XBP1s Protects Male Germ Stem Cell From LPS Damage By Regulating The Microenvironment

Male germ stem cells(mGSCs),also known as Spermatogonial stem cells(SSCs),are a kind of adult stem cells that can self-renew and differentiate in the basement membrane of the Seminole tubule and eventually differentiate into sperm.MGSCs can be used as an ideal model to study spermatogenesis,meiosis regulation,and cell reprogramming in vitro.X-box Binding protein 1(XBP1),as the downstream of the ER stress pathway IRE1α-XBP1,is very conserved in both single-celled organisms and advanced animals,and it is mainly involved in the regulation of cancer,metabolic diseases and other diseases.In addition,XBP1 is involved in spermatogenesis and the control of sperm quality and quantity.Therefore,exploring the functional mechanism of XBP1 in m GSCs can provide theoretical guidance for improving animal genetics and breeding,animal disease models and species conservation.In this study,we studied the effect of XBP1 on the proliferation of m GSCs in goats and the function of XBP1 through immune regulation.The experimental results are as follows:1.LPS induced male reproductive dysfunction,during which we found that XBP1 is up-regulation.Importantly,in the LPS induced reproductive dysfunction model,germ cells other than SSCs died while SSCs were retained.We found that XBP1 expression in SSCs was significantly higher than that in other germ cells by immunohistochemical staining.Further,immunofluorescence staining was used to find that XBP1 could co-locate with SSCs and macrophages.Based on the above results,we believed that XBP1 protected SSCs from LPS damage through direct action and indirect immune reaction.2.PCDH-CMV-MCS-EF1-XBP1s-GFP-T2A-PURO and CD513B-U6-XBP1-GFPT2A-PURO lentiviral vectors were combined with VSVG and PAX2 to transfect m GSCs,and the cells with stable XBP1s-overexpressing and XBP1-interfering were obtained.The effects of XBP1 s on the proliferation of goat spermatogonial stem cells(g SSCs)were investigated by growth curve mapping,q RT-PCR,western blotting,Ed U labeling,and testicular output tube transplantation.The results showed that overexpression of XBP1 s significantly promoted the proliferation of g SSCs,while interference of XBP1 s significantly inhibited the proliferation of gSSCs.Therefore,it is clear that XBP1 s can promote the proliferation of g SSCs.3.Since XBP1 can co-express with CD11 b in LPS model,we speculated that XBP1 may play an important role in SSCs survival through immune regulation of macrophages.Therefore,we overexpressed or interfered with Xbp1 s in mouse mononuclear macrophages(RAW264.7),screened out stable cell lines,received supernatant,and co-cultured with g SSCs.Then we detected that the RAW264.7 supernatant overexpressing XBP1 s promoted the proliferation of g SSCs.Meanwhile,RAW264.7’s supernatant that interferes with XBP1 s can inhibit the proliferation of g SSCs.ELISA and q RT-PCR showed that the expression of RAW264.7 proinflammatory factor IL-6 was up-regulated in overexpressed XBP1 s cells,while the expression of RAW264.7 proinflammatory factor IL-6 was down-regulated in interfered XBP1 s cells.At the same time,transcriptome sequencing showed that IL-6 and IL-6R were up-regulated in XBP1s-overexpressing m GSCs,and down-regulated in XBP1 sinterfering m GSCs.These results indicated that XBP1 s played an important role in maintaining SSCs proliferation by participating in immune regulation.In conclusion,we determined the effect of XBP1 s on the proliferation of m GSCs in goats and revealed the function of XBP1 s in regulating the proliferation of m GSCs in goats by participating in the immune response.The goat m GSCs were transfected with gene modification vectors,and the target genes of XBP1 s were verified and screened by comprehensive analysis in vitro and in vivo.This study provides a new scientific basis for studying the mechanism of self-renewal and spermatogenesis of goat m GSCs.It also provides a new target for the breeding of high quality livestock and the prevention and control of reproductive disorders.