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Papers by khosro pajooh

Research paper thumbnail of Dynamics of Interaction Between Ram Sperm with Plasmid Carrying Human Lysozyme Gene in SMGT

Research paper thumbnail of Techniques for augmentation of exogenous DNA uptake by ovine spermatozoa

Iranian journal of veterinary research, 2016

Sperm mediated gene transfer can be an inexpensive and simple method in animal transgenesis; howe... more Sperm mediated gene transfer can be an inexpensive and simple method in animal transgenesis; however its efficiency is poor, mainly due to the spermatozoa's lesser uptake of exogenous DNA. In the present study, the effects of lipofection and other augmentation techniques, such as sperm freezing and spermatozoa treatment with triton X100 and DMSO, on exogenous DNA uptake by sheep spermatozoa and motility of sperms with plasmid uptake were evaluated. In the first experiment, ram sperms were incubated with a complex of rhodamine labeled plasmid (p-EGFP) and Lipofectamine 2000(TM). In the second, spermatozoa were treated with Triton X-100(TM) or DMSO or were frozen without cryoprotectant. The results indicated that there was no significant difference (P<0.05) in the transfection rates and in the uptake intensity of lipofected sperms with 300 and 600 ng of plasmid in comparison with control group, i.e. transfected without lipofectamine. Furthermore, lipofection could not improve s...

Research paper thumbnail of Transfection of bovine spermatogonial stem cells in vitro

Iranian journal of veterinary research, 2017

Spermatogonial stem cells (SSCs) are the only stem cells in adults that can transfer genetic info... more Spermatogonial stem cells (SSCs) are the only stem cells in adults that can transfer genetic information to the future generations. Considering the fact that a single SSC gives rise to a vast number of spermatozoa, genetic manipulation of these cells is a potential novel technology with feasible application to various animal species. The aim of this study was to evaluate enhanced green fluorescent protein (EGFP) gene transfection into bovine SSCs via liposome carrier and assess the best incubation day in uptake exogenous gene by SSCs. Transfection efficiency of EGFP gene with lipofectamine 2000 was determined in days following each three day of transfection (day 4, 6 and 8 of the culture) by fluorescent microscope. Results showed that the transfected cells through lipofection increased significantly (P<0.05) in each three days of transfection in comparison with those of the control groups. The transfected SSCs were higher in comparison with those of the free exogenous gene carrie...

Research paper thumbnail of The Protective Effect of Curcumin on the Proliferation and Colonization of Spermatogonial Stem Cells in Gamma-Irradiated Rats

Journal of Advanced Biomedical Sciences

Background & Objective: One of the side effects of radiotherapy can be damage to spermatogonial s... more Background & Objective: One of the side effects of radiotherapy can be damage to spermatogonial stem cells that may lead to spermatogenesis disorders and sterility. Protective effects of curcumin on normal cells against radiotherapy side effects have already been shown. In the current study, the protective effects of curcumin on the spermatogonial stem cells against gamma radiation were evaluated. Materials & Methods: This study was done on 50 adult rats in 10 experimental groups. Four groups were injected 0, 25, 50, or 100mg/kg of curcumin in 1ml olive oil for 15 days intraperitoneally, then exposed to radiation at 2 Gy on the next day. Also, four groups were treated like above but without radiation; and two groups as control with and without radiation. The day after radiation, all of the rats were euthanatized, their testes were removed, and they underwent enzymatic digestion to co-culture spermatogonial stem cells. After 12 days, the colonization of spermatogonial stem cells was ...

Research paper thumbnail of Expression of fluorescent reporter protein was not obtained in ovine embryos produced through in vitro fertilization-sperm mediated gene transfer (IVF-SMGT)

Small Ruminant Research, 2016

Sperm mediated gene transfer (SMGT) has been reported to be a powerful tool for producing transge... more 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.

Research paper thumbnail of The effect of combining vitamin E and C on the viability improvement of transfected ovine spermatogonial stem cells after cryopreservation and thawing

TURKISH JOURNAL OF VETERINARY AND ANIMAL SCIENCES, 2017

The present study aimed to identify whether vitamin E and C and their combination in basal freezi... more 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.

