Chronic demyelination interferes with normal spermatogenesis in cuprizone-intoxicant C57/BL 6 mice: An experimental study.

Background: Due to myelin and axonal insults in multiple sclerosis individuals, motor coordination problems and endocrine imbalance may develop. Objective: This study aims to evaluate the role of chronic demyelination on the hypothalamic-pituitary-gonadal axis in the mouse model of multiple sclerosis. Materials and Methods: 20 adult C57/BL6 male mice were divided into 2 groups (n = 10/each) as follows: the control group (CONT) received a regular diet for 17 wk; and the experimental group (cuprizone [CPZ]) was fed with 0.2% CPZ for 12 wk and, then CPZ was withdrawn for 5 wk. Serum testosterone, histopathology of the brain and testis, and sperm analysis were evaluated. Results: The hypothalamic myelin content was significantly decreased in the arcuate nucleus following the 12 wk of CPZ consumption compared to the CONT group, and the statistical difference remained until 17 wk. Testosterone levels declined significantly in the CPZ group compared to the CONT group in the 12 th and 17 th wk. A significant decrease was observed in the height of the seminiferous epithelium and the interstitial tissue area, and the number of seminiferous epithelial cells in the CPZ group compared to the CONT group in the 12 th and 17 th wk. The sperm count, motility, and viability in the CPZ group significantly decreased compared to the CONT group in the 12 th and 17 th wk of the study. Conclusion: Chronic demyelination induced by CPZ intoxication, maybe through damage to the hypothalamus arcuate nucleus, leads to the hypothalamic-pituitary-gonadal axis disturbance and damage to the testis and spermatogenesis subsequently.

Learning Towards Maturation of Defined Feeder-free Pluripotency Culture Systems: Lessons from Conventional Feeder-based Systems.

Pluripotent stem cells (PSCs) are widely recognized as one of the most promising types of stem cells for applications in regenerative medicine, tissue engineering, disease modeling, and drug screening. This is due to their unique ability to differentiate into cells from all three germ layers and their capacity for indefinite self-renewal. Initially, PSCs were cultured using animal feeder cells, but these systems presented several limitations, particularly in terms of Good Manufacturing Practices (GMP) regulations. As a result, feeder-free systems were introduced as a safer alternative. However, the precise mechanisms by which feeder cells support pluripotency are not fully understood. More importantly, it has been observed that some aspects of the need for feeder cells like the optimal density and cell type can vary depending on conditions such as the developmental stage of the PSCs, phases of the culture protocol, the method used in culture for induction of pluripotency, and intrinsic variability of PSCs. Thus, gaining a better understanding of the divergent roles and necessity of feeder cells in various conditions would lead to the development of condition-specific defined feeder-free systems that resolve the failure of current feeder-free systems in some conditions. Therefore, this review aims to explore considerable feeder-related issues that can lead to the development of condition-specific feeder-free systems.

Ultrastructural study: in vitro and in vivo differentiation of mice spermatogonial stem cells.

Mouse testicular tissue is composed of seminiferous tubules and interstitial tissue. Mammalian spermatogenesis is divided into three stages: spermatocytogenesis (mitotic divisions) in which spermatogonial stem cells (SSCs) turn into spermatocytes, followed by two consecutive meiotic divisions in which spermatocytes form spermatids. Spermatids differentiate into spermatozoa during spermiogenesis. Various factors affect the process of spermatogenesis and the organization of cells in the testis. Any disorder in different stages of spermatogenesis will have negative effects on male fertility. The aim of the current study was to compare the in vitro and in vivo spermatogenesis processes before and after transplantation to azoospermic mice using ultrastructural techniques. In this study, mice were irradiated with single doses of 14 Gy 60Co radiation. SSCs isolated from neonatal mice were cultured in vitro for 1 week and were injected into the seminiferous tubule recipient's mice. Testicular cells of neonatal mice were cultured in the four groups on extracellular matrix-based 3D printing scaffolds. The transplanted testes (8 weeks after transplantation) and cultured testicular cells in vitro (after 3 weeks) were then processed for transmission electron microscopy studies. Our study's findings revealed that the morphology and ultrastructure of testicular cells after transplantation and in vitro culture are similar to those of in vivo spermatogenesis, indicating that spermatogenic cell nature is unaltered in vitro.

