17HSD Type Research Articles

Expression of 17 beta-hydroxysteroid dehydrogenase in human granulosa cells: correlation with follicular size, cytochrome P450 aromatase activity and oestradiol production.

The aim of this study was to examine the expression and regulation of type 1 17 beta-hydroxysteroid dehydrogenase (type 1 17-HSD) enzyme protein and mRNA, and 17-HSD activity in human granulosa cells. The cells were obtained from patients taking part in an in vitro fertilization programme. The cells from each patient were divided into two groups: cells obtained from preovulatory follicles (LGC = granulosa cells from large follicles > or = 18 mm in diameter), and cells from other visible follicles (SGC = granulosa cells from small follicles, less than 15 mm in diameter). The identity of 17-HSD enzyme protein expressed in human granulosa cells with placental cytosolic 17-HSD (type 1 17-HSD) was assessed by immunoblot analysis using polyclonal antibodies, and the enzyme was immunolocalized in the cytoplasm of granulosa cells. Type 1 17-HSD protein concentration, 17-HSD and cytochrome P450 aromatase (P450arom) activities and oestradiol (OE2) production in cells from LGC were significantly lower than the corresponding values obtained in SGC in the same patient (paired t-test). The type 1 17-HSD protein concentration, 17-HSD activity and P450arom activity were 140 +/- 16% (mean +/- S.E.M.), 121 +/- 22% and 113 +/- 26% higher in cells from SGC, which was also reflected in a 70 +/- 12% higher OE2 production in these cells. In freshly isolated cells from LGC or SGC, a high correlation between 17-HSD and P450arom activities was observed (r = 0.93, P < 0.001). In long-term cultured cells, type 1 17-HSD was stably expressed at least until day 9, while P450arom expression decreased. In addition, treatments with gonadotrophins did not affect type 1 17-HSD protein concentration and 17-HSD activity. In contrast to this, both P450arom activity and OE2 production were significantly increased (P < 0.05). The data, therefore, suggest that type 1 17-HSD and P450arom are expressed in parallel during the latest stages of follicular maturation but, in cultured granulosa-luteal cells, the enzymes are regulated by distinct mechanisms.

Characterization of 17β-hydroxysteroid dehydrogenase type 1 in choriocarcinoma cells: regulation by basic fibroblast growth factor

17β-Hydroxysteroid dehydrogenase type 1 (17-HSD type 1) is a steroidogenic enzyme catalyzing reversible interconversion of estradiol and estrone. 17-HSD type 1 is actively expressed in human placenta. We characterized 17-HSD type 1 expression and its regulation by basic fibroblast growth factor (bFGF) in JAR, JEG-3 and BeWo choriocarcinoma cell lines. Based on Southern and Northern analysis, as well as measurement of catalytic activity and immunoreactive protein, all the choriocarcinoma cell lines contained and expressed the gene coding for 17-HSD type 1, identical to that of normal human cells. However, the cell lines showed marked quantitative differences in the levels of expression of the enzyme, being lowest in JAR cells and highest in BeWo cells, as measured by immunofluorometric assay, Northern analysis and catalytic activity. These differences in the basal level of expression were most probably not based on any sequence differences in the putative proximal promoter area of the gene in different cell lines, since no dissimilarities were observed in the 806 bp region upstream from the transcription start site of 1.3 kb mRNA coding for 17-HSD type 1 except for frequent polymorphism characteristic of normal human cells using polymerase chain reaction/single-strand conformation polymorphism (PCR-SSCP) analysis. The reductive (estrone → estradiol) activity was about 4–7 times higher compared with the oxidative activity (estradiol → estrone) in all the cell lines studied, indicating that in these choriocarcinoma cell lines, 17-HSD activity favours estradiol formation. Treatment with 30 ng/ml of basic fibroblast growth factor (bFGF) for 5 days stimulated irnmunoreactive 17-HSD type 1 concentrations 4.5-fold and 3.5-fold above the control level in JAR and JEG-3 cells, respectively, whereas no stimulatory effects were seen in BeWo cells. Similarly, concentrations of 17-HSD type 1 1.3 kb mRNA and 17-HSD catalytic activity increased in JAR and JEG-3 cells after stimulation with bFGF, whereas no effects were seen in BeWo cells. We suggest that bFGF may have a regulatory role in placental estrogen metabolism via its effects on 17-HSD type 1. Keywords: Fibroblast growth factor; Placenta; Choriocarcinoma; Steroidogenesis; Estradiol

