CRISPR Screen Identifies the RNA-Binding Protein Eef1a1 as a Key Regulator of Myogenesis.

Skeletal muscle myogenesis hinges on gene regulation, meticulously orchestrated by molecular mechanisms. While the roles of transcription factors and non-coding RNAs in myogenesis are widely known, the contribution of RNA-binding proteins (RBPs) has remained unclear until now. Therefore, to investigate the functions of post-transcriptional regulators in myogenesis and uncover new functional RBPs regulating myogenesis, we employed CRISPR high-throughput RBP-KO (RBP-wide knockout) library screening. Through this approach, we successfully identified Eef1a1 as a novel regulatory factor in myogenesis. Using CRISPR knockout (CRISPRko) and CRISPR interference (CRISPRi) technologies, we successfully established cellular models for both CRISPRko and CRISPRi. Our findings demonstrated that Eef1a1 plays a crucial role in promoting proliferation in C2C12 myoblasts. Through siRNA inhibition and overexpression methods, we further elucidated the involvement of Eef1a1 in promoting proliferation and suppressing differentiation processes. RIP (RNA immunoprecipitation), miRNA pull-down, and Dual-luciferase reporter assays confirmed that miR-133a-3p targets Eef1a1. Co-transfection experiments indicated that miR-133a-3p can rescue the effect of Eef1a1 on C2C12 myoblasts. In summary, our study utilized CRISPR library high-throughput screening to unveil a novel RBP, Eef1a1, involved in regulating myogenesis. Eef1a1 promotes the proliferation of myoblasts while inhibiting the differentiation process. Additionally, it acts as an antagonist to miR-133a-3p, thus modulating the process of myogenesis.

Exploring Multi-Tissue Alternative Splicing and Skeletal Muscle Metabolism Regulation in Obese- and Lean-Type Pigs.

Alternative splicing (AS) is a crucial mechanism in post-transcriptional regulation, contributing significantly to the diversity of the transcriptome and proteome. In this study, we performed a comprehensive AS profile in nine tissues obtained from Duroc (lean-type) and Luchuan (obese-type) pigs. Notably, 94,990 AS events from 14,393 genes were identified. Among these AS events, it was observed that 80% belonged to the skipped exon (SE) type. Functional enrichment analysis showed that genes with more than ten AS events were closely associated with tissue-specific functions. Additionally, the analysis of overlap between differentially alternative splicing genes (DSGs) and differentially expressed genes (DEGs) revealed the highest number of overlapped genes in the heart and skeletal muscle. The novelty of our study is that it identified and validated three genes (PYGM, MAPK11 and CAMK2B) in the glucagon signaling pathway, and their alternative splicing differences were highly significant across two pig breeds. In conclusion, our study offers novel insights into the molecular regulation of diverse tissue physiologies and the phenotypic differences between obese- and lean-type pigs, which are helpful for pig breeding.

Mogroside V alleviates the heat stress-induced disruption of the porcine oocyte in vitro maturation.

Heat stress (HS) is a stressor that negatively affect female reproduction. Specially, oocytes are very sensitive to HS. It has been demonstrated that some active compounds can protect oocyte from HS. We previously found that Mogroside V (MV), extracted from Siraitia grosvenorii (Luo Han Guo), can protect oocyte from many kinds of stresses. However, how MV alleviates HS-induced disruption of oocyte maturation remains unknown. In this study, we treated the HS-induced porcine oocytes with MV to examine their maturation and quality. Our findings demonstrate that MV can effectively alleviate HS-induced porcine oocyte abnormal cumulus cell expansion, decrease of first polar body extrusion rate, spindle assembly and chromosome separation abnormalities, indicating MV attenuates oocyte mature defects. We further observed that MV can effectively alleviate HS-induced cortical granule distribution abnormality and decrease of blastocyst formation rate after parthenogenesis activation. In addition, MV treatment reversed mitochondrial dysfunction and lipid droplet content decrease, reduced reactive oxygen species levels, early apoptosis and DNA damage in porcine oocytes after HS. Collectively, this study suggests that MV can effectively protect porcine oocytes from HS.