Optimizing Immature Testicular Tissue and Cell Transplantation Results: Comparing Transplantation Sites and Scaffolds.

For patients who had testicular tissue cryopreserved before receiving gonadotoxic therapies, transplantation of testicular tissues and cells has been recommended as a potential therapeutic option. There are no studies that indicate the generation of sperm after human immature testicular tissue (ITT) or spermatogonial stem cells (SSCs) transplantation. The use of releasing scaffolds and localized drug delivery systems as well as the optimizing transplantation site can play an effective role in increasing the efficiency and improving the quality of testicular tissue and cell transplantation in animal models. Current research is focused on optimizing ITT and cell transplantation, the use of releasing scaffolds, and the selection of the right transplantation site that might restore sperm production or male infertility treatment. By searching the PubMed and Google Scholar databases, original and review papers were collected. Search terms were relevant for SSCs and tissue transplantation. In this review, we'll focus on the potential advantages of using scaffolds and choosing the right transplantation site to improve transplantation outcomes.

In vitro differentiation of primed human induced pluripotent stem cells into primordial germ cell-like cells.

BACKGROUND: Previous studies have shown significant results in the differentiation of mouse-induced pluripotent stem cells (miPSCs) into primordial germ cell-like cells (PGCLCs) and that human iPSCs (hiPSCs) can also differentiate into PGCLCs; however, the efficiency of PGCLC induction from hiPSCs is < 5%. In this study, we examined a new protocol to differentiate hiPSCs into PGCLCs. METHODS AND RESULTS: hiPSCs-derived embryoid bodies (EBs) were exposed to differentiate inducing factors, bone morphogenetic protein 4 (BMP4), and retinoic acid (RA) for 6 days. Cell differentiation was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence (IF) studies. Our results showed increased expression of the PRDM1 gene on the first day of differentiation. On other days, DAZL, VASA, and STRA8 genes increased, and the expression of PRDM1, NANOG, and OCT4 genes decreased. The expression of VASA, C-KIT, and STRA8 proteins was confirmed by IF. A flow cytometry analysis revealed that ~ 60% of differentiated cells were VASA- and STRA8-positive. CONCLUSION: EB formation and constant exposure of EBs to BMP4 and RA lead to the differentiation of hiPSCs into PGCLCs.

Genetic and Epigenetic Evaluation of Human Spermatogonial Stem Cells Isolated by MACS in Different Two and Three-Dimensional Culture Systems.

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: In 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 ß1, 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.

Plasma-Rich in Growth Factors Ameliorates Detrimental Effects of Cryopreservation on Human Sperm: A Prospective Study.

Objective: Sperm cryopreservation results in damage to membrane integrity, sperm viability, sperm motility, and DNA structure. We aimed to evaluate the effect of plasma rich in growth factors (PRGF) on sperm parameters during the freeze-thaw process. Materials and Methods: In the first phase of this prospective study, after sperm preparation, 10 normozoospermic specimens were cryopreserved by rapid freezing with different concentrations of PRGF including 0, 1, 5, and 10% to find the optimum dose. Sperm motility and viability were assessed in this phase. In the second phase of the study, based on the results of first phase, 25 normal sperm samples were frozen with 1% PRGF. All sperm parameters including motility, viability, acrosome reaction, and DNA integrity were assessed before freezing and after thawing. Results: The rates of progressive and total sperm motility and viability were significantly higher in 1% PRGF compared to control, 5%, and 10% PRGF in the first phase (P<0.05). Supplementation of freezing medium with 1% PRGF could significantly improve all sperm parameters including sperm motility, viability, normal morphology, acrosome integrity, chromatin structure, chromatin integrity, DNA denaturation, and DNA fragmentation in comparison with the control group. Conclusion: It appears that the supplementation of freezing medium with 1% PRGF could protect human sperm parameters during cryopreservation.

