Human corpus luteum: Presence of epidermal growth factor

receptors and binding characteristics

Ramchandra R. Ayyagari, M.D., and Firyal S. Khan-Dawood, Ph.D.

Chicago, Illinois

Epidermal growth factor receptors are present in many reproductive tissues but have not been

demonstrated in the human corpus luteum. To determine the presence of epidermal growth factor

receptors and its binding characteristics, we carried out studies on the plasma cell membrane fraction of

seven human corpora lutea (days 16 to 25) of the menstrual cycle. Specific epidermal growth factor

receptors were present in human corpus luteum. Insulin, nerve growth factor, and human chorionic

gonadotropin did not competitively displace epidermal growth factor binding. The optimal conditions for

corpus luteum-epidermal growth factor receptor binding were found to be incubation for 2 hours at 4° C

with 500 µg plasma membrane protein and 140 femtomol 1251-epidermal growth factor per incubate. The

number (mean ± SEM) of epidermal growth factor binding sites was 12.34 ± 2.99 x 10- 19 mol/µg

protein; the dissociation constant was 2.26 ± 0.56 x 10- 9 mol/L; the association constant was

0.59 ± 0.12 x 109 L/mol. In two regressing corpora lutea obtained on days 2 and 3 of the menstrual

cycle, there was no detectable specific epidermal growth factor receptor binding activity. Similarly no

epidermal growth factor receptor binding activity could be detected in ovarian stromal tissue. Our findings

demonstrate that specific receptors for epidermal growth factor are present in the human corpus luteum.

The physiologic significance of epidermal growth factor receptors in human corpus luteum is unknown, but

epidermal growth factor may be involved in intragonadal regulation of luteal function. (AM J OBSTET

GYNECOL 1987;156:942-6.)

Key words: Epidermal growth factor, corpus luteum, epidermal growth factor receptors

Epidermal growth factor (EGF) receptors have been reported in human reproductive tissues such as the placenta,'· 2 chorion,2 and uterus' but not in normal ovaries. EGF, a heat-stable, single-chain polypeptide with a molecular weight of 6045 daltons, stimulates cellular growth, and there is some evidence that it af­fects steroidogenesis in ovarian,4· 5 testicular,6 and cho­riocarcinoma tissues in vitro. 7 Progesterone secretion by the JEG-3 choriocarcinoma cell line is stimulated by EGF.7 EGF inhibited gonadotropin stimulation of tes­tosterone production in Leydig cells and follicle-stim­ulating hormone stimulation of luteinizing hormone­receptor induction in granulosa cells.6

• 7 This would sug­

gest that EGF receptors may be present in the ovary to produce a biologic response. However, there are no published data on the existence of EGF receptors in ovarian tissue. Since EGF is a growth factor and ap­pears to affect steroidogenesis in granulosa and Leydig

From the Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Illinois College of Med­icine.

Supported in part by a grant from the American College of Obstetri­cians and Gynecologists, district VI, Illinois section.

Received for publication October 3, 1986; accepted October 30, 1986. Reprint requests: Dr. Firyal S. Khan-Dawood, Department of Ob­

stetrics and Gynecology, University of Illinois College of Medicine, 840 S. Wood St., Chicago, IL 60612.

942

cells,4·6 these effects are probably mediated through a receptor mechanism and therefore EGF receptors may be present in ovarian tissues. Thus we carried out stud­ies to determine if EGF receptors are present in the human corpus luteum of the menstrual cycle and ovar­ian stroma and to obtain the binding characteristics if such receptors are present.

Material and methods

Reagents. Mouse EGF and nerve growth factor were obtained from Collaborative Research Inc., Lexington, Massachusetts. 1251 was obtained from Amersham Corp., Arlington Heights, Ill. (specific activity 16. 7 mCi/mg iodine). Human chorionic gonadotropin and insulin were gifts from the National Pituitary Agency, Baltimore, Maryland.

