Prolactin and progesterone effect on specific estradiol binding in uterine and mammary tissues invitro

Vol. 55, No. 4, 1973

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PROLACTIN AND PROGESTERONEEFFECT ON SPECIFIC ESTRADIOL BINDING IN UTERINE AND MAMMARYTISSUES IN VITROt Benjamin S. Leung, Ph.D. and Gordon H. Sasaki, M.D. Department of Surgery, University of Oregon Medical School, Portland, Oregon 97201 Received October

26,1973

SUMMARY Specific estradiol binding (SEB~ in uterine and breast tissues was measured by tissue incorporation of ~H-estradiol in the presence and in the absence of an estrogen competitor, nafoxidine hydrochloride. SEB in explants from uterus during pregnancy was much lower than that during lactation or from uterus of young rats. The presence of prolactin (5 pg/ml) in organ culture increases SEB capacity in these estrogen target tissues, and progesterone (I ~g/ml) appears to offset this prolactin-induced increase of SEB. These results represent the f i r s t in v i t r o demonstration of prolactin regulation on estradiol binding in estrogen target tissues. The possible mechanisms of interaction among these hormones are discussed. Introduction Abundant evidence demonstrates that the f i r s t event in estrogen action is the spontaneous binding of estrogen to the cytoplasmic estrogen receptor (ER), a subunit of which is subsequently translocated to the nucleus ( I ) .

The in-

teraction of nuclear estrogen receptor at the gene site is believed to trigger a series of biochemical processes leading to estrogen-induced RNA and protein synthesis (2,3).

Attention in the past decade has focused largely on the se-

quential events following the binding of estrogen to estrogen receptor.

Direct

evidence for a regulatory mechanism c o n t r o l l i n g the synthesis and/or degradation of receptor protein is lacking.

Circumstantial evidence suggesting such a re-

gulatory mechanism has been obtained by Sarff and Gorski (4) who reported that after a single injection of estradiol (0.I ~g) into i n t a c t immature rats the cytoplasmic ER decreased by 50% followed by a replenishment beginning about six hours after estrogen i n j e c t i o n , and by 16 hours the estrogen receptor level reaches control values.

The replenishment of ER is blocked by cycloheximide or actino-

t Preliminary results reported at the IX Acta Endocrinologica Congress, Oslo, Norway, June 1973.

Copyright ©1973 by Academic Press, Inc. All rights o f reproduction in any form reserved.

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mycin D when they are injected within two hours pOst-estradiol administration. ER replenishment cannot be demonstrated in an in v i t r o incubation of uteri with 3H-estradiol (4).

Laumas et al. (5) indicated recently that low levels of es-

tradiol pre-treatment for 2,4, or 6 hours to intact immature mice produce a significant increase in ~H-estradiol uptake in uterus of the mouse. Since these experiments were not repeated with hypophysectomized rats, i t is not certain i f this increase of ER capacity is a direct effect of estrogen on the uterus or an effect brought forth by p i t u i t a r y hormones such as prolactin. In this report, we present our preliminary results of organ culture demonstrating that prolactin potentiates the specific estrogen binding (SEB), in both uterus and mammary explants of rats during pregnancy and lactation. Materials and Methods Abdominal mammary glands, uterus and diaphragm were excised from pregnant or lactating Sprague-Dawley rats. prepared using a Stadie

Thin slices (0.5 mm) of mammary tissue were

Riggs apparatus.

Explants (I-2 mm) from these tissues

were placed in ice-cold chemically defined medium 199 (Gibco Co.) containing p e n i c i l l i n G (lO0 units/ml) unt il ready to be used. Culture solution E contains insulin (5 ~g/ml), p e n i c i l l i n (lO0 units/ml) and 3H-estradiol (l x lO-1°M)* dissolved in medium 199. Solution U has, in addition, nafoxidine hydrochloride (l x lO-s M)**.

