Prenatal exposure to Di(2-ethylhexyl) phthalate and high-fat diet synergistically disrupts gonadal function in male mice†.

Prenatal exposure to Di (2-ethylhexyl) phthalate (DEHP) impairs the reproductive system and causes fertility defects in male offspring. Additionally, high-fat (HF) diet is a risk factor for reproductive disorders in males. In this study, we tested the hypothesis that prenatal exposure to a physiologically relevant dose of DEHP in conjunction with HF diet synergistically impacts reproductive function and fertility in male offspring. Female mice were fed a control or HF diet 7 days prior to mating and until their litters were weaned on postnatal day 21. Pregnant dams were exposed to DEHP or vehicle from gestational day 10.5 until birth. The male offspring's gross phenotype, sperm quality, serum hormonal levels, testicular histopathology, and testicular gene expression pattern were analyzed. Male mice born to dams exposed to DEHP + HF had smaller testes, epididymides, and shorter anogenital distance compared with those exposed to HF or DEHP alone. DEHP + HF mice had lower sperm concentration and motility compared with DEHP mice. Moreover, DEHP + HF mice had more apoptotic germ cells, fewer Leydig cells, and lower serum testosterone levels than DEHP mice. Furthermore, testicular mRNA expression of Dnmt1 and Dnmt3a was two to eight-fold higher than in DEHP mice by qPCR, suggesting that maternal HF diet and prenatal DEHP exposure additively impact gonadal function by altering the degree of DNA methylation in the testis. These results suggest that the combined exposure to DEHP and high-fat synergistically impairs reproductive function in male offspring, greater than exposure to DEHP or HF diet alone.

Nod1-dependent NF-kB activation initiates hematopoietic stem cell specification in response to small Rho GTPases.

Uncovering the mechanisms regulating hematopoietic specification not only would overcome current limitations related to hematopoietic stem and progenitor cell (HSPC) transplantation, but also advance cellular immunotherapies. However, generating functional human induced pluripotent stem cell (hiPSC)-derived HSPCs and their derivatives has been elusive, necessitating a better understanding of the developmental mechanisms that trigger HSPC specification. Here, we reveal that early activation of the Nod1-Ripk2-NF-kB inflammatory pathway in endothelial cells (ECs) primes them to switch fate towards definitive hemogenic endothelium, a pre-requisite to specify HSPCs. Our genetic and chemical embryonic models show that HSPCs fail to specify in the absence of Nod1 and its downstream kinase Ripk2 due to a failure on hemogenic endothelial (HE) programming, and that small Rho GTPases coordinate the activation of this pathway. Manipulation of NOD1 in a human system of definitive hematopoietic differentiation indicates functional conservation. This work establishes the RAC1-NOD1-RIPK2-NF-kB axis as a critical intrinsic inductor that primes ECs prior to HE fate switch and HSPC specification. Manipulation of this pathway could help derive a competent HE amenable to specify functional patient specific HSPCs and their derivatives for the treatment of blood disorders.

Host-specific signatures of the respiratory microbiota in domestic animals.

While the importance of respiratory microbiota in maintaining respiratory health is increasingly recognized, we still lack a comprehensive understanding of the unique characteristics of respiratory microbiota specific to individual hosts. This study aimed to address this gap by analyzing publicly available 16S rRNA gene datasets from various domestic animals (cats, dogs, pigs, donkeys, chickens, sheep, and cattle) to identify host-specific signatures of respiratory microbiota. The findings revealed that cattle and pigs exhibited the highest Shannon diversity index and observed features, indicating a greater microbial variety compared to other animals. Discriminant analysis demonstrated distinct composition of respiratory microbiota across different animals, with no overlapping abundant taxa. The linear discriminant analysis effect size highlighted prevalent host-specific microbiota signatures in different animal species. Moreover, the composition and diversity of respiratory microbiota were significantly influenced by various factors such as individual study, health status, and sampling sites within the respiratory tract. While associations between host and respiratory microbiota have been uncovered, the relative contributions of host and environment in the selection of respiratory microbiota and their impact on host fitness remain unclear. Further investigations involving diverse hosts are necessary to fully comprehend the significance of host-microbial coevolution in maintaining respiratory health.

