Prenatal exposure to an environmentally relevant mixture of Canadian Arctic contaminants decreases male reproductive function in an aging rat model.

Elevated levels of organochlorines (OC) have been reported in Inuit populations in the Arctic. We hypothesized that prenatal exposure to a Canadian Arctic OC mixture adversely affects male reproductive function and health with age. Sprague-Dawley female rats (F0) were gavaged with an environmentally relevant concentration of an Arctic OC mixture or corn oil (Control) during mating with untreated males until parturition (F1 litters). After postnatal day (PND) 90, the weights of the OC F1 males differed dramatically relative to Controls (P<0.05; n=10) and they exhibited respiratory distress. Except for possible thinning of the alveolar barrier, histological observation of the lungs revealed no apparent pathology to explain the respiratory distress. At PND 365, OC F1 males had reduced relative reproductive organ weights and lower sperm quality than Controls (P<0.05). At PND 90, OC F1 males were subfertile (P<0.05), but were infertile at PND 365. In conclusion, environmentally relevant prenatal OC exposure reduced reproductive function and health in aging male rats, providing new insight into the effects of early-life exposures to these contaminants.

[Morphometry of pulmonary tissue: From manual to high throughput automation]. / Morphométrie du tissu pulmonaire : de la méthode manuelle à l'automatisation à haut débit.

Weibel's research has shown that any alteration of the pulmonary structure has effects on function. This demonstration required a quantitative analysis of lung structures called morphometry. This is possible thanks to stereology, a set of methods based on principles of geometry and statistics. His work has helped to better understand the morphological harmony of the lung, which is essential for its proper functioning. An imbalance leads to pathophysiology such as chronic obstructive pulmonary disease in adults and bronchopulmonary dysplasia in neonates. It is by studying this imbalance that new therapeutic approaches can be developed. These advances are achievable only through morphometric analytical methods, which are increasingly precise and focused, in particular thanks to the high-throughput automation of these methods. This review makes a comparison between an automated method that we developed in the laboratory and semi-manual methods of morphometric analyzes. The automation of morphometric measurements is a fundamental asset in the study of pulmonary pathophysiology because it is an assurance of robustness, reproducibility and speed. This tool will thus contribute significantly to the acceleration of the race for the development of new drugs.

Surface Polysaccharide Mutants Reveal that Absence of O Antigen Reduces Biofilm Formation of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is a Gram-negative bacterium belonging to the Pasteurellaceae family and the causative agent of porcine pleuropneumonia, a highly contagious lung disease causing important economic losses. Surface polysaccharides, including lipopolysaccharides (LPS) and capsular polysaccharides (CPS), are implicated in the adhesion and virulence of A. pleuropneumoniae, but their role in biofilm formation is still unclear. In this study, we investigated the requirement for these surface polysaccharides in biofilm formation by A. pleuropneumoniae serotype 1. Well-characterized mutants were used: an O-antigen LPS mutant, a truncated core LPS mutant with an intact O antigen, a capsule mutant, and a poly-N-acetylglucosamine (PGA) mutant. We compared the amount of biofilm produced by the parental strain and the isogenic mutants using static and dynamic systems. Compared to the findings for the biofilm of the parental or other strains, the biofilm of the O antigen and the PGA mutants was dramatically reduced, and it had less cell-associated PGA. Real-time PCR analyses revealed a significant reduction in the level of pgaA, cpxR, and cpxA mRNA in the biofilm cells of the O-antigen mutant compared to that in the biofilm cells of the parental strain. Specific binding between PGA and LPS was consistently detected by surface plasmon resonance, but the lack of O antigen did not abolish these interactions. In conclusion, the absence of the O antigen reduces the ability of A. pleuropneumoniae to form a biofilm, and this is associated with the reduced expression and production of PGA.

