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Biology of Reproduction 67, 1285-1296 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Neonatal Exposure to Genistein Induces Estrogen Receptor (ER){alpha} Expression and Multioocyte Follicles in the Maturing Mouse Ovary: Evidence for ERß-Mediated and Nonestrogenic Actions

Wendy N. Jeffersona,c, John F. Couseb,c, Elizabeth Padilla-Banksa, Kenneth S. Korachb, and Retha R. Newbold1,a

a Developmental Endocrinology Section, Laboratory of Molecular Toxicology, Environmental Toxicology Program, b Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, Environmental Diseases and Medicine Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 c Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27605


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Outbred CD-1 mice were treated neonatally on Days 1–5 with the phytoestrogen, genistein (1, 10, or 100 µg per pup per day), and ovaries were collected on Days 5, 12, and 19. Ribonuclease protection assay analysis of ovarian mRNA showed that estrogen receptor ß (ERß) predominated over ER{alpha} in controls and increased with age. Genistein treatment did not alter ERß expression, however, ER{alpha} expression was higher on Days 5 and 12. ERß was immunolocalized in granulosa cells, whereas ER{alpha} was immunolocalized in interstitial and thecal cells. Genistein treatment caused a dramatic increase in ER{alpha} in granulosa cells. Genistein-treated ERß knockout mice showed a similar induction of ER{alpha}, which is seen in CD-1 mice, suggesting that ERß does not mediate this effect. Similar ER{alpha} induction in granulosa cells was seen in CD-1 mice treated with lavendustin A, a tyrosine kinase inhibitor that has no known estrogenic actions, which suggests that this property of genistein may be responsible. As a functional analysis, genistein-treated mice were superovulated and the number of oocytes was counted. A statistically significant increase in the number of ovulated oocytes was observed with the lowest dose, whereas a decrease was observed with the two higher doses. This increase in ovulatory capacity with the low dose coincided with higher ER{alpha} expression. Histological evaluations on Day 19 revealed a dose-related increase in multioocyte follicles (MOFs) in genistein-treated mice. Tyrosine kinase inhibition was apparently not responsible for MOFs because they were not present in mice that had been treated with lavendustin; however, ERß must play a role, because mice lacking ERß showed no MOFs. These data taken together demonstrate alterations in the ovary following neonatal exposure to genistein. Given that human infants are exposed to high levels of genistein in soy-based foods, this study indicates that the effects of such exposure on the developing reproductive tract warrant further investigation.

follicular development, granulosa cells, mechanisms of hormone action, oocyte development, steroid hormone receptors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Greater awareness of the resulting consequences of human and wildlife exposure to natural and synthetic estrogens has inspired the emerging field of environmental endocrine disruption. Several laboratories, including our own, have characterized the teratogenic and carcinogenic effects of gestational and neonatal exposure to 17ß-estradiol and the synthetic estrogen, diethylstilbestrol (DES), in rodents and humans alike [1–3]. In recent years, similar investigations have been undertaken of less potent but more ubiquitous estrogenic substances: namely, the plant-derived phytoestrogens. Perhaps the impetus for these investigations is the awareness that phytoestrogens such as genistein and coumestrol are known to be present in the human diet and are likely substantial components of vegetarian diets. Furthermore, childhood exposure to high levels of genistein and other phytoestrogens may occur through soy-based infant formulas and foods that are often specifically marketed for children [4, 5]. In fact, isoflavone concentrations in commercially available soy-based infant formulas can far exceed amounts found in the average diet of adult vegetarians in the United States [6]. It is estimated that infants who consume a diet of soy-based formulas are exposed to 6–9 mg per kg per day of soy isoflavones, more than 65% of which is genistein, whereas adults who consume a diet with modest amounts of soy isoflavones may be exposed to approximately 1 mg per kg per day [6].

Still, scientific data supporting the benefits of dietary phytoestrogens are insufficient. Reported beneficial effects of phytoestrogen exposure include a reduction in the risk of N-methyl-N-nitrosurea-induced mammary cancer in rats [7] and a lowering of cholesterol in humans [8]. Equally lacking are convincing laboratory studies describing possible detrimental effects of phytoestrogen exposure. However, we have recently reported that neonatal exposure to genistein leads to the induction of uterine adenocarcinoma in mice [9], similar to effects previously described following neonatal DES exposure. Hilakivi-Clarke et al. [10] have also described a higher incidence of mammary tumors in female rats following prenatal exposure to genistein. In humans, the possible detrimental effects of a vegetarian diet consumed during pregnancy include a higher incidence of hypospadias in boys, perhaps due to abnormally high maternal levels of soy isoflavone [11]. Therefore, the effects of either accidental or intended exposure to dietary phytoestrogens is a growing public health concern.

Estrogens are known to regulate multiple cell functions in target tissues, including growth and differentiation via nuclear receptor-mediated pathways. Furthermore, aberrant temporal or overstimulation of the estrogen signaling pathway during development has long been known to result in multiple, long-term abnormalities in the reproductive tract, including neoplasia [1–3]. Until recently, the majority of estrogen actions were believed to be mediated via a single form of nuclear estrogen receptor (ER). However, the discovery of a second ER, termed ERß (to differentiate it from the original, now termed ER{alpha}), has prompted a reevaluation of the physiology and toxicology of the estrogen signaling system. The biological significance of two ER subtypes remains unclear, but it may explain the selective and diverging actions of estrogens that occur in various target tissues. Whereas ER{alpha} appears to be the predominant ER form in the Müllerian-derived structures of the female reproductive tract [12–15], easily detectable levels of both ERs are present in the gonads of both sexes [16–18]. In the reproductive tract of late gestational and neonatal mice, ER{alpha} immunoreactivity is localized to the stromal and epithelial cells of the uterus and the interstitium of the ovary [17, 19], whereas ERß immunoreactivity is limited to the ovarian granulosa cells, with little to no detectable expression in the uterus [18].

