0013-7227/78/01O2-OOOl$O2.0O/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 102, No. 1
Printed in U.S.A.
Transition of the Rabbit Corpus Luteum to Estrogen Dependence during Early Luteal Development* JOSEPHINE B. MILLERf AND P. LANDIS KEYES Department of Physiology and The Reproductive Endocrinology Program, Department of Pathology, The University of Michigan, Ann Arbor, Michigan 48109 ABSTRACT. The early development of the rabbit corpus luteum shifts by day 6 postcoitum from a period of estrogen independence to one of estrogen dependence. In this report, the nature of this transition to estrogen dependence has been explored. Ectopic corpora lutea were established in rabbits by autotransplanting preovulatory follicles to the kidney 6.5-8.0 h after mating (day 0). At this time, the rabbits were bilaterally ovariectomized. To determine whether estradiol might be produced transiently by the developing corpora lutea or by an extraovarian source, estradiol concentrations were measured in peripheral blood and in renal blood. The concentration of estradiol in peripheral blood did not vary significantly through day 4 of development, and, on each day, averaged less than 1.2 pg/ml. No significant differences were observed between the concentrations of estradiol in peripheral blood, in blood draining the kidney bearing ectopic corpora lutea, or in blood from the contralateral kidney. Thus, the early development of ectopic corpora lutea occurs in an ex-
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RECENT report (1) from this laboratory has shown that the rabbit corpus luteum can develop and secrete progesterone for several days after ovulation in the absence of ovarian follicles. The observations that 1) ovarian follicles are the major, if not the only source of ovarian estradiol (3) and that 2) estradiol has been recognized as the luteotropic hormone in this species (3, 4) suggest that newly formed corpora lutea are not dependent on estradiol. For the ectopic corpus luteum developing in the absence of ovaries, the period of apparent autonomy ends around day 6 postcoitum, at which time corpora lutea begin to regress in the absence of exogenous estradiol (1). Received February 14, 1977. * This research was presented in part at the Seventh Annual Meeting of the Society for the Study of Reproduction, Ottawa, Ontario, Canada, 1974 (Abstract 54); supported by NIH Program Project Grant HD 08333. t Requests for reprints and correspondence should be addressed to: Josephine B. Miller, Ph.D., Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota 55901.
tremely low estradiol environment. When silastic capsules containing estradiol were inserted into these rabbits on day 3 or 5, serum progesterone rose steadily to ~6 ng/ml by day 10. If estradiol implants were not inserted until day 6 or 7, serum progesterone began to level off by day 5 and then to fall. This decline in progesterone was reversed within 24 h after insertion of the implants, suggesting that no substantial lag period is required for the expression of estradiol action. Therefore, days 5-6 represent a critical period for the corpus luteum, during which estradiol must be present for continued luteal development and secretion of progesterone. Estradiol binding to the cytosol and nuclear fractions from ectopic corpora lutea was low on day 3, increased significantly by day 5, and had fallen by day 6. Thus, the day of highest estradiol binding corresponds to the day which marks the transition from estrogen independence to estrogen dependence. (Endocrinology 102: 31, 1978)
In the present experiments, the apparent transition of the corpus luteum from an estrogen-independent to an estrogen-dependent tissue has been investigated to determine: 1) whether estradiol might be produced transiently by the developing corpus luteum or by an extraovarian source such as the adrenals; 2) whether a lag period exists for the action of estradiol to be expressed as has been reported for estrogen stimulation of ovalbumin synthesis in the chick oviduct (5); and 3) whether the requirement for estradiol around day 6 postcoitum coincides with increased levels of estradiol receptor in the corpus luteum.
Materials and Methods Animals Sexually mature New Zealand White rabbits, weighing approximately 4 kg, were caged individually in a room maintained at 72-76 F with 14 h of light each day. Teklad rabbit chow (6 ounces) and water were provided daily. 31
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MILLER AND KEYES
Endo i ' 1978 Vol 102 < N o l
10% glycerol, and 0.02% sodium azide, pH 7.5). Approximately 40 mg luteal tissue from two (days Female rabbits, which showed behavioral estrus, 4, 5, and 6) or three rabbits (day 3) were pooled were mated twice and 6.5-8 h later, sodium pentofor each determination. The homogenate was cenbarbital anesthesia was administered iv. The rabtrifuged at 1200 X g for 10 min at 4 C to yield a bits were ovariectomized through a midventral innuclear pellet and a low speed supernatant. The cision and the large preovulatory follicles (usually nuclear pellet was resuspended in 2.0 ml phosphate six to eight) were removed from both ovaries and buffer and DNA was determined in a 0.3-ml aliquot autotransplanted beneath the kidney capsule as described previously (1). These transplanted folli- by the method of Burton (10). Nuclear binding sites were determined by the cles develop into corpora lutea which secrete suffimethod of Clark et al. (11) with modification. Phoscient quantities of progesterone to maintain pregphate buffer (1.0 ml) was added to the remaining nancy (6). The day of mating and follicle autotransnuclear suspension which was then centrifuged at plantation was designated day 0. 1500 X g for 10 min at 4 C. The supernatant was discarded and the nuclear pellet washed two more times with 3.0 ml phosphate buffer and the pellet Silastic implants of estradiol resuspended in 0.8 ml phosphate buffer. Aliquots Estradiol implants were made from Silastic med- of this nuclear suspension were incubated with ical grade tubing (0.132 inches id; 0.183 inches od) either 10~8 M [3H]estradiol or 10~8 M [3H]estradiol which was packed with crystalline estradiol and containing 10~6 M diethylstilbestrol to determine sealed. The preparation and characteristics of these total and non-specific exchangeable sites, respecimplants have been previously described (7). When tively. After incubation at 37 C for 30 min in a indicated, estradiol implants were inserted sc at shaking water bath to allow exchange of [3H]estrathe base of the neck. diol, the tubes were placed on ice for 5-10 min and 3.0 ml ice-cold phosphate buffer were added to each tube. The contents of the tubes were mixed Radioimmunoassay for progesterone and 17/3-es- on a vortex mixer and the tubes centrifuged at 1500 tradiol X g for 10 min at 4 C. The bound [3H]estradiol was Progesterone concentrations were determined in extracted from the final washed nuclear pellet with petroleum ether extracts of serum using a double 3.0 ml absolute ethanol and counted in 10 ml toluantibody radioimmunoassay which was developed ene-based scintillation fluid (28 g PPO in 8 pints by Niswender (8), and has been validated for use toluene). DNA was determined in the extracted in rabbits (7). This assay has a sensitivity (defined pellet, and the results expressed as disintegrations as 2 SD from the 100% binding point of the assay) per min of specific estradiol bound per jug DNA. Cytosol binding sites were determined by a direct of approximately 8 pg and a 50% binding point of assay. The low speed (1,200 X g X 10 min) supernaapproximately 350 pg. Serum estradiol concentratant was centrifuged on a Beckman type 40A rotor tions were determined using a radioimmunoassay developed by England et al. (9) as modified by at 39,000 rpm (approximately 100,000 X g) for 60 Holt et al. (7). All samples for estradiol determi- min at 4 C to yield a high speed supernatant (cynation were extracted with benzene and then chro- tosol). Aliquots of each cytosol preparation (usually were incubated for 24 h at 4 C matographed on Sephadex LH-20 columns using a at three ndilutions) 3 with 10~ M [ H]estradiol and 10"u M [3H]estradiol benzene-methanol (9:1, vol/vol) solvent system. 8 This assay has a sensitivity of approximately 0.2 containing 10~ M diethylstilbestrol to determine total and non-specific binding, respectively. In some pg and a 50% binding point of approximately 10 experiments, 10~n M [3H]estradiol containing 10~9 PgM diethylstilbestrol was used to determine nonspecific binding. After incubation, 1.0 ml dextrancharcoal (0.0125% dextran T80 and 0.125% Norit Determination of nuclear or cytosol fraction bind- A in phosphate buffer) was added to each incubaing of estradiol tion tube to adsorb free hormone. After centrifuEctopic corpora lutea were removed from the gation for 10 min at 800 X g at 4 C, the supernatant kidney on days 3,4, 5, and 6, then cleaned, weighed, was counted in 10 ml toluene-based scintillation and homogenized by hand with all-glass tissue ho- fluid. Results are expressed as specific (i.e., total mogenizers in 1.0 ml iced sodium phosphate buffer less non-specific binding) disintegrations per min (10 mM sodium phosphate, 10 raM thioglycerol, bound per jug DNA.
