Alpha-linolenic acid alleviates the detrimental effects of lipopolysaccharide during in vitro ovine oocyte development.

During the transition period and early lactation of ruminants with higher production, the reproductive organs are exposed to various stressors, like inflammation stimulators such as lipopolysaccharides (LPS), as a consequence of high concentrate consumption. In this study, we aimed to determine the probable potential of α-linolenic acid (ALA) in alleviating LPS-induced effects in ovine oocytes in vitro as well as the underlying controlling mechanisms. Different concentrations of LPS (0, 0.01, 0.1, 1, and 10 µg/mL) were added to the oocyte maturation medium to evaluate its effect on oocyte developmental competence. Likewise, different concentrations of ALA (0, 10, 50, 100, and 200 µM/mL) were added to the maturation medium to define its effects on oocyte developmental competence. Accordingly, a combination of ALA and LPS in a dose-dependent manner was added to the maturation medium to elucidate their effect on oocyte developmental competence and uncover any possible potential of ALA to alleviate the detrimental effect induced by the presence of LPS. The expressions of candidate genes were measured in mature oocytes treated either with ALA, LPS, or ALA plus LPS. Adding LPS to the maturation medium decreased the cleavage rate of the treated oocytes, and those oocytes reached the blastocyst stage at a lower rate. Adding ALA to the maturation medium in the presence of LPS alleviated the detrimental effects of LPS in a dose-dependent manner, which ultimately led to higher cleavage and blastocyst formation. A higher expression of Trim26, GRHPR, NDUFA, PGC-1α, SOD, CS, SDH, p53, and CAT was observed in LPS-treated oocytes compared with the ALA and control groups. Additionally, CS and CAT transcripts were down-regulated in oocytes in LPS plus ALA-treated group compared to that of the LPS-treated group. These findings revealed that ALA has the potential to alleviate the detrimental effects induced by LPS on in ovine oocytes during maturation in vitro. Thus, LPS-detrimental effect and ALA-preventing mechanisms seem to be regulated through the expression of genes involved in mitochondrial biogenesis and function, oxidative stress, and antioxidant systems.

Suicide gene therapy using allogeneic adipose tissue-derived mesenchymal stem cell gene delivery vehicles in recurrent glioblastoma multiforme: a first-in-human, dose-escalation, phase I clinical trial.

BACKGROUND: Glioblastoma multiforme (GBM) is associated with remarkably poor prognosis, and its treatment is challenging. This investigation aimed to evaluate the safety of suicide gene therapy using allogeneic adipose tissue-derived mesenchymal stem cells (ADSCs) carrying herpes simplex virus-thymidine kinase (HSV-TK) gene for the first time in patients with recurrent GBM. METHODS: This study was a first-in-human, open-label, single-arm, phase I clinical trial with a classic 3 + 3 dose escalation design. Patients who did not undergo surgery for their recurrence were included and received this gene therapy protocol. Patients received the intratumoral stereotactic injection of ADSCs according to the assigned dose followed by prodrug administration for 14 days. The first dosing cohort (n = 3) received 2.5 × 105 ADSCs; the second dosing cohort (n = 3) received 5 × 105 ADSCs; the third dosing cohort (n = 6) received 10 × 105 ADSCs. The primary outcome measure was the safety profile of the intervention. RESULTS: A total of 12 patients with recurrent GBM were recruited. The median follow-up was 16 (IQR, 14-18.5) months. This gene therapy protocol was safe and well tolerated. During the study period, eleven (91.7%) patients showed tumor progression, and nine (75.0%) died. The median overall survival (OS) was 16.0 months (95% CI 14.3-17.7) and the median progression-free survival (PFS) was 11.0 months (95% CI 8.3-13.7). A total of 8 and 4 patients showed partial response and stable disease, respectively. Moreover, significant changes were observed in volumetric analysis, peripheral blood cell counts, and cytokine profile. CONCLUSIONS: The present clinical trial, for the first time, showed that suicide gene therapy using allogeneic ADSCs carrying the HSV-TK gene is safe in patients with recurrent GBM. Future phase II/III clinical trials with multiple arms are warranted to validate our findings and further investigate the efficacy of this protocol compared with standard therapy alone. TRIAL REGISTRATION: Iranian Registry of Clinical Trials (IRCT), IRCT20200502047277N2. Registered 8 October 2020, https://www.irct.ir/ .

Aflatoxin B1 impairs in vitro early developmental competence of ovine oocytes.

