progesterone modulation of estrogen-stimulated uterine biosynthetic events and estrogen receptor...

Molecular and Cellular Endocrinology, 8 (1977) 121-134 0 Elsevier/North-Holland Scientific Publishers, Ltd.

PROGESTERONE MODULATION OF ESTROGEN-STIMULATED UTERINE

BIOSYNTHETIC EVENTS AND ESTROGEN RECEPTOR LEVELS

Hemlata S. BHAKOO and Benita S. KATZENELLENBOGEN

Department of Physiology and Biophysics, University of Illinois, and School of Basic Medical Sciences, University of Illinois College of Medicine, Urbana, Ill. 61801, U.S.A.

Received 28 January 1977; accepted 19 April 1977

The effect of progesterone on estrogen-stimulated biosynthetic events - namely, glucose metabolism, fluid imbibition, DNA synthesis, and replenishment of estrogen receptor - is studied in uteri of immature (22-24dayold) rats and immature estrogen-primed rats.

Pretreatment with progesterone alone (2 mg s.c./rat) for up to 48 h does not markedly influence the subsequent uterine response to estrogen in terms of 2-h deoxyglucose metabolism, 3-h uterine wet weight or 24-h DNA synthesis rate. Moreover, progesterone alone increases uterine glucose metabolism and the rate of DNA synthesis, as does estradiol (5 pg), although the magnitude of the responses obtained is lower with progesterone. However, simultaneous exposure to progesterone plus estradiol (E + P) for over 12 h results in a decreased uterine responsiveness to subsequent estrogen as monitored by glucose metabolism and uterine wet weight stimulation. This decreased responsiveness to estrogen after exposure to E + P cor- relates with a decreased content of uterine cytosol estrogen receptor.

After treatment with E (5 ng) plus P (0.5 or 2 mg), or E alone, the initial movement of the estrogen receptor into the nucleus is similar, but by 12 h after E + P the rate of reappearance of the cytosol receptor is slower and the levels of cytosol receptor are depressed, reaching only 50-60% of that seen after E alone. This depression of cytosol receptor replenishment is most marked with the steroid progesterone, although exposure to estradiol(5 rg) plus high doses of dihydrotestosterone or testosterone (2 mg), but not low doses (0.5 mg), results in slightly depressed levels of cytosol receptor at 12-48 h. Cortisol (0.5 or 2 mg) is without any effect.

Administration of either progesterone or the inhibitor cycloheximide or actinomycin D at zero time (along with the estradiol) or up to 4--6 h following estradiol injection is markedly effective in decreasing the levels of estradiol cytoplasmic receptor (to approx. 40-50% of the control, E alone, level by 12 h) in 3day estrogen-primed rat uteri. The striking similarity in the time course and extent of the cycloheximide-, actinomycin D-, and progesterone-sensitive inhibition of estrogen receptor replenishment suggests that progesterone may influence uterine sensitivity to estrogen by interfering with the de novo synthesis of new estrogen receptors.

Keywords: uterus; DNA synthesis; deoxyglucose metabolism; cycloheximide; actinomy- tin D.

Estrogen and progesterone are two steroid hormones which play major roles in modulating the growth, differentiation, and function of the female reproductive tract. Many of their synergistic and antagonistic actions have been well character-

121

122 H.S. Bhakoo and B.S. Katzenellenbogen

ized (Astwood, 1938; Knobil, 1952; Yochim and DeFoe, 1962) while others are still being discovered. However, the basis of their synergistic or antagonistic inter-

actions remains unclear. Our finding of an inhibitory effect of progesterone on the relative rate of in-

duced protein (IP) synthesis (Bhakoo and Katzenellenbogen, 1977) led us to inves-

tigate whether progesterone had additional antagonistic effects on other estrogen- stimulated biosynthetic events - glucose metabolism, fluid imbibition, DNA syn-

thesis, replenishment of estrogen receptor - in the immature and estrogen-primed rat uterus. Our studies indicate that simultaneous treatment with progesterone plus

estrogen results in a decreased responsiveness to subsequent estrogen in terms of

glucose metabolism and fluid imbibition, and that this decreased responsiveness cor-

relates with a decreased content of uterine cytosol estrogen receptor. Further, the similarity of the time course and extent of inhibition of estrogen receptor replenish-

ment after progesterone or either cycloheximide or actinomycin D suggests that progesterone influences uterine sensitivity to estrogen by interfering with the de

novo synthesis of new estrogen receptors.

