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Research Article |
Developmental Endocrinology and Endocrine Disruptor Section,3 Laboratory of Molecular Toxicology, National Institute of Environmental Health Sciences, NIH/DHHS, Research Triangle Park, North Carolina 27709
Department of Environmental and Molecular Toxicology,4 North Carolina State University, Raleigh, North Carolina 27605
Department of Biology,5 Syracuse University, Syracuse, New York 13210
ABSTRACT
Early in ovarian differentiation, female mouse germ cells develop in clusters called oocyte nests or germline cysts. After birth, mouse germ cell nests break down into individual oocytes that are surrounded by somatic pregranulosa cells to form primordial follicles. Previously, we have shown that mice treated neonatally with genistein, the primary soy phytoestrogen, have multi-oocyte follicles (MOFs), an effect apparently mediated by estrogen receptor 2 (ESR2, more commonly known as ERbeta). To determine if genistein treatment leads to MOFs by inhibiting breakdown of oocyte nests, mice were treated neonatally with genistein (50 mg/kg per day) on Days 1–5, and the differentiation of the ovary was compared with untreated controls. Mice treated with genistein had fewer single oocytes and a higher percentage of oocytes not enclosed in follicles. Oocytes from genistein-treated mice exhibited intercellular bridges at 4 days of age, long after disappearing in controls by 2 days of age. There was also an increase in the number of oocytes that survived during the nest breakdown period and fewer oocytes undergoing apoptosis on Neonatal Day 3 in genistein-treated mice as determined by poly (ADP-ribose) polymerase (PARP1) and deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end-labeling (TUNEL). These data taken together suggest that genistein exposure during development alters ovarian differentiation by inhibiting oocyte nest breakdown and attenuating oocyte cell death.
developmental biology, environment, mechanisms of hormone action, ovary
INTRODUCTION
During normal ovarian differentiation, female germ cells are found as clusters or nests of cells. Recently, these nests were found to have characteristics of germline cysts found in female invertebrates and almost all male animal species [1, 2]. Cysts are formed when oogonia undergo a series of incomplete cell divisions shortly after their formation, resulting in clusters of connected cells that subsequently enter meiosis and are called oocytes [1]. After birth, mouse germline cysts or nests break down into individual oocytes (nest breakdown) that become surrounded by somatic pregranulosa cells to form primordial follicles [3]. During the process of nest breakdown, a subset of oocytes in each nest dies with only a third of the initial number of oocytes surviving [3]. The key observation that a subpopulation of oocytes of the nest die suggests that there are two cell types in the germ cell nests: 1) cells that survive, differentiate, and become functional oocytes and 2) cells that support or protect the oocytes and eventually die.
Selective cell death of some oocytes in the nest may involve regulation of cell survival and/or programmed cell death pathways. Steroid hormones have been implicated in the regulation of cell death in many tissues [4–10]. In the adult mammalian ovary, where many follicles undergo atresia (cell death), estrogen has been shown to protect granulosa cells from death [11]. In contrast, in the nervous system, there is evidence that estrogen promotes cell death [12]. It is currently not known if estrogen treatment regulates the number of oocytes that die during nest breakdown.
Estrogenic compounds can induce a variety of effects on reproductive organs, including abnormal morphology and neoplasia. One effect is the appearance of multioocyte follicles (MOFs) in the adult ovary [13]. In contrast to normal ovaries, ovaries from adult female mice treated as neonates with the major endogenous estrogen 17ß-estradiol (E2) or the synthetic estrogen diethylstilbestrol (DES) have an increased occurrence of follicles with more than one oocyte [14–16]. Although this effect of estrogenic compounds has been known for many years, the origin of MOFs remains unknown. One possibility is that estrogen treatment during the time of neonatal ovarian differentiation results in oocyte nests that persist and become surrounded by granulosa cells, resulting in MOFs in the adult ovary. Alternatively, nest breakdown may occur normally, but the migration of granulosa cells around individual oocytes may be affected. A third possibility is that two or more follicles fused together. If MOFs are the result of incomplete nest breakdown induced by estrogens, other chemicals that exert estrogenic activity could also cause this effect.
