| Objective: In the testis of mammals,the blood-testis barrier near the basement membrane(BM)of seminiferous epithelium is crucial for spermatogenesis.BTB is formed by adjacent Sertoli cells,dividing seminiferous epithelium into basal compartment near the basement membrane and adluminal compartment,providing an immune barrier for spermatogenesis.BM is constituted by type ? collagen,laminins,heparin sulfate proteoglycan and nidogen.Type ? collagen is an important component of basement membrane.In the testis of rodents,type ? collagen is mainly a three-helix structure composed of three ? chains.Collagens in basement membrane can be hydrolyzed by matrix metalloproteinase(MMP)and release a variety of bioactive substances(such as tumor suppressant,endostatin,angiogenesis inhibitor,etc.),which are involved in the regulation of cell proliferation,adhesion and apoptosis through integrin receptors on the surface of target cell membrane.C-terminal of type ? collagen protein ?3 chain in rat testis can be hydrolyzed by MMP-9 into protein fragments of28 k Da,called NC1 domain.Studies have shown that NC1 recombinant protein treatment in primary Sertoli cells of rats can lead to the abnormal Sertoli cell-cell interface tight junction and the disrutped distribution of proteins related to the basal ectoplasmic specialization,thus affecting the function of BTB.The results of subsequent studies showed that the injection of NC1 overexpression plasmid into rat testis resulted in the destruction of the BTB barrier function and the exuviation of germ cells from seminiferous epithelium,which involved abnormal cytoskeletal arrangement and the abnormal distribution of tight junction proteins.However,the mechanism of NC1 affects the BTB function and spermatogenesis is unclear,nor is it clear in rats with such disorders.This study was to explore how NC1 influenced spermatogenesis and the BTB,and in rat models to explore whether NC1 associated with abnormal spermatogenesis.Methods: 1.Primary Sertoli cells were transfected with NC1 overexpressed plasmid in vitro,and the expression and activation of Rho A and Cdc42 were detected by western blot and immunoprecipitation.2.NC1 overexpressed plasmid and/or dominant negative mutant of Cdc42,namely Cdc42 T17 N were used to transfect primary Sertoli cells in vitro.We conduct the following experiments as follows.(1)Western blot and immunoprecipitation were used to detect the expression and activation of Cdc42.(2)Sertoli cell permeability was determined at different time points using Trans-epithelial resistance(TER).(3)NC1 overexpressed plasmid and/or Cdc42 T17 N were used to transfect rat primary Sertoli cells in vitro,and western blotting and immunoprecipitation were used to detect extracellular specific structures and tight junction proteins,western blotting was used to detect related signaling proteins as well.(4)NC1 overexpressed plasmid and/or Cdc42 T17 N were used to transfection rat primary Sertoli cells in vitro,and the expression and distribution of actin regulatory protein were detected by immunoprecipitation and immunofluorescence,while actin bundling assay and actin polymerization assay were used to detect the bundling and polymerization ability of F-actin.(5)NC1 overexpressed plasmid and/or Cdc42 T17 N were used to transfect rat primary Sertoli cells in vitro.Tubulin composition and regulatory protein expression and distribution were detected by immunoprecipitation and immunofluorescence.(6)MT spin-down assay and MT polymerization assay were used to detect the polymerization ability of MT.3.Primary Sertoli cells were transfected with NC1 overexpressed plasmid in vitro,and the Cdc42-GTPase was detected by immunoprecipitation and western blot after adding Akt1/2 specific activator SC79,and the distribution and expression of junction proteins,signaling proteins,microfilamentes and microtubules-related proteins were detected by immunofluorescence and immunoprecipitation.4.The chemotherapeutic drug busulfan was used to construct the rat spermatogenic disorder model.(1)The morphological changes of spermatogenic tubules in each group were detected by HE staining.(2)Biotin was used to detect whether the rat spermatogenic dysfunction model induced by busulfan was accompanied by impaired BTB barrier function.