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.
International Journal of Biological Macromolecules, Apr 1, 2023
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 in uencing 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 spermatogonial cells and other cells of the different stages of spermatogenesis were analysed by immunocytochemistry, real-time PCR, electron microscopy and morphological staining. Results The results showed that colonies with positive spermatogenesis markers were observed in both culture systems. The presence of different levels of spermatogenesis in two groups media was con rmed, but morphological spermatozoa were observed only in hybrid hydrogel group. Conclusions Based on the results of this paper, it can be concluded that an agarose and extracellular matrix-based scaffold which maintains the mechanical strength of the scaffold during culture and also increases the biological activity of the scaffold, can support from all physiological activities of SSCs such as survival, proliferation and differentiation.
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.
Background: In the males, Spermatogonial Stem Cells (SSCs) contribute to the production of sex ce... more Background: In the males, Spermatogonial Stem Cells (SSCs) contribute to the production of sex cells and fertility. In vitro SSCs culture can operate as an effective strategy for studies on spermatogenesis and male infertility treatment. Cell culture in a three-dimensional (3D) substrate, relative to a two-dimensional substrate (2D), creates better conditions for cell interaction and is closer to in vivo conditions. In the present study, in order to create a 3D matrix substrate, decellularized testicular matrix (DTM) was used to engender optimal conditions for SSCs culture and differentiation. Materials and Methods: After, testicular cells enzymatic extraction from testes of brain-dead donors, the SSCs were proliferated in a specific culture medium for four weeks, and after confirming the identity of the colonies derived from the growth of these cells, they were cultured on a layer of DTM as well as in 2D condition with a differentiated culture medium. In the Sixth week since the initiation of the differentiation culture, the expression of pre meiotic (OCT4 & PLZF), meiotic (SCP3 & BOULE) and post meiotic (CREM & Protamine-2) genes were measured in both groups. Results: The results indicated that the expression of pre meiotic, meiotic and post meiotic genes was significantly higher in the cells cultured on DTM (P ≤ 0.001). Conclusion: SSCs culture in DTM with the creation of ECM and similar conditions with in vivo can be regarded as a way of demonstrating spermatogenesis in vitro, which can be adopted as a treatment modality for male infertility.
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 ...
Application of a three-dimensional (3D) culture system for in vitro proliferation and differentia... more Application of a three-dimensional (3D) culture system for in vitro proliferation and differentiation of human spermatogonial stem cells (SSCs) is a useful tool for the investigation of the spermatogenesis process and the management of male infertility particularly in prepubertal cancer patients. The main purpose of this study was to investigate the proliferation of human SSCs co-cultured with Sertoli cells in soft agar culture system (SACS) supplemented by Laminin and growth factors. Testicular cells were isolated from testes of brain-dead patients and cultured in two-dimensional (2D) culture system for 3 weeks. After 3 weeks, functional SSCs were evaluated by xenotransplantation and also identification of cells was assessed by immunocytochemistry, flow cytometry, and RT-PCR. Then, SSCs and Sertoli cells were transferred to the upper layer of SACS for 3 weeks. After 3 weeks, the number of colonies and the expression of specific SSCs and Sertoli cell markers, as well as apoptotic genes were evaluated. Our results showed that transplanted SSCs, migrated into the basement membrane of seminiferous tubules of recipient mice. The expression of PLZF, α6-Integrin, and Vimentin proteins in SSCs and Sertoli cells were observed in 2D and 3D culture systems. The expression rate of PLZF, α6-Integrin, Bcl2, and colony number in SACS supplemented by Laminin and growth factors group were significantly higher than nonsupplemented groups (P ≤ 0.01), but the expression rate of c-kit and Bax in supplemented group were significantly lower than non-supplemented groups (P ≤ 0.05). This 3D co-culture system decreased apoptosis and increased propagation of human SSCs. Therefore, this designed system can be utilized to increase the proliferation of human SSCs in prepubertal male cancer and azoospermic men to obtain an adequate SSCs number to outotransplant success and in vitro spermatogenesis.
