Bisphenol A and 17α-ethinylestradiol-induced transgenerational gene expression differences in the brain-pituitary-testis axis of medaka, Oryzias latipes†.

Endocrine disrupting chemicals (EDCs), such as bisphenol A (BPA) and 17α-ethinylestradiol (EE2), can have far reaching health effects, including transgenerational abnormalities in offspring that never directly contacted either chemical. We previously reported reduced fertilization rates and embryo survival at F2 and F3 generations caused by 7-day embryonic exposure (F0) to 100 µg/L BPA or 0.05 µg/L EE2 in medaka. Crossbreeding of fish in F2 generation indicated subfertility in males. To further understand the mechanisms underlying BPA or EE2-induced adult onset and transgenerational reproductive defects in males, the present study examined the expression of genes regulating the brain-pituitary-testis (BPT) axis in the same F0 and F2 generation male medaka. Embryonic exposure to BPA or EE2 led to hyperactivation of brain and pituitary genes, which are actively involved in reproduction in adulthood of the F0 generation male fish, and some of these F0 effects continued to the F2 generation (transgenerational effects). Particularly, the F2 generation inherited the hyperactivated state of expression for kisspeptin (kiss1 and kiss2) and their receptors (kiss1r and kiss2r), and gnrh and gnrh receptors. At F2 generation, expression of DNA methyltransferase 1 (dnmt1) decreased in brain of the BPA treatment lineage, while EE2 treatment lineage showed increased dnmt3bb expression. Global hypomethylation pattern was observed in the testis of both F0 and F2 generation fish. Taken together, these results demonstrated that BPA or EE2-induced transgenerational reproductive impairment in the F2 generation was associated with alterations of reproductive gene expression in brain and testis and global DNA methylation in testis.

Effects of the environmental estrogenic contaminants bisphenol A and 17α-ethinyl estradiol on sexual development and adult behaviors in aquatic wildlife species.

Endocrine disrupting chemicals (EDCs), including the mass-produced component of plastics, bisphenol A (BPA) are widely prevalent in aquatic and terrestrial habitats. Many aquatic species, such as fish, amphibians, aquatic reptiles and mammals, are exposed daily to high concentrations of BPA and ethinyl estradiol (EE2), estrogen in birth control pills. In this review, we will predominantly focus on BPA and EE2, well-described estrogenic EDCs. First, the evidence that BPA and EE2 are detectable in almost all bodies of water will be discussed. We will consider how BPA affects sexual and neural development in these species, as these effects have been the best characterized across taxa. For instance, such chemicals have been in many cases reported to cause sex-reversal of males to females. Even if these chemicals do not overtly alter the gonadal sex, there are indications that several EDCs might demasculinize male-specific behaviors that are essential for attracting a mate. In so doing, these chemicals may reduce the likelihood that these males reproduce. If exposed males do reproduce, the concern is that they will then be passing on compromised genetic fitness to their offspring and transmitting potential transgenerational effects through their sperm epigenome. We will thus consider how diverse epigenetic changes might be a unifying mechanism of how BPA and EE2 disrupt several processes across species. Such changes might also serve as universal species diagnostic biomarkers of BPA and other EDCs exposure. Lastly, the evidence that estrogenic EDCs-induced effects in aquatic species might translate to humans will be considered.

Developmental exposure to bisphenol A (BPA) alters sexual differentiation in painted turtles (Chrysemys picta).

Environmental chemicals can disrupt endocrine signaling and adversely impact sexual differentiation in wildlife. Bisphenol A (BPA) is an estrogenic chemical commonly found in a variety of habitats. In this study, we used painted turtles (Chrysemys picta), which have temperature-dependent sex determination (TSD), as an animal model for ontogenetic endocrine disruption by BPA. We hypothesized that BPA would override TSD and disrupt sexual development. We incubated farm-raised turtle eggs at the male-producing temperature (26°C), randomly assigned individuals to treatment groups: control, vehicle control, 17ß-estradiol (E2, 20ng/g-egg) or 0.01, 1.0, 100µgBPA/g-egg and harvested tissues at hatch. Typical female gonads were present in 89% of the E2-treated "males", but in none of the control males (n=35). Gonads of BPA-exposed turtles had varying amounts of ovarian-like cortical (OLC) tissue and disorganized testicular tubules in the medulla. Although the percentage of males with OLCs increased with BPA dose (BPA-low=30%, BPA-medium=33%, BPA-high=39%), this difference was not significant (p=0.85). In all three BPA treatments, SOX9 patterns revealed disorganized medullary testicular tubules and ß-catenin expression in a thickened cortex. Liver vitellogenin, a female-specific liver protein commonly used as an exposure biomarker, was not induced by any of the treatments. Notably, these results suggest that developmental exposure to BPA disrupts sexual differentiation in painted turtles. Further examination is necessary to determine the underlying mechanisms of sex reversal in reptiles and how these translate to EDC exposure in wild populations.

