In this article, we focused on the impact of precisely chemically modified FLI maturation medium enriched with fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), insulin-like growth factor 1 (IGF1), and polyvinyl alcohol (PVA) and its potential to improve the efficiency of in vitro production of porcine embryos. We hypothesized that enhancing the composition of the maturation medium could result in an elevated production of embryos in vitro and can affect EGA. FLI medium resulted in a significantly higher rate of oocyte blastocyst maturation and formation compared to the control DMEM medium. In addition, immunocytochemical labelling confirmed the detection of UBF in 4-cell FLI parthenogenic embryos, suggesting similarities with natural embryo development. Through RNAseq analysis, upregulated genes present in 4-cell FLI embryos were found to play key roles in important biological processes such as cell proliferation, cell differentiation, and transcriptional regulation. Based on our findings, we demonstrated the positive influence of FLI medium in the evaluation of in vitro embryo production, EGA detection, transcriptomic and proteomic profile, which was confirmed by the positive activation of the embryonal genome in the 4-cell stage of parthenogenetically activated embryos.
- MeSH
- Blastocyst drug effects metabolism MeSH
- Fertilization in Vitro MeSH
- Fibroblast Growth Factor 2 * pharmacology MeSH
- Insulin-Like Growth Factor I * pharmacology MeSH
- Culture Media * chemistry pharmacology MeSH
- Leukemia Inhibitory Factor * pharmacology MeSH
- Oocytes MeSH
- Swine embryology genetics MeSH
- Proteomics MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
Innumerable similarities in reproductive cyclicity and hormonal alterations highlight the considerable utility of the mare to study aspects of follicular dynamics and reproductive function in view of the largely constricted, human research subjects. The bi-directional communication between the growing oocyte and the surrounding somatic cells embodies the hallmark of mammalian follicular development, partially mediated by extracellular vesicles (EVs) encapsulated with microRNAs (miRNAs) and present in the follicular fluid (FF). Here, we aimed to decipher the dynamics of the miRNAs in EVs from equine FF aspirated in vivo during different stages of follicular development, namely, predeviation (PreDev; 18-20 mm), deviation (Dev; 22-25 mm), postdeviation (PostDev; 26-29 mm), preovulatory (PreOV; 30-35 mm), and impending ovulation (IMP; ∼40 mm). Approximately 176 known miRNAs were found in all groups with 144 mutually detected among all groups. Cluster analysis exhibited 15 different expression patterns during follicular development. Among these patterns, a group of 22 miRNAs (including miR-146b-5p, miR-140, and miR-143) exhibited a sharp reduction in expression from the PreDev until the PreOV stage. Another cluster of 23 miRNAs (including miR-106b, miR-199a-5p, and miR-125a-5p) exhibited a stable expression pattern at the PreDev stage until the PostDev stage, with a significant increase at the PreOV stage followed by a significant decrease at the IMP stage. In conclusion, this study provides greater insights into the stage-specific expression dynamics of FF EV-miRNAs during equine follicular development, which may propose novel approaches to improve ART and provide new biomarkers to facilitate the assessment of ovarian pathophysiological conditions.
- MeSH
- Extracellular Vesicles * genetics metabolism MeSH
- Follicular Fluid metabolism MeSH
- Horses MeSH
- Humans MeSH
- MicroRNAs * metabolism MeSH
- Ovarian Follicle metabolism MeSH
- Ovulation genetics MeSH
- Mammals MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
Seminal plasma (SP) provides essential nutrients, transport, and protection to the spermatozoa during their journey through the male and female reproductive tracts. Extracellular vesicles (EVs) are one of the main components of the SP with several biomolecular cargoes, including miRNAs, that can influence spermatozoa functions and interact with the cells of the female reproductive tract. This study aimed to isolate, characterize, and identify the miRNA expression profiles in the SP-EVs isolated from fertile (F) and subfertile (S) rabbit bucks that could serve as fertility biomarkers. In this study, the methods to isolate and identify EVs including exosomes, from SP of 3 F and S bucks have been developed. Ultracentrifugation and size exclusion chromatography analysis were using to isolate EVs from SP of F and S males that were qualitative and quantitively characterised using transmission electron microscopy, nanoparticle tracking analysis and western blotting. In addition, total RNA, including miRNA, was isolated, sequenced and identified from SP-EVs samples. Different SP-EVs concentrations (8.53 × 1011 ± 1.04 × 1011 and 1.84 × 1012 ± 1.75 × 1011 particles/mL of SP; P = 0.008), with a similar average size (143.9 ± 11.9 and 115.5 ± 2.4 nm; P = 0.7422) in F and S males, respectively was observed. Particle size was not significantly correlated with any kinetic parameter. The concentration of SP-EVs was positively correlated with the percentage of abnormal forms (r = 0.94; P < 0.05) and with the percentage of immotile spermatozoa (r = 0.88; P < 0.05). Small-RNA-seq analysis identified a total of 267 and 244 expressed miRNAs in the F and S groups, respectively. Two miRNAs (let-7b-5p and let-7a-5p) were the top most abundant miRNAs in both groups. Differential expression analysis revealed that 9 miRNAs including miR-190b-5p, miR-193b-5p, let-7b-3p, and miR-378-3p, and another 9 miRNAs including miR-7a-5p, miR-33a-5p, miR-449a-5p, and miR-146a-5p were significantly up- and downregulated in the F compared to the S group, respectively. The SP from F and S rabbit males contains EVs with different miRNA cargo correlated with spermatogenesis, homeostasis, and infertility, which could be used as biomarkers for male fertility and potential therapies for assisted reproductive technologies.