INTRODUCTION AND OBJECTIVES: Platelet rich plasma (PRP) is a novel therapeutic increasingly promo... more INTRODUCTION AND OBJECTIVES: Platelet rich plasma (PRP) is a novel therapeutic increasingly promote for various indications, including several within urology. PRP is a platelet concentrate extracted from whole blood via proprietary separator gel systems. Prevailing theories regarding efficacy suggest that local delivery of trophic factors will promote tissue regeneration. Detailed quantitative analysis of delivered factors is essential and prior investigations have failed to adequately delineate profiles. We sought to characterize PRP cytokine profiles across a spectrum of donors using a single proprietary system. METHODS: Whole blood was collected from 10 healthy volunteers age 20-65 using standard technique into 11 or 22cc PRP tubes from Eclipse Aesthetics (The Colony, Texas). Tubes were centrifuged until separated. Concentrated PRP (platelet layer mixed with a minority of the platelet poor layer) was sent immediately for 200 Human Biomarker Testing (Ray Biotec; Norcross, Georgia). Platelet concentration was determined for each sample. RESULTS: The ten highest factors and their primary roles are demonstrated (Table 1). Across the cohort, relative to whole blood, platelet concentration was 1.91 fold higher in the final product. Platelet concentration in PRP obtained from 22cc tubes was significantly higher than 11cc tubes (p[.037). At p[.05, heat mapping reveals 31 trophic factors responsible for driving variability in expression between 22cc and 11cc samples (Table 2). CONCLUSIONS: The majority of the most concentrated cytokines are growth factors and cell adhesion molecules, correlating with the presumed mechanism of PRP. Collection tube characteristics may affect platelet concentration and the level of trophic factor expression. These findings underscore the difficulty of producing a standardized PRP product and suggest a need for further investigation into isolation of the highly expressed factors and optimization of collection.
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...
Manganese inhibits oxidative stress damage. The aim of this study was to investigate the protecti... more Manganese inhibits oxidative stress damage. The aim of this study was to investigate the protective role of manganese on testis structure and sperm parameters in adult mice exposed to formaldehyde (FA). Twenty adult male NMRI mice were selected and randomly divided into four groups: (i) control; (ii) sham; (iii) 'FA'-exposed group; and (iv) 'FA and manganese chloride'exposed group. The FA-exposed groups received 10 mg kg À1 FA daily for 14 days, and manganese chloride was just injected intraperitoneally 5 mg kg À1 on 2nd weeks. Mice were sacrificed, and spermatozoa were collected from the cauda of the right epididymis and analysed for count, motility, morphology and viability. The other testicular tissues were weighed and prepared for histological examination upon removal. Seminiferous tubules, lumen diameters and epithelium thickness were also measured. The findings revealed that FA significantly reduced the testicular weight, sperm count, motility, viability and normal morphology compared with control group (P 0.05). In addition, seminiferous tubules atrophied and seminiferous epithelial cells disintegrated in the FA group in comparison with the control group (P 0.05). However, manganese improved the testicular structure and sperm parameters in FA-treated mice testes (P 0.05). According to the results, manganese may improve and protect mice epididymal sperm parameters and testis structure treated with FA respectively.
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.
International Journal of Biological Macromolecules, Apr 1, 2023
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 in uencing 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 spermatogonial cells and other cells of the different stages of spermatogenesis were analysed by immunocytochemistry, real-time PCR, electron microscopy and morphological staining. Results The results showed that colonies with positive spermatogenesis markers were observed in both culture systems. The presence of different levels of spermatogenesis in two groups media was con rmed, but morphological spermatozoa were observed only in hybrid hydrogel group. Conclusions Based on the results of this paper, it can be concluded that an agarose and extracellular matrix-based scaffold which maintains the mechanical strength of the scaffold during culture and also increases the biological activity of the scaffold, can support from all physiological activities of SSCs such as survival, proliferation and differentiation.
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.
Background: In the males, Spermatogonial Stem Cells (SSCs) contribute to the production of sex ce... more Background: In the males, Spermatogonial Stem Cells (SSCs) contribute to the production of sex cells and fertility. In vitro SSCs culture can operate as an effective strategy for studies on spermatogenesis and male infertility treatment. Cell culture in a three-dimensional (3D) substrate, relative to a two-dimensional substrate (2D), creates better conditions for cell interaction and is closer to in vivo conditions. In the present study, in order to create a 3D matrix substrate, decellularized testicular matrix (DTM) was used to engender optimal conditions for SSCs culture and differentiation. Materials and Methods: After, testicular cells enzymatic extraction from testes of brain-dead donors, the SSCs were proliferated in a specific culture medium for four weeks, and after confirming the identity of the colonies derived from the growth of these cells, they were cultured on a layer of DTM as well as in 2D condition with a differentiated culture medium. In the Sixth week since the initiation of the differentiation culture, the expression of pre meiotic (OCT4 & PLZF), meiotic (SCP3 & BOULE) and post meiotic (CREM & Protamine-2) genes were measured in both groups. Results: The results indicated that the expression of pre meiotic, meiotic and post meiotic genes was significantly higher in the cells cultured on DTM (P ≤ 0.001). Conclusion: SSCs culture in DTM with the creation of ECM and similar conditions with in vivo can be regarded as a way of demonstrating spermatogenesis in vitro, which can be adopted as a treatment modality for male infertility.
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 ...
