Localization and variation of TRAIL and its receptors in human placenta during gestation

The localization of TRAIL and its receptors in human placenta was studied under light microscopy using immunohistochemistry method. The variation of TRAIL and its receptors with development was also detected by in situ semi-quantification. The syncytiotrophoblast, cytotrophoblast, stromal cells and the capillary endothelium cells in human placenta all appeared to be TRAIL immunoreactive and the immunoreactive material was distributed on membrane and in cytoplasm with negative nuclei. During whole gestation there was no obvious variation of the staining of TRAIL. Although DR4, DR5, DcR1 and DcR2 can also be detected in the placenta throughout pregnancy, DR4 and DR5 staining increased with development whereas DcR1 and DcR2 staining decreased. Interestingly, at the beginning of the gestation DR4 and DR5 staining distributed on the cytotrophoblast mainly, whereas DcR1 and DcR2 mainly located in the syncytiotrophoblast cells. Collectively, these results suggest that human placenta may not only produce TRAIL but also be a TRAIL target organ, and that TRAIL/TRAILR system could take part in the self-homeostasis of placenta during whole gestation.     

Differential localization of heat shock proteins 90, 70, 60 and 27 in human decidua and placenta during pregnancy

Little is known about the localization of heat shock proteins (HSPs) in the decidua and placenta during the course of normal pregnancy. In this study, we have examined the localization of the HSPs in decidual and placental tissues obtained from women during the first, second and third trimesters of pregnancy (five in each trimester) by immunohistochemistry using highly specific antisera. HSPs 90, 70, 60 and 27 were detected in decidual stromal cells during each trimester. The intensity of staining did not change during gestation for HSPs 60 and 27, whereas it decreased with advancing gestation for HSPs 90 and 70. HSPs 90 and 60 were localized primarily in the nucleus, whereas HSP 70 was present equally in the nucleus and the cytoplasm; HSP 27 was primarily in the cytoplasm. In the placenta, HSPs 90, 70 and 60 were localized in cytotrophoblast, syncytiotrophoblast, intermediate trophoblast, Hofbauer and endothelial cells. HSPs 90 and 60 were localized primarily in the nucleus, while HSP 70 was in the nucleus and the cytoplasm. In the placenta, HSP 27 was detected only in the intermediate trophoblast and syncytiotrophoblast cells and only in the first two trimesters. These results indicate that there are striking differences in the subcellular localization of HSPs in the decidua and the placenta during normal pregnancy.     

Characterization and modulation of sex steroid metabolizing activity in normal human keratinocytes in primary culture and HaCaT cells

Skin, the largest organ of the human body, synthesizes active sex steroids from adrenal C19 precursor steroids. Normal human breast epidermal keratinocytes in primary culture were used to evaluate the enzymatic activities responsible for the formation and degradation of active androgens and estrogens during keratinocyte differentiation. Enzymatic activities, including 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase (3beta-HSD), 17beta-hydroxysteroid dehydrogenase (17beta-HSD), 5alpha-reductase and 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) were measured using [3H] steroids as substrates. After 10-60 days in culture, no 3beta-HSD activity was detected, but all other activities were measured, demonstrating the ability of keratinocytes to convert androstenedione (4-DIONE) into the potent androgen dihydrotestosterone (DHT). Furthermore, marked changes in enzymatic activity were observed during cell differentiation: 17beta-HSD was first detected during the third week of culture, the level of activity reaching a peak during the fourth week. This peak was followed by a progressive decrease during keratinization. On the other hand, 5alpha-reductase and 3alpha-HSD activities were first detected during the fourth week of culture. The enzymatic activities involved in the formation and degradation of sex steroids were also characterized in the immortalized human keratinocyte cell line HaCaT. It was then found that HaCaT cells possess a pattern of steroid metabolizing enzymes similar to that of human epidermal keratinocytes in culture. Since glucocorticoids are known to exert potent pharmacological effects on the skin, the effect of dexamethasone (DEX) on cell proliferation and enzymatic activities was determined using HaCaT cells. DEX causes a 55% decrease in HaCaT cell proliferation (IC50: 10nM) whereas DEX caused a three- to five-fold stimulation of oxidative 17beta-HSD activity in intact cells in culture (ED50: 30 nM) and this stimulatory effect was competitively blocked by the glucocorticoid antagonist RU486. A four-fold increase in type 2 17beta-HSD mRNA levels was also observed as measured by real-time PCR, correlating with the increase in oxidative activity. No effect of DEX on the other enzymatic activities (3beta-HSD, 5alpha-reductase, and 3alpha-HSD) was observed. Since increased levels of inflammatory cytokines have been detected in some skin diseases then these cytokines might play a role in the differentiation of keratinocytes. In this regard, we found that interleukin-4 (IL-4) induced the expression of 3beta-HSD in HaCaT cells, thus allowing the cells to produce a different set of sex steroids from adrenal C19 precursors. The present data thus indicate that HaCaT cells are a useful model to further study the regulation of the enzymes involved in the metabolism of sex steroids in keratinocytes.     

