Branching morphogenesis of the mouse mammary gland after exposure to benzophenone-3.

Pubertal mammary branching morphogenesis is a hormone-regulated process susceptible to exposure to chemicals with endocrine disruptive capacity, such as the UV-filter benzophenone-3 (BP3). Our aim was to assess whether intrauterine or in vitro exposure to BP3 modified the branching morphogenesis of the female mouse mammary gland. For this, pregnant mice were dermally exposed to BP3 (0.15 or 50 mg/kg/day) from gestation day (GD) 8.5 to GD18.5. Sesame oil treatment served as control. Changes of the mammary glands of the offspring were studied on postnatal day 45. Further, mammary organoids from untreated mice were cultured under branching induction conditions and exposed for 9 days to BP3 (1 × 10-6 M, 1 × 10-9 M, or 1 × 10-12 M with 0.01% ethanol as control) to evaluate the branching progression. Mice that were exposed to BP3 in utero showed decreased mRNA levels of progesterone receptor (PR) and WNT4. However, estradiol and progesterone serum levels, mammary histomorphology, proliferation, and protein expression of estrogen receptor alpha (ESR1) and PR were not significantly altered. Interestingly, direct exposure to BP3 in vitro also decreased the mRNA levels of PR, RANKL, and amphiregulin without affecting the branching progression. Most effects were found after exposure to 50 mg/kg/day or 1 × 10-6 M of BP3, both related to sunscreen application in humans. In conclusion, exposure to BP3 does not impair mammary branching morphogenesis in our models. However, BP3 affects PR transcriptional expression and its downstream mediators, suggesting that exposure to BP3 might affect other developmental stages of the mammary gland.

Preceding anti-spike IgG levels predicted risk and severity of COVID-19 during the Omicron-dominant wave in Santa Fe city, Argentina.

The SARS-CoV-2 Omicron variant has increased infectivity and immune escape compared with previous variants, and caused the surge of massive COVID-19 waves globally. Despite a vast majority (~90%) of the population of Santa Fe city, Argentina had been vaccinated and/or had been infected by SARS-CoV-2 when Omicron emerged, the epidemic wave that followed its arrival was by far the largest one experienced in the city. A serosurvey conducted prior to the arrival of Omicron allowed to assess the acquired humoral defences preceding the wave and to conduct a longitudinal study to provide individual-level real-world data linking antibody levels and protection against COVID-19 during the wave. A very large proportion of 1455 sampled individuals had immunological memory against COVID-19 at the arrival of Omicron (almost 90%), and about half (48.9%) had high anti-spike immunoglobulin G levels (>200 UI/ml). However, the antibody titres varied greatly among the participants, and such variability depended mainly on the vaccine platform received, on having had COVID-19 previously and on the number of days elapsed since last antigen exposure (vaccine shot or natural infection). A follow-up of 514 participants provided real-world evidence of antibody-mediated protection against COVID-19 during a period of high risk of exposure to an immune-escaping highly transmissible variant. Pre-wave antibody titres were strongly negatively associated with COVID-19 incidence and severity of symptoms during the wave. Also, receiving a vaccine shot during the follow-up period reduced the COVID-19 risk drastically (15-fold). These results highlight the importance of maintaining high defences through vaccination at times of high risk of exposure to immune-escaping variants.

Psychological Factors Associated with Functional Disability in Patients with Hip and Knee Osteoarthritis.

