Uterine Patterning, Endometrial Gland Development, and Implantation Failure in Mice Exposed Neonatally to Genistein
2020; National Institute of Environmental Health Sciences; Volume: 128; Issue: 3 Linguagem: Inglês
10.1289/ehp6336
ISSN1552-9924
AutoresWendy N. Jefferson, Elizabeth Padilla‐Banks, Alisa A. Suen, Lindsey J. Royer, Sharon M. Zeldin, Ripla Arora, Carmen J. Williams,
Tópico(s)Effects and risks of endocrine disrupting chemicals
ResumoVol. 128, No. 3 ResearchOpen AccessUterine Patterning, Endometrial Gland Development, and Implantation Failure in Mice Exposed Neonatally to Genistein Wendy N. Jefferson, Elizabeth Padilla-Banks, Alisa A. Suen, Lindsey J. Royer, Sharon M. Zeldin, Ripla Arora, and Carmen J. Williams Wendy N. Jefferson Address correspondence to Wendy N. Jefferson, NIEHS/NIH/DHHS, P.O. Box 12233, MD E4-05, Research Triangle Park, NC 27709 USA. Email: E-mail Address: [email protected] Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, USA , Elizabeth Padilla-Banks Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, USA , Alisa A. Suen Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, USA , Lindsey J. Royer Department of Obstetrics, Gynecology, and Reproductive Biology, Institute for Quantitative Health Science and Engineering, College of Human Medicine, Michigan State University, East Lansing, Michigan, USA , Sharon M. Zeldin Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, USA , Ripla Arora Department of Obstetrics, Gynecology, and Reproductive Biology, Institute for Quantitative Health Science and Engineering, College of Human Medicine, Michigan State University, East Lansing, Michigan, USA , and Carmen J. Williams Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, USA Published:18 March 2020CID: 037001https://doi.org/10.1289/EHP6336Cited by:4AboutSectionsPDF Supplemental Materials ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmail AbstractBackground: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 17β-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/EHP6336IntroductionDevelopmental exposure to estrogenic chemicals is associated with abnormalities in the female reproductive tract that lead to infertility and cancer in women and in mouse models (Reed and Fenton 2013). Phytoestrogens, or plant-derived estrogenic compounds, are a group of environmentally relevant chemicals that exert estrogenic activity. A major source of phytoestrogens in the diet is the isoflavones found in soy (Kurzer and Xu 1997). Survey studies estimating the amount of soy protein and isoflavones consumed per day by Asian adults suggest an intake of 6–11g of soy protein of which 25–50mg is isoflavones (Messina et al. 2006), an approximate isoflavone exposure of <1mg/kg per day. In a small study (n=194) of adult Asian-American women, the participants were found to have serum circulating levels of isoflavones of <100 nM (Wu et al. 2004). The most prevalent isoflavone in human exposures is genistein (GEN), which makes up approximately 65% of the isoflavone content in soy products (Adlercreutz and Mazur 1997; Kurzer and Xu 1997). In a small study of British women (n=100) split into four groups ranging from no soy intake to high soy intake, the participants were found to have circulating levels of GEN from 14.3 to 378 nM (Verkasalo et al. 2001). A much higher exposure to isoflavones occurs in human infants fed soy-based infant formulas, with estimates of around 10mg/kg per day in one small study (Setchell et al. 1997). The high intake of soy isoflavones in human infants most likely results in higher exposure rates than in adults. Supporting this statement, a small study of human infants fed exclusively soy-based infant formula had serum circulating levels of GEN (1–10μM), which is at least 10-fold higher than any natural exposure of adults (Cao et al. 2009; Setchell et al. 1997).A mouse model of developmental phytoestrogen exposure was described previously where neonatal CD-1 female pups were exposed to GEN at 50mg/kg per day by subcutaneous injection (Doerge et al. 2002). This dosing strategy produced serum circulating levels that closely approximated the levels measured in a study with human infants consuming soy-based infant formula (Cao et al. 2009). In this model system, female mice exposed neonatally to GEN were infertile (Jefferson et al. 2005), and 35% of the mice developed uterine cancer later in life (Newbold et al. 2001). A hallmark of this cancer is abnormal cellular differentiation characterized by distinct basal cell populations that express