In vitro cytotoxicity of folate-silica-gold nanorods on mouse acute lymphoblastic leukemia and spermatogonial cells

Objective: The purpose of this study was to evaluate in vitro cytotoxicity of gold nanorods [GNRs] on the viability of spermatogonial cells [SSCs] and mouse acute lymphoblastic leukemia cells [EL4s]

Materials and Methods: In this experimental study, SSCs were isolated from the neonate mice, following enzymatic digestion and differential plating. GNRs were synthesized, then modified by silica and finally conjugated with folic acid to form F-Si-GNRs. Different doses of F-Si-GNRs [25, 50, 75, 100, 125 and 140 microM] were used on SSCs and EL4s. MTT [3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide] proliferation assay was performed to examine the GNRs toxicity. Flow cytometry was used to confirm the identity of the EL4s and SSCs. Also, the identity and functionality of SSCs were determined by the expression of specific spermatogonial genes and transplantation into recipient testes. Apoptosis was determined by flow cytometry using an annexin V/propidium iodide [PI] kit

Results: Flow cytometry showed that SSCs and EL4s were positive for Plzf and H-2kb, respectively. The viability percentage of SSCs and EL4s that were treated with 25, 50, 75, 100, 125 and 140 microM of F-Si-GNRs was 65.33 +/- 3.51%, 60 +/- 3.6%, 51.33 +/- 3.51%, 49 +/- 3%, 30.66 +/- 2.08% and 16.33 +/- 2.51% for SSCs and 57.66 +/- 0.57%, 54.66 +/- 1.5%, 39.66 +/- 1.52%, 12.33 +/- 2.51%, 10 +/- 1% and 5.66 +/- 1.15% for EL4s respectively. The results of the MTT assay indicated that 100 microM is the optimal dose to reach the highest and lowest level of cell death in EL4s and in SSCs, respectively

Conclusion: Cell death increased with increasing concentrations of F-Si-GNRs. Following utilization of F-Si-GNRs, there was a significant difference in the extent of apoptosis between cancer cells and SSCs

Isolation, culture and characterization of human sertoli cells by flow cytometry: development of procedure

Background: the sertoli cells in the testis create unique and safe environment to protect seminiferous tubules from auto antigens and invading pathogens. These cells produce the survival factor of the blood-testis barrier and produce special materials such as androgen binding proteins and contribute to the coordinated action of spermatogenesis. Given that the sertoli cells play an essential role in spermatogenesis and the lack of these cells leads to the disruption of spermatogenesis, it is necessary to investigate the behavior and performance of these cells. To achieve this goal, the cells must first be extracted. The aim of this study was to develop a procedure to isolate, culture, and characterize human sertoli cells

Methods: in order to isolate the sertoli cells of azoospermia patients who underwent [testicular sperm extraction] TESE surgery, washing up and multi_stage enzyme digestion of single cells, culture on petri dishes impregnated with datura stramonium lectin agglutinin [DSA] were done and then the cells were passaged for several times and isolated. For more purification, flow cytometry method with FSH receptor antibody was used. Immunocytochemistry assays and Elisa test for identification of these cells were employed

Results: the purification method resulted in more than 97% purity. The nature of sertoli cells was confirmed by morphology evaluation, detecting anti-mullerian hormone in sertoli cell culture media and the presence of FSH receptor on sertoli cells

Conclusion: this study introduced and applied a method to isolate, culture, and purify human sertoli cells with high purity which made possible further researches on these cells

expression of TLR2 and TLR3 in sertoli cells of azoospermic patients

Objective: Toll-like receptors [TLRs] on Sertoli cells are thought to have essential roles in sperm protection. This study was conducted to investigate the expression of TLR2 and TLR3 in Sertoli cells of men with azoospermia

Materials and Methods: In this experimental study, testicular biopsies were taken from ten azoospermic men. Following enzymatic dissociation, the samples were moved to lectin coated petri dishes. After a few passages, all cells were cultivated and Seroli cells were sorted by flow cytometry. To confirm Sertoli cell purification, alkaline phosphatase activity [ALP] and immunohistochemistry assays were employed. The expression of TLR2 and TLR3 at the transcript and protein levels was examined with real-time quantitative reverse transcription-polymerase chain reaction [RT-QPCR] and western blot, respectively

Results: Isolation, purification and cultivation of human Sertoli cells were performed successfully. Efficacy of purification of Sertoli cells by fluorescence-activated cell sorting [FACS] sorter was 97%. The type of cultured cells was confirmed by vimentin and follicle-stimulating hormone [FSH] receptor markers. Furthermore, the existence of anti- Mullerian hormone in culture was confirmed. RT-PCR showed that both genes were expressed in Sertoli cells. Consistently, proteins of both were also expressed in Sertoli cells. Moreover, QPCR showed that the relative expression of TLR3 transcripts was significantly higher than TLR2 in Sertoli cells. Although both genes are expressed in fibroblast cells, their level of expression was significantly lower than in Sertoli cells

