Guoquan Wu

Background: Cumulus cells (CCs) are well known to play a regulatory role in driving meiotic resumption and orchestrating the maturation of porcine oocytes. However, their role in oocyte development under vitrification stress remains unclear. Results: In this study, whole-transcriptome sequencing was used to compare the expression differences of circular RNAs (circRNAs), long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and messenger RNAs (mRNAs) in CCs derived from fresh and vitrified porcine cumulus-oocyte complexes (COCs) following in vitro maturation. The results revealed 2,603 differentially expressed (DE) mRNAs (DEmRNAs), 27 DEmiRNAs, 511 DElncRNAs, and 89 DEcircRNAs. Gene ontology analysis of these RNAs or their target genes revealed enrichment in numerous processes, including cytoskeleton organization, cell cycle, phosphorylation, apoptosis, and stress granule assembly. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed their involvement in pathways, including mitogen-activated protein kinase (MAPK) signaling, cell senescence, oocyte meiosis, actin cytoskeleton regulation, ErbB signaling, and cell cycle. Additionally, a putative ceRNA network was constructed involving 27 lncRNAs, 8 circRNAs, 5 miRNAs, and 49 mRNAs, and miR-9843-3p, miR-425-5p, miR-140-5p, miR-451, and miR-664-5p were identified as potential candidate regulators within this network. Four mRNAs, four miRNAs, four lncRNAs and four circRNAs were screened from the constructed predicted ceRNA network and subjected to qRT-PCR validation. Their expression trends showed high consistency with RNA-seq results, supporting the expression patterns identified by transcriptome sequencing and bioinformatic analyses. Further enrichment analysis of mRNAs involved in the constructed predicted ceRNA network revealed their predominant enrichment in important cellular pathways, including Wnt signaling, Hippo signaling, ErbB signaling, MAPK signaling, and mTOR signaling. Conclusion: These findings indicate the impact of vitrification on transcriptional regulation in CCs, which may provide candidate molecular clues for optimizing vitrification technology for porcine COCs.

Vitrification is an efficient approach to preserve porcine germinal vesicle (GV)-stage oocytes, yet cryo-induced damage remains a major limitation to subsequent embryonic development. Although previous studies have investigated transcriptomic and proteomic changes in oocytes and cumulus cells (CCs) following vitrification, the metabolic alterations underlying cryoinjury are poorly understood. In this study, metabolomic profiling of vitrified oocytes and CCs following in vitro maturation (IVM) was carried out identify key differential metabolites. Functional validation was further conducted by supplementing candidate metabolites during oocyte maturation. In vitrified oocytes, 19 metabolites were significantly upregulated, and 44 were significantly downregulated. Integrated metabolomic and transcriptomic profiling demonstrated that significantly altered metabolites and genes converged on biological pathways governing energy metabolism, glutathione metabolism, and glycerophospholipid metabolism. In CCs derived from vitrified oocytes, 70 metabolites exhibited marked upregulation, while 21 showed significantly downregulation. Integrated analysis profiling demonstrated that significantly altered metabolites and genes were converged on pathways such as glutathione metabolism and glycerophospholipid metabolism. Notably, oocytes and CCs shared 28 differentially expressed metabolites, among which cysteinyl-glycine (Cys-Gly) was significantly reduced in both oocytes and CCs following vitrification. Supplementing IVM with Cys-Gly improved vitrified oocyte quality, as evidenced by reduced oxidative stress, suppressed excessive apoptosis and aberrant autophagy, enhanced embryonic development, and increased cumulus expansion. Collectively, these findings provide a comprehensive metabolic profile of porcine GV oocytes and CCs after vitrification, offering new insights into the mechanisms of oocyte cryoinjury. Targeting key metabolic pathways or supplementing specific metabolites, such as Cys-Gly, may represent a promising strategy to improve the viability of vitrified porcine oocytes.

