The combined effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and the phytoestrogen genistein on steroid hormone secretion,AhR and ERβ expression and the incidence of apoptosis in granulosa cells of medium porcine follicles

Low doses of endocrine disrupting chemicals (EDCs) used in combination may act in a manner different from that of individual compounds. The objective of the study was to examine in vitro effects of low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 100 pM) and genistein (500 nM) on: 1) progesterone (P₄) and estradiol (E₂) secretion (48 h); 2) dynamic changes in aryl hydrocarbon receptor (AhR) mRNA and protein expression (1, 3, 6, 24 and 48 h); 3) dynamic changes in estrogen receptor β (ERβ) mRNA and protein expression (1, 3, 6, 24 and 48 h); and 4) induction of apoptosis in porcine granulosa cells derived from medium follicles (3, 6 and 24 h). TCDD had no effect on P₄ or E₂ production, but potentiated the inhibitory effect of genistein on P₄ production. In contrast to the individual treatments which did not produce any effects, TCDD and genistein administered together decreased ERβ and AhR protein expression in granulosa cells. Moreover, the inhibitory effect of TCDD on AhR mRNA expression was abolished by genistein. The treatments did not induce apoptosis in the cells. In summary, combined effects of low concentrations of TCDD and genistein on follicular function of pigs differed from that of individual compounds. The results presented in the current paper clearly indicate that effects exerted by low doses of EDCs applied in combination must be taken into consideration when studying potential risk effects of EDCs on biological processes.     

The effects analysis of two neonicotinoid insecticides on in vitro maturation of porcine oocytes using hanging drop monoculture method

Acetamiprid (ACE) and imidacroprid (IMI) are known neonicotinoid insecticides with strong affinities for the insect-selective nicotinic acetylcholine receptor. These provide insect control by hyperstimulating insect nerves and are used for agricultural pest management. However, it has also been reported that ACE and IMI affect mammalian reproductive function. We determined the effects of ACE and IMI on the in vitro maturation of porcine oocytes. Significant decreases in nuclear maturation rates were observed in the ACE or IMI-exposed groups. Also, in matured oocytes from the ACE or IMI-exposed groups, irregular chromosomes were observed. Our results suggest that ACE and IMI exposure was detrimental to porcine oocytes and the extent of the effects depends on the concentration of exposure.     

Na?ve-like conversion enhances the difference in innate in vitro differentiation capacity between rabbit ES cells and iPS cells

Quality evaluation of pluripotent stem cells using appropriate animal models needs to be improved for human regenerative medicine. Previously, we demonstrated that although the in vitro neural differentiating capacity of rabbit induced pluripotent stem cells (iPSCs) can be mitigated by improving their baseline level of pluripotency, i.e., by converting them into the so-called “naïve-like” state, the effect after such conversion of rabbit embryonic stem cells (ESCs) remains to be elucidated. Here we found that naïve-like conversion enhanced the differences in innate in vitro differentiation capacity between ESCs and iPSCs. Naïve-like rabbit ESCs exhibited several features indicating pluripotency, including the capacity for teratoma formation. They differentiated into mature oligodendrocytes much more effectively (3.3–7.2 times) than naïve-like iPSCs. This suggests an inherent variation in differentiation potential in vitro among PSC lines. When naïve-like ESCs were injected into preimplantation rabbit embryos, although they contributed efficiently to forming the inner cell mass of blastocysts, no chimeric pups were obtained. Thus, in vitro neural differentiation following naïve-like conversion is a promising option for determining the quality of PSCs without the need to demonstrate chimeric contribution. These results provide an opportunity to evaluate which pluripotent stem cells or treatments are best suited for therapeutic use.     

