Estetrol Increases Progesterone Genetic Response without Triggering Common Estrogenic Effects in Endometriotic Cell Lines and Primary Cultures.

Estetrol (E4), a natural estrogen produced by the human fetal liver, is actively studied for menopause and breast cancer treatment. It has low side effects and preferential estrogen receptor alpha (ERα) affinity. There are no data about its effects on endometriosis, a common gynecological disease affecting 6-10% of cycling women, generating painful pelvic lesions and infertility. Current combined hormone treatment (progestins and estrogens) is safe and efficient; nevertheless, one-third of patients develop progesterone (P4) resistance and recurrence by reducing P4 receptors (PRs) levels. We aimed to compare E4 and 17ß-estradiol (E2) effects using two human endometriotic cell lines (epithelial 11Z and stromal Hs832 cells) and primary cultures from endometriotic patients. We evaluated cell growth (MTS), migration (wound assay), hormone receptors levels (Western blot), and P4 response by PCR array. Compared to E2, E4 did not affect cell growth or migration but increased estrogen receptor alpha (ERα) and PRs, and reduced ERß. Finally, the incubation with E4 improved the P4 gene response. In conclusion, E4 increased PRs levels and genetic response without inducing cell growth or migration. These results suggest that E4 might be useful for endometriosis treatment avoiding P4 resistance; however, evaluating its response in more complex models is required.

Molecular Characterization of Embryos with Different Buoyancy Levels in the Yellowtail Kingfish (Seriola lalandi).

The buoyancy of eggs and embryos is associated with successful development in pelagic fish. Buoyancy is the result of oocyte hydration, which depends on the osmotic force exerted by free amino acids (FAA) generated by yolk proteolysis, and cathepsins are the main enzymes involved in this process. Seriola lalandi is a pelagic fish whose farming has been hampered by development failure that have been partially attributed to decreased buoyancy of embryos. Therefore, the aim of this study was to compare the mRNA expression and activity of cathepsins B, D, and L, as well as the FAA content in floating and low-floating embryos at different developmental stages. The chosen stages were eggs, morula, blastula, gastrula and 24 h embryos. Complementary assessments showed that there were no differences attributed to buoyancy status in embryo and oil droplet diameters, as well as the transcriptional status at any developmental stage. Cathepsin B did not show differences in mRNA expression or activity related to buoyancy at any stage. Cathepsin D displayed higher transcript and activity levels only in low-floating eggs compared with those floating. Cathepsin L showed higher expression in floating eggs and 24 h embryos compared with that of low-floating, but the activity of this enzyme was higher in floating eggs and morula. Total FAA content constantly decreased throughout development in floating embryos, but it was always higher than low-floating embryos until gastrula stage. In 24 h embryos floating and low-floating embryos share similar quantities of FAA. In summary, differences in the expression and activity of cathepsins between floating and low-floating embryos could be revealed at specific embryonic stages, suggesting different functions of these enzymes throughout development. Besides 24 h embryos, FAA content seems to be a decisive factor for buoyancy of embryos during early development of S. lalandi. Overall, considering the main role of cathepsins and FAA in buoyancy acquisition process and therefore in both embryo quality and viability, our study identifies good marker candidates to evaluate embryo quality in the farming of this species.

Embryo Buoyancy and Cell Death Gene Expression During Embryogenesis of Yellow-Tail Kingfish Seriola lalandi.

In pelagic fish, embryo buoyancy is a noteworthy aspect of the reproductive strategy, and is associated with overall quality, survival, and further developmental success. In captivity, the loss of buoyancy of early embryos correlates with high mortality that might be related to massive cell death. Therefore, the aim of this study was to evaluate under captivity conditions the expression of genes related to the apoptosis process during the early embryonic development of the pelagic fish Seriola lalandi, and its relationship to the buoyancy of embryos. The relative expression of bcl2, bax-like, casp9, casp8, and casp3 was evaluated by RT-qPCR and FasL/Fas protein levels by western blot in five development stages of embryos sorted as floating or low-floating. All genes examined were expressed in both floating and low-floating embryos up to 24 h of development. Expression of the pro-apoptotic factors bax, casp9, casp8, and casp3 was higher in low-floating as compared with floating embryos in a developmental stage-specific manner. In contrast, there was no difference in expression of bcl2 between floating and low-floating embryos. Fas protein was detected as a single band in floating embryos without changes in expression throughout development; however, in low-floating embryos, three higher intensity reactive bands were detected in the 24-h embryos. Interestingly, FasL was only detected at 24-h in floating embryos, whereas in low-floating samples this ligand was present at all stages, with a sharp increase as development progressed. Cell death, as evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, was highly increased in low-floating embryos as compared to floating embryos throughout all developmental stages, with the highest levels observed during the gastrula stage and at 24 h. The results of this study suggest that an increase in cell death, probably associated with the intrinsic and extrinsic apoptosis pathways, is present in low-floating embryos that might explain their lower developmental potential under captivity conditions.

