Placental 17β-hydroxysteroid oxidoreductase, lactate dehydrogenase and malate dehydrogenase during the latter half of pregnancy in the mouse

J. Steroid Biochem. Moiec. Biol. Vol. 46, No. 1, pp. 61-67, 1993 Printed in Great Britain. All rights reserved

0960-0760/93 $6.00 + 0.00 Copyright © 1993 Pergamon Press Ltd

PLACENTAL 17//-HYDROXYSTEROID OXIDOREDUCTASE, LACTATE DEHYDROGENASE AND MALATE DEHYDROGENASE DURING THE LATTER HALF OF PREGNANCY IN THE MOUSE CHARLESH. BLOMQUIST,*HuGH C. HENSLEIGH,DONNA L. BLOCKand LINDAA. Department of Obstetrics and Gynecology, Ramsey Clinic and St Paul-Ramsey Medical-Center, St Paul, MN 55101 and Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, School of Medicine, University of Minnesota, Minneapolis, MN 55455, U.S.A. (Received 21 October 1992; accepted 26 February 1993)

S m m l r y - - T h e specific activity of 17~-hydroxysteroid oxidoreductase (17-HOR) with estradiol-17~ (E2), estrone (El) and testosterone (T), as well as that of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) were measured in homogenates of CF-1 mouse placenta during the latter half of pregnancy. 17-HOR activity with E2 and T increased over 100-fold between days 9 and 12, and 3- to 4-fold between days 15 and 19, with no further change to day 21. In contrast, activity with E1 increased 39-fold between days 9 and 12, 3.8-fold between days 15 and 19 but then decreased between days 19 and 21. The E2/T activity ratio was constant while the E2/EI ratio increased between days 9 and 21. LDH increased 2-fold between days 9 and 12 with no further increase to day 19. MDH was constant from day 9 to 19. Activity with E2 was inhibited by T, 5=-dihydrotestosterone (5=-DHT) and DHA but not by El, androstenedione (A) or 20=-dihydroprogesterone. Activity with T was inhibited by E2, 5cz-DHT and DHA, but not by A. In contrast, activity with El was inhibited by A and DHA but not by E2, T or 5=-DHT. The results suggest placental 17-HOR is developmentally regulated. Although the results are also suggestive of multiple forms of 17-HOR, a single enzyme with an ordered kinetic mechanism cannot be ruled out.

INTRODUCTION

tochemically in rat placenta from midgestation to term [10, 11]. Activity with either E2 or T increases during the latter half of pregnancy, suggestive of developmental regulation of this enzyme, as well. The pattern of development of 17-HOR in mouse placenta has not been characterized but there have been findings which suggest it may differ from that in rat. For example, Wu and co-workers[12, 13] quantitated 17-HOR in mouse and rat preimplantation embryos by direct assay and reported that in mouse embryos oxidative or dehydrogenase activity (E2 to El) increases between days 1 and 5, while reductase activity (El to E2) decreases over the same time period. In contrast, both activities decrease in rat preimplantation embryos. Further evidence that developmental regulation of placental estrogenmetabolizing enzymes may differ between mouse and rat comes from the work of Hobkirk et al. [14]. They showed that estrogen sulfotransferase in mouse placenta increases approx. 10fold between days 12 and 18 of pregnancy, but is undetectable in rat placenta over the same interval. On the basis of these observations, this

17~-Hydroxysteroid oxidoreductase (17-HOR) activitieswith estradiol-17// (E2) and estrone (El), as well as testosterone (T) and androstenedione (A), catalyze important steps in steroidogenesis in ovary and placenta. The relative roles of these organs in hormone synthesis, and shifts in the patterns of steroid production during pregnancy differ among animal species. During the latter half of pregnancy in the rat and mouse, the placenta is the principal source of steroid precursors for maternal ovarian estrogen formation [I-4]. Recent studies have established that in rat placenta cholesterol side-chain cleavage cytochrome P450 and 17u-hydroxylase/ 17,20-1yase cytochrome P450 are developmentally regulated at the level of mRNA and protein synthesis [4-6]. 17-HOR activities are wide-spread in rat and mouse tissues [7-9] and have been detected his*To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, St PauI-Ramsey Medical Center, 640 Jackson Street, St Paul, MN 55101, U.S.A. 61

