0013-7227/78/1024-1176$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society

Vol. 102, No. 4 Printed in U.S.A.

Role of Intraluteal Estrogen in the Regulation of the Rat Corpus Luteum during Pregnancy* GEULA GIBORIf AND P. LANDIS KEYES Reproductive Endocrinology Program, Departments of Pathology and Physiology, The University of Michigan, Ann Arbor, Michigan 48109 and Department of Physiology, The University of Illinois at the Medical Center, Chicago, Illinois 60680 ABSTRACT. The pronounced aromatizing ability of the rat corpus luteum and the ability of estradiol to maintain luteal progesterone synthesis suggest that estrogen formed within the luteal cell might act locally to maintain luteal function. To examine this hypothesis, rats were treated with either estradiol (100 /ig/day), high or low levels of testosterone via Silastic capsules (20 cm or 1 cm in length), or dihydrotestosterone (20cm capsule) after hypophysectomy and hysterectomy on day 12 of pregnancy. Hypophysectomy and hysterectomy caused serum progesterone and androgen levels, estradiol concentrations in the corpora lutea, and the content of estradiol receptor in luteal cell nuclei to decrease significantly, and caused the cessation of luteal growth. The daily administration of 100 /ig estradiol or of high levels of testosterone via the 20-cm Silastic

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URING the second half of pregnancy, the rat corpus luteum is regulated by placental hormones (1), one of which is rat placental luteotropin (2, 3). However, reports by Takayama and Greenwald (4) and by Gibori et al. (5) have provided convincing evidence that estrogens can directly stimulate luteal growth and maintain progesterone secretion after hypophysectomy and hysterectomy on day 12 of pregnancy. Inasmuch as relatively large amounts of estradiol were required to achieve luteotropic effects, Gibori et al. (5) proposed that such injections were necessary to maintain normal intraluteal concentrations of estradiol. Waynforth et al. (6) and Elbaum and Keyes (7) have clearly shown that rat corpora lutea have high concentra-

Received June 14, 1977. * This work was presented in part at the 1977 FASEB Meeting, Chicago, Illinois (Abstract 222). It was supported by NIH Grant HD-07127 and by CRB and BRSG grants from University of Illinois to G.G. f To whom requests for reprints should be addressed: Department of Physiology, University of Illinois at the Medical Center, P.O. Box 6998, 901 S. Wolcott Street, Chicago, Illinois 60680.

capsule increased the estradiol concentration in the corpora lutea dramatically, maintained serum progesterone from day 12 through day 15 at concentrations similar to those in pregnant, sham-operated animals, and increased the nuclear content of estradiol receptor in the corpora lutea. Treatment with the small testosterone capsule maintained the serum androgen and progesterone levels, estradiol concentrations in the corpora lutea, and luteal growth at levels observed in pregnant, sham-operated animals. Treatment with the 20-cm dihydrotestosterone capsule did not sustain progesterone secretion and luteal growth. These results suggest that estrogen formed by aromatization within the corpora lutea may play an important role in the regulation of luteal function during pregnancy in the rat. (Endocrinology 102: 1176, 1978)

tions of estradiol during pregnancy. A pathway for local accumulation of estradiol in rat corpora lutea has been revealed in the demonstration of aromatization of testosterone to estradiol by corpora lutea in vivo (8, 9) and in vitro (7). In the present investigation, we have examined the hypothesis that estradiol formed within luteal tissue by aromatization of testosterone can act locally to maintain progesterone synthesis and normal luteal function.