Aflatoxin is considered as one of the most harmful mycotoxins in the world to be found in human food and animal feed. Previous reports have revealed that Aflatoxin B1 (AFB1) may disrupt gamete development through epigenetic modifications as well as promotion of oxidative stress, excessive autophagy, and apoptosis. Therefore, in this study we aimed to address the effects of AFB1 on the meiotic and cytoplasmic maturation, internal reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm), blastocyst formation as well as mRNA alterations for the apoptotic (BAX and Caspase3), anti-apoptotic (BCL2), and DNA methyltransferase (DNMTs) genes in ovine oocytes. To accomplish this, maturation of oocytes was performed in presence of increasing AFB1 concentrations (0, 10, 50, and 100 µM). Meiotic and cytoplasmic maturation, intracellular ROS level, and ΔΨm were evaluated following 24 h of IVM. Embryonic cleavage and blastocyst formation following fertilization were also assessed. We also investigated alterations of BAX, BCL2,Caspase3, DNMT1, DNMT3a, and DMT3b mRNA levels in mature oocytes. In the presence of 50 and 100 µM AFB1, the number of oocytes reaching the metaphase II stage decreased and the oocytes presented with lower intracellular levels of GSH (P < 0.05). Furthermore, intracellular ROS production in matured oocytes reached the highest-level following exposure to 50 and 100 µM of AFB1 (P < 0.05). Reduction of ΔΨm was clearly evident in the AFB1-treated groups (P < 0.05). Rates of cleavage and blastocyst formation decreased in the presence of AFB1 as compared with those recorded in the Control group (P < 0.05). Apoptosis-related gene analysis in AFB1 treated groups (10 and 50 µM) revealed a higher abundance of the BAX and Caspase3 genes, and a lower abundance of the BCL2 gene as compared with the Control group (P < 0.05). Additionally, our data showed that relative abundances of DNMT3b gene decreased in the 10 µM group when compared to the Control group (P < 0.05). We showed that exposure of oocytes to AFB1 leads to a reduced nuclear and cytoplasmic maturation that may ultimately impair the embryonic development in the sheep oocyte. Furthermore, alterations in DNA methylation and initiation of apoptosis through excessive ROS generation could be a prime molecular mechanism responsible for the disruption of oocyte developmental competence in the presence of AFB1 in the ovine model.

In Vivo and In Vitro Evaluation of Bull Semen Processed with Zinc (Zn) Nanoparticles.

Defective sperms cause fertilization failure under both in vivo and in vitro conditions. Therefore, providing optimal conditions during semen storage is a prerequisite for maintaining viability. The current study investigated bull semen quality in vitro and in vivo when zinc (Zn) nanoparticles were used as antioxidant during semen processing and cryopreservation. In total, 32 ejaculates were collected from four Holstein bulls. All ejaculates were pooled and diluted with Bioxcell-extender containing 0 (control group), 10-6, 10-5, 10-4, 10-3, and 10-2 M of Zn nanoparticles. Several physical and biochemical sperm parameters were determined after freeze-thawing process. In vitro embryo development rate and pregnancy rate were monitored after in vitro fertilization or artificial insemination using semen treated with Zn nanoparticles. Plasma membrane integrity was improved (P < 0.05) in bull semen treated with 10-6 M (69.3%), and 10-2 (62.4%) of Zn nanoparticles compared to untreated group (51.3%). In addition, proportions of live spermatozoa with active mitochondria were increased (P < 0.05) in semen supplemented with Zn nanoparticles at concentration of 10-6 M (67.3%), and 10-2 (85.3%) compared to control group (49.8%). Moreover, the level of MDA was lower (P < 0.05) in semen with Zn nanoparticles at 10-6 M (2.97 mol/mL) and 10-2 (2.7 mol/mL) concentrations than control semen samples (3.77 mol/mL). However, sperm total and progressive motility, sperm viability, DNA fragmentation, and pregnancy rate were not affected by treatment of semen with Zn nanoparticles. On the other hand, supplementation of in vitro maturation media with 10-6 M Zn nanoparticles has increased blastocyst rate (P < 0.05) compared to other experimental groups, while addition of Zn nanoparticles-treated sperm during in vitro fertilization did not affect embryo development rate. In conclusion, supplementation of Zn nanoparticles to semen has improved its quality without affecting embryo development rate in vitro. However, in vitro embryo development rate was increased when Zn nanoparticles were supplemented to IVM media. This support the notion of Zn nanoparticles beneficial action on improving bovine gametes quality without affecting pregnancy rate.

Transcriptional profile of ovine oocytes matured under lipopolysaccharide treatment in vitro.

Lipopolysaccharide (LPS) derived from gram negative bacteria cell wall is known to cause ruminal acidosis and/or infectious diseases such as metritis and mastitis which has a significant negative impact on the reproductive performance. This study aimed to investigate the effect of LPS on oocyte maturation and subsequent development in vitro. Ovine cumulus oocyte complexes (COCs) were matured in a medium supplemented with 0 (control), 0.01, 0.1, 1 and 10 µg/mL LPS. Nuclear maturation, cleavage and blastocyst rate, mitochondrial membrane potential (ΔΨm), intracellular reactive oxygen species (ROS) content and changes to the transcript abundance were evaluated. In case of the maturation rate, the percentage of oocytes reaching the MII stage was lower following exposure to 10 µg/mL LPS in comparison to the control group (P < 0.05). Moreover, the blastocyst rate decreased in case of 1 and 10 µg/mL LPS when compared to the control group (P < 0.05). ROS overproduction accompanied by a decreased ΔΨm were recorded in LPS treated oocytes in comparison to the control group (P < 0.05). The 3' tag digital gene expression profiling method revealed that 7887 genes were expressed while only seven genes exhibited changes in the transcript abundance following exposure to LPS. Tripartite motif containing 25 (TRIM25), Tripartite motif containing 26 (TRIM26), Zona Pellucida glycoprotein 3 (ZP3), Family with sequence similarity 50-member A (FAM50A), Glyoxalate and hydroxy pyruvate reductase (GRHPR), NADH ubiquinase oxireductase subunit A8 (NDUFA8) were down-regulated (P < 0.05), while only Centrin 3 (CETN3) was up-regulated (P < 0.05). Our findings show that LPS has undesirable effects on the maturation competence of ovine oocytes and subsequent embryo development. In addition, the transcriptomic profiling results may shed more light on the molecular mechanisms of LPS-induced infertility in ruminants.