Patients and tissues. Ovarian tissues were obtained from nine premenopausal women who gave informed consent and were undergoing bilateral tubal ligation or hysterectomy with salpingo-oophorectomy. All patients had had regular menstrual cycles for at least 6 months before surgery. Of the nine patients, seven were in the luteal phase of the menstrual cycle, which was further confirmed by their plasma progesterone levels and his­tologic endometrial dating.8 All tissues were kept on ice and transported immediately to the laboratory .where the corpus luteum was further dissected clean of the

Volume 156 Number 4

3

:s ! 2.5 e Q.

~ 2

1 ~ 1.5

~ u.. 1 (!) 0.9 ~ 0.8

.. d:~ ~ &:~

0.3 0.2 0.1

____ ..._ __ 4•c

22°c

00 ........ ~2_,_4 ........ ~5_._5 ........ ~10-'-1~2 ......... 14__._1~6 ......... 18__._2~0 ......... 22 ......... 2~4 ......... 26 ........ 2~8 ......... 30_._.32

INCUBATION TIME (hours)

Fig. I. Effect of incubation time on corpus luteum EGF re­ceptor binding to 1251-mouse EGF at incubation temperatures of 4° C and 22° C. Binding was maximum with 1 hour incu­bation at 22° C or 2 hours at 4° C. Values given are mean ± SEM (n = 4).

'2 ·;; 0 Ci Cl

~ 0

! 0 z

4

3

5 2 ID u.. (!)

~ 1 .L "' N

45 10 15 20 22 25 30 35 37 40

TEMPERATURE (°C)

Fig. 2. Effect of incubation temperatures on corpus luteum EGF receptor binding to 1251-mouse EGF. The incubation time was 2 hours. Maximum binding was obtained at 4° C. Values given are mean ± SEM (n = 4).

remaining ovarian tissues if it had not been obtained intraoperatively by luteectomy. A small piece of each corpus luteum was taken for histologic section and ex­amination to further date the corpus luteum.9 The cor­pus luteum (n = 7), ovarian stromal tissues (n = 4), and two regressing corpora lutea on days 2 and 3 of the cycle were processed individually for plasma cell membrane preparations and receptor studies as de­scribed below.

Receptor preparation. All tissues were washed with chilled physiologic saline solution until clear of blood. All tissue manipulations were carried out at 4° C. A small piece of corpus luteum (0.5 gm) was minced with scissors and homogenized in 2.5 ml 10 mmol/L Tris-HCl buffer at pH 7.0, containing 250 mmol/L su­crose and 1 mmol/L CaCl2 (homogenization buffer) with a Tekmar tissue homogenizer (Tissumizer, Tek-

Corpus luteum epidermal growth factor receptors 943

c 0.9 "iii 0 0.8 Q. Cl 0.7 ~ 0 0.6 .E c 0.5 z ::::> 0.4 0 ID

0.3 u.. <!) w 0.2 E l. 0.1 "' ~

0 0 200 400 600 800 1000 1200

CORPUS LUTEUM PLASMA MEMBRANE PROTEIN (ug)

Fig. 3. Effect of increasing amounts of corpus luteum plasma membrane protein on its binding to 1251-mouse EGF. Maxi­mum binding was obtained with 500 µ.g plasma membrane protein. Values given are mean ± SEM (n = 4).

1

~ 0.9 -0 li 0.8

~0.7 ...... ~ 0.6

~05 c . z ::::> 0.4 0 a:i 0.3 LL ~ w 0.2 E l. 0.1

It) N

200

CONCENTRATION OF 1251-mEGF ADDED

Cfmol)

Fig. 4. Effect of the concentration of 1251-mouse EGF added on its binding to EGF receptors in human corpus luteum. The protein content of corpus luteum plasma membrane used was about 500 µ.g per assay tube. Incubation was carried out at 4° C for 2 hours. The specific activity of the 1251-mouse EGF used was 100 to 200 µ.Ci/mg. Binding of 1251-mouse EGF to EGF receptors in human corpus luteum increased with in­creasing concentrations of 1251-mouse EGF to reach a maxi­mum with 140 fmol of 1251-mouse EGF. Values given are mean ± SEM (n = 4).

mar Co., Cincinnati, Ohio) at a setting of 60 using 3 bursts of 10 seconds each with 20-second pauses in between. The homogenates were centrifuged at 800 g at 4° C for 15 minutes. The 800 g pellet was stored at - 70° C for future nuclear studies. The 800 g super­natant was further centrifuged at 100,000 g at 4° C for 60 minutes to obtain the plasma membrane fraction.

