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

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.

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A comparison of methods for preparing enriched populations of bovine spermatogonia

Reproduction Fertility and Development ◽

10.1071/rd08129 ◽

2009 ◽

Vol 21 (3) ◽

pp. 393 ◽

Cited By ~ 41

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.

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Propagation of bovine spermatogonial stem cells in vitro

Reproduction ◽

10.1530/rep-07-0419 ◽

2008 ◽

Vol 136 (5) ◽

pp. 543-557 ◽

Cited By ~ 90

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.

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

Biology of Reproduction ◽

10.1095/biolreprod28.5.1217 ◽

1983 ◽

Vol 28 (5) ◽

pp. 1217-1223 ◽

Cited By ~ 18

Author(s):

Tatsuji Haneji ◽

Mamiko Maekawa ◽

Yoshitake Nishimune

Keyword(s):

Type A ◽

In Vitro Differentiation ◽

Serum Free ◽

Free Media ◽

Type A Spermatogonia

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

Journal of Endocrinology ◽

10.1677/joe.0.0890257 ◽

1981 ◽

Vol 89 (2) ◽

pp. 257-NP ◽

Cited By ~ 1

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.

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Isolation, characterization, in vitro expansion and transplantation of Caspian trout (Salmo caspius) type a spermatogonia

General and Comparative Endocrinology ◽

10.1016/j.ygcen.2019.113341 ◽

2020 ◽

Vol 289 ◽

pp. 113341

Author(s):

Samaneh Poursaeid ◽

Mohammad-Reza Kalbassi ◽

Seyedeh-Nafiseh Hassani ◽

Hossein Baharvand

Keyword(s):

Type A ◽

In Vitro Expansion ◽

Type A Spermatogonia

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Retinoids Induce Differentiation of Type A Spermatogonia In Vitro: Organ Culture of Mouse Cryptorchid Testes

Journal of Nutrition ◽

10.1093/jn/113.6.1119 ◽

1983 ◽

Vol 113 (6) ◽

pp. 1119-1123 ◽

Cited By ~ 11

Author(s):

Tatsuji Haneji ◽

Mamiko Maekawa ◽

Yoshitake Nishimune

Keyword(s):

Organ Culture ◽

Type A ◽

In Vitro Organ Culture ◽

Type A Spermatogonia

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Hormones and the differentiation of type A spermatogonia in mouse cryptorchid testes incubated in vitro

Journal of Endocrinology ◽

10.1677/joe.0.0940043 ◽

1982 ◽

Vol 94 (1) ◽

pp. 43-NP ◽

Cited By ~ 20

Author(s):

Tatsuji Haneji ◽

Yoshitake Nishimune

Keyword(s):

Cell Differentiation ◽

Germ Cell ◽

Cyclic Adenosine Monophosphate ◽

Type A ◽

Adenosine Monophosphate ◽

Testicular Germ Cell ◽

Adult Mice ◽

Germ Cell Differentiation ◽

Type A Spermatogonia

In order to study hormonal effects on testicular germ cell differentiation, especially on type A spermatogonia, artificially induced cryptorchid testes of adult mice were cultured in a medium containing testosterone, dihydrotestosterone, tri-iodothyronine, dibutyryl 3′: 5′ cyclic adenosine monophosphate, human chorionic gonadotrophin, LH, FSH, insulin and transferrin. These substances, with the exception of FSH, showed no stimulatory effect on the differentiation of type A spermatogonia. However, FSH activated cell division in type A spermatogonia and stimulated them to differentiate, while LH showed neither the promotion of differentiation nor a synergistic effect on FSH-mediated germ cell differentiation.

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Maintenance of adult mouse type A spermatogonia in vitro: influence of serum and growth factors and comparison with prepubertal spermatogonial cell culture

Reproduction ◽

10.1530/rep.0.1240791 ◽

2002 ◽

pp. 791-799 ◽

Cited By ~ 50

Author(s):

LB Creemers ◽

K den Ouden ◽

AM van Pelt ◽

DG de Rooij

Keyword(s):

Germ Cells ◽

Culture Medium ◽

Somatic Cells ◽

Calf Serum ◽

Type A ◽

Dividing Cells ◽

Deficient Mice ◽

Type A Spermatogonia

The culture of spermatogonial cells under well-defined conditions would be an important method for elucidating the mechanisms involved in spermatogenesis and in establishing tissue regeneration in vivo. In this study, a serum-free culture system was established, with type A spermatogonia isolated from adult vitamin A-deficient mice. At days 1, 3 and 7 of culture, the viability and proliferation of cells were monitored. The viability of the cells decreased by day 7 to 10% of the cells present. Proliferation occurred mainly during day 1, when 1% of the germ cells was proliferating. Co-labelling for a germ cell marker (heat shock protein-90alpha, Hsp90alpha), and a marker used to detect dividing cells (bromodeoxyuridine, BrdU), showed that this proliferation was restricted to germ cells. In an attempt to improve these parameters, medium containing fetal calf serum (FCS) was used. Viability was not influenced by serum, but proliferation was markedly enhanced. However, after day 7 of incubation with FCS, co-immunolocalization for Hsp90alpha and BrdU showed a preferential proliferation of somatic cells. Comparison of cultures of adult cells with cultures of prepubertal germ cells, commonly used in studies of spermatogenesis, showed that prepubertal germ cells are twice as viable. In addition, a different proliferation profile was observed, with a peak at day 3. Here, a distinct proliferation of somatic cells was also noted. The results from the present study indicate that the origin of isolated germ cells partly determines culture outcome and that cultures of prepubertal germ cells may not be representative for adult spermatogenesis. Moreover, adding FCS to the culture medium invokes the risk of profound and undesirable effects on cell composition, also underlining the need for identification of germ cells during culture.

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