Glyphosate-based herbicide worsens alterations induced by cafeteria diet on rat uterus.

Exposure to glyphosate-based herbicides (GBH) and consumption of cafeteria (CAF) diet, which are widespread in Western society, seem to be associated with endometrial hyperplasia (EH). Here, we aimed to evaluate the effects of a subchronic low dose of GBH added to the CAF diet on the rat uterus. Female Wistar rats were fed from postnatal day (PND)21 until PND240 with chow (control) or CAF diet. Since PND140, rats also received GBH (2 mg of glyphosate/kg/day) or water through food, yielding four experimental groups: control, CAF, GBH, and CAF+GBH. On PND240, CAF and CAF+GBH animals showed an increased adiposity index. With respect to the control group, no changes in the serum levels of 17ß-estradiol and progesterone were found. However, progesterone levels were higher in the CAF+GBH group than in the CAF and GBH groups. In the uterus, both studied factors alone and in combination induced morphological and molecular changes associated with EH. Furthermore, the addition of GBH provoked an increased thickness of subepithelial stroma in rats fed with the CAF diet. As a consequence of GBH exposure, CAF+GBH rats exhibited an increased density of abnormal gland area, considered preneoplastic lesions, as well as a reduced PTEN and p27 expression, both tumor suppressor molecules that inhibit cell proliferation, with respect to control rats. These results indicate that the addition of GBH exacerbates the CAF effects on uterine lesions and that the PTEN/p27 signaling pathway seems to be involved. Further studies focusing on the interaction between unhealthy diets and environmental chemicals should be encouraged to better understand uterine pathologies.

Epigenetic disruption of placental genes by chronic maternal cafeteria diet in rats.

Maternal diet has impact on reproduction, fetal development and offspring behavior, although molecular mechanisms remained unknown. Our aims were to assess (1) the effects of a cafeteria (CAF) diet (western diet habits) on female reproductive performance, fetal and placental parameters on gestational day 21 and litter size and pup weight at birth; and (2) placental messenger RNA (mRNA) expression and epigenetic regulation of Insulin-Like Growth Factor (Igf) and Vascular Endothelial Growth Factor (Vegf) and their receptors. Female Wistar rats were fed with control or CAF diet from weaning until parturition. At week 14 after diets started, females were mated and half of the animals were euthanized on gestational day 21 to evaluate reproductive parameters including the pregnancy rate, number of corpora lutea, implantation sites and resorption sites. Moreover, fetal weight and length, placental weight, and placental index were recorded. Placentas were collected for mRNA quantification and DNA methylation analysis. The remaining animals were allowed to give birth and the number and weight of the pups were evaluated. CAF diet did not affect reproductive performance or fetal weight and length. However, CAF-fed animals showed a decrease in placental weight and index and the pups exhibited a low birth weight. Additionally, we found an upregulation of Igf2 and a down regulation of Vegf placental mRNA expression in CAF dams, associated with methylation status changes of their promoters. We conclude that female chronic CAF diet consumption impairs feto-placental development and could be explained by an epigenetic disruption of Igf and Vegf systems.

Aberrant Hoxa10 gene methylation as a mechanism for endosulfan-induced implantation failures in rats.