Transcriptomic Analysis of mRNA Expression Profiles in the Microglia of Mouse Brains Infected with Rabies Viruses of Varying Virulence.

Rabies is a lethal encephalitis caused by the rabies virus (RABV) with a fatality rate near 100% after the onset of clinical symptoms in humans and animals. Microglia are resident immune cells in the central nervous system. Few studies have been conducted on the functional role of microglia in RABV infection. Here, we performed a transcriptomic analysis of mRNA expression profiles in the microglia of mouse brains intracerebrally infected with RABV. We successfully isolated single microglial cells from the mouse brains. The survival rate of dissociated microglial cells was 81.91%-96.7%, and the purity was 88.3%. Transcriptomic analysis revealed 22,079 differentially expressed mRNAs identified in the microglia of mouse brains infected with RABV strains (rRC-HL, GX074, and CVS-24) of varying virulence at 4 and 7 days post-infection (dpi) compared to the control group. The numbers of DEGs versus the control at 4 and 7 dpi in mice infected with rRC-HL, GX074, and CVS-24 were 3622 and 4590, 265 and 4901, and 4079 and 6337. The GO enrichment analysis showed that response to stress, response to external stimulus, regulation of response to stimulus, and immune system process were abundant during RABV infection. The KEGG analysis indicated that the Tlr, Tnf, RIG-I, NOD, NF-κB, MAPK, and Jak-STAT signaling pathways were involved in RABV infection at both 4 and 7 dpi. However, some phagocytosis and cell signal transduction processes, such as endocytosis, p53, phospholipase D, and oxidative phosphorylation signaling pathways, were only expressed at 7 dpi. The involvement of the Tnf and Tlr signaling pathways prompted us to construct a protein-protein interaction (PPI) network of these pathways. The PPI revealed 8 DEGs, including Mmp9, Jun, Pik3r1, and Mapk12. Notably, Il-1b interacted with Tnf and Il-6 with combined scores of 0.973 and 0.981, respectively. RABV causes significant changes in mRNA expression profiles in the microglia in mice. 22,079 differentially expressed mRNAs were identified in the microglia of mice infected with RABV strains of varying virulence at 4 and 7 dpi. The DEGs were evaluated using GO, KEGG, and PPI network analysis. Many immune pathways were up-regulated in RABV-infected groups. The findings will help elucidate the microglial molecular mechanisms of cellular metabolism dysregulated by RABV and may provide important information for investigating RABV pathogenesis and therapeutic methods.

Integrative metabolomic and transcriptomic analysis reveals difference in glucose and lipid metabolism in the longissimus muscle of Luchuan and Duroc pigs.

Luchuan pig, an obese indigenous Chinese porcine breed, has a desirable meat quality and reproductive capacity. Duroc, a traditional western breed, shows a faster growth rate, high feed efficiency and high lean meat rate. Given the unique features these two porcine breeds have, it is of interest to investigate the underlying molecular mechanisms behind their distinctive nature. In this study, the metabolic and transcriptomic profiles of longissimus dorsi muscle from Duroc and Luchuan pigs were compared. A total of 609 metabolites were identified, 77 of which were significantly decreased in Luchuan compared to Duroc, and 71 of which were significantly elevated. Most differentially accumulated metabolites (DAMs) upregulated in Luchuan were glycerophospholipids, fatty acids, oxidized lipids, alcohols, and amines, while metabolites downregulated in Luchuan were mostly amino acids, organic acids and nucleic acids, bile acids and hormones. From our RNA-sequencing (RNA-seq) data we identified a total of 3638 differentially expressed genes (DEGs), 1802 upregulated and 1836 downregulated in Luchuan skeletal muscle compared to Duroc. Combined multivariate and pathway enrichment analyses of metabolome and transcriptome results revealed that many of the DEGs and DAMs are associated with critical energy metabolic pathways, especially those related to glucose and lipid metabolism. We examined the expression of important DEGs in two pathways, AMP-activated protein kinase (AMPK) signaling pathway and fructose and mannose metabolism, using Real-Time Quantitative Reverse Transcription PCR (qRT-PCR). Genes related to glucose uptake, glycolysis, glycogen synthesis, fatty acid synthesis (PFKFB1, PFKFB4, MPI, TPI1, GYS1, SLC2A4, FASN, IRS1, ULK1) are more activated in Luchuan, while genes related to fatty acid oxidation, cholesterol synthesis (CPT1A, HMGCR, FOXO3) are more suppressed. Energy utilization can be a decisive factor to the distinctive metabolic, physiological and nutritional characteristics in skeletal muscle of the two breeds we studied. Our research may facilitate future porcine breeding projects and can be used to reveal the potential molecular basis of differences in complex traits between various breeds.

