Endocrinol. Japon. 1967, 14(3), 251～258

BIOSYNTHESIS OF IN IN VITRO PERFUSION

OF THE HUMAN PLACENTA

TETSUYANAKAYAMA, KIYOSHIARAI, TORU TABEI, TAKUMIYANAIHARA, KAZUO SATOH, KEIJI NAGATOMI AND YOSHIHISAFUJITA Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, Tokyo

SYNOPSIS

Perfusions of the human term placenta with radioactive ,

, DHEA, , and -17ƒÀ as precursors

were carried out and the formation of estrogens was investigated. After per-

fusion for three hours with human whole blood, added with human chorionic

and cofactors, radioactive metabolites in the perfusate were studied. The radioactive metabolites were purified by chromatography at each

step of purification. Estrogens were measured by the Kober reaction, and the

radioactivity was counted by the gas flow counter. These experiments demon-

strated that C-19 were aromatized in a high yield by the perfused

human placenta to and estradiol-17ƒÀ in the ratio of approximately 3

to 1, but that it was not capable of converting to . It was also

noted that there was no conversion of estradiol-17ƒÀ to estriol. In the perfusion

study with DHEA, which is considered to be an important intermediate to

estriol in the feto-placental unit during pregnancy, 10.7% of the initial radio-

activity was recovered as a polar phenolic compound which behaved like 16-

epiestriol on preliminary identification. Furthermore, neither progesterone nor

cholesterol converted to estrogens in the human perfused placenta.

The human placenta is an abundant source estradiol-17ƒÀ to estriol, when HCG was added of production in late pregnancy. It to the perfusing fluid. He also identified tenta- is well known that as human pregnancy ad- tively 16-epiestriol in the perfusate. Andro- vances urinary excretion of estriol* increases stenedione has been shown to be converted at a rate greater than that of estrone and es- to estrogens in a system consisting of human

tradiol-17ƒÀ. Levitz et al. (1956) were unable placental microsomes (Ryan, 1959a). Path- to detect any evidence of conversion of es- ways of estrogen biosynthesis from C-19 tradiol-17ƒÀ to estriol in the perfused human steroids in the human placenta, have been

placenta. Troen (1961) demonstrated that the elucidated to some extent (Bagget, 1959; human placenta was capable of converting Dorfman and Gual, 1959; Ryan, 1959b). In

Received for publication. July 26, 1967. *The following abbrivations are used throughout this paper: Androstenedione: androst-4-ene-3 , 17-

dione (DHEA): 3ƒÀ-hydroxyandrost-5-en-17-one Testosterone: 17ƒÀ-

hydroxyandrost-4-en-3-one Progesterone: pregn-4-ene-3, 20-dione Cholesterol: cholesta-5-en-3ƒÀ-ol

Estradiol-17ƒÀ estra-1, 3, 5(10)-triene-3, 17ƒÀ-diol Estrone: 3-hydroxyestra-1, 3, 5(10)-trien-17-one

Estriol: estra-1, 3, 5(10)-triene-3, 16ƒ¿, 17ƒÀ-triol 16-epiestriol: estra-1, 3, 5(10)-triene-3, 168, 17ƒÀ-triol Endocrinol. Japon. 252 NAKAYAMA et al. Sept. 1967

the opinion of Little and Shaw (1961), pro- (Amersham, CFA 237, Batch 2,

gesterone is probably an intermediate in the 1ƒÊCi/28ƒÊg) synthesis of estrone and estradiol-17ƒÀ in this Radiochemical purity of these steroids was check-

organ. They have also demonstrated the con- ed by paper chromatography in the Bush system

version of progesterone to 17ƒ¿-hydroxypro- A.

gesterone in the placental homogenate. 2) Reference standards Though the conversion of cholesterol to Estradiol-17ƒÀ, estrone, estriol and 16-epiestriol

has been documented by Solo- were obtained from commercial sources. Purity- mon (1954 and 1960), no attempt has been of these standards was checked by paper chro-

made to observe the synthesis of estrogen matography and thin layer chromatography.

