A comparison of methods for preparing enriched populations of bovine spermatogonia

2009 ◽  
Vol 21 (3) ◽  
pp. 393 ◽  
Author(s):  
Muren Herrid ◽  
Rhonda J. Davey ◽  
Keryn Hutton ◽  
Ian G. Colditz ◽  
Jonathan R. Hill
Keyword(s):  
Cell Sorting ◽  
Gradient Centrifugation ◽  
Fold Increase ◽  
Type A ◽  
Datura Stramonium ◽  
Percoll Gradient ◽  
Isolated Cells ◽  
Antibody Staining ◽  
Type A Spermatogonia

The objective of the present study was to identify an efficient and practical enrichment method for bovine type A spermatogonia. Four different enrichment methods were compared: differential plating on laminin- or Datura stramonium agglutinin (DSA)-coated flasks, percoll-gradient isolation, magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). The isolated cells were characterised with Dolichos biflorus agglutinin (DBA) lectin staining for type A spermatogonia and vimentin-antibody staining for Sertoli cells. A 2 × 2 factorial design was used to investigate the enrichment efficiency on laminin and DSA. In the laminin-enrichment groups, 2 h incubation in plates coated with 20 μg mL–1 laminin yielded a 3.3-fold increase in DBA-positive cells in the adherent fraction, while overnight incubation in flasks coated with 20 μg mL–1 DSA produced a 3.6-fold increase in the non-adherent fraction. However, the greatest enrichment (5.3-fold) of DBA-positive cells was obtained after 2 h incubation in control flasks (coated with bovine serum albumin). Percoll-gradient centrifugation yielded a 3-fold increase in DBA-positive cells. MACS results showed a 3.5- to 5-fold enrichment while FACS produced a 4-fold increase in DBA-positive cells. It is concluded that differential plating is a better method of recovering large numbers of type A spermatogonia for germ cell transplantation, while MACS or FACS can provide highly enriched viable type A spermatogonia for in vitro culture. Further, the combination of differential plating and other enrichment techniques may increase the purification efficiency of type A spermatogonia.

scholarly journals Meagre Argyrosomus regius (Asso, 1801) Stem Spermatogonia: Histological Characterization, Immunostaining, In Vitro Proliferation, and Cryopreservation

Animals ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 851 ◽  
Author(s):  
Rosa Zupa ◽  
Nicola A. Martino ◽  
Giuseppina Marzano ◽  
Maria E. Dell’Aquila ◽  
Aldo Corriero
Keyword(s):  
Gradient Centrifugation ◽  
Type A ◽  
Single Type ◽  
In Vitro Proliferation ◽  
Argyrosomus Regius ◽  
In Captivity ◽  
Active Proliferation ◽  
Aquaculture Production ◽  
Type A Spermatogonia

The meagre, Argyrosomus regius, is a valued fish species of which aquaculture production might be supported by the development of a stem germ cell xenotransplantation technology. Meagre males were sampled at a fish farm in the Ionian Sea (Italy) at the beginning and end of the reproductive season. Small and large Type A undifferentiated spermatogonia were histologically identified in the germinal epithelium. Among the tested stemness markers, anti-oct4 and anti-vasa antibodies labeled cells likely corresponding to the small single Type A spermatogonia; no labeling was obtained with anti-GFRA1 and anti-Nanos2 antibodies. Two types of single A spermatogonia were purified via density gradient centrifugation of enzymatically digested testes. Testes from fish in active spermatogenesis resulted in a more efficient spermatogonial stem cell (SSC) yield. After cell seeding, meagre SSCs showed active proliferation from Day 7 to Day 21 and were cultured up to Day 41. After cryopreservation in dimethyl-sulfoxide-based medium, cell viability was 28.5%. In conclusion, these results indicated that meagre SSCs could be isolated, characterized, cultured in vitro, successfully cryopreserved, and used after thawing. This is a first step towards the development of a xenotransplantation technology that might facilitate the reproduction of this valuable species in captivity.