Other researchers have also reported the presence of ERß in the human ovary [20, 21]. There are numerous descriptions of detectable ER{alpha} transcripts in mouse and human oocytes [22, 23]. The divergent expression patterns of ER{alpha} and ERß in the developing and adult reproductive tract indicate the complexity of the estrogen signaling system and suggest that the two receptors likely play different physiological roles. This is further supported by the diverging transactivational activities and varied binding affinities of the two receptors for different ligands [24, 25]. For example, in vitro transactivational assays have shown that 4-hydroxytamoxifen acts as a partial agonist when bound to ER{alpha}, but as a full antagonist when it interacts with ERß [26]. Furthermore, 17ß-estradiol exhibits a similar in vitro binding affinity for both ERs, whereas several synthetic and naturally occurring xenoestrogens exhibit a binding preference for one of the two receptors [24, 25].

Genistein has long been recognized to have significant estrogenic activity in both in vivo and in vitro assays, including the induction of estrogen-regulated genes, hypertrophy, and cell proliferation in the rodent uterus [27, 28]. Furthermore, ERß has been shown to exert a 20-fold higher relative binding affinity for genistein than does ER{alpha} [25]. A similar binding preference for ERß has been reported for additional phytoestrogens, including coumestrol and naringenin [25]. However, the role of ER{alpha} and ERß, and the extent to which each contributes to the actions and possible toxicity of genistein exposure has only recently come under investigation [24, 25]. An additional confounding factor unique to the study of genistein is its well-described ability to inhibit tyrosine-specific kinases. This property of genistein was first described in 1987, in which Akiyama et al. showed in vitro that genistein was able to inhibit the tyrosine-kinase activity of the epidermal growth factor receptor via competitive inhibition for ATP binding [29]. Since this report, genistein has now been marketed as a reagent to effectively inhibit tyrosine-specific protein kinases in laboratory studies [30]. More recently, genistein's ability to inhibit tyrosine-specific kinases has been shown to be independent of its ER-mediated hormonal activities in vitro [31, 32].

Herein, we describe a syndrome of effects of neonatal genistein exposure on the maturing CD-1 mouse ovary that may be categorized as follows: a biochemical effect as the induction of ectopic expression of ER{alpha} in granulosa cells, a morphological effect as the induction of multioocyte follicles (MOFs) in the ovary, and a functional effect as the altered ovarian response to superovulation treatment. Furthermore, using the gene-targeted ER{alpha}-null (i.e., {alpha}ER knockout, or {alpha}ERKO) and ERß-null (ßERKO) mice as well as the nonestrogenic tyrosine-kinase inhibitor (lavendustin A), we were able to differentiate the estrogenic and tyrosine-kinase inhibitory properties of genistein and ascribe these properties to different ultimate effects in the ovary.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

All animal procedures complied with an animal care protocol that was approved by the National Institute of Environmental Health Sciences (NIEHS). Adult CD-1 (Crl:CD-1 [ICR] BR) mice were obtained from Charles River Breeding Laboratories (Raleigh, NC) and bred to male mice of the same strain in the breeding facility at NIEHS (Research Triangle Park, NC). Vaginal plug detection was considered Day 0 of pregnancy. Pregnant mice were housed under controlled lighting (12L:12D) and temperature (21°–22°C) conditions. Mice were provided with National Institutes of Health 31 laboratory mouse chow and fresh water ad libitum. Pregnant mice delivered their young at 19 days of gestation, at which time female pups were pooled together and redistributed to eight females per dam. Pups were treated on Days 1–5 with genistein (Sigma Chemical Company, St. Louis, MO) by s.c. injection at doses of 1, 10, or 100 µg per pup per day in corn oil (day of birth = Day 1). These doses are approximately 0.5, 5, or 50 mg per kg per day (estimating 2 g/pup). All CD-1 experiments consisted of 16 mice per treatment group per age. A second group of CD-1 mice were generated as described above and treated with the tyrosine kinase inhibitor, lavendustin A (Sigma), at doses of 1 or 10 µg per pup per day (0.5 or 5 mg per kg per day) on Days 1–5 (eight mice per group). The doses of lavendustin A chosen for this study were based on a study that showed tyrosine kinase inhibition in vivo at a dose of 1.3 mg per kg per day [33].

The {alpha}ERKO mice on a background of C57BL6 and ßERKO mice on a background of C57BL6/129J have been described previously [34] and were obtained from the breeding colony at NIEHS. Heterozygous breeding pairs from both knockout lines were used to generate pups for treatment. On the day of birth, female pups of each line were pooled together and redistributed to foster CD-1 mothers to avoid differences in maternal behaviors between the two ERKO lines. Pups were treated with genistein as described above.

In all experiments, mice were killed on either Day 5, 12, or 19 (except ERKO mice, which were killed on Day 19 only). For RNA or protein extraction, the reproductive tract plus gonads were collected into cold PBS, and the ovaries were carefully dissected from oviducts, frozen on dry ice, and pooled together per treatment group. Approximately eight mice per treatment were pooled on Days 5 and 12, and four mice per treatment were pooled on Day 19. For histological and immunohistochemical evaluations, the reproductive tract and gonads were dissected and immediately fixed in buffered formalin for 6 h at 4°C, transferred to 70% ethyl alcohol at 4°C, followed by routine paraffin embedding.

RNA Isolation and Ribonuclease Protection Assay

Total RNA was extracted from each sample of pooled tissue using Trizol reagent (Gibco-BRL, Grand Island, NY) according to the manufacturer's protocol. The concentration of each preparation was determined by A260 measurement, and the integrity was determined by visualization following 1% agarose gel electrophoresis. All RNA preparations were stored at -70°C until further use.