from cholesterol by perfusion of the human 3) Gonatropin (Teikoku Mfg. Co.,

placenta in vitro. Ltd.) was used as HCG. NADH was purchased Perfusion of the whole organ appears to from the Sigma Chemical Co. All reagents were

be a more physiological approach to its analytical grade and all solvents were redistilled

metabolic and synthetic activities than does before use. the study of homogenates, slices or perfused

isolated cotyledons. Technique of perfusion

The present investigation was undertaken The human placentas were obtained at normal

to examine the question of hydroxylation of term delivery. The two umbilical arteries and

estradiol-17ƒÀ or estrone at C-16 and also of the umbilical vein were cannulated by the

aromatization of the neutral steroids (andro- Nelaton catheters and flushed out via arteries

stenedione, testosterone and DHEA) by means with 300ml of heparinized saline. The placenta.

of in vitro perfusion of the human placenta. was attached to the perfusion apparatus, which

In addition, the metabolism of progesterone is illustrated diagrammatically in Figure 1. The

and cholesterol was also studied. perfusion fluid was circulated at a rate of 80 to 100ml/min., while being continuously oxygen-

MATERIALS AND METHODS ated by rotating filmoxygenator. A pump of DeBakey type gave a pulsatile movement with

Materials adjustable pressure and speed. The pressure

1) Radioactive steroids as tracers varied from 100 to 110mmHg. The whole circuit

Perfusion I: Androstenedione-4-14C: was kept in an airconditioned cabinet maintained

5ƒÊCi/200ƒÊg at 37•Ž. As perfusate, 150ml of human whole

(New England, NEC 136, Lot blood was used. The above-mentioned

No. 134-64-22, 1ƒÊCi/13ƒÊg) precursors dissolved in 0.2ml of propylene

Perfusion II: Testosterone-4-14C: 5ƒÊCi/200ƒÊg glycol, 6,000 I.U. of human chorionic gonado- (Amersham, CFA 129, Batch 7, tropin, 15mg of NADH, and 250mg of ox-

1ƒÊCi/13ƒÊg) ytetracycline were added to the perfusate. The

Perfusion III: Dehydroepiandrosterone-4-14C: perfusion time was three hours. Preliminary per- 3ƒÊCi/200ƒÊg fusions were performed to demonstrate that

(New England, NEC 206, Lot under the conditions used there was no histo- No. 134-98a-46b, 1ƒÊCi/7.09ƒÊg) logical evidence of rupture of blood vessels or

Perfusion IV: Progesterone-4-14C: 5ƒÊCi/200ƒÊg destruction of cells and nuclei. Histochemical

(Amersham, CFA 148, Batch 16, examinations of the placenta were made after 1ƒÊCi/12ƒÊg) the perfusion experiment, and the activities of

Perfusion V: Cholesterol-4-14C: 10ƒÊCi/150ƒÊg) acid phosphatase, alkaline phosphatase, succinic

(Amersham, CFA 128, Batch 43, dehydrogenase, nonspecific esterase and 3ƒÀ- 1ƒÊCi/15ƒÊg) hydroxysteroid dehydrogenase were assessed. Perfusion VI: Estradiol-17ƒÀ-16-14C: 1ƒÊCi/200ƒÊg Vol.14, No.3 ESTROGEN FORMATION IN THE PLACENTA 253

Measurement of radioactivity and determination the extraction of plasma without hydrolysis was

of estrogens carried out three times with the same volume of

These procedures were carried out as described an ethyl acetate-ether mixture (1: 1). The residue

elsewhere (Nakayama et al., 1966a). was dissolved in 70% methanol, stored at -15•Ž

overnight, and lipid was removed by refrigerated

Formation of derivatives centrifuge. In perfusion V, the residue of 70%

Estrogens were methylated with dimethyl methanol was partitioned between pentane and

sulfate according to Brown (1955). 90% methanol. The 90% methanol fraction was

subjected to the separation of the phenolic frac-

Chromatography tion (Ryan and Smith, 1965). The pentane-solu-

Paper chromatography was developed on ble fraction contained unchanged labelled cho-