Dietary supplementation with jasmine flower residue improves meat quality and flavor of goat.

Jasmine flower residue (JFR) is a by-product retained in the production process of jasmine tea and can be used as an unconventional feed due to its rich nutrient value. This study aimed to evaluate the effects of the addition of JFR to the diet of goats on their meat quality and flavor. Twenty-four castrated Nubian male goats were randomly divided into two groups and fed a mixed diet containing 10% JFR (JFR, n = 12) or a conventional diet (CON, n = 12) for 45 days. Meat quality and flavor were measured at the end of the treatment. The addition of JFR to the diet could reduce the shear force of the longissimus dorsi muscle, as well as, the cross-sectional area and diameter of muscle fibers, indicating that the addition of JFR improved meat quality. JFR also increased the content of glutamic acid and ω-3 polyunsaturated fatty acid (C18:3n3 and C20:5N3) and reduced the content of C24:1 and saturated fatty acid (C20:0 and C22:0). In addition, the use of JFR increased the content of acetaldehyde and hexanal in the meat. Furthermore, JFR introduced new volatile components in the meat. The umami, saltiness, and richness of the meat also improved. In conclusion, the addition of jasmine flower residue to the diet can improve the meat quality and flavor of goat.

Mogroside-rich extract from Siraitia grosvenorii fruits protects against heat stress-induced intestinal damage by ameliorating oxidative stress and inflammation in mice.

Global warming makes humans and animals more vulnerable to heat stress. Heat stress can cause multiorgan dysfunction, especially in the intestine, primarily via oxidative stress and inflammation. Mogroside-rich extract (MGE) is the active ingredient of Siraitia grosvenorii and has significant antioxidant and anti-inflammatory activity. However, whether MGE can alleviate the intestinal damage caused by heat stress has not been explored. In this study, mice were given 600 mg kg-1 MGE followed by exposure to high temperature (40 °C for 2 h per day), and the structures and molecular changes in the ileum were examined. Our findings showed that body weight was decreased by heat stress, while the activity of serum superoxide dismutase (SOD) was increased. We further found that heat stress impaired the intestinal barrier by reducing the number of goblet cells and mRNA levels of the tight junction proteins zona occludens protein 1 (ZO-1), Occludin (OCLD) and recombinant mucin 2 (MUC2 mucin), but it increased the mRNA level of trefoil factor 3 (TFF3). Interestingly, MGE treatment reversed these changes. Furthermore, heat stress increased the activity of SOD in the intestine, downregulated the expression of the oxidative stress-related genes glutathione peroxidase 1 (GPX1), SOD2 and nuclear factor erythroid 2-related factor 2 (NRF2), and upregulated the expression of catalase (CAT). Moreover, heat stress increased tumor necrosis factor-α (TNF-α) levels in the intestine and upregulated the expression of the inflammation-related genes interleukin 10 (IL-10), TNF-α, Interferon-γ (IFN-γ), toll like receptor 4 (TLR4) and nuclear factor-kappa B (NF-kB). However, MGE treatment effectively reduced TNF-α levels and restored the normal activity of SOD and normal mRNA levels for both oxidative stress-related and inflammation-related genes. In summary, our results showed that MGE can protect against heat stress-induced intestinal damage by ameliorating inflammation and oxidative stress.

Resveratrol ameliorates the defects of meiotic maturation in lipopolysaccharide exposed porcine oocytes.

