Superior performance of biocomposite nanoparticles PLGA-RES in protecting oocytes against vitrification stimuli.

Irreversible cryogenic damage caused by oocyte vitrification limits its widespread use in female fertility preservation. In recent years, nanoparticles (NPs) have gained great attention as potential alternatives in protecting oocytes against cryoinjuries. In this paper, a novel composite nanoparticle, poly (lactic-co-glycolic acid)-resveratrol (PLGA-RES) was designed to improve the biocompatibility and sustained release properties by encapsulating natural antioxidant RES into PLGA NPs. Firstly, biotoxicity and oxidation resistance of PLGA-RES were determined, and the results showed that PLGA-RES had nontoxic effect on oocyte survival during in vitro maturation (IVM) (97.08% ± 0.24% vs. 98.89% ± 1.11%, p > 0.05). Notably, PLGA-RES even increased maturation (65.10% ± 4.11% vs. 52.85% ± 2.87%, p < 0.05) and blastocyst rate (56.13% ± 1.36% vs. 40.91% ± 5.85%, p < 0.05). Moreover, the reduced reactive oxygen species (ROS) level (13.49 ± 2.30 vs. 34.07 ± 3.30, p < 0.01), increased glutathione (GSH) (44.13 ± 1.57 vs. 37.62 ± 1.79, p < 0.01) and elevated mitochondrial membrane potential (MMP) levels (43.10 ± 1.81 vs. 28.52 ± 1.25, p < 0.01) were observed in oocytes treated with PLGA-RES when compared with that of the control group. Subsequently, the role of PLGA-RES played in oocytes during vitrification was systematically evaluated. The results showed that the addition of PLGA-RES during vitrification and thawing significantly improved the survival rate (80.42% ± 1.97% vs. 75.37% ± 1.3%, p < 0.05). Meanwhile, increased GSH (15.09 ± 0.86 vs. 14.51 ± 0.78, p < 0.01) and mitochondrial membrane potential (22.56 ± 3.15 vs. 6.79 ± 0.60, p < 0.01), decreased reactive oxygen species levels (52.11 ± 2.95 vs. 75.41 ± 7.23, p < 0.05) and reduced mitochondrial abnormality distribution rate (25.00% ± 0.29% vs. 33.33% ± 1.15%, p < 0.01) were assessed in vitrified MII oocytes treated with PLGA-RES. Furthermore, transcriptomic analyses demonstrated that PLGA-RES participated in endocytosis and PI3K/AKT/mTOR pathway regulation, which was verified by the rescued expression of ARRB2 and ULK3 protein after PLGA-RES treatment. In conclusion, PLGA-RES exhibited potent antioxidant activity, and could be used as an efficacious strategy to improve the quality of vitrified oocytes.

Integrated transcriptomics and proteomics assay identifies the role of FCGR1A in maintaining sperm fertilization capacity during semen cryopreservation in sheep.

