In order to establish a stable in vitro culture platform for chicken small intestine three-dimensional (3D) organoids, in this study, crypt cells were collected from the small intestine of 18-day-old embryos of AA broilers. On the basis of the L-WRN conditioned medium, we optimized the culture conditions of chicken small intestinal organoids by adjusting the proportions of nicotinamide, N-acetylcysteine, LY2157299, CHIR99021, Jagged-1, FGF, and other cytokines to select the medium suitable for the long-term stable growth of the organoids. The optimization results showed that the addition of 1.5 µmol/L CHIR99021 significantly improved the organoid formation efficiency and organoid diameter. When 0.5 µmol/L Jagged-1 was added, a small amount of bud-like tissue appeared in organoids. After the addition of 50 ng/mL FGF-2, the rate of organoid germination was significantly increased. The 1.5 µmol/L CHIR99021, 0.5 µmol/L Jagged-1, and 50 ng/mL FGF-2 added in the medium can cooperate with each other to improve the formation and speed up the proliferation and differentiation of organoids, while improving the stemness maintenance of cells. The morphology, cell types, and culture characteristics of chicken small intestinal organoids were studied by HE staining, transmission electron microscopy, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), indirect immunofluorescence, and immunohistochemistry. The results showed that the 3D organoids of the chicken small intestine cultured in vitro were morphologically consistent with the chicken intestinal tissue and contained differentiated epithelial cells. In summary, we successfully established an in vitro culture system for chicken small intestinal organoids, providing a new method for the subsequent research on chicken intestinal physiology, pathology, and host-pathogen interaction mechanism and the development of relevant drugs.
Animals ; Organoids/metabolism* ; Intestine, Small/drug effects* ; Chickens ; Cell Culture Techniques/methods* ; Culture Media ; Chick Embryo ; Tissue Culture Techniques/methods*

Objective: The aim of this study was to explore the ototoxicity of toluene in the early development of zebrafish embryos/larvae. Methods: Zebrafish were utilized to explore the ototoxicity of toluene. Locomotion analysis, immunofluorescence, and qPCR were used to understand the phenotypes and molecular mechanisms of toluene ototoxicity. Results: The results demonstrated that at 2 mmol/L, toluene induced zebrafish larvae death at 120 hours post fertilization (hpf) at a rate of 25.79% and inhibited the rate of hatching at 72 hpf. Furthermore, toluene exposure inhibited the distance travelled and average swimming velocity of zebrafish larvae while increasing the frequency of movements. As shown by fluorescence staining of hair cells, toluene inhibited the formation of lateral line neuromasts and middle line 1 (Ml Conclusion This study indicated that toluene may affect the development of both the inner ear and lateral line systems in zebrafish, while the lateral line system may be more sensitive to toluene than the inner ear.
Animals ; Ear, Inner/growth & development* ; Embryo, Nonmammalian/drug effects* ; Gene Expression Regulation, Developmental/drug effects* ; Hair Cells, Auditory/metabolism* ; Lateral Line System/growth & development* ; Locomotion/drug effects* ; Ototoxicity/physiopathology* ; Toluene/toxicity* ; Zebrafish

Animals ; Embryo, Nonmammalian ; drug effects ; metabolism ; Ethanol ; toxicity ; RNA, Messenger ; metabolism ; Resveratrol ; pharmacology ; Zebrafish ; Zebrafish Proteins ; metabolism

OBJECTIVETo investigate the pro-angiogenic effects of paeoniflorin (PF) in a vascular insufficiency model of zebrafish and in human umbilical vein endothelial cells (HUVECs).

METHODSIn vivo, the pro-angiogenic effects of PF were tested in a vascular insufficiency model in the Tg(fli-1:EGFP)y1 transgenic zebrafish. The 24 h post fertilization (hpf) embryos were pretreated with vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor II (VRI) for 3 h to establish the vascular insufficiency model and then post-treated with PF for 24 h. The formation of intersegmental vessels (ISVs) was observed with a fluorescence microscope. The mRNA expression of fms-like tyrosine kinase-1 (flt-1), kinase insert domain receptor (kdr), kinase insert domain receptor like (kdrl) and von Willebrand factor (vWF) were analyzed by real-time polymerase chain reaction (PCR). In vitro, the pro-angiogenic effects of PF were observed in HUVECs in which cell proliferation, migration and tube formation were assessed.

RESULTSPF (6.25-100 μmol/L) could rescue VRI-induced blood vessel loss in zebrafish and PF (25-100 μmol/L), thereby restoring the mRNA expressions of flt-1, kdr, kdrl and vWF, which were down-regulated by VRI treatment. In addition, PF (0.001-0.03 μmol/L) could promote the proliferation of HUVECs while PF stimulated HUVECs migration at 1.0-10 μmol/L and tube formation at 0.3 μmol/L.

CONCLUSIONPF could promote angiogenesis in a vascular insufficiency model of zebrafish in vivo and in HUVECs in vitro.

