Bone Marrow Stem Cells To Explore The Possibility Of Horizontal Differentiation In The Testis

Testis, as a reproductive organ in male mammals, composes of two main parts: seminiferous tubules for spermatogenesis and interstitial tissue for secreting androgen which keep the male characters. Testis has a relatively immune microenvironment, named as niche, there mainly are three types of cells--Leydig cells, sertoli cells and germ cells. In details, Leydig cells lies among the seminiferous tubules, which can secret androgen to maintain the hormone environment; Sertoli cells build the blood-testis barrier with the tight junction to provide the special environment without immunity, which is very important for spermatogenesis. Recent studies have demonstrated that Sertoli cells can provide a common survival and differentiated environment for spermatogonial stem cells (SSC), furthermore, sertoli cells not only can recognize SSC of same species in testis, but also can recognize SSC of other species, which is permitted to go through the blood-testis barrier to arrive at the basement membrane, then proliferate and differentiate. But, whether the niche provided by sertoli cells or the character of germ cells themselves play a key role in the progress of spermatogenesis was yet known.On the other hand, recent years the studies concerning stem cells mostly concentrated on embryonic stem cells (ES) and adult stem cells(AS). Although ES with a totipotent potential showed very perspective future, its applications still remains controversial for ethical reasons; By contrast, BMSC can be easily obtained, have not ethical question. Moreove, recent studies demonstrated that bone marrow stem cells (BMSC) have plasticity. BMSC are capable oftransdifferentiating into several tissue-forming cells, such as bone, cartilage, fat, muscle, liver, kidney, heart, and even brain cells. Thus it is possible that BMSC may transdifferentiate into spermatogonia in the environment of mice testis. If yes, it means that the niche provided by sertoli cells play more important role in spermatogenesis than spermatogonia themselves; otherwise the spermatogonia seems more important in the process.To address above question, our experiments start with extracting BMSC of mice, the using techniques of injection via rete testis and tail vein, respectively, donor BMSC from HSL+/- mice were transplanted into recipient testis of wild type mice that endogenous SSC had been eliminated by busulfan treatment. Meanwhile, the SSC transplantation as positive control. Finally, homing, proferating and differentiating of BMSC in the seminiferous tubes of recipient mice were checked as monitoration of the success in the transplantation.As a result, 80 days after the transplant operation, spermatogenesis of the donor stem cells could be observed clearly and the male have reproduced progeny after mating with normal female. This result demonstrated that the extraneous SSC could adversely go through the blood-testis barrier to arrive at the basement membrane, then proliferate and differentiate into male germ cells. The success of control transplantation with spermatogonia demonstrated that our technique for the cell transplantation was reliable in the research.The cell transplantation experiments included two methods: one is that transplant donor BMSC into recipient testis via rete testis, the other is that transplant donor BMSC into recipient testis via tail vein. The results of usingtechnique of injection via rete testis showed that extraneous SSC could adversely go through the blood-testis barrier to arrive at the basement membrane, then proliferate and differentiate, that is to say, can transdifferentiate into spermatogonia. furthermore, the fluorescent result from in situ PCR showed that all the germ cells were donor derived. The results of using injection technique via tail vein showed that within 6M after transplanting, the epithelium of recipient seminiferous tubules is still remained very thin, means extraneous BMSC did not arrive at the basement membrane, therefore not proliferated and differentiated yet.It demonstrated that sertoli cells not only could recognize endoneous SSC in testis, but also recongnize extraneous BMSC via rete testis transplantion, moreover, sertoli cells permit these cells going through the blood-testis barrier to arrive at the basement menbrane, then proliferate and differentiate. But extraneous BMSC via blood transplantion could not arrive at the basement membrane.Our experiments using techniques of injection via rete testis and tail vein probe how BMSC differentiate in the testicular niche and its transdifferentiation potential. Unlike the transplantation via rete testis, we found that extraneous BMSC via blood transplantion could not approach to the basement menbrane and reproliferate, indicating that there was something blocking BMSC riching to the seminisferous tubules. In conclusion, our results clearly revealed that BMSC kept potential to transdefferentiate into germ cells induced by testicular niche and the niche played a more important role than stem cells themselves inthe transplantation. The present study is very helpful for addressing the basic questions about adult stem cell plasticity and impeling clinic practice concerned with male fertility.