Mini Bioreactor Can Support In Vitro Spermatogenesis of Mouse Testicular Tissue.

Objective: It was in the early 20th century when the quest for in vitro spermatogenesis started. In vitro spermatogenesis is critical for male cancer patients undergoing gonadotoxic treatment. Dynamic culture system creates in vivo-like conditions. In this study, it was intended to evaluate the progression of spermatogenesis after testicular tissue culture in mini-perfusion bioreactor. Materials and Methods: In this experimental study, 12 six-day postpartum neonatal mouse testes were removed and fragmented, placed on an agarose gel in parallel to bioreactor culture, and incubated for 8 weeks. Histological, molecular and immunohistochemical evaluations were carried out after 8 weeks. Results: Histological analysis suggested successful maintenance of spermatogenesis in tissues grown in the bioreactor but not on agarose gel, possibly because the central region did not receive sufficient oxygen and nutrients, which led to necrotic or degenerative changes. Molecular analysis indicated that Plzf, Tekt1 and Tnp1 were expressed and that their expression did not differ significantly between the bioreactor and agarose gel. Immunohistochemical evaluation of testis fragments showed that PLZF, SCP3 and ACRBP proteins were expressed in spermatogonial cells, spermatocytes and spermatozoa. PLZF expression after 8 weeks was significantly lower (P<0.05) in tissues incubated on agarose gel than in the bioreactor, but there was no significant difference between SCP3 and ACRBP expression among the bioreactor and agarose gel culture systems. Conclusion: This three-dimensional (3D) dynamic culture system can provide somewhat similar conditions to the physiological environment of the testis. Our findings suggest that the perfusion bioreactor supports induction of spermatogenesis for generation of haploid cells. Further studies will be needed to address the fertility of the sperm generated in the bioreactor system..

A decade of molecular preimplantation genetic diagnosis of 350 blastomeres for beta-thalassemia combined with HLA typing, aneuploidy screening and sex selection in Iran.

BACKGROUND: Preimplantation genetic diagnosis (PGD) has been developed to detect genetic disorders before pregnancy which is usually done on blastomeres biopsied from 8-cell stage embryos obtained from in vitro fertilization method (IVF). Here we report molecular PGD results for diagnosing of beta thalassemia (beta-thal) which are usually accompanied with evaluating chromosomal aneuploidies, HLA typing and sex selection. METHODS: In this study, haplotype analysis was performed using short tandem repeats (STRs) in a multiplex nested PCR and the causative mutation was detected by Sanger sequencing. RESULTS: We have performed PGDs on 350 blastomeres from 55 carrier couples; 142 blastomeres for beta-thal only, 75 for beta-thal and HLA typing, 76 for beta-thal in combination with sex selection, and 57 for beta-thal and aneuploidy screening. 150 blastomeres were transferable, 15 pregnancies were happened, and 11 babies born. We used 6 markers for beta-thal, 36 for aneuploidy screening, 32 for sex selection, and 35 for HLA typing. To our knowledge combining all these markers together and the number of STR markers are much more than any other studies which have ever done. CONCLUSIONS: PGD is a powerful diagnostic tool for carrier couples who desire to have a healthy child and wish to avoid medical abortion.

Selecting motile, non-apoptotic and induced spermatozoa for capacitation without centrifuging by MACS-Up method.

