Altered ovarian reserve in Ewe lambs exposed to a glyphosate-based herbicide.

Glyphosate-based herbicides (GBHs) are considered endocrine disruptors that affect the female reproductive tract of rats and ewe lambs. The present study aimed to investigate the impact of neonatal exposure to a low dose of a GBH on the ovarian follicular reserve of ewe lambs and the response to a gonadotropic stimulus with porcine FSH (pFSH). To this end, ewe lambs were orally exposed to an environmentally relevant GBH dose (1 mg/kg/day) or vehicle (Control) from postnatal day (PND) 1 to PND14, and then some received pFSH (50 mg/day) between PND41 and 43. The ovaries were dissected, and follicular types and gene expression were assessed via RT-PCR. The treatments did not affect the body weight of animals, but pFSH increased ovarian weight, not observed in GBH-exposed lambs. GBH-exposed lambs showed decreased Estrogen receptor-alpha (56%), Progesterone receptor (75%), Activin receptor II (ACVRII) (85%), and Bone morphogenetic protein 15 (BMP15) (88%) mRNA levels. Control lambs treated with pFSH exhibited downregulation of Follistatin (81%), ACVRII (77%), BMP15 (93%), and FSH receptor (FSHr) (72%). GBH-exposed lambs treated with pFSH displayed reduced ACVRII (68%), BMP15 (81%), and FSHr (50%). GBH-exposed lambs also exhibited decreased Anti-Müllerian hormone expression in primordial and antral follicles (27%) and (54%) respectively) and reduced Bone morphogenetic protein 4 (31%) expression in primordial follicles. Results suggest that GBH disrupts key follicular development molecules and interferes with pFSH action in ovarian receptors, decreasing the ovarian reserve. Future studies should explore whether this decreased ovarian reserve impairs adult ovarian function and its response to superovulation stimuli.

A Reliable Method for Quantifying Plasma Cell-Free DNA Using an Internal Standard Strategy: Evaluation in a Cohort of Non-Pregnant and Pregnant Women.

The use of plasma cell-free DNA (cfDNA) as a useful biomarker in obstetric clinical practice has been delayed due to the lack of reliable quantification protocols. We developed a protocol to quantify plasma cfDNA using an internal standard strategy to overcome difficulties posed by low levels and high fragmentation of cfDNA. cfDNA was isolated from plasma samples of non-pregnant (NP, n = 26) and pregnant (P, n = 26) women using a commercial kit and several elution volumes were evaluated. qPCR parameters were optimized for cfDNA quantification, and several quantities of a recombinant standard were evaluated as internal standard. Absolute quantification was performed using a standard curve and the quality of the complete method was evaluated. cfDNA was eluted in a 50-µl volume, actin-ß (ACTB) was selected as the target gene, and qPCR parameters were optimized. The ACTB standard was constructed and 1000 copies were selected as internal standard. The standard curve showed R2 = 0.993 and E = 109.7%, and the linear dynamic range was defined between 102 and 106 ACTB copies/tube. Repeatability and reproducibility in terms of CV were 19% and up to 49.5% for ACTB copies per milliliter of plasma, respectively. The range of cfDNA levels was 428-18,851 copies/mL in NP women and 4031-2,019,363 copies/mL in P women, showing significant differences between the groups. We recommend the application of internal standard strategy for a reliable plasma cfDNA quantification. This methodology holds great potential for a future application in the obstetric field.

Disruption of the developmental programming of the gonad of the broad snouted caiman (Caiman latirostris) after in ovo exposure to atrazine.

Environmental exposure to agrochemicals during early stages of development can induce subtle alterations that could permanently affect normal physiology. Previously, we reported that in ovo exposure to atrazine (ATZ) disrupts testicular histoarchitecture in postnatal caimans (Caiman latirostris). To assess whether such alterations are the result of disruption of gonadal developmental programming, this study aimed to evaluate the expression of histofunctional biomarkers (VASA, ER, PR, PCNA, and aromatase) and genes involved in gonadal development and differentiation (amh, sox-9, sf-1 and cyp19-a1) in the gonads of male and female caiman embryos and to assess the effect of ATZ exposure on these biomarkers and genes in the gonads of male embryos. Our results suggest that amh, aromatase and sox-9 play a role in sex determination and gonadal differentiation. In male caiman embryos, ATZ exposure increased aromatase expression and altered the temporal expression pattern of amh and sox-9 evidencing an ATZ-induced disruption of gonadal developmental programming. Since the effects of ATZ are consistent across all vertebrate classes, the ATZ-mediated disruptive effects here observed could be present in other vertebrate species.

