Applications of single-cell RNA sequencing in spermatogenesis and molecular evolution

Spermatogenic cell heterogeneity is determined by the complex process of spermatogenesis differentiation.However,effectively revealing the regulatory mechanisms underlying mammalian spermatogenic cell development and differentiation via traditional methods is difficult.Advances in technology have led to the emergence of many single-cell transcriptome sequencing protocols,which have partially addressed these challenges.In this review,we detail the principles of 10x Genomics technology and summarize the methods for downstream analysis of single-cell transcriptome sequencing data.Furthermore,we explore the role of single-cell transcriptome sequencing in revealing the heterogeneity of testicular ecological niche cells,delineating the establishment and disruption of testicular immune homeostasis during human spermatogenesis,investigating abnormal spermatogenesis in humans,and,ultimately,elucidating the molecular evolution of mammalian spermatogenesis.

Single-cell RNA sequencing reveals atlas of dairy goat testis cells

Single-cell RNA sequencing(scRNA-seq)is useful for exploring cell heterogeneity.For large animals,however,little is known regarding spermatogonial stem cell(SSC)selfrenewal regulation,especially in dairy goats.In this study,we described a high-resolution scRNA-seq atlas derived from a dairy goat.We identified six somatic cell and five spermatogenic cell subtypes.During spermatogenesis,genes with significantly changed expression were mainly enriched in the Notch,TGF-β,and Hippo signaling pathways as well as the signaling pathway involved in the regulation of stem cell pluripotency.We detected and screened specific candidate marker genes(TKTL1 and AES)for spermatogonia.Our study provides new insights into goat spermatogenesis and the development of testicular somatic cells.

Depletion of stearoyl-CoA desaturase(scd) leads to fatty liver disease and defective mating behavior in zebrafish

Stearyl coenzyme A desaturase(SCD), also known as delta-9 desaturase, catalyzes the rate-limiting step in the formation of monounsaturated fatty acids.In mammals, depletion or inhibition of SCD activity generally leads to a decrease in triglycerides and cholesteryl esters. However, the endogenous role of scd in teleost fish remains unknown. Here, we generated a zebrafish scd mutant(scd-/-) to elucidate the role of scd in lipid metabolism and sexual development. Gas chromatography-mass spectrometry(GC-MS) showed that the scd-/- mutants had increased levels of saturated fatty acids C16:0 and C18:0, and decreased levels of monounsaturated fatty acids C16:1 and C18:1. The mutant fish displayed a short stature and an enlarged abdomen during development. Unlike Scd-/ -mammals, the scd-/- zebrafish showed significantly increased fat accumulation in the whole body,especially in the liver, leading to hepatic mitochondrial dysfunction and severe cell apoptosis.Mechanistically, srebf1, a gene encoding a transcriptional activator related to adipogenesis,acc1 and acaca, genes involved in fatty acid synthesis, and dgat2, a key gene involved in triglyceride synthesis, were significantly upregulated in mutant livers to activate fatty acid biosynthesis and adipogenesis. The scd-/- males exhibited defective natural mating behavior due to defective genital papillae but possessed functional mature sperm. All defects in the scd-/- mutants could be rescued by ubiquitous transgenic overexpression of scd. In conclusion, our study demonstrates that scd is indispensable for maintaining lipid homeostasis and development of secondary sexual characteristics in zebrafish.

Transcription factor Dmrt1 triggers the SPRY1-NF-κB pathway to maintain testicular immune homeostasis and male fertility

Bacterial or viral infections,such as Brucella,mumps virus,herpes simplex virus,and Zika virus,destroy immune homeostasis of the testes,leading to spermatogenesis disorder and infertility.Of note,recent research shows that SARS-CoV-2 can infect male gonads and destroy Sertoli and Leydig cells,leading to male reproductive dysfunction.Due to the many side effects associated with antibiotic therapy,finding alternative treatments for inflammatory injury remains critical.Here,we found that Dmrt1 plays an important role in regulating testicular immune homeostasis.Knockdown of Dmrt1 in male mice inhibited spermatogenesis with a broad inflammatory response in seminiferous tubules and led to the loss of spermatogenic epithelial cells.Chromatin immunoprecipitation sequencing(ChIP-seq)and RNA sequencing(RNA-seq)revealed that Dmrt1 positively regulated the expression of Spry1,an inhibitory protein of the receptor tyrosine kinase(RTK)signaling pathway.Furthermore,immunoprecipitation-mass spectrometry(IP-MS)and co-immunoprecipitation(Co-IP)analysis indicated that SPRY1 binds to nuclear factor kappa B1(NF-κB1)to prevent nuclear translocation of p65,inhibit activation of NF-κB signaling,prevent excessive inflammatory reaction in the testis,and protect the integrity of the blood-testis barrier.In view of this newly identified Dmrt1-Spry1-NF-κB axis mechanism in the regulation of testicular immune homeostasis,our study opens new avenues for the prevention and treatment of male reproductive diseases in humans and livestock.