Single neonatal estrogen implant sterilizes female animals by decreasing hypothalamic KISS1 expression.

Reproductive sterilization by surgical gonadectomy is strongly advocated to help manage animal populations, especially domesticated pets, and to prevent reproductive behaviors and diseases. This study explored the use of a single-injection method to induce sterility in female animals as an alternative to surgical ovariohysterectomy. The idea was based on our recent finding that repetitive daily injection of estrogen into neonatal rats disrupted hypothalamic expression of Kisspeptin (KISS1), the neuropeptide that triggers and regulates pulsatile secretion of GnRH. Neonatal female rats were dosed with estradiol benzoate (EB) either by daily injections for 11 days or by subcutaneous implantation of an EB-containing silicone capsule designed to release EB over 2-3 weeks. Rats treated by either method did not exhibit estrous cyclicity, were anovulatory, and became infertile. The EB-treated rats had fewer hypothalamic Kisspeptin neurons, but the GnRH-LH axis remained responsive to Kisspeptin stimulation. Because it would be desirable to use a biodegradable carrier that is also easier to handle, an injectable EB carrier was developed from PLGA microspheres to provide pharmacokinetics comparable to the EB-containing silicone capsule. A single neonatal injection of EB-microspheres at an equivalent dosage resulted in sterility in the female rat. In neonatal female Beagle dogs, implantation of an EB-containing silicone capsule also reduced ovarian follicle development and significantly inhibited KISS1 expression in the hypothalamus. None of the treatments produced any concerning health effects, other than infertility. Therefore, further development of this technology for sterilization in domestic female animals, such as dogs and cats is worthy of investigation.

Estrogen receptor alpha signaling in dendritic cells modulates autoimmune disease phenotype in mice.

Estrogen is a disease-modifying factor in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) via estrogen receptor alpha (ERα). However, the mechanisms by which ERα signaling contributes to changes in disease pathogenesis have not been completely elucidated. Here, we demonstrate that ERα deletion in dendritic cells (DCs) of mice induces severe neurodegeneration in the central nervous system in a mouse EAE model and resistance to interferon beta (IFNß), a first-line MS treatment. Estrogen synthesized by extragonadal sources is crucial for controlling disease phenotypes. Mechanistically, activated ERα directly interacts with TRAF3, a TLR4 downstream signaling molecule, to degrade TRAF3 via ubiquitination, resulting in reduced IRF3 nuclear translocation and transcription of membrane lymphotoxin (mLT) and IFNß components. Diminished ERα signaling in DCs generates neurotoxic effector CD4+ T cells via mLT-lymphotoxin beta receptor (LTßR) signaling. Lymphotoxin beta receptor antagonist abolished EAE disease symptoms in the DC-specific ERα-deficient mice. These findings indicate that estrogen derived from extragonadal sources, such as lymph nodes, controls TRAF3-mediated cytokine production in DCs to modulate the EAE disease phenotype.

Identification of Transcription Factor Binding Sites by Cleavage Under Target and Release Using Nuclease in Zebrafish.

Cleavage Under Targets and Release Using Nuclease (CUT&RUN) has emerged as a chromatin profiling strategy that excels traditional methods. Although CUT&RUN has been widely utilized in mammalian cells, its use in the zebrafish is at its early stages. In this study, we have developed a protocol to successfully perform CUT&RUN to map transcription factor (TF) binding sites in embryonic, adult tissues, and FACS-sorted zebrafish cells. We also provide a detailed workflow for the identification of predicted TF binding sites that can be utilized in any animal species. Altogether, our strategy will expand this invaluable tool to the zebrafish community, improving the epigenetic resolution that can be achieved in this model organism.

Early postnatal exposure to di(2-ethylhexyl) phthalate causes sex-specific disruption of gonadal development in pigs.