proteins normally restricted to the cervix and upper vagina (Suen et al. 2016, 2018). This phenotype is consistent with our findings of posteriorization in the oviduct of GEN-exposed mice (Jefferson et al. 2011). Reproductive tract posteriorization has been described in mouse models with deletions of important uterine patterning genes, most notably the Hoxa and Wnt gene families (Du and Taylor 2015; Franco et al. 2011; Hayashi et al. 2011). These data suggest that neonatal exposure to GEN alters female reproductive tract differentiation and leads to a molecular signature resembling patterning gene deletions.GEN-exposed mice exhibit complete infertility for multiple reasons, including abnormal function of the hypothalamic–pituitary–gonadal axis, leading to ovulation failure and abnormal estrous cycling, deficits in oviductal support of preimplantation embryo development, and failure of embryo development following implantation (Jefferson et al. 2005, 2009). Implantation is a complex process orchestrated by a carefully timed series of estrogen and progesterone signals (Lee et al. 2007; Spencer 2014). These hormonal signals drive endometrial proliferation, which is followed by endometrial differentiation events that create a short window of implantation during which the endometrium can support invasion by the embryo. Glands within the endometrial stroma produce factors required for implantation such as enzymes, transporters, and secreted proteins. One of the most important factors is leukemia inhibitory factor (LIF), without which implantation does not occur (Salleh and Giribabu 2014; Stewart et al. 1992). Upon embryo implantation, the uterine stroma undergoes decidualization, during which the stromal cells expand and differentiate to support the fetal–maternal interface required for proper embryo development.Complex hormonal signaling and precise timing of endometrial proliferation and differentiation events are crucial for successful embryo implantation and growth, but how neonatal GEN exposure disrupts this process is unknown. Here we comprehensively examined the mechanisms underlying neonatal GEN-exposure–induced deficits in uterine support for implantation.Material and MethodsAnimalsTimed-pregnant CD-1 mice were obtained from the in-house National Institutes of Health/National Institute of Environmental Sciences (NIH/NIEHS, Research Triangle Park, NC) breeding colony. Mice were handled under approved animal care and use protocols according to NIH/NIEHS guidelines. Mice were fed NIH-31 diet and housed in a temperature-controlled environment (21–22°C) with a 12-h light:12-h dark cycle. At birth, postnatal day 1 (PND1), pups were randomly standardized to 10 female pups per dam. Female pups were treated by subcutaneous injection of 0.02mL of corn oil [control (CON); Spectrum; Catalog No. CO136] or GEN (Sigma; Catalog No. G6649) dissolved in corn oil at 50mg/kg per day on PND1 through PND5, as described previously (Jefferson et al. 2005). The corn oil used in this study was confirmed to have no detectable estrogenic activity in a uterotrophic bioassay, as previously described (Jefferson et al. 2002). Uterine tissues were collected on PND5, 4 h after the last injection or the mice were weaned at PND21 and housed five per cage. Uteri were collected at various time points detailed in subsequent "Methods" sections. At 6–8 weeks of age, females were either ovariectomized for hormone response assays, superovulated and bred, or naturally bred for prepregnancy and early pregnancy end points. Of note, all females used for natural breeding time points in this study were 6 weeks of age. GEN-exposed mice do not become anovulatory until later in life (at ∼4 months of age) (Jefferson et al. 2005). Superovulation was accomplished by subcutaneous injection of 5 IU equine chorionic gonadotropin (eCG; Sigma; Catalog No. E4877) in 0.1mL saline followed 48 h later with 5 IU human chorionic gonadotropin (hCG; EMD Millipore; Catalog No. 230734) in 0.1mL saline. A small group of superovulated mice was used for serum collection 24 h post hCG treatment without breeding. For early pregnancy end points, superovulated females or naturally bred females were placed with proven fertile males and the presence of a vaginal plug was used as a determination of pregnancy and considered gestational day (GD) 0.5. For intact mice at 2 months of age, uteri were collected and the estrous cycle stage determined by vaginal cytology. The number of mice used for each experiment is detailed in each "Methods" section