High Mitochondrial Membrane Potential Research Articles

Enantioselective biomarkers of maize toxicity induced by hexabromocyclododecane based on submicroscopic structure, gene expression and molecular docking

Hexabromocyclododecane (HBCD), as a monitored chemical of the Chemical Weapons Convention, the Stockholm Convention and the Action Plan for New Pollutants Treatment in China, raises significant concerns on its impact of human health and food security. This study investigated enantiomer-specific biomarkers of HBCD in maize (Zea mays L.). Upon exposure to HBCD enantiomers, the maize root tip cell wall exhibited thinning, uneven cell gaps, and increased deposition on the cell outer wall. Elevated malondialdehyde (MDA) indicated lipid peroxidation, with higher mitochondrial membrane potential (MMP) inhibition in (+)-enantiomer treatments (47.2%–57.9%) than (−)-enantiomers (14.4%–37.4%). The cell death rate significantly increased by 37.7%–108.8% in roots and 16.4%–62.4% in shoots, accompanied by the upregulation of superoxide dismutase isoforms genes. Molecular docking presenting interactions between HBCD and target proteins, suggested that HBCD has an affinity for antioxidant enzyme receptors with higher binding energy for (+)-enantiomers, further confirming their stronger toxic effects. All indicators revealed that oxidative damage to maize seedlings was more severe after treatment with (+)-enantiomers compared to (−)-enantiomers. This study elucidates the biomarkers of phytotoxicity evolution induced by HBCD enantiomers, providing valuable insights for the formulation of more effective policies to safeguard environmental safety and human health in the future.

Enrichment of thawed boar spermatozoa with an intact membrane using Magnetic Activated Cell Sorting

Not all boar sperm samples survive cryopreservation well. A method of eliminating damaged sperm might enable more cryopreserved boar semen to be used for pig breeding. In this study we investigated the use of Magnetic Activated Cell sorting (MACS) to eliminate damaged sperm from thawed boar semen samples. The thawed samples were mixed with Dead cell removal particles and were applied to the column in a SuperMACS II. Different fractions were collected: Original sample (O), Flow-through (FT), and Eluate (E). Sperm membrane integrity, mitochondrial membrane potential and reactive oxygen species were evaluated by flow cytometry after staining with SYBR 14 and propidium iodide, or 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethylbenzimidazolylcarbocyanine iodide, or hydroethidine and dichlorodihydrofluorescein diacetate, respectively. The FT samples had increased membrane integrity, a greater proportion of sperm with high mitochondrial membrane potential and a greater proportion of sperm negative for hydrogen peroxide than O samples (P<0.0001), which in turn had increased membrane integrity than E samples (P <0.0001). However, differences were seen between boars. The FT samples had increased values of live, superoxide positive sperm than O samples (P <0.0001) and O samples had greater values than E samples (P <0.0001), while there was no effect of boar. Sperm quality was best in the FT fraction, comprising approximately 32% of the sperm sample. In conclusion, although there were differences between boars, MACS separation can improve sperm quality in thawed semen samples. It would be interesting to see if this improvement is reflected in fertility outcomes.

Mitochondria-Selective Dicationic Small-Molecule Ligand Targeting G-Quadruplex Structures for Human Colorectal Cancer Therapy.

Mitochondria are important drug targets for anticancer and other disease therapies. Certain human mitochondrial DNA sequences capable of forming G-quadruplex structures (G4s) are emerging drug targets of small molecules. Despite some mitochondria-selective ligands being reported for drug delivery against cancers, the ligand design is mostly limited to the triphenylphosphonium scaffold. The ligand designed with lipophilic small-sized scaffolds bearing multipositive charges targeting the unique feature of high mitochondrial membrane potential (MMP) is lacking and most mitochondria-selective ligands are not G4-targeting. Herein, we report a new small-sized dicationic lipophilic ligand to target MMP and mitochondrial DNA G4s to enhance drug delivery for anticancer. The ligand showed marked alteration of mitochondrial gene expression and substantial induction of ROS production, mitochondrial dysfunction, DNA damage, cellular senescence, and apoptosis. The ligand also exhibited high anticancer activity against HCT116 cancer cells (IC50, 3.4 μM) and high antitumor efficacy in the HCT116 tumor xenograft mouse model (∼70% tumor weight reduction).

Cryopreservation of ram semen: baicalein efficiency on oxidative stress, chromatin integrity, viability and motility post thaw.