- MeSH
- Extracellular Vesicles * metabolism MeSH
- Fertility genetics MeSH
- Infertility * veterinary MeSH
- Rabbits MeSH
- MicroRNAs * metabolism MeSH
- Semen MeSH
- Animals MeSH
- Check Tag
- Rabbits MeSH
- Male MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
The statement that fully-grown porcine oocytes (oocytes from follicles with diameter from 3 to 6 mm) are transcriptionally quiescent is not as strongly supported as it was before. Currently, we know that there is a difference between the transcription profile of germinal vesicle (GV) and metaphase II (MII) oocytes. The goal of our study was to compare the transcription profile of GV, germinal vesicle breakdown (GVBD), metaphase I (MI), and MII oocytes matured in the chemically defined medium FLI. Oocytes were sequenced, and the results were subsequently validated using quantitative reverse transcription polymerase chain reaction (RT-qPCR). We detected multiple differentially transcribed mRNAs, of which many were upregulated. Among them we found mRNAs necessary for protein production, mitochondrial functions and cytoplasmic maturation. Collectively, these data support the hypothesis that transcription activity in fully-grown porcine oocytes is necessary for key processes during their successful maturation in vitro in a chemically defined maturation medium.
- MeSH
- Cell Nucleus metabolism MeSH
- In Vitro Oocyte Maturation Techniques * veterinary methods MeSH
- RNA, Messenger genetics metabolism MeSH
- Oocytes * metabolism MeSH
- Swine MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
Complicated geometry in combination with surface treatment strongly deteriorates fatigue resistance of metallic dental implants. Mechanical properties of pure Ti grade 2, usually used for dental implant production, were shown to be significantly improved due to intensive grain refinement via Conform SPD. The increase of the tensile strength properties was accompanied by a significant increase in the fatigue resistance and fatigue endurance limit. However, the SLA treatment usually used for the implants' surface roughening, resulted in the fatigue properties and endurance limit decrease, while this effect was more pronounced for the ultrafine-grained comparing to the coarse-grained material when tested under tensile-tensile loading mode. The testing of the implants is usually provided under the bending mode. Even though different testing condition for the conventional specimens tests and implants testing was adopted, a numerical study revealed their comparable fatigue properties. The fatigue limit determined for the implants was 105% higher than the one for coarse-grained and only by 4 % lower than the one for ultrafine-grained Ti grade 2. Based on the obtained results, conventional specimens testing can be used for the prediction of the fatigue limit of the implants.
Although our knowledge regarding oocyte quality and development has improved significantly, the molecular mechanisms that regulate and determine oocyte developmental competence are still unclear. Therefore, the objective of this study was to identify and analyze the transcriptome profiles of porcine oocytes derived from large or small follicles using RNA high-throughput sequencing technology. RNA libraries were constructed from oocytes of large (LO; 3-6 mm) or small (SO; 1.5-1.9 mm) ovarian follicles and then sequenced in an Illumina HiSeq4000. Transcriptome analysis showed a total of 14,557 genes were commonly detected in both oocyte groups. Genes related to the cell cycle, oocyte meiosis, and quality were among the top highly expressed genes in both groups. Differential expression analysis revealed 60 up- and 262 downregulated genes in the LO compared with the SO group. BRCA2, GPLD1, ZP3, ND3, and ND4L were among the highly abundant and highly significant differentially expressed genes (DEGs). The ontological classification of DEGs indicated that protein processing in endoplasmic reticulum was the top enriched pathway. In addition, biological processes related to cell growth and signaling, gene expression regulations, cytoskeleton, and extracellular matrix organization were among the highly enriched processes. In conclusion, this study provides new insights into the global transcriptome changes and the abundance of specific transcripts in porcine oocytes in correlation with follicle size.