Sub-inhibitory concentrations of penicillin G induce biofilm formation by field isolates of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is a Gram-negative bacterium and causative agent of porcine pleuropneumonia. This is a highly contagious disease that causes important economic losses to the swine industry worldwide. Penicillins are extensively used in swine production and these antibiotics are associated with high systemic clearance and low oral bioavailability. This may expose A. pleuropneumoniae to sub-inhibitory concentrations of penicillin G when the antibiotic is administered orally. Our goal was to evaluate the effect of sub-minimum inhibitory concentration (MIC) of penicillin G on the biofilm formation of A. pleuropneumoniae. Biofilm production of 13 field isolates from serotypes 1, 5a, 7 and 15 was tested in the presence of sub-MIC of penicillin G using a polystyrene microtiter plate assay. Using microscopy techniques and enzymatic digestion, biofilm architecture and composition were also characterized after exposure to sub-MIC of penicillin G. Sub-MIC of penicillin G significantly induced biofilm formation of nine isolates. The penicillin G-induced biofilms contained more poly-N-acetyl-D-glucosamine (PGA), extracellular DNA and proteins when compared to control biofilms grown without penicillin G. Additionally, penicillin G-induced biofilms were sensitive to DNase which was not observed with the untreated controls. Furthermore, sub-MIC of penicillin G up-regulated the expression of pgaA, which encodes a protein involved in PGA synthesis, and the genes encoding the envelope-stress sensing two-component regulatory system CpxRA. In conclusion, sub-MICs of penicillin G significantly induce biofilm formation and this is likely the result of a cell envelope stress sensed by the CpxRA system resulting in an increased production of PGA and other matrix components.

Zinc as an agent for the prevention of biofilm formation by pathogenic bacteria.

AIMS: Biofilm formation is important for the persistence of bacteria in hostile environments. Bacteria in a biofilm are usually more resistant to antibiotics and disinfectants than planktonic bacteria. Our laboratory previously reported that low concentrations of zinc inhibit biofilm formation of Actinobacillus pleuropneumoniae. The aim of this study is to evaluate the effect of zinc on growth and biofilm formation of other bacterial swine pathogens. METHODS AND RESULTS: To determine the effect of zinc on biofilm formation, biofilms were grown with or without zinc in 96-well plates and stained with crystal violet. At micromolar concentrations (0-250 µ mol l(-1)), zinc weakly inhibited bacterial growth and it effectively blocked biofilm formation by A. pleuropneumoniae, Salmonella Typhymurium and Haemophilus parasuis in a dose-dependent manner. Additionally, biofilm formation of Escherichia coli, Staphylococcus aureus and Streptococcus suis was slightly inhibited by zinc. However, zinc did not disperse preformed biofilms. To determine whether zinc inhibits biofilm formation when poly-N-acetylglucosamine (PGA) is present, PGA was detected with the lectin wheat germ agglutinin. Only A. pleuropneumoniae and Staph. aureus biofilms were found to contain PGA. CONCLUSION: Zinc used at nonbactericidal concentrations can inhibit biofilm formation by several Gram-negative and Gram-positive bacterial swine pathogens. SIGNIFICANCE AND IMPACT OF STUDY: The antibiofilm activity of zinc could provide a tool to fight biofilms, and the nonspecific inhibitory effect may well extend to other important human and animal bacterial pathogens.

Southern Ocean frontal structure and sea-ice formation rates revealed by elephant seals.

Polar regions are particularly sensitive to climate change, with the potential for significant feedbacks between ocean circulation, sea ice, and the ocean carbon cycle. However, the difficulty in obtaining in situ data means that our ability to detect and interpret change is very limited, especially in the Southern Ocean, where the ocean beneath the sea ice remains almost entirely unobserved and the rate of sea-ice formation is poorly known. Here, we show that southern elephant seals (Mirounga leonina) equipped with oceanographic sensors can measure ocean structure and water mass changes in regions and seasons rarely observed with traditional oceanographic platforms. In particular, seals provided a 30-fold increase in hydrographic profiles from the sea-ice zone, allowing the major fronts to be mapped south of 60 degrees S and sea-ice formation rates to be inferred from changes in upper ocean salinity. Sea-ice production rates peaked in early winter (April-May) during the rapid northward expansion of the pack ice and declined by a factor of 2 to 3 between May and August, in agreement with a three-dimensional coupled ocean-sea-ice model. By measuring the high-latitude ocean during winter, elephant seals fill a "blind spot" in our sampling coverage, enabling the establishment of a truly global ocean-observing system.

Variations in behavior and condition of a Southern Ocean top predator in relation to in situ oceanographic conditions.

