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3β-hydroxysteroid dehydrogenase-isomerase activity in placentae from pregnancies complicated by steroid sulphatase deficiency
3/?-HYDROXYSTEROID DEHYDROGENASE-ISOMERASE ACTIVITY IN PLACENTAE FROM PREGNANCIES COMPLICATED BY STEROID SULPHATASE DEFICIENCY I. MARTON*and R. E. OAKEY Division of Steroid Endocrinology, Department of Chemical Pathology, School of Medicine, University of Leeds, Leeds, England SUMMARY The 3j-hydroxysteroid dehydrogenase-isomerase activity of preparations of human placentae was determined in ritro by measuring the conversion of [‘HI-pregnenolone to C3H]-progesterone. The mean activity (330 + 40 c.p.m./mg protein/30 min, mean f SD, II = 8) of placentae deficient in steroid sulphatase, previously stored at -20°C. was significantly less (P < 0.001, r-test) than that of control placentae which had been treated in an identical manner (600 + 60 c.p.m./mg protein/30 min. n = 15).However. when tested without freezing, the mean activity of the same control placentae was 2240 f 675 c.p.m./mg protein/30 min (II = 15) and the activity of a fresh sulphatase deficient placenta (2!70c.p.m./mg protein/30 min) was indistinguishable from that of the controls. 3/I_Hydroxysteroid dehydrogenaseisomerase activity therefore appears to be sensitive to freezing and thawing and the effect is more marked in sulphatase deficient placentae. This finding confirms the earlier reports of lower activities of steroid metabolising enzymes (other than sulphatase) in steroid sulphatase deficient placentae. The results from the deficient placenta studied fresh and after freezing imply that the lower 3B-hydroxysteroid dehydrogenase-isomerase activity in steroid sulphatase deficient placentae is due to the study of tissue which had been stored at -20°C rather than of fresh tissue. Mean 3/$hydroxysteroid dehydrogenase-isomerase activity of fresh placenta from male infants was significantly less (P < 0.05) than that of placentae where the fetus was female.
INTRODUIXION Placentae from pregnancies complicated by steroid sulphatase deficiency lack the ability to hydrolyse the 3B-yl sulphates of pregnenolone, dehydroepiandrosterone and oestrone. There is uncertainty whether
other placental enzymes which use steroids as substrates are also deficient to some degree. France and Liggins[ 1] and France, Seddon and Liggins[Z] found lower activities for 3j%hydroxysteroid dehydrogenaseisomerase and aromatase in placentae from two pregnancies complicated by sulphatase deficiency than in normal placentae, but concluded that enzymes other than sulphatase were unaffected. Oakey, Cawood and Macdonald[3] found that the activities of aromatase, 17/I-hydroxysteroid dehydrogenase and 3/?-hydroxysteroid dehydrogenase for dehydroepiandrosterone and prenenolone in a placenta from an affected pregnancy were substantially lower than in a control. These findings were confirmed in another ‘case [4] and also recognized for aromatase and for 3/?-hydroxysteroid dehydrogenase-isomerase plus aromatase [S]. In none of these instance could firm generalizations be made as investigations were usually restricted to the study of one or two affected placentae. It has now become possible to identify affected pregnancies before delivery on the basis of infusion tests [6], estimation of dehydroepiandrosterone sulphate in amniotic
* Present address; Department of Obstetrics, graduate Medical School, Budapest, Hungary.
fluid [S, 71 or simple urinary assays [S]. Such earlier diagnosis provides the opportunity to assess the activity of enzymes, e.g. 3/?-hydroxysteroid dehydrogenase-isomerase, in affected placentae on a larger scale than before. Consequently, we have measured the conversion of [“HI-pregnenolone to [3H]-progesterone by the microsomal fraction from eight placentae deficient in steroid sulphatase activity. These conversions were compared with those shown by 27 placentae obtained from unaffected pregnancies, SUBJECTS
Eight placentae were obtained from women whose pregnancies were complicated by steroid sulphatase deficiency (confirmed in vitro see below). Seven of these were stored at -20” before preparation of a microsomal fraction. The eighth was obtained immediately upon delivery and stored on ice for 2 h during transport to the laboratory. All infants were males; their birthweights, where recorded, were more than 2.6 kg. Only one had a spontaneous’vertex delivery, the remainder were delivered by lower segment caesarean section or with the aid of forceps. Maternal oestrogen excretion was < 12 pmo1/24 h in all cases. For comparative purposes 27 placentae were obtained immediately after delivery following essentially uncomplicated pregnancies. Twenty infants delivered spontaneously, three were delivered by forceps and four by caesarean section. There were 15 male and 12 female infants. All weighed more than 2.65 kg and all placentae weighed more than 0.5 kg.
