For the first time, alpha-ketoglutarate (AKG) was used to differentiate Spermatogonial stem cells... more For the first time, alpha-ketoglutarate (AKG) was used to differentiate Spermatogonial stem cells (SSCs) in the presence of BMP-4 and Retinoic acid. So, SSCs were isolated from testis of 3-6 day-old mice by enzymatic digestion by collagenase and trypsin. The Cell suspension was cultured for one week in DMEM/F12 and 20% FBS in presence of GDNF growth factor. The proliferated cells were divided between control and treatment groups. In the control group, the cells were cultured for three weeks in DMEM/F12 containing 10% FBS in presence of 10-6 M retinoic acid and 40 ng/ml of BMP-4. A dose of 0.1 M AKG was added to the treatment group. The presence of Sertoli cells in culture system was confirmed by positive reaction of vimentin immunocytochemistry. The colonies that appeared on Sertoli cells also showed positive alkaline phosphatase activity and Oct4 immunocytochemistry reaction. qRT-PCR studies showed that the expression of Acrosin and Sycp3 genes was low in two groups after 7 days of culture. 21 days after culture, in the treatment group, the expression of Acrosin and Sycp3 genes was significantly increased rather than control group (p≤0.05). Large number of early spermatids were observed in the treatment group based on TEM studies.
Many child cancer patients endure anticancer therapy containing alkylating agents before sexual m... more Many child cancer patients endure anticancer therapy containing alkylating agents before sexual maturity. Busulfan (BU), as an alkylating agent, is a chemotherapy drug, causing DNA damage and cytotoxicity in germ cells. In the present study, we aimed to investigate the protective effect of astaxanthin (AST), as a potent antioxidant and powerful reactive oxygen species (ROS) scavenger, on BU-induced toxicity in human spermatogonial stem cells. For this purpose, testes were obtained from four brain-dead donors. After tissue enzymatic digestions, testicular cells were cultured for 3 weeks for spermatogonial stem cell (SSC) isolation and purification. K562 cell line was cultured to survey the effect of AST on cancer treatment. The cultured SSCs and K562 cell line were finally treated with AST (10μM), BU (0.1nM), and AST+BU. The expression of NRF-2, HO-1, SOD2, SOD3, TP53, and apoptotic genes, including CASP9, CASP3, BCL2, and BAX, were assayed using real-time PCR. Moreover, ROS level in different groups and malondialdehyde level and total antioxidant capacity in cell contraction of SSCs were measured using ELISA. Data showed that AST significantly upregulated the expression of NRF-2 gene (P<0.001) and protein (P<0.005) and also significantly decreased the production of BU-induced ROS (P<0.001). AST activated the NRF-2/HO-1 pathway that could remarkably restrain BU-induced apoptosis in SSCs. Interestingly, AST upregulated the expression level of apoptosis genes in the K562 cell line. The results of this study indicated that AST reduces the side effects of BU on SSCs without interference with its chemotherapy effect on cancerous cells through modulation of the NRF-2/HO-1 and mitochondria-mediated apoptosis pathways.
Aim: MiRNA's-143 and -206 are powerful apoptotic regulators in cancer cells. This study aimed... more Aim: MiRNA's-143 and -206 are powerful apoptotic regulators in cancer cells. This study aimed to use miRNA-143- and 206-loaded poly(lactic- co-glycolic) acid (PLGA) nanoparticles conjugated with folic acid to induce apoptosis in the EL4 cancer cells. Materials &amp; methods: The therapy was conducted in six groups: treatment with both miRNAs simultaneously (mixed miRNAs), miRNA-206 treatment, miRNA-143 treatment, blank PLGA, blank polyethylenimine (PEI) and complex PEI-miRNAs. Results: In terms of viability, in mixed miRNAs no synergistic effect was observed on EL4 cell elimination. However, in the single miRNA-206 group, a stronger apoptotic effect was observed than the mixed miRNAs group and single miRNA-143 group alone. Conclusion: MiRNAs' apoptotic induction effects in cancer cells were found to be remarkable.