Di(2-ethylhexyl) phthalate (DEHP) is a chemical commonly used as a plasticizer to render polyvinyl chloride products more durable and flexible. Although exposure to DEHP has raised many health concerns due to the identification of DEHP as an endocrine disruptor, it is still used in consumer products, including polyvinyl chloride plastics, medical tubing, car interiors, and children's toys. To investigate the impact of early life exposure to DEHP on the ovary and testes, newborn piglets were orally dosed with DEHP (20 or 200 mg/kg/day) or vehicle control (tocopherol-stripped corn oil) for 21 days. Following treatment, ovaries, testes, and sera were harvested for histological assessment and measurement of steroid hormone levels. In male piglets, progesterone and pregnenolone levels were significantly lower in both treatment groups compared to control, whereas in female piglets, progesterone was significantly higher in the 20 mg group compared to control, indicating sex-specific effects in a non-monotonic manner. Follicle numbers and gene expression of steroidogenic enzymes and apoptotic factors were not altered in treated ovaries compared to controls. In DEHP-treated testes, germ cell migration was impaired and germ cell death was significantly increased compared to controls. Overall, the results of this study suggest that neonatal exposure to DEHP in pigs leads to sex-specific disruption of the reproductive system.

Single intranasal administration of 17ß-estradiol loaded gelatin nanoparticles confers neuroprotection in the post-ischemic brain.

Globally, ischemic stroke is a leading cause of death and adult disability. Previous efforts to repair damaged brain tissue following ischemic events have been hindered by the relative isolation of the central nervous system. We have developed a gelatin nanoparticle-mediated intranasal drug delivery system as an efficient, non-invasive method for delivering 17ß-estradiol (E2) specifically to the brain, enhancing neuroprotection, and limiting systemic side effects. Young adult male C57BL/6 J mice subjected to 30 min of middle cerebral artery occlusion (MCAO) were administered intranasal preparations of E2-GNPs, water soluble E2, or saline as control 1 h after reperfusion. Following intranasal administration of 500 ng E2-GNPs, brain E2 content rose by 5.24 fold (P<0.0001) after 30 min and remained elevated by 2.5 fold at 2 h (P<0.05). The 100 ng dose of E2-GNPs reduced mean infarct volume by 54.3% (P<0.05, n=4) in comparison to saline treated controls, demonstrating our intranasal delivery system's efficacy.

Meta-analysis of bovine respiratory microbiota: link between respiratory microbiota and bovine respiratory health.

Bovine respiratory microbiota plays a significant role in bovine respiratory health. We conducted a meta-analysis using publicly available 16S rRNA gene datasets from the respiratory tract to characterize respiratory microbiota in feedlot cattle. Our aims were to determine the factors that influence microbiota development and to assess the differences in microbiota composition and diversity between healthy calves and those that developed bovine respiratory disease (BRD). Our results showed that the overall composition and diversity of respiratory microbiota in cattle were significantly affected by study design, 16S rRNA hypervariable region sequenced, health status, time since arrival to the feedlot, sampling sites in the respiratory tract and antibiotic treatment. Assessment of diversity indices showed a statistically significant difference between the BRD-affected cattle and healthy control calves. Using multivariate network analysis and Spearman's correlation analyses, we further distinguished the taxa that were commonly associated with BRD when the day of arrival to the feedlot was added to the model. The probability of being identified as BRD was significantly correlated with days 7, 12 and 14 following the calf's arrival to the feedlot. These findings could help in proposing strategies to further evaluate the link between respiratory microbiota and bovine respiratory health.

Progesterone Receptor Serves the Ovary as a Trigger of Ovulation and a Terminator of Inflammation.

Ovulation is triggered by the gonadotropin surge that induces the expression of two key genes, progesterone receptor (Pgr) and prostaglandin-endoperoxide synthase 2 (Ptgs2), in the granulosa cells of preovulatory follicles. Their gene products PGR and PTGS2 activate two separate pathways that are both essential for successful ovulation. Here, we show that the PGR plays an additional essential role: it attenuates ovulatory inflammation by diminishing the gonadotropin surge-induced Ptgs2 expression. PGR indirectly terminates Ptgs2 expression and PGE2 synthesis in granulosa cells by inhibiting the nuclear factor κB (NF-κB), a transcription factor required for Ptgs2 expression. When the expression of PGR is ablated in granulosa cells, the ovary undergoes a hyperinflammatory condition manifested by excessive PGE2 synthesis, immune cell infiltration, oxidative damage, and neoplastic transformation of ovarian cells. The PGR-driven termination of PTGS2 expression may protect the ovary from ovulatory inflammation.