62

CHAgLESH. BLOMQUISTet al.

investigation was designed to quantitate and characterize 17-HOR activity with both E2 and El in homogenates of mouse placenta from day 9 to 21 of pregnancy. Additionally because of the primary role of mouse placenta in androgen formation during the latter half of pregnancy, the activity of mouse placental 17-HOR with T was also examined. As an initial approach to comparing developmental patterns of enzymes of steroidogenesis with those of intermediary metabolism, we also assayed lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) in placental homogenates at days 9, 12 and 19 of pregnancy.

ized by hand in a glass Dounce homogenizer containing ice-cold buffer composed of 0.25 M sucrose, 10 mM 2-mercaptocthanoi and 0.1 M Bicine, pH 9.0. Homogenates were then centrifuged at 1500g for 10 min. The supernatants were filtered through four layers of gauze and stored on ice until assayed for enzymatic activity within 2-4 h. Cytosol and microsomes

Cytosol and a particulate fraction enriched in microsomes were prepared by centrifugation of homogenates at 105,000g for 60rain. The supernatant from this step was taken as the cytosol. The pellet was resuspended in buffer and washed by repeat centrifugation, after MATERIAI~ AND MErHODS which it was suspended in buffer and designated microsomes. The protein content of homogenMaterials ares, cytosols and microsomes was quantitated Reagents and supplies were purchased by the method of Markwell et al. [15], with from the following source: [6,7-3HIE2 bovine serum albumin as the protein standard. (1.5 TBq/mmo!, 40 Ci/mmo!) and [2,4,6,7-31-!]E! (4.0 TBq/mmol, 108 Ci/mmol), Ametsham Enzyme assays Corp. (Arlington Heights, IL); [l,2-~I4Tr For 17-HOR activity measurements, 10-/~i (1.9 TBq/mmol, 52.5 Ci/mmol), Dupont NEN aliquots of homogenate, cytosol or microsomes Products (Boston, MA); Bicine (N,N-b'~2- were combined with 10/~1 of reaction mixture hydroxyethyl]glycine), Hepes (N-2-hydroxy- containing 1.0 mM NAD and 2.0/~M [JH]E2 or ethylpiperazine-N'-2-ethanesutfonic acid) and [3HIT in 0.08 M Bicine, pH 9.0, or 1.0mM bovine serum albumin, Sigma Chemical Co. (St NADH and 2.0#M [3I-1]E1 in 0.08 M Hepes, Louis, MO); NAD and NADH, Pharmacia pH 7.2. Reaction mixtures were incubated at LKB Biotechnology, Inc. (Piscataway, N J); 37°C in tightly-stoppered tubes for 30 rain after sodium pyruvate and oxaloacetic acid, Cal- which they were transferred in total to the biochem (San Diego, CA); silica gel HL plates, preadsorbant layer of silica gel HL plates and Analtech, Inc. (Newark, DE); Ecolume, ICN taken to dryness in a stream of air. Unlabeled Schwarz Mann (Cleveland, OH). E2 and E1 or T and A were added as carriers Animals

CF-1 mice were purchased from Charles River Laboratories (Wilmington, MA). All procedures were approved by the Animal Care and Use Committee of St Paul-Ramsey Medical Center. Female mice (19-21 g) were housed in an air-conditioned, controlled-temperature room and maintained on a 12-h light/dark cycle. They were placed individually with males and allowed to breed naturally. The p r ~ m ~ of a vaginal semen plug was t~ken as day 1 of pregnancy (term= 21-22 days). The females were then separated from the males for the remainder of the pregnancy. Placental homogenates

