TY - JOUR
T1 - Identification and IVC of spermatogonial stem cells in prepubertal buffaloes
AU - Yu, Xue
AU - Riaz, Hasan
AU - Dong, Ping
AU - Chong, Zhenlu
AU - Luo, Xuan
AU - Liang, Aixin
AU - Yang, Liguo
N1 - Funding Information:
This work was supported by International Cooperation key Project of China (No. OS2012ZR0216 ) and the Earmarked Fund for Modern Agro-industry Technology Research System (No. CARS-37-04B ).
PY - 2014/6
Y1 - 2014/6
N2 - Development of suitable selective marker for buffalo spermatogonial stem cells (SSCs), optimization of long-term IVC conditions, and their pluripotent retention capacity in buffaloes can be of prime importance in selective genetic modifications of this species. In the present study, we identified CDH1 as a specific marker for buffalo SSCs and revealed that it existed in two protein isoforms (large [135 kDa] and small [90 kDa] subunits) in the buffalo testis; furthermore, immunohistochemical analysis revealed that CDH1 expression was present in spermatogonia but absent in the somatic cells of 4-month-old buffalo testis. After 7 days of enrichment, expression of CDH1 was also detectable in IVC colonies (~53% enrichment efficiency by Fluorescence-activated cell sorting (FACS)). For long-term culture of SSCs, proliferation studies with different factors showed that combination of 20 ng/mL GDNF, 10 ng/mL FGF2, and 1000 U/mL LIF could significantly promote number of colonies (~two folds) and proliferation of buffalo SSCs (~three folds) compared with those of control or single-treatment groups; furthermore, addition of these combination growth factors significantly upregulated the messenger RNA level of spermatogonial-specific and pluripotency-related markers (BCL6B, GFRA1, and POU5F1), whereas downregulated receptor tyrosine kinase (KIT). For confirmation of their stem cell potential, Dolichos biflorus agglutinin-stained cells were identified in the basal membrane of seminiferous tubules of xenotransplanted mice testis. These findings indicate the identification of a new buffalo SSCs marker; furthermore, it may help in establishing long-term culture that would assist in genetic modification of these buffaloes.
AB - Development of suitable selective marker for buffalo spermatogonial stem cells (SSCs), optimization of long-term IVC conditions, and their pluripotent retention capacity in buffaloes can be of prime importance in selective genetic modifications of this species. In the present study, we identified CDH1 as a specific marker for buffalo SSCs and revealed that it existed in two protein isoforms (large [135 kDa] and small [90 kDa] subunits) in the buffalo testis; furthermore, immunohistochemical analysis revealed that CDH1 expression was present in spermatogonia but absent in the somatic cells of 4-month-old buffalo testis. After 7 days of enrichment, expression of CDH1 was also detectable in IVC colonies (~53% enrichment efficiency by Fluorescence-activated cell sorting (FACS)). For long-term culture of SSCs, proliferation studies with different factors showed that combination of 20 ng/mL GDNF, 10 ng/mL FGF2, and 1000 U/mL LIF could significantly promote number of colonies (~two folds) and proliferation of buffalo SSCs (~three folds) compared with those of control or single-treatment groups; furthermore, addition of these combination growth factors significantly upregulated the messenger RNA level of spermatogonial-specific and pluripotency-related markers (BCL6B, GFRA1, and POU5F1), whereas downregulated receptor tyrosine kinase (KIT). For confirmation of their stem cell potential, Dolichos biflorus agglutinin-stained cells were identified in the basal membrane of seminiferous tubules of xenotransplanted mice testis. These findings indicate the identification of a new buffalo SSCs marker; furthermore, it may help in establishing long-term culture that would assist in genetic modification of these buffaloes.
KW - Buffalo
KW - CDH1
KW - Proliferation
KW - Spermatogonial stem cell
UR - http://www.scopus.com/inward/record.url?scp=84899929783&partnerID=8YFLogxK
U2 - 10.1016/j.theriogenology.2014.03.002
DO - 10.1016/j.theriogenology.2014.03.002
M3 - Article
C2 - 24703765
AN - SCOPUS:84899929783
SN - 0093-691X
VL - 81
SP - 1312
EP - 1322
JO - Theriogenology
JF - Theriogenology
IS - 9
ER -