- MeSH
- Gene Regulatory Networks physiology MeSH
- Oocytes metabolism MeSH
- Oogenesis genetics MeSH
- Ovarian Follicle cytology MeSH
- Reverse Transcriptase Polymerase Chain Reaction MeSH
- Swine genetics growth & development MeSH
- Signal Transduction genetics MeSH
- Gene Expression Profiling MeSH
- Transcriptome * MeSH
- High-Throughput Nucleotide Sequencing MeSH
- Gene Expression Regulation, Developmental physiology MeSH
- Animals MeSH
- Check Tag
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Elevated summer temperature is reported to be the leading cause of stress in dairy and beef cows, which negatively affects various reproductive functions. Follicular cells respond to heat stress (HS) by activating the expression of heat shock family proteins (HSPs) and other antioxidants. HS is reported to negatively affect the bi-directional communication between the follicular cells and the oocyte, which is partly mediated by follicular fluid extracellular vesicles (EVs) released from surrounding cells. As carriers of bioactive molecules (DNA, RNA, protein, and lipids), the involvement of EVs in mediating the stress response in follicular cells is not fully understood. Here we used an in vitro model to decipher the cellular and EV-coupled miRNAs of bovine granulosa cells in response to HS. Moreover, the protective role of stress-related EVs against subsequent HS was assessed. For this, bovine granulosa cells from smaller follicles were cultured in vitro and after sub-confluency, cells were either kept at 37 °C or subjected to HS (42 °C). Results showed that granulosa cells exposed to HS increased the accumulation of ROS, total oxidized protein, apoptosis, and the expression of HSPs and antioxidants, while the viability of cells was reduced. Moreover, 14 and 6 miRNAs were differentially expressed in heat-stressed granulosa cells and the corresponding EVs, respectively. Supplementation of stress-related EVs in cultured granulosa cells has induced adaptive response to subsequent HS. However, this potential was not pronounced when the cells were kept under 37 °C. Taking together, EVs generated from granulosa cells exposed to HS has the potential to shuttle bioactive molecules to recipient cells and make them robust to subsequent HS.
- MeSH
- Apoptosis MeSH
- Extracellular Vesicles genetics metabolism pathology MeSH
- Granulosa Cells metabolism pathology MeSH
- Cattle Diseases epidemiology genetics prevention & control MeSH
- Ovarian Follicle metabolism pathology MeSH
- Heat Stress Disorders genetics physiopathology veterinary MeSH
- Heat-Shock Response * MeSH
- Gene Expression Regulation MeSH
- Cattle MeSH
- Gene Expression Profiling MeSH
- Animals MeSH
- Check Tag
- Cattle MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Brilliant cresyl blue (BCB) vital labelling is a powerful method for analyzing the quality of porcine cumulus-oocyte complexes. Our aim was to investigate the correlation between the selection of porcine oocytes using BCB labelling and selected intranuclear characteristics of porcine oocytes and parthenotes. Moreover, BCB labelling was correlated with the diameter of the oocyte and the developmental potential of the parthenotes. The following methods were used: BCB labelling, measurement of the diameter of the oocyte, parthenogenetic activation, immunocytochemistry, transmission electron microscopy, enucleation and relative protein concentration (RPC) analysis. We determined that the diameter of the oocytes in the BCB-positive (BCB+) group was significantly larger than in the BCB-negative (BCB-) group. Immediately after oocyte selection according to BCB labelling, we found significant difference in chromatin configuration between the analyzed groups. BCB+ oocytes were significantly better at maturation than BCB- oocytes. BCB+ embryos were significantly more competent at cleaving and in their ability to reach the blastocyst stage than BCB- embryos. Ultrastructural analyses showed that the formation of active nucleoli in the BCB+ group started at the 8-cell stage. Conversely, most BCB- embryos at the 8-cell and 16-cell stages were fragmented. No statistically significant difference in RPC in nucleolus precursor bodies (NPBs) between BCB+ and BCB- oocytes was found. We can conclude that BCB labelling could be suitable for assessing the quality of porcine oocytes. Moreover, the evaluation of RPC indicates that the quantitative content of proteins in NPB is already established in growing oocytes.
