Ameliorative effect of Stevia rebaudiana Bertoni on sperm parameters, in vitro fertilization, and early embryo development in a streptozotocin-induced mouse model of diabetes.

Diabetes mellitus (DM) is a common metabolic disease characterized by high blood sugar levels. It is well known that men with diabetes frequently experience reproductive disorders and sexual dysfunction. In fact, sperm quality has a significant effect on fertilization success and embryo development. The current study aimed to investigate the effect of Stevia rebaudiana hydroalcoholic extract on serum testosterone levels, sperm parameters, in vitro fertilization (IVF) success, and in vitro embryonic developmental potential to reach the blastocyst stage in a streptozotocin (STZ)-induced mouse model of diabetes. In this research, 30 male mice were distributed randomly into control, diabetic (streptozotocin 150 mg/kg) and diabetic + Stevia (400 mg/kg) groups. The results revealed a decrease in body and testis weight and elevated blood fasting blood sugar (FBS) levels in the diabetic group, compared with the control. However, Stevia treatment significantly increased body and testis weight, while serum FBS levels were decreased compared with the diabetic group. In addition, Stevia significantly increased blood testosterone levels compared with the diabetic group. Moreover, sperm parameters were improved considerably by Stevia treatment compared with the diabetic group. Furthermore, Stevia administration significantly promoted IVF success rate and in vitro development of fertilized oocytes compared with the diabetic group. In summary, our data indicated that Stevia enhanced sperm parameters, IVF success, and in vitro embryonic developmental competency in diabetic mice, probably because of its antioxidant effects. Therefore, Stevia could ameliorate sperm parameters that, in turn, increase fertilization outcomes in experimental-induced diabetes.

All-Trans Retinoic Acid-Preconditioned Mesenchymal Stem Cells Improve Motor Function and Alleviate Tissue Damage After Spinal Cord Injury by Inhibition of HMGB1/NF-κB/NLRP3 Pathway Through Autophagy Activation.

Spinal cord injury (SCI) is a significant public health issue that imposes numerous burdens on patients and society. Uncontrolled excessive inflammation in the second pathological phase of SCI can aggravate the injury. In this paper, we hypothesized that suppressing inflammatory pathways via autophagy could aid functional recovery, and prevent spinal cord tissue degeneration following SCI. To this end, we examined the effects of intrathecal injection of all-trans retinoic acid (ATRA)-preconditioned bone marrow mesenchymal stem cells (BM-MSCs) (ATRA-MSCs) on autophagy activity and the HMGB1/NF-κB/NLRP3 inflammatory pathway in an SCI rat model. This study demonstrated that SCI increased the expression of Beclin-1 (an autophagy-related gene) and NLRP3 inflammasome components such as NLRP3, ASC, Caspase-1, and pro-inflammatory cytokines IL-1ß, IL-18, IL-6, and TNF-α. Additionally, following SCI, the protein levels of key autophagy factors (Beclin-1 and LC3-II) and HMGB1/NF-κB/NLRP3 pathway factors (HMGB1, p-NF-κB, NLRP3, IL-1ß, and TNF-α) increased. Our findings indicated that ATRA-MSCs enhanced Beclin-1 and LC3-II levels, regulated the HMGB1/NF-κB/NLRP3 pathway, and inhibited pro-inflammatory cytokines. These factors improved hind limb motor activity and aided in the survival of neurons. Furthermore, ATRA-MSCs demonstrated greater beneficial effects than MSCs in treating spinal cord injury. Overall, ATRA-MSC treatment revealed beneficial effects on the damaged spinal cord by suppressing excessive inflammation and activating autophagy. Further research and investigation of the pathways involved in SCI and the use of amplified stem cells may be beneficial for future clinical use.

Medroxyprogesterone acetate attenuates demyelination, modulating microglia activation, in a cuprizone neurotoxic demyelinating mouse model.

Clinical data reported a reduction of Multiple sclerosis (MS) symptoms during pregnancy when progesterone levels are high. Medroxyprogesterone acetate (MPA) is a synthetic progestin contraceptive with unknown neuroprotective effects. This study investigated the effect of a contraceptive dose of MPA on microglia polarization and neuroinflammation in the neurotoxic cuprizone (CPZ)-induced demyelinating mouse model of MS. Mice received 1 mg of MPA weekly, achieving similar serum concentrations in human contraceptive users. Results revealed that MPA therapy significantly reduced the demyelination in the corpus callosum. In addition, MPA treatment induced a significant reduction in microglia M1-markers (iNOS, IL-1ß and TNF-α) while M2-markers (Arg-1, IL-10 and TGF-ß) were significantly increased. Moreover, MPA resulted in a significant decrease in the number of iNOS positive cells (M1), whereas TREM-2 positive cells (M2) significantly increased. Furthermore, MPA decreased the protein expression levels of NF-κB and NLRP3 inflammasome as well as mRNA expression levels of the downstream product IL-18. In summary, MPA reduces the level of demyelination and has an anti-inflammatory role in CNS demyelination by inducing M2 microglia polarization and suppressing the M1 phenotype through the inhibition of NF-κB and NLRP3 inflammasome. Our results suggest that MPA should be a suitable contraceptive pharmacological agent in demyelinating diseases.