Genistein, a soybean phytoestrogen, has previously been shown to have estrogenic activity in both in vitro and in vivo studies in mice and rats [17–21]. The estrogenic activity of this compound is of concern because human fetuses and infants can be exposed to genistein during critical periods of development through soy consumption of mothers during pregnancy and lactation [22] and through soy-based infant formulas and other soy products that children consume [23–25]. The concentrations of genistein and other isoflavones found in some of these soy-based products can far exceed the amount found in an adult diet. In fact, infants on soy-based infant formulas consume approximately 6–9 mg/kg per day of genistein compared to 1 mg/kg per day in an adult vegetarian diet [24]. Neonatal exposure to genistein at a dose of 50 mg kg–1 day–1 leads to an increased incidence of uterine adenocarcinoma in mice later in life [26]. In addition, the incidence of uterine tumors in the genistein treated mice (35%) was similar to the incidence in mice given an equal estrogenic dose of DES, 0.001 mg/kg per day (31%) [26]. The levels of genistein used in that study were subsequently shown to produce serum-circulating levels of genistein in mice similar to those found in infants consuming soy-based infant formulas [27]. Another study using rats verified these data by showing that a dose of 40 mg/kg per day also gave a serum circulating level of genistein similar to what is found in infants on soy-based formulas [28]. Recent studies have shown that developmental exposure to genistein can cause alterations in the development of the female reproductive tract of the rodent, including altered estrous cyclicity, altered ovarian function, subfertility, and infertility [29–32].
The effects of genistein on the developing ovary following neonatal exposure at doses of 0.5, 5, and 50 mg kg–1 day–1 included the presence of MOFs similar to those reported following neonatal exposure to E2 or DES [14–16, 33]. Another study exposing rats orally during perinatal life also showed the presence of MOFs supporting genistein's ability to alter ovarian morphology in another species [34]. Genistein has tyrosine kinase inhibitory activity in addition to its estrogenic activity [35]. However, mice treated with lavendustin (a tyrosine kinase inhibitor without estrogenic activity) did not develop MOFs, eliminating genistein's tyrosine kinase inhibitory action as a possible mode of action in forming MOFs [33]. Transgenic mouse models lacking estrogen receptor (ESR) 1 or 2 (more commonly known as ER
and ERß, respectively) have demonstrated that these receptor subtypes are the primary mediators of estrogen activity in both reproductive and nonreproductive tissues [36, 37]. Transgenic mice lacking Esr1 developed MOFs following neonatal genistein treatment, but mice lacking Esr2 did not show this phenotype, suggesting the effect of genistein is mediated specifically through ESR2 [33]. In addition, mice lacking ESR2 have been previously shown to have reduced fertility, more atretic follicles, and fewer corpora lutea, suggesting that some of the oocytes may not be healthy, and more oocytes are dying instead of being ovulated [36, 37]. ESR2 is expressed in ovarian granulosa cells of adult mice and has been detected as early as Day 1 in neonatal mice [38, 39]. All these data taken together suggest that ESR2 may play a critical role in normal ovarian differentiation and that chemicals with estrogenic activity, particularly those that bind preferentially to ESR2, may disrupt this process.
Previous studies of the effect of genistein on the ovary have examined immature and adult rodents, but nothing is known about the effects on younger mice. To further study the mechanisms involved in the formation of MOFs and possible disruption of the development of the ovary, the current study examines the effects of neonatal genistein treatment on ovarian differentiation, including oocyte nest breakdown, primordial follicle assembly, and follicle development.
MATERIALS AND METHODS
Animals and Neonatal Treatment
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 the National Institute of Environmental Health Sciences (NIEHS; Research Triangle Park, NC). Vaginal plug detection was considered Day 0.5 of pregnancy. Pregnant mice were housed under controlled lighting (12L:12D) and temperature (21–22°C) conditions. Mice were housed in polysulfone, ventilated cages (Technoplast, Inc., Exton, PA) and provided with National Institutes of Health (NIH) 31 laboratory mouse chow and fresh water ad libitum. The diet has been previously analyzed for genistein content [40]. All animal procedures complied with an approved NIEHS/NIH animal care protocol and the Syracuse University Institutional Animal Care and Use Committee.