Background: The sertoli cells in the testis create unique and safe environment to protect seminif... more Background: The sertoli cells in the testis create unique and safe environment to protect seminiferous tubules from auto antigens and invading pathogens. These cells produce the survival factor of the blood-testis barrier and produce special materials such as androgen binding proteins and contribute to the coordinated action of spermatogenesis. Given that the sertoli cells play an essential role in spermatogenesis and the lack of these cells leads to the disruption of spermatogenesis, it is necessary to investigate the behavior and performance of these cells. To achieve this goal, the cells must first be extracted. The aim of this study was to develop a procedure to isolate, culture, and characterize human sertoli cells. Methods: In order to isolate the sertoli cells of azoospermia patients who underwent (testicular sperm extraction) TESE surgery, washing up and multi_stage enzyme digestion of single cells, culture on petri dishes impregnated with datura stramonium lectin agglutinin (DSA) were done and then the cells were passaged for several times and isolated. For more purification, flow cytometry method with FSH receptor antibody was used. Immunocytochemistry assays and Elisa test for identification of these cells were employed. Results: The purification method resulted in more than 97% purity. The nature of sertoli cells was confirmed by morphology evaluation, detecting anti-mullerian hormone in sertoli cell culture media and the presence of FSH receptor on sertoli cells. Conclusion: This study introduced and applied a method to isolate, culture, and purify human sertoli cells with high purity which made possible further researches on these cells.
Spermatogonial stem cells (SSCs) are in the beginning of a complex process in which they transmit... more Spermatogonial stem cells (SSCs) are in the beginning of a complex process in which they transmit genetic information from generation to generation. Any failure in this process can result in infertility. It has been suggested that transplantation of spermatogonial stem cells, following their maintenance and culturing, may restore fertility in some infertile patients. Because fertility restoration through SSCs transplantation has been successfully achieved in animal experiments, we hope human studies can follow in the near future. The isolation and cultivation of SSCs help us study their biological characteristics and their application in therapeutic approaches. In this review, we studied spermatogenesis in rodents and humans. We also compared markers and different SSC culture systems in both.
Numerous scaffolds are developed in the field of testicular bioengineering. However, effectively ... more Numerous scaffolds are developed in the field of testicular bioengineering. However, effectively replicating the spatial characteristics of native tissue, poses a challenge in maintaining the requisite cellular arrangement essential for spermatogenesis. In order to mimic the structural properties of seminiferous tubules, the objective is to fabricate a biocompatible tubular scaffold. Following the decellularization process of the testicular tissue, validation of cellular remnants' elimination from the specimens is conducted using 4′,6‐diamidino‐2‐phenylindole staining, hematoxylin and eosin staining, and DNA content analysis. The presence of extracellular matrix (ECM) components is confirmed through Alcian blue, Orcein, and Masson's trichrome staining techniques. The electrospinning technique is employed to synthesize the scaffolds using polycaprolactone (PCL), extracted ECM, and varying concentrations of graphene oxide (GO) (0.5%, 1%, and 2%). Subsequently, comprehensive ev...
DOAJ (DOAJ: Directory of Open Access Journals), Aug 28, 2022
Objective: Epigenetic and genetic changes have important roles in stem cell achievements. Accordi... more Objective: Epigenetic and genetic changes have important roles in stem cell achievements. Accordingly, the aim of this study is the evaluation of the epigenetic and genetic alterations of different culture systems, considering their efficacy in propagating human spermatogonial stem cells isolated by magnetic-activated cell sorting (MACS). Materials and Methods: IIn this experimental study, obstructive azoospermia (OA) patient-derived spermatogonial cells were divided into two groups. The MACS enriched and non-enriched spermatogonial stem cells (SSCs) were cultured in the control and treated groups; co-culture of SSCs with Sertoli cells of men with OA, co-culture of SSCs with healthy Sertoli cells of fertile men, the culture of SSCs on PLA nanofiber and culture of testicular cell suspension. Gene-specific methylation by MSP, expression of pluripotency (NANOG, C-MYC and OCT-4), and germ cells specific genes (Integrin α6, Integrin β1, PLZF) evaluated. Cultured SSCs from the optimized group were transplanted into the recipient azoospermic mouse. Results: The use of MACS for the purification of human stem cells was effective at about 69% with the culture of the testicular suspension, being the best culture system. Upon purification, the germ-specific gene expression was significantly higher in testicular cell suspension and treated groups (P≤0.05). During the culture time, gene-specific methylation patterns of the examined genes did not show any changes. Our data from transplantation indicated the homing of the donor-derived cells and the presence of human functional sperm. Conclusion: Our in vivo and in vitro results confirmed that culture of testicular cell suspension and selection of spermatogonial cells could be effective ways for purification and enrichment of the functional human spermatogonial cells. The epigenetic patterns showed that the specific methylation of the evaluated genes at this stage remained constant with no alteration throughout the entire culture systems over time.