Comparison of immune responses to intranasal and intrapulmonary vaccinations with the attenuated Mycoplasma hyopneumoniae 168 strain in pigs

The aim of this study was to evaluate the immune responses to intranasal and intrapulmonary vaccinations with the attenuated Mycoplasma hyopneumoniae (Mhp) 168 strain in the local respiratory tract in pigs. Twenty-four pigs were randomly divided into 4 groups: an intranasal immunization group, an intrapulmonary immunization group, an intramuscular immunization group and a control group. The levels of local respiratory tract cellular and humoral immune responses were investigated. The levels of interleukin (IL)-6 in the early stage of immunization (P<0.01), local specific secretory IgA (sIgA) in nasal swab samples (P<0.01); and IgA- and IgG-secreting cells in the nasal mucosa and trachea were higher after intranasal vaccination (P<0.01) than in the control group. Interestingly, intrapulmonary immunization induced much stronger immune responses than intranasal immunization. Intrapulmonary immunization also significantly increased the secretion of IL-6 and local specific sIgA and the numbers of IgA- and IgG-secreting cells. The levels of IL-10 and interferon-γ in the nasal swab samples and the numbers of CD4⁺ and CD8⁺ T lymphocytes in the lung and hilar lymph nodes were significantly increased by intrapulmonary immunization compared with those in the control group (P<0.01). These data suggest that intrapulmonary immunization with attenuated Mhp is effective in evoking local cellular and humoral immune responses in the respiratory tract. Intrapulmonary immunization with Mhp may be a promising route for defense against Mhp in pigs.     

Anethole inhibits growth of recently emerged multidrug resistant toxigenic Vibrio cholerae O1 El Tor variant strains in vitro

To search natural compounds having inhibitory effect on bacterial growth is important, particularly in view of growing multidrug resistant (MDR) strains of bacterial pathogens. Like other bacterial pathogens, MDR Vibrio cholerae, the causative agent of diarrheal disease cholera, is becoming a great concern. As an approach of searching new antimicrobial agents, here, we show that anethole, a well-studied natural component of sweet fennel and star anise seeds, could potentially inhibit the growth of MDR O1 El Tor biotype, the ongoing 7th cholera pandemic variant strains of toxigenic V. cholerae. The minimum inhibitory concentration (MIC) of anethole against diverse O1 El Tor biotype strains is evaluated as 200 µg/ml. Moreover, the effect of anethole is bactericidal and exerts rapid-killing action on V. cholerae cells. This study is the first report which demonstrates that anethole, purified from natural compound, is a potent inhibitor of growth of toxigenic V. cholerae. Our data suggest that anethole could be a potential antimicrobial drug candidate, particularly against MDR V. cholerae mediated infections.     

Improvement of bacterial clearance and relief of clinical signs of Salmonella enterica serovar Typhimurium infection in pigs through upregulation of Th 1-specific responses by administration of a combination of two silicate minerals,biotite and bentonite

Biotite and bentonite are phyllosilicate minerals that were originally used in industrial applications. Several beneficial activities of them have recently been reported, especially regulation of the immune system and antimicrobial effects. Therefore, we investigated the immune-enhancing and bacterial clearance effects of a biotite and bentonite mixture (BBM) on experimental infection of Salmonella enterica serovar Typhimurium (S. Typhimurium) to determine whether the BBM could be used as an alternative antibiotic. We administered 1% or 2% BBM as a feed supplement. We then evaluated the bacterial clearance effects of the BBM against S. Typhimurium. We also evaluated the immune-enhancing effect of the BBM through several immunological experiments that included examination of the lysozyme activity, CD4⁺/CD8⁺ T lymphocyte ratio and the T-helper type 1 (Th 1) cytokine profile. The clinical signs of S. Typhimurium and the number of viable bacteria in feces and tissues were significantly decreased in both BBM groups, especially in the 2% BBM group. The BBM also markedly enhanced the lysozyme activity, CD4⁺/CD8⁺ T lymphocyte ratio and expression levels of IFN-γ and IL-12 in S. Typhimurium-challenged pigs. Therefore, the BBM could be a good candidate as an alternative antibiotic that improves Th 1-specific immune responses and the bacterial clearance effect.     

