Estrogen receptors in the human uterus

Estrogen receptors in the human uterus Cell-free

studies with

postpartum,

and nonpregnant

estradioLl7fl

SIMON DON Rochester,

patients,

uterine cytosol correlated

with

from pregnant, plasma

levels

R. S.

the use of human

HENDERSON,

SCHALCH, New

B.M.,

B.CH.,

M.A.

M.D.

York

Good competitive binding of estradiol-17/3 was observed in the uterine cytosol, or 105,OOOO g supernatant fraction, of nonpregnant uterine homogenates. The most eficacious binding is found in uteri exposed to low levels of endogenous estrogens; progressively less binding is observed in uteri exposed to estrogenic and progesteronelike compounds [oral contraceptives), in fiostpartum uteri, and in postmenopausal uteri. No competitive binding was observed in pregnant uteri except for one uterus which contained a missed abortion. The receptor apgears to be a protein and bears resemblance to the rat uterus receptor that has been extensively investigated. There is definite competition for the receptor by similar concentrations of diethylstilbestrol, much less by estrone and estriol, and none by higher concentrations of firogesterone and testosterone. It, therefore, seems that the receptor is more specific for estradiol than the other naturally occurring steroids tested. Estimations numbers of receptors per picomole of cytosol and the dissociation constants for the varying uterine states are determined. A modified competitive-protein-binding radioassay for plasma estradiol with the use of a human uterine macromolecule is also described.

use of a single subcutaneous injection of 50 PC of 6,7-3H estradiol-17/3 two hours before hysterectomy, showed that the radioactive content of tissues of the reproductive tract was well above that attributable to their plasma content. The follicular-phase uterus gave a greater uptake than the Iuteal phase, and proportionally more of the endometrial radioactivity was associated with nuclei in the follicular than the luteal phase. The vagina and cervix showed lower variable radioactivity. Wyss and his associates6 demonstrated estradiol receptors in the soluble fractions of human myometrial and endometrial homogenates. When these fractions were incubated with 6,7-3H estradiol-17/3, there was one peak of radioactivity on the sucrose density gradient (5s) in contrast to the rat and rabbit incubates that showed 2 peaks, one at 9.5 and 5s values. The lighter component of the animal fractions was shown

T H E M E c H A N I s M of estrogen binding and some of the characteristics of an estrogen receptor in the rat uterus have been partially elucidated. I-3 It is known that the subcellular distribution of labeled estradiol given in vivo is approximately 50 per cent in the nuclear myofibrillar fraction and 30 per cent in the cytosol, or 105,000 g supernatant fraction.4 The remaining estradiol is adherent to the cell wall. Studies on the human uteruq5 with the From the Department of Obstetrics and Gynecology and the Department of Medicine, University of Rochester, Strong Memorial Hospital. Supported by United States Public Health Service Research Grant AM 08943. y?ee;ived for publication October 28, ;fg;;pted

for publication

November

of

19, 762

Volwue Kumber

Estrogen receptors

112 6

Table I. Pregnant

patients

included

in uterus

763

in study

Age

Estimated week of Bestation

J. H.

40

17

Autolysed fetus, crown to rump length 6 cm., 12 weeks’ size. Endometrium: decidual reaction. Placenta degenerated. No evidence of vasculitis.

History of vasculitis, undetermined type. Prednisone, 20 mg. per day. Abortion and permanent sterilization.

C. B.

28

18

Immature placenta and 180 gram fetus 13 cm. crown to rump length, consistent with 17 weeks’ gestation.

History of menorrhagia controlled with progestational agents. Abortion and permanent sterilization.

S. G.

27

11

Placenta 11 weeks’ gestation, male crown t3 rump length 4 cm.

Permanent abortion.

sterilization

and

v.

L.

‘9

18

Female fetus, 11.5 cm. crown to rump length, immature placenta 16 to 17 weeks’ gestation

Permanent abortion.

sterilization

and

M.

K.