Structure and Expression of a New Complementary DNA Encoding the almost Exclusive 3β-Hydroxysteroid Dehydrogenase/Δ5-Δ4-lsomerase in Human Adrenals and Gonads

The 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta HSD) enzyme catalyzes the oxidation and isomerization of delta 5-3 beta-hydroxysteroid precursors into delta 4-ketosteroids, thus leading to the formation of all classes of steroid hormones. In addition, 3 beta HSD catalyzes the interconversion of 3 beta-hydroxy- and 3-keto-5 alpha-androstane steroids. Clinical observations in patients with 3 beta HSD deficiency as well as our recent data obtained by Southern blot analysis using a human placental 3 beta HSD cDNA (type I) as probe suggested the existence of multiple related 3 beta HSD isoenzymes. We now report the isolation and characterization of a second type of cDNA clone (arbitrarily designated type II) encoding 3 beta HSD after screening of a human adrenal lambda gt22A library. The nucleotide sequence of 1676 basepairs of human 3 beta HSD type II cDNA predicts a protein of 371 amino acids with a calculated molecular mass of 41,921 daltons, which displays 93.5% and 96.2% homology with human placental type I and rhesus macaque ovary 3 beta HSD deduced proteins, respectively. To characterize and compare the kinetic properties of the two isoenzymes, plasmids derived from pCMV and containing type I or type II 3 beta HSD full-length cDNA inserts were transiently expressed in HeLa human cervical carcinoma cells. In vitro incubation with NAD+ and 3H-labeled pregnenolone or dehydroepiandrosterone shows that the type I protein possesses a 3 beta HSD/delta 5-delta 4 isomerase activity higher than type II, with respective Km values of 0.24 vs. 1.2 microM for pregnenolone and 0.18 vs. 1.6 microM for dihydroepiandrosterone, while the specific activity of both types is equivalent. Moreover, incubation in the presence of NADH of homogenates from cells transfected with type I or type II 3 beta HSD indicates that dihydrotestosterone is converted into 5 alpha-androstane-3 beta, 17 beta-diol, with Km values of 0.26 and 2.7 microM, respectively. Ribonuclease protection assay using type I- and type II-specific cRNA probes revealed that type II transcripts are the almost exclusive 3 beta HSD mRNA species in the human adrenal gland, ovary, and testis, while type I transcripts correspond to the almost exclusive 3 beta HSD mRNA species in the placenta and skin and represent the predominantly expressed species in mammary gland tissue. The present data show for the first time that adrenals and gonads express a type of 3 beta HSD isoenzyme that is distinct from the type expressed in the placenta.(ABSTRACT TRUNCATED AT 400 WORDS)

Investigation of the wholesomeness of gamma irradiated fresh pork — Long-term animals feeding studies

17β-Hydroxysteroid dehydrogenase type 2 (17HSD type 2) catalyzes the inactivation of estradiol, testosterone and dihydrotestosterone into biologically less active 17-keto forms. Our recent Northern analysis indicated that the enzyme is expressed both in mouse placenta and fetus. The present data indicate that in the placenta the distribution of enzyme expression changes during pregnancy. In the choriovitelline placenta (day 8) 17HSD type 2 was expressed both in mural and polar giant cells. Later, on days 9–12.5, the mRNA was also detected in the junctional zone, and in late gestation (days 14.5–17.5), 17HSD type 2 mRNA was predominantly expressed only at the labyrinth region. In the fetus, 17HSD type 2 expression appears in the liver on day 11. At day 12 the expression was strongly increased in the liver, and at the same time moderate mRNA expression was also detected in the esophagus and intestine. In these tissues, high constitutive expression of 17HSD type 2 was then maintained throughout pregnancy. At later stages of development (days 15–16) the mRNA was, furthermore, detected in epithelial cells of the stomach, tongue, oropharynx and nasopharynx, as well as in the kidney. We conclude that the expression pattern of 17HSD type 2 in the developing placenta and fetus suggests a role for the enzyme in maintaining a barrier to the transfer of active 17-hydroxy forms of sex steroids between the fetus and maternal circulation.