Semen cryopreservation is a promising technology employed in preserving high-quality varieties in animal husbandry and is also widely applied in the human sperm bank. However, the compromised qualities, such as decreased sperm motility, damaged membrane structure, and reduced fertilization competency, have significantly hampered the efficient application of this technique. Therefore, it is imperative to depict various molecular changes found in cryopreserved sperm and identify the regulatory network in response to the cryopreservation stress. In this study, semen was collected from three Chinese Merino rams and divided into untreated (fresh semen, FS) and programmed freezing (programmed freezing semen, PS) groups. After measuring different quality parameters, the ultra-low RNA-seq and tandem mass tag-based (TMT) proteome were conducted in both the groups. The results indicated that the motility (82.63% ± 3.55% vs. 34.10% ± 2.90%, p < 0.05) and viability (89.46% ± 2.53% vs. 44.78% ± 2.29%, p < 0.05) of the sperm in the FS group were significantly higher compared to those in the PS group. In addition, 45 upregulated and 291 downregulated genes, as well as 30 upregulated and 48 downregulated proteins, were found in transcriptomics and proteomics data separately. Moreover, three integrated methods, namely, functional annotation and enrichment analysis, Pearson's correlation analysis, and two-way orthogonal partial least squares (O2PLS) analysis, were used for further analysis. The results suggested that various differentially expressed genes and proteins (DEGs and DEPs) were mainly enriched in leishmaniasis and hematopoietic cell lineage, and Fc gamma receptor Ia (FCGR1A) was significantly downregulated in cryopreserved sperm both at mRNA and protein levels in comparison with the fresh counterpart. In addition, top five genes (FCGR1A, HCK, SLX4, ITGA3, and BET1) and 22 proteins could form a distinct network in which genes and proteins were significantly correlated (p < 0.05). Interestingly, FCGR1A also appeared in the top 25 correlation list based on O2PLS analysis. Hence, FCGR1A was selected as the most potential differentially expressed candidate for screening by the three integrated multi-omics analysis methods. In addition, Pearson's correlation analysis indicated that the expression level of FCGR1A was positively correlated with sperm motility and viability. A subsequent experiment was conducted to identify the biological role of FCGR1A in sperm function. The results showed that both the sperm viability (fresh group: 87.65% ± 4.17% vs. 75.8% ± 1.15%, cryopreserved group: 48.15% ± 0.63% vs. 42.45% ± 2.61%, p < 0.05) and motility (fresh group: 83.27% ± 4.15% vs. 70.41% ± 1.07%, cryopreserved group: 45.31% ± 3.28% vs. 35.13% ± 2.82%, p < 0.05) were significantly reduced in fresh and frozen sperm when FCGR1A was blocked. Moreover, the cleavage rate of embryos fertilized by FCGR1A-blocked sperm was noted to be significantly lower in both fresh (95.28% ± 1.16% vs. 90.44% ± 1.56%, p < 0.05) and frozen groups (89.8% ± 1.50% vs. 82.53% ± 1.53%, p < 0.05). In conclusion, our results revealed that the downregulated membrane protein FCGR1A can potentially contribute to the reduced sperm fertility competency in the cryopreserved sheep sperm.

Metabolomic and Transcriptomic Changes Underlying the Effects of L-Citrulline Supplementation on Ram Semen Quality.

This study examined the effects of L-Cit supplementation on ram semen quality through metabolomics and transcriptomics. A total of 16 rams were randomly categorized into two groups. The control group was fed a basic diet, whereas the experimental group received feed supplemented with 12 g/d of L-Cit. Semen and blood were collected from the rams on days 0 and 72 to measure sugar, pyruvate, amino acid, and nontargeted metabolite contents. Additionally, hypothalamic and testicular tissues were collected for a transcriptomic analysis. We found 27 differential metabolites between the control and experimental groups, of which 21 were downregulated (p < 0.05) and 6 were upregulated (p < 0.05). Compared with the control group, xylose and pyruvate contents in seminal plasma increased by 43.86% and 162.71%, respectively (p < 0.01). Additionally, the levels of 11 amino acids showed a significant increase in seminal plasma (p < 0.01). Furthermore, 961 and 715 differentially expressed genes were detected in the hypothalamic and testicular tissues, respectively. The pathways of significant enrichment in the hypothalamus and testes were protein digestion, absorption, glycolysis/gluconeogenesis, and amino as well as nucleotide sugar metabolisms. In the present study, L-Cit improved protein synthesis and blood metabolism, consequently increasing the contents of most amino acids in ram seminal plasma. Specifically, the hypothalamus controlled the expression of glycolysis/gluconeogenesis-related genes in the testes through its metabolites released into the serum, thereby providing energy for sperm production, which led to a decrease in the sugar content of seminal plasma.

Proteomic profile of mouse oocytes after vitrification: A quantitative analysis based on 4D label-free technique.

Mature oocyte cryopreservation represents an important trend for future fertility preservation, however, the relatively low efficiency has hampered its clinical application. Proteomic profiling is a method of choice for the exploration of the molecular mechanism underlying cryoinjuries. Here, a systematic comparison of protein expression between fresh and vitrified oocytes was performed based on the 4D label-free technique, an informative method with high sensitivity. Our results indicated that the oocyte survival rate was significantly reduced after vitrification. Proteomic results showed that 32 proteins were up-regulated, while 77 proteins were down-regulated in vitrified oocytes compared with the fresh counterparts. Gene Ontology (GO) functional analysis revealed that differentially expressed proteins (DEPs) were involved in metabolism, mitochondrial function, cytoskeleton and other cell functions. Moreover, proteins that participated in signaling transduction mechanisms were the largest category based on Clusters of Orthologous Groups of protein/EuKaryotic Orthologous Groups (COG/KOG) functional classification. In addition, over-expressed DEPs were enriched for "nucleus", "protein binding", "membrane", "cytoplasm" as well as mitochondrial function. Furthermore, we discovered that the DEPs were clustered in pyruvate metabolism, citric acid (TCA) cycle and glucose metabolism by Protein-Protein Interaction (PPI) network evaluation. In conclusion, our data demonstrate that vitrification induces multi-level damages in oocytes, the dynamic proteomic profiling will provide systematic insights into uncovering the mechanism underlying cryoinjuries.

