromal cells. Types 1, 7, and 10 17beta-HSD, estrogen sulfotransferase type 1, and 11beta-HSD types 1 and 2 were found only in epithelial cells. The present results indicate that both epithelial and stromal cells in the mouse prostate play a role in local formation and inactivation of hormonal steroids.     

Characterization of type 12 17beta-hydroxysteroid dehydrogenase, an isoform of type 3 17beta-hydroxysteroid dehydrogenase responsible for estradiol formation in women

A novel 17beta-hydroxysteroid dehydrogenase (17beta-HSD) chronologically named type 12 17beta-HSD (17beta-HSD12), that transforms estrone (E1) into estradiol (E2) was identified by sequence similarity with type 3 17beta-HSD (17beta-HSD3) that catalyzes the formation of testosterone from androstenedione in the testis. Both are encoded by large genes spanning 11 exons, most of them showing identical size. Using human embryonic kidney-293 cells stably expressing 17beta-HSD12, we have found that the enzyme catalyzes selectively and efficiently the transformation of E1 into E2, thus identifying its role in estrogen formation, in contrast with 17beta-HSD3, the enzyme involved in the biosynthesis of the androgen testosterone in the testis. Using real-time PCR to quantify mRNA in a series of human tissues, the expression levels of 17beta-HSD12 as well as two other enzymes that perform the same transformation of E1 into E2, namely type 1 17beta-HSD and type 7 17beta-HSD, it was found that 17beta-HSD12 mRNA is the most highly expressed in the ovary and mammary gland. To obtain a better understanding of the structural basis of the difference in substrate specificity between 17beta-HSD3 and 17beta-HSD12, we have performed tridimensional structure modelization using the coordinates of type 1 17beta-HSD and site-directed mutagenesis. The results show the potential role of bulky amino acid F234 in 17beta-HSD12 that blocks the entrance of androstenedione. Overall, our results strongly suggest that 17beta-HSD12 is the major estrogenic 17beta-HSD responsible for the conversion of E1 to E2 in women, especially in the ovary, the predominant source of estrogens before menopause.     

Expression of enzymes involved in estrogen metabolism in human prostate.

There is evidence that estrogens can directly modulate human prostate cell activity. It has also been shown that cultured human prostate cancer LNCaP can synthesize the active estrogen estradiol (E2). To elucidate the metabolism of estrogens in the human prostate, we have studied the expression of enzymes involved in the formation and inactivation of estrogens at the cellular level. 17beta-Hydroxysteroid dehydrogenase (17beta-HSD) types 1, 2, 4, 7, and 12, as well as aromatase mRNA and protein expressions, were studied in benign prostatic hyperplasia (BPH) specimens using in situ hybridization and immunohistochemistry. For 17beta-HSD type 4, only in situ hybridization studies were performed. Identical results were obtained with in situ hybridization and immunohistochemistry. All the enzymes studied were shown to be expressed in both epithelial and stromal cells, with the exception of 17beta-HSD types 4 and 7, which were detected only in the epithelial cells. On the basis of our previous results, showing that 3beta-HSD and 17beta-HSD type 5 are expressed in human prostate, and of the present data, it can be concluded that the human prostate expresses all the enzymes involved in the conversion of circulating dehydroepiandrosterone (DHEA) to E2. The local biosynthesis of E2 might be involved in the development and/or progression of prostate pathology such as BPH and prostate cancer through modulation of estrogen receptors, which are also expressed in epithelial and stromal cells.     

Immunocytochemical localization of type 5 17beta-hydroxysteroid dehydrogenase in human reproductive tissues.

17beta-hydroxysteroid dehydrogenase (17beta-HSD) controls the last step in the formation of all androgens and all estrogens. At least six 17beta-HSD isoenzymes have been identified. The recently cloned Type 5 17beta-HSD transforms 4-dione into testosterone. To gain a better understanding of the role of this enzyme in reproductive tissues, we immunocytochemically localized the enzyme in human male and female reproductive organs. In the ovary of adult premenopausal women (25-40 years of age), immunostaining was found in corpus luteum cells. In the uterus, staining was detected only in the epithelial cells of the endometrium. Immunolabeling was also detected in the mammary gland, a positive reaction being detected in epithelial cells of acini and intralobular ducts as well as in the surrounding stromal cells. In the testis, strong staining was seen in the Leydig cells, and a weak but specific reaction was occasionally detected in Sertoli and germ cells. In the prostate, specific labeling was observed in alveoli and stromal fibroblasts. In alveoli, all the basal cells were generally labeled, whereas the luminal cells exhibited variations in immunoreactivity. In all the reproductive organs examined, specific staining was routinely detected in the walls of blood vessels, including the endothelial cells. These results indicate a cell-specific localization of Type 5 17beta-HSD in the different human reproductive organs, thus suggesting new mechanisms of local androgen and estrogen formation that may play an important physiological role.     

