Melatonin Promotes Differentiation of Human Spermatogonial Stem Cells Cultured on Three-Dimensional Decellularized Human Testis Matrix.

PURPOSE: The use of 3D (3-Dimensional) culture systems supported cell-to-cell and cell-to-extracellular matrix (ECM) interactions, proliferation, and differentiation of SSCs (Spermatogonial stem cells). The potential advantages of ECM-based scaffolds for in vitro spermatogenesis have been indicated in human and animal experiments. Furthermore, the strong antioxidant and anti-inflammatory activities of melatonin have improved in vitro manipulation of human SSCs in culture conditions. MATERIALS AND METHODS: SSCs were isolated from the testis of three dead-brain patients and then propagated for four weeks. The characterization of SSC colonies was done using real-time PCR (Polymerase chain reaction), ICC (Immunocytochemistry), and xenotransplantation to mice model. Decellularization of the human testis was performed using 0.3% sodium dodecyl sulfate (SDS) solution and 1% Triton X-100. Also, various characterizations of DTM (Decellularized testicular matrix ) were carried out using histological staining and DNA content analysis. The optimum dose of melatonin was selected by MTT (Methyl thiazol tetrazolium). SSCs were cultured in 4 groups: control, melatonin, ECM, and ECM-melatonin in a differentiation medium for four weeks. The expression of differentiation genes was evaluated by real-time polymerase chain reaction. In addition, the viability of cultured cells was assessed by MTT assay. RESULTS: The results of ICC and real-time PCR showed the expression of undifferentiated SSC markers (PLZF and GRFA1) in SSC colonies following the 2D culture of isolated SSCs. The presence of testicular ECM components after different staining methods; and the reduction of DNA content confirmed the proper decellularization process. Germ cell apoptosis significantly decreased in melatonin and ECM groups, and the higher viability of SSCs was seen in the ECM-melatonin group. The relative expression of GFRA1 and PRM2 decreased and increased in ECM and ECM-melatonin groups, respectively. CONCLUSION: Our study showed that the addition of melatonin to the human naturally-derived ECM scaffold could provide a suitable platform for inducing the differentiation and preserving the viability of SSCs.

Quercetin as a possible complementary therapy in multiple sclerosis: Anti-oxidative, anti-inflammatory and remyelination potential properties.

Multiple sclerosis (MS) is a complex autoimmune disorder of the central nervous system (CNS) which causes various symptoms such as fatigue, dyscoordination weakness and visual weakness. The intricacy of the immune system and obscure etiology are the main reasons for the lack of a definite treatment for MS. Oxidative stress is one of the most important key factors in MS pathogenesis. It can enhance inflammation, neurodegeneration and autoimmune-mediated processes, which can lead to excessive demyelination and axonal disruption. Recently, promising effects of Quercetin as a non-pharmacological anti-oxidant therapy have been reported in preclinical studies of MS disease. In this review, we provide a compendium of preclinical and clinical studies that have investigated the effects of Quercetin on MS disease to evaluate its potential utility as a complementary therapy in MS. Quercetin treatment in MS disease not only protects the CNS against oxidative stress and neuroinflammation, but it also declines the demyelination process and promotes remyelination potential. The present study clarifies the reported knowledge on the beneficial effects of Quercetin against MS, with future implication as a neuroprotective complementary therapy.

Differentiation of human primary testicular cells in the presence of SCF using the organoid culture system.