Postnatal exposure to a glyphosate-based herbicide interferes with the development and growth of the mammary gland of pre-pubertal Ewe lambs.

The aim of the present study was to evaluate whether early postnatal exposure to a glyphosate-based herbicide (GBH) alters pre-pubertal mammary development in Friesian lambs. To this end, from postnatal day 1-14, ewe lambs were exposed subcutaneously or orally to GBH (2 mg/kg bw/day) or vehicle (control) and mammary gland biopsies were obtained at 45 days of age. GBH-exposed lambs exhibited larger mammary ducts and less area occupied by terminal duct lobular units than controls, accompanied by an increase in the area of adipocytes in the mammary stroma. Lambs subcutaneously exposed to GBH showed increased protein expression of estrogen receptor alpha; however, both GBH-exposed groups had decreased mRNA expression of this receptor. Control lambs showed nuclear progesterone receptor (PR) protein expression, whereas GBH-exposed animals showed cytoplasmic PR expression; both GBH-exposed groups exhibited decreased mRNA expression of PR. GBH-exposed lambs also had decreased epithelial cell proliferation. Regarding insulin-like growth factors, both groups showed similar IGF-1 mRNA and protein expression but decreased expression of its receptor, and increased IGFBP5 expression. In addition, phosphorylated AKT was only observed in the mammary gland of control lambs. Our results show that early postnatal exposure to GBH, regardless of the exposure route, affects the IGF-1 system and the AKT/protein kinase B pathway, interfering with steroid hormone receptor expression and cell proliferation. This consequently modifies the growth and development of the pre-pubertal mammary gland of Frisian lambs.

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.

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.

Are glyphosate and glyphosate-based herbicides endocrine disruptors that alter female fertility?

Numerous evidences have alerted on the toxic effects of the exposure to glyphosate on living organisms. Glyphosate is the herbicide most used in crops such as maize and soybean worldwide, which implies that several non-target species are at a high risk of exposure. Although the Environmental Protection Agency (EPA-USA) has reaffirmed that glyphosate is safe for users, there are controversial studies that question this statement. Some of the reported effects are due to exposure to high doses; however, recent evidences have shown that exposure to low doses could also alter the development of the female reproductive tract, with consequences on fertility. Different animal models of exposure to glyphosate or glyphosate-based herbicides (GBHs) have shown that the effects on the female reproductive tract may be related to the potential and/or mechanisms of actions of an endocrine-disrupting compound. Studies have also demonstrated that the exposure to GBHs alters the development and differentiation of ovarian follicles and uterus, affecting fertility when animals are exposed before puberty. In addition, exposure to GBHs during gestation could alter the development of the offspring (F1 and F2). The main mechanism described associated with the endocrine-disrupting effect of GBHs is the modulation of estrogen receptors and molecules involved in the estrogenic pathways. This review summarizes the endocrine-disrupting effects of exposure to glyphosate and GBHs at low or "environmentally relevant" doses in the female reproductive tissues. Data suggesting that, at low doses, GBHs may have adverse effects on the female reproductive tract fertility are discussed.

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.

Correction to: Response to comments on: Perinatal exposure to a glyphosate-based herbicide impairs female reproductive outcomes and induces second-generation adverse effects in Wistar rats.

We report a mistake in the tables published in Milesi et al. (2019).

Neonatal exposure to a glyphosate-based herbicide alters the uterine differentiation of prepubertal ewe lambs.