scholarly journals Propagation of bovine spermatogonial stem cells in vitro

Reproduction ◽  
2008 ◽  
Vol 136 (5) ◽  
pp. 543-557 ◽  
Author(s):  
Pedro M Aponte ◽  
Takeshi Soda ◽  
Katja J Teerds ◽  
S Canan Mizrak ◽  
Henk J G van de Kant ◽  
...  
Keyword(s):  
Stem Cells ◽  
Growth Factor ◽  
Cultured Cells ◽  
Somatic Cells ◽  
Type A ◽  
Spermatogonial Stem Cells ◽  
Seminiferous Tubules ◽  
Type A Spermatogonia ◽  
Stimulation Of

The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. A specialized medium and several growth factors were tested to study thein vitrobehavior of bovine type A spermatogonia, a cell population that includes the SSCs and can be specifically stained for the lectin Dolichos biflorus agglutinin. During short-term culture (2 weeks), colonies appeared, the morphology of which varied with the specific growth factor(s) added. Whenever the stem cell medium was used, round structures reminiscent of sectioned seminiferous tubules appeared in the core of the colonies. Remarkably, these round structures always contained type A spermatogonia. When leukemia inhibitory factor (LIF), epidermal growth factor (EGF), or fibroblast growth factor 2 (FGF2) were added, specific effects on the numbers and arrangement of somatic cells were observed. However, the number of type A spermatogonia was significantly higher in cultures to which glial cell line-derived neurotrophic factor (GDNF) was added and highest when GDNF, LIF, EGF, and FGF2 were all present. The latter suggests that a proper stimulation of the somatic cells is necessary for optimal stimulation of the germ cells in culture. Somatic cells present in the colonies included Sertoli cells, peritubular myoid cells, and a few Leydig cells. A transplantation experiment, using nude mice, showed the presence of SSCs among the cultured cells and in addition strongly suggested a more than 10 000-fold increase in the number of SSCs after 30 days of culture. These results demonstrate that bovine SSC self-renew in our specialized bovine culture system and that this system can be used for the propagation of these cells.

scholarly journals Senescence of canine biotinylated erythrocytes: increased autologous immunoglobulin binding occurs on erythrocytes aged in vivo for 104 to 110 days

Blood ◽  
1993 ◽  
Vol 82 (11) ◽  
pp. 3469-3473 ◽  
Author(s):  
JA Christian ◽  
AH Rebar ◽  
GD Boon ◽  
PS Low
Keyword(s):  
Life Span ◽  
Cell Sorting ◽  
Gradient Centrifugation ◽  
Magnetic Cell Sorting ◽  
Autologous Plasma ◽  
Low Degree ◽  
La Jolla ◽  
Immunoglobulin Binding

Abstract We have evaluated senescence related changes in canine red blood cells (RBCs) using the biotinylation system, where RBCs are labeled in vivo with biotin at the beginning of their life span, and retrieved from circulation on immobilized avidin at the end of their life span. This approach avoids the controversial use of density gradient centrifugation to collect presumably old RBCs. Furthermore, the dog is an appropriate model for human RBC senescence because it has a low degree of random RBC loss and a similarly long RBC life span (approximately 110 days). Two dogs had 97% to 100% of their circulating RBCs biotinylated by infusion of N-hydroxysuccinimido biotin (Clontech, Palo Alto, CA; Calbiochem, La Jolla, CA) dissolved in dimethyl sulfoxide. At postbiotinylation days 104 and 107 for one dog and day 110 for the other dog, biotinylated RBCs were isolated by magnetic cell sorting and analyzed for the presence of autologous IgG using 125I- labeled sheep-antidog IgG (SAD IgG). On all 3 days, there were at least three times more SAD IgG molecules per RBC on senescent biotinylated RBCs than on control (unfractionated) RBCs (day 104: 11,677 v 3,399; day 107: 6,710 v 2,115; day 110: 6,042 v 1,838 molecules of SAD IgG per senescent v control RBC). Furthermore, it is unlikely that an immune response to the conjugated biotin had been elicited, because fresh in vitro biotinylated RBCs that were incubated in autologous plasma (taken after exposure to circulating biotinylated RBCs for 113 days) and then exposed to the SAD IgG showed no increase in antibody binding over control (non-biotinylated) RBCs (1,431 v 1,378 cpm/10(8) biotinylated v control RBCs; P > .20). These results suggest that senescence of canine biotinylated RBCs is characterized by binding of autologous IgG and that antibiotin antibodies do not contribute to this process.