Anti-obesity activity of diglyceride containing conjugated linoleic acid in C57BL/6J ob/ob mice

This study was to investigate the anti-obesity effects of diglyceride (DG)-conjugated linoleic acid (CLA) containing 22% CLA as fatty acids in C57BL/6J ob/ob male mice. There were four experimental groups including vehicle control, DG, CLA, and DG-CLA. The test solutions of 750 mg/kg dose were orally administered to the mice everyday for 5 weeks. CLA treatments significantly decreased mean body weight in the obese mice throughout the experimental period compared to the control (p < 0.01). All test solutions significantly decreased the levels of triglyceride, glucose and free fatty acids in the serum compared with control (p < 0.05). The levels of total cholesterol were also significantly reduced in DG and DG-CLA groups compared with the control group (p < 0.05). CLA significantly decreased weights of renal and epididymal fats compared with the control (p < 0.05). DG and DG-CLA also significantly decreased the epididymal fat weights compared with the control (p < 0.05). A remarkable decrease in the number of lipid droplets and fat globules was observed in the livers of mice treated with DG, CLA, and DG-CLA compared to control. Treatments of DG and CLA actually increased the expression of peroxisome proliferator-activated receptor gamma. These results suggest that DG-CLA containing 22% CLA have a respectable anti-obesity effect by controlling serum lipids and fat metabolism.     

Knockout mouse production assisted by Blm knockdown

Production of knockout mice using targeted embryonic stem cells (ESCs) is a powerful approach for investigating the function of specific genes in vivo. Although the protocol for gene targeting via homologous recombination (HR) in ESCs is already well established, the targeting efficiency varies at different target loci and is sometimes too low. It is known that knockdown of the Bloom syndrome gene, BLM, enhances HR-mediated gene targeting efficiencies in various cell lines. However, it has not yet been investigated whether this approach in ESCs is applicable for successful knockout mouse production. Therefore, we attempted to answer this question. Consistent with previous reports, Blm knockdown enhanced gene targeting efficiencies for three gene loci that we examined by 2.3–4.1-fold. Furthermore, the targeted ESC clones generated good chimeras and were successful in germline transmission. These data suggest that Blm knockdown provides a general benefit for efficient ESC-based and HR-mediated knockout mouse production.     

Sexing of Dog Sperm by Fluorescence In Situ Hybridization

Effective preselection of sex has been accomplished in several species of livestock and also in humans using the flow cytometric sperm sorting method. A guaranteed high sorting accuracy is a key prerequisite for the widespread use of sperm sexing. The standard validation method is flow cytometric remeasurement of the DNA content of the sexed sperm. Since this method relies on the same instrument that produced the original sperm separation, it is not truly independent. Therefore, to be able to specifically produce either male or female offspring in the dog, we developed a method of direct visualization of sex chromosomes in a single sperm using fluorescence in situ hybridization (FISH) as a validation method. Denaturation of canine spermatozoa by immersion in 1 M NaOH for 4 min yielded consistent hybridization results with over 97% hybridization efficiency and a good preservation of sperm morphology. There was no significant difference between the theoretical ratio (50:50) and the observed ratio of X- and Y-chromosome-bearing spermatozoa in any of the three dogs. In addition, the mean purities of flow-sorted sex chromosomes in spermatozoa of the three dogs were 90.8% for the X chromosome fraction and 89.6% for the Y chromosome fraction. This sorting was evaluated by using the dual color FISH protocol. Therefore, our results demonstrated that the FISH protocol worked reliably for both unsorted and sexed sperm samples.     

Epidermal growth factor improves developmental competence and embryonic quality of singly cultured domestic cat embryos

This study examined the influence of EGF on the expression of EGF receptors (EGFR) and developmental competence of embryos cultured individually versus those cultured in groups. Cat oocytes were in vitro matured and fertilized (IVM/IVF), and cleaved embryos were randomly assigned to one of seven culture conditions: one group each in which embryos were subjected to group culture supplemented with or without 5 ng/ml EGF and five groups in which embryos were subjected to single-embryo culture supplemented with EGF (0, 5, 25, 50 or 100 ng/ml). Morulae, blastocysts and hatching blastocysts were assessed at days 5 and 7; post IVF, respectively, and total blastocyst cell numbers were assessed at day 7. Relative mRNA expressions of EGFR of 2–4-cell embryos, 8–16-cell embryos, morulae and blastocysts cultured in groups or singly with or without EGF supplementation were examined. OCT3/4 and Ki67 in blastocysts derived from the group or single-embryo culture systems with or without EGF supplementation were localized. A higher rate of embryos cultured in groups developed to blastocysts than individually incubated cohorts. Although EGF increased blastocyst formation in the single-embryo culture system, EGF did not affect embryo development in group culture. Expression levels of EGFR decreased in morulae and blastocysts cultured with EGF. An increased ratio of Ki67-positive cells to the total number of cells in the blastocyst was observed in singly cultured embryos in the presence of EGF. However, EGF did not affect the expression of OCT3/4. These findings indicate that EGF enhanced developmental competence of cat embryos cultured singly by stimulating cell proliferation and modulating the EGFR expression at various developmental stages.     

