Ronald Hobkirk

Steroid sulfation: Current concepts

Formation of steroid sulfates is catalyzed by sulfotransferase enzymes that are widely distributed and frequently of high specificity. Steroid sulfates cannot be described as being active hormones, but some serve in tissue sites as precursors of active steroids formed by enzymic cleavage of the sulfate group by sulfatase enzymes. There is increasing evidence that intracellular sulfation and desulfation can play a role in regulating the availability of active steroid hormones near target sites. There are strong indications for this regulation in the uterine compartment, in the liver, and in mammary cancer cells.

Nuclear receptors for progesterone and estradiol in the guinea pig uterine compartment during gestation

Because progesterone suppresses myometrial contractility, the assumption is often made that the withdrawal of this steroid is a prerequisite for parturition. However, steroid patterns in maternal blood of the guinea pig do not consistently change with impending parturition and it has been claimed that progesterone does not suppress guinea pig myometrial contraction. The present study investigated progesterone and estrogen nuclear receptor binding in myometrium, endometrium, and chorion between 32 days of gestation and delivery at 67-71 days. Binding characteristics and behavior during sedimentation in sucrose density gradients were typical of the steroid hormone receptor family. Decreased progestin binding occurred in the myometrium, from a high of 1600 fmol/mg DNA at 49-51 days to a low of 450 fmol/mg DNA (P < 0.01) on the day of detectable pubic relaxation. This decrease commenced at 60-63 days just before the onset of relaxation. A similar, though less well defined change occurred in endometrium. Estradiol nuclear receptor binding in myometrium remained at about 350 fmol/mg DNA from 32 days until 1-2 days pre-partum when it increased to about 650 fmol/mg DNA (P < 0.05). Estradiol binding in endometrium showed an inconsistent pattern and chorion binding for both progestin and estradiol was low and unremarkable. We conclude that there is a potential for decreased progesterone effect in myometrium at about one week before delivery and increased estrogen action in that tissue immediately before delivery.

The metabolism of estrone and estradiol-17β and their 3-sulfates by female guinea pig liver microsomes

The metabolism, by female guinea pig liver microsomes of estrogen 3-sulfates (estrone-3-sulfate and 17beta-estradiol-3-sulfate) was compared to that of the unconjugated estrogens, estrone and estradiol-17beta. Metabolites identified indicated that 16beta-hydroxylated products (16beta judrpxuestrpme amd 16 epiestriol) arose mainly from the free estrogens while 16alpha-hydroxy steroid sulfates (16alpha hydroxyestrone-3-sulfate and estriol-3-sulfates and estriol-3-sulfate) were predominantly formed from the sulfated estrogens. These results show that the sulfate moiety at position 3 of the steroids directs 16-hydroxylation from the beta to the alpha configuration.

Progesterone metabolism by guinea pig intrauterine tissues.

Progesterone metabolism by guinea pig amnion, chorion, myometrium, and endometrium was studied at the following gestational stages. Day 45 represents mid-gestation, about 5 days before strong chorion interaction between the entire surface of the chorion and the uterus; days 57-58, 1-2 days after chorion attachment, and 2-3 days before the onset of pubic symphysis relaxation; days +1-+6, 1-6 days after the onset of pubic symphysis relaxation, i.e. within 1 week of parturition. The high metabolic activity of chorion exceeded that by amnion at all stages. Metabolism by endometrium and myometrium was always low. Conversion of progesterone by amnion significantly decreased (P < 0.05) between days 57-58 and days +1-+6. Progesterone metabolites produced by chorion and amnion were identified by TLC, HPLC, and capillary GC/MS. Both tissues converted progesterone to three major products during 60-min incubations. These were 5 alpha-pregnane-3,20-dione, 3 alpha-hydroxy-5 alpha-pregnan-20-one, and 3 beta-hydroxy-5 alpha-pregnan-20-one. The metabolite pattern differed between the two tissues. Three-minute incubations with chorion resulted in a significantly higher proportion of 3 alpha-hydroxy-4-pregnen-20-one (P < 0.01) and 5 alpha-pregnane-3,20-dione (P < 0.025) than at 60 min. The production of 3 beta-hydroxy-5 alpha-pregnen-20-one by chorion decreased (P < 0.05) between days 50-51 and 57-58. The ratio of 3 alpha-hydroxy-5 alpha-pregnan-20-one to 3 beta-hydroxy-5 alpha-pregnan-20-one increased (P < 0.05) between days 45 post-relaxation. The marked conversion of progesterone by chorion, or the formation of one or more of its metabolites, may serve to influence uterine function prior to delivery.