The generation and use of the riboprobes used in this study have been previously described [16]. Antisense riboprobes were generated from linearized templates using Maxiscript reagents (Ambion, Austin, TX), the appropriate RNA polymerase (T3 or T7), and incorporation of [32P]CTP (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer's protocol. The full-length mouse ER{alpha} antisense riboprobe was 490 nucleotides (nt) and produced a specific protected fragment of 366 nt [16]. The mouse ERß antisense riboprobe was 318 nt in full length, and generated a protected fragment of 262 nt [16]. An antisense riboprobe specific for mouse cyclophilin, used to equate loading among lanes, was generated from the template pTRI-Cyclophilin (Ambion) at a full-length of 165 nt, and produced a protected fragment of 103 nt.

All RNase protection assay (RPA) reactions consisted of 5 x 104 cpm of each of the three probes, 10 µg of sample RNA, and yeast tRNA (for a final total RNA equal to 25 µg), which were mixed and ethanol-precipitated at -70°C for a minimum of 3 h to a maximum of overnight. The resulting pellets were then processed through the RPA using Hybspeed ribonuclease protection assay reagents (Ambion) according to the manufacturer's protocol. Final analysis of protected fragments was carried out by electrophoresis on a 1.5-mm thick gel of 6% bis-acrylamide, 8.3 M urea, and 1x Tris-borate EDTA (Novex, San Diego, CA), which was then fixed, dried, and exposed to a PhosphorImager screen followed by exposure to x-ray film. Quantitation of the protected fragments from each sample was carried out using a Molecular Dynamics PhosphorImager Storm 860 and ImageQuant software (Sunnyvale, CA).

Protein Isolation and ER{alpha} Western Blotting

Nuclear protein was isolated from samples of pooled ovaries collected at 19 days of age using the N-PER kit (Pierce, Rockford, IL), and then sample concentration was determined using the bicinchoninic acid kit (Pierce) according to the manufacturer's protocol. For Western blot analysis, a total of 25 µg per sample was electrophoresed on a 10% Bis-Tris gel (Novex) and transferred to nitrocellulose. The gel was then stained with Simply blue (Invitrogen, Grand Island, NY) to ensure the efficiency of the transfer. The blot was then washed in Tris-buffered saline with 1% Tween-20 (TBS-T) at pH 7.4, blocked with 10% BSA for 30 min at room temperature, and then allowed to incubate with anti-ER{alpha} antibody (E1396; Sigma) at a dilution of 1:5000 for 1 h at room temperature. Specific immunoreactivity was then detected using the enhanced chemiluminescence detection reagents (Amersham) according to the manufacturer's protocol. Following detection, the blot was then stained with India ink to ensure equal loading and efficient transfer.

Estrogen Receptor Immunohistochemistry

Ovarian tissues were embedded in paraffin and serially cut in 4 µm sections. Tissue sections from a minimum of four mice per treatment for each time point were randomly selected and immunostained for ER{alpha} and ERß. For ER{alpha} immunodetection, tissue sections were deparaffinized in xylene, hydrated in a series of graded ethanols, and washed in 1x Automation Buffer (AB; Fisher Scientific, Norcross, GA). Sections were then treated with 3% hydrogen peroxide to eliminate endogenous peroxidase. Following washes in AB, sections were placed in coplin jars in citrate buffer pH 8.0 (Biocare Medical, Walnut Creek, CA) and placed into a decloaker (Biocare Medical) at a setting of 5 min for antigen retrieval. Following the antigen retrieval step, sections were rinsed with distilled water and then treated according to the instructions provided with the mouse-on-mouse kit (Vector Laboratories, Burlingame, CA). Briefly, sections were incubated in blocking solution for 1 h at room temperature. Sections were rinsed with Tris-buffered saline pH 7.4 and then incubated with anti-mouse ER{alpha} (Oncogene Science, Manhasset, NY) diluted 1:250 in diluent from the kit for 30 min. Negative controls were run on adjacent tissue sections with preimmune serum or without the primary antibody (buffer only). Sections were then incubated with biotinylated goat anti-mouse and avidin-biotin complex following the instructions in the kit. Visualization of the peroxidase was carried out by covering the sections with diaminobenzidine (DAB; Sigma) at 0.5 mg/ml in AB containing 0.01% hydrogen peroxide for 10 min. Sections were rinsed in distilled water, counterstained with hematoxylin, dehydrated in a graded series of ethanols and xylenes, and coverslipped for evaluation with light microscopy.

Adjacent ovarian tissue sections were also immunostained for ERß using techniques previously described [18]. Briefly, sections were deparaffinized and rehydrated, endogenous peroxidase was eliminated, and antigen retrieval was performed as described for ER{alpha} above. Following the antigen retrieval step, tissue sections were rinsed with water and then with AB pH 6.8. All AB used for the detection of ERß had a pH of 6.8 (lower pH was found to enhance the signal of this protein). Sections were then incubated with 10% BSA in AB for 20 min, then incubated overnight at 4°C using rabbit anti-mouse ERß at a dilution of 1:200 in AB pH 6.8 (Oncogene Science). Negative controls of adjacent tissue sections were incubated with preimmune serum at the same dilution or no primary antibody (buffer only). Sections were then rinsed and incubated with biotinylated goat anti-rabbit (Vector Laboratories) at a concentration of 1:500 for 1 h followed by ExtrAvidin Peroxidase (Sigma) for 30 min. Protein detection was visualized with DAB as described for ER{alpha}. Sections were rinsed in distilled water, counterstained with hematoxylin, dehydrated, coverslipped, and evaluated using light microscopy.

Morphological Evaluation

Morphological evaluations of ovarian sections were carried out on ovaries from animals of each treatment group at 19 days of age. For each animal, three sections (6 µm) were prepared from both ovaries from different depths and stained with hematoxylin and eosin according to standard laboratory procedures [35]. Ovaries from eight mice per treatment group were analyzed for the presence of MOFs and atretic follicles with light microscopy. The presence of one MOF in a single ovarian section categorized a mouse as positive for MOFs. The number of atretic follicles was reported as the number per ovary section.

