Isolation of subtype spermatogonia in juvenile rats

Abstract

The present study was aimed at finding an effective method to isolate and purify the subtype of type A spermatogonial stem cells (SSCs) in juvenile rats. Testes from 9-days-old rats were used to isolate germ cells by using two-step enzymatic digestion. The expression of c-kit in the testes of the rats was immunohistochemically detected. After isolation, cell suspension was enriched further by discontinuous density gradient centrifugation. Then type A1–A4 spermatogonia was isolated from the purified spermatogonia with c-kit as the marker by using fluorescence-activated cell sorting (FACS). Electron microscopy was used to observe their ultrastructure. Finally, highly purified and viable subtype of SSCs was obtained. Cells separation with discontinuous density gradient centrifugation significantly increased the concentration of c-kit positive cells [(18.65±1.69)% after the centrifugation versus (3.16±0.84)% before the centrifugation, P<0.01]. Furthermore, the recovery and viability were also high [(65.9±1.24)% and (85.6±1.14)%]. It is concluded that FACS with c-kit as the marker in combination with discontinuous density gradient centrifugation can well enrich type A1–A4 spermatogonia from the testes of 9-days-old rats.

Keywords

spermatogonia / subtype / FACS

References

[1] HuckinsC.. The spermatogonial stem cell population in adult rats. II. A radioautographic analysis of their cell cycle properties. Cell Tissue Kinet, 1971, 4(4): 313-334

[2] ClermontY., MaugerA.. Existence of a spermatogonial chalone in the rat testis. Cell Tissue Kinet, 1974, 7(2): 165-172

[3] DettinL., RavindranathN., HofmannM. C., et al.. Morphological characterization of the spermatogonial subtypes in the neonatal mouse testis. Biol Reprod, 2003, 69(5): 1565-1571

[4] HuckinsC., OakbergE. F.. Morphological and quantitative analysis of spermatogonia in mouse testes using whole mounted seminiferous tubules, I. The normal testes. Anat Rec, 1978, 192(4): 519-528

[5] Lacham-KaplanO.. In vivo and in vitro differentiation of male germ cells in the mouse. Reproduction, 2004, 128(2): 147-152

[6] MalkovM., FisherY., DonJ.. Developmental schedule of the postnatal rat testis determined by flow cytometry. Biol Reprod, 1998, 59(1): 84-92

[7] van PeltA. M., MorenaA. R., van Dissel-EmilianiF. M., et al.. Isolation of the synchronized A spermatogonia from adult vitamin A-deficient rat testes. Biol Reprod, 1996, 55(2): 439-444

[8] RossiP., DolciS., SetteC., et al.. Molecular mechanisms utilized by alternative c-kit gene products in the control of spermatogonial proliferation and sperm-mediated egg activation. Andrologia, 2003, 35(1): 71-78

[9] DolciS., WilliamsD. E., ErnstM. K., et al.. Requirement for mast cell growth factor for primordial germ cell survival in culture. Nature, 1991, 352(6338): 809-811

[10] GodinI., DeedR., CookeJ., et al.. Effects of the steel gene product on mouse primordial germ cells in culture. Nature, 1991, 352(6338): 807-809

[11] BroudyV. C.. Stem cell factor and hematopoiesis. Blood, 1997, 90(4): 1345-1364

[12] Besmer P, Manova K, Duttlinger R et al. The kit-ligand (steel factor) and its receptor c-kit/W: pleiotropic roles in gametogenesis and melanogenesis. Dev Suppl, 1993: 125–137

[13] PackerA. I., BesmerP., BachvarovaR. F.. Kit ligand mediates survival of type A spermatogonia and dividing spermatocytes in postnatal mouse testes. Mol Reprod Dev, 1995, 42(3): 303-310

[14] KisselH., TimokhinaI., HardyM. P., et al.. Point mutation in kit receptor tyrosine kinase reveals essential roles for kit signaling in spermatogenesis and oogenesis without affecting other kit responses. EMBO J, 2000, 19(6): 1312-1326