DNA methylation is a well-established epigenetic mechanism controlling gene expression. Environmental chemicals, such as pesticides have been shown to alter DNA methylation. We have previously shown that the insecticide endosulfan impairs female fertility in rats by increasing the rate of preimplantation embryo losses. In this study, we evaluated whether early postnatal exposure to endosulfan affects long-term transcriptional regulation of Homeobox A10 (Hoxa10) gene, which is a key marker of endometrial receptivity. Female rats were neonatally exposed to 6 or 600 µg/kg/day (ENDO6 and ENDO600, respectively) of endosulfan and uterine samples collected on gestational day (GD) 5. Hoxa10 protein and mRNA levels were assessed by immunohistochemistry and quantitative real-time PCR (qRT-PCR), respectively. In silico analysis of enzyme-specific restriction sites and predicted transcription factors were performed to investigate the methylation status of the regulatory regions of Hoxa10 gene by methylation-sensitive restriction enzymes-PCR technique. The expression of the DNA methyltransferases (Dnmts) was also evaluated. ENDO600 showed a decreased uterine Hoxa10 expression at protein and transcript level, while ENDO6 decreased only the level of transcripts, during the receptive stage. In addition, endosulfan increased levels of Dnmt3a and Dnmt3b. Dysregulation of DNA methylation patterns of Hoxa10 regulatory regions was detected in ENDO6- and ENDO600-treated rats. All these results suggest that aberrant DNA methylation in Hoxa10 gene could be an underlining mechanism contributing to explain endosulfan-induced preimplantation losses.

Disruption of developmental programming with long-term consequences after exposure to a glyphosate-based herbicide in a rat model.

Glyphosate-based herbicides (GBHs) have been associated with endocrine disrupting effects on reproductive organs. We examined whether postnatal exposure to GBH affects developmental programming of the uterus with long-term consequences. Female Wistar pups received vehicle (control) or GBH (2 mg of glyphosate/kg/day) from postnatal day (PND) 1 to PND7, where the developing uterus is highly sensitive to endocrine disruption. Short-, mid- and long-term effects were evaluated on PND8, PND120 and PND600, respectively. GBH induced hyperplasia and epigenetic alterations in the uterus of neonatal females (PND8). DNA hypermethylation, enrichment of H3K9me3 and reductions of H3K27me3 at regulatory regions of the morphoregulatory gene Hoxa10 resulted in gene downregulation. In young adult females (PND120), GBH increased 17ß-estradiol (E2) and decreased progesterone (P4) serum levels, altering estrous cyclicity. Aged females (PND600) exposed to GBH developed leiomyoma and pre-neoplastic glandular lesions in the uterus. Vaginal rhabdomyosarcoma and intrahepatic bile duct adenoma were also observed. In conclusion, neonatal exposure to GBH altered the expression and induced hypermethylation of the Hoxa10 gene in uterine tissue at early life, and increased E2/P4 ratio serum level at middle-age. We propose that epigenetic reprogramming of Hoxa10 in association with hormonal imbalance could be among the possible mechanisms underlying the long-term adverse effects detected in GBH-exposed rats.

AHR agonistic effects of 6-PN contribute to potential beneficial effects of Hops extract.

Hops (Humulus lupulus) is used as an alternative to hormone replacement therapy due to the phytoestrogen, 8-prenylnaringenin (8-PN). To examine the potential risks/benefits of hops extract and its compounds (8-PN and 6-prenylnaringenin, 6-PN), we aimed to evaluate the estrogen receptor α (ERα) and aryl hydrocarbon receptor (AHR) signaling pathways in human endometrial cancer cells. Hops extract, 8-PN and 6-PN showed estrogenic activity. Hops extract and 6-PN activated both ERα and AHR pathways. 6-PN increased the expression of the tumor suppressor gene (AHRR), and that of genes involved in the estrogen metabolism (CYP1A1, CYP1B1). Although 6-PN might activate the detoxification and genotoxic pathways of estrogen metabolism, hops extract as a whole only modulated the genotoxic pathway by an up-regulation of CYP1B1 mRNA expression. These data demonstrate the relevant role of 6-PN contained in the hops extract as potential modulator of estrogen metabolism due to its ERα and AHR agonist activity.

Altered uterine angiogenesis in rats treated with a glyphosate-based herbicide.

