Developmental estrogen exposure in mice disrupts uterine epithelial cell differentiation and causes adenocarcinoma via Wnt/ß-catenin and PI3K/AKT signaling.

Tissue development entails genetically programmed differentiation of immature cell types to mature, fully differentiated cells. Exposure during development to non-mutagenic environmental factors can contribute to cancer risk, but the underlying mechanisms are not understood. We used a mouse model of endometrial adenocarcinoma that results from brief developmental exposure to an estrogenic chemical, diethylstilbestrol (DES), to determine causative factors. Single-cell RNA sequencing (scRNAseq) and spatial transcriptomics of adult control uteri revealed novel markers of uterine epithelial stem cells (EpSCs), identified distinct luminal and glandular progenitor cell (PC) populations, and defined glandular and luminal epithelium (LE) cell differentiation trajectories. Neonatal DES exposure disrupted uterine epithelial cell differentiation, resulting in a failure to generate an EpSC population or distinguishable glandular and luminal progenitors or mature cells. Instead, the DES-exposed epithelial cells were characterized by a single proliferating PC population and widespread activation of Wnt/ß-catenin signaling. The underlying endometrial stromal cells had dramatic increases in inflammatory signaling pathways and oxidative stress. Together, these changes activated phosphoinositide 3-kinase/AKT serine-threonine kinase signaling and malignant transformation of cells that were marked by phospho-AKT and the cancer-associated protein olfactomedin 4. Here, we defined a mechanistic pathway from developmental exposure to an endocrine disrupting chemical to the development of adult-onset cancer. These findings provide an explanation for how human cancers, which are often associated with abnormal activation of PI3K/AKT signaling, could result from exposure to environmental insults during development.

Unexpected nuclear hormone receptor and chromatin dynamics regulate estrous cycle dependent gene expression.

Chromatin changes in response to estrogen and progesterone are well established in cultured cells, but how they control gene expression under physiological conditions is largely unknown. To address this question, we examined in vivo estrous cycle dynamics of mouse uterus hormone receptor occupancy, chromatin accessibility and chromatin structure by combining RNA-seq, ATAC-seq, HiC-seq and ChIP-seq. Two estrous cycle stages were chosen for these analyses, diestrus (highest estrogen) and estrus (highest progesterone). Unexpectedly, rather than alternating with each other, estrogen receptor alpha (ERα) and progesterone receptor (PGR) were co-bound during diestrus and lost during estrus. Motif analysis of open chromatin followed by hypoxia inducible factor 2A (HIF2A) ChIP-seq and conditional uterine deletion of this transcription factor revealed a novel role for HIF2A in regulating diestrus gene expression patterns that were independent of either ERα or PGR binding. Proteins in complex with ERα included PGR and cohesin, only during diestrus. Combined with HiC-seq analyses, we demonstrate that complex chromatin architecture changes including enhancer switching are coordinated with ERα and PGR co-binding during diestrus and non-hormone receptor transcription factors such as HIF2A during estrus to regulate most differential gene expression across the estrous cycle.

Methionine metabolism is essential for SIRT1-regulated mouse embryonic stem cell maintenance and embryonic development.

Methionine metabolism is critical for epigenetic maintenance, redox homeostasis, and animal development. However, the regulation of methionine metabolism remains unclear. Here, we provide evidence that SIRT1, the most conserved mammalian NAD+-dependent protein deacetylase, is critically involved in modulating methionine metabolism, thereby impacting maintenance of mouse embryonic stem cells (mESCs) and subsequent embryogenesis. We demonstrate that SIRT1-deficient mESCs are hypersensitive to methionine restriction/depletion-induced differentiation and apoptosis, primarily due to a reduced conversion of methionine to S-adenosylmethionine. This reduction markedly decreases methylation levels of histones, resulting in dramatic alterations in gene expression profiles. Mechanistically, we discover that the enzyme converting methionine to S-adenosylmethionine in mESCs, methionine adenosyltransferase 2a (MAT2a), is under control of Myc and SIRT1. Consistently, SIRT1 KO embryos display reduced Mat2a expression and histone methylation and are sensitive to maternal methionine restriction-induced lethality, whereas maternal methionine supplementation increases the survival of SIRT1 KO newborn mice. Our findings uncover a novel regulatory mechanism for methionine metabolism and highlight the importance of methionine metabolism in SIRT1-mediated mESC maintenance and embryonic development.