Pregnant mice delivered pups at 19.5 days of gestation; pups were separated according to sex, pooled together, and then randomly standardized to eight female pups per litter. Male pups were used in another experiment. Female pups were treated on Days 1–5 with genistein (Sigma Chemical Company, St. Louis, MO) by subcutaneous injection at 50 mg/kg per day in corn oil (approximately 100 µg/pup per day) or left untreated as controls. This dose and route of exposure of genistein has been previously shown in our laboratory to induce a high incidence of MOFs and to produce serum circulating levels of genistein of 6.8 ± 1.4 µM [26, 33]; this level is similar to that seen in infants consuming soy-based infant formulas, 1–5 µM [24, 27].
Whole Mount Immunohistochemistry and Fluorescence Microscopy
Mice treated as described previously were killed on Days 2, 3, 4, 5, and 6 by decapitation (eight mice per treatment group per age). These ages were chosen because nest breakdown and primordial follicle assembly occur during the first several days after birth [3]. The reproductive tract was removed, and the ovaries were carefully dissected away from the remainder of the reproductive tract in cold PBS. Ovaries were fixed in 5% EM-grade paraformaldehyde in PBS for 1 h followed by several washes in 5% BSA, 0.1% Triton X-100 in PBS. Whole ovaries were immunostained as previously described [1, 3, 41]. The STAT3 (C20) antibody (Santa Cruz Biotechnology, La Jolla, CA) was used at a dilution of 1:500 [41]. Propidium iodide or Toto-3 (Molecular Probes, now part of Invitrogen, Carlsbad, CA) was used to label nuclei. As a negative control, immunohistochemistry was performed without primary antibody (data not shown). Samples were imaged on a Zeiss Pascal Confocal microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY).
Analysis of Oocyte Nest Breakdown, Primordial Follicle Assembly, and Follicle Development
Whole ovaries labeled with the germ cell marker STAT3 as described previously were examined for percent single oocytes relative to the total number of oocytes to assess oocyte nest breakdown as previously described [1, 3]. STAT3 has been shown to be a specific marker for germ cells [41]. Briefly, the number of individual oocytes relative to the number of oocytes in nests was determined by examining two regions per ovary. For each region, a single confocal section was examined. In addition, for each of these regions, a stack of 10 sections, 1 micron apart centered around the single section, was obtained. This stack of sections was used to determine if oocytes in the center section were associated with oocyte nests above or below the plane of focus. Seven to eight mice per treatment group per age were analyzed. For primordial follicle assembly and development, the number of each type of follicle per region was determined by examining four representative, confocal sections at least 20 µm apart and determining the total number of each type of follicle per region. Two regions were examined per ovary, and there were seven to eight mice per treatment group. For primordial follicle assembly, oocytes were considered unassembled if granulosa cells did not completely surround them (Fig. 1A). Follicles were classified as follows: primordial (oocyte surrounded by several granulosa cells with flattened nuclei; Fig. 1B), primary (oocyte surrounded by one layer of granulosa cells with cuboidal nuclei; Fig. 1C), or secondary (oocyte surrounded by more than one layer of granulosa cells).
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Determination of Germ Cell Number
The number of oocytes per region was determined by counting the number of oocytes in four representative, confocal sections at least 20 µm apart and determining the total number of oocytes per region. Two regions were examined per ovary. Ovaries from seven to eight mice per treatment group were analyzed on Postnatal Days 2, 4, and 6.
Transmission Electron Microscopy
Ovaries from control and genistein-treated mice at 4 days of age were dissected in cold PBS, fixed in 2.5% glutaraldehyde (Electron Microscopy Sciences, Fort Washington, PA) in Jones Phosphate Buffer (65 mM NaCl, 2.5 mM KCl, 3.5 mM NaH2PO4, 1.5 mM Na2HPO4, pH 7.2) and processed for transmission electron microscopy at the State University of New York Upstate Medical University Pathology department EM facility. Samples were postfixed in 1% OsO4, dehydrated through an ethanol series, equilibrated in propylene oxide, and embedded in Araldite 502 (Electron Microscopy Sciences). Thin sections were stained with uranyl acetate and lead citrate. For each data point, four sections from one ovary, each 20 µm apart, were examined. The samples were analyzed with a FEI Tecnai BioTwin 12 transmission electron microscope and images recorded with a AMT Advantage Plus CCD camera.