PURPOSE: Development of organoids using human primary testicular cells has remained a challenge due to the complexity of the mammalian testicular cytoarchitecture and culture methods. In this study, we generated testicular organoids derived from human primary testicular cells. Then, we evaluated the effect of stem cell factor (SCF) on cell differentiation and apoptosis in the testicular organoid model. METHODS: The testicular cells were harvested from the three brain-dead donors. Human spermatogonial stem cells (SSCs) were characterized using immunocytochemistry (ICC), RT-PCR and flow cytometry. Testicular organoids were generated from primary testicular cells by hanging drop culture method and were cultured in three groups: control group, experimental group 1 (treated FSH and retinoic acid (RA)), and experimental group 2 (treated FSH, RA and SCF), for five weeks. We assessed the expression of SCP3 (Synaptonemal Complex Protein 3) as a meiotic gene, PRM2 (Protamine 2) as a post-meiotic marker and apoptotic genes of Bax (BCL2-Associated X Protein) and Bcl-2 (B-cell lymphoma 2), respectively by using RT-qPCR. In addition, we identified the expression of PRM2 by immunohistochemistry (IHC). RESULTS: Relative expression of SCP3, PRM2 and Bcl-2 were highest in group 2 after five weeks of culture. In contrast, BAX expression level was lower in experimental group 2 in comparison with other groups. IHC analyses indicated the highest expression of PRM2 as a postmeiotic marker in group 2 in comparison to 2D culture and control groups but not find significant differences between experimental group 1 and experimental group 2 groups. Morphological evaluations revealed that organoids are compact spherical structures and in the peripheral region composed of uncharacterized elongated fibroblast-like cells. CONCLUSION: Our findings revealed that the testicular organoid culture system promote the spermatogonial stem cell (SSC) differentiation, especially in presence of SCF. Developed organoids are capable of recapitulating many important properties of a stem cell niche.

Advances of three-dimensional (3D) culture systems for in vitro spermatogenesis.

The loss of germ cells and spermatogenic failure in non-obstructive azoospermia are believed to be the main causes of male infertility. Laboratory studies have used in vitro testicular models and different 3-dimensional (3D) culture systems for preservation, proliferation and differentiation of spermatogonial stem cells (SSCs) in recent decades. The establishment of testis-like structures would facilitate the study of drug and toxicity screening, pathological mechanisms and in vitro differentiation of SSCs which resulted in possible treatment of male infertility. The different culture systems using cellular aggregation with self-assembling capability, the use of different natural and synthetic biomaterials and various methods for scaffold fabrication provided a suitable 3D niche for testicular cells development. Recently, 3D culture models have noticeably used in research for their architectural and functional similarities to native microenvironment. In this review article, we briefly investigated the recent 3D culture systems that provided a suitable platform for male fertility preservation through organ culture of testis fragments, proliferation and differentiation of SSCs.

In vitro spermatogenesis in artificial testis: current knowledge and clinical implications for male infertility.

Men's reproductive health exclusively depends on the appropriate maturation of certain germ cells known as sperm. Certain illnesses, such as Klinefelter syndrome, cryptorchidism, and syndrome of androgen insensitivity or absence of testis maturation in men, resulting in the loss of germ cells and the removal of essential genes on the Y chromosome, can cause non-obstructive azoospermia. According to laboratory research, preserving, proliferating, differentiating, and transplanting spermatogonial stem cells or testicular tissue could be future methods for preserving the fertility of children with cancer and men with azoospermia. Therefore, new advances in stem cell research may lead to promising therapies for treating male infertility. The rate of progression and breakthrough in the area of in vitro spermatogenesis is lower than that of SSC transplantation, but newer methods are also being developed. In this regard, tissue and cell culture, supplements, and 3D scaffolds have opened new horizons in the differentiation of stem cells in vitro, which could improve the outcomes of male infertility. Various 3D methods have been developed to produce cellular aggregates and mimic the organization and function of the testis. The production of an artificial reproductive organ that supports SSCs differentiation will certainly be a main step in male infertility treatment.

Generation of Haploid Spermatids on Silk Fibroin-Alginate-Laminin-Based Porous 3D Scaffolds.