Widespread enhancer activation via ERα mediates estrogen response in vivo during uterine development.

Little is known regarding how steroid hormone exposures impact the epigenetic landscape in a living organism. Here, we took a global approach to understanding how exposure to the estrogenic chemical, diethylstilbestrol (DES), affects the neonatal mouse uterine epigenome. Integration of RNA- and ChIP-sequencing data demonstrated that ∼80% of DES-altered genes had higher H3K4me1/H3K27ac signal in close proximity. Active enhancers, of which ∼3% were super-enhancers, had a high density of estrogen receptor alpha (ERα) binding sites and were correlated with alterations in nearby gene expression. Conditional uterine deletion of ERα, but not the pioneer transcription factors FOXA2 or FOXO1, prevented the majority of DES-mediated changes in gene expression and H3K27ac signal at target enhancers. An ERα dependent super-enhancer was located at the Padi gene locus and a topological connection to the Padi1 TSS was documented using 3C-PCR. Chromosome looping at this site was independent of ERα and DES exposure, indicating that the interaction is established prior to ligand signaling. However, enrichment of H3K27ac and transcriptional activation at this locus was both DES and ERα-dependent. These data suggest that DES alters uterine development and consequently adult reproductive function by modifying the enhancer landscape at ERα binding sites near estrogen-regulated genes.

Regulator of G-protein signaling 2 (RGS2) suppresses premature calcium release in mouse eggs.

During oocyte maturation, capacity and sensitivity of Ca(2+) signaling machinery increases dramatically, preparing the metaphase II (MII)-arrested egg for fertilization. Upon sperm-egg fusion, Ca(2+) release from IP3-sensitive endoplasmic reticulum stores results in cytoplasmic Ca(2+) oscillations that drive egg activation and initiate early embryo development. Premature Ca(2+) release can cause parthenogenetic activation prior to fertilization; thus, preventing inappropriate Ca(2+) signaling is crucial for ensuring robust MII arrest. Here, we show that regulator of G-protein signaling 2 (RGS2) suppresses Ca(2+) release in MII eggs. Rgs2 mRNA was recruited for translation during oocyte maturation, resulting in ∼ 20-fold more RGS2 protein in MII eggs than in fully grown immature oocytes. Rgs2-siRNA-injected oocytes matured to MII; however, they had increased sensitivity to low pH and acetylcholine (ACh), which caused inappropriate Ca(2+) release and premature egg activation. When matured in vitro, RGS2-depleted eggs underwent spontaneous Ca(2+) increases that were sufficient to cause premature zona pellucida conversion. Rgs2(-/-) females had reduced litter sizes, and their eggs had increased sensitivity to low pH and ACh. Rgs2(-/-) eggs also underwent premature zona pellucida conversion in vivo. These findings indicate that RGS2 functions as a brake to suppress premature Ca(2+) release in eggs that are poised on the brink of development.

Mediator complex component MED13 regulates zygotic genome activation and is required for postimplantation development in the mouse.

Understanding factors that regulate zygotic genome activation (ZGA) is critical for determining how cells are reprogrammed to become totipotent or pluripotent. There is limited information regarding how this process occurs physiologically in early mammalian embryos. Here, we identify a mediator complex subunit, MED13, as translated during mouse oocyte maturation and transcribed early from the zygotic genome. Knockdown and conditional knockout approaches demonstrate that MED13 is essential for ZGA in the mouse, in part by regulating expression of the embryo-specific chromatin remodeling complex, esBAF. The role of MED13 in ZGA is mediated in part by interactions with E2F transcription factors. In addition to MED13, its paralog, MED13L, is required for successful preimplantation embryo development. MED13L partially compensates for loss of MED13 function in preimplantation knockout embryos, but postimplantation development is not rescued by MED13L. Our data demonstrate an essential role for MED13 in supporting chromatin reprogramming and directed transcription of essential genes during ZGA.