Cell Death Assessment
For poly (ADP-ribose) polymerase (PARP1) labeling, whole mount immunohistochemistry was performed on ovaries collected from control and genistein-treated mice on Days 1–6 (six to eight mice per treatment group per age). Whole ovaries were stained sequentially, first with STAT3 antibody followed by anti-rabbit Alexa 568 secondary antibody, to label all oocytes (as described previously) and then with antibody against the apoptosis-specific cleaved form of PARP1 directly labeled with Alexa 488 at a dilution of 1:100 (New England Biolabs, Ipswich, MA). The number of PARP1-positive oocytes was determined as a percent of the total number of oocytes in four representative, confocal sections in each mouse ovary at least 20 µm apart.
For terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL), ovaries were collected from control and genistein-treated mice on Days 2, 3, and 4 and fixed in cold 10% neutral buffered formalin (four mice per treatment group). Tissues were changed to cold 70% ethanol, embedded in paraffin, and sectioned at 6 µm. Sections were deparaffinized and rehydrated, and detection of apoptotic cells was carried out using the TUNEL apoptosis detection kit following the instructions provided in the kit (Upstate, Lake Placid, NY) with the following exceptions. The secondary antibody used was ExtrAvidin Peroxidase (Sigma) at a dilution of 1:50 for 30 min at room temperature followed by NovaRED following the kit instructions (Vector Laboratories, Burlingame, CA). The sections were then counterstained with hematoxylin, dehydrated, and coverslipped. A minimum of two nonadjacent sections from three mice per age per treatment group were stained and counted using this method. The percentage of apoptotic oocytes was determined, averaged per mouse, and then averaged for each treatment group for each age.
Statistical Analysis
A two-way ANOVA was conducted using main effects of hormone and day on percentage follicle type, percentage single oocytes, number of oocytes, percentage TUNEL-positive cells, and percentage PARP1-positive oocytes. PROC GLM of SAS 9.1 (SAS Institute Inc., Cary, NC) was used to calculate the least-squares means and test specific hypotheses for hormone effects on each day. A level of P < 0.05 was considered significant.
RESULTS
Primordial Follicle Assembly Was Disrupted Following Neonatal Genistein Treatment
To examine the effects of neonatal genistein treatment on primordial follicle assembly and development, we determined the number of unassembled oocytes and primordial and primary follicles in genistein-treated and control mice at 4 days of age using ovary whole mounts immunostained with STAT3 antibody and propidium iodide. The percent of unassembled follicles was significantly higher in the genistein-treated mice (73.4 ± 3.7%) compared to controls (56.7 ± 2.9%), while the percent of primordial and primary follicles was significantly lower in the genistein-treated mice (Fig. 1D). Thus, primordial follicle assembly was disrupted in genistein-treated mice.
Genistein Treatment Retards Oocyte Nest Breakdown
Although the rate of primordial follicle assembly was reduced following genistein treatment, it was not clear if this was due to a problem of granulosa cell migration around the oocytes or a deficiency in oocyte nest breakdown. To determine this, once again we examined ovary whole mounts immunostained for STAT3 and propidium iodide. Figure 2 shows a Day 4 control ovary (A–C) and a Day 4 genistein-treated ovary (D–F). The control ovaries had a very high percentage of single oocytes (44%), similar to previously reported data [1, 3]. In contrast, the genistein-treated ovaries had fewer single oocytes (21.2%), and several large oocyte nests were still apparent. A summary of the percentage of single oocytes in each treatment group can be seen in Figure 3. There were significantly fewer single oocytes at 4, 5, and 6 days of age following neonatal genistein treatment; the largest difference was at 6 days of age, where 57% of oocytes are single in control ovaries and only 36% of oocytes in the genistein-treated group were single. While the percentage of single oocytes is decreased in treated ovaries, the overall number of oocytes increases because of decreased apoptosis. Not only the percentage but also the actual number of single oocytes in the treated group decreased. These results support the idea that genistein inhibits the process of oocyte nest breakdown.
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To determine if the oocytes were still connected by intercellular bridges (as germline nests would be), we used transmission electron microscopy to measure the frequency of intercellular bridges connecting oocytes. In a previous study, the frequency of bridges declined rapidly in the CD-1 strain of mice shortly after birth, becoming virtually undetectable by 3 days of age [1, 3]. We did not detect any intercellular bridges in control mice at 4 days of age in this study (0 out of 325 oocytes). However, in genistein-treated mice, the frequency of bridges was 0.5% (3 out of 633 oocytes), supporting the idea that genistein treatment is inhibiting nest breakdown. Examples of electron micrographs from a Day 1 control ovary (Fig. 4A) and a Day 4 genistein-treated ovary (Fig. 4B) are shown.