Background Spermatogenesis refers to the differentiation of spermatogonial stem cells (SSCs) loca... more Background Spermatogenesis refers to the differentiation of spermatogonial stem cells (SSCs) located in the basal part of seminiferous tubules into haploid spermatozoa. Prerequisites for in vitro spermatogenesis include an extracellular matrix, a culture medium containing factors influencing the survival, proliferation and differentiation of spermatogenic cells, and testicular somatic cells that play a supporting role for SSCs. Accordingly, in this paper, the potential of co-culture of Sertoli cells and SSCs embedded in a hybrid hydrogel of agarose and laminin as one of the main components of the extracellular matrix of the basal compartment of seminiferous tubules in the presence of culture medium containing growth factors was investigated. Methods After three–week conventional culture of the cellular suspension of enzymatic digestion of human testicular tissue, the cells were cultured in agarose hydrogel alone or hybrid hydrogel (agarose/laminin) for 74 days. Then, the presence of...
Objective Spermatogonial stem cells (SSCs), as unipotent stem cells, are responsible for the prod... more Objective Spermatogonial stem cells (SSCs), as unipotent stem cells, are responsible for the production of sperm throughout the male’s life. Zinc finger and BTB domain containing 16 (ZBTB16/PLZF) genes provide various functions in the cell development, signaling pathway, growth regulatory and differentiation. Here, we aimed to investigate expression of the PLZF germ cell gene marker in testis, SSCs, pluripotent embryonic stem cells (ES cells) and ES-like cells of mouse testis. Materials and Methods In this experimental study, we examined the expression of the PLZF germ cell marker in the testis section and testicular cell culture of neonate and adult mice by immunohistochemistry (IMH), immunocytochemistry (ICC) and Fluidigm Real-Time polymerase chain reaction (PCR). Results IMH data indicated that the PLZF protein was localized in the neonate testis cells of the tubules center as well as the basal compartment of adult testis seminiferous tubules. Counting PLZF IMH-positive cells in ...
Objective Toll-like receptors (TLRs) on Sertoli cells are thought to have essential roles in sper... more Objective Toll-like receptors (TLRs) on Sertoli cells are thought to have essential roles in sperm protection. This study was conducted to investigate the expression of TLR2 and TLR3 in Sertoli cells of men with azoospermia. Materials and Methods In this experimental study, testicular biopsies were taken from ten azoospermic men. Following enzymatic dissociation, the samples were moved to lectin coated petri dishes. After a few passages, all cells were cultivated and Seroli cells were sorted by flow cytometry. To confirm Sertoli cell purification, alkaline phosphatase activity (ALP) and immunohistochemistry assays were employed. The expression of TLR2 and TLR3 at the transcript and protein levels was examined with real-time quantitative reverse transcription-polymerase chain reaction (RT-QPCR) and western blot, respectively. Results Isolation, purification and cultivation of human Sertoli cells were performed successfully. Efficacy of purification of Sertoli cells by fluorescence-ac...
PURPOSE Generating functional gametes for patients with male infertility is of great interest. We... more PURPOSE Generating functional gametes for patients with male infertility is of great interest. We investigated different cultural systems for proliferation of SSCs derived from obstructive azoospermic patients. Materials and Methods: Testicular cells were obtained from men with obstructive azoospermia. after enzymatic digestion process, cells assigned to various groups: culture of SSCs in the dish without cover (control group), co-culture of SSCs with infertile Sertoli cells (I), co-culture of SSCs with fertile Sertoli cells (II), culture of SSCs on nanofiber (covered with laminin) (III), culture of testicular cell suspension (IV). Then cells were cultured and evaluated colony formation, gene-specific methylation by MSP, quantitative genes expression of pluripotency (Nanog, C-Myc, Oct-4) and specific germ cell (Integrin α6, Integrin β1, PLZF) genes in five different culture systems. Results: Our findings indicate a significant increase in the number and diameter of colonies in IV ...