Glyphosate-based herbicides (GBHs) are the agrochemicals most used around the globe. However, they might have adverse effects on human and animal health. Previously, we showed that female rats neonatally exposed to GBHs exhibit altered expression of morphogenetic molecules and biomarkers of uterine development. We also observed a reduction in the size of implantation sites, altered expression of decidualization-related molecules, and increased post-implantation losses. Since decidualization comprises morphogenetic, biochemical and vascular changes, here we investigated the effects of neonatal GBH exposure on uterine angiogenesis in neonatal and pregnant rats. To achieve this, Wistar female rats were exposed to saline solution or GBH (2 mg glyphosate/kg-bw/day) on post-natal days (PND) 1, 3, 5 and 7. On PND8, uterine samples were collected for developmental studies. On PND90, the remaining females were mated and in the morning of gestational day (GD) 9, the implantation sites were collected. Angiogenesis-related molecules and cells involved in this process were identified and/or measured by immunohistochemistry or RT-PCR. On PND8, GBH-treated rats showed increased vascular endothelial growth factor (VEGF) expression and decreased Notch1, inducible nitric oxide synthase (iNOS) and Angiopoietin-2 (Ang2) mRNA levels. Vascular area, vessel diameter, endothelial cell proliferation, VEGF and Nestin protein expression, and VEGF, Notch1, iNOS and cyclooxygenase-2 (Cox-2) genes were downregulated in implantation sites of exposed females, while Ang2, VEGF receptor 1 and interleukin-10 (IL-10) were increased. Mast cells and macrophages were increased on PND8 and GD9 of treated rats. The increased Transforming growth factor-beta expression in the antimesometrial zone and IL-10 mRNA expression suggest that the M2 type is the predominant population of macrophages on implantation sites. In conclusion, neonatal GBH exposure alters the expression of angiogenesis-related molecules at neonatal uterine development and decidual reaction, suggesting altered vascular support. These alterations might contribute to the increased post-implantation losses observed in GBH-treated rats.

Epigenetic Changes Associated With Exposure to Glyphosate-Based Herbicides in Mammals.

Glyphosate is a phosphonomethyl amino acid derivative present in a number of non-selective and systemic herbicides. During the last years the use of glyphosate-based herbicide (GBH) has been increasing exponentially around the world, including Argentina. This fact added to the detection of glyphosate, and its main metabolite, amino methylphosphonic acid (AMPA), in environmental matrices such as soil, sediments, and food, has generated great concern about its risks for humans, animals, and environment. During the last years, there were controversy and intense debate regarding the toxicological effects of these compounds associated with the endocrine system, cancer, reproduction, and development. The mechanisms of action of GBH and their metabolites are still under investigation, although recent findings have shown that they could comprise epigenetic modifications. These are reversible mechanisms linked to tissue-specific silencing of gene expression, genomic imprinting, and tumor growth. Particularly, glyphosate, GBH, and AMPA have been reported to produce changes in global DNA methylation, methylation of specific genes, histone modification, and differential expression of non-coding RNAs in human cells and rodents. Importantly, the epigenome could be heritable and could lead to disease long after the exposure has ended. This mini-review summarizes the epigenetic changes produced by glyphosate, GBHs, and AMPA in humans and rodents and proposes it as a potential mechanism of action through which these chemical compounds could alter body functions.

Evaluation of Development of the Rat Uterus as a Toxicity Biomarker.

The developing uterus is highly sensitive to a brief exposure to different substances, in particular those with endocrine-disrupting activity. Thus, exposure to environmental, nutritional, chemical, and other xenobiotic factors affecting signaling events during critical organizational periods can alter the normal course of uterine development with lasting consequences. In this chapter, we provide an experimental protocol to evaluate the development of the rat uterus as a toxicity biomarker at two different developmental time points: (1) the neonatal period, on postnatal day (PND) 8, and (2) the prepubertal period, on PND21. In this experimental approach, we propose to assess: (1) uterine morphology and cytodifferentiation, (2) uterine cell proliferation, and (3) the expression of proteins involved in uterine organogenetic differentiation. All these morphological and molecular markers are useful tools to determine the consequences of exposure to toxicants with the potential to disrupt the uterine development.

Perinatal exposure to glyphosate or a glyphosate-based formulation disrupts hormonal and uterine milieu during the receptive state in rats.