Expression of aromatase and 17beta-hydroxysteroid dehydrogenase types 1, 7 and 12 in breast cancer. An immunocytochemical study

It is known that there is a local biosynthesis of estradiol (E2) in breast carcinoma. The steroidogenic enzymes involved in E2 formation are aromatase which transforms testosterone into E2 and androstenedione into estrone (E1) and reductive 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) which convert E1 into E2. Using immunocytochemistry, we have studied the expression of aromatase and the three reductive 17beta-HSDs 17beta-HSD types 1, 7 and 12 in 41 specimens of female human breast carcinoma and adjacent non-malignant tissues. These results were correlated with the estrogen receptor alpha (ERalpha) and beta (ERbeta), progesterone receptor, androgen receptor, CDC47 and c-erb B-2 expressions and with the tumor stages. Aromatase was found in 58%, 17beta-HSD type 7 in 47% and 17beta-HSD type 12 in 83% of the breast cancer specimens. The 17beta-HSD type 1 could be detected in only one tumor. A significant correlation was observed between the aromatase, 17beta-HSD type 7 and 17beta-HSD type 12 expression, as well as between each of the two enzymes 17beta-types 7 and 12 and the ERbeta expression. The expression of 17beta-HSD type 12 was significantly higher in breast carcinoma specimens than in normal tissue. There was also a significant association of CDC 47 expression with ERbeta, AR and 17beta-HSD type 12. The results indicate that aromatase, 17beta-HSD type 7 and 17beta-HSD type 12, but not 17beta-HSD type 1, are commonly expressed in human breast cancer. Moreover, the high expression of both 17beta-HSD type 12 and ERbeta in breast carcinoma cells may play a role in the development and/or progression of breast cancer.     

Cloning and characterization of human form 2 type 7 17beta-hydroxysteroid dehydrogenase, a primarily 3beta-keto reductase and estrogen activating and androgen inactivating enzyme

Type 7 17beta-HSD catalyzes the transformation of estrone (E1) into estradiol (E2) and dihydrotestosterone (DHT) into 5alpha -androstane-3beta,17beta-diol (3beta-diol) as well as zymosterone into zymosterol. This suggests that in addition to cholesterol metabolism, the enzyme could play a critical role in estrogen-sensitive cells, since it inactivates DHT that generally shows antagonistic effect in the cells, while producing active E2 for cell proliferation. In this report, we describe the cloning and characterization of a second form of type 7 17beta-HSD (17beta-HSD7_2) that shares 95.6% identity with 17beta-HSD7_1. Using a 7.5kb genomic DNA fragment of 17beta-HSD7_1 as probe, we have obtained 7 BAC clones: three clones containing the 17beta-HSD7_1 gene and four containing the 17beta-HSD7_2 gene. The corresponding 17beta-HSD7_2 cDNA fragments of the coding region were obtained by amplification using RT-PCR and subcloned into pCMV expression vector and stably transfected into human embryonic kidney (HEK-293) cells. The overexpressed 17beta-HSD7_2 catalyzes efficiently the transformation of E1 into E2 and of DHT into 3beta-diol. Ribonuclease protection assays (RPA) indicate that 17beta-HSD7_2 is expressed in the liver, prostate, uterus and placenta. FISH mapping using the 7.5kb genomic DNA fragment as well as 2 BAC clones of each form allowed us to map the 17beta-HSD7_1 gene on chromosome band 1q23, and 17beta-HSD7_2 on band 10p11.2. These results contrast with a previous report that the 17beta-HSD7_1 gene was mapped to chromosomal band 10p11.2. This newly identified form of 17beta-HSD7 could have a significant role by modulating active hormone levels in estrogen-sensitive cells or tissues.     