24

8

Placenta, 8 weeks’ gestation. crown to rump length.

Permanent abortion.

sterilization

and

B. O’B.

33

6 Hr. postpartum

Patient

Pathologic diagnosis

Reason far

Fetus,

fetus,

2.4 cm.

Delivered of 3,260 gram girl. Post-cesarean section uterus and cervix with remnant of lower uterine segment placenta accreta.

to be due to nonspecific serum proteins which could be removed with washing. However, after washing the human tissue, there was still a peak of radioactivity sedimentary at the 5S value, suggesting that the human specific-binding protein has the same sedimentation rate as that of the nonspecificbinding serum proteins. No specific uptake was observed with the same amount of testosterone-l ,2-3H or progesterone 1 7a-3H. The receptor protein was demonstrated in both young and postmenopausal women. HHhnel? also presented evidence for specific receptors in the soluble fractions of the human uterine homogenates with a much higher affinity for estradiol than extracts from skeletal muscle. The specific estradiol binding is shown to be inhibited by SHgroup blocking reagents and by competing estrogenic compounds. This study was designed to examine the estradiol competitive-binding characteristics of soluble fractions of human myometrial, endometrial, and placental homogenates in the pregnant state and full-thickness uterine homogenates in the postpartum, nonpregnant, and postmenopausal states with the use

and other operation

Postpartum hysterectomy postpartum hemorrhage placenta accreta.

factors

for due to

of tritiated estradiol. By this means, an examination of the in vitro reactions between estradiol and the “estrogen receptors” is made. In each patient studied, pre- and postoperative estradiol-1 t/3 levels were determined so that they could be correlated with the estradiol competitive-binding curve of that particular cytosol. Materials

and

methods

A Tri Carb liquid scintillation spectrometer, Model 314AX (Packard Instrument Co., Downers Grove, Illinois), was used for radioactive measurements and had an efficiency of 32 per cent for tritium. Materials used in the competitive-proteinbinding radioassay for estrogen and the cytosol curves are described by Corker and Exley.s 6.7-3H-Estradiol-17/3 (specific activity 40 c. per millimole) from New England Nuclear Co. was checked for radiochemical purity by thin-layer chromatography (aluminum oxide) and found to be chromatographically pure.

Uterine total

collection and preparation.

of 12 human

uteri

were obtained,

A

each

764

Henderson

and

March 15, 1972 :\Iu. J. Obstet. Gynecol.

Schalch

Table II. Nonpregnant

patients

included

in study

Pathologic diagnosis

Preoperative diagnosis

Estradiol (ng./lOO ml.)

Age

J. P.

51

Menorrhagia, relaxation

B. S.

28

Pelvic relaxation, desire for permanent sterilization

Total vaginal hysterectomy, proliferative endometrium

6

Ovulen

J. B.

26

Uterine descensus, desire for permanent sterilization

Total abdominal hysterectomy, endometrium irregular shedding

5

Ovral

17.0

B. R.

34

Pelvic relaxation, desire for permanent sterilization

Endometrium early secretory phase, total vaginal hysterectomy

IUCD (Lippes loop)

43.0

A. V.

29

Pelvic relaxation, desire for permanent sterilization

Endometrium of secretory type with progesterone effect, total vaginal hysterectomy

Depo-Provera, 200 mg., 2 months previously

38.5

V. H.

38

Menorrhagia, fibroid uterus

Uterus with proliferative type endometrium and leiomyoma of myometriurn, total abdominal hysterectomy

Ovulen 21 contains and 0.05 mg. of ethinyl

Bilateral salpingo-oophorectomy, total vaginal hysterectomy, atrophic endometrium, myometrium no pathologic diagnosis