Whatman No. 1 filter paper in the Bush's solvent lesterol.

systems (A, B 1 and C)(Bush, 1961). Localization Before separation of the phenolic fraction,

of the standard estrogens was made by spraying known amount of estrone, estradiol-17ƒÀ, estriol

with potassium ferric cyanide-ferric chloride and 16-epiestriol were added to the extract as

reagent (Eberlein and Bongiovanni, 1958). Ģ4-3 carriers. The residue was dissolved in benzene-

Ketosteroids were visualized by ultraviolet light. petroleum ether (1: 1), from which the "estriol" Gradient elution partition chromatography fraction was extracted with distilled water and

on Celite column according to Engel et al. (1961) then the "estrone-estradiol" fraction was extract-

was employed. ed with 0.4N NaOH (Brown, 1955; Roy and

Alumina column chromatography of the Brown, 1960). The remainder of the extraction

methylated phenolic fractions was carried out was considered to contain the "neutral" fraction.

according to the Brown's method (1955). The phenolic fraction from perfusions II, IV,

Thin layer chromatography was developed in V and VI were separated by paper chromato-

cyclohexane and ethyl acetate (1: 1),(1: 2). graphy in the Bush systems B 1 and C, and by

The standard estrogens and their derivatives were thin layer chromatography in cyclohexane-ethyl

visualized by spraying with sulfuric acid and acetate (1: 1). The methylated derivatives of

heating at 100•Ž for 10mins. estrogens thus separated, were further purified

on alumina column chromatography (Brown,

Recrystallization to constant specific activity 1955). The phenolic fractions from other per-

The samples were recrystallized as the parent fusions (I and III) were separated by gradient

compound, using alternating or different solvent elution partition chromatography on a Celite

systems. An allowable limit for the establishment column according to Engel et al. (1961); as the

of radiochemical purity was 5•`8% difference in stationary phase 90% methanol was used, and

the specific activity of the last two sets of crystals. 2, 2, 4-trimethylpentane and 1, 2-dichlorethane

These criteria are in agreement with those report- were used as the mobile phase. Subsequent to

ed by Axelrod et al. (1965). these separations and the formation of deriva-

tives, paper chromatography in the Bush systems

Extraction and purification B 1 and C, and thin layer chromatography in

Immediately after perfusion, the perfusate was cyclohexane-ethyl acetate (1: 1),(1: 2) were collected and centrifuged at 3,000 rpm for 15 employed for further purification. After the mins. Plasma was separated and the red blood purification by chromatographies, eluted sam- cells were then suspended in saline. The suspen- ples were added with standard carriers and re- sion was centrifuged and the supernatant was crystallized as the parent compounds from dif- added to the plasma. It was felt that hydrolysis ferent solvents. "Neutral" fractions from each perfusion ex was not necessary in this case since other studies - have shown that the vast bulk of the recovered periment were tentatively separated by paper steroids would be non-conjugated. Accordingly chromatography in the Bush system A. Endocrinol. Japon. 254 NAKAYAMA et al. Sept. 1967