Animals were sacrificed by cervical dislocation. Placentas were carefully diueeted from fetuses and membranes, weighed and homogen-

in 30/~1 of ethanol and also taken to dryness The plates were developed at room temperature in benzene acetone (4:1) and air dried. Steroids were located by a light spraying with water and the plates again allowed to dry. Steroid-containing spots were scraped into plastic vials containing 10 ml of Ecolume for liquid scintillation counting. Product formation was quantitated on the basis of cpm in product steroid as a percentage of total cpm recovered in substrate and product, as described previously [16] and then expressed as nmol of product/rag protein- 30 rain. Assays were run in duplicate at each of 2 or 3 dilutions of protein. Reaction mixtures lacking homogenate were run as controls. LDH and MDH were assayed spectrophotometrically in 10 x 10mm cuvets with 3.0-mi reaction mixtures containing homogenate, 0.18M Hepes, pH 7.2, 0.22raM NADH and

63

Placental 17p-hydroxysteroidoxidoreductase 1.0 mM pyruvate (LDH) or 0.2 mM oxaloacetic acid (MDH). Absorbance at 340nm was recorded at 30-s intervals for 5 min following the initiation of reaction by the addition of substrate. Reaction rates were converted to /~mol/mg protein, min on the basis of a molar absorbance for N A D H of 6.22 × 103.

Data analysis Mean values were compared by Student's t-test or analysis of variance in combination with the Newman-Keuls multiple comparisons test. Apparent Michaelis constants were estimated graphically by direct linear plots of initial velocity as a function of substrate concentration, as described previously [16].

17-HOR activity of placental homogenates Specific activities with E2, El and T on days 9, 12, 15, 19 and 21 of pregnancy are shown in Fig. 1. Activity with each substrate was at

120~-E2-.~E1 40~-uP';O.m, I,i ~"~ ":~gl :::"f-)"F''P":~' EI 26.0 'P'°~'m"P":°'~jm' I psO.m IPSO.Ol

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15

19

l

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RESULTS

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Pregnancy

12 15 19 21 Day of Fig. 2. E2/EI and E2/T specificactivityratios for placental homogenateson days 9, 12, 15, 19 and 21 of pregnancy.The valuesare the mean + SE for homogenatesfromthe number of animals indicated in parenthvses.

the limit of detection on day 9 and increased markedly between days 9 and 12. The relative increases were 136-fold (0.16+0.10 t o 21.22 + 10.40 nmol/mg ; 30 min), 174-fold (0.08 + 0.06 to 13.95 + 7.77 nmol/mg • 30 rain) and 39-fold (0.04 + 0.02 to 1.36 + 0.53 nmol/ rag. 30 rain) for E2, T and El, respectively. A second increase was observed in each case between days 15 and 19, with relative increases of 3.6-, 4.1- and 3.8-fold for activity with E2, T and El. Between days 19 and 21, dehydrogenase activity with E2 or T was constant, while reductase activity with E1 decreased (P ~<0.01). When specific activity ratios were compared (Fig. 2), the E2/EI ratios on days 15 and 19 were significantly larger than that on day 9. In contrast, E2/T ratios were constant between days 9 and 21. To assess if these increases reflected developmental regulation of 17-HOR or were increases in a constitutive activity related to placental growth, placental weight and protein yield in homogenates on days 9, 12, 15, 19 and 21 were compared. As shown in Fig. 3, placental weight increased approx. 5-fold between days 9 and 19 and was then essentially constant to day 21. The yield of protein per g of tissue did not vary between days 9 and 21.

Fig. 1. 1 7 - H O R activities with E2, El and T of mouse Subcellular distribution of 17-HOR phtaental homolanates on days 9, 12, 15, 19 and 21 of Figure 4 shows 17-HOR specific activities prellnancy.The valuesate the mean + SE for homoganates from the number of animals indicated in parentheses. with E2 and E1 ofcytosol and microsomes from

SBMB46/1--F

CHARLES H. BLOMQUIST et al.