Angiogenesis Inducing Agents ; pharmacology ; therapeutic use ; Animals ; Animals, Genetically Modified ; Cells, Cultured ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Embryo, Nonmammalian ; Glucosides ; pharmacology ; therapeutic use ; Human Umbilical Vein Endothelial Cells ; drug effects ; physiology ; Humans ; Monoterpenes ; pharmacology ; therapeutic use ; Neovascularization, Physiologic ; drug effects ; Phytotherapy ; Vascular Diseases ; drug therapy ; pathology ; Zebrafish

Oligodendrocytes (OLs) are myelinating glial cells that form myelin sheaths around axons to ensure rapid and focal conduction of action potentials. Here, we found that an axonal outgrowth regulatory molecule, AATYK (apoptosis-associated tyrosine kinase), was up-regulated with OL differentiation and remyelination. We therefore studied its role in OL differentiation. The results showed that AATYK knockdown inhibited OL differentiation and the expression of myelin genes in vitro. Moreover, AATYK-deficiency maintained the proliferation status of OLs but did not affect their survival. Thus, AATYK is essential for the differentiation of OLs.
Animals ; Animals, Newborn ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Cell Differentiation ; drug effects ; physiology ; Cell Proliferation ; drug effects ; genetics ; Cells, Cultured ; Cuprizone ; toxicity ; Demyelinating Diseases ; chemically induced ; metabolism ; pathology ; Embryo, Mammalian ; Gene Expression Regulation, Developmental ; genetics ; Ki-67 Antigen ; metabolism ; Mice ; Mice, Inbred C57BL ; Myelin Basic Protein ; metabolism ; Myelin Proteolipid Protein ; metabolism ; Myelin Sheath ; drug effects ; metabolism ; Oligodendroglia ; drug effects ; metabolism ; Protein-Tyrosine Kinases ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley

The increased levels of intracellular reactive oxygen species (ROS) in granulosa cells (GCs) may affect the pregnancy results in women with polycystic ovary syndrome (PCOS). In this study, we compared the in vitro fertilization and embryo transfer (IVF-ET) results of 22 patients with PCOS and 25 patients with tubal factor infertility and detected the ROS levels in the GCs of these two groups. Results showed that the PCOS group had significantly larger follicles on the administration day for human chorionic gonadotropin than the tubal factor group (P < 0.05); however, the number of retrieved oocytes was not significantly different between the two groups (P > 0.05). PCOS group had slightly lower fertilization, cleavage, grade I/II embryo, clinical pregnancy, and implantation rates and higher miscarriage rate than the tubal factor group (P > 0.05). We further found a significantly higher ROS level of GCs in the PCOS group than in the tubal factor group (P < 0.05). The increased ROS levels in GCs caused GC apoptosis, whereas NADPH oxidase 2 (NOX2) specific inhibitors (diphenyleneiodonium and apocynin) significantly reduced the ROS production in the PCOS group. In conclusion, the increased ROS expression levels in PCOS GCs greatly induced cell apoptosis, which further affected the oocyte quality and reduced the positive IVF-ET pregnancy results of women with PCOS. NADPH oxidase pathway may be involved in the mechanism of ROS production in GCs of women with PCOS.
Abortion, Spontaneous ; epidemiology ; Acetophenones ; therapeutic use ; Adult ; Apoptosis ; drug effects ; Embryo Transfer ; Female ; Fertilization in Vitro ; Granulosa Cells ; metabolism ; Humans ; NADPH Oxidases ; antagonists & inhibitors ; Onium Compounds ; therapeutic use ; Oocyte Retrieval ; Oxidative Stress ; Polycystic Ovary Syndrome ; drug therapy ; Pregnancy ; Pregnancy Rate ; Reactive Oxygen Species ; metabolism

As the number of young cancer survivors increases, quality of life after cancer treatment is becoming an ever more important consideration. According to a report from the American Cancer Society, approximately 810,170 women were diagnosed with cancer in 2015 in the United States. Among female cancer survivors, 1 in 250 are of reproductive age. Anticancer therapies can result in infertility or sterility and can have long-term negative effects on bone health, cardiovascular health as a result of reproductive endocrine function. Fertility preservation has been identified by many young patients diagnosed with cancer as second only to survival in terms of importance. The development of fertility preservation technologies aims to help patients diagnosed with cancer to preserve or protect their fertility prior to exposure to chemo- or radiation therapy, thus improving their chances of having a family and enhancing their quality of life as a cancer survivor. Currently, sperm, egg, and embryo banking are standard of care for preserving fertility for reproductive-age cancer patients; ovarian tissue cryopreservation is still considered experimental. Adoption and surrogate may also need to be considered. All patients should receive information about the fertility risks associated with their cancer treatment and the fertility preservation options available in a timely manner, whether or not they decide to ultimately pursue fertility preservation. Because of the ever expanding number of options for treating cancer and preserving fertility, there is now an opportunity to take a precision medicine approach to informing patients about the fertility risks associated with their cancer treatment and the fertility preservation options that are available to them.
Adult Stem Cells ; Cell Culture Techniques ; Cryopreservation/*methods ; *Embryo, Mammalian ; Female ; Fertility Preservation/*methods ; Humans ; Neoplasms/drug therapy/*therapy ; *Oocytes ; Ovarian Follicle/drug effects/metabolism/transplantation ; *Ovary/transplantation ; Ovulation Induction/methods ; Precision Medicine

