Impact of media supplements FGF2, LIF and IGF1 on the genome activity of porcine embryos produced in vitro.

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.

Meiotic and developmental competence of growing pig oocytes derived from small antral follicles is enhanced in culture medium containing FGF2, LIF, and IGF1 (FLI medium).

BACKGROUND: Oocytes of large animal species isolated from small ovarian follicles (< 2 mm) are less competent to support early embryonic development after in vitro maturation and fertilization than their counterparts isolated from medium-sized and preovulatory follicles. This study aimed to assess the effect of a new maturation medium containing FGF2, LIF, and IGF1 (FLI medium) on the meiotic and developmental competence of pig cumulus-oocytes complexes (COCs) derived from the small and medium-sized follicles. METHODS: The growing oocytes were isolated from 1 to 2 (small follicle; SF) and the fully-grown ones from 3 to 6 (large follicle; LF) mm follicles and matured in a control M199 medium with gonadotropins and EGF and the FLI medium enriched by the triplet of growth factors. The matured oocytes were parthenogenetically activated and cultured to the blastocyst stage. Chromatin configuration before and during the culture and MAP kinase activity were assessed in the oocytes. Finally, the expression of cumulus cell genes previously identified as markers of oocyte quality was assessed. RESULTS: The maturation and blastocyst rates of oocytes gained from LF were significantly higher than that from SF in the control medium. In contrast, similar proportions of oocytes from LF and SF completed meiosis and developed to blastocysts when cultured in FLI. Most of the oocytes freshly isolated from SF possessed germinal vesicles with fine filaments of chromatin (GV0) or chromatin surrounding the nucleolus (GVI; 30%); the oocytes from LF were mainly in GVI (or GVII) exhibiting a few small lumps of chromatin beneath the nuclear membrane. When cultured in the FLI medium for 16 h, an acceleration of the course of maturation in oocytes both from SF and LF compared to the control medium was observed and a remarkable synchrony in the course of chromatin remodeling was noticed in oocytes from SF and LF. CONCLUSIONS: This work demonstrates that the enrichment of culture medium by FGF2, LIF, and IGF1 can enhance the meiotic and developmental competence of not only fully-grown, but also growing pig oocytes and significantly thus expanding the number of oocytes available for various assisted reproductive technology applications.

Characterization and identification of extracellular vesicles-coupled miRNA profiles in seminal plasma of fertile and subfertile rabbit bucks.

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.

Fatigue Properties of Spring Steels after Advanced Processing.

This article deals with the effect of strain-assisted tempering (SAT) on the fatigue properties of 54SiCr6 steel used for spring steel wires in a wide variety of automotive applications, including coil springs. This steel spring wire is extremely strong, having a high elastic limit and yield point, giving the steel excellent energy accumulation and fatigue properties. This combination opens up new possibilities in helical and cylindrical coil spring design, resulting in the reduction of both size and weight. Lightweight coil springs lead to improvements in fuel consumption, stability and vehicle traction. A large plastic deformation and SAT were applied to enhance the yield point of the study material. Improvements in the static and cyclic properties of steel springs were investigated using tensile tests and 3PB fatigue tests at ambient temperature. In addition, an advanced laser shock peening (LSP) process was employed to increase the fatigue resistance of the SAT material. The results presented here show great improvements in the static and fatigue properties over commercial steel treatment. The material quality of the wires was evaluated to be insufficient for further processing with cold coiling.

Porcine oocytes matured in a chemically defined medium are transcriptionally active.

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.

Dynamics of extracellular vesicle-coupled microRNAs in equine follicular fluid associated with follicle selection and ovulation.

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.

Small-extracellular vesicles and their microRNA cargo from porcine follicular fluids: the potential association with oocyte quality.