Several studies have shown an association between psychosocial variables and functional capacity in chronic pain processes such as osteoarthritis. The aim of this study was to test a structural equations model that shows the predictive weight of certain variables such as catastrophizing, self-efficacy and kinesiophobia on functional pain and WOMAC subscales scores of pain and physical function of older patients diagnosed with hip and knee osteoarthritis. We also assessed the specific weight of age in terms of the factors. The study was conducted on a sample of 170 patients (142 women and 28 men mean age, 74.44 years range, 50-96 years). The main variables evaluated were WOMAC subscales scores of pain and physical function, self-efficacy, catastrophizing and kinesiophobia. To assess these variables, we used the Spanish validated version of the Western Ontario and McMaster Universities questionnaire, the Chronic Pain Self-Efficacy Scale, the Pain Catastrophizing Scale and the Tampa Scale for Kinesiophobia, respectively. We tested a structural equations model (IBM SPSS Amos version 22). The results showed the predominant predictive weight (both direct and indirect) of catastrophizing while simultaneously ruling out the relevance of age as a predictor of WOMAC subscales scores of pain and physical function. This study provides data of interest on the explanatory mechanisms that underlie the direct and inverse relationships between the studied psychological variables.

Long-term effects of in ovo exposure to an environmentally relevant dose of atrazine on the thyroid gland of Caiman latirostris.

The increased incidence of human thyroid disorders, particularly in women, suggests that the exposure to endocrine-disrupting compounds (EDCs) together with sex-related factors could play a role in thyroid dysregulation. Since the herbicide atrazine (ATZ) is an environmental EDC suspected to behave as a thyroid disruptor, and Caiman latirostris is a crocodilian species highly sensitive to endocrine disruption that can be exposed to ATZ, this study aimed to describe the histoarchitecture and sexually dimorphic features of the thyroid gland of C. latirostris, and to determine the long-term effects of in ovo exposure to an environmentally relevant dose of ATZ (0.2 ppm) on its thyroid gland and growth. Control caimans showed no sexual dimorphisms. In contrast, ATZ-exposed caimans showed altered embryo growth but an unaltered temporal pattern of development and a sexually dimorphic response in the body condition index growth curves postnatally, which suggests a female-related increase in fat storage. Besides, both male and female exposed caimans showed increases in the size of the thyroid stromal compartment, content of interstitial collagen, and follicular hyperplasia, and decreases in the expression of androgen receptor in the follicular epithelium. ATZ-exposed females, but not males, also showed evidences of thyroid enlargement, colloid depletion, increased follicular epithelial height and increased presence of microfollicular structures. Our results demonstrate that prenatal exposure of caimans to ATZ causes thyroid disruption and that females were more vulnerable to ATZ than males. The effects were organizational and observed long after exposure ended. These findings alert on ATZ side-effects on the growth, metabolism, reproduction and development of non-target exposed organisms, particularly females.

Bisphenol A and benzophenone-3 exposure alters milk protein expression and its transcriptional regulation during functional differentiation of the mammary gland in vitro.

The plastic monomer and plasticizer bisphenol A (BPA), and the UV-filter benzophenone-3 (BP3) have been shown to have estrogenic activities that could alter mammary gland development. Our aim was to analyze whether BPA or BP3 direct exposure affects the functional differentiation of the mammary gland using an in vitro model. Mammary organoids were obtained and isolated from 8 week-old virgin female C57BL/6 mice and were differentiated on Matrigel with medium containing lactogenic hormones and exposed to: a) vehicle (0.01% ethanol); b) 1 × 10-9 M or 1 × 10-6 M BPA; or c) 1 × 10-12 M, 1 × 10-9 M or 1 × 10-6 M BP3 for 72 h. The mRNA and protein expression of estrogen receptor alpha (ESR1) and progesterone receptor (PR) were assessed. In addition, mRNA levels of PR-B isoform, glucocorticoid receptor (GR), prolactin receptor (PRLR) and Stat5a, and protein expression of pStat5a/b were evaluated at 72 h. The mRNA and protein expression of milk proteins and their DNA methylation status were also analyzed. Although mRNA level of PRLR and GR was similar between treatments, mRNA expression of ESR1, total PR, PR-B and Stat5a was increased in organoids exposed to 1 × 10-9 M BPA and 1 × 10-12 M BP3. Total PR expression was also increased with 1 × 10-6 M BPA. Nuclear ESR1 and PR expression was observed in all treated organoids; whereas nuclear pStat5a/b alveolar cells was observed only in organoids exposed to 1 × 10-9 M BPA and 1 × 10-12 M BP3. The beta-casein mRNA level was increased in both BPA concentrations and 1 × 10-12 M BP3, which was associated with hypomethylation of its promoter. The beta-casein protein expression was only increased with 1 × 10-9 M BPA or 1 × 10-12 M BP3. In contrast, BPA exposure decreased alpha-lactalbumin mRNA expression and increased DNA methylation level in different methylation-sensitive sites of the gene. Also, 1 × 10-9 M BPA decreased alpha-lactalbumin protein expression. Our results demonstrate that BPA or BP3 exposure alters milk protein synthesis and its transcriptional regulation during mammary gland differentiation in vitro.