A second hit by PFOS exposure exacerbated developmental defects in medaka embryos with a history of ancestral BPA exposure.

Bisphenol-A (BPA), a known endocrine-disrupting chemical (EDC) in plastics and resins, has been found to induce heritable health effects in fish and mammals, affecting directly exposed individuals and indirectly their progenies in subsequent generations. It is not clearly understood if subsequent generations of the BPA-exposed ancestors have increased sensitivity to the second hit by the chemicals of emerging concern. To understand this, the present study examined the effects of developmental exposure to perfluorooctanesulfonic acid (PFOS), which has been a global contaminant recently, in embryos whose ancestors were exposed to BPA. Two lineages of medaka (Oryzias latipes) were established: 1) the BPA lineage in which the F0 generation was exposed to 10 ug/L BPA during early development and 2) the control lineage with no BPA exposure in the F0 generation. These lineages were raised up to the F4 generation without further exposure. The embryos of the F4 generation were exposed to PFOS at 0, 0.002, 0.02, 0.2, 2, and 20 mg/L concentrations. Early developmental defects resulting in mortality, delayed hatching, teratogenic phenotypes, and altered gene expression were examined in both lineages. The expression level of genes encoding DNA methyltransferases and genes responsible for oxidative stress defense were determined. Following environmentally relevant PFOS exposure, organisms with a history of BPA exposure displayed significant changes in all categories of developmental defects mentioned above, including increased expression of genes related to oxidative stress, compared to individuals without BPA exposure. The present study indicates that a history of ancestral BPA exposure can alter sensitivity to developmental disorders following the second hit by PFOS exposure. The variable of ancestral BPA exposure should be considered in mechanistic, medical, and regulatory toxicology, and can also be applied to holistic environmental equity research.

The Conflict between Regulatory Agencies over the 20,000-Fold Lowering of the Tolerable Daily Intake (TDI) for Bisphenol A (BPA) by the European Food Safety Authority (EFSA).

BACKGROUND: The European Food Safety Authority (EFSA) recommended lowering their estimated tolerable daily intake (TDI) for bisphenol A (BPA) 20,000-fold to 0.2 ng/kg body weight (BW)/day. BPA is an extensively studied high production volume endocrine disrupting chemical (EDC) associated with a vast array of diseases. Prior risk assessments of BPA by EFSA as well as the US Food and Drug Administration (FDA) have relied on industry-funded studies conducted under good laboratory practice protocols (GLP) requiring guideline end points and detailed record keeping, while also claiming to examine (but rejecting) thousands of published findings by academic scientists. Guideline protocols initially formalized in the mid-twentieth century are still used by many regulatory agencies. EFSA used a 21st century approach in its reassessment of BPA and conducted a transparent, but time-limited, systematic review that included both guideline and academic research. The German Federal Institute for Risk Assessment (BfR) opposed EFSA's revision of the TDI for BPA. OBJECTIVES: We identify the flaws in the assumptions that the German BfR, as well as the FDA, have used to justify maintaining the TDI for BPA at levels above what a vast amount of academic research shows to cause harm. We argue that regulatory agencies need to incorporate 21st century science into chemical hazard identifications using the CLARITY-BPA (Consortium Linking Academic and Regulatory Insights on BPA Toxicity) nonguideline academic studies in a collaborative government-academic program model. DISCUSSION: We strongly endorse EFSA's revised TDI for BPA and support the European Commission's (EC) apparent acceptance of this updated BPA risk assessment. We discuss challenges to current chemical risk assessment assumptions about EDCs that need to be addressed by regulatory agencies to, in our opinion, become truly protective of public health. Addressing these challenges will hopefully result in BPA, and eventually other structurally similar bisphenols (called regrettable substitutions) for which there are known adverse effects, being eliminated from all food-related and many other uses in the EU and elsewhere. https://doi.org/10.1289/EHP13812.

