Viability Of Testicular Cells Research Articles

Comparison of Different Extraction Procedures for Isolating Bacteriostatic and Anti-pseudorabies Virus Components from Rodgersia Sambucifolia (Hemsl)

Medicinal plants are considered a repository of bioactive ingredients. Rodgersia sambucifolia (Hemsl), a herbaceous perennial originating from East Asia, has demonstrated bacteriostatic and antiviral properties. The aim of this study was to identify an effective extraction procedure for isolating potential bacteriostatic and antiviral compounds from the plant. The crude extracts were obtained using ethanol, methanol or acetone, and stepwise extracted with ethyl acetate, n-butanol or water. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and diameters of the inhibition zone were evaluated for bacteriostatic efficacy. These tests were carried out with Enterococcus faecalis, Enterococcus faecium, Shigella dysenteriae, Streptococcus lactis, Staphylococcus aureus, and Streptococcus lactis. The antiviral activities of prevention, treatment, and neutralization, utilizing pseudorabies virus, were evaluated using swine testicular cell viability. The active ingredients from the best bioactivity extraction procedure were analyzed using liquid chromatography-mass spectrometry (LC-MS). When ranked highest to lowest, the bacteriostatic and antiviral effects were obtained in the order of the crude extracts prepared from ethanol, methanol, and acetone. Bacteriostatic and antiviral effects were highest in the stepwise extraction of ethyl acetate, followed by the extraction of n-butanol and water. The main compounds identified by LC-MS in the ethyl acetate phase in descending order of content were: bergenin, palmitic acid, baicalein, linoleic acid, chrysin, γ-linolenic acid, catechin gallate, catechin, ursolic acid, and baicalin. This study provides a research basis for the development of bacteriostatic and antiviral drugs from traditional Chinese herbal medicines.

DNA integrity and viability of testicular cells from diverse wild species after slow freezing or vitrification.

Cryopreservation of testicular tissues offers new possibilities to protect endangered species, genetically valuable individuals or even the fertility potential of prepubertal individuals who have died unexpectedly. However, the use of this technique still remains a challenge. In this study, slow freezing and vitrification of testicular tissue was investigated to find out which cryopreservation method could better preserve the viability and DNA integrity of testicular germ cells in diverse wild species. Testes were obtained post-mortem from 18 artiodactyls (wild boar, roe deer, dwarf goat, mhor gazelle, European mouflon, African forest buffalo, Malayan tapir, dorcas gazelle, Iberian ibex, gnu, red river hog), 5 primates (colobus monkey, capuchin monkey, mandrill), 8 carnivores (gray wolf, Persian leopard, binturong, European mink, American black bear, suricata), and 2 rodents (Patagonian mara). The testicles belonged to adult individuals and were cut into small pieces and cryopreserved by needle immersed vitrification or uncontrolled slow freezing using a passive cooling device. After warming or thawing, testicular tissues were enzymatically digested and two germ cell types were differentiated based on their morphology: rounded cells (spermatogonia, spermatocytes, and early spermatids) and elongated cells (elongated spermatids and spermatozoa). Cell viability was assessed by SYBR-14/propidium iodide while DNA fragmentation by TUNEL assay with fluorescence microscope. Our preliminary results revealed that our uncontrolled slow freezing method better preserved the viability and DNA integrity of elongated cells than vitrification. Such trend was observed in all species, being significant in artiodactyls, carnivores, and primates. Similarly, the viability and DNA integrity of rounded cells was also better maintained in primates by uncontrolled slow freezing, while in carnivores, vitrification by needle immersion showed better results in this type of cells. In artiodactyls and rodents both techniques preserved the viability of rounded cells in a similar manner, although the DNA integrity of these cells was greater after needle immersed vitrification in artiodactyls. In conclusion, the effectiveness of each cryopreservation method is affected by the phylogenetic diversity between species and cell type.

Localization and in silico-based functional analysis of miR-202 in bull testis.