Due to its high efficiency and safety, oocyte vitrification finds broad application in many fields of life sciences, such as clinical assisted reproduction and conservation of animal genetic resources. However, vitrification may cause cellular damage and reduce the quality of oocytes and their cumulus cells (CCs), which could be closely related to disorders in lipid metabolism. At present, the impact of vitrification upon the lipid profile of oocytes and CCs has not been systematically elucidated. In this study, we used porcine germinal vesicle cumulus–oocyte complexes (COCs) as a model to analyze their lipid characteristics after vitrification and in vitro maturation (IVM), utilizing untargeted lipid metabolomics. Our results showed that an overall count of 37 down-regulated and 8 up-regulated differential lipids was identified in the vitrified oocytes. Pathway analysis confirmed the enrichment in glycerophospholipid metabolism and fat digestion and absorption, etc. Combined with transcriptomic analysis, three enriched pathways were revealed, including the AMPK signaling pathway, metabolic pathways, and fatty acid elongation. On the other hand, a total of four down-regulated and eight up-regulated differential lipids were detected in the vitrified CCs. Pathway enrichment implicated autophagy, glycerophospholipid metabolism, etc. A joint analysis of metabolomic and transcriptomic data revealed four enrichment pathways, including cholesterol metabolism, fat digestion and absorption, regulation of lipolysis in adipocytes, and metabolic pathways. Notably, the supplementation of lysophosphatidylcholine during IVM attenuated oxidative stress, enhanced mitochondrial activity, and enhanced the viability and embryonic development of cryopreserved porcine oocytes. The results indicate that vitrification alters lipids in oocytes and CCs, and the supplementation of lipids plays a role in improving the quality of vitrified oocytes.

The preservation of porcine oocytes is critically important for advancing superior breeds and conserving genetic resources in pig production. Vitrification has gained traction as a preferred alternative to slow freezing for porcine oocytes because of its effectiveness in reducing ice crystal formation, yet it can still negatively affect oocyte quality, compromising their in vitro maturation (IVM) and later embryonic development. Long non-coding RNAs (lncRNAs) have proven to be key players in numerous biological processes, such as oocyte growth, maturation, and early embryogenesis. Despite this, the effects of vitrified porcine germinal vesicle (GV) oocytes, particularly regarding IVM and the dynamic expression patterns of lncRNAs during embryonic development, remain largely unclear. To address this gap, this study conducted lncRNA sequencing at the metaphase II (MII), parthenogenetic 4-cell embryo, and parthenogenetic blastocyst stages sourced from both fresh and vitrified GV oocytes. This method enabled us to ascertain the impact of vitrification on lncRNA expression throughout oocyte maturation and embryonic development. Results identified 773 differentially expressed lncRNAs (DELs) at the MII stage, 1973 at the parthenogenetic 4-cell, and 1192 at the parthenogenetic blastocyst. Enrichment analysis of forecasted target genes revealed their involvement in key regulatory pathways associated with the cell cycle, meiosis, stress response, and metabolic activity. Overall, this study provides a comprehensive overview of lncRNA expression during oocyte maturation and embryonic development following porcine GV oocyte vitrification, thereby shedding light on the molecular mechanisms behind vitrification-induced damage.

Pseudorabies virus (PRV) is a highly pathogenic agent that adversely impacts swine populations, leading to considerable economic losses within the Chinese pig industry. Furthermore, the potential for PRV to transmit across species from pigs to other hosts has attracted significant attention. In light of this, it is essential to identify efficacious antiviral agents for the treatment of PRV infection. In this study, we investigated the antiviral properties of Diltiazem HCl (DTZ) against PRV infection in susceptible cell lines. Our results demonstrated that DTZ significantly inhibited PRV infection in both PK15 and Vero cells. Moreover, this chemical compound exhibited antiviral activity against both variant and classical strains of PRV, as well as herpes simplex virus type 1 (HSV-1). Time-of-addition assays showed that DTZ exerted its inhibitory effect through specific interference with the virus replication process. Subsequent transcriptomic analysis via RNA sequencing indicated that the calcium signaling pathway might be involved in the antiviral properties of DTZ against PRV infection. Specifically, treatment with EGTA or calcium ion (Ca²⁺)-free medium inhibited PRV infection; this inhibitory effect was substantially mitigated upon the reintroduction of CaCl₂. In summary, DTZ effectively suppressed PRV infection in vitro, demonstrating its potential as an antiviral agent against PRV infections.