Superovulation Study

As a functional analysis of the ovary, the ovulatory capacity of neonatally genistein-treated mice was determined using methods previously described [36]. CD-1 mice treated neonatally with genistein as described above were weaned at 21 days of age. On Day 22, all mice received a single s.c. injection of 2.2 eCG (Sigma) followed 48–52 h later with 3.2 IU hCG (Sigma). The animals were then killed 16–20 h after the hCG injection and the oviduct was removed and placed in M-2 medium (Specialty Media, Lavallette, NJ) supplemented with 0.3% hyaluronidase (Sigma). The oocyte/cumulus mass was surgically extracted from the oviduct, and the oocytes were counted after enzymatic disassociation from the surrounding cumulus. The data shown are the sum of the two experiments, each of which included eight mice per treatment group.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Genistein Induction of ER{alpha} in the Maturing Ovary

Figure 1 shows the results of RPAs for ER{alpha} and ERß mRNA in the ovaries of untreated prepubertal mice, indicating that ERß transcripts were the predominant receptor transcript expressed in the ovaries at all ages examined, with a pronounced increase occurring between 5 and 12 days of age. ER{alpha} mRNA was also detectable at all ages examined, but it exhibits relatively stable levels on Days 5 and 12 and an apparent decrease at 19 days of age. However, at 5 days of age, mice exposed to the lowest dose of genistein (Gen-1) exhibited an almost 3-fold increase in the level of ER{alpha} mRNA; this pattern was no longer apparent in similarly treated mice at 12 and 19 days of age (Fig. 2A). A similar increase in ER{alpha} mRNA levels was observed in the middle genistein group (Gen-10), with levels peaking at 12 days of age. ERß mRNA showed a similar expression pattern as that of ER{alpha} on Day 5 with increases in the Gen-1 and Gen-10 dose group; this increase was no longer apparent at 12 and 19 days of age (Fig. 2A). The highest genistein treatment group (Gen-100) showed a decrease in ER{alpha} and ERß mRNA at 5 days of age compared with controls (Fig. 2B), but this difference also became less apparent by 12 and 19 days of age (Fig. 2B).



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  		FIG. 1. RPA results of ER{alpha} and ERß in ovaries of control mice throughout development. Total ovarian RNA from mice 5, 12, and 19 days of age was assayed for ER expression by RPA. ER{alpha} is expressed on Day 5 and decreases over the next two time points. ERß is highly expressed in the ovary at all ages examined and shows an increase with age. Note that ERß is the predominant ER subtype in the ovary at all ages examined. Each sample was normalized to cyc and the data are presented as %cyc



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  		FIG. 2. RPA results of ER{alpha} and ERß mRNA in the ovary throughout development following neonatal genistein exposure. Total ovarian RNA from vehicle and genistein-treated mice of 5, 12, and 19 days of age was assayed for ER expression by RPA. Each sample was normalized to cyc as a percentage, then fold control was calculated by using the normalized value of ER{alpha} or ERß divided by its own age-matched control value. ER{alpha} and ERß mRNA are present in all samples. ER{alpha} mRNA increases on Day 5 following the lowest dose of genistein, and ER{alpha} increases on Day 12 following Gen-10 treatment (A). ERß appears to be higher at 5 days of age in the Gen-1 and Gen-10 groups, but this increase is less apparent by 12 and 19 days of age. At the highest dose of genistein (Gen-100), ER{alpha} and ERß appear to be lower at 5 days of age, but this difference is also less apparent at 12 and 19 days of age (B)

To correlate the observed changes in ER transcript levels to changes in protein levels, Western blot and immunohistochemistical (IHC) analysis for ER{alpha} and IHC for ERß were carried out on Day 19. In agreement with the genistein-induced increase in ER{alpha} mRNA levels shown in Figure 2, a correlating increase in ER{alpha} protein in the ovary was indicated by Western blot (Fig. 3A). As expected, ER{alpha} immunoreactivity was easily detectable and localized to the interstitial and thecal cells, and was not detectable in granulosa cells of the ovary in control mice (Fig. 3, B and C). Although ER{alpha} was localized in the interstitial and thecal cells of genistein-treated mice as in controls, IHC demonstrated a striking induction of expression of ER{alpha} in the granulosa cells (Fig. 3, D–G). Once again, as with the mRNA, the strongest induction of ER{alpha} protein was observed in the Gen-1 and Gen-10 dose groups (Fig. 3, D and E). The highest treatment group (Gen-100) also showed ER{alpha} expression in the granulosa cells, but the pattern of staining appeared to be more mosaic, with some cells staining darker than others (Fig. 3H).



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  		FIG. 3. A) ER{alpha} Western blot of nuclear protein from ovaries collected at 19 days of age. An increase in ER{alpha} protein appears following the lowest dose of genistein (lane 2) compared with controls (lane 1). An apparent decrease in ER{alpha} appears in the highest dose of genistein (lane 4) compared with controls. Lane 1, control; lane 2, Gen-1; lane 3, Gen-10; lane 4, Gen-100. B–G) ER{alpha} immunohistochemistry on CD-1 Day 19 ovaries following neonatal exposure to genistein. B and C) Controls, D and E) Gen-1, F) Gen-10, G) Gen-100. ER{alpha} can be seen predominantly in the interstitial and thecal cells of the ovary in control animals. For genistein-treated mice, ER{alpha} can be seen in the interstitial, thecal, and granulosa cells. A multioocyte follicle can be seen in the Gen-10 and Gen-100 photographs. Bar = 100 µm

Immunohistochemistry detection of ERß in control and genistein-treated mice indicated strong expression in the granulosa cells at all ages examined, which is in agreement with previous reports (Fig. 4, A and B). With the exception of a slight increase in ERß immunoreactivity in the Gen-1 group (Fig. 4, C and D), no obvious changes in the localization of ERß were observed.