Bull fertility is pivotal to the prosperity of the cattle industry worldwide. miR‐202 has been shown to be gonad specific and to have key roles in gonad function in different species. To further understand the involvement of miR‐202 in bull reproduction, this study aimed to establish its localization in bovine testicular tissue and to identify putative biological functions using bioinformatics approaches. We assessed the miR‐202 expression in paraffin‐embedded tissue samples collected form an abattoir using in situ hybridization. miR‐202 was present in Sertoli cells and in germ cells at different stages of development. Using available databases, a total of 466 predicted gene targets of miR‐202 were identified. Functional annotation revealed that miR‐202 target genes were mainly associated with protein modification and phosphorylation processes as well as longevity regulating pathway. Moreover, genes in the longevity regulating pathway mapped to PI3K/Akt/mTOR pathway which is involved in promoting proliferation of testicular cells and spermatogenesis. These findings suggest that miR‐202 plays important roles in regulating proliferation and viability of testicular cells including somatic and germ cells.

Using clinically derived human tissue to 3-dimensionally bioprint personalized testicular tubules for in vitro culturing: first report

To study the feasibility and spermatogenic potential of 3-dimensional (3D) bioprinting personalized human testicular cells derived from a patient with nonobstructive azoospermia (NOA). A human testicular biopsy from a single donor with NOA was dissociated into single cells, expanded invitro, and 3D bioprinted into tubular structures akin to the seminiferous tubule using AGC-10 bioink and an RX1 bioprinter with a CENTRA coaxial microfluidic printhead from Aspect Biosystems. Three-dimensional organoid cultures were used as a nonbioprinted invitro control. Academic medical center. A 31-year-old man with NOA with testis biopsy demonstrating Sertoli cell-only syndrome. Three-dimensional bioprinting and invitro culturing of patient-derived testis cells. Cellular viability after printing was determined, along with the expression of phenotypic and spermatogenic functional genetic markers after 12 days of invitro culture. Testicular cultures were expandable invitro and generated sufficiently large numbers for 3D bioprinting at 35 million cells per mL of bioink. Viability 24 hours after printing was determined to be 93.4% ± 2.4%. Immunofluorescence staining for the phenotype markers SRY-Box transcription factor 9, insulin-like 3, actin alpha 2 smooth muscle, and synaptonemal complex protein 3 after 12 days was positive, confirming the presence of Sertoli, Leydig, peritubular myoid, and meiotic germ cells. Reverse transcription qualitative polymerase chain reaction analysis showed that after 12 days in spermatogenic media, the bioprints substantially up-regulated spermatogenic gene expression on par with nonbioprinted controls and showed a particularly significant improvement in genes involved in spermatogonial stem cell maintenance: inhibitor of deoxyribonucleic acid binding 4 by 365-fold; fibroblast growth factor 3 by 94,152-fold; stem cell growth factor receptor KIT by twofold; stimulated by retinoic acid 8 by 125-fold; deleted in azoospermia-like by 114-fold; synaptonemal complex protein 3 by sevenfold; zona pellucida binding protein by twofold; transition protein 1 by 2,908-fold; and protamine 2 by 11-fold. This study demonstrates for the first time the feasibility of 3D bioprinting adult human testicular cells. We show that the bioprinting process is compatible with high testicular cell viability and without loss of the main somatic phenotypes within the testis tissue. We demonstrate an increase in germ cell markers in the 3D bioprinted tubules after 12 days of invitro culture. This platform may carry future potential for disease modeling and regenerative opportunities in a personalized medicine framework.

An Efficient In Vitro Culture System To Amplify Spermatogonia Stem Cell Markers

Introduction: Proliferation of spermatogonial stem cells (SSCs) can be a treatment for infertile men. Here, we design an efficient method based on culturing in the presence of Sertoli cells to improve the expression level of some specific spermatogonia stem cell genes during two weeks post culture. Materials and Methods: Cells were derived from neonatal (2-6 days old) mice testes and were cultured in DMEM medium with FBS. The colonization of cultured SSCs in days 4, 7, and 14 of culture was counted via phase-contrast microscope and Image J software. Methyl thiazolyl tetrazolium (MTT) test was performed to evaluate the viability of cultured SSCs in days 3, 7, and 14 of culture. The expression level and the alteration pattern of specific spermatogonial markers, i.e., Stra8, DAZL, and Piwill2 was examined via real-time polymerase chain reaction (PCR) during two weeks post culture. Results: The number and the diameters of colonies showed a significant increase in cultured cells. MTT results proved the higher viability of testicular cells during the culture period. The results of ALP staining detected a positive reaction in spermatogonia colonies. Real-time PCR data showed that culturing SSCs in the presence of interstitial cells of the testis, amplified the level and alteration pattern of specific spermatogonia stem cells genes beneficial in the enrichment of SSCs propagation. Conclusion: Providing a similar culture environment to testicular niche increases viability, forms SSCs colonies, and regulates the level and alteration pattern of spermatogonia stem cell genes.