Baicalein (B) has potential antioxidant properties, but it has not been tested as a ram semen extender. This study aimed to assess the impact of B on various sperm parameters and determine its potential influence on semen quality after the freeze-thawing process. During the breeding season, ejaculates were obtained from four rams with the aid of an artificial vagina. The collected mixed semen samples were divided into four groups: control (C; 0), B0.5 (0.5 mM), B1 (1 mM), and B2 (2 mM). After semen extension, the samples were loaded into 0.25 mL straws and stored for 2 h at 4°C prior to freezing in liquid nitrogen vapor and thawed in a water bath at 37°C. Among the groups, B0.5 demonstrated the highest progressive motility results, while B1 and B2 exhibited reduced motility (p < 0.05). In terms of high mitochondrial membrane potential, plasma membrane and acrosome integrity, and viability, B0.5 showed significantly superior outcomes to the other B groups (p < 0.05), although it was not significantly better than C. B1 displayed the highest plasma membrane integrity levels (p < 0.05). Notably, B2 displayed the lowest total antioxidant status levels among the groups (p < 0.05). The findings of this study suggested that the in vitro spermatological characteristics of ram spermatozoa such as progressive motility and chromatin integrity can be protected from the freeze-thawing process by using the 0.5 mM dose of baicalein as a semen extender. The treatment of sperm freezing might benefit from further in-depth research on the role of B in the improvement of cryoinjury and its underlying processes.

Supplementation of Extender with Melatonin Improves the Motility, Mitochondrial Membrane Potential, and Fertilization Ability of Cryopreserved Brown-Marbled Grouper Sperm.

Sperm cryopreservation is a valuable tool for breeding, conservation, and genetic improvement in aquatic resources, while oxidative damage will cause a decline in sperm quality during this progress. Melatonin (MT), a natural antioxidant hormone, is used as an additive in sperm cryopreservation to reduce cellular damage from oxidative stress. Here, we aimed to investigate the effect of adding MT to the freezing medium in sperm cryopreservation of brown-marbled grouper (Epinephelus fuscoguttatus). Different concentrations of MT (0, 0.1, 0.25, and 0.5 mg/mL) were tested. We evaluated sperm motility, viability, apoptosis, mitochondrial membrane potential (MMP), and fertilization ability to assess the effects of MT supplementation. Our results demonstrated that the addition of MT to the extender improved the post-thaw motility, MMP, and fertilization ability of brown-marbled grouper sperm. The total motility, curvilinear velocity, straight linear velocity, and average path velocity in MT-treated groups (0.1 and 0.25 mg/mL) exhibited significantly higher values than that of the control group. A higher MMP (p < 0.05) was observed in the group treated with 0.25 mg/mL MT, suggesting that supplementation of MT in the extender might be able to protect mitochondrial membrane integrity effectively. Regarding fertilizing ability, 0.25 mg/mL MT yielded a significantly higher hatching rate than the control. An adverse effect was found with the concentration of MT up to 0.5 mg/mL, suggesting the possible toxicity of a high-dose addition. In this study, we optimized the sperm cryopreservation protocol of brown-marbled grouper, which might be valuable for sperm cryopreservation and sample commercialization of groupers and other fish.

In Vitro Effects of Charged and Zwitterionic Liposomes on Human Spermatozoa and Supplementation with Liposomes and Chlorogenic Acid during Sperm Freezing.

Semen handling and cryopreservation induce oxidative stress that should be minimized. In this study, human semen was supplemented during cryopreservation with formulations of handmade liposomes and chlorogenic acid (CGA), an antioxidant compound. Zwitterionic (ZL), anionic (AL), and cationic (CL) liposomes were synthesized and characterized. Three aliquots of swim-up-selected sperm were incubated with ZL, AL, and CL (1:10,000), respectively. The percentages of sperm with progressive motility, high mitochondrial membrane potential (MMP; JC-1), double-stranded DNA (dsDNA acridine orange), and acrosome integrity (Pisum sativum agglutinin) were assessed. Then, human semen was frozen using both 1:10,000 ZL and CGA as follows: freezing medium/empty ZL (EL), freezing medium/empty ZL/CGA in the medium (CGA + EL), freezing medium/CGA loaded ZL (CGA), freezing medium (CTR). The same sperm endpoints were evaluated. ZL were the most tolerated and used for semen cryopreservation protocols. All the supplemented samples showed better endpoints versus CTR (p < 0.001). In particular, spermatozoa from the CGA and CGA + EL A samples showed increased motility, dsDNA, and acrosome integrity versus CTR and EL (p < 0.001; motility EL vs. CGA + EL p < 0.05). ZL and CGA can improve post-thaw sperm quality, acting on both cold shock effect management and oxidative stress. These findings open new perspectives on human and animal reproduction.

