R. Hobkirk

Estrogen sulfotransferase activity in guinea pig uterus and chorion

An estrogen sulfotransferase (ST) is detectable in high speed supernatants of pregnant guinea-pig uterus and shows maximum activity between about 47 and 55 days of gestation, with a decrease toward term. No appreciable activity was apparent in the non-pregnant state or before at least 43 days of pregnancy. A considerably higher ST activity is present in chorion as early as 30 days of gestation, and this also decreases toward term. The two STs exhibit similar KM (0.1-0.13 microM with estrone as substrate) and pI (5.8) values, as well as similar specificities. Estradiol-17 beta and estriol are sulfurylated 82 and 6% that of estrone at equimolar concn. Neither p-nitrophenol nor several neutral steroids are substrates for the enzymes. Enzyme activity is poorly expressed in the absence of thiol groups, the presence of monothioglycerol stimulating uterine and chorion enzymes by 5- and 15-fold, respectively. Stimulation is also observed in the presence of Mg2+, Ca2+ or Mn2+. Chromatofocusing on a poly buffer ion-exchanger from pH 7.4 to 4.0 resulted in elution of a sharp peak of enzyme activity, at pH = 5.8, from both tissues provided that the eluting buffer contained thiol groups and 0.25 M sucrose. This single step resulted in at least a 35- to 100-fold increase in specific activity. The partially purified enzyme from chorion exhibited a KM for estrone of 0.13 microM.

Partial characterization of steroid sulfohydrolase and steroid sulfotransferase activities in purified porcine leydig cells

Subcellular fractions of purified pig Leydig cells from 7 different animals have been investigated with respect to their abilities to catalyze the sulfation of several steroids and the hydrolysis of the sulfated forms of these same steroids. Considerable estrone sulfate sulfohydrolase of pH optimum 7.5 and high apparent Km was found to be concentrated in the 105,000 g pellet but no evidence was obtained, in any subcellular fraction, for the presence of any activity toward the 3-sulfate of pregnenolone, dehydroepiandrosterone (DHA) or delta 5-androstene-3 beta,17 beta-diol (androstenediol). Cytosolic sulfotransferase activity toward estrone, pregnenolone, DHA and androstenediol was present in each animal. The activity toward these 4 substrates was eluted from a gel filtration column as a single peak of apparent molecular weight 43 KDa. Upon chromatofocusing, a sharp estrogen sulfotransferase peak of apparent pI 6.1 and pH optimum 9.5, was clearly separated from the neutral steroid sulfotransferase which eluted over a more acidic pH range in a manner suggestive of the presence of several isozymes. This latter, which exhibited a wide pH optimum range between 6 and 8.5, was most active toward androstenediol, and least active toward pregnenolone. The estrogen sulfotransferase exhibited Michaelis-Menten kinetics (apparent Km = 4 microM). The neutral steroid sulfotransferase activity increased in velocity with increasing androstenediol or DHA concentration up to 1 microM beyond which considerable substrate inhibition occurred. It appears from these data that neutral steroid sulfates synthesized in the pig Leydig cell are not subject to enzymic desulfation in the same cells.

Estrone sulfatase activity in guinea pig tissues.

Estrone sulfatase activity is widespread in guinea pig tissues. Whole homogenates of adult testis, uterus, lung, adrenal, amnion, ovary, chorion, small intestine, placenta, spleen, kidney and liver exhibit approximately descending order of specific activity. Certain properties, including pH requirement, lack of inhibition by inorganic sulfate and magnitude of estimated Km values, are similar to that for arylsulfatase C of rat liver. Of the subcellular fractions prepared from guinea pig tissues, microsomes exhibit the highest specific activity although considerable enzyme activity remains associated with large cellular fragments sedimenting at 750 g. The sulfatase activity is readily inhibited by inorganic phosphate even when substrate concentration satisfied zero order kinetics. Rat liver arylsulfatase C is not inhibited under these conditions. Sensitivity of the guinea pig enzyme activity to inhibition by a variety of steroids and related compounds, is markedly less than for rat liver. Diethylstilbestrol(DES) strongly inhibits the rat liver enzyme but has little effect on the guinea pig liver system. Guinea pig testicular activity is suppressed to a degree intermediate between these extremes by increasing DES concentration. In guinea pig lung, kidney, and possibly liver, elevated fetal enzyme activities decrease from neonatal to adult life. Testicular activity appears to follow the opposite trend. Uterine enzyme activity is not markedly affected by pregnancy.