Lipopolysaccharide (LPS), a significant virulence factor of gram-negative bacteria, adversely affects female reproduction, especially the maturation and early embryonic development of oocytes, through inducing of inflammatory and oxidative stress-associated toxic responses. Resveratrol (Res), a potent antioxidant, exhibits many beneficial effects on the maturation and developmental competence of oocytes. However, it is unclear whether Res can restore LPS-induced defects in the maturation of oocytes during meiosis. In this study, we used porcine oocytes to explore the protective effects of Res and its underlying mechanism against the toxic impacts of LPS exposure on meiotic maturation and developmental competence of oocytes during meiosis. The oocytes were randomly assigned to a control, LPS-exposed or Res-supplemented group. Nuclear and cytoplasmic maturation was assessed after 26 h (MI) or 44 h (MII) of in vitro maturation (IVM). Our results showed that 10 µM Res significantly improved the rates of oocyte maturation and blastocyst formation after exposure to 15 µg/mL LPS. In addition, Res preserved the normal spindle/chromosome structure and maintained acetylated tubulin levels, actin polymerization and cortical granules (CGs) distribution. Additionally, Res protected mitochondrial content and function, scavenges reactive oxygen species (ROS), and reduced DNA damage and apoptosis in LPS-exposed oocytes. Furthermore, inhibition of SIRT1 by its specific inhibitor EX527 suppressed the recovery of ROS levels, mitochondrial content, and spindle/chromosome structure by Res supplementation. In summary, this study shows that Res can alleviate the impacts of LPS-induced toxicity on meiosis in porcine oocytes by upregulating SIRT1, which ameliorates oxidative stress and increasing mitochondrial content.

Mogroside-Rich Extract From Siraitia grosvenorii Fruits Ameliorates High-Fat Diet-Induced Obesity Associated With the Modulation of Gut Microbiota in Mice.

Siraitia grosvenorii is a kind of medicinal food plant. The mogroside-rich extract (MGE) of its fruits can effectively ameliorate obesity, but the underlying mechanisms remain underexplored. In this study, we aimed to determine whether MGE can ameliorate obesity by protecting against the divergences of gut microbiota. Mice were challenged with a high-fat diet (HFD) and treated with MGE by oral gavage. Then, the characteristics of the gut microbiota were determined by 16S rDNA analysis. Our findings showed that MGE could significantly reduce body weight gain and fat tissue weight of the mice fed with HFD. Moreover, MGE markedly attenuated fatty liver, and improved glucose tolerance and insulin sensitivity. We further found that the gut microbiota structures were disturbed by HFD feeding. In particular, the abundance of Firmicutes was increased and the abundance of Bacteroidetes was decreased, resulting in an increased proportion of Firmicutes to Bacteroidetes (F/B), which contributes to obesity. Interestingly, the abnormal proportion of F/B of HFD feeding mice was restored to the level of control mice by MGE treatment. Additionally, the abundances of obesogenic microbiota, such as Ruminiclostridium and Oscillibacter were also decreased after MGE treatment. In summary, our findings demonstrate that MGE can modulate gut microbiota in obese mice and shed new light on how it alleviates obesity.

2-Mercaptoethanol promotes porcine oocyte maturation in vitro by maintaining autophagy homeostasis.

2-Mercaptoethanol (2-ME) is often used as an antioxidant to optimize culture systems for in vitro oocyte maturation in livestock. However, the relationship between 2-ME and autophagy has not yet been elucidated. In this study, we hypothesized that 2-ME can promote porcine oocyte maturation in vitro by maintaining autophagy homeostasis. To test this hypothesis, we explored the effects of 2-ME on the maturation of porcine oocytes exposed to an autophagy activator (rapamycin) or an autophagy inhibitor (3-methyladenine, i.e., 3-MA) in vitro. Rapamycin-induced autophagy over-activation significantly increased autophagy- and apoptosis-related gene expression, oxidative stress, apoptosis rates, abnormal mitochondrial redistribution, and significantly decreased oocyte first polar body extrusion (PBE) rates, spindle/chromosome integrity and developmental competence. 3-MA-mediated autophagy inhibition exerted similar effects on all these parameters except the expression of genes that promote autophagy and inhibit apoptosis. Importantly, 2-ME supplementation significantly attenuated the detrimental effects of rapamycin and 3-MA. Interestingly, we observed that 44 h of coincubation with rapamycin/3-MA and 2-ME restored autophagy homeostasis in vitro. In conclusion, our study confirmed that 2-ME promotes porcine oocyte maturation and embryo development in vitro by maintaining autophagy homeostasis and lays a foundation for further research on the underlying mechanism.