In newly improved MACS-Up method, magnetic field has been applied to separate non-apoptotic spermatozoa directly from the neat semen. The spermatozoa during passing through a viscous layer, located on the neat semen, contacted with progesterone and induced for the capacitation. Then, a clean population of non-apoptotic, and capacitated spermatozoa were selected in the pure culture media. Selected spermatozoa may be useful for use in ART. The 80 semen samples from normozoospermic individuals were divided separately into 4 attempts. Semen analysis, SCSA (sperm chromatin structure assay), FLICA (fluorescein-labelled inhibitors of caspase) methods, immunoassay of phosphorylation of tyrosine residues of sperm proteins, nuclear DNA integrity, caspase 3 activity and sperm capacitation rate were all performed for evaluation of sperm parameters respectively. To examine all aspects, the MACS-Up method compared with DGC (density gradient centrifuging) and MACS-DGC methods separately. This method can isolate non-apoptotic spermatozoa directly from the neat semen, which has similar performance compared to the MACS-DGC method. Movement and passing spermatozoa through the viscous layer, and contact with progesterone, significantly induced spermatozoa for capacitation compared with the control group. Also, the MACS-Up in comparison with routine DGC method could select spermatozoa with significantly higher total and progressive motility, DNA integrity, induced sperm population for capacitation and normal morphology. MACS-Up can be developed as an effective, short-time, and ease of performing method and used practically to select functional spermatozoa as novel sperm selection procedure. However, for clinical use of MACS-Up, all clinical aspects of this method should be considered and evaluated.

Alterations of miR-16, miR-let-7a and their target genes expression in human blastocysts following vitrification and re-vitrification.

Embryo cryopreservation is a widely used technique in infertility management and today is an essential part of assisted reproductive technology (ART). In some cases, re-vitrification can be applied to good quality supernumerary warmed embryos that have not been transferred in the present cycle. However, there is no study about re-vitrification impact on microRNA and gene expression in human embryos. The purpose of this study is to evaluate miR-16, miR-let7a and target genes expression in in vitro produced human blastocysts following re-vitrification.Day3 embryos obtained from ICSI cycles of fertile couples referring for family balancing program were biopsied and cultured individually. On the fourth day (post-ICSI) male ones (choices of their parents) were transferred and the females (good quality embryos) were donated for research. Donated embryos were cultured to blastocyst stage and assigned to three groups: fresh, vitrified and re-vitrification. Embryos were vitrified on Cryotech carriers. Then blastocysts of three groups were individually assessed for expression of miR-16, miR-let7a and target genes.The results showed that re-vitrification of human blastocysts did not affect the ability to re-expand in culture. In addition, significant decrease was observed in miR-16 and miR-let7a expression in re-vitrified group compared to fresh (p < 0.05). A significant upregulation of the target genes ITGß3 and BCL-2 in re-vitrified and vitrified embryos was observed compared to the fresh group (p < 0.05). The expression of BAX as a pro-apoptotic gene showed a significant decrease in re-vitrification group comparing with the fresh one (P < 0.05).The results of this research indicated that re-vitrification of embryos changes the expression of miR-16, miR-let-7a and their target genes. These alterations include increased expression of BCl-2 and ITGß3 genes which play important roles in embryo survival and implantation, respectively. Clinical proof of these effects requires further research.

Comparison of two methods for prolong storage of decellularized mouse whole testis for tissue engineering application: An experimental study.

BACKGROUND: Biological scaffolds are derived by the decellularization of tissues or organs. Various biological scaffolds, such as scaffolds for the liver, lung, esophagus, dermis, and human testicles, have been produced. Their application in tissue engineering has created the need for cryopreservation processes to store these scaffolds. OBJECTIVE: The aim was to compare the two methods for prolong storage testicular scaffolds. MATERIALS AND METHODS: In this experimental study, 20 male NMRI mice (8 wk) were sacrificed and their testes were removed and treated with 0.5% sodium dodecyl sulfate followed by Triton X-100 0.5%. The efficiency of decellularization was determined by histology and DNA quantification. Testicular scaffolds were stored in phosphate-buffered saline solution at 4°C or cryopreserved by programmed slow freezing followed by storage in liquid nitrogen. Masson's trichrome staining, Alcian blue staining and immunohistochemistry, collagen assay, and glycosaminoglycan assay were done prior to and after six months of storage under each condition. RESULTS: Hematoxylin-eosin staining showed no remnant cells after the completion of decellularization. DNA content analysis indicated that approximately 98% of the DNA was removed from the tissue (p = 0.02). Histological evaluation confirmed the preservation of extracellular matrix components in the fresh and frozen-thawed scaffolds. Extracellular matrix components were decreased by 4°C-stored scaffolds. Cytotoxicity tests with mouse embryonic fibroblast showed that the scaffolds were biocompatible and did not have a harmful effect on the proliferation of mouse embryonic fibroblast cells. CONCLUSION: Our results demonstrated the superiority of the slow freezing method for prolong storage of testicular scaffolds.