Artificially Increasing Cortical Tension Improves Mouse Oocytes Development by Attenuating Meiotic Defects During Vitrification.

Oocyte cryopreservation demonstrates great benefits in the conservation of animal germplasm resources and assisted reproductive technology. However, vitrification causes damages in oocytes, which would lead to the decrease of oocyte quality, and embryonic development post fertilization. Cytoskeleton plays an important role in regulating cell shape, organelle migration, cell division and mechanical signal transduction. Cortical tension is a reflection of the physiological state and contractile ability of cortical cytoskeleton. Appropriate cortical tension is prerequesite for normal oocyte meiosis. In the present study, oocyte cortical tension was examined by evaluating the levels of cortical tension-related protein pERM (Phospho-Ezrin/Radixin/Moesin) and pMRLC (Phospho-Myosin Light Chain 2). We found that the cortical tension of vitrified oocytes was decreased. Increasing cortical tension of vitrified oocytes by adding 10 µg/ml ConA during in vitro culture could significantly improve the polar body extrusion rate and embryo development. Furthermore, increasing the cortical tension could improve spindle positioning, maintain kinetochore-microtubule (KT-MT) attachment, strengthen spindle assembly checkpoint (SAC) activity, and reduce the aneuploidy rate in vitrified oocytes. In conclusion, vitrification induced a remarkable decrease in cortical tension, and increasing the cortical tension could rescue the meiosis defect and improve oocyte quality.

Identifying the heat resistant genes by multi-tissue transcriptome sequencing analysis in Turpan Black sheep.

Heat stress not only affects the physical condition but also affects reproductive performance in sheep. A thorough understanding of the molecular and physiological mechanisms underlying heat stress would certainly improve livestock productivity and provide genetic evaluation ways for heat resistant breeds selection. In this study, 85 Turpan Black sheep, a breed exhibited excellent heat resistance from long-term artificial selection, and 85 heat sensitive Kazakh sheep in Turpan basin were tested for physiological and reproductive performance from July to August in summer. The results showed that the estrus rate was significantly higher in Turpan Black sheep (P < 0.05), while the heart rate and respiratory rate of Turpan Black sheep are significantly lower than that of Kazakh sheep (P < 0.05). Furthermore, to clarify genes participated in heat stress response, the pituitary, ovarian and hepatic tissues from three Turpan Black sheep and three Kazakh sheep were subjected to RNA-seq. The results indicated that 32, 49 and 69 genes were up-regulated, and 39, 60 and 145 genes were down-regulated in pituitary, ovarian and hepatic tissues in Turpan Black sheep compared with that of the Kazakh sheep, respectively. KEGG and gene set enrichment analysis showed that the differentially expressed genes were mainly involved in signal transduction pathways. In particular, the differentially expressed genes in hepar were enriched in the energy metabolism pathway, while the differentially expressed genes in ovarian tissue were enriched in the ovarium steroidogenesis pathway. In conclusion, our results implied that the pituitary-ovary axis might include hepar as downstream targeted organism in heat resistant regulation. Under heat stress, the signals released from pituitary would impact steroidogenesis in ovary, and further alter energy metabolism in hepar. As we know, this is the first comparative study to investigate the gene expression in multi-tissue in sheep under heat stress.

Time-delay signature concealing electro-optic chaotic system with multiply feedback nonlinear loops.

