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Effects of estrogenic compounds on neonatal oocyte development
Reproductive Toxicology 34 (2012) 51–56
Contents lists available at SciVerse ScienceDirect
Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
Effects of estrogenic compounds on neonatal oocyte development Jenna R. Karavan, Melissa E. Pepling ∗ Syracuse University, Syracuse, NY, United States
a r t i c l e
i n f o
Article history: Received 8 September 2011 Received in revised form 20 January 2012 Accepted 17 February 2012 Available online 3 March 2012 Keywords: Oocyte development Cyst breakdown Primordial follicle assembly Follicle development Estrogens
a b s t r a c t In the mouse, oocytes develop in germline cysts that undergo breakdown resulting in primordial follicles, consisting of a single oocyte surrounded by granulosa cells. During this process, approximately two-thirds of the oocytes die. Exposure of female mice to environmental estrogens can alter oocyte development, limiting the number of primordial follicles that can be used for reproduction. Here we asked whether exposure to synthetic estrogens, diethylstilbestrol, ethinyl estradiol and bisphenol A affected perinatal oocyte development. Neonatal mice were injected with a low or high dose of each compound on postnatal days (PND) 1–4 and ovaries analyzed on PND5. Cyst breakdown, oocyte survival and follicle development were altered. The percentage of single oocyte was reduced from 84% in controls to 50–75%. The oocyte number per section was increased from 8 to 12–16. Follicle activation was reduced with 62% primordial follicles in controls to over 80% in most cases. © 2012 Elsevier Inc. All rights reserved.
1. Introduction The incidence of reproductive disorders among women has increased significantly in the United States in recent years from 6.7 million (10% of the population) in 1995 [1] to 7.3 million (12% of the population) in 2002 [2]. Many female reproductive disorders stem from disruption of normal oocyte development. The entire population of oocytes is formed early in development when primordial follicles each consisting of one oocyte and several granulosa cells are assembled [3]. Prior to follicle formation the oocytes develop in germ cell cysts consisting of oocytes connected by intercellular bridges that form through division of oogonia followed by incomplete cytokinesis [4]. In the fetal mouse, the oocytes within these cysts begin to separate a few days before birth and become enclosed by granulosa cells [5]. During cyst breakdown many oocytes die and only one-third of the oocytes in these germ line cysts survive to become enclosed in primordial follicles [6]. This primordial follicle pool is incredibly important for fertility because it represents the total population of oocytes available to a female during her reproductive lifetime. Estrogens can affect neonatal oocyte development. Exposure of neonates to estrogenic compounds such as estradiol (E2 ) [7], phytoestrogens like genistein [8] or with synthetic estrogens including diethylstilbestrol (DES) [9] or bisphenol-A (BPA) [10] significantly increases the number of multiple oocyte follicles (MOFs) in ovaries
∗ Corresponding author at: Department of Biology, Syracuse University, 348 Life Sciences Complex, 107 College Place, Syracuse, NY 13244, United States. Tel.: +1 315 443 4541; fax: +1 315 443 2012. E-mail address: [email protected] (M.E. Pepling). 0890-6238/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.reprotox.2012.02.005
of exposed animals when they are examined as adults. The oocytes in a MOF are believed to be remnants of a germ cell cyst that did not properly separate and instead the oocytes became enclosed together in one follicle. Indeed, neonatal exposure to E2 or genistein reduced cyst breakdown [11,12]. These findings led to the proposal of a model where during normal development high levels of estradiol maintain oocytes in cysts [11]. Late in fetal development estradiol levels drop allowing oocytes to separate. When neonates are exposed to exogenous estrogens cyst breakdown is blocked and MOFs are formed. A similar model was proposed earlier where high levels of steroid hormones would prevent follicle assembly during gestation [13]. However, in that study only progesterone was found to block follicle assembly. However, while estradiol did not block follicle assembly, it did block follicle activation. The endocrine disrupting effects of three synthetic estrogens, DES, BPA and ethinyl estradiol (EE) have been investigated. DES was the first synthetic estrogen produced and many pregnant women were treated with DES in their first trimester during the 1940s–1970s in order to prevent miscarriage or spontaneous abortion [14]. This exposure affected both the pregnant mother that was treated as well as her offspring and caused detrimental effects on fertility as well as increased risks for several types of cancer. EE is one of the main active ingredients in many contraceptive pills. BPA is used to make polycarbonate plastic compounds and is found in baby bottles, water bottles and other household items and is also used as a lining in food and beverage containers. Studies have shown adverse affect of BPA on the reproductive tracts of males and females [15]. For example, in female mouse embryos, BPA treatment caused defects in meiosis [16]. The effects of DES, BPA and EE on cyst breakdown and primordial follicle assembly have not been thoroughly investigated. Kim and
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colleagues [17] found that in mice, neonatal DES treatment reduced follicle activation and also reduced the number of primordial follicles formed suggesting that follicle assembly was also reduced. In contrast, a study looking at the effects of neonatal BPA and DES exposure in rats found that while the number of primordial follicles was reduced it was due to an increased number of activated follicles [18]. Here we examine the effects of 2 different doses of DES, BPA and EE specifically on cyst breakdown and associated oocyte loss as well as on primordial follicle recruitment. 2. Materials and methods 2.1. Animals The CD1 outbred mouse strain was obtained from Charles River Laboratories (Wilmington, MA, USA). CD1 females were mated with males and checked daily for vaginal plugs. The morning a vaginal plug was detected was designated as 0.5 days post-coitum (dpc). Birth usually occurred at 19.5 dpc and was designated as postnatal day (PND) 1. Ovaries were collected from CD1 neonates at PND5 in 1× phosphate-buffered saline (PBS). 