In vitro production of sperm is a desirable idea for fertility preservation in azoospermic men and prepubertal boys suffering from cancer. In this study, a biocompatible porous scaffold based on a triad mixture of silk fibroin (SF), alginate (Alg), and laminin (LM) is developed to facilitate the differentiation of mouse spermatogonia stem cells (SSCs). Following SF extraction, the content is analyzed by SDS-PAGE and stable porous 3D scaffolds are successfully prepared by merely Alg, SF, and a combination of Alg-SF, or Alg-SF-LM through freeze-drying. Then, the biomimetic scaffolds are characterized regarding the structural and biological properties, water absorption capacity, biocompatibility, biodegradability, and mechanical behavior. Neonatal mice testicular cells are seeded on three-dimensional scaffolds and their differentiation efficiency is evaluated using real-time PCR, flow cytometry, immunohistochemistry. Blend matrices showed uniform porous microstructures with interconnected networks, which maintained long-term stability and mechanical properties better than homogenous structures. Molecular analysis of the cells after 21 days of culture showed that the expression of differentiation-related proteins in cells that are developed in composite scaffolds is significantly higher than in other groups. The application of a composite system can lead to the differentiation of SSCs, paving the way for a novel infertility treatment landscape in the future.

Protective effects of human amniotic membrane derived mesenchymal stem cells (hAMSCs) secreted factors on mouse spermatogenesis and sperm chromatin condensation following unilateral testicular torsion.

Testicular torsion is considered a urological disorder that requires immediate detorsion surgery. Ischemia/reperfusion (I/R) injury after testicular torsion detorsion causes of drastic impairment of spermatogenesis and infertility. Cell-free-based approaches seem to be a promising strategy to prevent I/R injury, they have more stable biological properties, and they contain paracrine factors of mesenchymal stem cells. The purpose of this study was to evaluate the protective effects of human amniotic membrane derived mesenchymal stem cells (hAMSCs) secreted factors on mouse sperm chromatin condensation and spermatogenesis improvement after I/R injury. hAMSCs were isolated and characterized by RT- PCR and flow cytometry, preparation of hAMSCs secreted factors was performed. Forty male mice were randomly divided into 4 groups: sham-operated, torsion detorsion, torsion detorsion+ intratesticular injection of DMEM/F-12, and torsion detorsion+ intratesticular injection of hAMSCs secreted factors. After one cycle of spermatogenesis, the mean number of germ cells, Sertoli, Leydig, myoid as well as tubular parameters, Johnson score, and spermatogenesis indexes were evaluated by H& E and PAS stainings. Sperm chromatin condensation and relative expression of c-kit and prm 1 genes were assessed by aniline blue staining and real-time PCR, respectively. The mean number of spermatogenic cells, Leydig, myoid, Sertoli, spermatogenesis parameters, Johnson score, as well as germinal epithelial height and diameters of seminiferous tubules decreased significantly after I/R injury. The thickness of basement membrane and percentage of sperm with excessive histone significantly increased, while the relative expression of c-kit and prm 1 significantly decreased in torsion detorsion group (p 0.001). hAMSCs secreted factors remarkably restored normal sperm chromatin condensation, spermatogenesis parameters and histomorphometric organization of seminiferous tubules via intratesticular injection (p 0.001). Thus, hAMSCs secreted factors may potentially salvage torsion-detorsion-induced infertility.

In vitro complete differentiation of human spermatogonial stem cells to morphologic spermatozoa using a hybrid hydrogel of agarose and laminin.

Spermatogenesis refers to the differentiation of the spermatogonial stem cells (SSCs) located in the base seminiferous tubules into haploid spermatozoa. Prerequisites for in vitro spermatogenesis include an extracellular matrix (ECM), paracrine factors, and testicular somatic cells which play a supporting role for SSCs. Thus, the present study evaluated the potential of co-culturing Sertoli cells and SSCs embedded in a hybrid hydrogel of agarose and laminin, the main components of the ECM. Following the three-week conventional culture of human testicular cells, the cells were cultured in agarose hydrogel or agarose/laminin one (hybrid) for 74 days. Then, immunocytochemistry, real-time PCR, electron microscopy, and morphological staining methods were applied to analyze the presence of SSCs, as well as the other cells of the different stages of spermatogenesis. Based on the results, the colonies with positive spermatogenesis markers were observed in both culture systems. The existence of the cells of all three phases of spermatogenesis (spermatogonia, meiosis, and spermiogenesis) was confirmed in the two groups, while morphological spermatozoa were detected only in the hybrid hydrogel group. Finally, a biologically improved 3D matrix can support all the physiological activities of SSCs such as survival, proliferation, and differentiation.