Post475
I. MARTONand
476 MATERIALS
AND METHODS
Reagents and chemicals were A.R. grade (B.D.H. Chemicals Ltd., Poole, Dorset) unless otherwise speciPBD (2-(4’-t-butyl phenyl)-S-(4”fied. Butyl biphenyly1)1,3,4-oxadiazole) was purchased from Ciba (ARL) Ltd., Duxford, Cambridge; Nicotinamide adenosine dinucleotide (NADP), Triton X-l 14, PPO (2,5diphenyloxazole), and dimethyl POPOP (l,4-bis 2(4-methyl-5-phenyl-oxazolyl)benzene) from Sigma London Chemical Co. Ltd., Poole, Dorset. Non-radioactive steroids were obtained from Steraloids Ltd., Croydon, Surrey. [7a-3H]-pregnenolone (SA 18 Cilmmol), [4-14C]-progesterone (SA 58.8 mCi/ mmol) and [7c(-3H]-dehydroe.piandrosterone sulphate, sodium salt (SA 4.6 mCi/mmol) were supplied by the Radiochemical Centre, Amersham, Berks. The purity of all steroids was examined by paper chromatography in an appropriate solvent system and found to be satisfactory. Preparation
of placental
tissue
Tissue from seven of the eight cases of steroid sulphatase deficiency was received deep frozen from other hospitals. Each sample was thawed slowly and used to prepare a microsomal fraction. Placentae were also obtained within 30 min of delivery after 27 pregnancies without biochemical complications and 1 complicated by steroid sulphatase deficiency. From each of these 27 control and 1 deficient tissues, two centrally located cotyledons were cut, freed from membranes and washed with NaCl solution (0.9% w/v). One cotyledon from each was stored at -20°C for direct comparison with stored deficient placentae; the other cotyledon was processed without delay. Preparation
of placental microsome fractions
The tissue, either fresh or after thawing, was suspended in Tris-HCl buffer (0.2 mol/l, pH 7.3, fortified with sucrose, 0.25 mol/l, 2 ml buffer/g tissue) and homogenized for 3 periods of 10 set, with 30 set intervals for cooling using an MSE homogenizer (MSE Ltd., Buckingham Gate, London SWI). Cell debris and nuclei were removed by centrifuging at 800s for 10min. The supernatant was centrifuged again at 10,000 g for 30 min. This supernatant, referred to subsequently as the microsomal fraction- was used for measurement of enzyme activity. The protein content of the supernatant was determined by the method of Lowry, Roseborough, Farr and Randal1[9] using bovine serum albumin as reference material and the concentration adjusted with buffer to 1 mg/lOO~. of 3jChydroxysteroid Estimation isomerase activity for pregnenolone
dehydrogenase-
The microsomal fraction was incubated with [3H]-pregneno10ne. Steroids, including C3H]-progesterone formed during incubation, were extracted from the incubation mixture with ether and treated with
R. E. OAKEY acetylating reagent thereby facilitating the separation of progesterone from remaining substrate, which was (20-0x0-5acetate converted to pregnenolone pregnen-3gyl acetate). C3H]-Progesterone was isolated by paper chromatography and the yield of this metabolite, corrected for manipulative loss by reverse isotope dilution technique, was calculated. In detail, Tris-HC1 buffer (0.2 mol/l, pH 7.3, 3 ml), NADP (50nmol in 100 ~1 buffer), [3H]-pregneno10ne (4 x lo4 c.p.m., in 50 ~1 ethanol) and microsomal fraction (100~1 containing 1 mg protein) were mixed and incubated at 37°C for 30 min. Acetone (2 ml) containing [‘4C]-progesterone (3 x IO3c.p.m., 400 pmol) was added to stop the enzyme reactions and the mixture was placed in boiling water for 10 min. After cooling the steroids were extracted into ether (2 x 3 ml). The ether was evaporated and the residue treated with acetylating reagent (0.5 ml pyridine, 0.5 ml acetic anhydride) for 8 h at 60°C. The reagent remaining was evaporated and the residue was mixed with progesterone and pregnenolone (0.5 ~01 of each), and chromatographed on paper in the solvent system light methanol-water SO-100°C); petroleum-(b.p. (100:96:4, by vol.). Radioactive progesterone (R, 0.32), clearly separated from pregnenolone acetate (RF 0.80), was located and eluted. A portion was used for measurement of the 3H/‘4C and hence calculation of the yield of product. The remainder was set aside for assessment of the purity of the progesterone by recrystallization. A sample of supernatant which had been boiled was included in each batch of incubations. The quantity of [3H]-progesterone formed was calculated from the amount of [3H]-progesterone isolated, corrected for manipulative loss assessed from the loss of [‘4C]-progesterone. The enzyme activity was expressed as the quantity of [3H]-progesterone formed per mg protein in 30 min. Estimation of steroid sulphatase activity
The rate of hydrolysis of C3H]-dehydroepiandrosterone sulphate was measured. Microsomal fraction (100 ~1 containing 1 mg protein), Tris-HCl buffer (0.2 mol/l, pH 7.3, 3 ml), NADP (50nmol in 100 ~1 buffer) and [3H]-dehydroepiandrosterone sulphate (2.3 x lo4 c.p.m., 6.8 nmol in 100~1 buffer) were mixed and incubated at 37°C for 30min. The enzyme reaction was stopped by heating the mixture at 100°C for 10 min. After cooling, the mixture was shaken with toluene (3 x 3ml) to extract any [‘HIdehydroepiandrosterone produced. The toluene was evaporated and the [“H ] determined. Incubations of boiled supernatant were included to serve as analytical controls. Enzyme activity was expressed as the amount of ether soluble tritium released per mg protein in 30 min. Liquid scintillation counting