Novel Antioxidant Humanin Analogue Restores Bull Semen Cryosurvivability

The current study was intended to determine the effects of humanin analogue (HNG) supplementation on semen freezability and oxidative stress parameters of cryopreserved bull spermatozoa. A total of 24 semen ejaculates, from Frieswal breeding bulls, were selected for the experiment. Samples with ≥70% progressive motility, ≥500 million spermatozoa/mL, and &gt;2 mL volume were utilized for further processing, supplementation, and cryopreservation. Each sample was divided into four equal groups and the experiment consisted of three treatment groups and one control group. In the control group, the spermatozoa were frozen in an extender without HNG, while in the other three groups, HNG supplementation was done at a concentration of 2, 10, and 20 μM, respectively. The fortification of HNG at a concentration of 10 and 20 µM significantly (p &lt;0.05) improved the percent post-thaw motility and kinetic variables as assessed by CASA, viability, acrosome- and plasma membrane-integrity as compared to 2 µM and control group. The supplementation showed significant (p &lt;0.05) controlled oxidative stress as exhibited by a reduction in lipid peroxidation and reactive oxygen species levels. The supplemented groups also exhibited significant (p &lt;0.05) improvements in total antioxidant levels, increased mean percentage of spermatozoa exhibiting high mitochondrial membrane potential, DNA integrity, acrosome-intact noncapacitated spermatozoa, and zona binding ability. In conclusion, the supplementation of humanin analogue at 10 and 20 µM significantly improved the post-thaw semen quality, functional parameters, and zona binding ability and controlled the oxidative stress in cryopreserved spermatozoa. Thus, the supplementation of its analogue may potentially prove to be a valuable approach for the improvement of semen freezability in crossbred Frieswal bulls.

Novel Apoplastic Antifreeze Proteins of Deschampsia antarctica as Enhancer of Common Cell Freezing Media for Cryobanking of Genetic Resources, a Preliminary Study.

Antifreeze proteins (AFPs) are natural biomolecules found in cold-adapted organisms that lower the freezing point of water, allowing survival in icy conditions. These proteins have the potential to improve cryopreservation techniques by enhancing the quality of genetic material postthaw. Deschampsia antarctica, a freezing-tolerant plant, possesses AFPs and is a promising candidate for cryopreservation applications. In this study, we investigated the cryoprotective properties of AFPs from D. antarctica extracts on Atlantic salmon spermatozoa. Apoplastic extracts were used to determine ice recrystallization inhibition (IRI), thermal hysteresis (TH) activities and ice crystal morphology. Spermatozoa were cryopreserved using a standard cryoprotectant medium (C+) and three alternative media supplemented with apoplastic extracts. Flow cytometry was employed to measure plasma membrane integrity (PMI) and mitochondrial membrane potential (MMP) postthaw. Results showed that a low concentration of AFPs (0.05 mg/mL) provided significant IRI activity. Apoplastic extracts from D. antarctica demonstrated a cryoprotective effect on salmon spermatozoa, with PMI comparable to the standard medium. Moreover, samples treated with apoplastic extracts exhibited a higher percentage of cells with high MMP. These findings represent the first and preliminary report that suggests that AFPs derived from apoplastic extracts of D. antarctica have the potential to serve as cryoprotectants and could allow the development of novel freezing media.

Arap aygırlarında spermatolojik parametrelerin sezonun farklı dönemlerine göre değişimlerinin değerlendirilmesi

In this study, determining of the variations on spermatological characteristics of Arabian stallion semen in the different periods of the breeding season is aimed. The periods have been described as the beginning of the season (BS: 15th February – 15th March), mid-season (MS: 15th April – 15th May), and end of the season (ES: 30th May – 30th June). A total of 7 Arabian stallions that were 5-10 years old, with body condition scores between 3-3.5 and without any problems in terms of general and reproductive health, were enrolled in the study. In total, three semen samples were collected from each stallion, once in each period. Then, these samples were evaluated in terms of volume, concentration, total motility (TMOT), progressive motility (PMOT), viability, high mitochondrial membrane potential (HMMP), acrosome integrity (AI), capacitation index (Ci), and lipid peroxidation (LPO) parameters, which were analyzed by computer assisted sperm analysis and flowcytometry devices. As a result, the differences in volume, concentration, TMOT, and PMOT between the BS, MS, and ES periods were statistically insignificant (p&gt;0.05). The lowest viability values were determined in the BS period (p

Pyrroloquinoline Quinone Improves Ram Sperm Quality through Its Antioxidative Ability during Storage at 4 °C.