Daily exposure to phthalates and alkylphenols alters miR biogenesis and expression in mice ovaries.

Reproductive hormone imbalance in infertile women is correlated to high levels of phthalates and alkylphenols, which are among endocrine-disrupting chemicals (EDCs). Previous studies have shown that they interfere with gene expression by deregulating levels of microRNAs (miRs), small non-coding RNAs targeting mRNAs encoding enzymes in the hormone biosynthesis pathway. However, this effect depends on the target organ, dose and whether or not they are alone or in mixtures. Our goal was to study whether the biosynthesis, and a specific group of miRs targeting mRNAs encoding enzymes in steroid hormone biosynthesis, are deregulated in the ovaries of female mice chronically exposed to a mixture of three phthalates (DEHP+DBP+BBP) and two alkylphenols (NP+OP) at a human environmentally relevant dose. We performed qPCR and Western blot assays along with a bioinformatics approach and found that this mixture modified the biogenesis machinery of miRs, inducing an increase in the mRNA levels of Drosha and Dicer1 and DROSHA protein levels. In addition, we found changes in the precursor and mature forms of miR-96-5p, miR-200b-3p, miR-365-3p, miR-378a-3p and miR-503-5p which target steroidogenic pathway enzymes. Finally, using primary granulosa cell culture, we confirmed that miR-200b-3p targets Cyp19a1, transcript encoding CYP19A1, the enzyme that produces estradiol (E2). These results indicate that chronic exposure to phthalates and alkylphenols mixture alters the biogenesis of ovary miRs and increases the expression of miRs implicated in the control of steroidal hormone synthesis in female mice, thus contributing to reproductive pathologies.

Antigen retrieval by citrate solution improves western blot signal.

In the present work, we describe and evaluate an additional step to the standard western blot protocol to increase signal strength after revealing. Weak or absence of signal is a common issue in western blot protocol leading to unexpected results. In our Antigen Retrieval for Western Blot Method (ARWB method), after transfer, the membrane was incubated in a citrate buffer following normal antigen retrieval procedure used for immunohistochemistry. Later, standard protocol was performed in order to reveal and compare with unexposed membranes to this antigen retrieval step. Signal in bands obtained by the modified protocol resulted significantly higher (in all 13 antibodies analyzed) compared to standard protocol. Some bands were only visible after citrate incubation. This method is a simple and economical way to improve results in western blot analysis. •The ARWB method significantly increases band's density in all antibodies analyzed.•Protein localization does not influence the efficacy of the ARWB method since membrane and citoplasmatic proteins bands increase their signal in a similar way after the protocol is performed.•This ARWB method is simple, safe, economical and undoubtedly helpful in immunoblotting for proteins with weak signal.

Combined proteomic and miRNome analyses of mouse testis exposed to an endocrine disruptors chemicals mixture reveals altered toxicological pathways involved in male infertility.

The increase in male idiopathic infertility has been associated with daily exposure to endocrine disruptors chemicals (EDCs). Nevertheless, the mechanisms of action in relation to dysregulating proteins and regulatory microRNAs are unknown. We combined proteomic and miRNome analyses of mouse testis chronically exposed to low doses of a define mixture of EDCs [phthalates: bis(2-ethylhexyl), dibutyl and benzyl-butyl; 4-nonylphenol and 4-tert-octylphenol], administered in the drinking water from conception until adulthood (post-natal Day 60/75) and compared them with no-exposed control mice. We analysed fertility parameters and global changes in the patterns of mice testis proteome by 2D electrophoresis/mass spectrometry, along with bioinformatic analyses of dysregulated microRNAs, and their association with published data in human infertile patients. We detected a decrease in the potential fertility of exposed mice associated with changes in the expression of 18 proteins (10 up-regulated, 8 down-regulated). Functional analysis showed that 89% were involved in cell death. Furthermore, we found a group of 23 microRNAs/isomiRs (down-regulated) correlated with six of the up-regulated target proteins (DIABLO, PGAM1, RTRAF, EIF4E, IVD and CNDP2). Regarding this, PGAM1 up-regulation was validated by Western blot and mainly detected in Sertoli cells. Some of these microRNA/protein dysregulations were reported in human testis with spermatogenic failure. Overall, a chronic exposure to EDCs mixture in human males could potentially lead to spermatogenic failure through changes in microRNA expression, which could post-transcriptionally dysregulate mRNA targets that encode proteins participating in cell death in testicular cells. Finally, these microRNA/protein dysregulations need to be validated with other EDCs mixtures and concentrations.