below.Mice with conditional deletion of Foxa2 in the uterus were generated by crossing Foxa2-floxed mice (Stock No. 022620; Jackson Laboratory) with Pgr-cre mice (from F. DeMayo, NIEHS, Research Triangle Park, NC) (Soyal et al. 2005). Mice that were flox/flox, cre– served as wild-type controls (Foxa2 WT), whereas littermates that were flox/flox, cre+ were lacking Foxa2 in the uterus (Foxa2 cKO). Housing and weaning were as described above. At 2 months of age, Foxa2 WT and Foxa2 cKO female mice were bred to Foxa2 WT males until the detection of a vaginal plug. Only vaginal plug–positive mice were used in the experiments described below.Whole-Mount ImmunofluorescenceUteri were collected from female mice (CON and GEN; n=10 per group) at 26 d of age (prior to establishment of estrous cyclicity) and immediately fixed in dimethyl sulfoxide:methanol (1:4 ratio) and stored at −20°C until processing. Immunofluorescence was carried out, as previously described (Arora et al. 2016). In brief, tissues were blocked using 2% powdered milk and 1% Triton™ X-100 in phosphate buffered saline (PBS) for 2 h at room temperature (RT). Primary antibodies for mouse CDH1 (Clontech; Catalog No. M108) and FOXA2 (Abcam; Catalog No. ab108422) were diluted (1:200) in block, and uteri were incubated for 5 d at 4°C. Uteri were washed (PBS + 1% Triton) six times for 30 min each and incubated with secondary antibodies, fluorescently conjugated Alexa Fluor IgGs (1:500), donkey anti-rabbit (Invitrogen; Catalog No. A31572), and goat anti-rat (Invitrogen; Catalog No. A21247) for 2 d at 4°C. Uteri were washed (1% Triton™ X-100 in PBS) six times for 30 min each, dehydrated in methanol, and incubated overnight in 3% hydrogen peroxide diluted in methanol. Uteri were washed in 100% methanol twice for 15 min each and cleared overnight using benzyl alcohol:benzyl benzoate, 1:2 ratio. Uteri were imaged using a Leica SP8 TCS confocal microscope with white-light laser, using a 10× air objective with z-stacks 7μm apart. Full uterine horns were imaged using tile scans and tiles were merged using the mosaic merge function of the Leica software (version 3.5.5; Leica Microsystems).Image AnalysisLeica immunofluorescence software files (Leica) were analyzed using Imaris (version 9.2; Bitplane Imaris). Surfaces were created in surpass three-dimensional mode for the CDH1+ lumen signal and the FOXA2+ glandular signal. Gland numbers were determined by number of disconnected components in the FOXA2+ gland surface, as previously described (Arora et al. 2016). Images of the surfaces and the two-dimensional optical slices were captured using the snapshot function.Hormone Response AssaysThe treatment regimen for the hormone response assays is depicted in Figures 1A and 2A. Female mice (CON and GEN) were ovariectomized at 6 weeks of age and allowed to recover for 10–14 d to clear endogenous hormones. The mice were then treated by subcutaneous injection of 0.1mL of corn oil vehicle (Veh) or one of the following hormone regimens using 17β-estradiol (E2; Sigma; Catalog No. E8875) or progesterone (P4; Sigma; Catalog No. P0130) or a combination of the two as follows: a) E2 group; 25μg/mouse for 2 h or 24 h (n=5 per group); b) E2-3X group; E2 25μg/mouse for three daily injections collected 24 h after the last injection for uterine weight (n=5 per group); c) E2 + P4 group; E2 6.7 ng/mouse for 3 d, rest for 2 d, E2 100 ng/mouse + P4 40mg/mouse for 3 d, collected 24 h after last injection (n=5 per group); d) Decidua group; E2 + P4 regimen but 24 h after last dose the uterine horn was infused with corn oil to initiate decidual response, as previously described (Paria et al. 1999). Uteri were collected either 2 h after oil infusion or continued E2 + P4 for 8 consecutive d and then uteri were collected 24 h after the last injection for uterine weight (n=20 per group); and e) P4 group; 40mg/mouse for 6 h or 24 h (n=5 per group). All uteri collected for molecular hormone response assays were frozen at −80°C until further use.Figure 1. Uterine response to estradiol in genistein (GEN)-exposed mice. (A) Schematic depicting treatment and collection timing for 17β-estradiol (E2) experiments. Each arrow represents a single injection of the chemical indicated above the arrows. (B) Uterine wet weight (in grams) is plotted for each time point [Vehicle (Veh), E2-2h, E2-24h, and E2-3X] for both control (CON) and GEN-exposed mice. (C) Real-time RT-PCR of select E2-regulated genes 2 h post E2 treatment in CON and GEN-exposed mice. (D) Real-time RT-PCR of select