Purification and Characterization of Gonocytes Isolated from DsRed Transgenic Pigs
Date Issued
2012
Date
2012
Author(s)
Chang, Chia-Chun
Abstract
Gonocytes have been known to be derived from primordial germ cells and to give rise to undifferentiated spermatogonia, or spermatogonial stem cells (SSCs), which establish and maintain spermatogenesis within the postnatal testes. Mouse SSCs were demonstrated as that they can long-term proliferate in vitro without losing the ability of differentiating into sperm after transplantation. Besides, a few SSCs can automatically reprogram to a pluripotent status which is similar to those of embryonic stem cells (ESCs). Due to these special features, SSC is a valuable cell model for epigenetic and reproductive studies, and will be a novel platform for transgenic animal production as well.
Establishment of significantly enriched population of SSCs is a critical step that will facilitate the researches of their molecular and biological properties. Despite of the lack of specific surface marker, the weak adhesion ability and the large size of gonocytes suggested the possibility to purify gonocytes by differential plating and cell sorting. Most cells of our DsRed transgenic pigs, in which the transgene of DsRed fluorescent protein was driven by the ubiquitous CAG promoter, stably represent fluorescence. However, to our surprise, in all the male germ cells, the expression levels of DsRed were found to be much lower than those found in most of somatic cells. Hence, gonocytes, which were isolated from pigs between 4 to 6 weeks of age, were purified by differential plating following by cell sorting sequentially. The high forward scatter (FSC) and weak fluorescence cell population was collected, and the germ cells were labeled with an antibody targets a germ cell specific marker, VASA. The purity of gonocytes was significantly elevated from 1.0 ± 0.3% to 90.4 ± 1.6% (p<0.01), which is also significantly higher than the other ones (80.9 ± 2.6%) selected by FSC only (p<0.01).
PLZF is a transcriptional regulator essential for self-renewal and maintenance of SSCs. Here in this study, immunostain was performed on testes sections from pigs at each 2, 4, 6 and 8 weeks of age, respectively. The ratio of PLZF positive germ cells appeared to be increased with ages and the relation between PLZF expression and germ cell homing was observed, suggesting PLZF is a marker for porcine SSCs. For the purpose to analyze the stem cell potential of purified gonocytes, the expression of PLZF was detected by immunocytochemistry. There were 67.2 ± 7.3% of VASA positive cells, found in those cells purified after cell sorting, and these cells were also shown positive to PLZF, indicating the stemness of them.
Overall, a two-step purification method for preparing gonocyte-enriched testicular cells from single testis has been successfully established after this present study. The highly pure fluorescent gonocytes might include a specific germ cell population, which can derive to SSCs. As a consequence, this gonocyte-enriched cell population could be an excellent model for further studies related to both characterization of primitive porcine germ cells and the germ cell fate determination.
Subjects
Gonocyte
Spermatogonial stem cell
transgenic pig
Type
thesis
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