The male reproductive toxicity after 5-Fluorouracil exposure: DNA damage, oxidative stress, and mitochondrial dysfunction in vitro and in vivo.

5-Fluorouracil (5-FU), a chemotherapeutic drug used to treat a variety of cancers, can enter the environment through different routes, causing serious public health and environmental concerns. It has been reported that 5-FU exposure adversely affects male reproductive function, and its effects on this system cannot be avoided. In this study, using western blotting and quantitative polymerase chain reaction studies, we found that 5-FU promoted testicular injury by inducing oxidative stress, which was accompanied by the inhibition of nuclear factor erythroid 2-related factor 2/antioxidant response element signaling. Accumulation of reactive oxygen species (ROS) aggravated 5-FU-mediated mitochondrial dysfunction and apoptosis in murine cell lines and testes, indicating oxidative stress and mitochondrial-dependent apoptotic signaling play crucial roles in the damage of spermatogenic cells caused. N-Acetyl-L-cysteine, an antioxidant that scavenges intracellular ROS, protected spermatogenic cells from 5-FU-induced oxidative damage and mitochondrial dysfunction, revealing the important role of ROS in testicular dysfunction caused by 5-FU. We found that 5-FU exposure induces testicular cell apoptosis through ROS-mediated mitochondria pathway in mice. In summary, our findings revealed the reproductive toxicological effect of 5-FU on mice and its mechanism, provided basic data reference for adverse ecological and human health outcomes associated with 5-FU contamination or poisoning.

Emerging roles of ferroptosis in male reproductive diseases.

Ferroptosis is a type of programmed cell death mediated by iron-dependent lipid peroxidation that leads to excessive lipid peroxidation in different cells. Ferroptosis is distinct from other forms of cell death and is associated with various diseases. Iron is essential for spermatogenesis and male reproductive function. Therefore, it is not surprising that new evidence supports the role of ferroptosis in testicular injury. Although the molecular mechanism by which ferroptosis induces disease is unknown, several genes and pathways associated with ferroptosis have been linked to testicular dysfunction. In this review, we discuss iron metabolism, ferroptosis, and related regulatory pathways. In addition, we analyze the endogenous and exogenous factors of ferroptosis in terms of iron metabolism and testicular dysfunction, as well as summarize the relationship between ferroptosis and male reproductive dysfunction. Finally, we discuss potential strategies to target ferroptosis for treating male reproductive diseases and provide new directions for preventing male reproductive diseases.

BMP4 is insufficient to differentiate umbilical cord mesenchymal stem cells into germ cell-like cells in vitro.

OBJECTIVES: Mesenchymal stem cell (MSC)-based therapies are expected to restore the fertility of infertile patients. In addition to MSC-derived paracrine effects to improve reproductive function, the differentiation of MSCs into germ cell (GC)-like cells is still a promising method to repair the injured reproductive system. The aim of this study was to examine the effect and potential mechanism of BMP4 in inducing umbilical cord MSC (UcMSC) transdifferentiation into GC-like cells. MATERIAL AND METHODS: UcMSCs were isolated, cultured and identified by flow cytometry and multilineage differentiation assays. After induction with 12.5 ng/mL BMP4 for 21 days, UcMSCs were collected for further examination. Immunofluorescence was used to detect the expression of Prdm1 and Prdm14; RT-PCR and RNA sequencing were used to detect differential gene expression (DEGs). RESULTS: The morphology of UcMSCs became large and flat after treatment with BMP4; the expression of GC-related genes (OCT4, Prdm1, Ifitm3 and Stella) was significantly downregulated, and further immunofluorescence results also confirmed the significant downregulation of Prdm1 in UcMSCs with BMP4 induction, while the expression of Prdm14 was significantly upregulated. The results of RNA sequencing and further analysis revealed no explicit correlation between BMP4 induction and the differentiation of UcMSCs into GC-like cells based on the 662 screened DEGs in UcMSCs with or without BMP4 induction. CONCLUSIONS: The differentiation of MSCs into GC-like cells is rather complex, and BMP4 alone is insufficient to induce UcMSCs to differentiate into GC-like cells, regardless of protein level or gene expression level.

