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Zheng Y.,Northwest University, China | Zheng Y.,Key Laboratory for Biotechnology of Agriculture Ministry of China | Tian X.,Northwest University, China | Tian X.,Key Laboratory for Biotechnology of Agriculture Ministry of China | And 8 more authors.
Journal of Assisted Reproduction and Genetics | Year: 2013

Purpose: To study the effects of serum and growth factors on propagation of porcine male germline stem cells (MGSCs) in vitro and develop a culture system for these stem cells. Methods: Fresh testicular cells from neonatal piglets were obtained by mechanical dissociation and collagenase-trypsin digestion. After differential plating, non-adhering cells were cultured in media supplemented with different concentrations of serum (0, 1 %, 2 %, 5 %, 10 %). After 10 days of primary culture, the cells were maintained in media supplemented with different concentrations of growth factors (basic fibroblast growth factor and epidermal growth factor at 1, 5, 10 ng/ml). The number of MGSC-derived colonies with different sizes was determined in each treatment to assess the effects of serum concentrations and growth factors. Results: The number of MGSC-derived colonies was significantly higher in the presence of 1 % rather than 10 % fetal bovine serum (FBS). Basic fibroblast growth factor (bFGF) at 1, 5 ng/ml and epidermal growth factor (EGF) at 5, 10 ng/ml significantly promoted colony formation. Immunocytochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and xenotransplantation assays demonstrated the presence of functional stem cells in cultured cell population. Conclusions: In vitro propagation of porcine MGSCs could be maintained in the presence of 1 % FBS and supplementation of growth factors for 1 month. © 2013 Springer Science+Business Media New York. Source


Zheng Y.,Northwest University, China | Zheng Y.,Key Laboratory for Biotechnology of Agriculture Ministry of China | Zhang Y.,Northwest University, China | Zhang Y.,Key Laboratory for Biotechnology of Agriculture Ministry of China | And 8 more authors.
Reproduction | Year: 2014

Spermatogenesis, an elaborate and male-specific process in adult testes by which a number of spermatozoa are produced constantly for male fertility, relies on spermatogonial stem cells (SSCs). As a sub-population of undifferentiated spermatogonia, SSCs are capable of both self-renewal (to maintain sufficient quantities) and differentiation into mature spermatozoa. SSCs are able to convert to pluripotent stem cells during in vitro culture, thus they could function as substitutes for human embryonic stem cells without ethical issues. In addition, this process does not require exogenous transcription factors necessary to produce induced-pluripotent stem cells from somatic cells. Moreover, combining genetic engineering with germ cell transplantation would greatly facilitate the generation of transgenic animals. Since germ cell transplantation into infertile recipient testes was first established in 1994, in vivo and in vitro study and manipulation of SSCs in rodent testes have been progressing at a staggering rate. By contrast, their counterparts in domestic animals, despite the failure to reach a comparable level, still burgeoned and showed striking advances. This review outlines the recent progressions of characterization, isolation, in vitro propagation, and transplantation of spermatogonia/SSCs from domestic animals, thereby shedding light on future exploration of these cells with high value, as well as contributing to the development of reproductive technology for large animals. © 2014 Society for Reproduction and Fertility. Source


Zheng Y.,Northwest University, China | Zheng Y.,Key Laboratory for Biotechnology of Agriculture Ministry of China | He Y.,Northwest University, China | He Y.,Key Laboratory for Biotechnology of Agriculture Ministry of China | And 12 more authors.
Reproduction, Fertility and Development | Year: 2014

Gonocytes are important for the study of spermatogenesis. Identification and isolation of gonocytes has been reported in rodents but not in pigs due to a lack of molecular markers for gonocytes. The objective of this study was to identify THY1 expression in porcine testicular tissue and subsequently utilise THY1 as a marker to isolate and enrich porcine gonocytes from testes of newborn piglets. Immunohistochemical analysis showed that THY1 was expressed in gonocytes. Double-immunofluorescent analysis of THY1 and ZBTB16 indicated that THY1 and ZBTB16 were partially co-localised in gonocytes. Double- immunofluorescent analysis of both THY1 and GATA4 suggested that THY1 + cells were not Sertoli cells. Magnetic-activated cell sorting of THY1+ cells yielded a cell population with an enrichment of UCHL1+ gonocytes 3.4-fold of that of the unsorted testicular cell population. Western blot and quantitative reverse transcription-polymerase chain reaction analyses confirmed that the selected THY1+ fraction had a higher expression of UCHL1 than the unsorted cells. In conclusion, the study demonstrated that THY1 is a surface marker of gonocytes in testes of pre-pubertal boars and could be utilised to identify and isolate porcine gonocytes. The findings will also facilitate culture and manipulation of male germline stem cells. © CSIRO 2014. Source

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