1 mg. of Ethynodiol estradiol. Depo-Provera

diacetate contains

of cycle

Parity

Patient

pelvic

Day

Contraception

6 years postmenopausal

16

Unknownno menses

4

and 0.1 mg. of mestranol. medroxyprogesterone acetate

being transferred from the operating room to the laboratory in crushed ice, immediately after hysterectomy. All hysterectomies took place between 8 A.M. and 12 noon. Five uteri contained intact pregnancies (Table I) ; one was 6 hours postpartum, and the other uteri were in varying states as shown in Table II. All pregnant uteri were opened, and 2 Gm. full-thickness sections of endometrium and placenta were separately glass homogenized at 2O C. with 6 ml. of Tris buffer and subsequently centrifuged for 15 minutes at 0’ C. (5,000 g) . The supernatant was then recentrifuged at 94,000 g for 90 minutes at 0’ C. The myometrium was similarly treated except 4 Gm. of tissue was taken and given three 10 second full-speed bursts on a Sorval homogenizer with 12 ml. of Tris buffer as the tissue was too fibrous to be glass homogenized. The nonpregnant uteri were

None

None

21

9.87

Ovral contains 0.5 mg. of norgesnel (aqueous suspension).

opened, and a 4 Gm. full-thickness block containing endometrium and myometrium was excised, as it was impractical to separate endometrium from myometrium, and homogenized in the homogenizer with 12 ml. of Tris buffer. Plasma from two patients, one pregnant subject (C. B.) and one nonpregnant subject (A. V.), was treated in a manner similar to that of the endometrial and placental samples to demonstrate that the blood contained within the uterus was not responsible for the receptor action demonstrated by the uterine homogenates. Competitive--inhibition-binding curves and protein content were determined on all fresh supernatants. The remainder was stored in small aliquots at -15O C.; if good binding was demonstrated, the cytosol was used for estradiol-17p assays.

Plasma extraction, chromatography, and radioassay. The plasma extraction and com-

Volume

1 I:!

Number

6

petitive-protein-binding method of Corker and Exleys was duplicated, except that the soluble uterine macromolecule used was obtained from human uteri in the follicular phase and not from 7-day-pregnant rabbits. Preliminary studies showed that the rabbit gave a lower uptake of radioactive estradiol-1 7/3 and less pronounced competitive inhibition than the human cytosol. The extraction tubes were all inoculated with 600 counts per minute (c.p.m.) (6.81 pg., specific activity 40 c per millimole) of estradiol-17p evaporated to dryness, so that recovery could be determined. Plasma extractions were performed on 0.2 ml. in nonpregnant, 0.1 ml. in pregnant, and 1 ml. in postmenopausal patients. Two other extractions were performed with a plasma:ether volume ratio of 1:5. Aluminum oxide thin-layer chromatography was used for final separation, the unknown areas of estradiol-17,B being located by spotting 20 kg of estradiol on the peripheral lanes of each plate; the band was identified with an ultraviolet light hand scanner. This band was extrapolated across the lanes containing the unknowns. The areas containing the unknown estradiol fractions were then eluted with 3 ml. of ethylacetate, the eluants being diluted or not, depending on the estimated estradiol value, and then evaporated to dryness in the reaction tubes. A duplicate series for 6 tubes was then set up for a standard curve, containing 0, 20, 50, 100, 200, and 300 pg., respectively, of unlabeled estradiol evaporated to dryness. Then, to each tube, containing known or unknown amounts of estradiol, 0.23 ml. of Tris bufier was added, containing 62.65 pg. (about 3,500 c.p.m.) of 6,7-3H-estradiol17p. After cooling to 4O C., 50 ~1 of cold uterine cytosol was added. The tubes containing these reaction mixtures were then equilibrated, after shaking, at 40’ C. for about 16 hr. (overnight). After equilibration, I ml. of dextran-coated charcoal suspension was added and incubated at 4O C. for 10 minutes. The mixture was then centrifuged, the supernatant being decanted and dissolved in 10 ml. of toluene

Estrogen

ioto

receptors

IdO

in uterus

260

765

360

PICOGRAM

Fig.