Semen cryopreservation results in the differential remodeling of the molecules presented in sperm, and these alterations related to reductions in sperm quality and its physiological function have not been fully understood. Given this, this study aimed to investigate the cryoinjury mechanism of goat sperm by analyzing changes of the metabolic characteristics in sperm during the cryopreservation process. The ultra-high-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) technique was performed to explore metabolite profiles of fresh sperm (C group), equilibrated sperm (E group), and frozen–thawed sperm (F group). In total, 2570 metabolites in positive mode and 2306 metabolites in negative mode were identified, respectively. After comparative analyses among these three groups, 374 differentially abundant metabolites (DAMs) in C vs. E, 291 DAMs in C vs. F, and 189 DAMs in E vs. F were obtained in the positive mode; concurrently, 530 DAMs in C vs. E, 405 DAMs in C vs. F, and 193 DAMs in E vs. F were obtained in the negative mode, respectively. The DAMs were significantly enriched in various metabolic pathways, including 31 pathways in C vs. E, 25 pathways in C vs. F, and 28 pathways in E vs. F, respectively. Among them, 65 DAMs and 25 significantly enriched pathways across the three comparisons were discovered, which may be tightly associated with sperm characteristics and function. Particularly, the functional terms such as TCA cycle, biosynthesis of unsaturated fatty acids, sphingolipid metabolism, glycine, serine and threonine metabolism, alpha-linolenic acid metabolism, and pyruvate metabolism, as well as associated pivotal metabolites like ceramide, betaine, choline, fumaric acid, L-malic acid and L-lactic acid, were focused on. In conclusion, our research characterizes the composition of metabolites in goat sperm and their alterations induced by the cryopreservation process, offering a critical foundation for further exploring the molecular mechanisms of metabolism influencing the quality and freezing tolerance of goat sperm. Additionally, the impacts of equilibration at low temperature on sperm quality may need more attentions as compared to the freezing and thawing process.

Using two-dimensional electrophoresis along with mass spectroscopy, we have investigated how the cryopreservation process affected the protein profile of goat ejaculated sperm. In this study, five bucks were used for semen collection. After removal of seminal plasma, the Tris-based extender containing glycerol and egg yolk was used to freeze semen. The results indicated that the post-thaw sperm quality showed a significant reduction compared with fresh sperm. The numbers of protein spots acquired in fresh and post-thaw sperm were 2926 ± 57 and 3061 ± 81, respectively. Twenty-two different abundant proteins (DAPs) were identified between fresh sperm and frozen–thawed sperm (≥3.0-folds, p < 0.05). The abundances of 19 proteins were significantly higher in the fresh sperm than the post-thaw sperm. The results of the gene ontology annotation showed the primary location of the DAPs on sperm cytoskeleton, protein complex, cytoplasm, and mitochondria. In addition, these proteins were mainly involved in ion binding, small molecular metabolic processes, structure molecule activity, guanosine triphosphatase activity, oxidoreductase activity, and protein complex assembly. The interaction networks among these DAPs demonstrated that they may play roles in oxidoreductase activity, structure, acrosomal function, and motility of sperm. Collectively, the proteome of goat sperm was altered during the cryopreservation process, demonstrating that protein modification induced by cryopreservation may be associated with the reduced quality of goat sperm after thawing.

In this study, the effects of trehalose and 1, 3-cyclohexanediol (1, 3-CHD) on the ultrastructure of frozen-thawed ram sperm were assessed and compared. In the control group, sperm were frozen without trehalose and 1, 3-CHD. In the trehalose group, 100 mM trehalose was used for sperm cryopreservation. In the cyclohexanediol group, the freezing extender contained 100 mM 1, 3-CHD. The transmission electron microscope (TEM) was used to observe the ultrastructural alterations of sperm. For verification of the TEM results, the plasma membrane and acrosome integrity of ram frozen sperm was assessed. Three fertility-proven rams were used in this study. Semen collection was repeated 6 times. The collected semen was pooled to preclude the individual difference each time. The sperm collected from a representative ram were used for ultrastructural observation. The TEM results indicated extensive and severe cryoinjuries on the main organelles of ram frozen sperm. Some alterations in plasma membrane, including detachment, rupture, dilation, or loss, appeared in post-thaw sperm. The bending shape and leakage of genetic materials were also observed in the nucleus. In addition, the outer acrosome membrane in some frozen sperm was broken or partly lost. Further, leakage of the inner contents of acrosomes also occurred. Sperm mitochondria was negatively influenced by cryopreservation. With 1, 3-CHD or trehalose, the percentage of sperm with normal acrosomes was 62% or 64%, and it was significantly higher than that of the control (41.51%, p < 0.05). However, different from trehalose, 1, 3-CHD cannot efficiently protect the post-thaw integrity of the plasma membrane (48.09% vs. 26.92%, p < 0.05). The TEM results were consistent with the quality assessment of frozen-thawed sperm. Collectively, trehalose and 1, 3-CHD can mitigate cryoinjuries on sperm ultrastructure. The cryoprotective effects of trehalose on sperm plasma membrane are superior to 1, 3-CHD. Sperm plasma membrane is the most sensitive to cryoinjuries, followed by acrosomes, mitochondria, and nuclei.