Heme Oxygenase-1 Regulates Zearalenone-Induced Oxidative Stress and Apoptosis in Sheep Follicular Granulosa Cells.

Zearalenone (ZEA) is a common non-steroidal estrogenic mycotoxin found in a range of animal feeds and poses a serious threat to the reproductive health of farm animals and humans. However, the mechanism underlying ZEA-induced reproductive toxicity in sheep remains unknown. Granulosa cells are crucial for egg maturation and the fertility of female sheep. In this study, we aimed to examine the impact of different ZEA concentrations on sheep follicular granulosa cells and to elucidate the potential molecular mechanism underlying ZEA-induced toxicity using transcriptome sequencing and molecular biological approaches. Treating primary sheep follicular granulosa cells with different concentrations of ZEA promoted the overproduction of reactive oxygen species (ROS), increased lipid peroxidation products, led to cellular oxidative stress, decreased antioxidant enzyme activities, and induced cell apoptosis. Using transcriptome approaches, 1395 differentially expressed genes were obtained from sheep follicular granulosa cells cultured in vitro after ZEA treatment. Among them, heme oxygenase-1 (HMOX1) was involved in 11 biological processes. The protein interaction network indicated interactions between HMOX1 and oxidative and apoptotic proteins. In addition, N-acetylcysteine pretreatment effectively reduced the ZEA-induced increase in the expression of HMOX1 and Caspase3 by eliminating ROS. Hence, we suggest that HMOX1 is a key differential gene involved in the regulation of ZEA-induced oxidative stress and apoptosis in follicular granulosa cells. These findings provide novel insights into the prevention and control of mycotoxins in livestock.

Establishment of Bactrian Camel Induced Pluripotent Stem Cells and Prediction of Their Unique Pluripotency Genes.

Induced pluripotent stem cells (iPSCs) can differentiate into all types of cells and can be used in livestock for research on biological development, genetic breeding, and in vitro genetic resource conservation. The Bactrian camel is a large domestic animal that inhabits extreme environments and holds value in the treatment of various diseases and the development of the local economy. Therefore, we transferred four mouse genes (Oct4, Sox2, Klf4, and c-Myc) into Bactrian camel fetal fibroblasts (BCFFs) using retroviruses with a large host range to obtain Bactrian camel induced pluripotent stem cells (bciPSCs). They were comprehensively identified based on cell morphology, pluripotency gene and marker expression, chromosome number, transcriptome sequencing, and differentiation potential. The results showed the pluripotency of bciPSCs. However, unlike stem cells of other species, late formation of stem cell clones was observed; moreover, the immunofluorescence of SSEA1, SSEA3, and SSEA4 were positive, and teratoma formation took four months. These findings may be related to the extremely long gestation period and species specificity of Bactrian camels. By mining RNA sequence data, 85 potential unique pluripotent genes of Bactrian camels were predicted, which could be used as candidate genes for the production of bciPSC in the future. Among them, ASF1B, DTL, CDCA5, PROM1, CYTL1, NUP210, Epha3, and SYT13 are more attractive. In conclusion, we generated bciPSCs for the first time and obtained their transcriptome information, expanding the iPSC genetic information database and exploring the applicability of iPSCs in livestock. Our results can provide an experimental basis for Bactrian camel ESC establishment, developmental research, and genetic resource conservation.

circAMN1-Mediated Ferroptosis Regulates the Expulsion of Placenta in Trophoblast Cells.

After delivery, the death of trophoblast cells can promote the expulsion of the placenta. Ferroptosis, an iron-dependent programmed cell death, is involved in mammalian development. Circular RNAs are associated with placental development; however, it is unclear whether circular RNAs regulate the expulsion of fetal membranes through ferroptosis. The gene expression profiles in the tail vein blood of Holstein cows with normal and retained placentas were investigated using RNA sequencing and a GSE214588 dataset. circAMN1 and SLC39A8 expression was significantly downregulated in the blood of cows with a retained placenta, whereas miR-205_R-1 expression was significantly upregulated. We validated erastin-induced ferroptosis in trophoblast cells. Transfection with si-circAMN1 and miR-205_R-1 mimic reduced intracellular total iron, Fe2+, and glutathione disulfide levels; increased intracellular glutathione levels and glutathione/glutathione disulfide; and enhanced cell viability in these cells. In contrast, transfection with pcDNA3.1 circAMN1 and an miR-205_R-1 inhibitor promoted ferroptosis. As an miR-205_R-1 sponge, circAMN1 regulated the expression of SLC39A8 to control erastin-induced ferroptosis and regulated the proliferation, invasion, and migration of trophoblast cells. Our findings provide a theoretical basis for studying the mechanism by which programmed cell death regulates fetal membrane expulsion and indicate its potential as a therapeutic target for placenta retention.