1. Competitive-binding inhibition curves of progesterone (closed diamond), testosterone (open hiamond), estriol (open square), estrone (open and diethylstilbestrol (closed square) circle), compared with estradiol (closed circle). Each point represents duplicates of increasing amounts of added nonlabeled substances expressed as a percentage of the supernatant radioactivity when no nonlabe!ed substance was added.

scintillator mixture. A standard curve was then drawn up from the results of the tubes containing known amounts of estradiol, and the unknowns were calculated from the curve and expressed as nanograms per 100 ml. of plasma after correction for recovery.

Recovery, accuracy, precision, and practicability. The mean recovery of 34 samples, calculated by adding 600 c.p.m. (6.81 pg.) of labeled estradiol, was 54 + 12 per cent (SD.), the range being 30 to 80 per cent. This compared well with other methods, considering that most others use 5 times as much plasma.“-I1 The extraction of estradiol-17P from water or plasma also gave results similar to those of Corker and Exleys; the mean recovery of standards (200 to 1,000 pg.) from plasma was 95 + 5 per cent (SD.), and the mean recovery from water was 98 + 10 per cent (SD.). All samples and standard curves were run in duplicate.

766

Henderson

and

Schalch Am.

March J. Obstet.

15, 1972 Gynecol.

D”r Cycle

20 50

100

1;o

2io

2;o

3do

PICOGRAM OF UNLABELED178 ESTRADIOL Fig. 2. Competitive-binding curves of uterine cystosol from nonpregnant patients. Each point represents the mean of duplicate runs and is corrected to 1 mg. per 50 PL of cytosol. The highest protein value for cytosol was 720 ,Icg per 50 gL; and the lowest, 220 mg. per 50 KL of cytosol. The crosshatching denotes the premenopause.

The specificity of the method was tested by running curves with the use of increasing amounts of estriol, estrone, and diethylstilbestrol in a way similar to that described for estradiol. The results show (Fig. 1) that estriol and estrone did compete for radioactive estradiol-17P, but the competition was less than that of estradiol itself. Diethylstilbestrol was, however, shown to be more competitive than estradiol while progesterone and testosterone gave no competition. The coefficient of variation of 4.2 per cent and the mean blank value in terms of 100 ml. of plasma was 0.8 ng. per 100 ml. One person can perform 6 sample determinations in duplicate in two days. Uterine cytosols. The competitive inhibi-

tion-binding curves of all the uterine cytosol were examined in duplicate runs with the use of a series of tubes as in the estradiol assay containing 0, 20, 50, 100, 200, and 300 pg. of unlabeled estradiol standards, 50 ~1 of cytosol being added to each tube. The different plots in pregnant, puerperal, missed abortal, menopausal, and nonpregnant uteri with or without exogenous estrogen and progesterone for contraception are seen in Figs. 2 and 3. Plots for placenta and endometrium alone are seen in Fig. 4. Competitive binding properties of the cytosol were extremely sensitive to temperature, and, if allowed to remain at room temperature for more than 3 hours, these properties were lost. Also, boiling promptly destroyed

Volume Number

112 6

Estrogen

receptors

in uterus

767

7001

\

Missed

AB (J.H.)

control

Pkegnhlbod

O-0

I, , 20 50

I

100

I

150

(0.)

I

I

I

200

250

300

PICOGRAM OF UNLABELED17/i?ESTRADIOL Fig. 3. Competitive-binding curves of myometrial cytosol from pregnant patients. The missedabortion and 6 hour postpartum cytosol is also included; only these two values are corrected for protein. The crosshatching denotes pregnancy.

100 150 200 250 300 PICOGRAMOF UNLABELED17fiESTRADlOL(STANDARDS) 20

50

Fig. 4. Competitive-binding curves for placental tients. Crosshatching denotes nonbinding. any competitive binding property of the cytosols, making it likely that these binding properties were due to one or more proteins.* *Protein determinations of the made by Dr. Patrick Frengley with of Lowry and associates.9

cytosol the use

fractions were of the method

and endometrial

cytosol from pregnant

pa-

Effect of charcoal on cytosol. To show that the charcoal-dextran suspension had no detrimental effect on the uterine cytosol per se, it was incubated in one case with freshly prepared uterine cytosd in a proportion of 2 : 1 for 1 hour. No change, apart

768

Henderson

and

March Am. J. Obstet.