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Neonatal Oocyte Death Was Reduced with Genistein Treatment
Using ovaries whole mount immunostained for STAT3 and propidium iodide, we counted the total number of oocytes on Postnatal Days 2, 4, and 6 (Fig. 5). We found significantly more oocytes at Postnatal Days 4 and 6 in treated mice, supporting the idea that genistein treatment increases oocyte survival. We counted oocytes in representative regions of ovaries; therefore, a change in ovary size would affect the total number of oocytes. However, we did not observe any differences in ovary size with genistein treatment that might impact the overall number of oocytes.
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Since genistein treatment led to increased oocyte numbers, we wanted to determine if genistein affected oocyte programmed cell death. To assess apoptosis, the percentage of PARP1-positive and TUNEL-positive oocytes were compared between control and genistein-treated mice throughout the treatment period. Representative ovary sections immunostained with PARP1 are shown in Figure 6B (Day 3 control) and Figure 6C (Day 3 genistein-treated), and representative ovary sections stained for TUNEL are shown in Figure 6E (Day 3 control) and Figure 6F (Day 3 genistein-treated). A summary of the percentage of PARP1-positive cells at 2, 3, and 4 days of age is shown in Figure 6A, and the percentage of TUNEL-positive cells at 2, 3, and 4 days of age is shown in Figure 6D. There were differences between control and genistein-treated ovaries using either method to detect cell death. The most significant difference was at 3 days of age, where there was a significant decrease in the percentage of oocytes undergoing apoptosis following neonatal genistein treatment as assessed by both PARP1 and TUNEL. In contrast, there was a significant increase in apoptosis using the TUNEL method at 2 days of age; however, this was not observed using PARP1 labeling. This suggests that genistein may initially induce or speed up the process of cell death of some oocytes. Since TUNEL and PARP1 measure apoptosis at different stages, this may explain the discrepancy between the two and further supports the idea that this effect is transient, occurring for only a brief time directly following the first treatment. These data, taken together with the overall increase in oocyte numbers at 4 and 6 days of age (Fig. 5), support the idea that genistein treatment influences oocyte survival.
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DISCUSSION
Previous studies have shown that neonatal exposure to the phytoestrogen genistein alters ovarian morphology later in life, including the presence of MOFs [33]. Several possibilities for the process by which these abnormal follicles form exist. The granulosa cells could improperly enclose more than one oocyte, or the process of oocyte nest breakdown could be disrupted. In addition, it is possible that two follicles could fuse. Therefore, we examined the differentiation of the ovary during the time of neonatal genistein treatment. The data from this study show that there is a much higher percentage of unassembled oocytes following genistein treatment as well as many fewer single oocyte follicles. In addition, there were still intercellular bridges connecting the oocytes in the genistein-treated mice, while none were apparent in the control mice by 4 days of age. This indicates that MOFs resulted from incomplete breakdown of oocyte nests, leaving the pregranulosa cells multiple oocytes to surround during the differentiation process.
The mechanism by which oocyte nest breakdown normally occurs is not fully understood. In addition, the mechanism by which genistein disrupts this process is not known, although ESR2 (ERß) has been implicated as having a role [33]. We propose that during normal ovarian differentiation, exposure of fetal oocytes to maternal estrogen keeps the oocytes in nests. Shortly after birth, the level of estrogen drops, and initiation of nest breakdown is triggered (Fig. 7). Therefore, nest breakdown is inhibited when neonatal oocytes are exposed to estrogenic compounds such as genistein. Previous work from others on the presence of MOFs following specific windows of exposure to DES supports this hypothesis; mice treated during the first few days after birth develop MOFs, while mice treated later than Day 10 do not [16]. In addition, mice treated prenatally with DES also had fewer MOFs than mice treated neonatally [42]. This work was also replicated in vitro by the same laboratory, suggesting that neonatal mouse ovaries are highly susceptible to forming MOFs in the presence of exogenous estrogens, while other stages of development are less sensitive [15, 16]. These data support our hypothesis that estrogen is responsible for maintaining oocyte nests early in development, but after oocyte nest breakdown has occurred, MOFs can no longer be formed. The fact that prenatal exposure to DES also does not induce many MOFs (although other abnormalities occur) also suggests that exogenous estrogen during pregnancy only mimics the normal process of inhibiting oocyte nest breakdown during this period of development.