UDPGA-dependent estrogen glucuronyltransferase of guinea-pig uterus: Assay, temporal relationships in pregnancy and some characteristics

Abstract The UDPGA-dependent estrogen glucuronyltransferase (GT) of guinea-pig uterus increases sharply at about 50 days of gestation to specific activity (SA) values which are, on average, 40-fold those during the first 7 weeks. The enzyme is endometrial and exhibits optimal activity at pH 8. Fetal, immature and adult non-pregnant animals possess low activity, or none at all. The uterine microsomal GT is activated by Mn2+, Ca2+ and Mg2+ and by low concentrations of the amphoteric detergent, Miranol H2M, but not by cholate, deoxycholate, Cutscum or Triton detergents. Miranol does not alter the kM (estrone) of the enzyme. The SA of non-activated lever microsomal GT is 50-fold greater than that from uterus. The latter conjugates estrone and the isomeric estradiols but not estriol, testosterone, dehydroisoandrosterone, cortisol or p-nitrophenol. Activity toward estrone is inhibited by estradiol but not by estriol or p-nitrophenol. Liver GT conjugates estrone, estradiol, estriol, testosterone and p-nitrophenol. Uterine GT has apparent KM values of 1.6 μM (estrone) and 0.2 mM (UDPGA). Liver microsomal activity shows a km of 2.4 μM for estrone. GT activity toward estrone is not present in guinea-pig placenta but is considerable in chorion and variable in amnion. It is speculated that the behaviour of the uterine enzyme is consistent with a control mechanism for uterine estrogen levels in late pregnancy.

Early urinary conjugated metabolites of intravenously injected [6,7-3H]-estradiol-17β in the human subject

Abstract [6,7-3H]-Estradiol-17β (E2) was administered intravenously into four non-pregnant women (three by acute injection; one by 30 min infusion). Urine was collected at short time intervals within 3 h and was analyzed by established procedures for labelled metabolites. At 10 min after injection the main metabolite was estradiol-17-glucosiduronate (E217G), accompanied in at least one subject by a minor amount of estradiol-3-glucosiduronate (E23G) and no other conjugate. The excretion of E217G ceased within 1 h (acute injection of E2) or 2–3 h (30 min infusion). After 10 min estrone-3-glucosiduronate (E13G) excretion commenced, and continued through the study period as did that of E2G. Estriol-16-glucosiduronate (E316G) appeared in identifiable form between 20 and 40 min, and “estrogen sulfates” between 40 and 60 min. The amounts of E316G and estrone-3-sulfate were highly variable between subjects, as was the case for a number of other, as yet unidentified, labelled metabolites. It is suggested that E217G is a direct, and perhaps “unique” metabolite of peripherally injected E2 in the human.

16-Hydroxylation of estrogens in pigmented and albino guinea-pigs.

Abstract Metabolites of injected [3H]-estrone-3-sulfate were investigated in urine, gall bladder bile, liver and kidney of mature pigmented and albino male and non-pregnant female guinea pigs (English Shorthair variety). Considerable formation of estriol, 16α-hydroxyestrone and 16β-hydroxyestrone disulfates, as well as estriol and 16α-hydroxyestrone monosulfates occurred in pigmented females. Monoglucuronides and monosulfates of estrone and estradiol-17β (estradiol) were also observed. No 16-hydroxysteroid monosulfates were detected in pigmented males but disulfates were present in urine, to a lesser extent than for females. No hydroxysteroid monosulfates were found in albinos of either sex and, although small amounts of a disulfate-like fraction occurred in urine, the steroid moieties were not identified. Pigmented females formed lesser amounts of hydroxysteroids from injected [3H]-estrone or estradiol than from estrone sulfate. These metabolites were not detected in the other animal groups. Qualitative patterns of estrone and estradiol monoglucuronides and monosulfates were similar in pigmented and albino animals, regardless of the precursor injected.

Estrone sulfate sulfohydrolase in the developing brain and pituitary of rat, mouse and guinea pig.