Expression of Bcl-2 and p53 at the fetal-maternal interface of rhesus monkey

To study the apoptosis and its mechanism at the fetal-maternal interface of early gestation, localization of apoptotic cells in the implantation sites of the rhesus monkey on day 17, 19, 28 and 34 of pregnancy were first examine by using the TUNEL technique. The expression of Ki67, a molecular marker of proliferating cells, and two apoptotic proteins, B cell lymphoma/leukaemia-2 (Bcl-2) and P53, were then studied by immunohistochemistry. Apoptotic nuclei were observed mainly in the syncytiotrophoblast. Ki67 was confined almost exclusively to cytotrophoblasts. The localization of Bcl-2 protein follows that of the apoptotic nuclei and its expression level increased as the development of the placenta progressed on. P53 was detected to some extent in cytotrophoblasts and syncytiotrophoblast covering the basal feet of the anchoring villi during the late stage of placentation. Based on these observations, it might be suggested that Bcl-2 could be possible to play an interesting role in limiting degree of nuclear degradation and sustaining cell suvival in the multi-nucleated syncytiotrophoblast cells during early pregnancy, and P53 could also be essential in regulating the trophoblastic homeostasis by controlling its proliferation or apoptosis.     

Subcellular localization of gonadotropins and testosterone in the developing fetal rat testis.

Gonadotropins and testosterone were immunocytochemically localized in the fetal rat testes 16-18 days of gestation with the unlabeled antibody-peroxidase anti-peroxidase complex technique. Maximum staining for gonadotropins with antiserum to the beta chain of human chorionic gonadotropin (anti-hCGbeta) occurred at 16 days gestation in the seminferous tubule and 17 days gestation in interstitial (Leydig) cells. Anti hCGbeta sites were on the plasma membranes at the luminal aspects of Sertoli cells at 16 days gestation. In addition, intracellular hCGbeta sites were evident including the nucleus, nucleolus, ribosomes, some vesicles, lysosomes and centrioles. The stain for hCGbeta disappeared rapidly and by 17 days was limited to patches in the cytoplasm and nuclei. In the fetal testes, staining for anti-testosterone binding sites was most intense at 18 days of gestation either in lipid droplets or on nuclei of Leydig and Sertoli cells. Very little testosterone stain was observed before 18 days of gestation. These findings agree with physiologic data that suggest that gonadotropins bind to receptors and stimulate testicular development and the capacity for testosterone production.     

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.     

11 beta-Hydroxysteroid dehydrogenase activity of the human placenta during pregnancy

A method for the quantitative estimation of 11 beta-hydroxysteroid dehydrogenase activity (11 beta-HSD; EC.1.1.146) in human placental homogenates is described. This method is based on the separation of cortisol and cortisone by high performance liquid chromatography after extraction from homogenates incubated in the presence of cortisol and NADP. 11 beta-HSD activity (pmol/g wet weight per min) averaged 900 +/- 150 (mean +/- SEM) at 10 +/- 2 weeks of gestation, 915 +/- 35 at 17 +/- 2 weeks and 790 +/- 42 at 40 +/- 2 weeks, thus supporting the view that the placenta is an effective barrier to materno-fetal cortisol transfer throughout gestation.     