Inhibition Of Collagen And ADP-Induced Platelet Aggregation By Plasma Fibronectin

1981 ◽  
Author(s):  
D G Moon ◽  
J E Kaplan
Keyword(s):  
Gradient Centrifugation ◽  
Fold Increase ◽  
Lag Time ◽  
Collagen Type I ◽  
Type I ◽  
Plasma Fibronectin ◽  
Aggregation Rate ◽  
Tail Tendon ◽  
Induced Aggregation

Platelets contain fibronectin a glycoprotein with an established affinity for collagen. This observation has led other investigators to postulate that fibronectin is the platelet collagen receptor. The much greater concentration of fibronectin in the plasma surrounding platelets, however, has led us to suggest that plasma fibronectin may bind to collagen and competitively inhibit the platelet- collagen interaction. Rat platelets were isolated by Stractan density gradient centrifugation and aggregated with acid-solubilized rat tail tendon collagen (Type I) in a Payton 300B Aggregometer. Fibronectin was twice purified by affinity chromatography with gelatin linked to CNBr- activated Sepharose 4B. Simultaneous addition of 50 μg fibronectin and 25 μg collagen to platelets suspended in Tyrodes solution at 37°C resulted in a 2-fold increase in lag time and a 30% decrease in aggregation rate as compared to control values. When collagen was preincubated in Tyrodes solution for 12 minutes at 26°C without platelets to allow for prior fibrillogenesis, the addition of 50 μg fibronectin with the platelets resulted in <20% increase in lag time and a 20-30% decrease in aggregation rate. In a separate series of experiments, fibronectin was also found to inhibit ADP-induced aggregation. In this case, the initial rate of aggregation was comparable with and without fibronectin, but this maximal rate was maintained for a shorter period in the presence of fibronectin. Thus, fibronectin reduced the in vitro aggregation response to two different physiological stimuli. Our data supports previous studies which indicate that fibronectin reduces the reactivity of platelets with collagen and provides evidence of a role for fibronectin in modulating platelet responses in the absence of collagen.

Isolation and Culture of Single Cell Types from Rat Liver

2016 ◽  
Vol 201 (4) ◽  
pp. 253-267 ◽  
Author(s):  
Qidi Zhang ◽  
Ying Qu ◽  
Zhenghong Li ◽  
Qingqing Zhang ◽  
Mingyi Xu ◽  
...  
Keyword(s):  
Single Cell ◽  
Rat Liver ◽  
Gradient Centrifugation ◽  
Cell Types ◽  
Ethylene Glycol Tetraacetic Acid ◽  
High Yield ◽  
Isolated Cells ◽  
Liver Sinusoidal Endothelial Cells ◽  
Pathology Of The Liver