E2-regulated genes 24 h post E2 treatment in CON and GEN-exposed mice. Mean±SEM is plotted along with individual values for all panels. Different letters indicate significance at p≤0.05 using Kruskal-Wallis test followed by Dunn's test (n=3–5 per group). Note: RT-PCR, reverse transcription polymerase chain reaction; SEM, standard error of the mean.Figure 2. Decidual response in GEN-exposed mice. (A) Schematic depicting treatment and collection timing for decidua experiments (top) and progesterone (P4) experiments (bottom); events prior to the dashed line are the same for each group. Each arrow represents a single injection of the chemical indicated above the arrows. Uterine tissue collection time points are indicated by an arrow. (B) Uterine weight (in grams) in non–oil-infused horn and oil-infused horn with decidual response in CON and GEN-exposed mice. (C) Real-time RT-PCR of select decidual response genes without oil infusion or 2 h following oil infusion. (D) Real-time RT-PCR of select progesterone-regulated genes 6 h and 24 h after P4 treatment. Mean±SEM is plotted along with individual values for all panels. Different letters indicate significance at p≤0.05 using Kruskal-Wallis test followed by Dunn's test (n=7–17 per group for A; n=3–5 per group for B and C). Note: CON, control; GEN, genistein; RT-PCR, reverse transcription polymerase chain reaction; SEM, standard error of the mean.Hormone AssaysEight-week-old female mice (CON or GEN) were superovulated, as described above in the "Animals" section; half of the mice were bred to proven male breeders and pregnancy confirmed by vaginal plug the next day (n=5–7 mice per group). Blood was collected from the vena cava 48 h after eCG (no hCG; nonpregnant) or 24 h after hCG from vaginal plug–positive mice (pregnant); uteri were also collected and frozen at −80°C at both time points. Blood was allowed to clot for 1 h at RT and then centrifuged at 8,000×g at 4°C for 10 min to isolate serum. E2 and P4 serum levels were measured using respective radioimmunoassay kits (Diagnostic Systems Laboratory: P4, Catalog No. DSL 3400; E2, Catalog No. DSL 4400) according to the manufacturer's instructions.Early Pregnancy and LIF RescueEight-week-old female mice (CON or GEN) were bred to proven male breeders (2:1) until vaginal plug detection (GD0.5). Uteri were collected on GD1.5, GD3.5, GD4.5, or GD5.5 and frozen at −80°C until RNA isolation (n=4 per group). For GD4.5 or GD5.5, vaginal plug–positive mice were given a tail vein injection of Evan's blue dye (1% in 0.1mL saline) 2–3 min prior to being euthanized to visualize implantation sites; only mice that had confirmed blue implantation sites were used for further analysis. For LIF rescue, vaginal plug–positive CON and GEN females were treated with either saline or LIF (BioLegend; Catalog No. 554008; 10mg/mouse) in 0.1mL saline by intraperitoneal injection on GD3.5 (n=9–12 per group). On GD5.5, pregnant mice were given a tail vein injection of Evan's blue dye and the number of implantation sites were counted; GD9.5 implantation sites were counted without blue dye injection (n=6–7 per group). Implantation sites were counted and imaged. As a positive control for the LIF rescue, 8-week-old female Foxa2 WT or Foxa2 ut-cKO mice (n=5–7 mice per group) were bred to Foxa2 WT males until vaginal plug detection, as previously described (Jeong et al. 2010); LIF injections on GD3.5 rescued implantation failure in Foxa2 ut-cKO mice on GD5.5 as visualized by Evan's blue dye.Histopathology and ImmunohistochemistryUterine tissues collected from PND5 pups, intact mice at 2 months of age, and pregnant mice on GD5.5 were fixed in 10% neutral buffered formalin [0.29 M monosodium phosphate (NaH2PO4-H2O), 0.24 M disodium phosphate (Na2HPO4-7H2O), 10% formaldehyde] overnight at 4°C, which was then changed to ice cold 70% ethanol (n=3–5 per group). The tissues were processed, embedded in paraffin, and sectioned at 6μm. Tissue sections were stained with either hematoxylin and eosin (H&E) or Masson's trichrome stain using standard protocols or immunostained using standard protocols (Carson and Hladik 2009; Munro 1971). KRT14 immunostaining was performed using the standard avidin–biotin–peroxidase technique previously reported (Suen et al. 2018). In brief, rabbit polyclonal anti-KRT14 antibody (BioLegend; Catalog No. PRB-155P; concentration 0.8mg/mL) was used as the primary antibody and biotinylated donkey anti-rabbit IgG (Jackson ImmunoResearch; 1:500 dilution) was used as the secondary, and the complex was