• Journal of Animal Reproduction and Biotechnology
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• v.35 no.1
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• pp.21-27
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• 2020

Somatic cell nuclear transfer derived embryonic stem cells (NT-ESCs) have significant advantages in various fields such as genetics, embryology, stem cell science, and regenerative medicine. However, the poor establishment of NT-ESCs hinders various research. Here, we applied fasudil, a Rho-associated kinase (ROCK) inhibitor, to develop somatic cell nuclear transfer (SCNT) embryos and establish NT-ESCs. In the study, MII oocytes were isolated from female B6D2F1 mice and performed SCNT with mouse embryonic fibroblasts (MEFs). The reconstructed NT-oocytes were activated artificially, and cultured to blastocysts in KSOM supplemented with 10 μM fasudil. Further, the blastocysts were seeded on inactivated MEFs in embryonic stem cell medium supplemented with 10 μM fasudil. A total of 26% of embryos formed into blastocysts in the fasudil treated group, while this ratio was 44% in the fasudil free control group. On the other hand, 30% of blastocysts were established NT-ESCs after exposure of fasudil, which was significantly higher than the control group (10%). The results suggest that fasudil reduced blastocyst development after SCNT due to inhibition of 2 cell cleavage while improved the establishment of NT-ESCs through the anti-apoptotic pathway.

• Journal of Korean Neurosurgical Society
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• v.60 no.4
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• pp.417-423
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• 2017

Objective : Repair of sensorial nerve defect is an important issue on peripheric nerve surgery. The aim of the present study was to determine the effects of sensory-motor nerve bridging on the denervated dermatomal area, in rats with sensory nerve defects, using a neural cell adhesion molecule (NCAM). Methods : We compared the efficacy of end-to-side (ETS) coaptation of the tibial nerve for sural nerve defect repair, in 32 Sprague-Dawley rats. Rats were assigned to 1 of 4 groups : group A was the sham operated group, group B rats had sural nerves sectioned and buried in neighboring muscles, group C experienced nerve sectioning and end-to-end (ETE) anastomosis, and group D had sural nerves sectioned and ETS anastomosis was performed using atibial nerve bridge. Neurological evaluation included the skin pinch test and histological evaluation was performed by assessing NCAM expression in nerve terminals. Results : Rats in the denervated group yielded negative results for the skin pinch tests, while animals in the surgical intervention groups (group C and D) demonstrated positive results. As predicted, there were no positively stained skin specimens in the denervated group (group B); however, the surgery groups demonstrated significant staining. NCAM expression was also significantly higher in the surgery groups. However, the mean NCAM values were not significantly different between group C and group D. Conclusion : Previous research indicates that ETE nerve repair is the gold standard for peripheral nerve defect repair. However, ETS repair is an effective alternative method in cases of sensorial nerve defect when ETE repair is not possible.

• Clinical and Experimental Reproductive Medicine
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• v.44 no.3
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• pp.119-125
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• 2017

Objective: In vitro culture of preimplantation embryos is improved by grouping embryos together in a drop of media. Individually cultured embryos are deprived of paracrine factors; with this in mind, we investigated whether the addition of a single embryo-secreted factor, interleukin-6 (IL-6), could improve the development of individually cultured embryos. Methods: Mouse embryos were cultured individually in $2{\mu}L$ of G1/G2 media in 5% oxygen and supplemented with a range of doses of recombinant mouse or human IL-6. Results: Mouse IL-6 increased hatching at doses of 0.01 and 10 ng/mL compared to the control (93% and 93% vs. 78%, p< 0.05) and increased the total number of cells at a dose of 0.1 ng/mL compared to the control ($101.95{\pm}3.36$ vs. $91.31{\pm}3.33$, p< 0.05). In contrast, the highest dose of 100 ng/mL reduced the total number of cells ($79.86{\pm}3.29$, p< 0.05). Supplementation with human IL-6 had a different effect, with no change in hatching or total cell numbers, but an increase in the percentage of inner cell mass per embryo at doses of 0.1, 1, and 100 ng/mL compared to the control ($22.9%{\pm}1.1%$, $23.3%{\pm}1.1%$, and $23.1%{\pm}1.1%$ vs. $19.5%{\pm}1.0%$, p< 0.05). Conclusion: These data show that IL-6 improved mouse embryo development when cultured individually in complex media; however, an excess of IL-6 may be detrimental. Additionally, these data indicate that there is some cross-species benefit of human IL-6 for mouse embryos, but possibly through a different mechanism than for mouse IL-6.