Attenuation of aquaporin-3 may be contributing to low sperm motility and is associated with activated caspase-3 in asthenozoospermic individuals.

Aquaporins play a crucial in transportation of water and solutes across cell membranes but their roles in male fertility are controversial. This study aimed to determine association of the expression level of aquaporin-3 (AQP3) and caspase-3 (CASP3) activity with sperm motility in asthenozoospermic individuals. Thirty-five asthenozoospermic and 35 normozoospermic individuals, participated in this study. Sperm chromatin structure assay (SCSA) for estimating of the DNA-damaged spermatozoa and Fluorescein-labelled inhibitors of caspases for assessment of active CASP3 were used by flow cytometry. Gene and protein expressions of AQP3 and CASP3 were assessed by real-time PCR and flow cytometry respectively. The AQP3 gene expression level in asthenozoospermic individuals was significantly lower than that of normozoospermic group whereas it was higher for the CASP3 gene expression (p < .01). The SCSA data in asthenozoospermic was significantly higher than that of normozoospermic group (p < .01). There was a negative and significant correlation between attenuated AQP3 protein level with activated CASP3 and SCSA in the asthenozoospermic group. We showed that the attenuated AQP3 level may contribute to low sperm motility via reducing glycerol for energy production in sperm tails of asthenozoospermia. Increasing CASP3 activity could indirectly show the status of active apoptosis in individuals with asthenozoospermia.

Formation of organoid-like structures in the decellularized rat testis.

Objectives: In testis, the extracellular matrix (ECM) in addition to the supportive role for cells in the seminiferous epithelium, is also essential for the accurate functioning of these cells. Thus, using a decellularized testicular ECM (DTECM), as a scaffold for three-dimensional (3D) culture of testicular cells can mimic native ECM for studying in vitro spermatogenesis. Materials and Methods: The rat testis was decellularized via perfusion of 0.5% sodium dodecyl sulfate (SDS) for 48 hr, followed by 1% Triton X-100 for 6 hr, and then 1% DNase I for 1 hr. The efficiency of decellularization was evaluated by histology, immunohistochemistry (IHC), scanning electron microscopy (SEM), and MTT test. The prepared scaffolds were recellularized with testicular cells and cultured and assessed with hematoxylin-eosin (H&E) staining after two weeks. Results: Based on the H&E image, no trace of cell components could be observed in DTECM. IHC images demonstrated collagen types I and IV, laminin, and fibronectin were preserved. Masson's trichrome and alcian blue staining revealed that collagen and glycosaminoglycans (GAGs) were retained, and the SEM image indicated that 3D testicular architecture remained after the decellularization process. Based on the results of the MTT test, DTECM was cytocompatible, and H&E images represented that DTECM supports testicular cell arrangements in seminiferous tubule-like structures (STLSs) and organoid-like structures (OLSs). Conclusion: The results showed that the applied protocol successfully decellularized the testis tissue of the rat. Therefore, these scaffolds may provide an appropriate vehicle for in vitro reconstruction of the seminiferous tubule.

Effect of Acoustic Cavitation on Mouse Spermatogonial Stem Cells: Colonization and Viability.