Sperm motility is an important factor in the migration of sperm from the uterus to the oviduct. During sperm preservation in vitro, sperm generates excessive ROS that damages its function. This study aims to investigate whether the addition of pyrroloquinoline quinone (PQQ) to the diluted medium could improve chilled ram sperm quality, and then elucidates the mechanism. Ram semen was diluted with Tris-citric acid-glucose (TCG) medium containing different doses of PQQ (0 nM, 10 nM, 100 nM, 1000 nM, 10,000 nM), and stored at 4 °C. Sperm motility patterns, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential, reactive oxygen species (ROS) levels, malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, and ATP levels were measured after preservation. Furthermore, the expressions of NADH dehydrogenase 1 (MT-ND1) and NADH dehydrogenase 6 (MT-ND6) in sperm were also detected by western blotting. In addition, sperm capacitation and the ability of sperm to bind to the zona pellucina were also evaluated. It was observed that the addition of PQQ significantly (p < 0.05) improved ram sperm motility, membrane integrity, and acrosome integrity during preservation. The percentage of sperm with high mitochondrial membrane potential in the PQQ treatment group was much higher than that in the control. In addition, supplementation of PQQ also decreased the sperm MDA and ROS levels, while increasing ATP levels. Interestingly, the levels of MT-ND1 and MT-ND6 protein in sperm treated with PQQ were also higher than that of the control. Furthermore, the addition of 100 nM PQQ to the medium decreased ROS damage in MT-ND1 and MT-ND6 proteins. The addition of 100 nM PQQ significantly (p < 0.05) increased protein tyrosine phosphorylation in ram sperm after induced capacitation. Furthermore, the value of the sperm-zona pellucida binding capacity in the 100 nM PQQ treatment group was also much higher than that of the control. Overall, during chilled ram- sperm preservation, PQQ protected ram sperm quality by quenching the ROS levels to reduce ROS damage and maintain sperm mitochondrial function, and preserved the sperm's high ability of fertilization.

Selective Antitumor Effect and Lower Toxicity of Mitochondrion-Targeting Derivatization of Triptolide.

Triptolide has a significant antitumor activity, but its toxicity limits its clinical application. As the mitochondrion-targeting strategy showed an advantage in selective antitumor effect based on the higher mitochondrial membrane potential (MMP) in tumor cells than normal cells, the lipophilic cations triphenylphosphonium and E-4-(1H-indol-3-yl vinyl)-N-methylpyridinium iodide (F16) were selected as targeting carriers for structural modification of triptolide. The derivatives bearing F16 generally retained most antitumor activities, overcame its inhibition plateau phenomena, and enhanced its selective antitumor effect in lung cancer. The representative derivative F9 could accumulate in the mitochondria of NCI-H1975 cells, inducing apoptosis and a dose-dependent increase in intracellular reactive oxygen species and reducing MMP. Moreover, no effects were observed in normal cells BEAS-2B. In vivo studies showed that the developmental, renal, and liver toxicities of F9 to zebrafish were significantly lower than those of triptolide. This study provides a promising idea to relieve the toxicity problem of triptolide.