Impact of gestational low protein diet and postnatal bisphenol A exposure on chemically induced mammary carcinogenesis in female offspring rats.

This study evaluated the effect of gestational low protein diet (LPD) and/or postnatal bisphenol A (BPA) exposure on mammary gland development and carcinogenesis in female offspring. Pregnant Sprague-Dawley rats were fed a normal protein diet (NPD, 17% protein) or LPD (6% protein). At weaning, female offspring were distributed in four groups (NPD, LPD, NPD + BPA, and LPD + BPA) and received vehicle or BPA in drinking water (0.1%), during postnatal day (PND) 21 to 51. On PND 51, some female offspring were euthanized or received a single dose of 7,12-dimethylbenzoanthracene (DMBA, 30 mg/kg, i.g.) and were euthanized on PND 250. On PND 51, neither gestational LPD nor postnatal BPA exposure, individually or in combination, significantly altered the development of mammary gland tree, mean number of terminal structures or estrogen receptor beta (ER-ß), proliferating cell nuclear antigen (PCNA) or caspase-3 protein expression in the mammary tissue. A significant reduction in mammary epithelial area (%) was observed in both LPD groups and a significant increase in ER-α protein expression was detected only in LPD group. In LPD + BPA group was observed a significant increase in both fat pad area (%) and in mean number of mammary epithelial cells positive for progesterone receptor (PR). On PND 250, the groups that received BPA presented lower latency and higher tumor incidence and tumor multiplicity and LPD + BPA group more aggressive tumors. These findings suggest that postnatal BPA exposure associated with gestational LPD is able to induce morphological changes in the mammary gland and increase susceptibility to mammary carcinogenesis.

Temperature- vs. estrogen-induced sex determination in Caiman latirostris embryos: Both females, but with different expression patterns of key molecules involved in ovarian development.

Caiman latirostris is a species with temperature dependent sex determination (TSD), which implies that the incubation temperature of the eggs is the main factor that determines the sex during a thermo-sensitive period (TSP). However, estrogens play a critical role in this process. The administration of 17ß-estradiol (E2) previous to TSP overrides the effects of male incubation temperature, producing phenotypic females. This effect has been defined as sex reversal or estrogen-induced sex determination (E2SD). The aim of the present study is to describe similarities and differences in the effects of TSD and E2SD treatment conditions on ovary development. Our results show that the two treatment conditions studied are able to produce different ovaries. Treatment with E2 modified the expression pattern of estrogen receptor alpha and progesterone receptor, and expression of the enzyme aromatase. Moreover, in E2SD females, the proliferation/apoptosis dynamic was also altered and high expression of TAp63 was observed suggesting the presence of greater DNA damage in germ cells. To the best of our knowledge, this is the first report that describes the morphology of the female gonad of C. latirostris in three stages of embryonic development and shows the expression of TAp63 during the gonad development of a reptile. It is important to emphasize that the changes demonstrated in E2SD female gonads of embryos show that environmental compounds with proven estrogenic activity alter the follicular dynamics of C. latirostris in neonatal as much as in juvenile animals, endangering their reproductive health and possibly bringing consequences to ecology and evolution.

