The aim of the present study was to evaluate the effects of quercetin as an antioxidant supplement on frozen-thawed ram epididymal sperm quality. Quercetin is a type of flavonoid antioxidant that is found in plants, with the ability to scavenge free radicals. Twenty testicles from mature rams were collected from a nearby slaughterhouse immediately after slaughter. Epididymal spermatozoa were recovered from the caudal of epididymides by injecting Bracket and Oliphant's (BO) medium retrogradely through the ductus deferens and extended with a tris egg-yolk-based extender and supplemented with 0, 5, 10, 15, 20, and 50 µg/mL quercetin. Following equilibration, the straws were frozen, and then plunged into liquid nitrogen. After thawing, optimized concentrations of quercetin were defined based on their viabilities and used to assess fertilization and developmental potential. The results showed that the viability of frozen-thawed spermatozoa significantly increased by using 5 and 10 µg/mL quercetin in the freezing extender. However, total and progressive motility of frozen-thawed spermatozoa were not affected by 5 and 10 µg/mL quercetin in comparison with control (0 µg/mL). The mean number of zygote, morula, and blastocyst stage embryos increased significantly by using 5 and 10 µg/mL quercetin compared with other frozen-thawed treatments(P <0.05). However, the blastocyst rate of fresh sperm was significantly higher (P <0.05). In conclusion, to improve the quality of frozen-thawed ram epididymal spermatozoa, 5 and 10 µg/mL quercetin appears to be an attractive option. Further studies are suggested to understand the synergistic effect of quercetin with other antioxidants to improve the ram freezing-thawing process.
The present study aimed to identify whether vitamin E and C and their combination in basal freezing medium improved the viability of postthaw transfected spermatogonial stem cells (SSCs). For this purpose, SSCs were harvested by enzymatic digestion twice. After enrichment and culture on Sertoli cell feeder layer, SSCs were characterized by using alkaline phosphatase staining and expression of oct-4 and c-kit genes as specific stem cell markers. As to the transfection of SSCs, different concentrations of DNA (0.2, 0.4, and 0.8 µg) and turbofect (0.4 and 0.8 µL) in 100 µL of medium were studied. The results showed that cryopreservation of SSCs in the presence of 25 µg/mL vitamin E and 10 µg/mL vitamin C could increase cell viability and expression of antiapoptotic genes. The best combination of DNA and turbofect for transfection of SSCs in 100 µL of medium was 0.8 µg and 0.4 µL, respectively. However, SSCs indicated lower transfection efficiency compared with Sertoli cells (~30% vs. 70%, respectively). Cryopreservation with the addition of vitamin E and C in the basal freezing medium could increase cell viability of transfected SSCs as well. The findings of this study also suggest the need for further research regarding improvement of the transfection efficiency of SSCs.
Sperm mediated gene transfer (SMGT) has been reported to be a powerful tool for producing transgenic animals on a mass scale using spermatozoa as vectors for exogenous DNA. In this study the possibility of using in vitro fertilization (IVF)-SMGT to produce transgenic ovine embryos was investigated for the first time. For this purpose, sperm were obtained from the epididymal testicle areas of 4 rams and different concentration of DNA (0.4, 0.8 and 1.6 μg) and TurboFect (0.25, 0.5, 1 and 2 μg) were used for sperm transfection (1×10 6). Enhanced-GFP-expressing vector pEGFP-N1 was used as the carrier. In order to evaluate the performance of transgenic sperm, in vitro fertilization technology was used. After the preparing oocytes received from the ovaries of slaughterhouse origin, oocytes with more than three layers of granulosa and uniform cytoplasm were selected and matured in TCM-199 medium containing 10% fetal calf serum, follicle stimulating hormone (FSH) (5μg/ml), β-17 estradiol (1μg/ml) and sodium pyruvate 0.81 (Mm). Bracket and Oliphant's (BO) medium and modified Charles Rosenkrans medium with amino acids (mCR2aa) were used for in vitro fertilization and culture, respectively. Results showed that transfected sperm with different concentrations of DNA and TurboFect carrier were unable to transfer the GFP gene to in vitro matured oocytes as the GFP gene was not expressed in neither zygotes nor morula stage embryos. Further optimization for SMGT improvement such as different transfection reagent and method like electroporation, using antioxidants in transfection medium to overcome the apoptosis and also separation of transfected sperm from untransfected ones before insemination have been suggested. Research highlights Transfected sperm with different concentrations of DNA and TurboFect carrier were unable to transfer the GFP gene to in vitro matured ovine oocytes. By increasing the amount of DNA, the expression of GFP gene was improved in fibroblast cells. The mean number of zygote and morula stage embryos significantly decreased, by increasing the amount of TurboFect.