Lipid transfer proteins involved in plant-pathogen interactions and their molecular mechanisms.

Nonspecific lipid transfer proteins (LTPs) are small, cysteine-rich proteins that play numerous functional roles in plant growth and development, including cutin wax formation, pollen tube adhesion, cell expansion, seed development, germination, and adaptation to changing environmental conditions. LTPs contain eight conserved cysteine residues and a hydrophobic cavity that provides a wide variety of lipid-binding specificities. As members of the pathogenesis-related protein 14 family (PR14), many LTPs inhibit fungal or bacterial growth, and act as positive regulators in plant disease resistance. Over the past decade, these essential immunity-related roles of LTPs in plant immune processes have been documented in a growing body of literature. In this review, we summarize the roles of LTPs in plant-pathogen interactions, emphasizing the underlying molecular mechanisms in plant immune responses and specific LTP functions.

Protein glycosylation changes during systemic acquired resistance in Arabidopsis thaliana.

N-glycosylation, an important post-translational modification of proteins in all eukaryotes, has been clearly shown to be involved in numerous diseases in mammalian systems. In contrast, little is known regarding the role of protein N-glycosylation in plant defensive responses to pathogen infection. We identified, for the first time, glycoproteins related to systemic acquired resistance (SAR) in an Arabidopsis thaliana model, using a glycoproteomics platform based on high-resolution mass spectrometry. 407 glycosylation sites corresponding to 378 glycopeptides and 273 unique glycoproteins were identified. 65 significantly changed glycoproteins with 80 N-glycosylation sites were detected in systemic leaves of SAR-induced plants, including numerous GDSL-like lipases, thioglucoside glucohydrolases, kinases, and glycosidases. Functional enrichment analysis revealed that significantly changed glycoproteins were involved mainly in N-glycan biosynthesis and degradation, phenylpropanoid biosynthesis, cutin and wax biosynthesis, and plant-pathogen interactions. Comparative analysis of glycoproteomics and proteomics data indicated that glycoproteomics analysis is an efficient method for screening proteins associated with SAR. The present findings clarify glycosylation status and sites of A. thaliana proteins, and will facilitate further research on roles of glycoproteins in SAR induction.

Comparison of rapid freezing versus vitrification for human sperm cryopreservation using sucrose in closed straw systems.

Rapid freezing and vitrification using sucrose are two simple and cost-effective sperm cryopreservation methods. However, it is still unclear which method is better and what the optimal concentration of sucrose is. This study aimed to determine the optimal sucrose concentration for human sperm cryopreservation and compare the cryoprotective effects of rapid freezing versus vitrification using different closed straw systems in terms of sperm motility and DNA integrity. Our data showed that: (1) The optimal sucrose concentration for vitrification was 0.25 mol/l among the tested 0, 0.125, 0.25 and 0.5 mol/l concentrations; (2) Sperm total motility and progressive motility were cryopreserved significantly better by rapid freezing than vitrification in standard 0.5 ml cryostraws (P < 0.05); and (3) Sperm total motility and progressive motility were cryopreserved significantly better by vitrification in the straw-in-straw system than rapid freezing in the standard 0.5 cryostraw (P < 0.05), but no difference was found in sperm nuclear DNA fragmentation level between the two cryopreservation methods (P > 0.05). It was concluded that sucrose at 0.25 mol/l concentration is suitable for human sperm rapid freezing and vitrification, and sperm cryopreservation can be achieved by rapid freezing using closed standard 0.5 ml straws or by vitrification using the novel straw-in-straw system made of standard 0.25 and 0.5 ml straws.

Studies on the basic issues relevant to sperm cryopreservation in humans.