BACKGROUND: Ovarian follicular fluids (FFs) contain several kinds of regulatory factors that maintain a suitable microenvironment for oocyte development. Extracellular vesicles (EVs) are among the factors that play essential roles in regulating follicle and oocyte development through their cargo molecules that include microRNAs (miRNAs). This study aimed to investigate small-EV (s-EV) miRNAs in porcine FFs and their potential association with oocyte quality. METHODS: Individual aspirated oocytes were stained with lissamine green B stain (LB), a vital stain for oocyte quality, and each oocyte was classified as high-quality (unstained; HQ) or low-quality (stained; LQ). FFs corresponding to oocytes were pooled together into HQ and LQ groups. Small-EVs were isolated from FFs, characterized, and their miRNA cargo was identified using the Illumina NovaSeq sequencing platform. Additionally, s-EVs from the HQ and LQ groups were utilized to investigate their effect on oocyte development after co-incubation during in vitro maturation. RESULTS: A total of 19 miRNAs (including miR-125b, miR-193a-5p, and miR-320) were significantly upregulated, while 23 (including miR-9, miR-206, and miR-6516) were downregulated in the HQ compared to the LQ group. Apoptosis, p53 signaling, and cAMP signaling were among the top pathways targeted by the elevated miRNAs in the HQ group while oocyte meiosis, gap junction, and TGF-beta signaling were among the top pathways targeted by the elevated miRNAs in the LQ group. The supplementation of small-EVs during maturation does not affect the oocyte developmental rates. However, LQ s-EVs increase the proportion of oocytes with homogeneous mitochondrial distribution and decrease the proportion of heterogeneous distribution. CONCLUSION: Our findings indicated that FF-EVs contain different miRNA cargos associated with oocyte quality and could affect the mitochondrial distribution patterns during oocyte maturation.

Extended Continuous Cooling Transformation (CCT) Diagrams Determination for Additive Manufacturing Deposited Steels.

Continuous cooling transformation (CCT) diagrams are widely used when heat treating steels and represent which type of phase will occur in a material as it is cooled at different cooling rates. CCT diagrams are constructed on the basis of dilatometry measurements on relatively small testing samples (cylindrical shape with diameter of 4mm and length of 11 mm in this study). The main aim of this work was to demonstrate the possibility of evaluating the tensile test properties using mini-tensile tests from miniature volumes (1.4 × 10-7 m3 for one sample) subsequent to determination of the CCT diagram and to extend a standard CCT diagram with information about strength, ductility and the estimated value of the work-hardening coefficient. Mini-tensile tests (MTT) were recently developed due to the low availability of experimental material and have already been successfully used for local mechanical property characterization of metals. CCT diagrams were constructed for 42CrMo4 steel prepared by the laser-directed energy deposition (L-DED) process, for commercially available 42CrMo4 steel conventionally manufactured (for comparison of traditional processing and AM preparation) and for H13 tool steel deposited by the selective laser melting (SLM) process.

The Role of MAPK3/1 and AKT in the Acquisition of High Meiotic and Developmental Competence of Porcine Oocytes Cultured In Vitro in FLI Medium.

The developmental potential of porcine oocytes cultured in vitro was remarkably enhanced in a medium containing FGF2, LIF and IGF1 (FLI) when compared to a medium supplemented with gonadotropins and EGF (control). We analyzed the molecular background of the enhanced oocyte quality by comparing the time course of MAPK3/1 and AKT activation, and the expression of genes controlled by these kinases in cumulus-oocyte complexes (COCs) cultured in FLI and the control medium. The pattern of MAPK3/1 activation in COCs was very similar in both media, except for a robust increase in MAPK3/1 phosphorylation during the first hour of culture in the FLI medium. The COCs cultured in the FLI medium exhibited significantly higher activity of AKT than in the control medium from the beginning up to 16 h of culture; afterwards a deregulation of AKT activity occurred in the FLI medium, which was not observed in the control medium. The expression of cumulus cell genes controlled by both kinases was also modulated in the FLI medium, and in particular the genes related to cumulus-expansion, signaling, apoptosis, antioxidants, cell-to-cell communication, proliferation, and translation were significantly overexpressed. Collectively, these data indicate that both MAPK3/1 and AKT are implicated in the enhanced quality of oocytes cultured in FLI medium.

Fatigue properties of UFG Ti grade 2 dental implant vs. conventionally tested smooth specimens.

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.

Damage tolerant design of additively manufactured metallic components subjected to cyclic loading: State of the art and challenges.