We investigated the effects of perinatal exposure to a glyphosate-based herbicide (GBH) or glyphosate alone (Gly) on female fertility and the hormonal and uterine milieu during the preimplantation period. F0 pregnant rats orally received a GBH or Gly in a dose of 2 mg of glyphosate/kg/day from gestational day (GD) 9 until weaning. F1 females were evaluated to determine the reproductive performance on GD19; and the sex steroid serum levels, the expression of estrogen receptor alpha (ERα), progesterone receptor (PR) and implantation-related genes on GD5 (preimplantation period). GBH and Gly induced preimplantation losses in F1 rats. GBH and Gly groups exhibited higher 17ß-estradiol serum levels, without changes in progesterone. Both compounds increased the uterine ERα protein expression, with no differences at transcript level; and only Gly decreased PR mRNA expression. Also, GBH and Gly downregulated Hoxa10 and Lif genes, with no difference in Muc1 and Areg expression. To conclude, perinatal exposure to a GBH or Gly disrupted critical hormonal and uterine molecular targets during the receptive state, possibly associated with the implantation failures. Overall, similar results were found in GBH- and Gly-exposed rats, suggesting that the active principle might be the main responsible for the deleterious effects.

Glyphosate-based herbicide induces hyperplastic ducts in the mammary gland of aging Wistar rats.

Glyphosate-based herbicide (GBH) exposure is known to have adverse effects on endocrine-related tissues. Here, we aimed to determine whether early postnatal exposure to a GBH induces long-term effects on the rat mammary gland. Thus, female Wistar pups were injected with saline solution (Control) or GBH (2 mg glyphosate/kg/day) on postnatal days (PND) 1, 3, 5 and 7. At 20 months of age, mammary gland samples were collected to determine histomorphological features, proliferation index and the expression of steroid hormone receptors expression, by immunohistochemistry, and serum samples were collected to assess 17ß-estradiol (E2) and progesterone (P4) levels. GBH exposure induced morphological changes evidenced by a higher percentage of hyperplastic ducts and a fibroblastic-like stroma in the mammary gland. GBH-treated rats also showed a high expression of steroid hormone receptors in hyperplastic ducts. The results indicate that early postnatal exposure to GBH induces long-term alterations in the mammary gland morphology of aging female rats.

Acute uterine effects and long-term reproductive alterations in postnatally exposed female rats to a mixture of commercial formulations of endosulfan and glyphosate.

Endosulfan and glyphosate are widely used pesticides and have been associated to reproductive disorders. We examine the acute and long-term effects of postnatal exposure to commercial formulations of endosulfan (EF), glyphosate (glyphosate-based herbicide, GBH) and a mixture of both pesticides (MIX). After birth, female pups of Wistar rats received saline solution (CONTROL), EF (600 µg/kg of b.w/day), GBH (2 mg/kg of b.w/day) or a mixture (at the same doses) from postnatal day (PND) 1 to PND7. The uterine histology and expression of Hoxa10, estrogen (ERα) and progesterone (PR) receptors were evaluated on PND8. Reproductive performance was evaluated on gestational day 19. GBH and MIX rats showed an increment of 1) the incidence of luminal epithelial hyperplasia, 2) PR and Hoxa10 expression. EF modified ERα and Hoxa10 expression. During adulthood, MIX and GBH rats showed higher post-implantation losses while EF alone produced an increase of pre-implantation losses. We showed that the co-administration of both pesticides produced acute uterine effects and long-term deleterious reproductive effects that were similar to those induced by GBH alone. We consider important to highlight the necessity to evaluate the commercial pesticide mixture as a more representative model of human exposure to a high number of pesticides.

Epigenetic disruption of estrogen receptor alpha is induced by a glyphosate-based herbicide in the preimplantation uterus of rats.

