ABSTRACT The chromatoid body (CB) is a hallmark of haploid male germ cells, but how it selects and regulates RNAs has remained unclear. Here we define the RNA landscape of the CB by transcriptome and small RNA profiling of isolated CBs throughout mouse round spermatid differentiation. We find that the CB selectively concentrates pachytene piRNAs, retrotransposon transcripts, specific mRNA isoforms and intron-retaining mRNAs, revealing extensive RNA sorting within this cytoplasmic germline condensate. The enrichment of transposable element transcripts together with PIWI–piRNA complexes identifies the CB as a surveillance center that may safeguard genome integrity in post-meiotic haploid cells. Unexpectedly, mRNA recruitment is not determined by predicted piRNA targeting. Instead, transcript localization is encoded by a combinatorial set of intrinsic sequence and structural features that accurately predict CB enrichment at isoform resolution. These findings establish the CB as a dynamic post-transcriptional regulatory compartment that integrates genome surveillance, RNA quality control and selective transcript sorting, uncovering general principles through which biomolecular condensates shape cell-specific transcriptomes.

In brief:Temporally regulated translation is critical for late steps of spermatogenesis due to transcriptional silencing during chromatin condensation. This study shows that the function of the cytoplasmic granule, the late chromatoid body, is connected to translational regulation in condensing spermatids. Abstract:Spermatogenesis culminates in a dramatic morphological transformation, including a tight compaction of the chromatin and nuclear reshaping that largely silences transcription. Due to transcriptional silencing, the production of sperm-specific proteins needed for the morphological transformation requires active storage and translational regulation of mRNAs transcribed in earlier cell types. The germline-specific ribonucleoprotein (RNP) granule, the chromatoid body (CB), accumulates RNAs and has a role in RNA regulation in early haploid cells (round spermatids). In late haploid cells (elongating spermatids), the CB is transformed into the so-called late-CB, whose function in RNA regulation has remained elusive. Here we characterized the function of the late-CB by identifying proteins and RNAs interacting with the known late-CB marker, testis-specific serine/threonine-protein kinase 2 (TSSK2). We showed that TSSK2 and the late-CB associate with translation initiation factors and ribosomal proteins. Furthermore, we revealed an association of TSSK2 with a specific set of mRNAs that are enriched in polysome fractions in elongating spermatids, supporting the role of the late-CB in temporally regulated translation. These results link the function of the late-CB to RNA regulation during late spermatogenesis for the first time, providing important novel information about the RNA regulatory processes required for spermatogenesis and male fertility.

To explore the impact of epigenetic modifications on egg-laying traits in geese, we employed genome-wide bisulfite sequencing (WGBS) to analyze DNA methylation patterns in pituitary tissues of high-(HYP) and low-yield (LYP) Sichuan White geese. We achieved high-quality sequencing data (mean 19.09 Gb raw reads, 15.49 Gb clean reads, 79.1% unique mapping rate) with a bisulfite conversion efficiency of 99.88%. Comparative analysis revealed 2394 differentially methylated regions (DMRs) and 422 differentially methylated genes (DMGs) between HYP and LYP groups. We identified five key differentially methylated candidate genes (BMPER, INHA, NMBR, NK3R, and DSG2) linked to egg-laying traits in Sichuan White geese. Integrated GO and KEGG enrichment analysis conducted to explore the role of regulatory networks of epigenetic modification on egg-laying traits in Sichuan White geese identified multiple metabolic pathways associated with egg-laying traits (promoting egg transport, ovulation, and yolk protein synthesis and secretion), thus providing a basis for subsequent functional verification.