2.2. Whole-mount antibody staining of neonatal ovaries Ovaries were fixed in 5.3% EM grade formaldehyde in PBS (Ted Pella Inc., Redding, CA, USA) overnight at 4 ◦ C. The ovaries were then washed several times in 0.1% Triton X-100 in PBS (PT) and then in PT + 5% bovine serum albumin (BSA) at room temperature to block nonspecific binding. Immunostaining was performed as previously described [19]. Whole ovaries were labeled with STAT-3 (C20) antibody (Santa Cruz Biotechnology, La Jolla, CA, USA), diluted 1:500 in PT + 5% BSA. The secondary antibody, goat anti-rabbit Alexa 488 (Invitrogen, Carlsbad, CA, USA), was diluted 1:200 in PT + 5% BSA. Nuclei were labeled with propidium iodide (PI) (Molecular Probes) at 5 g/ml. Ovaries were mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA). 2.3. Confocal microscopy Ovaries were analyzed using an LSM 710 confocal microscope (Carl Zeiss Microimaging, Inc., Thornwood, NY, USA). The confocal images were taken by imaging two separate areas in each ovary, with four planar Images 20 m apart in each area to give a total of eight sections per ovary. The number of oocytes was determined by counting the number of oocytes in each image. Cyst breakdown was assessed by counting the number of single oocytes and comparing this to the number of oocytes that were still in cysts. In order to determine whether oocytes were in cysts or not, for each of the eight sections, a z-stack of images each 1 m apart was obtained with 5 images above the section and 5 images below the section being analyzed. This allowed us to determine whether an oocyte was part of a germline cyst above or below the plane of focus. Finally, follicle development was quantified in each of the eight sections by determining the stage of development of each follicle. The oocytes were classified as unassembled if they were not entirely surrounded by granulosa
cells and if they were in contact with another oocyte. Single oocytes enclosed in follicles fell into three categories: primordial, primary, or secondary. A single oocyte surrounded by flattened, crescent-shaped granulosa cells defined a primordial follicle. A primary follicle consisted of a single oocyte enclosed in one layer of cuboidal granulosa cells. Transitional follicles with some flattened granulosa cells and some cuboidal granulosa cells were also classified as primary follicles. An oocyte surrounded by multiple layers of cuboidal granulosa cells was classified as a secondary follicle. 2.4. Preparation and delivery of estrogenic compounds CD1 neonates were subcutaneously injected with a total volume of 50 l of either peanut oil (Wegmans Food Markets, Rochester, NY, USA) or a mixture consisting of an estrogenic compound dissolved in peanut oil. Three different estrogenic compounds were used: diethylstilbestrol (DES) (Acros Organics, Morris Plains, NJ, USA), ethinyl estradiol (EE) (Sigma–Aldrich, St. Louis, MO, USA), and bisphenol-A (BPA) (Sigma–Aldrich). Two concentrations of each compound were used: (1) 5 mg per kg of body weight per day (mg/kg/d) or 10 g per pup and (2) 50 mg/kg/d or 100 g per pup. An average pup weight was used to determine the chemical concentration given and was not adjusted for any increase in body weight during the 4 days of injections. Solutions of each compound were prepared by first dissolving in absolute ethanol (Pharmco-AAPER, Brookfield, CT, USA) then added to the peanut oil. The ethanol was allowed to evaporate before use. Neonates were injected daily from PND1 to PND4. On PND5, ovaries were collected and fixed for immunocytochemistry. 2.5. Measurement of programmed cell death For poly ADP-ribose polymerase (PARP) labeling, whole mount immunocytochemistry was performed on ovaries from mice treated on PND 1 and 2 with peanut oil or with 50 mg/kg/d DES, EE or BPA and collected on PND 3. Ovaries were labeled with PARP antibody (Abcam, Cambridge, MA, USA), diluted 1:100 in PT + 5% BSA. The whole mount antibody staining procedure was followed except nuclei were labeled with Toto-3 (Invitrogen) diluted 1:2000. 2.6. Statistical analyses of cyst breakdown, total number of oocytes, and follicle development One-way ANOVA was implemented in order to determine statistical significance using SPSS, version 17. The Tukey post hoc test was used to compare the control group with each treatment group to investigate effects on cyst breakdown, total number of oocytes, and follicle development with p values less than 0.05 considered significant.
3. Results 3.1. Cyst breakdown in mice injected with estrogenic compounds Estradiol as well as the phytoestrogen, genistein block cyst breakdown [11,12]. To determine if synthetic estrogens had
Fig. 1. Effects of estrogenic compounds on cyst breakdown. Percent single oocytes in mice injected with peanut oil and with two concentrations of DES, EE, or BPA from PND 1 to 4 and collected on PND5. Data are presented as the mean ± SEM. Asterisks indicate a significance difference in comparison to mice injected with peanut oil (one way ANOVA, p < 0.05; n = 8–12 ovaries per group).
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Fig. 2. Confocal sections demonstrating the effects of estrogenic compounds on cyst breakdown and oocyte number. (A)–(C) Confocal section of an ovary from a control pup injected with peanut oil from PND 1 to 4 and collected on PND5 labeled with (A) Stat3 antibody (green) to visualize oocytes, (B) propidium iodide (red) to visualize nuclei and (C) overlay. Most oocytes have separated and are individually enclosed in primordial follicles (arrowheads) though some small 2 cell cysts are still present (white circle) (D)–(F) confocal section of an ovary from a pup injected with 100 g DES from PND 1 to 4 and collected on PND5 labeled with (D) Stat3 antibody (green) to visualize oocytes, (E) propidium iodide (red) to visualize nuclei and (F) overlay. (G)–(I) Confocal section of an ovary from a pup injected with 100 g EE from PND 1 to 4 and collected on PND5 labeled with (G) Stat3 antibody (green) to visualize oocytes, (H) propidium iodide (red) to visualize nuclei and (I) overlay. (J)–(L) Confocal section of an ovary from a pup injected with 100 g BPA from PND 1 to 4 and collected on PND5 labeled with (J) Stat3 antibody (green) to visualize oocytes, (K) propidium iodide (red) to visualize nuclei and (L) overlay. All three chemicals delayed cyst breakdown resulting in large cysts (white circles). Scale bar, 20 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Effects of estrogenic compounds on oocyte number. Number of oocytes per section in mice injected with peanut oil and with two concentrations of DES, EE, or BPA from PND 1 to 4 and collected on PND5. Data are presented as the mean ± SEM. Asterisks indicate a significance difference in comparison to mice injected with peanut oil (one way ANOVA, p < 0.05; n = 8–12 ovaries per group).