944 Ayyagari and Khan-Dawood

100

; 0 110

!!! !!! ~ 8 ,. 0 .. .. g 7 E . ..,-~

80

50

_ ... --o HCG

-~ -4-2.5 5 10 25 50 100 250 500 1000

UNLABELLED PEPTIDE (nmol)

Fig. 5. Specificity of corpus luteum EGF receptor binding. Only increasing amounts of unlabeled EGF but not insulin, nerve growth factor (NGF), and human chorionic gonado­tropin (HCC) inhibited EGF receptor binding to 1251-mouse EGF. The binding shown when unlabeled EGF was added represent specific binding after subtracting for nonspecific binding. Bo = Specific binding of 1251-mouse EGF in the ab­sence of any unlabeled peptide and was taken to be 100%. B = binding of '"'I-mouse EGF in the presence of the un­labeled peptide. The molecular weights of the peptides used for calculation were EGF, 6045; HCG, 40,000; insulin, 6000; and nerve growth factor (mouse submandibular gland), 130,000.

The cytosol fraction in the supernatant was stored at - 70° C for further studies. The 100,000 g pellets were resuspended in 1.5 ml Tris-HCI buffer, pH 7.0, con­taining 5 mmol/L CaCl2 , 75 mmol/L NaCl, and 0.5% bovine serum albumin (incubation buffer). An aliquot was taken for protein determination by the method of Lowry et al. '0

Receptor binding studies. Purified mouse EGF was used as the standard. 125I-mouse EGF was prepared by iodinating mouse EGF with 125I (specific activity 16.7 mCi/mg iodine; Amersham Corp.) according to the chloramine-T method." Free unincorporated iodine was removed by absorption with H ycel resin beads (Hycel Inc., Houston, Texas). The radioiodinated mix­ture was purified by gel filtration on a Sephadex G-25 column (0.9 by 17 cm). The purified 125I-mouse EGF gave a specific activity of 100 to 200 µCi/mg.

Fifty-microliter aliquots of the plasma membrane preparation from the corpora lutea or ovarian stroma, containing 500 µg protein, were incubated with 25 ng unlabeled mouse EGF in 50 µI incubation buffer and 50 µI 125I-mouse EGF (about 60,000 cpm) for 2 hours at 4° C. The final incubation volume was 200 µI. The reaction was terminated by the addition of 500 µl chilled 10 mmol/L Tris-HCI buffer, pH 7.0. The mix­ture was then centrifuged at 4000 g for 30 minutes, the supernatant was aspirated, and the pellets were counted for 5 minutes in a Beckman -y-counter (Beck­man Instruments, Inc., Palo Alto, California) with a

-C')

io T""

x -Q)

3

2.5

2

~ 1.5. LL ....... "O c: ::J

~ 1

0.5

April 1987 Am J Obstet Gynecol

Ka=0.82x 109umol

Kd= 1.22x 1 o-9mol/L

Number of Binding Sites=3x 1 o-19mol/µg

o..___....___..~__._~_._~~~.....___.~_.

0 10 20 30 40 50 60 70 80

Bound EGF(x 10-13)mol/L

Fig. 6. Characteristic Scatchard plot of corpus luteum (day 17 of the cycle) EGF receptor binding to 1251-mouse EGF. The Scatchard plot of the saturation binding showed curvilinearity, indicating negative cooperativity. The initial slope was used to calculate the apparent kd and k,.

counting efficiency of 73. 7% for 1251. Binding studies were performed in the presence and absence of unla­beled mouse EGF.

