ABSTRACT. Apoptotic cell death has recently been suggested to be the underlying mechanism of ovarian follicle atresia. To study the regulation of follicle.
0013.7227/93/1335-2204$03.00/0 Endocrinology Copyright 0 1993 by The Endocrine
Estrogens Granulosa HAKAN
BILLIGt,
Vol. 133, No. 5 Printed in U.S.A.
Society
Inhibit and Androgens Cell Apoptosis* ITSUKO
FURUTAS,
Division of Reproductive Biology, Department Medicine, Stanford, California 94305-5317
AND
AARON
of Gynecology
Enhance
Ovarian
J. W. HSUEHS and Obstetrics,
Stanford
University
School of
ABSTRACT Apoptotic cell death has recently been suggested to be the underlying mechanism of ovarian follicle atresia. To study the regulation of follicle cell apoptosis by sex steroids, we have analyzed ovarian DNA fragmentation, the hallmark of apoptosis, in rats treated with estrogens and androgens. Immature rats were hypophysectomized and implanted with diethylstilbestrol (DES) capsules. Two days later, DES implants were removed in some animals, followed by treatment with estrogens with or without androgens. The extent of ovarian apoptotic DNA fragmentation was analyzed by autoradiography of size-fractionated DNA labeled at 3’-ends by [32P]dideoxy-ATP. After DES withdrawal, ovarian weight decreased and DNA fragmentation increased in a time-dependent manner. In granulosa cells, an increase in apoptotic DNA fragmentation was seen 12 h after withdrawal of DES implants, followed by a 25fold increase at 48 h. In situ analysis of DNA fragmentation on histological sections of ovaries, using a nonisotopic labeling of DNA by digoxigenin-dideoxy-UTP also demonstrated that apoptosis -induced by DES withdrawal is confined to the granulosa cells in early antral and preantral follicles. No increase in DNA breakdown was detected in thecal cells and interstitial tissues or granulosa cells of primordial
and primary follicles. In contrast, replacement with DES (0.5 mg twice daily) or estradiol benzoate (3 mg daily) completely prevented the observed ovarian weight loss and increases in granulosa cell apoptosis. Treatment with estradiol benzoate (0.003-3 mg/day) dose dependently suppressed the apoptosis seen 2 days after removal of DES implants. Furthermore, the antiatretogenic effect of estrogen was blocked by treatment with testosterone (0.5 mg twice daily), which increased ovarian apoptotic DNA fragmentation and decreased ovarian weight in DES-treated animals in a time-dependent manner. Also, in situ examination showed that androgen treatment increased apoptosis in the granulosa cells in a subpopulation of early antral and preantral follicles. The specificity of testosterone action was further demonstrated by the lack of effect of progesterone and cortisol on ovarian apoptosis. These data suggest that sex steroids play an important role in the regulation of ovarian apoptotic cell death, with estrogens preventing apoptosis and androgens antagonizing the effect of estrogens. These data provide the basis for future studies on the role of sex steroid hormones in follicular atresia and the regulation of endonuclease activity by steroid hormones. (Endocrinology 133: 2204-2212, 1993)
I
nucleosomal fragmentation of cellular DNA (20-23), a hallmark of apoptotic cell death (24). In addition, treatment with epidermal growth factor or basic fibroblast growth factor suppressesthe spontaneous onset of apoptosis in cultured preovulatory rat follicles (21). With the establishment of a sensitive and quantitative method to analyze the presenceof apoptotic DNA fragmentation in small amounts of DNA from ovarian cells and tissues (20, 21), it has been possible to correlate increased apoptotic DNA fragmentation with decreased follicular estrogen content and cytochrome I’450 aromatase mRNA levels in atretic follicles (23). Becausethe effects of estrogens and androgens on follicle atresia have only been suggested based on morphological criteria, the molecular changes induced by these sex steroids in the follicles are unknown. Using a quantitative DNA end-labeling gel fractionation and a qualitative in situ technique, the present study found, for the first time, regulation of apoptotic DNA fragmentation in the granulosa cells of hypophysectomized rats treated with estrogensand androgens in vivo.
