Biosynthesis Of Estriol Research Articles

Role of OAT4 in Uptake of Estriol Precursor 16α-Hydroxydehydroepiandrosterone Sulfate Into Human Placental Syncytiotrophoblasts From Fetus.

Estriol biosynthesis in human placenta requires the uptake of a fetal liver-derived estriol precursor, 16α-hydroxydehydroepiandrosterone sulfate (16α-OH DHEAS), by placental syncytiotrophoblasts at their basal plasma membrane (BM), which faces the fetal circulation. The aim of this work is to identify the transporter(s) mediating 16α-OH DHEAS uptake at the fetal side of syncytiotrophoblasts by using human placental BM-enriched vesicles and to examine the contribution of the putative transporter to estriol synthesis at the cellular level, using choriocarcinoma JEG-3 cells. Organic anion transporter (OAT)-4 and organic anion transporting polypeptide 2B1 proteins were enriched in human placental BM vesicles compared with crude membrane fraction. Uptake of [(3)H]16α-OH DHEAS by BM vesicles was partially inhibited in the absence of sodium but was significantly increased in the absence of chloride and after preloading glutarate. Uptake of [(3)H]16α-OH DHEAS by BM vesicles was significantly inhibited by OAT4 substrates such as dehydroepiandrosterone sulfate, estrone-3-sulfate, and bromosulfophthalein but not by cyclosporin A, tetraethylammonium, p-aminohippuric acid, or cimetidine. These characteristics of vesicular [(3)H]16α-OH DHEAS uptake are in good agreement with those of human OAT4-transfected COS-7 cells as well as forskolin-differentiated JEG-3 cells. Estriol secretion from differentiated JEG-3 cells was detected when the cells were incubated with 16α-OH DHEAS for 8 hours but was inhibited in the presence of 50 μM bromosulfophthalein. Our results indicate that OAT4 at the BM of human placental syncytiotrophoblasts plays a predominant role in the uptake of 16α-OH DHEAS for placental estriol synthesis.

Transport of the placental estriol precursor 16α-hydroxy-dehydroepiandrosterone sulfate (16α-OH-DHEAS) by stably transfected OAT4-, SOAT-, and NTCP-HEK293 cells

16α-Hydroxy-dehydroepiandrosterone sulfate (16α-OH-DHEAS) mainly originates from the fetus and serves as precursor for placental estriol biosynthesis. For conversion of 16α-OH-DHEAS to estriol several intracellular enzymes are required. However, prior to enzymatic conversion, 16α-OH-DHEAS must enter the cells by carrier mediated transport. To identify these carriers, uptake of 16α-OH-DHEAS by the candidate carriers organic anion transporter OAT4, sodium-dependent organic anion transporter SOAT, Na+-taurocholate cotransporting polypeptide NTCP, and organic anion transporting polypeptide OATP2B1 was measured in stably transfected HEK293 cells by LC–MS–MS. Furthermore, the study aimed to localize SOAT in the human placenta. Stably transfected OAT4-HEK293 cells revealed a partly sodium-dependent transport for 16α-OH-DHEAS with an apparent Km of 23.1±5.1μM and Vmax of 485.0±39.1pmol/mgprotein/min, while stably transfected SOAT- and NTCP-HEK293 cells showed uptake only under sodium conditions with Km of 319.0±59.5μM and Vmax of 1465.8±118.8pmol/mgprotein/min for SOAT and Km of 51.4±9.9μM and Vmax of 1423.3±109.6pmol/mgprotein/min for NTCP. In contrast, stably transfected OATP2B1-HEK293 cells did not transport 16α-OH-DHEAS at all. Immunohistochemical studies and in situ hybridization of formalin fixed and paraffin embedded sections of human late term placenta showed expression of SOAT in syncytiotrophoblasts, predominantly at the apical membrane as well as in the vessel endothelium.In conclusion, OAT4, SOAT, and NTCP were identified as carriers for the estriol precursor 16α-OH-DHEAS. At least SOAT and OAT4 seem to play a functional role for the placental estriol synthesis as both are expressed in the syncytiotrophoblast of human placenta.

Expression and cyclic variability of CYP3A4 and CYP3A7 isoforms in human endometrium and cervix during the menstrual cycle.

CYP3A4, a cytochrome p450 (p450) isoform metabolizes estrogens, whereas CYP3A7, a fetal liver p450 isoform, is involved in estriol biosynthesis. The goal of this study was to evaluate expression of these enzymes in human uterine tissue during the proliferative and secretory phases of the menstrual cycle. Endometrium and cervix specimens were collected from women undergoing hysterectomy (n = 36). Total mRNA was extracted, quantified, and reverse-transcription polymerase chain reaction (RT-PCR) was carried out using consensus primers for CYP3A. The 453 base pairs PCR product was hybridized with specific internal oligonucleotide probes for CYP3A4 or CYP3A7 end labeled with (32)P gamma-ATP. The relative intensity of hybridization was determined by autoradiography. Expression of CYP3A7 in endometrium was significantly greater (approximately 10-fold) in the proliferative phase compared with the secretory phase (p < 0.05). CYP3A4 expression was comparable between the two phases. Expression of both enzymes was minimal in the cervix. Fluorescence in situ hybridization of paraffinized sections indicated localized expression of CYP3A enzymes in the glandular epithelium as well as the stroma. Comparison of relative fluorescence intensity indicated differential expression of CYP3A7 in various phases of the menstrual cycle. These results suggest that CYP3A expression in the endometrium of premenopausal women may vary depending on the menstrual cycle phase.

