title: In vitro culture and morphological characterization of prepubertal buffalo (Bubalus bubalis) putative spermatogonial stem cell.
abstract: OBJECTIVE
  Spermatogonial stem cells (SSCs) have the unique ability both to self-renew and to produce progeny that undergo differentiation to spermatozoa. The present study has been carried out to develop a method to purify and enrich the pure populations of spermatogonial stem cell like cells in buffalo.
  
  
  METHODS
  The spermatogonial cells were isolated from testes of 3-7 month old buffalo calves and disaggregated by double enzymatic digestion. Mixed population of isolated cells were then plated on Datura stramonium agglutinin (DSA) lectin coated dishes for attachment of Sertoli cells. The desired cells were obtained from suspension medium after 18 h of incubation and then loaded on discontinuous density gradient using percoll (20-65 %) and different types of spermatogonia cells were obtained at interface of each layer. These cells were cultured in vitro.
  
  
  RESULTS
  Spermatogonial cells isolated have spherical outline and two or three eccentrically placed nucleoli, created a colony after proliferation during first week or immediately after passage. After 7-10 days of culture, the resulted developed colonies of spermatogonial cells expressed the spermatogonial specific genes like Plzf and VASA; and other pluripotency related markers viz. alkaline phosphtase, DBA, CD9, CD90, SSEA-1, OCT-4, NANOG and REX-1.
  
  
  CONCLUSIONS
  Our results show that the isolated putative spermatogonial stem cells exhibit the expression of pluripotency related and spermatogonial specific genes. This study may help to establish a long term culture system for buffalo spermatogonia.
authors.affiliation: Animal Biotechnology Centre, National Dairy Research Institute, Karnal, 132001, India.
authors.forename: S
authors.initials: S
authors.lastname: Kala
authors.forename: R
authors.initials: R
authors.lastname: Kaushik
authors.forename: K P
authors.initials: KP
authors.lastname: Singh
authors.forename: P H
authors.initials: PH
authors.lastname: Kadam
authors.forename: M K
authors.initials: MK
authors.lastname: Singh
authors.forename: R S
authors.initials: RS
authors.lastname: Manik
authors.forename: S K
authors.initials: SK
authors.lastname: Singla
authors.forename: P
authors.initials: P
authors.lastname: Palta
authors.forename: M S
authors.initials: MS
authors.lastname: Chauhan
mesh_terms: D000818:Animals; D002020:Buffaloes; D002417:Cattle; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D019070:Cell Lineage; D002478:Cells, Cultured; D008297:Male; D012708:Sertoli Cells / Q000166:cytology / Q000378:metabolism; D013093:Spermatogonia / Q000166:cytology*; D013234:Stem Cells / Q000166:cytology*; D013737:Testis / Q000166:cytology / Q000378:metabolism