Bacillus licheniformis ameliorates Aflatoxin B1-induced testicular damage by improving the gut-metabolism-testis axis.

Global aflatoxin B1 (AFB1) contamination is inevitable, and it can significantly damage testicular development. However, the current mechanism is confusing. Here, by integrating the transcriptome, microbiome, and serum metabolome, we comprehensively explain the impact of AFB1 on testis from the gut-metabolism-testis axis. Transcriptome analysis suggested that AFB1 exposure directly causes abnormalities in testicular inflammation-related signalling, such as tumor necrosis factor (TNF) pathway, and proliferation-related signalling pathways, such as phosphatidylinositide 3-kinases-protein kinase B (PI3K-AKT) pathway, which was verified by immunofluorescence. On the other hand, we found that upregulated inflammatory factors in the intestine after AFB1 exposure were associated with intestinal microbial dysbiosis, especially the enrichment of Bacilli, and enrichment analysis showed that this may be related to NLR family pyrin domain containing 3 (NLRP3)-mediated NOD-like receptor signalling. Also, AFB1 exposure caused blood metabolic disturbances, manifested as decreased hormone levels and increased oxidative stress. Significantly, B. licheniformis has remarkable AFB1 degradation efficiency (> 90%). B. licheniformis treatment is effective in attenuating gut-testis axis damage caused by AFB1 exposure through the above-mentioned signalling pathways. In conclusion, our findings indicate that AFB1 exposure disrupts testicular development through the gut-metabolism-testis axis, and B. licheniformis can effectively degrade AFB1.

Cyanidin-3-O-glucoside Mitigates the Ovarian Defect Induced by Zearalenone via p53-GADD45a Signaling during Primordial Follicle Assembly.

Zearalenone (ZEN) is well known as a kind of endocrine disruptor whose exposure is capable of causing reproductive toxicity in animals. Cyanidin-3-O-glucoside (C3G) is a derivative of cyanidin and owns multiple biofunctions, and prior efforts have suggested that C3G has therapeutic actions for reproductive diseases. In this article, a ZEN exposure model during primordial follicle assembly was constructed using the in vitro culture platform of neonatal mouse ovaries. We investigated the protective effect of C3G on ZEN-induced ovarian toxicity during primordial follicle assembly in mice, as well as its potential mechanism. Interestingly, we observed that C3G could effectively protect the ovary from ZEN damage, mainly by restoring primordial follicle assembly, which upregulated the expression of LHX8 and SOHLH1 proteins and relieved ZEN-induced DNA damage. Next, to explore the mechanism by which C3G rescued ZEN-induced injury, we performed RNA sequencing (RNA-seq). The bioinformatic analysis illustrated that the rescue pathway of C3G was associated with p53-Gadd45a signaling and cell cycle. Then, western blotting and flow cytometry results revealed that C3G restored the expression levels of cyclin-dependent kinase 6 (CDK6) and cyclin D2 (CCND2) and regulated the ovarian cell cycle to normal. In conclusion, our findings manifested that C3G could alleviate ZEN-induced primordial follicle assembly impairment by restoring the cell cycle involved in p53-GADD45a signaling.

Induced differentiation of primordial germ cell like cells from SOX9+ porcine skin derived stem cells.

Understanding the mechanisms behind porcine primordial germ cell like cells (pPGCLCs) development, differentiation, and gametogenesis is crucial in the treatment of infertility. In this study, SOX9+ skin derived stem cells (SOX9+ SDSCs) were isolated from fetal porcine skin and a high-purity SOX9+ SDSCs population was obtained. The SOX9+ SDSCs were induced to transdifferentiate into PGCLCs during 8 days of cultured. The results of RNA-seq, western blot and immunofluorescence staining verified SDSCs have the potential to transdifferentiate into PGCLCs from aspects of transcription factor activation, germ layer differentiation, energy metabolism, and epigenetic changes. Both adherent and suspended cells were collected. The adherent cells were found to be very similar to early porcine primordial germ cells (pPGCs). The suspended cells resembled late stage pPGCs and had a potential to enter meiotic process. This SDSCs culture-induced in vitro model is expected to provide suitable donor cells for stem cell transplantation in the future.