64

p <0.01

I=

t

1001,.

I

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I

--,-

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homogenates on days 10 and 19. Day 10 placenta was used because cytosolic activity was barely detectable on day 9. Activity increased in both subcellular fractions between days 10 and 19. With either E2 or E1 as substrate, microsomal specific activity was approx. 15-fold greater than that of cytosol on both days. The E2/E1 ratio on day 19 exceeded that on day 10 for both cytosol and microsomes, 16.3 + 5.7 vs 8.0 _+ 4.8 and 11.5 + 5.6 vs 7.1 _+ 2.5. Although microsomal 17-HOR specific activity was higher than cytosol, approx. 70% o f the total activity with either E2 or E1 was recovered in the cytosol.

LDH and MDH in placental homogenates 10 9

12

15

19

21

Oey of Preom.~ Fig. 3. Placental weight and protein yield per g of tissue fresh weight on days 9, 12, 15, 19 and 21 of pregnancy.The values are mean + SE for the number of animals indicated in parentheses.

As another approach to assessing possible developmental regulation o f 17-HOR, the activities o f the putative constitutive enzymes L D H and M D H were measured in homogenates of day 9, 12 and 19 placentas (Fig. 5). L D H specific activity increased < 2-fold between days 9 and 12 and then was constant to day 19. M D H specific activity did not vary between days 9 and 19, results which would be expected with constitutive enzymes.

Kinetic properties of 17-HOR and inhibition by steroids F..2-~E1

T

140

To estimate K , values for E2, T and N A D (data not shown), samples of placental hom-

E1 --~F..2

2

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10.0

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(6)

(4)

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12,0

0.0

8.0

0.4 0.2

1.0-

T

4.0

(I)

10

(4)

2_

((I)

(4)

10

10

Day of Pm0nm,~ Fig. 4. 17-HORspecificactivitiesof micrmomesand cytosole from day 10 and 19 placentas. The values are the mean 4- SE ofdata from the number of animals indicatedin parentheses. *Differentfrom day 10 value at P ~0.01.

II

-,.-

2.01

2.0

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I

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4.0"

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oey o~pm0m.cy Fig. 5. LDH and MDH sim:ific acfiqties of plaamtsl

h o ~ on days 9, 12and t9of ~ , ~ are tile ~ ± S E of homolmmm ~ the n ~ animals indicated in parentheses.

of

Placental 17fl-hydroxysteroid oxidoreduotase

ogenate (days 16-t8) were assayed in duplicate at pH 9.0 with either 0.5 mM N A D and 0.5, 1.0, 2.0, 4.0 or 8.0/~M [3I-I]E2 or [3H]T, or 1.0/~M steroid and 0.2, 0.4, 1.0, 2.0 or 10.0 mM NAD. In initial experiments it was determined that aotivity was optimal for either steroid at pH 9-10. Apparent K, values were 0.8/~M for E2 and 1.0 ~M for T. The Ks for NAD was 0.8 mM with either substrate. At 5.0 mM coenzyme the NAD/NADP activity ratio with E2 was 2.24 + 0.49 (n = 5). For the conversion of E1 to E2, activity was essentially constant between pH 6.2 and 8.2. At pH 7.2 the K,, for El was 4.3/~M. The N A D H / N A D P H activity ratio (5.0 mM coenzyme) was 2.26 + 0.19 (n = 5). Because estimates of Km values for different substrates with homogenates are difficult to interpret with regard to the number of enzyme forms present, the susceptibility of activity with E2, T and El to inhibition by C~9 and C21 steroids was also examined. Dehydrogenase activity with E2 was inhibited by both T and 5u-dihydrotestosterone (5a-DHT), 20a-dihydroprogesterone (20a-DHP) was ineffective (Fig. 6). When various 17B-hydroxysteroids and 17ketosteroids were compared at an inhibitor/substrate ratio of 100:1, activity with E2 was inhibited by T, 5a-DHT and DHA but not by E1 or A. The latter steroid had a slight activating effect (Fig. 7). Similarly, activity with T was inhibited by 5a-DHT, DHA and E2 but not by A or E1 (Fig. 7). In marked contrast with these results, activity with E1 was inhibited by