Testicular cancer is the most common cancer affecting men in reproductive age, and cisplatin is o... more Testicular cancer is the most common cancer affecting men in reproductive age, and cisplatin is one of the major helpful chemotherapeutic agents for treatment of this cancer. In addition, exposure of testes cancer cells to cisplatin could potentially eliminate tumour cells from germ cells in patients. The aim of this study was to evaluate the effect of cisplatin on viability of mouse acute lymphoblastic leukaemia cell line (EL‐4) and neonatal mouse spermatogonial cells in vitro. In this study, the isolated spermatogonial stem cells (SSC) and EL‐4 were divided into six groups including control (received medium), sham (received DMSO in medium) and experimental groups which received different doses of cisplatin (0.5, 5, 10 and 15 μg ml−1). Cells viability was evaluated with MTT assay. The identity of the cultured cells was confirmed by the expression of specific markers. Our finding showed that viability of both SSC and EL‐4 cells was reduced with the dose of 15 μg/ml when compared to the control group (P ≤ 0.05). Also, the differences between the IC50 in doses 10 and 15 μg/ml at different time were significant (P ≤ 0.05). The number of TUNEL‐positive cells was increased, and the BAX and caspase‐3 expressions were upregulated in EL4 cells for group that received an effective dose of cisplatin). In conclusion, despite the dramatic effects of cisplatin on both cells, spermatogonial stem cells could form colony in culture.
Illicit drug use can be an important cause of male infertility. The aim of this study was to inve... more Illicit drug use can be an important cause of male infertility. The aim of this study was to investigate the effects of an Iranian illicit drug, Kerack, on sperm parameters, testicular structure and CatSper genes expression of mice. In this study, 25 male mice were divided into five groups consisting of control, sham and three experimental groups. All animal in experimental groups were addicted to Kerack for 7 days. These experimental groups include experimental I which was given Kerack at a dose of 5 mg/kg, experimental II, 35 mg/kg and experimental III, 70 mg/kg, intraperitoneally twice a day for a period of 35 days. Mice were then sacrificed and spermatozoas were removed from cauda epididymis and analyzed for count, motility, morphology (normal/abnormal) and viability. Right testes were removed, weighed and processed for light microscopic studies whereas left testes removed were subjected to total mRNA extraction for using in real-time PCR (RT-PCR). The results were analyzed by p...
For the first time, alpha-ketoglutarate (AKG) was used to differentiate Spermatogonial stem cells... more For the first time, alpha-ketoglutarate (AKG) was used to differentiate Spermatogonial stem cells (SSCs) in the presence of BMP-4 and Retinoic acid. So, SSCs were isolated from testis of 3-6 day-old mice by enzymatic digestion by collagenase and trypsin. The Cell suspension was cultured for one week in DMEM/F12 and 20% FBS in presence of GDNF growth factor. The proliferated cells were divided between control and treatment groups. In the control group, the cells were cultured for three weeks in DMEM/F12 containing 10% FBS in presence of 10-6 M retinoic acid and 40 ng/ml of BMP-4. A dose of 0.1 M AKG was added to the treatment group. The presence of Sertoli cells in culture system was confirmed by positive reaction of vimentin immunocytochemistry. The colonies that appeared on Sertoli cells also showed positive alkaline phosphatase activity and Oct4 immunocytochemistry reaction. qRT-PCR studies showed that the expression of Acrosin and Sycp3 genes was low in two groups after 7 days of culture. 21 days after culture, in the treatment group, the expression of Acrosin and Sycp3 genes was significantly increased rather than control group (p≤0.05). Large number of early spermatids were observed in the treatment group based on TEM studies.
Many child cancer patients endure anticancer therapy containing alkylating agents before sexual m... more Many child cancer patients endure anticancer therapy containing alkylating agents before sexual maturity. Busulfan (BU), as an alkylating agent, is a chemotherapy drug, causing DNA damage and cytotoxicity in germ cells. In the present study, we aimed to investigate the protective effect of astaxanthin (AST), as a potent antioxidant and powerful reactive oxygen species (ROS) scavenger, on BU-induced toxicity in human spermatogonial stem cells. For this purpose, testes were obtained from four brain-dead donors. After tissue enzymatic digestions, testicular cells were cultured for 3 weeks for spermatogonial stem cell (SSC) isolation and purification. K562 cell line was cultured to survey the effect of AST on cancer treatment. The cultured SSCs and K562 cell line were finally treated with AST (10μM), BU (0.1nM), and AST+BU. The expression of NRF-2, HO-1, SOD2, SOD3, TP53, and apoptotic genes, including CASP9, CASP3, BCL2, and BAX, were assayed using real-time PCR. Moreover, ROS level in different groups and malondialdehyde level and total antioxidant capacity in cell contraction of SSCs were measured using ELISA. Data showed that AST significantly upregulated the expression of NRF-2 gene (P<0.001) and protein (P<0.005) and also significantly decreased the production of BU-induced ROS (P<0.001). AST activated the NRF-2/HO-1 pathway that could remarkably restrain BU-induced apoptosis in SSCs. Interestingly, AST upregulated the expression level of apoptosis genes in the K562 cell line. The results of this study indicated that AST reduces the side effects of BU on SSCs without interference with its chemotherapy effect on cancerous cells through modulation of the NRF-2/HO-1 and mitochondria-mediated apoptosis pathways.
