Effect of Human Chorionic Gonadotropin on the Destructive Effects of Nandrolone Decanoate on Sperm Parameters of Testes in Adult Rats

Background & aim: Nandrolone decanoate is one of the most drug Anabolic-androgenic steroids (AAS). On the other hand, human chorionic gonadotropin (hCG) induce secretion of testosterone and increased androgen production. The main aim of this study investigated the effects of mentioned hormone on destructive effects of drug AAS in cells and tissues of male reproductive system. Methods: In the present laboratory-experimental study, male Wistar rats were divided into 5 groups of three. Treated groups received 10 mg/kg/weekly of Nandrolone (Nd) for eight weeks, hormone group (H) rats received 500 IU weekly (IM or intra muscular)  of hCG for 8 weeks, Nandrolone plus hCG group (Nd – H) received Nandrolone solvent or peanut oil as a vehicle or sham (Sh) and Control (CO) without any injection. Sperm parameters such as motility, count and morphology were evaluated after 8 weeks by light microscopic. In addition, percentage of sperm viability was prepared using Eosin-nigrosin. Moreover, testes, Epydidim tail, prostate and seminal vesicle were weighted by laboratory scales. Statistical analysis was carried out by one-way ANOVA. Results: The percentage of progressive motile sperm was decreased in Nd and Nd - H groups in comparison to the control and sham groups. The percentage of normal sperm morphology was not significantly decreased in treated groups compared to the control and sham groups. In addition, the results of sperm morphology indicated that a high percentage of abnormal sperm morphology (Tailless and Coil) was seen in the Nd experimental group. Additionally, the viability percentage was significantly decreased in the Nd group in comparison to the other groups. The testes weight was significantly decreased in the Nd group compared to the control and sham groups. Conclusion: The hCG had positive effects on the destructive effects of Nandrolone on sperm parameters of adult rats and decreased negative effects of Nandrolone

Three-dimensional electrospun gelatin scaffold coseeded with embryonic stem cells and sertoli cells: A promising substrate for in vitro coculture system

In this study, we present an electrospun gelatin (EG) scaffold to mimic the extracellular matrix of the testis. The EG scaffold was synthesized by electrospinning and crosslinked with glutaraldehyde vapor to decrease its water solubility and degradation rate. The scanning electron microscope micrographs showed the homogenous morphology of randomly aligned gelatin fibers. The average diameter of gelatin fibers before and after crosslinking was approximately 180 and 220 nm, respectively. Modulus, tensile strength, and elongation at break values were as 161.8 ± 24.4 MPa, 4.21 ± 0.54 MPa, and 7.06 ± 2.12 MPa, respectively. The crosslinked EG showed 75.2 ± 4.5 weight loss after 14 days with no changes in the pH value of degradation solution. Cytobiocompatibility of the EG for sertoli cells and embryonic stem cells (ESCs) was determined in vitro. Sertoli cells were isolated from mouse testis and characterized by immunostaining and flow cytometry. The effects of EG on proliferation and attachment of both sertoli cells and ESCs were examined. The EG scaffolds exhibited no cytotoxicity for sertoli and ESCs. Both sertoli and ESCs were well attached and grown on EG. Coculture of sertoli and ESCs on EG showed better ESCs adhesion compared with ESCs alone. Our findings indicate the potential of EG as a substrate for proliferation, adhesion, and coculture of sertoli and ESCs and may be considered as a promising engineered microenvironment for in vitro coculture system with the aim of guiding stem cells differentiation toward sperm-producing cells. © 2019 Wiley Periodicals, Inc

Effect of combination of Melatonin and All-Trans retinoic acid on maturation, fertilization and embryonic development of immature mouse oocytes