Testicular Tissue Vitrification: a Promising Strategy for Male Fertility Preservation.

Destruction of spermatogonial stem cells in juvenile men survivors of pediatric cancers leads to infertility as a side effect of gonadotoxic therapies. Sperm freezing before cancer treatment is commonly used in the clinic for fertility preservation, but this method is not applicable for prepubertal boys due to the lack of mature sperm. In these cases, cryopreservation of testicular tissues is the only option for fertility preservation. Although controlled slow freezing (CSF) is the most common procedure for testicular tissue cryopreservation, vitrification can be used as an alternative method. Controlled vitrification has prevented cell damage and formation of ice crystals. Procedures were done easily and quickly with a brief exposure time to high concentration of cryoprotectants without expensive equipment. Different studies used vitrification of testicular tissues and they assessed the morphology of seminiferous tubules, apoptosis, and viability of spermatogonial cells. Transplantation of vitrified testicular tissue into infertile recipient mice as well as in vitro culture of vitrified tissues was done in previous studies and their findings showed complete spermatogenesis and production of mature sperm. Review articles usually have compared controlled slow freezing with vitrification. In this review, we focused only on the vitrification method and its results. Despite promising results, many studies have been done for finding an optimal cryopreservation protocol in order to successfully preserve fertility in prepubertal boys.

Evaluation of Apoptosis-related Genes and Hormone Secretion Profiles Using Three Dimensional Culture System of Human Testicular Organoids.

BACKGROUND: In reproductive biology, testicular organoids can be used to treat infertility and to study testicular development and spermatogonial stem cells (SSCs) differentiation. Generating organoid from primary cells is challenging. In this study, testicular organoids were created using human primary testicular cells and evaluated the apoptotic gene expression and hormone secretion profiles of the organoids. MATERIALS AND METHODS: Primary human testicular cells were isolated using 2-step enzymatic digestion from three brain-dead donors. Immunocytochemistry and flow cytometry analyses were performed to confirm human SSCs. Isolated cells were cultured in three experimental groups: control group (2 dimensional (2D)), group 1 (organoid culture after 2D culture), and group 2 (organoid culture immediately after enzymatic digestion). Testicular organoids were cultured in DMEM/F-12 media supplemented with follicle-stimulating hormone (FSH) and fetal bovine serum (FBS) for four weeks. After 24 hours and four weeks of culture, reverse transcription quantitative real-time PCR (RT-qPCR) was used to investigate the relative expression of apoptotic genes (caspase 3, 9, Bax, and Bcl-2). At 24 hours, two weeks, and four weeks after culture, enzyme-linked immunoassay (ELISA) was used to determine the testosterone and inhibin B concentrations. Light microscopy and toluidine blue staining were also used for morphological analysis. RESULTS: RT-qPCR results revealed that pro-apoptotic (caspase 3, 9, Bax) gene expression levels were highest in group 2 after 24 h and four weeks of culture. In contrast, the expression level of Bcl-2 (anti-apoptotic) was lower in group 2 compared to other groups. The hormone secretion levels decreased in a time-dependent manner during the cultivation. According to morphological evaluations, testicular organoids are compact, spherical structures with two to three elongated cells organized along their border. CONCLUSION: Our findings revealed that the testicular organoid culture system maintained hormonal secretory abilities, demonstrating the function of Sertoli and Leydig cells in the absence of testis-specific environments.

Combination effects of mesenchymal stem cells transplantation and anodal transcranial direct current stimulation on a cuprizone-induced mouse model of multiple sclerosis.