SRY induced TCF21 genome-wide targets and cascade of bHLH factors during Sertoli cell differentiation and male sex determination in rats.

Male sex determination is initiated through the testis-determining factor SRY that promotes Sertoli cell differentiation and subsequent gonadal development. The basic helix-loop-helix (bHLH) gene Tcf21 was identified as one of the direct downstream targets of SRY. The current study was designed to identify the downstream targets of TCF21 and the potential cascade of bHLH genes that promote Sertoli cell differentiation. A modified ChIP-Chip comparative hybridization analysis identified 121 direct downstream binding targets for TCF21. The gene networks and cellular pathways potentially regulated by these TCF21 targets were identified. One of the main bHLH targets for TCF21 was the bHLH gene scleraxis (Scx). An embryonic ovarian gonadal cell culture was used to examine the functional role of Sry, Tcf21, and Scx to promote an in vitro sex reversal and induction of Sertoli cell differentiation. SRY and TCF21 were found to induce the initial stages of Sertoli cell differentiation, whereas SCX was found to induce the later stages of Sertoli cell differentiation associated with pubertal development using transferrin gene expression as a marker. Therefore, a cascade of SRY followed by TCF21 followed by SCX appears to promote, in part, Sertoli cell fate determination and subsequent differentiation. The current observations help elucidate the initial molecular events involved in the induction of Sertoli cell differentiation and testis development.

Potassium perchlorate effects on primordial germ cells of developing medaka larvae.

Perchlorate is a chemical compound commonly used in military artillery and equipment. It has been detected in drinking water, air, soil, and breast milk. Exposure of humans to perchlorate can occur in the theater of war and areas adjacent to military training grounds. A high concentration of perchlorate has been found to affect reproduction in vertebrates, including fish. However, whether environmental concentrations of perchlorate can affect primordial germ cells (PGCs), the founders of sperm and eggs, is not clearly understood. In the present study, we examined the effects of 0, 10, 100, and 1000 µg/L potassium perchlorate exposure on the embryonic development of medaka and their PGCs. Perchlorate exposure delayed hatching time, reduced heartbeat, inhibited migration of PGCs, and increased developmental deformities in the larvae. The 10 and 20 mg/L concentrations of perchlorate were lethal to embryos, whereas vitamin C co-treatment (1 mg/L) completely blocked perchlorate-induced mortality. RNA-seq analysis of isolated PGCs showed a non-linear pattern in expression profiles of differentially altered genes. Significantly upregulated genes were found in PGCs from the 10 and 1000 µg/L groups, whereas the 100 µg/L groups showed the highest number of significantly downregulated genes. Gene ontology analysis predicted differentially expressed genes to be involved in proteolysis, metabolic processes, peptides activity, hydrolase activity, and hormone activity. Among the cellular components, extracellular, intracellular, sarcoplasmic, and 6-phosphofructokinase and membrane-bounded processes were affected. Ingenuity Pathway Analysis of PGC transcriptomes revealed thyroid hormone signaling to be affected by all concentrations of perchlorate. The present results suggested that perchlorate affected the development of medaka larvae and vitamin C was able to ameliorate perchlorate-induced embryo mortality. Additionally, perchlorate altered the global transcriptional network in PGCs in a non-linear fashion suggesting its potential effects on developing germ cells and fertility.

SRY directly regulates the neurotrophin 3 promoter during male sex determination and testis development in rats.

Neurotrophin 3 (Ntf3) is expressed in Sertoli cells and acts as a chemo-attractant for cell migration from the mesonephros into the developing testis, a process critical to the early morphological events of testis cord formation. The male sex-determining gene Sry initiates the process of testicular development. Sox9 is a key regulator of male sex determination and is directly regulated by SRY. Information on other downstream target genes of SRY is limited. The current study demonstrates an interaction of SRY with the Ntf3 promoter both in vitro and in vivo. The Ntf3 promoter in both rat and mouse contains at least one putative SRY binding site in the -0.6 kb promoter region. In a luciferase reporter assay system, both SRY and SOX9 stimulated the Ntf3 promoter in vitro through an interaction with this SRY-binding motif. In an immunoprecipitation-based pull-down assay, recombinant SRY protein bound the Ntf3 promoter fragment containing an intact SRY binding site, whereas the same protein did not interact with the fragment containing a mutated SRY motif. Specific antibodies against SRY were used in a chromatin immunoprecipitation (ChIP) assay of embryonic testis and were found to precipitate the Ntf3 promoter region. The SRY ChIP assay confirmed the direct interaction between SRY and the Ntf3 promoter in vivo during male sex determination. Observations suggest that SRY physically interacts with the Ntf3 promoter during male sex determination to coordinate cell migration in the testis to form testis cords.

