Culture conditions for maintaining the survival and mitotic activity of rainbow trout transplantable type A spermatogonia

2007 ◽  
Vol 75 (3) ◽  
pp. 529-537 ◽  
Author(s):  
Shinya Shikina ◽  
Shoko Ihara ◽  
Goro Yoshizaki
Keyword(s):  
Rainbow Trout ◽  
Mitotic Activity ◽  
Type A ◽  
Culture Conditions ◽  
Type A Spermatogonia

scholarly journals Stem cell activity of type A spermatogonia is seasonally regulated in rainbow trout†

2017 ◽  
Vol 96 (6) ◽  
pp. 1303-1316 ◽  
Author(s):  
Mana Sato ◽  
Makoto Hayashi ◽  
Goro Yoshizaki
Keyword(s):  
Stem Cell ◽  
Rainbow Trout ◽  
Cell Activity ◽  
Type A ◽  
Stem Cell Activity ◽  
Type A Spermatogonia

Establishment of novel monoclonal antibodies for identification of type A spermatogonia in teleosts†

2019 ◽  
Vol 101 (2) ◽  
pp. 478-491 ◽  
Author(s):  
Makoto Hayashi ◽  
Kensuke Ichida ◽  
Sakiko Sadaie ◽  
Misako Miwa ◽  
Ryo Fujihara ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Monoclonal Antibodies ◽  
Rainbow Trout ◽  
Cell Surface ◽  
Germ Cell ◽  
Cell Transplantation ◽  
Germ Cells ◽  
Type A ◽  
Germ Cell Transplantation ◽  
Type A Spermatogonia

AbstractWe recently established a germ cell transplantation system in salmonids. Donor germ cells transplanted into the body cavity of recipient embryos migrate toward and are incorporated into the recipient gonad, where they undergo gametogenesis. Among the various types of testicular germ cells, only type A spermatogonia (A-SG) can be incorporated into the recipient gonads. Enriching for A-SG is therefore important for improving the efficiency of germ cell transplantation. To enrich for A-SG, an antibody against a cell surface marker is a convenient and powerful approach used in mammals; however, little is known about cell surface markers for A-SG in fish. To that end, we have produced novel monoclonal antibodies (mAbs) against cell-surface molecules of rainbow trout (Oncorhynchus mykiss) A-SG. We inoculated mice with living A-SG isolated from pvasa-GFP transgenic rainbow trout using GFP-dependent flow cytometry. By fusing lymph node cells of the inoculated mice with myeloma cells, we generated 576 hybridomas. To identify hybridomas that produce mAbs capable of labeling A-SG preferentially and effectively, we screened them using cell ELISA, fluorescence microscopy, and flow cytometry. We thereby identified two mAbs that can label A-SG. By using flow cytometry with these two antibodies, we could enrich for A-SG with transplantability to recipient gonads from amongst total testicular cells. Furthermore, one of these mAbs could also label zebrafish (Danio rerio) spermatogonia. Thus, we expect these monoclonal antibodies to be powerful tools for germ cell biology and biotechnology.

scholarly journals Production of functional eggs and sperm from in vitro-expanded type A spermatogonia in rainbow trout

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Yoshiko Iwasaki-Takahashi ◽  
Shinya Shikina ◽  
Masaya Watanabe ◽  
Akira Banba ◽  
Masaru Yagisawa ◽  
...  
Keyword(s):  
Rainbow Trout ◽  
Type A ◽  
Type A Spermatogonia

Short-term in vitro culturing improves transplantability of type A spermatogonia in rainbow trout (Oncorhynchus mykiss )

2013 ◽  
Vol 80 (9) ◽  
pp. 763-773 ◽  
Author(s):  
Shinya Shikina ◽  
Kazue Nagasawa ◽  
Makoto Hayashi ◽  
Maki Furuya ◽  
Yoshiko Iwasaki ◽  
...  
Keyword(s):  
Rainbow Trout ◽  
Oncorhynchus Mykiss ◽  
Type A ◽  
Short Term ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Type A Spermatogonia

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.