Materials and Methods General Pregnant rats were obtained from Holtzman and were maintained at 76 F on a daily light: dark cycle of 14:10 h, with free access to Teklad rat chow and water. The day sperm were found in the vaginal smear was designated day 1 of gestation. Operations A clean but not aseptic technique was used for all operations, with ether as anesthesia. Hypophysectomy was performed by the transauricular method by using a stereotaxic instrument (10). Completeness of removal of the pituitary was

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ESTROGEN ACTION IN RAT CORPUS LUTEUM judged by the recovery of the pituitary at operation and by the absence of any fragments in the fossa at autopsy. Rats incompletely hypophysectomized J. were not included in experiments. Hysterectomy or hysterectomy-ovariectomy was performed through ^ a midline abdominal incision. Sham operations consisted of laparotomy and penetration of the ear drum by the aspirating syringe without removal of •> the pituitary. Autopsy 4

On day 15 of pregnancy, each rat was killed by an overdose of ether. The pituitary fossa of hypophysectomized rats was carefully examined. The ovaries were removed and the corpora lutea were excised and trimmed of all adhering non-luteal tis^ sue under a stereoscopic dissecting microscope. The corpora lutea and the non-luteal tissues, consisting of all the ovarian tissue which remained after the * corpora lutea of pregnancy were removed, were weighed on a torsion balance and immediately frozen for the subsequent determination of estradiol content. Fresh corpora lutea were used for estradiol k receptor assays. Hormone treatments Estradiol-17/? was dissolved in sesame oil and 100 jtig were injected daily in a volume of 0.25 ml sc from day 12 through day 14. Control rats were injected daily with the same volume of sesame oil. > Silastic capsules were prepared from Dow Corn, ing, 602-305 Silastic medical grade tubing (3.18-mm od) and filled with crystalline steroid as described by Legan et al. (11). Some capsules were designed to provide surface areas of 2000 mm2 and were filled with either testosterone or dihydrotestosterone (DHT). Other capsules with a surface area of 100 mm2 were filled with a 1:1 mixture of cholesterol >y and testosterone. The release rates for testosterone from capsules with these dimensions are 0.7 mg/day/1000 mm2 (12). The release rate of DHT is similar to that of testosterone (Vernon Gay, personal communication). Control rats received • either empty or cholesterol-filled implants. Capsules were implanted on day 12 immediately after * operations and were maintained through day 15. No evidence of adhesions or infections was noted at autopsy. Blood samples *

Blood samples (0.5 ml) were taken serially by puncture of the jugular vein through the unbroken skin by using a 26 gauge, 0.5-in. needle with the rat

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under light ether anesthesia. The blood was allowed to clot, then it was stored for several hours at 4 C, centrifuged, and the serum stored at —20 C. Progesterone RIA Serum progesterone was quantified after petroleum ether extraction by using procedures reported previously (13). 17p-Estradiol RIA Estradiol in ovarian tissues was quantified after benzene extraction of homogenates and chromatography of extracts on Sephadex LH-20 columns as described previously (7). RIA for testosterone (immunoreactive androgen) Immunoreactive androgen was quantified in benzene-hexane (1:1) extracts of serum by using RIA procedures described by Niswender et al. (14) and by Payne et al. (15). The antiserum was produced in rabbits against testosterone-lla-hemisuccinate conjugated to bovine serum albumin, and is highly specific for testosterone. DHT is the only steroid tested which cross-reacted significantly with the antiserum (14). Because we have made no attempts to compare testosterone activities in chromatographed vs. non-chromatographed extracts of serum in these rats, the term "immunoreactive androgen" will be used for these determinations. Estradiol receptor assay On day 15, corpora lutea were excised from hypophysectomized-hysterectomized rats treated with either a 20-cm testosterone implant, a 20-cm empty implant, or 100 jug estradiol daily. The corpora lutea were homogenized in 0.01 M Tris buffer, 0.0015 M EDTA, and 0.25 M sucrose, pH 7.4. The estradiol receptor content in luteal cell nuclei was measured by a nuclear exchange assay as described by Richards (16). In brief, an 800 X g crude nuclear pellet was obtained and resuspended in 2 ml buffer. Aliquots of the suspension were added to six tubes (300 jul/tube), each containing 1 ml buffer and 10 nM [2,4,6,7-3H]estradiol (100 Ci/mmol). Two of the tubes also contained 1 JUM diethylstilbestrol for the determination of non-specific binding. After incubation (at 37 C for 1 h) and centrifugation, nuclei were resuspended in 1 ml buffer and sedimented through a sucrose cushion by centrifugation at 75,000 X g for 25 min at 4 C. Nuclei were resuspended in 1 ml distilled water, and aliquots (300 /xl) were taken for measurement of radioactivity in a

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GIBORI AND KEYES

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liquid scintillation spectrometer and for measurement of DNA (17).