Approximately equal amounts of explants from these tissues were

separately transferred to the following flasks containing 25 ml each of (a) solution E, (b) solution U, (c) solution E with ovine prolactin (5 ~g/ml)*** or other hormones, and (d) solution U with prolactin or other hormones. After incubation at 37°C in a 5% C02-air chamber (Sheldon) and with continual agitation (60-80 cycles/min), approximately 12-15 explants from each flask were removed at various time intervals and washed three times with 15 ml ice-cold Krebs• ~H-estradiol, New England Nuclear, NET 317 Estradiol-178-2,4,6,7-3H(N) I05 Ci/mM • * Nafoxidine hydrochloride or Ull,lO0 was a g i f t from the Upjohn Company, Kalamazoo, Michigan, U.S.A. • ** Ovine prolactin, NIH-P-510,courtesy of the National Institutes of Art hri t i s and Metabolic Diseases.

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20.0]

18,o::1. - NAFOXIDINE

14,0>12,0? ×

z-

iO.O -

8.0-

6,0

&

+ NAFOXIDI

NE

4,0

2,0

0

1

2

3

4

HOURS OF INCUBATION

Figure l : Specific e~tradiol binding (SEB) in uterine explants. Conditions for in~rporation of H-estradiol by uterine tissue (35 day old rats) in the presence of Nafoxidine hydrochloride (solid points) and in i t s absence (open points) are as described by Jensen et al. (6). Explants were dried and radioa c t i v i t y estimated according to Materials and Methods. Each point represents the mean of t r i p l i c a t e s . SEB is calculated by the difference of radioactivity incorporated (open point - solid point). Ringers-Henseleit solution, pH 7.3.

Washed explants were divided equally into 3

groups, dried overnight at 80% in aluminum cups and weighed afterward (about 3-5 mg). After the dried explants were wetted with a drop of d i s t i l l e d water, they were solubilized with soluene at room temperature for 12 hours.

Radioac-

t i v i t y w a s estimated in I0 ml Liquifluor-toluene with a Beckman LS-250 Liquid S c i n t i l l a t i o n system.

Efficiency was approximately 30%. The r a d i o a c t i v i t y of

explants from solution E minus that from solution U represents the SEB capacity. Results Nafoxidine hydrochloride has been found to compete with estrogen for the estrogen receptor binding sites.

Making use of this fact, a simple technique

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z~ UTERUS (35 DAYS)

12,0 "G

i0,0

m ~ ×

z~ 8,0

/"/////

/

/. '/

1

/•

UTERUS

(LACTATING 16 DAYS)

/,/' ~:

~ o

6,0

4,0

// •

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&

•

/

.

.

......... O

UTERUS (PREGNANT16 DAYS)

I "'" "'0 /0 ,'"

2,0

"'~'-~"

/~'" ..~ "~-'~0

~

1

B ~

"''-[3

° ~

BREAST (LACTATION) DIAPHRAGM

9

2

3

4

HOURSOF INCUBATION

Figure 2: T~me course of SEB in estrogen target and non-target tissues after exposure to H-estradiol with or without Nafo×idine. Measurementof SEB is according to Figure I. Each point represents average of triplicates. by Jensen (6) was used to measure specific estrogen binding (SEB) in uterine and mammary tissues.

Explants were incubated up to four hours in solutions

containing 3H-estradiol and 3H-estradiol plus Nafoxidine.

The difference in

3H-estradiol incorporation represents the SEB capacity (Figure l ) . In Figure 2, the SEB values of estrogen target and non-target tissues from rats during different physiological states were compared. Uterine tissue of young rats (35 days old) has the highest SEB value.

In lactating animals,

uterine tissue has a much higher SEB than breast tissue.

Diaphragm, a non-

target tissue for estrogen does not demonstrate SEB capacity.

In mid or late

pregnancy, a similar relationship but lower SEB is observed for uterine and breast tissues. A series of experiments were performed by the addition of prolactin to explants of uterus and breast from late pregnant rats (Table l ) .

In these experi-

ments, explants were pre-incubated in culture medium 199 for various periods of time in the presence or absence of prolactin.