visualized using 3-diaminobenzidine (DAB) chromagen (Dako). FOXA2 immunostaining was performed following heat-induced antigen retrieval using a Decloaker (Biocare Medical) with 1× ethylenediaminetetraacetic acid pH 8.5 for 15 min at 110°C. Endogenous peroxide was quenched using 3% hydrogen peroxide for 15 min. Sections were incubated with Rodent Block M (BioCare Medical) for 20 min at RT. Rabbit monoclonal anti-FOXA2 (Cell Signaling; Catalog No. 8186S; concentration 1mg/mL) was applied for 1 h at RT. Tissues were then incubated with Rabbit on Rodent Horse Radish Peroxidase (HRP) Polymer (BioCare Medical; Catalog No. RMR622) for 30 min at RT. Complexes were visualized using DAB.Protein Isolation and ImmunoblottingPND5 or GD3.5 frozen uterine tissues (n=4 per group) were pulverized on dry ice, and 20mg of crushed tissue was homogenized by hand-held homogenizer using 200μL of tissue protein extraction reagent (T-PER™; Invitrogen). Samples were centrifuged at 10,000 rpm for 5 min and the total protein extract was removed from the pellet. Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher; Catalog No. 1861281) was added at 1:100 to prevent protein degradation. The protein concentration was determined using the Qubit protein assay (Life Technologies; Catalog No. Q33212). Twenty micrograms of total protein was electrophoresed on a 4–20% Tris-glycine sodium dodecyl sulfate gel (Bio-Rad) and transferred to a polyvinylidene fluoride membrane (Bio-Rad) using a Trans-Blot turbo (Bio-Rad) for 7 min. Blots were blocked with 5% milk in Tris buffered saline (TBS; Bio-Rad; Catalog No. 1706435) plus 0.1% Tween-20 (TBS-T; Sigma; Catalog No. P1379) for 1 h at RT and then primary antibodies applied overnight at 4°C. The primary antibodies were diluted in 5% milk in TBS-T at the following concentrations: anti-FOXA2 (0.20μg/mL); anti-SOX17 (1.0μg/mL; R&D; Catalog No. AF1924); anti-WNT4 (0.5μg/mL; R&D; Catalog No. AF475). Blots were washed 3×15 min in TBS-T and the appropriate peroxidase-conjugated secondary antibody diluted in 1% milk in TBS-T was applied for 1 h at RT: anti-rabbit (0.032μg/mL; Jackson Immunoresearch; 711-035-152); anti-goat (0.032μg/mL; Jackson Immunoresearch; 805-035-180); anti-mouse (1:10,000; GE Healthcare; Catalog No. NA931V). Blots were washed 3×15 min with TBS-T. Blots were incubated with Super Signal West Femto Chemiluminescent Substrate (Thermo Fisher; Catalog No. 34095) and visualized using the ChemiDoc Touch Imaging System (Bio-Rad). All blots were stripped using Restore Western Blot Stripping Buffer (Thermo Fisher; Catalog No. 21059). Beta-actin immunoblotting was performed as a loading control, as described above, using anti-β-actin (anti-ACTB; 2μg/mL; Sigma; Catalog No. A1978) and then HRP-conjugated anti-mouse (1:10,000; GE Healthcare; Catalog No. NA931V). ACTB blots were visualized using Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher; Catalog No. 34087) and the ChemiDoc Touch Imaging System (Bio-Rad).RNA Isolation and MicroarrayFrozen uteri from CON- and GEN-exposed mice from PND5, PND22, hormone response assays, and early pregnancy were pulverized on dry ice and total RNA isolated using the Qiagen RNeasy kit and DNaseI cleanup kit on the column (Qiagen; Catalog Nos. 74104 and 79254) (n=3–5 per group). For real-time reverse transcription polymerase chain reaction (real-time RT-PCR), 1μg RNA was reverse transcribed to make complementary DNA (cDNA) using the First Strand Synthesis kit (Invitrogen; Catalog No. 11904) following the manufacturer's instructions. Real-time RT-PCR was performed using 20 ng cDNA, 2X SYBR green (Thermo Fisher; Catalog No. 4367659), and primers designed using Primer 3 Express (Koressaar and Remm 2007) using an Applied Biosystems Step One Plus real-time PCR machine with standard settings (Thermo Fisher). A list of primers (Sigma) can be found in the Supplemental Material in Excel File 1.For microarray analysis, total RNA was isolated from uterine tissues (n=4 per group) using the RNeasy Mini Kit and RNase free DNase set (Qiagen; Catalog Nos. 74104 and 1080901). Microarray was performed using Agilent Whole Mouse Genome 4×44 multiplex format oligo arrays (Agilent Technologies), as previously described (Jefferson et al. 2011). Differentially expressed genes were determined by the Genomics Suite Gene Expression workflow of Partek software package (version 6.6; Partek). To identify differentially expressed probes, a raw data cutoff of <10 and log2-transformed analysis of variance (ANOVA) with unadjusted p<0.05 was applied to determine