The pattern of estrone sulfate sulfohydrolase (estrogen sulfatase) development in the brain of rat, mouse and guinea pig has been established by assaying whole homogenates. Activity was measurable in each species from the fetal state to adulthood. Maximum brain content was reached at about 20 days of age in rat, 14 days in mouse and 15 days in guinea pig. A considerable decrease occurred between 14 days and adulthood in mouse and lesser decreases were seen in rat and guinea pig. The subcellular distribution of enzyme in rat and mouse brain appeared to change from the immature to the adult state. No major differences in enzyme activity occurred between the sexes at any age. Tissue concentration of enzyme in the hypothalamic-preoptic area of rat and mouse was similar to that in the remainder of the brain. In guinea pig the brain concentration was slightly lower than that of the hypothalamic-preoptic region. Sulfatase content of the pituitary was low in all 3 species but the tissue concentration was considerably higher than that of brain, particularly in rat and mouse. Apparent Km values for brain sulfatase were in the range 6-17 microM, with no striking sex difference. Apparent Kms for pituitary sulfatase of immature rat and guinea pig were similar to those for brain in the same animals but that for mouse pituitary (0.9 microM) was much lower. It is unlikely that brain or pituitary sulfatase is by itself, a major factor in making available potentially active estrogen for use during differential sex development in these species.

Formation of androgen conjugates by porcine granulosa cells.

The relative abilities of isolated and recombined theca and granulosa cells, derived from medium-sized porcine ovarian follicles, to synthesize androgens and estrogens were compared. Isolated thecal preparations produced large amounts of immunoreactive androstenedione and testosterone. When theca was co-cultured with granulosa cells, accumulation of both these androgens was markedly less. Though the co-cultures produced significantly higher amounts of estradiol, this increase did not account for the reduced androgen production. To determine if the lesser androgen accumulation in the combined cultures was due to metabolism by granulosa cells of androgens to other metabolites, the fate of [3H]androstenedione or [3H]testosterone was followed. Whenever granulosa cells were present in the incubation dishes, most of the radioactivity remained in the aqueous fraction after ether extraction. Examination of the aqueous fraction by DEAE-Sephadex A25 demonstrated the presence of androgen sulphates. The results suggest that granulosa cells are the site at which follicular formation of androgen sulphates takes place.

Development and control of guinea-pig liver estrone sulfate 16α-hydroxylase☆

Abstract Estrone sulfate 16α-hydroxylase activity is undetectable in liver microsomes from fetal guinea-pigs of the English Shorthair variety. Within 2 days of birth, considerable activity is present in both sexes of pigmented and albino animals. In the pigmented group, maximum activity occurs during the second week of life, with the mean values for each of the first 4 weeks, in both sexes, significantly higher than for the corresponding mature (> 12-week-old) animals. Immature levels in the albino group are also significantly higher than those of mature albinos. Pigmented females of all ages possess significantly higher activities than do their male counterparts. There are no such sex-related differences in albinos. Pigmented animals of all ages exhibit higher activities than do their albino counterparts. Castration of either sex, pigmented or albino, results in increased enzyme activities as compared with intact or sham-operated controls. Gestation leads to maternal enzyme values which are significantly above those of non-pregnant females, whether pigmented or albino. Beyond the first few days of life, total liver microsomal cytochrome P450 shows no significant change with age, gestation or pigmentation. These data support the conclusion that estrone sulfate 16α-hydroxylase activity in the guinea-pig is markedly diminished, following sexual maturity, by presently unknown factors. This holds for both pigmented and albino animals but the decrease is greater in the latter. This decrease can be reversed by castration in either sex, or by pregnancy and could possibly relate to gonadal-pituitary relationships as demonstrated by others for rat liver hydroxylases.

Chromatofocusing of mammalian estrone sulfate sulfohydrolase activity

Estrone sulfate sulfohydrolase (estrogen sulfatase) activity was solubilized by treatment with Triton X-100 from 105,000 g pellets of guinea pig uterus, testis and brain, as well as from rat liver and human placenta. The solubilized forms were subjected to chromatofocusing in the fast protein liquid chromatography (FPLC) system and on conventional columns packed in our laboratory. The guinea pig tissue pattern was complex. Uterus showed peaks of activity with apparent pIs of 9.11 and 7.6; testis contained 3 peaks with pIs of 9.18, 8.7 and 7.5; brain possessed peaks with pIs of 9.28 and 8.6. In each case the major activity peak was that with pI greater than 9. Rat liver activity chromatofocused as a single peak of apparent pI = 6.87 and the human placental enzyme also showed a single, though broad, peak, of pI = 6.57. This suggests not only that the guinea pig enzyme(s) differs markedly from those of rat liver and human placenta, but that there may be qualitative differences between the forms in the three guinea pig tissues. Chromatofocusing behaviour was not independent of the specific exchange resins and ampholytes utilized. The recovered enzyme activity was fairly stable and it seems that chromatofocusing could be a useful step in purification of the guinea pig enzyme(s), particularly the main form possessing a pI greater than 9.