Cell survival after cryopreservation of dissociated testicular cells from feline species

Testicular cell suspension (TCS) can be cryopreserved for male germ-line preservation and fertility restoration. We aimed to validate a cryopreservation protocol for TCS of domestic cat to be applied in endangered felids species. Testis tissue from adult domestic cats was enzymatically dissociated and spermatogenic cells were enriched. The resulting TCS was diluted in 7.5% or 15% Me2SO based medium. Slow and fast freezing methods were tested. We examined the effects of freezing approaches using two combinations of fluorescent dyes: Calcein-AM with Propidium iodide (C/PI) and SYBR14 with Propidium iodide (S/PI). Ploidy analysis of domestic cat fresh TCS revealed that the majority of testicular cells were haploid cells. Based on microscopic observation, two size populations (12.3 ± 2.3 μm and 20.5 ± 4 μm in diameter) were identified and presumed to be mainly spermatids and spermatocytes, respectively. Both evaluation methods proved higher viability of aggregated cells before and after cryopreservation compared with single cells, and superiority of low concentration of Me2SO (7.5%) in association with slow freezing to preserve viability of testicular cells. However, S/PI resulted in a more precise evaluation compared with the C/PI method. The combination of 7.5% Me2SO-based medium with slow freezing yielded post thaw viability of S/PI labeled aggregated (49.8 ± 20%) and single cells (31.5 ± 8.1%). Comparable results were achieved using testes of a Cheetah and an Asiatic golden cat. In conclusion, TCS from domestic cat can be successfully cryopreserved and has the potential to support fertility restoration of endangered felids species.

Optimizing methods for human testicular tissue cryopreservation and spermatogonial stem cell isolation.

Cryopreservation of testicular tissue before cancer therapy for fertility preservation in prepubertal boys with cancer is of great interest in reproductive medicine. Isolation of spermatogonial stem cells (SSCs) from cryopreserved tissues would be a suitable cell source to re-establish spermatogenesis after cancer therapy. We herein establish optimized protocols for cryopreservation of human testicular tissue and isolation of SSCs from cryopreserved tissue. We developed a freezing protocol that provided high testicular cell viability and supported structural integrity and tubular epithelium coherence similar to fresh tissue. Then, we established a protocol that allowed efficient isolation of functional SSCs from cryopreserved tissues. Isolated cells were found on the testicular basement membrane after xenotransplantation. Our results demonstrated the preservation of testicular tissue structure and high cell viability with efficient isolation of SSCs after testicular cryopreservation, which is promising for future therapeutic applications in fertility preservation.

