Effects of ovarian varicose vein on mitochondrial structure, malondialdehyde and prooxidants - Antioxidants balance in rat ovaries Efectos de las Venas Ováricas Varicosas sobre la Estructura Mitocondrial, Niveles de Malondialdehído y Balance Prooxidantes-antioxidantes en Ovarios de Ratas

Oxidative stress is increased in varicose veins. Many studies have implicated oxidative stress in the pathogenesis of infertility causing diseases of the female reproductive tract. The aim of this study was to determine whether varicocele can cause raised levels of reactive oxygen species and denaturation of mitochondrial structure in ovaries of female rats or not. In each experimental study, 15 weaning-age female rats were divided equally in 3 groups: Unilateral Varicose Vein (A), Sham (B) and Control (C) groups. Mitochondrial structure and malondialdehyde levels as a product of lipid peroxidation and Prooxidants-Antioxidants Balance were evaluated 60 days after intervention in proestrus stage. Comparisons between groups were made by the measured test. After 2 months, our results showed that mitochondrial structure ultra-structurally was denatured with histologic examination, malondialdehyde and prooxidants-antioxidants balance levels of left ovaries increased significantly in varicocele group compared to control and sham groups (P�0.05). In the right side, malondialdehyde increased significantly, but in prooxidantsantioxidants balance levels, there is no significant differences between groups. The data of control and sham groups were the same. These findings may support the concept that increased levels of malondialdehyde and PAB in varicocele may cause negative effects on fertility, so using antioxidants maybe useful. © 2015, International Journal of Morphology. All rights reserved

A comparison between the colony formation of adult mouse spermatogonial stem cells in Co cultures with sertoli and STO (mouse embryonic fibroblast cell line)

Objective: The aim of this study was to compare the colony formation of spermatogonial stem cells (SSCs) on Sertoli and STO (Mouse embryonic fibroblast cell line) feeder cell layers during a two-week period. Materials and Methods: Initially, sertoli cells and SSCs were isolated from adult mouse testes using a two-step enzymatic digestion and lectin immobilization. Characteristics of the isolated cells were immunocytochemically confirmed by examining for the presence of Oct-4, CDH1, promyelocytic leukaemia zinc finger factor (PLZF), SSC C-kit, and the distribution of Sertoli cell vimentin. SSCs were then cultured above the Sertoli, STO and the control (without co-culture) separately for two weeks. In all three groups, the number and diameter of colonies were evaluated using an invert microscope on the 3rd, 7th, 10th and 14th day. β1 and α6-integrin m-RNA expressions were assessed using a reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. Furthermore, Oct-4 m RNA expression was assessed using real time PCR. Statistical analysis was performed using ANOVA; and the paired two-sample t test and Tukey's test were used as post-hoc tests for the data analysis of the three sertoli, STO and control cocultures. Results: At the four specified time points, our results showed significant differences (p<0.05) in colony numbers and diameters among the sertoli, STO and control groups. The number and diameter of colonies increased more rapidly in the sertoli coculture than in the other two Our results at all four time points also showed significant differences (p<0.05) in the mean colony numbers and diameters between the three groups, with the Sertoli coculture having the highest mean values for colony numbers and diameters. The RT-PCR results, after two-weeks of culturing, showed that β1-integrin was expressed in all three groups co-cultures, but α6-integrin was not expressed. Additionally, based on real time PCR results, the three genes (β1-integrin, α6-integrin, Oct-4) mentioned were also expressed in all three co cultures groups. Conclusion: Based on the optimal effects of Sertoli feeder cells on spermatogonial stem cells in a co culture system, as also confirmed by several other studies, their use is suggested to achieve better colonization of SSCs

Mouse and human spermatogonial stem cells

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

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 β1, 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

Optimization of decellularized human placental macroporous scaffolds for spermatogonial stem cells homing