Aim: MiRNA's-143 and -206 are powerful apoptotic regulators in cancer cells. This study aimed... more Aim: MiRNA's-143 and -206 are powerful apoptotic regulators in cancer cells. This study aimed to use miRNA-143- and 206-loaded poly(lactic- co-glycolic) acid (PLGA) nanoparticles conjugated with folic acid to induce apoptosis in the EL4 cancer cells. Materials &amp; methods: The therapy was conducted in six groups: treatment with both miRNAs simultaneously (mixed miRNAs), miRNA-206 treatment, miRNA-143 treatment, blank PLGA, blank polyethylenimine (PEI) and complex PEI-miRNAs. Results: In terms of viability, in mixed miRNAs no synergistic effect was observed on EL4 cell elimination. However, in the single miRNA-206 group, a stronger apoptotic effect was observed than the mixed miRNAs group and single miRNA-143 group alone. Conclusion: MiRNAs' apoptotic induction effects in cancer cells were found to be remarkable.
Background: The sertoli cells in the testis create unique and safe environment to protect seminif... more Background: The sertoli cells in the testis create unique and safe environment to protect seminiferous tubules from auto antigens and invading pathogens. These cells produce the survival factor of the blood-testis barrier and produce special materials such as androgen binding proteins and contribute to the coordinated action of spermatogenesis. Given that the sertoli cells play an essential role in spermatogenesis and the lack of these cells leads to the disruption of spermatogenesis, it is necessary to investigate the behavior and performance of these cells. To achieve this goal, the cells must first be extracted. The aim of this study was to develop a procedure to isolate, culture, and characterize human sertoli cells. Methods: In order to isolate the sertoli cells of azoospermia patients who underwent (testicular sperm extraction) TESE surgery, washing up and multi_stage enzyme digestion of single cells, culture on petri dishes impregnated with datura stramonium lectin agglutinin (DSA) were done and then the cells were passaged for several times and isolated. For more purification, flow cytometry method with FSH receptor antibody was used. Immunocytochemistry assays and Elisa test for identification of these cells were employed. Results: The purification method resulted in more than 97% purity. The nature of sertoli cells was confirmed by morphology evaluation, detecting anti-mullerian hormone in sertoli cell culture media and the presence of FSH receptor on sertoli cells. Conclusion: This study introduced and applied a method to isolate, culture, and purify human sertoli cells with high purity which made possible further researches on these cells.
Spermatogonial stem cells (SSCs) are in the beginning of a complex process in which they transmit... more Spermatogonial stem cells (SSCs) are in the beginning of a complex process in which they transmit genetic information from generation to generation. Any failure in this process can result in infertility. It has been suggested that transplantation of spermatogonial stem cells, following their maintenance and culturing, may restore fertility in some infertile patients. Because fertility restoration through SSCs transplantation has been successfully achieved in animal experiments, we hope human studies can follow in the near future. The isolation and cultivation of SSCs help us study their biological characteristics and their application in therapeutic approaches. In this review, we studied spermatogenesis in rodents and humans. We also compared markers and different SSC culture systems in both.