Expression of breast cancer resistance protein (BCRP/ABCG2) in human placenta throughout gestation and at term before and after labor

Breast cancer resistance protein, BCRP, is a multidrug resistance protein that is highly expressed in the human placenta. In cancer tissues, this protein actively extrudes a wide variety of chemically and structurally unrelated chemotherapeutic drugs and other compounds. Studies in mice have shown that in the absence of BCRP activity in the placenta, there is a 2-fold increase in the uptake in BCRP substrates into fetus. This suggests that in the placenta, BCRP extrudes compounds that would otherwise cross the syncytiotrophoblast cells into fetal circulation. The purpose of this study was to examine the expression and localization of BCRP in the human placenta throughout gestation. Tissues from 6-13, 16-19, 24-29, 32-35, and 38-41 weeks of gestation were used. Real time RT-PCR analysis demonstrated that the mRNA levels of BCRP in the placenta do not change significantly as gestation progressed. However, Western blot analysis revealed that the protein levels increased towards the end of gestation. We demonstrated that BCRP is localized to the syncytiotrophoblast of the placenta and in some fetal blood vessels within the placenta. Tissues from the early stages of pregnancy (6-13 weeks) showed fewer BCRP positive blood vessels than term tissues (38-41 weeks).     

Immunocytochemical localization of progesterone-binding protein (PBP) in guinea-pig placental tissue

Summary The cellular localization of progesterone-binding protein (PBP) in the guinea-pig placenta was studied by use of immunocytochemical procedures. Within the chorioallantoic placenta, a strong positive reaction was observed in the interlobar and marginal trophoblast from the third week of gestation to term. PBP was localized in the cytoplasm of the syncytiotrophoblast, and the nuclei were never stained. At the ultrastructural level, the immunoreaction was associated with the rough endoplasmic reticulum, the Golgi apparatus and the perinuclear space. No deposits were seen in any other cell organelles. This localization strongly suggests that the interlobar syncytium is related to PBP synthesis. In the labyrinth, a weak immunoreaction was observed by light microscopy around some blood lacunae. At the ultrastructural level the dense deposits were localized in vesicles located near the maternal lacunae.The distribution of PBP was also studied by light microscopy in other tissues from pregnant guinea-pig. No PBP, or PBP-like material, was detected inside cells from liver, muscle, heart, lung, kidney, ovary, and uterus. A weak immunoreaction for PBP was detected in vascularized zones of these organs.These observations strongly suggest that PBP, a protein related to gestation in the guinea-pig, is elaborated by the placental tissue of this hystricomorph rodent. PBP is the first steroid-binding plasma protein shown to be of extrahepatic origin.     

Modulation of apelin and APJ receptor in normal and preeclampsia-complicated placentas

Apelin is an endogenous ligand of the human orphan receptor APJ. This peptide is produced through processing from the C-terminal portion in the pre-pro-protein consisting of 77 amino acid residues and exists in multiple molecular forms. Although the main physiological functions of apelin have not yet been clarified, it is known that apelin is involved in the regulation of blood pressure, blood flow and central control of body fluid homeostasis in different organs. Since human placenta is a tissue where vasculogenesis, blood pressure and flow are dramatically important to allow a normal embryonic and fetal growth and development, the aim of the present study was to investigate the immunohistochemical distribution of apelin and APJ in normal placentas throughout pregnancy and in preeclampsia-complicated placentas. Specifically, we observed that in normal placentas the expression levels of apelin decreased from the first to the third trimester of gestation in both cytotrophoblast and syncytiotrophoblast cells and in the stroma of placental villi, in contrast with increased expression levels of APJ in the cytoplasm of cytotrophoblast cells and in the cytoplasm of endothelial cells of normal placenta samples. In contrast, in preeclampsia-complicated pregnancies, we observed a very strong increase of expression levels of both apelin and APJ receptor in all the placental compartments, cytotrophoblast, syncytiotrophoblast and stroma with a particular increase in endothelial cells inside preeclamptic placental villi. Our data seem to indicate an important role of apelin and APJ in the regulation of fetal development through a correct regulation of human placenta formation during pregnancy. Moreover, the strong expression levels of apelin and APJ in preeclamptic placentas, suggest their possible involvement in the onset of this pathology.     

Expression of 17beta-hydroxysteroid dehydrogenases in mesophilic and extremophilic yeast

17beta-hydroxysteroid dehydrogenases (17beta-HSDs) are enzymes responsible for reversible interconversions of biologically active 17-hydroxy and inactive 17-keto steroids. We have performed a survey of 17beta-HSD activity in yeast. Constitutive 17beta-HSD activity was found in three mesophilic yeast species: Candida tropicalis, Cryptococcus tsukubaensis, and Saccharomyces cerevisiae as well as in three extremophilic black yeast species: Hortaea werneckii, Trimmatostroma salinum, and Phaeotheca triangularis, indicating that 17beta-HSD activity is widely distributed among yeast. In extremophilic black yeast, NaCl modulated enzyme activity. Enzymes resembling 17beta-HSD from the filamentous fungus Cochliobolus lunatus were detected in Trimmatostroma salinum and Phaeotheca triangularis. Sequences with identity to the Saccharomyces cerevisiae YBR159w gene were not observed in other yeast species possessing a similar enzyme activity. The results suggest the existence of at least three different types of 17beta-HSD in yeast.     