Results Serum progesterone Hypophysectomy and hysterectomy on day 12 of pregnancy and treatment with "vehicle" only (either empty implant, cholesterol implant, or oil) resulted in a precipitous reduction (~75%) in serum progesterone (Fig. 1) as compared to sham-operated pregnant controls. However, the maintenance of serum progesterone at concentrations of approximately 25 ng/ml indicates that the corpora lutea continued to secrete progesterone for at least 3 days after hypophysectomy and hysterectomy, as reported earlier by Rothchild et al. (18). Treatment with testosterone via large or small implants maintained serum progesterone at concentrations similar to those in shamoperated pregnant animals (Fig. 1). Serum progesterone also remained elevated in rats treated with estradiol, although concentrations were lower (P < 0.05) on day 14 than in sham-operated animals. In contrast, the administration of DHT, a non-aromatizable anl20r

Kndo • 1978 Vol 102 • No 4

drogen, did not prevent the decline in serum progesterone after hypophysectomy and hysterectomy (Fig. 1). Testosterone treatment had no effect in rats hypophysectomized, hysterectomized, and ovariectomized on day 12 (Fig. 1). Serum immunoreactive androgen The insertion of a large (2000 mm2) Silastic implant on day 12 caused a marked elevation in serum immunoreactive androgens measured in serum on day 15 (Table 1). Androgen levels in rats with the small implant were slightly elevated above that in sham-operated, pregnant rats, whereas serum concentrations in hypophysectomized-hysterectomized rats treated with "vehicle" only declined below values in sham-operated rats. Luteal weight On day 15 of pregnancy, the mean luteal weight in hypophysectomized-hysterectomized rats treated with the vehicle only was reduced by almost 50% compared to shamoperated controls (Table 2). Estradiol (100 /ig/day) or testosterone treatment via both large and small testosterone-filled implants allowed the corpora lutea to grow as much as in intact pregnant rats, whereas the 20-cm DHT implant was no more effective than the vehicle. Weight of the non-luteal ovarian tissues On day 15 of pregnancy, in sham-operated TABLE 1. Serum immunoreactive androgen activity

Ovariectomy L . Plus T2, 12

13

14

Treatment

Serum testosterone (ng/ml)

Day of "Pregnancy"

FIG. 1. Effects of estradiol, testosterone (Ti and T20), or DHT on serum progesterone concentrations after hypophysectomy and hysterectomy on day 12 of pregnancy. Sham-operated rats were pregnant. Ovariectomized rats were triply operated on day 12. (For details of treatments, see footnote, Table 2). Each point is the mean ± SE. Range in number of observations (rats) for each group: sham-operated, 10-11 rats; Ti, 6-11; T20, 6-8; estradiol, 5; DHT, 5-9; vehicle, 13-19 (days 14 and 15), 4 (day 13); ovariectomy, 3-4; day 12, 12. Breaks in some curves on day 13 indicate that no determinations were made on that day.

Sham-operated, day 12 of pregnancy 1.8 ± 0.2° Hypophysectomy and hysterectomy day 12 plus: Empty Silastic capsule (20 cm) 0.4 ±0.1 20-cm (T20) testosterone-filled cap- 32.5 ± 2.6 sule 1-cm (Ti) testosterone-filled capsule 3.1 ± 0.3 0 Mean ± SE; n = four per treatment group. Testosterone (immunoreactive androgen) was determined on samples obtained on day 15 of pregnancy (sham-operated animals) or on equivalent day 15 of pregnancy in hypophysectomized-hysterectomized rats. Sham-operated rats were pregnant.