Schalch

15, 1972 Gynrcol.

SCATCHARD PLOTS

n

I i

I 2

I j

I d

AMOUNT

I 5

0

B.R. IUCD B.S. O.C.

0

V.H. NO Rx

0

J.B.

O.C.

A

AL’.

1 M

I

I

I

b

i

8

9

10

BOUND (10“’ molar)

Figs. 5 and 6. Scatchard plots to determine the dissociation constants and the number of binding sites. The bound and free estradiol were calculated from Figs. 2 and 3. Again all results were corrected to give values for 1 mg. per 50 @L. The relationship used was: (bound)/(free) = l/K (binding site) - (bound). K - l/slope. (binding sites) = X intercept. (From Toft and associates.l*) Fig. 5 denotes the nonpregnant cytosol; and Fig. 6, the missed-abort& postpartum, and menopausal cytosol. The dissociation constants are expressed in moles. from the dilutional aspect, resulted, and, after an overnight standard curve assay, the curve was similar to that of the untreated cytosol. Collection of blood. Ten cubic centimeters of venous blood was drawn between one and three hours preoperatively on all sub-

jects and on the majority at various times postoperatively (Table III), placed in heparinized tubes, and immediately centrifuged. The plasma was stored at -IO0 C.

cycle, 5 and 6 days, respectively, but which were exposed to exogenous estrogensin the form of oral contraceptives. The shapes of these curves were less steep than the firstmentioned curve, and this occurrence may well be related to the highest estrogen blood levels of 20.2 and 17.0 ng., respectively, in these patients. The uterine cytosol from Patients A. V. and B. R. showed almost identical curves at decreased levels of uptake. The former pa-

to await

tient,

estradiol

determinations.

Results

Nonpreguant uterine cytosol. The competitive binding curves of the nonpregnant uteri are shown in Fig. 2. The most dramatic curve is displayed by the cytosol from the fourth

day of the cycle;

this patient

was not

exposed to exogenous estrogen, and the plasma estradiol was relatively low (9.87 ng. per 100 ml.). The next highest curves were shown by uteri from similar positions in the

with

a blood

estradiol

level of 38.5 ng.

per 100 ml., was exposed to prolonged depoprogesterone therapy. The reason for this high level is unexplained but may have represented breakthrough ovulation. The latter patient was at the sixteenth day in the cycle and had an estradiol value of 43.0 ng. per 100 ml. and a uterus which contained an intrauterine contraceptive device (Lippes loop “D”). Finally, the cytosol from the postmenopausal patient, J. P., gave a much flatter

Volume Sumbrr

112 6

Estrogen

0.31

receptors

in uterus

769

SCATCHARDPLOTS

0

J.H. Missed

A

B.O’B.

+

J.P. Menop.

‘--.&----e-s-

-O-A-

2

1

AB

P.P

- - - +

3

4

AMOUNT BOUND(IC%olar) Fig. 6. For legend,seeoppositepage.

Table III. Patient

Pregnant patients’ estradiol-17/3 determinations (nanograms per 100 ml.) Preoperatively

J. H.

375

C. B.

820

S. G.

921

v. L.