similar effects on oocyte development, neonatal mice were exposed to DES, EE, or BPA and cyst breakdown was analyzed. All estrogens tested significantly reduced the percent of single oocytes except for the lower concentration of BPA (Fig. 1). Mice injected with peanut oil alone had 84.2% single oocytes. In mice treated with 10 g DES, the percent of single oocytes decreased significantly to 55.3% while the percent of single oocytes decreased slightly more to 50.8% in mice treated with the higher concentration of DES. Mice treated with the lower concentration of EE also had a significant reduction in the percent of single oocytes at 62.6% and treatment with the higher concentration was only slightly lower at 61.2% single oocytes. The percent of single oocytes decreased slightly to 75.4% with for the lower concentration of BPA but was not significant while the higher BPA concentration caused a significant drop to 68.6%. Fig. 2 shows representative confocal sections from control ovaries (A–C), and ovaries treated with the higher concentrations of DES (D–F), EE (G–I) and BPA (J–L). While the control ovaries still had some 2 cell cysts, the treated ovaries had not only 2 cell cysts but larger cysts as well. 3.2. Total number of oocytes in mice injected with estrogenic compounds During the perinatal period, normally, two thirds of the oocytes are lost as the cysts break apart into individual oocytes [6]. Previously, neonates injected with genistein were shown to have more surviving oocytes while estradiol injections had no effect on oocyte number [11,12]. To determine if synthetic estrogens affected oocyte survival, the total number of oocytes per confocal section was determined for mice injected with peanut oil alone and for mice injected with DES, EE, or BPA. The higher concentrations of DES and EE and both concentrations of BPA significantly increased oocyte survival as shown in Fig. 3. These treatment groups had 12.3, 12.5, 13.0, and 15.8 oocytes per confocal section, respectively, while mice injected with peanut oil had an average of 8.0 oocytes per section. Ovaries from neonates treated with 10 g DES/day and 10 g EE/day, had 8.8 and 10.2 oocytes per section respectively, which was not significantly different from controls (Fig. 3). To determine if the increase in oocyte number was due to a reduction in apoptosis, the percent of PARP positive oocytes was assessed in PND3 mice treated with peanut oil or with 50 mg/kg/d DES, EE or BPA on PND 1 and 2. PND3 was chosen because in our previous studies we found that programmed cell death was reduced
Fig. 4. Effects of estrogenic compounds on programmed cell death. Percent of PARP positive oocytes per section in mice injected with peanut oil or with 50 mg/kg/d DES, EE, or BPA on PND 1–2 and collected on PND3. Data are presented as the mean ± SEM. Asterisks indicate a significance difference in comparison to mice injected with peanut oil (one way ANOVA, p < 0.05; n = 6 ovaries per group).
at PND3 with genistein treatment [12]. DES, EE and BPA treated mice all had a decreased percentage of PARP positive oocytes (6.4, 6.9 and 8.1%) as compared to oil treated mice (11.9%) (Fig. 4). 3.3. Follicle development in mice injected with estrogenic compounds After follicles form, some start to develop immediately. To determine whether synthetic estrogen exposure could affect follicle development, the percent of primordial, primary and secondary follicles were determined in ovaries from mice injected with peanut oil or with DES, EE, or BPA. Control mice injected with peanut oil alone had 62.3% primordial follicles, 27.9% primary follicles, and 9.7% secondary follicles (Fig. 5). Exposure to the higher concentrations of DES and EE and both high and low concentrations of BPA significantly increased the percent of primordial follicles to 82.6%, 82.7%, 89.5% and 81%, respectively. Treatment with the lower concentration of DES increased the number of primary follicles to 37.8% and decreased the number of secondary follicles to 0%, and treatment with the higher concentration of DES decreased the percentage of primary follicles to 16% and the percentage of secondary follicles to 1.4%. However, these changes were not
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Fig. 5. Effects of estrogenic compounds on follicle development. Percent primordial, primary and secondary follicles in mice injected with peanut oil and with two concentrations of DES, EE, or BPA from PND 1 to 4 and collected on PND5. Data are presented as the mean ± SEM. Asterisks indicate a significance difference in comparison to mice injected with peanut oil (one way ANOVA, p < 0.05; n = 8–12 ovaries per group).
significant. A similar trend was observed for both concentrations of EE. Mice injected with the lower concentration of EE had 34.6% primary follicles and 0.8% secondary follicles. Mice injected with the higher concentration of EE had 16.9% primary follicles and 0.4% secondary follicles. While treatment with both concentrations of BPA did lower the percentage of primary follicles, this effect was significant only for the higher concentration of BPA, which had 10.5% primary follicles. Treatment with both concentrations of BPA also lowered the percentage of secondary follicles to 0.8% and 0%, but this effect was not significant in both instances. 4. Discussion This study finds that synthetic estrogens, DES, BPA and EE delay cyst breakdown and associated oocyte loss as well as follicle activation. Oocyte number increased and follicle activation decreased in a dose dependent manner with all three chemicals. The increase in oocyte number was associated with a decrease in programmed cell death. However, while treatment with each chemical reduced cyst breakdown, the higher dose had only a slightly greater effect for DES and BPA and no change at all for EE. It is not clear why the higher doses did not have a greater effect. It is likely that estrogens are only one of many factors controlling oocyte development and that other compensatory mechanisms contribute to normal cyst breakdown and associated oocyte loss. Normally, as cysts break down, some oocytes are lost. Here we found that synthetic estrogens reduced the number of oocytes lost. Previously, some estrogens and some modes of exposure have affected neonatal oocyte numbers in rodents while others have not. Treatment of neonates with injections of genistein reduced oocyte death [12] while genistein treatment of neonatal ovaries in culture did not alter oocyte number [11]. This suggests that different routes of exposure may have different effects. However, estradiol exposure by neonatal injection or in organ culture had no effect on oocyte survival [11]. It may be that genistein as well as the chemicals used in this study have a different mechanism of action depending on the route of exposure while estradiol does not. Another study investigating the effects of neonatal DES injection in mice found fewer follicles at PND5 by no difference with controls at PND10 [17]. However, this study did not count unassembled oocytes. Work investigating effects of BPA and DES on neonatal rats, did not find any effect on oocyte survival [18]. These two studies did use lower concentrations of estrogen than were used in the present study which may account for the difference. While the
