| Spermatogonial stem cells(SSCs),as primitive germ cells in mammalian malespermatogenesis system, are of utmost importance in study on spermatogenesis andproduction of transgenic animals. In rodents, the identification and isolation of SSCs areprogressive. However, due to a lack of specific markers, there is little development incorresponding research on livestocks. In present study, we assumed that THY1could be amarker of SSCs in pigs.The analysis of Immunohistochemistry and Double-immunofluorescence weredevelopped to comfirm the hypothesis.THY1antibody was used to isolate SSCs throughMagnetic Activated Cell Sorting (MACS).UCHL1was used to identify SSCs after sorted.Differential plating and seminiferous tubule-culture were conducted to isolate and purifySSCs. The results of test followed:（1）The immunohistochemistry of porcine testis sections collected from5d,3mo pigsdemonstrated that THY1was specifically expressed in SSCs. The THY1+cells showed sparsedistribution and large size in nucleus. However, the particular stain was restricted at one sideof the THY1+cell membrane. To investigate the expression of THY1in porcine testis, weemployed the Double-Immunofluorescence between THY1and PLZF, which showed that asubpopulation of PLZF+cells could also express THY1. Double-Immunofluorescencebetween THY1and the specific marker of sertoli cells, GATA4further characterized thecell-type specificity of THY1expression. The results showed that the expression of THY1was different from that of GATA4through the appearance of stained cells, whichdemonstrated that THY1+cells were not sertoli cells.So THY1can be a candicate marker ofSSCs in pigs.(2) THY1antibody was used to purify SSCs collected from10d pigs. UCHL1, a markerfor SSCs in boars, was used to determine the isolation efficiency.The results revealed thatafter isolation through MACS, the ratio of purpose cells was higher in sorted fraction than thatin presorted fraction in appearance. The analysis of immunocytochemistry showed that theisolation with THY1antibody could enrich54.97±2.66﹪of UCHL1-positive cells, comparedwith merely15.29±3.03﹪of those in unsorted fraction, Collectively, the isolation withTHY1antibody by MACS could enrich SSCs of pigs.The culture of positive cells for shortterm revealed that there were colony formed.Western Blot and qRT-PCR were carried out to determine the expression of UCHL1in THY1+cells and in unsorted cells, respectively. Theanalysis of Western Blot revealed that the expression of UCHL1in THY1+cells wassignificantly greater than that in unsorted cells. Similarly, the results of qRT-PCR showed thatthe expression of UCHL1in THY1+fraction was approximately40-fold greater than that inunsorted fraction.(3) The results showed that SSCs with high vitality could be obtained via the two methods.SSCs population enriched to36.9%and40.7%respectively, via differential plating orseminiferous tubule-culture. Both it techniques can be used for isolation and purification ofporcine SSCs. |