Bisphenol A disrupts the temporal pattern of histofunctional changes in the female reproductive tract of Caiman latirostris.

Recently, we have described the ontogeny of histofunctional differentiation changes in the oviduct of Caiman latirostris. The expression of estrogen receptor alpha and progesterone receptor shows that the caiman oviduct could be a target of the action of xenoestrogens such as the widely environmentally present Bisphenol A (BPA), early in life. The aims of this study were: to complement oviduct characterization by establishing the ontogenetic changes in androgen receptor (AR) expression and assessing the effects of early postnatal exposure to 17-ß-estradiol (E2) or BPA on the histofunctional features of the oviduct. AR was expressed in all the stages studied. The spatial pattern of AR immunostaining changed from neonatal to juvenile caimans. In the luminal epithelium, changes were at the subcellular level, from cytoplasmic to nuclear. In the subepithelium, although both cytoplasmic and nuclear AR expression was observed, changes were mainly at tissue level, from the subepithelial compartment to the outer muscular layer. The oviduct was highly sensitive to E2 and BPA at the early postnatal developmental stage. E2- and BPA-exposed caimans showed increased luminal epithelial height and higher proliferative activity. Changes in histomorphological features (measured by a scoring system), steroid hormone receptors, collagen remodeling and muscle-associated proteins suggest a precocious oviduct histofunctional differentiation in E2- and BPA-exposed caimans. The modification of the temporal pattern of oviductal biomarkers suggests that organizational changes could impair C. latirostris reproductive health later in life. The alterations in the caiman female reproductive tract exposed to BPA highlight the importance of preserving aquatic environments from plastic pollution.

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.

Effects of neonatal exposure to a glyphosate-based herbicide on female rat reproduction.

In this study, we investigated whether neonatal exposure to a glyphosate-based herbicide (GBH) alters the reproductive performance and the molecular mechanisms involved in the decidualization process in adult rats. Newborn female rats received vehicle or 2 mg/kg/day of a GBH on postnatal days (PND) 1, 3, 5 and 7. On PND90, the rats were mated to evaluate (i) the reproductive performance on gestational day (GD) 19 and (ii) the ovarian steroid levels, uterine morphology, endometrial cell proliferation, apoptosis and cell cycle regulators, and endocrine pathways that regulate uterine decidualization (steroid receptors/COUP-TFII/Bmp2/Hoxa10) at the implantation sites (IS) on GD9. The GBH-exposed group showed a significant increase in the number of resorption sites on GD19, associated with an altered decidualization response. In fact, on GD9, the GBH-treated rats showed morphological changes at the IS, associated with a decreased expression of estrogen and progesterone receptors, a downregulation of COUP-TFII (Nr2f2) and Bmp2 mRNA and an increased expression of HOXA10 and the proliferation marker Ki67(Mki67) at the IS. We concluded that alterations in endometrial decidualization might be the mechanism of GBH-induced post-implantation embryo loss.

Neonatal exposure to xenoestrogens impairs the ovarian response to gonadotropin treatment in lambs.

Bisphenol A (BPA) and diethylstilbestrol (DES) are xenoestrogens, which have been associated with altered effects on reproduction. We hypothesized that neonatal xenoestrogen exposure affects the ovarian functionality in lambs. Thus, we evaluated the ovarian response to exogenous ovine FSH (oFSH) administered from postnatal day 30 (PND30) to PND32 in female lambs previously exposed to low doses of DES or BPA (BPA50: 50 µg/kg per day, BPA0.5: 0.5 µg/kg per day) from PND1 to PND14. We determined: i) follicular growth, ii) circulating levels of 17ß-estradiol (E2), iii) steroid receptors (estrogen receptor alpha, estrogen receptor beta, and androgen receptor (AR)) and atresia, and iv) mRNA expression levels of the ovarian bone morphogenetic protein (BMPs) system (BMP6, BMP15, BMPR1B, and GDF9) and FSH receptor (FSHR). Lambs neonatally exposed to DES or BPA showed an impaired ovarian response to oFSH with a lower number of follicles ≥2 mm in diameter together with a lower number of atretic follicles and no increase in E2 serum levels in response to oFSH treatment. In addition, AR induction by oFSH was disrupted in granulosa and theca cells of lambs exposed to DES or BPA. An increase in GDF9 mRNA expression levels was observed in oFSH-primed lambs previously treated with DES or BPA50. In contrast, a decrease in BMPR1B was observed in BPA0.5-postnatally exposed lambs. The modifications in AR, GDF9, and BMPR1B may be associated with the altered ovarian function due to neonatal xenoestrogen exposure in response to an exogenous gonadotropin stimulus. These alterations may be the pathophysiological basis of subfertility syndrome in adulthood.