Human fetal liver estrogen 16 alpha-hydroxylase: precursor specificity, kinetic parameters, and in vitro regulation.

The properties of human fetal liver (HFL) estrogen 16 alpha-hydroxylase (16 alpha-OHase) were studied in microsomal preparations and hepatocytes maintained in culture. A specific assay was developed for the determination of estrogen 16 alpha-OHase activity based on the enzymatic release of tritium from the C-16 alpha position of stereospecifically labeled 16 alpha-3H-labeled C18-steroids, viz. 17 beta-[16 alpha-3H]estradiol, 17 beta-[16 alpha-3H]estradiol 3-sulfate, [16 alpha-3H]estrone, and [16 alpha-3H]estrone sulfate. The percentage of tritium at the C-16 alpha position of 17 beta-[16 alpha-3H]estradiol was 92.5%. There was no kinetic isotope effect in the 16 alpha-hydroxylation of 17 beta-[16 alpha-3H]estradiol. HFL hepatocyte and microsomal 16 alpha-OHase activity was linear with incubation time for at least 2 h, with a hepatocyte number up to 6.7 X 10(6) cells/ml and a microsomal protein concentration up to 1 mg/ml. The apparent Km of 16 alpha-OHase for estrone sulfate (E1S) was greater than that for either 17 beta-estradiol (E2) or estrone (E1; 2.9-6.4 microM vs. 0.70-0.84 microM). The maximum velocity of HFL 16 alpha-OHase also was greater with E1S or 17 beta-estradiol 3-sulfate than with either E1 or E2, and E1S was the most efficient substrate. The apparent temperature optimum for the microsomal enzyme was 37 C, and the apparent pH optimum was 7.0. 16 alpha-Hydroxylation of E1S by HFL microsomes was inhibited noncompetitively by E1. The biosynthesis of estriol from E2 by fetal liver microsomes does not require an intermediate oxidation step of E2 to E1 as appears to be the case in vivo in the human adult; this was demonstrated by the formation of [17 alpha-3H]estriol from [17 alpha-3H] E2 in incubations with HFL microsomes. A number of growth factors, hormones, and xenobiotics were preincubated with hepatocytes for 24 or 72 h to test for stimulation/inhibition of 16 alpha-OHase activity. 16 alpha-OHase activity was stimulated by dexamethasone, forskolin, (Bu)2cAMP, cholera toxin, 1,2-benzanthracene, and phenobarbital. Diethylstilbestrol, E2, and progesterone did not alter hepatocyte 16 alpha-OHase activity, except when very high concentrations of E2 and progesterone were used, when they became inhibitory; other hormones and growth factors did not alter the basal levels of the enzyme.

In vitro production of estriol from four steroid precursors by rabbit tissues.

The production of estriol was demonstrated in intact leukocytes and homogenates of liver, adrenals and ovaries of young adult female rabbits from 17 beta-estriol, testosterone, androst-4-ene-3,17-dione and dehydroepiandrosterone. Androst-4-ene-3,17-dione constituted the best substrate in liver and adrenals and the poorest substrate for estriol production in lymphocytes. Estriol was least readily transformed into estriol in liver and adrenals, while it proved to be the best substrate in ovaries. Phenobarbital administration to rabbits resulted in 49-fold and 23-fold increases in the transformation of androst-4-ene-3,17-dione and dehydroepiandrosterone, respectively, into estriol by lymphocytes. The results indicate that the enzymes involved in estriol biosynthesis in the various tissues respond differently to phenobarbital.

Synthesis of 16α- 3H androgen and estrogen substrates for 16α-hydroxylase

The synthesis of 16α- 3H androgens and estrogens is described. 1-( 3H)-Acetic acid in the presence of zinc dust reacts with 16α-bromo-17-ketosteroids to produce 16α- 3H-17-ketosteroids. This chemical reaction was used to prepare 16α- 3H-dehydroepiandrosterone (I) and 16α- 3H-estrone acetate (XI) from 16α-bromo-dehydroepiandrosterone (X) and from 16α-bromo-estrone acetate (XII), respectively. Using appropriate microbiological techniques, it was possible to convert these radiolabelled substrates into 16α- 3H-androstenedione (II) and 16α- 3H-estradiol-17β (VII). 16α- 3H-Estrone (VI) was obtained by the chemical hydrolysis of 16α- 3H-estrone acetate. The label distribution as determined by microbiological 16α-hydroxylations indicated a specific labelling of 77% for androgens and 65% for estrogens in the 16α position. These substrates can be used for measuring the 16α hydroxylase activity, an important step in the biosynthesis of estriol (VIII) and estetrol (IX).