Numerous scaffolds are developed in the field of testicular bioengineering. However, effectively ... more Numerous scaffolds are developed in the field of testicular bioengineering. However, effectively replicating the spatial characteristics of native tissue, poses a challenge in maintaining the requisite cellular arrangement essential for spermatogenesis. In order to mimic the structural properties of seminiferous tubules, the objective is to fabricate a biocompatible tubular scaffold. Following the decellularization process of the testicular tissue, validation of cellular remnants' elimination from the specimens is conducted using 4′,6‐diamidino‐2‐phenylindole staining, hematoxylin and eosin staining, and DNA content analysis. The presence of extracellular matrix (ECM) components is confirmed through Alcian blue, Orcein, and Masson's trichrome staining techniques. The electrospinning technique is employed to synthesize the scaffolds using polycaprolactone (PCL), extracted ECM, and varying concentrations of graphene oxide (GO) (0.5%, 1%, and 2%). Subsequently, comprehensive ev...
DOAJ (DOAJ: Directory of Open Access Journals), Aug 28, 2022
Objective: Epigenetic and genetic changes have important roles in stem cell achievements. Accordi... more Objective: Epigenetic and genetic changes have important roles in stem cell achievements. Accordingly, the aim of this study is the evaluation of the epigenetic and genetic alterations of different culture systems, considering their efficacy in propagating human spermatogonial stem cells isolated by magnetic-activated cell sorting (MACS). Materials and Methods: IIn this experimental study, obstructive azoospermia (OA) patient-derived spermatogonial cells were divided into two groups. The MACS enriched and non-enriched spermatogonial stem cells (SSCs) were cultured in the control and treated groups; co-culture of SSCs with Sertoli cells of men with OA, co-culture of SSCs with healthy Sertoli cells of fertile men, the culture of SSCs on PLA nanofiber and culture of testicular cell suspension. Gene-specific methylation by MSP, expression of pluripotency (NANOG, C-MYC and OCT-4), and germ cells specific genes (Integrin α6, Integrin β1, PLZF) evaluated. Cultured SSCs from the optimized group were transplanted into the recipient azoospermic mouse. Results: The use of MACS for the purification of human stem cells was effective at about 69% with the culture of the testicular suspension, being the best culture system. Upon purification, the germ-specific gene expression was significantly higher in testicular cell suspension and treated groups (P≤0.05). During the culture time, gene-specific methylation patterns of the examined genes did not show any changes. Our data from transplantation indicated the homing of the donor-derived cells and the presence of human functional sperm. Conclusion: Our in vivo and in vitro results confirmed that culture of testicular cell suspension and selection of spermatogonial cells could be effective ways for purification and enrichment of the functional human spermatogonial cells. The epigenetic patterns showed that the specific methylation of the evaluated genes at this stage remained constant with no alteration throughout the entire culture systems over time.
Background Spermatogenesis refers to the differentiation of spermatogonial stem cells (SSCs) loca... more Background Spermatogenesis refers to the differentiation of spermatogonial stem cells (SSCs) located in the basal part of seminiferous tubules into haploid spermatozoa. Prerequisites for in vitro spermatogenesis include an extracellular matrix, a culture medium containing factors influencing the survival, proliferation and differentiation of spermatogenic cells, and testicular somatic cells that play a supporting role for SSCs. Accordingly, in this paper, the potential of co-culture of Sertoli cells and SSCs embedded in a hybrid hydrogel of agarose and laminin as one of the main components of the extracellular matrix of the basal compartment of seminiferous tubules in the presence of culture medium containing growth factors was investigated. Methods After three–week conventional culture of the cellular suspension of enzymatic digestion of human testicular tissue, the cells were cultured in agarose hydrogel alone or hybrid hydrogel (agarose/laminin) for 74 days. Then, the presence of...
Objective Spermatogonial stem cells (SSCs), as unipotent stem cells, are responsible for the prod... more Objective Spermatogonial stem cells (SSCs), as unipotent stem cells, are responsible for the production of sperm throughout the male’s life. Zinc finger and BTB domain containing 16 (ZBTB16/PLZF) genes provide various functions in the cell development, signaling pathway, growth regulatory and differentiation. Here, we aimed to investigate expression of the PLZF germ cell gene marker in testis, SSCs, pluripotent embryonic stem cells (ES cells) and ES-like cells of mouse testis. Materials and Methods In this experimental study, we examined the expression of the PLZF germ cell marker in the testis section and testicular cell culture of neonate and adult mice by immunohistochemistry (IMH), immunocytochemistry (ICC) and Fluidigm Real-Time polymerase chain reaction (PCR). Results IMH data indicated that the PLZF protein was localized in the neonate testis cells of the tubules center as well as the basal compartment of adult testis seminiferous tubules. Counting PLZF IMH-positive cells in ...