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  		FIG. 4. ERß immunohistochemistry on CD-1 Day 19 ovaries following neonatal exposure to genistein. A and B) Control, C and D) Gen-1, E) Gen-10, F) Gen-100. ERß can be seen predominantly in granulosa cells of the ovary in control animals. For genistein-treated mice, a slight increase in ERß can be seen in granulosa cells. A multioocyte follicle can be seen in the Gen-10 and Gen-100 photographs. Bar = 100 µm

Genistein Induction of ER{alpha} in Granulosa Cells Is Not ERß-Mediated

As described earlier, genistein is known to possess both ER-mediated estrogenic activity and ER-independent tyrosine-kinase inhibitory properties. To gain insight into the pathway by which genistein elicits ectopic induction of ER{alpha} in the granulosa cells of the maturing ovary, we carried out two experiments. The first study involved the treatment and evaluation of mice lacking either ER{alpha} ({alpha}ERKO) or ERß (ßERKO) to determine whether this effect was due to the estrogenic properties of genistein and, if so, which receptor is involved. Wild-type (C57BL/6), {alpha}ERKO, and ßERKO animals were exposed to genistein as neonates and evaluated at 19 days of age, similar to those studies on CD-1 mice described above. The C57BL/6 wild-type and ßERKO mice treated with vehicle exhibited the same ER expression pattern as the CD-1 controls described earlier (i.e., ER{alpha} being localized to the interstitial and thecal cells and ERß being limited to the granulosa cells; data not shown). Also in agreement with the results in the CD-1 mice, wild-type C57BL/6 mice exposed to genistein exhibited the induction of ER{alpha} in the granulosa cells (Fig. 5A). It is interesting that genistein induction of ER{alpha} in granulosa cells was preserved in ßERKO mice (Fig. 5B), indicating that this effect of genistein exposure is not dependent on the presence of functional ERß. Because {alpha}ERKO mice lack immunoreactivity for ER{alpha} [37], we are not able to evaluate the role that ER{alpha} may have in this genistein effect; however, we can report that neonatal genistein exposure had no effect on ERß expression in the granulosa cells of the {alpha}ERKO ovary.



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  		FIG. 5. ER{alpha} immunohistochemistry at 19 days of age in genistein-treated ßERKO mice and lavendustin A-treated CD-1 mice. A) Wild -type Gen-10, B) ßERKO Gen-10, C) lavendustin 1 µg/pup/day, D) lavendustin 10 µg/pup/day. Note the localization of ER{alpha} in granulosa cells in all panels

With evidence that genistein induction of ER{alpha} in granulosa cells was not ERß-dependent, we undertook a second study to evaluate the possibility that this effect may be attributed to the tyrosine-kinase inhibitory action of genistein. In this experiment, neonatal CD-1 mice were exposed to either genistein as before, or lavendustin A, a specific tyrosine-kinase inhibitor with no documented estrogenic activity [31, 33]. Ovaries from these mice were collected and evaluated at 19 days of age for ER{alpha} immunoreactivity. As shown in Figure 5, C and D, ovaries from mice exposed to the lower dose of lavendustin A exhibited no apparent differences in ER{alpha} expression or localization when compared with those of control animals. In contrast, ovaries from animals exposed to the high dose of lavendustin A exhibited an appreciable induction of ER{alpha} immunoreactivity in the granulosa cells, although not as robust as that observed in the genistein-treated mice described above (Fig. 5D). The lavendustin A treatment had no apparent effect on ERß levels in the ovary (data not shown). Therefore, the genistein induction of ER{alpha} in granulosa cells is independent of functional ERß and may be in part attributed to the tyrosine-kinase inhibitory property of genistein.

Genistein Induction of Multioocyte Follicles in the Ovary

Ovaries from genistein-exposed mice exhibited no obvious gross changes compared with those of control animals. However, microscopic evaluation indicated a remarkable induction of MOFs at 19 days of age in animals exposed to genistein as neonates (Table 1). In contrast, among a total of eight control mice, no MOFs were observed at any age examined. Furthermore, a dose-related increase in the incidence of MOFs was observed, with 75% of those females exposed to the highest dose of genistein exhibiting at least one MOF. Along with a dose-related increase in the number of animals exhibiting MOFs was a corresponding increase in the number of MOFs per ovary (Table 1). In fact, an ovary of the Gen-100 group exhibited eight MOFs in a single section (not shown). An example of a section of one of the genistein-treated ovaries can be seen in Figure 6, which shows a number of MOFs in the same section, as well as example of a triovular follicle. Also observed in genistein-exposed animals at 19 days of age was a higher incidence in atretic intermediate and large follicles in the Gen-10 group compared with controls (4.5 ± 0.4 per ovary section in controls vs. 9.1 ± 1.0 in the Gen-10 group; Gen-1 group 5.6 ± 0.3).


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  		TABLE 1. Incidence of multioocyte follicles following neonatal treatment with genistein or lavendustin A



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  		FIG. 6. Morphology of MOFs following neonatal genistein treatment. Shown is a representative ovary (C57/Bl6) at 19 days of age following neonatal treatment with genistein 100 µg/pup/day, illustrating the presence of several MOFs (asterisks). Shown in the inset is an example of three oocytes within a single follicle, with the nuclei of two of these being clearly visible

Multioocyte follicles most often possessed two distinct germ cells, but animals exposed to the highest genistein dose exhibited MOFs with as many as four oocytes in a single follicle. Oocytes of MOFs were consistently similar in size and each appeared relatively healthy when viewed with light microscopy. Multioocyte follicles appeared most similar to preantral-stage follicles, possessing multiple layers of granulosa cells and an intact theca. No indications of a disruption in the basement membrane of the MOF were observed.