OBJECTIVES: The mechanical index has long been one of the main criteria used to assess the safety limits for therapeutic medical applications. However, the safety of the mechanical index parameter is considered to be unknown in male fertility, which has a very significant role in vitro conditions. In this study, the effect of cavitation interactions due to mechanical index regions was evaluated on spermatogonial stem cells. METHODS: The acoustic pressure and mechanical index equations at the low intensities and the intended frequency were modeled and solved. The mechanical index average of 40 kHz frequency was selected as subthreshold, 0.70, and above the cavitation threshold. Neonatal spermatogonial stem cells were cultured. Spermatogonial stem cells are stimulated by low-level ultrasound for 5 days and colonization and viability evaluated on the seventh day. RESULTS: Based on modeling, the mechanical index average was chosen as 0.40, 0.51, 0.75, and 0.89. The mechanical index of 0.40 and 0.89 resulted in a number of colonies of 93 ± 4 and 32 ± 4, respectively. An increase in colony diameter could be observed for a 0.40 mechanical index during all days of the culture that in the culture on the seventh day had the largest average colony diameter of 174.05 ± 1.22 µm in comparison with other groups (p < 0.05). The cell viability was not significantly different among the groups. CONCLUSION: The results suggest that a low-intensity ultrasound of 40 kHz with a 0.40 mechanical index can be effective in increasing the proliferation and colonization of spermatogonia in stem cells during culture.

Effect of fullerene nanoemulsion on the repair of wrinkles induced by UVB radiation: A c57bl6 mice model.

INTRODUCTION: The effect of fullerene nanoemulsion on skin wrinkle repair in an animal model was evaluated using ultrasonic images processing. METHODS: Wrinkles were created in C57BL6 mice during 35 days of UVB radiation. Then, to investigate the therapeutic effect of fullerene nanoemulsions, mice were divided into three groups of control, UVB radiation, and treatment with fullerene nanoemulsion. Stable fullerene nanoemulsions were prepared using shear equalization. The therapeutic effect of fullerene nanoemulsion was investigated by extracting the skin thickness and mechanical parameters. Histology studies were performed to confirm the reliability of the treatment. RESULTS: A significant decrease was observed in the thickness of the epidermis and dermis layers (43% and 36%), Young modulus (27%), and the shear modulus (20%) of the skin on day 28 of the fullerene nanoemulsion treatment. Skin stiffness obtained by tensiometry on day 28 of the treatment showed a 48% reduction in the treatment group compared with the control group. Histological results confirmed the effect of fullerene nanoemulsions on wrinkle repair. CONCLUSION: The healing effect of fullerene nanoemulsion in wrinkle repair was confirmed. To study the skin repair, parameters including Young modulus, the shear modulus, and skin layer thickness can be calculated using ultrasonic images processing.

Non-invasive evaluation of elasticity of skin with the processing of ultrasound images during ultraviolet radiation: An animal photoaging model.

OBJECTIVE: The aim of this study was to provide a non-invasive imaging method to evaluate the physical and mechanical parameters as a novelty method during skin photoaging. METHODS: In order to evaluate the process of skin damage, 25 mice (C57BL6) were exposed to UVB radiation (0.03 mW/cm2 ), 5 times a week for 5 weeks. The thickness of the epidermal and dermal layers was measured weekly from the ultrasound images (40 MHz). The elastic parameters of the skin were estimated from the processing of the sequential ultrasound images with the motion detection algorithm during the injury generation process. RESULTS: The thickening, Young modulus, and shear modulus of the dermal and epidermal layers during the UVB damage process significantly increased during the 5-week study period (P < .05). In addition, the percentage of changes in the thickness of the epidermal layer (0.22 ± 0.01 mm in day 0 to 0.37 ± 0.02 mm in day 35) and dermal layer (0.57 ± 0.05 mm in day 0 to 0.90 ± 0.08 mm in day 35) increased by 68% and 57%, respectively. Furthermore, Young modulus (154.41 ± 8.8 kPa) was 11 times more than that of non-irradiated skin (14.90 ± 2.2 kPa) and the shear modulus (2.33 ± 0.04 kPa) was 2.2 times more than non-irradiated skin (1.06 ± 0.04 kPa). CONCLUSION: With processing the sequential ultrasound images and extracting the thickening, the elasticity of the skin layers can detect skin lesions by UVB radiation.

The mouse testis tissue culture could resume spermatogenesis as same as in vivo condition after human spermatogonial stem cells transplantation.

