Triptolide exposure triggers testicular vacuolization injury by disrupting the Sertoli cell junction and cytoskeletal organization via the AKT/mTOR signaling pathway.

BACKGROUND: Despite the known reproductive toxicity induced by triptolide (TP) exposure, the regulatory mechanism underlying testicular vacuolization injury caused by TP remains largely obscure. METHODS: Male mice were subjected to TP at doses of 15, 30, and 60 µg/kg for 35 consecutive days. Primary Sertoli cells were isolated from 20-day-old rat testes and exposed to TP at concentrations of 0, 40, 80, 160, 320, and 640 nM. A Biotin tracer assay was conducted to assess the integrity of the blood-testis barrier (BTB). Transepithelial electrical resistance (TER) assays were employed to investigate BTB function in primary Sertoli cells. Histological structures of the testes and epididymides were stained with hematoxylin and eosin (H&E). The expression and localization of relevant proteins or pathways were assessed through Western blotting or immunofluorescence staining. RESULTS: TP exposure led to dose-dependent testicular injuries, characterized by a decreased organ coefficient, reduced sperm concentration, and the formation of vacuolization damage. Furthermore, TP exposure disrupted BTB integrity by reducing the expression levels of tight junction (TJ) proteins in the testes without affecting basal ectoplasmic specialization (basal ES) proteins. Through the TER assay, we identified that a TP concentration of 160 nM was optimal for elucidating BTB function in primary Sertoli cells, correlating with reductions in TJ protein expression. Moreover, TP exposure induced changes in the distribution of the BTB and cytoskeleton-associated proteins in primary Sertoli cells. By activating the AKT/mTOR signaling pathway, TP exposure disturbed the balance between mTORC1 and mTORC2, ultimately compromising BTB integrity in Sertoli cells. CONCLUSION: This investigation sheds light on the impacts of TP exposure on testes, elucidating the mechanism by which TP exposure leads to testicular vacuolization injury and offering valuable insights into comprehending the toxic effects of TP exposure on testes.

Single-cell RNA sequencing analysis to evaluate antimony exposure effects on cell-lineage communications within the Drosophila testicular niche.

The increasing production and prevalence of antimony (Sb)-related products raise concerns regarding its potential hazards to reproductive health. Upon environmental exposure, Sb reportedly induces testicular toxicity during spermatogenesis; moreover, it is known to affect various testicular cell populations, particularly germline stem cell populations. However, the cell-cell communication resulting from Sb exposure within the testicular niche remains poorly understood. To address this gap, herein we analyzed testicular single-cell RNA sequencing data from Sb-exposed Drosophila. Our findings revealed that the epidermal growth factor receptor (EGFR) and WNT signaling pathways were associated with the stem cell niche in Drosophila testes, which may disrupt the homeostasis of the testicular niche in Drosophila. Furthermore, we identified several ligand-receptor pairs, facilitating the elucidation of intercellular crosstalk involved in Sb-mediated reproductive toxicology. We employed scRNA-seq analysis and conducted functional verification to investigate the expression patterns of core downstream factors associated with EGFR and WNT signatures in the testes under the influence of Sb exposure. Altogether, our results shed light on the potential mechanisms of Sb exposure-mediated testicular cell-lineage communications.

Single-cell transcriptome characteristics of testicular terminal epithelium lineages during aging in the Drosophila.