Responses by marine top predators to environmental variability have previously been almost impossible to observe directly. By using animal-mounted instruments simultaneously recording movements, diving behavior, and in situ oceanographic properties, we studied the behavioral and physiological responses of southern elephant seals to spatial environmental variability throughout their circumpolar range. Improved body condition of seals in the Atlantic sector was associated with Circumpolar Deep Water upwelling regions within the Antarctic Circumpolar Current, whereas High-Salinity Shelf Waters or temperature/salinity gradients under winter pack ice were important in the Indian and Pacific sectors. Energetic consequences of these variations could help explain recently observed population trends, showing the usefulness of this approach in examining the sensitivity of top predators to global and regional-scale climate variability.

Immunoelectron microscopic localization of three key steroidogenic enzymes (cytochrome P450(scc), 3 beta-hydroxysteroid dehydrogenase and cytochrome P450(c17)) in rat adrenal cortex and gonads.

The biosynthesis of steroid hormones in endocrine steroid-secreting glands results from a series of successive steps involving both cytochrome P450 enzymes, which are mixed-function oxidases, and steroid dehydrogenases. So far, the subcellular distribution of steroidogenic enzymes has been mostly studied following subcellular fractionation, performed in placenta and adrenal cortex. In order to determine in situ the intracellular distribution of some steroidogenic enzymes, we have investigated the ultrastructural localization of the three key enzymes: P450 side chain cleavage (scc) which converts cholesterol to pregnenolone; 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) which catalyzes the conversion of 3 beta-hydroxy-5-ene steroids to 3-oxo-4-ene steroids (progesterone and androstenedione); and P450(c17) which is responsible for the transformation of C(21) into C(19) steroids (dehydroepiandrosterone and androstenedione). Immunogold labeling was used to localize the enzymes in rat adrenal cortex and gonads. The tissues were fixed in 1% glutaraldehyde and 3% paraformaldehyde and included in LR gold resin. In the adrenal cortex, both P450(scc) and 3 beta-HSD immunoreactivities were detected in the reticular, fascicular and glomerular zones. P450(scc) was exclusively found in large mitochondria. In contrast, 3 beta-HSD antigenic sites were mostly observed in the endoplasmic reticulum (ER) with some gold particles overlying crista and outer membranes of the mitochondria. P450(c17) could not be detected in adrenocortical cells. In the testis, the three enzymes were only found in Leydig cells. Immunolabeling for P450(scc) and 3 beta-HSD was restricted to mitochondria, while P450(c17) immunoreactivity was exclusively observed in ER. In the ovary, P450(scc) and 3 beta-HSD immunoreactivities were found in granulosa, theca interna and corpus luteum cells. The subcellular localization of the two enzymes was very similar to that observed in adrenocortical cells. P450(c17) could also be detected in theca interna cells of large developing and mature follicles. As observed in Leydig cells, P450(c17) immunolabeling could only be found in the ER. These results indicate that in different endocrine steroid-secreting cells P450(scc), 3 beta-HSD and P450(c17) have the same association with cytoplasmic organelles (with the exception of 3 beta-HSD in Leydig cells), suggesting similar intracellular pathways for biosynthesis of steroid hormones.

Androgen formation and metabolism in the pulmonary epithelial cell line A549: expression of 17beta-hydroxysteroid dehydrogenase type 5 and 3alpha-hydroxysteroid dehydrogenase type 3.

Surfactant synthesis within developing fetal lung type II cells is affected by testosterone and 5alpha-dihydrotestosterone (5alpha-DHT). The pulmonary epithelial cell line A549, isolated from a human lung carcinoma, like normal lung type II cell, produces disaturated phosphatidylcholines and has been widely used for studying the regulation of surfactant production. Androgen receptor has been detected in A549 cells; however, the capacity of these cells for androgen synthesis and metabolism has not been investigated at molecular level. This study was undertaken to identify the steroidogenic enzymes involved in the formation and metabolism of androgens from adrenal C19 steroid precursors in A549 cells. When cultured in the presence of normal FCS, A549 intact cells converted DHEA to androstenediol, androstenedione principally to testosterone, and 5alpha-DHT to 5alpha-androstane 3alpha,17beta-diol. High levels of 17beta-hydroxysteroid dehydrogenase (HSD) and 3alpha-HSD activities were detected in both cytosol and microsomes isolated from homogenates. Analysis of A549 RNA indicated the presence of 17beta-HSD type 4 and type 5, and of 3alpha-HSD type 3 messenger RNAs. Very low levels of 3beta-HSD type 1 and 5alpha-reductase type 1 messenger RNAs and activities were detected. With regard to active androgen formation, there was little or no capacity for the conversion of DHEA to 5alpha-DHT. In contrast, androstenedione was rapidly transformed to testosterone. The pattern of steroid metabolism was not affected by the use of charcoal-stripped FCS or by the synthetic glucocorticoid dexamethasone. Together, our findings show that A549 cells express a pattern of steroid metabolism in which 17beta-HSD type 5 and 3alpha-HSD type 3 are the predominant enzymes. The level of androgens is regulated at the level of catalysis in intact cells such that the intracellular level of testosterone is stabilized, whereas 5alpha-DHT is rapidly inactivated by reduction to 3alpha,17beta-diol. This pattern of androgen metabolism has implications for the relative importance of testosterone and 5alpha-DHT in normal lung development and surfactant production.