Follicle autotransplantation
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LUTEAL TRANSITION TO ESTROGEN DEPENDENCE Determination of nuclear binding of estradiol by in vitro uptake Ectopic corpora lutea were removed on day 5 from four ovariectomized Dutch Belted rabbits. The corpora lutea from two rabbits were pooled for each incubation. Each corpus luteum was divided in half and each half then placed in one of two groups. The tissue in one group was incubated in 1.0 ml phosphate buffer containing 1(T9 M [3H]estradiol and the second group in buffer containing 10"9 M [3H]estradiol plus 10~7 M diethylstilbestrol. After a 30-min incubation at 4 C, each incubation vial was placed in a Dubnoff metabolic shaker and incubated for an additional 30 min at 37 C. The vials were placed on ice for 5-10 min, the tissue removed, blotted on filter paper, and homogenized by hand with all-glass tissue homogenizers in 1.0 ml ice-cold phosphate buffer. The homogenate was centrifuged at 1200 X g for 10 min at 4 C to yield a nuclear pellet and a low speed supernatant. The nuclear pellet was washed four times with 4.0 ml phosphate buffer to remove free estradiol. The bound [3H]estradiol was extracted from the final washed nuclear pellet with 3.0 ml absolute ethanol and counted in 10 ml toluene-based scintillation fluid. DNA was determined in the extracted pellet and the results expressed as disintegrations per min of specific (i.e., diethylstilbestrol-inhibitable) binding per /ig DNA. The low speed superantant was recentrifuged as previously described to obtain a high speed cytosol fraction. After dextran-charcoal adsorption of free hormone, aliquots (0.3 ml) of the cytosol fraction were counted in 10 ml toluenebased scintillation fluid to determine the amount of residual estradiol binding. Exp 1: Serum estradiol derived from a) extraovarian sources or b) from ectopic luteal tissue, a) Estradiol concentrations were determined in blood samples (6.0 ml) taken from the marginal ear vein on days 1,2,3, and 4 from each of six ovariectomized rabbits with ectopic corpora lutea. b) Nineteen ovariectomized rabbits with ectopic corpora lutea were anesthetized with sodium pentobarbital iv on day 3, 4, 5, or 6; blood samples (6.0 ml) were drawn from the marginal ear vein and from both renal veins. When taking blood samples from the renal vein, the needle (21 gauge) was placed near the kidney and above the entry of the adrenal vein into the left renal vein. In these experiments, corpora lutea were allowed to develop in the right kidney in a few animals. This was done to control for possible elevations in estradiol due to adrenal vein backflow, since the adrenal vein enters the
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vena cava directly on the right side but enters the renal vein on the left side. Exp 2: Effect of delayed estradiol administration on serum progesterone. Eighteen rabbits were mated, ovariectomized, and the preovulatory follicles transplanted to the left kidney. A single Silastic capsule containing estradiol was implanted sc on days 3, 5, 6, and 7 into each of two, five, four, and seven rabbits, respectively. Beginning on day 1, blood samples (3.0 ml) were taken daily from the marginal ear vein of each rabbit for progesterone determination. At laparotomy on day 10, a renal vein sample (5.0 ml) was taken and the corpora lutea removed from the kidney, cleaned, and weighed on a torsion balance to the nearest 0.5 mg. Four additional ovariectomized rabbits with ectopic corpora lutea were used to determine the rise in serum estradiol levels after estradiol treatment. Blood samples (6.0 ml) were taken from the marginal ear vein for estradiol determinations on days 5, 6, and 7, and 0.5, 1, 2, 4, and 24 h after insertion of a single estradiol implant on day 7. Exp 3: Nuclear and cytosol estradiol binding in developing ectopic corpora lutea. The binding of (3H]estradiol was determined in both nuclear and cytosol fractions of ectopic corpora lutea removed on days 3, 4, 5, and 6 from ovariectomized rabbits which were not treated with estradiol. In another experiment, ectopic corpora lutea were incubated with [3H]estradiol to determine whether estradiol could be concentrated in the nuclear fraction, since other studies demonstrate that the cytosol estradiol-receptor complex can be translocated to the nucleus of estrogen target tissues (12, 13). To determine the ability of other steroids to compete for estradiol binding, aliquots of the cytosol fraction prepared from day 5 ectopic corpora lutea removed from three Dutch Belted rabbits were incubated with 10"9 M [3H]estradiol or in 10~9 M [3H]estradiol containing either 10~6 M diethylstilbestrol, progesterone, 20a-dihydroprogesterone, or testosterone. Inhibition by cold steroid is expressed as a percentage of diethylstilbestrol inhibition. Treatment of blood samples Blood samples were allowea to clot for approximately 24 h at 4 C then were centrifuged at 800 X g for 20 min at 4 C. The serum was stored separately for estradiol and progesterone determination. All serum samples from an individual rabbit were an-
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MILLER AND KEYES
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E33 from peripheral blood G3 from kidney with corpora lutea ES from kidney without corpora lutea
alyzed in the same assay and each assay included samples from both control and experimental animals.
Statistics
Endo • 1978 Vol 102 • No 1
02
Data obtained on each day from individual animals in the same group were pooled and means and SE were calculated. The statistical test employed was Student's t test. A P value of less than 0.05 was considered significant.
Results
5
day 3
day 4
day 5
day 6
DAYS AFTER FOLLICLE AUTOTRANSPLANTATION
FIG. 2. Peripheral and renal vein estradiol concentrations in anesthetized, ovariectomized rabbits with ectopic corpora lutea. Vertical bars indicate mean ± SE (P > 0.05 between groups on each day).