Aging is a complex biological process leading to impaired functions, with a variety of hallmarks. In the testis of Drosophila, the terminal epithelium region is involved in spermatid release and maturation, while its functional diversity and regulatory mechanism remain poorly understood. In this study, we performed single-cell RNA-sequencing analysis (scRNA-seq) to characterize the transcriptomes of terminal epithelium in Drosophila testes at 2-, 10 and 40-Days. Terminal epithelium populations were defined with Metallothionein A (MtnA) and subdivided into six novel sub-cell clusters (EP0-EP5), and a series of marker genes were identified based on their expressions. The data revealed the functional characteristics of terminal epithelium populations, such as tight junction, focal adhesion, bacterial invasion, oxidative stress, mitochondrial function, proteasome, apoptosis and metabolism. Interestingly, we also found that disrupting genes for several relevant pathways in terminal epithelium led to male fertility disorders. Moreover, we also discovered a series of age-biased genes and pseudotime trajectory mediated state-biased genes during terminal epithelium aging. Differentially expressed genes during terminal epithelium aging were mainly participated in the regulation of several common signatures, e.g. mitochondria-related events, protein synthesis and degradation, and metabolic processes. We further explored the Drosophila divergence and selection in the functional constraints of age-biased genes during aging, revealing that age-biased genes in epithelial cells of 2 Days group evolved rapidly and were endowed with greater evolutionary advantages. scRNA-seq analysis revealed the diversity of testicular terminal epithelium populations, providing a gene target resource for further systematic research of their functions during aging.

L(1)10Bb serves as a conservative determinant for soma-germline communications via cellular non-autonomous effects within the testicular stem cell niche.

The testicular stem cell niche is the central regulator of spermatogenesis in Drosophila melanogaster. However, the underlying regulatory mechanisms are unclear. This study demonstrated the crucial role of lethal (1) 10Bb [l(1)10Bb] in regulating the testicular stem cell niche. Dysfunction of l(1)10Bb in early-stage cyst cells led to male fertility disorders and compromised cyst stem cell maintenance. Moreover, the dysfunction of l(1)10Bb in early-stage cyst cells exerted non-autonomous effects on germline stem cell differentiation, independently of hub signals. Notably, our study highlights the rescue of testicular defects through ectopic expression of L(1)10Bb and the human homologous protein BUD31 homolog (BUD31). In addition, l(1)10Bb dysfunction in early-stage cyst cells downregulated the expression of spliceosome subunits in the Sm and the precursor RNA processing complexes. Collectively, our findings established l(1)10Bb as a pivotal factor in the modulation of Drosophila soma-germline communications within the testicular stem cell niche.

Exposure to Titanium Dioxide Nanoparticles Leads to Specific Disorders of Spermatid Elongation via Multiple Metabolic Pathways in Drosophila Testes.

Titanium dioxide nanoparticles (TiO2 NPs) have been extensively utilized in various applications. However, the regulatory mechanism behind the reproductive toxicity induced by TiO2 NP exposure remains largely elusive. In this study, we employed a Drosophila model to assess potential testicular injuries during spermatogenesis and conducted bulk RNA-Seq analysis to elucidate the underlying mechanisms. Our results reveal that while prolonged exposure to lower concentrations of TiO2 NPs (0.45 mg/mL) for 30 days did not manifest reproductive toxicity, exposure at concentrations of 0.9 and 1.8 mg/mL significantly impaired spermatid elongation in Drosophila testes. Notably, bulk RNA-seq analysis revealed that TiO2 NP exposure affected multiple metabolic pathways including carbohydrate metabolism and cytochrome P450. Importantly, the intervention of glutathione (GSH) significantly protected against reproductive toxicity induced by TiO2 NP exposure, as it restored the number of Orb-positive spermatid clusters in Drosophila testes. Our study provides novel insights into the specific detrimental effects of TiO2 NP exposure on spermatid elongation through multiple metabolic alterations in Drosophila testes and highlights the protective role of GSH in countering this toxicity.

Distribution characteristics of mitochondria-rich segments in the epididymis.

The epididymis is a highly specialized tissue that plays vital roles in sperm maturation and storage. The spatio-temporal repertoire of epididymal cells and their gene expression in the epididymis remain less characterized. With the help of single-cell RNA sequencing (scRNA-seq), Shi et al., reveal a spatio- and segment-specific distribution pattern of mitochondria that adds another layer of complexity to our understanding of the epididymis. They unexpectedly find a higher abundance of mitochondria and mitochondrial transcription in the corpus and cauda compared to the caput of epididymis, which are believed to be responsible for providing the energy necessary for sperm maturation and motility.

Structure and molecular basis of spermatid elongation in the Drosophila testis.