65

[ ] E2---~EI m

[ ] T---~A

i lm 100

SO

A

DHA

T

E1

F..2

Fig. 7. Effects of A, DHA, 5a-DHT, T, El and E2 on 17-HOR activity with F_,2 or T. Reaction mixtures (20/41 total volume) containing homogenate (day 16-18), 0.5 mM NAD, 1.0 [JH]E2 or [~H]T and 100~M steroid additive in 0.08 M Bicine, pH 9.0, were incubated at 37°C and analyzed as described under Materials and Methods. Control assays lacked steroid additive. The values are the mean ± SE of three separate experiments in which samples were assayed in duplicate. *Different from control at P ~ 0.01; **different from control at P < 0.05.

the 17-ketosteroids A and DHA but not by the 17fl-hydroxy compounds E2, T or 5a-DHT (Fig. 8). DISCUSSION

BoRe e t a/.[10] detected 17-HOR activity with E2 and T histochemicaUy in day 12 rat

E1 ---~F~

T

lf~

a~

I

.L

100

v-

SO

10

20

90

40

80

[&z-OHT, Tor20(x-DHP] (pmol/I) Fig. 6. Inhibition of placental 17-HOR activity with E2 by T, 5a-DHT and 20a-DHP. Reaotion mixtures (20/~1 total volume) containing 12.2/~g of protein, 1.0#M [3I-I]E2, 0.5 mM NAD and 0, 5.0, 25.0 or 50.0/~M T (O), 5a-DHT (@) or 20~-DHP (Q) in 0.05 M Bicine, pH 9.0, were incubated at 37°C for 30rain and then ,n~lyzed by thin layer chromatography as described under Materials and Methods. The values are the mean ± SD of duplicate assays.

Ph A

~

&~l~fr

T

!~

Fig. 8. Effects of A, DHA, 5a-DHT, T and E2 on 17-HOR activity with El. Reaction mixtures (20/AI total volume) contained homogenate, 0.5 mM NADH, 1.0 pM [3H]E! and 100/~M steroid additive in 0.08M Helms, FH 7.2. were incubated and analyzed as described in Fig. 7. The values are the mean ± SE of two (E2 as inhibitor) or three separate experiments in which samples were a ~ y e d in duplicate. *Different from control at P ~g0.01.

66

CHARLESH. BLOMQUISTet aL

placenta. In a subsequent histochemical study, also of rat placenta, Ferguson and Christie [11] found that activity with E2 and T is detectable at trace levels on day 10 1/2, increases to the 1 + to 2 + level by day 17 1/2 and then to the 3 + level by day 20 1/2. The results presented here indicate a similar two-phase pattern of development occurs in mouse placenta. This biphasic pattern in which relative increases in 17-HOR greatly exceed the rate of placental growth, and differ markedly from the behavior of the putative constitutive enzymes LDH and MDH, is suggestive of developmental regulation of 17HOR. Whether a single enzyme or multiple forms of 17-HOR are present in mouse placenta remains to be established. Ghraf e t a l . [7] presented results which suggest not only that there are multiple forms of 17-HOR in rat tissues but that there are differences among tissues in their regulation, as well. They did not present data on placenta. Bogovich and Payne[17] separated and partially purified from rat testes a 17//hydroxysteroid dehydrogenase and a 17-ketosteroid reductase with steroid specificities similar to those reported here. Wu and coworkors[12, 13] observed differing developmental patterns for 17-HOR activity with E2 and E1 in preimplantation mouse embryo and Milewich etal. [8, 9] reported differences in E2/E1 activity ratios among tissues of adult male and female mice. In the present study the similar Km values for E2 and T, the constancy of the E2/T activity ratio, the inhibition of activity with E2 by T, 50e-DHT and DHA, but not by El, A or 20a-DHP, and the inhibition of activity with T by E2, 50c-DHT and DHA but not by A, are consistent with a form of 17-HOR reactive as a dehydrogenase with both Cis and C~9 17~hydroxysteroids, but with a low affinity for E1 and A as well as C2~ steroids. Reductase activity with El, however, appears to have a distinctly different specificity, with an extremely low affinity for 17//-hydroxysteroids. These specificity differences, the changing E2/E1 activity ratios and the presence of activity in both cytosol and microsomes are suggestive of multiple forms of 17-HOR. However, the differences in steroid specificity shown in Figs 7 and 8 could also reflect the presence of a single enzyme with an ordered kinetic mefdmnism in which steroid substrates do not bind to free enzyme but in which 17~-hydroxysteroids bind only to an enzyme-NAD complex and 17-ketosteroids only to an enzyme-NADH complex.