Allicin protects porcine oocytes against LPS-induced defects during maturation in vitro.

Lipopolysaccharide (LPS), the main virulence factor of gram-negative bacteria, severely impairs the function of the female reproductive system. It has especially harmful effects on oocytes and subsequent embryonic development. The use of active plant substances to ameliorate the damage caused by LPS exposure is a strategy worthy of attention. In this study, porcine oocytes were used to investigate the protective effects and underlying mechanisms of allicin, an extract derived from garlic, on LPS-exposed oocytes in vitro. Our data indicated that supplementation with 1 µM allicin significantly attenuated the LPS-mediated reductions in the first polar body extrusion rate and the subsequent blastocyst formation rate. Allicin also mitigated the abnormalities in spindle assembly, chromosome alignment, actin polymerization, and cortical granule distribution caused by LPS exposure. Furthermore, allicin restored reactive oxygen species (ROS), early apoptosis and autophagy to normal physiological levels in LPS-exposed oocytes. In conclusion, our findings confirm that allicin can protect oocytes against LPS-induced damage. The results of this study will help promote the application of plant-derived bioactive substances to ameliorate oocyte maturation defects.

Insufficient pyruvate in culture medium arrests mouse embryos at the first cleavage stage associated with abnormal epigenetic modifications.

Energy is essential for early embryogenesis, and fertilized eggs can successfully develop to blastocyst in in vitro culture medium with an appropriate energy supply. Conversely, embryonic development is negatively affected by a suboptimal energy supply. We previously observed that a low level of pyruvate greatly arrests mouse embryos at the 2-cell stage. However, how methylation modifications are affected at this specific stage remains unknown. In this study, we found that mouse embryos could timely develop to the 4-cell stage in K+simplex optimized medium (KSOM) with control level of pyruvate, but embryos were significantly arrested at the 2-cell stage when pyruvate was reduced to 0.2-fold of the control level. Moreover, the fluorescence intensities of 5 mC, H3K4me2, H3K9me2 and H3K27me2 in the 2-cell stage embryos of the 0.2-fold pyruvate group were notedly lower than those of the control group, but N6-methyladenosine (m6A) fluorescence intensity was higher, suggesting that global genomic DNA, histone and m6A methylation modifications are disrupted with low levels of pyruvate. Consistently, the mRNA levels of genes related to DNA methylation, histone methylation and m6A modifications were also disturbed in the 2-cell stage embryos cultured with low levels of pyruvate. In summary, our findings demonstrate that insufficient pyruvate in culture medium results in mouse embryonic developmental arrest, at least in part due to defects in methylation modifications.

Mogroside-rich extract from Siraitia grosvenorii fruits protects against the depletion of ovarian reserves in aging mice by ameliorating inflammatory stress.

Mogroside-rich extract (MGE), the main bioactive component of dried Siraitia grosvenorii fruit, has long been used as a natural sweetener and traditional Chinese medicine. This extract possesses various types of pharmacological activities, such as anti-inflammatory, antioxidative, hypoglycemic and hypolipemic activities. Moreover, we recently revealed that MGE has beneficial effects on female reproduction. Increasing maternal age leads to a rapid reduction in female fertility; in particular, it dramatically decreases ovarian function. Nevertheless, whether MGE can alleviate ovarian aging and the underlying mechanisms have not yet been explored. In this study, mice were treated with MGE by supplementation in drinking water from 10 to 44 weeks of age. Then, ovarian function and molecular changes were determined. Our findings showed that MGE treatment protected aged mice from estrous cycle disorder. Moreover, MGE treatment significantly increased the ovarian reserves of aged mice. RNA-seq data showed that MGE upregulated the expression of genes related to gonad development, follicular development, and hormone secretion in ovarian tissue. Additionally, inflammatory stress was induced, as indicated by upregulation of inflammation-related gene expression and elevated TNF-α levels in the ovarian tissues of aged mice; however, MGE treatment attenuated inflammatory stress. In summary, our findings demonstrate that MGE can ameliorate age-related estrous cycle disorder and ovarian reserve decline in mice, possibly by alleviating ovarian inflammatory stress.