Spermatid elongation is a crucial event in the late stage of spermatogenesis in the Drosophila testis, eventually leading to the formation of mature sperm after meiosis. During spermatogenesis, significant structural and morphological changes take place in a cluster of post-meiotic germ cells, which are enclosed in a microenvironment surrounded by somatic cyst cells. Microtubule-based axoneme assembly, formation of individualization complexes and mitochondria maintenance are key processes involved in the differentiation of elongated spermatids. They provide important structural foundations for accessing male fertility. How these structures are constructed and maintained are basic questions in the Drosophila testis. Although the roles of several genes in different structures during the development of elongated spermatids have been elucidated, the relationships between them have not been widely studied. In addition, the genetic basis of spermatid elongation and the regulatory mechanisms involved have not been thoroughly investigated. In the present review, we focus on current knowledge with regard to spermatid axoneme assembly, individualization complex and mitochondria maintenance. We also touch upon promising directions for future research to unravel the underlying mechanisms of spermatid elongation in the Drosophila testis.

A Novel Extract From Ginkgo biloba Inhibits Neuroinflammation and Maintains White Matter Integrity in Experimental Stroke.

Ginkgo biloba L. leaf extract (GBE) has been added in many commercial herbal formulations such as EGb 761 and Shuxuening Injection to treat cardiovascular diseases and stroke worldwide. However, the comprehensive effects of GBE on cerebral ischemia remained unclear. Using a novel GBE (nGBE), which consists of all the compounds of traditional (t)GBE and one new compound, pinitol, we investigated its effect on inflammation, white matter integrity, and long-term neurological function in an experimental stroke model. Both transient middle cerebral artery occlusion (MCAO) and distal MCAO were conducted in male C57/BL6 mice. We found that nGBE significantly reduced infarct volume at 1, 3, and 14 days after ischemia. Sensorimotor and cognitive functions were superior in nGBE treated mice after MCAO. nGBE inhibited the release of IL-1ß in the brain, promoted microglial ramification, and regulated the microglial M1 to M2 phenotype shift at 7 days post injury. In vitro analyses showed that nGBE treatment reduced the production of IL-1ß and TNFα in primary microglia. Administration of nGBE also decreased the SMI-32/MBP ratio and enhanced myelin integrity, thus exhibiting improved white matter integrity at 28 days post stroke. These findings demonstrate that nGBE protects against cerebral ischemia by inhibiting microglia-related inflammation and promoting white matter repair, suggesting that nGBE is a promising therapeutic strategy for long-term recovery after stroke.

A Drosophila model of gestational antimony exposure uncovers growth and developmental disorders caused by disrupting oxidative stress homeostasis.

The toxic heavy metal antimony (Sb) is ubiquitous in our daily lives. Various models have shown that Sb induces neuronal and reproductive toxicity. However, little is known about the developmental toxicity of Sb exposure during gestation and the underlying mechanisms. To study its effects on growth and development, Drosophila stages from eggs to pupae were exposed to different Sb concentrations (0, 0.3, 0.6 and 1.2 mg/mL Sb); RNA sequencing was used to identify the underlying mechanism. The model revealed that prenatal Sb exposure significantly reduced larval body size and weight, the pupation and eclosion rates, and the number of flies at all stages. With 1.2 mg/mL Sb exposure in 3rd instar larvae, 484 genes were upregulated and 694 downregulated compared to controls. Biological analysis showed that the disrupted transcripts were related to the oxidative stress pathway, as verified by reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) and glutathione (GSH) intervention experiments. Sb exposure induced oxidative stress imbalance could be rectified by chelation and antioxidant effects of NAC/GSH. The Drosophila Schneider 2 (S2) model further demonstrated that NAC and GSH greatly ameliorated cell death induced by Sb exposure. In conclusion, gestational Sb exposure disrupted oxidative stress homeostasis, thereby impairing growth and development.

Single-cell RNA-sequencing reveals the transcriptional landscape of ND-42 mediated spermatid elongation via mitochondrial derivative maintenance in Drosophila testes.