570

M. K.

310

B. O’B.

3 Hr.

6 Hr.

Weeks’ gestation (by fiathology)

4 Days

67.3

7.6

17

83.1

11.2

11

40.0

a.5

20.7

10.3

108

345 213 85.3

6 Days

24 Hr. 105

90.0

100.0

curve with a reduced uptake compared with that of the other curves but was still higher than the plasma “cytosol” curve which was a straight line. Pregnant uterine cytosol. The truly pregnaat uterine cytosol, as contrasted with the postpartum and missed-abortal cytosol, showed no competitive binding, and the plots were all of low uptake (Fig. 3) . The lowest uptakes came from the 17 and 18 weeks’ gestation uteri and were insignificantly different from the uptake shown by plasma of a pregnant patient. The plasma estradiol determinations ranged from 570 to 921 ng. per 100 ml., except for a value of 210 ng. per 100 ml. in M. K., who was 8 weeks pregnant. Estradiol competitive-binding plots

8.8

10.2

12

ia

5.0

a

6 Hours post -.___ partum

were also determined for endometriodecidual and placental cytosol from Patients C. B., S. G., and V. L. These are shown in Fig. 4 and are practically identical with the combined myometrial and endometrial plots of the same patients, demonstrated in Fig. 3. Competitive binding was shown by the missed abortal and postpartal uteri, respectively (Fig. 3) . It is interesting that binding in both casesoccurred in spite of relatively high estrogen levels (Table III) and that the curves were of the low uptake type, being situated between the postmenopausaland the depo-progesterone uteri as shown in Fig. 2. The Scatchard plots. A more rigorous

770

Henderson

and

March Am. J. Obstet.

Schalch

analysis was made of the cytosol which showed competitive-binding curves, by subjecting them to Scatchardl” plots, the results of which are seen in Figs. 5 and 6. The Scatchard plot enables one to make definite statements about the receptor as to the number of binding sites and their dissociation constants. Most of the plots were not straight lines and gave curves at their lower ends. According to Rosenthal,ll these probably represent more than one receptor or a system with two or more groups of noninteracting binding sites with differing dissociation constants. The upper straight part of the curve is, therefore, extrapolated in several cases so that numbers of binding sites and dissociation constants can be calculated. According to Toft and colleagues,12 geometric analysis shows that the X intercept equals the number of binding sites and the reciprocal of the slope to be equal to the dissociation constant. Significant deductions can only be made when the Scatchard plot is truly a straight line, but, in spite of this, the concentration of binding sites in Fig. 5 ranges from 3.4 to 9.5 x lo-lo M per milligram of protein in 50 ~1 of cytosol. Fig. 6 shows the Scatchard plots of the cytosol giving rather poor binding curves; the range of binding site concentrations is 1.6 to 3.5 x 1O-1o M per milligram of protein in 50 ~1 of cytosol. The dissociation constants in both figures are scattered, ranging between 4.75 x lo-l2 to 1.05 x la-lo M. Plasma estradiol-17/K The pregnant patients’ plasma demonstrated a rapid clearance of estradiol in the immediate postabortal-hysterectomy period (Table III) . In two patients, C. B. and V. L., umbilical venous cord blood was obtained on opening the hysterectomy specimen and showed an increased plasma level of estradiol, 900 and 923 ng. per 100 ml., respectively, compared with that of the mixed venous blood, 820 and 570 ng. per 100 ml., respectively. In contrast, the uterine venous plasma obtained from C. B. showed a slightly lower level of estradiol (720 ng. per milliliter) as compared with forearm mixed venous samples.

15, 1972 Gynecol.