Stathmin-1 Regulates Hematopoietic Stem Cell Mitochondrial and Cellular Function

Mitochondrial function is important to control HSC quiescence and self-renewal. HSCs are known to have low oxidative phosphorylation (OXPHOS), and low mitochondrial reactive oxygen species, however, studies have shown that HSCs have high mitochondrial membrane potential (MMP), which supports their function (Hinge et al 2020; Mansell et al 2021). Microtubules are important for normal mitochondrial function, acting as the main pathway through which mitochondria transport occurs. Stathmin 1 (Stmn1) is a cytoplasmic phosphoprotein that regulates microtubule dynamics via the promotion of microtubule catastrophe or the sequestration of free alpha/beta-tubulin dimers. It is highly expressed in normal hematopoietic stem cells (HSCs) and neural tissues. Also known as oncoprotein 18 (OP18) or leukemia-associated protein 18 (LAP18), Stmn1 is overexpressed in multiple cancers, including both myeloid and lymphoid leukemias. Despite its robust expression in normal HSCs and its elevated expression in leukemias, little is known about Stmn1's role in either normal or malignant HSCs. Herein, we found that Stmn1 is a critical regulator of HSC mitochondrial and cellular function. To determine the role of Stmn1 in HSCs, we first assessed HSC numbers and function in Stmn1 -/-mice. We determined HSC and progenitor numbers, as well as peripheral blood lineages, in young adult Stmn1 -/-mice and WT littermates. Compared to WT, Stmn1 -/- mice have mild anemia and thrombocytopenia, however, HSC numbers are similar. Despite normal HSC numbers, Stmn1 -/-HSCs had markedly impaired repopulating activity in competitive transplantation experiments (10% vs. 60% chimerism at 24 weeks p&amp;lt;0.0001). Similar defects in repopulating activity were found in non-competitive transplants and competitive intra-tibial transplants (done to circumvent any potential homing defects). In addition, Stmn1 -/- HSCs formed fewer colonies when plated in methocult, and were not capable of serial replating. Finally, Stmn1 -/- mice had delayed hematopoietic recovery following a single dose of the chemotherapeutic agent 5-fluorouracil (5-FU). We next assessed the cycling and apoptosis of HSCs. Stmn1 -/- HSCs are hyper-quiescent at baseline, although they can be stimulated to cycle in response to cytokine stimulation (1.3% vs. 3.4% S/G 2/M phase at baseline and 3.4% vs. 7% S/G 2/M phase following stimulation). In addition, they have elevated levels of apoptosis compared to WT (18.5% vs. 9.90%). Consistent with its role as a microtubule destabilizer, Stmn1 expression in HSCs coincides with tubulin expression by confocal immunofluorescent microscopy, and Stmn1 -/- HSCs have decreased tubulin compared to WT HSCs. To gain further insight into their impaired function, we next performed RNA-seq on sorted Stmn1 -/- and WT HSCs. Differential pathway analysis showed alterations in genes associated with cellular metabolism, particularly mitochondrial function (OXPHOS pathway) in the Stmn1 -/- HSCs compared to WT. Indeed, measurement of mitochondrial function using the Seahorse analyzer showed that Stmn1 -/- HSCs and progenitors have impaired maximal respiratory capacity compared to WT (251.29 vs 411.84 pmol/min). In addition, Stmn1 -/- HSCs showed reduced mitochondrial membrane potential and increased mtROS as assessed by flow-based assays. Finally, the mitochondria of Stmn1 -/- HSCs showed markedly abnormal cristae morphology by TEM, consistent with their impaired function, and Stmn1 -/- HSCs have decreased mitochondria number compared to WT (medium 12.8 vs 17.3 per 2D slice cell; p&amp;lt;0.0250). As elevated ROS can impair HSC function, we assessed Stmn1 -/- HSCs following treatment with MitoTEMPO, a specific scavenger of mitochondrial superoxide. Treatment of Stmn1 -/- mice with 4mg/Kg of MitoTEMPO or vehicle control for two weeks resulted in improved HSC colony formation and serial replating capacity in Methocult. Together, these data identify Stmn1 as a critical regulator of HSC function and suggest a model whereby Stmn1 promotes normal microtubule dynamics and mitochondrial health in HSCs. Ongoing experiments are aimed at understanding Stmn1's role in regulating mitochondrial quality control processes such as mitophagy and fission in both normal and malignant HSCs, and in elucidating the effects of Stmn1 loss on leukemic cell growth and viability.

Enhancement of Mitochondrial Membrane Potential and Pro-Survival Pathways with Azole Compound C7 Resulting in Expansion of Hematopoietic Stem and Progenitor Cells