Conclusion: This study confirmed expression of TLR2 and TLR3 in human Sertoli cells. This may be an indicator of their roles in developing immunity against pathogens as well as allo- and auto-antigens or viral antigens in seminiferous tubules

In vitro development of embryos from experimentally Kerack-addicted Mice

Background: Prenatal drug exposure, as a common public health concern, is associated with an increased risk of adverse effects on early embryo development

Objective: To investigate the in vitro development of - embryo from experimentally Kerack-addicted mice

Materials and Methods: Twenty-five female mice were studied in five groups: control, vehicle, and three experimental groups of Kerack-dependent mice [I, II, and III] which received different doses of Kerack for 14 days. After the establishment of addiction model [7 days], experimental groups I, II, and III were given Kerack intraperitoneally at the doses of 5, 35, and 70 mg/kg, twice a day for a period of 7 days, respectively. The vehicle group received normal saline and lemon juice whilst the control group just received water and food. Morulae were obtained through oviduct flashing. The survived embryos were cultured in T6+ 5mg/ml bovine serum albumin. The developmental rates up to hatched stage daily and embryo quality [differential staining and Tunnel staining] were also assessed

Results: The developmental potential of embryos obtained from the addicted mother was significantly decreased in comparison with control group. There was a significant reduction in the rate of blastocyst formation in the high dose Kerack dependent group. However, in addicted mice there was reduction in the total cell number [40.92% vs. 65.08% in control] and, inner cell mass percentage [17.17% vs. 26.15% in control] while apoptotic cells numbers were increased [7.17 vs. 1.46 in control] [p<0.05]

Conclusion: The Kerack addiction during pregnancy retards preimplantation development and induces apoptosis

Role of somatic testicular cells during mouse spermatogenesis in three-dimensional collagen gel culture system

Spermatogonial stem cells [SSCs] are the only cell type that can restore fertility to an infertile recipient following transplantation. Much effort has been made to develop a protocol for differentiating isolated SSCs in vitro. Recently, three-dimensional [3D] culture system has been introduced as an appropriate microenvironment for clonal expansion and differentiation of SSCs. This system provides structural support and multiple options for several manipulation such as addition of different cells. Somatic cells have a critical role in stimulating spermatogenesis. They provide complex cell to cell interaction, transport proteins and produce enzymes and regulatory factors. This study aimed to optimize the culture condition by adding somatic testicular cells to the collagen gel culture system in order to induce spermatogenesis progression. In this experimental study, the disassociation of SSCs was performed by using a two-step enzymatic digestion of type I collagenase, hyaluronidase and DNase. Somatic testicular cells including Sertoli cells and peritubular cells were obtained after the second digestion. SSCs were isolated by Magnetic Activated Cell Sorting [MACS] using GDNF family receptor alpha-1 [Gfr Alpha -1] antibody. Two experimental designs were investigated. 1. Gfr Alpha -1 positive SSCs were cultured in a collagen solution. 2. Somatic testicular cells were added to the Gfr Alpha -1 positive SSCs in a collagen solution. Spermatogenesis progression was determined after three weeks by staining of synaptonemal complex protein 3 [SCP3]-positive cells. Semi-quantitative Reverse Transcription PCR was undertaken for SCP3 as a meiotic marker and, Crem and Thyroid transcription factor-1 [TTF1] as post meiotic markers. For statistical analysis student t test was performed Testicular supporter cells increased the expression of meiotic and post meiotic markers and had a positive effect on extensive colony formation. Collagen gel culture system supported by somatic testicular cells provides a microenvironment that mimics seminiferous epithelium and induces spermatogenesis in vitro

[Effects of kerack used in addict Iranian people on fertility of adult mice]

Infertility is one of the most serious social problems. Illicit drug use can be an important cause of male factor infertility. Kerack which its use is rising up in Iran refers to a high purity street-level heroin [heroin Kerack]. Heroin Kerack used in Iran is an opioid and has harmful effects on body organs. The aim of this study is to investigate the effects of Kerack used in Iran on fertility adult mice. In this study, 25 male mice were divided into five groups [control, sham and three experimental]. Experimental groups of Kerack-dependent mice [received ascending dose of Kerack for seven days] were divided into three categories, experimental I, II and III. Experimental I was given Kerack at a dose of 5 mg/kg, experimental II 35 mg/kg and experimental III 70 mg/kg, intraperitoneally twice a day for a period of 35 days. The sham group received normal saline and lemon juice [2.6 micro l/ml] whilst the control group just received water and food. Mice were then scarified and sperm removed from cauda epididymis were analyzed for sperm count, motility, morphology [normal/abnormal] and viability. Testes were also removed, weighed and processed for light microscopic studies. The results showed that fertility were significantly decreased in addicted mice compared with control groups [P

Autologous transplantation of adult mice spermatogonial stem cells into gamma irradiated testes