A novel time-delay signature (TDS) concealing electro-optic (EO) chaotic system with multiply feedback nonlinear loops is proposed and analyzed by numerical simulation. The proposed system employs mutual injection structure implemented by two asymmetric branches named as multiply feedback nonlinear loop which introduces an extra nonlinear factor to the system dynamic equation. The complexity of the chaos system is increased by introducing this multiply feedback nonlinear loop. The permutation entropy (PE) of the proposed system is improved to higher than 0.96 when feedback strength (ß) equals 5. The proposed system can enter to chaos regime with a small ß (ß = 0.8). The TDS is concealed effectively due to the extra nonlinear factor introduced by multiply feedback nonlinear loop. Meanwhile, key-space of the proposed system is about 1012 times that of the classical EO system because more tunable time delay parameters are introduced. Furthermore, the performance of a secure communication system based on the proposed chaotic system is discussed, and the simulation results show that the system is sensitive to time delay parameters and robust to feedback strength, which proves the proposed system is suitable for secure communication.

Effect of functional single nucleotide polymorphism g.-572 A > G of apolipoprotein A1 gene on resistance to ketosis in Chinese Holstein cows.

The ketosis has negative effects on the high-yielding dairy cows during early lactation. Apolipoprotein A1 (APOA1) is a component of high-density lipoprotein. However, the association of APOA1 gene with ketosis, and the molecular mechanisms of expression of APOA1 gene are not fully understood in dairy cows. In this study, expression of APOA1 in the liver and blood was investigated by RT-qPCR and immunohistochemistry, and genetic variation in the 5'-flanking region of the AOPA1 gene was also screened and identified. In addition, correlation of the single nucleotide polymorphisms (SNPs) of APOA1 gene with blood ketone characters, and activity of APOA1 promoter were analyzed in dairy cows. The results showed that ApoA1 protein was expressed in the liver, and the mRNA level of APOA1 was significantly higher in the cows with ketosis comparing to the healthy cows. In addition, a novel SNP (g.-572 A > G) in the core promoter of the APOA1 gene was identified between base g.-714 and g.-68 through transient transfection in both HepG2 cell and FFb cell, and luciferase report assay. Moreover, there was lower concentration of blood ß-hydroxybutyrate in cows with genotype GG comparing to the cows with genotypes AA and AG. This study reported for the first time that the genetic variant g.-572 A > G in the core promoter region of APOA1 gene was associated with the ketosis in Chinese Holstein cows, and g.-572 A > G may be used as a genetic marker for ketosis prevention.

Effects of early pregnancy on expression of interferon-stimulated gene 15, STAT1, OAS1, MX1, and IP-10 in ovine liver.

Interferon-tau (IFNT) regulates maternal recognition during early pregnancy in ruminants. The liver can serve as a hematopoietic organ, and it has immune functions. This study hypothesized whether mRNA and proteins of interferon-stimulated genes (ISGs) induced by early pregnancy are upregulated in maternal liver. Therefore, we determined the expression of interferon-stimulated gene 15-kDa protein (ISG15), 2',5'-oligoadenylate synthetase 1 (OAS1), myxovirus resistance protein 1 (MX1), interferon-gamma-inducible protein 10 (IP-10), and signal transducer and activator of transcription 1 (STAT1) in maternal livers during early pregnancy in sheep. Ovine livers were sampled on day 16 of the estrous cycle, and days 13, 16, and 25 of pregnancy, and expression of ISGs was detected by quantitative real-time PCR, Western blot, and immunohistochemistry analysis. Our results showed that there were increases in expression of the mRNA and proteins of ISG15, OAS1, IP-10, STAT1, and MX1 during early pregnancy. STAT1 protein was limited to the hepatocytes, and endothelial cells of proper hepatic arteries and hepatic portal veins. In conclusion, the upregulation of ISG15, OAS1, IP-10, STAT1, and MX1 proteins may be implicated in maternal hepatic immune adjustment and other functions during early pregnancy in sheep.

Expression of melatonin receptors and CD4 in the ovine thymus, lymph node, spleen and liver during early pregnancy.