Analysis of Toxic Effects of Fluoride on Ovine Follicular Granulosa Cells Using RNA-Seq.

Fluoride is abundant in the environment and is an essential trace element in living organisms. However, prolonged excessive fluoride intake can lead to fluorosis, which poses a threat to the reproductive health of animals and humans. Although previous research has mainly focused on animal models, the impact of fluoride on ovine follicular granulosa cells (GCs) has not been comprehensively elucidated. This study employed RNA-Seq technology to elucidate the toxic effects of fluoride on ovine follicular GCs and its mechanism of action. Culturing primary ovine follicular GCs in vitro and subjecting them to fluoride treatment revealed 3218 differentially expressed genes (DEGs), with 2278 upregulated and 940 downregulated. Significantly, this study unveiled fluoride's induction of endoplasmic reticulum (ER) stress in cells, triggering a cascade involving the PERK pathway factor ATF4, leading to cell death via DDIT3/CHOP activation and the subsequent upregulation of CHAC1, ATF3, ERO1α, and TRIB3. These findings provide crucial insights into the toxicity of fluoride in ovine, offering a foundation for mitigating fluoride-related losses in the farming industry.

Screening and Mechanism Study of Three Antagonistic Drugs, Oxysophoridine, Rutin, and Phellodendrine, against Zearalenone-Induced Reproductive Toxicity in Ovine Oocytes.

Zearalenone (ZEN) is a common fungal toxin with reproductive toxicity in various grains. It poses a serious threat to ovine and other animal husbandry industries, as well as human reproductive health. Therefore, investigating the mechanism of toxicity and screening antagonistic drugs are of great importance. In this study, based on the natural compound library and previous Smart-seq2 results, antioxidant and anti-apoptotic drugs were selected for screening as potential antagonistic drugs. Three natural plant compounds (oxysophoridine, rutin, and phellodendrine) were screened for their ability to counteract the reproductive toxicity of ZEN on ovine oocytes in vitro using quantitative polymerase chain reaction (qPCR) and reactive oxygen species detection. The compounds exhibited varying pharmacological effects, notably impacting the expression of antioxidant (GPX, SOD1, and SOD2), autophagic (ATG3, ULK2, and LC3), and apoptotic (CAS3, CAS8, and CAS9) genes. Oxysophoridine promoted GPX, SOD1, ULK2, and LC3 expression, while inhibiting CAS3 and CAS8 expression. Rutin promoted SOD2 and ATG3 expression, and inhibited CAS3 and CAS9 expression. Phellodendrine promoted SOD2 and ATG3 expression, and inhibited CAS9 expression. However, all compounds promoted the expression of genes related to cell cycle, spindle checkpoint, oocyte maturation, and cumulus expansion factors. Although the three drugs had different regulatory mechanisms in enhancing antioxidant capacity, enhancing autophagy, and inhibiting cell apoptosis, they all maintained a stable intracellular environment and a normal cell cycle, promoted oocyte maturation and release of cumulus expansion factors, and, ultimately, counteracted ZEN reproductive toxicity to promote the in vitro maturation of ovine oocytes. This study identified three drugs that antagonize the reproductive toxicity of ZEN on ovine oocytes, and compared their mechanisms of action, providing data support and a theoretical basis for their subsequent application in the ovine breeding industry, reducing losses in the breeding industry, screening of ZEN reproductive toxicity antagonists and various toxin antagonists, improving the study of ZEN reproductive toxicity mechanisms, and even protection of human reproductive health.

