Bovine Follicular Fluid Derived Extracellular Vesicles Modulate the Viability, Capacitation and Acrosome Reaction of Bull Spermatozoa

While follicular fluid (FF) is known to enhance the functional properties of spermatozoa, the role of FF-derived extracellular vesicles (EVs) in this respect is unknown. We hypothesized that bovine FF EVs convey signals to spermatozoa supporting sperm viability, inducing sperm capacitation and acrosome reaction. In this study, the effects of bovine FF EVs on sperm functions are evaluated. Irrespective of the size of the follicles which FF EVs had originated from, they were capable of supporting sperm viability, inducing capacitation and acrosome reaction. These effects were specific to the source of bovine FF EVs, as human-cell-line-derived or porcine FF EVs did not affect spermatozoa viability or induced capacitation and acrosome reaction. A minimum of 5 × 105 EVs/mL was adequate to maintain sperm viability and induce capacitation and acrosome reaction in spermatozoa. Interestingly, with FF EV trypsin treatment, FF EVs lost their ability to support sperm functions. In conclusion, this study demonstrates that bovine FF EVs can support spermatozoa function and may contribute to a favorable periconceptional microenvironment. This is an important aspect of the interactions between different sexes at the earliest stages of reproduction and helps to understand molecular mechanisms modulating processes such as sperm competition and female cryptic choice.

Trypsin induces an aversive response in zebrafish by PAR2 activation in keratinocytes

Previously we have shown that trypsin, a protein typically involved in digestion, is released from gills of both fresh and saltwater fishes into surrounding water under stress or injury. We have also shown that each species produces trypsin with different specific activities. In this report, using zebrafish as a model, we identified that trypsin induces an aversive response in zebrafish larvae and adult zebrafish. Since Protease-Activated Receptor 2 (PAR2) responds to trypsin, we tested whether the aversive response is dependent on the activation of PAR2 located on the zebrafish skin cells. Zebrafish larvae treated separately with neomycin and zinc sulfate also showed aversive response indicating neuromast, and olfactory cells are not involved in this aversion. Cultured keratinocytes from zebrafish showed a response to trypsin. Zebrafish larvae subjected to knockdown of par2a also exhibited reduced escape response. Similarly, par2a-deficient mutant larvae displayed no response to trypsin. Since it has been shown that stress activates PAR2 and sends signals to the brain as shown by the increased c-fos expression, we tested c-fos expression in adult zebrafish brains after trypsin treatment of adults and found enhanced c-fos expression by qRT-PCR. Taken together, our results show that the trypsin activates PAR2 on keratinocytes signaling the brain, and this pathway of trypsin-induced escape response will provide a unique communication mechanism in zebrafish. Furthermore, since PAR2 activation also occurs in pain/pruritus sensing, this model might be useful in elucidating components of signaling pathways in pain/pruritus.

A simple method for isolation of rest of trypsinized stem cells with magnetic beads

Till now, there is no laboratory in the world, which makes thoughts that there may be still stem cells remaining in the flasks after the trypsin treatment. The user checks the presence of stem cells during trypsinization with the microscope whether the whole population of stem cells has been collected. Magnetic beads are being used around the world to isolate different kinds of cells like CD4, CD8, CD34, and other cells. One of the advantages of magnetic isolation is that they can be used to isolate cells from solutions containing even a low number of targeted cells. We conducted the experiments to see whether all remaining MSC can be obtained from a trypsin-treated flask with magnetic beads isolation. During the magnetic isolation with CD90 specific Magnetic beads, it was found under the microscope that there are huge numbers of cells being attached to beads. The results indicate that there are still a huge number of rest cells in the trypsinized flask but most of the users think that they have isolated all cells, which is not true and they are throwing away valuable stem cells. The magnetic beads can be used to isolate the rest of MSC because for many applications, there is a need of the largest number of stem cells for conducting studies like flow cytometry, molecular testing along with pre-clinical and clinical studies.

Effects of trypsin on aggregation, disaggregation and aggregate morphology of red blood cells in autologous plasma and serum

We study the influence of trypsin on aggregation, disaggregation, and aggregate morphology of RBCs in autologous plasma and serum. The effect of trypsin on the surface charge of red blood cells and the aggregation of glutaraldehyde fixed cells after treatment with the enzyme was also studied. RBC aggregation was studied by means of an aggregometer and microscopic observations. The results obtained in this study indicate that trypsin treatment increases RBCs aggregation in autologous plasma and serum. The disaggregation of erythrocytes after trypsin treatment considerably decreased in autologous plasma and serum. Increase in the strength of red blood cell aggregates was observed in autologous plasma and serum. The microscopic images of RBCs aggregates indicate the formation of globular (pathologic) structures of aggregates in autologous plasma and serum. Trypsin decrease the surface charge of RBCs. In autologous plasma and serum, the cup shapes of RBCs appear. The control RBCs fixed with glutaraldehyde were not aggregated after their placement in autologous plasma. At the same time, red blood cells pretreated with trypsin and fixed with glutaraldehyde interact with each other in autologous plasma. The physiological significance of glycoproteins of erythrocyte surface for RBCs aggregation was discussed.