Length increase of the human alpha -globin 3'-untranslated region disrupts stability of the pre-mRNA but not that of the mature mRNA.

Polyadenylation increases the stability of mRNA molecules. By studying the effect of the length of 3'-untranslated region (UTR) on mRNA levels, we have found that alpha-globin pre-mRNA is stabilized by a mechanism that does not modulate the half-life of mature mRNA. The insertion of DNA fragments of various unrelated sequences into the 3'-UTR of the human alpha-globin gene strongly reduces mRNA abundance upon transfection into choriocarcinoma JEG-3 cells. We found an inverse relationship between mRNA levels and the length of the introduced fragments. In fact, mRNA levels as low as 1% were observed after inserting a 477-nucleotide (nt) fragment, whereas inserting a fragment of 86 nt at the same position had no effect on mRNA accumulation. DNA insertion induced no change in transcription rate or in half-life of mature mRNA. Semi-quantitative reverse transcription-polymerase chain reaction revealed that inserting a 477-nt fragment in the 3'-UTR resulted in decreased levels of nuclear pre-mRNA in proportion to that observed for mature mRNA. In contrast, the insertion of the 477-nt exogenous DNA in the last intron had no effect on mRNA levels despite the presence of intronic sequences in the pre-mRNA. This shows that the reduction of pre-mRNA level was not due to the insertion of putative ribonuclease cleavage sites or the insertion of a segment DNA that reduces the elongation efficiency. Taken together, our results strongly support the existence of a pre-mRNA stabilizing mechanism that can be disrupted by increasing the length of the 3'-UTR. The fact that the half-life of mature mRNA is not affected by DNA insertion is compatible with a pre-mRNA-specific stabilizing mechanism that acts specifically before polyadenylation.

Localization of type 1 17beta-hydroxysteroid dehydrogenase mRNA and protein in syncytiotrophoblasts and invasive cytotrophoblasts in the human term villi.

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) play a key role in the synthesis of sex steroids. The hallmark of this family of enzymes is the interconversion, through their oxydoreductive reactivity at position C17, of 17-keto- and 17beta-hydroxy-steroids. Because this reaction essentially transforms steroids having low binding activity for the steroid receptor to their more potent 17beta-hydroxysteroids isoforms, it is crucial to the control of the physiological activities of both estrogens and androgens. The human placenta produces large amounts of progesterone and estrogens throughout pregnancy. The placental type 1 17beta-HSD enzyme (E17beta-HSD) catalyzes the reduction of the low activity estrogen, estrone, into the potent estrogen, estradiol. We studied the cell-specific expression of type 1 17beta-HSD in human term placental villous tissue by combining in situ hybridization to localize type 1 17beta-HSD mRNA with immunohistochemistry using an antibody against human placental lactogen, a trophoblast marker. Immunolocalization of E17beta-HSD was also performed. To ascertain whether other steroidogenic enzymes are present in the same cell type, cytochrome P450 cholesterol side-chain cleavage (P450scc), P450 aromatase, and type 1 3beta-hydroxysteroid dehydrogenase (3beta-HSD) were also localized by immunostaining. Our results showed that the syncytium is the major steroidogenic unit of the fetal term villi. In fact, type 1 17beta-HSD mRNA and protein, as well as P450scc, P450 aromatase, and 3beta-HSD immunoreactivities were found in these cells. In addition, our results revealed undoubtedly that extravillous cytotrophoblasts (CTBs), e.g. those from which cell columns of anchoring villous originate, also express the type 1 17beta-HSD gene. However, CTBs lying beneath the syncytial layer, e.g. those from which syncytiotrophoblasts develop, contained barely detectable amounts of type 1 17beta-HSD mRNA as determined by in situ hybridization. These findings, along with those from other laboratories confirm the primordial role of the syncytium in the synthesis of steroids during pregnancy. In addition, our results indicate for the first time that CTBs differentiating along the invasive pathway contain type 1 17beta-HSD mRNA.