MP41-20 DEVELOPMENTAL POTENTIAL OF VITRIFIED MOUSE TESTICULAR TISSUE AFTER ECTOPIC TRANSPLANTATION

You have accessJournal of UrologyStem Cell Research1 Apr 2017MP41-20 DEVELOPMENTAL POTENTIAL OF VITRIFIED MOUSE TESTICULAR TISSUE AFTER ECTOPIC TRANSPLANTATION Nazila Yamini, Gholamreza Pourmand, Fardin Amidi, Mojdeh Salehnia, Nahid Ataei Nejad, and Seyed Mohammad Hossein Noori Mougahi Nazila YaminiNazila Yamini More articles by this author , Gholamreza PourmandGholamreza Pourmand More articles by this author , Fardin AmidiFardin Amidi More articles by this author , Mojdeh SalehniaMojdeh Salehnia More articles by this author , Nahid Ataei NejadNahid Ataei Nejad More articles by this author , and Seyed Mohammad Hossein Noori MougahiSeyed Mohammad Hossein Noori Mougahi More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1288AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Cryopreservation of immature testicular tissue should be considered as an important factor for fertility preservation in young boys with cancer. The objective of this study is to investigate whether immature testicular tissue of mice can be successfully cryopreserved using a simple vitrification procedure to maintain testicular cell viability, proliferation, and differentiation capacity. METHODS In this experimental study, immature mice testicular tissue fragments(0.5-1 mm2) were vitrified-warmed in order to assess the effect of vitrification on testicular tissue cell viability. Trypan blue staining was used to evaluate developmental capacity. Vitrified tissue (n=42) and fresh (control, n=42) were ectopically transplanted into the same strain of mature mice (n=14) with normal immunity. After 4 weeks, the graft recovery rate was determined. Hematoxylin and eosin (H&E) staining was used to evaluate germ cell differentiation, immunohistochemistry staining by proliferating cell nuclear antigen (PCNA) antibody, and terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay for proliferation and apoptosis frequency. RESULTS Vitrification did not affect the percentage of cell viability. Vascular anastomoses was seen at the graft site. The recovery rate of the vitrified graft did not significantly differ with the fresh graft. In the vitrified graft, germ cell differentiation developed up to the secondaryspermatocyte, which was similar to fresh tissue. Proliferation and apoptosis in the vitrified tissue was comparable to the fresh graft. CONCLUSIONS Vitrification resulted in a success rates similar to fresh tissue (control) in maintaining testicular cell viability and tissue function. These data provided further evidence that vitrification could be considered an alternative for cryopreservation of immature testicular tissue. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e543 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Nazila Yamini More articles by this author Gholamreza Pourmand More articles by this author Fardin Amidi More articles by this author Mojdeh Salehnia More articles by this author Nahid Ataei Nejad More articles by this author Seyed Mohammad Hossein Noori Mougahi More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Developmental Potential of Vitrified Mouse Testicular Tissue after Ectopic Transplantation.

Cryopreservation of immature testicular tissue should be considered as an important factor for fertility preservation in young boys with cancer. The objective of this study is to investigate whether immature testicular tissue of mice can be successfully cryopreserved using a simple vitrification procedure to maintain testicular cell viability, proliferation, and differentiation capacity. In this experimental study, immature mice testicular tissue fragments (0.5-1 mm²) were vitrified-warmed in order to assess the effect of vitrification on testicular tissue cell viability. Trypan blue staining was used to evaluate developmental capacity. Vitrified tissue (n=42) and fresh (control, n=42) were ectopically transplanted into the same strain of mature mice (n=14) with normal immunity. After 4 weeks, the graft recovery rate was determined. Hematoxylin and eosin (H&E) staining was used to evaluate germ cell differentiation, immunohistochemistry staining by proliferating cell nuclear antigen (PCNA) antibody, and terminal deoxynucleotidyl transferase (TdT) dUTP Nick- End Labeling (TUNEL) assay for proliferation and apoptosis frequency. Vitrification did not affect the percentage of cell viability. Vascular anastomoses was seen at the graft site. The recovery rate of the vitrified graft did not significantly differ with the fresh graft. In the vitrified graft, germ cell differentiation developed up to the secondary spermatocyte, which was similar to fresh tissue. Proliferation and apoptosis in the vitrified tissue was comparable to the fresh graft. Vitrification resulted in a success rates similar to fresh tissue (control) in maintaining testicular cell viability and tissue function. These data provided further evidence that vitrification could be considered an alternative for cryopreservation of immature testicular tissue.

Effect of newborn bovine serum on cryopreservation of adult bovine testicular tissue

Bovine serum is widely used for cryopreservation of various cells and tissues. However, its cryoprotective effects on the cells and tissues are ambiguous and controversial. To test the effects of newborn calf serum (NCS) on cryopreservation of bovine testis tissue, NCS of 0%, 5%, 10% and 20% (v/v) was added into minimum essential medium + 10% dimethyl sulphoxide (DMSO)-based medium according to our previous report. Interestingly, the testicular cell viabilities and spermatogonia percentages from four groups were very close. The results indicated that an increase in the concentration of NCS in freezing medium to 20% has no significant effect on survival of both testicular cells and spermatogonia, and 10% DMSO-based freezing medium can maintain the testicular cell viability and spermatogonia percentage at a relatively high level (83.4 ± 0.7 and 56.5 ± 2.2 respectively). Taken together, NCS is dispensable for cryopreservation of adult bovine testis tissue. Our results provide an evidence for cutting down the costs in cryopreservation research of bovine testis tissue by reducing or giving up the use of serum.