Cross-species analysis of transcriptome emphasizes a critical role of TNF-α in mediating MAP2K7/AKT2 signaling in zearalenone-induced apoptosis.

Zearalenone (ZEN) is a widespread and transgenerational toxicant that can cause serious reproductive health risks, which poses a potential threat to global agricultural production and human health; its estrogenic activity can lead to reproductive toxicity through the induction of granulosa cell apoptosis. Herein, comparative transcriptome analysis, single-cell transcriptome analysis, and weighted gene co-expression network analysis (WGCNA) combined with gene knockout in vivo and RNA interference in vitro were used to comprehensively describe the damage caused by ZEN exposure on ovarian granulosa cells. Comparative transcriptome analysis and WGCNA suggested that the tumor necrosis factor (TNF)-α-mediated mitogen-activated protein kinase 7 (MAP2K7)/ AKT serine/threonine kinase 2 (AKT2) axis was disordered after ZEN exposure in porcine granulosa cells (pGCs) and mouse granulosa cells (mGCs). In vivo gene knockout and in vitro RNA interference verified that TNF-α-mediated MAP2K7/AKT2 was the guiding signal in ZEN-induced apoptosis in pGCs and mGCs. Moreover, single-cell transcriptome analysis showed that ZEN exposure could induce changes in the TNF signaling pathway in offspring. Overall, we concluded that the TNF-α-mediated MAP2K7/AKT2 axis was the main signaling pathway of ZEN-induced apoptosis in pGCs and mGCs. This work provides new insights into the mechanism of ZEN toxicity and provides new potential therapeutic targets for the loss of livestock and human reproductive health caused by ZEN.

Aflatoxin B1 disrupts testicular development via the cell cycle-related Ras/PI3K/Akt signaling in mice and pig.

Aflatoxins B1 (AFB1), a type I carcinogen widely present in the environment, not only poses a danger to animal husbandry, but also poses a potential threat to human reproductive health, but its mechanism is still unclear. To address this question, multi-omics were performed on porcine Sertoli cells and mice testis. The data suggest that AFB1 induced testicular damage manifested as decreased expression of GJA1, ZO1 and OCCLUDIN in mice (p < 0.01) and inhibition of porcine Sertoli cell proliferation. Transcriptomic analysis suggested changes in noncoding RNA expression profiles that affect the cell cycle-related Ras/PI3K/Akt signaling pathway after AFB1 exposure both in mice and pigs. Specifically, AFB1 caused abnormal cell cycle of testis with the characterization of decreased expressions of CCNA1, CCNB1 and CDK1 (p < 0.01). Flow cytometry revealed that the G2/M phase was significantly increased after AFB1 exposure. Meanwhile, AFB1 downregulated the expressions of Ras, PI3K and AKT both in porcine Sertoli cell (p < 0.01) and mice testis (p < 0.01). Metabolome analysis verified the alterations in the PI3K/Akt signaling pathway (p < 0.05). Moreover, the joint analysis of metabolome and microbiome found that the changes of metabolites were correlated with the expression of flora. In conclusion, we have demonstrated that AFB1 impairs testicular development via the cell cycle-related Ras/PI3K/Akt signaling.

Multi-omics analysis reveals that iron deficiency impairs spermatogenesis by gut-hormone synthesis axis.