(3)Western blot was used to detect the expression of MMP-9,NC1,Akt,p-Akt and Cdc42-GTPase in the rat spermatogenic disorder model.(4)The distribution of NC1,?1-integrin,Laminin-?3,ZO-1,occludin,MT and F-actin in rat spermatogenic disorder model was detected by immunofluorescence.5.The MMP-9 inhibitor JNJ0966 injecting into rat's testis can alleviate spermatogenic disorder and BTB injury induced by busulfan through reducing the generation of NC1.Meanwhile,the intratesticular transfection group was set up with NC1 overexpressed plasmid.(1)Western blotting was used to detect the changes of MMP-9 activity and NC1 expression.(2)Changes in spermatogenesis were observed by HE staining.(3)Biotin method to detect BTB permeability.(4)Immunofluorescence observation of junction protein distribution and F-actin and microtubule skeleton arrangement in frozen section of testicular tissue.(5)F-actin in vivo assay and microtubule ratio-spin down assay were used to detect actin and microtubule polymerization in testicular tissues of each group.Results: 1.Overexpression NC1 affected primary Sertoli cell barrier function in rats by up-regulating Cdc42-GTPase,but Cdc42 T17 N could inhibit the barrier function disruption caused by NC1 overexpression.2.NC1 overexpression affected the junction protein(occludin?CAR?ZO-1?N-cadherin??-catenin)distribution in primary Sertoli cells of rats through activation of Cdc42,but did not affect its expression.Cdc42 T17 N inhibited the distribution of junction protein caused by NC1 overexpression,while NC1 overexpression activated rp S6 and inhibited Akt,but Cdc42 T17 N had no significant effect on rp S6 and Akt.3.Cdc42 T17 N can inhibit Eps8 expression and F-actin distribution changes caused by NC1 overexpression,and can also inhibit the decreased bundling and polymerization ability of F-actin caused by NC1 overexpression.4.Cdc42T17 N can inhibit the abnormal distribution of microtubule regularoty and constituent proteins caused by NC1 overexpression,as well as the reduction of tubulin polymerization ability.5.Cdc42 T17 N by overexpression of NC1 and its effect on linker protein distribution can be inhibited by SC79.6.The effect of overexpression of NC1 on the distribution of F-actin and MT skeletal tissues of Sertoli cells could be inhibited by SC79.7.The spermatogenesis was obviously abnormal in the model of spermatogenesis induced by busulfan.8.BTB barrier function was impaired in rat spermatogenic dysfunction model induced by busulfan.9.The expression levels of NC1 and MMP-9were significantly increased in the rat spermatogenic dysfunction model induced by low dose busulfan.10.Abnormal distribution of NC1,?1-integrin,laminin-?3,ZO-1,occludin,MT,and F-actin in rat spermatogenic disorder model induced by small dose of busulfan.11.By reducing NC1,MMP-9 inhibitor can alleviate spermatogenic disorder and BTB injury induced by busulfan.12.JNJ0966 can relieve the abnormal distribution of spermatogenic tubules junction proteins(laminin-?3,ZO-1),F-actin and MT in rat model of spermatogenic disturbance,and this effect can be antagonized by NC1 overexpression plasmid transfected in testis.13.JNJ0966 can alleviate the decreased F-actin and microtubule aggregation in testicular tissues in the model of busulfan spermatogenic disorder,and this effect can be antagonized by intratesticular transfection of NC1 overexpression plasmid.Conclusion: 1.NC1 can activate the Cdc42 in Sertoli cells of rats by down-regulating the activity of Akt pathway,thereby changing the distribution of junction proteins in Sertoli cells.Meanwhile,by down-regulating the Eps8 and EB1 expression,it can affect the arrangement of F-actin and MT,and thus regulate the barrier function of Sertoli cells.2.The spermatogenic dysfunction model in rats was accompanied by BTB functional damage and increased NC1 expression.Inhibition of MMP-9 activity could improve the aggregation and distribution of BTB connective protein and F-actin and MT in testicular tissues by reducing NC1 level,thus relieving the BTB functional damage and spermatogenic disorder caused by low dose of busulfan. |
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