Tissue- and site-specific gene expression of type 2 17beta-hydroxysteroid dehydrogenase: in situ hybridization and specificenzymatic activity studies in human placental endothelial cells of the arterial system.

Progesterone and estradiol are the most potent human sex steroid hormones of placental origin and are essential to the maintenance of pregnancy, the timing of parturition, the maturation of many fetal organs, and the preparation of the maternal reproductive system. Naturally, regulatory mechanisms must be in place to coordinate the synthesis and inactivation of these two hormones. We have previously shown that the highest levels of type 1 and type 2 17beta-hydroxysteroid dehydrogenase (17betaHSD) messenger ribonucleic acids (mRNAs) occur in the placenta, particularly in the villi. However, in contrast to type 1 17betaHSD mRNA, type 2 17betaHSD mRNA was not detectable in cell cultures of human cytotrophoblasts or syncytiotrophoblasts. Using in situ hybridization, we unequivocally identified endothelial cells as the only cell type expressing the type 2 17betaHSD gene in fetal villi. Moreover, type 2 17betaHSD mRNA was specifically detected in the endothelial cells of the arterial system, and at higher levels in the villi compared with endothelial cells of the cord arteries when the two tissue sections were cohybridized. In fact, both mRNA levels and enzymatic activity are at their highest levels in arterial endothelial cells. In conclusion, the endothelial cells of the villous arterioles are the primary site of type 2 17betaHSD gene expression. This suggests a regulatory role for these cells in the control of progestin, androgen, and estrogen levels during pregnancy, thus opening a whole new way of viewing regionalization and localization of steroidogenesis in the human villi.

Separation by thin-layer chromatography of the most common androgen-derived C19 steroids formed by mammalian cells.

Several methods have been developed in the past for the separation and identification of closely related steroid hormones. Although these methods are effective, most of them use HPLC-derived systems and are expensive, laborious, or time-consuming. In the course of our studies of the metabolism of dehydroepiandrosterone and androstenedione in tissues, we have modified a previously published technique in such a way that in one TLC step we can separate most of the androgen C19 steroid derivatives produced by mammalian cells. We have used this modified technique for the past 2 years with considerable success and reproducible results, and we find it to be rapid and relatively inexpensive.

Modulation of 17alpha-hydroxylase/17,20-lyase activity of guinea pig cytochrome P450c17 by site-directed mutagenesis.

Microsomal cytochrome P450c17 (17a-hydroxylase/17,20-Lyase) catalyzes two reactions in the delta5 and delta4 pathways leading to the production of C19 steroids. Transient expression of human, bovine, porcine, rat, and mouse P450c17 cDNAs showed that the protein has 17alpha-hydroxylase and 17,20-Lyase activities, converting pregnenolone and progesterone into delta5- and delta4-Cl9 steroids, respectively, although the rat and mouse proteins have a preferential pathway toward the delta4 steroids. The guinea pig (gp) P450c17 shares 46% to 70% amino acid identity with the corresponding proteins of other species, and further characterization indicated that the guinea pig enzyme only converts progesterone to androstenedione. In this study, we have tried to identify amino acid(s) in the gpP450c17 that governs such a steroid specificity. Among the various mutants that we have created, change of the arginine (R) residue at position 200 to an asparagine (N) (R200N) in the gpP450c17 protein increased reactivity toward pregnenolone compared with the wild-type enzyme. Pregnenolone was converted into 17alpha-hydroxypregnenolone and dehydroepiandrosterone. However, this gain occurred at the expense of the 17,20-lyase activity toward 17alpha-hydroxyprogesterone. The R200N mutation in the gpP450c17 protein introduced a potential N-linked glycosylation site (200Asn-X-Thr202); however, substitution of the Thr202 residue by an asparagine (R200N/T202N), which abolishes the site, did not change the preference of the gpP450c17 mutant for pregnenolone. Furthermore, introduction of a putative glycosylation site at amino acid 185 in the gpP450c17 enzyme did not alter substrate specificity. The properties of the amino acid were also investigated, and neither the charge nor the size of the sidechain elicited change in the substrate specificity of gpP450c17. Thus, our results demonstrate that the mutation of arginine to asparagine at position 200 changes the substrate specificity of the gpP450c17 enzyme.