To determine the effects of time on corpus luteum EGF receptor binding, experiments were carried out as above at 22° C and 4° C incubation temperature with varying incubation times ranging from 30 minutes through 4 hours. To determine the optimal incubation temperature, EGF receptor binding studies were per­formed with 2 hours of incubation and at 4° C, 22° C, and 37° C. Experiments were then undertaken with increasing amounts of plasma membrane preparation from 250 to 1000 µg protein per tube and incubation for 2 hours at 4° C. To determine saturability of EGF receptor binding with 125I-mouse EGF, incuba­tions were carried out with increasing quantities of 125I-mouse EGF, from 20 to 180 femtomol, with a constant amount (500 µg) of plasma membrane pro­tein. Specificity of the corpus luteum plasma membrane binding to 125I-mouse EGF was checked against human chorionic gonadotropin, nerve growth factor from mouse submandibular gland, and insulin. Having elu­cidated the optimal conditions for corpus luteum plasma membrane binding to 125I-mouse EGF, binding

Volume 156 Number 4

Corpus luteum epidermal growth factor receptors 945

Table I. Binding characteristics of EGF receptor in seven human corpora lutea of the menstrual cycle

Patient Day of No. cycle* k, (Limo/) k/moifL)

l 16 0.82 x 109 1.22 x 2 17 0.32 x 109 3.13 x 3 20 1.7 x 109 0.59 x 4 21 3.6 x 109 0.28 x 5 22 0.1 x 109 10 x 6 24 0.46 x 109 2.17 x 7 25 3.28 x 109 0.31 x

All Lu teal 0.59 ± 0.12 x 109 2.26 ± 0.56 x

*Based on endometrial and corpus luteal histologic dating.

studies were performed on the plasma membranes of seven different corpora lutea, two regressing corpora lutea, and seven ovarian stromal tissues separately.

All binding data reported are for specific binding obtained from the differences between total and non­specific binding determined in parallel in the presence of excess unlabeled mouse EGF. The EGF receptor­specific binding to 1251-mouse EGF was analyzed by Scatchard plots12 for each individual tissue specimen and the respective binding parameters, including the number of binding sites, the association constant (k,) and the dissociation constant (kd) were determined.

Results

Effect of time and temperature on EGF receptor binding. Fig. 1 shows the effect of increasing incubation time on EGF receptor binding to 1251-mouse EGF at 4° C and 22° C. Maximum binding was obtained with 1 hour incubation at 22° C, but at 4° C it was 2 hours. At both temperatures, binding subsequently declined with increasing time of incubation.

The effect of incubation temperature on EGF recep­tor binding to 1251-mouse EGF is shown in Fig. 2. Bind­ing was maximum at 4° C and decreased thereafter at 22° C and 37° C.

When increasing amounts (protein) of corpus luteum plasma membrane were used, a linear increase in bind­ing of 1251-mouse EGF was observed (Fig. 3). We chose 500 µg corpus luteum plasma membrane protein to obtain adequate binding and to have sufficient tissue for Scatchard analysis.

The effect of adding increasing concentrations of radioactive ligand (1 251-mouse EGF) is shown in Fig. 4. With 500 µg plasma membrane protein and an incu­bation time of 2 hours at 4° C, binding of 1251-mouse EGF to its receptors in human corpus luteum increased with increasing concentrations of 1251-mouse EGF to reach a maximum when 140 fmol 1

251-mouse EGF was used.