Bone marrow mesenchymal stem cell-derived exosomal microRNAs target PI3K/Akt signaling pathway to promote the activation of fibroblasts

BACKGROUND Fibroblast plays a major role in tendon-bone healing.Exosomes derived from bone marrow mesenchymal stem cells(BMSCs)can activate fibroblasts and promote tendon-bone healing via the contained microRNAs(miRNAs).However,the underlying mechanism is not comprehensively understood.Herein,this study aimed to identify overlapped BMSC-derived exosomal miRNAs in three GSE datasets,and to verify their effects as well as mechanisms on fibroblasts.AIM To identify overlapped BMSC-derived exosomal miRNAs in three GSE datasets and verify their effects as well as mechanisms on fibroblasts.METHODS BMSC-derived exosomal miRNAs data(GSE71241,GSE153752,and GSE85341)were downloaded from the Gene Expression Omnibus(GEO)database.The candidate miRNAs were obtained by the intersection of three data sets.TargetScan was used to predict potential target genes for the candidate miRNAs.Functional and pathway analyses were conducted using the Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)databases,respectively,by processing data with the Metascape.Highly interconnected genes in the protein-protein interaction(PPI)network were analyzed using Cytoscape software.Bromodeoxyuridine,wound healing assay,collagen contraction assay and the expression of COL I andα-smooth muscle actin positive were applied to investigate the cell proliferation,migration and collagen synthesis.Quantitative real-time reverse transcription polymerase chain reaction was applied to determine the cell fibroblastic,tenogenic,and chondrogenic potential.RESULTS Bioinformatics analyses found two BMSC-derived exosomal miRNAs,has-miR-144-3p and hasmiR-23b-3p,were overlapped in three GSE datasets.PPI network analysis and functional enrichment analyses in the GO and KEGG databases indicated that both miRNAs regulated the PI3K/Akt signaling pathway by targeting phosphatase and tensin homolog(PTEN).In vitro experiments confirmed that miR-144-3p and miR-23b-3p stimulated proliferation,migration and collagen synthesis of NIH3T3 fibroblasts.Interfering with PTEN affected the phosphorylation of Akt and thus activated fibroblasts.Inhibition of PTEN also promoted the fibroblastic,tenogenic,and chondrogenic potential of NIH3T3 fibroblasts.CONCLUSION BMSC-derived exosomes promote fibroblast activation possibly through the PTEN and PI3K/Akt signaling pathways,which may serve as potential targets to further promote tendon-bone healing.

Correction of intense laser–plasma interactions by QED vacuum polarization in collision of laser beams

The influence of vacuum polarization effects on the interactions of multiple ultra-intense lasers with plasmas is discussed. The nonlinear paraxial monochromatic model of the interactions has been improved by considering the Heisenberg–Euler Lagrangian density of two laser processes. Comparing the corrections of vacuum polarization effects in the collision of laser beams with one generated by a single intense laser, we find that the former has a higher order of magnitude correction. The laser collision also produces variations in the propagation direction and polarization direction of the lasers propagating in the plasma. In addition, the strong-field quantum electrodynamic(QED) effects can be enhanced by increasing the laser intensity or frequency difference, or by adjusting the incident angles of the two laser beams.