DNA methylation reprogramming in medaka fish, a promising animal model for environmental epigenetics research.

DNA methylation is a major epigenetic modification that undergoes dramatic changes in two epigenetic reprogramming windows during development: first in preimplantation embryos and second in primordial germ cell (PGC) specification. In both windows, DNA methylation patterns are reprogrammed genome-wide, and the majority of inherited methylation marks are erased, generating cells with broad developmental potential. Recent studies reported that the reprogramming of genome methylation in medaka is similar to human and mouse, suggesting that medaka may serve as a suitable biomedical model for comparative studies focused on the epigenetic and transgenerational inheritance of phenotypic traits. In this mini review, we will discuss how somatic and germ cells in post-fertilization stage embryos are epigenetically reprogrammed in mammals and fishes with a particular focus on DNA methylation dynamics.

Distinct expression patterns of seven crucial microRNAs during early embryonic development in medaka (Oryzias latipes).

MicroRNAs (i.e. miRNAs) are small non-coding RNAs that play essential modulation roles in embryonic development in vertebrates. Paternal and maternal miRNAs contribute to the development of post-fertilization embryo and zygotic genome activation. The pattern of expression and their roles in embryonic development of medaka are not clearly understood. The present study, therefore, examined a temporal expression of seven miRNAs, ola-let-7a, ola-miR-202-3p, ola-miR-126-3p, ola-miR-122, ola-miR-92a, ola-miR-125a-3p and ola-miR-430a in sperm, oocytes, and embryos during early developmental stages. Three unique expression patterns of miRNAs were observed. ola-let7a, ola-miR-202-3p and ola-miR-126-3p showed both paternal and maternal expression, and ola-miR-122, ola-miR-92a, ola-miR-125a-3p showed maternal expression only. The expression of six out of seven miRNAs significantly decreased after maternal-zygotic transition (MZT), whereas ola-miR-430a expression initiated only after MZT. The temporal dynamic expression of these miRNAs suggests their potential roles in early embryogenesis and genome-zygotic activation in medaka.

Developmental abnormalities and epigenetic alterations in medaka (Oryzias latipes) embryos induced by triclosan exposure.

Triclosan (TCS), an antibacterial and antifungal agent present in some consumer products, has been detected in the environment at varying concentrations. TCS exposure has been found to cause developmental abnormalities and endocrine disruption in various species of fish. It is not clearly understood whether TCS exposure causes epigenetic alterations in developing embryos and their germ cells. In the present study, we examined the effects of TCS exposure (0, 50, 100 and, 200 µg/L) on embryonic development and primordial germ cells (PGCs), which are precursors of sperm and eggs, in medaka (Oyzias latipes). Developmental TCS exposure from 8 h post-fertilization through 15 days post-fertilization (dpf) resulted in several developmental abnormalities, including enlarged yolk sac, decreased head trunk angle (HTA), and severe edema in the pericardial region. The male ratio increased in the 100 µg/L TCS exposure group, which was negatively correlated with the expression of cyp19ala (a gene encoding aromatase) and arα (androgen receptor alpha). Developmental 50 µg/L TCS exposure resulted in global hypomethylation in the whole body but not in the isolated PGCs. Expression of the gene encoding DNA methyltransferases (dnmt1 and dnmt3aa) was decreased by 50 µg/L TCS exposure both in the whole body and PGCs. TCS altered the expression of genes encoding enzymes involved in DNA methylation and demethylation in PGCs, suggesting epigenetic effects on germ cells. The present results demonstrate that the embryos exposed to the tested concentrations of TCS develop deformities during the early life stages and that the TCS within this range possesses endocrine disrupting properties potential enough to alter sex ratios of developing embryos.

Transcriptome analysis of testis reveals the effects of developmental exposure to bisphenol a or 17α-ethinylestradiol in medaka (Oryzias latipes).