N ALL mammalian speciesstudied, atresia is a prominent feature of ovarian follicular development (l-3). Lessthan 0.1% of all follicles present at birth mature and ovulate, whereas more than 99.9% of the follicles undergo atresia. Although follicle atresia has been found throughout female reproductive life, the hormonal mechanism behind the massive cell death is largely unknown. Earlier studies have shown that gonadotropins (4, 5) and gonadal steroids (6, 7) modulate the incidence of atresia in the ovary. Treatment with estrogens increases follicular growth, ovarian weight, and the mitotic index of granulosa cells (8-11). In contrast, androgens causedeterioration of ovarian follicles by increasing the number of pyknotic granulosa cells and degenerated oocytes and decreasing ovarian weight in estrogen-treated hypophysectomized rats (7, 12-15). Indeed, an increased androgen to estrogen ratio is found in follicular fluid of atretic follicles (16, 17), and estrogen production is invariably decreasedin atretic follicles (18, 19). Recent studies have suggested that follicle atresia in chicken, porcine, and rodent ovaries is associatedwith inter-
Materials
Received July 20, 1993. *This work was supported by NIH Grant HD-13527. t Supported in part by Fogarty Fellowship F05-TW04559-01 from the NIH and Swedish Medical Research Council Grant B93-10380. $ On leave from the Department of Obstetrics and Gynecology, Hokkadio University, Sapporo, Japan. 5 To whom requests for reprints should be addressed.
Hormones
and Methods
and animals
Estradiol benzoate, testosterone, progesterone, cortisol, and diethylstilbestrol (DES) were obtained from Sigma Chemical Co. (St. Loius, MO). Steroids were dissolved in ethanol, diluted further in corn oil (5% ethanol and 95% corn oil), and injected in a loo-p1 dose. 2204
SEX
STEROIDS
MODULATE
APOPTOSIS
IN OVARIAN
FOLLICLES
2205
Immature female Sprague-Dawley rats were obtained from Johnson Laboratories (Bridgeview, IL). At 22 days of age, the animals were hypophysectomized and implanted with Silastic tubing (15 mm) containing DES. The animals were delivered on the second postoperative day and were given physiological saline with 5% glucose ad libitum.
fractioned on a 2% agarose gel, transferred and incubated with digoxigenin antibody phatase for color reaction.
Steroid
Experiments were repeated at least three times, and representative autoradiograms are presented. Quantitative data represent the mean + SEM of three to six separate gel runs. Statistical evaluation was performed by analysis of variance, followed by Student-Newman-Keuls test. P < 0.05 was considered significant (28).
treatment
and tissue preparation
On day 24 of age, DES implants were removed, and animals were treated SC every 12 h with DES, testosterone, cortisol, or progesterone every 12 h. Some rats were treated with estradiol benzoate every 24 h. The animals were killed at the indicated times, and ovaries were isolated and weighed. One ovary from each animal was snap-frozen in a dry ice-ethanol bath, and the follicles from the contralateral ovary were punctured to isolate granulosa cells. After centrifugation at 250 X g for 3 min, the granulosa cells were snap-frozen before storage at -70 C.
DNA isolation
and analysis
Cellular DNA was isolated as previously described (21, 25) and quantitated spectrophotometrically at 260 nm. Aliquots of DNA (500 ng) from each sample were labeled at 3’-ends with [32P]dideoxy-ATP (ddATP; 3000 Ci/mmol; Amersham, Arlington Heights, IL) using terminal transferase (25 U/sample; Boehringer Mannheim, Indianapolis, IN), as previously described (26). Labeled DNA (150 ng) preparations were fractionated through 2% agarose gels by electrophoresis (6.5 V/ cm). Gels were dried for 2 h in a slab gel dryer without heat and exposed to Kodak X-Omat film (Eastman Kodak, Rochester, NY) at -70 C. After autoradiography, portions of each lane corresponding to DNA less than 15 kilobases (kb) were isolated and counted in a p-counter for quantitation of the degree of internucleosomal DNA fragmentation.