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Neonatal treatment with testosterone and progesterone has also been found to result in increased MOFs [16, 43]. For testosterone, the effect is likely due to its conversion to estrogen because inhibition of this conversion process suppresses the effect of testosterone treatment [43]. In a recent study in rats, neonatal progesterone treatment was found to reduce primordial follicle assembly, while both progesterone and estrogen treatment reduced the transition from primordial to primary follicle in the initial wave of folliculogenesis [44]; however, oocyte nest breakdown was not specifically examined. This study also showed reduced neonatal oocyte programmed cell death in progesterone treated mice. Together these data suggest that the steroid hormone balance may also be important for proper ovarian differentiation and development.
Recent work examining aromatase (Cyp19a1)-deficient mice also contributes to our knowledge of the effects of estrogens on the developing ovary. Aromatase (CYP19A1) is the enzyme necessary for the conversion of testosterone into estrogen. Thus, aromatase-deficient mice lack endogenous estrogen. Adult female aromatase knockout mice have fewer primordial follicles than wild-type animals, and estrogen treatment of adults does not alleviate this effect, suggesting that estrogen may play a role earlier, during the formation of primordial follicles that occurs neonatally [45].
In the ovaries of normal adult female mice, follicles consist of one oocyte surrounded by one or more layers of granulosa cells; follicles with more than one oocyte are rarely found (less than 1% in the Swiss strain [46]). However, the percent of animals with MOFs as well as the percent MOFs per total follicles varies widely depending on mouse strain [15, 16]. These data suggest that genetic modifiers may play a role in ovarian differentiation, and in turn the activity of these modifiers may be altered by exogenous influences. MOFs have also been reported in 98% of human ovaries at a frequency of 0.6% to 2.44% per ovary and up to 8% of follicles collected for IVF [47, 48]. The MOFs observed in mice and humans have been postulated to be remnants of oocyte clusters that did not separate and become enclosed in follicles during neonatal primordial follicle assembly [15, 16, 47]. It is unclear whether genistein treatment alters oocyte quality. There is evidence in mice that oocytes derived from MOFs have a reduced fertilization rate [49], although in humans no difference has been found [48].
The molecular mechanisms by which genistein caused these effects on the developing ovary are just beginning to be elucidated. Previously, ESR2 (ERß) was found to be involved in the formation of MOFs present in immature mice treated with genistein [33]. In that study, immature mice lacking ESR2 did not have MOFs when treated with genistein as neonates. This finding is consistent with the hypothesis that genistein is acting as an estrogen through ESR2 to disrupt ovarian differentiation. ESR2 has been detected in the ovary early in development by RT-PCR, Western blotting, and immunohistochemistry [38, 39], suggesting the possibility that genistein acts directly on the ovary through ESR2. While ESR2 is expressed predominantly in the granulosa cells later in development, the expression of ESR2 in the oocytes themselves very early during development cannot be ruled out. In either case, genistein acts as an estrogen interacting with ESR2 to send a signal that ultimately inhibits oocyte nest breakdown. Further evidence for estrogen's direct effects on the developing ovary comes from an in vitro study showing that neonatal ovaries grown in culture and then transplanted into a host (under the kidney capsule) develop MOFs in the presence of estrogens [14].
Several other genes have been implicated in neonatal ovarian differentiation. For example, mice lacking bone morphogenetic factor 15 (Bmp15) or growth differentiation factor 9 (Gdf9) have an increased number of MOFs as well as other defects of ovarian differentiation [50]. Both proteins are members of the transforming growth factor beta (TGFß) superfamily; they are oocyte-secreted factors expressed early in ovarian differentiation [51]. In contrast, transgenic overexpression of another member of the TGFß family, inhibin alpha, disrupts normal ovarian development and ovaries exhibit MOFs [52]. Mice lacking a basic helix-loop-helix protein called factor in the germline alpha (FIG1A) lack the ability to form primordial follicles [53]. Therefore, it appears that there are many factors that contribute to proper differentiation of the ovary during the neonatal time period and that disruption of any of these appears to cause permanent alterations in ovarian morphology and possibly function later in life.