Ovarian tissue is critical for goose reproduction. This study aimed to investigate gene regulation by DNA methylation in relation to the reproductive traits of geese. We performed whole-genome bisulfite sequencing (WGBS) on ovarian tissues from Sichuan white geese (high-laying-rate group: HLRG, ♀ = 3; low-laying-rate group: LLRG, ♀ = 3) during the laying period. The results showed a higher level of hypermethylated differentially methylated regions (DMRs) in the HLRG, indicating a higher overall methylation level compared to the LLRG. In total, we identified 2831 DMRs and 733 differentially methylated genes (DMGs), including 363 genes with upregulated methylation. These DMGs were significantly enriched in pathways related to microtubule function (GO:0005874; GO:0000226), GnRH secretion, thyroid hormone signaling, ECM-receptor interaction, and PI3K-Akt signaling. Integration with RNA-seq data identified eight overlapping genes between DMGs and differentially expressed genes (DEGs), with five genes (CUL9, MEGF6, EML6, SYNE2, AK1BA) exhibiting a correlation between hypomethylation and high expression. EML6, in particular, emerged as a promising candidate, potentially regulating follicle growth and development in Sichuan white geese. Future studies should focus on further verifying the role of the EML6 gene. In conclusion, this study provides important insights into the regulatory mechanisms of DNA methylation influencing reproductive traits in geese, offering novel candidate markers for future goose breeding programs.

Multi-omics has helped elucidate the gene expression patterns and genomic variations closely associated with economically significant traits in geese. Despite the substantial genomic data generated through extensive goose studies, a unified platform for integrating these datasets is lacking. To address this gap, we introduced the Goose Multi-omics Database (GMD), which is accessible at http://goosedb.com/. The GMD is a comprehensive resource enabling streamlined search, analysis, and visualization of genetic information through a unified interface, providing insights into phenotypic traits, gene sequences, structures, expression profiles, genomic variations, gene families, homology, and collinearity. Equipped with robust analytical tools such as GBrowse and BLAST, the GMD facilitates rapid access to target gene information, significantly enhancing the efficiency and productivity of genomic research. By serving as a versatile and intuitive online repository, the GMD offers transformative potential for advancing goose biology, fostering multi-omics investigations, and integrating cutting-edge methodologies such as deep learning to accelerate discoveries in goose genomics.

In eukaryotes, the nucleocytoplasmic export of bulk poly(A)+-mRNAs through the nuclear pore complex is mediated by the ubiquitously expressed NXT1-NXF1 heterodimer. In humans, NXT1 has an X-chromosomal paralog, NXT2, which exhibits testis-enriched expression, suggesting a role in spermatogenesis. Here, we report the in vivo interaction of NXT2 with crucial components of the nuclear export machinery, including NXF1, the testis-specific NXF1 paralogs NXF2 and NXF3, and nuclear pore complex proteins. Binding to NXF2 and NXF3 is mediated by the NTF2-like domain of NXT2. By identifying infertile men with loss-of-function variants in NXT2 and NXF3, we link the impaired NXT2-NXF activity to disturbed germ cell development. The predominant absence of germ cells in men with NXT2 deficiency indicates its critical function already during fetal or first steps of germ cell development. In contrast, loss of NXF3 affects later stages of spermatogenesis, resulting in quantitatively and qualitatively impaired sperm production.

OBJECTIVES:This review highlights the current knowledge of polysaccharide from Lilii Bulbus, including the extraction, purification, structure, structure modification, biological activities and application, which will hopefully provide reference for further research and development of polysaccharide from Lilii Bulbus. MATERIALS AND METHODS:Literature searches were conducted on the following databases: Pubmed, ACS website, Elsevier, Google Scholar, Web of Science and CNKI database. Keywords such as "Lilii Bulbus", "polysaccharide", "preparation", "biological activities" and "application" were used to search relevant journals and contents, and some irrelevant contents were excluded. RESULTS:In general, the study of Lilium Bulbus polysaccharide extraction and purification, structure characterization and biological activity has made substantial progress, these findings highlight the lilium brownii polysaccharide enormous potential in biomedical applications, of lilium brownii polysaccharide laid a solid foundation for further research. DISCUSSION AND CONCLUSIONS:However, it should be noted that the relevant mechanism of the effective effect of lily bulb polysaccharide still needs to be worked on by researchers. These findings highlight the great potential of lily polysaccharides in biomedical applications, and lay a solid foundation for further research on lily polysaccharides.