Metformin Therapy Attenuates Pro-inflammatory Microglia by Inhibiting NF-κB in Cuprizone Demyelinating Mouse Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic disorder characterized by reactive gliosis, inflammation, and demyelination. Microglia plays a crucial role in the pathogenesis of MS and has the dynamic plasticity to polarize between pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes. Metformin, a glucose-lowering drug, attenuates inflammatory responses by activating adenosine monophosphate protein kinase (AMPK) which suppresses nuclear factor kappa B (NF-κB). In this study, we indirectly investigated whether metformin therapy would regulate microglia activity in the cuprizone (CPZ)-induced demyelination mouse model of MS via measuring the markers associated with pro- and anti-inflammatory microglia. Evaluation of myelin by luxol fast blue staining revealed that metformin treatment (CPZ + Met) diminished demyelination, in comparison to CPZ mice. In addition, metformin therapy significantly alleviated reactive microgliosis and astrogliosis in the corpus callosum, as measured by Iba-1 and GFAP staining. Moreover, metformin treatment significantly downregulated the expression of pro-inflammatory associated genes (iNOS, H2-Aa, and TNF-α) in the corpus callosum, whereas expression of anti-inflammatory markers (Arg1, Mrc1, and IL10) was not promoted, compared to CPZ mice. Furthermore, protein levels of iNOS (pro-inflammatory marker) were significantly decreased in the metformin group, while those of Trem2 (anti-inflammatory marker) were increased. In addition, metformin significantly increased AMPK activation in CPZ mice. Finally, metformin administration significantly reduced the activation level of NF-κB in CPZ mice. In summary, our data revealed that metformin attenuated pro-inflammatory microglia markers through suppressing NF-κB activity. The positive effects of metformin on microglia and remyelination suggest that it could be used as a promising candidate to lessen the incidence of inflammatory neurodegenerative diseases such as MS.

The effect of adding Rosmarinic and Ascorbic acids to vitrification media on fertilization rate of the mice oocyte: An experimental study.

BACKGROUND: Oocytes vitrification is a pivotal step for the widespread and safekeeping of animal genetic resources. Oocytes endure notable morphological and functional damage during cryopreservation. Oxidative stress is one of the adverse effects that vitrification imparts on oocytes. OBJECTIVE: In the present study, we investigated the antioxidant effect of Rosmarinic and Ascorbic acids on the quality and fertilizing ability of frozen-thawed mice oocyte. MATERIALS AND METHODS: In this experimental study, germinal vesicle oocytes obtained from two-months-old (30-40gr) NMRI mice were randomly divided into four groups. The basic cryoprotectants were 7.5% (v/v) ethylene glycol+7.5% (v/v) Propanediol as an equilibration media. Vitrification medium contained 15% (v/v) ethylene glycol+15% (v/v) propanediol, and 0.5 M sucrose. In the first group (Control), nothing was added to vitrification mediums, whereas, in the second and third groups, 0.5 mmol/L of Ascorbic acid and 105 µmol/L of Rosmarinic acid were added into vitrification medium, respectively. The cumulative concentration of Rosmarinic and Ascorbic acids were added to group 4. Mouse oocytes were vitrified and preserved for one month. The thawed oocytes were transferred into the α-MEM medium (Alpha Minimum Essential Medium) and maintained in this medium for 24 hr, to be matured and reach the metaphase II stage. RESULTS: The addition of Rosmarinic and Ascorbic acids to the vitrification solution improved the survival, maturation of Germinal vesicles, fertilization rate, and finally development to 4-cell stage. Maturation rates to 4-cell stage for Ascorbic acid, Rosmarinic acid, and both of them together were 80%, 80.76%, and 86.61%, respectively. CONCLUSION: These results indicate that the addition of a cumulative concentration of 0.5 mmol/L Ascorbic acid and 105 µmol/L of Rosmarinic acid to the cryopreservation solution for the mouse immature oocytes would be of significant value (p < 0.01).

Combined Effect of Retinoic Acid and Basic Fibroblast Growth Factor on Maturation of Mouse Oocyte and Subsequent Fertilization and Development.