As a pineal gland hormone, melatonin acts through its receptors to modulate the immune system. The immune system is composed of primary and secondary organs, and immune organs are adapted to the presence of the fetal alloantigen during pregnancy. However, it is unclear whether melatonin affects maternal immune organs during early pregnancy in sheep. In this study, the ovine thymus, lymph node, spleen and liver were sampled at day 16 of the oestrous cycle, and at days 13, 16 and 25 of pregnancy. The expression of melatonin receptor 1A (MT1), melatonin receptor 1B (MT2) and cluster of differentiation 4 (CD4) was detected by quantitative real-time polymerase chain reaction, Western blot and immunohistochemistry experiments. Our results showed that during early pregnancy there was an upregulation of MT1 mRNA and protein in the thymus, lymph node and liver, and there was a downregulation in the spleen. The expression of MT2 mRNA and protein was increased in the thymus but decreased in the spleen and liver, and there was no significant change in the lymph node during early pregnancy. CD4 protein was upregulated in the thymus, lymph node and liver, but there were no significant changes in the spleen during early pregnancy. In conclusion, early pregnancy induces tissue-specific expression of MT1, MT2 and CD4, which may be due to the different functions of the thymus, lymph node, spleen and liver. Further, melatonin is involved in immune regulation of the maternal thymus, lymph node, spleen and liver during early pregnancy in sheep.

Population Structure, and Selection Signatures Underlying High-Altitude Adaptation Inferred From Genome-Wide Copy Number Variations in Chinese Indigenous Cattle.

Copy number variations (CNVs) have been demonstrated as crucial substrates for evolution, adaptation and breed formation. Chinese indigenous cattle breeds exhibit a broad geographical distribution and diverse environmental adaptability. Here, we analyzed the population structure and adaptation to high altitude of Chinese indigenous cattle based on genome-wide CNVs derived from the high-density BovineHD SNP array. We successfully detected the genome-wide CNVs of 318 individuals from 24 Chinese indigenous cattle breeds and 37 yaks as outgroups. A total of 5,818 autosomal CNV regions (683 bp-4,477,860 bp in size), covering ~14.34% of the bovine genome (UMD3.1), were identified, showing abundant CNV resources. Neighbor-joining clustering, principal component analysis (PCA), and population admixture analysis based on these CNVs support that most Chinese cattle breeds are hybrids of Bos taurus taurus (hereinafter to be referred as Bos taurus) and Bos taurus indicus (Bos indicus). The distribution patterns of the CNVs could to some extent be related to the geographical backgrounds of the habitat of the breeds, and admixture among cattle breeds from different districts. We analyzed the selective signatures of CNVs positively involved in high-altitude adaptation using pairwise Fst analysis within breeds with a strong Bos taurus background (taurine-type breeds) and within Bos taurus×Bos indicus hybrids, respectively. CNV-overlapping genes with strong selection signatures (at top 0.5% of Fst value), including LETM1 (Fst = 0.490), TXNRD2 (Fst = 0.440), and STUB1 (Fst = 0.420) within taurine-type breeds, and NOXA1 (Fst = 0.233), RUVBL1 (Fst = 0.222), and SLC4A3 (Fst=0.154) within hybrids, were potentially involved in the adaptation to hypoxia. Thus, we provide a new profile of population structure from the CNV aspects of Chinese indigenous cattle and new insights into high-altitude adaptation in cattle.

Differential expression of T helper cytokines in the liver during early pregnancy in sheep.

Liver plays important roles in the innate and adaptive immunity, and contributes to the maternal immune adjustments during pregnancy in mice and rats. T helper 1 (Th1) and Th2 cytokines are related to immune response. However, expression of Th1 and Th2 cytokines in maternal livers is unclear during early pregnancy in sheep. In this study, livers were collected on day 16 of the estrous cycle and on days 13, 16 and 25 of pregnancy (n = 6 for each group) in ewes, and qRT-PCR, western blot and immunohistochemistry were used to analyze the expression of Th1 and Th2 cytokines in the livers. Our results showed that interferon-gamma (IFN-γ), interleukin (IL)-2, IL-4, IL-6 and IL-10 were downregulated, and IL-5 was upregulated in the livers during early pregnancy. Furthermore, there was no effect for early pregnancy on expression of TNF-ß in the livers, and the IFN-γ protein was limited to the endothelial cells of the proper hepatic arteries and portal veins. In conclusion, early pregnancy exerted its effect on the liver to regulate the Th cytokines expression, but there was no evident shift from Th1 to Th2 cytokines, which may be necessary for the maternal hepatic immune adjustments during early pregnancy in sheep.