Experimental in vitro transmission of Babesia sp. (EU1) by Ixodes ricinus

Babesia sp. (EU1), first characterized in 2003, has been implicated in human cases of babesiosis in Italy, Austria and Germany. It has been identified in roe deer and in its suspected tick vector, Ixodes ricinus, in several European countries. The aim of the present study was to validate the competence of I. ricinus as a vector of Babesia sp. (EU1) via experimental infections. For this purpose, a parasite strain isolated from roe deer was cloned in sheep erythrocytes. After experimental infections, parasite DNA was successfully amplified by PCR in both eggs and larvae originating from infected I. ricinus females and in the salivary glands of females exposed to Babesia sp. (EU1) as nymphs. We also demonstrate that infected females were able to transmit parasite DNA during a new blood meal. Together with previous epidemiological studies, these results validate I. ricinus as a competent vector for Babesia sp. (EU1).     

Porphyromonas gulae 41-kDa fimbriae induced osteoclast differentiation and cytokine production

Porphyromonas gulae is considered to be associated with canine periodontitis. We have previously reported that the P. gulae American Type Culture Collection (ATCC) 51700 comprised 41-kDa fimbriae. The purpose of the present study was to demonstrate the roles of 41-kDa fimbrial protein in periodontal disease. In this study, we examined the involvement of the 41-kDa fimbrial protein in osteoclast differentiation and cytokine production in murine macrophages. Furthermore, alveolar bone resorption induced by P. gulae infection in rats was evaluated. To estimate osteoclast differentiation, bone marrow cells and MC3T3-G2/PA6 cells were cultured with or without the 41-kDa fimbrial protein for 7 days. BALB/c mouse peritoneal macrophages were stimulated with the 41-kDa fimbrial protein, and the levels of interleukin (IL)-1β and tumor necrosis factor (TNF)-α production were determined by enzyme-linked immunosorbent assay. Osteoclast differentiation was significantly enhanced by treatment with the 41-kDa fimbrial protein in a dose-dependent manner. The total area of pits formed on the dentine slices with osteoclasts incubated with the 41-kDa fimbrial protein was significantly greater than that of the control. The purified 41-kDa fimbrial protein induced IL-1β and TNF-α production in BALB/c mouse peritoneal macrophages after 6 hr of incubation in a dose-dependent manner. The bone loss level in rats infected with P. gulae was significantly higher than that of the sham-infected rats. These results suggest that P. gulae 41-kDa fimbriae play important roles in the pathogenesis of periodontal disease.     

RNAi-mediated knockdown of INHBB increases apoptosis and inhibits steroidogenesis in mouse granulosa cells

Inhibins are members of the TGFβ superfamily and act as suppressors of follicle stimulating hormone (FSH) secretion from pituitary glands via a negative feedback mechanism to regulate folliculogenesis. In this study, the INHBB gene was knocked down by three RNAi-Ready pSIREN-RetroQ-ZsGreen vector- mediated recombinant plasmids to explore the effects of INHBB silencing on granulosa cell (GC) cell cycle, apoptosis and steroid production in vitro. Quantitative real-time polymerase chain reaction, Western blot, flow cytometry and ELISA were performed to evaluate the role of INHBB in the mouse GC cell cycle, apoptosis and steroid production in vitro. The results showed that the relative mRNA and protein expression of INHBB in mouse GCs can be significantly reduced by RNAi with pshRNA-B1, pshRNA-B2 and pshRNA-B3 plasmids, with pshRNA-B3 having the best knockdown efficiency. Downregulation of the expression of INHBB significantly arrests cells in the G1 phase of the cell cycle and increases the apoptosis rate in GCs. This was further confirmed by downregulation of the protein expressions of Cyclin D1, Cyclin E and Bcl2, while the protein expression of Bax was upregulated. In addition, specific downregulation of INHBB markedly decreased the concentration of estradiol and progesterone, which was further validated by the decrease in the mRNA levels of CYP19A1and CYP11A1. These findings suggest that inhibin βB is important in the regulation of apoptosis and cell cycle progression in granulosa cells. Furthermore, the inhibin βB subunit has a role in the regulation of steroid hormone biosynthesis. Evidence is accumulating to support the concept that inhibin βB is physiologically essential for early folliculogenesis in the mouse.     