Germline-dependent transmission of male reproductive traits induced by an endocrine disruptor, di-2-ethylhexyl phthalate, in future generations.

In males, defective reproductive traits induced by an exposure to an endocrine disruptor are transmitted to future generations via epigenetic modification of the germ cells. Interestingly, the impacted future generations display a wide range of heterogeneity in their reproductive traits. In this study, the role that the Y chromosome plays in creating such heterogeneity is explored by testing the hypothesis that the Y chromosome serves as a carrier of the exposure impact to future generations. This hypothesis implies that a male who has a Y chromosome that is from a male that was exposed to an endocrine disruptor will display a more severe reproductive phenotype than a male whose Y chromosome is from an unexposed male. To test this hypothesis, we used a mouse model in which F1 generation animals were exposed prenatally to an endocrine disruptor, di-2-ethylhexyl phthalate (DEHP), and the severity of impacted reproductive traits was compared between the F3 generation males that were descendants of F1 males (paternal lineage) and those from F1 females (maternal lineage). Pregnant dams (F0 generation) were exposed to the vehicle or 20 or 200 µg/kg/day of DEHP from gestation day 11 until birth. Paternal lineage F3 DEHP males exhibited decreased fertility, testicular steroidogenic capacity, and spermatogenesis that were more severely impaired than those of maternal lineage males. Indeed, testicular transcriptome analysis found that a number of Y chromosomal genes had altered expression patterns in the paternal lineage males. This transgenerational difference in the DEHP impact can be attributed specifically to the Y chromosome.

Prenatal exposure to an environmentally relevant phthalate mixture disrupts testicular steroidogenesis in adult male mice.

Endocrine disrupting chemicals (EDCs) in the environment are considered to be a contributing factor to the decline in the sperm quality. With growing evidence of the harmful effects of EDCs on the male reproductive system, we tested the hypothesis that prenatal exposure to an environmentally relevant phthalate mixture adversely affects reproductive outcomes and androgen synthesis. In this study, an environmentally relevant composition of phthalates (15% DiNP, 21% DEHP, 36% DEP, 15% DBP, 8% DiBP, and 5% BBzP) that were detected in urine samples of pregnant women in Illinois, United States, was used. Pregnant CD-1 mice (F0) were orally dosed with a vehicle or the phthalate mixtures (20 µg/kg/day, 200 µg/kg/day, 200 mg/kg/day, or 500 mg/kg/day) from gestational day 10.5 to the day of birth. Then, the indices of the reproductive function of the F1 males born to these dams were assessed. Those male mice prenatally exposed to the phthalate mixture had smaller gonads, prostates and seminal vesicles, especially in the 20 µg/kg/day and 500 mg/kg/day phthalate mixture groups, compared to the controls. Importantly, at the age of 12 months, those prenatally exposed mice had significantly lower serum testosterone concentrations accompanied by the decreased mRNA expression of testicular steroidogenic genes (StAR, Cyp11, and Cyp17) and impaired spermatogenesis. Taken together, this study found that prenatal exposure to environmentally relevant doses of a phthalate mixture caused a life-long impact on the reproduction in male mice.

Sanitary pads and diapers contain higher phthalate contents than those in common commercial plastic products.

Sanitary pads and diapers are made of synthetic plastic materials that can potentially be released while being used. This study measured the amounts of volatile organic compounds (VOCs) (methylene chloride, toluene, and xylene) and phthalates (DBP, DEHP, DEP, and BBP) contained in sanitary pads and diapers. In sanitary pads, 5,900- and 130-fold differences of VOC and phthalate concentrations were seen among the brands. In the diapers, 3- and 63-fold differences of VOC and phthalate concentrations were detected among the brands. VOC concentrations from the sanitary pads and diapers were similar to that of the residential air. However, phthalate concentrations of sanitary pads and diapers were significantly higher than those found in common commercial plastic products. As sanitary pads and diapers are in direct contact with external genitalia for an extended period, there is a probability that a considerable amount of VOCs or phthalates could be absorbed into the reproductive system.

Synergetic action between the rumen microbiota and bovine health.