Increased urinary estriol excretion without change in blood estriol concentration resulting from bed rest in the third trimester of pregnancy in the human.

Placing normally pregnant women at bed rest during the third trimester significantly increased the amount of total estrogen excreted into the urine to 122% of the control value, but the concentration of total serum estriol was not significantly altered. Estriol biosynthesis, secretion, conjugation, metabolism, and excretion are regulated by many poorly understood factors, but for purposes of clinical evaluation of the well-being of the infant, bed rest would appear to be another circumstance under which more reliable information can be obtained by blood instead of urine analysis.

ESTROGEN METABOLISM IN THE POLYCYSTIC OVARY SYNDROME; EFFECT OF OVARIAN WEDGE RESECTION AND CLOMIPHENE

Abstract Estrone and estradiol-17β secretion and interconversion were normal in patients with the polycystic ovary syndrome. Radioactive evidence is presented for abnormal estriol biosynthesis in this disorder. The influence of bilateral ovarian wedge resection and clomiphene on estrogen excretion, production, and metabolism is described. The two forms of therapy were found to have diverse effects on these parameters.

臍帯血中17-KS分画に関する研究

In recent years, new theory concerned upon the estriol biosynthesis in the pregnant was developed remarkably. Diczfalusy and others demonstrated that DHA from foetal adrenal was the main precurssor of estriol in the pregnant. Their approaches for this problem were tried mainly with in vitro incubation and circulation studies. In this study, the approach was tried with the determination of steroids in cord blood, maternalblood, maternal urine and urine of newborn. Androstanedion, Δ4-androstenedione, DHA, androsterone and etiocholanolone in cord blood were deter minated by new method, which was cosisted of following procedure.1) 3s Hydrolysis (free steroid extraction, hydrolysis with β-glucuronidase in phosphatete buffer and transesterification with methyl-green and acetic acid.2) Florisil column chromatography.3) Thin layer chromatography.4) Colorimetry following Lieberman, colour development with modified zimmerman reaction.The results of this determination were as following.In two cases, five androgens in cord blood and maternal peripheral blood, eight fractions of estrogens and nine fractions of 17-Ketosteroids in maternal urine, were determined. Also nine fractions of 17-Ketosteroids and four fractions of estrogens were determined in pooled urine of newborn.The results of these determination were as followings.1) DHA level in cord blood was higher than that in maternal blood. But DHA level in maternal blood was cosiderably high. DHA in cord blood: 88.4μg/100ml, DHA in maternal blood: 37.3μg/100 ml It should be indicated that, thinking of maternal blood volume, maternal DHA is important for estriol biosynthesis.2) Although DHA level in maternal urine was high, but DHA level in urine of newborn was low. Urinary DHA in mother: 1111.3μg/day, Urinary DHA in newborn: 10.050.0μg/day By this results, it is difficult to think that all estriol should be the product of foetal DHA.3) Urinary estriol in early post-natal days was relatively high and then decreased to 10 and less μg in seven days of newborn. This indicates that the stored estriol was excreted in post-natal days. By these results, it should be clear that estriol biosynthesis is done not only in foetoplacentar unit, but also in maternalplacentar unit. It is claimed that steroids metabolism in pregnancy, as was seen in estriol biosynthesis, must be examined not as foeto-placentar unit but as foeto-placentar-maternal relation.

Estrogen metabolism in the polycystic ovary syndrome: Effect of ovarian wedge resection and clomiphene

Estrone and estradiol-17β secretion and interconversion were normal in patients with the polycystic ovary syndrome. Radioactive evidence is presented for abnormal estriol biosynthesis in this disorder. The influence of bilateral ovarian wedge resection and clomiphene on estrogen excretion, production, and metabolism is described. The two forms of therapy were found to have diverse effects on these parameters.

ESTRIOL BIOSYNTHESIS BY SOW OVARY.

ESTRIOL BIOSYNTHESIS BY SOW OVARY.

Pettenkofer chromogens in the maternal and fetal circulations: Detection of 3β, 16α-dihydroxy-androst-5-en-17-one in umbilical cord blood

At least part of the precursors for the biosynthesis of estriol may reach the placenta from the maternal or the fetal circulations. This possibility led us to estimate 3β-hydroxy-Δ 5-steroids in blood from the umbilical artery and vein and in maternal peripheral blood. Solvolyzed plasma extracts from 12 separate collections were chromatographed on alumina and the fractions examined by means of the modified Pettenkofer reaction. The most abundant of these compounds in maternal plasma was dehydroepiandrosterone. Its concentration in the fetal circulation, although more than double the maternal concentration, was smaller than that of another Pettenkofer chromogen, 16α-hydroxy-dehydroepiandrosterone (3β, 16α-dihydroxyandrost-5-en-17-one), heretofore unrecognized in fetal blood. The differences between the plasmatic concentrations in the umbilical artery and vein suggest a fetal contribution of 16α-hydroxydehydroepiandrosterone to the placenta, presumably to play a role as precursor for the biosynthesis of estriol.