Lack of involvement of retinoic acid receptor α in retinoid-induced skin irritation in hairless mice

It has been proposed that RARγ, the major retinoic acid receptor (RAR) subtype in skin, mediates retinoid-induced skin irritation. However, RARα is also found in skin, and its role in retinoid-induced skin irritation has not been tested. In this study, RAR subtype-specific agonists and antagonists were used to test the possible contribution of RARα to retinoid-induced skin irritation. Female hairless mice were treated topically on the dorsal skin for 5 days with various retinoids over a 2-log dose range, and cutaneous toxicity was scored by semiquantitative visual observations of skin flaking and abrasions daily up to 3 days post-treatment. Three RARα-selective agonists were ≥ 100-fold less potent as skin irritants than the structurally-related RAR pan-agonist, TTNPB. Skin irritation potency decreased in the following order: TTNPB > >Am580 > AGN 193835 > > 193836 and correlated with RARβ and/or RARγ binding affinity rather than RARα binding affinity. TTNPB-induced skin irritation was blocked in a dose-dependent fashion by co-treatment with the RAR pan-antagonist AGN 193109 but was not blocked by co-treatment with the RARα-specific antagonist AGN 194301. In contrast, skin irritation induced by the RARα-selective agonist AGN 193835 was almost completely blocked by co-treatment with AGN 193644, an RAR β/gg-selective antagonist. These data demonstrate that RARα is not significantly involved in mediating retinoid-induced skin irritation in mice and suggest that RARα-selective agonists may have reduced mucocutaneous side effects relative to other retinoids.     

Recombinant Gallid herpesvirus 2 with interrupted meq genes confers safe and efficacious protection against virulent field strains

Gallid herpesvirus 2 (GaHV-2) continuously evolves, which reduces the effectiveness of existing vaccines. To construct new GaHV-2 candidate vaccines, LMS, which is a virulent GaHV-2 field strain isolated from diseased chicken flocks in Southwest China in 2007, was modified such that both copies of its meq oncogene were partially deleted. The resulting virus, i.e., rMSΔmeq, was characterized using PCR and sequencing. To evaluate the safety and protective efficacy of rMSΔmeq, specific pathogen-free (SPF) chickens were inoculated with 2000 plaque forming units (pfu) and 20,000 pfu of rMSΔmeq immediately after hatching. All birds grew well during the experimental period, and none of the challenged chickens developed Marek’s disease-associated lymphoma. In addition, the rMSΔmeq- and CVI988/Rispens-vaccinated SPF chickens were challenged with 1000 pfu and 5000 pfu of the representative virulent GaHV-2 Md5 strain and 1000 pfu of the variant GaHV-2 strains LCC or LTS. The results showed that the rMSΔmeq strain provided complete protection, which was similar to that provided by the CVI988/Rispens vaccine (protective index (PI) of 95.5) when challenged with a conventional dose of the Md5 strain. However, rMSΔmeq provided a PI of 90.9 when challenged with 5000 pfu of the Md5 strain, which was significantly higher than that provided by the CVI988/Rispens vaccine (54.5). rMSΔmeq provided a PI of 86.4 against LCC, which was equal to that provided by the CVI988/Rispens vaccine (81.8). In addition, rMSΔmeq provided a PI of 100 against LTS, which was significantly higher than that provided by the CVI988/Rispens vaccine (68.2). Altogether, the rMSΔmeq virus provided efficient protection against representative and variant GaHV-2 strains. In conclusion, the rMSΔmeq virus is a safe and effective vaccine candidate for the prevention of Marek's disease and is effective against the Chinese variant GaHV-2 strains.     

An AANAT/ASMT transgenic animal model constructed with CRISPR/Cas9 system serving as the mammary gland bioreactor to produce melatonin‐enriched milk in sheep

Melatonin as a potent antioxidant exhibits important nutritional and medicinal values. To produce melatonin‐enriched milk will benefit the consumers. In this study, a sheep bioreactor which generates melatonin‐enriched milk has been successfully developed by the technology that combined CRISPR/Cas9 system and microinjection. The AANAT and ASMT were cloned from pineal gland of Dorper sheep (Ovis aries). The in vitro studies found that AANAT and ASMT were successfully transferred to the mammary epithelial cell lines and significantly increased melatonin production in the culture medium compared to the nontransgenic cell lines. In addition, the Cas9 mRNA, sgRNA, and the linearized vectors pBC1‐AANAT and pBC1‐ASMT were co‐injected into the cytoplasm of pronuclear embryos which were implanted into ewes by oviducts transferring. Thirty‐four transgenic sheep were generated with the transgenic positive rate being roughly 35% which were identified by Southern blot and sequencing. Seven carried transgenic AANAT, two carried ASMT, and 25 carried both of AANAT and ASMT genes. RT‐PCR and Western blot demonstrated that the lambs expressed these genes in their mammary epithelial cells and these animals produced melatonin‐enriched milk. This is the first report to show a functional AANAT and ASMT transgenic animal model which produce significantly high levels of melatonin milk compared to their wild‐type counterparts. The advanced technologies used in the study laid a foundation for generating large transgenic livestock, for example, the cows, which can produce high level of melatonin milk.     