present study investigated exposure by subcutaneous injection, the route of exposure of humans to endocrine disruptors would likely be oral. It is unclear what oral doses would cause comparable effects and will be the subject of future investigation. One study compared the effects oral exposure versus subcutaneous injection of the phytoestrogen genistein and found that the route of exposure did not matter [20]. We found differences in response to the chemicals used here for both cyst breakdown and oocyte number. DES and EE significantly reduced cyst breakdown at both concentrations tested while the reduction in cyst breakdown was only significant with the higher dose of BPA. Conversely, both concentrations of BPA increased oocyte number per section while only the higher doses of DES and EE resulted in this response. This supports of the idea of different mechanisms of action for each chemical. We found that DES, EE and BPA reduced follicle activation and development resulting in more primordial follicles and fewer primary and secondary follicles at PND5. Several studies have found that estrogenic compounds delayed the development of the first wave of primordial follicles into primary follicles in rats and mice [5,13,17] while others have found no effect [21]. These variations may be due to different mechanisms of action depending on the chemical compound used and also species and strain differences. In addition, some studies have shown estrogens to have the opposite effect on follicle development promoting follicle activation [5,18,22]. In the hamster, relatively low levels of estradiol promoted follicle development while higher concentrations had no effect [22]. This suggests that concentration may dictate the effect an estrogen has on follicle development. Supporting this, Rodriguez and colleagues used a relatively low concentration of DES (20 g/kg-d) compared to the current study and found that primordial follicle activation was increased while here we found follicle activation decreased [18]. There may also be species and strain effects. Previously, our lab found that estradiol treatment increased the percent of primordial follicles relative to all follicles in the CD1 mouse strain while in the C57BL/6 strain, the percent of primordial follicles decreased [5]. Classic estrogen signaling takes place through two nuclear estrogen receptors (ERs), ER␣ or ER [23]. Both nuclear ERs are expressed in the neonatal ovary [21]. Estrogen can also signal by several other mechanisms including through membrane receptors [24]. At least one membrane receptor, GPR30, has been identified and there are likely several others [25]. The receptors that are used by estrogens to impact neonatal oocyte development are unclear.
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Genistein and DES have been shown cause an increase in MOFs through ER [8,17]. DES has been shown to activate both ER␣ and ER in tissue culture and neonatal exposure to selective agonists of each receptor also cause MOFs [26]. The doses used in our experiments are relatively high. We chose to compare the same two concentrations of each chemical, 5 mg/kg/d and 50 mg/kg/d though each varies in its lowest observed adverse effect concentration as well as estrogenicity in other studies. All three chemicals affected neonatal oocyte development. Effects of lower doses of each chemical on cyst breakdown and follicle formation need to determined as well as the specific mechanism of action of each chemical. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments We thank Robin Jones for help with statistics. This work was partially supported by National Science Foundation grant, IOB0613895 (MEP), Syracuse University Bridge Funds (MEP) and the Ruth Meyer Undergraduate Scholarship program (JRK). References [1] Stephen EH, Chandra A. Use of infertility services in the United States: 1995. Fam Plann Perspect 2000;32:132–7. [2] Stephen EH, Chandra A. Declining estimates of infertility in the United States: 1982–2002. Fertil Steril 2006;86:516–23. [3] Hirshfield AN. Development of follicles in the mammalian ovary. Int Rev Cytol 1991;124:43–101. [4] Pepling ME, Spradling AC. Female mouse germ cells form synchronously dividing cysts. Development 1998;125:3323–8. [5] Pepling ME, Sundman EA, Patterson NL, Gephardt GW, Medico Jr L, Wilson KI. Differences in oocyte development and estradiol sensitivity among mouse strains. Reproduction 2010;139:349–57. [6] Pepling ME, Spradling AC. The mouse ovary contains germ cell cysts that undergo programmed breakdown to form follicles. Dev Biol 2001;234:339–51. [7] Iguchi T, Takasugi N, Bern HA, Mills KT. Frequent occurrence of polyovular follicles in ovaries of mice exposed neonatally to diethylstilbestrol. Teratology 1986;34:29–35. [8] Jefferson WN, Couse JF, Padilla-Banks E, Korach KS, Newbold RR. Neonatal exposure to genestein induces estrogen receptor (ER)␣ expression and multioocyte follicles in the maturing mouse ovary: evidence for ER-mediated and nonestrogenic actions. Biol Reprod 2002;67:1285–96.
[9] Iguchi T, Fukazawa Y, Uesugi Y, Taksugi N. Polyovular follicles in mouse ovaries exposed neonatally to diethylstibestrol in vivo and in vitro. Biol Reprod 1990;43:478–84. [10] Suzuki A, Sugihara A, Uchida K, Sato T, Ohta Y, Katsu Y, et al. Developmental effects of perinatal exposure to bisphenol-A and diethylstilbestrol on reproductive organs in female mice. Reprod Toxicol 2002;16:107–16. [11] Chen Y, Jefferson WN, Newbold RR, Padilla-Banks E, Pepling ME. Estradiol, progesterone, and genistein inhibit oocyte nest breakdown and primordial follicle assembly in the neonatal mouse ovary in vitro and in vivo. Endocrinology 2007;148:3580–90. [12] Jefferson W, Newbold R, Padilla-Banks E, Pepling M. Neonatal genistein treatment alters ovarian differentiation in the mouse: inhibition of oocyte nest breakdown and increased oocyte survival. Biol Reprod 2006;74:161–8. [13] Kezele P, Skinner MK. Regulation of ovarian primordial follicle assembly and development by estrogen and progesterone: endocrine model of follicle assembly. Endocrinology 2003;144:3329–37. [14] Giusti RM, Iwamoto K, Hatch EE. Diethylstilbestrol revisited: a review of the long-term health effects. Ann Intern Med 1995;122:778–88. [15] Richter CA, Birnbaum LS, Farabollini F, Newbold