Enzyme purification and the development of eDNA probes will be necessary to resolve this question. The biphasic increases in mouse placental 17-HOR occur in concert with increases in maternal serum levels of androgens and estrogens [I, 18, 19]. Placental 17-HOR may play a role in affecting steroid levels within the maternal-fetal-placental unit. During the latter half of pregnancy, the placenta is the major site of formation of androgen precursors for ovarian estrogen formation [1-4]. The increasing capacity of the placenta for the conversion of T to A correlates well with the fact that androstenedione is the major C,9 steroid product [1, 2]. Because mouse placenta lacks aromatase, it is not a site of de novo estrogen formation [20, 21]. However Hobkirk e t a l . [14], on the basis of their observation of marked increases in estrogen sulfotransferase activity, proposed that the placenta may control fetal exposure to E2 by converting it to the 3-sulfate. The findings of this investigation that 17-HOR activity with E2 increases and also that the apparent capacity of the placenta for reaction in the oxidative direction exceeds reductase activity, thus favoring E! formation, lend further support to this concept. Thus 17-HOR may function along with estrogen sulfotransferase to regulate E2 levels within the developing mouse fetus. Acknowledgements--The authors are grateful to Dr Joseph P. Holt, Jr for his encouragement, to Ms Linda SackeRLundeen and Mr Wayne Lundeen for help with the figures and to Ms Mary Gunderson, Ms Margaret Roddy and Mr Herb Crandull and the staff of the Animal Care Facility for skilful technical assistance. This work was supported by Grants 8458 and 8485 from the Ramsey Foundation. REFERENCES I. Soares M. J. and Ta~amantes F.: Midpresmncy elevation of serum andrmtenedione levels in the C~H/HeN mouse: Placental origin. Da/ocr/no/ogy 113 (1983) 1408-1412. 2. Jackson J. A. and Albrecht E. D.: The development of placental andrmtenedione and testosterone production and their utilization by the ovary for ammati~tion to estroi~ during fat pregnancy. Biol. Reprod. 33 (1985) 451-457. 3. Sridarlm R. and Gibori G.: Placcmtal-ovarian relationship in the control of teatmtarone m~-tion in the rat. P/acenta II (1987) 327-333. 4. Wlmdutw M. L., Johnson D. C., ghan I., Ezkaltein B. and Gibori G.: Phteentul secretion of androgetm in the rat. Rm/ocr/ao/ogy 119 (1986) 2642-2648. 5. Gibori G., Khan I., Warshaw M. L., McLean M. P., Puryear T. K., Nelson S., Durkee T. J., A~aar S., Steimehneider A. and Rao M. C.: PIacvntal,~ieflwd refftlators and the complex control of luteal cell fenction. Recent Prof. Horm. Res. 44 (1988) 377-429.