Ex-vivo expansion of hematopoietic stem &amp; progenitor cells (HSPC) could improve hematopoietic recovery and reduce post-transplant complications, especially for umbilical cord blood (UCB) grafts where limited total cell numbers result in delayed engraftment. Freshly-thawed UCB mononucleated cells (MNCs) or purified CD34 + cells were cultured in animal component free medium supplemented with C7 and a basal cytokine cocktail. The effects of the expansion protocol were measured with phenotypic, in-vitro, and in-vivo functional assays. A range of molecular techniques were also performed to elucidate the mechanisms of action of C7. In previous studies, expansion cultures initiated with UCB-MNCs and supplemented with C7 resulted in significant expansions of UCB HSPC over 11 days and these grafts demonstrated elevated and sustained multi-lineage engraftment of human cells in primary NSG mice (Bari et al., 2018). In UCB-CD34+ enriched cultures, the addition of C7 to a basal cytokine cocktail also boosted absolute CD45+CD34(BR)+CD38-CD45RA- progenitor expansion by 283±29.4 fold over 11 days which was 1.86±0.12 fold higher than control cultures (p&amp;lt;0.001; n=6). Furthermore, C7 induced significant expansion of primitive HSPC in UCB-MNC defined by phenotype CD45+CD34(BR)+CD38-CD45RA-CD90+CD49f+ by more than 633±8.5 fold, which is 7.5±0.16 fold higher compared to control cultures (p&amp;lt;0.01; n=3). The addition of C7 to CD34+ cells from mobilized peripheral blood (PB) and bone marrow (BM) also resulted in a significant expansion of HSPCs (p&amp;lt;0.05; n=3) (Fig 1A). When expanded UCB-CD34+ were engrafted in NSG mice, NSG PB analysis at week 3 and week 28 post-transplantation showed significantly higher (p&amp;lt;0.01) human CD45+ cell engraftment in C7 expanded graft (16.4%±2.6) than cytokine expanded graft (7.8%±0.9). NSG BM analysis at week 31 also exhibited similar trends for human CD45+ cell engraftment in C7 expanded grafts with multilineage reconstitution (p&amp;lt;0.01; n=6). Significantly enhanced CD45+ cell viability (p&amp;lt;0.01; n=3) was observed in C7 cultures compared to control cultures. Mitochondrial health was also improved as confirmed by lower mitochondrial superoxides and higher mitochondrial membrane potential in C7-cultured UCB-MNCs (p&amp;lt;0.05; n=3) that is reflective of reduced reactive oxygen species (ROS) (Fig 1B). As C7 is a chemical analogue of a p38 inhibitor (SB203580), p-p38 (T180/Y182) was significantly reduced in C7-expanded CD34+ culture compared to controls. Significant increases in HIF-1α in C7 expanded CD34+ cells were also observed, potentially indicative of a hypoxic state induced by C7 to benefit HSPC together with reduced levels of ROS. Kinase inhibition profiling studies on C7 identified CK1δ, a kinase involved in Wnt and p53 signalling pathways, as one of the activation targets directly activated by C7. CK1δ was boosted in C7-expanded cells and this was further validated with western blots on C7 expanded pooled CD34+. CK1δ plays a putative role in p53 activation which is stipulated to promote HSC quiescence favouring long-term maintenance of HSPCs. Western blot analysis also revealed that p53 and BCL-2 were increased with C7 expanded CD34+. GSK3β was also boosted in C7 expanded pooled CD34+ cultures possibly as a result of CK1δ signalling leading to increased proliferation via the Wnt signalling pathway. Multiple safety studies were conducted on C7 to ensure its safety in clinical applications. This included the BIOMAP phenotypic safety and toxicology tests on human primary cells, AMES test and fluctuation assays for bacteria and SAFETYscan47 TM In Vitro Pharmacological Profiling for biochemical safety, all of which were negative for any cytotoxicity or off-targets associated with acute toxicity. No changes were detected in cytogenetics and fluorescence in situ hybridization with UCB-MNC for major leukemia-related genetics as well when they were cultured with C7. As a small molecule capable of significant HSPC expansion from both UCB and PB sources, C7 is promising for the future of HSPC transplantation. The mechanism behind C7 is likely to be a multi-pronged one with p38 inhibition, increase in HIF-1α, CK1δ activation, improved mitochondrial health, and induction of pro-survival pathways for expanding HSPC populations.

297 Evaluation of Semen Characteristics During Pubescent Maturation After Prenatally Induced Stress of Brahman Bulls

Abstract The objective of this study was to determine if prenatal stress impacted semen characteristics during pubescent maturation of Brahman bulls (n = 23). Prenatal stress was achieved by transporting Brahman dams for 2 h on d 60, 80, 100, 120, and 140 (± 5 d) of gestation. Semen from sexually maturing males born to transported (PNS; n = 11) or non-transported (CON; n = 12) dams was classified based on motility and concentration as prepubertal (&amp;lt; 10% and &amp;lt; 50 million), peripubertal (≥ 10% or ≥ 50 million), pubertal (≥ 10% and ≥ 50 million), or sexually mature (≥ 30% or ≥ 500 million). Sperm characteristics were determined by staining sperm with specific dyes [viability - Propidium Iodide and SYBR-14; Mitochondrial membrane potential (MMP) – JC-1; DNA damage – Aracidine orange; ROS –conversion of H2DCFDA to DCF and conversion of hydroethidine to ethidium] and analysis on an Amnis FlowSight flow cytometer. Treatment differences were tested by SAS procedures. Analysis of repeated measures (PROC MIXED) and chi-square (PROC FREQ) compared semen characteristics and percent pubertal versus mature, respectively. There was a weak tendency (P = 0.14) for more control bulls to reach puberty (100%) compared with PNS bulls (82%), but no difference (P = 1.0) in percentage reaching sexual maturity by 22 months of age (82% and 82%). Scrotal circumference (P &amp;lt; 0.01) and motility [at collection (P &amp;lt; 0.01) and post-thaw (P = 0.05)] increased as bulls matured but was not impacted by treatment (P &amp;gt; 0.86) or treatment by time (P &amp;gt; 0.60). Sperm viability (at collection, post-thawing, and post-stress test) was not impacted by treatment (P &amp;gt; 0.20), time (P &amp;gt; 0.53), or treatment by time (P &amp;gt; 0.50). Proportion of viable sperm with high MMP was not impacted by treatment (P = 0.70) or treatment by time (P = 0.49), but was increased (P = 0.03) in early peripubertal collections compared with pubertal or mature collections. Proportion of sperm with damaged DNA was decreased in PNS (1.6 ± 0.3%; P = 0.04) versus CON (2.4 ± 0.2%) but not impacted by time (P = 0.47) or treatment by time (P = 0.41). Percentage of sperm with elevated ROS (H2O2 and •O2) was not impacted by treatment (P &amp;gt; 0.33), time (P &amp;gt; 0.17), or treatment by time (P &amp;gt; 0.41). Prenatal stress tended to impact the proportion of bulls that reached puberty but had no impact on the percentage that reached sexual maturity by 22 months of age. Prenatal stress also impacted the proportion of sperm with DNA damage, but did not impact scrotal circumference, motility, MMP, or ROS. Stage of sexual maturity impacted scrotal circumference, motility, and MMP, but did not impact ROS. In summary, PNS may impact the ability to reach puberty, but did not impact sperm characteristics from prepuberty to sexual maturity.

