Evaluation of the effect of mitoquinone on functional parameters, DNA structure, and genes expression related to the apoptotic and antioxidants of human sperm after freezing-thawing.

OBJECTIVE: Sperm freezing is considered as an effective way in assisted reproductive technology (ART) programs, it has detrimental effects on sperm function, due to the production of reactive oxygen species (ROS). This study aimed to investigate the potential of Mitoquinone (MitoQ) in inhibiting the production of mitochondrial ROS during sperm freezing. METHODS: A total of 20 human normozoosperm samples were collected for this study. The samples were divided into four groups, each containing different concentrations of MitoQ (0, 0.2, 2, and 20 nM), and then subjected to the freezing process. After thawing, the sperm suspensions were evaluated for parameters including motility, morphology, acrosome integrity, adenosine triphosphate (ATP) level, intracellular ROS, viability, chromatin packaging, DNA denaturation, DNA fragmentation, as well as the expression of antioxidants (GPX, SOD) and apoptotic (Bax, Bcl2) genes. RESULTS: The results showed that total and progressive mobility of sperms significantly increased in the 2 nM group, while significantly decreased in the 20 nM group (p ≤ 0.05). Sperm morphology did not significantly improve across all the tested concentrations (p ≥ 0.05). Intracellular ROS levels showed a significant decrease and increase in the concentrations of 2 and 20 nM, respectively (p ≤ 0.05). Furthermore, a significant increase was observed in viability, ATP, acrosome integrity, chromatin packaging, and non-denatured and non-fragmented DNA after treatment with 2 nM of MitoQ, compared with the control group (p ≤ 0.05). Regarding gene expressions, the relative expressions of oxidative stress genes were increased in the 2 nM group and decreased in the 20 nM group (p ≤ 0.05), while no significant difference was observed in the expressions of apoptotic genes compared with the control group (p ≥ 0.05). All the comparisons were made with respect to the control group. CONCLUSION: Adding the optimal concentration of MitoQ (2 nM) to the sperm freezing medium not only improves sperm functional parameters and reduces DNA damages, but also stimulates the expression of antioxidant genes, leading to even greater benefits for sperm cryopreservation.

The Effect of The Conditioned Medium from Human Embryonic Stem Cells on Mouse Oocytes In Vitro Maturation.

OBJECTIVE: Some reports have indicated that conditioned medium from growing mouse embryonic stem cells (ESCs) provides a supportive condition for small follicles growing, oocyte maturation, and following embryo growth. The aim of this study is assessing in vitro maturation (IVM) and consequent in vitro fertilization (IVF) outcome of immature mouse oocytes using human embryonic stem cells conditioned medium (HESCM). MATERIALS AND METHODS: In this experimental study, 240 germinal vesicle (GV) oocytes were took from NMRI female mice, aged 4-6 weeks, 48 hours before injection of 5 IU pregnant mare serum gonadotropin (PMSG). 120 GV oocytes without cumulus cells were cultured in each of the groups. 120 GV were cultured in HESCM as test groups and also 120 GV cultured in human embryonic stem cells medium (HESM) as control groups. After evaluating the metaphase II (MII) oocyte maturation rate at 8, 16 and 24 hours, the MII oocytes subsequently were fertilized in vitro and the two-cell embryo development rate was recorded at days 1, 2, and 3. Statistical analysis was performed by using the generalized estimating equations (GEE) method that calculated their rate ratio. RESULTS: Our data indicated there are significant differences between the maturation rates in HESCM and HESM (P=0.004), also the two-cell embryo development was significant between two culture media (P=0.00). CONCLUSION: Similar to some other studies, the secretome of the HESCM showed a significant impact on the IVM outcomes in mice.

Chitosan/silk fibroin/nitrogen-doped carbon quantum dot/α-tricalcium phosphate nanocomposite electrospinned as a scaffold for wound healing application: In vitro and in vivo studies.

A highly porous nanofibrous network that can functionalize antibacterial and therapeutic agents can be considered a suitable option for skin wound healing. In this study, α-tricalcium phosphate (α-TCP)/nitrogen-doped carbon quantum dots (N-CQDs) nanocomposite was synthesized and then applied to the fabrication of novel chitosan (CS)/silk fibroin (SF)/N-CQDs/α-TCP wound dressing via electrospinning system. The prepared nanomaterials were well characterized using X-ray diffraction, Fourier-transform infrared, scanning and transmission electron microscopes analyses, and antibacterial assay. Furthermore, nanofibers were evaluated regarding their physical properties, such as tensile behavior, water uptake capacity, and water contact angle. The results reveal that CS/SF/N-CQDs/α-TCP showed lower MIC values against E. coli and S. aureus (1.45 ± 0.26 mg/mL and 1.59 ± 0.12 mg/mL) compared to other synthesized materials. Also, in-vitro investigations were performed, and the MTT assay on the HFF cell line revealed that CS/SF/N-CQDs/α-TCP nanofiber could possess good biocompatibility. Interestingly, the scratch test proved that faster cell migration and proliferation occurred in the presence of CS/SF/N-CQDs/α-TCP 73.23 ± 2.71 %). Finally, we examined the wound healing ability of CS/SF/N-CQDs/α-TCP nanofiber using an animal model. The results confirmed that produced nanofiber could efficiently promote wound closure by 96.73 ± 1.25 % in 12 days. Histopathological analyses verified accelerated re-epithelization and well-structured epidermis in CS/SF/N-CQDs/α-TCP nanofiber-treated group. Based on our findings, the CS/SF/N-CQDs/α-TCP nanofiber with excellent antimicrobial properties is highly suitable for wound healing and skin tissue regeneration applications.