Epithelial membrane protein 2 (EMP2) deficiency alters placental angiogenesis, mimicking features of human placental insufficiency.

Epithelial membrane protein-2 (EMP2) is a tetraspan protein predicted to regulate placental development. Highly expressed in secretory endometrium and trophectoderm cells, previous studies suggest that it may regulate implantation by orchestrating the surface expression of integrins and other membrane proteins. In order to test the role of EMP2 in pregnancy, mice lacking EMP2 (Emp2-/- ) were generated. Emp2-/- females are fertile but have reduced litter sizes when carrying Emp2-/- but not Emp2+/- fetuses. Placentas of Emp2-/- fetuses exhibit dysregulation in pathways related to neoangiogenesis, coagulation, and oxidative stress, and have increased fibrin deposition and altered vasculature. Given that these findings often occur due to placental insufficiency resulting in an oxygen-poor environment, the expression of hypoxia-inducible factor-1 alpha (HIF-1α) was examined. Placentas from Emp2-/- fetuses had increased total HIF-1α expression in large part through an increase in uterine NK (uNK) cells, demonstrating a unique interplay between uNK cells and trophoblasts modulated through EMP2. To determine if these results translated to human pregnancy, placentas from normal, term deliveries or those complicated by placental insufficiency resulting in intrauterine growth restriction (IUGR) were stained for EMP2. EMP2 was significantly reduced in both villous and extravillous trophoblast populations in IUGR placentas. Experiments in vitro using human trophoblast cells lines indicate that EMP2 modulates angiogenesis by altering HIF-1α expression. Our results reveal a novel role for EMP2 in regulating trophoblast function and vascular development in mice and humans, and suggest that it may be a new biomarker for placental insufficiency. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

CaV3.2 T-type channels mediate Ca²âº entry during oocyte maturation and following fertilization.

Initiation of mouse embryonic development depends upon a series of fertilization-induced rises in intracellular Ca(2+). Complete egg activation requires influx of extracellular Ca(2+); however, the channels that mediate this influx remain unknown. Here, we tested whether the α1 subunit of the T-type channel CaV3.2, encoded by Cacna1h, mediates Ca(2+) entry into oocytes. We show that mouse eggs express a robust voltage-activated Ca(2+) current that is completely absent in Cacna1h(-/-) eggs. Cacna1h(-/-) females have reduced litter sizes, and careful analysis of Ca(2+) oscillation patterns in Cacna1h(-/-) eggs following in vitro fertilization (IVF) revealed reductions in first transient length and oscillation persistence. Total and endoplasmic reticulum (ER) Ca(2+) stores were also reduced in Cacna1h(-/-) eggs. Pharmacological inhibition of CaV3.2 in wild-type CF-1 strain eggs using mibefradil or pimozide reduced Ca(2+) store accumulation during oocyte maturation and reduced Ca(2+) oscillation persistence, frequency and number following IVF. Overall, these data show that CaV3.2 T-type channels have prev8iously unrecognized roles in supporting the meiotic-maturation-associated increase in ER Ca(2+) stores and mediating Ca(2+) influx required for the activation of development.

Calcium influx-mediated signaling is required for complete mouse egg activation.