Table 1. Conversion of C-19 steroids in perfusion of human placenta

RESULTS

In perfusions I, II and III, the evidence for the conversion of C-19 steroids (androstenedi- one-4-14C in perfusion I, testosterone-4-14C in perfusion II and DHEA-4-14C in perfusion III) was presented (Table 1). It can be seen that these androgens are easily aromatized to estrogens in almost the same amount in each perfusion. In perfusion I, the initial extract of the perfusate contained the radioactivity of 1,- 281,800 cpm, and 84.8% of which were found in the phenolic fraction. Following gradient Fig. 2. Celite column chromatogram of the phenolic elution partition chromatography of this frac- fraction in perfusion I. Solid lines indicate 14C tion, 43.8% was found in the estrone fraction measurements. Dotted lines indicate weight of and 20.0% in the estradiol-17ƒÀ fraction. the standard carriers. PI; estrone, PII; estradiol- There could be found only 1.5% and 0.8•“ 17ƒÀ, PIII; 16-epiestriol, PIV; estriol. The gradient with dichloroethane was appleid at the tube of the radioactivity in the 16-epiestriol and No. 28. estriol fractions (Fig. 2). Each radioactive peak was then purified by paper chromato- Table 2. Specific activities of the graphy. The radiochemical purity of the radioactive products in perfusionI estrone and estradiol-17ƒÀ fractions was check- ed by the specific activity obtained as cpm

per ƒÊg Kober chromogen, and then standard carriers of 8.26mg of estrone and 6.35mg of Fig. 1. Schematic diagram of the perfusion ap- estradiol-17ƒÀ were added and recrystallized paratus. to a constant specific activity (Table 2). How- Vol.14, No.3 ESTROGEN FORMATION IN THE PLACENTA 255

ever, the specific activity in the estriol and 16-epiestriol fractions fell so rapidly in the steps of purification that no conversion of the perfused to those substances could be demonstrated. The crude extract of the perfusion fluid from perfusion II contained 589,000 cpm, and 76.9% of the radioactivity was recovered from the "estrone-estradiol" fraction while 4.6% from the "estriol" fraction. Each frac- tion was further separated by paper chromato-

graphy; 47.2% was found in the estrone frac- tion and 15.9% in the estradiol-17ƒÀ fraction. In this experiment, methylated derivatives were made and then purified on alumina Fig. 3. Celite column chromatogram of the "es- column chromatography. No marked change trone-estradiol" fraction in perfusion III. Solid in the specific activity was observed in these lines indicate 14C measurements. Dotted lines indicate weight of the standard carriers. PI; procedures (12,300 cpm/ƒÊg to 10,300 cpm/ƒÊg unknown peak, PII; estrone, PIII; estradiol-17ƒÀ, in estrone and 13,000 cpm/ƒÊg to 10,100 cpm/ PIv; 16-epiestriol, Pv; estriol. The gradient μg in estradiol-17β). The identities of radio- with dichloroethane was applied at the tube active materials in the "estriol" fraction was No. 28. not established. The conversion of DHEA-4-14C, a C-19 Table 3. Specific activities of the radioactive steroid which is considered to be an important products in peaks II, III (Fig. 3) and IV intermediate to estriol during pregnancy, was (Fig. 4) in perfusion III. studies in perfusion III. The initial extract of the perfusate contained 475,750 cpm. The conversion of DHEA into phenolic steroids occurred in a high yield similar to perfusions using other C-19 steroids. The three distinct

peaks appeared on the Celite column chro- matogram of the "estrone-estradiol" fraction

(Fig. 3). The specific activities of estrone, estradiol-17ƒÀ and estriol expressed as cpm

perƒÊg Kober chromogen are shown in Table the 6 or 15 position by our method, so that 3. The radioactive metabolites recovered in contaminations in the 16-epiestriol fraction the estrone and estradiol-17ƒÀ fractions be- were not completely excluded. haved exactly as the standard estrone and In perfusions IV and V, in which proges- estradiol-17ƒÀ. The radiochemical purity of terone-4-14C (perfusion IV) and cholesterol- the estriol fraction was not confirmed. 4-14C(perfusion V) were added as precursors, It was noted that more than 10% of the no conversion to the phenolic steroids could radioactivity was found in the 16-epiestriol be demonstrated. fraction (PIII in Fig. 4), and this radioactive In perfusion IV, the initial extract contained metabolite behaved exactly as the added car- 963,900 cpm, but there could be found only rier standard. The constant specific activity 1% of the radioactivity in the phenolic frac- of this 16-epiestriol is presented in Table 4. tion. And no estrogen could be identified. But, it was difficult to separate 16-epiestriol In perfusion V, more than 80% of the from estradiol-17ƒÀ with hydroxyl group at radioactivity was found in the pentane-soluble Endocrinol. Japon. 256 NAKAYAMA et al. Sept. 1967