Exp 1: Serum estradiol derived from extrathese same implants inserted on day 0 were ovarian sources or ectopic corpora lutea 18.0 ±1.4 pg/ml (n = 15). This suggests that Serum estradiol concentrations in ovariec- the insertion of a capsule containing estradiol tomized rabbits with ectopic corpora lutea did causes a rapid elevation in serum estradiol not differ through day 4 of development and which is maintained for at least 10 days. on each day averaged less than 1.2 pg/ml The effect of estradiol administration on serum (Fig. 1). No significant difference was days 3, 5, 6, and 7 on serum progesterone found on day 3, 4, 5, or 6 among estradiol concentrations in animals with ectopic corconcentrations in peripheral blood, in blood pora lutea is shown in Fig. 3. When implants draining the kidney bearing ectopic corpora were inserted on day 3 or 5, progesterone lutea, or in blood draining the contralateral concentrations rose throughout the experikidney (Fig. 2). ment and by day 10, reached levels of 5.9 ng/ml and 6.0 ± 1 . 1 ng/ml (means ± SE). Exp 2: Effect of delayed administration of These values do not differ significantly from estradiol on serum progesterone progesterone concentrations on day 10 (4.6 ± 0.5 ng/ml) in rabbits which had estradiol imThe rise in serum estradiol after insertion plants inserted on day 0 (1). In contrast, if of estradiol implants sc on day 7 into ovariestradiol implants were inserted on day 6 or ectomized rabbits with ectopic corpora lutea 7, mean progesterone concentrations were was very rapid. Before implant insertion, lower on day 10 (3.5 ± 0.7 ng/ml and 3.1 ± mean (±SE) serum estradiol levels were very 0.5 ng/ml, respectively) than in rabbits with low ( 0.05 between days).
Exp 3: Estradiol binding by nuclei and cytosol of developing ectopic corpora lutea Figure 4 shows the binding of estradiol to nuclear and cytosol fractions of ectopic cor-
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LUTEAL TRANSITION TO ESTROGEN DEPENDENCE
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(4)
n-5
n-2
r/
g
T / E 2 Implant day5
E 2 Implant
t
day 3
UJ 02
1
/
T/
T/I
o o , UJ I
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0
I
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UJ n-4 UJ O O3
E 2 Implant day?
tr
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tr 1 I
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9
O
DAYS AFTER FOLLICLE
1
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AUTOTRANSPLANTATION
FIG. 3. Serum progesterone concentrations in rabbits with ectopic corpora lutea after insertion of estradiol implants. Capsules containing estradiol were inserted on days 3, 5, 6, and 7 as indicated. Parentheses represent a value of n which differs from that indicated on the graph.
pora lutea removed on days 3, 4, 5, and 6. Diethylstilbestrol-inhibitable binding of estradiol to cytosol of ectopic corpora lutea increased significantly from day 3 to day 5. Nuclear binding also increased during this time. Incubation of ectopic corpora lutea removed on day 5 with [3H]estradiol resulted in diethylstilbestrol-inhibitable binding of [3H]estradiol to the nuclear fraction (415 and 269 dpm//xg DNA). The radioactivity which remained in the cytosol fractions prepared from such incubated tissues was not distinguishable from background radioactivity. The binding of 10~9 M [3H]estradiol to cytosol fractions of day 5 ectopic corpora lutea was inhibited by 1000-fold excess diethylstilbestrol and partially inhibited by testosterone, but was not inhibited by 1000-fold excess progesterone or 20a-dihydroprogesterone (Table 1). Discussion These experiments support the concept that the development of newly formed corpora lutea and the secretion of progesterone are at first not dependent upon estradiol, but after
5-6 days postcoitum the corpora lutea develop an obligate dependence upon estradiol. The acquisition of dependence occurs simultaneously with the appearance of estrogen receptor in the corpus luteum (see Figs. 3 and 4). In a recent publication (1), we concluded that estrogen from ovarian follicles was not essential for the early development of corpora lutea, although we could not exclude the possibility that estrogen might be available from other sources (e.g., adrenals or the developing corpus luteum itself). In the present experiments, we found no evidence of elevated estradiol levels in serum on day 1, 2, 3, or 4 postcoitum. The levels of estradiol observed were uniformly low (~1 pg/ml compared with estradiol levels of 10 pg/ml in rabbits with ovaries), and indistinguishable from estradiol levels in ovariectomized rabbits (1). Since the concentration of progesterone in blood from kidneys bearing ectopic corpora lutea is 7- to 13-fold higher than in peripheral blood (1), we expected to find a similar gradient for estradiol if ectopic corpora lutea produced the hormone. However, we found no evidence of estradiol production by the developing cor-
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MILLER AND KEYES
36 6Or
NUCLEAR
T 40
0.05 for all other days). The small number at the bottom of each bar represents the number of observations. Each observation was made using luteal tissue removed from two to three rabbits. TABLE 1. Binding of [3H]estradiol (10~9 M) to cytosol fractions from day 5 ectopic corpora lutea: Effect of ^ ^ steroids and diethylstilbestrol Steroid" Progesterone 20a-Dihydroprogesterone Testosterone Diethylstilbestrol a
% Diethylstilbestrol inhibition 0 0 52 100
Steroid or diethylstilbestrol (10~6 M).
pora lutea. These results are consistent with the observation of Telegdy and Savard (14) and that of Elbaum and Keyes (15) that the fully developed rabbit corpus luteum does not produce estradiol. Thus, we infer that these levels of estradiol are too low to support the continued development of the corpora lutea beyond the period of apparent autonomy, and that the early development of isolated corpora lutea in these experiments takes place in an extremely low estradiol environment. These
Endo i > 1978 Vol 102 < N o l
observations are essentially in agreement with those of Hilliard and Eaton (16), who found extremely low concentrations of estradiol in ovarian venous blood for the first 3 days after ovulation. These experiments show that the level and profile of serum progesterone in rabbits with an estradiol implant inserted on days 3 or 5 postcoitum are essentially the same as those in rabbits with an estradiol implant inserted on the day of mating and follicle autotransplantation (1). However, if insertion of the implant is delayed until day 6 or 7, serum progesterone levels off or declines, but these effects are reversed within 24 h after estradiol treatment. Without estradiol treatment, serum progesterone reaches castrate levels by days 9-10 (1). These results suggest that days 5-6 represent a critical period during which estradiol must be present for the corpus luteum to develop further and to survive. Furthermore, it is clear that estradiol treatment commencing on day 6 or 7 "rescues" the waning corpus luteum, as indicated by the increase in serum progesterone, and the results suggest that no substantial lag period exists for the expression of estradiol action. The patterns of estradiol binding to cytosol and nuclear fractions from ectopic corpora lutea indicate an increasing capacity to bind estradiol as the tissue becomes estrogen dependent. Estradiol binding to cytosol fractions was low on day 3, had increased significantly by day 5, and had fallen by day 6 (Fig. 4). Thus, the day of highest estradiol binding corresponds to the day which marks the transition from estrogen independence to estrogen dependence. Although circulating estradiol levels were low and relatively unchanging during the first 4 days of luteal development, the low level of cytosol estrogen binding on day 3 might be explained by occupancy of available receptor by estrogens released with the preovulatory LH surge (16). However, one would expect that prior occupancy of receptor would be reflected in high nuclear binding on day 3. This was not observed (Fig. 4), suggesting either that ectopic corpora lutea had little estrogen "receptor" at this time or were unable to translocate the estrogen-receptor com-