Estrogen sulfotransferase and 17β-hydroxysteroid dehydrogenase activities in guinea-pig chorion through gestation

Estrogen sulfotransferase (EST) activity measured under optimal in vitro conditions in the 105,000 g cytosols (HSS) of homogenized intrauterine tissues (amnion, chorion, endometrium, decidua basalis and placenta) from guinea-pigs at the 50th day of gestation indicated that the highest specific activity occurred in the chorion. EST activity in the chorion increased from day 34 (early gestation) to peak around day 45 (mid-gestation), before significantly decreasing around day 50 and further declining to barely detectable levels beyond day 60 (late gestation, the onset of parturition). 17 beta-Hydroxysteroid dehydrogenase (17 beta-HSD) activity in the chorion was almost completely membrane associated. The specific activity of the 17 beta-HSD reduction reaction in the 105,000 g pellet was 2.5-fold higher at mid-gestation than at late gestation, while the specific activity of the 17 beta-HSD oxidation reaction was 1.7-fold higher at mid-gestation as compared with late gestation. When intact pieces of chorion tissue from mid- and late gestation were incubated with 5 nM [3H]estradiol (E2), approx. 80% of the recovered free estrogen was E1 (estrone). Only chorion from animals at the onset of parturition were able to produce detectable amounts of E2 from 5 nM [3H]E1. Under the same experimental conditions the ratio of estradiol sulfate (E2S) to estrone sulfate (E1S) isolated from the media and methanol washes of late gestation chorion tissue was 3-4 times greater than for the day 45 tissue.

Metabolites of estradiol-17β in guinea pig uterus late in pregnancy

The metabolism of estradiol-17beta by the guinea pig uterus late in pregnancy was studied in vivo and in vitro. Whole uteri were examined for estrogen metabolites one hour following an intravenous injection of [3H]-estradiol-17beta or uterine sections were examined after incubation for one hour at 37 degrees C in medium containing [3H]-estradiol-17beta. In both instances uterine tissue metabolized estradiol-17beta to five products; estrone, estrone-3-sulfate, 17beta-estradiol-3-sulfate, estrone-3-glucuronide and 17beta-estradiol-3-glucuronide. Of the total radioactive products 11-43% were glucuronides, 17-26% were sulfates and 4-17% was estrone. These results indicate that the guinea pig uterus actively transforms estradiol-17beta into glucuronides and sulfates late in pregnancy.

Microscopic and biochemical analysis of the viability and permeability of guinea pig amnion and chorion laeve in vitro

Tissue viability and permeability of guinea pig amnion and chorion leave were analyzed microscopically and biochemically. The vital dyes T1111 and fluorescein diacetate were used to locate and determine the integrity of cell plasma membranes in early and late tissue in vitro using confocal laser scanning microscopy and scanning electron microscopy. Early amnion and chorion laeve were each found to contain a single epithelial cell layer, composed of membrane-intact cells. In contrast, plasma membrane lesions were present throughout the epithelium of late amnion. Late chorion laeve contained both regions of intact and damaged epithelial cells on its maternal side. There was also a layer of membrane-intact squamous cells on the fetal side of late chorion laeve. ATP measurements confirmed that early fetal membranes were viable after incubation in isotonic salt solutions at physiological pH. Late amnion was depleted of ATP stores while late chorion laeve retained its capacity for generating energy. These viability markers indicate that late guinea pig amnion is not a viable tissue in vitro, while late chorion laeve is a viable but probably degenerating tissue. Confocal X-Z scans were used to trace the movement of T1111 through the tissue as an indication of permeability to free solutes. Whereas dye will permeate across the main thickness of early amnion and chorion leave, it did not pass between cells, but was blocked, presumably by a line of tight junctions. Late amnion was characterized by the complete permeability to T1111. Late chorion leave contained regions where solute migration was blocked, but overall was a permeable tissue. These results provide an important context for the interpretation of molecular movement across fetal membranes.