Then, a two-hour incorpora-

tion of 3H-estradiol was performed as described above. SEB represented by

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TABLE 1 HORMONAL EFFECT ON SPECIFIC ESTROGEN BINDING

EXPLANTS

HOURS

HORMONES

INCUBATION

ADDED

DPM X i0

/5 MG TISSUE

E

U

E-U

I i

UTERUS

I

14.6

12,8

1,8

(MID-PREGNANCY)

I

20,2

13,7

6,5

IP

26,9

13.0

13,9

UTERUS

I

12,6

14,6

(LATE PREGNANCY)

I

7,2

3,9

0 3.3

IP

41,7

5,4

36.3

BREAST

I

16,9

14,7

2,2

(LATE PREGNANCY)

I

9,6 +0,7

9,0 ± i , 0

0,6

IP

12,5 ±3,2

9,1 -+0,i

3,4

I

0,4±0,1

0,3~0,i

0,1

I

1,8±0,2

1.7±0.2

0,1

IP

1,9~0,0

1,8+0,i

0,1

DIAPHRAGM

I = INSULIN IN MEDIUM

199

P = PROLACTIN

Table I: Explants of breast or uterus were preincubated for various periods of time in medium 199 containing insulin ( I ) , and p e n i c i l l i n in the presence or absence of prolactin (P). A two-hour uptake of ~H-estradiol was performed according to conditions shown in Figure I.

E-U column in Table 1 increased invariably for uterine and breast explants in the presence of prolactin as compared to controls. t i n , up to ten-fold lower ER binding was observed.

In the absence of prolac-

The prolactin-induced in-

crease of SEB is tissue specific and does not occur in diaphragm explants exposed to prolactin. Figures 3 and 4 show the SEB in uterine explants from pregnant and lactating rats. done.

In these experiments, however, no pre-incubation with prolactin was

Explants were incubated at the s t a r t in the presence or absence of

prolactin in solution E (containing 3H-estradiol) or solution U (containing 3H-estradiol plus Nafoxidine) as described in Methods. 1184

During the f i r s t

two

BIOCHEMICAl AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 55, No. 4, 1973

~-

6,0

5,0 ? x

+

7.

4,0

~ ~ ~ .

i Ill'-

....

~ z m

3,0

~

2+(1

,,

//

.

~

.

o

•

.

~

+P

, d'

_p

+-.

"

"''" "~ P + Pr

] ,"

~

1,0

i 0

i

I

I

I

i

,

2

3

4

5

6

HOURS OF INCUBATION

F i _ _ ~ 3 : Prolactin (P) and progesterone (Pr) effects on SEB in uterine exp-~t~ (late pregnancy). SEB is evaluated under conditions described in Materials and Methods Eachpoint represents the mean of triplicates. Progesterone (l ~g/mli and prolactin (5 pg/ml) were used. hours of incubation, SEB was one to several fold higher in the presence of prolactin than that without prolactin.

Again, explants from diaphragm did not

demonstrate this effect of prolactin (date not shown). Progesterone (l ~g/ml) inhibits the prolactin

potentiation of SEB in uterine explants.

Discussion The presence of ER in uterine tissue has been well documented (I-4). Only recently, the presence of ER in breast tissues of rodents during lactation was reported (7,8).

By using similar methods, such as the sucrose

gradient and dextran coated charcoal techniques, we confirmed these findings (9). Our in vitro results demonstrate that prolactin stimulates and progesterone inhibits SEB in uterine and mammary explants.

These observations

correspond with the ER binding capacity in these tissues during different hormonal changes in the animal. during pregnancy (lO).

In rats, prolactin levels are elevated

At parturition, plasma prolactin levels are extremely

high and remain elevated during lactation (lO-ll).

I t is known that pro-

gesterone levels are high during pregnancy, but fall sharply just prior to

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5,0 7 x

4,0

E 3.0

2.0 <

1,0 u

I

I

i

I

I

I

1

2

3

4

5

6

HOURS OF INCUBATION

Figure 4 : Prolactin (P) and progesterone (Pr) effects on SEB in uterine explant~ (8 day l a c t a t i o n ) . Conditions for SEB are the same as in Figure 3.

delivery and remain r e l a t i v e l y low during lactation.