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LUTEAL TRANSITION TO ESTROGEN DEPENDENCE plex to the nucleus. When ectopic corpora lutea removed on day 5 were incubated with estradiol, binding was increased in the nuclear fraction prepared from this tissue and was decreased in the cytosol fraction. This suggests that, at least by day 5, the cytosol receptor is able to move into the nucleus once it binds estradiol and is consistent with the hypothesis that the ectopic corpus luteum is behaving as a classical estrogen target tissue. Recently, we have found a 400% increase in binding of estradiol to luteal cytosol fractions between days 3 and 6 of pseudopregnancy (unpublished observation). This observation indicates that the increase in receptor observed in ectopic corpora lutea resembles the change in receptor found in normal, in situ, corpora lutea. Because of the small amount of tissue available during the early days of development, we were unable to characterize carefully the estrogen binding seen in ectopic corpora lutea. Thus, the binding pattern observed during the first 6 days of development indicate relative rather than absolute changes. However, for several reasons, we propose that this binding is characterized by high affinity, low capacity, and specificity, suggesting similarity to the estrogen receptor described in in situ corpora lutea (17, 18). First, the dissociation constant for the estrogen-receptor complex in pseudopregnant rabbit corpora lutea is reported to be 2.3 X 10"u M (18). Thus, we used 10~" M [3H]estradiol in our binding assays to minimize the possibility of low affinity binding to non-specific sites (i.e., albumin or /^-globulins) which have been demonstrated in the rat uterus to have dissociation constants from 10"4-10"8 M for estradiol (19). Second, the binding of 10~u M [3H]estradiol can be completely inhibited by 100-fold excess unlabeled estradiol or diethylstilbestrol, which suggests that this binding is of low capacity. Third, we have found that even at a very high concentration (10~6 M), progesterone and 20a-dihydroprogesterone, the major steroids secreted by the rabbit corpus luteum (20), do not inhibit cytosol binding of 10~9 M [3H] estradiol, and testosterone inhibits only partially (Table 1). The partial inhibition of estradiol binding
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by testosterone was not observed by Scott and Rennie (18) and our results cannot be explained at this time. However, in recent experiments with in situ corpora lutea, testosterone was also observed to inhibit estradiol binding but was approximately 100-fold less potent than the estrogens (unpublished observation). From this study, it is not possible to determine whether the increased estradiol binding on day 5 is due to an increased number of binding sites or to a change in binding site affinity. However, changes in estradiol binding observed during development or with aging have not been found to be due to changes in receptor affinity (Reference 21 and personal communications from B. Katzenellenbogen, J. Roth, and D. Toft). In conclusion, the following events are proposed to account for the early development of the corpus luteum and its transition to an estrogen-dependent tissue. After a preovulatory LH surge which initiates luteinization, the newly formed corpora lutea have an innate ability to develop and secrete progesterone for several days without a requirement for either pituitary gonadotropic hormones (22, 23) or ovarian estradiol, the major luteotropic hormone. However, by approximately day 5 postcoitum, further development of the corpus luteum is arrested and progesterone synthesis begins to decrease in the absence of estradiol which originates from the developing ovarian follicles. In the absence of estradiol at this stage, the corpus luteum dies prematurely. Coincident with this transition to estrogen dependence is the appearance of a cytosol substance with high affinity for estradiol which is presumed to be the estrogen receptor. References 1. Miller, J. B., and P.L. Keyes, Progesterone synthesis in developing rabbit corpora lutea in the absence of follicular estrogens, Endocrinology 97: 83, 1975. 2. Mills, T. M., P. J. A. Davies, and K. Savard, Stimulation of estrogen synthesis in rabbit follicles by luteinizing hormone, Endocrinology 88: 857, 1971. 3. Robson, J. M., Maintenance by oestrin of the luteal function in hypophysectomized rabbits, J Physiol 90: 435, 1937. 4. Hilliard, J., Corpus luteum function in guinea pigs, hamsters, rats, mice, and rabbits, Biol Reprod 8: 203, 1973. 5. O'Malley, B. W., W. L. McGuire, P. O. Kohler, and S. G. Korenman, Studies on the mechanism of steroid hormone
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6.
7.
8.
9.
10.
11.
12. 13. 14.
MILLER AND KEYES regulation of synthesis of specific .proteins. Recent Prog Horm Res 25: 105, 1969. Keyes, P. L., and D. T. Armstrong, Development of corpora lutea from follicles autotransplanted under the kidney capsule in rabbits, Endocrinology 85: 423, 1969. Holt, J. A., P. L. Keyes, J. M. Brown, and J. B. Miller, Premature regression of corpora lutea in pseudopregnant rabbits following the removal of polydimethylsiloxane capsules containing 17/?-estradiol, Endocrinology 97: 76, 1975. Niswender, G. D., Influence of the site of conjugation on the specificity of antibodies to progesterone, Steroids 22: 413, 1973. England, B. G., G. D. Niswender, and A. R.-Midgley, Jr., Radioimmunoassay of estradiol-17/? without chromatography, J Clin Endocrinol Metab 38: 42, 1974. Burton, K., A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid, Biochem J %2: 315, 1956. Clark, J. H., P. S. Campbell, and E. J. Peck, Jr., Receptorestrogen complex in the nuclear fraction of the pituitary and hypothalamus of male and female immature rats, Neuroendocrinology 77: 218, 1972. Jensen, E. V., and E.R. DeSombre, Mechanism of action of the female sex hormones, Ann Rev Biochem 41: '203, 1972. O'Malley, B. W., and A. R. Means, Female steroid hormones and target cell nuclei, Science 183: 610, 1974. Telegdy, G., and K. Savard, Steroid formation in vitro in rabbit ovary, Steroids 8: 685, 1966.
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15. Elbaum, D. J., and P. L. Keyes, Synthesis of 17/S-estradiol by isolated ovarian tissues of the pregnant rat: aromatization in the corpus luteum, Endocrinology 99: 573, 1976. 16. Hilliard, J., and L. W. Eaton, Jr., Estradiol-17/?, progesterone, and 20a-hydroxypregn-4-en-3-one in rabbit ovarian venous plasma. II. From mating through implantation, Endocrinology 89: 522, 1971. 17. Lee, C, P. L. Keyes, and H. I. Jacobson, Estrogen receptor in the rabbit corpus luteum, Science 173: 1032, 1971. 18. Scott, R. S., and P. I. C. Rennie, An estrogen receptor in the corpora lutea of the pseudopregnant rabbit, Endocrinology 89: 297, 1971. 19. Ellis, J. D., and H. J. Ringold, The uterine estrogen receptor: A physiochemical study, In McKerns, K. W. (ed.), The Sex Steroids, Appleton-Century-Crofts, New York, 1971, p. 73. 20. Savard, K., The biochemistry of the corpus luteum, Biol Reprod 8: 183, 1973. 21. Feherty, P., D. M. Robertson, H. B. Waynforth, and A. E. Kellie, Changes in the concentration of high-affinity oestradiol receptors in rat uterine supernatant preparations during the oestrous cycle, pseudopregnancy, maturation, and after ovariectomy, Biochem J 120: 837, 1970. 22. Firor, W. M., Hypophysectomy in pseudopregnant rabbits, AmJPhysiol 104: 204, 1933. 23. Smith, P. E., and W. E. White, The effect of hypophysectomy on ovulation and corpus luteum formation in the rabbit, JAMA 97: 1861, 1931.