Mammalian fertilization is accompanied by oscillations in egg cytoplasmic calcium (Ca(2+)) concentrations that are critical for completion of egg activation. These oscillations are initiated by Ca(2+) release from inositol 1,4,5-trisphosphate (IP(3))-sensitive intracellular stores. We tested the hypothesis that Ca(2+) influx across the plasma membrane was a requisite component of egg activation signaling, and not simply a Ca(2+) source for store repletion. Using intracytoplasmic sperm injection (ICSI) and standard in vitro fertilization (IVF), we found that Ca(2+) influx was not required to initiate resumption of meiosis II. However, even if multiple oscillations in intracellular Ca(2+) occurred, in the absence of Ca(2+) influx, the fertilized eggs failed to emit the second polar body, resulting in formation of three pronuclei. Additional experiments using the Ca(2+) chelator, BAPTA/AM, demonstrated that Ca(2+) influx is sufficient to support polar body emission and pronucleus formation after only a single sperm-induced Ca(2+) transient, whereas BAPTA/AM-treated ICSI or fertilized eggs cultured in Ca(2+)-free medium remained arrested in metaphase II. Inhibition of store-operated Ca(2+) entry had no effect on ICSI-induced egg activation, so Ca(2+) influx through alternative channels must participate in egg activation signaling. Ca(2+) influx appears to be upstream of CaMKIIγ activity because eggs can be parthenogenetically activated with a constitutively active form of CaMKIIγ in the absence of extracellular Ca(2+). These results suggest that Ca(2+) influx at fertilization not only maintains Ca(2+) oscillations by replenishing Ca(2+) stores, but also activates critical signaling pathways upstream of CaMKIIγ that are required for second polar body emission.

Transducin-like enhancer of split-6 (TLE6) is a substrate of protein kinase A activity during mouse oocyte maturation.

Fully grown oocytes in the ovary are arrested at prophase of meiosis I because of high levels of intraoocyte cAMP that maintain increased levels of cAMP-dependent protein kinase (PKA) activity. Following the luteinizing hormone surge at the time of ovulation, cAMP levels drop, resulting in a reduction in PKA activity that triggers meiotic resumption. Although much is known about the molecular mechanisms of how PKA activity fluctuations initiate the oocyte's reentry into meiosis, significantly less is known about the requirement for PKA activity in the oocyte after exit from the prophase I arrest. Here we show that although PKA activity decreases in the oocyte upon meiotic resumption, it increases throughout meiotic progression from the time of germinal vesicle breakdown (GVBD) until the metaphase II (MII) arrest. Blocking this meiotic maturation-associated increase in PKA activity using the pharmacological inhibitor H89 resulted in altered kinetics of GVBD, defects in chromatin and spindle dynamics, and decreased ability of oocytes to reach MII. These effects appear to be largely PKA specific because inhibitors targeting other kinases did not have the same outcomes. To determine potential proteins that may require PKA phosphorylation during meiosis, we separated oocyte protein extracts on an SDS-PAGE gel, extracted regions of the gel that had corresponding immune reactivity towards an anti-PKA substrate antibody, and performed mass spectrometry and microsequencing. Using this approach, we identified transducin-like enhancer of split-6 (TLE6)-a maternal effect gene that is part of the subcortical maternal complex-as a putative PKA substrate. TLE6 localized to the oocyte cortex throughout meiosis in a manner that is spatially and temporally consistent with the localization of critical PKA subunits. Moreover, we demonstrated that TLE6 becomes phosphorylated in a narrow window following meiotic resumption, and H89 treatment can completely block this phosphorylation when added prior to GVBD but not after. Taken together, these results highlight the importance of oocyte-intrinsic PKA in regulating meiotic progression after the prophase I arrest and offer new insights into downstream targets of its activity.

Neonatal phytoestrogen exposure alters oviduct mucosal immune response to pregnancy and affects preimplantation embryo development in the mouse.

Treatment of neonatal mice with the phytoestrogen genistein (50 mg/kg/day) results in complete female infertility caused in part by preimplantation embryo loss in the oviduct between Days 2 and 3 of pregnancy. We previously demonstrated that oviducts of genistein-treated mice are "posteriorized" as compared to control mouse oviducts because they express numerous genes normally restricted to posterior regions of the female reproductive tract (FRT), the cervix and vagina. We report here that neonatal genistein treatment resulted in substantial changes in oviduct expression of genes important for the FRT mucosal immune response, including immunoglobulins, antimicrobials, and chemokines. Some of the altered immune response genes were chronically altered beginning at the time of neonatal genistein treatment, indicating that these alterations were a result of the posteriorization phenotype. Other alterations in oviduct gene expression were observed only in early pregnancy, immediately after the FRT was exposed to inflammatory or antigenic stimuli from ovulation and mating. The oviduct changes affected development of the surviving embryos by increasing the rate of cleavage and decreasing the trophectoderm-to-inner cell mass cell ratio at the blastocyst stage. We conclude that both altered immune responses to pregnancy and deficits in oviduct support for preimplantation embryo development in the neonatal genistein model are likely to contribute to infertility phenotype.