MATERIALS AND METHODS

Animals and hormone administration Studies utilized immature female Holtzman (Holtzman Co., Madison, Wis.) rats,

21-24 days old, or rats (20 days old) which received estradiol-170 (5 pg S.C. in

saline/rat) once daily for 3 days prior to assay. Animals received progesterone (P, 2 mg S.C. in 0.2 ml of sesame oil), estradiol-170 (E, 5 I-18 S.C. in 0.5 ml of 0.15 M NaCl containing 1% ethanol) or both (E t P; injected at different sites subcutaneously)

for various periods of time. The control animals received either sesame oil and/or saline. At designated time intervals, the animals were killed by decapitation, and

their uteri were excised, stripped of all surrounding fatty tissue, and assayed as described below.

Assay of 2-deoxy-D-glucose phosphorylation Glucose metabolism was studied by quantitating the conversion of 2-deoxy-D-

glucose to 2-deoxy-D-glucose-6-POe (Smith and Gorski, 1968; Gorski and Raker, 1973). At various times after exposure to E, P, or E + P, uterine tissue (2 uteri per flask, duplicate flasks per time point) was incubated at 37°C with 1 PCi 2-deoxy-D- glucose-l-[‘4C] (53 mCi/mmol; New England Nuclear) in 2.0 ml Eagle’s HeLa medium (Difco). After 30 min the tissue was rinsed in cold 0.9% NaCl, homogenized in 5% perchloric acid, and further processed for determination of unmetabol- ized 2-deoxyglucose and 2-deoxyglucose-6-PO4 radioactivity using the Dowex Ag2- X8 resin (BioRad) adsorption method exactly as described by Gorski and Raker

(1973).

Progesterone modulation of uterine biosynthetic events 123

Assay of (3H]methyl-thymidine incorporation into DNA The rate of [3H]thymidine incorporation into DNA was determined by the

method described by Kaye et al. (1972). Uterine tissue (2 uteri per flask) from ex-

perimental or control animals was incubated at 37°C with 1 PCi [3H]methyl-

thymidine (20 Ci/mmol; Schwarz-Mann) in 2.0 ml Eagle’s HeLa medium. After 1 h of incubation the tissue was rinsed in cold’0.9% NaCl and homogenized in 0.3 M

cold perchloric acid. The washed perchloric acid precipitable material was then

hydrolyzed at 90°C with 0.5 N HCI. Aliquots of hydrolysate were counted to mea- sure the incorporated [3H]thymidine and the total DNA content was measured

with the diphenylamine reagent containing acetaldehyde as described (Burton,

1956; Katzenellenbogen and Greger, 1974).

Determination of nuclear or cytosol binding sites by /3HJestradiol exchange At indicated times after exposure to P, E, P t E, or vehicle control, uteri (3 per

group in immature rats, or 2 per group in 3-day estradiol-primed rats) were excised and thoroughly rinsed with 20 ml of iced 0.01 M Tris HCl-0.0015 M EDTA, pH

7.4 at 25°C (TE buffer), and then homogenized with Kontes all-glass tissue grinders in 1.0 ml of iced 0.01 M Tris HCl-0.0015 M EDTA-0.02% sodium azide, pH 7.4 at

25°C (TEA buffer). Nuclear binding sites were determined in the SOOg X 20 min

washed nuclear pellet by the exchange method of Anderson et al. (1972) with slight modification (Katzenellenbogen, 1975). The cytosol exchange assay of Kat- zenellenbogen et al. (1973) was performed on the 180,OOOg X 60 min supernatant (cytosol) and was used to determine specific binding sites, both empty sites and sites previously filled with estrogen. To eliminate any variations due to tissue size, the protein content of the cytosol was determined according to the method of Lowry (1951) and the DNA content in the pellets was determined by a moditica- tion of the method of Burton (1956) (Katzenellenbogen and Greger, 1974); the

cytosol receptor content was expressed as pmol/pg protein, and the nuclear receptor concentration as pmol/pg DNA.

Determination of progesterone-, cycloheximide-, and actinomycin D-sensitive time course of inhibition of estrogen cytosol receptor replenishment

The time course of the progesterone-, cycloheximide-, and actinomycin D-

sensitive inhibition of estradiol cytosol receptor replenishment was determined in 3- day estradiol-primed immature rats. At 24 h following the last estradiol-priming injection, the animals received another estradiol injection. In some experiments progesterone (2 mg/rat, experimentals) or oil (0.2 ml, controls) was injected at vari-

ous times after estrogen. In other experiments the animals received either cycloheximide or actinomycin D (both from Sigma; 200 pg/rat i.p. at zero time and at 6 h in experimental) or saline (control) at designated time periods after estradiol.

A single i.p. injection of 200 pg cycloheximide was found to block protein synthesis (as measured by L-[14C]leucine incorporation into trichloroacetic acid precipitable material) almost 100% for up to 6 h. Reinjection of cycloheximide every


5-6 h maintained protein synthesis almost 100% inhibited for 12 h (as reported

previously by Sarff and Gorski, 1971) and hence this regimen was followed in the

studies reported here. A single injection of actinomycin D (200 pg) was found to block approx. 80% of [3H]uridine incorporation into RNA over a 12 h period. Injection of actinomycin D at zero and 6 h further reduced [3H]uridine incorporation into RNA to 10% of control and showed no significant effect on total protein

synthesis for up to 12 h. In all experiments the animals were killed at 12 h following the estrogen injec-

tion. Uteri were excised, thoroughly rinsed with cold TE buffer, and homogenized

in Kontes all-glass tissue grinders in 1.1 ml of TEA buffer as described above. The cytosol exchange assay was performed on the 180,OOOg X 60 min supernatant

(cytosol) according to the method of Katzenellenbogen et al. (1973).

RESULTS

Effect of progesterone (P) on estradiol (El-induced increases in the phosphorylation of 2-deoxyglucose

Estradiol has been shown to increase the rate of metabolic conversion of 2-

deoxy-D-[‘4C]glucose to 2-deoxyglucose-6-PO4 in the uterus. The time course of estrogen effects on 2-deoxyglucose-6-PO4 formation is seen in fig. 1A. Estrogen elicits an enhanced rate of 2-deoxyglucose phosphorylation at 2 -4 h that is 3-4-

fold that of the control, followed by another wave of enhanced 2-deoxyglucose

phosphorylation at 24 h, as reported by Gorski and Raker (1973). Progesterone

alone (fig. 1A) does not evoke an enhanced rate of formation of 2-deoxy-D-[ 14C]- glucose-6-PO4 until 6 h, and a peak is seen at 24 h. Coadministration of progester-

one with estradiol (P + E; fig. 1A) results in a pattern of 2-deoxyglucose-6-phosphate formation that appears to be the additive result of E and P alone.

The effect of pretreatment with E, P, or E + P on the subsequent ability of estradiol to elicit an enhanced 2-h deoxyglucose response is seen in fig. 1B. Following pretreatment with estradiol (E + E) for 2-6 h, a second estradiol injection is able to evoke little, if any, additional deoxyglucose metabolism; however, it is apparent that after estradiol pretreatment for over 12 h, the rate of production of DGP is high in response to a second E injection (5-7-fold that of the control). Pretreat- ment with P (P + E) has little effect on the subsequent responsiveness to E injection. However, pretreatment with E t P results in a decreased responsiveness to a second E injection, when compared with the response evoked after E pretreatment alone (compare E + P + E vs. E -+ E, fig. 1B).

Effect of progesterone on estradiol-induced increase in uterine wet weight Fig. 2 shows the time course of the effect of E, P, or E + P on uterine wet weight

(panel A) and the effect of pretreatment with P (panel B), E or E f P (panel C) on the subsequent ability of estradiol to elicit an increase in uterine wet weight. As


IIll 1

’ 246 12 I I IIIII I I I I I

24 36 40 246 12 24 36 46

HOURS HOURS Fig. 1. Time course of the effect of estradiol (E), progesterone (P), and estradiol plus progesterone (E + P) on the metabolic conversion of 2-deoxy-D-[ 14C]glucose to 2-deoxy-D-glucose-6- phosphate (panel A), and the effect of a prior injection of E, P, or E + P on the subsequent ability of estradiol to elicit an enhanced phosphorylation of 2-deoxy-D-glucose[ 14C] (panel B). Immature female rats (2 per group) were given either estrogen or progesterone or E + P for designated periods of time (panel A) or E, P, or E + P for designated periods of time followed by estradiol (5 pg) for 2 h (panel B). Uteri were excised and incubated with 2-deoxy-D-glucose-l- [ 1 4C] in Eagle’s HeLa medium for 30 min at 37°C and then assayed for 2-deoxy-D-glucose-6- phosphate (DGP) formation as described in Methods. Each point represents a mean of at least 3-4 groups i- SEM.

seen in fig. 2A, a single injection of E or E + P results in a rapid (maximal by 3-6 h) increase in uterine wet weight with little change beyond the first 3-6 h. P alone

does not increase uterine wet weight until after 24 h. Pretreatment with P for 3-4 h (fig. 2B) does not affect the ability of a subse-

quent E injection to elicit a significant increase in 3 h uterine wet weight (P -+ E vs. oil -+ E). After pretreatment with E (fig. 2C; E -+ E), there is a significant response

to the second estradiol injection by 12 h, with increasing responsiveness at 24 h and 41 h after the initial estradiol injection; in contrast, after pretreatment with E + P

there is a considerably diminished response to the second estradiol injection (compare E + P -+ E vs. E + E).

Effect of P on the estradiol-induced increase in DNA synthesis rate Earlier studies have shown (Mueller, 1960; Kaye et al., 1972) that estradiol in-

creases the rate of [3H]methyl-thymidine incorporation into newly synthesized DNA maximally by 24 h in the immature rat uterus. Fig. 3 shows the time course of the effects of E or P alone on [3H] thymidine incorporation into newly synthes-


320

260

8oo 3 I I2 I 24 1 41 I 80 03 ’ 12 I 24 , 41 I

TIME OF SECOND (E, or SALINE) INJECTION- HOURS

-I

Fig. 2. Time course of the effect of estradiol (E), progesterone (P), or estradiol plus progesterone (E + P) on uterine wet weight (panel A) and the effect of a prior injection of E, P, or E + P on the subsequent ability of estradiol to elicit an increase in uterine wet weight (panels B and C). Immature, Zlday-old rats (4 per group, 2 separate experiments) were injected S.C. with 5 c(g E, 2 mg P, or E + P or control vehicle at zero time and at 3, 12, 24 or 41 h thereafter, rats received a second injection of either 5 pg E (pane!s B and C) or saline (control vehicle; panel A). Uterine wet weight was then determined at 3 h after the second injection. Values represent the mean f SEM. The control (saline/oil injected) uterine wet weight was 39.5 1 f 2.02 mg.

ized DNA and the effect of P pretreatment for various periods of time followed by estradiol for 24 h. While pretreatment with P for 8-72 h does not markedly influence the increased incorporation into DNA elicited by estradiol (P -+ E; DNA synthesis rate monitored at 24 h after the E injection; compare with E alone for 24 h), P itself induces a marked stimulation of DNA synthesis rate by 24-36 h when given alone. (It is interesting to note that a single injection of P results in a marked depression of the DNA synthesis rate, to 25% of the control, by 72 h, as does estradiol [compare 0 h vs. 72 h values on E and P curves] .) The estrogen curve in fig. 3 further confirms a prevrous observation (Kaye, 1972) on the time course of L3H]-


8 16 24 36 4Ei.72

HOURS

Fig. 3. Time course of the effect of estradiol (E) or progesterone (P) alone on [3H]methyl- thymidine incorporation into newly synthesized DNA, and of progesterone pretreatment on subsequent estradiol-induced increase (P + E) in [ 3H] methyl-thymidine incorporation into DNA. Immature female rats (2 per group) received either progesterone in oil for the designated period of time followed by estradiol (5 pg/rat) in saline for 24 h prior to sacrifice (P + E), or they received progesterone or estradiol alone at zero time and were sacrificed at the indicated times. The uteri were excised and incubated with [3H]methyl-thymidine in Eagle’s HeLa me dium for 1 h at 37°C. At the end of the incubation the uteri were washed 3 times with saline and assayed for [3H]thymidine incorporation in newly synthesized DNA as described in Methods. The data is expressed as % of control cpm/pg DNA (no treatments). Each point represents a mean of 3-4 groups f SEM.

thymidine incorporation into newly synthesized DNA, namely, that the rate of

DNA synthesis is maximal by 24-36 h after estradiol.

Effect of progesterone on the levels of estradiol receptor in immature and estrogen- primed uteri; studies on steroid specificity of the effect

Since pretreatment of immature rats with E + P (as opposed to E alone) resulted

in a decreased responsiveness to subsequent estrogen (as monitored by 2 h deoxyglucose metabolism or 3 h uterine wet weight), it was of interest to determine whether the effect was mediated by a change in the levels of estrogen receptor. As

shown previously (Bhakoo and Katzenellenbogen, 1977) P (2 mg/rat) does not move any detectable quantities of estrogen receptor from the cytoplasmic to the nuclear fractions of the immature rat uterus over a 41 h period (as determined by nuclear and cytosol exchange assays) while E (5 pg) is fully able to translocate almost all of the estrogen receptor sites. Treatment with E + P (fig. 4) also results in the initial movement of most of the estrogen receptor into the nucleus, but at later times, beyond 12 h, the replenishment of the cytosol receptor is depressed after treatment with E t P; after E alone, the levels of cytosol receptor return to levels

H.S. Bhakoo and B.S. Katzenellenbogen

1.2 , , I I I

-

“I 3 12 24 41

I’ ’ I I

I I 2.0

CYTOSOL _J

E P I .6

I

41

HOURS Fig. 4. Content of specific estrogen binding sites present in nuclear (upper panel) and cytosol (lower panel) fractions of the immature rat uterus as a function of time after a single injection of estradiol (5 pg s.c./rat; E) or progesterone (2 mg s.c./rat; P) or estradiol plus progesterone (E + P) or saline and oil (control uteri) as determined by the nuclear and cytosol exchange assay described in Materials and Methods. Each point represents the mean of two closely correspond- ing determinations in duplicate per point (3 uteri/group) and is corrected for nonspecific binding. Cytosol exchange data is after 24 h of exchange, and hence represents ‘total cytosol sites’. Total cytosol sites differ little from unfilled cytosol sites determined by direct binding at 0-4°C. The vertical bars represent f SEM.

above the control (preinjection level) by 24 h and remain elevated until at least 41

h, whereas after P + E the rate of reappearance of the cytosol receptor is slower and

levels above the control are never attained (compare E + P vs. E cytosol curves).

The effect of other steroid hormones on the level of cytosol estrogen receptor is

seen in fig. 5. Simultaneous exposure to estradiol plus 2 mg of either P, dihydro-

testosterone, or testosterone results in levels of cytosol receptor at 12,24, and 41 h

that are significantly below those of uteri exposed to estradiol alone. Cortisol is

without any effect. The depression with P is much more marked than that seen


.5

L Control _,-P

ti K 1.0

d

:: = 0.5 0

01 I I I I

12 24 36 48

HOURS Fig. 5. The effect of various steroidal hormones on the level of the cytosol estrogen receptor. Unprimed, immature rats (3/group) received 5 pg of estradiol (E) S.C. in saline plus 2 mg of either progesterone(P), testosterone(T), dihydrotestosterone (DHT), or cortisol (CORT.) suspen- sion in 0.2 ml sesame oil (injected S.C. at another site) together at zero time or 5 fig E plus 0.2 ml oil (E curve) at zero time. Uteri were excised at indicated times thereafter and the level of estradiol receptor in the cytosol was determined by the cytosol [ 3H]estradiol exchange assay. Each point represents the mean plus range of two separate determinations. The control level of receptor is that found in the immature rat uterus receiving no treatments (solid horizontal line).

with either androgen. Likewise, P at 0.5 mg is as effective as 2 mg P while’0.5 mg of T or DHT is not able to depress cytosol estrogen receptor levels (data not shown).

Further, this depression of estradiol cytosol receptor per uterus cannot be attributed to differences in ute-rine size due to these treatments as uterine weights in all experimental groups do not differ significantly over the 48 h period.

In the 3-day estrogen-primed rat uterus, a similar inhibitory effect of E + P on the replenishment of the cytosol estrogen receptor is observed (fig. 6). This figure shows the time course of the effect of P, E, E + P, and oil alone on the content and distribution of the uterine estradiol receptor between nuclear and cytoplasmic fractions in 3-day estrogen-primed immature rats. Estradiol, by 1 h, translocates most of the estradiol cytoplasmic receptors into the nuclear compartment. Thereafter, the estradiol binding sites are replenished in the cytoplasm and reach maximal levels by 12 h after the estradiol injection and remain at this level until 41 h while the nuclear receptor levels have decreased to the control level by 12 h. However, administration of P along with E results in a depressed replenishment of the estradiol cyto-