Bisphenol-A and Nonylphenol Induce Apoptosis in Reproductive Tract Cancer Cell Lines by the Activation of ADAM17.

Endocrine-disruptor chemicals (EDCs), such as bisphenol A (BPA) and nonylphenol (NP), have been widely studied due to their negative effects on human and wildlife reproduction. Exposure to BPA or NP is related to cell death, hormonal deregulation, and cancer onset. Our previous studies showed that both compounds induce A Disintegrin And Metalloprotease 17 (ADAM17) activation. Here, we show that BPA and NP induce apoptosis in prostate and ovary cancer cell lines, in a process dependent on ADAM17 activation. ADAM17 knockdown completely prevented apoptosis as well as the shedding of ADAM17 substrates. Both compounds were found to induce an increase in intracellular calcium (Ca2+) only in Ca2+-containing medium, with the NP-treated cells response being more robust than those treated with BPA. Additionally, using a phosphorylated protein microarray, we found that both compounds stimulate common intracellular pathways related to cell growth, differentiation, survival, and apoptosis. These results suggest that BPA and NP could induce apoptosis through ADAM17 by activating different intracellular signaling pathways that may converge in different cellular responses, one of which is apoptosis. These results confirm the capacity of these compounds to induce cell apoptosis in cancer cell lines and uncover ADAM17 as a key regulator of this process in response to EDCs.

Exposición a Bisfenol A (BPA) en mujeres embarazadas y su relación con la obesidad en sus hijos: Revisión Sistemática / Pregnant Women's Exposure to Bisphenol A (BPA) and its Relation to their Children's Obesity: A systematic Review / Exposição ao bisfenol a (BPA) em mulheres engravidadas e a sua relação com a obesidade em seus filhos: uma revisão sistemática

Resumen Introducción: El bisfenol A (BPA) es un contaminante químico no persistente que altera el funcionamiento normal del sistema endocrino. Se sugiere que la exposición prenatal a BPA se asocia con la obesidad en la descendencia. Objetivo: Revisar la literatura sobre la exposición al BPA en mujeres embarazadas y su relación con la obesidad en sus hijos. Metodología: Revisión sistemática de acuerdo a la guía PRISMA, donde se realizaron búsquedas en las bases de datos Pubmed, ScienceDirect, Clinical Key, Medline, Ebsco y Scielo y el motor de búsqueda Google Scholar hasta 30 de abril de 2017 por dos investigadores independientes que utilizaron iguales términos de búsqueda. Se incluyeron estudios prospectivos de cohorte realizados que midieron el BPA en la orina materna. Resultados: Se incluyeron 5 estudios con tamaños de muestra que varían entre 297 y 757 binomios madre e hijo, se encontró asociación positiva entre la exposición prenatal a BPA con la circunferencia de cintura en niños de cuatro años β: 0.28 (IC95%:0.01 a 0.57) y el índice de masa grasa β: 0.31 (IC95%: 0.01 a 0.60) en dos de los estudios. Además, se observaron asociaciones positivas y/o negativas no significativas con índice de masa corporal y su puntaje Z, porcentaje de grasa, sobrepeso/obesidad, peso y talla al nacer, porcentaje de masa grasa. Conclusión: Los resultados del cuerpo existente de estudios epidemiológicos de cohorte, limita las afirmaciones sobre un vínculo causal entre la exposición prenatal BPA y la obesidad postnatal.

Abstract Introduction: Bisphenol A (BPA) is a non-persistent chemical pollutant which alters the normal functioning of the endocrine system. It is suggested that prenatal exposure is related to descendant obesity. Objective: Review literature on pregnant women's exposure to BPA and the relation to their children's obesity. Methodology: Systematic review in accordance with PRISMA guidelines. Searches were conducted on databases including Pubmed, ScienceDirect, Clinical Key, Medline, Ebsco and Scielo and Google Scholar search engine until April 30, 2017 by two independent researchers that used the same search terms. Prospective cohort conducted studies were included because they measured BPA in maternal urine. Results: Five studies were included with sample sizes ranging from 297 to 757 mother-child binomials. The review found a positive association between prenatal BPA exposure with 4-year-old children's waist circumference β: 0.28 (95% CI :0.01 to 0.57) and the fat mass index β: 0.31 (95%CI: 0.01 to 0.60) in two of the studies. non-significant positive and/or negative associations where observed with body mass index z-scores, overweight/ obesity, weight and size at birth, body mass percentage. Conclusion: The results of cohort epidemiological studies constrain statements regarding a causal link between prenatal BPA exposure and postnatal obesity.