Spironolactone-related inhibitors of type II 17beta-hydroxysteroid dehydrogenase: chemical synthesis, receptor binding affinities, and proliferative/antiproliferative activities.

The family of 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyzes the formation and inactivation of testosterone (T), dihydrotestosterone (DHT), and estradiol (E2), thus playing a crucial role in the regulation of active steroid hormones in target tissues. Among the five known 17beta-HSD enzymes, type II catalyzes the oxidation of E2 into estrone (E1), T into androstenedione, DHT into androstanedione, and 20alpha-dihydroprogesterone into progesterone. Specific inhibitors are thus an interesting means to study the regulation and to probe the structure of type II 17beta-HSD. In this context, we have efficiently synthesized a series of 7alpha-thioalkyl and 7alpha-thioaryl derivatives of spironolactone that inhibit type II 17beta-HSD. These new C19-steroidal inhibitors possess two important pharmacophores, namely 17-spiro-gamma-lactone and a bulky side-chain at the 7alpha-position. It was found that a para-substituted benzylthio group at the 7alpha-position enhances the inhibitory potency of spironolactone derivatives on type II 17beta-HSD. In fact, the compound with a para-hydroxy-benzylthio group showed an IC50 value of 0.5 microM against type II 17beta-HSD, whereas the compound with a para-[2-(1-piperidinyl)-ethoxy]-benzylthio group inhibited this enzyme with an IC50 value of 0.7 microM. The latter inhibitor is more selective than the former because it did not show any inhibitory potency against P450 aromatase as well as any affinity towards four steroid receptors (AR, PR, GR, ER). As a result, this inhibitor did not show any proliferative effect on androgen-sensitive Shionogi cells and estrogen-sensitive ZR-75-1 cells. These findings contribute to a better knowledge of the structure of type II 17beta-HSD and offer an interesting tool to study the regulation of this enzyme in several biological systems.     

Localization of type 5 17β-hydroxysteroid dehydrogenase mRNA in mouse tissues as studied by in situ hybridization

The mouse enzyme type 5 17-hydroxysteroid dehydrogenase (17-HSD) catalyzes the conversion of androstenedione to testosterone and, to a lesser degree, the conversion of estrone to estradiol. In order to determine the exact sites of action of type 5 17-HSD, we studied the cellular localization of the mRNA of the enzyme in mouse tissues by using in situ hybridization. Specific hybridization signal was found in the liver, ovary, adrenal cortex, and kidney. In the liver of mice of both sexes, a strong signal was observed in all hepatocytes. In the ovary, specific labeling was detected in the granulosa and theca interna cells in growing follicles and in luteal cells. In the female adrenal cortex, intense labeling was restricted to the zona reticularis, whereas no type 5 17-HSD mRNA expression could be found in the male adrenal cortex. In the kidney of mice of both sexes, type 5 17-HSD mRNA was expressed in epithelial cells in both the proximal and distal convoluted tubules. The data indicate that androgens and estrogens are formed via the action of type 5 17-HSD in specific cell types in the liver, ovary, adrenal cortex, and kidney.This work was supported by Genome Canada and Genome Québec.     

Quantitative aspects of anin situ hybridization procedure for detecting mRNAs in cells using 96-well microplates

The universal quantitation of the DNA hybridization reaction has been a goal sought by many researchers. Part of this search has been the need to develop a rapid, sensitive, easy-to-perform, and quantitative method to measure the abundance of specific mRNAs directly within cells. Conventionally mRNA detection can be done by advanced quantitativein situ hybridization (ISH) using either image analysis or fluorescencein situ hybridization (FISH), or indirectly by extraction of mRNA from cells or tissue and using Northern blot or quantitative polymerase chain reaction (PCR). We examined the quantitative nature of probe binding to intracellular mRNA in a sensitive and easy-to-use nonisotopic method of ISH previously developed in our laboratories. The method is applicable to isolated primary cells or cells in culture. The procedural details are very simple, with cells being centrifuged into 96-well microplates, fixed with formalin, and pretreated with Triton X-100 and Nonidet P-40 before photobiotin-labeled cDNA probes are applied. Biotin from the hybridization of probe to target is detected using multiple applications of streptavidin and biotinylated alkaline phosphatase and visualized by thep-nitrophenyl phosphate conversion method. The quantitative parameters of the ISH procedure were determined by measuring the levels of expression of erythropoietin (EPO) mRNA and its translated protein in transfected COS-7 cells. There is a log-linear relationship between the levels of signal obtained in the ISH reaction in 96-well microplates and the EPO protein levels measured by enzyme-linked immunosorbent assay (ELISA). This demonstrated relationship is important in the standardization and use of these procedures to measure quantitatively mRNAs within cells.     