Regulation of cytochrome P450 cholesterol side-chain cleavage, 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase type 1 and estradiol-17 beta-hydroxysteroid dehydrogenase mRNA levels by calcium in human choriocarcinoma JEG-3 cells.

In human placenta the cytochrome P450 side-chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase type 1 (3 beta-HSD-1) convert cholesterol and pregnenolone producing progesterone, whereas 17 beta-hydroxysteroid dehydrogenase type 1 (17 beta-HSD-1) mediates the interconversion of estrone and estradiol. We have examined the effects of calcium on phorbol ester- and cAMP-induced P450scc, 3 beta-HSD-1 and 17 beta-HSD-1 mRNAs in human JEG-3 cells. A23187 increased in a dose-dependent fashion in the 1.3 kb 17 beta-HSD-1 mRNA whereas a weaker increase followed by a gradual depletion effect of A23187 was observed on 3 beta-HSD-1 mRNA. No significant effect of A23187 on P450scc mRNA was observed. Using 0.50 microM of A23187 the induction of 3 beta-HSD-1 and 17 beta-HSD-1 mRNAs was maximum within about 6 h whereas P450scc mRNA levels stayed unaffected throughout the time-course period. The action of A23187 was synergistic on cAMP-stimulated 17 beta-HSD-1 mRNA levels, while in a dose-dependent manner A23187 progressively depleted 3 beta-HSD-1 and P450scc mRNA abundance probably by activation of a calcium-/calmodulin-dependent phosphodiesterase. On the phorbol 12-myristate, 13-acetate (PMA)-stimulated 3 beta-HSD-1, 17beta-HSD-1 and P450scc mRNA levels only the lowest concentration of A23187 potentialized the PMA effect on the 17 beta-HSD-1 mRNA levels. Using thapsigargin (TG), a cell-permeable sesquiterpene lactone that releases calcium by inhibiting sarco/endoplasmic reticular calcium-ATPase, our data indicated the presence in JEG-3 cells of TG-sensitive and TG-insensitive calcium-ATPases regulating 3 beta-HSD-1 and 17 beta-HSD-1 mRNA levels. These results emphasized the complexity of calcium contribution with the protein kinase A and C pathways in the regulation of P450scc, 3 beta-HSD-1 and 17 beta-HSD-1 mRNA levels. In addition, the different sensitivity of these genes to calcium suggest they could be activated by different subclasses of PKCs.

Expression of the genes for 3 beta-hydroxysteroid dehydrogenase type 1 and cytochrome P450scc during syncytium formation by human placental cytotrophoblast cells in culture and the regulation by progesterone and estradiol.

We have investigated the expression of cholesterol side-chain cleavage cytochrome P450 (P450scc) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) type 1 genes during human trophoblast differentiation in culture and the modulation of their steady-state mRNA levels by steroids. During the first 24 to 48 h after plating, mononucleated cells aggregated, forming colonies. After 60 h in culture, cell diameters were increased and nuclei appeared centrally distributed within large cells, consistent with syncytiotrophoblast formation. During these striking morphological changes in culture the expression and activity levels of 3 beta-HSD type 1 and P450scc increased significantly as isolated cytotrophoblasts progressed to a differentiated state, with P450scc and 3 beta-HSD type 1 mRNAs activities being more abundant in cells cultured for 48 to 72 h. In the same culture, however, the amount of 3 beta-HSD protein decreased during the first 12 to 24 h by 50% compared with freshly isolated trophoblasts but remained at these levels throughout the culture period. The specific activity of the 3 beta-HSD as determined with pregnenolone or dehydroepiandrosterone was similar but increased with time as syncytiotrophoblast was formed in vitro. These observations provide additional evidence that the expression of these two progesterone-synthesizing enzymes is coincident and that they reach their maximum steady-state mRNA levels at a time when syncytium formation occurs in vitro. Incubation of trophoblast cells with progesterone or estradiol increased the abundance of P450scc and 3 beta-HSD type 1 mRNAs but had no significant effect on the amount of 3 beta-HSD protein. These observations of the regulation of 3 beta-HSD type 1 mRNA levels by steroids suggest a complex relationship of the mechanisms regulating transcription/mRNA processing and transduction of the 3 beta-HSD type 1 gene.