In eukaryotes, the nucleocytoplasmic export of bulk poly(A)+-mRNAs through the nuclear pore complex is mediated by the ubiquitously expressed NXT1-NXF1 heterodimer. In humans, NXT1 has an X-chromosomal paralog, NXT2, which exhibits testis-enriched expression, suggesting a role in spermatogenesis. Here, we report the in vivo interaction of NXT2 with crucial components of the nuclear export machinery, including NXF1, the testis-specific NXF1 paralogs NXF2 and NXF3, and the nuclear pore complex proteins NUP93 and NUP214. Further, NXT2's NTF2-like domain mediates binding to NXF2 and NXF3. By identifying infertile men with loss-of-function variants in NXT2 and NXF3, we link the impaired NXT2-NXF activity to disturbed germ cell development. The predominant absence of germ cells in men with NXT2 deficiency indicates its critical function already during fetal or first steps of germ cell development. In contrast, loss of NXF3 affects later stages of spermatogenesis resulting in quantitatively and qualitatively impaired sperm production. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was carried out within the frame of the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) funded Clinical Research Unit Male Germ Cells (CRU326, project no. 329621271, grants to F.T. and N.N.). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Ethics Committees/Institutional Review Board of the University of Muenster (Ref. No. Muenster: 2012-555-f-S and 2010-578-f-S) gave ethical approval for this work. The Ethics Committee/Institutional Review Board of Nijmegen University gave ethical approval for this work (NL50495.091.14 version 5.0). The Ethics committee of Newcastle upon Tyne Hospitals NHS Foundation Trust (Newcastle, UK) gave ethical approval for this work (REC ref. 18/NE/0089). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Sequencing data of the MERGE study is available upon request to the corresponding author. Access to this data is limited for each case and specific consent of the respective samples. Sequencing data from the Nijmegen cohort have been deposited in the European Genome-phenome Archive(EGA) under the accession code EGAS00001005417 [https://ega-archive.org/studies/EGAS00001005417] and will be made available upon reasonable request for academic use and within the limitations of the provided informed consent by the corresponding author upon acceptance. Every request will be reviewed by the Newcastle University Male Infertility Genomics Data Access Committee; the researcher will need to sign a data access agreement after approval. All other data supporting the findings of this study are available from the corresponding author upon reasonable request. AlphaFold2 structure accession code for NXT2 is AF-Q9NPJ8-F1 [https://alphafold.ebi.ac.uk/entry/Q9NPJ8]. The mass spectrometry proteomics data have been deposited the ProteomeXchange Consortium via the PRIDE56 partner repository with the dataset identifier PXD052904 [https://www.ebi.ac.uk/pride/] and will be released upon acceptance of the manuscript. Genetic variants identified in NXT2 and NXF3 were submitted to ClinVar (SCV005043065-SCV005043067; [https://www.ncbi.nlm.nih.gov/clinvar/]). Source data are provided with this paper

Transmissible gastroenteritis virus (TGEV) is an important pathogen that can cause changes in the expression profile of cell miRNA. In this study, PCP could treat piglets infected with TGEV by in vivo experiment. High-throughput sequencing technology was used to detect that 9 miRNAs were up-regulated and 17 miRNAs were down-regulated during PCP inhibition of TGEV infection in PK15 cells. Meanwhile, miR-181 was related to the target genes of key proteins of apoptosis pathway. PK15 was treated with different PCP after transfection of miR-181 mimic or inhibitor. Real-time PCR was used to detect the effect of TGEV replication, electron microscopy, TEM and Hoechst fluorescence staining were used to detect the biological function of the treated cells, and western blot (WB) was used to detect the expression of key signaling factors cyt C, caspase 9 and P53 in the apoptotic signaling pathway. The results showed that compared with the control group, 250 μg/mL PCP significantly inhibited the replication of TGEV gRNA and gene N ( P < 0.01). PK15 cells in PCP groups (250 and 125 μg/mL) had uniform cell morphology and a small number of floating cells by microscopic, no typical virus structure was observed in 250 μg/mL PCP group under TEM, and apoptosis staining showed that 250 μg/mL PCP significantly reduced the number of apoptotic cells. PCP may inhibit TGEV-induced apoptosis through Caspase-dependent mitochondrial pathway after transfection of miR-181. The above results provided a theoretical basis for further research on the mechanism of PCP anti-TGEV.