Endocrine disrupting chemicals (EDCs) can induce abnormalities in organisms via alteration of molecular pathways and subsequent disruption of endocrine functions. Bisphenol A (BPA) and 17α-ethinylestradiol (EE2) are ubiquitous EDCs in the environment. Many aquatic organisms, including fish, are often exposed to varying concentrations of BPA and EE2 throughout their lifespan. Both BPA and EE2 can activate estrogenic signaling pathways and cause adverse effects on reproduction via alteration of pathways associated with steroidogenesis. However, transcriptional pathways that are affected by chronic exposure to these two ubiquitous environmental estrogens during embryonic, larval, and juvenile stages are not clearly understood. In the present study, we examined transcriptional alterations in the testis of medaka fish (Oryzias latipes) chronically exposed to a low concentration of BPA or EE2. Medaka were exposed to BPA (10 µg/L) or EE2 (0.01 µg/L) from 8 h post-fertilization (as embryos) to adulthood 50 days post fertilization (dpf), and transcriptional alterations in the testis were examined by RNA sequencing (RNA-seq). Transcriptomic profiling revealed 651 differentially expressed genes (DEGs) between BPA-exposed and control testes, while 1475 DEGs were found between EE2-exposed and control testes. Gene ontology (GO) analysis showed a significant enrichment of "intracellular receptor signaling pathway", "response to steroid hormone" and "hormone-mediated signaling pathway" in the BPA-induced DEGs, and of "cilium organization", "microtubule-based process" and "organelle assembly" in the EE2-induced DEGs. Pathway analysis showed significant enrichment of "integrin signaling pathway" in both treatment groups, and of "cadherin signaling pathway", "Alzheimer disease-presenilin pathway" in EE2-induced DEGs. Single nucleotide polymorphism (SNP) and insertion-deletion (Indel) analysis found no significant differences in mutation rates with either BPA or EE2 treatments. Taken together, global gene expression differences in testes of medaka during early stages of gametogenesis were responsive to chronic BPA and EE2 exposure.

Effects of early life stage exposure of largemouth bass to atrazine or a model estrogen (17α-ethinylestradiol).

Endocrine disrupting contaminants are of continuing concern for potentially contributing to reproductive dysfunction in largemouth and smallmouth bass in the Chesapeake Bay watershed (CBW) and elsewhere. Exposures to atrazine (ATR) have been hypothesized to have estrogenic effects on vertebrate endocrine systems. The incidence of intersex in male smallmouth bass from some regions of CBW has been correlated with ATR concentrations in water. Fish early life stages may be particularly vulnerable to ATR exposure in agricultural areas, as a spring influx of pesticides coincides with spawning and early development. Our objectives were to investigate the effects of early life stage exposure to ATR or the model estrogen 17α-ethinylestradiol (EE2) on sexual differentiation and gene expression in gonad tissue. We exposed newly hatched largemouth bass (LMB, Micropterus salmoides) from 7 to 80 days post-spawn to nominal concentrations of 1, 10, or 100 µg ATR/L or 1 or 10 ng EE2/L and monitored histological development and transcriptomic changes in gonad tissue. We observed a nearly 100% female sex ratio in LMB exposed to EE2 at 10 ng/L, presumably due to sex reversal of males. Many gonad genes were differentially expressed between sexes. Multidimensional scaling revealed clustering by gene expression of the 1 ng EE2/L and 100 µg ATR/L-treated male fish. Some pathways responsive to EE2 exposure were not sex-specific. We observed differential expression in male gonad in LMB exposed to EE2 at 1 ng/L of several genes involved in reproductive development and function, including star, cyp11a2, ddx4 (previously vasa), wnt5b, cyp1a and samhd1. Expression of star, cyp11a2 and cyp1a in males was also responsive to ATR exposure. Overall, our results confirm that early development is a sensitive window for estrogenic endocrine disruption in LMB and are consistent with the hypothesis that ATR exposure induces some estrogenic responses in the developing gonad. However, ATR-specific and EE2-specific responses were also observed.

Developmental and epigenetic effects of Roundup and glyphosate exposure on Japanese medaka (Oryzias latipes).