For determination
of 3/I-hydroxysteroid
dehydro-
3/?-HSD in sulphatase deficiency
genase-isomerase activity, radioactive steroids were dissolved in toluene (5 ml containing butyi PBD 0.4% w/v and methanol 2% v/v). Tritium and [“Cl were measured by liquid scintillation spectrometry (Model SL30 Intertechnique Ltd., Brighton, Sussex) at efficiencies of 32% and 57”/0respectively. On counting 3H in the presence of 14C a correction was made for those counts derived from 14C which appear in the 3H channel. Crossover from 3H to 14C channel was negligible. At least 10,000 counts of each isotope were collected. For determination of steroid sulphatase activity tritiated steroids were dissolved in buffer (100 ~1) and counted in 8 ml of xylene containing Triton X-l 14 (20%) PPO (0.3%) and methyl POPOP (0.2%) at an efficiency of 36%. RESULTS
Estimation of 3/l-hydroxysteroid isomerase activity
dehydrogenase-
Preliminary experiments demonstrated that the yield of [3H]-progesterone from the quantity of substrate selected was directly proportional to incubation time, up to 40min, and to the protein content, up to 1.5 mg. Incubation was therefore carried out for 30min with 1 mg of protein at pH 7.3 and in the presence of NADP (50 nmol). Repetitive estimation of the activity of portions from the same placental preparation, stored at -20°C showed that the analytical precision (expressed as a coefficient of variation) was < 10%. The effectiveness of the purification procedure is illustrated by the yield of [3H]-progesterone obtained on incubation of boiled microsomal fractions, which was always less than 0.1% (~40 c.p.m./mg protein). Additional evidence was provided by the stability of the ‘H/“C ratio of the isolated progesterone during recrystallisation. The ratios found were: starting ma-
Stored at -
XPC
477
terial 2.56; crystals: 1st crop 2.48, 2nd crop 2.51, 3rd crop 2.46; mother liquors: 1st liquor 2.50, 2nd liquor 2.62, 3rd liquor 2.61. Stability of 3b-hydroxysteroid dehydrogenaseisomerase activity on storage at - 20°C
Since most samples of sulphatase deficient placentae had been stored for various periods at -2O”C, it was necessary to assess whether there was deterioration of the enzyme activity during prolonged storage under these conditions. Portions of fresh placentae from an unaffected and a deficient pregnancy were stored at - 20°C. After I, 2 and 3 months the tissue was thawed, microsomal fractions were prepared and the enzyme activity measured. For the sulphatase deficient placenta the yields were 400,395 and 400 c.p.m./mg protein/30 min whilst the yields from the control placenta were 640, 650 and 630 c.p.m./mg protein/30 min. Therefore, during prolonged storage at -20°C there was no further deterioration of 3/I-hydroxysteroid dehydrogenaseisomerase activity, in either sulphatase deficient or control tissue. 3B-Hydroxysteroid dehydrogenase-isomerase activity from sulphatase deficient and control placentae The enzyme activity of preparations of 8 placentae from sulphatase deficient pregnancies and I5 control placentae in which the baby was male (all after storage at -20°C) are shown in Fig. 1. The mean activity ( f SD) for preparations of sulphatase deficient placentae (330 * 40 c.p.m./mg protein/30 min) was significantly less (P < 0.05) than the mean value for the unaffected controls (600 k 60 c.p.m./mg protein/30 min). However, when microsomal fractions from the same 15 control placentae and 1 deficient placenta were prepared without freezing, the enzyme activity
Fresh .
Fig. 1. Yield of C3H]-progesterone (c.p.m./mg protein/30 min) from [“HI-pregnenolone, by 1O$OOg supernatant from sulphatase deficient placentae, 0; from control placentae associated with male infants, A; and from control placentae associated with female infants, B.
1. MARTON and R. E. OAKEY
478
for the controls (2240 f 675 c.p.m./mg protein/30 min) was indistinguishable from that of sulphatase deficient tissue (2 I70 c.p.m./mg protein/30 min). although after freezing and thawing, the enzyme activity of this particular tissue was only 350 c.p.m./mg protein/30 min. These results demonstrate that the process of freezing and thawing causes lower activity of placental 3/I-hydroxysteroid dehydrogenase-isomerase activity and this effect is more evident in sulphatase deficient placentae. l@ct
of the sex qf the
dehydrogenase-isomerase
fetus 011 3/?-hydro.u_vsteroid actiuit)
During the course of this work 3/&hydroxysteroid dehydrogenase-isomerase activity was measured in microsomal fractions from 12 placentae where the baby was female and from 15 where the baby was male (Fig. 1). When measured on preparations derived from tissue which had not been frozen, the mean enzyme activity of placentae associated with female babies (3480 rf: 760 c.p.m./mg protein/30 min) was significantly higher (P < 0.05) than that from placentae where the baby was male (2240 + 670c.p.mJmg protein/30min). However, a/fter storage of the tissue at -2O”C, enzyme activity was diminished in both to 710 f 80 and 600 + 60 c.p.m./mg groups protein/30 mill respectively and no significant difference between the mean values was apparent. effect of freezing
and thawing
011 steroid