Recently, we reported

that ER binding capacity of breast tissues during pregnancy is low; and i t increases during lactation and reaches i t s maximum during late lactation (9). This ER binding capacity in mammary gland remains high for two or three days after p a r t u r i t i o n but decreases to the basal level as in the adult quiescent state a few days following separation of the young from the mother. lar finding was also reported by W i t t l i f f

and colleagues (12, 13).

A simiER

binding in the uterus follows similar patterns of a l t e r a t i o n during these physiological changes (Leung, unpublished data).

Thus, at high levels of pro-

l a c t i n and high levels of progesterone during pregnancy, ER binding is low; at high levels of prolactin and low levels of progesterone during l a c t a t i o n , ER binding is high; and at low levels of prolactin and low levels of progesterone during involution and quiescent state, ER binding capacity is low.

I t is not

certain, however, i f the high level of uterine ER in immature or young rats is related to elevated levels of endogenous prolactin and low levels of progesterone. Progesterone does not compete with estrogen at the ER binding sites (13,14). Thus, i t is u n l i k e l y that either the low ER value in pregnancy (9,13) or the progesterone-inhibition of SEB in v i t r o reported here is a r e s u l t of the competi-

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tive binding between estrogen and progesterone.

Our results favor another

possible role of progesterone; that i s , i t serves as a feedback control in situ for the prolactin-stimulated estrogen binding.

I f this is true, the many anta-

gonistic actions of estrogen and progesterone may be explained. The potentiation of SEB by prolactin may arise from one or a combination of the following situations: (a) by the increase in cytoplasmic estrogen pool through an increase in the permeability of the plasma membrane, (b) by the activation of an as yet unknown precursor of ER, (c) by the increase in de novo ER synthesis, and (d) by the maintenance of ER i n t e g r i t y for estrogen binding.

Experiments are in progress to elucidate these p o s s i b i l i t i e s .

In conclusion, we have demonstrated that prolactin stimulates estrogen binding in target tissues. gesterone.

The stimulatory effect is

counteracted by pro-

The exact nature of the estrogen binding induced by prolactin and

the mechanisms of interaction among prolactin, progesterone and estrogen, require further experimentation. Acknowledgements This research was supported by grants from the Oregon Division of the American Cancer Society. We thank J.S. Leung for his technical assistance. References I. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13. 14.

Jensen, E.V, and DeSombre, E.R., Ann Rev Biochem 41 ;203, 1972. Hamilton, T.H., in the Biochemistry of Steroid Hormone Action, Smellie, R.M.S. ed., Academic Press, New York 1971, pp 49-84. Baulieu, E.E., Wira, C.R., Milgrom, E., and Raynaud-Jammet, C., in Karolinska Symposia on Research Methods in Reproductive Endocrinology, 5th Symposium, Stockholm 1972, Diczfalusy, E. ed., pp 396-419. Sarff, M. and Gorski, J., Biochemistry 10:2557, 1971. Laumas, V., Farooq, A., and Laumas, K.R., J Steroid Biochem 3:871, 1972. Jensen, E.V., DeSombre, E.R., and Jungblut, P.W., in Endogenous Factors Influencing Host Tumor Balance, Wissler, R.W., Dao, T.L., Wood, S., J r . , eds., University of Chicago Press, Chicago 1967, pp 15-30. W i t t l i f f , J.L., Gardner, D.G., Battema, W.L. and Gilbert, P.J., Biochem Biophys Res Conm 48:119, 1972. Shyamala, G. and Nandi, S., Endocrinology 91:861, 1972. Leung, B.S. and Sasaki, G.H., Acta Endocr (Kbh) Suppl 177:26, 1973 (Abstract). Bast, J.D. and Melampy, R.M., Endocrinology 91:1499, 1972. Ford, J.J. and Melampy, R.M., Endocrinology 93:540, 1973. Beers, P.C. and W i t t l i f f , J.L., Fed Proc 32:651, 1973 (Abstract). Gardner, D.G. and W i t t l i f f , J.L., Biochemistry (In press). Toft, D., Shyamala, G. and Gorski, J., Proc Natl Acad Sci 57:1740, 1967. 1187