The exposure to endocrine-disrupting compounds (EDCs), such as glyphosate-based herbicides (GBHs), during early life might alter female fertility. The aim of the present study was to evaluate the effects of neonatal exposure to a GBH on sheep uterine development. To achieve this, Friesian ewe lambs were exposed to GBH (2 mg/kg of body weight/day; n = 12) or vehicle (controls; n = 10) through s.c. injections, from postnatal day (PND) 1 to PND14; on PND45, the uteri were obtained to evaluate histomorphological and molecular parameters. Morphological parameters were determined by picrosirius-hematoxylin staining. Protein expression of Ki67 (as a cell proliferation marker), p27, and molecules involved in uterine organogenetic differentiation was measured by immunohistochemistry. We also determined the mRNA expression of the IGF molecular pathway by RT-PCR. Although histomorphology was not modified, the uteri of GBH-exposed ewe lambs showed lower cell proliferation, together with higher p27 protein expression. In addition, the uteri of GBH-exposed ewe lambs showed increased gene expression of insulin-like growth factor binding protein 3 (IGFBP-3), decreased expression of ERα in the luminal (LE) and glandular (GE) epithelia and in the subepithelial stroma (SS), and lower PR expression in the LE but higher in the GE and SS. In addition, GBH treatment decreased the uterine expression of Wnt5a in the GE, of Wnt7a in the SS, of ß-catenin in the LE and GE, of Hoxa10 in the SS, and of Foxa2 in the GE as compared with controls. In conclusion, neonatal exposure to GBH decreased cell proliferation and altered the expression of molecules that control proliferation and development in the uterus. All these changes might have adverse consequences on uterine differentiation and functionality, affecting the female reproductive health of sheep. GBH may be responsible for uterine subfertility, acting as an EDC.

Perinatal exposure to bisphenol A (BPA) impairs neuroendocrine mechanisms regulating food intake and kisspetin system in adult male rats. Evidences of metabolic disruptor hypothesis.

Bisphenol A (BPA) is a compound used in the polymerization of plastic polycarbonates. It is an endocrine disruptor and it has been postulated to be an obesogen. Our objective was to determine the influence of perinatal exposure to BPA on body weight, hormone levels, metabolic parameters and hypothalamic signals that regulate food intake and kisspeptin system in adult male rats. Male rats were exposed to 50 µg/kg/day of BPA or vehicle from day 9 of gestation to weaning in the drinking water. Since weaning, they were fed with control or high fat diet for 20 weeks. Perinatal exposure to BPA impaired glucose homeostasis, induced obesity and increased food intake in adult male rats altering hypothalamic signals, partially mimicking and/or producing an exacerbation of the effects of feeding fat diet. We also observed an increase in kisspeptin expression by BPA exposure. Evidences shown in this work support the metabolic disruptor hypothesis for BPA.

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.

Effect of neonatal exposure to endosulfan on myometrial adaptation during early pregnancy and labor in rats.

Proper myometrial adaptation during gestation is crucial for embryo implantation, pregnancy maintenance and parturition. Previously, we reported that neonatal exposure to endosulfan alters uterine development and induces implantation failures. The present work investigates the effects of endosulfan exposure on myometrial differentiation at the pre-implantation period, and myometrial activation during labor. Newborn female rats were s.c. injected with corn oil (vehicle) or 600 µg/kg/day of endosulfan (Endo600) on postnatal days (PND) 1, 3, 5 and 7. On PND90, the rats were mated to evaluate: i) the myometrial differentiation on gestational day 5 (GD5, pre-implantation period), by assessment myometrial histomorphology, smooth muscle cells (SMCs) proliferation, and expression of proteins involved in myometrial adaptation for embryo implantation (steroid receptors, Wnt7a and Hoxa10); ii) the timing of parturition and myometrial activation during labor by determining the uterine expression of contraction-associated genes (oxytocin receptor, OTXR; prostaglandin F2α receptor, PTGFR and connexin-43, Cx-43). Endosulfan decreased the thickness of both myometrial layers, with a concomitant decrease in the collagen remodeling. Blood vessels relative area in the interstitial connective tissue between muscle layers was also decreased. Endo600 group showed lower myometrial proliferation in association with a downregulation of Wnt7a and Hoxa10. Although in all females labor occurred on GD23, the exposure to endosulfan altered the timing of parturition, by inducing advancement in the initiation of labor. This alteration was associated with an increased uterine expression of OTXR, PTGFR and Cx-43. In conclusion, neonatal exposure to endosulfan produced long-term effects affecting myometrial adaptation during early pregnancy and labor. These alterations could be associated with the aberrant effects of endosulfan on the implantation process and the timing of parturition.

Neonatal exposure to a glyphosate-based herbicide alters the histofunctional differentiation of the ovaries and uterus in lambs.