chromatography, and 1,083 cpm/ƒÊg after the alumina column chromatography). However, bulk of the radioactive metabolite in the "estriol" fraction was so different from the

standard estriol with a decrease in its specific activity during the steps of purification that no identification of radioactive estriol could be made. Table 5 summarizes the results obtained from perfusions of the human placenta under

Table 5. Conversion of radioactive steroids in perfusion of human placenta

Fig. 4. Celite column chrolt,i ograrn of the "es-

triol" fraction. Solid lines indicate 14C measure- ments. Dotted lines indicate weight of the

standard carriers. PI; estrone, PII; estradiol- 17ƒÀ, PIII; 16-epiestriol, Pry; estriol. The gradient

with dichioroethane was applied at the tube No. 26.

Table 4. 16-epiestriol like radioactive product

(peak III in Fig. 4)in each step of purification the same conditions. This reveals a similar extent of aromatization for the three C-19 steroid precursors (androstenedione, testo- sterone and DHEA) with values ranging be- tween 50.1% and 63.8% of the initial radio- activity recovered. No conversion to estriol was demonstrated from those C-19 steroids and from estradiol-17ƒÀ. It is interesting to note that the ratio of fraction, and only 14.2% was in the 90•“ estrone to estradiol-17ƒÀ was approximately methanol fraction. In the phenolic fraction, 2.5 to 1 when C-19 steroid was used as a there could be found only 0.1 % of the initial precursor, whereas 11.6 to 1 when estradiol- count. Following the separation procedure 17ƒÀ was used. by thin layer chromatography, identifications It is of great interest that the high yield of three estrogens in this fraction were not of 16ƒÀ-hydroxylated metabolite was formed established. from DHEA, while no epiestriol was formed Eighty one percent of the initial radioacti- from other C-19 precursors. vity was included in the "estrone-estradiol" No conversion of progesterone and cho- fraction when estradiol-17ƒÀ-16-14C was used lesterol to estrogens was demonstrated. as a precursor (perfusion VI). Following the separation by paper chromatography, 60.3•“ DISCUSSION of the radioactivity was found in the estrone fraction. Sequential chromatography of the The aromatization of C-19 steroids, the estrone fraction before and after methylation formation of estrogens, were investigated in caused no significant change in the specific the human placenta. With respect to these activity (1,105 cpm/ƒÊg after the first paper results it is evident that a full term placenta Vol.14, No.3 ESTROGEN FORMATION IN THE PLACENTA 257

possesses an extremely high capacity of a- Heinrichs et al. (1966) and Nakayama et al. romatization. This suggests to us that the (1967) showed by the in vitro experiment that principal route of placental estrogen synthesis the fetal liver had 16ƒ¿-hydroxylase of DHEA. may consist of aromatization of steroid pre- Nakayama et al. (1967) also reported that cursors reaching the placenta via the fetal the fetal adrenal could convert DHEA to and/or the maternal circulation. The conver- 16ƒ¿-hydroxylated DHEA in an incubation sion of neutral steroids such as testosterone, experiment. An assumption may be made androstenedione and DHEA to estrone and that the 16ƒ¿-hydroxylated DHEA thus formed estradiol-17ƒÀ by placental preparation in vitro reaches the placenta, where it is aromatized is remarkably efficient, as first demonstrated to estriol. Our results from in vivo experiment, by Ryan (1958). Indeed, recent in vitro in which double isotopes were used in the