Characterization of the guinea pig 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4-isomerase expressed in the adrenal gland and gonads

The guinea pig adrenal gland, analogous to the human, possesses the capacity to synthesize C(19) steroids. In order to further understand the control of guinea pig adrenal steroidogenesis we undertook the characterization of the guinea pig 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3beta-HSD) expressed in the adrenal gland. A cDNA clone encoding guinea pig 3beta-HSD isolated from a guinea pig adrenal library is predicted to encode a protein of 373 amino acid residues and 41,475Da. Ribonuclease protection assay suggests that this cDNA corresponds to the predominant, if not the sole, mRNA species detectable in total RNA from the guinea pig adrenal gland, ovary and testis. The guinea pig 3beta-HSD shows a similar affinity for both pregnenolone and dehydroepiandrosterone, and in addition, a 17beta-HSD type II-like activity was also observed. A phylogenetical analysis of the 3beta-HSD gene family demonstrates that the guinea pig is in a parallel branch to the myomorpha group supporting the hypothesis that the guinea pig lineage has branched off after the divergence among primates, artiodactyls and rodents, suggesting the paraphyly of the order rodentia.     

印记基因Dlk1在小鼠胚胎发育中的表达分析

目的:研究印记基因Dlk1在小鼠胚胎发育过程中的动态表达模式,以揭示Dlk1与胚胎发育的关系。方法:通过半定量PCR和定量PCR分析Dlk1在小鼠胚胎发育E8.5～E19.5的基因表达模式,并选取Dlk1表达量最高的时期进行胚胎切片原位杂交和组织定量PCR分析。结果:在小鼠胚胎发育E8.5～E15.5时,Dlk1的表达逐渐升高,在E15.5时表达量达到最高;E15.5～E19.5时,Dlk1表达有所下降,但仍然维持较高水平。E15.5切片原位杂交显示,垂体、肺脏、软骨、舌和背侧肌肉组织中Dlk1表达较高,组织定量PCR实验进一步证实了原文杂交的结果。结论:Dlk1在小鼠胚胎发育中后期持续表达,并呈现一定的组织特异性,对胚胎发育可能起重要的调节作用。     

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.     

Independent differentiation of mammotropes and somatotropes in the chicken embryonic pituitary gland

It has been reported that mammotropes in a rodent pituitary gland are derived from somatotropes via somatomammotropes (SMTs), cells that produce both growth hormone (GH) and prolactin (Prl). However, no studies have been done on the transdifferentiation of somatotropes in the chicken pituitary gland. In this study, in order to determine the origin of mammotropes, we studied detail property of appearance of chicken somatotropes, mammotropes and pit-1 cells and then evaluated the existence of SMTs in the chicken embryonic pituitary gland. Immunohistochemical analysis revealed that GH-immunopositive (GH-ip) cells appeared on embryonic day (E) 14 and were mainly distributed in the caudal lobe, while Prl-immunopositive (Prl-ip) cells appeared in the cephalic lobe of the pituitary gland on E16. In situ hybridization (ISH) and RT-PCR analysis showed that expression of GH and Prl mRNA starts at E12 in the caudal lobe and at E14 in the cephalic lobe respectively. Pit-1 mRNA was first detected on E5 by RT-PCR, and pit-1 mRNA-expressing cells were found in the cephalic lobe on E8. Then with the ontogeny of the chicken, these cells spread into both lobes. Using a double staining method with ISH and immunohistochemistry, we could not detect the existence of SMTs in the chicken embryonic pituitary gland even in the marginal region of either lobe. These results suggest that chicken somatotropes and mammotropes independently appear in different lobes of pituitary gland and that transdifferentiation from somatotropes to mammotropes is not the central route for differentiation of mammotropes in the embryonic chicken pituitary gland.