- MeSH
- Staining and Labeling methods MeSH
- Blastocyst chemistry cytology metabolism MeSH
- Cell Nucleus chemistry ultrastructure MeSH
- Embryo, Mammalian chemistry cytology ultrastructure MeSH
- Nuclear Proteins metabolism MeSH
- Microscopy, Confocal MeSH
- Oocytes chemistry cytology metabolism MeSH
- Oxazines chemistry MeSH
- Swine MeSH
- Reproducibility of Results MeSH
- Microscopy, Electron, Transmission MeSH
- Cell Size MeSH
- Animals MeSH
- Check Tag
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
The serine proteases, tissue- and urokinase-type plasminogen activators (PLAT and PLAU) and their inhibitors SERPINE1/2 are regulators of plasminogen to plasmin conversion. They are widely expressed in ovarian tissues, including granulosa and cumulus cells, and their expression is regulated by gonadotropins. The aim of this work was to assess the effect of serine protease inhibitors (aprotinin and AEBSF) and SERPINE1/2 on FSH-induced cumulus cell expansion, the production of prostaglandin E2 (PGE2) and retention of hyaluronic acid (HA) in expanding cumulus. The serine protease activity proved to be essential for the production of PGE2 and also for the retention of HA; the inhibition of plasminogen activators by SERPINE1/2 had the same effect. Collectively, these data indicate that plasmin is required for proper function of expanding cumulus cells in vitro and presumably also in vivo in the pre-ovulatory follicles.
- MeSH
- Aprotinin pharmacology MeSH
- Dinoprostone metabolism MeSH
- Follicle Stimulating Hormone pharmacology MeSH
- Plasminogen Activator Inhibitor 1 pharmacology MeSH
- Serine Proteinase Inhibitors pharmacology MeSH
- Cumulus Cells cytology drug effects metabolism MeSH
- Hyaluronic Acid metabolism MeSH
- Oocytes cytology drug effects metabolism MeSH
- Swine MeSH
- Serpin E2 pharmacology MeSH
- Sulfones pharmacology MeSH
- Animals MeSH
- Check Tag
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
The maturation of mammalian oocytes in vitro can be stimulated by gonadotropins (follicle-stimulating hormone, FSH) or their intrafollicular mediator, epidermal growth factor (EGF)-like peptide-amphiregulin (AREG). We have shown previously that in pig cumulus-oocyte complexes (COCs), FSH induces expression and the synthesis of AREG that binds to EGF receptor (EGFR) and activates the mitogen-activated protein kinase 3/1 (MAPK3/1) signaling pathway. However, in this study we found that FSH also caused a rapid activation of MAPK3/1 in the cumulus cells, which cannot be explained by the de novo synthesis of AREG. The rapid MAPK3/1 activation required EGFR tyrosine kinase (TK) activity, was sensitive to SRC proto-oncogene non-receptor tyrosine kinase (SRC)-family and protein kinase C (PKC) inhibitors, and was resistant to inhibitors of protein kinase A (PKA) and metalloproteinases. AREG also induced the rapid activation of MAPK3/1 in cumulus cells, but this activation was only dependent on the EGFR TK activity. We conclude that in cumulus cells, FSH induces a rapid activation of MAPK3/1 by the ligand-independent transactivation of EGFR, requiring SRC and PKC activities. This rapid activation of MAPK3/1 precedes the second mechanism participating in the generation and maintenance of active MAPK3/1-the ligand-dependent activation of EGFR depending on the synthesis of EGF-like peptides.
- MeSH
- Transcriptional Activation MeSH
- Amphiregulin metabolism MeSH
- ErbB Receptors metabolism MeSH
- Extracellular Signal-Regulated MAP Kinases genetics MeSH
- Follicle Stimulating Hormone pharmacology MeSH
- In Vitro Oocyte Maturation Techniques MeSH
- Cells, Cultured MeSH
- Cumulus Cells cytology drug effects metabolism MeSH
- Mitogen-Activated Protein Kinase 1 genetics MeSH
- Mitogen-Activated Protein Kinase 3 genetics MeSH
- Oocytes cytology drug effects metabolism MeSH
- Swine MeSH
- Signal Transduction drug effects MeSH
- src-Family Kinases metabolism MeSH
- Animals MeSH
- Check Tag
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