Multiple sclerosis (MS) has no absolute treatment, and researchers are still exploring to introduce promising therapy for MS. Transcranial direct current stimulation (tDCS), is a safe, non-invasive procedure for brain stimulating which can enhance working memory, cognitive neurohabitation and motor recovery. Here, we evaluated the effects of tDCS treatment and Mesenchymal stem cells (MSCs) transplantation on remyelination ability of a Cuprizone (CPZ)-induced demyelination mouse model. tDCS significantly increased the motor coordination and balance abilities in CPZ + tDCS and CPZ + tDCS + MSCs mice in comparison to the CPZ mice. Luxol fast blue (LFB) staining showed that tDCS and MSCs transplantation could increase remyelination capacity in CPZ + tDCS and CPZ + MSCs mice compared to the CPZ mice. But, the effect of tDCS with MSCs transplantation on remyelination process was larger than each of treatment alone. Immunofluorescence technique indicated that the numbers of Olig2+ cells were increased by tDCS and MSCs transplantation in CPZ + tDCS and CPZ + MSCs mice compared to the CPZ mice. Interestingly, the combination effect of tDCS and MSCs was larger than each of treatment alone on Oligodendrocytes population. MSCs transplantation significantly decreased the TUNEL+ cells in CPZ + MSCs and CPZ + tDCS + MSCs mice in comparison to the CPZ mice. Also, the combination effects of tDCS and MSCs transplantation was much larger than each of treatment alone on increasing the mRNA expression of BDNF and Sox2, while decreasing P53 as compared to CPZ mice. It can be concluded that the combination usage of tDCS and MSCs transplantation enhance remyelination process in CPZ-treated mice by increasing transplanted stem cell homing, oligodendrocyte generation and decreasing apoptosis.

Improvement of Rat Sperm Chromatin Integrity and Spermatogenesis with Omega 3 following Bleomycin, Etoposide and Cisplatin Treatment.

The objective of this study is to examine the effects of omega 3 treatment on rat sperm chromatin condensation, DNA damage and spermatogenesis after bleomycin, etoposide and cisplatin (BEP) treatment. In this experimental study, 40 male rats were divided into four groups: Control, BEP, Omega 3 and BEP + Omega 3. Sperm chromatin condensation and DNA damage were assessed using aniline blue and acridine orange staining, respectively. Results show that the mean percentage of sperms with excessive histone and DNA damage was significantly increased in the BEP group after 9 weeks as compared to control group (p˂0.001). While, in the BEP + Omega 3 group, the mean percentage of sperm with excessive histone and DNA damage was decreased significantly compared with BEP group (p˂0.001). The testicular histomorphometric analysis indicated that omega 3 has a significant effect on the mean number of spermatogonia, primary spermatocytes, leydig cells and testicular histology properties following BEP treatment. The mean count of aforementioned cells significantly increased after omega 3 treatment compared with the BEP group (p˂0.001). Our data indicated omega 3 may be had beneficial effect for improving chromatin condensation, DNA damage during spermatogenesis and testicular histomorphic properties following BEP treatment.

Application of platelet-rich plasma (PRP) improves self-renewal of human spermatogonial stem cells in two-dimensional and three-dimensional culture systems.

Spermatogonial stem cells (SSCs) are very sensitive to chemotherapy and radiotherapy, so male infertility is a great challenge for prepubertal cancer survivors. Cryoconservation of testicular cells before cancer treatment can preserve SSCs from treatment side effects. Different two-dimensional (2D) and three-dimensional (3D) culture systems of SSCs have been used in many species as a useful technique to in vitro spermatogenesis. We evaluated the proliferation of SSCs in 2D and 3D culture systems of platelet-rich plasma (PRP). testicular cells of four brain-dead patients cultivated in 2D pre-culture system, characterization of SSCs performed by RT-PCR, flow cytometry, immunocytochemistry and their functionality assessed by xenotransplantation to azoospermia mice. PRP prepared and dosimetry carried out to determine the optimized dose of PRP. After preparation of PRP scaffold, cytotoxic and histological evaluation performed and SSCs cultivated into three groups: control, 2D culture by optimized dose of PRP and PRP scaffold. The diameter and number of colonies measured and relative expression of GFRa1 and c-KIT evaluated by real-time PCR. Results indicated the expression of PLZF, VASA, OCT4, GFRa1 and vimentin in colonies after 2D pre-culture, xenotransplantation demonstrated proliferated SSCs have proper functionality to homing in mouse testes. The relative expression of c-KIT showed a significant increase as compared to the control group (*: p < 0.05) in PRP- 2D group, expression of GFRa1 and c-KIT in PRP scaffold group revealed a significant increase as compared to other groups (***: p < 0.001). The number and diameter of colonies in the PRP-2D group showed a considerable increase (p < 0.01) as compared to the control group. In PRP- scaffold group, a significant increase (p < 0.01) was seen only in the number of colonies related to the control group. Our results suggested that PRP scaffold can reconstruct a suitable structure to the in vitro proliferation of SSCs.