Intracellular regulation of 17 beta-hydroxysteroid dehydrogenase type 2 catalytic activity in A431 cells.

There is growing evidence that various isoforms of 17 beta-hydroxysteroid dehydrogenase (17-HSD) are regulated at the level of catalysis in intact cells. A number of investigators have proposed that the NAD(P)/NAD(P)H ratio may control the direction of reaction. In a previous study, we obtained evidence that A431 cells, derived from an epidermoid carcinoma of the vulva, are enriched in 17-HSD type 2, a membrane-bound isoform reactive with C18 and C19 17 beta-hydroxysteroids and 17-ketosteroids. The present investigation was undertaken to confirm the presence of 17-HSD type 2 in A431 cells and to assess intracellular regulation of 17-HSD at the level of catalysis by comparing the activity of homogenates and microsomes with that of cell monolayers. Northern blot analysis confirmed the presence of 17-HSD type 2 mRNA. Exposure of cells to epidermal growth factor resulted in an increase in type 2 mRNA and, for microsomes, increases in maximum velocity (Vmax) with no change in Michaelis constant (Km) for testosterone and androstenedione, resulting in equivalent increases in the Vmax/Km ratio consistent with the presence of a single enzyme. Initial velocity data and inhibition patterns were consistent with a highly ordered reaction sequence in vitro in which testosterone and androstenedione bind only to either an enzyme-NAD or an enzyme-NADH complex respectively. Microsomal dehydrogenase activity with testosterone was 2- to 3-fold higher than reductase activity with androstenedione. In contrast, although cell monolayers rapidly converted testosterone to androstenedione, reductase activity with androstenedione or dehydroepiandrosterone (DHEA) was barely detectable. lactate but not glucose, pyruvate or isocitrate stimulated the conversion of androstenedione to testosterone by monolayers, suggesting that cytoplasmic NADH may be the cofactor for 17-HSD type 2 reductase activity with androstenedione. However, exposure to lactate did not result in a significant change in the NAD/NADH ratio of cell monolayers. It appears that within A431 cells 17-HSD type 2 is regulated at the level of catalysis to function almost exclusively as a dehydrogenase. These findings give further support to the concept that 17-HSD type 2 functions in vivo principally as a dehydrogenase and that its role as a reductase in testosterone formation by either the delta 4 or delta 5 pathway is limited.

Isolation and characterization of a stereospecific 3beta-hydroxysteriod sulfotransferase (pregnenolone sulfotransferase) cDNA.

In contrast to humans, who possess a hydroxysteroid sulfotransferase (HSST), namely, DHEA sulfotransferase (DHEA-ST), that displays broad substrate specificities, HSSTs of the guinea pig show a high substrate stereoselectivity, as shown by the recent cloning of a chiral-specific 3alpha-hydroxysteroid sulfotransferase. Herein, we report the cloning and expression of the substrate and chiral-specific pregnenolone sulfotransferase (PREG-ST). Transfection of the pCMV expression vector containing PREG-ST cDNA in transformed human embryonal kidney (293) cells showed that the expressed enzyme selectively catalyzes the 3beta-hydroxysteroid substrate. It converts pregnenolone to pregnenolone sulfate most efficiently, whereas dehydroepiandrosterone and epiandrosterone were transformed at a much lower rate, and androsterone, a 3alpha-hydroxysteroid, was not significantly metabolized (30-fold lower). Thus, the enzyme was identified as pregnenolone sulfotransferase. DNA analysis predicts a protein of 287 amino acids with a calculated molecular mass of 34,199 daltons. Alignment of the amino acid sequence with other sulfotransferases indicated that guinea pig pregnenolone sulfotransferase shares 75 and 80% homology with human DHEA sulfotransferase and rat hydroxysteroid dehydrogenase, respectively. RNA blot analysis using guinea pig liver, intestine, adrenal, kidney, epididymis, testis, and lung showed a single RNA species at 1.3 kb is expressed in liver, intestine, and kidney. Guinea pig 3beta-hydroxysteroid sulfotransferase is thus different from that in humans, who possess two mRNA species of 1.3 and 1.8 kb.