Previously, we have shown that perinatal exposure to a glyphosate-based herbicide (GBH) induces implantation failures in rats. Estrogen receptor alpha (ERα) is critical for successful implantation. ERα transcription is under the control of five promoters (E1, OT, O, ON, and OS), which yield different transcripts. Here, we studied whether perinatal exposure to a GBH alters uterine ERα gene expression and prompts epigenetic modifications in its regulatory regions during the preimplantation period. Pregnant rats (F0) were orally treated with 350 mg glyphosate/kg bw/day through food from gestational day (GD) 9 until weaning. F1 females were bred, and uterine samples were collected on GD5 (preimplantation period). ERα mRNA levels and its transcript variants were evaluated by RT-qPCR. Enzyme-specific restriction sites and predicted transcription factors were searched in silico in the ERα promoter regions to assess the methylation status using the methylation-sensitive restriction enzymes-PCR technique. Post-translational modifications of histones were studied by the chromatin immunoprecipitation assay. GBH upregulated the expression of total ERα mRNA by increasing the abundance of the ERα-O transcript variant. In addition, different epigenetic changes were detected in the O promoter. A decrease in DNA methylation was observed in one of the three sites evaluated in the O promoter. Moreover, histone H4 acetylation and histone H3 lysine 9 trimethylation (H3K9me3) were enriched in the O promoter in GBH-exposed rats, whereas H3K27me3 was decreased. All these alterations could account for the increase in ERα gene expression. Our findings show that perinatal exposure to a GBH causes long-term epigenetic disruption of the uterine ERα gene, which could be associated with the GBH-induced implantation failures.

Glyphosate-based herbicide enhances the uterine sensitivity to estradiol in rats.

In a previous work, we detected that postnatal exposure to a glyphosate-based herbicide (GBH) alters uterine development in prepubertal rats causing endometrial hyperplasia and increasing cell proliferation. Our goal was to determine whether exposure to low-dose of a GBH during postnatal development might enhance the sensitivity of the uterus to an estrogenic treatment. Female Wistar pups were subcutaneously injected with saline solution (control) or GBH using the reference dose (2 mg/kg/day, EPA) on postnatal days (PND) 1, 3, 5, and 7. At weaning (PND21), female rats were bilaterally ovariectomized and treated with silastic capsules containing 17ß-estradiol (E2, 1mg/ml) until they were two months of age. On PND60, uterine samples were removed and processed for histology, immunohistochemistry and mRNA extraction to evaluate: i) uterine morphology, ii) uterine cell proliferation by the detection of Ki67, iii) the expression of the estrogen receptors alpha (ESR1) and beta (ESR2), and iv) the expression of WNT7A and ß-catenin. GBH-exposed animals showed increased luminal epithelial height and stromal nuclei density. The luminal and glandular epithelium were markedly hyperplastic in 43% of GBH-exposed animals. GBH exposure caused an increase in E2-induced cell proliferation in association with an induction of both ESR1 and ESR2. GBH treatment decreased membranous and cytoplasmic expression of ß-catenin in luminal and glandular epithelial cells and increased WNT7A expression in the luminal epithelium. These results suggest that early postnatal exposure to a GBH enhances the sensitivity of the rat uterus to estradiol, and induces histomorphological and molecular changes associated with uterine hyperplasia.

Induction of uterine hyperplasia after cafeteria diet exposure.

Our aim was to evaluate whether chronic administration of CAF affects the uterus and induces the morphological and molecular changes associated with endometrial hyperplasia. Female Wistar rats exposed to CAF from weaning for 20 weeks displayed increased energy intake, body weight and fat depots, but did not develop metabolic syndrome. The adult uteri showed an increase in glandular volume fraction and stromal area. The epithelial proliferation rate and protein expression of oestrogen receptor alpha (ERα) were also increased. The CAF diet enhanced leptin serum levels and the long form of leptin receptor (Ob-Rb) mRNA expression in the uterus. No changes were detected in either insulin serum levels or those of insulin growth factor I (IGF-I) mRNA expression. However the levels of IGF-I receptor (IGF-IR) mRNA were lower in CAF-fed animals. Overall, the results indicate that our rat model of the CAF diet produces morphological and molecular changes associated with uterine hyperplasia and could predispose to endometrial carcinogenesis.