Resumo Introdução: O bisfenol A (BPA) é um contaminante químico não persistente que altera o funcionamento normal do sistema endócrino. Se sugere que a exposição pré-natal se associa com a obesidade na descendência. Objetivo: Revisar a literatura sobre a exposição ao BPA em mulheres engravidadas e a sua relação com a obesidade em seus filhos. Metodologia: Revisão sistemática de acordo com a guia PRISMA. Se realizaram pesquisas nas bases de dados Pubmed, ScienceDirect, Clinical Key, Medline, Ebsco e Scielo e o motor de pesquisa Google Scholar até o 30 de Abril de 2017 por dois investigadores independentes que utilizaram os mesmos termos de busca. Se incluíram estudos prospectivos de coorte realizados que calcularam o BPA na urina materna. Resultados: Se incluíram 5 estudos com tamanhos de amostra entre 297 e 757 binômios mãe e filho, se encontrou associação positiva entre a exposição pré-natal a BPA com a circunferência de cintura em meninos de quatro anos β: 0.28 (IC95%:0.01 a 0.57) e o índice de massa de gordura β: 0.31 (IC95%: 0.01 a 0.60) em dois dos estudos. Se enxergaram associações positivas e/ou negativas não significativas com índice de massa corporal e a sua pontuação Z, porcentagem de gordura, sobrepeso/obesidade, peso e dimensão ao nascer, porcentagem de massa de gordura. Conclusão: Os resultados de estudos epidemiológicos de coorte, limita as afirmações sobre um vínculo causal entre a exposição pré-natal BPA e a obesidade pós-natal.

Reproductive Alterations in Chronically Exposed Female Mice to Environmentally Relevant Doses of a Mixture of Phthalates and Alkylphenols.

Endocrine-disrupting chemicals (EDCs) are exogenous compounds that modify hormone biosynthesis, causing adverse effects to human health. Among them, phthalates and alkylphenols are important due to their wide use in plastics, detergents, personal care products, cosmetics, and food packaging. However, their conjoint effects over reproductive female health have not been addressed. The aim of this work was to test the effect of chronically exposed female mice to a mixture of three phthalates [bis (2-ethylhexyl), dibutyl, and benzyl butyl] and two alkylphenols (4-nonylphenol and 4-tert-octylphenol) from conception to adulthood at environmentally relevant doses. These EDCs were administered in two doses: one below the minimal risk dose to cause adverse effects on human development and reproduction [1 mg/kg body weight (BW)/d of the total mixture] and the other one based on the reference value close to occupational exposure in humans (10 mg/kg BW/d of the total mixture). Our results show that both doses had similar effects regarding the uterus and ovary relative weight, estrous cyclicity, serum levels of progesterone and 17ß-estradiol, and expression of key elements in the steroidogenesis pathway (acute steroidogenic regulatory protein and CYP19A1). However, only the 1-mg/kg BW/d dose delayed the onset of puberty and the transition from preantral to antral follicles, whereas the 10-mg/kg BW/d dose decreased the number of antral follicles and gonadotropin receptor expression. In addition, we observed changes in several fertility parameters in exposed females and in their progeny (F2 generation). In conclusion, our results indicate that chronic exposure to a complex EDC mixture, at environmentally relevant doses, modifies reproductive parameters in female mice.

Editor's Highlight: Differential Effects of Exposure to Single Versus a Mixture of Endocrine-Disrupting Chemicals on Steroidogenesis Pathway in Mouse Testes.

Endocrine-disrupting chemicals (EDCs) generate reproductive dysfunctions affecting the biosynthesis of steroid hormones and genes of the steroidogenic pathway. EDCs effects are mainly reported as a result of exposure to single compounds. However, humans are environmentally exposed to a mixture of EDCs. Herein, we assess chronic exposure to single alkylphenols and phthalates versus a mixture in mouse testes histology and steroidogenesis. Pregnant mice were exposed through drinking water to: 0.3 mg/kg-body weight (BW)/d of each phthalate (bis (2-ethylhexyl) phthalate, dibutyl phthalate, benzyl butyl phthalate), 0.05 mg/kg-BW/d of each alkylphenol (4-nonylphenol, 4-tert-octylphenol), or their mixture, covering from 0.5 postcoital day to weaning, continuing in the male offspring each exposure until adulthood (60-days old). Body and relative testis weight were increased in mixture-exposed mice along with histological alterations. Intratesticular testosterone (T) changed only in mice exposed to DBP, whereas estradiol (E2) levels were altered in all groups (except benzyl butyl phthalate). mRNA levels of genes encoding hormones of the steroid pathway (Cyp11a1, Hsd3b1, Cyp17a1, and Cyp19a1), cholesterol transporters (Star), and transcriptional factors (Sp1) showed that mice exposed to single or mixed compounds had alterations in at least 2 transcripts. However, none of the different types of exposure induced changes in all transcripts. In addition, changes at the mRNA or protein levels with single compounds were not always the same as those with a mixture. In conclusion, the effects of a chronic exposure to a mixture of EDCs on the expression of genes and proteins of the steroidogenic pathway and hormonal status were different from those exposed to single EDC.

