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 4ºC 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 4ºC-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. Key words: Cryopreservation, Testis, Scaffold, Mouse.&nbsp

Up-regulation of CatSper genes family by selenium

<p>Abstract</p> <p>Background</p> <p>CatSper1-4 are a unique family of sperm cation channels, which are exclusively expressed in the testis and play an important role in sperm motility and male fertility. Despite their vital role in male fertility, almost nothing is known about the factors regulating their expression. Here, we investigated the effects of selenium (Se) on the expression of CatSper genes and sperm parameters in aging versus young male mice.</p> <p>Methods</p> <p>Forty 11-13 months aging male mice and forty 2-3 months young adult male mice were used. The animals were divided in two experimental groups: first group including aging males and second group comprising of young adult males, both treated with Se. The experimental groups were injected intra-peritoneally with Se (0.2 mg/kg) daily, for up to 5 weeks. Two other groups, aging and young adult mice without Se treatment were used as controls. All the animals were rapidly sacrificed by cervical dislocation on the days 21, 28, 35 and 42 after Se treatment. Subsequently, the morphology of the collected sperms was analyzed, and one of the testes from each mouse used for semi-quantitative RT-PCR. The significancy of the data was analyzed using ANOVA.</p> <p>Results and Discussion</p> <p>Our data revealed that there was a significant up-regulation of CatSper genes in the experimental groups compared to the control ones. Furthermore, the results of sperm analysis showed that the sperm parameters were improved in the aging as well as young adult male mice following Se treatment.</p> <p>Conclusion</p> <p>Se treatment in the aging subjects could up-regulate the expression of CatSper genes, and therefore results in elevation of sperm motility. Furthermore, Se treatment improved sperm parameters, especially morphology and viability rates.</p

Detection of genetic variation in sample of Iranian proofed Holstein cattle by using microsatellite marker

This study describes genetic variation among samples of Iranian Holstein cattle (Bos taurus) by using microsatellite markers. Semen samples of individuals were taken followed by DNA extraction. A panel of13 microsatellites was used for evaluation of 13 loci in 68 Holstein proofed bulls. Mean value for allele per locus detected is 6.615, ranging from 10 (SPS115) to 4 (ETH3). All the microsatellite DNA markers showed high polymorphism and displayed a relatively high level of genetic variation as estimated by allelic diversity and heterozygosity. Estimated heterozygosities ranged from 1.000 (BM2113, TGLA122, TGLA126, ETH3, MGTG4B, SPS115, TGLA227 and INRAO23) to 0.633 (SPS113) with mean value of 0.946. All the loci showed deviation from Hardy- Weinberg equilibrium (p &lt; 0.001), polymorphism information content (PIC) calculated for each marker exceeded 0.6 and the mean value of Shannon information index was estimated to be 1.606. Obtained results showing heterozygousity can be useful for the development of breeding strategies for genetic improvement in Iranian Holstein cattle.Keywords: Genetic variability, polymorphism, microsatellite loci, Bos taurus, Iranian Holstein cattle, DN

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&lt;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&lt;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

Effect of donor cells concentration on colonization of human spermatogonial stem cells in recipient mouse testes.

Exogenesis (cross-species) germ cell transplantation provides an opportunity to investigate fundamental aspects of spermatogenesis. In this study, testis biopsies of patients with maturation arrest of spermatogenesis during a one year ago were first minced mechanically into small pieces and then Spermatogonial Stem Cells (SSCs) and Sertoli cells isolated by the two- step enzymatic digestion, were plated and grown on DSA-Lectin coated dishes in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal calf serum. Transplantation of human spermatogonial cells into mouse recipient testis was performed on day 7 (before colony formation) and 2 weeks after culturing (colony formation). The effects of different concentrations of spermatogonial cell on quantity of transplantation and percent of colonized seminiferous tubules were assayed during 8 weeks after transplantation. The result showed that SSCs can be observed on the basement membrane of the seminiferous tubules in place of spermatogonial stem cells and proliferation occurs about 4 weeks after transplantation. The difference in donor cells concentration had more effect on colonization of mouse recipient testis (p<0.05). It will be an alternative approach for the repopulation of infertile seminiferous tubules and preservation of fertility, in the futur

Evaluation of Physical Parameters of Skin by Consecutive Ultrasonic Image Processing During Ultraviolet Radiation in an Animal Model of Wrinkled Skin

BACKGROUND AND OBJECTIVE: Skin aging is divided into two categories of intrinsic and extrinsic aging. Skin aging due to repeated exposure to ultraviolet radiation (extrinsic aging) is different from aging caused by time (intrinsic aging). The appearance of wrinkles caused by sunlight is due to subcutaneous fat atrophy and reduced production of collagen and elastin, thereby altering the biomechanical properties of the skin tissue. This study was conducted to investigate the skin damage caused by ultraviolet B (UVB) radiation by consecutive ultrasonic image processing with high resolution. METHODS: In this experimental study, we evaluated the skin injury process among 25 C57BL/6 mice in healthy group (zero dose), and case group exposed to UVB radiation at 0.03 milliwatts per square centimeter (5 times a week for 5 weeks) due to differences in skin characteristics. Physical parameters of dermal and epidermal layers were measured and evaluated weekly from day 7 to day 35 using ultrasonic image processing. FINDINGS: The thickness of the dermal and epidermal layers obtained by ultrasonic processing during the process of ultraviolet radiation injury in the mouse model significantly increased during the 5 – week study (p < 0.05). In addition, the percentage of changes in the thickness of the epidermis layer (from 0.22±0.01 mm on day zero to 0.37 ± 0.02 mm on the thirty-fifth day) and the dermal layer (from 0.57 ± 0.05 on day zero to 0.90 ± 0.08 mm on the thirty-fifth day) showed 68% and 57% increase, respectively. CONCLUSION: The results showed that UVB radiation increased the thickness of the skin layers

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

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

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