Host-rumen-microbe interactions are essential components of many physiological processes, and therefore can affect ruminant health. Classical knowledge of rumen microbiology is based on culture-dependent methodologies, which only account for 10-20% of the rumen bacterial communities. While, the advancement in DNA sequencing and bioinformatics platforms provide novel approaches to investigate the composition and dynamics of the rumen microbiota. Recent studies demonstrated that the ruminal ecosystem is highly diverse and harbors numerous microbial communities. The composition of these microbial communities are affected by various environmental factors such as nutrition and different management strategies. Disturbance in the microbial ecology of the rumen is associated with the development of various diseases. Despite the flow of recent rumen-based studies, rumen microbiota is still not fully characterized. This review provides an overview of recent efforts to characterize rumen microbiota and its potential role in rumen health and disease. Moreover, the recent effects of dietary interventions and probiotics on rumen microbiota are discussed.

Prenatal Exposure to DEHP Induces Neuronal Degeneration and Neurobehavioral Abnormalities in Adult Male Mice.

Phthalates are a family of synthetic chemicals that are used in producing a variety of consumer products. Di-(2-ethylhexyl) phthalate (DEHP) is an widely used phthalate and poses a public health concern. Prenatal exposure to DEHP has been shown to induce premature reproductive senescence in animal studies. In this study, we tested the hypothesis that prenatal exposure to DEHP impairs neurobehavior and recognition memory in her male offspring and we investigated one possible mechanism-oxidative damage in the hippocampus. Pregnant CD-1 female mice were orally administered 200 µg, 500 mg, or 750 mg/kg/day DEHP or vehicle from gestational day 11 until birth. The neurobehavioral impact of the prenatal DEHP exposure was assessed at the ages of 16-22 months. Elevated plus maze and open field tests were used to measure anxiety levels. Y-maze and novel object recognition tests were employed to measure memory function. The oxidative damage in the hippocampus was measured by the levels of oxidative DNA damage and by Spatial light interference microscopic counting of hippocampal neurons. Adult male mice that were prenatally exposed to DEHP exhibited anxious behaviors and impaired spatial and short-term recognition memory. The number of hippocampal pyramidal neurons was significantly decreased in the DEHP mice. Furthermore, DEHP mice expressed remarkably high levels of cyclooxygenase-2, 8-hydroxyguanine, and thymidine glycol in their hippocampal neurons. DEHP mice also had lower circulating testosterone concentrations and displayed a weaker immunoreactivity than the control mice to androgen receptor expression in the brain. This study found that prenatal exposure to DEHP caused elevated anxiety behavior and impaired recognition memory. These behavioral changes may originate from neurodegeneration caused by oxidative damage and inflammation in the hippocampus. Decreased circulating testosterone concentrations and decreased expression of androgen receptor in the brain also may be factors contributing to the impaired neurobehavior in the DEHP mice.

Porcine intestinal lymphoid tissues synthesize estradiol.

Estradiol (17ß-estradiol) is synthesized primarily in the gonads of both sexes and regulates the development and function of reproductive organs. Recently, we reported that intestinal lymphocyte homeostasis is regulated by estradiol synthesized de novo in the endothelial cells of the high endothelial venules (HEVs) of mesenteric lymph nodes and Peyer's patches in mice. This observation prompted us to hypothesize that HEVs of intestinal lymphoid tissues are sites of estradiol synthesis across species. In this study, we examined whether estradiol is synthesized in the intestinal lymphoid tissues of adolescent piglets. Comparisons of estradiol levels in blood and tissue showed that estradiol concentrations in mesenteric lymph nodes and Peyer's patches were significantly higher than the level in serum. Reverse transcription polymerase chain reaction showed that porcine intestinal lymphoid tissues express mRNAs for steroidogenic enzymes (StAR, 17ß-Hsd,3ß-Hsd, Cyp17a1, and Cyp19a1), and immunohistochemical results in ilial tissue showed expression of aromatase (CYP19) in Peyer's patch-localized endothelial cells of HEVs. When mesenteric lymph node and Peyer's patch tissues were cultured in vitro, they produced estradiol. Taken together, the results indicate that mesenteric lymph nodes and Peyer's patches are sites of estradiol synthesis in adolescent piglets.