sulphatase
activity
Steroid sulphatase activity of microsomal fractions from samples of 8 sulphatase deficient and 27 unaffected placentae after storage at -20°C and from 1 defi-’ cient and 27 normal fresh placentae was measured. The mean activity (*SD) in preparations of frozen was 970 _+ 736 c.p.m./mg deficient placentae protein/30min whilst that shown by the single defiwithout freezing was tested cient placenta 1102c.p.m./mg protein/30 min; that of boiled tissue was 620 + 460 c.p.m./mg protein/30 min (II = 6). These mean valuks were significantly less (P < 0.001) than mean values from control placentae tested fresh (17020 + 2070 c.p.m./mg protein/30 min II = 6) or after freezing (I 4720 f 920 c.p.m./mg protein/30 min II = 27). The mean values for control placentae tested fresh or after freezing are significantly different (P = 0.01) indicating that storage at -20°C and thawing has a small deleterious effect on sulphatase activity. When the activities of the fresh and thawed sappIes from each of the six control placentae were compared by a paired t-test, the loss of activity due to treatment was still significant (P < 0.02). There was no difference in sulphatase activity in placentae associated with male or female fetuses. DISCUSSION
The results reported here demonstrate
that the ac-
tivity of 3/Ghydroxysteroid dehydrogenase-isomerase for pregnenolone in placentae deficient in steroid sulphatase is significantly less than that found in normal placentae. provided measurements are made after storage of the tissue at -20’C. This finding confirms, on a larger number of placentae, our earlier results [3,4]. It may also provide an explanation of the lower activities than normal of steroid metabolising enzymes which were found in steroid sulphatase deficient tissue previously subjected to - 20°C [Z-5]. When the 3/Ghydroxysteroid dehydrogenaseisomerase activity was measured without freezing, the single sample of tissue deficient in steroid sulphatase which was available showed 3P-hydroxysteroid dehydrogenase isomerase activity equal to that of control tissue. If this is confirmed in subsequent studies and also applies to other placental enzymes involved in the conversion of androgen sulphates to estrogens then it appears that only one enzyme or complex~steroid sulphatase-is deficient. Thus it would be expected that mutation of only one gene would be required to cause this condition. This gene is apparently located on the short arm of the X-chromosome. adjacent to those controlling the development of X-linked ichthyosis and to the Xg” blood group locus [lo]. A search for associations between steroid sulphatase deficiency and other inherited abnormalities may therefore be of value in mapping the locus of associated genes. Mean placental 3P-hydroxysteroid dehydrogenaseisomerase activity associated with male infants was significantly less than that associated with females, The reason for such a difference is not clear. It may be due to the presence of different quantities of endogenous steroids which act as inhibitors of the enzyme activity, for example testosterone or androstenedione [I I]. A significant difference in the mean concentration of testosterone in cord blood from male and female infants has been established [12]. but differences linked to fetal sex in the concentrations of other steroids have not been reported. A report of differences in progesterone concentrations in umbilical vein blood, associated with fetal sex, in the rhesus monkey [13] has not been confirmed in the human [1416]. Our finding of differences in 3p-HSD activity associated with fetal sex therefore requires independent corroboration. Ackrlowledger,lrrltsWe
thank the Hungarian Ministry of Health for financial support (to I.M.) and the many Biochemists and Obstetricians who assisted with the supply of tissue and urine.
REFERENCES I. France J. T. and L&ins G. C.: Placental sulfatase dkficiency. J. ch. Endocr. Met& 29 (1969) 1X-141. 2. France J. T.. Seddon R. J. and Liggins c. C.: A study of pregnancy with low estrogen production due to placental sulfatase deficiency. J. clh. Endocr. Metah. 36 (1973) 1-9.
3P-HSD in sulphatase deficiency 3. Oakey R. E., Cawood M. L. and Macdonald R. R.: Biochemical and clinical observations in a pregnancy with placental sulphatase and other enzyme deficiencies. Clitr. Eftdocr. 3 (1974) 131-148. 4. Prooth S.: A study of placental sulphatase deficiency: activities of placental enzymes involved in estrogen biosynthesis. M.Sc. thesis University of Leeds 1974. 5. Osathanondh R., Carrick J., Ryan K. J. and Tulchinsky D.: Placental sulfatase deficiency: A case study. J. c/i!!. Entlocr. M~rah. 43 (1976) 208-214. 6. Tabei T. and Heinrichs W. L.: Diagnosis of placental sulfatase deficiency Aar. J. Ohsrut. G_rnrc. It4 (1976) 4094 14. 7. Braunstein G. D., Ziel F. H.. Allen A.. Van de Velde R. and Wade M. E.: Prenatal diagnosis of placental steroid sulfatase deficiency. rlnt. J. Ohsrcr. Grnec: 126 (1976) 716-719. 8. Oakey R. E.: Placental sulphatase deficiency: Anteparturn differential diagnosis from foetal adrenal hypoplasia. Clirt. Endorr. 9 (i 978) Xi-XX. 9. Lowry 0. H.. Roseborough N. J.. Farr A. L. and Randall R. J.: Protein measurement with the Folin phenol reagent. 6. hiol. chcrn. 193 ( 1951) 265-275.