Búsqueda del plásmido de virulencia de Salmonella en aislamientos clínicos colombianos / Search of the Salmonella virulence plasmid in Colombian clinical isolates

Introducción: Salmonella es un bacilo Gram negativo, patógeno intracelular que invade células intestinales no fagocíticas y macrófagos en un proceso complejo que requiere múltiplesgenes. El plásmido de virulencia de Salmonella “spv” contiene el gen spvR cuya función es aumentarsu virulencia produciendo invasión sistémica en ratones.Métodos: Se buscó el gen spvR de Salmonella spp en aislamientos clínicos humanos provenientes de pacientes con sintomatología sistémica (fiebre tifoidea) y no sistémica (diarrea), con el fin de comparar la presencia del gen con la clínica del paciente. El gen spvR se buscó en 55 aislamientosde S. Typhi de pacientes con fiebre tifoidea, 20 de S. Enteritidis y 20 de S. Typhimurium, aislados de pacientes con diarrea. Se realizó PCR a partir de ADN genómico y plasmídico. Los productos sesecuenciaron y las secuencias obtenidas de material genómico y plasmídico se compararon utilizando análisis bioinformáticos Resultados: El 100 % de los aislamientos de S. Typhifueron negativos para spvR, mientras que 75 % de losaislamientos de S. Enteritidis y 40 % de S. Typhimurium,presentaron amplificación del gen spvR, tanto en material genómico como plasmídico. Se evidenció tambiénla presencia de secuencias plasmídicas homólogas al gen spvR en regiones cromosomales de los serotiposestudiados.Conclusiones: Los resultados muestran que la ausenciadel plásmido de virulencia de Salmonella no está relacionadacon la infección sistémica, pues no fue encontrado en S. Typhi productora de fiebre tifoidea. Se confirmótambién que existe material cromosomal homólogo al genspvR del plásmido de virulencia en el cromosoma de los serotipos S. Enteritidis y S. Typhimurium, pero no seencontró material similar en el cromosoma de S. Typhi.

Introduction: Salmonella is a Gram negative bacillus,intracellular pathogen that invades nonphagocytic intestinal cells and macrophages in a complex process that requires multiple genes.The salmonella virulence plasmid “spv” contains the spvR gene whose function is to increase its virulence producing systemic invasion in mice. Methods: We search for spvR gene in human clinicalisolates of Salmonella spp from patients with systemic infection (typhoid fever) and with nonsystemic infection (diarrheal), in order to comparethe presence of the gene to clinical outcome. spvR was searched in 55 clinical isolates of S. Typhi from typhoid fever patients, 20 of S. Enteritidisand 20 of S. Typhimurium isolates frompatients with diarrheal. PCR from chromosomal and plasmid DNA was performed. The PCR productswere sequenced and the sequences obtained from genomic and plasmid materials were compared using bioinformatics analysis.Results: 55 (100 %) isolates of S. Typhi were negativefor spvR. 15 (75 %) isolates of S. Enteritidis and 8 (40 %) of S. Typhimurium were positive forspvR both from genomic and plasmid material. These results show that clinical isolates of S.Typhi considered more virulent not showed spvR, and that diarrhea-producing serotypes S. Enteritidisand S. Typhimurium in 75 and 40 % respectively were positive for the gene, questioning the role of virulence of the gene in strains producing human infections. It also showed the presenceof plasmid gene sequences homologous to chromosomal regions in the studied serotypes.Conclusion: Results show that the absence of the plasmid of Salmonella’s virulence is not relatedwith the systemic outcome in the clinical isolates of Salmonella Typhi that were studied. There was also confirmed that exists chromosomal material homologous to the gene spvR virulenceplasmid in the chromosome of serotypes S. Enteritidis and S. Typhimurium but similar material wasn’t found in S. Typhi chromosome.