InstructionPlant polysaccharides are important components of Chinese herbal medicine. Many polysaccharides, such as Astragalus polysaccharide and Lycium barbarum polysaccharide, have important functions such as tumor growth inhibition, antioxidant, anti-aging and immune regulation.MethodsLiterature searches were conducted on the following databases: Pubmed, ACS website, Elsevier, Google Scholar, Web of Science and CNKI database. Keywords such as “plant polysaccharides”, “apoptosis”, “miRNA”, “apoptosis pathway” and “regulation of apoptosis” were used to search relevant journals and contents, and some irrelevant contents were excluded. In this review, the effects of plant polysaccharides on cell apoptosis and how to regulate miRNA expression were reviewed, which provided the basis for further research on the mechanism of polysaccharide action.ResultsPlant polysaccharides regulated apoptosis probably by regulating the expression of mirnas, and the expression of certain downstream signaling factors of miRNA were changed. Some of these factors could directly act on apoptosis-related signals, activate or inhibit signaling pathways, and thus regulate cell apoptosis.Discussion and conclusionIn this review, we discussed the regulatory effects of plant polysaccharides on apoptosis and their possible molecular mechanisms. However, most of the current studies lack clinical experimental data, so it is still necessary to further study the specific mechanism of plant polysaccharides affecting cell miRNA expression and apoptosis, so that plant polysaccharides can be more widely used in clinical and practical aspects.

Transmissible gastroenteritis virus (TGEV) could cause diarrhea, vomiting, dehydration and even death in piglets, miRNA played an important role in the interaction between virus and cell. The study aimed to investigate the impact of miR-17 on the polysaccharide of Polygonum Cillinerve (PCP) in combating TGEV. miR-17 was screened and transfection validation was performed by Real-time PCR. The function of miR-17 on PK15 cells infected with TGEV and treated with PCP was investigated by DCFH-DA loading probe, JC-1 staining and Hoechst fluorescence staining. Furthermore, the effect of miR-17 on PCP inhibiting TGEV replication and apoptosis signaling pathways during PCP against TGEV infection was measured through Real-time PCR and Western blot. The results showed that miR-17 mimic and inhibitor could be transferred into PK15 cells and the expression of miR-17 significantly increased and decreased respectively compared with miR-17 mimic and inhibitor (P < 0.05). A total 250 μg/mL of PCP could inhibit cells apoptosis after transfection with miR-17. PCP (250 μg/mL and 125 μg/mL) significantly inhibited the decrease in mitochondrial membrane potential induced by TGEV after transfection with miR-17 (P < 0.05). After transfection of miR-17 mimic, PCP at concentrations of 250 μg/mL and 125 μg/mL significantly promoted the mRNA expression of P53, cyt C and caspase 9 (P < 0.05). Compared with the control group, the replication of TGEV gRNA and gene N was significantly inhibited by PCP at concentrations of 250 μg/mL and 125 μg/mL after transfection of both miR-17 mimic and inhibitor (P < 0.05). PCP at 62.5 μg/mL significantly inhibited the replication of gene S following transfection with miR-17 inhibitor (P < 0.05). These results suggested that PCP could inhibit the replication of TGEV and apoptosis induced by TGEV by regulating miR-17.

The growth of skeletal muscle involves complex developmental processes that play an important part in the determinization of pork quality. The investigation of skeletal muscle mRNA or miRNA profiles is especially important for finding molecular approaches to improve meat quality in pig breeding. Therefore, we studied the transcriptome (mRNA and miRNA) profiles of skeletal muscle with RNA-Seq in three developmental stages of pigs: 65-day embryonic (E65), postnatal 0 days (natal) and 10 months (adult). We found 10,035, 9050 and 4841 differentially expressed (DE) genes for natal vs. E65, adult vs. E65 and adult vs. natal, 55, 101 and 85 DE miRNA for natal vs. E65, adult vs. E65 and adult vs. natal, respectively. In addition, the target genes of DE miRNA that was in a negative correlation with the corresponding miRNA in the same comparison group were selected for enrichment analysis. Gene Ontology terms were mainly classified into developmental processes. Pathway analysis revealed enrichment in the Rap1 signaling pathway, citrate cycle and oxidative phosphorylation and carbon. Finally, RT-PCR was employed for validating the level of expression of 11 DE miRNA and 14 DEGs. The transcriptome profiles of skeletal muscle from the different developmental stages of the Bamei pigs were obtained. From these data, hundreds of DE miRNA and mRNA, and the miRNA–mRNA regulatory network can provide valuable insights into further understanding of key molecular mechanisms and improving the meat quality in pig breeding.