Background and Objective: With respect to the antioxidant role of melatonin and retinoic acid, it seems to be effective both in the maturation and embryonic development. This study was done to investigate the effect of combination of melatonin and All-Trans retinoic acid (RA) on maturation, fertilization and embryonic development of immature mouse oocytes. Methods: In this experimental study, cumulus - oocyte complex (COCs) were recovered from 4-6 week old female mice NMRI and were divided into 6 maturation medium groups including control, sham, experiment 1(melatonin 100 nM, 1 and 2 µM), experiment 2 (retinoic acid 1, 2, 4, 6 µM), experiment 3 (melatonin 2 µM+RA 4 µM), experiment 4 (Mel 100nM + retinoic acid 4µM). The maturation rate was recorded after 24 hours of culture in a humidified atmosphere of 5% CO2 at 37°C. The matured oocytes were fertilized with sperm. Fertilization and embryonic development rates to the blastocyst stage were recorded. Results: Maturation rate in the control and sham groups were 50.6% and 49.4%, respectively. Maturation rate were 54.3%, 54.8%, 59.9% in melatonin group with concentrations of 100 nM, 1 and 2 µM, respectively. Maturation rate were 51.6%, 51%, 59% and 49.6% in t-RA group with concentrations of 1, 2, 4, 6 μM. Maturation rate were 60.4% and 54.2% in the experiment 3 and 4 groups, respectively. The maturation rates in the melatonin 2 µM, retinoic acid 4 µM and experiment 3 significantly increased in compare to control (P<0.05). The embryonic development rate in the melatonin with 100nM concentration and 4 µM of retinoic acid increased significantly compared to controls (P<0.05). Although, embryonic development rate in experiment 3 was higher than control, but lower in compare to melatonin 100 nM and the retinoic acid 4 µM. The embryonic development rate in experiment 4 significantly increased in compare to control (P<0.05). Conclusion: Combination of melatonin and All-Trans retinoic acid in medium culture increase maturation rate and improved embryonic development in dose dependent manner

In Vitro Maturation and Embryo Development to blastocyst Mouse Germinal Vesicle Oocytes after Vitrification

Abstract Background & aim: Vitrification is a simple and ultra rapid technique for the conservation of fertility. Improving pregnancy rate associate with the use of cryopreserved oocytes would be an important advanced in human assisted reproductive technology (ART). The purpose of this study was to evaluate survival, oocytes maturation and embryo development to the blastocyst stage after vitrification of oocytes germinal vesicle-stage and multi stage Methods: In the present experimental study, germinal vesicle oocytes with or without cumulus cells were transferred to vitrification solution containing 30% (v/v) ethylene glycol, 18% (w/v) Ficoll-70, and 0.3 M sucrose, either by single step or in a step-wise way. After vitrification and storage in liquid nitrogen, the oocytes were thawed and washed twice in culture medium TCM119, and then subjected to in vitro maturation, fertilization, and culture. Data analysis was performed by using One-way variance and Tukey tests. Results: Oocytes survival, metaphase 2 stage oocyte maturation, fertilization and embryo formed blastocyst in vitrification methods multistage were significantly higher than the single step procedure (P<0/05) Conclusion: The Germinal vesicle stage oocytes vitrified with cumulus cells and stepwise procedure had positive effect on the survival, maturation and developmental rate on blastocyst compared to oocytes without cumulus cell and single step procedure. Key words: Germinal Vesicle Oocyte, Blastocyst, Vitrification, Ethylene glyco

Melatonin ameliorates testes against forced treadmill exercise training on spermatogenesis in rats

INTRODUCTION: It is well documented that some forced exercises can have bad effects on the genital system. Melatonin is a potent antioxidant that is effective in reducing the physical stress

Matrigel enhances differentiation of human adipose tissue-derived stem cells into dopaminergic neuron