Fig. 5 shows the competition of unlabeled mouse EGF, nerve growth factor, human chorionic gonado-

No. of binding sites Cooperativeness

(moll µ,g protein) demonstrated

10-9 3.0 x 10-19 Negative cooperativeness 10-9 19.29 x 10-19 Negative cooperativeness 10-9 10.43 x 10-19 Negative cooperativeness 10-9 13.4 x 10-19 Negative cooperativeness 10-9 37.3 x 10-19 Negative cooperativeness 10-9 14.5 x 10-19 Negative cooperativeness 10-9 7.62 x 10-19 Negative cooperativeness 10-9 12.34 ± 2.99 x 10-19

tropin, and insulin on corpus luteum EGF receptor binding to 1251-mouse EGF. Unlabeled EGF competi­tively displaced 1251-mouse EGF and inhibited its bind­ing to corpus luteum EGF receptors. Insulin, nerve growth factor, and human chorionic gonadotropin in high concentrations failed to compete for bind­ing to corpus luteum plasma membrane binding to 1251-mouse EGF.

With increasing concentrations of 1251-mouse EGF in the incubation mixture, specific binding to EGF recep­tors reached maximum binding at about 150 fmol of 1251-mouse EGF. Scatchard plot of this binding data was curvilinear (Fig. 6), which suggests the presence of either two sets of noninteracting binding sites or one set of binding sites with an interaction between occu­pied and unoccupied sites indicating negative cooper­ativity. When excess unlabeled EGF (500 µg) was added, dissociation of already bound 12'1-EGF was en­hanced more than dilution by buffer alone, thus sug­gesting negative cooperativity.

The mean ( ± SEM) of ke calculated from the indi­vidual dissociation constants (Table I) obtained from Scatchard plots of the seven corpora lutea was 2.26 ± 0.56 x 10- 9 mol/L. The total number of avail­able binding sites as calculated from the intercept on the abscissa was 12.34 ± 2.99 x 10- 19 mol/µg protein. k, was 0.59 ± 0.12 x 109 L/mol.

Two regressing corpora lutea obtained on days 2 and 3 of the menstrual cycle showed no binding activity of their plasma membrane preparations to 1251-mouse EGF. Similarly, ovarian stromal tissues (n = 4) showed no specific EGF receptors.

Comment

Our findings show for the first time the presence of a specific binding activity for EGF on the plasma cell membrane preparations from human corpora lutea that fulfills the criteria of a receptor for EGF. EGF receptors were not detectable in regressing corpora lu­tea and ovarian stromal tissue. The specificity of EGF receptors on the human corpus luteum is attested

946 Ayyagari and Khan-Dawood

to by the failure of insulin, nerve growth factor, and human chorionic gonadotropin to compete with 125I-mouse EGF binding to the corpus luteum EGF receptor.

The optimal conditions for corpus luteum EGF re­ceptor binding were found to be incubation for 2 hours at 4° C with 500 µg plasma membrane protein and 140 fmol 125I-mouse EGF per incubate. The binding char­acteristics of EGF receptors in human corpus luteum are of a lower magnitude than those reported for the human placenta.2· 13 We obtained a k. of 0.6 x 109

L/mol in corpus luteum EGF receptors that is lower than values obtained for human placenta. The number of EGF receptor binding sites in human corpus luteum is 12.3 x l0- 19 mol/µg protein, which is about three orders of magnitude less than in the human placenta 13

but only 20 times less than in the chorion.2 However, the studies with the placenta and membranes were per­formed at an incubation temperature of 22° C. Scat­chard analysis of corpus luteum EGF receptors gave a curvilinear plot in all seven fresh corpora lutea studied. This was confirmed to be the result of negative coop­erativity rather than two sets of noninteracting binding sites, a finding that is similar to that observed with pla­cental EGF receptors2

· 13 and insulin receptors. 11

The physiologic and biologic significance of EGF re­ceptors is unclear. Two possible effects may include luteal cell growth differentiation and modulation of luteal cell steroidogenesis. EGF is a mitogenic polypep­tide and EGF receptor expression is related to differ­entiation capacity in both normal and transformed ker­atinocytes.15 It is therefore tempting to postuiate that EGF receptor expression in the corpus luteum is related to differentiation capacity of the luteal cells. With re­spect to the rossible role of EGF on luteal steroido­genesis, EGF appears to affect steroidogenesis in ovar­ian,1· 5 testicular,6 and choriocarcinoma tissues in vitro. 7