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Vol. 102, No. 1
Printed in U.S.A.
Transition of the Rabbit Corpus Luteum to Estrogen Dependence during Early Luteal Development* JOSEPHINE B. MILLERf AND P. LANDIS KEYES Department of Physiology and The Reproductive Endocrinology Program, Department of Pathology, The University of Michigan, Ann Arbor, Michigan 48109 ABSTRACT. The early development of the rabbit corpus luteum shifts by day 6 postcoitum from a period of estrogen independence to one of estrogen dependence. In this report, the nature of this transition to estrogen dependence has been explored. Ectopic corpora lutea were established in rabbits by autotransplanting preovulatory follicles to the kidney 6.5-8.0 h after mating (day 0). At this time, the rabbits were bilaterally ovariectomized. To determine whether estradiol might be produced transiently by the developing corpora lutea or by an extraovarian source, estradiol concentrations were measured in peripheral blood and in renal blood. The concentration of estradiol in peripheral blood did not vary significantly through day 4 of development, and, on each day, averaged less than 1.2 pg/ml. No significant differences were observed between the concentrations of estradiol in peripheral blood, in blood draining the kidney bearing ectopic corpora lutea, or in blood from the contralateral kidney. Thus, the early development of ectopic corpora lutea occurs in an ex-
A
RECENT report (1) from this laboratory has shown that the rabbit corpus luteum can develop and secrete progesterone for several days after ovulation in the absence of ovarian follicles. The observations that 1) ovarian follicles are the major, if not the only source of ovarian estradiol (3) and that 2) estradiol has been recognized as the luteotropic hormone in this species (3, 4) suggest that newly formed corpora lutea are not dependent on estradiol. For the ectopic corpus luteum developing in the absence of ovaries, the period of apparent autonomy ends around day 6 postcoitum, at which time corpora lutea begin to regress in the absence of exogenous estradiol (1). Received February 14, 1977. * This research was presented in part at the Seventh Annual Meeting of the Society for the Study of Reproduction, Ottawa, Ontario, Canada, 1974 (Abstract 54); supported by NIH Program Project Grant HD 08333. t Requests for reprints and correspondence should be addressed to: Josephine B. Miller, Ph.D., Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota 55901.
tremely low estradiol environment. When silastic capsules containing estradiol were inserted into these rabbits on day 3 or 5, serum progesterone rose steadily to ~6 ng/ml by day 10. If estradiol implants were not inserted until day 6 or 7, serum progesterone began to level off by day 5 and then to fall. This decline in progesterone was reversed within 24 h after insertion of the implants, suggesting that no substantial lag period is required for the expression of estradiol action. Therefore, days 5-6 represent a critical period for the corpus luteum, during which estradiol must be present for continued luteal development and secretion of progesterone. Estradiol binding to the cytosol and nuclear fractions from ectopic corpora lutea was low on day 3, increased significantly by day 5, and had fallen by day 6. Thus, the day of highest estradiol binding corresponds to the day which marks the transition from estrogen independence to estrogen dependence. (Endocrinology 102: 31, 1978)
In the present experiments, the apparent transition of the corpus luteum from an estrogen-independent to an estrogen-dependent tissue has been investigated to determine: 1) whether estradiol might be produced transiently by the developing corpus luteum or by an extraovarian source such as the adrenals; 2) whether a lag period exists for the action of estradiol to be expressed as has been reported for estrogen stimulation of ovalbumin synthesis in the chick oviduct (5); and 3) whether the requirement for estradiol around day 6 postcoitum coincides with increased levels of estradiol receptor in the corpus luteum.
Materials and Methods Animals Sexually mature New Zealand White rabbits, weighing approximately 4 kg, were caged individually in a room maintained at 72-76 F with 14 h of light each day. Teklad rabbit chow (6 ounces) and water were provided daily. 31
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32
MILLER AND KEYES
Endo i ' 1978 Vol 102 < N o l
10% glycerol, and 0.02% sodium azide, pH 7.5). Approximately 40 mg luteal tissue from two (days Female rabbits, which showed behavioral estrus, 4, 5, and 6) or three rabbits (day 3) were pooled were mated twice and 6.5-8 h later, sodium pentofor each determination. The homogenate was cenbarbital anesthesia was administered iv. The rabtrifuged at 1200 X g for 10 min at 4 C to yield a bits were ovariectomized through a midventral innuclear pellet and a low speed supernatant. The cision and the large preovulatory follicles (usually nuclear pellet was resuspended in 2.0 ml phosphate six to eight) were removed from both ovaries and buffer and DNA was determined in a 0.3-ml aliquot autotransplanted beneath the kidney capsule as described previously (1). These transplanted folli- by the method of Burton (10). Nuclear binding sites were determined by the cles develop into corpora lutea which secrete suffimethod of Clark et al. (11) with modification. Phoscient quantities of progesterone to maintain pregphate buffer (1.0 ml) was added to the remaining nancy (6). The day of mating and follicle autotransnuclear suspension which was then centrifuged at plantation was designated day 0. 1500 X g for 10 min at 4 C. The supernatant was discarded and the nuclear pellet washed two more times with 3.0 ml phosphate buffer and the pellet Silastic implants of estradiol resuspended in 0.8 ml phosphate buffer. Aliquots Estradiol implants were made from Silastic med- of this nuclear suspension were incubated with ical grade tubing (0.132 inches id; 0.183 inches od) either 10~8 M [3H]estradiol or 10~8 M [3H]estradiol which was packed with crystalline estradiol and containing 10~6 M diethylstilbestrol to determine sealed. The preparation and characteristics of these total and non-specific exchangeable sites, respecimplants have been previously described (7). When tively. After incubation at 37 C for 30 min in a indicated, estradiol implants were inserted sc at shaking water bath to allow exchange of [3H]estrathe base of the neck. diol, the tubes were placed on ice for 5-10 min and 3.0 ml ice-cold phosphate buffer were added to each tube. The contents of the tubes were mixed Radioimmunoassay for progesterone and 17/3-es- on a vortex mixer and the tubes centrifuged at 1500 tradiol X g for 10 min at 4 C. The bound [3H]estradiol was Progesterone concentrations were determined in extracted from the final washed nuclear pellet with petroleum ether extracts of serum using a double 3.0 ml absolute ethanol and counted in 10 ml toluantibody radioimmunoassay which was developed ene-based scintillation fluid (28 g PPO in 8 pints by Niswender (8), and has been validated for use toluene). DNA was determined in the extracted in rabbits (7). This assay has a sensitivity (defined pellet, and the results expressed as disintegrations as 2 SD from the 100% binding point of the assay) per min of specific estradiol bound per jug DNA. Cytosol binding sites were determined by a direct of approximately 8 pg and a 50% binding point of assay. The low speed (1,200 X g X 10 min) supernaapproximately 350 pg. Serum estradiol concentratant was centrifuged on a Beckman type 40A rotor tions were determined using a radioimmunoassay developed by England et al. (9) as modified by at 39,000 rpm (approximately 100,000 X g) for 60 Holt et al. (7). All samples for estradiol determi- min at 4 C to yield a high speed supernatant (cynation were extracted with benzene and then chro- tosol). Aliquots of each cytosol preparation (usually were incubated for 24 h at 4 C matographed on Sephadex LH-20 columns using a at three ndilutions) 3 with 10~ M [ H]estradiol and 10"u M [3H]estradiol benzene-methanol (9:1, vol/vol) solvent system. 8 This assay has a sensitivity of approximately 0.2 containing 10~ M diethylstilbestrol to determine total and non-specific binding, respectively. In some pg and a 50% binding point of approximately 10 experiments, 10~n M [3H]estradiol containing 10~9 PgM diethylstilbestrol was used to determine nonspecific binding. After incubation, 1.0 ml dextrancharcoal (0.0125% dextran T80 and 0.125% Norit Determination of nuclear or cytosol fraction bind- A in phosphate buffer) was added to each incubaing of estradiol tion tube to adsorb free hormone. After centrifuEctopic corpora lutea were removed from the gation for 10 min at 800 X g at 4 C, the supernatant kidney on days 3,4, 5, and 6, then cleaned, weighed, was counted in 10 ml toluene-based scintillation and homogenized by hand with all-glass tissue ho- fluid. Results are expressed as specific (i.e., total mogenizers in 1.0 ml iced sodium phosphate buffer less non-specific binding) disintegrations per min (10 mM sodium phosphate, 10 raM thioglycerol, bound per jug DNA.