RR, Rubin BS, Talsness CE, et al. In vivo effects of bisphenol A in laboratory rodent studies. Reprod Toxicol 2007;24:199–224. [16] Susiarjo M, Hassold TJ, Freeman E, Hunt PA. Bisphenol A exposure in utero disrupts early oogenesis in the mouse. PLoS Genet 2007;3:e5. [17] Kim H, Hayashi S, Chambon P, Watanabe H, Iguchi T, Sato T. Effects of diethylstilbestrol on ovarian follicle development in neonatal mice. Reprod Toxicol 2009;27:55–62. [18] Rodriguez HA, Santambrosio N, Santamaria CG, Munoz-de-Toro M, Luque EH. Neonatal exposure to bisphenol A reduces the pool of primordial follicles in the rat ovary. Reprod Toxicol 2010;30:550–7. [19] Murphy K, Carvajal L, Medico L, Pepling ME. Expression of Stat3 in germ cells of developing and adult mouse ovaries and testes. Gene Expression Patterns 2005;5:475–82. [20] Jefferson WN, Doerge D, Padilla-Banks E, Woodling KA, Kissling GE, Newbold R. Oral exposure to genistin, the glycosylated form of genistein, during neonatal life adversely affects the female reproductive system. Environ Health Perspect 2009;117:1883–9. [21] Chen Y, Breen K, Pepling ME. Estrogen can signal through multiple pathways to regulate oocyte cyst breakdown and primordial follicle assembly in the neonatal mouse ovary. J Endocrinol 2009;202:407–17. [22] Wang C, Roy SK. Development of primordial follicles in the hamster: role of estradiol-17beta. Endocrinology 2007;148:1707–16. [23] Dahlman-Wright K, Cavailles V, Fuqua SA, Jordan VC, Katzenellenbogen JA, Korach KS, et al. International union of pharmacology. LXIV. Estrogen receptors. Pharmacol Rev 2006;58:773–81. [24] Heldring N, Pike A, Andersson S, Matthews J, Cheng G, Hartman J, et al. Estrogen receptors: how do they signal and what are their targets. Physiol Rev 2007;87:905–31. [25] Revankar CM, Cimino DF, Sklar LA, Arterburn JB, Prossnitz ER. A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science (New York, NY) 2005;307:1625–30. [26] Nakamura T, Katsu Y, Watanabe H, Iguchi T. Estrogen receptor subtypes selectively mediate female mouse reproductive abnormalities induced by neonatal exposure to estrogenic chemicals. Toxicology 2008;253:117–24.
Contents lists available at SciVerse ScienceDirect
Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
Effects of estrogenic compounds on neonatal oocyte development Jenna R. Karavan, Melissa E. Pepling ∗ Syracuse University, Syracuse, NY, United States
a r t i c l e
i n f o
Article history: Received 8 September 2011 Received in revised form 20 January 2012 Accepted 17 February 2012 Available online 3 March 2012 Keywords: Oocyte development Cyst breakdown Primordial follicle assembly Follicle development Estrogens
a b s t r a c t In the mouse, oocytes develop in germline cysts that undergo breakdown resulting in primordial follicles, consisting of a single oocyte surrounded by granulosa cells. During this process, approximately two-thirds of the oocytes die. Exposure of female mice to environmental estrogens can alter oocyte development, limiting the number of primordial follicles that can be used for reproduction. Here we asked whether exposure to synthetic estrogens, diethylstilbestrol, ethinyl estradiol and bisphenol A affected perinatal oocyte development. Neonatal mice were injected with a low or high dose of each compound on postnatal days (PND) 1–4 and ovaries analyzed on PND5. Cyst breakdown, oocyte survival and follicle development were altered. The percentage of single oocyte was reduced from 84% in controls to 50–75%. The oocyte number per section was increased from 8 to 12–16. Follicle activation was reduced with 62% primordial follicles in controls to over 80% in most cases. © 2012 Elsevier Inc. All rights reserved.
1. Introduction The incidence of reproductive disorders among women has increased significantly in the United States in recent years from 6.7 million (10% of the population) in 1995 [1] to 7.3 million (12% of the population) in 2002 [2]. Many female reproductive disorders stem from disruption of normal oocyte development. The entire population of oocytes is formed early in development when primordial follicles each consisting of one oocyte and several granulosa cells are assembled [3]. Prior to follicle formation the oocytes develop in germ cell cysts consisting of oocytes connected by intercellular bridges that form through division of oogonia followed by incomplete cytokinesis [4]. In the fetal mouse, the oocytes within these cysts begin to separate a few days before birth and become enclosed by granulosa cells [5]. During cyst breakdown many oocytes die and only one-third of the oocytes in these germ line cysts survive to become enclosed in primordial follicles [6]. This primordial follicle pool is incredibly important for fertility because it represents the total population of oocytes available to a female during her reproductive lifetime. Estrogens can affect neonatal oocyte development. Exposure of neonates to estrogenic compounds such as estradiol (E2 ) [7], phytoestrogens like genistein [8] or with synthetic estrogens including diethylstilbestrol (DES) [9] or bisphenol-A (BPA) [10] significantly increases the number of multiple oocyte follicles (MOFs) in ovaries
∗ Corresponding author at: Department of Biology, Syracuse University, 348 Life Sciences Complex, 107 College Place, Syracuse, NY 13244, United States. Tel.: +1 315 443 4541; fax: +1 315 443 2012. E-mail address: [email protected] (M.E. Pepling). 0890-6238/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.reprotox.2012.02.005
of exposed animals when they are examined as adults. The oocytes in a MOF are believed to be remnants of a germ cell cyst that did not properly separate and instead the oocytes became enclosed together in one follicle. Indeed, neonatal exposure to E2 or genistein reduced cyst breakdown [11,12]. These findings led to the proposal of a model where during normal development high levels of estradiol maintain oocytes in cysts [11]. Late in fetal development estradiol levels drop allowing oocytes to separate. When neonates are exposed to exogenous estrogens cyst breakdown is blocked and MOFs are formed. A similar model was proposed earlier where high levels of steroid hormones would prevent follicle assembly during gestation [13]. However, in that study only progesterone was found to block follicle assembly. However, while estradiol did not block follicle assembly, it did block follicle activation. The endocrine disrupting effects of three synthetic estrogens, DES, BPA and ethinyl estradiol (EE) have been investigated. DES was the first synthetic estrogen produced and many pregnant women were treated with DES in their first trimester during the 1940s–1970s in order to prevent miscarriage or spontaneous abortion [14]. This exposure affected both the pregnant mother that was treated as well as her offspring and caused detrimental effects on fertility as well as increased risks for several types of cancer. EE is one of the main active ingredients in many contraceptive pills. BPA is used to make polycarbonate plastic compounds and is found in baby bottles, water bottles and other household items and is also used as a lining in food and beverage containers. Studies have shown adverse affect of BPA on the reproductive tracts of males and females [15]. For example, in female mouse embryos, BPA treatment caused defects in meiosis [16]. The effects of DES, BPA and EE on cyst breakdown and primordial follicle assembly have not been thoroughly investigated. Kim and