Impairment of sperm efficiency in mice following short-term nano-titanium dioxide exposure: An experimental study.

BACKGROUND: Titanium dioxide nanoparticles (TiO 2 NPs) are widely used in many compounds. Recent evidence has displayed some cytotoxic effects of TiO 2 NPs on male reproduction. OBJECTIVE: The effects of TiO 2 NP administration on sperm parameters and chromatin and seminiferous histopathology of male mice were investigated. MATERIALS AND METHODS: In this experimental study, 32 NMRI male mice (35 ± 3 gr, 8-12-week-old) were divided into four groups (n = 8/each): treated groups were fed orally with 2.5 (group I), 5 (group II) and 10 (group III) mg/kg/day TiO 2 NPs for 40 days and the control group received phosphate buffered saline. Sperm parameters, DNA integrity and chromatin quality were assessed using chromomycin A3, aniline blue, toluidine blue staining and TUNEL. Hematoxylin eosin staining was performed to measure spermatogenic cells and the total diameter of seminiferous tubules. Also, sex hormone and malondyaldehyde levels were measured. RESULTS: Abnormal sperm tails rose in group III (28.87 ± 4.91) in comparison with the control group (12.75 ± 3.95). However, chromomycin A3 staining and TUNEL showed higher levels in group III in comparison with the control group, whereas aniline blue and toluidine blue staining showed no differences. A significantly lower spermatogenesis index and lumen parameters were observed in group III. Leydig cell numbers, cellular diameters and the area of the seminiferous tubules were lower in the treated groups. The testosterone level was also lower in these groups and the percentage of malondyaldehyde in the seminal fluid was higher. CONCLUSION: Exact mechanisms of TiO 2 NPs are not clear; however, cytotoxic and genotoxic effects of TiO 2 NPs may relate to oxidative stress. Given their widespread use, TiO 2 NPs should be a public health focus of attention.

Effects of phosalone plant pesticide on sperm parameters and sexual hormone levels in Wistar rats: An experimental study.

BACKGROUND: Phosalone is an organophosphate insecticide, applied to control of plant pests. This compound has various side effects because it acts as an acetyl cholinesterase enzyme inhibitor. OBJECTIVE: To investigate the effects of phosalone on the sperm parameters of and levels of sex hormones in adult male rats. MATERIALS AND METHODS: In this experimental study, 16 adult (8-12 wk) male Wister rates (weighing 220-280 gr) were randomly assigned into 4 groups (n = 4/each). Group 1 (control) received only routine adequate water and food; Group 2, 3, and 4 received different low doses of phosalone (60, 90, and 120 mg/kg respectively). The rats were weighed and anesthetized after 48 days. Sperm parameters including number, motility, and viability as well as sex hormones (such as Luteinizing Hormone, Follicle Stimulating Hormone, and testosterone) were evaluated and compared after removing the epididymis tail. RESULTS: Our results showed that phosalone decreased sperm motility, viability, and number in a dose-dependent manner. The level of FSH and LH was increased, and testosterone was decreased. Also, depending on the dose, phosalone decrease sperm motility and viability (p ≤ 0.001), while the level of FSH and LH was increased and testosterone was decreased (p = 0.861). CONCLUSION: Phosalone has negative effects on reproductive indices in male rats and can cause serious damage and decrease the number and sperms motility. It can also cause infertility due to changing the concentration of hormones.

A Simple Technique for Three-Dimensional Imaging and Segmentation of Brain Vasculature U sing Fast Free-of-Acrylamide Clearing Tissue in Murine.

OBJECTIVE: Fast Free-of-Acrylamide Clearing Tissue (FACT) is a recently developed protocol for the whole tissue three-dimensional (3D) imaging. The FACT protocol clears lipids using sodium dodecyl sulfate (SDS) to increase the penetration of light and reflection of fluorescent signals from the depth of cleared tissue. The aim of the present study was using FACT protocol in combination with imaging of auto-fluorescency of red blood cells in vessels to image the vasculature of a translucent mouse tissues. MATERIALS AND METHODS: In this experimental study, brain and other tissues of adult female mice or rats were dissected out without the perfusion. Mice brains were sliced for vasculature imaging before the clearing. Brain slices and other whole tissues of rodent were cleared by the FACT protocol and their clearing times were measured. After 1 mm of the brain slice clearing, the blood vessels containing auto-fluorescent red blood cells were imaged by a z-stack motorized epifluorescent microscope. The 3D structures of the brain vessels were reconstructed by Imaris software. RESULTS: Auto-fluorescent blood vessels were 3D imaged by the FACT in mouse brain cortex. Clearing tissues of mice and rats were carried out by the FACT on the brain slices, spinal cord, heart, lung, adrenal gland, pancreas, liver, esophagus, duodenum, jejunum, ileum, skeletal muscle, bladder, ovary, and uterus. CONCLUSION: The FACT protocol can be used for the murine whole tissue clearing. We highlighted that the 3D imaging of cortex vasculature can be done without antibody staining of non-perfused brain tissue, rather by a simple autofluorescence.