Follicle autotransplantation
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LUTEAL TRANSITION TO ESTROGEN DEPENDENCE Determination of nuclear binding of estradiol by in vitro uptake Ectopic corpora lutea were removed on day 5 from four ovariectomized Dutch Belted rabbits. The corpora lutea from two rabbits were pooled for each incubation. Each corpus luteum was divided in half and each half then placed in one of two groups. The tissue in one group was incubated in 1.0 ml phosphate buffer containing 1(T9 M [3H]estradiol and the second group in buffer containing 10"9 M [3H]estradiol plus 10~7 M diethylstilbestrol. After a 30-min incubation at 4 C, each incubation vial was placed in a Dubnoff metabolic shaker and incubated for an additional 30 min at 37 C. The vials were placed on ice for 5-10 min, the tissue removed, blotted on filter paper, and homogenized by hand with all-glass tissue homogenizers in 1.0 ml ice-cold phosphate buffer. The homogenate was centrifuged at 1200 X g for 10 min at 4 C to yield a nuclear pellet and a low speed supernatant. The nuclear pellet was washed four times with 4.0 ml phosphate buffer to remove free estradiol. The bound [3H]estradiol was extracted from the final washed nuclear pellet with 3.0 ml absolute ethanol and counted in 10 ml toluene-based scintillation fluid. DNA was determined in the extracted pellet and the results expressed as disintegrations per min of specific (i.e., diethylstilbestrol-inhibitable) binding per /ig DNA. The low speed superantant was recentrifuged as previously described to obtain a high speed cytosol fraction. After dextran-charcoal adsorption of free hormone, aliquots (0.3 ml) of the cytosol fraction were counted in 10 ml toluenebased scintillation fluid to determine the amount of residual estradiol binding. Exp 1: Serum estradiol derived from a) extraovarian sources or b) from ectopic luteal tissue, a) Estradiol concentrations were determined in blood samples (6.0 ml) taken from the marginal ear vein on days 1,2,3, and 4 from each of six ovariectomized rabbits with ectopic corpora lutea. b) Nineteen ovariectomized rabbits with ectopic corpora lutea were anesthetized with sodium pentobarbital iv on day 3, 4, 5, or 6; blood samples (6.0 ml) were drawn from the marginal ear vein and from both renal veins. When taking blood samples from the renal vein, the needle (21 gauge) was placed near the kidney and above the entry of the adrenal vein into the left renal vein. In these experiments, corpora lutea were allowed to develop in the right kidney in a few animals. This was done to control for possible elevations in estradiol due to adrenal vein backflow, since the adrenal vein enters the
33
vena cava directly on the right side but enters the renal vein on the left side. Exp 2: Effect of delayed estradiol administration on serum progesterone. Eighteen rabbits were mated, ovariectomized, and the preovulatory follicles transplanted to the left kidney. A single Silastic capsule containing estradiol was implanted sc on days 3, 5, 6, and 7 into each of two, five, four, and seven rabbits, respectively. Beginning on day 1, blood samples (3.0 ml) were taken daily from the marginal ear vein of each rabbit for progesterone determination. At laparotomy on day 10, a renal vein sample (5.0 ml) was taken and the corpora lutea removed from the kidney, cleaned, and weighed on a torsion balance to the nearest 0.5 mg. Four additional ovariectomized rabbits with ectopic corpora lutea were used to determine the rise in serum estradiol levels after estradiol treatment. Blood samples (6.0 ml) were taken from the marginal ear vein for estradiol determinations on days 5, 6, and 7, and 0.5, 1, 2, 4, and 24 h after insertion of a single estradiol implant on day 7. Exp 3: Nuclear and cytosol estradiol binding in developing ectopic corpora lutea. The binding of (3H]estradiol was determined in both nuclear and cytosol fractions of ectopic corpora lutea removed on days 3, 4, 5, and 6 from ovariectomized rabbits which were not treated with estradiol. In another experiment, ectopic corpora lutea were incubated with [3H]estradiol to determine whether estradiol could be concentrated in the nuclear fraction, since other studies demonstrate that the cytosol estradiol-receptor complex can be translocated to the nucleus of estrogen target tissues (12, 13). To determine the ability of other steroids to compete for estradiol binding, aliquots of the cytosol fraction prepared from day 5 ectopic corpora lutea removed from three Dutch Belted rabbits were incubated with 10"9 M [3H]estradiol or in 10~9 M [3H]estradiol containing either 10~6 M diethylstilbestrol, progesterone, 20a-dihydroprogesterone, or testosterone. Inhibition by cold steroid is expressed as a percentage of diethylstilbestrol inhibition. Treatment of blood samples Blood samples were allowea to clot for approximately 24 h at 4 C then were centrifuged at 800 X g for 20 min at 4 C. The serum was stored separately for estradiol and progesterone determination. All serum samples from an individual rabbit were an-
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MILLER AND KEYES
34
E33 from peripheral blood G3 from kidney with corpora lutea ES from kidney without corpora lutea
alyzed in the same assay and each assay included samples from both control and experimental animals.
Statistics
Endo • 1978 Vol 102 • No 1
02
Data obtained on each day from individual animals in the same group were pooled and means and SE were calculated. The statistical test employed was Student's t test. A P value of less than 0.05 was considered significant.
Results
5
day 3
day 4
day 5
day 6
DAYS AFTER FOLLICLE AUTOTRANSPLANTATION
FIG. 2. Peripheral and renal vein estradiol concentrations in anesthetized, ovariectomized rabbits with ectopic corpora lutea. Vertical bars indicate mean ± SE (P > 0.05 between groups on each day).