Comment The results clearly show that a uterine estradiol receptor can be demonstrated by this method in the human nonpregnant state but not in the pregnant state. It is, therefore, concluded that the receptor, if present in pregnancy, probably has all its binding sites occupied and thus cannot be demonstrated by this method. However, it does appear possible that a receptor can be demonstrated in the case of the postpartal and postabortal cytosol in spite of relatively high estrogen levels of 570 ng. per 100 ml. or more. The effect of endogenous and exogenous estrogen causes a general decrease in receptor identification, which appears to be greatest early in the cycle, when the blood estrogens are low. The postmenopausal uterine receptors are probably maintained at a lower numerical level but still appear to display base-line function as far as the estradiol uptake and competitive binding are concerned. The cytosol from pregnant uteri demonstrated a greater degree of estradiol uptake corresponding with the shortness of the gestational period, but no conclusions can be drawn as numbers are very small and the uteri were studied only up to 18 weeks’ gestation. The basic biochemical mechanism of estradiol action on the human uterus is unknown, but this study shows that the degree of binding and the availability of receptor sites may depend on the plasma estradiol level, except in the postmenopausal uterus where absolute numbers of receptors are probably reduced, but tissue estradiol levels would be necessary to confirm this. The Scatchard plots in this study gave a rough idea of the concentration of binding sites, the mean of 4 of the 8 being 3.75 x 10-*O M per milligram of protein in 50 ~1 of cytosol. The results are compared with those of Gorski and co-workers1 who found the concentration of binding sites to be 8.9 x IO-l3 M per uterus or about 20,000 molecules per cell in the oophorectomized rat with a dissociation constant of 7.6 x lo-lo M. The modified-estradiol competitive-pro-

Volume Number

112 6

tein-binding radioassay gave nonpregnantblood-level results which in general agreed with those of Mikhail and associates13 using a radioimmunoassay method and Korenman and colleagueP using a radioligand assay. The pregnant estradiol levels compared favorably with Munson and co-workerP and Smith and ArilG who used a gas-liquid chromatography electron capture method. This study shows a radical difference between the competitive-binding ability of pregnant and nonpregnant uteri, and some correlation between degree of binding and plasma estrogen level can be made. An obvious extension of this study would be an analysis of the precise changes in the re-

Estrogen

receptors

in uterus

771

ceptor during the menstrual cycle and also in patients oophorectomized recently and those oophorectomized several days previously. The future study of estradiol and other reproductive steroid receptors in various end organs and also those structures such as the hypothalamus whose action is modified by these steroids, as part of feedback mechanisms, is of the utmost importance in clucidating the mechanisms of reproductive endocrinology. The authors are very grateful to Mrs. Doris Commons for her help in preparation of this manuscript and to Miss Delores Shock for her excellent technical assistance.

REFERENCES

1. Gorski, J., Taft, D., Shyamala, G., Smith, D., and Notides, A.: Recent Progr. Horm. Res. 24: 45, 1968. 2. Jensen, E. V., and DeSombre, E. R.: Biothem. J. 115: 28, 1969. 3. Notides, A. C.: Endocrinology 87: 987, 1970. 4. Noteboom, W. D., and Gorski, J.: Arch. Biochem. Biophys. 111: 559, 1965. 5. Brush, M. G., Taylor, R. W., and King, R. J. B.: J. Endocrinol. 39: 599, 1967. 6. Wyss, R. H., Heinrichs, W. L., and Herrman, W. L.: J. Clin. Endocrinol. 28: 1227, 1968. 7. H&hnel, R.: Steroids 17: 105, 1971. 8. Corker, C. S., and Exley, D.: Steroids 15: 469, 1970. 9. Lowry, 0. H., Rosenbrough, J. J., Farr, A. L., and Randall, R. J.: Biochem. J. 193: 256, 1951.

10. Scatchard, G.: Ann. N. Y. Arad. Sri. 51: 650, 1949. 11. Rosenthal, M. E.: Anal. Biochem. 20: 525, 1967. 12. Taft, D., Shyamala, G., and Gorski, J.: Proc. Nat. Acad. Sci. 57: 1740, 1967. 13. Mikhail. G.. Wu. C. H.. Ferin. M.. and Vande kield, R. i.: Steroids 15: ‘333, ‘1970. 14. Korenman, S. G., Perrin, L. E., and McCallum, T. P.: J. Clin. Endocrinol. 29: 879. 1969.

15. Munson, A. K., Mueller, J. R., and Yannone, M. E.: AM. J. OBSTET. GYNECOL. 108: 340, 1970. 16. Smith, 0. W., and Ari, K.: J, Clin. Endocrinol. 23: 1141, 1963.