Rapid freezing and vitrification are becoming popular for sperm freezing in humans; however, basic and critical issues relevant to sperm cryopreservation remain to be resolved. The aims of the present study were to study the effects of osmolality of freezing medium, sperm concentrations, thawing methods, and sugars (sucrose and trehalose) on sperm motility and DNA integrity by rapid freezing using 0.5 ml standard straws loaded with 100 µl sperm each. The results showed that (1) the post-thaw recovery rates of total motility and progressive motility of sperm cryopreserved in freezing medium containing 0.25 M sucrose with 442 mOsm/kg osmolality were significantly higher (p < 0.05) than that of sperm cryopreserved in freezing medium containing 0.25 M sucrose with 536 mOsm/kg osmolality (36.5 ± 2.8% and 36.9 ± 1.7% versus 30.4 ± 1.9% and 30.3 ± 2.9%, respectively), (2) cryopreservation of both total and progressive motilities was not significantly affected (p > 0.05) by sperm concentrations in the range from 5 to 20 × 106 sperm/ml, (3) thawing method 37°C for 2 min was better than 42°C for 15 s in terms of post-thaw recovery rates of both total and progressive motilities (p < 0.05), (4) 0.25 M trehalose was better than 0.25 M sucrose in cryopreserving both total and progressive motilities (p < 0.05), and (5) sperm nuclear DNA is relatively resistant to the changes of the above factors compared with sperm motility. It was concluded that human sperm can be best cryopreserved by rapid freezing using 0.25 M sucrose or trehalose with osmolality 442 to 457 mOsm/kg at high sperm concentration followed by thawing at 37°C. Trehalose is a stronger cryoprotectant than sucrose for sperm cryopreservation.

Protective Effect of Sucrose and Antioxidants on Cryopreservation of Sperm Motility and DNA Integrity in C57BL/6 Mice.

Currently the most popular mouse sperm freezing medium is R18SM3+MTG containing 18% raffinose, 3% skim milk, and 0.5 mM monothioglycerol (MTG), but there is no information available about whether MTG and other antioxidants can cryoprotect mouse sperm DNA integrity. It is also uncertain if sucrose can be used successfully for sperm cryopreservation. In this report we compared the cryoprotective effects of sucrose and raffinose, as well as the antioxidants MTG, reduced glutathione (GSH), and quercetin on sperm motility, DNA integrity, and fertility in the C57BL/6J mouse strain. Results show that: (1) 10% sucrose in the presence of 3% skim milk and 0.5 mM MTG (S10SM3+MTG) was as effective as R18SM3+MTG (p > 0.05) in cryopreserving sperm motility (21.0% ± 4.0% vs. 19.0% ± 3.6%), DNA integrity (8.1% ± 1.5% vs. 9.0% ± 1.5% TUNEL positive), fertilization rate (48.3% ± 7.5% vs. 45.0% ± 7.9%), and pup birth rate (36.7% ± 10.0% vs. 37.7% ± 3.5%); (2) Supplementation of freezing medium with MTG (0.5 mM), GSH (0.5 mM), or quercetin (25 µM) had a significant (p < 0.05) cryoprotective effect on sperm motility recovery rate compared to that of controls (MTG, 32.7% ± 10.7% vs. 19.4% ± 4.2%; GSH, 34.3% ± 3.7% vs. 24.5% ± 1.8%; quercetin, 36.3% ± 3.3% vs. 25.1% ± 3.6%); and (3) Cryopreservation significantly increased sperm DNA fragmentation level (16.4% ± 2.3% in R18SM3 and 14.6% ± 2.5% in S10SM3) compared to that of fresh sperm (3.0% ± 2.0%); however, supplementation with MTG (0.5 mM), GSH (0.5 mM), or quercetin (25 µM) significantly (p < 0.05) decreased the sperm DNA fragmentation level (MTG, 8.1% ± 1.5%; GSH, 9.0% ± 1.5%; and quercetin, 8.3% ± 1.3%). It was concluded that sucrose can be used as effectively as raffinose for mouse sperm cryopreservation, and supplementation of MTG, GSH, or quercetin at an appropriate concentration can help cryoprotect sperm motility and DNA integrity.