Objective Toll-like receptors (TLRs) on Sertoli cells are thought to have essential roles in sper... more Objective Toll-like receptors (TLRs) on Sertoli cells are thought to have essential roles in sperm protection. This study was conducted to investigate the expression of TLR2 and TLR3 in Sertoli cells of men with azoospermia. Materials and Methods In this experimental study, testicular biopsies were taken from ten azoospermic men. Following enzymatic dissociation, the samples were moved to lectin coated petri dishes. After a few passages, all cells were cultivated and Seroli cells were sorted by flow cytometry. To confirm Sertoli cell purification, alkaline phosphatase activity (ALP) and immunohistochemistry assays were employed. The expression of TLR2 and TLR3 at the transcript and protein levels was examined with real-time quantitative reverse transcription-polymerase chain reaction (RT-QPCR) and western blot, respectively. Results Isolation, purification and cultivation of human Sertoli cells were performed successfully. Efficacy of purification of Sertoli cells by fluorescence-ac...
PURPOSE Generating functional gametes for patients with male infertility is of great interest. We... more PURPOSE Generating functional gametes for patients with male infertility is of great interest. We investigated different cultural systems for proliferation of SSCs derived from obstructive azoospermic patients. Materials and Methods: Testicular cells were obtained from men with obstructive azoospermia. after enzymatic digestion process, cells assigned to various groups: culture of SSCs in the dish without cover (control group), co-culture of SSCs with infertile Sertoli cells (I), co-culture of SSCs with fertile Sertoli cells (II), culture of SSCs on nanofiber (covered with laminin) (III), culture of testicular cell suspension (IV). Then cells were cultured and evaluated colony formation, gene-specific methylation by MSP, quantitative genes expression of pluripotency (Nanog, C-Myc, Oct-4) and specific germ cell (Integrin α6, Integrin β1, PLZF) genes in five different culture systems. Results: Our findings indicate a significant increase in the number and diameter of colonies in IV ...
Testicular cancer is the most common cancer affecting men in reproductive age, and cisplatin is o... more Testicular cancer is the most common cancer affecting men in reproductive age, and cisplatin is one of the major helpful chemotherapeutic agents for treatment of this cancer. In addition, exposure of testes cancer cells to cisplatin could potentially eliminate tumour cells from germ cells in patients. The aim of this study was to evaluate the effect of cisplatin on viability of mouse acute lymphoblastic leukaemia cell line (EL‐4) and neonatal mouse spermatogonial cells in vitro. In this study, the isolated spermatogonial stem cells (SSC) and EL‐4 were divided into six groups including control (received medium), sham (received DMSO in medium) and experimental groups which received different doses of cisplatin (0.5, 5, 10 and 15 μg ml−1). Cells viability was evaluated with MTT assay. The identity of the cultured cells was confirmed by the expression of specific markers. Our finding showed that viability of both SSC and EL‐4 cells was reduced with the dose of 15 μg/ml when compared to the control group (P ≤ 0.05). Also, the differences between the IC50 in doses 10 and 15 μg/ml at different time were significant (P ≤ 0.05). The number of TUNEL‐positive cells was increased, and the BAX and caspase‐3 expressions were upregulated in EL4 cells for group that received an effective dose of cisplatin). In conclusion, despite the dramatic effects of cisplatin on both cells, spermatogonial stem cells could form colony in culture.
Illicit drug use can be an important cause of male infertility. The aim of this study was to inve... more Illicit drug use can be an important cause of male infertility. The aim of this study was to investigate the effects of an Iranian illicit drug, Kerack, on sperm parameters, testicular structure and CatSper genes expression of mice. In this study, 25 male mice were divided into five groups consisting of control, sham and three experimental groups. All animal in experimental groups were addicted to Kerack for 7 days. These experimental groups include experimental I which was given Kerack at a dose of 5 mg/kg, experimental II, 35 mg/kg and experimental III, 70 mg/kg, intraperitoneally twice a day for a period of 35 days. Mice were then sacrificed and spermatozoas were removed from cauda epididymis and analyzed for count, motility, morphology (normal/abnormal) and viability. Right testes were removed, weighed and processed for light microscopic studies whereas left testes removed were subjected to total mRNA extraction for using in real-time PCR (RT-PCR). The results were analyzed by p...
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Papers by Morteza Koruji