Roundup and other glyphosate-based herbicides are the most commonly used herbicides in the world, yet their effects on developing fish embryos are not clearly understood. The present study, therefore, examined developmental teratogenic effects and adult-onset reproductive effects of exposure to environmentally relevant concentrations of glyphosate and Roundup in Japanese medaka fish (Oryzias latipes). Hd-rR strain medaka embryos were exposed to 0.5 mg/L glyphosate, 0.5 mg/L and 5 mg/L Roundup (glyphosate acid equivalent) for the first 15 days of their embryonic life and then allowed to sexually mature without further exposure. Whole body tissue samples were collected at 15 days post fertilization (dpf) and brain and gonad samples were collected in mature adults. Hatching success and phenotypic abnormalities were recorded up until 15 dpf. Roundup (0.5 mg/L) and glyphosate decreased cumulative hatching success, while glyphosate exposure increased developmental abnormalities in medaka fry. Expression of the maintenance DNA methyltransferase gene Dnmt1 decreased, whereas expression of methylcytosine dioxygenase genes (Tet1, Tet2 and Tet3) increased in fry at 15 dpf suggesting that epigenetic alterations increased global DNA demethylation in the developing fry. Fecundity and fertilization efficiency were not altered due to exposure. Among the reproduction-related genes in the brain, kisspeptin receptor (Gpr54-1) expression was significantly reduced in females exposed to 0.5 mg/L and 5 mg/L Roundup, and Gpr54-2 was reduced in the 0.5 mg/L Roundup treatment group. No change in expression of these genes was observed in the male brain. In the testes, expression of Fshr and Arα was significantly reduced in medaka exposed to 0.5 mg/L Roundup and glyphosate, while the expression of Dmrt1 and Dnmt1 was reduced in medaka exposed to 0.5 mg/L glyphosate. No change in expression of these genes was observed in the ovaries. The present study demonstrates that Roundup and its active ingredient glyphosate can induce developmental, reproductive, and epigenetic effects in fish; suggesting that ecological species, mainly fish, could be at risk for endocrine disruption in glyphosate and Roundup-contaminated water bodies.

Bisphenol A and 17α-ethinylestradiol-induced transgenerational differences in expression of osmoregulatory genes in the gill of medaka (Oryzias latipes).

Embryonic bisphenol A (BPA) and 17α-ethinylestradiol (EE2) exposure can have far reaching health effects in fish, including adult onset transgenerational reproductive abnormalities, anxiety, and cardiac disorders. It is unknown whether these two environmental estrogens can induce transgenerational abnormalities in the gill. The present study examined transgenerational effects of BPA or EE2 exposure on genes that are critical for osmoregulation in fish. Medaka (Oryzias latipes) embryos were exposed to either BPA (100 µg/L) or EE2 (0.05 µg/L) for the first 7 days of embryonic development and never thereafter for the remainder of that generation (F0) and in subsequent generations of this study (F1, F2, and F3). Expression of osmoregulatory genes (NKAα1a, NKAα1b, NKAα1c, NKAα3a, NKAα3b, NKCC1a, and CFTR) were examined in gills of the first-generation (F0) adults which were directly exposed as embryo and in the fourth-generation adults (F3), which were never exposed to either of these environmental estrogens. Significant alterations in expression of osmoregulatory genes were observed in both F0 and F3 generations. Within the F0 generation, a sex-specific expression pattern was observed with a downregulation of osmoregulatory genes in males and an upregulation of osmoregulatory genes in females. At the F3 generation, this pattern reversed with the majority of the osmoregulatory genes upregulated in males and downregulated in females, suggesting that exposure to BPA and EE2 during embryonic development induced transgenerational impairment in molecular events associated with osmoregulatory functions in subsequent generations. These adverse outcomes may have impacts on physiological functions related to osmoregulation of fish inhabiting contaminated aquatic environments.

Estrogen receptor 1 expression and methylation of Esr1 promoter in mouse fetal prostate mesenchymal cells induced by gestational exposure to bisphenol A or ethinylestradiol.