Melatonin regulates dihydrotestosterone formation via its membrane receptor in the epididymal epithelial cells of sheep.

Both melatonin and androgen, which affect sperm fertility, are the important factors in epididymis of male animal. In the present study, we confirmed that melatonin regulates the formation of dihydrotestosterone (DHT) in sheep epididymides. Here, we investigated the localization and the expression levels of melatonin keys synthases AANAT and HIOMT, membrane receptors MT1 and MT2, and nuclear receptor RORα in sheep epididymides and testes. We also cultured epididymal epithelial cells and treated them with different concentrations of melatonin (10-11-10-7 M) and luzindole (10-5 M) and 4P-PDOT (10-5 M) to investigate whether melatonin is involved in the regulation of DHT formation and whether these effects are mediated through its receptor pathways. The results showed that AANAT, HIOMT, MT1, MT2, and RORα were differentially expressed between sheep epididymides and testes. In addition, melatonin is involved in mediating the formation of DHT in epididymal epithelial cells, and its influence on DHT is at least partially regulated by the melatonin receptor pathway. Our findings showed that melatonin regulates the functions of the testes and epididymides through an autocrine mechanism and regulates the formation of androgen in sheep epididymides via the receptor pathway. These results provide a basis for further exploring the regulatory mechanisms of melatonin in animal reproduction.

Smart-seq2 Technology Reveals a Novel Mechanism That Zearalenone Inhibits the In Vitro Maturation of Ovine Oocytes by Influencing TNFAIP6 Expression.

Zearalenone (ZEN), a non-steroidal estrogenic fungal toxin widely present in forage, food, and their ingredients, poses a serious threat to animal and human reproductive health. ZEN also threatens ovine, a major source of human food and breeding stock. However, the mechanisms underlying the impact of ZEN on the in vitro maturation (IVM) of ovine oocytes remain unclear. This study aimed to elucidate these mechanisms using the Smart-seq2 technology. A total of 146 differentially expressed genes were obtained, using Smart-seq2, from sheep oocytes cultured in vitro after ZEN treatment. ZEN treatment inhibited RUNX2 and SPP1 expression in the PI3K signaling pathway, leading to the downregulation of THBS1 and ultimately the downregulation of TNFAIP6; ZEN can also decrease TNFAIP6 by reducing PTPRC and ITGAM. Both inhibit in vitro maturation of ovine oocytes and proliferation of cumulus cells by downregulating TNFAIP6. These findings provide data and a theoretical basis for elucidating ZEN's toxicity mechanisms, screening therapeutic drugs, and reducing ZEN-related losses in the ovine industry.

miRNA-150_R-1 mediates the HIF-1/ErbB signaling pathway to regulate the adhesion of endometrial epithelial cells in cows experiencing retained placenta.

Retained placenta (RP) refers to reproductive disorders caused by the failure of fetal membranes to be expelled 12 h after delivery in dairy cows. Postpartum adhesion of the fetal membranes to the uterus causes diseases such as mastitis or endometritis, which threatening the profitability of the dairy industry. Emerging evidence suggests that micro RNAs (miRNAs) play crucial roles in various processes, such as the occurrence and progression of fetal membranes discharge. However, the molecular mechanisms of miRNAs in RP remain unknown. In this study, we performed RNA-sequencing to characterize the expression profiles of mRNAs and miRNAs in caudal vein blood samples of postpartum Holstein cows whose fetal membranes were discharged normally or retained to identify RP-related genes and evaluate their molecular mechanisms. We identified 44 differentially expressed miRNAs (19 upregulated and 25 downregulated) and 706 differentially expressed mRNAs (325 upregulated and 381 downregulated) in the RP group compared to the normal fetal membranes discharge group. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis revealed that differentially expressed mRNAs were mainly enriched in the extracellular matrix, cell adhesion, and autoimmunity-related biological processes or pathways. Further analyses using RNA-sequencing, a dual luciferase reporter system, quantitative reverse transcription-PCR, immunofluorescence, and western blotting verified that endothelial PAS domain protein 1 (EPAS1) is regulated by miR-150_R-1 in endometrial epithelial cells. We demonstrated the relationship between EPAS1 and RP and confirmed that EPAS1 is upregulated in the blood and placenta of cows that experience RP. Further, we proposed a model of the miRNA-mRNA negative regulatory network mediated by the HIF-1/ErbB signaling pathway to show its regulatory role in RP.