The adult Leydig cells (ALCs), originated from stem Leydig cells (SLCs), can secrete testosterone which is essential for germ cell development and sexual behavior maintenance. As a synthetic compound, ethane dimethane sulfonate (EDS), a well-known alkylating agent, has been reported to specifically ablate ALCs. In this study, EDS was verified to ablate differentiated pig LCs by experiments. Subsequently, the primary isolated pig LCs (containing SLCs and differentiated LCs) and EDS-treated LCs (almost exclusively SLCs) were collected for RNA-seq 4,904 genes and 15 miRNAs were differently expressed between the two groups. Down-regulated genes in the EDS-treated group were mainly related to steroid hormone biosynthesis. The highest up-regulation miRNAs was miR-205 after EDS treatment. Additionally, miR-205 was expressed more highly in pig SLCs clones compared with differentiated LCs. Through qRT-PCR, western blot (WB), TUNEL, EDU and flow cytometry, miR-205 was found to induce cell apoptosis, but did not affect proliferation or differentiation in both TM3 and GC-1spg mouse cell lines. Through luciferase reporter assays and WB, RAP2B was identified as a target gene of miR-205. Besides, overexpression of miR-205 inhibited the expressions of PI3K, Akt and p-AKT. All these findings were helpful for elucidating the regulation mechanism in pig LCs.

In this study, the adjuvant activity of ophiopogon polysaccharide liposome (OPL) was investigated. The effects of OPL on the splenic lymphocyte proliferation of mice were measured in vitro. The results showed that OPL could significantly promote lymphocyte proliferation singly or synergistically with PHA and LPS and that the effect was better than ophiopogon polysaccharide (OP) at most of concentrations. The adjuvant activities of OPL, OP and mineral oil were compared in BALB/c mice inoculated with inactivated PPV in vivo. The results showed that OPL could significantly enhance lymphocyte proliferation, increase the proportion of CD4(+) and CD8(+) T cells, improve the HI antibody titre and specific IgG response, and promote the production of cytokines, and the efficacy of OPL was significantly better than that of OP. In addition, OPL significantly improved the cellular immune response compared with oil adjuvant. These results suggested that OPL possess superior adjuvanticity and that a medium dose had the best efficacy. Therefore, OPL can be used as an effective immune adjuvant for an inactivated PPV vaccine. (C) 2015 Published by Elsevier B.V.

Objectives. The adjuvant activity of Epimedium polysaccharide-propolis flavone liposome (EPL) was investigated in vitro and in vivo. Methods. In vitro, the effects of EPL at different concentrations on splenic lymphocytes proliferation and mRNA expression of IFN- γ and IL-6 were determined. In vivo, the adjuvant activities of EPL, EP, and mineral oil were compared in BALB/c mice through vaccination with inactivated porcine circovirus type 2 (PCV2) vaccine. Results. In vitro, EPL promoted lymphocytes proliferation and increased the mRNA expression of IFN- γ and IL-6, and the effect was significantly better than EP at all concentrations. In vivo, EPL significantly promoted the lymphocytes proliferation and the secretion of cytokines and improved the killing activity of NK cells, PCV2-specific antibody titers, and the proportion of T-cell subgroups. The effects of EPL were significantly better than EP and oil adjuvant at most time points. Conclusion. EPL could significantly improve both PCV2-specific cellular and humoral immune responses, and its medium dose had the best efficacy. Therefore, EPL would be exploited in an effective immune adjuvant for inactivated PCV2 vaccine.