There have been few reports on the simultaneous isolation of multiple liver cell populations thus far. As such, this study was aimed at establishing a protocol for the simultaneous separation of hepatocytes (HCs), hepatic stellate cells (HSCs), liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs) from the rat liver and assessing the in vitro culture of these cells. Single-cell suspensions from the liver were obtained by ethylene glycol tetraacetic acid/collagenase perfusion. After low-speed centrifugal separation of HCs, pronase was added to the nonparenchymal cell fraction to eliminate the remaining HCs. Subsequently, HSCs, LSECs and KCs were purified by two steps of density gradient centrifugation using Nycodenz and Percoll in addition to selective attachment. Pronase treatment increased the HSC yield (1.5 ± 0.2 vs. 0.7 ± 0.3 cells/g liver, p < 0.05) and improved LSEC purity (93.6 ± 3.6 vs. 82.5 ± 5.6%, p < 0.01). The isolated cells could also be cultured in vitro. LSEC apoptosis began on day 3 and reached a maximum on day 7. A few surviving LSECs began proliferating and split to form a cobblestone, sheet-like appearance on day 14. The LSECs on day 14 lost fenestrations but retained scavenger function. Thus, viable and purified liver cells were obtained with a high yield from the rat liver using the developed method, which may be useful for studying the physiology and pathology of the liver in the future.

Analysis of the hematopoietic potential of muscle-derived cells in mice

Blood ◽  
2002 ◽  
Vol 100 (2) ◽  
pp. 721-723 ◽  
Author(s):  
Hartmut Geiger ◽  
Jarrod M. True ◽  
Barry Grimes ◽  
Elizabeth J. Carroll ◽  
Roger A. Fleischman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Muscle Tissue ◽  
Cell Sorting ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Muscle Stem Cells ◽  
Antibody Staining ◽  
Stem And Progenitor Cells

Abstract Cells in murine muscle have been reported to differentiate into hematopoietic stem and progenitor cells and thus repopulate the hematopoietic system of an irradiated animal. This activity was attributed to muscle stem cells. We used an in vitro and in vivo approach to identify the hematopoietic repopulating activity found in muscle tissue of mice by antibody staining and cell sorting. We confirmed existence of a hematopoietic repopulating cell in muscle tissue, but the data strongly suggest that repopulation is due not to muscle stem cells but to hematopoietic cells present in muscle tissue. Unexpectedly, the blood-forming cells were enriched in muscle relative to their frequency in peripheral blood.

scholarly journals In Vitro Differentiation of Type a Spermatogonia from Mouse Cryptorchid Testes in Serum-Free Media

1983 ◽  
Vol 28 (5) ◽  
pp. 1217-1223 ◽  
Author(s):  
Tatsuji Haneji ◽  
Mamiko Maekawa ◽  
Yoshitake Nishimune
Keyword(s):  
Type A ◽  
In Vitro Differentiation ◽  
Serum Free ◽  
Free Media ◽  
Type A Spermatogonia

scholarly journals Isolation and Characterization of Highly Pure Type A Spermatogonia From Sterlet (Acipenser ruthenus) Using Flow-Cytometric Cell Sorting

2021 ◽  
Vol 9 ◽  
Author(s):  
Xuan Xie ◽  
Tomáš Tichopád ◽  
Galina Kislik ◽  
Lucie Langerová ◽  
Pavel Abaffy ◽  
...  
Keyword(s):  
Germ Cells ◽  
Cell Sorting ◽  
Developmental Stages ◽  
Type A ◽  
Cell Populations ◽  
Forward Scatter ◽  
Flow Cytometric ◽  
Flow Cytometric Cell ◽  
Type A Spermatogonia

Sturgeons are among the most ancient linages of actinopterygians. At present, many sturgeon species are critically endangered. Surrogate production could be used as an affordable and a time-efficient method for endangered sturgeons. Our study established a method for identifying and isolating type A spermatogonia from different developmental stages of testes using flow cytometric cell sorting (FCM). Flow cytometric analysis of a whole testicular cell suspension showed several well-distinguished cell populations formed according to different values of light scatter parameters. FCM of these different cell populations was performed directly on glass slides for further immunocytochemistry to identify germ cells. Results showed that the cell population in gate P1 on a flow cytometry plot (with high forward scatter and high side scatter parameter values) contains the highest amount of type A spermatogonia. The sorted cell populations were characterized by expression profiles of 10 germ cell specific genes. The result confirmed that setting up for the P1 gate could precisely sort type A spermatogonia in all tested testicular developmental stages. The P2 gate, which was with lower forward scatter and side scatter values mostly, contained type B spermatogonia at a later maturing stage. Moreover, expressions of plzf, dnd, boule, and kitr were significantly higher in type A spermatogonia than in later developed germ cells. In addition, plzf was firstly found as a reliable marker to identify type A spermatogonia, which filled the gap of identification of spermatogonial stem cells in sterlet. It is expected to increase the efficiency of germ stem cell culture and transplantation with plzf identification. Our study thus first addressed a phenotypic characterization of a pure type A spermatogonia population in sterlet. FCM strategy can improve the production of sturgeons with surrogate broodstock and further the analysis of the cellular and molecular mechanisms of sturgeon germ cell development.