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colleagues [17] found that in mice, neonatal DES treatment reduced follicle activation and also reduced the number of primordial follicles formed suggesting that follicle assembly was also reduced. In contrast, a study looking at the effects of neonatal BPA and DES exposure in rats found that while the number of primordial follicles was reduced it was due to an increased number of activated follicles [18]. Here we examine the effects of 2 different doses of DES, BPA and EE specifically on cyst breakdown and associated oocyte loss as well as on primordial follicle recruitment. 2. Materials and methods 2.1. Animals The CD1 outbred mouse strain was obtained from Charles River Laboratories (Wilmington, MA, USA). CD1 females were mated with males and checked daily for vaginal plugs. The morning a vaginal plug was detected was designated as 0.5 days post-coitum (dpc). Birth usually occurred at 19.5 dpc and was designated as postnatal day (PND) 1. Ovaries were collected from CD1 neonates at PND5 in 1× phosphate-buffered saline (PBS). 2.2. Whole-mount antibody staining of neonatal ovaries Ovaries were fixed in 5.3% EM grade formaldehyde in PBS (Ted Pella Inc., Redding, CA, USA) overnight at 4 ◦ C. The ovaries were then washed several times in 0.1% Triton X-100 in PBS (PT) and then in PT + 5% bovine serum albumin (BSA) at room temperature to block nonspecific binding. Immunostaining was performed as previously described [19]. Whole ovaries were labeled with STAT-3 (C20) antibody (Santa Cruz Biotechnology, La Jolla, CA, USA), diluted 1:500 in PT + 5% BSA. The secondary antibody, goat anti-rabbit Alexa 488 (Invitrogen, Carlsbad, CA, USA), was diluted 1:200 in PT + 5% BSA. Nuclei were labeled with propidium iodide (PI) (Molecular Probes) at 5 g/ml. Ovaries were mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA). 2.3. Confocal microscopy Ovaries were analyzed using an LSM 710 confocal microscope (Carl Zeiss Microimaging, Inc., Thornwood, NY, USA). The confocal images were taken by imaging two separate areas in each ovary, with four planar Images 20 m apart in each area to give a total of eight sections per ovary. The number of oocytes was determined by counting the number of oocytes in each image. Cyst breakdown was assessed by counting the number of single oocytes and comparing this to the number of oocytes that were still in cysts. In order to determine whether oocytes were in cysts or not, for each of the eight sections, a z-stack of images each 1 m apart was obtained with 5 images above the section and 5 images below the section being analyzed. This allowed us to determine whether an oocyte was part of a germline cyst above or below the plane of focus. Finally, follicle development was quantified in each of the eight sections by determining the stage of development of each follicle. The oocytes were classified as unassembled if they were not entirely surrounded by granulosa
cells and if they were in contact with another oocyte. Single oocytes enclosed in follicles fell into three categories: primordial, primary, or secondary. A single oocyte surrounded by flattened, crescent-shaped granulosa cells defined a primordial follicle. A primary follicle consisted of a single oocyte enclosed in one layer of cuboidal granulosa cells. Transitional follicles with some flattened granulosa cells and some cuboidal granulosa cells were also classified as primary follicles. An oocyte surrounded by multiple layers of cuboidal granulosa cells was classified as a secondary follicle. 2.4. Preparation and delivery of estrogenic compounds CD1 neonates were subcutaneously injected with a total volume of 50 l of either peanut oil (Wegmans Food Markets, Rochester, NY, USA) or a mixture consisting of an estrogenic compound dissolved in peanut oil. Three different estrogenic compounds were used: diethylstilbestrol (DES) (Acros Organics, Morris Plains, NJ, USA), ethinyl estradiol (EE) (Sigma–Aldrich, St. Louis, MO, USA), and bisphenol-A (BPA) (Sigma–Aldrich). Two concentrations of each compound were used: (1) 5 mg per kg of body weight per day (mg/kg/d) or 10 g per pup and (2) 50 mg/kg/d or 100 g per pup. An average pup weight was used to determine the chemical concentration given and was not adjusted for any increase in body weight during the 4 days of injections. Solutions of each compound were prepared by first dissolving in absolute ethanol (Pharmco-AAPER, Brookfield, CT, USA) then added to the peanut oil. The ethanol was allowed to evaporate before use. Neonates were injected daily from PND1 to PND4. On PND5, ovaries were collected and fixed for immunocytochemistry. 2.5. Measurement of programmed cell death For poly ADP-ribose polymerase (PARP) labeling, whole mount immunocytochemistry was performed on ovaries from mice treated on PND 1 and 2 with peanut oil or with 50 mg/kg/d DES, EE or BPA and collected on PND 3. Ovaries were labeled with PARP antibody (Abcam, Cambridge, MA, USA), diluted 1:100 in PT + 5% BSA. The whole mount antibody staining procedure was followed except nuclei were labeled with Toto-3 (Invitrogen) diluted 1:2000. 2.6. Statistical analyses of cyst breakdown, total number of oocytes, and follicle development One-way ANOVA was implemented in order to determine statistical significance using SPSS, version 17. The Tukey post hoc test was used to compare the control group with each treatment group to investigate effects on cyst breakdown, total number of oocytes, and follicle development with p values less than 0.05 considered significant.
3. Results 3.1. Cyst breakdown in mice injected with estrogenic compounds Estradiol as well as the phytoestrogen, genistein block cyst breakdown [11,12]. To determine if synthetic estrogens had
Fig. 1. Effects of estrogenic compounds on cyst breakdown. Percent single oocytes in mice injected with peanut oil and with two concentrations of DES, EE, or BPA from PND 1 to 4 and collected on PND5. Data are presented as the mean ± SEM. Asterisks indicate a significance difference in comparison to mice injected with peanut oil (one way ANOVA, p < 0.05; n = 8–12 ovaries per group).