We evaluated structural and functional changes of fresh and frozen-thawed adult mouse spermatogonial stem cells following auto-transplantation into gamma-irra-diated testes. In this experimental research, the right testes from adult mice [n=25] were collected, then Sertoli and spermatogonial cells were isolated using two-step enzymatic digestion, lectin immobilization and differential plating. Three weeks after cultivation, the Bromodeoxyuridine [BrdU]-labeled spermatogonial cells were transplanted, via rete testis, into the other testis of the same mouse, which had been irradiated with 14Gy. The mice were transplanted with: fresh cells [control 1], fresh cells co-cultured with Sertoli cells [control 2], the frozen-thawed cells [experimental 1] and frozen-thawed cells co-cultured with Sertoli cells [experimental 2]. The morphological changes between different transplanted testes groups were compared in 8 weeks after transplantation. The statistical significance between mean values was determined by Kruskal Wallis and one-way analysis of variance in efficiency of transplantation. The statistical analysis revealed significant increases in the mean percentage of testis weight and normal seminiferous tubules following spermatogonial stem cells transplantation in the recipient's testes. The normal seminiferous tubules percentage in the co-culture system with fresh cells and frozen-thawed groups were more than those in non-transplanted and fresh cell transplanted groups [p

Comparison of neonatal and adult mice-derived sertoli cells in support of expansion of mouse spermatogonial stem cells in vitro

Background: This study compared neonatal and adult mice-derived Sertoli cells [NSCs and ASCs] to examine the influence of feeder cells derived from donors of different ages on the maintenance of mouse spermatogonial stem cells [SSCs] in vitro

Materials and Methods: SSCs were derived from the testes of six-day-old mice. They were subsequently transferred to Sertoli cells which were isolated by datura stramonium agglutinin [DSA] lectin from neonatal and adult mice for five days

Results: The numbers of spermatogonial colonies, the numbers of cells per colony, and cloning efficiency were assessed in presence of NSCs and ASCs. The expression of alpha 6-and beta 1-integrin-positive cells was evaluated. Moreover, the functionality of the cells was assessed by their transplantation into the testes of busulfan-induced infertile mice. Colony efficiency assay showed that the number of colonies derived from single spermatogonial cells were significantly higher on NSCs. Additionally, the transplantation of dissociated colonies into the testes of busulfan-induced infertile mice showed their migration to the seminiferous basal membrane

Conclusion: These results show that NSCs may provide a more favorable microenvironment in comparison with ASCs for in vitro culture of spermatogonial colonies

rare anatomical variant of ansa cervicalis: case report

A rare variant of ansa cervicalis was discovered during dissection of the neck in a male cadaver. The superior root of the Ansa cervicalis was formed by the C1 ventral ramus. It accompanied with the vagus nerve in place of the hypoglossal nerve and descended into the carotid sheath with it. Moreover the C1 ventral ramus formed from the sheath and joined the inferior root from C2 and C3 ventral rami

[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

[ comparison between the colony formation of adult mouse spermatogonial stem cells in co cultures with sertoli and STO [mouse embryonic fibroblast cell line]]

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. 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. beta1 and alpha6 -integrin m-RNA expressions were assessed using a reverse transcription polymerase chain reaction [RT-PCR] and realtime 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. 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 beta1 -integrin was expressed in all three groups cocultures, but alpha6 -integrin was not expressed. Additionally, based on real time PCR results, the three genes [beta1 -integrin, alpha6 -integrin, Oct-4] mentioned were also expressed in all three co cultures groups. 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

Assessment of Epidermal growth factor [EGF] effects on development of vitrified mouse morulae to the blastocyst stage

Many investigators are interested in finding the new cultural systems that can support the in vitro development of pre-implantation embryos better. Previous studies suggested that growth factors such as epidermal growth factor [EGF] are important in pre-implantation embryo development and implantation process. On the other hand, it is very important to support post thaw development of frozen embryos. The purpose of this study was to determine if the developmental potential of mouse morulae survived after vitrification could be increased using medium containing EGF. Mouse morulae were divided into vitrified and non-vitrified groups. Vitrification procedure was carried out using a combination of 40% ethylene glycol, 30% ficoll and 0.5 M sucrose [EFS40] as cryoprotectant. The embryos were warmed rapidly using 0.5 M sucrose. The survived embryos were cultured either on T6 or T6+EGF media. Accordingly, the embryos of the non-vitrified group were also cultured. The developmental rates in all groups were daily recorded and compared statistically using Chi-square test. The results showed that after 4 days of culture, the developmental potential of non-vitrified embryos cultured on T6+EGF was significantly increased. There was no significant difference between vitrified embryos cultured on T6 and T6+EGF media. In conclusion, the developmental potential of vitrified-warmed embryos does not increase in the medium containing EGF, even though there was significant increased developmental potential of non-vitrified embryos after culture on medium containing EGF. It is needed to do more study about the changes which will probably happen on the embryo EGF receptors following vitrification