Exposure to atrazine and endosulfan alters oviductal adenogenesis in the broad-snouted caiman (Caiman latirostris).

The molecular pathways involved in oviductal adenogenesis are highly conserved among vertebrates. In this work, we study the histomorphological changes and molecular pathways involved in Caiman latirostris oviductal adenogenesis and the effects of in ovo exposure to environmentally relevant doses of endosulfan (END) and atrazine (ATZ) on these processes. To this end, the histomorphological changes at epithelial and subepithelial compartments, the protein expressions of ß-catenin and Wnt-7a, and the gene expression of metalloproteinases (MMPs) and its inhibitors (TIMPs) were evaluated as biomarkers of oviductal adenogenesis in prepubertal juvenile C. latirostris. Exposure to END altered adenogenesis-related epithelium characteristics and mRNA expression of MMP2, MMP9, and TIMP1. Exposure to ATZ increased the width of the subepithelial stroma with loosely arranged collagen fibers and increased ß-catenin expression in buds (invaginated structures that precede glands). The results demonstrate that in ovo exposure to ATZ and END alters oviductal adenogenesis at tissue, cellular, and molecular levels. An altered oviductal adenogenesis could impair fertility, raising concern on the effects of pesticide pollution in wildlife and domestic animals.

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.

Plasma cell-free DNA as a monitoring tool for high-risk pregnancies associated with antiphospholipid syndrome.

INTRODUCTION: Despite thromboprophylaxis, women with antiphospholipid syndrome (APS) face high-risk pregnancies due to proinflammatory and prothrombotic states. This highlights the need for new monitoring and prognostic tools. Recent insights into the pathophysiological role of neutrophil activation and extracellular trap (NET) formation in this syndrome led to the exploration of plasma cell-free DNA (cfDNA), a derivative of NETosis, as a promising biomarker. MATERIALS AND METHODS: cfDNA was isolated and quantified from plasma samples of healthy pregnant women (control group, HC) and women with APS (APS group). We assessed the physiological variability of cfDNA across the three trimesters in HC. Levels of cfDNA were compared between APS and HC by gestational trimester. ROC curve analysis was performed to evaluate the efficacy of cfDNA levels for classifying APS patients. Furthermore, cfDNA levels in pregnant women with APS with obstetric complications were compared to those from uncomplicated pregnancies. RESULTS: Among HC, cfDNA significantly increased in the third trimester compared to the first and second. Elevated cfDNA levels in APS compared to HC were observed in the first and second trimesters. First-trimester cfDNA levels demonstrated the highest classification ability to discriminate between APS and HC patients (AUC: 0.906). Among APS, those with complicated pregnancies (fetal growth restriction, preeclampsia, placenta accreta) exhibited significantly elevated cfDNA levels in the second trimester. CONCLUSIONS: Elevated levels of cfDNA in pregnant women with APS, particularly among those with obstetric complications, supports further investigation into the potential of cfDNA as a valuable tool in the obstetric management of women with APS.

Glyphosate modifies the gene expression and migration of trophoblastic cells without altering the process of angiogenesis or the implantation of blastocysts in vitro.