479
IO. Shapiro L. J.. Weiss R., Buxman M. M., Vidgoff J., Dimond R. L., Roller J. A. and Wells R. S.: Enzymatic basis of typical X-linked ichthyosis. Lancer ii (19’78) 756757. I 1. Townsley J. D.: inhibition of placenta1 3fi-hydroxysteroid dehydrogena~ by naturally occurring steroids.. Acta atdocr. Copedt. 79 (I 975) 740-748. 12. Forest M. G., Cathiard A. M. and Bertrand J. A.: Evidence of testicular activity in early infancy. J. c/in. Ernlocr. Mrtuh. 37 (1973) 148-151. 13. Hagemenas F. C. and Kittinger G. W.: In influence of fetal sex on plasma progesterone levels. f+docrinolqy_t 91 (1972) 2.53-256. 14. Hagemenas F. C. and Kittinger G. W.: The influence of fetal sex on the levels of plasma progesterone in the human fetus. .I. &I?. Ettducr. Mrrah. 36 (1973) 389-391. 15. Tulchinsky D. and Okada D. M.: Hormones in human pregnancy IV. Plasma progesterone. Ant. J. Ohsrer. Gyttec. 121 (1975) 293-299. 16. Antonipillai 1. and Murphy B. E. P.: Serum oestrogens and progesterone in mother and infant at delivery. Br. .I. Ohsrrt. G,~nurc. 84 (I 977) 179- 185.
INTRODUIXION Placentae from pregnancies complicated by steroid sulphatase deficiency lack the ability to hydrolyse the 3B-yl sulphates of pregnenolone, dehydroepiandrosterone and oestrone. There is uncertainty whether
other placental enzymes which use steroids as substrates are also deficient to some degree. France and Liggins[ 1] and France, Seddon and Liggins[Z] found lower activities for 3j%hydroxysteroid dehydrogenaseisomerase and aromatase in placentae from two pregnancies complicated by sulphatase deficiency than in normal placentae, but concluded that enzymes other than sulphatase were unaffected. Oakey, Cawood and Macdonald[3] found that the activities of aromatase, 17/I-hydroxysteroid dehydrogenase and 3/?-hydroxysteroid dehydrogenase for dehydroepiandrosterone and prenenolone in a placenta from an affected pregnancy were substantially lower than in a control. These findings were confirmed in another ‘case [4] and also recognized for aromatase and for 3/?-hydroxysteroid dehydrogenase-isomerase plus aromatase [S]. In none of these instance could firm generalizations be made as investigations were usually restricted to the study of one or two affected placentae. It has now become possible to identify affected pregnancies before delivery on the basis of infusion tests [6], estimation of dehydroepiandrosterone sulphate in amniotic
* Present address; Department of Obstetrics, graduate Medical School, Budapest, Hungary.
fluid [S, 71 or simple urinary assays [S]. Such earlier diagnosis provides the opportunity to assess the activity of enzymes, e.g. 3/?-hydroxysteroid dehydrogenase-isomerase, in affected placentae on a larger scale than before. Consequently, we have measured the conversion of [“HI-pregnenolone to [3H]-progesterone by the microsomal fraction from eight placentae deficient in steroid sulphatase activity. These conversions were compared with those shown by 27 placentae obtained from unaffected pregnancies, SUBJECTS
Eight placentae were obtained from women whose pregnancies were complicated by steroid sulphatase deficiency (confirmed in vitro see below). Seven of these were stored at -20” before preparation of a microsomal fraction. The eighth was obtained immediately upon delivery and stored on ice for 2 h during transport to the laboratory. All infants were males; their birthweights, where recorded, were more than 2.6 kg. Only one had a spontaneous’vertex delivery, the remainder were delivered by lower segment caesarean section or with the aid of forceps. Maternal oestrogen excretion was < 12 pmo1/24 h in all cases. For comparative purposes 27 placentae were obtained immediately after delivery following essentially uncomplicated pregnancies. Twenty infants delivered spontaneously, three were delivered by forceps and four by caesarean section. There were 15 male and 12 female infants. All weighed more than 2.65 kg and all placentae weighed more than 0.5 kg.
Post475
I. MARTONand
476 MATERIALS
AND METHODS
Reagents and chemicals were A.R. grade (B.D.H. Chemicals Ltd., Poole, Dorset) unless otherwise speciPBD (2-(4’-t-butyl phenyl)-S-(4”fied. Butyl biphenyly1)1,3,4-oxadiazole) was purchased from Ciba (ARL) Ltd., Duxford, Cambridge; Nicotinamide adenosine dinucleotide (NADP), Triton X-l 14, PPO (2,5diphenyloxazole), and dimethyl POPOP (l,4-bis 2(4-methyl-5-phenyl-oxazolyl)benzene) from Sigma London Chemical Co. Ltd., Poole, Dorset. Non-radioactive steroids were obtained from Steraloids Ltd., Croydon, Surrey. [7a-3H]-pregnenolone (SA 18 Cilmmol), [4-14C]-progesterone (SA 58.8 mCi/ mmol) and [7c(-3H]-dehydroe.piandrosterone sulphate, sodium salt (SA 4.6 mCi/mmol) were supplied by the Radiochemical Centre, Amersham, Berks. The purity of all steroids was examined by paper chromatography in an appropriate solvent system and found to be satisfactory. Preparation
of placental
tissue