Determination of the Effect of Epigallocatechin Gallate on Oxidative Stress, Apoptosis and Sperm Quality in Rabbit Semen

Objectives;In this study, it was aimed to determine the effect of Epigallocatechin gallate (EGCG) on the cryopreservation of rabbit semen. &#x0D; Material and metod; For this purpose, semen was collected from six male New Zealand rabbits. Collected semen were divided into 5 groups as control, 25 µM, 50 µM, 100 µM and 200 µM. Semen samples were reconstituted and frozen after adding EGCG. Frozen straws were then thawed. &#x0D; Results; After freezing and thawing, total motility, progressive motility, and rate of fast and medium speed sperm were significantly higher in the 50 µM group than in the other groups. Static sperm ratio was found to be lower in the 50 µM group than in the other groups. When the flow cytometry results were examined, there was no statistical difference between the groups in terms of the ratio of dead and live sperm. However, the rate of dead sperm with acrosome damage was found to be the lowest in the 50 µM group. High mitochondrial membrane potential sperm ratio was found higher in 50 µM and 100 µM groups compared to other groups (p

Bull Sperm SWATH-MS-Based Proteomics Reveals Link between High Fertility and Energy Production, Motility Structures, and Sperm-Oocyte Interaction.

The prediction of male or semen fertility potential remains a persistent challenge that has yet to be fully resolved. This work analyzed several in vitro parameters and proteome of spermatozoa in bulls cataloged as high- (HF; n = 5) and low-field (LF; n = 5) fertility after more than a thousand artificial inseminations. Sperm motility was evaluated by computer-assisted sperm analysis. Sperm viability, mitochondrial membrane potential (MMP) and reactive oxygen species (mROS) of spermatozoa were assessed by flow cytometry. Proteome was evaluated by the SWATH-MS procedure. Spermatozoa of HF bulls showed significantly higher total motility than the LF group (41.4% vs 29.7%). Rates of healthy sperm (live, high MMP, and low mROS) for HF and LF bull groups were 49% and 43%, respectively (p > 0.05). Spermatozoa of HF bulls showed a higher presence of differentially abundant proteins (DAPs) related to both energy production (COX7C), mainly the OXPHOS pathway, and the development of structures linked with the motility process (TPPP2, SSMEM1, and SPAG16). Furthermore, we observed that equatorin (EQTN), together with other DAPs related to the interaction with the oocyte, was overrepresented in HF bull spermatozoa. The biological processes related to protein processing, catabolism, and protein folding were found to be overrepresented in LF bull sperm in which the HSP90AA1 chaperone was identified as the most DAP. Data are available via ProteomeXchange with identifier PXD042286.

Energy (and Reactive Oxygen Species Generation) Saving Distribution of Mitochondria for the Activation of ATP Production in Skeletal Muscle.

Exercise produces oxidants from a variety of intracellular sources, including NADPH oxidases (NOX) and mitochondria. Exercise-derived reactive oxygen species (ROS) are beneficial, and the amount and location of these ROS is important to avoid muscle damage associated with oxidative stress. We discuss here some of the evidence that involves ROS production associated with skeletal muscle contraction and the potential oxidative stress associated with muscle contraction. We also discuss the potential role of H2O2 produced after NOX activation in the regulation of glucose transport in skeletal muscle. Finally, we propose a model based on evidence for the role of different populations of mitochondria in skeletal muscle in the regulation of ATP production upon exercise. The subsarcolemmal population of mitochondria has the enzymatic and metabolic components to establish a high mitochondrial membrane potential when fissioned at rest but lacks the capacity to produce ATP. Calcium entry into the mitochondria will further increase the metabolic input. Upon exercise, subsarcolemmal mitochondria will fuse to intermyofibrillar mitochondria and will transfer the mitochondria membrane potential to them. These mitochondria are rich in ATP synthase and will subsequentially produce the ATP needed for muscle contraction in long-term exercise. These events will optimize energy use and minimize mitochondria ROS production.