Another point of regulation in ovarian differentiation is the natural process of oocyte cell death. During oocyte nest breakdown, approximately two-thirds of the oocytes die by apoptosis [3]. We have shown in the current study that oocyte cell death is initially increased at 2 days of age using the TUNEL method, but this appears to be transient since the PARP1 method did not show this difference. PARP1 measures apoptosis earlier in the process, and by 24 h after the first treatment, there is no difference in the number of oocytes entering apoptosis in the genistein-treated ovaries compared to the controls. However, apoptosis is attenuated on Day 3 following neonatal genistein treatment by both detection methods, and there is also an increase in the overall total number of oocytes. This suggests that cell survival and/or cell death pathways are altered following neonatal genistein treatment. In addition, estrogens have been shown to alter survival, usually inhibiting cell death and enhancing cell survival [11, 12]. In the developing ovary, compelling evidence from two existing transgenic mouse strains supports the idea that the B-cell leukemia/lymphoma 2 (BCL2) family of proteins may be involved in regulating apoptosis during oocyte nest breakdown. Both mice overexpressing Bcl2 (a cell survival gene) and mice lacking Bax (a cell death gene) have increased numbers of oocytes during neonatal life [54, 55]. In addition, adult females with a targeted disruption of Bcl2 have fewer oocytes and follicles with either a degenerating oocyte or no oocyte at all [56].
Over the past few years, research on phytoestrogens, like genistein, has increased. There are mixed results suggesting some beneficial effects as well as some adverse effects, depending on the timing of exposure, dose level, and endpoint examined. Some studies show that exposure to genistein early in life prevents carcinogen induced mammary gland cancer [57, 58], while others show increased mammary gland cancer occurs following treatment during specific developmental windows [59]. Others have shown improved cholesterol synthesis rates of human infants consuming soy-based formulas [60]. On the other hand, vegetarian diets usually contain high levels of soy, and recent epidemiology reports have shown an association of a vegetarian diet during pregnancy with an increased incidence of hypospadias in the male offspring [61] and an increase in autoimmune disease and the use of allergy medicines in children fed soy-based infant formulas [62]. In addition to genistein, neonatal exposure to another environmental estrogen, bisphenol A, has been shown to cause MOFs in mice [42]. This finding further supports the idea that a compound's estrogenic activity can cause altered ovarian differentiation.
In conclusion, we have shown that genistein alters ovarian differentiation during neonatal development. Ovaries from neonatal mice treated with genistein have more oocytes not enclosed in follicles, more oocytes persisting in nests, and retention of oocyte intercellular bridges. Retention of intercellular bridges between oocytes also demonstrates that genistein inhibits oocyte nest breakdown in neonatal mice. In addition, neonatal genistein treatment influenced oocyte survival as shown by decreased oocyte apoptosis and increased oocyte numbers. All these data, taken together, show that ovarian differentiation is a complex and multifaceted process. Disruption in any of these pathways can lead to alterations in the normal progression of ovarian development and subsequent normal ovarian function.
ACKNOWLEDGMENTS
We thank Judith Emmen, John Vandenbergh, Gerda Breitwieser, Scott Erdman, and Eleanor Maine for helpful comments on the manuscript. We thank Maureen Barzca for assistance with electron microscopy. Special thanks to Tom Starmer, Raymond Collins, and Nancy Hughes for assistance with statistical analysis.
FOOTNOTES
1 This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences. 
2 Correspondence: Wendy Jefferson, NIEHS, 111 Alexander Dr., Research Triangle Park, NC 27709. FAX: 919 541 4634; jeffers1{at}niehs.nih.gov
Received: 21 July 2005.
First decision: 18 August 2005.
Accepted: 28 September 2005.
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E. E Nilsson, J. Stanfield, and M. K Skinner Interactions between progesterone and tumor necrosis factor-{alpha} in the regulation of primordial follicle assembly. Reproduction, December 1, 2006; 132(6): 877 - 886. [Abstract] [Full Text] [PDF]
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E. Padilla-Banks, W. N. Jefferson, and R. R. Newbold Neonatal Exposure to the Phytoestrogen Genistein Alters Mammary Gland Growth and Developmental Programming of Hormone Receptor Levels Endocrinology, October 1, 2006; 147(10): 4871 - 4882. [Abstract] [Full Text] [PDF]
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