Valproic Acid Enhance Reprogramming of Bactrian Camel Cells through Promoting the Expression of Endogenous Gene c-Myc and the Process of Angiogenesis.

BACKGROUND AND OBJECTIVES: Induced pluripotent stem cells (iPSCs) are usually generated by reprogramming differentiated cells throughthe introduction of specific transcription factors, but this is a difficult and inefficient process. Valproic acid (VPA) is a histone deacetylase inhibitor that significantly improves the efficiency of iPSC generation. But its role and mechanism are still unclear. METHODS AND RESULTS: We transduced Bactrian camel fetal fibroblasts (BCFFs) with retroviruses carrying defined factors (OCT4, SOX2, KLF4, c-MYC and EGFP; OSKMG) in the presence of VPA. Cells were collected (Day 7) and analyzed using RNA-seq technology. Afterwards, different groups of cells and transcriptomics results were detected by PCR and qRT-PCR technology. The results showed that VPA promoted the expression of the endogenous gene c-Myc and inhibited cell proliferation; at the same time, it promoted the expression of VEGF and other genes related to angiogenesis. CONCLUSIONS: When VPA is added to the culture medium, only the cells that have begun to reprogram can break the G2/M repression through the expression of the endogenous gene c-Myc, and use the nutrients and space in the culture dish to proliferate normally, which can achieve the purpose of directly improving the efficiency of reprogramming. Another new discovery for Bactrian camels, VPA significantly increased the expression of VEGFC and other genes, promoting the transformation of fibroblasts to endothelial cells (different from the mesenchymal-to-epithelial transition process of other species) to accelerate the early induction of Bactrian camels iPSc process. Overall, this study proved the new mechanism of VPA in enhancing the induction of pluripotency from the transcriptome level.

Melatonin protects against lipopolysaccharide-induced epididymitis in sheep epididymal epithelial cells in vitro.

Melatonin has protective effects against inflammation but its role in epididymitis is unknown. We addressed this in the present study using lipopolysaccharide (LPS)-stimulated sheep epididymal epithelial cells as an in vitro inflammation model. We found that interleukin (IL)-1ß, IL-6, tumor necrosis factor α, and cyclooxygenase (COX)-2 mRNA levels; COX-2 and Toll-like receptor (TLR)-4 protein levels; and nuclear factor (NF)-κB p65 phosphorylation were increased by LPS treatment. These effects were reversed in a dose-dependent manner by melatonin (10-11-10-7 M). Quantitative reverse transcription PCR and immunofluorescence analyses showed that the melatonin receptors MT1 and MT2 were expressed in sheep epididymal epithelial cells. The inhibitory effect of melatonin on inflammation was abrogated by the MT1 and MT2 receptor antagonist luzindole and the MT2 ligand 4-phenyl-2-propanamide tetraldehyde. Thus, melatonin exerted anti-inflammatory effect in epididymal epithelial cells by inhibiting TLR4/NF-κB signaling, suggesting its potential as an effective drug for the treatment of epididymitis in sheep.

Comparative Profiling of MicroRNAs Reveals the Underlying Toxicological Mechanism in Mice Testis Following Carbon Ion Radiation.

This study investigated the toxicity of heavy ion radiation to mice testis by microRNA (miRNA) sequencing and bioinformatics analyses. Testicular indices and histology were measured following enterocoelia irradiation with a 2 Gy carbon ion beam, with the testes exhibiting the most serious injuries at 4 weeks after carbon ion radiation (CIR) exposure. Illumina sequencing technology was used to sequence small RNA libraries of the control and irradiated groups at 4 weeks after CIR. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses implicated differential miRNAs in the regulation of target genes involved in metabolism, development, and reproduction. Here, 8 miRNAs, including miR-34c-5p, miR-138, and 6 let-7 miRNA family members previously reported in testis after radiation, were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to validate miRNA sequencing data. The differentially expressed miRNAs described here provided a novel perspective for the role of miRNAs in testis toxicity following CIR.