Metastasis-associated protein 3 (MTA3) regulates G2/M progression in proliferating mouse granulosa cells.

Metastasis-associated protein 3 (MTA3) is a constituent of the Mi-2/nucleosome remodeling and deacetylase (NuRD) protein complex that regulates gene expression by altering chromatin structure and can facilitate cohesin loading onto DNA. The biological function of MTA3 within the NuRD complex is unknown. Herein, we show that MTA3 was expressed highly in granulosa cell nuclei of all ovarian follicle stages and at lower levels in corpora lutea. We tested the hypothesis that MTA3-NuRD complex function is required for granulosa cell proliferation. In the ovary, MTA3 interacted with NuRD proteins CHD4 and HDAC1 and the core cohesin complex protein RAD21. In cultured mouse primary granulosa cells, depletion of endogenous MTA3 using RNA interference slowed cell proliferation; this effect was rescued by coexpression of exogenous MTA3. Slowing of cell proliferation correlated with a significant decrease in cyclin B1 and cyclin B2 expression. Granulosa cell populations lacking MTA3 contained a significantly higher percentage of cells in G2/M phase and a lower percentage in S phase compared with control cells. Furthermore, MTA3 depletion slowed entry into M phase as indicated by reduced phosphorylation of histone H3 at serine 10. These findings provide the first evidence to date that MTA3 interacts with NuRD and cohesin complex proteins in the ovary in vivo and regulates G2/M progression in proliferating granulosa cells.

Uterine Patterning, Endometrial Gland Development, and Implantation Failure in Mice Exposed Neonatally to Genistein.

BACKGROUND: Embryo implantation relies on precise hormonal regulation, associated gene expression changes, and appropriate female reproductive tract tissue architecture. Female mice exposed neonatally to the phytoestrogen genistein (GEN) at doses similar to those in infants consuming soy-based infant formulas are infertile due in part to uterine implantation defects. OBJECTIVES: Our goal was to determine the mechanisms by which neonatal GEN exposure causes implantation defects. METHODS: Female mice were exposed to GEN on postnatal days (PND)1-5 and uterine tissues collected on PND5, PND22-26, and during pregnancy. Analysis of tissue weights, morphology, and gene expression was performed using standard histology, confocal imaging with three-dimensional analysis, real-time reverse transcription polymerase chain reaction (real-time RT-PCR), and microarrays. The response of ovariectomized adults to 17ß-estradiol (E2) and artificial decidualization were measured. Leukemia inhibitory factor (LIF) injections were given intraperitoneally and implantation sites visualized. Gene expression patterns were compared with curated data sets to identify upstream regulators. RESULTS: GEN-exposed mice exhibited reduced uterine weight gain in response to E2 treatment or artificial decidualization compared with controls; however, expression of select hormone responsive genes remained similar between the two groups. Uteri from pregnant GEN-exposed mice were posteriorized and had reduced glandular epithelium. Implantation failure was not rescued by LIF administration. Microarray analysis of GEN-exposed uteri during early pregnancy revealed significant overlap with several conditional uterine knockout mouse models, including Foxa2, Wnt4, and Sox17. These models exhibit reduced endometrial glands, features of posteriorization and implantation failure. Expression of Foxa2, Wnt4, and Sox17, as well as genes important for neonatal uterine differentiation (Wnt7a, Hoxa10, and Msx2), were severely disrupted on PND5 in GEN-exposed mice. DISCUSSION: Our findings suggest that neonatal GEN exposure in mice disrupts expression of genes important for uterine development, causing posteriorization and diminished gland function during pregnancy that contribute to implantation failure. These findings could have implications for women who consumed soy-based formulas as infants. https://doi.org/10.1289/EHP6336.