Sex- and age-associated differences in episodic-like memory and transcriptional regulation of hippocampal steroidogenic enzymes in rats.

The aim of this study was to evaluate the episodic-like memory (ELM) and the transcriptional regulation of the enzymes involved in hippocampal allopregnanolone synthesis in young adult and middle-aged male and female rats. Young adult males, but not middle-aged ones, showed a good performance in the ELM task. In contrast, neither young nor middle-aged females were able to discriminate the spatial order in which the objects were presented. In females, aging decreased the transcription of steroidogenic-related genes. In addition, the mRNA levels of 5α-reductase-1 were higher and the methylation of its promoter was lower in young adult females than in males, suggesting an epigenetic control. Further studies are needed to establish correlations between ELM and the transcriptional regulation of hippocampal steroidogenic enzymes. Our results contribute to the knowledge of sex differences in gene expression, methylation and memory during aging.

Neonatal exposure to a glyphosate-based herbicide alters uterine decidualization in rats.

We investigated whether defective modulation of uterine signaling may cause decidualization failure in rats neonatally exposed to a glyphosate-based herbicide (GBH). Female pups received vehicle or 2mg/kg of GBH from postnatal day (PND) 1 to PND7. On PND8 and PND21, Wnt5a and ß-catenin expression was evaluated in uterine samples. On gestational day (GD) 9, Wnt5a, Wnt7a and ß-catenin expression and Dkk1 and sFRP4 mRNA were evaluated on implantation sites. On PND8, GBH-exposed rats showed increased Wnt5a and ß-catenin expression in luminal epithelium (LE), whereas on PND21, they showed increased Wnt5a and ß-catenin expression in subepithelial stroma but decreased ß-catenin expression in glandular epithelium. On GD9, GBH-exposed rats showed decreased Wnt5a and Wnt7a expression in the antimesometrial zone and LE respectively, without changes in ß-catenin expression, while Dkk1 and sFRP4 were up- and down-regulated respectively. We concluded that neonatal GBH exposure may lead to embryo losses by disturbing uterine signaling.

Uterine ERα epigenetic modifications are induced by the endocrine disruptor endosulfan in female rats with impaired fertility.

High ERα activity may disrupt the window of uterine receptivity, causing defective implantation. We investigated whether implantation failures prompted by endosulfan are associated with aberrant ERα uterine expression and DNA methylation status during the pre-implantation period. ERα-dependent target genes that play a crucial role in the uterine receptivity for embryo attachment and implantation were also investigated. Newborn female rats received corn oil (vehicle, Control), 6 µg/kg/d of endosulfan (Endo6) or 600 µg/kg/d of endosulfan (Endo600) on postnatal days (PND) 1, 3, 5, and 7. On PND90, females were made pregnant and on gestational day 5 (GD5, pre-implantation period) uterine samples were collected. ERα expression was assessed at protein and mRNA levels by immunohistochemistry and real time RT-PCR, respectively. ERα transcript variants mRNA containing alternative 5'-untranslated regions (5'UTRs) were also evaluated. We searched for predicted transcription factors binding sites in ERα regulatory regions and assessed their methylation status by Methylation-Sensitive Restriction Enzymes-PCR technique (MSRE-PCR). The expression of the ERα-dependent uterine target genes, i.e. mucin-1 (MUC-1), insulin-like growth factor-1 (IGF-1), and leukemia inhibitory factor (LIF), was assessed by real time RT-PCR. Both doses of endosulfan increased the expression of ERα and its transcript variants ERα-OS, ERα-O, ERα-OT and ERα-E1. Moreover, a decreased DNA methylation levels were detected in some ERα regulatory regions, suggesting an epigenetic up-regulation of it transcription. ERα overexpression was associated with an induction of its downstream genes, MUC-1 and IGF-1, suggesting that endosulfan might alter the uterine estrogenic pathway compromising uterine receptivity. These alterations could account, at least in part, for the endosulfan-induced implantation failures.