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ESTROGEN ACTION IN RAT CORPUS LUTEUM TABLE 2. Effect of androgens and estradiol on the weight of corpora lutea and the weight of non-luteal ovarian tissue in rats hypophysectomized and hysterectomized on day 12 of pregnancy

Treatment"

Sham operations Hypophysectomyhysterectomy plus: Vehicle Estradiol Ta, T, DHT

Luteal wt, day 15 (mg/corpus luteum)

Non-luteal tissue, day 15 (mg/two ovaries)

3.8 ±0.1 (12)*

2.0 3.5 4.0 3.5 1.9

± 0.1 ±0.3 ±0.2 ±0.2 ±0.1

(20) (5) (8) (10) (9)

29.2 ± 0.2 (11)

23.8 ± 21.7 ± 21.3 + 19.8 + 20.6 ±

1.6 (16) 2.3 (5) 1.3 (8) 0.8 (6) 2.0 (9)

"Vehicle refers to an empty implant, a cholesterol implant, or oil injection; estradiol, 100 /xg in oil injected on days 12, 13, and 14; T20, Silastic capsule 20 cm in length, filled with testosterone and implanted sc on day 12; T|, Silastic capsule, 1 cm in length, filled with a mixture of testosterone: cholesterol (1:1) and implanted sc on day 12; DHT, Silastic capsule, 20 cm in length, filled with DHT and implanted sc on day 12. Sham-operated rats were pregnant. h Mean ± SE (number of animals).

animals, the non-luteal component of the ovaries weighed 29 mg/two ovaries and contained visible follicles (Table 2). Three days after hypophysectomy-hysterectomy on day 12 of pregnancy, the non-luteal tissue appeared pale, weighed less than controls, and had no visible large follicles. Treatment with estradiol, testosterone, or DHT did not prevent atrophy of the extraluteal tissue(s). Luteal estradiol concentrations The estradiol concentration in corpora lutea of rats hypophysectomized and hysterectomized on day 12 of pregnancy and treated with vehicle only was 7-fold lower than in shamoperated animals on day 15 of pregnancy (Fig. 2). Estradiol or testosterone (T20) treatment greatly increased the luteal estradiol concentration above that in sham-operated animals. Administration of testosterone via the smaller implant maintained the luteal estradiol concentration at values similar to those in shamoperated animals. Nuclear estradiol receptor content in corpora lutea By the third day after hypophysectomy and

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hysterectomy, the nuclear content of estradiol receptor had declined below that in shamoperated controls (Table 3). Testosterone (T20) treatment partially maintained nuclear receptor content in the corpora lutea.

Discussion The results reported here confirm the observations of Takayama and Greenwald (4) and of Gibori et al. (5) of luteotropic effects of 17/?-estradiol in the rat. We have also extended these observations by demonstrating that testosterone can completely mimic the luteotropic effects of estradiol. For reasons 200 100 e 50

o s 10 M

UJ

o

Q>

5

Sham- Vehicle Estradiol Operated Injected Injected

Large (T 2 0 )

Small (T,)

Testosterone

Implant

Testosterone

Implant

FIG. 2. Effects of estradiol or testosterone treatment on luteal estradiol concentrations in rats hypophysectomized and hysterectomized on day 12 of pregnancy. Sham-operated rats were pregnant. For details of treatments, see footnote, Table 2. Each bar represents the mean ± SE. Number of observations (rats): sham-operated, 8; vehicle, 15; estradiol-injected, 5; T2n implant, 8; Ti implant, 10. TABLE 3. Luteal cell nuclear content of estradiol receptor in rats hypophysectomized-hysterectomized on day 12 of pregnancy Treatment

cpm//xg DNA

Sham operated Hypophysectomized-hysterectomized plus: Empty Silastic capsule 20-cm (T20) testosterone-filled capsule

135 ± 10" 18 ± 5 84 ± 6

" Mean ± SE; n = two observations; each observation represents receptor determination from a pool of corpora lutea obtained from five to eight rats. All determinations were made on day 15 of pregnancy (sham-operated animals) or on equivalent day 15 of pregnancy in hypophysectomized-hysterectomized rats.