Considering that research has mainly focussed on how excessive iron supplementation leads to reproductive cytotoxicity, there is a lack of in-depth research on reproductive system disorders caused by iron deficiency. To gain a better understanding of the effects of iron deficiency on the reproductive system, especially spermatogenesis, we first constructed a mouse model of iron deficiency. We employed multi-omic analysis, including transcriptomics, metabolomics, and microbiomics, to comprehensively dissect the impact of iron deficiency on spermatogenesis. Moreover, we verified our findings in detail using western blot, immunofluorescence, immunohistochemistry, qRT-PCR and other techniques. Microbiomic analysis revealed altered gut microbiota in iron-deficient mice, and functional predictive analysis showed that gut microbiota can regulate spermatogenesis. The transcriptomic data indicated that iron deficiency directly alters expression of meiosis-related genes. Transcriptome data also revealed that iron deficiency indirectly regulates spermatogenesis by affecting hormone synthesis, findings confirmed by metabolomic data, western blot and immunofluorescence. Interestingly, competing endogenous RNA networks also play a vital role in regulating spermatogenesis after iron deficiency. Taken together, the data elucidate that iron deficiency impairs spermatogenesis and increases the risk of male infertility by affecting hormone synthesis and promoting gut microbiota imbalance.

Comparative transcriptomic analysis reveals that TPX2 and AURXA are involved in porcine PCV2 infection.

Porcine circovirus type 2 (PCV2) has been a notorious killer for the pig industry, causing substantial economic losses worldwide. However, its pathogenesis is still poorly understood. Comparative transcriptomic analysis and weighted gene co-expression network analysis (WGCNA) were performed in different porcine tissues after PCV2 infection. Our comparative transcriptomic analysis obtained 40 key differentially expressed genes (DEGs), and our WGCNA identified 458 hub genes. Significantly, both TPX2 microtubule nucleation factor (TPX2) and Aurora kinase A (AURKA) are included in these key DEGs and hubs genes. Our gene ontology (GO) analysis indicated that the key DEGs and hub genes participated in cell cycle regulation and immune response. The expressive levels of TPX2 and AURKA went down in the spleen but up in the kidneys after infection with PCV2. We conclude that TPX2 and AURKA played an essential role in PCV2 infection.

Maternal Zearalenone exposure impacted ovarian follicle formation and development of suckled offspring.

Zearalenone (ZEN) is a secondary metabolite, which is mainly produced by Fusarium fungi and exists in various feeds and agricultural products. Recently, an increasing amount of data has shown that ZEN, as an estrogen-like hormone, can have harmful effects on the female reproductive system, especially on oogenesis and folliculogenesis. Breast milk is considered to be the ideal form of nutrition for infants; however, there are some records of contaminants in food, such as mycotoxins, which may be transferred from maternal blood to milk. In this study, we investigated the toxic effects of breast milk on folliculogenesis in offspring following maternal ZEN exposure. Our results showed that maternal ZEN exposure significantly inhibited the process of primordial follicle (PF) assembly and reduced the number of PFs in suckled offspring's ovaries. In addition, RNA-seq analysis showed that RIG-I-like receptor (RLRs) signaling pathways were activated after exposed to ZEN, which increased the expression levels of DNA damage (γ-H2AX, RAD51, and PARP1) and apoptosis related protein (BAX/BCL2 and Caspase-3). Finally, ZEN exposure interfered with follicular development, as evidenced by the reduced percentages of oocyte maturation and embryonic development when the offspring grew to adolescence. It is worth noting that maternal ZEN exposure disrupted the tri-methylation levels of H3K4, H3K9, and H3K27 in the offspring's oocytes. Our results indicated that maternal ZEN exposure affected ovarian development in offspring through the breast milk, which may be detrimental to their reproductive capability in adult life.

Inflammatory cytokines as key players of apoptosis induced by environmental estrogens in the ovary.