Developmentally Programmed Tankyrase Activity Upregulates ß-Catenin and Licenses Progression of Embryonic Genome Activation.

Embryonic genome activation (EGA) is orchestrated by an intrinsic developmental program initiated during oocyte maturation with translation of stored maternal mRNAs. Here, we show that tankyrase, a poly(ADP-ribosyl) polymerase that regulates ß-catenin levels, undergoes programmed translation during oocyte maturation and serves an essential role in mouse EGA. Newly translated TNKS triggers proteasomal degradation of axin, reducing targeted destruction of ß-catenin and promoting ß-catenin-mediated transcription of target genes, including Myc. MYC mediates ribosomal RNA transcription in 2-cell embryos, supporting global protein synthesis. Suppression of tankyrase activity using knockdown or chemical inhibition causes loss of nuclear ß-catenin and global reductions in transcription and histone H3 acetylation. Chromatin and transcriptional profiling indicate that development arrests prior to the mid-2-cell stage, mediated in part by reductions in ß-catenin and MYC. These findings indicate that post-transcriptional regulation of tankyrase serves as a ligand-independent developmental mechanism for post-translational ß-catenin activation and is required to complete EGA.

Effects of endocrine disruptors on obesity.

Environmental chemicals with hormone-like activity can disrupt the programming of endocrine signalling pathways that are established during perinatal life and result in adverse consequences that may not be apparent until much later in life. Increasing evidence implicates developmental exposure to environmental hormone mimics with a growing list of adverse health consequences in both males and females. Most recently, obesity has been proposed to be yet another adverse health effect of exposure to endocrine disrupting chemicals (EDCs) during critical stages of development. Obesity is quickly becoming a significant human health crisis because it is reaching epidemic proportions worldwide, and is associated with chronic illnesses such as diabetes and cardiovascular disease. In this review, we summarize the literature reporting an association of EDCs and the development of obesity, and further describe an animal model of exposure to diethylstilbestrol that has proven useful in studying mechanisms involved in abnormal programming of various oestrogen target tissues during differentiation. Together, these data suggest new targets (i.e. adipocyte differentiation and mechanisms involved in weight homeostasis) of abnormal programming by EDCs, and provide evidence that support the scientific term 'the developmental origins of adult disease'. The emerging idea of an association of EDCs and obesity expands the focus on obesity from intervention and treatment to include prevention and avoidance of these chemical modifiers.

Estradiol, progesterone, and genistein inhibit oocyte nest breakdown and primordial follicle assembly in the neonatal mouse ovary in vitro and in vivo.

In developing mouse ovaries, oocytes develop as clusters of cells called nests or germ cell cysts. Shortly after birth, oocyte nests dissociate and granulosa cells surround individual oocytes forming primordial follicles. At the same time, two thirds of the oocytes die by apoptosis, but the link between oocyte nest breakdown and oocyte death is unclear. Although mechanisms controlling breakdown of nests into individual oocytes and selection of oocytes for survival are currently unknown, steroid hormones may play a role. Treatment of neonatal mice with natural or synthetic estrogens results in abnormal multiple oocyte follicles in adult ovaries. Neonatal genistein treatment inhibits nest breakdown suggesting multiple oocyte follicles are nests that did not break down. Here we investigated the role of estrogen signaling in nest breakdown and oocyte survival. We characterized an ovary organ culture system that recapitulates nest breakdown, reduction in oocyte number, primordial follicle assembly, and follicle growth in vitro. We found that estradiol, progesterone, and genistein inhibit nest breakdown and primordial follicle assembly but have no effect on oocyte number both in organ culture and in vivo. Fetal ovaries, removed from their normal environment of high levels of pregnancy hormones, underwent premature nest breakdown and oocyte loss that was rescued by addition of estradiol or progesterone. Our results implicate hormone signaling in ovarian differentiation with decreased estrogen and progesterone at birth as the primary signal to initiate oocyte nest breakdown and follicle assembly. These findings also provide insight into the mechanism of multiple oocyte follicle formation.