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GIBORI AND KEYES

which will be given below, we believe that the effects of testosterone are, in reality, attributable to the actions of estradiol formed within the luteal tissue. The significance of this work lies in the demonstration of a pathway for local accumulation of estradiol in the corpus luteum and in the demonstration of a possible role for intraluteal estradiol in the regulation of the rat corpus luteum. Several arguments can be advanced as evidence that estradiol is the ultimate active hormone in rats treated with testosterone. First, estradiol alone maintained luteal function and growth, even though serum progesterone was inexplicably lower on day 14 than in control pregnant rats. Estrogen receptors have been identified in rat luteal tissue (16), and diethylstilbestrol-inhibitable nuclear binding of estradiol has been confirmed in this study. The inability of the large testosterone implant to maintain fully nuclear estradiol receptor content may be related to the residual but waning effects of lactogenic hormones after hypophysectomy and hysterectomy. These hormones seem to be necessary for the maintenance of estradiol receptor in luteal tissue (19). The possibility that testosterone interacts with the estrogen receptor seems remote because excess testosterone did not inhibit significantly the binding of [3H]estradiol (16). A second argument is that DHT had no discernible luteotropic effects. Although measurements of this hormone were not made, the release rate from Silastic capsules is similar to that of testosterone which was present in high concentrations in serum of rats with the 20-cm testosterone implant. Gay (20) has reported that DHT is rapidly converted in vivo to a metabolite which is suspected to be 5a-androstane-3a,17/?-diol, but this metabolite is also a strong androgen. Therefore, it seems reasonable to conclude that the tropic effect of testosterone is not via known androgen metabolites. The possibility remains, however, that testosterone itself may have direct actions in rat luteal tissue. Finally, the recent demonstration that estradiol formation by rat corpora lutea can be enhanced as much as 400-fold in the presence of testosterone in vitro (7) and the marked elevation of intralu-

Endo i > 1978 Vol 102 t No 4

teal estradiol in the presence of the large testosterone implant (Fig. 2) strongly indicate a functional luteal aromatase system. Gibori and Kraicer (8) have reported that ectopic corpora lutea form estrogen from testosterone implanted adjacent to the corpora lutea. The estrogen was not derived from other ovarian tissues because the rats were ovariectomized. In view of the marked ability of ovarian follicles to synthesize androgens and estrogens, a possible contribution by this tissue to the estrogen content of corpora lutea in normal rats cannot be neglected. Although the non-luteal portion of the ovary atrophied after hypophysectomy (Table 2), ovarian follicles may have formed estradiol in the presence of testosterone. In immature rats, aromatase activity in follicles is dependent on FSH (21), but this relationship has not been established in pregnant rats. Recent studies have shown the ectopic corpora lutea secrete adequate quantities of progesterone for the maintenance of pregnancy in rats ovariectomized on day 13 of pregnancy (22, 23). These results indicate that the non-luteal tissues of the ovary do not produce hormones or hormone precursors during the second half of pregnancy that are critical for continued function of these isolated corpora lutea. The failure of corpora lutea to function normally after hysterectomy at midpregnancy (24) argues strongly that the placenta and/or fetus exert the major regulatory influence on the corpora lutea. It is well known that the placenta is the source of a lactogenic hormone and that this hormone stimulates luteal function (1, 2). However, Gibori et al. (5) have reported that the normal increase in serum progesterone at the beginning of the second half of pregnancy requires the actions of both placental luteotropin (serum from day 12 pregnant rats) and 17/?-estradiol. This implies that a placental hormone other than placental luteotropin might be responsible for stimulation of estradiol production by the corpora lutea. A recently reported rat chorionic, LH-like hormone (25) might maintain luteal estradiol concentrations through direct actions on the luteal tissue because, during the first half of pregnancy, LH seems to be required for the

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Role of intraluteal estrogen in the regulation of the rat corpus luteum during pregnancy.

0013-7227/78/1024-1176$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society Vol. 102, No. 4 Printed in U.S.A. Role of Intraluteal Estroge...
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