Application of a three-dimensional (3D) culture system for in vitro proliferation and differentia... more Application of a three-dimensional (3D) culture system for in vitro proliferation and differentiation of human spermatogonial stem cells (SSCs) is a useful tool for the investigation of the spermatogenesis process and the management of male infertility particularly in prepubertal cancer patients. The main purpose of this study was to investigate the proliferation of human SSCs co-cultured with Sertoli cells in soft agar culture system (SACS) supplemented by Laminin and growth factors. Testicular cells were isolated from testes of brain-dead patients and cultured in two-dimensional (2D) culture system for 3 weeks. After 3 weeks, functional SSCs were evaluated by xenotransplantation and also identification of cells was assessed by immunocytochemistry, flow cytometry, and RT-PCR. Then, SSCs and Sertoli cells were transferred to the upper layer of SACS for 3 weeks. After 3 weeks, the number of colonies and the expression of specific SSCs and Sertoli cell markers, as well as apoptotic genes were evaluated. Our results showed that transplanted SSCs, migrated into the basement membrane of seminiferous tubules of recipient mice. The expression of PLZF, α6-Integrin, and Vimentin proteins in SSCs and Sertoli cells were observed in 2D and 3D culture systems. The expression rate of PLZF, α6-Integrin, Bcl2, and colony number in SACS supplemented by Laminin and growth factors group were significantly higher than nonsupplemented groups (P ≤ 0.01), but the expression rate of c-kit and Bax in supplemented group were significantly lower than non-supplemented groups (P ≤ 0.05). This 3D co-culture system decreased apoptosis and increased propagation of human SSCs. Therefore, this designed system can be utilized to increase the proliferation of human SSCs in prepubertal male cancer and azoospermic men to obtain an adequate SSCs number to outotransplant success and in vitro spermatogenesis.
INTRODUCTION AND OBJECTIVES: Platelet rich plasma (PRP) is a novel therapeutic increasingly promo... more INTRODUCTION AND OBJECTIVES: Platelet rich plasma (PRP) is a novel therapeutic increasingly promote for various indications, including several within urology. PRP is a platelet concentrate extracted from whole blood via proprietary separator gel systems. Prevailing theories regarding efficacy suggest that local delivery of trophic factors will promote tissue regeneration. Detailed quantitative analysis of delivered factors is essential and prior investigations have failed to adequately delineate profiles. We sought to characterize PRP cytokine profiles across a spectrum of donors using a single proprietary system. METHODS: Whole blood was collected from 10 healthy volunteers age 20-65 using standard technique into 11 or 22cc PRP tubes from Eclipse Aesthetics (The Colony, Texas). Tubes were centrifuged until separated. Concentrated PRP (platelet layer mixed with a minority of the platelet poor layer) was sent immediately for 200 Human Biomarker Testing (Ray Biotec; Norcross, Georgia). Platelet concentration was determined for each sample. RESULTS: The ten highest factors and their primary roles are demonstrated (Table 1). Across the cohort, relative to whole blood, platelet concentration was 1.91 fold higher in the final product. Platelet concentration in PRP obtained from 22cc tubes was significantly higher than 11cc tubes (p[.037). At p[.05, heat mapping reveals 31 trophic factors responsible for driving variability in expression between 22cc and 11cc samples (Table 2). CONCLUSIONS: The majority of the most concentrated cytokines are growth factors and cell adhesion molecules, correlating with the presumed mechanism of PRP. Collection tube characteristics may affect platelet concentration and the level of trophic factor expression. These findings underscore the difficulty of producing a standardized PRP product and suggest a need for further investigation into isolation of the highly expressed factors and optimization of collection.
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...
Manganese inhibits oxidative stress damage. The aim of this study was to investigate the protecti... more Manganese inhibits oxidative stress damage. The aim of this study was to investigate the protective role of manganese on testis structure and sperm parameters in adult mice exposed to formaldehyde (FA). Twenty adult male NMRI mice were selected and randomly divided into four groups: (i) control; (ii) sham; (iii) 'FA'-exposed group; and (iv) 'FA and manganese chloride'exposed group. The FA-exposed groups received 10 mg kg À1 FA daily for 14 days, and manganese chloride was just injected intraperitoneally 5 mg kg À1 on 2nd weeks. Mice were sacrificed, and spermatozoa were collected from the cauda of the right epididymis and analysed for count, motility, morphology and viability. The other testicular tissues were weighed and prepared for histological examination upon removal. Seminiferous tubules, lumen diameters and epithelium thickness were also measured. The findings revealed that FA significantly reduced the testicular weight, sperm count, motility, viability and normal morphology compared with control group (P 0.05). In addition, seminiferous tubules atrophied and seminiferous epithelial cells disintegrated in the FA group in comparison with the control group (P 0.05). However, manganese improved the testicular structure and sperm parameters in FA-treated mice testes (P 0.05). According to the results, manganese may improve and protect mice epididymal sperm parameters and testis structure treated with FA respectively.
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Papers by Morteza Koruji