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

The objective of this study was to compare the expression of Col1a1, Col1a2, and procollagen I in the seminiferous tubules of immature and adult mice and to characterize the cellular expression pattern of procollagen I in germ cells during spermatogenesis in order to provide necessary groundwork for further functional studies in the process of spermatogenesis. Microarray analysis demonstrated that Col1a1 and Col1a2 were abundantly expressed in the seminiferous tubules of 6-day-old mice compared with 60-day-old mice, and the expression levels of Col1a1 and Col1a2 mRNA were validated using a semi-quantitative RT-PCR assay. Western blot analysis further confirmed that procollagen I was expressed at a higher level in the seminiferous tubules of 6-day-old mice compared with 60-day-old mice. Immunohistochemical analysis revealed that type A spermatogonia were positive for procollagen I in the testis of 6-day-old mice, whereas Sertoli cells were negative for this protein. Thein vivoprocollagen I staining in type A spermatogonia was corroborated in spermatogonia exhibiting a high potential for proliferation and the ability to form germ cell colonies inin vitroculture. Moreover, procollagen I was also detected in type A spermatogonia, intermediate spermatogonia, type B spermatogonia, and preleptotene spermatocytes in the adult mouse testes, but positive staining disappeared in more differentiated germ cell lineages detaching from the basement membrane, including leptotene spermatocytes, pachytene spermatocytes, round spermatids and elongated spermatids. These data suggest that Col1a1, Col1a2 and procollagen I are associated with type A spermatogonia and play a potential role in mediating the detachment and migration of germ cells during spermatogenesis.

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

Animals ◽

10.3390/ani10050851 ◽

2020 ◽

Vol 10 (5) ◽

pp. 851 ◽

Cited By ~ 2

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.

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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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Vitamin A and Follicle-Stimulating Hormone Synergistically Induce Differentiation of Type A Spermatogonia in Adult Mouse Cryptorchid Testesin Vitro

Endocrinology ◽

10.1210/endo-114-3-801 ◽

1984 ◽

Vol 114 (3) ◽

pp. 801-805 ◽

Cited By ~ 23

Author(s):

TATSUJI HANEJI ◽

MAMIKO MAEKAWA ◽

YOSHITAKE NISHIMUNE

Keyword(s):

Vitamin A ◽

Adult Mouse ◽

Follicle Stimulating Hormone ◽

Type A ◽

Type A Spermatogonia ◽

Stimulating Hormone

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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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IN VITRO CULTURE OF WHEAT TISSUES. I. CALLUS FORMATION, ORGAN REDIFFERENTIATION AND SINGLE CELL CULTURE

Canadian Journal of Genetics and Cytology ◽

10.1139/g69-037 ◽

1969 ◽

Vol 11 (2) ◽

pp. 294-304 ◽

Cited By ~ 73

Author(s):

T. Shimada ◽

T. Sasakuma ◽

K. Tsunewaki

Keyword(s):

Cell Culture ◽

Growth Factors ◽

Single Cell ◽

Common Wheat ◽

Callus Formation ◽

Casein Hydrolysate ◽

Callus Growth ◽

Callus Cell ◽

Basal Media

Callus induction, organ formation from callus and single callus cell culture have been tried in wheat. Though kinetin showed no effects, supplements of 2,4-D (1~10mg/1) or IAA (50mg/1) to the basal media induced calluses from seedling roots of einkorn, emmer and common wheats, and from stem pieces of common wheat. The best callus growth was obtained when casein hydrolysate (1g/1) or coconut milk (1%) was added to the basal media. Callus growth was also vigorous when 2,4-D (0.5~2.0mg/1) was added. Root formation from callus took place in all kinds of tested media, except those containing no growth factors or supplemented with 2,4-D at high concentrations (1~5mg/1). Shoot formation occurred in six cases and no growth factors were found to be specifically effective on shoot differentiation. Two plants were restored and reached maturity. Calluses of common wheat consisted of eudiploid and aneuploid cells at almost the same frequencies. The great majority of aneuploid cells had 42 ± 3 chromosomes. The restored plants showed normal chromosome constitution. Single callus-cell suspensions were obtained by the liquid culture of seeds in a shaker. A filtrate of the single cell suspension was plated on a solid agar medium, and some colonies were formed. However, plating efficiency was very low and colony growth was slow.

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