BACKGROUND: In this experimental study, germinal vesicle (GV) oocytes obtained from two-months-old NMRI mice were randomly divided into control, sham and three experimental groups. The basic culture medium was α-MEM supplemented with 10% fetal bovine serum (FBS), 50 mg/l streptomycin, 60 mg/l penicillin and 10 ng/ ml epidermal growth factors. Each of the experimental groups received one of the following treatments: RA (2 µM), bFGF (20 ng/ml) or combination of RA and bFGF with the indicated concentrations. After 24 hours, capacitated spermatozoa were added to in vitro matured oocytes. Five hours later, the oocytes were cultured in fresh droplets of M2 medium for 24 hours and assessed for cleavage to the two-cells stage. MATERIALS AND METHODS: In this experimental study, germinal vesicle (GV) oocytes obtained from two-months-old NMRI mice were randomly divided into control, sham and three experimental groups. The basic culture medium was α-MEM supplemented with 10% fetal bovine serum (FBS), 50 mg/l streptomycin, 60 mg/l penicillin and 10 ng/ ml epidermal growth factors. Each of the experimental groups received one of the following treatments: RA (2 µM), bFGF (20 ng/ml) or combination of RA and bFGF with the indicated concentrations. After 24 hours, capacitated spermatozoa were added to in vitro matured oocytes. Five hours later, the oocytes were cultured in fresh droplets of M2 medium for 24 hours and assessed for cleavage to the two-cells stage. RESULTS: As compared with the control group, the rate of maturation was significantly increased in the RA (P<0.001) and bFGF+RA (P<0.02) groups with 58 ± 10 and 57 ± 3.46, respectively. The rate of maturation was significant in the RA (P<0.02) and bFGF+RA (P<0.03) groups, in comparison with the bFGF group. The bFGF+RA group had higher rate (83 ± 1.52) of two-cells development, than control (33 ± 1, P<0.001). CONCLUSION: Our findings showed beneficial effects of 2 µM RA and 20 ng/ml bFGF combination on mouse oocyte IVM.

Post-treatment of melatonin with CCl4 better reduces fibrogenic and oxidative changes in liver than melatonin co-treatment.

Therapeutic effects of melatonin (MEL) in targeting CCl4 -induced liver fibrosis has been widely known, but there is no study comparing oxidative and fibrogenic changes in co- and post-treatment of MEL with CCl4 , which was further aimed in this experiment. Male SD rats were injected with CCl4 (1 mL/kg/i.p./daily) dissolved 1:1 in olive oil for 1 month. Some animals received MEL (20 mg/kg/i.p./daily) diluted in 1 mL PBS in combination with CCl4 (co-treatment), and some rats were treated with MEL, beginning with injection of the last dose of CCl4 for one month (post-treatment). The groups were control, CCl4 , CCl4 -co vehicle, CCl4 -post vehicle, post-CCl4 , MEL co-treatment, and MEL post-treatment. MEL post-treatment group showed significantly lower lipid deposition, serum malondialdehyde (MDA), serum alanine aminotransferase (ALT), and liver hydroxyproline. This group also had low expressions of Bax and transforming growth factor-ß1 (TGF-ß1). MEL post-treatment group revealed higher sera levels of albumin, superoxide dismutase (SOD) and glutathione peroxidase (GPx). Expression levels of metalloproteinase-13 (MMP-13) and Bcl2 was also higher in this group (P ≤ 0.05 vs co-treatment). Results of the present study indicated that MEL post-treatment is more powerful in reduction of CCl4 -induced liver fibrosis through reduction of oxidative stress and maintenance of matrix balance.

Embryonic Stem Cell Conditioned Medium Supports In Vitro Maturation of Mouse Oocytes.

BACKGROUND: This study aimed to investigate the maturation and fertilization rates of immature mouse oocytes using Embryonic Stem Cell Conditioned Medium (ESCM). METHODS: Germinal Vesicle (GV) stage oocytes were observed in 120 NMRI mice, aged 4-6 weeks. GV oocytes with or without cumulus cells were subjected to IVM in either ESCM, Embryonic Stem Cell Growth Medium (ESGM), or α-minimum essential medium (α-MEM). After recording the Metaphase II (MII) oocyte maturation rate, the oocytes were fertilized in vitro. The fertilization success rate was recorded after 24 hr. The embryos were maintained in potassium Simplex Optimization Medium (KSOM) for 96 hr and allowed to grow until the blastocyst stage. After recording developmental competence, they were transferred into the uteri of pseudopregnant mice and their birth rates were recorded. RESULTS: No significant difference existed between the maturation rates in α-MEM (68.18%) and ESCM (64.67%; p>0.05), whereas this rate was significantly higher for both α-MEM and ESCM compared to ESGM (32.22%; p<0.05). A significant difference in IVF success rate existed for oocytes grown in α-MEM (69.44%), ESCM (61.53%), and ESGM (0%). A significantly higher developmental competence was observed at the blastocyst stage for oocytes grown in α-MEM (51.2%) compared to ESCM (35%; p<0.05). 17 days after embryo transfer into the uteri of pseudopregnant mice, there was a nonsignficant (p>0.05), similar birth rate between α-MEM and ESCM (47 vs. 40%). CONCLUSION: ESCM is an effective medium for preantral follicle growth, oocyte maturation, and subsequent embryo development.