Placental 17~-hydroxysteroid oxidoreductaze 6. Durkee T. J., McLean M. P., Hales D. B., Payne A. H., Waterman M. R., Khan I. and Gibori G.: P4501~, and P450scc gene expresion and restdation in the rat placenta. E,ndocrmo/ogy 138 (1992) 1309-1317. 7. Ghraf R., Vetter U. and ScJariefers H.: Organ specificity in the sexual differentiation of the 17~-hydroxysteroid dehyd~roge~__~ activities of the rat. Hoppe-Seyler's Z. Physiol. Chem. 3 ~ (1974) 543-550. 8. Milewich L., Garcia R. L. and Gerrity L. W.: Steroid sulfatase and 17fl-hydroxysteroid oxidoreductaee activities in mouse tissues. J. Steroid Biochem. 21 (1984) 529-538. 9. Milewich L., Garcia R. L. and Gerrity L W.: 17flHydroxysteroid oxidoreductase: A ubiquitous enzyme. Interconversion of estrone and estradiol- 17p in BALB/c mouse tissues. Metabol~m 34 (1985) 938-944. 10. Botte V., Materazzi G. and Chieffi G.: Histoehemical distribution of 3-hydroxysteroid dehydrogenase and 17,,- and 17~-hydroxysteroid dehydrogenases in the placenta and foetal membranes of the rat. J. Endocr. 34 (1966) 179-183. 11. Fergnmn M. M. and Christie G. A.: Distribution of hydroxysteroid dehydrogenases in the placentae and foetal membranes of various mammals. J. Endocr. 38 (1967) 291-306. 12. Wu J. T. and Lin G. M.: The presence of 17/~-hydroxysteroid dehydrogermse activity in preimplantation rat and mouse blastocysts. J. Exp. Zoo. 7.20 (1982) 121-124. 13. Wu J. T. and Matsumoto P. S.: Changing 17flhydroxysteroid dchydrogenase activity in preimplantation rat and mouse embryos. Biol. Reprod. 32 (1985) 561-566.

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14. Hobkirk R., Cardy C. A., Saidi F., Kennedy T. G. and Girard L.: Development and characteristics of an oestrosen sulphotransferase in placenta and uterus of the prelpumt mouse. Biochem. J. 216 (1983) 451-457. 15. MarkweH M. K., Haas S. M., Tolbert N. E. and Bieber L. L.: Protein determination in membrane and lipoprotein samples: Manual and automated procedures. Met& Enzym. 72 (1981) 296-303. 16. Blomquist C. H., Lindemann N. J. and Hakauson E. Y.: Inhibition of 17~-hydroxysteroid dehydrosenase (17flHSD) activities of human placenta by steroids and non-steroidal hormone agnnists and antagonists. Stero/d~ 43 (1984) 571-586. 17. Bogovich K. and Payne A.: Purification of rat testicuiar mierosomal 17-ketosteroid reductase. Evidence that 17ketusteroid reductase and 17p-hydroxysteroid dehydro8enase are distinct enzymes. J. Biol. Chem. 255 (1980) 5552-5559. 18. Barkley M. S., Michael S. D., Geschwind I. I. and Bradford G. E.: Plasma testosterone during pregnancy in the mouse. Endocrino/ogy 100 (1977) 1472-1475. 19. Barkley M. S., Geschwind I. I. and Bradford G. E.: The gestational pattern of estradiol, testosterone and progesterone secretion in selected strains of mice. Biol. Reprod. 20 (1979) 733-738. 20. Vinson G. P. and Chester-Jones I.: The capacity of mouse fetus and placenta to synthesize steroids from progesterone in vitro. Gen. Comp. Endocr. 4 (1964) 415-419. 21. Riembesa R., Marchut M. and Warehol A.: Formation and metaboSsm of progesterone by the mouse placenta in vitro. J. Steroid Biochem. 2 (1971) !11-119.