Variations in phytoestrogen content between different mill dates of the same diet produces significant differences in the time of vaginal opening in CD-1 mice and F344 rats but not in CD Sprague-Dawley rats.

BACKGROUND: The optimum test diet and rodent species/strain for evaluating endocrine-disrupting compounds (EDCs) are critical. OBJECTIVES: We conducted studies to evaluate rodent species sensitivity and the effects of diets varying in phytoestrogen content on the time of vaginal opening (VO) in CD-1 mice, Fischer 344 (F344) rats, and CD Sprague-Dawley (S-D) rats. METHODS: Mice were weaned on postnatal day (PND) 15 and rats on PND19 and randomly assigned to control or test diets. Body weights, food consumption, and time of VO were recorded. RESULTS: The time of VO was significantly advanced in F344 rats fed diets containing daidzein and genistein, whereas these same diets did not advance VO in S-D rats. When animals were fed the AIN-76A diet spiked with genistein, time of VO was significantly advanced at all doses in CD-1 mice, at the two highest doses in F344 rats, and at the highest dose in S-D rats. The time of VO in F344 rats was more highly correlated with the phytoestrogen content than with the total metabolizable energy (ME) of 12 diets. CONCLUSIONS: The S-D rat is less sensitive to dietary phytoestrogens compared with the F344 rat or the CD-1 mouse, suggesting that the S-D rat is not the ideal model for evaluating estrogenic activity of EDCs. The profound effects of dietary phytoestrogens on the time of VO, an estrogen-sensitive marker, indicate that a standardized open-formula phytoestrogen-free diet containing a low ME level should be used to optimize the sensitivity of estrogenic bioassays.

Disruption of the developing female reproductive system by phytoestrogens: genistein as an example.

Studies in our laboratory have shown that exposure to genistein causes deleterious effects on the developing female reproductive system. Mice treated neonatally on days 1-5 by subcutaneous injection of genistein (0.5-50 mg/kg) exhibited altered ovarian differentiation leading to multioocyte follicles (MOFs) at 2 months of age. Ovarian function and estrous cyclicity were also disrupted by neonatal exposure to genistein with increasing severity observed over time. Reduced fertility was observed in mice treated with genistein (0.5, 5, or 25 mg/kg) and infertility was observed at 50 mg/kg. Mammary gland and behavioral endpoints were also affected by neonatal genistein treatment. Further, transgenerational effects were observed; female offspring obtained from breeding genistein treated females (25 mg/kg) to control males had increased MOFs. Thus, neonatal treatment with genistein at environmentally relevant doses caused adverse consequences on female development which is manifested in adulthood. Whether adverse effects occur in human infants exposed to soy-based products such as soy infant formulas is unknown but the neonatal murine model may help address some of the current uncertainties since we have shown that many effects obtained from feeding genistin, the glycosolated form of genistein found in soy formula, are similar to those obtained from injecting genistein.

Perinatal exposure to environmental estrogens and the development of obesity.

Dietary substances and xenobiotic compounds with hormone-like activity can disrupt the programming of endocrine signaling pathways that are established during perinatal differentiation. The consequences of this disruption may not be apparent until later in life but increasing evidence implicates developmental exposure to environmental hormone-mimics with a growing list of adverse health effects including reproductive problems and increased cancer risks. Obesity has recently been proposed to be yet another adverse health consequence of exposure to endocrine disrupting substances during development. There is a renewed focus on identifying contributions of environmental factors to the development of obesity since it is reaching worldwide epidemic proportions, and this disease has the potential to overwhelm healthcare systems with associated illnesses such as diabetes and cardiovascular disease. Here, we review the literature that proposes an association of perinatal exposure to endocrine disrupting chemicals, in particular those with estrogenic activity, with the development of obesity later in life. We further describe an animal model of developmental exposure to diethylstilbestrol (DES) to study mechanisms involved in programming for obesity. Our experimental data support the idea that adipocytes and the mechanisms involved in weight homeostasis are novel targets of abnormal programming of environmental estrogens, some of which are found in our foods as naturally occurring substances or inadvertently as contaminants.