• Journal of Microbiology and Biotechnology
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• v.29 no.8
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• pp.1230-1239
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• 2019

Scutellaria baicalensis Georgi has been widely used in China for treatment of various diseases. This study investigated the effect of Scutellaria baicalensis Georgi extracts (SBE) against Coxsackievirus B3 (CVB3)-induced myocarditis in vitro and in vivo. In vitro, Hela cells and primary myocardial cells were infected with CVB3 and treated with SBE ($50-800{\mu}g/ml$) and ribavirin ($200{\mu}M$) for 48 h and then determined by CCK8 assay. Real-time PCR and western blotting assays were performed. In vivo, a myocarditis model was induced in male BALB/c mice by injecting CVB3 suspension intraperitoneally for three times, followed by treatment with SBE (400 and 200 mg/kg) and ribavirin (100 mg/kg) for 28 days. SBE ameliorated the cytotoxicity of CVB3 in Hela cells, especially at $400{\mu}g/ml$ (39.93% vs 65.67%, p < 0.05) without influencing cell growth and also significantly reduced CVB3 replication in primary myocardial cells. The levels of AKT, ERK, and p38 were increased after CVB3 infection. SBE could downregulate the expressions of AKT and p38. In vivo, the mortality rate from CVB3 reached to 66.67%, while 10.00% and 23.33% of this came after 400 and 200 mg/kg SBE treatment, respectively (p < 0.05). The CVB3 replication was obviously reduced after SBE administration from day 5. Similarly, the levels of AKT, ERK, and p38 mRNAs and proteins were increased, and SBE suppressed the expression of AKT and p38. Our study indicates that SBE is a promising potent antiviral agent against CVB3-induced myocarditis by inhibition of virus replication via depressing AKT and p38 expressions.

• BMB Reports
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• v.52 no.9
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• pp.572-576
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• 2019

Bisphosphonates are the mainstay of therapy worldwide for osteoporosis. However, bisphosphonates also have limitations. The objective of this study was to determine the role of miR-101-3p/Rap1b signal pathway in osteoclast differentiation after treatment with bisphosphonates. Our results revealed that miR-101-3p was an important regulator in bisphosphonates treated-osteoclasts. When miR-101-3p was down-regulated in bone marrow-derived macrophage-like cells (BMMs), the development of mature osteoclasts was promoted, and vice versa. However, alendronate decreased multinucleated cell number regardless of whether miR-101-3p was knocked down or over-expressed. TRAP activity assay confirmed the above results. Luciferase assay indicated that miR-101-3p was a negative regulator of Rap1b. Western blot analysis revealed that protein expression level of Rap1b in BMMs transfected with OV-miR-101-3p was lower than that in BMMs transfected with an empty vector. Rap1b overexpression increased TRAP-positive multinucleated cells, while Rap1b inhibition decreased the cell numbers. In vivo data showed that miR-101-3p inhibited osteoclast differentiation in ovariectomized mice while overexpressed of Rap1b blocked the differentiation. Taken together, our data demonstrate that miR-101-3p/Rap1b signal pathway plays a key role in osteoclast differentiation after treatment with bisphosphonates.

• Clinical and Experimental Reproductive Medicine
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• v.46 no.2
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• pp.67-75
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• 2019

Objective: Sperm cryopreservation has been widely used in assisted reproductive technology, as it offers great potential for the treatment of some types of male infertility. However, cryopreservation may result in changes in membrane lipid composition and acrosome status, as well as reductions in sperm motility and viability. This study aimed to evaluate sperm DNA fragmentation damage caused by conventional freezing using the sperm chromatin dispersion test. Methods: In total, 120 fresh human semen samples were frozen by conventional methods, using SpermFreeze Solution as a cryoprotectant. Routine semen analysis and a Halosperm test (using the Halosperm kit) were performed on each sample before freezing and after thawing. Semen parameters and sperm DNA fragmentation were compared between these groups. Results: There was a significant decrease in sperm progressive motility, viability, and normal morphology after conventional freezing (32.78%, 79.58%, and 3.87% vs. 16%, 55.99%, and 2.55%, respectively). The sperm head, midpiece, and tail defect rate increased slightly after freezing. Furthermore, the DNA fragmentation index (DFI) was significantly higher after thawing than before freezing (19.21% prior to freezing vs. 22.23% after thawing). Significant increases in the DFI after cryopreservation were observed in samples with both normal and abnormal motility and morphology, as well as in those with normal viability. Conclusion: Conventional freezing seems to damage some sperm parameters, in particular causing a reduction in sperm DNA integrity.

• The Korean Journal of Physiology and Pharmacology
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• v.25 no.1
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• pp.69-77
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• 2021

Propofol infusion syndrome characterized by rhabdomyolysis, metabolic acidosis, kidney, and heart failure has been reported in long-term propofol use for sedation. It has been reported that intracellular adenosine triphosphate (ATP) is reduced in rhabdomyolysis. The study aims to investigate the protective effect of ATP against possible skeletal muscle damage of propofol in albino Wistar male rats biochemically and histopathologically. PA-50 (n = 6) and PA-100 (n = 6) groups of animals was injected intraperitoneally to 4 mg/kg ATP. An equal volume (0.5 ml) of distilled water was administered intraperitoneally to the P-50, P-100, and HG groups. One hour after the administration of ATP and distilled water, 50 mg/kg propofol was injected intraperitoneally to the P-50 and PA-50 groups. This procedure was repeated once a day for 30 days. The dose of 100 mg/kg propofol was injected intraperitoneally to the P-100 and PA-100 groups. This procedure was performed three times with an interval of 1 days. Our experimental results showed that propofol increased serum CK, CK-MB, creatinine, BUN, TP I, ALT, AST levels, and muscle tissue MDA levels at 100 mg/kg compared to 50 mg/kg and decreased tGSH levels. At a dose of 100 mg/kg, propofol caused more severe histopathological damage compared to 50 mg/kg. It was found that ATP prevented propofol-induced muscle damage and organ dysfunction at a dose of 50 mg/kg at a higher level compared to 100 mg/kg. ATP may be useful in the treatment of propofol-induced rhabdomyolysis and multiple organ damage.