Undoubtedly, a better understanding and the further development of approaches for damage tolerant component design of AM parts are among the most significant challenges currently facing the use of these new technologies. This article presents a thorough overview of the workshop discussions. It aims to provide a review of the parameters affecting the damage tolerance of parts produced by additive manufacturing (shortly, AM parts) with special emphasis on the process parameters intrinsic to the AM technologies, the resulting defects and the residual stresses. Based on these aspects, basic concepts are reviewed and critically discussed specifically for AM materials: Criteria for damage tolerant component design;Criteria for the determination of fatigue and fracture properties;Strategies for the determination of the fatigue life in dependence of different manufacturing conditions;Methods for the quantitative characterization of microstructure and defects;Methods for the determination of residual stresses;Effect of the defects and the residual stresses on the fatigue life and behaviour. We see that many of the classic concepts need to be expanded in order to fit with the particular microstructure (grain size and shape, crystal texture) and defect distribution (spatial arrangement, size, shape, amount) present in AM (in particular laser powder bed fusion). For instance, 3D characterization of defects becomes essential, since the defect shapes in AM are diverse and impact the fatigue life in a different way than in the case of conventionally produced components. Such new concepts have immediate consequence on the way one should tackle the determination of the fatigue life of AM parts; for instance, since a classification of defects and a quantification of the tolerable shapes and sizes is still missing, a new strategy must be defined, whereby theoretical calculations (e.g. FEM) allow determining the maximum tolerable defect size, and non-destructive testing (NDT) techniques are required to detect whether such defects are indeed present in the component. Such examples show how component design, damage and failure criteria, and characterization (and/or NDT) become for AM parts fully interlinked. We conclude that the homogenization of these fields represents the current challenge for the engineer and the materials scientist.

Adaptive and Biological Responses of Buffalo Granulosa Cells Exposed to Heat Stress under In Vitro Condition.

The steroidogenesis capacity and adaptive response of follicular granulosa cells (GCs) to heat stress were assessed together with the underlying regulating molecular mechanisms in Egyptian buffalo. In vitro cultured GCs were exposed to heat stress treatments at 39.5, 40.5, or 41.5 °C for the final 24 h of the culture period (7 days), while the control group was kept under normal conditions (37 °C). Comparable viability was observed between the control and heat-treated GCs at 39.5 and 40.5 °C. A higher release of E2, P4 and IGF-1 was observed in the 40.5 °C group compared with the 39.5 or 41.5 °C groups. The total antioxidant capacity was higher in response to heat stress at 39.5 °C. At 40.5 °C, a significant upregulation pattern was found in the expression of the stress resistance transcripts (SOD2 and NFE2L2) and of CPT2. The relative abundance of ATP5F1A was significantly downregulated for all heat-treated groups compared to the control, while TNFα was downregulated in GCs at 39.5 °C. Expression analyses of stress-related miRNAs (miR-1246, miR-181a and miR-27b) exhibited a significant downregulation in the 40.5 °C group compared to the control, whereas miR-708 was upregulated in the 39.5 and 40.5 °C groups. In conclusion, buffalo GCs exhibited different adaptive responses, to the different heat stress conditions. The integration mechanism between the molecular and secretory actions of the GCs cultured at 40.5 °C might provide possible insights into the biological mechanism through which buffalo GCs react to heat stress.

Inhibition of miR-152 during In Vitro Maturation Enhances the Developmental Potential of Porcine Embryos.

Oocyte developmental competence is regulated by various mechanisms and molecules including microRNAs (miRNAs). However, the functions of many of these miRNAs in oocyte and embryo development are still unclear. In this study, we managed to manipulate the expression level of miR-152 during oocyte maturation to figure out its potential role in determining the developmental competence of porcine oocytes. The inhibition (Inh) of miR-152 during oocyte maturation does not affect the MII and cleavage rates, however it significantly enhances the blastocyst rate compared to the overexpression (OvExp) and control groups. Pathway analysis identified several signaling pathways (including PI3K/AKT, TGFß, Hippo, FoxO, and Wnt signaling) that are enriched in the predicted target genes of miR-152. Gene expression analysis revealed that IGF1 was significantly up-regulated in the Inh group and downregulated in the OvExp group of oocytes. Moreover, IGF1R was significantly upregulated in the Inh oocyte group compared to the control one and IGFBP6 was downregulated in the Inh oocyte group compared to the other groups. Blastocysts developed from the OvExp oocytes exhibited an increase in miR-152 expression, dysregulation in some quality-related genes, and the lowest rate of blastocyst formation. In conclusion, our results demonstrate a negative correlation between miR-152 expression level and blastocyst rate in pigs. This correlation could be through targeting IGF system components during oocyte development.

Extracellular Vesicles as Mediators of Environmental and Metabolic Stress Coping Mechanisms During Mammalian Follicular Development.