Decellularized scaffolds have been found to be excellent platforms for tissue engineering applications. The attempts are still being made to optimize a decellularization protocol with successful removal of the cells with minimal damages to extracellular matrix components. We examined twelve decellularization procedures using different concentrations of Sodium dodecyl sulfate and Triton X-100 (alone or in combination), and incubation time points of 15 or 30 min. Then, the potential of the decellularized scaffold as a three-dimensional substrate for colony formation capacity of mouse spermatogonial stem cells was determined. The morphological, degradation, biocompatibility, and swelling properties of the samples were fully characterized. The 0.5/30 SDS/Triton showed optimal decellularization with minimal negative effects on ECM (P � 0.05). The swelling ratios increased with the increase of SDS and Triton concentration and incubation time. Only 0.5/15 and 30 SDS showed a significant decrease in the SSCs viability compared with other groups (P < 0.05). The SSCs colony formation was clearly observed under SEM and H&E stained slides. The cells infiltrated into the subcutaneously implanted scaffold at days 7 and 30 post-implantation with no sign of graft rejection. Our data suggest the 0.5/30 SDS/Triton as an excellent platform for tissue engineering and reproductive biology applications. Figure not available: see fulltext. © 2021, The Author(s)

Stem cells research and its applications: A review

Research in developmental biology has led to the discovery of different types of stem cells (totipotent, pluripotent and multipotent stem cells) that can give rise to multiple tissue types. This review summarizes a description about the stem cell concept, different type of stem cells and their potential applications. The face of extraordinary advances in the prevention, diagnosis and treatment of human diseases, devastating illnesses such as heart disease, diabetes, cancer and diseases of the nervous system, continue to deprive people of health, independence and well-being has been reviewed in this study. Stem cell research leading to prospective therapies in reparative medicine has the potential to affect the lives of millions of people around the world and there is a good reason to be optimistic. The road towards the development of an effective cell-based therapy for widespread use is long and involves overcoming numerous technical, legislative, ethical and safety issues. © 2011 Asian Network for Scientific Information

Recruiting testicular torsion introduces an azoospermic mouse model for spermatogonial stem cell transplantation

Purpose: To investigate the long-term effect of testicular torsion on sperm parameters and testis structure in order to introduce a novel mice azoospermic model for spermatogonial stem cell transplantation. Materials and Methods: Unilateral testicular torsion was created. The animals were divided into two groups each containing 15 mice. They underwent 2 and 4 hours of unilateral testicular ischemia, respectively. All animals in this experiment were aged matched. The experimental (n = 5) groups were studied 2, 4 and 10 weeks after testicular ischemia reperfusion. Moreover, the left testes and epididymis were removed for sperm analysis and for weight and histopathological evaluation. Finally isolated spermatogonial stem cells were transplanted in the testes that underwent 2 hours of ischemia reperfusion, two weeks post-surgery. Results: All the investigated parameters demonstrated a sharp decline at 2, 4 and 10 weeks after testicular torsion, whereas 2-hour ischemia was found to be less injurious in testicular tissue structure. Two months after xenotransplantation, the transplanted cells were localized in the basal of the seminiferous tubules of the recipient ischemic testes. Conclusion: Torsion can cause permanent azoospermia in mouse. Also Testicular torsion 2 weeks after the 2 hours ischemia reperfusion may prove useful for recipient preparation for SSCs transplantation in mouse

Isolation and proliferation of spermatogonial cells from ghezel sheep

Background: Sheep industry has taken steps toward transforming itself into a more efficient and competitive field. There are many varieties of sheep breeds in the world that each of them serves a useful purpose in the economies of different civilizations. Ghezel sheep is one of the Iranian important breeds that are raised for meat, milk and wool. Field of spermatogonial cell technologies provides tools for genetic improvement of sheep herd and multiple opportunities for research. Spermatogonial cells are the only stem cells capable of transmitting genetic information to future generations. Methods: This study was designed to extend the technique of isolation and in vitro proliferation of spermatogonial cells in Ghezel sheep. Results: Isolated cells were characterized further by using specific markers for type A spermatogonia, including PLZF. Also, sertoli cells were characterized by vimentin which is a specific marker for sertoli cells. After 10 days of co-culture, viability rates of the cells was above 94.7, but after the freezing process the viability rates were 74 percent. Conclusion: In this study, a standard method for isolation and in vitro proliferation of spermatogonial stem cells in Ghezel sheep was developed. © 2018, Avicenna Journal of Medical Biotechnology. All rights reserved