Shipping duration and temperature influence the characteristics of cryopreserved horse semen stored in different shipping devices for up to 14 days

This study aimed to investigate the effects of storing horse semen either in a dry shipper (≤ −150 °C) or on dry ice (≤ −78 °C) for up to 14 days. A total of 264 frozen semen straws from male horses (n = 8) stored in liquid nitrogen were transferred on day 0 (d0) to a dry shipper or a dry ice styrofoam box. On d1, d3, d7, d10, and d14, straws from the dry shipper and dry ice were returned to the liquid nitrogen container. Semen was evaluated by CASA for total (TMot), progressive motility (PMot) and sperm velocity parameters, by fluorescence microscopy for percentage of membrane-intact sperm (SYBR14/PI), high mitochondrial membrane potential (HMMP; JC1) and DNA fragmentation. Temperature inside the containers was monitored continuously. Until d7, no changes were observed in TMot, PMot, and membrane-intact spermatozoa. Thereafter, all three parameters decreased in semen stored on dry ice but not in a dry shipper (time p < 0.001, time x shipping device p < 0.001). The HMMP decreased continuously over time in both containers with a more pronounced decrease on dry ice compared to the dry shipper (shipping device p < 0.01, time p < 0.001, time x device p < 0.001). The DNA fragmentation increased on d10–14 on dry ice and d14 in the dry shipper (time p < 0.001, time x device p < 0.01). In conclusion, frozen horse semen can be safely stored for up to 7 days on dry ice. Sperm DNA integrity and HMMP, however, were adversely affected after 14 days in both shipping devices.

Negative correlations of mitochondrial DNA copy number in commercial frozen bull spermatozoa with the motility parameters after thawing

The purpose of the current study was to investigate the relationship between mitochondrial content of commercial frozen-thawed bull spermatozoa and motility. Firstly, mitochondrial DNA copy number per spermatozoon (MDCN), mitochondrial content (MC), the percentage of spermatozoa with high mitochondrial membrane potential (HMMP), intracellular reactive oxygen species (ROS) and motility parameters of frozen-thawed spermatozoa derived from five bulls were determined by using qPCR, flow cytometry and CASA, respectively, and analyzed the relationships. Results showed that all parameters examined, including MDCN, MC, HMMP, ROS and motility indicators, significantly differed among frozen spermatozoa from different bulls. Both MDCN and MC were negatively correlated with HMMP and motility indicators, but positively with ROS, of course, whereas there was a highly positive relationship between MDCN and MC. Secondly, when MDCN and MC were examined in frozen spermatozoa prepared at different points in the lives of four bulls, those did not correlate overall throughout their lives (1.3–14.3 years old), but did correlate significantly in two sires. From these results, we conclude that MDCN and MC of frozen spermatozoa differ among sires, and are negatively correlated with HMMP and sperm motility parameters, probably due to mitochondrial oxidative stress resulted in the presence of ROS, demonstrating that these appear to be useful markers to assess sires’ spermatozoa. It should be noted that the MDCN and MC of bull spermatozoa may not vary overall with the age of the sire, whereas those changes with age in some individuals and may affect sperm motility.

Analysis of changes in the morphological structures of sperm during preservation of liquid boar semen in two different seasons of the year

Animal fertility is the result of a combination of genetic, physiological and environmental factors. Assessment of semen quality plays a key role in determining the reproductive performance of boars. The aim of the study was to determine the effect of two seasons of the year on the morphology, morphometry, cell membrane integrity and mitochondrial activity of sperm during storage of liquid boar semen at 17 °C. The study was carried out using 20 boars. Four ejaculates were collected from each boar in each of two seasons of the year. All tests were carried out five times: at 1, 24, 48, 96 and 168 h during storage. The results showed that sperm in ejaculates collected in summer are more sensitive to storage conditions than those from ejaculates obtained in winter. The percentage of sperm with morphological defects was shown to increase with the storage time of the diluted ejaculates, particularly between 96 and 168 h of preservation. In summer, the percentage of sperm with an intact cell membrane and the percentage with high mitochondrial membrane potential are much lower than in winter, at every hour of semen preservation. In the case of boars used for artificial insemination, it is worth taking into account the season when the semen is collected, especially if it is to be stored in liquid form. Assessment of sperm cell structures during storage of liquid semen should be implemented at insemination stations and should be carried out more often in summer.