During spermatogenesis, mitochondria extend along the whole length of spermatid tail and offer a structural platform for microtubule reorganization and synchronized spermatid individualization, that eventually helps to generate mature sperm in Drosophila. However, the regulatory mechanism of spermatid mitochondria during elongation remains largely unknown. Herein, we demonstrated that NADH dehydrogenase (ubiquinone) 42 kDa subunit (ND-42) was essential for male fertility and spermatid elongation in Drosophila. Moreover, ND-42 depletion led to mitochondrial disorders in Drosophila testes. Based on single-cell RNA-sequencing (scRNA-seq), we identified 15 distinct cell clusters, including several unanticipated transitional subpopulations or differentiative stages for testicular germ cell complexity in Drosophila testes. Enrichments of the transcriptional regulatory network in the late-stage cell populations revealed key roles of ND-42 in mitochondria and its related biological processes during spermatid elongation. Notably, we demonstrated that ND-42 depletion led to maintenance defects of the major mitochondrial derivative and the minor mitochondrial derivative by affecting mitochondrial membrane potential and mitochondrial-encoded genes. Our study proposes a novel regulatory mechanism of ND-42 for spermatid mitochondrial derivative maintenance, contributing to a better understanding of spermatid elongation.

Single-cell RNA sequencing reveals cell landscape following antimony exposure during spermatogenesis in Drosophila testes.

Antimony (Sb), is thought to induce testicular toxicity, although this remains controversial. This study investigated the effects of Sb exposure during spermatogenesis in the Drosophila testis and the underlying transcriptional regulatory mechanism at single-cell resolution. Firstly, we found that flies exposed to Sb for 10 days led to dose-dependent reproductive toxicity during spermatogenesis. Protein expression and RNA levels were measured by immunofluorescence and quantitative real-time PCR (qRT-PCR). Single-cell RNA sequencing (scRNA-seq) was performed to characterize testicular cell composition and identify the transcriptional regulatory network after Sb exposure in Drosophila testes. scRNA-seq analysis revealed that Sb exposure influenced various testicular cell populations, especially in GSCs_to_Early_Spermatogonia and Spermatids clusters. Importantly, carbon metabolism was involved in GSCs/early spermatogonia maintenance and positively related with SCP-Containing Proteins, S-LAPs, and Mst84D signatures. Moreover, Seminal Fluid Proteins, Mst57D, and Serpin signatures were highly positively correlated with spermatid maturation. Pseudotime trajectory analysis revealed three novel states for the complexity of germ cell differentiation, and many novel genes (e.g., Dup98B) were found to be expressed in state-biased manners during spermatogenesis. Collectively, this study indicates that Sb exposure negatively impacts GSC maintenance and spermatid elongation, damaging spermatogenesis homeostasis via multiple signatures in Drosophila testes and therefore supporting Sb-mediated testicular toxicity.

[Relationship between species distribution and habitat effective temperature in subalpine meadow of Mt. Xiaowutai].

By the methods of sugar-inversion, plant community field survey and laboratory analysis, this paper measured the effective temperature of habitats, relative coverage of species, and aboveground biomass in subalpine meadow of Mt.Xiaowutai, and analyzed the relationship between species distribution and habitat effective temperature in landscape scale. The results showed that among 97 vascular plant species registered, the distribution patterns of about 36 % of total species were influenced by the effective temperature of the habitats, and the relative coverage of about 20% of them correlated significantly with the effective temperature. Eight species showed a negative correlation between their relative coverage and effective temperature, and eleven species had a positive correlation between their relative coverage or aboveground biomass and effective temperature. The eleven species with a positive correlation had certain drought resistance feature or a tendency distributing towards warmer habitats. Concerning the effectiveness of species feature applied in the analysis, relative coverage could be used to determine the relationship between species distribution and effective temperature efficiently, if the species frequency ranged from 90 % to 20 %. But, for the dominant species with their frequency over 90 %, aboveground biomass seemed to be more suitable for the analysis.