Exp 1: Serum estradiol derived from extrathese same implants inserted on day 0 were ovarian sources or ectopic corpora lutea 18.0 ±1.4 pg/ml (n = 15). This suggests that Serum estradiol concentrations in ovariec- the insertion of a capsule containing estradiol tomized rabbits with ectopic corpora lutea did causes a rapid elevation in serum estradiol not differ through day 4 of development and which is maintained for at least 10 days. on each day averaged less than 1.2 pg/ml The effect of estradiol administration on serum (Fig. 1). No significant difference was days 3, 5, 6, and 7 on serum progesterone found on day 3, 4, 5, or 6 among estradiol concentrations in animals with ectopic corconcentrations in peripheral blood, in blood pora lutea is shown in Fig. 3. When implants draining the kidney bearing ectopic corpora were inserted on day 3 or 5, progesterone lutea, or in blood draining the contralateral concentrations rose throughout the experikidney (Fig. 2). ment and by day 10, reached levels of 5.9 ng/ml and 6.0 ± 1 . 1 ng/ml (means ± SE). Exp 2: Effect of delayed administration of These values do not differ significantly from estradiol on serum progesterone progesterone concentrations on day 10 (4.6 ± 0.5 ng/ml) in rabbits which had estradiol imThe rise in serum estradiol after insertion plants inserted on day 0 (1). In contrast, if of estradiol implants sc on day 7 into ovariestradiol implants were inserted on day 6 or ectomized rabbits with ectopic corpora lutea 7, mean progesterone concentrations were was very rapid. Before implant insertion, lower on day 10 (3.5 ± 0.7 ng/ml and 3.1 ± mean (±SE) serum estradiol levels were very 0.5 ng/ml, respectively) than in rabbits with low ( 0.05 between days).
Exp 3: Estradiol binding by nuclei and cytosol of developing ectopic corpora lutea Figure 4 shows the binding of estradiol to nuclear and cytosol fractions of ectopic cor-
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LUTEAL TRANSITION TO ESTROGEN DEPENDENCE
V
35
(4)
n-5
n-2
r/
g
T / E 2 Implant day5
E 2 Implant
t
day 3
UJ 02
1
/
T/
T/I
o o , UJ I
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0
I
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UJ n-4 UJ O O3
E 2 Implant day?
tr
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tr 1 I
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O
DAYS AFTER FOLLICLE
1
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AUTOTRANSPLANTATION
FIG. 3. Serum progesterone concentrations in rabbits with ectopic corpora lutea after insertion of estradiol implants. Capsules containing estradiol were inserted on days 3, 5, 6, and 7 as indicated. Parentheses represent a value of n which differs from that indicated on the graph.
pora lutea removed on days 3, 4, 5, and 6. Diethylstilbestrol-inhibitable binding of estradiol to cytosol of ectopic corpora lutea increased significantly from day 3 to day 5. Nuclear binding also increased during this time. Incubation of ectopic corpora lutea removed on day 5 with [3H]estradiol resulted in diethylstilbestrol-inhibitable binding of [3H]estradiol to the nuclear fraction (415 and 269 dpm//xg DNA). The radioactivity which remained in the cytosol fractions prepared from such incubated tissues was not distinguishable from background radioactivity. The binding of 10~9 M [3H]estradiol to cytosol fractions of day 5 ectopic corpora lutea was inhibited by 1000-fold excess diethylstilbestrol and partially inhibited by testosterone, but was not inhibited by 1000-fold excess progesterone or 20a-dihydroprogesterone (Table 1). Discussion These experiments support the concept that the development of newly formed corpora lutea and the secretion of progesterone are at first not dependent upon estradiol, but after
5-6 days postcoitum the corpora lutea develop an obligate dependence upon estradiol. The acquisition of dependence occurs simultaneously with the appearance of estrogen receptor in the corpus luteum (see Figs. 3 and 4). In a recent publication (1), we concluded that estrogen from ovarian follicles was not essential for the early development of corpora lutea, although we could not exclude the possibility that estrogen might be available from other sources (e.g., adrenals or the developing corpus luteum itself). In the present experiments, we found no evidence of elevated estradiol levels in serum on day 1, 2, 3, or 4 postcoitum. The levels of estradiol observed were uniformly low (~1 pg/ml compared with estradiol levels of 10 pg/ml in rabbits with ovaries), and indistinguishable from estradiol levels in ovariectomized rabbits (1). Since the concentration of progesterone in blood from kidneys bearing ectopic corpora lutea is 7- to 13-fold higher than in peripheral blood (1), we expected to find a similar gradient for estradiol if ectopic corpora lutea produced the hormone. However, we found no evidence of estradiol production by the developing cor-
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MILLER AND KEYES
36 6Or
NUCLEAR
T 40
0.05 for all other days). The small number at the bottom of each bar represents the number of observations. Each observation was made using luteal tissue removed from two to three rabbits. TABLE 1. Binding of [3H]estradiol (10~9 M) to cytosol fractions from day 5 ectopic corpora lutea: Effect of ^ ^ steroids and diethylstilbestrol Steroid" Progesterone 20a-Dihydroprogesterone Testosterone Diethylstilbestrol a
% Diethylstilbestrol inhibition 0 0 52 100
Steroid or diethylstilbestrol (10~6 M).
pora lutea. These results are consistent with the observation of Telegdy and Savard (14) and that of Elbaum and Keyes (15) that the fully developed rabbit corpus luteum does not produce estradiol. Thus, we infer that these levels of estradiol are too low to support the continued development of the corpora lutea beyond the period of apparent autonomy, and that the early development of isolated corpora lutea in these experiments takes place in an extremely low estradiol environment. These
Endo i > 1978 Vol 102 < N o l
observations are essentially in agreement with those of Hilliard and Eaton (16), who found extremely low concentrations of estradiol in ovarian venous blood for the first 3 days after ovulation. These experiments show that the level and profile of serum progesterone in rabbits with an estradiol implant inserted on days 3 or 5 postcoitum are essentially the same as those in rabbits with an estradiol implant inserted on the day of mating and follicle autotransplantation (1). However, if insertion of the implant is delayed until day 6 or 7, serum progesterone levels off or declines, but these effects are reversed within 24 h after estradiol treatment. Without estradiol treatment, serum progesterone reaches castrate levels by days 9-10 (1). These results suggest that days 5-6 represent a critical period during which estradiol must be present for the corpus luteum to develop further and to survive. Furthermore, it is clear that estradiol treatment commencing on day 6 or 7 "rescues" the waning corpus luteum, as indicated by the increase in serum progesterone, and the results suggest that no substantial lag period exists for the expression of estradiol action. The patterns of estradiol binding to cytosol and nuclear fractions from ectopic corpora lutea indicate an increasing capacity to bind estradiol as the tissue becomes estrogen dependent. Estradiol binding to cytosol fractions was low on day 3, had increased significantly by day 5, and had fallen by day 6 (Fig. 4). Thus, the day of highest estradiol binding corresponds to the day which marks the transition from estrogen independence to estrogen dependence. Although circulating estradiol levels were low and relatively unchanging during the first 4 days of luteal development, the low level of cytosol estrogen binding on day 3 might be explained by occupancy of available receptor by estrogens released with the preovulatory LH surge (16). However, one would expect that prior occupancy of receptor would be reflected in high nuclear binding on day 3. This was not observed (Fig. 4), suggesting either that ectopic corpora lutea had little estrogen "receptor" at this time or were unable to translocate the estrogen-receptor com-