Genistein Induction of Multioocyte Follicles in the Ovary Is ERß-Mediated

To gain insight into the pathway by which genistein exposure results in the appearance of MOFs in the ovary, we once again evaluated ovaries from genistein-exposed {alpha}ERKO and ßERKO mice as well as CD-1 mice exposed to lavendustin A. In contrast to CD-1 mice, a small percentage of wild-type C57BL/6 mice treated with vehicle only exhibited MOFs, indicating this to be an underlying phenotype in this strain, as previously reported [38]. However, increasing doses of neonatal genistein exposure led to similar increases in the occurrence of MOFs in wild-type C57BL/6 mice, with animals of the Gen-10 and Gen-100 groups exhibiting an 82% and 100% incidence, respectively (Table 1). Also similar to the results in CD-1 mice was a higher frequency of MOFs per ovary with the higher genistein doses, as one C57BL/6 female of the Gen-50 000 group exhibited 10 MOFs in a single ovarian section. A similar dose-response to genistein-induction of MOFs was exhibited in exposed {alpha}ERKO mice, indicating this effect of genistein to be independent of functional ER{alpha}. In contrast, the ovaries of genistein-exposed ßERKO mice indicated a dramatic decrease in the incidence of MOFs, with only 1 of a total of 12 treated mice exhibiting at least one MOF. Females exposed to either dose of lavendustin A exhibited no incidence of MOFs (Table 1). Therefore, induction of MOFs by neonatal genistein exposure appears to be due to an ERß-mediated mechanism rather than inhibition of tyrosine-specific kinases.

Evaluation of Genistein on Ovarian Function by Superovulation

To gain some insight of the effect that neonatal genistein exposure may have on the functional capacity of the ovary, control and neonatally exposed mice of 22–23 days of age were superovulated and evaluated for oocyte yield. As shown in Table 2, we observed divergent effects of genistein exposure among the three doses used. Whereas females exposed to the two highest doses of genistein exhibited a below normal oocyte yield (not statistically significant from controls), females exposed to the lowest dose of genistein exhibited a statistically significant increase (P < 0.05) in the number of ovulated oocytes. Table 2 shows the combined results of two separate superovulation trials, both of which produced similar results.


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  		TABLE 2. Number of oocytes ovulated following superovulation in mice treated neonatally with genistein


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the current study, we evaluated a series of effects of neonatal genistein exposure on the maturing mouse ovary that may be categorized as follows: a biochemical effect as the stimulation of ectopic expression of ER{alpha} in granulosa cells, a morphological effect as the induction of MOF in the ovary, and a functional effect as the altered ovarian response to superovulation treatment. Furthermore, through the use of ERKO mice and the nonestrogenic tyrosine-kinase inhibitor, lavendustin A, we have determined that these effects rely on more than one response mechanism of genistein. Whereas the induction of MOFs in the ovary appears to be an ERß-mediated effect of genistein, the stimulation of ectopic ER{alpha} expression in granulosa cells may be attributed to tyrosine-kinase inhibitory actions of genistein. Furthermore, the two effects of neonatal genistein exposure on the ovary are not directly related, as illustrated by the ability of genistein to induce MOFs in the {alpha}ERKO ovary.

In agreement with previous reports, we have shown that transcripts for both ER{alpha} and ERß are detectable in the prepubertal mouse ovary, and expression of each is limited to the thecal/interstitial and granulosa cells, respectively [17, 18]. Furthermore, ERß is clearly the predominant form expressed during the neonatal period and increases more than 2-fold between the ages of 5 and 12 days, similar to findings in the rat ovary [39], whereas ER{alpha} expression in the ovary remains low and relatively stable during this same period in mice (as shown herein) and rats alike [39]. Genistein exposure had no discernible effect on ERß expression levels or pattern in the maturing ovary. These findings are similar to that of Ikeda et al. [40] in which neonatal treatment of mice with estradiol benzoate also had no effect on ERß expression [40]. In contrast, we observed a dramatic induction of ER{alpha} expression in granulosa cells following neonatal genistein exposure. We found it interesting that the greatest rise in ER{alpha} mRNA levels was the 3-fold increase seen at Day 5 (Gen-1 dose), within 24 h of the final genistein treatment, whereas the peak level of ER{alpha} immunoreactivity in granulosa cells was not observed until 19 days of age. Such a delay between the appearance of mRNA and detectable protein levels has been shown for other transcripts during oogenesis and spermatogenesis [41, 42].

Our laboratory recently reported a similar delay in the ontogeny of ERß in the developing testis of the mouse [18]. It was interesting that genistein induction of ER{alpha} expression in granulosa cells exhibited an inverse dose response in that the most abundant expression of ER{alpha} was observed in the ovaries of mice exposed to the lowest dose of neonatal genistein. However, this dose-response relationship is not atypical in the study of xenoestrogen effects, as similar findings of low-dose effects on other reproductive parameters following prenatal or neonatal exposure to estrogenic compounds such as DES, bisphenol A, and methoxychlor have also been reported [43–46].

The mechanism by which neonatal genistein exposure results in the stimulation of ER{alpha} expression in granulosa cells remains to be fully elucidated. Transcriptional regulation of the ER{alpha} gene is not well understood [47]. Recent studies have shown that regulatory sequences of the human and rodent ER{alpha} genes are highly complex units, possessing as many as seven exons in the 5'-untranslated sequences [47]. Kos et al. [48] recently reported that the mouse ER{alpha} gene is transcribed from at least five distinct promoters, all of which result in transcripts encoding the same functional 66-kDa protein. In addition, several studies have reported positive autoregulation of the ER{alpha} promoter via ligand-bound ER{alpha} in vitro [49–51], and have shown that multiple partial estrogen response elements within the human ER{alpha} promoter allow for ER binding and up-regulation of receptor expression [49]. Therefore, it is plausible that genistein induction of ER{alpha} expression in granulosa cells observed here is mediated via the activation of ER{alpha} on its own promoter, or perhaps via ERß acting on the ER{alpha} promoter. However, neither 17ß-estradiol nor DES have been reported to cause a similar induction of ER{alpha} expression in granulosa cells of the maturing ovary, arguing against the possibility of a direct receptor-mediated effect of genistein on the ER{alpha} promoter. Furthermore, ovaries of the maturing ßERKO female were also susceptible to genistein induction of ER{alpha} expression in granulosa cells, indicating that the underlying mechanism is not dependent on functional ERß. Because {alpha}ERKO mice by definition lack functional ER{alpha}, we were not able to evaluate the role that ER{alpha} may have in autoregulation.