• Journal of Korean Neurosurgical Society
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• v.64 no.3
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• pp.386-405
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• 2021

Recent advancements in basic research on the process of secondary neurulation and increased clinical experience with caudal spinal anomalies with associated abnormalities in the surrounding and distal structures shed light on further understanding of the pathoembryogenesis of the lesions and led to the new classification of these dysraphic entities. We summarized the changing concepts of lesions developed from the disordered secondary neurulation shown during the last decade. In addition, we suggested our new pathoembryogenetic explanations for a few entities based on the literature and the data from our previous animal research. Disordered secondary neurulation at each phase of development may cause corresponding lesions, such as failed junction with the primary neural tube (junctional neural tube defect and segmental spinal dysgenesis), dysgenesis or duplication of the caudal cell mass associated with disturbed activity of caudal mesenchymal tissue (caudal agenesis and caudal duplication syndrome), failed ingression of the primitive streak to the caudal cell mass (myelomeningocele), focal limited dorsal neuro-cutaneous nondisjunction (limited dorsal myeloschisis and congenital dermal sinus), neuro-mesenchymal adhesion (lumbosacral lipomatous malformation), and regression failure spectrum of the medullary cord (thickened filum and filar cyst, low-lying conus, retained medullary cord, terminal myelocele and terminal myelocystocele). It seems that almost every anomalous entity of the primary neural tube may occur in the area of secondary neurulation. Furthermore, the close association with the activity of caudal mesenchymal tissue in secondary neurulation involves a wider range of surrounding structures than in primary neurulation. Although the majority of the data are from animals, not from humans and many theories are still conjectural, these changing concepts of normal and disordered secondary neurulation will provoke further advancements in our management strategies as well as in the pathoembryogenetic understanding of anomalous lesions in this area.

• Clinical and Experimental Reproductive Medicine
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• v.47 no.4
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• pp.293-299
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• 2020

Objective: The goal of the present study was to investigate the rate of chromosomal aneuploidies in surplus embryos after sex determination at the cleavage stage. Then, the same chromosomal aneuploidies were evaluated in blastocysts after extended culture. Methods: Sixty-eight surplus embryos were biopsied at the cleavage stage and incubated for an additional 3 days to allow them to reach the blastocyst stage. The embryos were reanalyzed via fluorescence in situ hybridization (FISH) to examine eight chromosomes (13, 15, 16, 18, 21, 22, X, and Y) in both cleavage-stage embryos and blastocysts. Results: Although the total abnormality rate was lower in blastocysts (32.35%) than in cleavage-stage embryos (45.58%), the difference was not significant (p=0.113). However, when we restricted the analysis to autosomal abnormalities, we observed a significant difference in the abnormality rate between the cleavage-stage embryos (44.11%) and the blastocysts (17.64%, p=0.008). A higher rate of sex chromosomal abnormalities was also observed in cleavage-stage embryos (29.4%) than in blastocysts (14.70%, p=0.038). Conclusion: The data indicated that embryo biopsy should be conducted at the blastocyst stage rather than the cleavage stage. The results also emphasized that examination of common chromosomal aneuploidies apart from sex selection cycles can be conducted in the blastocyst stage with the FISH method.

• Clinical and Experimental Reproductive Medicine
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• v.49 no.1
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• pp.40-48
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• 2022

Objective: As the associations of sperm DNA fragmentation with morphology have not been examined in detail, this study aimed to investigate the relationship between abnormalities of morphological details and DNA integrity in human sperm. Methods: In this cross-sectional study, men from infertile couples were enrolled at Hue Center for Reproductive Endocrinology and Infertility, Vietnam. Conventional semen parameters, including morphological details, were analyzed following the World Health Organization 2010 criteria. Sperm DNA fragmentation was evaluated using a sperm chromatin dispersion assay. The relationships and correlations between semen parameters, sperm morphology, and the type of halosperm and the DNA fragmentation index (DFI) were analyzed. Results: Among 130 men in infertile couples, statistically significant differences were not found in the sperm halo type between the normal and abnormal sperm morphology groups. The percentage of round-head spermatozoa was higher in the DFI >15% group (16.98%±12.50%) than in the DFI ≤15% group (13.13% ±8.82%), higher values for amorphous heads were found in the DFI >15% group, and lower values for tapered heads were observed in the DFI ≤15% group; however, these differences were not statistically significant. Small-halo sperm and the DFI were positively correlated with round-head sperm (r=0.243, p=0.005 and r=0.197, p=0.025, respectively). Conclusion: The rate of general sperm morphological abnormalities in semen analysis was not related to sperm DNA integrity. However, round sperm heads were closely associated with sperm DNA fragmentation.