Abstract
We have developed a new method that allows isolation of highly purified type A spermatogonia from prepubertal rats. The procedure is based on the maximal release of spermatogonia from the seminiferous epithelium obtained by the complete enzymatic digestion of the tubular basal lamina, followed by removal of contaminating somatic cells through adhesion to plastic dishes coated with the lectin Datura stramonium agglutinin and fractionation on a discontinuous Percoll gradient. The cell suspension obtained contains up to 85% type A spermatogonia. Besides morphological criteria, the identification of germ cells and somatic cells has been performed by means of immunocytochemical markers, such as c-kit receptor, which is present only in germ cells, and vimentin, which is present only in somatic cells. All type A spermatogonia isolated were c-kit positive, thus suggesting that c-kit receptor is present in both undifferentiated and differentiating type A spermatogonia. Preliminary culture experiments demonstrate that spermatogonia survival in vitro was significantly improved by the addition of 10% fetal calf serum or horse serum to the culture medium; however, optimal culture conditions remain to be established. In vitro studies on isolated spermatogonia may provide a significant contribution toward elucidation of the mechanisms regulating spermatogonial proliferation and differentiation.
| Original language | English |
|---|---|
| Pages (from-to) | 708-717 |
| Number of pages | 10 |
| Journal | Journal of Andrology |
| Volume | 17 |
| Issue number | 6 |
| Publication status | Published - 1996 |
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Keywords
- c-kit
- cell culture
- cell separation
- Type A spermatogonia
ASJC Scopus subject areas
- Endocrinology
Cite this
Isolation of highly purified type A spermatogonia from prepubertal rat testis. / Morena, Anna Rita; Boitani, Carla; Pesce, Maurizio; De Felici, Massimo; Stefanini, Mario.
In: Journal of Andrology, Vol. 17, No. 6, 1996, p. 708-717.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Isolation of highly purified type A spermatogonia from prepubertal rat testis
AU - Morena, Anna Rita
AU - Boitani, Carla
AU - Pesce, Maurizio
AU - De Felici, Massimo
AU - Stefanini, Mario
PY - 1996
Y1 - 1996
N2 - We have developed a new method that allows isolation of highly purified type A spermatogonia from prepubertal rats. The procedure is based on the maximal release of spermatogonia from the seminiferous epithelium obtained by the complete enzymatic digestion of the tubular basal lamina, followed by removal of contaminating somatic cells through adhesion to plastic dishes coated with the lectin Datura stramonium agglutinin and fractionation on a discontinuous Percoll gradient. The cell suspension obtained contains up to 85% type A spermatogonia. Besides morphological criteria, the identification of germ cells and somatic cells has been performed by means of immunocytochemical markers, such as c-kit receptor, which is present only in germ cells, and vimentin, which is present only in somatic cells. All type A spermatogonia isolated were c-kit positive, thus suggesting that c-kit receptor is present in both undifferentiated and differentiating type A spermatogonia. Preliminary culture experiments demonstrate that spermatogonia survival in vitro was significantly improved by the addition of 10% fetal calf serum or horse serum to the culture medium; however, optimal culture conditions remain to be established. In vitro studies on isolated spermatogonia may provide a significant contribution toward elucidation of the mechanisms regulating spermatogonial proliferation and differentiation.
AB - We have developed a new method that allows isolation of highly purified type A spermatogonia from prepubertal rats. The procedure is based on the maximal release of spermatogonia from the seminiferous epithelium obtained by the complete enzymatic digestion of the tubular basal lamina, followed by removal of contaminating somatic cells through adhesion to plastic dishes coated with the lectin Datura stramonium agglutinin and fractionation on a discontinuous Percoll gradient. The cell suspension obtained contains up to 85% type A spermatogonia. Besides morphological criteria, the identification of germ cells and somatic cells has been performed by means of immunocytochemical markers, such as c-kit receptor, which is present only in germ cells, and vimentin, which is present only in somatic cells. All type A spermatogonia isolated were c-kit positive, thus suggesting that c-kit receptor is present in both undifferentiated and differentiating type A spermatogonia. Preliminary culture experiments demonstrate that spermatogonia survival in vitro was significantly improved by the addition of 10% fetal calf serum or horse serum to the culture medium; however, optimal culture conditions remain to be established. In vitro studies on isolated spermatogonia may provide a significant contribution toward elucidation of the mechanisms regulating spermatogonial proliferation and differentiation.
KW - c-kit
KW - cell culture
KW - cell separation
KW - Type A spermatogonia
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UR - http://www.scopus.com/inward/citedby.url?scp=0030456865&partnerID=8YFLogxK
M3 - Article
C2 - 9016402
AN - SCOPUS:0030456865
VL - 17
SP - 708
EP - 717
JO - Journal of Andrology
JF - Journal of Andrology
SN - 0196-3635
IS - 6
ER -