OBJECTIVE: The introduction of alternative systems in vivo is very important for cancer patients who are treated with gonadotoxic treatment. In this study, we examine the progression of the spermatogenesis process after human spermatogonial stem cell (SSCs) transplantation in vivo and in tissue culture conditions. MATERIALS AND METHODS: Human SSCs were obtained from a Testicular Sperm Extractions (TESE) sample, and characterization of these cells was confirmed by detecting the promyelocytic leukemia zinc finger (PLZF) protein. These cells, after being labeled with Di-alkyl Indocarbocyanine (DiI), were transplanted to adult azoospermia mouse testes treated with Busulfan 40mg/kg. The host testicular tissue culture was then considered a test group and in vivo transplant a control group. After 8 weeks, immunohistochemical, morphometric and molecular studies were performed. RESULTS: The results of morphometric studies indicated that the mean number of spermatogonia, spermatocytes, and spermatids in the test groups was significantly lower than in the control group (P<0.05) and most of the cells responded positively to DiI tracing. Immunohistochemical study in both groups revealed expression of PLZF, Synaptonemal complex protein 3 (SCP3) and Acrosin Binding Protein (ACRBP) proteins in spermatogonial cells, spermatocyte and spermatozoa, respectively. Also, PLZF, Transition Protein 1 (TP1) and Tektin-1 (Tekt1) human-specific genes had a significant difference in the between test groups and control groups (P<0.05) in molecular studies. CONCLUSION: These results suggest that the conditions of testicular tissue culture after transplantation of SSCs can support spermatogenesis resumption, as well as in an in vivo condition.

Characterization of embryonic stem-like cells derived from mouse spermatogonial stem cells following low-intensity ultrasound treatment.

OBJECTIVE: Spermatogonial stem cells (SSCs) are able to form embryonic stem-like cells (ES-like cells) and embryonic bodies (EBs). Low-intensity ultrasound stimulation (LIUS) has positive effects on the growth and differentiation of the different cells. In this study, we tried to investigate the effects of LIUS on SSC differentiation to ES-like cells. MATERIALS AND METHODS: SSCs were isolated from neonatal mice and their identification was confirmed by tracking of PLZF, Oct-4, and C-Kit proteins. The SSCs and Sertoli cells were co-cultured in DMEM/F12 supplemented with 15% FBS and LIF. SSCs stimulated by LIUS with 200mW/CM2 intensity. Characterization of obtained ES-like cells was confirmed with Sox2, Oct-4, and SSEA-1 immunofluorescence staining. Also, real-time PCR was performed to analyse the expression of c-Myc and Nanog genes in ES-Like Cells and Stra8, Piwil2 and Plzf genes in SSCs after 21 days of the in vitro culture. RESULTS: Our results showed c-Kit, PLZF and Oct-4 proteins were expressed positively in SSCs and Sox2, Oct-4, SSEA-1 in the ES-like cells by immunocytochemistry. The results of flow cytometry showed a significant increase in expression of c-Myc and Nanog in ES-like cells compared to SSCs (p<.05), whereas the Stra8, Piwil2, and Plzf became down-regulated during 21 days of culture. ES-like markers cell SSEA-1, Sox2 and Oct-4 were increased in the LIUS group compared to the control group (p<.05). CONCLUSION: The results indicated that ES-like cells with pluripotency characteristics were derived from SSCs.

The Epigenetic Assessment of Human Spermatogenic Cells Derived from Obstructive Azoospermic Patients in Different Culture Systems.

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 were 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 colony formation, gene-specific methylation (by MSP), quantitative genes expression of pluripotency (Nanog, C-Myc, Oct-4) and specific germ cell (Integrin α6, Integrin ß1, PLZF) genes were evaluated in five different culture systems. RESULTS: Our findings indicate a significant increase in the number and diameter of colonies in IV group in compare to control group and other groups. Expression of germ specific genes in IV group were significantly increased (P ≤ 0.05) and levels of expression of pluripotency genes were significantly decreased in this group (P ≤ 0.05) compared with other groups. Gene-specific pattern of methylation of examined genes showed no changes in culture systems during the culture era. CONCLUSION: A microenvironment capable of controlling the proliferation of cell colonies can be restored by testicular cell suspension.