Transport properties of cultured branchial epithelia from freshwater rainbow trout: a novel preparation with mitochondria-rich cells

2000 ◽  
Vol 203 (10) ◽  
pp. 1523-1537 ◽  
Author(s):  
M. Fletcher ◽  
S.P. Kelly ◽  
P. Part ◽  
M.J. O'Donnell ◽  
C.M. Wood
Keyword(s):  
Rainbow Trout ◽  
Flux Ratio ◽  
Culture Conditions ◽  
Chloride Cells ◽  
Pavement Cells ◽  
Ratio Criterion ◽  
Peg 4000 ◽  
Tight Epithelia ◽  
Gill Filaments

A new double-seeded insert (DSI) technique is described for culture of branchial epithelial preparations from freshwater rainbow trout on filter supports. DSI epithelia contain both pavement cells and mitochondria-rich (MR) cells (15.7+/−2.5 % of total cell numbers). MR cells occur singly or in clusters, are voluminous, open apically to the ‘external environment’ and exhibit ultrastructural characteristics similar to those found in the ‘chloride cells’ of freshwater fish gills. After 6–9 days in culture with Leibovitz's L-15 medium on both surfaces (symmetrical conditions), transepithelial resistance (TER) stabilized at values as high as 34 k capomega cm(2), indicative of electrically ‘tight’ epithelia. The density of MR cells, the surface area of their clusters and transepithelial potential (TEP; up to +8 mV basolateral positive, mean +1.9+/−0.2 mV) were all positively correlated with TER. In contrast, preparations cultured using an earlier single-seeded insert (SSI) technique contained only pavement cells and exhibited a negligible TEP under symmetrical conditions. Na(+)/K(+)-ATPase activities of DSI preparations were comparable with those in gill filaments, but did not differ from those of SSI epithelia. Replacement of the apical medium with fresh water to mimic the in vivo situation (asymmetrical conditions) induced a negative TEP (−6 to −15 mV) and increased permeability to the paracellular marker PEG-4000. Under symmetrical conditions, unidirectional Na(+) and Cl(−) fluxes were in balance, and there was no active transport by the Ussing flux ratio criterion. Under asymmetrical conditions, there were large effluxes, small influxes and evidence for active Cl(−) uptake and Na(+) extrusion. Unidirectional Ca(2+) fluxes were only 0.5-1.0 % of Na(+) and Cl(−) fluxes; active net Ca(2+) uptake occurred under symmetrical conditions and active net extrusion under asymmetrical conditions. Thus, DSI epithelia exhibit some of the features of the intact gill, but improvements in culture conditions are needed before the MR cells will function as true freshwater ‘chloride cells’.

Studies of Sl/Sld in equilibrium with +/+ mouse aggregation chimaeras. II. Effect of the steel locus on spermatogenesis

Development ◽  
1988 ◽  
Vol 102 (1) ◽  
pp. 117-126 ◽  
Author(s):  
H. Nakayama ◽  
H. Kuroda ◽  
H. Onoue ◽  
J. Fujita ◽  
Y. Nishimune ◽  
...  
Keyword(s):  
Mast Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Cross Sections ◽  
Sertoli Cells ◽  
Type A ◽  
Precursor Cells ◽  
Intrinsic Defect ◽  
Serial Sections ◽  
Type A Spermatogonia

Mutant mice of Sl/Sld genotype are deficient in melanocytes, erythrocytes, mast cells and germ cells. Deficiency of melanocytes, erythrocytes and mast cells is not attributable to an intrinsic defect in their precursor cells but to a defect in the tissue environment that is necessary for migration, proliferation and/or differentiation. We investigated the mechanism of germ cell deficiency in male Sl/Sld mice by producing aggregation chimaeras from Sl/Sld and +/+ embryos. Chimaeric mice with apparent white stripes were obtained. Two of four such chimaeras were fertile and the phenotypes of resulting progenies showed that some Sl/Sld germ cells had differentiated into functioning sperms in the testis of the chimaeras. In cross sections of the testes of chimaeras, both differentiated and nondifferentiated tubules were observed. However, the proportions of type A spermatogonia to Sertoli cells in both types of tubules were comparable to the values observed in differentiated tubules of normal +/+ mice. We reconstructed the whole length of four tubules from serial sections. Differentiated and nondifferentiated segments alternated in a single tubule. The shortest differentiated segment contained about 180 Sertoli cells and the shortest nondifferentiated segment about 150 Sertoli cells. These results suggest that Sertoli cells of either Sl/Sld or +/+ genotype make discrete patches and that differentiation of type A spermatogonia does not occur in patches of Sl/Sld Sertoli cells.

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