Quercetin Attenuates Oxidative Damage Induced by Treatment of Embryonic Chicken Spermatogonial Cells with 4-Nitro-3-phenylphenol in Diesel Exhaust Particles

Quercetin, an antioxidant flavonoid, is considered beneficial for human and animal health. In this study, the protective effect of quercetin on oxidative damage to testicular cells was studied in embryonic chickens after treatment with 4-nitro-3-phenylphenol (PNMPP) derived from diesel exhaust particles. Testicular cells were challenged with PNMPP (10(-8)-10(-6) M) alone and in combination with quercetin for 48 h. The results showed that quercetin manifested no deleterious effect on spermatogonial cells up to 1.0 microg/ml. Exposure to PNMPP (10(-6) M) induced condensed nuclei and vacuolated cytoplasm and reductions in testicular cell viability and spermatogonial cell numbers (p<0.05). It also induced lipid peroxidation by an elevation of thiobarbituric acid reactive substances and decreased glutathione peroxidase activity and superoxide dismutase activity (p<0.05). Simultaneous supplementation with quercetin restored these parameters to the same levels as in the control. These data indicate that quercetin protects spermatogonial cells from oxidative damage in embryonic chickens intoxicated with PNMPP.

Protective Effect of Quercetin on the Reproductive Toxicity of 4-nitrophenol in Diesel Exhaust Particles on Male Embryonic Chickens

The 4-nitrophenol (PNP) in diesel exhaust particles (DEP) has been identified as a vasodilator and is a known degradation product of the insecticide parathion. In this study, the protective effect of quercetin, a potent oxygen free radical scavenger and metal chelator, against the oxidative damage of PNP on cultured testicular cells was studied in male embryonic chickens. Testicular cells from Day 18 embryos were cultured in serum-free McCoy's 5A medium and challenged with quercetin (1.0 microg/ml) alone or in combinations with PNP (10(-7)-10(-5) M) for 48 h. The oxidative damage was estimated by measuring cell viability, content of malondialdehyde (MDA), activity of superoxide dismutase (SOD) and glutathione peroxidation (GSH-Px) activity. The results showed that exposure to PNP (10(-5) M) induced condensed nuclei, vacuolated cytoplasm and a decrease in testicular cell viability and spermatogonial cell number. Exposure to PNP induced lipid peroxidation by elevation of the content of MDA. Exposure to PNP also decreased GSH-Px activity and SOD activity. However, simultaneous supplementation with quercetin restored these parameters to the same levels as the control. Consequently, PNP induced oxidative stress in spermatogonial cells, and dietary quercetin may attenuate the reproductive toxicity of PNP to restore the intracellular antioxidant system in the testicular cells of embryonic chickens.

Quercetin protects embryonic chicken spermatogonial cells from oxidative damage intoxicated with 3-methyl-4-nitrophenol in primary culture

Diesel exhaust particles (DEP) are considered to be one of the most important air pollutants. In this study, the protective effect of quercetin, an antioxidant flavonoid, on oxidative damage of testicular cells was studied by analysis of the intracellular antioxidant system of embryonic chickens after treatment with 3-methyl-4-nitrophenol (PNMC) derived from DEP. Testicular cells from 18-day-old embryos were cultured in serum-free McCoys’5A medium and challenged with PNMC (10 −7 to 10 −5 M) alone or in combinations with quercetin (1.0 μg/ml) for 48 h. Results showed that exposure to PNMC (10 −5 M) induced condensed nuclei and vacuolated cytoplasm, a decrease in testicular cell viability and spermatogonial cell number. Exposure to PNMC induced lipid peroxidation by an elevation of thiobarbituric acid reactive substances as well as decreasing glutathione peroxidation activity and superoxide dismutase activity. However, simultaneous supplementation with quercetin restored these parameters to the similar levels as the control. PNMC is therefore concluded to have induced the oxidative stress of the spermatogonial cells, which can be attenuated by combined quercetin treatment. Our results support the therapeutic use of quercetin in the prevention or treatment of the reproductive toxicity by environmental toxicant PNMC.