if there was a statistical difference between the means of the groups. The resulting significantly altered genes were subjected to a ±1.5-fold change cutoff and used to generate heat maps in Partek and were uploaded into the NextBio Correlation Engine ( http://www.nextbio.com/b/nextbio.nb; Illumina) for pathway enrichment and correlation to published studies. Published studies that exhibited high overlap with our data sets in NextBio and used to generate data are referenced in the relevant "Results" sections. All experiments were performed with at least three independent uterine samples from individual mice. Array data have been deposited in the Gene Expression Omnibus (GEO accession no. GSE138500).StatisticsAll statistical analyses were performed using GraphPad Prism (version 8.2.1; GraphPad Software). For uterine weight and serum hormone levels, a one-way ANOVA was performed, followed by Tukey's multiple comparison test. For real-time RT-PCR data with multiple comparisons, a Kruskal-Wallis test was performed, followed by uncorrected Dunn's test; each time point was independently collected. For real-time RT-PCR on uterine gland gene expression, a Mann-Whitney one-tailed test was performed (only testing for anticipated decrease). For the number of implantation sites per mouse in the LIF rescue experiment, a Kruskal-Wallis test was performed, followed by Dunn's test, and for the number of mice exhibiting pregnancy, a chi-square analysis was performed. For all tests, p≤0.05 was considered significant. All raw data for this manuscript can be found in the Supplemental Material in Excel File 2, with each tab containing the raw data used to generate a specific figure.ResultsEstrogen Response in GEN-Exposed MiceWe hypothesized that neonatal GEN exposure impaired implantation by disrupting estrogen signaling responses in the adult uterus. To test this idea, we performed a standard uterine wet weight assay on ovariectomized CON and GEN-exposed mice; the experimental design is depicted in Figure 1A. CON mice treated with E2 for 24 h (CON E2-24h) or E2 daily for 3 d (CON E2-3X) showed the expected significant increase in uterine wet weight compared with CON treated with corn oil vehicle (CON Veh) (Figure 1B). GEN-exposed mice also had a significant increase in uterine weight at these time points (GEN E2-24h and GEN E2-3X) compared with GEN Veh, but that response was dampened compared with CON mice (Figure 1B). Despite this dampened response, select estrogen-regulated genes (Hewitt et al. 2015) were not different between CON and GEN-exposed mice at 2 h or 24 h post E2 treatment (Figure 1C,D; see also Figure S1A,B). For example, Ltf exhibited the normal pattern of enhanced induction of expression following E2 treatment, with approximately 10-fold increases in both CON and GEN at 24 h and approximately 5,000-fold increases in both CON and GEN after 3 d of treatment (see Figure S1C). One exception was the overexpression of Ccnb2 in the GEN Veh group compared with CON Veh; however, there was no difference observed between the E2-treated CON and GEN groups.Decidualization and Progesterone Responses in GEN-Exposed MiceGiven that proper endometrial decidualization is required for successful implantation, we next tested whether neonatal GEN exposure impacted this response. Female CON and GEN-exposed mice were ovariectomized and then treated with a standard regimen of hormones and intrauterine oil infusion to elicit artificial decidualization; the experimental design is shown in Figure 2A (Paria et al. 1999). CON mice had the expected increase in uterine weight in the decidualized horn compared with the non–oil-infused horn (Figure 2B). Although GEN-exposed mice exhibited an increase in weight of the decidualized horn, the increase was significantly dampened compared with CON mice (Figure 2B). However, select characteristic gene expression changes induced by decidualization were not altered in GEN-exposed mice compared with CON mice (Figure 2C). In addition, well-characterized P4-responsive genes showed similar increases in both CON and GEN-exposed mice (Figure 2D).Serum Circulating Hormone Levels in GEN-Exposed MiceTo determine if implantation failure could result from alterations in circulating hormone levels, we measured serum levels of E2 and P4. Because the GEN-exposed mice were previously shown to have irregular cycles and only 50–60% of mice showed signs of pregnancy following vaginal plug detection (Jefferson et al. 2005), we m
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