Addition of D-penicillamine,hypotaurine, and epinephrine (PHE) mixture to IVF medium maintains motility and longevity of bovine sperm and enhances stable production of blastocysts in vitro

The present study aimed to establish an efficient system for bovine embryo production by in vitro fertilization (IVF) that can achieve stable normal fertilization and blastocyst developmental rates in any bull without optimization of the sperm concentration in IVF medium. We examined the effects of a PHE mixture (20 μM D-penicillamine, 10 μM hypotaurine and 1 μM epinephrine), theophylline (2.5 mM), and sperm concentration (1, 2 or 5 × 10⁶ cells/ml) on fertilization and blastocyst developmental rates. High cleavage rates (78.3 to 92.4%) and blastocyst developmental rates (31.9 to 62.0%) at day 7 were obtained in the presence of PHE and theophylline in IVF medium with a sperm concentration of 2 × 10⁶ cells/ml using sperm from 9 bulls. In addition, the synergistic effect of PHE and theophylline on normal fertilization (2 pronuclei) was clarified at 12 h after IVF with a sperm concentration of 1 × 10⁶ cells/ml. Moreover, high linearity, high flagellar beat cross frequency, and low amplitude of lateral head of motile sperm were found by computer-assisted sperm analysis. In conclusion, the combination of the PHE mixture and theophylline synergistically accelerates sperm motility and sperm penetration of bovine oocytes. Theophylline activates sperm motility with increasing intracellular cAMP. However, PHE prevents an excessive increase of cAMP and maintains sperm motility without hyperactivation. When the combination of PHE and theophylline is added to IVF medium at a sperm concentration of 2 × 10⁶ cells/ml, we can achieve stable normal fertilization and blastocyst development in any bull.     

Suspected natural lysosomal storage disease from ingestion of pink morning glory (Ipomoea carnea) in goats in northern Argentina

This study describes an occurrence of pink morning glory (Ipomoea carnea) intoxication in goats in northern Argentina. The clinical signs displayed by the affected animals were ataxia, lethargy, emaciation, hypertonia of the neck muscles, spastic paresis in the hind legs, abnormal postural reactions and death. The clinico-pathologic examination revealed that the affected animals were anemic and their serum level of aspartate aminotransferase was significantly increased. Cytoplasmic vacuolation in the Purkinje cells and pancreatic acinar cells was observed by histological examination. The neuronal lectin binding pattern showed a strong positive reaction to WGA (Triticum vulgaris), sWGA (succinylated T. vulgaris) and LCA (Lens culinaris). Although I. carnea is common in tropical regions, this is the first report of spontaneous poisoning in goats in Argentina.     

RNAi-mediated Knockdown of Xist Does Not Rescue the Impaired Development of Female Cloned Mouse Embryos