Tissue from seven of the eight cases of steroid sulphatase deficiency was received deep frozen from other hospitals. Each sample was thawed slowly and used to prepare a microsomal fraction. Placentae were also obtained within 30 min of delivery after 27 pregnancies without biochemical complications and 1 complicated by steroid sulphatase deficiency. From each of these 27 control and 1 deficient tissues, two centrally located cotyledons were cut, freed from membranes and washed with NaCl solution (0.9% w/v). One cotyledon from each was stored at -20°C for direct comparison with stored deficient placentae; the other cotyledon was processed without delay. Preparation
of placental microsome fractions
The tissue, either fresh or after thawing, was suspended in Tris-HCl buffer (0.2 mol/l, pH 7.3, fortified with sucrose, 0.25 mol/l, 2 ml buffer/g tissue) and homogenized for 3 periods of 10 set, with 30 set intervals for cooling using an MSE homogenizer (MSE Ltd., Buckingham Gate, London SWI). Cell debris and nuclei were removed by centrifuging at 800s for 10min. The supernatant was centrifuged again at 10,000 g for 30 min. This supernatant, referred to subsequently as the microsomal fraction- was used for measurement of enzyme activity. The protein content of the supernatant was determined by the method of Lowry, Roseborough, Farr and Randal1[9] using bovine serum albumin as reference material and the concentration adjusted with buffer to 1 mg/lOO~. of 3jChydroxysteroid Estimation isomerase activity for pregnenolone
dehydrogenase-
The microsomal fraction was incubated with [3H]-pregneno10ne. Steroids, including C3H]-progesterone formed during incubation, were extracted from the incubation mixture with ether and treated with
R. E. OAKEY acetylating reagent thereby facilitating the separation of progesterone from remaining substrate, which was (20-0x0-5acetate converted to pregnenolone pregnen-3gyl acetate). C3H]-Progesterone was isolated by paper chromatography and the yield of this metabolite, corrected for manipulative loss by reverse isotope dilution technique, was calculated. In detail, Tris-HC1 buffer (0.2 mol/l, pH 7.3, 3 ml), NADP (50nmol in 100 ~1 buffer), [3H]-pregneno10ne (4 x lo4 c.p.m., in 50 ~1 ethanol) and microsomal fraction (100~1 containing 1 mg protein) were mixed and incubated at 37°C for 30 min. Acetone (2 ml) containing [‘4C]-progesterone (3 x IO3c.p.m., 400 pmol) was added to stop the enzyme reactions and the mixture was placed in boiling water for 10 min. After cooling the steroids were extracted into ether (2 x 3 ml). The ether was evaporated and the residue treated with acetylating reagent (0.5 ml pyridine, 0.5 ml acetic anhydride) for 8 h at 60°C. The reagent remaining was evaporated and the residue was mixed with progesterone and pregnenolone (0.5 ~01 of each), and chromatographed on paper in the solvent system light methanol-water SO-100°C); petroleum-(b.p. (100:96:4, by vol.). Radioactive progesterone (R, 0.32), clearly separated from pregnenolone acetate (RF 0.80), was located and eluted. A portion was used for measurement of the 3H/‘4C and hence calculation of the yield of product. The remainder was set aside for assessment of the purity of the progesterone by recrystallization. A sample of supernatant which had been boiled was included in each batch of incubations. The quantity of [3H]-progesterone formed was calculated from the amount of [3H]-progesterone isolated, corrected for manipulative loss assessed from the loss of [‘4C]-progesterone. The enzyme activity was expressed as the quantity of [3H]-progesterone formed per mg protein in 30 min. Estimation of steroid sulphatase activity
The rate of hydrolysis of C3H]-dehydroepiandrosterone sulphate was measured. Microsomal fraction (100 ~1 containing 1 mg protein), Tris-HCl buffer (0.2 mol/l, pH 7.3, 3 ml), NADP (50nmol in 100 ~1 buffer) and [3H]-dehydroepiandrosterone sulphate (2.3 x lo4 c.p.m., 6.8 nmol in 100~1 buffer) were mixed and incubated at 37°C for 30min. The enzyme reaction was stopped by heating the mixture at 100°C for 10 min. After cooling, the mixture was shaken with toluene (3 x 3ml) to extract any [‘HIdehydroepiandrosterone produced. The toluene was evaporated and the [“H ] determined. Incubations of boiled supernatant were included to serve as analytical controls. Enzyme activity was expressed as the amount of ether soluble tritium released per mg protein in 30 min. Liquid scintillation counting
For determination
of 3/I-hydroxysteroid
dehydro-
3/?-HSD in sulphatase deficiency
genase-isomerase activity, radioactive steroids were dissolved in toluene (5 ml containing butyi PBD 0.4% w/v and methanol 2% v/v). Tritium and [“Cl were measured by liquid scintillation spectrometry (Model SL30 Intertechnique Ltd., Brighton, Sussex) at efficiencies of 32% and 57”/0respectively. On counting 3H in the presence of 14C a correction was made for those counts derived from 14C which appear in the 3H channel. Crossover from 3H to 14C channel was negligible. At least 10,000 counts of each isotope were collected. For determination of steroid sulphatase activity tritiated steroids were dissolved in buffer (100 ~1) and counted in 8 ml of xylene containing Triton X-l 14 (20%) PPO (0.3%) and methyl POPOP (0.2%) at an efficiency of 36%. RESULTS
Estimation of 3/l-hydroxysteroid isomerase activity
dehydrogenase-