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LUTEAL TRANSITION TO ESTROGEN DEPENDENCE plex to the nucleus. When ectopic corpora lutea removed on day 5 were incubated with estradiol, binding was increased in the nuclear fraction prepared from this tissue and was decreased in the cytosol fraction. This suggests that, at least by day 5, the cytosol receptor is able to move into the nucleus once it binds estradiol and is consistent with the hypothesis that the ectopic corpus luteum is behaving as a classical estrogen target tissue. Recently, we have found a 400% increase in binding of estradiol to luteal cytosol fractions between days 3 and 6 of pseudopregnancy (unpublished observation). This observation indicates that the increase in receptor observed in ectopic corpora lutea resembles the change in receptor found in normal, in situ, corpora lutea. Because of the small amount of tissue available during the early days of development, we were unable to characterize carefully the estrogen binding seen in ectopic corpora lutea. Thus, the binding pattern observed during the first 6 days of development indicate relative rather than absolute changes. However, for several reasons, we propose that this binding is characterized by high affinity, low capacity, and specificity, suggesting similarity to the estrogen receptor described in in situ corpora lutea (17, 18). First, the dissociation constant for the estrogen-receptor complex in pseudopregnant rabbit corpora lutea is reported to be 2.3 X 10"u M (18). Thus, we used 10~" M [3H]estradiol in our binding assays to minimize the possibility of low affinity binding to non-specific sites (i.e., albumin or /^-globulins) which have been demonstrated in the rat uterus to have dissociation constants from 10"4-10"8 M for estradiol (19). Second, the binding of 10~u M [3H]estradiol can be completely inhibited by 100-fold excess unlabeled estradiol or diethylstilbestrol, which suggests that this binding is of low capacity. Third, we have found that even at a very high concentration (10~6 M), progesterone and 20a-dihydroprogesterone, the major steroids secreted by the rabbit corpus luteum (20), do not inhibit cytosol binding of 10~9 M [3H] estradiol, and testosterone inhibits only partially (Table 1). The partial inhibition of estradiol binding
37
by testosterone was not observed by Scott and Rennie (18) and our results cannot be explained at this time. However, in recent experiments with in situ corpora lutea, testosterone was also observed to inhibit estradiol binding but was approximately 100-fold less potent than the estrogens (unpublished observation). From this study, it is not possible to determine whether the increased estradiol binding on day 5 is due to an increased number of binding sites or to a change in binding site affinity. However, changes in estradiol binding observed during development or with aging have not been found to be due to changes in receptor affinity (Reference 21 and personal communications from B. Katzenellenbogen, J. Roth, and D. Toft). In conclusion, the following events are proposed to account for the early development of the corpus luteum and its transition to an estrogen-dependent tissue. After a preovulatory LH surge which initiates luteinization, the newly formed corpora lutea have an innate ability to develop and secrete progesterone for several days without a requirement for either pituitary gonadotropic hormones (22, 23) or ovarian estradiol, the major luteotropic hormone. However, by approximately day 5 postcoitum, further development of the corpus luteum is arrested and progesterone synthesis begins to decrease in the absence of estradiol which originates from the developing ovarian follicles. In the absence of estradiol at this stage, the corpus luteum dies prematurely. Coincident with this transition to estrogen dependence is the appearance of a cytosol substance with high affinity for estradiol which is presumed to be the estrogen receptor. References 1. Miller, J. B., and P.L. Keyes, Progesterone synthesis in developing rabbit corpora lutea in the absence of follicular estrogens, Endocrinology 97: 83, 1975. 2. Mills, T. M., P. J. A. Davies, and K. Savard, Stimulation of estrogen synthesis in rabbit follicles by luteinizing hormone, Endocrinology 88: 857, 1971. 3. Robson, J. M., Maintenance by oestrin of the luteal function in hypophysectomized rabbits, J Physiol 90: 435, 1937. 4. Hilliard, J., Corpus luteum function in guinea pigs, hamsters, rats, mice, and rabbits, Biol Reprod 8: 203, 1973. 5. O'Malley, B. W., W. L. McGuire, P. O. Kohler, and S. G. Korenman, Studies on the mechanism of steroid hormone
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6.
7.
8.
9.
10.
11.
12. 13. 14.
MILLER AND KEYES regulation of synthesis of specific .proteins. Recent Prog Horm Res 25: 105, 1969. Keyes, P. L., and D. T. Armstrong, Development of corpora lutea from follicles autotransplanted under the kidney capsule in rabbits, Endocrinology 85: 423, 1969. Holt, J. A., P. L. Keyes, J. M. Brown, and J. B. Miller, Premature regression of corpora lutea in pseudopregnant rabbits following the removal of polydimethylsiloxane capsules containing 17/?-estradiol, Endocrinology 97: 76, 1975. Niswender, G. D., Influence of the site of conjugation on the specificity of antibodies to progesterone, Steroids 22: 413, 1973. England, B. G., G. D. Niswender, and A. R.-Midgley, Jr., Radioimmunoassay of estradiol-17/? without chromatography, J Clin Endocrinol Metab 38: 42, 1974. Burton, K., A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid, Biochem J %2: 315, 1956. Clark, J. H., P. S. Campbell, and E. J. Peck, Jr., Receptorestrogen complex in the nuclear fraction of the pituitary and hypothalamus of male and female immature rats, Neuroendocrinology 77: 218, 1972. Jensen, E. V., and E.R. DeSombre, Mechanism of action of the female sex hormones, Ann Rev Biochem 41: '203, 1972. O'Malley, B. W., and A. R. Means, Female steroid hormones and target cell nuclei, Science 183: 610, 1974. Telegdy, G., and K. Savard, Steroid formation in vitro in rabbit ovary, Steroids 8: 685, 1966.
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15. Elbaum, D. J., and P. L. Keyes, Synthesis of 17/S-estradiol by isolated ovarian tissues of the pregnant rat: aromatization in the corpus luteum, Endocrinology 99: 573, 1976. 16. Hilliard, J., and L. W. Eaton, Jr., Estradiol-17/?, progesterone, and 20a-hydroxypregn-4-en-3-one in rabbit ovarian venous plasma. II. From mating through implantation, Endocrinology 89: 522, 1971. 17. Lee, C, P. L. Keyes, and H. I. Jacobson, Estrogen receptor in the rabbit corpus luteum, Science 173: 1032, 1971. 18. Scott, R. S., and P. I. C. Rennie, An estrogen receptor in the corpora lutea of the pseudopregnant rabbit, Endocrinology 89: 297, 1971. 19. Ellis, J. D., and H. J. Ringold, The uterine estrogen receptor: A physiochemical study, In McKerns, K. W. (ed.), The Sex Steroids, Appleton-Century-Crofts, New York, 1971, p. 73. 20. Savard, K., The biochemistry of the corpus luteum, Biol Reprod 8: 183, 1973. 21. Feherty, P., D. M. Robertson, H. B. Waynforth, and A. E. Kellie, Changes in the concentration of high-affinity oestradiol receptors in rat uterine supernatant preparations during the oestrous cycle, pseudopregnancy, maturation, and after ovariectomy, Biochem J 120: 837, 1970. 22. Firor, W. M., Hypophysectomy in pseudopregnant rabbits, AmJPhysiol 104: 204, 1933. 23. Smith, P. E., and W. E. White, The effect of hypophysectomy on ovulation and corpus luteum formation in the rabbit, JAMA 97: 1861, 1931.
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