Mogroside V Alleviates Oocyte Meiotic Defects and Quality Deterioration in Benzo(a)pyrene-Exposed Mice.

Accumulating evidence has demonstrated that benzo(a)pyrene (BaP) exposure adversely affects female reproduction, especially oocyte meiotic maturation and subsequent embryo development. Although we previously found that mogroside V (MV), a major bioactive component of S. grosvenorii, can protect oocytes from quality deterioration caused by certain stresses, whether MV can alleviate BaP exposure-mediated oocyte meiotic defects remains unknown. In this study, female mice were exposed to BaP and treated concomitantly with MV by gavage. We found that BaP exposure reduced the oocyte maturation rate and blastocyst formation rate, which was associated with increased abnormalities in spindle formation and chromosome alignment, reduced acetylated tubulin levels, damaged actin polymerization and reduced Juno levels, indicating that BaP exposure results in oocyte nucleic and cytoplasmic damage. Interestingly, MV treatment significantly alleviated all the BaP exposure-mediated defects mentioned above, indicating that MV can protect oocytes from BaP exposure-mediated nucleic and cytoplasmic damage. Additionally, BaP exposure increased intracellular ROS levels, meanwhile induced DNA damage and early apoptosis in oocytes, but MV treatment ameliorated these defective parameters, therefore it is possible that MV restored BaP-mediated oocyte defects by reducing oxidative stress. In summary, our findings demonstrate that MV might alleviate oocyte meiotic defects and quality deterioration in BaP-exposed mice.

Mogroside V Protects Porcine Oocytes From Lipopolysaccharide-Induced Meiotic Defects.

Accumulating evidence has demonstrated that lipopolysaccharide (LPS) compromises female reproduction, especially oocyte maturation and competence. However, methods to protect oocyte quality from LPS-induced deterioration remain largely unexplored. We previously found that mogroside V (MV) can promote oocyte maturation and embryonic development. However, whether MV can alleviate the adverse effects of LPS exposure on oocyte maturation is unclear. Thus, in this study, we used porcine oocytes as a model to explore the effects of MV administration on LPS-induced oocyte meiotic defects. Our findings show that supplementation with MV protected oocytes from the LPS-mediated reduction in the meiotic maturation rate and the subsequent blastocyst formation rate. In addition, MV alleviated the abnormalities in spindle formation and chromosome alignment, decrease in α-tubulin acetylation levels, the disruption of actin polymerization, and the reductions in mitochondrial contents and lipid droplet contents caused by LPS exposure. Meanwhile, LPS reduced m6A levels in oocytes, but MV restored these epigenetic modifications. Furthermore, MV reduced reactive oxygen species (ROS) levels and early apoptosis in oocytes exposed to LPS. In summary, our study demonstrates that MV can protect oocytes from LPS-induced meiotic defects in part by reducing oxidative stress and maintaining m6A levels.

Low-level pyruvate inhibits early embryonic development and maternal mRNA clearance in mice.

Energy homeostasis and accomplishment of maternal-to-zygotic transition (MZT), which involves the timed processes of maternal mRNA clearance and zygotic genome activation (ZGA), are essential for mammalian embryogenesis. However, how energy substrates regulate maternal mRNA clearance and the underlying mechanisms have not yet been fully elucidated. Here, we found that mouse embryos were arrested at the 2-cell stage when the pyruvate level was reduced to one-fifth of the control level. Moreover, we observed that the mitochondrial contents and ROS levels were reduced. Interestingly, some maternal mRNA, including transcripts involved in the maternal factor-mediated mRNA decay (M-decay) pathway, was vastly degraded from 1-cell to 2-/4-cell embryos when cultured with control pyruvate levels, but the clearance of these transcripts was hindered when the pyruvate level was reduced. In contrast, some transcripts involved in the zygotic factor-mediated mRNA decay (Z-decay) pathway were vastly downregulated by the reduction in pyruvate. This effect was possibly due to a reduction in global transcription, as the embryos cultured with low-level pyruvate had lower transcription activity than embryos cultured with control pyruvate level. In summary, our findings demonstrate that low-level pyruvate inhibits maternal mRNA clearance, possibly by disrupting the M- and Z-decay pathways, extending our current understanding of the energy requirements of embryogenesis.