Experimental and numerical modeling on vacuum consolidation behavior of staged-filled soil slurry with prefabricated horizontal drain and flocculant

The vacuum-assisted prefabricated horizontal drain offers a promising method for strengthening soil slurry,allowing simultaneous filling and vacuum-dewatering via staged construction.However,there is limited research on the unique characteristics of staged filling.This study aims to investigate the vacuum consolidation process of staged-filled soil slurry through laboratory model tests and numerical simulations,also assessing the impact of anionic polyacrylamide.Comparative analyses are conducted between vacuum consolidation with and without anionic polyacrylamide,as well as self-weight consolidation without anionic polyacrylamide.Results reveal contour lines of excess pore pressure,water content,and soil strength forming an ellipse around the prefabricated horizontal drain board.During the consolidation process,a higher degree of consolidation,lower water content,and higher soil strength were observed closer to the prefabricated horizontal drain board.After treatment,the uppermost filling layer exhibits an average water content that was approximately 40%higher than the lower filling layer,and its average strength was about 60%lower.This discrepancy is primarily due to the absence of sealing on the top surface and the relatively short vacuum consolidation time caused by staged filling.The introduction of anionic polyacrylamide-induced flocculation significantly improves the initial consolidation rate but minimally affects the dewatering capacity of vacuum preloading.Using flocculant can enhance both the staged filling rate and soil strength(by 1e2 times).Additionally,employing a staggered arrangement between different prefabricated horizontal drain layers is advisable to prevent top-down penetration in areas with low soil strength.

Functional map of the macroglomerular complex of male Helicoverpaarmigera

The mechanism of sex pheromone reception in the male cotton bollworm Helicoverpa armigera has been extensively studied because it has become an important model system for understanding insect olfaction.However,the pathways of pheromone processing from the antenna to the primary olfactory center in H.armigera have not yet been clarified.Here,the physiology and morphology of male H.armigera olfactory sensory neurons(OSNs)were studied using single sensillum recording along with anterograde filling and intracellular recording with retrograde filling.OSNs localized in type A sensilla responded to the major pheromone component cis-11-hexadecenal,and the axonal terminals projected to the cumulus(Cu)of the macroglomerular complex(MGC).The OSNs in type B sensilla respondcd to the bchavioral antagonist cis-9-tetradecenal,and the axonal terminals projected to the dorsomedial anterior(DMA)unit of the MGC.In type C sensilla,there were 2 OSNs:one that responded to cis-9-tetradecenal and cis-11-hexadecenol with the axonal terminals projecting to the DMA,and another that responded to the secondary pheromone components cis-9-hexadecenal and cis-9-tetradecenal with the axonal terminals projecting to the dorsomedial posterior(DMP)unit of the MGC.Type A and type B sensilla also housed the secondary OSNs,which were silent neurons with axonal terminals projected to the glomerulus G49 and DMP.Overall,the neural pathways that carry information on attractiveness and aversiveness in response to female pheromone components in H.armigera exhibit distinct projections to the MGC units.

Neuronal architecture and functional mapping of the taste center of larval Helicoverpa armigera(Lepidoptera:Noctuidae)

The sense of taste plays a crucial role in herbivorous insects by discriminating nutrients from complex plant metabolic compounds.The peripheral coding of taste has been thoroughly studied in many insect species,but the central gustatory pathways are poorly described.In the present study,we characterized single neurons in the gnathal ganglion of Helicoverpa armigera larvae using the intracellular recording/staining technique.We identified different types of neurons,including sensory neurons,interneurons,and motor neurons.The morphologies of these neurons were largely diverse and their arborizations seemingly covered the whole gnathal ganglion.The representation of the single neurons responding to the relevant stimuli of sweet and bitter cues showed no distinct patterns in the gnathal ganglion.We postulate that taste signals may be processed in a manner consistent with the principle of population coding in the gnathal ganglion of H.armigera larvae.

Experimental study on geosynthetic-reinforced sand fill over marine clay with or without deep cement mixed soil columns under different loadings

Geosynthetics and deep cement mixed(DCM)soil columns have been widely used to improve soft soil grounds in many countries and regions.This paper presents an experimental study on a geosynthetic-reinforced sand fill over marine clay with or without DCM columns under different loadings.Two tests were conducted on the sand fill reinforced with fixed-end and free-end geosynthetics over marine clay under three-stage local loading to investigate the effects of the boundary conditions of geosynthetic reinforcement on reducing settlements.It is observed that the fixed-end geosynthetic sheet is more effective in reducing settlements than the free-end condition under identical local loading.Another test was conducted on the fixed-end geosynthetic-reinforced sand fill over the marine clay improved by DCM columns under single-stage uniform loading.The vertical stresses on the marine clay and on the DCM columns,as well as the tensile strains of the geosynthetic sheet in the overlying sand fill,were measured.The results revealed that the stress concentration ratio increases with an increase in consolidation settlements,and the maximum tensile strain of the geosynthetic sheet occurs near the edge rather than at the center of the top surface of the DCM columns.