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Fig. 2. Confocal sections demonstrating the effects of estrogenic compounds on cyst breakdown and oocyte number. (A)–(C) Confocal section of an ovary from a control pup injected with peanut oil from PND 1 to 4 and collected on PND5 labeled with (A) Stat3 antibody (green) to visualize oocytes, (B) propidium iodide (red) to visualize nuclei and (C) overlay. Most oocytes have separated and are individually enclosed in primordial follicles (arrowheads) though some small 2 cell cysts are still present (white circle) (D)–(F) confocal section of an ovary from a pup injected with 100 g DES from PND 1 to 4 and collected on PND5 labeled with (D) Stat3 antibody (green) to visualize oocytes, (E) propidium iodide (red) to visualize nuclei and (F) overlay. (G)–(I) Confocal section of an ovary from a pup injected with 100 g EE from PND 1 to 4 and collected on PND5 labeled with (G) Stat3 antibody (green) to visualize oocytes, (H) propidium iodide (red) to visualize nuclei and (I) overlay. (J)–(L) Confocal section of an ovary from a pup injected with 100 g BPA from PND 1 to 4 and collected on PND5 labeled with (J) Stat3 antibody (green) to visualize oocytes, (K) propidium iodide (red) to visualize nuclei and (L) overlay. All three chemicals delayed cyst breakdown resulting in large cysts (white circles). Scale bar, 20 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Effects of estrogenic compounds on oocyte number. Number of oocytes per section in mice injected with peanut oil and with two concentrations of DES, EE, or BPA from PND 1 to 4 and collected on PND5. Data are presented as the mean ± SEM. Asterisks indicate a significance difference in comparison to mice injected with peanut oil (one way ANOVA, p < 0.05; n = 8–12 ovaries per group).
similar effects on oocyte development, neonatal mice were exposed to DES, EE, or BPA and cyst breakdown was analyzed. All estrogens tested significantly reduced the percent of single oocytes except for the lower concentration of BPA (Fig. 1). Mice injected with peanut oil alone had 84.2% single oocytes. In mice treated with 10 g DES, the percent of single oocytes decreased significantly to 55.3% while the percent of single oocytes decreased slightly more to 50.8% in mice treated with the higher concentration of DES. Mice treated with the lower concentration of EE also had a significant reduction in the percent of single oocytes at 62.6% and treatment with the higher concentration was only slightly lower at 61.2% single oocytes. The percent of single oocytes decreased slightly to 75.4% with for the lower concentration of BPA but was not significant while the higher BPA concentration caused a significant drop to 68.6%. Fig. 2 shows representative confocal sections from control ovaries (A–C), and ovaries treated with the higher concentrations of DES (D–F), EE (G–I) and BPA (J–L). While the control ovaries still had some 2 cell cysts, the treated ovaries had not only 2 cell cysts but larger cysts as well. 3.2. Total number of oocytes in mice injected with estrogenic compounds During the perinatal period, normally, two thirds of the oocytes are lost as the cysts break apart into individual oocytes [6]. Previously, neonates injected with genistein were shown to have more surviving oocytes while estradiol injections had no effect on oocyte number [11,12]. To determine if synthetic estrogens affected oocyte survival, the total number of oocytes per confocal section was determined for mice injected with peanut oil alone and for mice injected with DES, EE, or BPA. The higher concentrations of DES and EE and both concentrations of BPA significantly increased oocyte survival as shown in Fig. 3. These treatment groups had 12.3, 12.5, 13.0, and 15.8 oocytes per confocal section, respectively, while mice injected with peanut oil had an average of 8.0 oocytes per section. Ovaries from neonates treated with 10 g DES/day and 10 g EE/day, had 8.8 and 10.2 oocytes per section respectively, which was not significantly different from controls (Fig. 3). To determine if the increase in oocyte number was due to a reduction in apoptosis, the percent of PARP positive oocytes was assessed in PND3 mice treated with peanut oil or with 50 mg/kg/d DES, EE or BPA on PND 1 and 2. PND3 was chosen because in our previous studies we found that programmed cell death was reduced
Fig. 4. Effects of estrogenic compounds on programmed cell death. Percent of PARP positive oocytes per section in mice injected with peanut oil or with 50 mg/kg/d DES, EE, or BPA on PND 1–2 and collected on PND3. Data are presented as the mean ± SEM. Asterisks indicate a significance difference in comparison to mice injected with peanut oil (one way ANOVA, p < 0.05; n = 6 ovaries per group).
at PND3 with genistein treatment [12]. DES, EE and BPA treated mice all had a decreased percentage of PARP positive oocytes (6.4, 6.9 and 8.1%) as compared to oil treated mice (11.9%) (Fig. 4). 3.3. Follicle development in mice injected with estrogenic compounds After follicles form, some start to develop immediately. To determine whether synthetic estrogen exposure could affect follicle development, the percent of primordial, primary and secondary follicles were determined in ovaries from mice injected with peanut oil or with DES, EE, or BPA. Control mice injected with peanut oil alone had 62.3% primordial follicles, 27.9% primary follicles, and 9.7% secondary follicles (Fig. 5). Exposure to the higher concentrations of DES and EE and both high and low concentrations of BPA significantly increased the percent of primordial follicles to 82.6%, 82.7%, 89.5% and 81%, respectively. Treatment with the lower concentration of DES increased the number of primary follicles to 37.8% and decreased the number of secondary follicles to 0%, and treatment with the higher concentration of DES decreased the percentage of primary follicles to 16% and the percentage of secondary follicles to 1.4%. However, these changes were not
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Fig. 5. Effects of estrogenic compounds on follicle development. Percent primordial, primary and secondary follicles in mice injected with peanut oil and with two concentrations of DES, EE, or BPA from PND 1 to 4 and collected on PND5. Data are presented as the mean ± SEM. Asterisks indicate a significance difference in comparison to mice injected with peanut oil (one way ANOVA, p < 0.05; n = 8–12 ovaries per group).
