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Abstract
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Background: Spermatogonial stem cells (SSCs) in the testis are crucial for transferring genetic information to the
next generation. Successful transplantation of SSCs to infertile men is an advanced therapeutic application in reproductive
biology research.
Methods: In this experimental research, both in vitro and in vivo characterization of undifferentiated and differentiated
SSCs were performed by morphology—immunocytochemistry (ICC), immunohistochemistry (IMH), Fluidigm
Real-Time polymerase chain reaction (RT-PCR) and flow cytometry analysis. The isolated SSCs were finally microinjected
into the rete testis of busulfan-treated mice.
The compact undifferentiated and more loosely connected round differentiated SSCs were isolated during testicular
cell expansion from their specific feeder layer.
Results: ICC analysis indicated high and low expression levels of Zbtb16 in undifferentiated and differentiated germ
cells. Also, IMH analysis showed different expression levels of Zbtb16 in the two different germ stem cell populations
of the testicular tissue. While Fluidigm RT-PCR analysis indicated overexpression of the TAF4B germ cell gene, the
expression of DAZL, VASA, and Zbtb16 were down-regulated during the differentiation of SSCs (P < 0.05). Also, flow
cytometry analysis confirmed the significant downregulation of Itgb1 and Itga4 during differentiation. By transplantation
of SSCs into busulfan-treated NOD/SCID mice, GFP-labeled sperm cells developed.
Conclusions: In the current study, we performed a transplantation technique that could be useful for the future
microinjection of SSCs during infertility treatment and for studying in vivo differentiation of SSCs into sperm.
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