Isolation and proliferation of spermatogonial cells from ghezel sheep

Background: Sheep industry has taken steps toward transforming itself into a more efficient and competitive field. There are many varieties of sheep breeds in the world that each of them serves a useful purpose in the economies of different civilizations. Ghezel sheep is one of the Iranian important breeds that are raised for meat, milk and wool. Field of spermatogonial cell technologies provides tools for genetic improvement of sheep herd and multiple opportunities for research. Spermatogonial cells are the only stem cells capable of transmitting genetic information to future generations. Methods: This study was designed to extend the technique of isolation and in vitro proliferation of spermatogonial cells in Ghezel sheep. Results: Isolated cells were characterized further by using specific markers for type A spermatogonia, including PLZF. Also, sertoli cells were characterized by vimentin which is a specific marker for sertoli cells. After 10 days of co-culture, viability rates of the cells was above 94.7, but after the freezing process the viability rates were 74 percent. Conclusion: In this study, a standard method for isolation and in vitro proliferation of spermatogonial stem cells in Ghezel sheep was developed. © 2018, Avicenna Journal of Medical Biotechnology. All rights reserved

The effects of poly L-lactic acid nanofiber scaffold on mouse spermatogonial stem cell culture

Introduction: A 3D-nanofiber scaffold acts in a similar way to the extracellular matrix (ECM)/basement membrane that enhances the proliferation and self-renewal of stem cells. The goal of the present study was to investigate the effects of a poly L-lactic acid (PLLA) nanofiber scaffold on frozen-thawed neonate mouse spermatogonial stem cells (SSCs) and testis tissues. Methods: The isolated spermatogonial cells were divided into six culture groups: (1) fresh spermatogonial cells, (2) fresh spermatogonial cells seeded onto PLLA, (3) frozen-thawed spermatogonial cells, (4) frozen-thawed spermatogonial cells seeded onto PLLA, (5) spermatogonial cells obtained from frozen-thawed testis tissue, and (6) spermatogonial cells obtained from frozen-thawed testis tissue seeded onto PLLA. Spermatogonial cells and testis fragments were cryopreserved and cultured for 3 weeks. Cluster assay was performed during the culture. The presence of spermatogonial cells in the culture was determined by a reverse transcriptase polymerase chain reaction for spermatogonial markers (Oct4, GFRα-1, PLZF, Mvh(VASA), Itgα6, and Itgβ1), as well as the ultrastructural study of cell clusters and SSCs transplantation to a recipient azoospermic mouse. The significance of the data was analyzed using the repeated measures and analysis of variance. Results: The findings indicated that the spermatogonial cells seeded on PLLA significantly increased in vitro spermatogonial cell cluster formations in comparison with the control groups (culture of SSCs not seeded on PLLA) (P�0.001). The viability rate for the frozen cells after thawing was 63.00 ± 3.56. This number decreased significantly (40.00 ± 0.82) in spermatogonial cells obtained from the frozen-thawed testis tissue. Both groups, however, showed in vitro cluster formation. Although the expression of spermatogonial markers was maintained after 3 weeks of culture, there was a significant downregulation for some spermatogonial genes in the experimental groups compared with those of the control groups. Furthermore, transplantation assay and transmission electron microscopy studies suggested the presence of SSCs among the cultured cells. Conclusion: Although PLLA can increase the in vitro cluster formation of neonate fresh and frozen-thawed spermatogonial cells, it may also cause them to differentiate during cultivation. The study therefore has implications for SSCs proliferation and germ cell differentiation in vitro. © 2013 Eslahi et al