Extracellular vesicles are evolutionarily conserved nano-sized phospholipid membraned structures and released from virtually all types of cells into the extracellular space. Their ability to carry various molecular cargos (mRNA, miRNA, proteins, and lipids) from one cell to the other to exert functional impact on the target cells enables them to play a significant role in cell to cell communication during follicular development. As the molecular signals carried by extracellular vesicles reflect the physiological status of the cells of origin, they are expected to mediate any effect of environmental or metabolic stress on the follicualr cells and the growing oocyte. Recent studies have evidenced that reproductive cells exposed to various environmental stressors (heat and oxidative stress) released extracellular vesicles enriched with mRNA and miRNA associated with stress response mechanisms. Moreover, the metabolic status of post-calving cows could be well-reflected in the follicular extracellular vesicle's miRNA profile, which signified the potential role of extracellular cellular vesicle molecular signals in mediating the effect of metabolic stress on follicular and oocyte development. In the present review, the potential role of extracellular vesicles in mediating the effect of environmental and metabolic stress in various reproductive cells and oocytes are thoroughly discussed Moreover, considering the importance of extracellular vesicles in shuttling protective or rescuing molecular signals during stress, their potential usage as means of targeted delivery of molecules to mitigate the effect of stress on oocytes are addressed as the focus of future research.

Plasma extracellular vesicle miRNAs as potential biomarkers of superstimulatory response in cattle.

The ability to predict superstimulatory response would be a beneficial tool in assisted reproduction. Using small RNAseq technology, we profiled extracellular vesicle microRNA (EV-miRNA) abundance in the blood plasma of heifers exhibiting variable responses to superstimulation. Estrous synchronized crossbred beef heifers (n = 25) were superstimulated and blood samples were collected from each heifer on Day 7 of consecutive unstimulated (U) and superstimulated (S) cycles. A subset of high (H) and low (L) responders was selected depending on their response to superstimulation and EV-miRNA profiles were analysed at both time-points in each heifer. Approximately 200 known miRNAs were detected in each sample with 144 commonly detected in all samples. A total of 12 and 14 miRNAs were dysregulated in UH vs. UL and in SH vs. SL heifers, respectively. Interestingly, miR-206 and miR-6517 exhibited the same differential expression pattern in H compared to L heifers both before and after superstimulation. Pathway analysis indicated that circadian rhythm and signaling pathways were among the top pathways enriched with genes targeted by dysregulated miRNAs in H vs. L responding heifers. In conclusion, heifers with divergent ovarian responses exhibited differential expression of plasma EV-miRNAs which may be used as a potential biomarker to predict superstimulation response.

Extracellular vesicles shuttle protective messages against heat stress in bovine granulosa cells.

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.

Local Mechanical Properties and Microstructure of EN AW 6082 Aluminium Alloy Processed via ECAP-Conform Technique.

An ultrafine-grained EN AW 6082 aluminum alloy was prepared by continuous serve plastic deformation (i.e., thermo-mechanical equal channel angular pressing (ECAP)-Conform process). A miniaturized tensile testing technique was used for estimating local mechanical properties with the aim to reveal the inhomogeneity of elastic and plastic properties in a workpiece volume. These inhomogeneities may appear due to the irregular shear strain distribution in a Conformed wire. Miniaturized samples for tensile testing were cut from the Conformed workpiece. Elongation of miniaturized samples was measured with a 2D digital image correlation technique as the optical extensometer. Tensile test characteristics, such as the yield strength and ultimate tensile strength, were consequently compared with results of conventional and hardness tests. The microstructure of Conformed bars was studied in the cross-section perpendicular and parallel to the extrusion direction using scanning electron microscope (SEM) and electron backscatter diffraction (EBSD) analysis. The microstructure of samples exhibits pronounced inhomogeneity, which is reflected by the hardness and tensile test results. Estimated distinctions between peripheral and central parts of the Conformed wires are probably a consequence of the significant strain differences realized in the upper and bottom wire parts.

Global transcriptome analysis of porcine oocytes in correlation with follicle size.

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.

Strain Range Dependent Cyclic Hardening of 08Ch18N10T Stainless Steel-Experiments and Simulations.

This paper describes and presents an experimental program of low-cycle fatigue tests of austenitic stainless steel 08Ch18N10T at room temperature. The low-cycle tests include uniaxial and torsional tests for various specimen geometries and for a vast range of strain amplitude. The experimental data was used to validate the proposed cyclic plasticity model for predicting the strain-range dependent behavior of austenitic steels. The proposed model uses a virtual back-stress variable corresponding to a cyclically stable material under strain control. This internal variable is defined by means of a memory surface introduced in the stress space. The linear isotropic hardening rule is also superposed. A modification is presented that enables the cyclic hardening response of 08Ch18N10T to be simulated correctly under torsional loading conditions. A comparison is made between the real experimental results and the numerical simulation results, demonstrating the robustness of the proposed cyclic plasticity model.

The serine protease inhibitors and SERPINE1/2 disrupt prostaglandin E2 production and hyaluronic acid retention in FSH-stimulated pig cumulus-oocyte complexes.

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.