The present study aimed to optimize the in vitro culture and transfection efficiency of bovine primary spermatogonial stem cells (SSCs). To this end, SSCs were obtained from newborn Holstein bull calves by two-step enzymatic digestion. After enrichment and culture, SSCs were characterized by using alkaline phosphatase (AP) staining and expression of vasa and thy1 genes as specific bovine SSC markers. To evaluate the effect of antioxidants on vitality, colony formation, and the expression of pro-and anti-apoptotic genes of bovine SSCs, various concentrations of vitamin C (5, 10, 25 and 50 µg/mL) and Trolox (a water soluble α-tocopherol analogue) (12.5, 25, 50 and 100 µg/mL) were added to the SSC culture medium. The results showed that SSCs treated with 50 µg/mL of vitamin C or 25 µg/mL of Trolox individually could increase cell viability and colony formation significantly in comparison with other concentrations and the control group. Additionally, the expressions of bax (as a pro-apoptotic gene) and bcl2 (as an anti-apoptotic gene) were significantly lower and higher than the control group, respectively. To optimize the transfection condition, the effective dosages of vitamin C or Trolox, with various concentrations of two transfection reagents (X-tremeGENE HP and Turbofect) and DNA, at day 8 of culture, were studied. Results showed that 1 μl X-tremeGENE HP or 0.5 μl Turbofect and 2 μg of DNA are the best concentrations for transfecting SSCs. However, X-tremeGENE HP expressed more potential for transfecting SSCs in comparison with Turbofect. Besides, no difference was observed between the use of defined doses of vitamin C or Trolox. ____________________________________________________________________________________
Systemic Lupus Erythematosus (SLE) is a chronic human autoimmune disease, which is characterized by increased activity of B cells and production of antibodies against tissue antigens. It engages many tissues and organs, including joints, kidneys, heart, and the nervous system. Although the exact pathogenesis of SLE remains to be elucidated, it is suggested that genetic background plays a paramount role in SLE etiology. Increasing evidence is indicating an important role for interleukins in progression of SLE. Interleukins are a group of cytokines secreted by T helper cells, monocytes, macrophages, and B cells, which are involved in growth and differentiation of T and B cells. The expression level of interleukins is influenced greatly by genetic composition. Therefore, some polymorphisms can control the expression of interleukins. Consequently, genetic studies can shed light on our understanding of SLE nature. Therefore, in the present study, the researchers reviewed the roles of eight key interleukin polymorphisms and their effects on SLE pathogenic.
Abstract. Sperm-mediated gene transfer (SMGT) has been considered as an innovative device for transgenesis on a mass scale by taking advantage of live spermatozoa to transfer exogenous DNA. However, the fertilizing ability of transfected sperm cells and the poor reproducibility of this method are still matters of controversy. Hence, the current study was conducted to evaluate transfecting the enhanced green fluorescent protein (EGFP) as the source of exogenous DNA into bovine spermatozoa using a high-performance polymer reagent as well as assessing the fertilizing capacity of transfected sperm cells by in vitro fertilization (IVF). In the first experiment, three different concentrations of rhodamine-labeled DNA and high-performance polymer transfection reagent, X-tremeGENE HP, were used to transfect bovine spermatozoa. In the second experiment, IVF and fluorescence microscopy methods were utilized to assess the fertilizing capacity of sperm cells carrying exogenous DNA when X-tremeGENE HP was used either alone or with dimethyl sulfoxide (DMSO) treatment. Findings revealed that at 1 µL X-tremeGENE HP and 1 µg of DNA concentration, approximately one-third of total spermatozoa were transfected. However, following IVF and fluorescence microscopy, no EGFP expression was detected in zygotes and morula-stage embryos. Results of this study showed that, although X-tremeGENE HP could transfer EGFP to bovine spermatozoa, transfected sperm cells were unable to transfer foreign DNA to matured bovine oocytes. Under our experimental conditions, we hypothesized that the absence of the EGFP fluorescence signal in embryos could be due to the detrimental effects of transfection treatments on sperm cells' fertility performance as well as incompetency of IVF to produce transgenic embryos using transfected sperm cells.
The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. Rho kinase (ROCK) belongs to a family of serine/threonine kinases and involves in a wide range of fundamental cellular functions. The aim of the present study was to study the effect of ROCK inhibitor, Y-27632 (0.1-40 µM), during the primary culture of ovine SSCs. SSCs were collected from 3-5-month-old's lamb testes. The viability of SSCs, the apoptosis assay of SSCs, the intracellular reactive oxygen species (ROS) analysis, and the SSCs markers and apoptosis-related gene expressions were detected by MTT reduction assay, Annexin V-FITC/ Propidium Iodide (PI) dual staining, flow cytometry and real-time-PCR studies, respectively. Morphological analyses indicated that the 5-10 µM Y-27632 had an optimal effect on the number of presumptive SSCs colonies and the area covered by them after a 10 days culture. The cell viability, apoptosis and necrosis of SSCs after 10 days' culture were not affected in comparison with the control group, and the 20 µM of Y-27632 resulted in significantly decreased cell viability (P<0.05) and an increased necrosis of cells. On day 10 after culture, the expression of P53 was decreased with an increase from 0 to 10 µM in the Y-27632 dose. In the 20 µM Y-27632 group, the expressions of P53 and Bax were higher and the Bcl-2 was lower than other groups and these values were significantly different from 5 and 10 µM Y-27632 groups (P<0.05). The level of intracellular ROS was decreased with an increase in the Y-27632 dose from 5 to 20 µM in comparison with the control group. In conclusion, the present study demonstrated that Y-27632 at a concentration of 5-10 µM provided optimal culture conditions for the primary culture of ovine SSCs.
The genetic manipulation of spermatogonial stem cells (SSCs) can be used as an alternative to somatic cell nuclear transfer method for the production of transgenic animals. SSCs are now in vitro cultured and transplanted in sheep, however, there are no known protocol for DNA transfection of sheep SSCs. The aim of present study was to define the optimal transfection conditions of spermatogonial stem cells (SSCs) in early and late ovine SSC colony formation stages in culture. SSCs were isolated from the slaughterhouse ram testis tissue using a two-step enzymatic digestion process. Results showed that, 2 µl of DNA with 0.5 µg of Lipofectamin or Turbofect were able to transfect SSC colonies at late stage. Since the colonies of SSCs were not in logarithmic growth phase, around 15% of colonies were transfected and no significant difference between Lipofectamin or Turbofect was observed. However, more cells were transfected on early stages of SSC colony formation (7th days), especially when Turbofect was used (around 40 and 45% for Lipofectamin and Turbofect, respectively). Although the early stages SSCs were more suitable for transfection, but the formation of colonies were impaired on transfected cells.
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