Fetal/neonatal environmental estrogen exposures alter developmental programing of the prostate gland causing onset of diseases later in life. We have previously shown in vitro that exposures to 17ß-estradiol (E2) and the endocrine disrupting chemical bisphenol A, at concentrations relevant to human exposure, cause an elevation of estrogen receptor α (Esr1) mRNA in primary cultures of fetal mouse prostate mesenchymal cells; a similar result was observed in the fetal rat urogenital sinus. Effects of these chemicals on prostate mesenchyme in vivo are not well understood. Here we show effects in mice of fetal exposure to the estrogenic drug in mixed oral contraceptives, 17α-ethinylestradiol (EE2), at a concentration of EE2 encountered by human embryos/fetuses whose mothers become pregnant while on EE2-containing oral contraceptives, or bisphenol A at a concentration relevant to exposures observed in human fetuses in vivo. Expression of Esr1 was elevated by bisphenol A or EE2 exposures, which decreased the global expression of DNA methyltransferase 3A (Dnmt3a), while methylation of Esr1 promoter was significantly increased. These results show that exposures to the environmental estrogen bisphenol A and drug EE2 cause transcriptional and epigenetic alterations to expression of estrogen receptors in developing prostate mesenchyme in vivo.

Nano-fibre Integrated Microcapsules: A Nano-in-Micro Platform for 3D Cell Culture.

Nano-in-micro (NIM) system is a promising approach to enhance the performance of devices for a wide range of applications in disease treatment and tissue regeneration. In this study, polymeric nanofibre-integrated alginate (PNA) hydrogel microcapsules were designed using NIM technology. Various ratios of cryo-ground poly (lactide-co-glycolide) (PLGA) nanofibres (CPN) were incorporated into PNA hydrogel microcapsule. Electrostatic encapsulation method was used to incorporate living cells into the PNA microcapsules (~500 µm diameter). Human liver carcinoma cells, HepG2, were encapsulated into the microcapsules and their physio-chemical properties were studied. Morphology, stability, and chemical composition of the PNA microcapsules were analysed by light microscopy, fluorescent microscopy, scanning electron microscopy (SEM), Fourier-Transform Infrared spectroscopy (FTIR), and thermogravimetric analysis (TGA). The incorporation of CPN caused no significant changes in the morphology, size, and chemical structure of PNA microcapsules in cell culture media. Among four PNA microcapsule products (PNA-0, PNA-10, PNA-30, and PNA-50 with size 489 ± 31 µm, 480 ± 40 µm, 473 ± 51 µm and 464 ± 35 µm, respectively), PNA-10 showed overall suitability for HepG2 growth with high cellular metabolic activity, indicating that the 3D PNA-10 microcapsule could be suitable to maintain better vitality and liver-specific metabolic functions. Overall, this novel design of PNA microcapsule and the one-step method of cell encapsulation can be a versatile 3D NIM system for spontaneous generation of organoids with in vivo like tissue architectures, and the system can be useful for numerous biomedical applications, especially for liver tissue engineering, cell preservation, and drug toxicity study.

Atrazine induced transgenerational reproductive effects in medaka (Oryzias latipes).

Atrazine is presently one of the most abundantly used herbicides in the United States, and a common contaminant of natural water bodies and drinking waters in high-use areas. Dysregulation of reproductive processes has been demonstrated in atrazine exposed fish, including alteration of key endocrine pathways on hypothalamic-pituitary-gonadal (HPG) axis. However, the potential for atrazine-induced transgenerational inheritance of reproductive effects in fish has not been investigated. The present study examined the effects of early developmental atrazine exposure on transgenerational reproductive dysregulation in Japanese medaka (Oryzias latipes). F0 medaka were exposed to atrazine (ATZ, 5 or 50 µg/L), 17α-ethinylestradiol (EE2, 0.002 or 0.05 µg/L), or solvent control during the first twelve days of development with no subsequent exposure over three generations. This exposure overlapped with the critical developmental window for embryonic germ cell development, gonadogenesis, and sex determination. Exposed males and females of the F0 generation were bred to produce an F1 generation, and this was continued until the F2 generation. Sperm count and motility were not affected in F0 males; however, both parameters were significantly reduced in the males from F2 Low EE2 (0.002 µg/L), Low ATZ (5 µg/L), and High ATZ (50 µg/L) lineages. Fecundity was unaffected by atrazine or EE2 in F0 through F2 generations; however, fertilization rate was decreased in low atrazine and EE2 exposure lineages in the F2 generation. There were significant transgenerational differences in expression of the genes involved in steroidogenesis and DNA methylation. These results suggest that although early life exposure to atrazine did not cause significant phenotypes in the directly exposed F0 generation, subsequent generations of fish were at greater risk of reproductive dysfunction.