DIFFERENTIAL EFFECTS OF CYCLIC AMP ON DNA SYNTHESIS BY GERM CELLS AND NON-GERM CELLS IN MOUSE TESTICULAR CULTURE

1981 ◽  
Vol 89 (2) ◽  
pp. 257-NP ◽  
Author(s):  
YOSHITAKE NISHIMUNE ◽  
TATSUJI HANEJI ◽  
SHIRO AIZAWA
Keyword(s):  
Cyclic Amp ◽  
Dna Synthesis ◽  
Germ Cells ◽  
Type A ◽  
Dibutyryl Cyclic Amp ◽  
Differential Effects ◽  
Low Concentration ◽  
Type A Spermatogonia

The effect of dibutyryl cyclic AMP (dbcAMP) on DNA synthesis in mouse cryptorchid explants with only type A spermatogonia was examined in vitro. Low concentration of dbcAMP (0·08 mmol/l) stimulated DNA synthesis by germ cells but inhibited that by non-germ cells.

scholarly journals Differences in myeloperoxidase activity from neutrophilic polymorphonuclear leukocytes of differing density: relationship to selective exocytosis of distinct forms of the enzyme

Blood ◽  
1983 ◽  
Vol 61 (6) ◽  
pp. 1116-1124
Author(s):  
SO Pember ◽  
JM Jr Kinkade
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Human Peripheral Blood ◽  
Gradient Centrifugation ◽  
Fold Increase ◽  
Myeloperoxidase Activity ◽  
Percoll Density Gradient Centrifugation ◽  
Density Relationship ◽  
Neutrophilic Polymorphonuclear Leukocytes

Elicited murine neutrophilic polymorphonuclear leukocytes (PMN) were fractionated by Percoll density gradient centrifugation into high density (HD) and intermediate density (ID) populations. As described in the accompanying article HD- and ID-PMN appear to represent “resting” and “activated” cell populations, respectively. Consistent with this possibility, histochemical and biochemical evidence suggested that ID- PMN were degranulated compared to HD-PMN. Myeloperoxidase (MPO) in the ID-PMN population showed increased sensitivity to inhibition by 3-amino- 1,2,4-triazole, and HD-PMN exhibited a 2–3-fold increase in chloride and iodide oxidation per unit of MPO activity compared to ID-PMN. When HD-PMN were induced to degranulate in vitro, the remaining cell- associated MPO displayed enzymatic properties characteristic of the activity associated with ID-PMN. The mechanism of this phenomenon was also investigated in vitro using purified human peripheral blood PMN and the synthetic chemotactic peptide N-formyl-methionyl-leucyl- phenylalanine. Differences in cell-associated MPO activity were shown to be related to selective exocytosis of enzymatically and chromatographically distinct forms of the enzyme. These data indicate that, in addition to the well known selective exocytosis of specific and azurophilic granules induced by various agents, selectivity may also occur at the level of enzymatically distinct forms of a particular granule enzyme. Moreover, our observations provide further evidence that density differences may be utilized to fractionate and study the generation of functionally distinct subpopulations of PMN that arise in vivo as well as in vitro following exposure to various stimuli.

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