Preliminary experiments demonstrated that the yield of [3H]-progesterone from the quantity of substrate selected was directly proportional to incubation time, up to 40min, and to the protein content, up to 1.5 mg. Incubation was therefore carried out for 30min with 1 mg of protein at pH 7.3 and in the presence of NADP (50 nmol). Repetitive estimation of the activity of portions from the same placental preparation, stored at -20°C showed that the analytical precision (expressed as a coefficient of variation) was < 10%. The effectiveness of the purification procedure is illustrated by the yield of [3H]-progesterone obtained on incubation of boiled microsomal fractions, which was always less than 0.1% (~40 c.p.m./mg protein). Additional evidence was provided by the stability of the ‘H/“C ratio of the isolated progesterone during recrystallisation. The ratios found were: starting ma-
Stored at -
XPC
477
terial 2.56; crystals: 1st crop 2.48, 2nd crop 2.51, 3rd crop 2.46; mother liquors: 1st liquor 2.50, 2nd liquor 2.62, 3rd liquor 2.61. Stability of 3b-hydroxysteroid dehydrogenaseisomerase activity on storage at - 20°C
Since most samples of sulphatase deficient placentae had been stored for various periods at -2O”C, it was necessary to assess whether there was deterioration of the enzyme activity during prolonged storage under these conditions. Portions of fresh placentae from an unaffected and a deficient pregnancy were stored at - 20°C. After I, 2 and 3 months the tissue was thawed, microsomal fractions were prepared and the enzyme activity measured. For the sulphatase deficient placenta the yields were 400,395 and 400 c.p.m./mg protein/30 min whilst the yields from the control placenta were 640, 650 and 630 c.p.m./mg protein/30 min. Therefore, during prolonged storage at -20°C there was no further deterioration of 3/I-hydroxysteroid dehydrogenaseisomerase activity, in either sulphatase deficient or control tissue. 3B-Hydroxysteroid dehydrogenase-isomerase activity from sulphatase deficient and control placentae The enzyme activity of preparations of 8 placentae from sulphatase deficient pregnancies and I5 control placentae in which the baby was male (all after storage at -20°C) are shown in Fig. 1. The mean activity ( f SD) for preparations of sulphatase deficient placentae (330 * 40 c.p.m./mg protein/30 min) was significantly less (P < 0.05) than the mean value for the unaffected controls (600 k 60 c.p.m./mg protein/30 min). However, when microsomal fractions from the same 15 control placentae and 1 deficient placenta were prepared without freezing, the enzyme activity
Fresh .
Fig. 1. Yield of C3H]-progesterone (c.p.m./mg protein/30 min) from [“HI-pregnenolone, by 1O$OOg supernatant from sulphatase deficient placentae, 0; from control placentae associated with male infants, A; and from control placentae associated with female infants, B.
1. MARTON and R. E. OAKEY
478
for the controls (2240 f 675 c.p.m./mg protein/30 min) was indistinguishable from that of sulphatase deficient tissue (2 I70 c.p.m./mg protein/30 min). although after freezing and thawing, the enzyme activity of this particular tissue was only 350 c.p.m./mg protein/30 min. These results demonstrate that the process of freezing and thawing causes lower activity of placental 3/I-hydroxysteroid dehydrogenase-isomerase activity and this effect is more evident in sulphatase deficient placentae. l@ct
of the sex qf the
dehydrogenase-isomerase
fetus 011 3/?-hydro.u_vsteroid actiuit)
During the course of this work 3/&hydroxysteroid dehydrogenase-isomerase activity was measured in microsomal fractions from 12 placentae where the baby was female and from 15 where the baby was male (Fig. 1). When measured on preparations derived from tissue which had not been frozen, the mean enzyme activity of placentae associated with female babies (3480 rf: 760 c.p.m./mg protein/30 min) was significantly higher (P < 0.05) than that from placentae where the baby was male (2240 + 670c.p.mJmg protein/30min). However, a/fter storage of the tissue at -2O”C, enzyme activity was diminished in both to 710 f 80 and 600 + 60 c.p.m./mg groups protein/30 mill respectively and no significant difference between the mean values was apparent. effect of freezing
and thawing
011 steroid
sulphatase
activity
Steroid sulphatase activity of microsomal fractions from samples of 8 sulphatase deficient and 27 unaffected placentae after storage at -20°C and from 1 defi-’ cient and 27 normal fresh placentae was measured. The mean activity (*SD) in preparations of frozen was 970 _+ 736 c.p.m./mg deficient placentae protein/30min whilst that shown by the single defiwithout freezing was tested cient placenta 1102c.p.m./mg protein/30 min; that of boiled tissue was 620 + 460 c.p.m./mg protein/30 min (II = 6). These mean valuks were significantly less (P < 0.001) than mean values from control placentae tested fresh (17020 + 2070 c.p.m./mg protein/30 min II = 6) or after freezing (I 4720 f 920 c.p.m./mg protein/30 min II = 27). The mean values for control placentae tested fresh or after freezing are significantly different (P = 0.01) indicating that storage at -20°C and thawing has a small deleterious effect on sulphatase activity. When the activities of the fresh and thawed sappIes from each of the six control placentae were compared by a paired t-test, the loss of activity due to treatment was still significant (P < 0.02). There was no difference in sulphatase activity in placentae associated with male or female fetuses. DISCUSSION