Melatonin protects porcine oocyte in vitro maturation from heat stress

Melatonin is a pleiotropic molecule which plays an important role in animal reproductive activities. Because of the increased global warming, the impact of heat stress (HS) on stockbreeding has become an inevitable issue to be solved. To investigate the potential effects of melatonin on the in vitro maturation of porcine oocyte under the HS, a HS model for porcine oocyte maturation has been used in this study and the different concentrations of melatonin (10^?6–10^?9 m ) were also tested for their protective effects on oocytes. The polar body rate, the index of the nuclear maturation of the oocytes, and the cleavage rate as well as the blastocyst rate were measured to evaluate the developmental competence of the oocytes after parthenogenetic activation (PA). The results showed that HS [in vitro maturation (IVM) 20–24 hr, 42°C] significantly reduced the polar body rate of oocytes and the blastocyte rate of porcine PA embryos, while melatonin (10^?7 m ) application not only improved polar body rate and blastocyte rate, but also preserved the normal levels of steroid hormone which is disrupted by HS. The presence of melatonin (10^?7 m ) during the oocyte maturation under the HS reduced reactive oxygen species (ROS) formation, enhanced glutathione (GSH) production, inhibited cell apoptosis, and increased the gene expressions of SIRT1, AKT2, and Polg2. Importantly, the endogenously occurring melatonin of cumulus–oocyte complexes was significantly induced by HS. The results indicated that melatonin application effectively protected the oocytes from HS. These observations warranted the further studies in vivo regarding to improve the reproductive activities of animals under the global warming environment.     

Melatonin improves the reprogramming efficiency of murine‐induced pluripotent stem cells using a secondary inducible system

This study focused on the effect of melatonin on reprogramming with specific regard to the generation of induced pluripotent stem cells (iPSCs). Here, a secondary inducible system, which is more accurate and suitable for studying the involvement of chemicals in reprogramming efficiency, was used to evaluate the effect of melatonin on mouse iPSC generation. Secondary fibroblasts collected from all‐iPSC mice through tetraploid complementation were cultured in induction medium supplemented with melatonin at different concentrations (0, 10^?6, 10^?7, 10^?8, 10^?9, or 10^?10 m ) or with vitamin C (50 μg/mL) as a positive control. Compared with untreated group (0.22 ± 0.04% efficiency), 10^?8 (0.81 ± 0.04%), and 10^?9 m (0.83 ± 0.08%) melatonin supplementation significantly improved reprogramming efficiency (P < 0.05). Moreover, we verified that the iPSCs induced by melatonin treatment (MiPSCs) had the same characteristics as typical embryonic stem cells (ESCs), including expression of the pluripotency markers Oct4, Sox2, and Nanog, the ability to form teratomas and all three germ layers of the embryo, as well as produce chimeric mice with contribution to the germ line. Interestingly, only the melatonin receptor MT₂ was detected in secondary fibroblasts, while MiPSCs and ESCs expressed MT₁ and MT₂ receptors. Furthermore, during the early stage of reprogramming, expression of the apoptosis‐related genes p53 and p21 was lower in the group treated with 10^?9 m melatonin compared with the untreated controls. In conclusion, melatonin supplementation enhances the efficiency of murine iPSC generation. These beneficial effects may be associated with inhibition of the p53‐mediated apoptotic pathway.     

Treatment of porcine donor cells and reconstructed embryos with the antioxidant melatonin enhances cloning efficiency

This study was conducted to investigate the effect of melatonin during the culture of donor cells and cloned embryos on the in vitro developmental competence and quality of cloned porcine embryos. At concentrations of 10^?6 m or 10^?8 m , melatonin significantly enhanced the proliferation of porcine fetal fibroblasts (PFFs), and the blastocyst rate was significantly increased in the 10^?10 m melatonin‐treated donor cell group. Cloned embryo development was also improved in embryo culture medium that was supplemented with 10^?9 m or 10^?12 m melatonin. When both donor cells and cloned embryos were treated with melatonin, the cleavage rate and total cell number of blastocysts were not significantly affected; however, the blastocyst rate was increased significantly (20.0% versus 11.7%). TUNEL assays showed that combined melatonin treatment reduced the rate of apoptotic nuclei (3.6% versus 6.1%). Gene expression analysis of the apoptosis‐related genes BAX, BCL2L1, and p53 showed that the expression of BCL2L1 was significantly elevated 2.7‐fold relative to the control group, while the expression of BAX and p53 was significantly decreased by 3.7‐fold and 23.2‐fold, respectively. In addition, we detected the expression of two melatonin receptors (MT1 and MT2) in PFFs but not in porcine cloned embryos. We conclude that exogenous melatonin enhances the development of porcine cloned embryos and improves embryo quality by inhibiting p53‐mediated apoptotic pathway. The proliferation of PFFs may be mediated by receptor binding, but the beneficial effects of melatonin on embryonic development may be receptor‐independent, possibly through melatonin's ability to directly scavenge free radicals.     

Bisphenol A (BPA) pharmacokinetics with daily oral bolus or continuous exposure via silastic capsules in pregnant rhesus monkeys: Relevance for human exposures

We measured serum dBPA in non-pregnant and pregnant female rhesus monkeys, fetuses and amniotic fluid. dBPA was administered by a daily oral bolus or sc implantation of Silastic capsules; both resulted in daily average serum unconjugated dBPA concentrations of <1 ng/ml. We observed lower serum concentrations of unconjugated dBPA in pregnant females relative to pre-pregnancy values, and generally lower concentrations in fetal serum than in maternal serum. Differences in pharmacokinetics of dBPA were evident between pre-pregnancy, early and late pregnancy, likely reflecting changes in maternal, fetal and placental physiology. The serum ratio of conjugated to unconjugated dBPA after continuous sc release of dBPA was similar to values reported in human biomonitoring studies and markedly lower than with oral administration, suggesting oral bolus exposure is not an appropriate human exposure model. We report elsewhere that there were numerous adverse effects on fetuses exposed to very low serum dBPA in these studies.