Successful transplantation of spermatogonial stem cells into the seminiferous tubules of busulfan-treated mice

Hossein Azizi,Amirreza Niazi Tabar,Thomas Skutella

doi:10.1186/s12978-021-01242-4

Abstract

BackgroundSpermatogonial 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.MethodsIn 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.ResultsICC 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.ConclusionsIn 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.

Highlights

Spermatogonial stem cells (SSCs) in the testis are crucial for transferring genetic information to the generation
While Fluidigm Real-Time polymerase chain reaction (RT-PCR) analysis indicated overexpression of the TAF4B germ cell gene, the expression of Deleted in azoospermia-like (DAZL), VASA, and Zbtb16 were down-regulated during the differentiation of Spermatogonial Stem cell (SSC) (P < 0.05)
Successful SSC transplantation into infertile men is an advanced therapeutic application in reproductive biology 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

Summary

Introduction

Spermatogonial stem cells (SSCs) in the testis are crucial for transferring genetic information to the generation. Spermatogenesis is an organized complex process within the seminiferous tubules, which is started by undifferentiated spermatogonia located on the basement membrane of the seminiferous tubule [1]. During this process, regulated proliferation and differentiation of undifferentiated spermatogonia determine the long-term production of mature spermatozoa for the transmission of genetic information to the subsequent generation [2]. In vivo conditions for normal spermatogenesis process require regulatory mechanics mainly controlled by endocrine signals from testicular somatic cells such as peritubular myoid cells (PTCs), Leydig cells, and Sertoli cells (SCs) [4]. PModS (peritubular modifies Sertoli) is a modulator molecule for androgen-binding protein, inhibin, and transferrin by SCs [5,6,7]

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