Comparison of conditions for cryopreservation of testicular tissue from immature mice

Cryopreservation of immature testicular tissue could be considered as a major step in fertility preservation for young boys with cancer. In the present study, eight different freezing protocols were evaluated in immature mice testis. Testis from six-day-old mice were frozen using either 1,2-propanediol (PROH) or dimethylsulphoxide (DMSO: D) at 1.5 M. Different cooling rate curves were tested: (i) controlled slow protocol with seeding (CS+) or (ii) without seeding (CS-), (iii) controlled rapid protocol and (iv) non-controlled protocol. Cryodamage of seminiferous cords was semi-quantitatively determined, establishing a scoring of alterations. Cell viability and apoptosis induction were assessed on testicular cell suspensions immediately after digestion (D0) and after a 20-h culture period (D1). Cells recovered after digestion of 100 mg tissue and the rate of living and non-apoptotic cells were quantified at D0 and D1. A long-term culture (9 days) of testis pieces was carried out for the protocol offering the best survival. Testosterone production, intratubular cell proliferation and tubule growth were assessed. DMSO produced optimal results in the different cooling rate curves tested when compared with PROH. Optimal results were obtained for the DCS- procedure (P < 0.05). Testosterone production, tubule growth and cell proliferation of post-thaw pieces were similar to fresh samples. Testis freezing with 1.5 M DMSO in a CS- procedure was found to maintain not only immature testicular tissue architecture, but also viability of testicular cells, endocrine and partial exocrine functions of the testis. Semi-quantitative evaluation of seminiferous cord cryodamage can be effectively used to rapidly screen optimal freezing conditions and as a possible quality control in a human application.

Quercetin Protects Spermatogonial Cells from 2,4-D-Induced Oxidative Damage in Embryonic Chickens

Quercetin, an antioxidant flavonoid, is considered beneficial to human and animal health. In this study, the protective effects of quercetin in relation to oxidative damage of testicular cells were studied by analysis of the intracellular antioxidant system after treatment of embryonic chickens with hypoxanthine-xanthine oxidase (HX-XO) or 2,4-dichlorophenoxyacetic acid (2,4-D). Testicular cells from Day 18 embryos were challenged with quercetin alone or in combinations with HX-XO or 2,4-D for 48 h in culture. The results showed that quercetin manifested no deleterious effects on spermatogonial cells at concentrations up to 1.0 microg/ml. Exposure to HX-XO or 2,4-D (50 microg/ml) induced condensed nuclei and vacuolated cytoplasm and a decrease in testicular cell viability and spermatogonial cell number. Membrane integrity was damaged by elevated lactate dehydrogenase leakage. Exposure to HX-XO or 2,4-D also elicited lipid peroxidation by elevation of thiobarbituric acid reactive substances and decreased glutathione content and superoxide dismutase activity. However, simultaneous supplementation with quercetin restored these parameters to the levels in the controls. Consequently, HX-XO and 2,4-D induced oxidative stress in spermatogonial cells; however, dietary quercetin may attenuate the negative effects of environmental toxicants and restore the antioxidant system in testicular cells.

Effect of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on Testosterone Production in Isolated Murine Testicular Cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an endocrine disruptor that is known to have widespread effects on the reproductive system. We examined the effects of TCDD on testosterone production of primary cultured murine testicular cells. The cells, derived from the testes of ICR mice at age 65 days, were exposed to 10-2-104 pM TCDD for 3, 24, or 48 hr and treated with human chorionic gonadotropin for 6 hr to induce production of testosterone. At these concentrations of TCDD, the viability of testicular cells was not affected. No significant time- or TCDD concentration-dependent effects were observed on the secretion of testosterone. The data suggest that TCDD does not have a direct influence on testosterone production in the ICR mouse.