α-6 integrin expression in bovine spermatogonial cells purified by discontinuous Percoll density gradient.
The study of spermatogonial stem cells (SSCs) provides a model to better understand adult stem cell biology. Besides the biomedical potential to perform studies of infertility in many species, SSCs hold a promising application at animal transgenesis. Because stem cells are thought to be associated with basement membranes, expression of α-6 integrin has been investigated as a marker of type A spermatogonial cells, which are considered SSCs because of their undifferentiated status and self-renewal ability. In this manner, the aim of this study was to isolate type A SSCs from adult bulls by a two-step enzymatic procedure followed by a discontinuous Percoll density gradient purification and verify the expression of α-6 integrin by flow cytometry and real-time RT-PCR before and after Percoll purification. Spermatogonial cells were successfully obtained using the two-step enzymatic digestion. An average of 1 × 10(5) viable cells per gram of testis was isolated. However, the discontinuous Percoll did not purify isolated cells regarding α-6 integrin expression. Flow cytometry analysis demonstrated no differences in the α-6 integrin expression between cell samples before and after Percoll purification (p = 0.5636). The same was observed in the real-time PCR analysis (p > 0.05). In addition to α-6 integrin, the expression of GFRa-1 and PGP9.5, known bovine SSCs markers, was detected in all samples studied. Considering that Percoll can reduce cell viability, it is possible to conclude that Percoll density gradient is not suitable to purify bovine SSC, according to α-6 integrin expression.
Laboratory of In Vitro Fertilization, Cloning and Animal Transgenesis, Department of Animal Reproduction, School of Veterinary Medicine and Animal Sciences, University of Sao Paulo, Sao Paulo, SP, Brazil. flaviarob@usp.br
F R O
FR
de Barros
R A
RA
Worst
G C P
GC
Saurin
C M
CM
Mendes
M E O A
ME
Assumpção
J A
JA
Visintin
D000818:Animals; D002417:Cattle / Q000502:physiology*; D002469:Cell Separation / Q000379:methods / Q000662:veterinary; D002499:Centrifugation, Density Gradient / Q000379:methods / Q000662:veterinary*; D005786:Gene Expression Regulation / Q000502:physiology*; D016023:Integrins / Q000235:genetics / Q000378:metabolism*; D008297:Male; D011205:Povidone / Q000737:chemistry*; D012822:Silicon Dioxide / Q000737:chemistry*; D013093:Spermatogonia / Q000378:metabolism*
D016023:Integrins; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone