0013-7227/79/1044-1112$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society
Vol. 104, No. 4 Printed in U.S.A.
Estrogen-Modulated Uterine Gene Transcription in Relation to Decidualization* STANLEY R. GLASSERf AND SHIRLEY A. McCORMACK Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030 ABSTRACT. Three daily injections of 2 mg progesterone are required to sensitize the uterus of the ovariectomized rat to deciduogenic stimuli. The period of uterine sensitivity is terminated by 17/3-estradiol (E2). The mechanisms of sensitization and desensitization have been studied. The availability of DNA sequences in uterine chromatin for RNA chain initiation was assayed under conditions which eliminate chain elongation and reinitiations. A 500% increase in RNA initiation sites was stimulated and maintained by progesterone; this increase was closely correlated with the sensitivity of the uterus to decidualization. At anytime after three daily injections of 2 mg progesterone, the injection of E2 (0.2 jug) depressed the transcriptive activity of uterine chromatin below control levels within 4 h, but the uterus remained fully sensitive for 30 h. Desensitization then proceeded rapidly and was complete 40 h after E2. This refractive state (restricted transcription and ab-
I
sence of uterine sensitivity) persisted if injection of progesterone was continued. Progesterone withdrawal also desensitized the uterus and caused a depression in uterine chromatin transcription. The chronology of these events was different (restricted transcription and loss of uterine sensitivity occur simultaneously 28-36 h after the last progesterone injection) and suggested that E2 intervention and progesterone withdrawal operate by different mechanisms to desensitize the uterus. We suggest that E2 modulates progesterone-induced uterine sensitivity, primarily by the qualitative restriction of gene expression. The products of E2-altered transcription may regulate the transformation of a sensitive uterus to one receptive to the blastocyst and thus allow implantation before the loss of uterine sensitivity takes place. (Endocrinology 104: 1112, 1979)
T IS now recognized that decidualization is a correlate of ovoimplantation in the rat. The period of time during which stromal cells can be transformed to decidual cells, however, is short, and the uterus of the pregnant or pseudopregnant rat loses its ability to form deciduoma in response to trauma. In the intact pregnant or pseudopregnant rat, the loss of this uterine sensitivity is due to the intervention of estrogen on the afternoon of day 3 (1). The ovariectomized rat has provided a model which has shown 1) that uterine sensitivity, i.e. stromal cell transformation to decidual cells, is determined by progesterone (P) (2) and 2) that a minimum of 48 h of exposure to P is required to induce decidualization (3). The administration of estrogen to the P-sensitized rat will convert the sensitized uterus to one that is receptive to blastocyst implantation (period of receptivity), initiate implantation, and later result in the complete loss of uterine sensitivity and receptivity. The mechanisms by which P transforms the neutral uterus to a state sensitive to deciduogenic stimuli and the subsequent loss of that sensitivity due to the intervention of estrogen are not known. Preliminary data indicate that the preimplantation period of uterine mat-
uration is characterized by an increase in the template capacity of uterine chromatin and the synthesis of new RNA and, subsequently, protein species (2, 4-6). This evidence of P-enhanced gene expression prompted us to investigate the loss of uterine sensitivity in terms of estrogen-induced alterations in gene transcription. For this purpose, we employed a rigorous probe which provides an accurate measurement of the number of DNA sequences in chromatin available for initiation of RNA synthesis. The assay is performed under conditions which exclude chain elongation and reinitiations (7, 8). The present data will show that the differentiation of the uterus to a sensitive state is dependent on a specific P regimen and that the loss of uterine sensitivity is related to the repression of gene expression by the intervention of estrogen.
Received March 13, 1978. * This work was supported by NIH Research Grants HD-08671, HD-07495, and CA-20853. f To whom requests for reprints should be addressed.
RNA polymerase isolation
Materials and Methods Nucleoside triphosphates were obtained from P-L Biochemicals. [3H]UTP (15 Ci/mmol) was obtained from Schwarz/ Mann. Rifampicin and calf thymus DNA were purchased from Sigma. Escherichia coli paste (strain B) was purchased from Miles Laboratory, Inc. All other chemicals were analyzed reagent grade, obtained from the J. T. Baker Co.
E. coli RNA polymerase containing the a-subunit was isolated from late log E. coli B paste by a modified method of
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GENE TRANSCRIPTION AND DECIDUALIZATION Burgess (9), as described by Bautz and Dunn (10). Stored at -20 C in storage buffer (0.01 M Tris-HCl, pH 7.9; 0.01 mM dithiothreitol; 0.10 mM EDTA; and 50% glycerol) at a concentration of 10 mg/ml, the enzyme preparation, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was greater than 95% pure. RNA polymerase was assayed using calf thymus DNA as a template. The specific activity of the enzyme was 309 U/mg protein; this corresponded to 60% of the maximum specific activity of 453 U/mg protein obtained by Burgess (9). The specific activity of the enzyme was then used for calculating the amount of enzyme used in in vitro transcription assays. Isolation of nuclei and preparation of chromatin Nuclei were isolated and purified by our modification of the method of Blobel and Potter (11), as detailed in a previous publication (12). Chromatin was isolated and purified by a modification of the method of Spelsberg et al. (13), in which all centrifugation speeds were reduced by 20% while the time of centrifugation remained unaltered. RNA synthesis without reinitiation E. coli RNA polymerase was diluted for use with 1 mg/ml bovine serum albumin. As described by Tsai et al. (7), increasing amounts of enzyme (0-40 jug) were first incubated with 5 jug chromatin at 37 C for 5 min in 200 jul preincubation buffer containing 12.5 ,umol Tris-HCl (pH 7.9), 0.25 jumol MnCl2, 12.5 jixmol (NH4)2SO,i (pH 7.9), and 0.50 /xmol j6-mercaptoethanol. Under these conditions, stable initiation complexes of chromatin and RNA polymerase could be formed while RNA synthesis was prevented clue to lack of added nucleotide triphosphates. At the end of the preincubation period, RNA synthesis was initiated by the simultaneous addition of 37.5 nmol ATP, GTP, CTP, and [3H]UTP (115 cpm/pmol), rifampicin (10 /xg), and heparin (200 jug) in a volume of 50 jul, and incubation continued for an additional 15 min at 37 C. In the presence of rifampicin, enzyme which was not present in a preinitiation complex form was inhibited, and only the polymerase bound to DNA in a stable preinitiation complex could initiate a RNA chain. Reinitiation was thus eliminated, and the number of RNA chains synthesized gives a measure of the number of initiation sites on the chromatin. Under these conditions, the primary, high affinity initiation sites were saturated by RNA polymerase. RNA synthesis was stopped by the addition of cold 5% trichloroacetic acid containing 0.01 M sodium pyrophosphate. The RNA precipitate was collected on glass fiber filters (Reeve Angel 934-AH); its radioactivity was counted in a tolueneSpectrafluor (1000:242, vol/vol) scintillation cocktail using a Beckman LS-233 liquid scintillation spectrophotometer. The number of initiation sites was calculated at the point where the saturation curve underwent a change in slope and was independent of enzyme concentration (14). Weak (secondary) initiation sites were not included in these calculations. The number of RNA chains produced was calculated from the total amount and the average chain length of RNA synthesized (14).
1113
RNA isolation The RNA synthesized was incubated for 30 min at 37 C with 100 jug nuclease-free, predigested pronase B (Sigma) in an extraction buffer containing 0.01 M NaCl, 0.01 M EDTA, 0.01 M sodium acetate (pH 5.0), 200 jug heparin, and 0.5% sodium dodecyl sulfate. The presence of heparin during the in vitro incubation proved indispensable for preventing RNA chain degradation by chromatin-bound RNase (14, 15). Equal volumes of redistilled phenol (saturated with buffer) and chloroform were added together, and the preparation was shaken in a glass Erlenmeyer flask at room temperature for at least 15 min. The mixture was centrifuged at 10,000 X g for 10 min, and the aqueous phase was removed. Phenol and chloroform were added again, and the operation was repeated until there was no protein interface between the aqueous and phenolic phases. The aqueous fraction was removed, 50 jug purified transfer RNA were added as a carrier, and the RNA was precipitated with 2.5 vol 95% ethanol at -20 C for 18 h. Sucrose gradient length)
centrifugation (measurement of chain
RNA was suspended in 100 ,ul distilled H2O and layered on 4.9-ml gradients containing 5-20% sucrose (RNase-free) in RNA extraction buffer; the gradients were centrifuged at 45,000 rpm in a Beckman SW 50.1 rotor at 4 C for 2.5 h. Mammalian ribosomal RNA was used to calibrate the gradients according to molecular weight. Twenty-five fractions (0.2 ml each) were collected, and the amount of trichloroacetic acid-precipitable radioactivity was determined. The sedimentation coefficient of each fraction was determined by reference to the molecular weight markers, assuming a linear relationship between position in the gradient and sedimentation coefficient. The chain length in nucleotides was calculated by the equations of Spirin (16) and Cedar and Felsenfeld (17), from which the average chain length of the synthesized RNA was also calculated. Template activity measurement The capacity of each chromatin preparation to serve as a template for DNA-dependent RNA synthesis in vitro using E. coli RNA polymerase and DNA (chromatin) was determined under established assay conditions (13). Reactions were run at 37 C for 10 min with 1 jug chromatin DNA and 5 U enzyme. Under these conditions, the amount of template (chromatin DNA) was rate limiting, and the incorporation of radioactivity into an acid-soluble product was linear. Animals Virgin rats, derived from a Sprague-Dawley strain and weighing 125-150 g, were purchased locally (Texas Breeding Co.). The animals were housed in a controlled environment on a 14h light-10-h dark schedule with free access to food and water. After a 7-day period of standardization, females were placed with males of proven fertility and were bilaterally ovariectomized on the morning after finding a copulatory plug and vaginal sperm (day 0 of pregnancy). Castrate animals were not treated for 7-10 days, after which they received 2 mg sc progesterone daily. After a minimum of three injections (48 h), a
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decidual cell reaction (DCR) was induced in the left horn by introducing, at the oviducal end, a needle with a slight barb. The needle was passed intraluminally to the cervical end and was withdrawn against the antimesometrial wall. The right horn was not disturbed and seved as a control. These animals were maintained on P for 96 h, when the decidualized horn and the contralateral control horn (excluding the cervix) were weighed to the nearest 0.1 mg. Sensitivity (S) of the uterus was expressed as follows: D (wt of DCR horn) - C (wt of control horn) x 100 = S% C (wt of control horn) In certain experiments, a single injection of 0.2 jig 17/?-estradiol (E2) in oil was administered sc after the third injection of P. In other experiments (P withdrawal experiments), animals were given three daily injections of P, the fourth injection was omitted, and P was readministered from the fifth injection onward. Analysis of template activity, the initiation site assay, and chain length analysis required that at least six animals be pooled to provide sufficient control horn material.
Results Uterine stimulation of ovariectomized rats treated with 2 mg P daily for at least 48 h (Px3) resulted in an increase in the weight of the traumatized (D) horn (DCR) approximately 580% greater than contralateral horns (Fig. 1, groups A, B, D, and E). There was no significant decline in the magnitude of the response or in the number of animals responding through the 10 daily injections of P (PxlO; Fig. 1, group E). The administration of estrogen, given as a single 0.2/xg dose of E2, at any time after the third injection of P completes the basic sequence (P-P-PE2) required by the
GROUP
NUMBER OF PRE-TRAUMA INJECTIONS
DCR(MG)' 200
I
2 3 4 5 6 7 8 9
A
P
P p /T
B
P
P PEp/T
c
P
P PE P
D
P P P P P/T p p p p p p p
E F
1979 Endo Vol 104 , No 4
GLASSER AND McCORMACK
1114
P
10
400
P
P
P P P E P P P P P P
P / P
T
/ T
regardless of the time of day, the decidual response
remained unaltered compared with the Px3 animal at zero time (Fig. 2). After a single injection of 0.2 jug E2 to the Px3 animal, the uterine sensitivity to a deciduogenic stimulus did not differ significantly from the P control through the 30th hour. By the 36th hour, however, the uterine decidual response was reduced by 15-20%. Uterine sensitivity regressed extremely rapidly from that point. At 39 h, the DCR was only 25% of control level and 3 h later, the response was only 10% of the control. No decidual cells could be identified in histological sections of these uteri. Single injections of estriol (Fig. 2), ranging from 0.2-10.0 jiig, given after the third P injection 6OOr
•- - _
500
DCR
400
673 ±22 mg
% RESPONSE
s
600
583 590 130 574 579 96
/T
uterus for decidualization and implantation. All of the animals treated in this manner (Fig. 1, group B) responded positively if the decidual response was induced in the next 24 h. If uterine stimulation was delayed more than 48 h after the basic sequence was completed (Fig. 1, groups C and F), the decidual response was markedly reduced, with fewer than 15% of the animals challenged demonstrating any aspect of the decidual cell response. These experiments served only to establish the broad parameters defining uterine sensitivity. In an effort to define more clearly the course of development and the extent of loss of uterine sensitivity, the following experiments were performed. Within 1 h after the third P injection (1000 h; Px3) and every 3 h thereafter for 48 h, the left uterine horn of randomly selected Px3 rats was stimulated by needle scratch. Throughout that period,
>-
n
300
0 —0
P ONLY
•--• •—•
0.2-10 ug E 3 0.2 ug E 2
200 LJ
OVARIECTOMIZED RATS (7/GROUP) •OCR WEIGHED 96 HR POST-TRAUMA % RESPONSE s p ^ ] * 100 P
P PE = BASIC P + E SEQUENCE
P = PROGESTERONE (2 mq/doy) E = ESTRADIOL \7-0 (0.2 pg) T«NEEDLE SCRATCH TRAUMA
100
OF PSYCHOYOS
FIG. 1. The effect of a single injection of E2 (0.2 jug) on the sensitivity of the uterus to deciduogenic stimuli. Three daily injections of P (P P P) are required to sensitize the uterus. E2 then completes the basic sequence required for implantation (P P PE). Uterine stimulation (T) must then occur within 24 h. At 96 h after stimulation, the control (C) and decidual (D) horns are removed and weighed. The mean weight (milligrams of the decidualized horn (±SEM) for each group was: A, 673.4 ± 22.1; B, 680.6 ± 27.0; C, 173.3 ± 17.8; D, 676.3 ± 30.5; E, 682.4 ± 34.1; and F, 127.2 ± 26.9. The DCR is expressed as a function of these weights, e.g. S% = [(D - C)/C] X 100. The rats used in these experiments were bilaterally ovariectomized and remained untreated for 10 days before the start of P treatment.
Px3
24 30 (Px4) HOURS
36
42
48 (Px5)
FIG. 2. Uterine sensitivity (S% = [(D - C)/C] x 100) regression after the injection of E2. Three injections of P (Px3) are required to sensitize the uterus to a deciduogenic stimulus, which produces a 585% increase in weight (673 mg) in the control horn after 96 h. Sensitivity can be maintained by continued injection of P (Px4 and Px5). Intervention by E2 (0.2 /ig) caused a loss of uterine sensitivity first noted by the 36th h. Single injections of E3 (0.2-10 fig) were not effective in causing the loss of uterine sensitivity.
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1115
GENE TRANSCRIPTION AND DECIDUALIZATION
did not significantly diminish the uterine response. An alternative method of reducing uterine sensitivity is to withdraw progestational support. The fourth injection of P was thus withheld in an effort to distinguish whether the estrogen-induced loss of uterine sensitivity was a specific mechanism related to that hormone or a more general process involving derangement of P-E interrelationships. As shown in Fig. 3, after the third P injection, sensitivity to the DCR could be maintained for 24 h. If no P was given at that time, the uterus rapidly lost its ability to respond to a deciduogenic stimulus. By the 36th hour after the last P injection, uterine sensitivity had regressed to less than 10% of control values. Resumption of P treatment with the regular fifth (Px5) injection did not restore sensitivity. The uterus remained in this nonresponsive (refractory) state for at least 10 days, as long as P was continued (data not shown). Analysis of uterine chromatin isolated at various periods during a course of P injections indicated that an increasing percentage of the genome became available for transcription (Fig. 4A). The 12% increase between injections 1 and 2 is equivocal (P < 0.05), but the 40% increase between Px2 and Px3 is significant (P < 0.001). Decidualization provoked a further increase in template capacity. Nondecidualized tissue reflects a gradual decrease which, although not statistically significant, does reflect increasing variability among animals of this group. + 70
A Castrate 9
The same general pattern may be noted in pregnant animals (Fig. 4B). Day 0 designates the day vaginal sperm and a copulatory plug were noted. The template activity measured on day 0 derives from the high titers 600r
Q
P 500 DCR 400
673 ±22 mg
300
200 100 0L Px3
24 (Px4)
30
36
42
HOURS
48 (Px5)
FIG. 3. The effect of P withdrawal on the duration of uterine sensitivity [(D - C)/C] X 100. The uterus was developed to the sensitive stage by three injections of P (Px3). Withholding a single injection of P, e.g. Px4, resulted in a loss of uterine sensitivity within 6 h after the injection was missed. Resumption of P injection (Px5) did not restore uterine sensitivity.
B. Pregnont 9
+ 60
+ 70 + 60
+ 50 =°
+ 50 Non-DCR
o O
+ 40
o
3 + 30
fc
o + 20
S o
+ 10
s«
I
-10
2 3 4 5 Number Daily Injections Progesterone (2mg)
1
2 3 4 5 Day of Pregnoncy
J - 10
FIG. 4. A, Increase in the proportion of chromatin template available for transcription in P-sensitized uteri. Bilaterally ovariectomized rats untreated for 10 days were injected daily with 2 mg P. Groups of animals were killed daily, and uterine template capacity was assayed with purified E. coli RNA polymerase, as described in Materials and Methods. Under conditions of this assay, the template (isolated uterine chromatin DNA) was rate limiting and the incorporation of radioactivity into an acid-soluble product was linear (12). Zero point data are represented by the template activity of untreated castrate uteri. Uterine stimulation after Px3 produced an increase in template capacity which was not mimicked by the unstimulated uterus. B, Template capacity of uterine chromatin isolated from pregnant rats on successive days of pregnancy. Presence of vaginal sperm is designated day 0. There was a progressive increase in template capacity in both ligated and nonligated horns during the preimplantation period. Implantation (nonligated horn) produced a continued increase in transcriptive activity at the site of nidation, whereas the template capacity of a sterile (ligated) horn was depressed at that time. The assay for template activity is the same as used in A.
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Endo • 1979 Vol 104 • No 4
GLASSER AND McCORMACK
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of estrogen which characterize proestrus on the previous day. The increase in template availability which follows (Fig. 4B) also reflects increased P levels in the blood (4). The increase in template availability is most marked between days 3-4. Implantation stimulates a further increase in the transcriptive capacity of chromatin isolated from nidatory sites, but gene expression of chromatin from the nongravid horn appears to be immediately depressed to day 0 levels. It is now recognized that the simple measurement of template activity, as recorded above, may be a complicated function of a number of factors involved in transcription. For this reason, the changes in transcription stimulated by P (Fig. 4A) were reexamined by a rifampicin challenge assay which distinguishes between the number of RNA chain initiations and the rate of RNA chain propagation under conditions which eliminate reinitiation. The number of initiation sites available for RNA transcription on chromatin isolated from uteri of untreated (10 days) castrate rats was equivalent to 4,000 initiation sites/pg chromatin DNA (Fig. 5). The availability of DNA sequences in chromatin was increased by three injections of P to an average of 21,000 initiation sites/pg DNA and was maintained at this level by continued administration of that hormone. The number of RNA chains initiated in uterine chromatin was severely reduced by E2 (Fig. 5). Within 4 h 24
after estrogen was given to the Px3 animals, the number of initiation sites was reduced to 4000 and was maintained at that level by the continued injection of P. The curve representing the loss of uterine sensitivity (Fig. 2), represented in Fig. 5, shows that this phenomenon occurs approximately 36 h after the repression of gene expression. P withdrawal results in a similar restriction of gene expression (Fig. 6). Between 24-28 h after the last P injection (Px3), the number of RNA chains initiated per pg DNA fell by 80%. Unlike the response to E2, there was no displacement in time (Fig. 6) between the initiation site curve and the uterine sensitivity curve reproduced from Fig. 3. None of these results appears to be due to a change in the average length of the RNA transcript (Table 1), as determined by sucrose density gradient analysis. Discussion The uterus becomes sensitive to deciduogenic stimuli after the third daily injection of P (Px3); (Fig. 1, group A) and remains so for at least 10 days (Fig. 1, group E). The decline in uterine sensitivity in the P-treated ovariectomized rat has been described as gradual and dose dependent (18, 19). We were unable to demonstrate any 24
600
16
400
o
600
o
400
16
LJ
200
1 8
?
200
12 (P x 3)
24
36 (P x 4)
48 (P x 5 )
HOURS
12 (Px3)
24
36 ( P x 4)
48 ( P x 5)
HOURS
FIG. 5. The number of RNA polymerase-binding sites available for initiation of RNA synthesis on uterine chromatin prepared from Ptreated ovariectomized rats. Intervention by a single 0.2-/zg dose of E2 after Px3 resulted in a rapid and marked decrease in the number of RNA initiation sites. Continued injections of P (Px4 and Px5) did not reverse this response. The conditions for performing this probe of uterine transcription are described in Materials and Methods. These changes in the index of transcriptive activity are compared to the time course describing the loss of uterine sensitivity, as noted in Fig. 1.
FIG. 6. The influence of P withdrawal on the number of RNA initiation sites in uterine chromatin of P-treated ovariectomized rats. Withholding a single injection of P (Px4) after the animal was exposed to three injections of P (Px3) produced a precipitous decrease in RNA initiation sites within 6 h after the injection was missed. Resumption of P treatment (Px5) did not restore the transcriptive activity to Px3 levels. Assay methods are the same as described in Fig. 5. These changes in the number of chromatin sites available for initiation of RNA synthesis are compared to the time course describing the loss of uterine sensitivity after P withdrawal (Fig. 3; compare the data with Fig. 5). Note differences in response of uterine transcription and sensitivity between E2 intervention and P withdrawal.
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GENE TRANSCRIPTION AND DECIDUALIZATION TABLE 1. Size of RNA product (nucleotides) synthesized by uterine chromatin Treatment
RNA chain length (number of nucleotides ± SD)"
Untreated ovari-x
Px 3 +0.2 jig E2 (4 h) +0.2 /jig E2 (12 h) +0.2 /j-g E2 (24 h) P X 10
722 ± 57 812 ± 39 770 ± 71 1090 ± 274 798 ± 86 785 ± 24
" Mean of three analyses. These values are not significantly different from each other.
significant differences between the decidual responses due to our smaller dose of 2 mg P/day and those obtained in previous studies with 2.5 and 5.0 mg P/day (19). It is of interest to note that ovariectomized animals treated with a single sc dose of 3.5 mg Provera retain their sensitivity to DCR for at least 35 days (data not shown). For at least 24 h after Px3 animals were treated with E2 (0.2 jig), their DCRs were equal to those of rats treated with P alone (Fig. 1, group B). If uterine stimulation was delayed more than 48 h after E2, the uterus completely lost its sensitivity (Fig. 1, groups C and F). These data are in agreement with those of Meyers (19). As long as P was continued, the uterus remained refractory. We have been able to maintain the nonresponsive condition for at least 10 days. Figure 1 (group F) represents a group of animals who have been refractory for 7 days. The time course of the loss of uterine sensitivity is of interest. Challenging the uterus at 3-h intervals after E2 indicates that the DCR is neither diminished nor potentiated, regardless of time of day, until 36 h after E2, when there is a relatively small but significant decrease (Fig. 2). Uterine sensitivity deteriorates very rapidly thereafter, falling to 10-15% of P controls during the next 3 h. A similar response to E2 was found in relatively long term P-treated or diapause-like uteri (Px5 and PxlO; data not shown). As we would anticipate from studies of the uterotrophic effect of estriol (20), single doses of E3 (0.210 fig, sc) failed to provoke a loss of uterine sensitivity (Fig. 2). The inhibitory effect of E2 on uterine sensitivity does not appear to be due to an excess of estrogen per se (19, 21). The dose used is that which completes the P-estrogen sequence required to initiate implantation. As an alternative, the loss of uterine sensitivity could be due to a relative dimunition of effective P levels. We effected a transient reduction in P titers by withholding the intended fourth daily injection (Px4). Challenging uteri in this experiment revealed a very rapid loss of uterine sensitivity within 6 h of the missed injection (Fig. 3).
1117
Thus, as a result of P withdrawal, the uterus becomes insensitive to deciduogenic stimuli almost immediately. In contrast, as a result of E2 administration, almost 42 h elapse before the uterus loses sensitivity (Fig. 2). Uterine decidualization in the rat may be regarded as the differentiation of a tissue that has been conditioned by exposure to a specific hormonal regimen (2, 4). The consensus is that during the preimplantation period there is increased synthesis of RNA on days 2 and 4 of pregnancy. This synthesis is further enhanced in decidualized tissue but reduced in interimplantation site tissue (5). On day 4, this RNA is nuclear and reportedly characterized by the appearance of RNA species that are either absent or present in small amounts in the uterus on days 1 and 6 (22). Increased synthesis of nuclear RNA and the synthesis of unique or selectively synthesized RNA species are likely to be associated with increases in the template capacity of uterine chromatin. Conversely, the loss of uterine sensitivity could be related to restriction of transcription of the DNA template. As the first step in testing this hypothesis, we compared the template activity of uterine chromatin on various days of pregnancy and P-maintained pseudopregnancy. During the first 3 days after a successful mating, there is a gradual (16%) increase in the proportion of template available for transcription (Fig. 4B). The increase in template activity between days 3-4 (day of implantation) is marked. A still greater proportion of the template becomes available at the site of nidation, whereas tissue at intersite locations and in a sterile horn is severely restricted (Figs. 4B and 6). A more consonant model for these present studies is illustrated in Fig. 4A. P injection of ovariectomized rats produces a modest increase in template availability, which is more marked after the second injection. Decidualization (antimesometrial) results in a further steady increase in the proportion of available template, whereas there is a restrictive trend in nondecidualized tissue (mesometrial) but the difference is not statistically significant. The significant increase in rat uterine chromatin template capacity between pregnancy days 3-4 and after P injections 2 and 3 supports the thesis stated above (2, 22, 23) that decidualization can be induced only in tissue that has undergone biochemical differentiation by exposure to a specific (P) hormonal regimen (2, 4). The dose of E2 used in these studies induces the differentiation of the sensitive uterus, which becomes receptive to the activated blastocyst in the 40 h before it finally becomes refractive. Initiation of implantation signifies the expression of rather specific but as yet undefined genetic information which was not detected by our template assay. Simple template activity measurements may be a complicated function of both available RNA polymerase initiation sites, and the rate of RNA chain
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Endo • 1979 Vol 104 • No 4
GLASSER AND McCORMACK
1118
elongation (13). Changes in the composition and structure of chromatin induced by steroid hormones could potentially affect either parameter with similar results. For this reason, we utilized an assay which allows a more accurate measurement of the DNA sequences in chromatin available for initiation of RNA synthesis. This assay is performed under conditions which exclude chain elongation (13). Changes in the composition and structure of chromatin induced by steroid hormones could potentially affect either parameter with similar results, tained for at least 10 days by continued injection of the hormone. Within 4 h after the injection of 0.2 jug E2, the number of initiation sites was reduced to the control level and remained at this level with continued injection of P (Fig. 5). The parallelism between the curves that describe the loss of uterine sensitivity and the loss of transcriptive capacity suggest a relationship between these two functions that is mediated by an estrogen-induced transcriptional event. These data are in contrast to those derived from the P withdrawal experiments in which the loss of transcriptional activity and the loss of uterine sensitivity seem superimposed (Fig. 6). Comparison of these two methods of inhibiting uterine sensitivity suggests that the mechanism initiated by E2 is not the same as that produced by P withdrawal. Although both methods produce loss of uterine sensitivity, E2 restricts gene expression some 36 h before the loss of sensitivity, whereas the two phenomena occur together after withdrawal of P. Though no exact analog for the animal model described in this paper exists, there are sufficient data, albeit circumstantial (Figs. 4A, 5, and 6) to suggest that P acts at the level of transcription in the uterus. Unique to this animal model is the restriction of gene expression produced by the intervention of E2 (Fig. 5). The quantitative aspects of this restriction are significant, but we suggest that the most important aspect of this alteration in transcription may prove to be qualitative. The eventual products of the redirection in gene expression produced by E2 may be implicated as regulators of uterine differentiation. These postulated regulatory proteins could be involved in the E2-induced transformation of the sensitive uterus to a uterus receptive to the late blastocyst, in the initiation of implantation, and in the eventual loss of uterine sensitivity (Figs. 2 and 5). Whether these processes are correlated, coordinated, or dissociable, or whether each requires its own specific regulatory protein remain to be investigated. Acknowledgments We thank Ms. Gail Brady and Mr. Alan Gunn for their expert technical assistance. Thanks are also due to Drs. Robert J. Schwartz and David W. Bullock for their advice and encouragement.
References 1. Zeilmaker, G. H., Experimental studies on the effects of ovariectomy and hypophysectomy on blastocyst implantation in the rat, Acta Endocrinol (Kbh) 44: 355, 1963. 2. Glasser, S. R., The uterine environment in implantation and decidualization, In Balin, H., and S. R. Glasser (eds.), Reproductive Biology, Excerpta Medica, Amsterdam, 1972, p. 776. 3. Psychoyos, A., Hormonal control of ovo-implantation, VitamHorm 31: 201, 1973. 4. Glasser, S. R., and J. H. Clark, A determinant role for progesterone in the development of uterine sensitivity to decidualization and ovo-implantation, In Markert, C, and J. Papaconstantinou (eds.), The Developmental Biology of Reproduction, Academic Press, New York, 1975, p. 311. 5. Heald, P. J., J. E. O'Grady, A. O'Hare, and M. Vass, Changes in uterine RNA during early pregnancy in the rat, Biochim Biophys Acta 262: 66, 1972. 6. O'Grady, J. E., G. E. Moffat, L. McMinn, M. A. Vass, A. O'Hare, and P. J. Heald, Uterine chromatin template activity during early stages of pregnancy in the rat, Biochim Biophys Acta 407: 125, 1975. 7. Tsai, M.-J., H. C. Towle, S. E. Harris, and B. W. O'Malley, Effect of estrogen on gene expression in chick oviduct: comparative aspects of RNA chain initiation in chromatin using homologous us E. coli RNA polymerase, J Biol Chem 251: 1960, 1976. 8. Schwartz, R. J., M.-J. Tsai, S. Y. Tsai, and B. W. O'Malley, Effect of estrogen on gene expression in the chick oviduct. V. Changes in the number of RNA polymerase binding and initiation sites in chromatin, J Biol Chem 250: 5175, 1975. 9. Burgess, R. R., A new method for the large scale purification of Escherichia coli deoxyribonucleic acid-dependent ribonucleic acid polymerase, J Biol Chem 244: 6160, 1969. 10. Bautz, E. K. F., and J. J. Dunn, DNA-dependent RNA polymerase from phage T4 infected E. coli: an enzyme missing a factor required for transcription of T4 DNA, Biochem Biophys Res Commun 34: 230, 1969. 11. Blobel, G., and V. R. Potter, Nuclei from rat liver: isolation method that combines purity with high yield, Science 154: 1662, 1966. 12. Glasser, S. R., F. Chytil, and T. C. Spelsberg, Early effects of estradiol-17/? on the chromatin and activity of DNA dependent RNA polymerases (I and II) of the rat uterus, Biochem J130: 947, 1972. 13. Spelsberg, T. C, and L. S. Hnilica, Dissociation of RNA-histone complexes by DNA, Experentia 26: 136, 1970. 14. Tsai, M.-J., R. J. Schwartz, S. Y. Tsai, and B. W. O'Malley, Effects of estrogen on gene expression in the chick oviduct. IV. Initiation of RNA synthesis on DNA and chromatin, J Biol Chem 250: 5165, 1975. 15. Cox, R. F., Transcription of high-molecular-weight RNA from henoviduct chromatin by bacterial and endogenous form-B RNA polymerases, Eur J Biochem 39: 49, 1973. 16. Spirin, A., Some problems concerning the macromolecular structure of ribonucleic acids, Prog Nucl Acid Res 1: 301, 1963. 17. Cedar, H., and G. Felsenfeld, Transcription of chromatin in vitro, JMolBiolll: 237, 1973. 18. Yochim, J. M., and V. J. DeFeo, Hormonal control of the onset magnitude and duration of uterine sensitivity in the rat by steroid hormones in the ovary, Endocrinology 72: 317, 1963. 19. Meyers, K. P., Hormonal requirements for the maintenance of oestradiol-induced inhibition of uterine sensitivity in the ovariectomized rat, J Endocrinol 46: 341, 1970. 20. Anderson, J., J. H. Clark, and E. J. Peck, Jr., The relationship between nuclear receptor estrogen binding and uterotrophic responses, Biochem Biophys Res Commun 48: 1460,1972. 21. Psychoyos, A., Endocrine control of egg implantation, In Greep, R. O., and E. B. Astwood (eds.), Handbook of Physiology, vol. 2, Sect. 7, part 2, American Physiological Society, Washington DC, 1973, p. 187. 22. Heald, P. J., J. E. O'Grady, and G. E. Moffat, The incorporation of 3 H-uridine into nuclear RNA in the uterus of the rat during early pregnancy, Biochim Biophys Acta 281: 347, 1972.
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Vol. 104, No. 4 Printed in U.S.A.
Estrogen-Modulated Uterine Gene Transcription in Relation to Decidualization* STANLEY R. GLASSERf AND SHIRLEY A. McCORMACK Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030 ABSTRACT. Three daily injections of 2 mg progesterone are required to sensitize the uterus of the ovariectomized rat to deciduogenic stimuli. The period of uterine sensitivity is terminated by 17/3-estradiol (E2). The mechanisms of sensitization and desensitization have been studied. The availability of DNA sequences in uterine chromatin for RNA chain initiation was assayed under conditions which eliminate chain elongation and reinitiations. A 500% increase in RNA initiation sites was stimulated and maintained by progesterone; this increase was closely correlated with the sensitivity of the uterus to decidualization. At anytime after three daily injections of 2 mg progesterone, the injection of E2 (0.2 jug) depressed the transcriptive activity of uterine chromatin below control levels within 4 h, but the uterus remained fully sensitive for 30 h. Desensitization then proceeded rapidly and was complete 40 h after E2. This refractive state (restricted transcription and ab-
I
sence of uterine sensitivity) persisted if injection of progesterone was continued. Progesterone withdrawal also desensitized the uterus and caused a depression in uterine chromatin transcription. The chronology of these events was different (restricted transcription and loss of uterine sensitivity occur simultaneously 28-36 h after the last progesterone injection) and suggested that E2 intervention and progesterone withdrawal operate by different mechanisms to desensitize the uterus. We suggest that E2 modulates progesterone-induced uterine sensitivity, primarily by the qualitative restriction of gene expression. The products of E2-altered transcription may regulate the transformation of a sensitive uterus to one receptive to the blastocyst and thus allow implantation before the loss of uterine sensitivity takes place. (Endocrinology 104: 1112, 1979)
T IS now recognized that decidualization is a correlate of ovoimplantation in the rat. The period of time during which stromal cells can be transformed to decidual cells, however, is short, and the uterus of the pregnant or pseudopregnant rat loses its ability to form deciduoma in response to trauma. In the intact pregnant or pseudopregnant rat, the loss of this uterine sensitivity is due to the intervention of estrogen on the afternoon of day 3 (1). The ovariectomized rat has provided a model which has shown 1) that uterine sensitivity, i.e. stromal cell transformation to decidual cells, is determined by progesterone (P) (2) and 2) that a minimum of 48 h of exposure to P is required to induce decidualization (3). The administration of estrogen to the P-sensitized rat will convert the sensitized uterus to one that is receptive to blastocyst implantation (period of receptivity), initiate implantation, and later result in the complete loss of uterine sensitivity and receptivity. The mechanisms by which P transforms the neutral uterus to a state sensitive to deciduogenic stimuli and the subsequent loss of that sensitivity due to the intervention of estrogen are not known. Preliminary data indicate that the preimplantation period of uterine mat-
uration is characterized by an increase in the template capacity of uterine chromatin and the synthesis of new RNA and, subsequently, protein species (2, 4-6). This evidence of P-enhanced gene expression prompted us to investigate the loss of uterine sensitivity in terms of estrogen-induced alterations in gene transcription. For this purpose, we employed a rigorous probe which provides an accurate measurement of the number of DNA sequences in chromatin available for initiation of RNA synthesis. The assay is performed under conditions which exclude chain elongation and reinitiations (7, 8). The present data will show that the differentiation of the uterus to a sensitive state is dependent on a specific P regimen and that the loss of uterine sensitivity is related to the repression of gene expression by the intervention of estrogen.
Received March 13, 1978. * This work was supported by NIH Research Grants HD-08671, HD-07495, and CA-20853. f To whom requests for reprints should be addressed.
RNA polymerase isolation
Materials and Methods Nucleoside triphosphates were obtained from P-L Biochemicals. [3H]UTP (15 Ci/mmol) was obtained from Schwarz/ Mann. Rifampicin and calf thymus DNA were purchased from Sigma. Escherichia coli paste (strain B) was purchased from Miles Laboratory, Inc. All other chemicals were analyzed reagent grade, obtained from the J. T. Baker Co.
E. coli RNA polymerase containing the a-subunit was isolated from late log E. coli B paste by a modified method of
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GENE TRANSCRIPTION AND DECIDUALIZATION Burgess (9), as described by Bautz and Dunn (10). Stored at -20 C in storage buffer (0.01 M Tris-HCl, pH 7.9; 0.01 mM dithiothreitol; 0.10 mM EDTA; and 50% glycerol) at a concentration of 10 mg/ml, the enzyme preparation, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was greater than 95% pure. RNA polymerase was assayed using calf thymus DNA as a template. The specific activity of the enzyme was 309 U/mg protein; this corresponded to 60% of the maximum specific activity of 453 U/mg protein obtained by Burgess (9). The specific activity of the enzyme was then used for calculating the amount of enzyme used in in vitro transcription assays. Isolation of nuclei and preparation of chromatin Nuclei were isolated and purified by our modification of the method of Blobel and Potter (11), as detailed in a previous publication (12). Chromatin was isolated and purified by a modification of the method of Spelsberg et al. (13), in which all centrifugation speeds were reduced by 20% while the time of centrifugation remained unaltered. RNA synthesis without reinitiation E. coli RNA polymerase was diluted for use with 1 mg/ml bovine serum albumin. As described by Tsai et al. (7), increasing amounts of enzyme (0-40 jug) were first incubated with 5 jug chromatin at 37 C for 5 min in 200 jul preincubation buffer containing 12.5 ,umol Tris-HCl (pH 7.9), 0.25 jumol MnCl2, 12.5 jixmol (NH4)2SO,i (pH 7.9), and 0.50 /xmol j6-mercaptoethanol. Under these conditions, stable initiation complexes of chromatin and RNA polymerase could be formed while RNA synthesis was prevented clue to lack of added nucleotide triphosphates. At the end of the preincubation period, RNA synthesis was initiated by the simultaneous addition of 37.5 nmol ATP, GTP, CTP, and [3H]UTP (115 cpm/pmol), rifampicin (10 /xg), and heparin (200 jug) in a volume of 50 jul, and incubation continued for an additional 15 min at 37 C. In the presence of rifampicin, enzyme which was not present in a preinitiation complex form was inhibited, and only the polymerase bound to DNA in a stable preinitiation complex could initiate a RNA chain. Reinitiation was thus eliminated, and the number of RNA chains synthesized gives a measure of the number of initiation sites on the chromatin. Under these conditions, the primary, high affinity initiation sites were saturated by RNA polymerase. RNA synthesis was stopped by the addition of cold 5% trichloroacetic acid containing 0.01 M sodium pyrophosphate. The RNA precipitate was collected on glass fiber filters (Reeve Angel 934-AH); its radioactivity was counted in a tolueneSpectrafluor (1000:242, vol/vol) scintillation cocktail using a Beckman LS-233 liquid scintillation spectrophotometer. The number of initiation sites was calculated at the point where the saturation curve underwent a change in slope and was independent of enzyme concentration (14). Weak (secondary) initiation sites were not included in these calculations. The number of RNA chains produced was calculated from the total amount and the average chain length of RNA synthesized (14).
1113
RNA isolation The RNA synthesized was incubated for 30 min at 37 C with 100 jug nuclease-free, predigested pronase B (Sigma) in an extraction buffer containing 0.01 M NaCl, 0.01 M EDTA, 0.01 M sodium acetate (pH 5.0), 200 jug heparin, and 0.5% sodium dodecyl sulfate. The presence of heparin during the in vitro incubation proved indispensable for preventing RNA chain degradation by chromatin-bound RNase (14, 15). Equal volumes of redistilled phenol (saturated with buffer) and chloroform were added together, and the preparation was shaken in a glass Erlenmeyer flask at room temperature for at least 15 min. The mixture was centrifuged at 10,000 X g for 10 min, and the aqueous phase was removed. Phenol and chloroform were added again, and the operation was repeated until there was no protein interface between the aqueous and phenolic phases. The aqueous fraction was removed, 50 jug purified transfer RNA were added as a carrier, and the RNA was precipitated with 2.5 vol 95% ethanol at -20 C for 18 h. Sucrose gradient length)
centrifugation (measurement of chain
RNA was suspended in 100 ,ul distilled H2O and layered on 4.9-ml gradients containing 5-20% sucrose (RNase-free) in RNA extraction buffer; the gradients were centrifuged at 45,000 rpm in a Beckman SW 50.1 rotor at 4 C for 2.5 h. Mammalian ribosomal RNA was used to calibrate the gradients according to molecular weight. Twenty-five fractions (0.2 ml each) were collected, and the amount of trichloroacetic acid-precipitable radioactivity was determined. The sedimentation coefficient of each fraction was determined by reference to the molecular weight markers, assuming a linear relationship between position in the gradient and sedimentation coefficient. The chain length in nucleotides was calculated by the equations of Spirin (16) and Cedar and Felsenfeld (17), from which the average chain length of the synthesized RNA was also calculated. Template activity measurement The capacity of each chromatin preparation to serve as a template for DNA-dependent RNA synthesis in vitro using E. coli RNA polymerase and DNA (chromatin) was determined under established assay conditions (13). Reactions were run at 37 C for 10 min with 1 jug chromatin DNA and 5 U enzyme. Under these conditions, the amount of template (chromatin DNA) was rate limiting, and the incorporation of radioactivity into an acid-soluble product was linear. Animals Virgin rats, derived from a Sprague-Dawley strain and weighing 125-150 g, were purchased locally (Texas Breeding Co.). The animals were housed in a controlled environment on a 14h light-10-h dark schedule with free access to food and water. After a 7-day period of standardization, females were placed with males of proven fertility and were bilaterally ovariectomized on the morning after finding a copulatory plug and vaginal sperm (day 0 of pregnancy). Castrate animals were not treated for 7-10 days, after which they received 2 mg sc progesterone daily. After a minimum of three injections (48 h), a
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decidual cell reaction (DCR) was induced in the left horn by introducing, at the oviducal end, a needle with a slight barb. The needle was passed intraluminally to the cervical end and was withdrawn against the antimesometrial wall. The right horn was not disturbed and seved as a control. These animals were maintained on P for 96 h, when the decidualized horn and the contralateral control horn (excluding the cervix) were weighed to the nearest 0.1 mg. Sensitivity (S) of the uterus was expressed as follows: D (wt of DCR horn) - C (wt of control horn) x 100 = S% C (wt of control horn) In certain experiments, a single injection of 0.2 jig 17/?-estradiol (E2) in oil was administered sc after the third injection of P. In other experiments (P withdrawal experiments), animals were given three daily injections of P, the fourth injection was omitted, and P was readministered from the fifth injection onward. Analysis of template activity, the initiation site assay, and chain length analysis required that at least six animals be pooled to provide sufficient control horn material.
Results Uterine stimulation of ovariectomized rats treated with 2 mg P daily for at least 48 h (Px3) resulted in an increase in the weight of the traumatized (D) horn (DCR) approximately 580% greater than contralateral horns (Fig. 1, groups A, B, D, and E). There was no significant decline in the magnitude of the response or in the number of animals responding through the 10 daily injections of P (PxlO; Fig. 1, group E). The administration of estrogen, given as a single 0.2/xg dose of E2, at any time after the third injection of P completes the basic sequence (P-P-PE2) required by the
GROUP
NUMBER OF PRE-TRAUMA INJECTIONS
DCR(MG)' 200
I
2 3 4 5 6 7 8 9
A
P
P p /T
B
P
P PEp/T
c
P
P PE P
D
P P P P P/T p p p p p p p
E F
1979 Endo Vol 104 , No 4
GLASSER AND McCORMACK
1114
P
10
400
P
P
P P P E P P P P P P
P / P
T
/ T
regardless of the time of day, the decidual response
remained unaltered compared with the Px3 animal at zero time (Fig. 2). After a single injection of 0.2 jug E2 to the Px3 animal, the uterine sensitivity to a deciduogenic stimulus did not differ significantly from the P control through the 30th hour. By the 36th hour, however, the uterine decidual response was reduced by 15-20%. Uterine sensitivity regressed extremely rapidly from that point. At 39 h, the DCR was only 25% of control level and 3 h later, the response was only 10% of the control. No decidual cells could be identified in histological sections of these uteri. Single injections of estriol (Fig. 2), ranging from 0.2-10.0 jiig, given after the third P injection 6OOr
•- - _
500
DCR
400
673 ±22 mg
% RESPONSE
s
600
583 590 130 574 579 96
/T
uterus for decidualization and implantation. All of the animals treated in this manner (Fig. 1, group B) responded positively if the decidual response was induced in the next 24 h. If uterine stimulation was delayed more than 48 h after the basic sequence was completed (Fig. 1, groups C and F), the decidual response was markedly reduced, with fewer than 15% of the animals challenged demonstrating any aspect of the decidual cell response. These experiments served only to establish the broad parameters defining uterine sensitivity. In an effort to define more clearly the course of development and the extent of loss of uterine sensitivity, the following experiments were performed. Within 1 h after the third P injection (1000 h; Px3) and every 3 h thereafter for 48 h, the left uterine horn of randomly selected Px3 rats was stimulated by needle scratch. Throughout that period,
>-
n
300
0 —0
P ONLY
•--• •—•
0.2-10 ug E 3 0.2 ug E 2
200 LJ
OVARIECTOMIZED RATS (7/GROUP) •OCR WEIGHED 96 HR POST-TRAUMA % RESPONSE s p ^ ] * 100 P
P PE = BASIC P + E SEQUENCE
P = PROGESTERONE (2 mq/doy) E = ESTRADIOL \7-0 (0.2 pg) T«NEEDLE SCRATCH TRAUMA
100
OF PSYCHOYOS
FIG. 1. The effect of a single injection of E2 (0.2 jug) on the sensitivity of the uterus to deciduogenic stimuli. Three daily injections of P (P P P) are required to sensitize the uterus. E2 then completes the basic sequence required for implantation (P P PE). Uterine stimulation (T) must then occur within 24 h. At 96 h after stimulation, the control (C) and decidual (D) horns are removed and weighed. The mean weight (milligrams of the decidualized horn (±SEM) for each group was: A, 673.4 ± 22.1; B, 680.6 ± 27.0; C, 173.3 ± 17.8; D, 676.3 ± 30.5; E, 682.4 ± 34.1; and F, 127.2 ± 26.9. The DCR is expressed as a function of these weights, e.g. S% = [(D - C)/C] X 100. The rats used in these experiments were bilaterally ovariectomized and remained untreated for 10 days before the start of P treatment.
Px3
24 30 (Px4) HOURS
36
42
48 (Px5)
FIG. 2. Uterine sensitivity (S% = [(D - C)/C] x 100) regression after the injection of E2. Three injections of P (Px3) are required to sensitize the uterus to a deciduogenic stimulus, which produces a 585% increase in weight (673 mg) in the control horn after 96 h. Sensitivity can be maintained by continued injection of P (Px4 and Px5). Intervention by E2 (0.2 /ig) caused a loss of uterine sensitivity first noted by the 36th h. Single injections of E3 (0.2-10 fig) were not effective in causing the loss of uterine sensitivity.
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1115
GENE TRANSCRIPTION AND DECIDUALIZATION
did not significantly diminish the uterine response. An alternative method of reducing uterine sensitivity is to withdraw progestational support. The fourth injection of P was thus withheld in an effort to distinguish whether the estrogen-induced loss of uterine sensitivity was a specific mechanism related to that hormone or a more general process involving derangement of P-E interrelationships. As shown in Fig. 3, after the third P injection, sensitivity to the DCR could be maintained for 24 h. If no P was given at that time, the uterus rapidly lost its ability to respond to a deciduogenic stimulus. By the 36th hour after the last P injection, uterine sensitivity had regressed to less than 10% of control values. Resumption of P treatment with the regular fifth (Px5) injection did not restore sensitivity. The uterus remained in this nonresponsive (refractory) state for at least 10 days, as long as P was continued (data not shown). Analysis of uterine chromatin isolated at various periods during a course of P injections indicated that an increasing percentage of the genome became available for transcription (Fig. 4A). The 12% increase between injections 1 and 2 is equivocal (P < 0.05), but the 40% increase between Px2 and Px3 is significant (P < 0.001). Decidualization provoked a further increase in template capacity. Nondecidualized tissue reflects a gradual decrease which, although not statistically significant, does reflect increasing variability among animals of this group. + 70
A Castrate 9
The same general pattern may be noted in pregnant animals (Fig. 4B). Day 0 designates the day vaginal sperm and a copulatory plug were noted. The template activity measured on day 0 derives from the high titers 600r
Q
P 500 DCR 400
673 ±22 mg
300
200 100 0L Px3
24 (Px4)
30
36
42
HOURS
48 (Px5)
FIG. 3. The effect of P withdrawal on the duration of uterine sensitivity [(D - C)/C] X 100. The uterus was developed to the sensitive stage by three injections of P (Px3). Withholding a single injection of P, e.g. Px4, resulted in a loss of uterine sensitivity within 6 h after the injection was missed. Resumption of P injection (Px5) did not restore uterine sensitivity.
B. Pregnont 9
+ 60
+ 70 + 60
+ 50 =°
+ 50 Non-DCR
o O
+ 40
o
3 + 30
fc
o + 20
S o
+ 10
s«
I
-10
2 3 4 5 Number Daily Injections Progesterone (2mg)
1
2 3 4 5 Day of Pregnoncy
J - 10
FIG. 4. A, Increase in the proportion of chromatin template available for transcription in P-sensitized uteri. Bilaterally ovariectomized rats untreated for 10 days were injected daily with 2 mg P. Groups of animals were killed daily, and uterine template capacity was assayed with purified E. coli RNA polymerase, as described in Materials and Methods. Under conditions of this assay, the template (isolated uterine chromatin DNA) was rate limiting and the incorporation of radioactivity into an acid-soluble product was linear (12). Zero point data are represented by the template activity of untreated castrate uteri. Uterine stimulation after Px3 produced an increase in template capacity which was not mimicked by the unstimulated uterus. B, Template capacity of uterine chromatin isolated from pregnant rats on successive days of pregnancy. Presence of vaginal sperm is designated day 0. There was a progressive increase in template capacity in both ligated and nonligated horns during the preimplantation period. Implantation (nonligated horn) produced a continued increase in transcriptive activity at the site of nidation, whereas the template capacity of a sterile (ligated) horn was depressed at that time. The assay for template activity is the same as used in A.
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Endo • 1979 Vol 104 • No 4
GLASSER AND McCORMACK
1116
of estrogen which characterize proestrus on the previous day. The increase in template availability which follows (Fig. 4B) also reflects increased P levels in the blood (4). The increase in template availability is most marked between days 3-4. Implantation stimulates a further increase in the transcriptive capacity of chromatin isolated from nidatory sites, but gene expression of chromatin from the nongravid horn appears to be immediately depressed to day 0 levels. It is now recognized that the simple measurement of template activity, as recorded above, may be a complicated function of a number of factors involved in transcription. For this reason, the changes in transcription stimulated by P (Fig. 4A) were reexamined by a rifampicin challenge assay which distinguishes between the number of RNA chain initiations and the rate of RNA chain propagation under conditions which eliminate reinitiation. The number of initiation sites available for RNA transcription on chromatin isolated from uteri of untreated (10 days) castrate rats was equivalent to 4,000 initiation sites/pg chromatin DNA (Fig. 5). The availability of DNA sequences in chromatin was increased by three injections of P to an average of 21,000 initiation sites/pg DNA and was maintained at this level by continued administration of that hormone. The number of RNA chains initiated in uterine chromatin was severely reduced by E2 (Fig. 5). Within 4 h 24
after estrogen was given to the Px3 animals, the number of initiation sites was reduced to 4000 and was maintained at that level by the continued injection of P. The curve representing the loss of uterine sensitivity (Fig. 2), represented in Fig. 5, shows that this phenomenon occurs approximately 36 h after the repression of gene expression. P withdrawal results in a similar restriction of gene expression (Fig. 6). Between 24-28 h after the last P injection (Px3), the number of RNA chains initiated per pg DNA fell by 80%. Unlike the response to E2, there was no displacement in time (Fig. 6) between the initiation site curve and the uterine sensitivity curve reproduced from Fig. 3. None of these results appears to be due to a change in the average length of the RNA transcript (Table 1), as determined by sucrose density gradient analysis. Discussion The uterus becomes sensitive to deciduogenic stimuli after the third daily injection of P (Px3); (Fig. 1, group A) and remains so for at least 10 days (Fig. 1, group E). The decline in uterine sensitivity in the P-treated ovariectomized rat has been described as gradual and dose dependent (18, 19). We were unable to demonstrate any 24
600
16
400
o
600
o
400
16
LJ
200
1 8
?
200
12 (P x 3)
24
36 (P x 4)
48 (P x 5 )
HOURS
12 (Px3)
24
36 ( P x 4)
48 ( P x 5)
HOURS
FIG. 5. The number of RNA polymerase-binding sites available for initiation of RNA synthesis on uterine chromatin prepared from Ptreated ovariectomized rats. Intervention by a single 0.2-/zg dose of E2 after Px3 resulted in a rapid and marked decrease in the number of RNA initiation sites. Continued injections of P (Px4 and Px5) did not reverse this response. The conditions for performing this probe of uterine transcription are described in Materials and Methods. These changes in the index of transcriptive activity are compared to the time course describing the loss of uterine sensitivity, as noted in Fig. 1.
FIG. 6. The influence of P withdrawal on the number of RNA initiation sites in uterine chromatin of P-treated ovariectomized rats. Withholding a single injection of P (Px4) after the animal was exposed to three injections of P (Px3) produced a precipitous decrease in RNA initiation sites within 6 h after the injection was missed. Resumption of P treatment (Px5) did not restore the transcriptive activity to Px3 levels. Assay methods are the same as described in Fig. 5. These changes in the number of chromatin sites available for initiation of RNA synthesis are compared to the time course describing the loss of uterine sensitivity after P withdrawal (Fig. 3; compare the data with Fig. 5). Note differences in response of uterine transcription and sensitivity between E2 intervention and P withdrawal.
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GENE TRANSCRIPTION AND DECIDUALIZATION TABLE 1. Size of RNA product (nucleotides) synthesized by uterine chromatin Treatment
RNA chain length (number of nucleotides ± SD)"
Untreated ovari-x
Px 3 +0.2 jig E2 (4 h) +0.2 /jig E2 (12 h) +0.2 /j-g E2 (24 h) P X 10
722 ± 57 812 ± 39 770 ± 71 1090 ± 274 798 ± 86 785 ± 24
" Mean of three analyses. These values are not significantly different from each other.
significant differences between the decidual responses due to our smaller dose of 2 mg P/day and those obtained in previous studies with 2.5 and 5.0 mg P/day (19). It is of interest to note that ovariectomized animals treated with a single sc dose of 3.5 mg Provera retain their sensitivity to DCR for at least 35 days (data not shown). For at least 24 h after Px3 animals were treated with E2 (0.2 jig), their DCRs were equal to those of rats treated with P alone (Fig. 1, group B). If uterine stimulation was delayed more than 48 h after E2, the uterus completely lost its sensitivity (Fig. 1, groups C and F). These data are in agreement with those of Meyers (19). As long as P was continued, the uterus remained refractory. We have been able to maintain the nonresponsive condition for at least 10 days. Figure 1 (group F) represents a group of animals who have been refractory for 7 days. The time course of the loss of uterine sensitivity is of interest. Challenging the uterus at 3-h intervals after E2 indicates that the DCR is neither diminished nor potentiated, regardless of time of day, until 36 h after E2, when there is a relatively small but significant decrease (Fig. 2). Uterine sensitivity deteriorates very rapidly thereafter, falling to 10-15% of P controls during the next 3 h. A similar response to E2 was found in relatively long term P-treated or diapause-like uteri (Px5 and PxlO; data not shown). As we would anticipate from studies of the uterotrophic effect of estriol (20), single doses of E3 (0.210 fig, sc) failed to provoke a loss of uterine sensitivity (Fig. 2). The inhibitory effect of E2 on uterine sensitivity does not appear to be due to an excess of estrogen per se (19, 21). The dose used is that which completes the P-estrogen sequence required to initiate implantation. As an alternative, the loss of uterine sensitivity could be due to a relative dimunition of effective P levels. We effected a transient reduction in P titers by withholding the intended fourth daily injection (Px4). Challenging uteri in this experiment revealed a very rapid loss of uterine sensitivity within 6 h of the missed injection (Fig. 3).