Intracrinology and the skin

The skin, the largest organ in the human body, is composed of a series of androgen-sensitive components that all express the steroidogenic enzymes required to transform dehydroepiandrosterone (DHEA) into dihydrotestosterone (DHT). In fact, in post-menopausal women, all sex steroids made in the skin are from adrenal steroid precursors, especially DHEA. Secretion of this precursor steroid by the adrenals decreases progressively from the age of 30 years to less than 50% of its maximal value at the age of 60 years. DHEA applied topically or by the oral route stimulates sebaceous gland activity, the changes observed being completely blocked in the rat by a pure antiandrogen while a pure antiestrogen has no significant effect, thus indicating a predominant or almost exclusive androgenic effect. In human skin, the enzyme that transforms DHEA into androstenedione is type 1 3beta-hydroxysteroid dehydrogenase (type 1 3beta-HSD) as revealed by RNase protection and immunocytochemistry. The conversion of androstenedione into testosterone is then catalyzed in the human skin by type 5 17beta-HSD. All the epidermal cells and cells of the sebaceous glands are labelled by type 5 17beta-HSD. This enzyme is also present at a high level in the hair follicles. Type 1 is the 5alpha-reductase isoform responsible in human skin for the conversion of testosterone into DHT. In the vagina, on the other hand, DHEA exerts mainly an estrogenic effect, this effect having been demonstrated in the rat as well as in post-menopausal women. On the other hand, in experimental animals as well as in post-menopausal women, DHEA, at physiological doses, does not affect the endometrial epithelium, thus indicating the absence of DHEA-converting enzymes in this tissue, and avoiding the need for progestins when DHEA is used as hormone replacement therapy.     

Localization of erythrocyte/HepG2-type glucose transporter (GLUT1) in human placental villi

Summary The syncytiotrophoblast covering the surface of the placental villi contains the machinery for the transfer of specific substances between maternal and fetal blood, and also serves as a barrier. Existence of a facilitated-diffusion transporter for glucose in the syncytiotrophoblast has been suggested. Using antibodies to erythrocyte/HepG2-type glucose transporter (GLUT1), one isoform of the facilitated-diffusion glucose transporters, we detected a 50 kD protein in human placenta at term. By use of immunohistochemistry, GLUT1 was found to be abundant in both the syncytiotrophoblast and cytotrophoblast. Endothelial cells of the fetal capillaries also showed positive staining for GLUT1. Electron-microscopic examination revealed that GLUT1 was concentrated at both the microvillous apical plasma membrane and the infolded basal plasma membrane of the syncytiotrophoblast. Plasma membrane of the cytotrophoblast was also positive for GLUT1. GLUT1 at the apical plasma membrane of the syncytiotrophoblast may function for the entry of glucose into its cytoplasm, while GLUT1 at the basal plasma membrane may be essential for the exit of glucose from the cytoplasm into the stroma of the placental villi. Thus, GLUT1 at the plasma membranes of syncytiotrophoblast and endothelial cells may play an important role in the transport of glucose across the placental barrier.     

Pluripotency of 17beta-hydroxysteroid dehydrogenase from the filamentous fungus Cochliobolus lunatus

Cochliobolus lunatus 17beta-hydroxysteroid dehydrogenase (17beta-HSD) is pluripotent for several steroidal and nonsteroidal substrates. In the presence of NADPH the enzyme was found to reduce 3-keto groups of 4,5-dihydro steroids, 20-keto groups, and most efficiently, 17-keto groups of steroidal substrates. In addition, several quinones were accepted and found to be even better substrates as steroids due to their higher affinity for the enzyme-coenzyme complex and faster conversion of the enzyme-coenzyme-substrate complex into the corresponding products. As suggested by the competition studies quinones and 17-ketosteroids are converted by the same active center of the enzyme. For all tested substrates, the equilibrium ordered mechanism was established with NADPH binding first to the enzyme. According to our knowledge, the investigated 17beta-HSD is the first known fungal pluripotent enzyme of this type.     