Tissue preparation
and in situ DNA 3’.end
labeling
Analysis of DNA fragmentation in histological sections was carried out based on a previously described method (27), with modifications as described below, using a nonradioactive approach. Ovaries were fixed in 4% buffered formaldehyde and embedded in paraffin. Sections (4-6 pm) were mounted on coated slides (Vectabond, Vector Laboratories, Burlingame, CA), deparaffinized, hydrated, and treated with proteinaseK for 30 min at 37 C in a humidified chamber (Boehringer Mannheim; 10 fig/ml in 20 mM Tris and 2 rnM CaC12, pH 7.4), folibwed by three washes in Tris buffer (100 mM Tris and 150 rnM NaCl, nH 7.5). DNA 3’.end labeling with nonradioactive digoxigenin-ddUTP (dig-ddUTP) was performed after incubation for 10 min in terminal transferase buffer (200 rnM potassium cacodylate, 25 rnM Tris, 0.25 mg/ml BSA, and 5 mM CoC12, pH 6.6) at room temperature. Terminal transferase (1 U/pi; Boehringer Mannheim), dig-ddUTP (5 go; Boehringer Mannheim), and ddATP (45 uM: I’harmacia, Unosala, Sweden) were added in fresh buffer and incubated at 37 C in a humidified chamber for 1 h. The ddNTI’s were used to prevent “tailing” and to allow the incorporation of one nucleotide per free DNA 3’-ends, thus ensuring a more quantitative assay. After three washes in Tris buffer, the sections were incubated with blocking buffer [lo0 rnM Tris, 150 mM NaCl (pH 7.5), and 0.5% (wt/vol) blocking reagent; Boehringer Mannheim] for 30 min at room temperature before the addition of antidigoxigenin antibody conjugated to alkaline phosphatase (Boehringer Mannheim). After incubation with the antibody [Boehringer Mannheim; 1:lOOO in 0.5% (wt/vol) blocking reagent, 100 mM Tris, and 150 mM NaCl, pH 7.51 at room temperature for 2 h in a humidified chamber, the slides were washed three times in Tris buffer and finally equilibrated in alkaline phosphatase buffer (100 rnM Tris, 100 rnM NaCl, and 50 rnM MgCl*, pH 9.5) before the addition of substrates (337.5 pg/ml nitroblue tetrazolium and 175 pg/ml5-bromo4-chloro-3-inodyl-phosphate; Boehringer Mannheim) for alkaline phosphatase. After 3-12 h in the dark, the color reaction was terminated with 10 rnM Tris and 1 rnM EDTA, pH 8. When either terminal transferase or the antibody conjugate was omitted from the procedure or when only ddATP was used as substrate for the terminal transferase, no color reaction was detected. Also, incorporation of dig-ddUTP into DNA was verified on isolated apoptotic ovarian DNA using terminal transferase. The apoptotic banding pattern was detected after the labeled DNA was
to nitrocellulose membrane, conjugated to alkaline phos-
Data analysis
Results Effects of DES withdrawal
on ovarian DNA fragmentation
Immature female rats were hypophysectomized and implanted with DES capsules at 22 days of age. The effect of estrogen deprivation on the integrity of ovarian DNA was studied by removal of DES implants 2 days after surgery. Ovaries were obtained every 12 h after the removal of DES implants (Fig. 1). Total ovarian DNA was end labeled with [32P]ddATP for analysis of apoptotic internucleosomal fragmentation using gel electrophoresis, followed by autoradiography. A time-dependent increase in DNA fragmentation to lower mol wt, seen as DNA ladders (