significant. A similar trend was observed for both concentrations of EE. Mice injected with the lower concentration of EE had 34.6% primary follicles and 0.8% secondary follicles. Mice injected with the higher concentration of EE had 16.9% primary follicles and 0.4% secondary follicles. While treatment with both concentrations of BPA did lower the percentage of primary follicles, this effect was significant only for the higher concentration of BPA, which had 10.5% primary follicles. Treatment with both concentrations of BPA also lowered the percentage of secondary follicles to 0.8% and 0%, but this effect was not significant in both instances. 4. Discussion This study finds that synthetic estrogens, DES, BPA and EE delay cyst breakdown and associated oocyte loss as well as follicle activation. Oocyte number increased and follicle activation decreased in a dose dependent manner with all three chemicals. The increase in oocyte number was associated with a decrease in programmed cell death. However, while treatment with each chemical reduced cyst breakdown, the higher dose had only a slightly greater effect for DES and BPA and no change at all for EE. It is not clear why the higher doses did not have a greater effect. It is likely that estrogens are only one of many factors controlling oocyte development and that other compensatory mechanisms contribute to normal cyst breakdown and associated oocyte loss. Normally, as cysts break down, some oocytes are lost. Here we found that synthetic estrogens reduced the number of oocytes lost. Previously, some estrogens and some modes of exposure have affected neonatal oocyte numbers in rodents while others have not. Treatment of neonates with injections of genistein reduced oocyte death [12] while genistein treatment of neonatal ovaries in culture did not alter oocyte number [11]. This suggests that different routes of exposure may have different effects. However, estradiol exposure by neonatal injection or in organ culture had no effect on oocyte survival [11]. It may be that genistein as well as the chemicals used in this study have a different mechanism of action depending on the route of exposure while estradiol does not. Another study investigating the effects of neonatal DES injection in mice found fewer follicles at PND5 by no difference with controls at PND10 [17]. However, this study did not count unassembled oocytes. Work investigating effects of BPA and DES on neonatal rats, did not find any effect on oocyte survival [18]. These two studies did use lower concentrations of estrogen than were used in the present study which may account for the difference. While the
present study investigated exposure by subcutaneous injection, the route of exposure of humans to endocrine disruptors would likely be oral. It is unclear what oral doses would cause comparable effects and will be the subject of future investigation. One study compared the effects oral exposure versus subcutaneous injection of the phytoestrogen genistein and found that the route of exposure did not matter [20]. We found differences in response to the chemicals used here for both cyst breakdown and oocyte number. DES and EE significantly reduced cyst breakdown at both concentrations tested while the reduction in cyst breakdown was only significant with the higher dose of BPA. Conversely, both concentrations of BPA increased oocyte number per section while only the higher doses of DES and EE resulted in this response. This supports of the idea of different mechanisms of action for each chemical. We found that DES, EE and BPA reduced follicle activation and development resulting in more primordial follicles and fewer primary and secondary follicles at PND5. Several studies have found that estrogenic compounds delayed the development of the first wave of primordial follicles into primary follicles in rats and mice [5,13,17] while others have found no effect [21]. These variations may be due to different mechanisms of action depending on the chemical compound used and also species and strain differences. In addition, some studies have shown estrogens to have the opposite effect on follicle development promoting follicle activation [5,18,22]. In the hamster, relatively low levels of estradiol promoted follicle development while higher concentrations had no effect [22]. This suggests that concentration may dictate the effect an estrogen has on follicle development. Supporting this, Rodriguez and colleagues used a relatively low concentration of DES (20 g/kg-d) compared to the current study and found that primordial follicle activation was increased while here we found follicle activation decreased [18]. There may also be species and strain effects. Previously, our lab found that estradiol treatment increased the percent of primordial follicles relative to all follicles in the CD1 mouse strain while in the C57BL/6 strain, the percent of primordial follicles decreased [5]. Classic estrogen signaling takes place through two nuclear estrogen receptors (ERs), ER␣ or ER [23]. Both nuclear ERs are expressed in the neonatal ovary [21]. Estrogen can also signal by several other mechanisms including through membrane receptors [24]. At least one membrane receptor, GPR30, has been identified and there are likely several others [25]. The receptors that are used by estrogens to impact neonatal oocyte development are unclear.
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Genistein and DES have been shown cause an increase in MOFs through ER [8,17]. DES has been shown to activate both ER␣ and ER in tissue culture and neonatal exposure to selective agonists of each receptor also cause MOFs [26]. The doses used in our experiments are relatively high. We chose to compare the same two concentrations of each chemical, 5 mg/kg/d and 50 mg/kg/d though each varies in its lowest observed adverse effect concentration as well as estrogenicity in other studies. All three chemicals affected neonatal oocyte development. Effects of lower doses of each chemical on cyst breakdown and follicle formation need to determined as well as the specific mechanism of action of each chemical. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments We thank Robin Jones for help with statistics. This work was partially supported by National Science Foundation grant, IOB0613895 (MEP), Syracuse University Bridge Funds (MEP) and the Ruth Meyer Undergraduate Scholarship program (JRK). References [1] Stephen EH, Chandra A. Use of infertility services in the United States: 1995. Fam Plann Perspect 2000;32:132–7. [2] Stephen EH, Chandra A. Declining estimates of infertility in the United States: 1982–2002. Fertil Steril 2006;86:516–23. [3] Hirshfield AN. Development of follicles in the mammalian ovary. Int Rev Cytol 1991;124:43–101. [4] Pepling ME, Spradling AC. Female mouse germ cells form synchronously dividing cysts. Development 1998;125:3323–8. [5] Pepling ME, Sundman EA, Patterson NL, Gephardt GW, Medico Jr L, Wilson KI. Differences in oocyte development and estradiol sensitivity among mouse strains. Reproduction 2010;139:349–57. [6] Pepling ME, Spradling AC. The mouse ovary contains germ cell cysts that undergo programmed breakdown to form follicles. Dev Biol 2001;234:339–51. [7] Iguchi T, Takasugi N, Bern HA, Mills KT. Frequent occurrence of polyovular follicles in ovaries of mice exposed neonatally to diethylstilbestrol. Teratology 1986;34:29–35. [8] Jefferson WN, Couse JF, Padilla-Banks E, Korach KS, Newbold RR. Neonatal exposure to genestein induces estrogen receptor (ER)␣ expression and multioocyte follicles in the maturing mouse ovary: evidence for ER-mediated and nonestrogenic actions. Biol Reprod 2002;67:1285–96.
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