Natural and synthetic environmental estrogens (EEs), interfering with the physiological functions of the body's estrogens, are widespread and are rising much concern for their possible deleterious effects on human and animal health, in particular on reproduction. In fact, increasing evidence indicate that EEs can be responsible for a variety of disfunctions of the reproductive system especially in females such as premature ovarian insufficiency (POI). Because of their great structural diversity, the modes of action of EEs are controversial. One important way through which EEs exert their effects on reproduction is the induction of apoptosis in the ovary. In general, EEs can exert pro-and anti-apoptotic effects by agonizing or antagonizing numerous estrogen-dependent signaling pathways. In the present work, results concerning apoptotic pathways and diseases induced by representative EEs (such as zearalenone, bisphenol A and di-2-ethylhexyl phthalate), in ovaries throughout development are presented into an integrated network. By reviewing and elaborating these studies, we propose inflammatory factors, centered on the production of tumor necrosis factor (TNF), as a major cause of the induction of apoptosis by EEs in the mammalian ovary. As a consequence, potential strategies to prevent such EE effect are suggested.

Single-cell transcriptome dissection of the toxic impact of Di (2-ethylhexyl) phthalate on primordial follicle assembly.

Rationale: Accumulated evidence indicates that environmental plasticizers are a threat to human and animal fertility. Di (2-ethylhexyl) phthalate (DEHP), a plasticizer to which humans are exposed daily, can trigger reproductive toxicity by acting as an endocrine-disrupting chemical. In mammals, the female primordial follicle pool forms the lifetime available ovarian reserve, which does not undergo regeneration once it is established during the fetal and neonatal period. It is therefore critical to examine the toxicity of DEHP regarding the establishment of the ovarian reserve as it has not been well investigated. Methods: The ovarian cells of postnatal pups, following maternal DEHP exposure, were prepared for single cell-RNA sequencing, and the effects of DEHP on primordial follicle formation were revealed using gene differential expression analysis and single-cell developmental trajectory. In addition, further biochemical experiments, including immunohistochemical staining, apoptosis detection, and Western blotting, were performed to verify the dataset results. Results: Using single-cell RNA sequencing, we revealed the gene expression dynamics of female germ cells and granulosa cells following exposure to DEHP in mice. Regarding germ cells: DEHP impeded the progression of follicle assembly and interfered with their developmental status, while key genes such as Lhx8, Figla, and others, strongly evidenced the reduction. As for granulosa cells: DEHP likely inhibited their proliferative activity, and activated the regulation of cell death. Furthermore, the interaction between ovarian cells mediated by transforming growth factor-beta signaling, was disrupted by DEHP exposure, since the expression of GDF9, BMPR1A, and SMAD3 was affected. In addition, DNA damage and apoptosis were elevated in germ cells and/or somatic cells. Conclusion: These findings offer substantial novel insights into the reproductive toxicity of DEHP exposure during murine germ cell cyst breakdown and primordial follicle formation. These results may enhance the understanding of DEHP exposure on reproductive health.

Different Characteristics of Hepcidin Expression in IL-6+/+ and IL-6-/- Neurons and Astrocytes Treated with Lipopolysaccharides.

A region-specific regulation of inflammation on the expression hepcidin in the brain has been demonstrated, however, it remains unknown whether there is also a cell-specific regulation of inflammation on hepcidin in the brain. Here, we investigated the effects of lipopolysaccharides (LPS) on the expression of hepcidin mRNA and also the expression of IL-6 mRNA, the phosphorylation of STAT3 and the expression of ferroportin 1 (Fpn1) and ferritin light chain (Ft-L) proteins in neurons and astrocytes obtained from wild type (IL-6+/+) and IL-6 knockout (IL-6-/-) mice. We demonstrated that the responses of the expression of hepcidin and IL-6 mRNAs, the phosphorylation of STAT3, and the expression of Fpn1 protein to LPS in IL-6+/+ astrocytes and also the responses of the expression of hepcidin mRNA, the phosphorylation of STAT3 and the expression of Fpn1 protein to IL-6 in IL-6-/- astrocytes were much stronger than those in IL-6+/+ and IL-6-/- neurons. A significant increase in Ft-L was found in LPS-treated IL-6+/+ and IL-6-treated IL-6-/- astrocytes, but not in LPS-treated IL-6+/+ and IL-6-treated IL-6-/- neurons. Our findings provide in vitro evidence for the existence of a cell-specific regulation of LPS on the expression of hepcidin and also Ft-L in the brain.