Background: Therapy based stem cells have offered a novel therapeutic approach for the improvement of neurodegenerative diseases, specially Parkinson. Hence, developing a well-established culture model with appropriate stem cells is extremely crucial in regenerative engineering to provide efficient targeted cells. Human adult mesenchymal stem cells derived from adipose tissue (hADSCs) have emerged as a promising source of stem cells due to their unique potentials of self-renewal and differentiation into other stem cells. The purpose of this study was to investigate the differentiation capacity of hADSCs into dopaminergic and neuron-like cells in the 3D culture plate (Matrigel). Methods and materials: hADSCs were obtained from adipose tissues of patients and then characterized morphologically with flowcytometry. Isolated cells were harvested to perform differentiation on Matrigel and tissue culture plate (TCP) supplemented with induction factors. The survival rate of cells during neural induction was monitored by MTT. The expression of specific cell markers was analyzed by QRT-PCR and immunocytochemistry on days 2, 8 and 14. The level of released dopamine was measured using HPLC technique. Results: Matrigel had a positive effect on maintaining cell growth compared to those on TCP. Moreover, the number of TH and MAPII positive cells is substantially higher in Matrigel than in TCP. Sox2 and Nestin had a prominent expression in hADSCs within the first days of differentiation. The gene expression of neural markers such as TH, Nurr1, LMX1A and DAT was detected and increased after day 8. Moreover, the dopamine released in the cell harvested on Matrigel was greater than those seeded on TCP. Conclusions: Overall, hADSCs could generate dopaminergic cells, which suggest its strong capability to serve as a tool for Parkinson disease model in the regenerative medicine

Gelatin Electrospun Mat as a Potential Co-culture System for in Vitro Production of Sperm Cells from Embryonic Stem Cells

Engineering of 3D substrates with maximum similarity to seminiferous tubules would help to produce functional sperm cells in vitro from stem cells. Here, we present a 3D electrospun gelatin (EG) substrate seeded with Sertoli cells and determine its potential for guided differentiation of embryonic stem cells (ESCs) toward germline cells. The EG was fabricated by electrospinning, and its morphology under SEM, as well as cytobiocompatibility for Sertoli cells and ESCs, was confirmed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and cell attachment assay. Embryoid bodies (EBs) were formed from ESCs and co-cultured with Sertoli cells, induced with BMP4 for 3 and 7 consecutive days to induce the differentiation of EBs toward germline cells. The differentiation was investigated by immunocytochemistry (ICC), flow cytometry, and RT-PCR in four experimental groups of EBs (EBs cultured in gelatin-coated cell culture plates); Scaffold/EB (EBs cultured on EG); ESCs/Ser (EBs and Sertoli cells co-cultured on gelatin-coated cell culture plates without EG); and Scaffold/EB/Ser (EBs and Sertoli cells co-cultured on EG). All experimental groups exhibited a significantly increased MVH (germline-specific marker) and decreased c-KIT (stemness marker) expression when compared with the EB group. ICC and flow cytometry revealed that Scaffold/EB/Ser had the highest level of MVH and the lowest c-KIT expression at both 3 and 7 days postdifferentiation compared with other groups. RT-PCR results showed a significant increase in the germline marker (Dazl) and a significant decrease in the ESC stemness marker (Nanog) in Scaffold/EB compared to the EB group. The germline markers Gcna, Stella, Mvh, Stra8, Piwil2, and Dazl were significantly increased in Scaffold/EB/Ser compared to the Scaffold/EB group. Our findings revealed that the EG scaffold can provide an excellent substrate biomimicking the micro/nanostructure of native seminiferous tubules and a platform for Sertoli cell-EB communication required for growth and differentiation of ESCs into germline cells. Copyright © 2020 American Chemical Society

Differentiation of human mesenchymal stem cells (MSC) to dopaminergic neurons: A comparison between Wharton's Jelly and olfactory mucosa as sources of MSCs