Three-dimensional co-culture of human spermatogonial stem cells with Sertoli cells in soft agar culture system supplemented by growth factors and Laminin.

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 non-supplemented 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.

Protective effects of zinc on rat sperm chromatin integrity involvement: DNA methylation, DNA fragmentation, ubiquitination and protamination after bleomycin etoposide and cis-platin treatment.

Testicular cancer is one of the most common malignancy in young men, chemotherapy induced damage in cancerous cells as well as healthy tissue, and we decided to investigate recovery effect of zinc (Zn) on chemotherapy-induced complications in rat chromatin integrity and testicular histomorphometry. The male rats (n = 40) were treated with BEP at appropriate dose levels of BEP (0.75, 7.5, and 1.5 mg/kg) for 9 weeks, with or without Zn; testicular histology, sperm DNA methylation, ubiquitination, DNA fragmentation and protamination were further assessed through immunofluorescence. BEP treatment significantly increased ubiquitination, and DNA fragmentation, considerably reducing global DNA methylation and protamination (P < 0.001), resulting in degenerative changes in testicular structure. Zn restored normal DNA methylation, protamination and structure of male gonads, maintained spermatogonial stem cells, and significantly reduced the mean percentage of ubiquitination and sperm DNA fragmentation as compared with BEP group (P < 0.001). We found that supplementation of Zn following chemotherapy can improve chromatin integrity, testicular organization and spermatogenesis.

Proliferation and differentiation of mouse spermatogonial stem cells on a three-dimensional surface composed of PCL/gel nanofibers / Proliferación y diferenciación de células madre espermatogónicas de ratón en una superficie tridimensional compuesta de nanofibras PCL / gel

Spermatogonial stem cells (SSCs) have self-renewal and differentiation capacity essential for sperm production throughout the male reproductive life. The electrospun polycaprolactone/gelatin (PCL/Gel) nanofibrous scaffold mimics important features of the extracellular matrix (ECM), which can provide a promising technique for the proliferation and differentiation of SSCs in vitro. The goal of the present study was to investigate the effects of PCL/Gel nanofibrous scaffold on the propagation and differentiation of neonate mouse SSCs (mSSCs). mSSCs were enzymatically isolated, and the cells were purified by differential plating method and seeded on scaffold. After 2 weeks, viability, colony number and diameter, and expression of specific spermatogonial cell genes were investigated. After mSSCs propagation, the cells were cultivated in a differentiation medium on the scaffold for another 2 weeks, and differentiating cells were analyzed by real-time PCR. The number of mSSC colony (P<0.01) and expression levels of specific spermatogonial genes Plzf and Inga6 (P<0.01) and also differentiation genes c-Kit, Tp1 and Ptm1 (P<0.05) were higher in scaffold group compared with control during the culture period. We conclude that mSSCs can be expanded and can differentiate toward spermatid cells on PCL/Gel nanofibrous scaffold with improved developmental parameters.