Immunocytochemical localization of transforming growth factor α, epidermal growth factor and epidermal growth factor receptor in the cat endometrium and placenta

This study was undertaken to determine the immunocytochemical localization of transforming growth factor α, epidermal growth factor and epidermal growth factor receptor in the endometrium of ovariectomized cats treated with oestradiol-17β and/or progesterone and in the endometrium and placenta of pregnant cats. Specific immunostaining was observed for all three antibodies. Moderate immunostaining for transforming growth factor α was observed in the epithelium of ovariectomized and oestrogen-treated cats. Dark epithelial staining was observed throughout pregnancy. The epithelial cells in progesterone-treated and peri-implantation animals contained dense deposits of reaction product, which were not reduced in intensity when immunoabsorbed antiserum was used. For epidermal growth factor, light--moderate epithelial staining was observed in ovariectomized and steroid-treated animals, and this increased in pregnant cats. Stromal staining for both the transforming and the epidermal growth factors was limited in steroid-treated animals and increased as pregnancy continued. Dark staining for epidermal growth factor receptor was observed in the epithelium and stroma in all the animals studied. The tips of surface epithelial convolutions in the non-implantation sites were always more darkly stained than in other regions of the surface epithelium. Staining in the placental trophoblast was limited to the syncytiotrophoblast for the two growth factors and the cytotrophoblast for the receptor during most of pregnancy and was absent late in pregnancy. The placental maternal giant cells contained specific immunoreactivity for all the immunogens from the middle of pregnancy to term. This study demonstrates that the two growth factors and the epidermal growth factor receptor are present in the endometrium and placenta of cats and suggests that these growth factors may play an autocrine/paracrine role during reproduction     

Inhibitory effect of synthetic progestins, 4-MA and cyanoketone on human placental 3 beta-hydroxysteroid dehydrogenase/5----4-ene-isomerase activity

Human placental 3 beta-hydroxysteroid dehydrogenase/5----4-ene isomerase (3 beta-HSD) purified from human placenta transforms C-21 (pregnenolone and 17 alpha-hydroxy pregnenolone) as well as C-19 (dehydroepiandrosterone and androst-5-ene-3 beta, 17 beta-diol) steroids into the corresponding 3-keto-4-ene-steroids and is thus involved in the biosynthesis of all classes of hormonal steroids. Trilostane, epostane and cyanoketone are potent inhibitors of 3 beta-HSD with Ki values of approximately 50 nM. 4-MA, a well known 5 alpha-reductase inhibitor, is also a potent inhibitor of 3 beta-HSD with a Ki value of 56 nM. Synthetic progestin compounds such as promegestone and RU2323 show relatively strong inhibitory effects with Ki values of 110 and 190 nM, respectively. Cyproterone acetate, a progestin used in the treatment of hirsutism, acne and prostate cancer as well as norgestrel and norethindrone that are widely used as oral contraceptives also inhibit 3 beta-HSD activity at Ki values of 1.5, 1.7 and 2.5 microM, respectively.     

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.     

Structural and biochemical characterization of human orphan DHRS10 reveals a novel cytosolic enzyme with steroid dehydrogenase activity

To this day, a significant proportion of the human genome remains devoid of functional characterization. In this study, we present evidence that the previously functionally uncharacterized product of the human DHRS10 gene is endowed with 17beta-HSD (17beta-hydroxysteroid dehydrogenase) activity. 17beta-HSD enzymes are primarily involved in the metabolism of steroids at the C-17 position and also of other substrates such as fatty acids, prostaglandins and xenobiotics. In vitro, DHRS10 converts NAD+ into NADH in the presence of oestradiol, testosterone and 5-androstene-3beta,17beta-diol. Furthermore, the product of oestradiol oxidation, oestrone, was identified in intact cells transfected with a construct plasmid encoding the DHRS10 protein. In situ fluorescence hybridization studies have revealed the cytoplasmic localization of DHRS10. Along with tissue expression data, this suggests a role for DHRS10 in the local inactivation of steroids in the central nervous system and placenta. The crystal structure of the DHRS10 apoenzyme exhibits secondary structure of the SDR (short-chain dehydrogenase/reductase) family: a Rossmann-fold with variable loops surrounding the active site. It also reveals a broad and deep active site cleft into which NAD+ and oestradiol can be docked in a catalytically competent orientation.