In the JEG-3 choriocarcinoma cell line, EGF stimulates progesterone secretion7 but it inhibits testosterone pro­duction by Leydig cells.6 Whether EGF has a similar direct effect on luteal steroidogenesis remains to be investigated, although intrajugular venous infusion of EGF for 24 hours during the luteal phase of cycling ewes induced a rise in follicle-stimulating hormone and luteinizing hormone but had no effect on plasma pro­gesterone during the next 7 days. 16

In conclusion, we have demonstrated the presence of EGF receptors in human corpus luteum of the men­strual cycle. The binding characteristics include an ap­parent k. of 0.6 x 109 Limo! and a kd of 2.3 x 10-9

April 1987 Am J Obstet Gynecol

mol/L, with the number of binding sites being 12.3 x l0- 19 mol/µg protein. The physiologic roles of EGF and its receptors on the corpus luteum remain to be established but may include cell differentiation and luteinization, as well as luteal steroidogenesis.

We thank Dr. M. Yusoff Dawood for consultations, discussions, and tissue collection.

REFERENCES I. O'Keefe E, Hollenberg MD, Cuatrecasas P. Epidermal

growth factor characteristics of specific binding in mem­branes from liver, placenta and other target tissues. Arch Biochem Biophys 1974;164:518.

2. Rao ChV, Carman FR, Chegini N, Schultz GS. Binding sites for epidermal growth factor in human fetal mem­branes. J Clin Endocrinol Metab 1984;58: I 034.

3. Hofmann GE, Rao ChV, Barrows GH, Schultz GS, San­filippo JS. Binding sites for epidermal growth factor in human uterine tissues and leiomyomas. J Clin Endocrinol Metab I 984;58:880.

4. Knecht M, Catt K. Epidermal growth factor and gonad­otropin-releasing hormone inhibit cyclic AMP-dependent luteinizing hormone receptor formation in ovarian gran­ulosa cells. J Cell Biochem 1983;2 I :201.

5. Jones PBC, Welsh TH, Hsueh AJW. Regulation ofovarian progestin production by epidermal growth factor in cul­tured rat granulosa cells. J Biol Chem 1982;257: 11268.

6. Lloyd CE, Ascoli M. On the mechanisms involved in the regulation of the cell surface receptor for hCG and mEGF in cultured Leydig tumor cells. J Cell Biol 1983;96:52 l.

7. Bahn RS, Speeg KV, Ascoli M Jr, Rabin D. Epidermal growth factor stimulates production of progesterone in cultured human choriocarcinoma cells. Endocrinology 1980;107:2121.

8. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Fertil Steril 1950; I: 13.

9. Corner AW. The histological dating of the corpus luteum of menstruation. Am J Anat I 956;98:377.

10. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265.

11. Hunter FC, Greenwood WM, Glover JS. The preparation of 131 1-labelled human growth hormone of high specific radioactivity. Biochem J 1963;89: 114.

12. Scatchard G. The attraction of proteins for small mole­cules and ions. Ann NY Acad Sci 1949;5 I :660.

13. Carson SA, Chase R, Ulep E, Scommegna A, Benve­niste R. Ontogenesis and characteristics of epidermal growth factor receptors in human placenta. AM J OBSTET GYNECOL 1983;147:932.

14. De Meyts P, Roth], Neville DMJr, GavinJR Ill, Lesniak MA. Insulin interactions with its receptors: experimental evidence for negative cooperativity. Biochem Biophy Res Commun 1973;55:154.

15. Boonstra], de Laat SW, Ponec M. Epidermal growth fac­tor receptor expression related to differentiation capacity in normal and transformed keratinocytes. Exp Cell Res 1985;161:421.

16. Shaw G,Jorgensen GI, Tweedale R, Tennison M, Waters MJ. Effect of epidermal growth factor on reproductive function of ewes. J Endocrinol 1985; 107:429.