Based on our findings that induction of ER{alpha} in granulosa cells was unique to neonatal genistein exposure, we hypothesized that this effect may be due to genistein's tyrosine-kinase inhibitory properties. We therefore treated neonatal females with lavendustin A, a specific tyrosine-kinase inhibitor that possesses no known estrogenic activity. We found it interesting that treatments with lavendustin A also resulted in an induction of ER{alpha} expression in granulosa cells, although the level of ER{alpha} immunoreactivity was not as robust as that seen following genistein treatment. The reasons for the less dramatic effect of lavendustin A are unknown at this time. Because the inhibitory actions of both genistein and lavendustin A on tyrosine-specific kinases have been mostly characterized under in vitro conditions, it is difficult to determine the optimum dose required to achieve a similar effect in vivo for either compound. Certainly, the pharmacokinetics of the drugs and therefore the active amounts delivered to the ovary are likely to differ. Furthermore, because genistein has been shown to preferentially bind ERß, it is possible that this interaction may allow for a high concentration of genistein within ovarian granulosa cells, endowing ERß with the role of a chaperone for genistein rather than an active partner. Nonetheless, the induction of ER{alpha} expression in granulosa cells of the prepubertal ovary by both genistein and the nonestrogenic lavendustin A strongly indicates this effect to be related to the inhibition of tyrosine-specific kinases within these cells.

As discussed, ER{alpha} expression in ovarian granulosa cells is low to undetectable during folliculogenesis in the mature ovary. However, ER{alpha} expression rapidly increases following ovulation in luteinizing granulosa cells undergoing terminal differentiation to form the corpus luteum, with a coincidental decrease in ERß [14, 52]. These data indicate that ER{alpha} is the predominant receptor form involved in mediating estrogen actions in the rodent corpus luteum. Therefore, a possible mechanism for the induction of ectopic expression of ER{alpha} in granulosa cells by genistein may be via the stimulation of signaling pathways involved in the normal induction of ER{alpha} expression in the corpus luteum. Recent studies have shown that induction of ER{alpha} expression in rat corpus luteum and in primary cultures of luteinizing cells is highly dependent on prolactin signaling via the prolactin receptor [52, 53]. However, Frasor et al. [53] have recently shown that prolactin induction of ER{alpha} expression in luteinizing granulosa cells occurs via the intracellular Jak2/Stat5 pathway, which is highly dependent on a cascade of tyrosine phosphorylation events. Therefore, it might be expected that genistein would in fact block prolactin signaling via its ability to specifically inhibit tyrosine kinase activity. Still, alternate prolactin signaling pathways involving Src kinases, mitogen-activated protein kinases, and protein kinase C, have been reported [54] and may not be as susceptible to inhibition by genistein or lavendustin A.

An indirect mechanism by which neonatal genistein exposure may alter prolactin signaling in the ovary may be via the induction of alterations in the hypothalamic-pituitary axis. Although the pituitary lactotroph is a well-characterized estrogen target cell, the neuroendocrine mechanisms that regulate lactotroph function do not fully mature until after birth in the rodent [55], making these tissues highly susceptible to insults from xenoestrogen exposure. Neonatal exposure of female mice to DES has been shown to result in hyperprolactinemia in the weeks just following treatment [56, 57]. More recently, Khurana et al. [58] has shown that rats exposed to low doses of the xenoestrogens octylphenol and bisphenol A also exhibit hyperprolactinemia as early as 25 days of age. Although similar in vivo data for genistein have not yet been reported, Stahl et al. [59] recently demonstrated that genistein is able to stimulate cell proliferation and prolactin synthesis in PR1 cells, a pituitary-tumor derived cell line. These same studies showed that genistein was as potent as estradiol in stimulating prolactin synthesis, and this action of both hormones was inhibited by the complete ER antagonist, ICI-182,780, strongly indicating a common ER-mediated mechanism [59]. Therefore, it is conceivable that neonatal genistein exposure in mice has resulted in premature increases in serum prolactin levels, which in turn may have a luteotropic action on the granulosa cells of the ovary, including the induction of ER{alpha} expression.

An interesting consequence of the induction of ER{alpha} in granulosa cells following neonatal genistein treatment is the presence of both ER forms within the same cell type. This is in contrast to the natural expression pattern exhibited by most tissues in which ER{alpha} and ERß are limited to separate compartments of a tissue, with the exception of certain regions of the brain [60]. Therefore, this unique effect of genistein, resulting in coexpression of both ER forms, introduces the possibility of ER-heterodimer actions within the cell. ER-heterodimer formation has been shown to be possible for both the human [61] and mouse [62] receptor forms in vitro. These studies invariably agree that the ER-heterodimer behaves as a positive transcription factor in vitro, most often exhibiting transactivational activity that is reduced relative to the ER{alpha} homodimer, but above that of the ERß homodimer [61, 62]. However, the existence of the ER heterodimer complex and its role in estrogen signaling in vivo remains to be substantiated. Perhaps the model described in this paper could be used as a unique opportunity to study the in vivo consequences of ER{alpha}/ERß heterodimers and the possible alternate gene transcription produced through these complexes as opposed to the respective homodimers.