Research paper thumbnail of Dynamics of Interaction Between Ram Sperm with Plasmid Carrying Human Lysozyme Gene in SMGT

Research paper thumbnail of Techniques for augmentation of exogenous DNA uptake by ovine spermatozoa

Iranian journal of veterinary research, 2016

Sperm mediated gene transfer can be an inexpensive and simple method in animal transgenesis; howe... more Sperm mediated gene transfer can be an inexpensive and simple method in animal transgenesis; however its efficiency is poor, mainly due to the spermatozoa's lesser uptake of exogenous DNA. In the present study, the effects of lipofection and other augmentation techniques, such as sperm freezing and spermatozoa treatment with triton X100 and DMSO, on exogenous DNA uptake by sheep spermatozoa and motility of sperms with plasmid uptake were evaluated. In the first experiment, ram sperms were incubated with a complex of rhodamine labeled plasmid (p-EGFP) and Lipofectamine 2000(TM). In the second, spermatozoa were treated with Triton X-100(TM) or DMSO or were frozen without cryoprotectant. The results indicated that there was no significant difference (P<0.05) in the transfection rates and in the uptake intensity of lipofected sperms with 300 and 600 ng of plasmid in comparison with control group, i.e. transfected without lipofectamine. Furthermore, lipofection could not improve s...

Research paper thumbnail of Transfection of bovine spermatogonial stem cells in vitro

Iranian journal of veterinary research, 2017

Spermatogonial stem cells (SSCs) are the only stem cells in adults that can transfer genetic info... more Spermatogonial stem cells (SSCs) are the only stem cells in adults that can transfer genetic information to the future generations. Considering the fact that a single SSC gives rise to a vast number of spermatozoa, genetic manipulation of these cells is a potential novel technology with feasible application to various animal species. The aim of this study was to evaluate enhanced green fluorescent protein (EGFP) gene transfection into bovine SSCs via liposome carrier and assess the best incubation day in uptake exogenous gene by SSCs. Transfection efficiency of EGFP gene with lipofectamine 2000 was determined in days following each three day of transfection (day 4, 6 and 8 of the culture) by fluorescent microscope. Results showed that the transfected cells through lipofection increased significantly (P<0.05) in each three days of transfection in comparison with those of the control groups. The transfected SSCs were higher in comparison with those of the free exogenous gene carrie...

Research paper thumbnail of The Protective Effect of Curcumin on the Proliferation and Colonization of Spermatogonial Stem Cells in Gamma-Irradiated Rats

Journal of Advanced Biomedical Sciences

Background & Objective: One of the side effects of radiotherapy can be damage to spermatogonial s... more Background & Objective: One of the side effects of radiotherapy can be damage to spermatogonial stem cells that may lead to spermatogenesis disorders and sterility. Protective effects of curcumin on normal cells against radiotherapy side effects have already been shown. In the current study, the protective effects of curcumin on the spermatogonial stem cells against gamma radiation were evaluated. Materials & Methods: This study was done on 50 adult rats in 10 experimental groups. Four groups were injected 0, 25, 50, or 100mg/kg of curcumin in 1ml olive oil for 15 days intraperitoneally, then exposed to radiation at 2 Gy on the next day. Also, four groups were treated like above but without radiation; and two groups as control with and without radiation. The day after radiation, all of the rats were euthanatized, their testes were removed, and they underwent enzymatic digestion to co-culture spermatogonial stem cells. After 12 days, the colonization of spermatogonial stem cells was ...

Research paper thumbnail of Expression of fluorescent reporter protein was not obtained in ovine embryos produced through in vitro fertilization-sperm mediated gene transfer (IVF-SMGT)

Small Ruminant Research, 2016

Sperm mediated gene transfer (SMGT) has been reported to be a powerful tool for producing transge... more 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.

Research paper thumbnail of The effect of combining vitamin E and C on the viability improvement of transfected ovine spermatogonial stem cells after cryopreservation and thawing

TURKISH JOURNAL OF VETERINARY AND ANIMAL SCIENCES, 2017

The present study aimed to identify whether vitamin E and C and their combination in basal freezi... more 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.