Expression And Functions Of FOXC2 In Murine Spermatogonial Stem Cells

Continuous spermatogenesis from puberty to old age in male mammals relies on spermatogonial stem cells(SSCs).SSCs are the only adult stem cells in mammals that transmit genes to subsequent generations.SSCs have the ability to self-renew and generate a large number of differentiated germ cells.In order to maintain normal spermatogenesis,the self-renewal and differentiation of SSCs are precisely regulated by intrinsic gene expression and extrinsic signals.The delicate balance between self-renewal and differentiation is of great importance.Studying SSCs provides an excellent model to understand adult stem cells and may lead to treatment of male infertility and testicular germ cell tumors.In this study,the expression of forkhead box C2(FOXC2)in mouse testis was detected by double immunofluorescent staining.The results showed that the expression of FOXC2 in testis was restricted to a subpopulation of undifferentiated spermatogonia.With the development of testis,the proportion of FOXC2-expressing spermatogonia decreased in the undifferentiated spermatogonia.Whole-mount double immunofluorescent staining of seminiferous tubules revealed that FOXC2 was mainly expressed in Asingle(As)and Apaired(Apr)spermatogonia.FOXC2 was rarely expressed in Aalignede(Aal)spermatogonia and its expression was not detected in Aal-16 spermatogonia.Further double immunofluorescent staining results showed that FOXC2 was expressed in the same cells wih actual stem cell marker GFR?1.The results of 5-Bromo-2'-deoxyuridine(BrdU)labeling experiments showed that most of the FOXC2-expressing spermatogonia were BrdU negative.SSC culture provides an excellent method for studying SSC function in vitro.Immunocytochemistry staining of SSCs cultured in vitro showed that almost all cells expressed undifferentiated spermatogonia marker PLZF while only a portion of them expressed FOXC2,which was consistent with the testis immunofluorescence and whole-mount results.Knock-down of Foxc2 significantly affects SSC clump maintenance in vitro.At 10 days after infection with the Foxc2 shRNA lentivirus,there was a significant reduction in both the clump size and the number of SSCs compared with cells infected with the negative control shRNA lentivirus.Knock-down of Foxc2 also significantly reduced the ability of SSCs to reestablish spermatogenesis.SSCs infected with Foxc2 shRNA lentivirus and SSCs infected with control shRNA lentivirus were transplanted into the testes of the busulfan-treated recipient BDF1 mice,respectively.Two months later,the number of colonies contributed by SSCs infected with Foxc2 shRNA lentivirus in the recipient testes was only 10.42%compared to the control.Analysis of the seminiferous tubules showed that homing and self-renewal of SSCs were normal within the first 2 weeks after transplantation.However,at the 4th week,the ability of SSCs to reestablish spermatogenesis began to decline.At 6 weeks after transplantation,depletion of SSCs was observed.We examined the effects of Foxc2 knock-down on the expression of genes involved in regulating SSC self-renewal,homing and differention.RT-qPCR results showed that there were no significant changes in the genes associated with SSC self-renewal and homing.The levels of Nanos2 and Id4 that inhibit SSC differentiation were down-regulated,while the levels of Nanos3,Sox3,Rarg,Stra8 and c-Kit that promote SSC differentiation were up-regulated.These results indicated that SSCs exhibited the trend of differentiation after knock-down of Foxc2.Extrinsic stimuli treatment showed that FOXC2 was up-regulated by glial cell line-derived neurotrophic factor(GDNF)and down-regulated by retinoic acid(RA).Signaling pathway experiments showed that GDNF up-regulated Foxc2 expression through PI3K-Akt pathway.In conclusion,FOXC2 is a transcription factor that plays a critical role in the survival and fate decision of SSCs.FOXC2 is predominately expressed in As and Apr spermatogonia to safeguard the delicate balance between actual and potential stem cells.GDNF and RA locally produced by Sertoli cells of testis act in opposite ways to regulate FOXC2 expression and SSC property in postnatal germ cells.