(Longchampt et al., 1960; Ryan, 1958 and feto-placental compartments, revealed that 1959) and in vivo (Bolte et al., 1964a) studies estriol was formed from DHEA more effici- have shown that several C-19 steroids can be ently than androstenedione, and that 16ƒ¿- converted to estrogens in mid and late preg- hydroxylation might precede to aromati- nancy. The results of our study further indi- zation in the feto-placental unit (unpublished cated that the neutral ƒ¢4C-19 steroids such results.). as androstenedione, testosterone and ƒ¢5C-19 It should also be emphasized from our steroid such as DHEA, were easily aromatized data of perfusions I through III, that the by the perfused placenta to yield a similar ratio of the radioactivity recovered in the ratio of estrone and estradiol-17ƒÀ; there was phenolic fraction was always the same regard- always two or three times more estrone than less of the precursors used. Exclusively in estradiol-17ƒÀ in the perfusate. It is of interest perfusion III, where DHEA was added, 10.7 that ten times more estrone than estradiol-17ƒÀ of the radioactivity was found in the 16-epi- was found following perfusion with estradiol- estriol fraction, and it was suggested that 16ƒÀ- 17ƒÀ. hydroxylation occurred in the placenta. It is also noteworthy from this study that Whereas, 16ƒ¿-hydroxylation, which might there was no conversion of these neutral be essential for the formation of estriol from steroids to estriol. This lack of placental 16ƒ¿- DHEA, could not be demonstrated in the hydroxylation was also apparent when es- present investigation. Now the problem re- tradiol-17ƒÀ was perfused. This fact suggests mains of the origin of the neutral steroids that the fetus with placenta must be responsi- which can be aromatized to estrogens, parti- ble for the formation of estriol during preg- cularly estriol, in the placenta. nancy. It has been known, from the studies Cholesterol is found in a high concentration of Diczfalusy (1964), Bolte et al. (1964b and in the umbilical blood, placenta and maternal c) and Nakayama et al. (1965 and 1966b), peripheral blood. The most abundant neutral that the feto-placental compartments have steroid elaborated during pregnancy is pro- the capacity to convert DHEA or estradiol- gesterone, which is formed by the placenta 17ƒÀ to estriol. Recentely, Diczfalusy (1964), (Venning, 1938). And the conversion of cho- Solomon (1966) and Siiteri and MacDonald lesterol into progesterone by placental tissue