Prenatal Exposure to DEHP Induces Premature Reproductive Senescence in Male Mice.

Di-(2-ethylhexyl) phthalate (DEHP) is the most commonly used phthalate, and it is an endocrine-disrupting chemical. This study tested a hypothesis that prenatal exposure to DEHP lays the foundation for premature gonadal dysfunction and subsequent reproductive senescence in male mice. Pregnant female CD-1 mice were orally dosed with vehicle control (tocopherol-stripped corn oil) or with 20 µg/kg/day, 200 µg/kg/day, 500 mg/kg/day, or 750 mg/kg/day of DEHP from gestational day 11 to birth. Overall, the prenatal DEHP exposure did not cause any overt physical health problems in male offspring, as no significant differences in their body nor gonadal weight were seen up to the age of 23 months. However, an age- and dose-dependent gonadal dysfunction was observed. As early as 7 months of age, the 750 mg/kg/day group of mice exhibited significantly reduced fertility. At 19 months of age, 86% of the 750 mg/kg/day mice became infertile, whereas only 25% of the control mice were infertile. At this age, all of the DEHP-exposed mice had lower serum testosterone levels, higher serum estradiol levels, and higher LH levels compared with control mice. Histological evaluations showed that mice prenatally exposed to DEHP displayed a wide array of gonadal and epididymal abnormalities such as increased germ cell apoptosis, degenerative seminiferous tubules, oligozoospermia, asthenozoospermia, and teratozoospermia in comparison to age-matching control mice. In summary, this study shows that prenatal exposure to DEHP induces premature reproductive senescence in male mice.

Estradiol Synthesis in Gut-Associated Lymphoid Tissue: Leukocyte Regulation by a Sexually Monomorphic System.

17ß-estradiol is a potent sex hormone synthesized primarily by gonads in females and males that regulates development and function of the reproductive system. Recent studies show that 17ß-estradiol is locally synthesized in nonreproductive tissues and regulates a myriad of events, including local inflammatory responses. In this study, we report that mesenteric lymph nodes (mLNs) and Peyer's patches (Pps) are novel sites of de novo synthesis of 17ß-estradiol. These secondary lymphoid organs are located within or close to the gastrointestinal tract, contain leukocytes, and function at the forefront of immune surveillance. 17ß-estradiol synthesis was initially identified using a transgenic mouse with red fluorescent protein coexpressed in cells that express aromatase, the enzyme responsible for 17ß-estradiol synthesis. Subsequent immunohistochemistry and tissue culture experiments revealed that aromatase expression was localized to high endothelial venules of these lymphoid organs, and these high endothelial venule cells synthesized 17ß-estradiol when isolated and cultured in vitro. Both mLNs and Pps contained 17ß-estradiol with concentrations that were significantly higher than those of peripheral blood. Furthermore, the total amount of 17ß-estradiol in these organs exceeded that of the gonads. Mice lacking either aromatase or estrogen receptor-ß had hypertrophic Pps and mLNs with more leukocytes than their wild-type littermates, demonstrating a role for 17ß-estradiol in leukocyte regulation. Importantly, we did not observe any sex-dependent differences in aromatase expression, 17ß-estradiol content, or steroidogenic capacity in these lymphoid organs.

Extra-gonadal sites of estrogen biosynthesis and function.

Estrogens are the key hormones regulating the development and function of reproductive organs in all vertebrates. Recent evidence indicates that estrogens play important roles in the immune system, cancer development, and other critical biological processes related to human well-being. Obviously, the gonads (ovary and testis) are the primary sites of estrogen synthesis, but estrogens synthesized in extra- gonadal sites play an equally important role in controlling biological activities. Understanding non-gonadal sites of estrogen synthesis and function is crucial and will lead to therapeutic interventions targeting estrogen signaling in disease prevention and treatment. Developing a rationale targeting strategy remains challenging because knowledge of extra-gonadal biosynthesis of estrogens, and the mechanism by which estrogen activity is exerted, is very limited. In this review, we will summarize recent discoveries of extra-gonadal sites of estrogen biosynthesis and their local functions and discuss the significance of the most recent novel discovery of intestinal estrogen biosynthesis. [BMB Reports 2016; 49(9): 488-496].