41

HOURS Fig. 6. The effect of progesterone (P), estradiol (El, progesterone plus estradiol (E + P) or oil alone on the distr~bot~ofl and content of estradiol receptor in nuclear (upper panel) and cytosol (lower panel) fractions of 3-day ~s~adiol-primed rat uteri. ~mnla~ure Female rats (2~group~~ 20-21 days old, received 5 ~.rg .of cstradiol S.C. in sdine once daily for 3 days. At 24 h after the last in.jection of estradiol, the animals rcceivcd either P, E, E + P or oil alone far designated periods of time. IJteri were excised and assayed for nuclear receptor (upper panel) and for total cytosol receptor (lower panel) by exchange assays described in Materials and Methods. The data for nucIear receptor is expressed as pmal/r.cg protein. The lightly dashed horizontal line extend- ing across the upper and lower panels represents the &clear and cytosol receptor levels in pr~l~‘uteri at time zero. Each point represents the mean f SEM of 3-4 determinations.

pIasmic receptor up to 41 h (compare cytosol E vs. E t- P curves), although it does not influence the initial movement of the cytosolic receptor into the nuclear com- yartment. It is also clear that in the absence of estradiol treatment (oil, control curve), the levels of the cytosolic receptors in these primed uteri decline markedly by 41 h. The decline seems to be somewhat faster in the progesterone-treated group than in the oil controls.


____----,

v I O4

I I I I

2 4 6 8

E2 TIME OF INJECTION OF COMPOUND AFTER E, L I I I I

12 IO 8 6 4

HOURS OF EXPOSURE TO COMPOUND

Fig. 7. Time course of progesterone-, cycloheximide-, and actinomycin D-sensitive inhibition of estradiol cytosol receptor replenishment in estrogen-primed rat uteri. Immature rats, 20-21 days old (Ygroup), were given 5 pg of estradiol in saline S.C. once daily, for 3 days. At 24 h following the third estradiol injection they received another injection of estradiol (Ez; 5 pg s.c.). Animals were then given 200 pg each of cycloheximide (CYCLO) or actinomycin D (ACT. D) in saline or saline alone i.p. or 2 mg of progesterone (7) in oil S.C. or vehicle (control) alone at the indicated time at a different site. For longer periods of treatment with the inhibitors (12, 10 and 8 h) cycloheximide and actinomycin-D were reinjected (200 @g/rat) at 6 h after the first injection to inhibit protein and RNA synthesis up to a minimum of 12 h. All animals were sacrificed 12 h after the injection of estradiol; uteri were excised and assayed for cytosol receptor by [3H]estradiol exchange assay, as described under Materials and Methods. Each point represents the mean plus range of 2 determinations from separate experiments.

Progesterone-, cycloheximide-, and actinomycin D-sensitive time course of inhibition of estradiol receptor replenishment

Fig. 7 compares the time courses and magnitude of the effects of progesterone, cycloheximide, and actinomycin D on cytosol receptor replenishment by estradiol in 3-day estrogen-primed immature rat uteri. In all cases, animals received a 5 pg

estradiol injection at zero time and were then exposed to progesterone, or cycloheximide, or actinomycin D for periods of 4-12 h with cytosol receptor levels being determined at 12 h after the initial estradiol (Ez) injection.

It is seen that when progesterone or the inhibitors are given either at zero time (along with the E2) or up to 4-6 h following estradiol injection (exposure to compound at least 6-8 h), they are markedly effective in decreasing the levels of the estradiol cytoplasmic receptor (to approx. 40-50% of the control, E2 alone, level);


however, while exposure for 6-8 h gives maximal inhibition, exposure for only the last 4 h of the 12 h period gives little, if any, inhibition of receptor replenishment. The fact that cycloheximide and actinomycin D given within 4-6 h following estradiol are effective in blocking 50-60% of the total sites replenished by 12 h

strongly suggests that about 60% of the estradiol cytosolic receptors replenished by 12 h after a single injection of estradiol are due to de novo synthesis. The rest, approx. 40%, may be sites recycled from the nuclear compartment (Mester and Bau- lieu, 1975). The progesterone-sensitive time course of inhibition of replenishment

of estradiol binding capacity closely parallels that seen for cycloheximide or actino-

mycin D and suggests that the inhibitory effect of progesterone on the replenish-

ment of the estradiol cytosol receptor may be by interfering with receptor synthesis per se.