Microfracture Augmentation With Trypsin Pretreatment and Growth Factor–Functionalized Self-assembling Peptide Hydrogel Scaffold in an Equine Model

Background: Microfracture augmentation can be a cost-effective single-step alternative to current cartilage repair techniques. Trypsin pretreatment combined with a growth factor–functionalized self-assembling KLD hydrogel (“functionalized hydrogel”) has been shown to improve overall cartilage repair and integration to surrounding tissue in small animal models of osteochondral defects. Hypothesis: Microfracture combined with trypsin treatment and a functionalized hydrogel will improve reparative tissue quality and integration as compared with microfracture alone in an equine model. Study Design: Controlled laboratory study. Methods: Bilateral cartilage defects (15-mm diameter) were created on the medial trochlear ridge of the femoropatellar joints in 8 adult horses (16 defects total). One defect was randomly selected to receive the treatment, and the contralateral defect served as the control (microfracture only). Treatment consisted of 2-minute trypsin pretreatment of the surrounding cartilage, subchondral bone microfracture, and functionalized hydrogel premixed with growth factors (platelet-derived growth factor and heparin-binding insulin-like growth factor 1). After surgery, all horses were subjected to standardized controlled exercise on a high-speed treadmill. Clinical evaluation was conducted monthly, and radiographic examinations were performed at 2, 16, 24, 32, 40, and 52 weeks after defect creation. After 12 months, all animals were euthanized. Magnetic resonance imaging, arthroscopy, gross pathologic evaluation of the joint, histology, immunohistochemistry, and biomechanical analyses were performed. Generalized linear mixed models (with horse as random effect) were utilized to assess outcome parameters. When P values were <.05, pairwise comparisons were made using least squares means. Results: Improved functional outcome parameters were observed for the treatment group, even though mildly increased joint effusion and subchondral bone sclerosis were noted on imaging. Microscopically, treatment resulted in improvement of several histologic parameters and overall quality of repaired tissue. Proteoglycan content based on safranin O–fast green staining was also significantly higher in the treated defects. Conclusion: Trypsin treatment combined with functionalized hydrogel resulted in improved microfracture augmentation. Clinical Relevance: Therapeutic strategies for microfracture augmentation, such as those presented in this study, can be cost-effective ways to improve cartilage healing outcomes, especially in more active patients.

Study on antibacterial activity and self-assembly of ovalbumin-derived peptides

Antimicrobial activity and self-assembly of the modified TK913 peptide are described. We designed the peptides TK9Z1-4 and TKZ2-3 based on the TK913 sequence and prepared these peptides by Fmoc-SPPS. TKZ3 shows morphology change in the different concentration and potent antimicrobial activities. In addition, TKZ3 has stability in trypsin treatment.

Transcriptomic analysis reveals hub genes and subnetworks related to ROS metabolism in Hylocereus undatus through novel superoxide scavenger trypsin treatment during storage

Background: It was demonstrated in our previous research that trypsin scavenges superoxide anions. In this study, the mechanisms of storage quality improvement by trypsin were evaluated in H. undatus. Results: Trypsin significantly delayed the weight loss and decreased the levels of ROS and membrane lipid peroxidation. Transcriptome profiles of H. undatus treated with trypsin revealed the pathways and regulatory mechanisms of ROS genes that were up- or downregulated following trypsin treatment by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses. The current results showed that through the regulation of the expression of hub redox enzymes, especially thioredoxin-related proteins, trypsin can maintain low levels of endogenous active oxygen species, reduce malondialdehyde content and delay fruit aging. In addition, the results of protein-protein interaction networks suggested that the downregulated NAD(P)H and lignin pathways might be the key regulatory mechanisms governed by trypsin.Conclusions: Trypsin significantly prolonged the storage life of H. undatus through regulatory on the endogenous ROS metabolism. As a new biopreservative, trypsin is highly efficient, safe and economical. Therefore, trypsin possesses technical feasibility for the quality control of fruit storage.