Cystathionine ß-synthase is required for body iron homeostasis.

Cystathionine ß-synthase (CBS) catalyzes the transsulfuration pathway and contributes, among other functions, to the generation of hydrogen sulfide. In view of the exceptionally high expression of CBS in the liver and the common interleukin-6 pathway used in the regulatory systems of hydrogen sulfide and hepcidin, we speculate that CBS is involved in body iron homeostasis. We found that CBS knockout (CBS-/- ) mice exhibited anemia and a significant increase in iron content in the serum, liver, spleen, and heart, along with severe damage to the liver, displaying a hemochromatosis-like phenotype. A high level of hepatic and serum hepcidin was also found. A major cause of the systemic iron overload is the reduced iron usage due to suppressed erythropoiesis, which is consistent with an increase in interleukin-6 and reduced expression of erythropoietin. Importantly, in the liver, absence of CBS caused both a reduction in the transcriptional factor nuclear factor erythroid 2-related factor-2 and an up-regulation of hepcidin that led to a decrease in the iron export protein ferroportin 1. The resulting suppression of iron export exacerbates iron retention, causing damage to hepatocytes. Finally, administration of CBS-overexpressing adenovirus into CBS mutant mice could partially reverse the iron-related phenotype. CONCLUSION: Our findings point to a critical role of CBS in iron homeostasis of the body, and the liver in particular; it is likely that a hemochromatosis-like phenotype in patients can be induced by aberration not only in the expression of key molecules in the hepcidin pathway but also of those related to CBS. (Hepatology 2018;67:21-35).

Hepcidin Protects Neuron from Hemin-Mediated Injury by Reducing Iron.

Hemin plays a key role in mediating secondary neuronal injury after intracerebral hemorrhage (ICH) and the cell toxicity of hemin is thought to be due to iron that is liberated when hemin is degraded. In a recent study, we demonstrated the iron regulatory hormone hepcidin reduces brain iron in iron-overloaded rats. Therefore, we hypothesized that hepcidin might be able to reduce iron and then protect neurons from hemin or iron-mediated neurotoxicity in hemin-treated neuronal cells. Here, we tested the hypothesis and demonstrated that ad-hepcidin and hepcidin peptide both have the ability to suppress the hemin-induced increase in LDH release and apoptotic cell numbers, to reduce cell iron and ferritin contents, and to inhibit expression of transferrin receptor 1, divalent metal transporter 1, and ferroportin 1 in hemin-treated neurons. We conclude that hepcidin protects neuron from hemin-mediated injury by reducing iron via inhibition of expression of iron transport proteins.

Bi-directionally protective communication between neurons and astrocytes under ischemia.

The extensive existing knowledge on bi-directional communication between astrocytes and neurons led us to hypothesize that not only ischemia-preconditioned (IP) astrocytes can protect neurons but also IP neurons protect astrocytes from lethal ischemic injury. Here, we demonstrated for the first time that neurons have a significant role in protecting astrocytes from ischemic injury. The cultured medium from IP neurons (IPcNCM) induced a remarkable reduction in LDH and an increase in cell viability in ischemic astrocytes in vitro. Selective neuronal loss by kainic acid injection induced a significant increase in apoptotic astrocyte numbers in the brain of ischemic rats in vivo. Furthermore, TUNEL analysis, DNA ladder assay, and the measurements of ROS, GSH, pro- and anti-apoptotic factors, anti-oxidant enzymes and signal molecules in vitro and/or in vivo demonstrated that IP neurons protect astrocytes by an EPO-mediated inhibition of pro-apoptotic signals, activation of anti-apoptotic proteins via the P13K/ERK/STAT5 pathways and activation of anti-oxidant proteins via up-regulation of anti-oxidant enzymes. We demonstrated the existence of astro-protection by IP neurons under ischemia and proposed that the bi-directionally protective communications between cells might be a common activity in the brain or peripheral organs under most if not all pathological conditions.