DISCUSSION

These studies indicate that simultaneous treatment with progesterone plus estradiol results in a decreased responsiveness of the uterus to subsequent estrogen as

monitored by deoxyglucose metabolism and uterine wet weight, two characteristic

responses to estrogen. However, pretreatment with progesterone alone has no effect on the subsequent uterine response to estrogen, in terms of glucose metabolism,

uterine wet weight, or DNA synthesis rate, although the relative rate of induced protein synthesis is markedly diminished (Bhakoo and Katzenellenbogen, 1977).

Our study indicates that pretreatment with progesterone alone for up to 72 h does not have any marked influence on the estrogen-induced total uterine DNA synthesis (fig. 3). Earlier work by Tachi et al. (1972) in mature ovariectomized rats has shown that 12 h of progesterone pretreatment inhibits DNA synthesis and cell division in the uterine luminal epithelium. Since our studies are in the whole im-

mature uterine tissue, and since the luminal epithelium comprises a very small fraction of the whole uterine tissue, we are unable to detect these effects. Our results indicate that a single injection of progesterone stimulates the synthesis of DNA markedly by 24-36 h. However, both estrogen and progesterone show a drastic suppresssion of the DNA synthesis rate (to approx. 25% of control) by 72 h. Kaye et al. (1972) have reported earlier that three daily injections of progesterone inhibited the estradiol-induced increase in [ 3H] thymidine incorporation into DNA and also the mitotic indices.

Many of our findings appear to be related fo the effects of hormone treatments on the level of the estrogen receptor. Treatment with progesterone plus estradiol results in a diminished replenishment of the estradiol cytosol receptor in both the mature and immature estrogen-primed rat uterus when compared with the re-

plenishment seen after estradiol treatment alone. This phenomenon has been sub- stantiated recently by Hseuh et al. (1976) and others (Brenner et al., 1974;Pavlik and Coulson, 1976). Pretreatment with progesterone alone, however, does no: alter


the levels of cytosol estrogen receptor as measured in the whole uterus.

Previous studies by Sarff and Gorski (197 1) and others (Mester et al., 1974) have shown that the replenishment of the estrogen receptor in the immature rat uterus

is in part dependent on its cycloheximide-sensitive protein synthesis. Our data indicate that in immature 3-day estradiol-primed rats, the replenishment of estradiol

receptor in the cytoplasm in response to an estradiol injection by 12 h is dependent on its resynthesis (to approx. 60% of total) while the rest (approx. 40%) is not de-

pendent on protein or RNA synthesis and may possibly arise from reshuttling or recycling of estrogen receptor from the nuclear to the cytoplasmic compartment.

Our observation reported here shows a striking similarity in the time course and extent of the cycloheximide-, actinomycin D-, and progesterone-sensitive inhibition of receptor replenishment (fig. 7). Each of the three compounds appears to inhibit the

estradiol receptor replenishment to approximately the same extent (about 60%)

and each is still able to inhibit replenishment when given within 4-6 h after the estradiol injection. This leads us to hypothesize that progesterone may somehow se- lectively interfere with the de novo synthesis of the estradiol receptor, although some ‘recycled’ receptor is present and hence a diminished, albeit detectable,

response to subsequent estrogen is obtained. Several studies have suggested that progesterone may influence the binding of

estrogen in target tissues. In the human uterus (Brush et al., 1967; Evans and Hahnel, 1971) or monkey oviduct (Brenner et al., 1974) the cytoplasmic fraction binds

more estrogen during the follicular phase than during the luteal (progesterone- dominated) phase of the menstrual cycle. The rabbit uterus has a decreased ability to bind estrogen following pseudo-pregnancy (Jacobson et al., 1972) as does the

rat uterus during early ovoimplantation (Mester et al., 1974). The studies reported

here suggest that these phenomena of progesterone antagonism may, at least in

part, be explained by the effect of progesterone on modulating the level of estrogen

receptor and hence the ability of the uterus to respond to estrogenic hormones.

ACKNOWLEDGEMENT

These studies were supported by National Institutes of Health Grants USPH HD06726 and CA181 19 and Ford Foundation Training Grant 700-0333 (predoc- toral fellowship, H.S.B.).

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progesterone modulation of estrogen-stimulated uterine biosynthetic events and estrogen receptor...

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