(1966) proposed that DHEA sulfate formed has been demonstrated in vitro (Solomon in the fetal adrenal from placental pregneno- et al., 1954; Morrison et al., 1956; Solomon, lone was hydroxylated at C-16 in the fetal 1960) and also in vivo (Bloch, 1945). The tissue. Bolte et al. (1966) suggested from present study indicated that neither choleste- their in vivo experiment with previable per- rol-14C nor progesterone-14C, which had been fused fetus that the fetal liver at mid term circulated in the isolated human placenta, had the capacity of 16ƒ¿-hydroxylation of converted into estrogen. This fact suggests DHEA sulfate. Slaunwhite et al. (1965), that the placenta possesses little, if any, Endocrinol. Japon. 258 NAKAYAMA et al. Sept. 1967 ability to split the side chains of those Engel, L. L., C. B. Cameron, A. Stoffyn, J. A. steroids. These finding corroborate the recent Alexander, O. Klein and N. D. Trofimow view of the feto-placento-maternal compart- (1961). Anal. Biochem. 2, 114. ments in the biosynthesis and metabolism of Heinrichs, W. L., H. H. Feder and A. Colas estrogens, particularly in the formation of (1966). Steroids 7, 91. estriol, in pregnancy proposed by Diczfalusy Levitz, M., P. Condon and J. Dancis (1956). (1964) and Solomon (1966). Endocrinology 58, 376. Little, B. and A. Shaw (1961). Acta Endocrinol. ACKNOWLEDGEMENTS 36, 455. Longchampt, J. E., C. Gual, M. Ehrenstein and Grateful acknowledgement is made to Prof. R. I. Dorfman (1960). Endocrinology 66, 416. Takashi Kobayashi for his constant interest Morrison, G., R. A. Meigs and K. J. Ryan and guidance in this investigation. The authors (1965). Steroids 5, Suppl. II 177. wish to thank Dr. Philip Troen, Prof. of Nakayama, T., K. Arai, K. Satoh, T. Yanaihara, Medicine, University of Pittsburgh, Pa., for T. Tabei and K. Nagatomi (1965). Clin. Endo- his review of the manuscript and helpful sug- crinol. 13, 581.(In Japanese) gestions. Nakayama, T., K. Arai, K. Satoh, K. Nagatomi, Thanks are also due to Miss Kyoko T. Tabei and T. Yanaihara (1966a). Endocrinol. Chatani, Miss Yoko Takanezawa and Miss Japon. 13, 153. Chiiko Yanagioka for their skillful technical Nakayama, T., K. Arai, T. Yanaihara,, T. Tabei, assistance. K. Nagatomi and K. Satoh (1966b). The World of Obst. and Gynecol. 18, 248.(In Japanese) REFERENCES Nakayama, T., K. Arai, K. Satoh, T. Yanaihara, K. Nagatomi and Y. Fujita (1967). Endocrinol. Axelrod, L. R., C. Matthijssen, J. W. Goldzieher Japon. 14, 269. and J. E. Pullman (1965). Acta Endocrinol. 49, Roy, E. J. and J. B. Brown (1960). J. Endocrinol. Suppl. 99. 21, 9. Bagget, B.(1959). Endocrinology 64, 600. Ryan, K. J.(1958). Biochim. Biophys. Acta 27, Bloch, K.(1945). J. Biol. Chem. 157, 661. 658. Bolte, E., S. Mancuso, G. Eriksson, N. Wiqvist Ryan, K. J.(1959a). J. Biol. Chem. 234, 268. and E. Diczfalusy (1964a). Acta Endocrinol. Ryan, K. J.(1959b). Federation Proc. 17, 1382. 45, 535. Ryan, K. J. and O. W. Smith (1965). Recent Bolte, E., S. Mancuso, G. Eriksson, N. Wiqvist Progr. in Hormone Research 21, 367. and E. Diczfalusy (1964b). Ibid. 45, 560. Siiteri, P. K. and P. C. MacDonald (1966). J. Bolte, E., S. Mancuso, G. Eriksson, N. Wiqvist Clin. Endocrinol. 26, 751. and E. Diczfalusy (1964c). Ibid. 45, 576. Slaunwhite, W. R., M. A. Karsay, A. Hollmer, Bolte, E., N. Wiqvist and E. Diczfalusy (1966). A. A. Sandberg and K. Niswander (1965). Ibid. 52, 583. Steroids 5, Suppl. II 211. Brown, J. B.(1955). Biochem. J. 60, 185. Solomon, S., A. L. Lenz, R. Vande Wiele and Bush, I. E. The Chromatography of Steroids. S. Lieberman (1954). Abst. Amer. Chem. Soc. Pergamon Press, London (1961). 126th Meeting, 29C. Diczfalusy, E.(1964). Federation Proc. 23, 791. Solomon, S. The Placenta and Fetal Membranes, Dorfman, R. I. and G. Gual (1959). Biochemis- William and Wilkins Co. Baltimore, p.201 try of Steroids 4, 230. (1960). Eberlein, W. R. and A. M. Bongiovanni (1958). Solomon, S.(1966). J. Clin. Endocrinol. 26, 762. J. Clin. Endocrinol. 18, 300. Troen, P.(1961). Ibid. 21, 895. Venning, E. H.(1938). J. Biol. Chem. 126, 595..