Generation of estradiol within the pregnant guinea pig uterine compartment with special reference to the myometrium.

Between about 50 and 58 days of gestation, the guinea pig chorion becomes attached in its entirety to the uterine wall, suggesting a facilitation of transfer of agents such as steroids between these tissues. At a time between 59 and 64 days, relaxation of the pubic symphysis starts, and anywhere from 5 to 8 days after that event delivery takes place. The present in vitro study was undertaken to evaluate estrone sulfate as a substrate for local production of estradiol, via the action of estrogen sulfatase and 17-hydroxysteroid dehydrogenase, in chorion, endometrium and myometrium taken at four distinct stages of gestation, as follows: 50 days, representing pre-chorion attachment to the uterus (stage 50); 1 or 2 days before pubic symphysis relaxation (minus 1 day, or -1 day); 1 day following relaxation (+1 day); and 1-2 days before delivery (late, or L). At these same stages, the metabolite patterns formed from estradiol were evaluated for endometrium and myometrium. Each of the tissues behaved somewhat differently. Overall hydrolysis of estrone sulfate by endometrium and myometrium exceeded that by chorion. Generation of free steroid from estrone sulfate increased 3-fold in chorion between stages 50 and -1 and during this period estradiol production from estrone sulfate increased 9-fold and continued to rise until delivery. Cytosolic estrogen sulfotransferase activity of chorion decreased 7-fold between stages 50 and -1. This suggested a tissue environment geared to producing potentially active estradiol. However, myometrium converted very little estrone into estradiol until just before delivery despite the facile formation of estrone from estradiol at stages -1, +1 and L. The control of estrogen metabolism by interaction of tissues in the uterus and by some form of enzyme regulation in these tissues suggests a possible role for locally produced estrogen in the stages leading up to parturition.

Behavior of mouse placental and uterine estrogen sulfotransferase during chromatography and other procedures

The estrogen sulfotransferase activity of high-speed supernatants of mouse placenta and uterus behaves on conventional and high-performance liquid chromatographic gel filtration as an enzyme species with a molecular weight of the order of 30 000. This is so whether the cytosols are freshly prepared or have been stored at -20 degrees C before chromatography. The presence of thiol groups or EDTA has no effect on the elution pattern. The partially purified enzyme is extremely unstable and is poorly recovered by (NH4)2SO4 fractionation. Some stabilization can be achieved in the presence of 0.1 microM estradiol. Chromatofocusing of cytosols results in the elution of one or two sulfotransferase peaks, depending upon experimental conditions such as the presence or absence of thiol groups. These peaks act upon both estrone and estradiol as substrates. Chromatofocusing by fast protein liquid chromatography (FPLC) in the absence of thiol groups results in the elution of one sulfotransferase peak whose activity can be detected only when thiol groups are present during enzyme assay.

Transfer of steroidal and nonsteroidal compounds across guinea pig fetal membranes.

Transfer of steroidal and nonsteroidal compounds across guinea pig amnion and chorion laeve was investigated as a function of stage of gestation, tissue orientation, steroid specificity, and molecular size. Each fetal membrane was examined at early and late stages of gestation, before and after pubic symphysis relaxation. Early amnion was impermeable to macromolecules and small charged molecules while [3H]estrone and [3H]pregnenolone were transferred, the latter depending on tissue orientation and involving conjugation at the basolateral interface. After symphysis dilation, amnion transferred all substrates tested with the exception of BSA; the molecular weight cutoff was approximately 5,000. Unlike amnion, early chorion transferred both free and conjugated steroids as well as inorganic sulfate. Transfer of estrone involved conjugation and depended on tissue orientation. Transfer of [3H]estrone-sulfate, [3H]estrone-glucuronide, and [3H]pregnenolone-sulfate was similar despite selective deconjugating activity toward estrone-sulfate. Near term, chorion was impermeable to inorganic sulfate and transfer of estrone-glucuronide depended on tissue orientation, involving deconjugation in the maternal to fetal direction. At no stage of gestation did chorion transfer macromolecules. These results suggest that the transfer of free and conjugated steroids across fetal membranes is differentially regulated by tissue, its stage of development, and direction of transfer.