The use of exogenous carbon monoxide releasing molecules (CORMs) provides promise for clinical application; however, the hazard potential of CORMs in vivo remains poorly understood. The developmental toxicity of CORM-3 was investigated by exposure to concentrations ranging from 6.25 to 400 μmol/L during 4-144 h post fertilization. Toxicity endpoints of mortality, spontaneous movement, heart rate, hatching rate, malformation, body length, and larval behavior were measured. CORM-3 disrupted the progression of zebrafish larval development at concentrations exceeding 50 μmol/L, resulting in embryonic developmental toxicity.
Animals ; Carbon Monoxide ; pharmacology ; Cardiotonic Agents ; toxicity ; Dose-Response Relationship, Drug ; Embryo, Nonmammalian ; drug effects ; Embryonic Development ; drug effects ; Organometallic Compounds ; toxicity ; Zebrafish ; embryology ; metabolism

OBJECTIVETo investigate the effect of di-(2-ethylhexyl) phthalate (DEHP) exposure on the growth and development of placenta, uterine natural killer (uNK) cell number and angiogenesis at the maternal-fetal interface in pregnant mice.

METHODSFrom day 1 of pregnancy, pregnant mice were exposed daily to DEHP by oral gavage at 125, 250, or 500 mg/kg for 13 consecutive days. The uterine and placental tissues were then harvested for HE staining and immunohistochemistry to examine the effect of DEHP exposure on the growth and development of the placenta and angiogenesis and uNK cell number at the maternal-fetal interface.

RESULTSCompared with the control group, the mice exposed to 500 mg/kg DEHP, but not those exposed to 125 and 250 mg/kg, showed significantly reduced number of embryo implantation (P<0.05). DEHP exposure significantly increased the rate of abortion. DEHP exposure at 125, 250, and 500 mg/kg significantly and dose-dependently lowered the placental weight compared with that in the control group (0.0637±0.0133, 0.0587±0.0176, 0.0524±0.0183 g vs 0.0786±0.0143 g, respectively; P<0.01), and significantly reduced the total area of the placenta and area of spongiotrophoblasts. DEHP exposure resulted in a significant reduction in the number of fetal vascular branches, and collapse and atresia of blood vessels. The mice exposed to DEHP at 125, 250, and 500 mg/kg had significantly lowered numbers of uNK cells (83.2±10.3, 60.7±12.4, and 50.4±14.5/HP, respectively) as compared with the control group (105.1±14.2/HP) at the maternal-fetal interface (P<0.01).

CONCLUSIONDEHP exposure significantly affects the growth and development of the placenta in mice possibly by suppressing angiogenesis and reducing uNK cell number at the maternal-fetal interface during pregnancy.

Animals ; Diethylhexyl Phthalate ; adverse effects ; Embryo Implantation ; Female ; Fetal Blood ; Killer Cells, Natural ; cytology ; Maternal Exposure ; adverse effects ; Mice ; Neovascularization, Physiologic ; Placenta ; drug effects ; Placentation ; drug effects ; Pregnancy ; Uterus ; drug effects

Long-term gonadotropin-releasing hormone agonist (GnRHa) administration before in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) in infertile women with endometriosis or adenomyosis significantly enhanced the chances of pregnancy in both fresh and frozen embryo transfer cycles. We hypothesized that long-term GnRHa treatment might also be beneficial for the idiopathic repeated implantation failure (RIF) patients. In the 21 patients receiving GnRHa and hormone replacement therapy (G-HRT) protocols for frozen embryo transfer, their data were compared with those of the 56 of frozen/fresh cycles they had previously undergone (previous protocols). Comparison showed that the finial results were significantly better with G-HRT protocols than with their previous protocols, with pregnancy rate, clinical pregnancy rate, implantation rate and on-going pregnancy rate being 70%, 60%, 40% and 38% respectively with G-HRT protocols, against 17%, 11%, 6.3% and 5% with previous protocols. The results showed that hormonally controlled endometrial preparation with prior GnRHa suppression could be used for patients who had experienced repeated failures of IVF treatment despite having morphologically optimal embryos, and the treatment may help increase the receptivity of the endometrium in these patients.
Adult ; Embryo Implantation ; drug effects ; Embryo Transfer ; methods ; Female ; Gonadotropin-Releasing Hormone ; administration & dosage ; analogs & derivatives ; pharmacology ; therapeutic use ; Hormone Replacement Therapy ; methods ; Humans ; Pituitary Gland ; drug effects ; Pregnancy ; Sperm Injections, Intracytoplasmic ; methods