The generation of dopaminergic neurons from stem cells is a potential therapeutic approach to treat neurodegenerative disorders, such as Parkinson's disease. The current study aims to investigate the potential of two different types of mesenchymal stem cells derived from human Wharton's jelly and nasal cavity for differentiation into dopaminergic neurons. The differentiation capacities of both cell types were evaluated using real-time PCR, immunocytochemistry, flow cytometry and HPLC. Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) are noted for their capability to differentiate into mesodermal and non-mesodermal cells, including neurons. However, it was demonstrated that having the same neuroectodermal origin as the nervous system, the olfactory ectomesenchymal stem cells (OE-MSCs) expressed the neural marker MAP2 as well as dopaminergic markers such as tyrosine hydroxylase (TH), dopamine transporter (DAT) and PITX3 to a greater extent than the WJ-MSCs both at the level of mRNA and protein. Furthermore, quantitative flow cytometric evaluation of these markers at 12 days post-induction supported the above-mentioned results. Finally, the assessment of the functionality of differentiated cells and their ability to synthesize dopamine measured by HPLC revealed that the OE-MSC-derived dopaminergic cells released almost the same amount of dopamine as that secreted by WJ-MSC-derived cells. Thus it showed the difference in their functionality to be negligible. Overall, it may be concluded that higher proliferation and differentiation capacity of OE-MSCs, along with their easier harvestability and autologous transplantability compared with WJ-MSCs, makes them a better cell source for stem cell therapy of neurodegenerative disorders which are caused by degeneration of dopaminergic neurons. © 2019 Elsevier B.V

In vitro and in vivo evaluations of three-dimensional hydroxyapatite/silk fibroin nanocomposite scaffolds

In this study, three-dimensional hydroxyapatite/silk fibroin (HAp/SF) nanocomposite scaffolds were successfully prepared through layer solvent casting combined with the freeze-drying technique for tissue engineering applications. Various SF aqueous concentrations, ranging from 2.5 to 10, were used to control the physicochemical properties of the prepared scaffolds. Biologic responses of the rat bone marrow stromal cells (rBMSCs) to the HAp/SF scaffolds were examined by culturing the cells within them. In addition, biodegradation and biocompatibility of the scaffolds were evaluated in vitro and in vivo, respectively. Among the prepared scaffolds, HAp/SF-2.5 was the most brittle sample and showed porous structure with lowest mechanical properties. The average pore diameters were 350 ± 67 and 112 ± 89 μm and decreased with the increase in the SF concentration from 5 to 10, respectively. The pores formed in the scaffolds, made up of the 5 SF, were more uniform and regular than those of the scaffolds made up of 5 and 10 SF. The HAp/SF scaffolds did not change the rBMSCs viability and were not cytotoxic compared with the control sample. The scanning electron microscopy micrographs showed that the cells migrated into the pores and well attached to the scaffolds and their cytoplasm was extended in all directions, indicating a promising cell adhesion, high biocompatibility, and no cytotoxicity of the HAp/SF-5 nanocomposite scaffolds. Subcutaneous implantation of the HAp/SF-5 scaffolds in rat models suggested an excellent biocompatibility. All data obtained from this study suggest the potential use of the HAp/SF-5 for hard tissue engineering. © 2014 International Union of Biochemistry and Molecular Biology, Inc

Long-term preservation effects on biological properties of acellular placental sponge patches

Decellularization, preservation protocol and storage time influence the biomechanical and biological properties of allografts and xenografts. Here, we examined the consequences of storage time on the antibacterial, angiogenic and biocompatibility properties of the decellularized placental sponge (DPS) in vitro and in vivo. The DPS samples were preserved for one, three and six months at �20 °C. The decellularized scaffolds showed uniform morphology with interconnected pores compared with not decellularized sponges. Storage time did not interfere with collagen and vascular endothelial growth factor contents, and cytobiocompatibility for Hu02 fibroblast cells. Chorioallantoic membrane assay and subcutaneous implantation indicated a decreased new vessel formation and neovascularization in six months DPS sample compared with other experimental groups. The number of CD4+ and CD68+ cells infiltrated into the six months DPS on the implanted site showed a significant increase compared with one and three months sponges. The antibacterial activities and angiogenic properties of the DPS decreased over storage time. Three months preservation at �20 °C is suggested as the optimal storage period to retain its antibacterial activity and high stimulation of new vessel formation. This storage protocol could be considered for preservation of similar decellularized placenta-derived products with the aim of retaining their biological properties. © 2020 Elsevier B.V