The results reported here demonstrate
that the ac-
tivity of 3/Ghydroxysteroid dehydrogenase-isomerase for pregnenolone in placentae deficient in steroid sulphatase is significantly less than that found in normal placentae. provided measurements are made after storage of the tissue at -20’C. This finding confirms, on a larger number of placentae, our earlier results [3,4]. It may also provide an explanation of the lower activities than normal of steroid metabolising enzymes which were found in steroid sulphatase deficient tissue previously subjected to - 20°C [Z-5]. When the 3/Ghydroxysteroid dehydrogenaseisomerase activity was measured without freezing, the single sample of tissue deficient in steroid sulphatase which was available showed 3P-hydroxysteroid dehydrogenase isomerase activity equal to that of control tissue. If this is confirmed in subsequent studies and also applies to other placental enzymes involved in the conversion of androgen sulphates to estrogens then it appears that only one enzyme or complex~steroid sulphatase-is deficient. Thus it would be expected that mutation of only one gene would be required to cause this condition. This gene is apparently located on the short arm of the X-chromosome. adjacent to those controlling the development of X-linked ichthyosis and to the Xg” blood group locus [lo]. A search for associations between steroid sulphatase deficiency and other inherited abnormalities may therefore be of value in mapping the locus of associated genes. Mean placental 3P-hydroxysteroid dehydrogenaseisomerase activity associated with male infants was significantly less than that associated with females, The reason for such a difference is not clear. It may be due to the presence of different quantities of endogenous steroids which act as inhibitors of the enzyme activity, for example testosterone or androstenedione [I I]. A significant difference in the mean concentration of testosterone in cord blood from male and female infants has been established [12]. but differences linked to fetal sex in the concentrations of other steroids have not been reported. A report of differences in progesterone concentrations in umbilical vein blood, associated with fetal sex, in the rhesus monkey [13] has not been confirmed in the human [1416]. Our finding of differences in 3p-HSD activity associated with fetal sex therefore requires independent corroboration. Ackrlowledger,lrrltsWe
thank the Hungarian Ministry of Health for financial support (to I.M.) and the many Biochemists and Obstetricians who assisted with the supply of tissue and urine.
REFERENCES I. France J. T. and L&ins G. C.: Placental sulfatase dkficiency. J. ch. Endocr. Met& 29 (1969) 1X-141. 2. France J. T.. Seddon R. J. and Liggins c. C.: A study of pregnancy with low estrogen production due to placental sulfatase deficiency. J. clh. Endocr. Metah. 36 (1973) 1-9.
3P-HSD in sulphatase deficiency 3. Oakey R. E., Cawood M. L. and Macdonald R. R.: Biochemical and clinical observations in a pregnancy with placental sulphatase and other enzyme deficiencies. Clitr. Eftdocr. 3 (1974) 131-148. 4. Prooth S.: A study of placental sulphatase deficiency: activities of placental enzymes involved in estrogen biosynthesis. M.Sc. thesis University of Leeds 1974. 5. Osathanondh R., Carrick J., Ryan K. J. and Tulchinsky D.: Placental sulfatase deficiency: A case study. J. c/i!!. Entlocr. M~rah. 43 (1976) 208-214. 6. Tabei T. and Heinrichs W. L.: Diagnosis of placental sulfatase deficiency Aar. J. Ohsrut. G_rnrc. It4 (1976) 4094 14. 7. Braunstein G. D., Ziel F. H.. Allen A.. Van de Velde R. and Wade M. E.: Prenatal diagnosis of placental steroid sulfatase deficiency. rlnt. J. Ohsrcr. Grnec: 126 (1976) 716-719. 8. Oakey R. E.: Placental sulphatase deficiency: Anteparturn differential diagnosis from foetal adrenal hypoplasia. Clirt. Endorr. 9 (i 978) Xi-XX. 9. Lowry 0. H.. Roseborough N. J.. Farr A. L. and Randall R. J.: Protein measurement with the Folin phenol reagent. 6. hiol. chcrn. 193 ( 1951) 265-275.
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IO. Shapiro L. J.. Weiss R., Buxman M. M., Vidgoff J., Dimond R. L., Roller J. A. and Wells R. S.: Enzymatic basis of typical X-linked ichthyosis. Lancer ii (19’78) 756757. I 1. Townsley J. D.: inhibition of placenta1 3fi-hydroxysteroid dehydrogena~ by naturally occurring steroids.. Acta atdocr. Copedt. 79 (I 975) 740-748. 12. Forest M. G., Cathiard A. M. and Bertrand J. A.: Evidence of testicular activity in early infancy. J. c/in. Ernlocr. Mrtuh. 37 (1973) 148-151. 13. Hagemenas F. C. and Kittinger G. W.: In influence of fetal sex on plasma progesterone levels. f+docrinolqy_t 91 (1972) 2.53-256. 14. Hagemenas F. C. and Kittinger G. W.: The influence of fetal sex on the levels of plasma progesterone in the human fetus. .I. &I?. Ettducr. Mrrah. 36 (1973) 389-391. 15. Tulchinsky D. and Okada D. M.: Hormones in human pregnancy IV. Plasma progesterone. Ant. J. Ohsrer. Gyttec. 121 (1975) 293-299. 16. Antonipillai 1. and Murphy B. E. P.: Serum oestrogens and progesterone in mother and infant at delivery. Br. .I. Ohsrrt. G,~nurc. 84 (I 977) 179- 185.