Transcriptome analysis revealed differences in the microenvironment of spermatogonial stem cells in seminiferous tubules between pre-pubertal and adult buffaloes.

The microenvironment in the seminiferous tubules of buffalo changes with age, which affects the self-renewal and growth of spermatogonial stem cells (SSCs) and the process of spermatogenesis, but the mechanism remains to be elucidated. RNA-seq was performed to compare the transcript profiles of pre-pubertal buffalo (PUB) and adult buffalo (ADU) seminiferous tubules. In total, 17,299 genes from PUB and ADU seminiferous tubules identified through RNA-seq, among which 12,271 were expressed in PUB and ADU seminiferous tubules, 4,027 were expressed in only ADU seminiferous tubules, and 956 were expressed in only PUB seminiferous tubules. Of the 17,299 genes, we identified 13,714 genes that had significant differences in expression levels between PUB and ADU through GO enrichment analysis. Among these genes, 5,342 were significantly upregulated and possibly related to the formation or identity of the surface antigen on SSCs during self-renewal; 7,832 genes were significantly downregulated, indicating that genes in PUB seminiferous tubules do not participate in the biological processes of sperm differentiation or formation in this phase compared with those in ADU seminiferous tubules. Subsequently, through the combination with KEGG analysis, we detected enrichment in a number of genes related to the development of spermatogonial stem cells, providing a reference for study of the development mechanism of buffalo spermatogonial stem cells in the future. In conclusion, our data provide detailed information on the mRNA transcriptomes in PUB and ADU seminiferous tubules, revealing the crucial factors involved in maintaining the microenvironment and providing a reference for further in vitro cultivation of SSCs.

Constant light exposure causes oocyte meiotic defects and quality deterioration in mice.

Artificial light at night (ALAN) exposes us to prolonged illumination, that adversely affects female reproduction. However, it remains to be clarified how prolonged light exposure affects oocyte meiotic maturation and quality. To this end, we exposed female mice to a constant light (CL) of 250 lux for different durations. Our findings showed that CL exposure for 7 weeks reduced the oocyte maturation rate. Meanwhile, CL exposure caused greater abnormalities in spindle assembly and chromosome alignment and a higher rate of oocyte aneuploidy than the regular light dark cycle. CL exposure also induced oxidative stress and caused mitochondrial dysfunction, which resulted in oocyte apoptosis and autophagy. Notably, our results showed that CL exposure reduced the levels of α-tubulin acetylation, DNA methylation at 5 mC, RNA methylation at m6A and histone methylation at H3K4me2 but increased the levels of histone methylation at H3K27me2 in oocytes. In summary, our findings demonstrate that constant bright light exposure causes oocyte meiotic defects and reduces cytoplasmic quality. These results extend the current understanding of ALAN-mediated defects in female reproduction.

Mogroside V improves porcine oocyte in vitro maturation and subsequent embryonic development.

Oocyte in vitro maturation (IVM) plays a pivotal role in in vitro embryo production. However, the efficiency of IVM is still low and needs to be further improved. In the present study, we evaluated the beneficial effects of mogroside V, an extract derived from Siraitia grosvenorii, on oocyte IVM. Porcine cumulus-oocyte complexes were cultured in IVM medium supplemented or not supplemented with mogroside V for 40 h. We found that mogroside V supplementation increased the percentage of oocyte first polar body extrusion and improved subsequent blastocyst formation after parthenogenetic activation. Furthermore, mogroside V reduced the levels of reactive oxygen species (ROS) and increased the mRNA expression of oxidative stress-related genes (SOD, CAT and SIRT1). Moreover, mogroside V supplementation enhanced the mitochondrial content, mtDNA copy number, mitochondrial membrane potential (ΔΨm), ATP generation, and the relative mRNA expression of mitochondria-related genes (PGC-1α and TFAM). In summary, our findings demonstrate that mogroside V supplementation reduces intracellular ROS levels and enhances mitochondrial function to promote porcine oocyte IVM.