In mice, one of the major epigenetic errors associated with somatic cell nuclear transfer (SCNT) is ectopic expression of Xist during the preimplantation period in both sexes. We found that this aberrant Xist expression could be impeded by deletion of Xist from the putative active X chromosome in donor cells. In male clones, it was also found that prior injection of Xist-specific siRNA could significantly improve the postimplantation development of cloned embryos as a result of a significant repression of Xist at the morula stage. In this study, we examined whether the same knockdown strategy could work as well in female SCNT-derived embryos. Embryos were reconstructed with cumulus cell nuclei and injected with Xist-specific siRNA at 6–7 h after oocyte activation. RNA FISH analysis revealed that siRNA treatment successfully repressed Xist RNA at the morula stage, as shown by the significant decrease in the number of cloud-type Xist signals in the blastomere nuclei. However, blastomeres with different sizes (from “pinpoint” to “cloud”) and numbers of Xist RNA signals remained within single embryos. After implantation, the dysregulated Xist expression was normalized autonomously, as in male clones, to a state of monoallelic expression in both embryonic and extraembryonic tissues. However, at term there was no significant improvement in the survival of the siRNA-injected cloned embryos. Thus, siRNA injection was largely effective in repressing the Xist overexpression in female cloned embryos but failed to rescue them, probably because of an inability to mimic consistent monoallelic Xist expression in these embryos. This could only be achieved in female embryos by applying a gene knockout strategy rather than an siRNA approach.     

Gene expression profile by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the liver of wild-type (AhR+/+) and aryl hydrocarbon receptor-deficient (AhR-/-) mice

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is one of the most toxic environmental pollutants that cause various biological effects on mammals. The purpose of our study was to identify the genes involved in hepatotoxicity and hepatocarcinogenesis caused by TCDD. C57BL/6 (AhR+/+, wild type) and B6.129-AhR/J (AhR-/-, knock out) mice were injected i.p. with a single treatment of TCDD at the dose of 100 microg/kg body weight. Relative liver weight was significantly increased at 72 hr after TCDD treatment without an apparent histopathological change in AhR+/+ mice (p<0.05). TCDD treatment also significantly increased activity of serum alanine aminotransferase in AhR-/- mice (p<0.05). The liver was analyzed for gene expression profiles 72 hr later. As compared with AhR-/- mice, the expression of 51 genes (>3-fold) was changed in AhR+/+ mice; 28 genes were induced, while 23 genes were repressed. Most of the genes were associated with chemotaxis, inflammation, carcinogenesis, acute-phase response, immune responses, cell metabolism, cell proliferation, signal transduction, and tumor suppression. This study suggests that the microarray analysis of genes in the liver of AhR+/+ and AhR-/- mice may help to clarify the mechanism of AhR-mediated hepatotoxicity and hepatocarcinogenesis by TCDD.     

Spontaneous Rhabdomyosarcoma in a Common Marmoset ( Callithrix jacchus )

The common marmoset (Callithrix jacchus) is now widely used in various research fields, including toxicology. However, information about the background pathology of this species is scarce. Here, we report a case of rhabdomyosarcoma that spontaneously occurred in a common marmoset. A 44-month-old male common marmoset was euthanized due to bilateral hind limb paralysis. At necropsy, a 2×2×5-cm intramuscular mass was observed in the lower right back. Histologically, the mass was mainly composed of interlacing bundles of spindle-shaped tumor cells. Immunohistochemically, the tumor cells were positive for myogenin, desmin, vimentin and alpha-smooth muscle actin. Ultrastructurally, the tumor cells contained bundles of myofilaments with Z-band-like structures. Thus, the tumor was diagnosed as a rhabdomyosarcoma. To our knowledge, this is the first report of spontaneous rhabdomyosarcoma that was definitely diagnosed in the common marmoset.     

Histocytological specificities of adrenal cortex in the New World Monkeys, Aotus lemurinus and Saimiri boliviensis

The New World monkey Aotus spp. (night monkeys) are expected for use of valuable experimental animal with the close species of Saimiri spp. (squirrel monkeys). Saimiri is known to show spontaneous hypercortisolemia, although few reports in Aotus. We compared basic states of blood steroid hormones and histological structure of the adrenal glands in two monkeys. Serum cortisol and ACTH levels were statistically lower in Aotus than Saimiri. Conversely, Aotus adrenocortical area showed significant enlargement, especially at the zona fasciculata. Electron microscopic observation at Aotus fasciculata cells revealed notable accumulation of large lipid droplets and irregular shapes of the mitochondrial cristae. These results suggest potential differences in cellular activities for steroidogenesis between Aotus and Saimiri and experimental usefulness in adrenocortical physiology and pathological models.