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Thus, as a result of P withdrawal, the uterus becomes insensitive to deciduogenic stimuli almost immediately. In contrast, as a result of E2 administration, almost 42 h elapse before the uterus loses sensitivity (Fig. 2). Uterine decidualization in the rat may be regarded as the differentiation of a tissue that has been conditioned by exposure to a specific hormonal regimen (2, 4). The consensus is that during the preimplantation period there is increased synthesis of RNA on days 2 and 4 of pregnancy. This synthesis is further enhanced in decidualized tissue but reduced in interimplantation site tissue (5). On day 4, this RNA is nuclear and reportedly characterized by the appearance of RNA species that are either absent or present in small amounts in the uterus on days 1 and 6 (22). Increased synthesis of nuclear RNA and the synthesis of unique or selectively synthesized RNA species are likely to be associated with increases in the template capacity of uterine chromatin. Conversely, the loss of uterine sensitivity could be related to restriction of transcription of the DNA template. As the first step in testing this hypothesis, we compared the template activity of uterine chromatin on various days of pregnancy and P-maintained pseudopregnancy. During the first 3 days after a successful mating, there is a gradual (16%) increase in the proportion of template available for transcription (Fig. 4B). The increase in template activity between days 3-4 (day of implantation) is marked. A still greater proportion of the template becomes available at the site of nidation, whereas tissue at intersite locations and in a sterile horn is severely restricted (Figs. 4B and 6). A more consonant model for these present studies is illustrated in Fig. 4A. P injection of ovariectomized rats produces a modest increase in template availability, which is more marked after the second injection. Decidualization (antimesometrial) results in a further steady increase in the proportion of available template, whereas there is a restrictive trend in nondecidualized tissue (mesometrial) but the difference is not statistically significant. The significant increase in rat uterine chromatin template capacity between pregnancy days 3-4 and after P injections 2 and 3 supports the thesis stated above (2, 22, 23) that decidualization can be induced only in tissue that has undergone biochemical differentiation by exposure to a specific (P) hormonal regimen (2, 4). The dose of E2 used in these studies induces the differentiation of the sensitive uterus, which becomes receptive to the activated blastocyst in the 40 h before it finally becomes refractive. Initiation of implantation signifies the expression of rather specific but as yet undefined genetic information which was not detected by our template assay. Simple template activity measurements may be a complicated function of both available RNA polymerase initiation sites, and the rate of RNA chain
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Endo • 1979 Vol 104 • No 4
GLASSER AND McCORMACK
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elongation (13). Changes in the composition and structure of chromatin induced by steroid hormones could potentially affect either parameter with similar results. For this reason, we utilized an assay which allows a more accurate measurement of the DNA sequences in chromatin available for initiation of RNA synthesis. This assay is performed under conditions which exclude chain elongation (13). Changes in the composition and structure of chromatin induced by steroid hormones could potentially affect either parameter with similar results, tained for at least 10 days by continued injection of the hormone. Within 4 h after the injection of 0.2 jug E2, the number of initiation sites was reduced to the control level and remained at this level with continued injection of P (Fig. 5). The parallelism between the curves that describe the loss of uterine sensitivity and the loss of transcriptive capacity suggest a relationship between these two functions that is mediated by an estrogen-induced transcriptional event. These data are in contrast to those derived from the P withdrawal experiments in which the loss of transcriptional activity and the loss of uterine sensitivity seem superimposed (Fig. 6). Comparison of these two methods of inhibiting uterine sensitivity suggests that the mechanism initiated by E2 is not the same as that produced by P withdrawal. Although both methods produce loss of uterine sensitivity, E2 restricts gene expression some 36 h before the loss of sensitivity, whereas the two phenomena occur together after withdrawal of P. Though no exact analog for the animal model described in this paper exists, there are sufficient data, albeit circumstantial (Figs. 4A, 5, and 6) to suggest that P acts at the level of transcription in the uterus. Unique to this animal model is the restriction of gene expression produced by the intervention of E2 (Fig. 5). The quantitative aspects of this restriction are significant, but we suggest that the most important aspect of this alteration in transcription may prove to be qualitative. The eventual products of the redirection in gene expression produced by E2 may be implicated as regulators of uterine differentiation. These postulated regulatory proteins could be involved in the E2-induced transformation of the sensitive uterus to a uterus receptive to the late blastocyst, in the initiation of implantation, and in the eventual loss of uterine sensitivity (Figs. 2 and 5). Whether these processes are correlated, coordinated, or dissociable, or whether each requires its own specific regulatory protein remain to be investigated. Acknowledgments We thank Ms. Gail Brady and Mr. Alan Gunn for their expert technical assistance. Thanks are also due to Drs. Robert J. Schwartz and David W. Bullock for their advice and encouragement.
References 1. Zeilmaker, G. H., Experimental studies on the effects of ovariectomy and hypophysectomy on blastocyst implantation in the rat, Acta Endocrinol (Kbh) 44: 355, 1963. 2. Glasser, S. R., The uterine environment in implantation and decidualization, In Balin, H., and S. R. Glasser (eds.), Reproductive Biology, Excerpta Medica, Amsterdam, 1972, p. 776. 3. Psychoyos, A., Hormonal control of ovo-implantation, VitamHorm 31: 201, 1973. 4. Glasser, S. R., and J. H. Clark, A determinant role for progesterone in the development of uterine sensitivity to decidualization and ovo-implantation, In Markert, C, and J. Papaconstantinou (eds.), The Developmental Biology of Reproduction, Academic Press, New York, 1975, p. 311. 5. Heald, P. J., J. E. O'Grady, A. O'Hare, and M. Vass, Changes in uterine RNA during early pregnancy in the rat, Biochim Biophys Acta 262: 66, 1972. 6. O'Grady, J. E., G. E. Moffat, L. McMinn, M. A. Vass, A. O'Hare, and P. J. Heald, Uterine chromatin template activity during early stages of pregnancy in the rat, Biochim Biophys Acta 407: 125, 1975. 7. Tsai, M.-J., H. C. Towle, S. E. Harris, and B. W. O'Malley, Effect of estrogen on gene expression in chick oviduct: comparative aspects of RNA chain initiation in chromatin using homologous us E. coli RNA polymerase, J Biol Chem 251: 1960, 1976. 8. Schwartz, R. J., M.-J. Tsai, S. Y. Tsai, and B. W. O'Malley, Effect of estrogen on gene expression in the chick oviduct. V. Changes in the number of RNA polymerase binding and initiation sites in chromatin, J Biol Chem 250: 5175, 1975. 9. Burgess, R. R., A new method for the large scale purification of Escherichia coli deoxyribonucleic acid-dependent ribonucleic acid polymerase, J Biol Chem 244: 6160, 1969. 10. Bautz, E. K. F., and J. J. Dunn, DNA-dependent RNA polymerase from phage T4 infected E. coli: an enzyme missing a factor required for transcription of T4 DNA, Biochem Biophys Res Commun 34: 230, 1969. 11. Blobel, G., and V. R. Potter, Nuclei from rat liver: isolation method that combines purity with high yield, Science 154: 1662, 1966. 12. Glasser, S. R., F. Chytil, and T. C. Spelsberg, Early effects of estradiol-17/? on the chromatin and activity of DNA dependent RNA polymerases (I and II) of the rat uterus, Biochem J130: 947, 1972. 13. Spelsberg, T. C, and L. S. Hnilica, Dissociation of RNA-histone complexes by DNA, Experentia 26: 136, 1970. 14. Tsai, M.-J., R. J. Schwartz, S. Y. Tsai, and B. W. O'Malley, Effects of estrogen on gene expression in the chick oviduct. IV. Initiation of RNA synthesis on DNA and chromatin, J Biol Chem 250: 5165, 1975. 15. Cox, R. F., Transcription of high-molecular-weight RNA from henoviduct chromatin by bacterial and endogenous form-B RNA polymerases, Eur J Biochem 39: 49, 1973. 16. Spirin, A., Some problems concerning the macromolecular structure of ribonucleic acids, Prog Nucl Acid Res 1: 301, 1963. 17. Cedar, H., and G. Felsenfeld, Transcription of chromatin in vitro, JMolBiolll: 237, 1973. 18. Yochim, J. M., and V. J. DeFeo, Hormonal control of the onset magnitude and duration of uterine sensitivity in the rat by steroid hormones in the ovary, Endocrinology 72: 317, 1963. 19. Meyers, K. P., Hormonal requirements for the maintenance of oestradiol-induced inhibition of uterine sensitivity in the ovariectomized rat, J Endocrinol 46: 341, 1970. 20. Anderson, J., J. H. Clark, and E. J. Peck, Jr., The relationship between nuclear receptor estrogen binding and uterotrophic responses, Biochem Biophys Res Commun 48: 1460,1972. 21. Psychoyos, A., Endocrine control of egg implantation, In Greep, R. O., and E. B. Astwood (eds.), Handbook of Physiology, vol. 2, Sect. 7, part 2, American Physiological Society, Washington DC, 1973, p. 187. 22. Heald, P. J., J. E. O'Grady, and G. E. Moffat, The incorporation of 3 H-uridine into nuclear RNA in the uterus of the rat during early pregnancy, Biochim Biophys Acta 281: 347, 1972.
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