Another observed effect of neonatal genistein exposure was the induction of MOFs in the ovary. This effect exhibited the more typical dose-response curve, in contrast to the induction in ER{alpha} expression discussed above, suggesting that the underlying mechanisms likely differ. It has long been recognized that prenatal and neonatal exposure to DES also results in the appearance of MOFs in mice [38, 63, 64], yet the mechanism remains unclear. Iguchi et al. demonstrated the neonatal ovary to be uniquely susceptible to this effect of DES relative to the adult ovary; this effect was specific to estrogen action and was not induced by similar treatments with progesterone or testosterone, when combined with an aromatase-inhibitor [65, 66]. Furthermore, this effect of DES appears to be directly on the ovary, because neonatal ovaries exposed to DES in culture and then transplanted to adult hosts exhibit MOFs [66]. In this study we furthered these findings by demonstrating that ovaries of ßERKO females are completely resistant to this effect of genistein, whereas {alpha}ERKO mice exhibited an incidence of MOF comparable to wild-type mice following genistein exposure. The lack of MOFs in animals treated with lavendustin A indicates that inhibition of tyrosine-specific kinases does not likely play a role in this effect of genistein. Therefore, these data strongly indicate that ERß is critical to this action of genistein on the maturing mouse ovary.

It is interesting that MOFs in the mouse ovary does not appear to be unique to estrogen exposure. Recent descriptions of mice lacking GDF-9 or BMP-15 [67], both oocyte-secreted growth factors, or preliminary data from mice lacking IRS-1 [68], which is a member of the insulin and insulin growth factor signaling pathway, exhibit a higher incidence of MOFs. Also, mice that overexpress the inhibin-{alpha} gene [69] exhibit a higher incidence of MOFs. Therefore, the mechanisms underlying the development of MOFs are obviously complex and involve multiple pathways. It is likely that MOFs are the result of a failure of primary follicular cells to separate primordial oocytes during the initial stages of follicle organization. Perhaps genistein acts via ERß to alter the proper granulosa cell response to the oocyte-secreted growth factors mentioned above, or it may alter the expression of proteases or adhesive proteins involved in oocyte-granulosa cell interaction. Although the reported incidence of MOFs in humans ranges from 24% [70] to 85% [71] depending on the study, both reports agree that the proportion of total follicles in the human ovary that are multioocyte is less than 1%.

The final effect of neonatal genistein exposure on the maturing mouse ovary observed in this study was found following superovulation of exposed females with exogenous gonadotropins. It was interesting that mice treated with the lowest dose of genistein as neonates exhibited a greater ovulatory capacity compared with untreated controls, whereas animals treated with higher doses yielded fewer ovulated oocytes. The underlying mechanism for this biphasic effect of genistein is unclear. Because both the {alpha}ERKO and ßERKO models exhibit innate reductions in ovulatory efficiency following superovulation [34], these animals were not included in this portion of the study. It is interesting, however, to note that the genistein dose that induced the greatest levels of ER{alpha} expression in granulosa cells also produced the greatest number of oocytes following superovulation. Estrogen action is known to be an antiatretic factor in the ovary [72] and clearly facilitates ovulation as demonstrated by the ERKO mice. Therefore, perhaps the elevated expression of ER{alpha} within the granulosa cells has allowed for a greater number of follicles available for ovulation during the gonadotropin treatment. Iguchi et al. [66] showed that superovulation of mice following neonatal exposure to DES resulted in a decrease in the number of MOFs in the ovary, speculating that perhaps a portion of the MOFs were ovulated, although assays to determine this were not carried out. The possibility exists that a greater number of oocytes were present in the oviduct of the Gen-1 group because of the rupture of MOFs; however, our studies indicated that females exposed to the higher doses of genistein actually possessed a greater number of MOFs but a reduction in oocyte yield following superovulation, which suggests that this is not a likely possibility.

Because mice exposed to the lowest dose of genistein are ovulating more oocytes following stimulation, it is possible that these animals exhibit a premature depletion of oocytes, resulting in lower fertility earlier in life. In fact, our laboratory has shown that mice exposed to low doses of DES prenatally exhibit an greater number of corpora lutea at 2 mo of age with a subsequent decrease by 6 mo of age relative to controls [35]. Studies are currently underway in our laboratory to determine the effects of neonatal genistein exposure on fertility and the possibility that these mice will exhibit infertility at an earlier age than their control counterparts.

In summary, we have shown that neonatal genistein exposure produces multiple effects on the morphology and function of the mouse ovary. Furthermore, we have begun to elucidate the mechanisms by which genistein elicits such effects. The ectopic induction of ER{alpha} expression in the granulosa cells of the ovary appears to be associated more with the tyrosine-kinase inhibitory properties of genistein rather than its estrogen actions, although indirect effects secondary to estrogenization of the hypothalamic-pituitary axis cannot be ruled out. In contrast, the induction of MOFs in the ovary, which appears to be a direct effect and unrelated to the changes in ER{alpha} expression, is dependent on the presence of functional ERß within the ovary. Future investigations into the mechanisms of the diverse effects of genistein will prove invaluable in evaluating the possible effects of phytoestrogens on reproductive function.


   ACKNOWLEDGMENTS
 
We thank Dr. David Schomberg and Dr. Diane Klotz for their critical review of the manuscript. We also thank Norris Flagler (NIEHS) for expert photomicroscopy.


   FOOTNOTES
 
1 Correspondence: Retha R. Newbold, Mail Drop E4-02, NIEHS, 111 Alexander Drive, South Campus, P.O. Box 12233, Research Triangle Park, NC 27709. FAX: 919 541 4634; newbold1{at}niehs.nih.gov Back

Received: 11 March 2002.

First decision: 26 March 2002.

Accepted: 26 April 2002.


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 RESULTS
 DISCUSSION
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