Effects of a glyphosate-based herbicide on the uterus of adult ovariectomized rats.

Glyphosate is the active ingredient of several herbicide formulations. Different reports suggest that glyphosate-based herbicides (GBHs) may act as endocrine disruptors. We evaluated the potential estrogenic effects of a GBH formulation using the uterotrophic assay. Adult ovariectomized rats were sc injected for 3 consecutive days with: saline solution (vehicle control), 2.10-5  g E2 /kg/day (uterotrophic dose; UE2 ), 2.10-7  g E2 /kg/day (nonuterotrophic dose; NUE2 ), or 0.5, 5, or 50 mg GBH/kg/day of the. Twenty-four hours after the last injection, the uterus was removed and weighed and processed for histopathology and mRNA extraction. Epithelial cell proliferation and height and expression of estrogen-responsive genes were evaluated (estrogen receptors, ERα and ERß; progesterone receptor, PR; complement 3, C3). Uterine weight and epithelial proliferation were not affected by GBH. However, the luminal epithelial cell height increased at GBH0.5. ERα mRNA was downregulated by all GBH doses and E2 groups, whereas PR and C3 mRNA were diminished by GBH0.5. GBH5-, GBH50-, and UE2 -treated rats showed downregulated ERα protein expression in luminal epithelial cells, while the receptor was upregulated in the stroma. GBH upregulated ERß (GBH0.5-50) and PR (GBH5) expressions in glandular epithelial cells, similar effect to that of NUE2 group. These results indicate that, although the uterine weight was not affected, GBH modulates the expression of estrogen-sensitive genes. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1191-1201, 2017.

Neonatal exposure to a glyphosate based herbicide alters the development of the rat uterus.

Glyphosate-based herbicides (GBHs) are extensively used to control weeds on both cropland and non-cropland areas. No reports are available regarding the effects of GBHs exposure on uterine development. We evaluated if neonatal exposure to a GBH affects uterine morphology, proliferation and expression of proteins that regulate uterine organogenetic differentiation in rats. Female Wistar pups received saline solution (control, C) or a commercial formulation of glyphosate (GBH, 2mg/kg) by sc injection every 48h from postnatal day (PND) 1 to PND7. Rats were sacrificed on PND8 (neonatal period) and PND21 (prepubertal period) to evaluate acute and short-term effects, respectively. The uterine morphology was evaluated in hematoxylin and eosin stained sections. The epithelial and stromal immunophenotypes were established by assessing the expression of luminal epithelial protein (cytokeratin 8; CK8), basal epithelial proteins (p63 and pan cytokeratin CK1, 5, 10 and 14); and vimentin by immunohistochemistry (IHC). To investigate changes on proteins that regulate uterine organogenetic differentiation we evaluated the expression of estrogen receptor alpha (ERα), progesterone receptor (PR), Hoxa10 and Wnt7a by IHC. The GBH-exposed uteri showed morphological changes, characterized by an increase in the incidence of luminal epithelial hyperplasia (LEH) and an increase in the stromal and myometrial thickness. The epithelial cells showed a positive immunostaining for CK8, while the stromal cells for vimentin. GBH treatment increased cell proliferation in the luminal and stromal compartment on PND8, without changes on PND21. GBH treatment also altered the expression of proteins involved in uterine organogenetic differentiation. PR and Hoxa10 were deregulated both immediately and two weeks after the exposure. ERα was induced in the stromal compartment on PND8, and was downregulated in the luminal epithelial cells of gyphosate-exposed animals on PND21. GBH treatment also increased the expression of Wnt7a in the stromal and glandular epithelial cells on PND21. Neonatal exposure to GBH disrupts the postnatal uterine development at the neonatal and prepubertal period. All these changes may alter the functional differentiation of the uterus, affecting the female fertility and/or promoting the development of neoplasias.