Adverse pregnancy outcomes have been associated with the presence of glyphosate (G) in umbilical cord, serum, and urine samples from pregnant women. Our aim was to study the effect of G on blastocyst implantation using an in vitro mouse model, and the migration and acquisition of endothelial phenotype of the human trophoblastic HTR8/SVneo (H8) cells. In mouse blastocysts, no differences in attachment time and implantation outgrowth area were observed after G exposure. H8 cell migration was stimulated by 0.625 µM G without cytotoxicity. After 6 h, the mRNA expression of vascular endothelial growth factor (VEGF) and C-C motif chemokine ligand 2 (CCL2) was upregulated in H8 cells exposed to 1.25 µM G when compared vehicle-treated cells (p ≤ 0.05). No differences were observed in interleukin 11, VEGF receptor 1, and coagulation factor II thrombin receptor in H8 cells exposed to different concentrations of G for 6 h compared to the vehicle. Interestingly, exposure to G did not alter angiogenesis as measured by a tube formation assay. Taken all together, these results suggest that G exposure may contribute as a risk factor during pregnancy, due to its ability to alter trophoblast migration and gene expression.

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.

Neonatal expression of amh, sox9 and sf-1 mRNA in Caiman latirostris and effects of in ovo exposure to endocrine disrupting chemicals.

Caiman latirostris is a reptilian species that exhibits temperature-dependent sex determination (TSD). Male-to-female sex reversal can be achieved after in ovo estrogen/xenoestrogen exposure. This is known as hormone-dependent sex determination (HSD). The amh, sox9 and sf-1 genes are involved in sex determination, sex differentiation, and steroidogenesis. The aims of this study were: (a) to establish the expression patterns of amh, sox9 and sf-1 mRNA in the gonad-adrenal-mesonephros (GAM) complexes of neonatal TSD-male and TSD-female caimans, (b) to compare the expression of these genes between TSD-females and HSD-females (born from E2-exposed eggs incubated at the male-producing temperature) and (c) to evaluate whether in ovo exposure to a low dose of E2 or bisphenol A (BPA) or to a high dose of endosulfan (END) modifies amh, sox9 or sf-1 mRNA expressions in neonatal males. The mRNA expressions of amh, sox9 and sf-1 in GAM complexes from TSD-males and TSD-females and from HSD-females were quantitatively compared by RT-PCR. A sexually dimorphic pattern of amh and sox9 mRNA expression was found, with a higher expression in TSD-males than in TSD-females. sf-1 mRNA did not differ between TSD-males and TSD-females. HSD-females exhibited a higher expression of sox9 than TSD-females. In males, increased mRNA expression of sex-determining genes was observed after in ovo exposure to END. E2 decreased sox9 but increased sf-1 mRNA expression. Changes induced by BPA were evident although not significant. These results provide new insights into the potential mechanisms that lead to the gonadal histo-functional alterations observed in caimans exposed to contaminated environments.

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.

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.

Mammary gland development in male rats perinatally exposed to propiconazole, glyphosate, or their mixture.

This study aimed to assess whether perinatal exposure to propiconazole (PRO), glyphosate (GLY) or their mixture (PROGLY) alters key endocrine pathways and the development of the male rat mammary gland. To this end, pregnant rats were orally exposed to vehicle, PRO, GLY, or a mixture of PRO and GLY from gestation day 9 until weaning. Male offspring were euthanized on postnatal day (PND) 21 and PND60. On PND21, GLY-exposed rats showed reduced mammary epithelial cell proliferation, whereas PRO-exposed ones showed increased ductal p-Erk1/2 expression without histomorphological alterations. On PND60, GLY-exposed rats showed reduced mammary gland area and estrogen receptor alpha expression and increased aromatase expression, whereas PRO-exposed ones showed enhanced lobuloalveolar development and increased lobular hyperplasia. However, PROGLY did not modify any of the endpoints evaluated. In summary, PRO and GLY modified the expression of key molecules and the development of the male mammary gland individually but not together.