The aim of the present study was to compare the effect of oral and subcutaneous exposure to a glyphosate-based herbicide (GBH) on the female reproductive system, specifically in the ovaries and uterus of prepubertal lambs. To this end, ewe lambs were exposed to a s.c. (n: 5) or an oral (n: 5) environmentally relevant dose of GBH (2 mg/kg/day) or to vehicle (controls, n: 12), from postnatal day (PND) 1 to PND14. Serum glyphosate and aminomethylphosphonic acid (AMPA) concentrations were measured on PND15 and PND45. The ovaries and uterus were obtained and weighed on PND45. Ovarian follicular dynamics and uterine morphological features were determined by picrosirius-hematoxylin staining. The proliferation marker Ki67 was evaluated by immunohistochemistry in ovarian and uterine samples. Glyphosate but not AMPA was detected in serum of exposed lambs on PND15, whereas neither glyphosate nor AMPA were detected on PND45. Controls were negative for glyphosate and AMPA on PND15 and PND45. GBH exposure did not affect ovarian or uterine weight. However, on PND45, the ovary of GBH-exposed lambs showed altered follicular dynamics, increased proliferation of granulosa and theca cells, and decreased mRNA expression of FSHR and GDF9, whereas their uterus showed decreased cell proliferation but no alterations in the histomorphology or gene expression. In conclusion, GBH exposure altered the ovarian follicular dynamics and gene expression, and the proliferative activity of the ovaries and uterus of lambs. It is noteworthy that all the adverse effects found in the ovaries and uterus of both GBH-exposed groups were similar, independently of the administration route.

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.

Perinatal exposure to a glyphosate-based herbicide impairs female reproductive outcomes and induces second-generation adverse effects in Wistar rats.

Glyphosate-based herbicides (GBHs) are the most globally used herbicides raising the risk of environmental exposition. Here, we investigated whether perinatal exposure to low doses of a GBH alters the female reproductive performance, and/or induced second-generation effects related to congenital anomalies or growth alterations. Pregnant rats (F0) received a GBH through food, in a dose of 2 mg (GBH-LD: GBH-low dose group) or 200 mg (GBH-HD: GBH-high dose group) of glyphosate/kg bw/day from gestational day (GD) 9 until weaning. Body weight gain and vaginal canal-opening of F1 females were recorded. Sexually mature F1 females were mated to evaluate their reproductive performance by assessing the pregnancy rate, and on GD19, the number of corpora lutea, the implantation sites (IS) and resorption sites. To analyze second-generation effects on F2 offspring, we analyzed the fetal morphology on GD19, and assessed the fetal length and weight, and the placental weight. GBH exposure neither altered the body weight gain of F1 females, nor vaginal opening onset. Although all GBH-exposed F1 rats became pregnant, a lower number of IS was detected. F2 offspring from both GBH groups showed delayed growth, evidenced by lower fetal weight and length, associated with a higher incidence of small for gestational age fetuses. In addition, higher placental weight and placental index were found in F2 offspring from GBH-HD dams. Surprisingly, structural congenital anomalies (conjoined fetuses and abnormally developed limbs) were detected in the F2 offspring from GBH-HD group. In conclusion, perinatal exposure to low doses of a GBH impaired female reproductive performance and induced fetal growth retardation and structural congenital anomalies in F2 offspring.

Postnatal exposure to a glyphosate-based herbicide modifies mammary gland growth and development in Wistar male rats.

Our aim was to evaluate whether postnatal exposure to a glyphosate-based herbicide (GBH) modifies mammary gland development in pre- and post-pubertal male rats. From postnatal day 1 (PND1) to PND7, male rats were injected subcutaneously every 48 h with either saline solution (vehicle) or 2 mg GBH/kg·bw. On PND21 and PND60, mammary gland and blood samples were collected. Estradiol (E2) and testosterone (T) serum levels, mammary gland histology, collagen fiber organization, mast cell infiltration, proliferation index, and estrogen (ESR1) and androgen receptor (AR) expression levels were evaluated. At PND21, GBH-exposed male rats exhibited greater development of the mammary gland with increased stromal collagen organization and terminal end buds (TEBs) compared to control rats. At PND60, the number of TEBs remained high and was accompanied by an increase in mast cell infiltration, proliferation and ESR1 expression in GBH-exposed male rats. In contrast, no effects were observed in E2 and T serum levels and AR expression in both days studied. Our results showed that a postnatal subacute treatment with GBH induces endocrine-disrupting effects in the male mammary gland in vivo, altering its normal development.