Las células madre espermatogónicas (CME) tienen capacidad de auto renovación y diferenciación esenciales para la producción de esperma a lo largo de la vida reproductiva masculina. El «scaffold¼ nanofibroso de policaprolactona / gelatina (PCL / Gel) electrohilado imita características importantes de la matriz extracelular (MEC), que puede proporcionar una técnica prometedora para la proliferación y diferenciación de CME in vitro. El objetivo del presente estudio fue investigar los efectos del «scaffold¼ nanofibroso PCL / Gel en la propagación y diferenciación de CME de ratones neonatos (mSSC). Los mSSC se aislaron enzimáticamente y las células se purificaron mediante un método de siembra diferencial y se sembraron en un «scaffold¼. Después de 2 semanas, se investigaron la viabilidad, el número y el diámetro de las colonias y la expresión de genes específicos de células espermatogónicas. Después de la propagación de mSSC, las células se cultivaron en un medio de diferenciación en el «scaffold¼ durante otras 2 semanas, y las células se analizaron mediante PCR en tiempo real. El número de colonias mSSC (P <0,01) y los niveles de expresión de los genes espermatogónicos específicos Plzf e Inga6 (P <0,01) y también los genes de diferenciación c-Kit, Tp1 y Ptm1 (P <0,05) fueron mayores en el grupo de «scaffold¼ en comparación con el control durante el período de cultivo. Concluimos que los mSSC pueden expandirse y diferenciarse en células espermátidas en un «scaffold¼ de nanofibras PCL / Gel con parámetros de desarrollo mejorados.

In Vitro Spermatogenesis by Three-dimensional Culture of Spermatogonial Stem Cells on Decellularized Testicular Matrix.

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.

Effect of Zinc on Spermatogenesis and Sperm Chromatin Condensation in Bleomycin, Etoposide, Cisplatin Treated Rats.

OBJECTIVE: The incidence rate of testicular cancer among young males is high. Co-administration of bleomycin, etoposide and cisplatin (BEP) has increased survival rate of patients with testicular cancer. Although BEP is one of the most effective treatment for testicular cancer, but it severely affects the reproductive system that ultimately leads to infertility. In addition to its antioxidant activity, zinc has an important role in progression of spermiogenesis. This study aimed to evaluate the effect of zinc on sperm parameters, chromatin condensation and testicular structure after BEP treatment. MATERIALS AND METHODS: In this experimental study, 40 male rats were divided into 4 groups (control, BEP, BEP+ zinc and zinc) and examined for 2 spermatogenesis periods (i.e. 18 weeks). The rats in BEP and BEP+ zinc group were treated with BEP at appropriate doses (0.75, 7.5, and 1.5 mg/kg) for three cycles of three weeks. Zinc at a dose of 10 mg/kg/day was administered to BEP+ zinc and zinc groups. After 18 weeks, we assessed sperm parameters, and excessive histone in sperm chromatin using aniline blue staining, as well as testicular structure and germ line cells using periodic acid-Schiff staining. RESULTS: After BEP treatment, significant decreases were observed in normal sperm morphology, motility, and concentration, as well as alterations in rat sperm chromatin condensation and testicular tissue (P<0.001). Furthermore, after zinc consumption for 9 weeks, we observed significant improvements of sperm parameters and chromatin condensation as well as a significant retrieval of spermatogonia, leydig cells and tubular architecture (P<0.05). CONCLUSION: Zinc administration after chemotherapy with BEP in testicular cancer might be potentially useful in declining the off target consequence associated with oxidative stress.

The Protective Effects of Omega3 on Ubiquitination and Protamination of Rat Sperm after Bleomycin, Etoposide, and Cisplatin Treatment.

The purpose of this study is evaluation the protective effect of omega3 on rat sperm protamination and ubiquitination after treatment with Bleomycin, Etoposide and Cisplatin (BEP) drugs. In present study, 40 male rats were divided into four groups: Control, BEP, Omega3 and BEP + Omega3. Sperm protamination and ubiquitination were assessed using chromomycin A3 and immunofluorescence staining respectively. The mean percentage of ubiquitinated sperm in BEP group was significantly increased relative to control group (P < .001). But, the mean percentage of sperm protamination significantly decreased in BEP group relative to control group (P < .001). Rats in BEP + Omega3 group showed a significantly decreased in the mean percentage of sperm ubiquitination as compared to BEP group (P < .05) while, sperm protamination increased significantly relative to BEP group (P < .001). Administration of Omega3 after chemotherapy showed an improvement in sperm ubiquitination and protamination. Our data indicated that omega3 after chemotherapy may be beneficial for chromatin remodeling during spermatogenesis following BEP treatment.