Effects And Molecular Mechanisms Of MAPK Phosphatase-1 On Regulating The Blood-Testis Barrier

Orichitis may lead to male infertility,which affects harmfully male reproductive health and their families.When orichitis occurs,immunogenic germ cells are exposed to the immune antigens and attacked by their own immune systems,which usually results in irreversible testicular dysfunction.It has always been difficult to cure the immunological infertility in orchitis patients.The blood-testis barrier（BTB）plays important roles in isolating the recognition of sperm antigen,establishing and maintaining the testicular immune privilege status.It has been reported that BTB dysfunction caused by inflammation is associated with immunological infertility.occludin,the first identified integral membrane protein in the tight junction（TJ）,can seal the intercellular space of Sertoli cells and maintain the function of BTB.occludin could be regulated by mitogen-activated protein kinases（MAPKs）,and it is closely associated with disturbance of spermatogenesis and sub-fertility caused by inflammation.MAP phosphatase 1（MKP）-1 has been regarded as a crucial negative regulator of innate immune response by deactivating MAPK.However,it remains unknown about the roles of MKP-1 in controlling the seminiferous tubules when orichitis occurs.Additionally,the mechanisms whether MKP-1 affects the structure and function of BTB by regulating the phosphorylation of MAPK signaling pathway need to be clarified.In the present study,we explored the mechanisms of MKP-1 in regulating occludin function and elucidated the modulation function of MKP-1 in BTB dynamic especially under the condition of inflammation,using the in vitro system and the Mkp-l^-/-mice.In this study,we first compared the expression patterns of MKP-1 in different stages of mouse testes.We found that MKP-1 was expressed at a high level at young and adult mouse testes,and its expression was decreased at puberty.MKP-1 showed different distribution characteristics in the testes during mouse development.MKP-1 was preferentially localized in Sertoli cells and spermatids,and it was also presented in blood vessels and some interstitial cells.Notably,the level of MKP-1 expression was significantly up-regulated following LPS treatment.MKP-1 staining was strongly detected in nuclei and peri-nuclear regions of cytoplasm in the Sertoli cells,and it was presented at Sertoli cell tight junctions（TJs）at stages Ⅶ-Ⅷ after LPS treatment.Compared with the control,stage-specific expression and localization of MKP-1 were found in Sertoli cells in LPS-treated mice,indicating that MKP-1 might be involved in the BTB dysfunction during LPS-induced acute testis inflammation.Next,we examined the effect of MKP-1 on occludin in the Sertoli cells in vitro.In normal Sertoli cells,the expression of MKP-1 was very low.In response to LPS stimulus,MKP-1 was significantly induced via the dose-and time-dependent manner.Meanwhile,the higher expression of occludin was detected in the control group,and a significant decrease in occludin expression was observed in Sertoli cells by LPS incubation.We further explored the molecular mechanisms of MKP-1 induction in regulating of occludin.LPS-induced decrease of occludin was modulated by MKP-1 through p38 and IκBα molecules.Furthermore,MKP-1 could control p38 by direct interaction and thus it might modulate occludin via p38-MKP-1 complex.Moreover,the effect of MKP-1 on occludin localization was also observed.occludin was localized primarily to the cell-cell interface by ’string-of-beads’ pattern with weak cytoplasmic and nuclei staining in vehicle-treated Sertoli cells.After LPS treatment,clathrin-mediated endocytosis was increased,which led to the decline of occludin in Sertoli cell membrane.Additionally,knockdown of Mkp-1 by RNAi was found to induce a drastic increase of endocytosis,which resulted in impaired progression of occludin in cell membrane compared to the LPS groups.Taken together,our data indicated that MKP-1 was capable of not only affecting occludin expression but also modifying the distribution and function of occludin.Finally,we probed the role of MKP-1 in regulating mouse testis in vivo.We evaluated the phenotype of male reproductive system of Mkp-l^-/-mice by three aspects as follows:morphological observation,semen examination,and mating ability of mice.Histological analyses revealed that poly-nuclear giant cells and apoptosis were enhanced in the epithelium in Mkp-l^-/-mice,reflecting the dysfunction of spermatogenesis.The fertility of male mice was modestly,but it was significantly reduced in Mkp-l^-/-mice.In addition,sperm from Mkp-1-null mice exhibited the reduction of sperm count and sperm motility as well as an increase of the ratio of sperm deformity.Meanwhile,there was perturbed integrity and increased permeability of the BTB in Mkp-l^-/-mice.We also found that the ascending of anti-sperm antibodies（AsAb）in the blood and an enhancement of TNFa in testes,indicating the change of testes immunological environment in Mkp-1-null mice.Our data revealed the impaired progression and change of occludin localization in Mkp-l^-/-mice.We compared the expression of occludin and related signaling molecules in Mkp-l^-/-mice with wild type mice.MKP-1 could attenuate LPS-induced decrease of occludin by modulating the phosphorylation of p38.Meanwhile,MKP-1 was capable of affecting the localization of occludin via endocytosis.Considered together,our data indicated that MKP-1 could maintain testicular immune microenvironment and act as an important suppressor of innate response involved in the orichitis.Collectively,we have for the first time demonstrated the perturb of BTB,an increase of anti-sperm antibodies,and the reduced fertility in Mkp-l^-/-mice,indicating that MKP-1 could maintain testicular immune privilege status.Furthermore,we have revealed that MKP-1 was capable of affecting the expression and localization of occludin by interaction with p38 and IκBαmolecules.This study thus proved novel insights into mechanisms underlying the protective effect of MKP-1 in orichitis,and it lays scientific basis and offers new targets for the diagnosis and treatment of male infertility caused by orichitis.